Optical Biosensors Present And Future 1st

Edition Frances S Ligler download

https://ebookbell.com/product/optical-biosensors-present-and-
future-1st-edition-frances-s-ligler-2160510

Explore and download more ebooks at ebookbell.com

Here are some recommended products that we believe you will be
interested in. You can click the link to download.

Plasmonic Optical Fiber Biosensors Christophe Caucheteur Mdric Loyez

https://ebookbell.com/product/plasmonic-optical-fiber-biosensors-
christophe-caucheteur-mdric-loyez-50168824

Design Of Novel Biosensors For Optical Sensing And Their Applications

In Environmental Analysis 1st Ed Kun Yin

https://ebookbell.com/product/design-of-novel-biosensors-for-optical-
sensing-and-their-applications-in-environmental-analysis-1st-ed-kun-
yin-10805114

Optical Fiberbased Plasmonic Biosensors Santosh Kumar Niteshkumar

Subhash Agrawal

https://ebookbell.com/product/optical-fiberbased-plasmonic-biosensors-
santosh-kumar-niteshkumar-subhash-agrawal-47165916

Optical Guidedwave Chemical And Biosensors I 1st Edition Kim E

Sapsford Auth

https://ebookbell.com/product/optical-guidedwave-chemical-and-
biosensors-i-1st-edition-kim-e-sapsford-auth-4208320
Optical Guidedwave Chemical And Biosensors Ii 1st Edition Benjamin L
Miller Auth

https://ebookbell.com/product/optical-guidedwave-chemical-and-
biosensors-ii-1st-edition-benjamin-l-miller-auth-1687070

Biosensors And Biodetection Methods And Protocols Volume 1

Opticalbased Detectors 2nd Edition Avraham Rasooly

https://ebookbell.com/product/biosensors-and-biodetection-methods-and-
protocols-volume-1-opticalbased-detectors-2nd-edition-avraham-
rasooly-5880968

Smart Biosensor Technology Optical Science And Engineering 1st Edition

George K Knopf

https://ebookbell.com/product/smart-biosensor-technology-optical-
science-and-engineering-1st-edition-george-k-knopf-2347994

Spr Biosensor Based On Polymer Multimode Optical Waveguide And

Nanoparticle Signal Enhancement Johannagabriela Walter

https://ebookbell.com/product/spr-biosensor-based-on-polymer-
multimode-optical-waveguide-and-nanoparticle-signal-enhancement-
johannagabriela-walter-11054824

Optical Nonlinearities In Nanostructured Systems Carlos Torrestorres

https://ebookbell.com/product/optical-nonlinearities-in-
nanostructured-systems-carlos-torrestorres-46074278
 PREFACE

Since the birth of the field of optical biosensors, the pace of evolution of this
field has been swift. While myriad reports have appeared describing applications
and advancements in optical biosensor technology, few existing volumes are
dedicated to a synopsis of this field. Since the development of optical biosensors
mirrors the advances in the rapidly evolving telecommunications industry, we
deemed the time to be ripe for such an opus. In order to catch the wave of this
rapidly developing technology, we endeavored to focus both on the current state
of the art and on technologies that will influence tommorrow's state of the art.
We hope that this particular compendium of concepts will trigger new synapses
to foma in the brains of our readers and yield even more innovation in the years
to come. The history sections are included in order to recognize the contributions
of the giants upon whose shoulders we stand--and we thank them for their
creativity and pioneering spirit. These sections are comparatively short, not so as
to minimize such contributions, but so that this book actually gets published in a
single volume.

According to the thematic focus on Present and Furore technology, the book is
divided into two parts. In the first part, we compiled a list of the most
outstanding optical biosensor technologies, while in the second part, the editors
used their crystal ball to select the science we deem exciting and promising in
terms of potential impact on biosensors. The optical biosensor technologies
include two very different fiber optic biosensors, planar waveguides, and the
displacement flow sensors, as well as sensors based on time-resolved
fluorescence, electrochemiluminescence, surface plasmon resonance, resonant
mirrors, and interferometry. The science for future technology development
includes four different methods for producing new recognition elements (genetic
engineering of proteins, chemical synthesis, combinatorial selection of
nucleotide-based receptors, and molecular imprinting), two methods for
immobilizing receptors on biosensors (sol gels and semi-synthetic membranes),
two methods for producing very bright signals (PEBBLES and quantum dots),
and soft lithography for surface patterning and microfluidics. We have asked
leaders in each field to provide our readers with as thorough and objective a
chapter as possible; they and their colleagues have been very patient with our
nagging and nit picking and, as will be obvious to you, have put inordinant
amounts of time into providing a conscientious review of their field.

We tasked the authors to describe the underlying principles behind each

technology, enumerate the types of applications for which it has been tested,
provide their opinions about the advantages and disadvantages of their favorite

vii
Preface

biosensor (and the objectivity each has provided is admirable!), and philosophize
on the future developments using that particular biosensor. The last section is
intended to be fun for the readers as well as the authors; however, it is available
for any clever venture capitalist to peruse as well.

Finally, the editors intend this book to be a gift of gratitude to our colleagues in
this rapidly expanding field. We appreciate the open sharing of ideas, the
encouragement, and the competition that motivates us to greater effort. To work
in the field of optical biosensors, one must be curious about biochemistry,
chemistry, physics, and engineering and the possibilities ever present in the
cracks between the disciplines. While information overload is a serious threat,
boredom never is. Since it is absolutely impossible to be expert in all these
fields, it behooves us to join forces with those who are. But even more than the
ideas and accomplishments of our fellows, we delight in their personalities and
camaraderie.

Sincerely,

Fran and Chris

viii
Optical Biosensors: Present and Future
F.S. Ligler and C.A. Rowe Taitt (editors)
9 2002 Elsevier Science B.V. All fights reserved

CHAPTER 1

OPTRODE-BASED FIBER OPTIC BIOSENSORS

(BIO-OPTRODE)

ISRAEL BIRAN, PH.D. AND DAVID R. WALT, PH.D.

The Max Tishler Laboratory for Organic Chemistry

Department Of Chemistry
Tufts University, Medford, MA 02155 USA

Optrode-based fiber optic biosensors (bio-optrodes) are analytical devices incorporating

optical fibers and biological recognition molecules. Optical fibers are small and flexible
"wires" made out of glass or plastic that can transmit light signals, with minimal loss,
over long distances. The light signals are generated by a sensing layer, which is usually
composed of biorecognition molecules and dyes, coupled to the fiber end. Light is
transmitted through the optical fibers to the sensing layer where different optical
phenomena such as absorption or luminescence are used to measure the interactions
between the analyte and the sensing layer. Bio-optrodes can be used for remote
analytical applications including clinical, environmental, and industrial process
monitoring. In the last decade, due to the rapidly growing use of fiber optics for
telecommunication applications, new fiber optic technologies have been developed
resulting in high-quality and inexpensive optical fibers that can be used for bio-optrode
applications. Recent advancements in bio-optrode technologies include the development
of nanoscale bio-optrodes, enabling measurements inside single living cells, and the
development of multi-analyte and reagentless bio-optrodes. Although currently no bio-
optrodes are commercially available, it is expected that the development of advanced
bio-optrode technologies will lead to commercially available devices for various
analytical applications.
Biran and Walt

Figure 1. Schematic diagram of optrode system.

I. Principle of Operation

The word "optrode" is a combination of the words "optical" and "electrode" and
refers to a fiber optic based analytical device that can measure the concentration
of a specific chemical or a group of chemicals in a sample of interest. The basic
design of an optrode system is shown in Figure 1. The main components of an
optrode are: (a) a light source; (b) an optical fiber to both transmit the light and
act as the substrate for (c) the sensing material, which is usually immobilized to
the surface of the end face of the fiber; and (d) a detector to measure the output
light signal. Computers or microprocessors are used to control the optrode
instrumentation and are employed to analyze the output signals.

The "heart" of the optrode is the sensing element. When the sensing element
interacts with the analyte, it undergoes physico-chemical transformations that
change its optical properties. This transduction mechanism generates optical
signals that can be correlated to the analyte concentration. The optical signals are
measured by launching light from the light source through the optical fiber to the
fiber end, where the sensing element is immobilized. The same fiber (Figure 1),
or a different fiber (Figure 6), is used to guide the output light to the detector
 Optrode-based Fiber Optic Biosensors

Core(nl)

Cladding(n2) Jaclket

Figure 2. Schematic diagram of an optical fiber shows core and clad structure.

(e.g., spectrophotometer, fluorometer) where the reflected, emitted or absorbed

light is measured. Optrode biosensors or bio-optrodes are optrodes in which the
sensing elements are of biological origin. Biological sensing elements, such as
enzymes, nucleic acids, antibodies and cells, are immobilized on optical fibers
and used for specific recognition of many different analytes (Cunningham, 1998;
Kuswandi et al., 2001; Mehrvar et al., 2000; Wolfbeis, 2000). Since most
biological sensing elements and most analytes do not possess intrinsic spectral
properties, the biorecognition events are transduced to optical signals (e.g.
changes in fluorescence or absorbance) by coupling optically responsive reagents
to the sensing elements. For example, fluorescent dyes are used to label nucleic
acids and convert the biorecognition interaction between two complementary
DNA strands into a fluorescence signal. In another example, an indicator dye,
which is optically sensitive to changes in H + concentrations, is used to transduce
enzymatic activity that consumes or releases H § into an optical signal. The
signals are generated on the fiber optic face and transmitted by the optical fiber to
a remote measurement device. The small dimensions of bio-optrodes allow
measurement in very small sample volumes, which make them suitable for
various clinical applications (Meadows, 1996; Vo-Dinh and Cullum, 2000). Bio-
optrodes are also useful for different sensing applications in the industrial and
environmental fields (Rogers and Mascini, 1998; Rogers and Poziomek, 1996;
Marose et al., 1999; Mulchandani and Bassi, 1995; Scheper et al., 1996).

In this section, optical fibers, their basic characteristics, and the optical methods
used to transduce a biorecognition event to an optical signal are described. The
instrumentation employed in optrode biosensors, the biological sensing elements,
and the methods to immobilize them on the fiber optic surfaces are summarized.
Biran and Walt

l n~ L: ' 7 [Cladding ( a )

n2 . Cladding (b)

Core

(c)
[ "- - n~ "
Acceptance
cone
Core

Figure 3. Propagation of light through the optical fiber occurs when the total internal
reflection condition exists at the interface between the core, (nl), and clad, (n2) such that
nl > n2. (a) Light entering the fiber is totally internally reflected (TIR), if the light angle is
greater than the critical angle (pc. (b) Light will be partially reflected and partially
refracted, if the light angle is less then the critical angle ~oc.(c) Light will propagate in
TIR only when the entering light angle is within the acceptance cone angle (~0m)range.

1.1. O p t i c a l f i b e r c h a r a c t e r i s t i c s a n d use in b i o - o p t r o d e s

Optical fibers are small and flexible "wires" made out of glass or plastic that can
transmit light signals, with minimal loss, for long distances. Optical fibers are
remarkably strong, flexible and durable and therefore can be used in harsh and
hazardous environments. Optical fibers are non-electrical, which make them
highly suitable for applications where the presence of electric current is
detrimental (e.g., in-vivo monitoring inside a patient body). In the last decade,
due to the rapidly growing use of fiber optics for telecommunication applications,
new fiber optic technologies have been developed resulting in high-quality and
inexpensive optical fibers that can also be used for sensing applications. Optical
fibers can transmit multiple optical signals simultaneously, thereby offering
multiplexing capabilities for sensing.
 Optrode-based Fiber Optic Biosensors

Optical fibers consist of a core with a refractive index, n~, surrounded by a

cladding with a lower refractive index, n2 (Figure 2). The difference in the
refractive indices between the core and the cladding enables the core-clad
interface to effectively act as a mirror such that a series of internal reflections
transmits the light from one end of the fiber to the other as shown in Figure 3 (a).
Light undergoes total internal reflection (TIR) at the core-clad interface if two
basic conditions are fulfilled: (a) The light strikes the cladding at an angle greater
than the critical angle, (Pc,(Figure 3 (a) and 3 (b)). The critical angle is defined by
the ratio between the clad and the core refractive indices, as shown in Equation
(1):
sin (p~ - n 2 / n 1 (1)

(b) The angles of the light entering the fiber should be within the acceptance
cone as shown in Figure 3 (c). The acceptance cone angle, (am, depends on the
refractive indices of the core and the clad and also on the refractive index of the
medium from which the light enters the fiber, no.

sin(Pm = (2)
l't o

Another important parameter that defines the fiber's light collection efficiency is
the numerical aperture (NA). This parameter is related to the acceptance cone's
angle and is given by:

NA = n0sinfo m (3)

A high NA indicates a wide acceptance cone and better light gathering

capabilities of the fiber. A typical NA value for a high quality glass fiber is 0.55,
but fiber NAs as high as 0.66 or as low as 0.22 have been used for sensing.

Optical fibers are usually made out of plastic and glass and have many different
configurations, formats, shapes, and sizes. Glass fibers are the most commonly
used fibers in optrode biosensors. Glass optical fibers can transmit light in the
visible and near-infrared regions of the optical spectrum (400 n m < ~, < 700 nm)
and are therefore suitable for measuring fluorescence signals generated by most
fluorescent dyes. For applications in which light in the UV region is required,
quartz (pure silica) is used as the fiber's core material and doped silica (with a
lower refractive index) is used as the cladding material. For most fiber optic-
based biosensors, optical fibers with diameters ranging from 50 to 500 ~tm are
employed.
Biran and Walt

Figure 4. Optical fiber bundle fabrication and its use for imaging. (a) Fiber bundles are
constructed from thousands of individual single fibers that are fused together. (b)
Coherent bundles can be used for imaging (Pantano and Walt, 1995). Reprinted with
permission from the American Chemical Society.

Recently, fiber optic bundles (Figure 4(a)) comprising thousands of identical

single fibers each with a diameter of a few micrometers, were employed for bio-
optrodes. The fibers can be bundled in a coherent or random fashion. In coherent
fiber bundles, the position of each fiber on one end is identical to its position on
the other end. These fibers were originally designed for imaging applications as
shown in Figure 4(b) and are also often called "optical imaging fibers". Imaging
fibers are suitable for multi-analyte optrode biosensor design (Healey and Walt,
1995; Healey et al., 1997a; Michael et al., 1998; Steemers and Walt, 1999; Walt,
2000) since each small individual fiber in the bundle can carry its own light
signal from one end of the bundle to the other. Moreover, optical imaging fiber-
based biosensors can be used for sensing and imaging simultaneously, providing
remote spatial sensing capabilities (Walt, 1998).

1.2. Optical phenomena employed for biosensing in bio-optrodes

In bio-optrodes, dyes are coupled to the biological sensing element and transduce
the biorecognition events to an optically detectable signal. Different optical

10
 Optrode-based Fiber Optic Biosensors

phenomena, including fluorescence, luminescence and absorption, are employed

for monitoring these optical changes. In this section, the basic principles of these
phenomena and their use in bio-optrodes are described.

Fluorescence is commonly used in bio-optrodes. Fluorescence occurs when

molecules are excited at a specific wavelength and re-emit radiation at a lower
energy, i.e., a longer wavelength. The absorption of the excitation light promotes
the molecule's energy from its ground state to a higher energy state. The
molecule emits fluorescent light when it returns to the ground state. Each
fluorescent molecule has a unique fluorescence spectrum since the excitation and
emission occur only at distinct energy levels corresponding to particular
wavelengths. The characteristic fluorescence spectrum of particular molecules
allows multiple fluorescent dyes to be used simultaneously in a single analytical
assay. In fluorescence-based bio-optrodes, the fluorescence signals are measured
by transmitting the excitation light through an optical fiber and measuring the
light emission using a detector. Usually the increase or decrease in fluorescence
intensity is measured and then correlated to the analyte concentration. For
example, when a fluorescent-labeled antibody is used as the sensing element, the
fluorescence intensity is proportional to the amount of antigen (analyte) bound to
the optical fiber. One method for measuring fluorescence lifetime is frequency-
domain. In this method, sinusoidally modulated light is used to excite the
fluorescent molecule. The resulting emission light also oscillates at the same
frequency. The emission light is phase shifted (delayed) and demodulated with
respect to the excitation light because of the finite lifetime of fluorescence. The
phase shift is expressed as a phase angle from which the lifetime can be
determined using simple relationships between the modulation frequency and the
degree of demodulation. The concentration of analyte that induces changes in the
molecule's fluorescence lifetime can be determined by measuring phase angle
values (Thompson et al., 1996).

A decrease in fluorescence intensity due to quenching can also used for sensing.
In this case, the biorecognition event causes a decrease in fluorescence
(quenching) of the fluorescent reporter molecule. The fluorescence decrease is
related to the analyte concentration. For example, a dye that undergoes
fluorescence quenching when the pH decreases can be coupled to an enzymatic
reaction that converts a substrate into an acidic product and results in a pH drop.
Thus, the decrease in fluorescence can be correlated to the analyte concentration
(see also Section 1.4.1). Fluorescence quenching is also one manifestation of
another fluorescence phenomenon used for sensing in bio-optrodes -fluorescence
resonance energy transfer (FRET). This phenomenon occurs when two distinct
fluorophores are present. If the emission spectrum of one fluorophore overlaps
with the excitation spectrum of a second fluorophore, and the two fluorophores
are in proximity (<100/~ ), then the excited fluorophore (donor) can transfer
energy non-radiatively to the second fluorophore (acceptor). There are two types
of acceptors. Quenchers are acceptors that are not fluorescent and therefore

11
Biran and Walt

cause the donor simply to decrease its fluorescence emission intensity.

Acceptors can also be fluorescent dyes that accept the energy non-radiatively
from the donor, and then re-emit the energy at specific emission wavelength.
This energy transfer results in an increase in light emission by the acceptor and a
decrease in light emission from the donor. When an energy transfer pair of
fluorophores is used to label two interacting molecules (e.g., antibody-antigen,
enzyme-substrate), they can be used for sensing. Recently, both the donor and the
acceptor molecules were incorporated into single biological molecules such as
proteins (Hellinga and Marvin, 1998) and nucleic acids (e.g., molecular beacons)
(Tyagi and Kramer, 1996; Tyagi et al., 2000). When these sensing molecules are
in their native conformation, the donor and the acceptor are in proximity and
therefore low fluorescence signals from the donor are obtained. When the
molecule interacts with the analyte, conformational changes occur that separate
the donor and the acceptor molecules and cause an increase in the fluorescence
from the donor (see Section 3.3).

The most commonly used fluorescent molecules in bio-optrodes are organic

dyes. Recently self-fluorescent proteins have also been used. The sources of
these proteins are marine organisms such as the jellyfish Aequorea victoria that
produce the green fluorescent protein (GFP) (Chalfie et al., 1994). When GFP is
excited, it emits light at a lower energy and therefore at a higher wavelength.
GFP is highly fluorescent, with a quantum efficiency of approximately 80% and
is very stable to heat and pH (5.5-12). The GFP has been expressed in different
cell types (bacteria, yeast, mammalian, plant) and used as reporter gene for
different cellular events (Naylor, 1999). In order to allow monitoring of several
cellular events simultaneously, several GFP mutants have been developed each
with unique excitation and emission wavelengths. Cells expressing fluorescent
proteins, and also the purified proteins have been used for constructing different
bio-optrodes (see Sections 1.4.1 and 3.3).

Time-resolved fluorescence spectroscopy is another phenomenon used in bio-

optrodes. This method is based on the fluorescent molecule's excited state
lifetime. The light intensity emitted from a molecule excited by a short pulse of
light decays exponentially with time. The decay time pattern is unique for each
molecule and can be used for analytical purposes. Barker et al. (1999) used this
method to improve the performance of a bio-optrode for nitric oxide detection.

A different light emission phenomenon used in bio-optrodes is

chemiluminescence. In contrast to fluorescence, chemiluminescence is produced
when a chemical reaction yields an excited species that emits light as it returns to
its ground state. The use of chemiluminescence in biosensors, including fiber
optic-based biosensors, was recently reviewed (Aboul-Enein et al., 2000; Gubitz
et al., 2001). In many bio-optrodes, the chemiluminescence of luminol is used to
generate the light signal. The reaction between luminol and H2Oz produces a

12
 Optrode-based Fiber Optic Biosensors

Figure 5. Design of flow-cells incorporating bio-optrodes (Kuswandi et al., 2001).

Reproduced with permission of the Royal Society of Chemistry.

luminescence signal and is also catalyzed by certain ions or molecules (e.g.,

MnO42-, Iz, Cu2+). This reaction can be used, for example, in enzyme-based bio-
optrodes in which the enzymatic reaction generates H202 (see Section 3.3).
Enzymes such as horseradish peroxidase can also catalyze or induce a
chemiluminescence reaction by producing H2Oz. In addition, alkaline
phosphatase and 13-galactosidase can be used to label biological sensing elements
such as antibodies or nucleic acids. In the presence of a 1,2-dioxetane substrate
(Bronstein et al., 1996), these enzymes catalyze light formation proportional to
the analyte concentration. Chemiluminescence-based bio-optrodes are usually
used in conjunction with flow cells. An optical fiber with an immobilized sensing
element is placed inside the flow cell and transmits the light signals to the
detector (Figure 5).

Bioluminescence is a biological chemiluminescent reaction. Many organisms

produce bioluminescence for signaling, self-protection, mating, attracting prey
and finding food (Campbell and Sala-Newby, 1993). The bioluminescence
reaction is catalyzed by the enzyme luciferase and requires the presence of
oxygen. The bioluminescent substrate used in this reaction is called luciferin.
Different luciferin molecules are used by different organisms. For example,

13
Biran and Walt

aldehydes and flavins are used by bacteria and imidazolopyrazines are employed
by some fish and squid. B ioluminescence can be applied for analytical
measurements in two ways: (1) One can detect cellular events inside living cells
by fusing the luciferase gene (e.g., the luc gene coding for firefly luciferase or the
lux gene coding for the bacteria Vibrio fischeri luciferase) to the gene of interest.
The in-vivo activity of the selected gene can be detected by monitoring the
luminescence signal (LaRossa, 1998). (2) Alternatively, one can use purified
recombinant luciferase and synthetic luciferin substrates for ex-vivo detection
assays for analytes such as ATP, NADH and FMN (Blum et al., 1993). In bio-
optrodes, the cells or the purified enzymes are immobilized on the fiber tip and
the luminescence signals are transmitted through the fiber to the detector.

Absorption is a simpler process than fluorescence and has also been used in bio-
optrodes. Absorption is a process in which light energy is absorbed by an atom or
a molecule, promoting the molecule from the ground energy state to a higher
energy excited state. The resulting energy is dissipated non-radiatively (i.e.,
thermally) to the medium when the excited state relaxes to the ground state. The
absorbance changes are related to the concentration [C] via the Beer-Lambert
relationship:

A = log(Io/I)= e . [ C ] . l (4)

where A is the optical absorbance, and Io and I are the intensities of transmitted
light in the absence and presence of the absorbing species respectively, 1 is the
effective path length, and e is the molar absorption coefficient. In practice,
optical fibers are used to measure absorbance by transmitting light through the
fiber to the-sensing layer and measuring changes in the scattered light.
Alternatively, light is transmitted through one arm of bifurcated optical fiber to
the sensing region and reflected light signals are measured through the other arm
of the fiber (Figure 6 (b)). In a different configuration, two fibers are placed with
one fiber facing the other creating an optical cell in which the distance between
excitation and collection fiber is the pathlength.

1.3. Optrode biosensor (bio-optrode) design and instrumentation

Different bio-optrode system designs have been used and recently reviewed
(Kuswandi et al., 2001; Mehrvar et al., 2000) The design of bio-optrodes is
similar to chemical optrode design and two basic configurations are used: (a) a
single fiber is used to transmit the light from the light source to the sample region
and back to the detector, as shown in Figure 1, or (b) multiple fibers are used in
which one fiber is employed to transmit the light to the sample region and the
other fiber or fibers are used to transmit light from the sample region to the
detector, as shown in Figure 6 (a). For the second configuration, the most
common format is a bifurcated fiber. Bifurcated fibers are fabricated by fusing

14
 Optrode-based Fiber Optic Biosensors

(a) ) Lighi Sourcei

() )'------~.etec;or ]

(b)
,, , ight Source [

J ~--'--~Detector. [

(c)

Sensing layer

Figure 6. Design principle of a bio-optrode. (a) Two fibers: one carries light to the
sensing layer and one carries the signal to the detector. (b) Bifurcated fiber: the
biosensing layer is placed on the fused end of the fiber (c) The biosensing layer is placed
on the central fiber and the surrounding fibers are used to collect the light signals.

two fibers on one end leaving the other ends free. The sensing elements are
immobilized on the fused side and the other ends of the bifurcated fiber are
connected to the light source and to the detector as shown in Figure 6 (b). In a
different configuration, multiple fibers comprising one central fiber surrounded
by several fibers are employed. The central fiber carries the immobilized sensing
elements and is connected to the light source; the surrounding fibers collect the
output light signals and transmit them to the detector (Figure 6 (c)).

The light sources used for bio-optrodes should provide sufficient light intensity
within the sensor wavelength operating range. In addition, the light output should
be stable over long time periods since light fluctuations may add noise to the
measurement and reduce the sensor sensitivity. The different light sources used
in bio-optrodes and their characteristics are summarized in Table 1.

In most fiber optic biosensor systems, the light transmitted from the sensing
element (output light) is measured by using photon detection devices, which

15
Biran and Walt

Table 1. Li ;ht sources

i ii

Type Wavelength Characteristics

Ill
(nm) [] i i I Ill

Tungsten lamp IR/NIR, visible High power output, bulky, expensive,

Deuterium lamp 200-300 used together with wavelength
200-1000
....
selection device.
LEDs 470-1300 LOW power Output, high stability,
long life, robust, compact size,
inexpensive.
Laser (N2, Ar § He~ 377, ~188-568, Monochromatic, very high power
Ne) 633 output, directional, bulky, expensiv e .
Laser Diodes 800-904 High power output, long life, narrow
spectral band, inexpensive, compact
.size.

I
Table .2. Light detectors Ill l I

Detector t~'l~e Advantages Limitations

Photomultipliers (PMT) Sensitive, fast, low noise, Need t~orhigh power
internal amplification, voltage supply,
compact. destruction by over
exposure
Photodiodes (PD) Fast, robust, compact, High noise, no internal
inexpensive amplifier .
Charge-coupled devices Very sensitive, can be Slow, expensive, need
used for. imaging for a cooling system.
Avalanche photodiodes Lower noise than PD, fast, More expensive than
sensitive, can tolerate PD
intense illumination

absorb photons and convert them into electrical signals. Several photon detectors
are available as shown in Table 2.

1.4. Biological sensing elements

Bi0-optrodes are constructed by immobilizing biological recognition

components, such as enzymes, antibodies, nucleic acids, or cells to optical fibers.
In nature, interactions between biological molecules, such as receptor-ligand,
antibody-antigen or two complementaryDNA strands, are highly specific. Some
of these recognition molecules can be purified and used in fiber optic biosensors.
Moreover, by using genetic engineering, the original recognition element's
structure can be modified and designed for a specific analytical application
(Hellinga and Marvin, 1998). Biological sensing compounds can be divided into

16
 Optrode-based Fiber Optic Biosensors

two main categories based on their bioactivity: biocatalysts (enzymes and cells),
and bioaffinity molecules (antibodies, receptors, and nucleic acids).

1.4.1. Biocatalyst-based optrodes. Enzymes are proteins that selectively bind

and catalyze the conversion of a substrate to product. Enzymes are used as
sensing elements in bio-optrodes based on their ability to bind specific substrates
(e.g., the analyte) and catalyze their conversion into an optically detectable
product (Kuswandi et al., 2001). The optical signal obtained, e.g., absorbance or
fluorescence, is proportional to the product concentration and consequently, to
the analyte concentration. Products that possess intrinsically optical properties
can be measured directly, but the most common enzymatic reactions products,
such as H § ammonia, oxygen, carbon dioxide and hydrogen peroxide, do not
possess optical properties and are therefore measured indirectly by using
indicators (Wolfbeis, 1997). The indicators change their optical properties when
interacting with these products. For example, fluorescein is a pH indicator and its
emission intensity can be correlated to changes in H + concentration. Other
indicators employed in enzyme bio-optrodes were recently reviewed (Kuswandi
et al., 2001).

An interesting example that demonstrates the simple fabrication and function of

enzyme-optrodes is the one used for glucose detection based on the enzyme
glucose oxidase. Glucose oxidase catalyzes the oxidation of glucose with oxygen
to produce gluconolactone and H202.

Glucose oxidase
Glucose + 0 2 . v Gluconic acid +H202
Two approaches have been employed to determine the glucose concentration
with the enzyme: (1) measuring the amount of oxygen consumed in the
enzymatic reaction using a ruthenium complex as an indicator (Rosenzweig and
Kopelman, 1996a, 1996b) or (2) measuring the amount of H202 produced using a
chemiluminescence indicator (Marquette et al., 2000).

In many cases, a sequence of enzymatic reactions is required to detect a specific

analyte. In order to fabricate bio-optrodes for detection of such analytes, two or
three enzymes are immobilized together on the optical fiber in such a way that
sequential reactions can occur. The first enzyme catalyzes the conversion of the
analyte to a product that serves as a substrate for subsequent enzymatic reactions
that eventually convert the initial analyte to an optically detectable product
(Michel et al., 1998a, 1998b). Using this methodology, analytes that could not be
detected in a single reaction step can be detected. In addition, coimmobilizing
two enzymes can achieve signal amplification through enzyme recycling systems
as shown in Figure 7 (Zhang et al., 1997).

17
Biran and Walt

-.. - " ' . ~..iFiberfi~ . "..i..:"...."...".!ii.:I.......i.i......."ii[..i....................

. .

NADH~ S Pyruvate~ S H202

Biocatalytic
Layer

NAD
§ Lactate S L 02
~ ~ Bulk
NADH Pyruvate O~ Solution
Figure 7. Schematic diagram of signal amplification using a dual-enzyme bio-optrode.
Pyruvate is detected using lactate dehydrogenase (LDH) and lactate oxidase (LDO),
which are co-immobilized on a fiber optic tip. Pyruvate concentration is determined by
measuring NADH fluorescence. Pyruvate and NADH diffuse from the bulk solution into
the enzyme layer, LDH catalyzes the formation of lactate and NAD§ during the reduction
of pyruvate. LDO then catalyzes the regeneration of pyruvate causing additional
consumption of NADH by the LDH-catalyzed reaction. Thus, the signal obtained using a
dual-enzyme system is higher then when a single enzyme is used (Zhang et al., 1997).
Reprinted with permission from Elsevier Science.

Inhibition of enzymatic reactions can also be used as a sensing mechanism in

bio-optrodes (Freeman and Bachas, 1992). In this approach, the inhibitor is the
analyte and the measured signal is the decrease in enzymatic activity. One
example is detection of organophosphate and carbamate pesticides using an
enzyme inhibition-based optrode. The bio-optrode is based on the inhibition of
acetylcholinesterase (ACHE) by organophosphate pesticides. The enzyme is co-
immobilized together with a pH sensitive dye at the fiber's distal end. The
substrate acetylcholine is hydrolyzed by AChE causing a change in the local pH
and thereby the fluorescence signal. The inhibition of this reaction can be
correlated to the pesticide concentration in the sample (Doong and Tsai, 2001;
Hobel and Polster, 1992).

In living cells, cellular functions are carried out by enzymes that simultaneously
catalyze numerous biochemical reactions. Some enzymatic activities that occur in
cells have been applied for bio-optrode fabrication. Although enzymes can be
isolated and purified, their activity outside the cells is usually reduced compared
to their activity within the cells where they function in an optimum environment
containing all the necessary cofactors. Whole cell biocatalysts are particularly
advantageous when the detection is based on a sequence of multiple enzymatic

18
 Optrode-based Fiber Optic Biosensors

reactions. These enzymatic cascade reactions are very difficult and complicated
to accomplish ex-vivo by coimmobilizing the enzymes but are relatively
straightforward when employing whole cells. In practice, whole cells that
produce Unique or enhanced enzymatic activity and can transform the analyte
(substrate) into detectable products or cells that produce cellular responses such
as changes in oxygen consumption are immobilized on optical fibers (Preininger
et al., 1994). The methods for detecting the products in cell-based fiber optic
biosensors are similar to those employed in enzyme optrodes. In a more recent
approach, cells are genetically engineered to over-express specific enzymes
involved in the analytical measurement. An example of this approach is the use
of E. coli cells that were engineered to over-express the enzyme
organophosphorus hydrolase (OPH) on their outer cell membrane (Mulchandani
et al., 1998). This enzyme catalyzes the hydrolysis of organophosphorus
pesticides to form a chromophoric product that can absorb light at a specific
wavelength. The cell optrode is fabricated by immobilizing the cells on a
bifurcated fiber optic tip and using a photomultiplier detection system to measure
the light signals. Although the specificity of whole cell optrodes is reduced
compared to enzyme optrodes, cells are very simple to use and obtain (e.g.,
growing the cells for a few hours), and there is no need for purification steps,
which makes cell bio-optrodes inexpensive to assemble.

A different approach for sensing with whole cells, which does not directly
involve biocatalysis, is based on utilizing genetic responses and signal
transduction mechanisms in living cells (Daunert et al., 2000; Kohler et al., 2000;
Naylor, 1999). Cells may express a specific gene or set of genes when a specific
molecule (e.g. analyte) is present in the cell's environment. By fusing reporter
genes, encoding for optically detectable enzymes or proteins (e.g., luciferase, [3-
galactosidase, GFP) to the responsive gene, the genetic response is measured and
correlated to the analyte concentration. For a more detailed description of this
approach, see Chapter 10 in this book.

1.4.2. Bioaffinity-based optrodes. The natural high selectivity of antibodies,

receptors and nucleic acids make them very powerful sensing elements for
recognizing their binding partners. Such bioaffinity optrodes are used as probes
because the recognition reaction is essentially irreversible. The bio-optrode
sensing elements must be regenerated or recharged before the probe can be used
to make another measurement. In many cases, a probe-based bio-optrode
configuration involves the use of a permanent fiber optic and a disposable
sensing layer that can be placed on the fiber optic's distal end (Figure 8).

lmmuno/receptor optrodes are a major group of bioaffinity fiber optic biosensors

based on transducing antibody-antigen (analyte) interactions into an optical
signal that is proportional to the antigen concentration. Monoclonal antibodies
that can recognize a specific antigenic epitope region (i.e., a specific spatial

19
B iran and Walt

Figure 8. Configuration of probe-based bio-optrodes with disposable biosensing

elements. (a) Biorecognition sensing molecules immobilized on membrane, which is
held by a screw cap on the optical fiber tip. (b) Disposable glass slide with gel entrapped
enzyme. (c) Nylon membrane, with immobilized sensing molecules, attached to the fiber
using an O-ring (Kuswandi et al., 2001). Reproduced with permission of the Royal
Society of Chemistry.

structure on the antigen molecule) or polyclonal antibodies that recognize

different antigenic epitopes are used in immuno-optrodes. Several detection
schemes are employed; the simplest scheme involves the detection of
intrinsically fluorescent analytes such as polynuclear aromatic hydrocarbons
(PAHs) (Vo-Dinh, et al., 2000). Antibodies are immobilized on the fiber surface
and a fluorescence signal is obtained when the analyte (antigen) binds to the
optrode's surface as shown in Figure 9(a).

A competition assay is a more generalized detection scheme that can be applied

to any antibody antigen pair. The detection is based on competition for the
antibody binding site between the antigen present in the sample (analyte) and an
externally added fluorescent-labeled antigen as shown in Figure 9 (b). A known
concentration of fluorescent-labeled antigen is added and captured by an
antibody, which is immobilized on the optical fiber surface. The fluorescence
signal obtained is measured and set as the initial signal. To perform an analysis,
the same fluorescent-labeled antigen concentration is mixed with a sample
containing an unknown antigen concentration. When this mixture is analyzed
using the bio-optrode, the resulting fluorescence signal obtained is lower than the
initial signal because of competition with the labeled antigen in the sample. The
relative decrease in the initial signal is proportional to the analyte concentration
in the sample. Using this detection scheme, bio-optrodes for the detection of
different analytes have been developed (Wittmann et al., 1996; Zhao et al.,
1995).

20
 Optrode-based Fiber Optic Biosensors

(a)

(b)
Optical fiber ~-~ Optical

(c)
Optica Optical fiber

' ~ Self-fluorescingantigen 0 Antigen

Antibody ~ Fluorescentdye
,.

Figure 9. Schematic principle of immuno bio-optrodes. (a) Detection of intrinsically-

fluorescent molecules using immobilized antibodies. (b) Competition assay using a
fluorescent-labeled antigen. (c) Sandwich immunoassay using an immobilized antibody
and a fluorescent-labeled antibody.

The preferred detection scheme is the sandwich immunoassay, which involves

the use of two antibodies. The first antibody is immobilized to the fiber and used
to capture the antigen while a second antibody, which is labeled by a fluorescent
dye or enzyme, is used to generate the signal (Figure 9(c)).

The competition and sandwich assays require using labeled antigens or

antibodies. Fluorescent molecules and enzymes are employed for labeling using
different chemistries (Wortberg, 1997). Very low concentrations of enzymes can
be detected based on their enzymatic activity. The enzymes used for labeling,
such as alkaline phosphatase, catalyze the conversion of a non-fluorescent
substrate to a fluorescent product and can be detected by monitoring the
fluorescent signal generated (Michael et al., 1998). Other enzymes, such as
horseradish peroxidase, can catalyze chemiluminescence reactions and are
detected by monitoring the emitted light signals (Aboul-Enein et al., 2000; Diaz
et al., 1998; Gubitz et al., 2001; Spohn et al., 1995). Enzyme labeling is more
sensitive than fluorescent dye labeling since the signal is amplified by the
enzymatic reaction. Another new technology to increase the labeling efficiency

21
Biran and Walt

Figure 10. Principle of DNA fiber optic biosensors. (a) Single strand DNA probe
molecules, with a sequence complementary to the target DNA sequence, are immobilized
onto the fiber. (b) The fluorescent-labeled sample DNA molecules are first dehybridized
and the fiber is dipped into the sample solution. (c) After hybridization, the
complementary strands of the target DNA are attached to the probe DNA on the fiber and
a fluorescence signal is obtained.

of biological molecules was recently proposed and will be discussed in Chapter

17.

Nucleic acid-based optrodes are the second major group of bioaffinity-based

optrodes. Nucleic acid base pairing is used as the sensing mechanism in bio-
optrodes for nucleic acid detection. The presence of a specific DNA sequence,
the "target", among millions of other different sequences is detected by
hybridization to its complementary DNA sequence, the "probe", which is
immobilized on the optical fiber, as shown in Figure 10. In a typical assay, the
target DNA is first amplified and fluorescently labeled using fluorescent primers
and the polymerase chain reaction (PCR). The resulting fluorescent double
stranded DNA molecules are dehybridized (usually by heating) (Figure 10 (b))
and then allowed to rehybridize (by cooling) to the single strand DNA probe
molecules immobilized on the fiber surface (Figure 10 (c)). The excess DNA
molecules are washed away, and if the complementary target DNA sequence is
present in the sample, a fluorescence signal is detected (Ferguson, et al., 1996).
For example, the target sequence can be a unique sequence found only in specific

22
 Optrode-based Fiber Optic Biosensors

pathogenic bacteria (Iqbal et al., 2000; Pilevar et al., 1998). The target DNA can
be easily extracted from water, wastewater or clinical samples, and the presence
of pathogenic microorganisms can be determined by the bio-optrode. Recently,
new groups of nucleic acid molecules, such as aptamers (Lee and Walt, 2000)
and molecular beacons (Liu et al., 2000; Steemers et al., 2000), were
incorporated as sensing molecules into bio-optrodes. These different DNA
sensing schemes can be multiplexed by fabricating an array of hundreds to
thousands of probes as will described later in Section 3.2.

1.5. Sensing element immobilization

Immobilization of sensing biomolecules to the optical fiber is a key step in bio-

optrode development. A good immobilization method should be simple, fast and
durable but, more importantly, it should be gentle so the biological molecule
being immobilized can retain its biochemical activity. In addition, biological
recognition elements are often coimmobilized together with indicator dyes, so
that ideally the immobilization method should be suitable for both molecules. In
some cases, the recognition compounds are immobilized directly to the optical
fiber surface. Alternatively, the molecules are first immobilized on membranes,
such as cellulose acetate or polycarbonate that are later physically attached to the
optical fiber (Figure 8). There are three main methods for immobilizing a
biological sensing compound: adsorption/electrostatic, entrapment, and covalent
binding. A schematic representation of these methods is shown in Figure 11.

Adsorption immobilization methods involve adsorbing the sensing material onto

a solid surface or polymer matrix. Sensing materials can be adsorbed directly on
the fiber optic end. This immobilization method is very simple; however the
adsorbed molecules tend to gradually leach from the solid support, decreasing
sensing performance and/or lifetime. In order to overcome leaching problems, the
solid support surface may first be modified with complementary functional
groups. For example, a hydrophobic surface can be prepared to immobilize a
hydrophobic species. Electrostatic interaction can also be employed for
immobilization. This immobilization scheme is based on interaction between
oppositely charged molecules. For example, an optical-fiber surface can be
coated with a positively charged layer (i.e., using poly-L-lysine) that interacts
electrostatically with negatively charged recognition molecules (Figure 11 (a)).
The electrostatic immobilization method is very easy and highly reproducible but
may be affected by changes in the medium pH or by changes in other ion
concentrations.

Entrapment immobilization involves physical entrapment of sensing bio-

molecules within a porous matrix (Figure 11 (b)). The biomolecules are
suspended in a monomer solution, which is then polymerized to a gel causing the
molecules to be entrapped. Such polymers can be either thermally or

23
Biran and Walt

Figure 11. Schematic diagram of three different immobilization techniques employed in

biooptrodes. (a) Absorption/electrostatic. (b) Entrapment. (c) Covalent immobilization.

photochemically initiated and attached to the fiber surface by dip-coating

procedures (Healey et al., 1995). The immobilized molecules usually do not
leach out of the matrix and can retain their biochemical activity. Polyacrylamide
gels are most commonly used for entrapment immobilization, although agarose
and calcium alginate gels have also been used (Polyak et al., 2001). One
important limitation of this approach is the slow diffusion rates of the analytes
and products through the immobilization matrix, which increases the bio-optrode
response time.

Optically transparent sol-gel glasses are also used for biological sensing molecule
entrapment as described in Chapter 14 (Dunn et al., 1998, 2001; Jordan et al.,
1996). Sol-gel glasses are produced by hydrolysis and polycondensation of
organometallic compounds, such as tetraethyl orthosilicate (Si(OCH3)4). The
sensing biomolecules are added to the reaction mixture during the formation of
the sol or gel. Sol-gel glasses prepared by this method contain interconnected
pores formed by a three-dimensional SiO2 network. As a result, the biomolecules
and dyes are trapped but small analytes can readily diffuse in and out of the
pores. The main advantages of the sol-gel glass immobilization method are the
chemical, photochemical and mechanical stability of the immobilized layer.

24
 Optrode-based Fiber Optic Biosensors

Disadvantages of sol-gel glass immobilization are the slow response times in

aqueous media and the fragility of thin sol-gel glass films compared with
polymer films.

Functional groups in the sensing biomolecules can be covalently bound to

reactive groups on the surface of optical fibers allowing robust immobilization
(Figure 11 (c)). The fiber surface can be chemically modified using silanization
reactions (Weetall, 1993). For example, the tiber surface can be aminosilanized
to form amine functional groups on the fiber surface followed by reaction w i t h -
COOH groups on the enzyme or antibody. Amine-modified surfaces can also
covalently bind to the biomolecule's amine groups using bifunctional cross-
linkers such as glutaraldehyde. Covalent immobilization methods are usually
more complicated and time-consuming compared with the other immobilization
techniques, but are very reliable since the biomolecules and dyes are not likely to
leach out. It should be noted that covalent binding might change the biomolecule
activity. In some cases, if the binding occurs at crucial sites (e.g., an enzyme
active site or an antibody binding site), activity can be lost completely. To avoid
such inactivation, substrate, inhibitors and other effectors are often included in
the immobilization medium to protect the active or binding site of the
biomolecules. In recent years, new techniques have been developed which enable
the immobilized molecule's orientation on the sensing surface to be controlled
resulting in an increase in the immobilization efficiency (Sackmann, 1996).

A more generalized and widely used binding method involves the use of avidin-
biotin chemistry (Wilchek and Bayer, 1990). The fiber surface can be modified
with biotin groups and bind avidin-modified biomolecular conjugates or vice
versa. This method is very attractive since many biotin -~ or avidin-labeled
enzymes, antibodies and nucleic acids are commercially available.

2. History

Optical fiber-based biosensors evolved from chemical optrodes. The first optical
fiber-based chemical sensor was developed by Lubbers and Opitz (1975). Their
device was designed to measure CO2 and 02 and was used in biological fluids. A
few years later, biological molecules were coupled to the optical fiber-based
chemical sensors and bio-optrodes were formed. One of the first bio-optrodes
involved coupling the enzyme glucose oxidase to an Oz optrode to fabricate a
glucose biosensor (Arnold, 1985). In the following years, many bio-optrodes
with different recognition molecules were developed and reported in several
books (Blum et al., 1994; Wolfbeis, 1991), and reviews (Aboul-Enein et al.,
2000; Fraser, 1995; Mehrvar et al., 2000; Rabbany et al., 1994; Wolfbeis, 2000).
Although the bio-optrode basic configuration has not changed much from the one
proposed by Lubbers and Opitz (1975), new types of optical fibers, optical
instruments, biorecognition molecules and indicators have been integrated into

25
Brian and Walt

bio-optrodes. These materials, combined with new immobilization techniques

and advanced optical approaches, led to the development of more sophisticated,
selective and sensitive bio-optrodes. Advances in two fields influenced bio-
optrode development in the last decade. First, development of new fiber optic
technologies that were developed for telecommunication applications. Second,
advances in molecular biology techniques allow specific biorecognition
molecules to be designed. Integration of technologies from these two fields has
led to the development of advanced bio-optrode technologies such as multi-
analyte bio-optrodes, reagentless bio-optrodes and nano bio-optrodes.

3. Advanced Bio-Optrode Technologies and Applications

In this section, a few examples of new bio-optrode technologies and applications

will be described. Although many novel and interesting papers related to bio-
optrode developments have been published in recent years, we focus here on a
few examples that emphasize the diversity of existing bio-optrode technologies.
In addition, a few examples of bio-optrode applications in the industrial,
environmental and clinical fields will be described.

3.1. Nano bio-optrodes

One of the most exciting advances in bio-optrode development is the

miniaturization of sensors to submicron dimensions. Nanotechnology facilitates
research in this field and leads to development of new nano bio-optrodes (Cullum
and Vo-Dinh, 2000). The main importance of such biosensors is their ability to
monitor biomolecule concentrations inside a single living cell and thereby
expand our knowledge about complex intracellular process.

In order to prepare nano bio-optrodes, optical fibers a few nanometers in

diameter are fabricated. The fabrication process involves pulling optical fibers
with an initial diameter of a few microns using a modified micropipette puller
optimized for optical fiber pulling. After pulling, tapered fibers are formed with
typical distal end (tip) diameters of 20-80 nm. This technique was used by
Kopelman and coworkers to make a nano fiber optic chemical sensor for
monitoring intracellular pH inside living cells (Tan et al., 1992). Changes in pH
were measured by immobilizing a pH sensitive dye to the fiber tip. The same
design was used to prepare an enzyme-based nano bio-optrode for nitric oxide
detection (Barker et al., 1998). Fluorescently labeled cytochrome c', which
undergoes conformational changes in the presence of NO, was immobilized to
the fiber tip. Changes in NO concentrations were correlated to changes in the
energy transfer between cytochrome c' and the fluorescent dye.

26
 Optrode-based Fiber Optic Biosensors

Figure 12. Nano bio-optrodes. (a) Fabricating a nano fiber optic tip. An optical fiber is
heated and pulled and a tapered end with submicron diameter is formed. The tapered
fiber side walls are then coated with a thin metal layer, using thermal evaporation, in
order to prevent excitation light leakage. Biorecognition molecules can be immobilized
on the fiber tip (Vo-Dinh, et al., 2000). Reprinted with permission from Nature
Biotechnol. (b) Scanning force micrograph (SFM) of nano fiber (Vo-Dinh, et al., 2001).
Reprinted with permission from Elsevier Science.

An antibody-based nano bio-optrode for the fluorescent analyte benzo[a]pyrene

tetrol (BPT) was also fabricated for detection inside a single living cell (Vo-Dinh
et al., 2000). The nano bio-optrode was prepared by coating the tapered fiber's
outside walls with a thin silver, gold or aluminum layer using a vacuum
evaporator as shown in Figure 12 (a). In this system, the fiber is held at an angle
relative to the metal vapor, resulting in a coating on the side of the fiber and
leaving the tip uncoated. This coating prevents light leakage from the fiber's
walls and helps to get maximum light intensity to the fiber tip. The fiber's
uncoated tip surface was then silanized in order to covalently attach anti-BPT
antibodies. The final nano bio-optrode tip diameter was 200 to 300 nm. Bio-
optrodes of this size have several advantages over larger bio-optrodes including
fast response time and higher sensitivity. Using BPT nano bio-optrodes, BPT
concentrations as low as --300 zeptomoles were detected (Vo-Dinh et al., 2001).

27
Biran and Walt

Figure 13. Measurements inside a single live cell using a nano bio-optrode. (a) The
optical measurement system. (b) A nano bio-optrode inside a single cell (Vo-Dinh et al.,
2001). Reprinted with permission from Elsevier Science.

The optical measurement system used with the nano bio-optrode is shown in
Figure 13(a). Laser light is transmitted through the fiber and used to excite the
captured BPT molecules. Changes in fluorescence signals due to the presence of
bound BPT molecules are transmitted through the microscope objective and
measured using a PMT. Using this experimental set-up, BPT molecules inside
single living cells were measured. The fiber's tip was inserted into the cell
(Figure 13(b)) and incubated for 5 minutes inside the cells to allow the antibodies
to bind the antigen (BPT). The fiber was then removed from the cell and the
fluorescence signal obtained from the bound BPT was immediately measured.
Concentrations as low as 9.6 x 1011M were measured inside the cells.

The ability to measure concentrations of specific analytes inside single living

cells with nano bio-optrodes can lead to a better understanding of many cellular
processes such as transport mechanisms through cellular membranes, signal
transduction pathways, complex enzymatic reactions and even gene expression.

28
 Optrode-based Fiber Optic Biosensors

3.2. Multi-ana|yte sensing

One of the main challenges of any sensor device is to detect several analytes
simultaneously. Multi-analyte sensing is important for clinical, environmental,
and industrial analysis. For example, measuring the presence of proteins,
antibodies, DNA sequences, antibiotics, viruses and bacteria in single blood
samples can provide physicians with rapid and comprehensive information about
a patient's medical condition. Several approaches have been described for multi-
analyte bio-optrode fabrication (Anderson et al., 2000; Healey et al., 1997a; Li
and Walt, 1995; Michael et al., 1998).

The conventional approach to preparing multi-analyte sensors is to simply bundle

multiple individual optical sensors. In this approach to multi-analyte sensing,
several optical fibers are assembled, each containing a different immobilized bio-
recognition molecule on a single fiber bundle. This approach was used to
fabricate multi-analyte biosensors for detecting different DNA target sequences
simultaneously (Ferguson et al., 1996). Eight optical fibers, each with a different
immobilized DNA probe, were bundled together as shown in Figure 14 (a). The
bundled fiber's distal end was inserted into the sample solution containing a
fluorescein isothiocyanate -labeled oligonucleotide with a sequence
complementary to one of the probe sequences. The fluorescence signals were
measured from the fiber's proximal end. Figure 14 (b) shows that when only one
target sequence is present, a signal is obtained only from the fiber (bright signal)
that contains the complementary probe sequence, while the rest of the fibers in
the bundle do not respond. When several target sequences were present, signals
from several fibers carrying the complementary probes were observed (Figure 14
(b)). In different work, the specificity of this approach was demonstrated (Healey
et al., 1997b). Two probes were prepared, one that was complementary to the H-
Ras oncogene sequence and a second probe containing a similar sequence but
with a single base-pair mismatch. When the hybridization reaction was
performed at low temperature, both sequences hybridized to the probe, but at
high temperature, only the wild type sequence hybridized. This experiment
shows that these sensors can be used for point mutation detection.

The same sensor configuration can be applied for different sensing elements such
as antibodies, enzymes or whole cells. This approach, theoretically, is not limited
in the number of individual fibers (each with a different sensing chemistry) that
can be used simultaneously; however the array size grows as more sensing
elements are added.

An alternative approach involves fabrication of discrete sensing regions, each

containing different bio-sensing elements, at precise spatial locations on an
imaging fiber's distal end (Figure 15 (a)). The sensing regions are formed using
photopolymerization techniques (Pantano and Walt, 1995). The imaging fiber's
proximal end is first prefunctionalized with a polymerizable silane. The fiber is

29
Biran and Walt

Figure 14. Multianalyte bio-optrode for oligonucleotide detection. (a) Schematic

diagram of bio-optrode setup. Individual optical fibers, each with a specific immobilized
oligonucleotide probe sequence are bundled together. The fiber's distal end is incubated
with the sample and the signals obtained at the proximal end are measured using a CCD
detector. (b) Fluorescence images acquired after incubating the multianalyte bio-optrode
in solutions containing different target sequences. Image F show the bio-optrode response
to the presence of three different targets in the sample (Ferguson, et al., 1996). Reprinted
with permission from Nature Biotech.

then dipped into a solution containing monomer, cross-linker, indicators,

photoinitiator and the sensing biomolecules. Using a pinhole, light is focused
onto a small area (-30 ~tm in diameter) on the imaging fiber's proximal end.
Light travels through the imagin fiber, from the illuminated are at the proximal
end to the distal end. At the distal end, the light activates a photinitiator and the
polymer layer is formed only at the illuminated area (Figure 15(b)). For the
formation of the next sensing polymer, light is focused on a different area at the
proximal end and the fiber's distal end is dipped into a polymerization solution
containing different sensing biomolecules.

Initially this approach was used to fabricate a multi-analyte sensor for pH, CO2
and 02 by forming sensing regions with different fluorescent dyes on a single
optical imaging fiber face (Ferguson et al., 1997). Based on this initial work,
multi-analyte biosensors for detecting penicillin and pH were developed (Healey
and Walt, 1995; Healey et al., 1997a). This sensor incorporated two sensing
regions; in one region the enzyme penicillinase was immobilized together with a
pH indicator, and in the second region only the pH indicator was immobilized. In
the presence of penicillin, the penicillinase activity results in the formation of H §
and therefore a decrease in the local pH in the polymer's microenvironment. By
simultaneously monitoring pH changes in both sensing regions (with and without

30
 Optrode-based Fiber Optic Biosensors

Figure 15. Multianalyte bio-optrode with different biosensing elements immobilized in

polymers attached to an imaging fiber. (a) Setup of photopolymerization procedure used
to fabricate the bio-optrode. Reprinted from Pantano and Walt (1995) with permission
from the American Chemical Society. (b) Scanning force micrograph of immobilized
sensing polymer on an imaging fiber (Ferguson et al., 1997). Reprinted with permission
from Elsevier Science.

the enzyme), the changes related to the enzymatic activity can be discriminated
from pH changes in the bulk solution. Thus, this dual sensor is able to detect
penicillin and can account for changes in the solution pH (Figure 16). A similar
approach was used to fabricate glucose and 02 biosensors. The enzyme glucose
oxidase was used and the depletion of 02 in the presence of glucose was
monitored (Li and Walt, 1995). A separate sensor for 02 was also prepared on the
same imaging fiber. When the glucose sensor signals were compared with the
signals obtained from the sensing region that contained only the 02 indicator, the
concentration of glucose could be determined. Both biosensors can be used to
determine the analyte concentrations in different environments. In addition, they
can provide information about both the biochemical analytes and pH or 02
concentrations using a single imaging optical fiber. A possible future application
for such biosensors may be for in-vivo multi-analyte sensing, where early
changes in drug levels, glucose, 02, and pH are important.

31
B iran and Walt

Figure 16. Imaging fiber-based penicillin and pH bio-optrode. (a) Response of bio-
optrode, similar to the one described in Figure 15, with penicillin-sensitive polymer
regions (containing the enzyme penicillinase) and pH-sensitive polymer regions. When
the penicillin concentration is increased, only the fluorescence intensity from the
penicillin-sensitive polymer regions increases. (b) Bio-optrode response to penicillin
(solid squares) and pH (empty squares) are shown in the left plot. The difference between
the buffer pH and the microenvironmental pH at the penicillin-sensitive polymer is
shown in the right plot (Healey and Walt, 1995). Reprinted with permission from the
American Chemical Society.

In both of these approaches (sensor bundling or photopolymerization), when

more then twenty optical fibers or polymer regions are required, the bundle of
fibers becomes too big or the photopolymerization protocol becomes
complicated. A new approach that overcomes this limitation was recently
proposed (Michael et al., 1998; Walt, 2000). This approach is based on using the
unique characteristics of optical imaging fibers (see Section 1.1). Imaging fibers
consist of thousands of optical fibers coherently bundled together, with each
individual fiber maintaining its ability to carry its own light signal from one end
of the fiber to the other. Thus, by attaching a sensing material to the individual
fiber's distal end, an array of thousands of sensing elements can be constructed
on the tip of a single imaging fiber array. In practice, microwells are fabricated
on the end of each individual fiber by selectively etching the fiber cores. This

32
 Optrode-based Fiber Optic Biosensors

Figure 17. High-density multianalyte bio-optrode composed of microsphere array on an

imaging fiber. (a) Scanning force micrograph (SFM) of microwell array fabricated by
selectively etching the cores of the individual fibers composing the imaging fiber. (b) The
sensing microspheres are distributed in the microwell. (c) Fluorescence image of a DNA
sensor array with ~ 13,000 DNA probe microspheres. (d) Small region of the array
showing the different fluorescence responses obtained from the different sensing
microspheres (Walt, 2000). Reprinted with permission from the American Association
for the Advancement of Science.

process results in the formation of a high density microwell array on the imaging
fiber tip as shown in Figure 17(a). The sensing elements are prepared by
immobilizing fluorescent indicators and/or biorecognition molecules to the
microsphere surfaces. The microspheres and microwells are matched in size such
that the microspheres can be distributed into the microwells to form an array of
sensing elements (Figure 17(b)). When different biorecognition molecules are
immobilized on different microspheres, the array can be used to detect multiple
analytes. A CCD detector is used to monitor and spatially resolve the
fluorescence signals obtained from each microsphere (Figure 17(c) and (d)).
Imaging and data analysis software are used to calculate the analyte
concentrations.

These sensor arrays are prepared by randomly distributing the microspheres into
the wells. In order to allow multi-analyte sensing, the location of each sensing
microsphere must be determined. The microsphere registration process involves

33
Biran and Walt

Figure 18. Randomly ordered array bio-optrode. (a) Schematic representation of the
biorecognition elements immobilized on different sets of encoded microspheres (AP-
alkaline phosphatase). The microspheres were encoded using three different ratios of two
fluorescence dyes: indodicarbocyanine (DilC) and Texas red cadaverine (TRC), both
dyes are excited at 577 nm and emit at 670 nm and 610 nm respectively. (b) The three
rnicrospheres types are mixed and randomly distributed into the microwell array.
Fluorescence responses in the presence of the AP fluorogenic substrate, avidin-FITC and
biotin-FITC are shown on the top images. The identity of each microsphere was
determined by calculating the emission ratio 670 nm/610 nm obtained using 577 nm
excitation light (bottom images) (Michael et al., 1998). Reprinted with permission from
the American Chemical Society.

using one of several encoding/decoding schemes. When the microspheres are

prepared, each type of microsphere is modified such that it carries a unique
optical marker in addition to the biorecognition element. This marker can be a
fluorescent dye or a combination of several different fluorescent dyes. Different
markers are used for the different microsphere types, allowing each of the
microspheres carrying a certain type of biomolecule to be encoded with a unique
optical signature. For example, as shown in Figure 18, three types of
microspheres were prepared by immobilizing the enzyme alkaline phosphatase to
one group of microspheres, avidin to the second group, and biotin to the third
group (Figure 18(a)). Each type was encoded with different concentrations of

34
 Optrode-based Fiber Optic Biosensors

Figure 19. Molecular beacons (MB) structure. (a) The hairpin structure is formed due to
the complementary sequences near the 3' and 5' ends. The single strand "loop" contains
the probe sequence. In this configuration, the fluorophore and quencher are in proximity
and therefore no fluorescence signal is produced. (b) When a target sequence binds, the
MB structure changes causing separation of the fluorophore and quencher resulting in a
fluorescence signal change.

two fluorescent dyes. When the three microsphere types were mixed and
randomly distributed into the microwell array, their location could be determined
by applying the appropriate excitation and emission wavelengths to establish the
different fluorescent markers on each bead. This biosensor was used for multi-
analyte detection of fluorescein diphosphate, biotin-FITC and avidin-FITC, as
shown in Figure 18(b). For each analyte, several different microspheres
produced fluorescence emission signals, indicated by the bright spots on the array
images. These images demonstrate two main advantages of this technology. First,
the presence of replicates of each microsphere type provides statistically
significant results and reduces the possibility of both false negatives and false
positives. Second, averaging signals from many identical individual sensing
elements results in higher signal/noise ratios. This multi-analyte biosensor design
was also used to develop a DNA biosensor with the ability to detect 25 different
fluorescently labeled DNA sequences simultaneously (Ferguson et al., 2000).
Another biosensor, comprising microspheres with different immobilized
molecular beacons, was used to detect three different unlabeled DNA sequences
(Steemers et al., 2000). Recently, microspheres with immobilized antibodies

35
Biran and Walt

were used for simultaneous detection of the clinically important drugs digoxin
and theophylline (Szurdoki et al., 2001).

Multi-analyte bio-optrodes are in the first stages of research and development.

Due to their importance for many analytical applications, it is expected that
research efforts will continue to advance the capabilities of such sensors.

3.3. Reagentless bio-optrodes for homogeneous assay

One limitation common to many bio-optrode technologies is the need to add

external reagents to the analytical assay. For example, when antibodies are used
as recognition molecules in a sandwich assay, there is a need to add secondary
labeled antibody in order to measure the analyte concentration (Figure 9 (c)). The
same requirement applies to a competition assay where a labeled antigen is used
(Figure 9 (b)). Most nucleic acid bio-optrodes are based on pre-labeling the target
sequence with fluorescent dye. The necessity to add reagents complicates the
assay procedure and limits the acceptance of bio-optrodes as standard and simple
analytical tools. Therefore, many research efforts have concentrated on
developing bio-optrodes for "mix and measure" assays where no reagents are
added. In this section, several approaches for reagentless (also called
homogeneous) bio-optrode fabrication will be described.

One approach for reagentless bio-optrode fabrication is based on monitoring

conformational changes in the biorecognition molecule following analyte
binding. The conformational changes are usually detected using FRET as the
transduction mechanism. In one example, molecular beacons (MB) wereused to
detect unlabeled DNA sequences (Steemers et al., 2000). Molecular beacon
structures consist of single stranded DNA in a hairpin configuration with a
fluorophore and quencher attached to opposite termini (Tyagi and Kramer, 1996).
The molecule's 3' and 5' ends are complementary to one another and form the
hairpin structure. The probe sequence, which is complementary to the target
sequence, is located in the center (Figure 19(a)). In the absence of target, the
fluorophore and quencher are within the requisite energy transfer distance,
resulting in fluorescence quenching (Figure 19(a)). Upon target binding, a
conformational change occurs, the hairpin separates (denatures) and the
fluorescence signal increases (Figure 19(b)). Using an imaging fiber-based MB
bio-optrode, three different sequences from mutant genes related to cystic
fibrosis were simultaneous detected (Steemers et al., 2000). The multi-analyte
imaging fiber-based bio-optrode was prepared as previously described in Section
3.2. Each type of MB probe was immobilized to beads that were encoded with
unique optical signatures. The resulting three types of beads were randomly
distributed into a microwell array and used for the analysis of three different
target sequences simultaneously.

36
 Optrode-based Fiber Optic Biosensors

W 0 .

i .
. . .

.
. .

.
. . .

.
. .

.
. .

.
. . .

.
. .

.
. . .

.
. .

.
. . .

.
. .

.
. . .

.
. .

.
. . . . . . . . . . . . . . ._ .~.. - -_ _:, _. _ - ....

8
-5
N \ 2 0 0 nM Co
~Q

o %
CM
-IO
%
I'--
%
%
-15
J
I~uM H64C- CY3-a~poCA %
%
FIBER: 20OHM CORE SILICA 'o 2,,uM Co
-20 5% GEL, 0.7ram THICK
EXC514nm EM .57Ohm+

-25t .... i _ J __n ..... n ....... I , - I ......

0 t Z 3 4 5 6
TtME a MINUTES

Figure 20. Reagentless Co 2§ bio-optrode. The enzyme carbonic anhydrase was labeled
with a fluorescent donor and immobilized to the fiber optic distal end. When Co 2+ binds
to the enzyme, the donor's fluorescence is quenched and the signal decreases (Thompson
et al., 1996). Reprinted with permission from Elsevier Science.

In a similar manner, donor and acceptor molecules can be incorporated into

proteins and used as reporters for substrate binding events. In one approach, the
enzyme carbonic anhydrase, which binds metal ions with high affinity and
selectivity, was used to fabricate Zn 2§ Co 2§ and Cu 2§ bio-optrodes (Thompson et
al., 1996; Thompson and Jones, 1993). Donor molecules, such as Cy-5 or Cy-3
dyes, were bound to primary amines in the protein, using N-hydroxysuccinimide
esters of the dyes as modification reagents. The acceptor molecules in this case
were the Co 2+ and Cu 2§ analytes themselves, which exhibit weak d-d absorbance
bands at long wavelengths. Thus, upon analyte binding, a decrease in the donor
fluorescence was observed. The decrease was measured by monitoring the time-
dependent phase angle change at a fixed frequency upon binding of the metal ion.
Results for two different concentrations of Co 2§ are shown in Figure 20. The
fiber configuration included an entrapped enzyme in a polyacrylamide layer
immobilized to the tip of an optical fiber.

37
 Biran and Walt

(a) ct)P ...(a.~!...

AMP . . . . . . . . . . . . . . = ADP AND/OR 2 A D P ~ - - - ~ - --

.....~ - +
adenylLate kinase ~enylate. kinase ATP
-'~f~;--
phosphoc'zcadnc cz'~atinc

creatine kinase

AI'P + l~iferin + O 7 ----i-u~cifera~.-7---~ oxylur + AMP § PPi + CO z

lisht (~.,,~ ,~ 560 nm)

(b)
. . . . r . - ,.. ~,,
~me I

t20
0
: ........ ,,%- . . . . . .*.,.--,-,., ..%.:;.*-,.**.,,,-'.+.+ . . . . R
...1
-2SD o
o

.St

ff
1'l'~al= perk-",.l : 2.~ lurers
:,= 3"2
m e a n valttt= f ~ ) : 1(15, O ]R.LU
l.mmlllnJ d~vlmion (SD) : 5 . 6 3 ;RI.,IJ
~f e l i n e m ~ dnv~.|cm : 5.3r qS

O . . . . . . . .
O 4 ~ i~ t6 20 24 28 32 36
Number o4' a s s a y s

Figure 2 I. Reagentless bio-optrode for AMP, ADP and ATP detection. (a) Schematic of
the enzymatic reactions employed for the measurements. (b) Repetitive measurements of
ATP using controlled released of luciferin from acrylic microspheres. The light intensity
was measured after each injection of 25 pmol ATP. The self-contained bio-optrode
reproducibility over three hours (32 repetitive injections) is shown (Michel et al., 1998a).
Reprinted with permission from Elsevier Science.

A related approach for fabricating reagentless enzyme-based biosensors is based

on transducing conformational changes occuring upon substrate binding into
FRET signals. Proteins such as calmodulin, maltose binding protein and
phosphate binding protein undergo conformational changes upon substrate
binding and were used to prepare such biosensors (Hellinga and Marvin, 1998).
Using genetic engineering, two FRET fluorescent groups (acceptor and donor)
are bound to two different cysteine residues that are spatially located such that
conformational changes, due to analyte binding, result in a FRET signal change.

38
 Optrode-based Fiber Optic Biosensors

A different example for a reagentless enzyme-based bio-optrode was recently

described (Michel et al., 1998a). The sensor was designed to detect the three-
adenylate nucleotides (ATP, ADP, AMP) using a three-enzyme reaction
sequence. Three enzymes were used: adenylate kinase, creatine kinase and
luciferase. The enzymes were compartmentalized in such a way that the product
of the first reaction would be accessible to serve as the substrate for the
subsequent reactions shown in Figure 21 (a). The final indicator reaction for all
three analytes involves the luciferase reaction. In previous bio-optrode designs
the cosubstrate for this reaction, luciferin, was externally added to the flow cell.
In the new bio-optrode design, luciferin was incorporated into acrylic
microspheres. When the microspheres were immobilized together with the
enzymes on the fiber surface they slowly released the luciferin allowing
continuous detection for 3 hours (Figure 21 (b)). This approach is generic for the
controlled release of cosubstrates or cofactors, which can be used in different
enzyme-based bio-optrodes.

3.4. Environmental applications

Many bio-optrodes have been proposed for use in environmental applications

(Marty et al., 1998; Rogers and Gerlach, 1999; Rogers and Poziomek, 1996;
Schobel et al., 2000). For remote monitoring, only the fiber tip containing the
biorecognition element has to be located at the measurement site (e.g., lakes,
rivers, sewage streams) while the optical signal detection instrumentation can be
located in a protected location away from the site. Optical fibers are small in
diameter and flexible, and therefore can be located in places inaccessible to other
sensing devices. In addition, the optical fiber's durable structure protects it from
harsh environmental conditions. At present, environmental bio-optrodes are still
in the research and development stage with most of the research focused on
detection scheme development and optimization.

Several antibody-based bio-optrodes have been described for detecting pesticides

such as terbutryn (Bier et al., 1992), parathion (Eldefrawi et al., 1995) and
imazethapyr (Wong et al., 1993). One example is a bio-optrode for the detection
of 2,4-dichlorophenoxyacetic acid (2,4-D) in water (Wittmann et al., 1996). In
this system, an optical fiber with an immobilized analyte (2,4-D) was placed into
a flow cell. The assay procedure involved several steps: (1) The fiber was
incubated with fluorescently labeled monoclonal antibody for 2,4-D and the
initial (maximum) fluorescence signal was measured. The fiber was then washed
with buffer; (2) The sample was incubated with fluorescently labeled monoclonal
antibody for 2,4-D; (3) The fiber was incubated with the sample-labeled antibody
solution mixture; (4) The fiber was washed and the signal was measured. When a
high concentration of analyte (>1000 ~tg/L) was present in the sample, a low
signal was obtained because most of the antibodies were occupied with the
sample analyte and could not bind to the 2,4-D immobilized on the fiber. This
bio-optrode was used to measure concentrations ranging between 0.2-100 lxg~, a

39
Biran and Walt

concentration range suitable for environmental applications where the permitted

level of 2,4-D in drinking water cannot exceed 0.1 ~tg/L. The sensing layer could
be regenerated by washing with proteinase K. This procedure enabled the bio-
optrode to be used for more than eight weeks and more than 500 successive
measurements. Such bio-optrodes have the potential to be useful for on-line
analysis of drinking water and to serve as warning devices for hazardous
pesticide contamination.

Enzyme bio-optrodes for environmental applications have also been developed.

The most common approach employs enzyme inhibition as the sensing
mechanism. The inhibition of the enzyme acetylcholinesterase (ACHE) with its
substrate, acetylcholine, by an organophosphate pesticide was used in several
sensors (Doong and Tsai, 2001; Eldefrawi et al., 1995; Xavier et al., 2000), and
was also described in Section 1.4.1.

A different enzyme-based bio-optrode that uses a chemiluminescence reaction

for detection of phenolic compounds was recently described (Ramos et al., 2001).
This bio-optrode is based on the enhancement of the luminol-H202-horseradish
peroxidase chemiluminescence reaction by phenolic compounds. Using this bio-
optrode, p-iodophenol, p-coumaric acid, and 2-naphthol were detected in
concentrations as low as 0.83 ~VI, 15nM, and 48 nM respectively. The bio-
optrode was fabricated by entrapping the enzyme in a sol-gel layer; the gel was
prepared directly on the fiber tip. The assay was performed by inserting the fiber
with the immobilized enzyme into a test tube containing the analyte, luminol and
H202. The chemiluminescence intensity maximum at 5 min was the output
signal.

Whole cells were also used for environmental bio-optrode construction.

Recombinant E. coli cells over-expressing the enzyme organophosphorus
hydrolase were immobilized to an optical fiber and used to detect
organophosphate nerve agents, as was described above (Section 1.4.1)
(Mulchandani et al., 1998). The bio-optrode detection limits were 3 ~M for
paraoxon and parathion and 5 ~M for coumaphos. The sensor was stable for over
a 1-month period and used for over 75 repeated measurements.

Using a different approach, in which the cell's genetic response was used as the
sensing mechanism, a whole cell bio-optrode was used for detection of
naphthalene and salicylate (Heitzer et al., 1994; Ripp et al., 2001). The sensing
was performed by Pseudomonas fluorescens HK44 cells carrying a plasmid
containing a genetic fusion between the nahG gene, which is responsive (i.e.,
induced) to the presence of naphthalene and salicylate, and the IuxCDABE
reporter gene, coding for the enzyme luciferase. B ioluminescence was produced
when the cells were exposed to either naphthalene or salicylate. The cells were
immobilized onto the surface of a liquid light guide or an optical fiber by using
strontium alginate. The bio-optrode tip was placed in a measurement flow-cell

40
 Optrode-based Fiber Optic Biosensors

that simultaneously received a waste stream solution and a maintenance medium.

A rapid increase in bioluminescence was obtained when one of the analytes was
present in the waste stream. Real environmental samples of pollutant mixtures
containing naphthalene were tested using this system. High bioluminescence was
obtained when aqueous solutions saturated with JP-4 jet fuel or aqueous
leachates from contaminated soil were tested.

Using a similar approach, Ikariyama and coworkers (1997) used recombinant E.

coli cells carrying genes responsive to the presence of aromatic compounds fused
to the tuc (coding for firefly luciferase) reporter gene. The cells were
immobilized to an optical fiber and the remote sensing device was used to
measure aromatics in the part per billion concentration range.

Recently, a bio-optrode based on the same idea, was reported in which

recombinant E. coli cells produced bioluminescence in response to the presence
of genotoxic agents (Polyak et al., 2001). This bio-optrode was able to detect as
low as 25 ~tg/L of mitomycin C in less then two hours.

The main importance of genetic response based bio-optrodes for environmental

analysis is the information they provide about the bioavailability of the analytes.
This parameter is very important and helps to decide how to treat the polluted site
and which remediation strategies to employ.

3.5. Clinical applications

The development of bio-optrodes for clinical applications is another promising

field and is focused on two types of applications; (a) in-vivo detection inside a
patient, (b) ex-vivo detection when clinical samples are analyzed at the patient's
bedside. The in-vivo bio-optrodes would enable continuous monitoring of
important analyte concentrations and would dramatically improve clinical
procedures such as heart bypass surgery and critical care procedures in patients
with compromised respiratory conditions. The optical fiber's small diameter,
flexibility, nontoxic nature, durability and lack of direct electrical connections
make them highly suitable for in-vivo applications. Moreover, optical fibers have
already proven to be valuable for in-vivo clinical applications such as endoscopic
procedures and laser power transmission for surgical procedures. For example,
endoscopes are used in endoscopic surgery for gall bladder removal and for chest
and knee surgery. In principle, bio-optrodes can be coupled to such devices and
used to provide analytical information during endoscopic surgeries. At present,
such bio-optrodes (or other in-vivo chemical optical sensors) have not been
implemented because of blood compatibility problems in which a thrombus (clot)
forms around the sensor tip and affects the measurement accuracy.

The second clinical application for bio-optrodes is ex-vivo diagnostics, mainly in

critical care situations. Most diagnostic tests are presently performed in a

41
Biran and Walt

centralized laboratory. Samples must be collected with the attendant transport,

storage and chain-of-custody issues. The remote location of the laboratory delays
the medical diagnosis. In order to provide rapid diagnostic tests, analytical
devices, such as bio-optrodes, can be used to bring the laboratory closer to the
patient. These point-of care devices should be sensitive, selective, self contained
(no need to add reagents), and simple to operate. They should also be small in
size in order to be conveniently located near the patient. In addition, it is
preferable that the sampling unit in contact with the sample (e.g., blood, urine) be
disposable. B io-optrode devices of this type are still not commercially available,
but there are similar chemical-based fiber optic sensor devices used routinely in
clinics. In these devices, fluorescent dyes are use as indicators for monitoring
blood gases (PO2, PCOz) and pH. In one device, the immobilized dyes are
incorporated into a disposable apparatus that is inserted into an extracorporeal
blood circuit on one side and connected to a fiber bundle on the other (Owen,
1996). These sensors are mainly used to monitor blood gases during open-heart
surgery. Another device is used for a paracorporeal measurement at the patient's
bedside (Martin et al., 1994). The sensors are placed into an external tube
connected to an arterial blood line. Blood samples are periodically and
automatically pumped into the tube, analyzed by the sensors, and then returned to
the blood line. In this way, the blood can be monitored semi-continuously
without requiring blood samples to be taken from the patient. It should be
possible to incorporate bio-optrodes into such devices and use them to monitor
other clinically important analytes.

For both in-vivo and ex-vivo applications, the first step in bio-optrode
development is to establish sensitive sensing mechanisms that can be used to
recognize specific analytes in a complex sample such as blood, urine or other
human fluids. Many examples of bio-optrodes directed for clinical applications
have been proposed (Meadows, 1996; Vidal et al., 1996; Vo-Dinh and Cullum,
2000). Several glucose bio-optrodes, based on the enzyme-catalyzed reaction of
glucose with glucose oxidase have been prepared or proposed (Li and Walt,
1995; Marquette et al., 2000; Moreno-Bondi et al., 1990). A submicrometer
glucose bio-optrode has been prepared (Rosenzweig and Kopelman, 1996a,
1996b). In this bio-optrode, the consumption of molecular oxygen is measured by
the fluorescence quenching of ruthenium complexes and serves as a reporter for
glucose concentration. The enzyme and the indicator are immobilized in an
acrylic polymer support on the fiber tip. The bio-optrode response is very fast (2
seconds) and concentrations as low as 1 x 1015 mol were detected.

Recently, a bio-optrode for myoglobin was described (Hanbury et al., 1997). This
self-contained antibody-based bio-optrode is clinically important since it can
serve as a method for monitoring the extent of myocardial infarction. Myoglobin
was detected by immobilizing a fluorescently labeled monoclonal antibody in
polyacrylamide gel on the tip of an optical fiber (Figure 22 (a)). Cascade Blue
was used both as the fluorescent labeling agent and as a FRET donor molecule.

42
 Optrode-based Fiber Optic Biosensors

When myoglobin was captured by the antibody, fluorescence energy transfer

occurred between Cascade Blue and the myoglobin heme group (acceptor).
Fluorescence quenching of Cascade Blue was measured and correlated to the
myoglobin concentration (Figure 22 (b)). The polyacrylamide gel was optimized
to serve as a size selective filter allowing only low molecular mass molecules to
penetrate and interact with the antibodies. Using this approach, myoglobin
(16,500 Da) could be discriminated from hemoglobin (bigger than 70 kDa). The
size selection was necessary since antibodies for myoglobin can also bind
hemoglobin due to similar antigenic determinants. As shown in Figure 22 (b), a
significant response was obtained when the bio-optrode was incubated with
myoglobin but no response was obtained with hemoglobin (93 nmol/L). The
detection limit of this bio-optrode was 5 nmol/L (83 ~tg/L), which is near the
clinical decision limit for myocardial infarction diagnostics. The limitation of
using the gel layer is the increased response time due to the low diffusion rate
through the gel layer. In addition, when the gel layer was used, the bio-optrode
response was irreversible even when the bio-optrode was incubated in a solution
containing a high concentration of myoglobin antibodies (Figure 22 (b)).
Irreversible sensor responses could limit the use of this bio-optrode for
continuous monitoring applications.

Another example of a bio-optrode for heart related disease diagnostics is the D-

dimer antigen bio-0ptrode (Grant and Glass, 1999). D-dimer antigen is formed
when vascular occlusions are treated with a thrombolytic agent in order to lyse
the clot. This treatment involves inserting a microcatheter at the occlusion site
and injecting thrombolytic agents. Although thrombolytic therapy can help in
preventing strokes, it suffers from several limitations that bio-optrodes can help
to overcome. One limitation of this approach is that it is difficult to know if the
occlusion occurred from an atherosclerotic plaque or a thrombus. Using the bio-
optrode, initiation of D-dimer antigen formation following the injection of a
small amount of thrombolytic agent would indicate the presence of a thrombus
clot. If D-dimer antigens are not detected after the thrombolytic agent injection, it
would be an indication that the occlusion is caused by an atherosclerotic plaque
and alternative treatment would be required. In addition, in the case of a
thrombus clot, the bio-optrode can be used for on-line monitoring of the
thrombolytic agent dosage needed to dissolve the clot by monitoring the D-dimer
antigen formation during the procedure. The bio-optrode principle of operation is
similar to the myoglobin bio-optrode described above. In this case, the antibody
is labeled with fluorescein and fluorescence quenching is observed when D-
dimer antigen binds. The fluorescently labeled antibody is immobilized in a sol-
gel on an optical fiber tip. The bio-optrode was used to detect D-dimer antigen in
human plasma and in whole blood; the detected concentration range was 0.56 - 6
ktg/ml, which is within the clinically relevant range. The limitations of this bio-
optrode are its poor reversibility and the short lifetime of the immobilized
antibodies (four weeks).

43
Biran and Walt

Figure 22. Reagentless bio-optrode for myoglobin detection. (a) Bio-optrode setup. (b)
Myoglobin bio-optrode responses. The bio-optrode responses in PBS buffer (0-80 min.)
and after incubations with hemoglobin (Hb), myoglobin (Mb) and unlabeled myoglobin
antibody (Ab) are shown (Hanbury et al., 1997). Reprinted with permission from Clin.
Chem.

In addition to the bio-optrodes described above that are directed to in-vivo

applications, several bio-optrodes for point-of-care applications have also been
described. These bio-optrodes offer a miniature design and a fast response time
for analytes such as bilirubin (Li and Rosenzweig, 1997), cholesterol (Marazuela
et al., 1997), and D-amino acids (Zhang et al., 1995).

3.6. Industrial applications including bioprocess monitoring

Cell culture-based bioprocesses are very complex to control since they are
sensitive to minor changes in the chemical composition of the fermentation
medium. Therefore, tools for on-line monitoring of different analyte
concentrations during the bioprocess are highly desirable. Bio-optrodes offer

44
 Optrode-based Fiber Optic Biosensors

~o.o -I..............x'"I- x , , ,,, ~

I'
- . _ .

:113.0'

o
20.0

tj
gl
0 10.0" tb3

0.0 "~" ~-'"

0 50 100 I(~ 800 ~ 800 ~0 400
process U m e / b

"~ il-............I
&

.~ t.5.

1,0,
o
&&
o
o 0.5,

0.0 I
0 IIo tI~ 160 2o0
,r I

II~
. . . .

~I~
9 . . . .

~
9
2
. . . . w

pl'oceII UlXle/h

Figure 23. Five-channel FIA-based enzyme bio-optrode for continuous monitoring of

lactate, glucose, glutamine, ammonia and glutamate (from top to bottom) during animal
cell cultivation. Chemiluminescence measurements were employed to determine the
analyte concentrations using five optical fibers, each with a different immobilized
enzyme (Spohn et al., 1995). Reprinted with permission from Elsevier Science.

several advantages in such applications. The ability to use optical fibers directly
inside fermentors (in situ) eliminates the need to periodically remove samples for
analysis in a remote analytical laboratory. Once inside the fermentor, bio-
optrodes can be used for sensitive and selective on-line monitoring of different
analytes. Fermentation substrates and products such as proteins, antibodies and
antibiotics can be monitored. Other parameters related to the biological status of
the cells, such as cell viability and activity, can also be measured. The ability to
perform this measurement from a remote location (e.g., central control room)
without using wires offers another important advantage. The main obstacle,
which prevents wide use of in-situ bio-optrodes (or any type of biosensor) is the

45
Biran and Walt

need to sterilize the bio-optrode, which may damage the sensing biomolecules.
For this reason, most bio-optrodes for bioprocess monitoring use a flow system
in which a sample of the medium is taken from the fermentor and is delivered to
the sensor (Dremel et al., 1992; Marose et al., 1999; Mulchandani and Bassi,
1995; Scheper et al., 1996).

In one example, a FIA-based enzyme bio-optrode system was used for

simultaneous detection of five different analytes (Spohn et al., 1995). Glucose,
lactate, glutamate, glutamine, and ammonia were detected in samples that were
removed during animal cell cultivation. The system is based on
chemiluminescence detection and consists of five optical fibers, each with a
different immobilized enzyme. Each fiber is inserted into a different flow cell
and, when the sample is injected, each fiber's response is measured. The results
are combined and the concentrations of the different analytes are determined.
Figure 23 shows results from a 350-hour monitoring of an animal cell culture
medium. In a similar way, antibody-based or nucleic acid-based bio-optrodes can
be used to monitor different bioprocesses.

4. Advantages and Limitations of Bio-optrode Technology

B io-optrodes offer several advantages over other biosensing technologies based

on the unique characteristics of optical fibers. The optical fiber's small
dimensions, flexibility, and their ability to transmit optical signals for long
distances allows them to be used for remote sensing in places where other
biosensors cannot be used. In addition, their ability to function without any direct
electrical connection to the sample makes them safer than electrochemical
biosensors. B io-optrodes are intrinsically simpler than electrode-based biosensors
since no reference electrodes are needed. Moreover, the development of new
biorecognition molecules, such as those containing FRET-based dyes, enables
the fabrication of self-contained bio-optrodes where no addition of reagent is
needed. B io-optrodes based on imaging fibers offer additional advantages since
they allow multiplexing with rnulti-analyte sensing capabilities.

It is expected that as new optical technologies are developed for

telecommunication applications, they will be adopted for bio-optrodes. These
technologies include miniaturization of light sources, detectors and optical fiber
components (Kostov and Rao, 2000). Furthermore, new developments in the area
of nanotechnology should eventually enable development of new bio-optrodes at
the nanometer scales.

Nevertheless, bio-optrode technologies suffer from several drawbacks. Some of

these drawbacks are common to all biosensor devices, with the most difficult one
being the poor stability of the biological recognition molecules. Such molecules
tend to be sensitive to pH or temperature changes and therefore have short

46
 Optrode-based Fiber Optic Biosensors

lifetimes. Another important limitation is the high cost of some of the purified
biological sensing materials. Regeneration of sensing biomolecules is usually
problematic and, in most cases, fresh biorecognition molecules are required for
each assay. In addition, there are several problems related to the immobilization
process including loss in activity, leaching of reagents, and the decreased
response time due to slow diffusion of analytes through the irrkrnobilized layer.

Several other bio-optrode limitations are related to the nature of optical fibers.
Since light signals are the measured parameter, bio-optrodes are sensitive to
ambient light interference and precautions must be taken either to exclude light
or to employ optical designs with lock-in detection capabilities. In most bio-
optrodes, there is a need to use indicator dyes in order to transduce the
biorecognition events. The dyes have to be immobilized together with the
biomolecules and therefore complicate the bio-optrode fabrication. In addition,
the dyes may leach from the immobilization matrix or may lose their
characteristics because of photobleaching.

5. Potential for Improving Performance or Expanding Current Capabilities

As with any sensing or monitoring device, the ideal bio-optrode should be

specific, sensitive, simple to fabricate and use, well adapted to the measurement
environment (e.g., detect specific analytes in a complex sample), reliable and
self-contained. When used as a sensor, it should be operated in a continuous and
reversible manner. When used as a probe, it should include a simple and
disposable unit that contains the sensing molecules. In addition, for many
applications, bio-optrodes should be small, able to detect multiple analytes
simultaneously, and enable measurements in remote sites. At present, no bio-
optrode device has achieved all these ideal performance capabilities.
Nevertheless, based on new bio-optrode technologies currently under
development, it is expected that the next generation of bio-optrodes will come
closer to achieving these goals.

The development of new bio-optrode technologies and devices is highly

dependent on advances in several different fields. Advances in biology,
chemistry, materials science, optics, electrical engineering, mechanical
engineering, and computer engineering, are expected to inspire new bio-optrode
technology development. In this section, a few new key technologies and their
future impacts on the bio-optrode field are discussed.

5.1. New optical fibers and instrumentation

Optical fibers have attracted attention mainly due to their use in

telecommunications. New technologies have been developed for fabricating
optical fibers with very efficient light transmission capabilities. Fibers can

47
Biran and Walt

transmit extremely high amounts of data when used in both single or bundle
format. These characteristics will advance the development of real-time multi-
analyte bio-optrodes for various analytical applications.

Improved, smaller, and less expensive light sources and detectors are driving
consumer electronics. Integration of these components into bio-optrodes can lead
to miniaturization and commercialization of bio-optrode devices (Kostov and
Rao, 2000). Among the different possible light sources, light emitting diodes
(LEDs) are very attractive to use in bio-optrodes. LEDs are very small, cover the
entire visible spectrum, produce optical power in the range of 0.1-5 mW, and
have a very long life (100,000 hours) and low cost (-$2). Once LEDs at a
particular wavelength have been demonstrated and commercialized, laser diodes
are usually available within a few years. Laser diodes have higher power output
and are nearly monochromatic whereas LEDs have a relatively broad spectral
emission output. Another interesting new light source is the scintillation light
source that can be used as a high stability light source for the UV and blue region
(Potyrailo et al., 1998). These sources are based on long-lived radioisotopes in
scintillation crystals, which convert the radioactive emission (typically beta
particles) into light emission. These sources are extremely stable, can be used
without external power sources, and have an expected life of 20 years.

In recent years, new generations of miniaturized and improved light detectors,

such as photodiodes (PD) and photomultiplier tubes (PMT), have been developed
(Kostov and Rao, 2000). The most sensitive detectors are avalanche photodiodes.
CCD chips are also rapidly developing; bigger chips with higher signal/noise
ratios, wider dynamic ranges, and lower dark currents have been developed. In
addition, CCD detectors have been miniaturized and integrated into small
devices. Image intensifiers have been integrated into CCD cameras to increase
light detection sensitivity. Although CCD chip prices have been dramatically
reduced, scientific grade CCD cameras are still very expensive (-$20,000). A
competing technology to CCD is the complementary metal oxide semiconductor
(CMOS) technology. Recent developments in this technology have demonstrated
light detection capabilities similar to CCD detectors. The advantages of this
technology are lower cost, simpler fabrication process and the ability to use it for
very fast image acquisition (32,000 pictures per second) because frame transfers
are not required as all the processing is done on chip. Both CCD and CMOS
technologies will most likely be integrated into future bio-optrode devices.

5.2. New biological recognition elements

The "heart" of any bio-optrode is the biological recognition element that initiates
the detection process by its interaction with the analyte. Development of new
biological recognition elements will increase the number and types of analytes
that can be detected by bio-optrodes. New advances in molecular biology
techniques allow the design or selection of new recognition molecules. For

48
 Optrode-based Fiber Optic Biosensors

example, using phage display technology it is possible to screen and identify a

single chain antibody (scFv) with specificity for almost any analyte
(Hoogenboom et al., 1998). The recombinant scFv molecule is a smaller version
of an antibody molecule containing the antigen binding site. Genetically
modified bacteriophages, each presenting a unique scFv molecule on its surface,
are used in the screening process. Phage presenting scFvs with higher affinities to
the analyte are selected. The system is designed in such a way that the sequence
coding for the scFv presented in the selected phage can be readily identified. This
sequence is placed in an expression vector and E. coli cells are used to produce
large quantities of the selected scFv molecule. This process is very powerful
since it allows antibodies to be identified and isolated in a very short time
(Goldman et al., 2000). Moreover, once the antibody is found, it takes only a few
days to produce it in large quantities. In addition, a scFv molecule can be
specifically designed to be used in biosensor devices by adding immobilization
capabilities to the molecule at the genetic level. For example, scFvs were fused to
the CBD (cellulose binding domain) resulting in scFv molecules that can be
easily immobilized to a cellulose membrane (Berdichevsky et al., 1999).

Using molecular biology techniques, several other biorecognition molecules have

been designed. Genetic fusion between antibody molecules and the green
fluorescent protein resulted in self-fluorescent antibodies, also called
fluorobodies, and eliminates the need to label the antibody with a fluorescent
dye. Other genetically modified molecules designed for biosensing are described
in Chapter 10. Biological recognition elements isolated using combinatorial
approaches, such as aptamers, are described in detail in Chapter 12. Biomimetic
polymer materials for bio-optrode applications are described in Chapters 11 and
13.

5.3. Imaging and biosensing

The coupling of chemical and biosensing capabilities to optical fiber-based

imaging devices (e.g., endoscopes) is expected to attract much attention in the
future. Optical imaging fibers are used in endoscopes since they can carry images
from one end of the fiber to the other due to the coherent nature of the fibers.
These imaging capabilities can be utilized to simultaneously image and measure
local analyte concentrations with micron-scale resolution (Michael and Walt,
1999). The imaging fiber's distal face is coated with an analyte-sensitive layer
(typically a biorecognition molecule and fluorescent indicator), which produces a
microsensor array capable of spatially resolving analyte concentrations. The
concept is shown in Figure 24. For example, an acetylcholine imaging fiber bio-
optrode was fabricated by coating the imaging fibers with a polymer layer
containing the enzyme acetylcholinesterase and fluorescein (Bronk et al., 1995).
The fiber was used both to visualize the tobacco hornworm and to measure
acetylcholine release from the neural ganglion (Walt, 1998). The acetylcholine

49
B iran and Walt

Figure 24. Combined imaging and biosensing concept. The technique provides the
ability to both view tissue slices or individual cells and measure the release or
consumption of different analytes using fluorescence techniques.

release was measured followed electrical stimulation of the worm's sensory

nerve. First, the fiber was placed over the neural ganglion. A white light image
was taken to visualize the neural ganglion morphology. The imaging fiber bio-
optrode was then switched to the fluorescence mode and the neural ganglion was
stimulated. Higher intensity signals were obtained from the neural ganglion
regions that released acetylcholine. The ability to observe the location of
neurotransmitter release, with microscale spatial resolution, can provide a
powerful tool for neuroscience researchers. In a similar approach, one day the
technology may be used for in-vivo applications. For example, a suspected cancer
tumor could be examined based both on its morphology and its response to
specific antibodies immobilized on the imaging fiber tip.

5.4. Data analysis

New bio-optrode technologies are expected to provide a large amount of

analytical information from each measurement. For example, multi-analyte bio-
optrodes will measure the concentration of many different analytes
simultaneously. When such measurements are performed in continuous fashion
(e.g., multiple measurements every second), it will generate a high data volume.

50
 Optrode-based Fiber Optic Biosensors

(a)
~,7

F=5

0 i'~"'V'l" " ' I " ' " I ' '" |-~ ' " ]1 ' " I ' ' " I " ' ' I ' r" | '~'

0 20 40 61:) 80 100 120 140 160 160 200

tlme [sl
(b) 10 ' ~ S m M ..........
................. ,oo

r.,. ~ _ ~SmM

0 20 40 ~0 eo 100 120 140 180 180 2oo

time Is]

Figure 25. Penicillin detection using a FIA-based bio-optrode. (a) Fluorescence signals
obtained in response to different penicillin concentrations. (b) Fluorescence signals
obtained with different buffer ion concentrations and a fixed penicillin concentration (5
g/L). Neural network analysis was applied to analyze the relationships between these two
responses (Muller et al., 1997). Reprinted with permission from Elsevier Science.

In order to acquire, analyze, and save such high data volumes, sophisticated
software should be developed or adapted from other high volume applications.

The most significant computerized task is the data analysis because it may affect
the specificity, sensitivity, and reproducibility of the bio-optrode. Analysis of
biosensor measurements may be complicated due to the high variability in the
activity of biorecognition molecules and because the measurements are usually
performed in a complex sample matrix. It was previously shown that even in a
simple FIA biosensor system for measurement of a single analyte in bioprocess
samples, there is a need to use advanced computational analysis in order to
improve the sensitivity, selectivity, and reproducibility of the measurement
(Hitzmann et al., 1998). For example, in typical bioprocess samples, the pH or
ion concentrations change during cultivation, which may affect the biosensor's
enzyme activity or antibody binding properties. In addition, inhibitors and
proteases can be produced during the bioprocess, which will affect protein-based
bio-optrodes, or nucleases can be produced, which will affect nucleic acid-based

51
Biran and Walt

biosensors. In order to overcome these problems, multivariate evaluation

techniques such as neural networks have been used. Scheper and coworkers
(Muller et al., 1997) applied the neural network approach to improve the analysis
of signals obtained from a FIA bio-optrode for penicillin. The need to use the
neural network approach was due to the sensitivity of the measurement to
changes in buffer ion concentration (Figure 25). The neural network was used to
simultaneously evaluate the ion and penicillin concentrations from a single
measurement based on characteristic signal shape variations. The shape
characteristics were thought to be useful because in a preliminary experiment,
multiple measurements of the same sample showed reproducible signal shapes.
The results from this neural network showed errors of less then 11% for six
different penicillin concentrations measured at five different ion concentrations.
This simple example demonstrates the power of such computing techniques in
the analysis of bio-optrode signals. It is clear that such techniques will be
essential for analyzing signals from multi-analyte bio-optrodes. Advanced
computational methods have recently been used for the analysis of chemical
sensor arrays (Jurs et al., 2000), and it is expected that they will be adapted to the
analysis of bio-optrode measurements. The generation of high amounts of
information from future multi-analyte bio-optrodes is expected to shift the
emphasis from signal measurement to data analysis.

6. References

Aboul-Enein, H. Y., R. I. Stefan, J. F. van Staden, X. R. Zhang, A. M. Garcia-

Campana and W. R. G. Baeyens, 2000, Critical Rev. Anal. Chem. 30,
271.
Anderson, G. P., K. D. King, K. L. Gaffney and L. H. Johnson, 2000, Biosens.
Bioelectron. 14, 771.
Arnold, M. A., 1985, Anal. Chem. 57, 565.
Barker, S. L. R., R. Kopelman, T. E. Meyer and M. A. Cusanovich, 1998, Anal.
Chem. 70, 971.
Barker, S. L. R., H. A. Clark, S. F. Swallen, R. Kopelman, A. W. Tsang and J. A.
Swanson, 1999, Anal. Chem. 71, 1767.
Berdichevsky, Y., E. Ben-Zeev, R. Lamed and I. Benhar, 1999, J. Immunol.
Meth. 228, 151.
Bier, F. F., W. Stocklein, M. Bocher, U. Bilitewski and R. D. Schmid, 1992,
Sens. Actuators B-Chem. 7, 509.
Blum, L. J., S. M. Gautier and P. R. Coulet, 1993, J. Biotechnol. 31,357.
B lum, L. J., S. M. Gautier and P. R. Coulet, 1994, In Food B iosensor Analysis,
Eds., G. Wagner and G. G. Guilbault, Marcel Dekker, New-York, pp.
101.
Bronk, K. S., K. L. Michael, P. Pantano and D. R. Walt, 1995, Anal. Chem. 67,
2750.

52
Other documents randomly have
different content
per guida il lume che traspariva dal riassunto presidenziale, e dove
parve a loro che inclinasse al sì eglino dissero sì, e dove al no, senza
tanto beccarsi il cervello, dissero no.
In meno di un'ora fu finita ogni cosa; la più parte di loro metteva
doppio tempo a desinare: neppure una delle formalità volute dalla
legge fu trascurata: vennero tutte eseguite puntualmente, chè il
manuale sapevano a mena dito.
I dodici rientrarono nella sala, dove li accoglie un silenzio inquieto,
foriero della tempesta. Il presidente domanda al capo dei giurati,
conforme alla usanza, qual sia il resultato della deliberazione; questi
con la mano sulla parte dove il cuore ha la gente, pronunzia la
formula sacramentale: «Sul mio onore e sulla mia coscienza la
deliberazione dei giurati è questa....» e la lesse, la quale sonò
alternativamente ora affermativa ed ora negativa con questa
ragione, che condannò senza pietà Felicina, e rimandò assoluto prete
Liborio.
Il presidente, in ordine al verdetto dei giurati, dichiarato prima
assoluto don Liborio, decretò si ponesse subito in libertà, non senza
ammonirlo di procedere un'altra volta meglio avvisato negli atti di
carità: anco questa ha i suoi confini; anzi per dirgliela in rima,
conciossiachè il presidente desse talora una capata nella poesia, gli
allegò la sentenza dello abate Pietro:

. . . . . . . . . . . e quando eccede,
Cangiata in vizio la virtù si vede.

E il diavolo rise. Quanto a Felicina, giudicata colpevole di netto, il

pubblico ministero chiese con bellissimo garbo alla Corte
l'applicazione della pena ai termini dell'articolo 531 del Codice
penale.
— E che porta questo articolo? — domandavano così per curiosità
l'uno all'altro gli astanti, ed anco i giurati.
— Ma! — rispose uno, tirando su una presa di tabacco —
semplicemente la morte.
— La morte! — I giurati saltarono su come i diavoli di Germania
scattano fuori dalle scatole, e si misero paura scambievolmente:
parecchi di loro per quel dì non pranzarono; due, il giorno di poi
ebbero a purgarsi; in altro modo non sapevano piangere. Le fanciulle
infeste a Felicina si dispersero a mo' di colombe pel sopravvenire del
falco; e tanto più volentieri mi valgo di questa comparazione, in
quanto che ho avuto luogo di osservare come gli uccelli cari a
Venere, non sieno punto, secondo la opinione universale, miti, al
contrario rissanti spesso fra loro a colpi di becco, ovvero di ale a mo'
che le femmine dispettose costumano co' gomiti.
Ai consiglieri della Corte non fece caldo nè freddo; nell'animo loro la
sentenza, come la nebbia, lasciò il tempo che aveva trovato: di
cotesta maniera arrosti tutti i giorni ne cuocono; all'odore dello
strinato ci sono avvezzi.
Chi trionfò davvero fu il procuratore del re; quando tornò a casa
pareva Ezio reduce dai gelidi Trioni con due corbelli di alloro: la
bambina che gli si fece incontro giù per le scale egli si pose a
cavalcioni sul collo, segno di sterminata allegrezza, perchè non
dimenticava mai la sua gravità, anco quando era col berretto da
notte e la veste da camera. A mensa si cantò da sè l'epinicio, ovvero
l'inno del trionfo; per celebrare degnamente la vittoria volle una
bottiglia di Chianti, proprio di quello del Ricasoli. Cotesto vino, che
ha il colore ed anco il gusto del sangue rappreso, piace ai procuratori
del re; anche il boia lo beve per le pasque.
La folla spulezzò in un attimo per cavarne i numeri, ed essere in
tempo per giocarli prima che chiudesse il banco del lotto. Solo una
vecchia tentennava il capo, e le gridava dietro: «Dove vai senza
giudizio? Numeri buoni saranno quelli che piglierò quando le
taglieranno la testa!»
A Fabrizio non fu mestieri interporre ricorso in Cassazione per
Felicina; ricondotta in carcere la prese il delirio e non la lasciò più:
poco prima dell'agonia, secondochè per ordinario succede, tornò a
rischiararla nella sua pienezza la luce dello intelletto, e se ne valse
per raccontare punto per punto le infamie del prete traditore ed
omicida. Innocente ella era, e gli uomini le posero per lapide al suo
sepolcro una sentenza di morte.
E pure questa sentenza troppo più che alla Felicina tornò maluriosa a
Fabrizio, il quale appena fu pronunziata declinava il capo nelle mani,
nè più si mosse, finchè gli uscieri vennero ad avvertirlo, che stavasi
per chiudere il Tribunale; da quella via lo consolarono dicendogli: «Si
faccia animo, se ha perso questa, ne vincerà un'altra!» Egli si destò,
e gli parve essersi rinnovata in lui la leggenda dei sette dormenti;
l'uragano gli aveva devastato lo spirito: amore, affetti, generose
aspirazioni, ogni cosa dispersa; vibrò truce lo sguardo al cielo, e
parve Giuliano l'apostata, quando raccolto nel cavo della mano il
proprio sangue lo gettava in alto a sfida del galileo. Giù per le scale
del Tribunale fu udito borbottare:
— Caligola era un moderato.... già che ci era doveva desiderare che
non i Romani, bensì tutti i viventi avessero un capo solo.... umanità!
umanità! non vali una corda che t'impicchi.
E don Liborio? Ah! il cielo non abbandona mai i suoi divoti, come
disse colui che rubò la corona alla Madonna degli Angioli. Nelle prime
ore della notte, che tiene dietro a cotesto giorno lugubre, mentre
don Liborio si confortava di cibo e di bevanda, ecco fu bussato
discretamente alla sua porta, dalla quale, schiusa cheta cheta simile
alla bocca di una volpe che sbadigli, entrò un prete umile in vista,
che, salutato appena don Liborio, così gli favellò:
— Qui non tira vento buono per lei; su si levi subito e non perda un
minuto di tempo; troppo ci costa di fatiche e di danaro, perchè noi
non evitiamo il caso di cominciare da capo.
— Ma voi chi siete?
— Io? Miri; — e gli mostrò un foglio alla vista del quale a don Liborio
cascarono i frasconi: era un mandato del vescovo: di protervo si fece
mogio, e si lasciò condurre come un montone. Trasferito pel
momento in luogo sicuro; più tardi, a diligenza dei reverendi padri di
Gesù, giunse a Roma inavvertito, dove presentatosi al cardinale
penitenziere, dopo essersi sentito raccontare a parte a parte una
lunga storia, che sapeva, cioè quella dei suoi delitti, venne
sottoposto a penitenza asprissima. Anche qui gli valsero le arti della
ipocrisia; e sì che i suoi compagni in chierica se ne intendevano;
tanto è, gli riuscì essere liberato dal carcere. Vagò pel mondo, ma
preceduto da per tutto dagli avvisi dei gesuiti, non potè fare di troppi
civanzi. Io lo incontrai in Corsica mercante. Mercante di che? Ve la
dò a indovinare in cento. Mercante di messe. Mercante di messe?
dite voi. Mercante di messe, dico io. Ecco come. I preti di Francia,
gesuiti o non gesuiti (imperciocchè voi dobbiate ficcarvi bene nella
mente che i preti sono come i fagiuoli, ve ne ha dei bianchi, dei
rossi, dei turchi, coll'occhio, ma in fondo sono tutti fagiuoli),
costumano pigliare dai devoti a dire più messe che possono, e che
non possono. In Francia il seme di Voltaire ha generato più preti, che
quello del Giappone bachi in Lombardia. E tuttavia non sopperiscono
alla richiesta di messe; taluno ha facoltà di binare [27], ma pochi:
potrebbero ternare e quadernare senza facoltà, ma non usa, forse in
grazia dello antico proverbio: Che non ci è putta nè ladrone che non
abbia qualche devozione; insomma, siccome in questo mondo se ne
ha a vedere di tutti i colori, così ci stanno anco dei preti, a cui non
basta l'animo appropriarsi il danaro delle messe senza celebrarle: per
le quali cose essi hanno trovato un mezzo termine, onde uscirne pel
rotto della cuffia, accollando la celebrazione delle messe agli
impresari. E questo si capisce; ma se l'accollo fanno alla pari, dove il
guadagno? E se con ribasso come evitano il peccato? Adagio; fanno
lo accollo alla pari, e ci guadagnano sopra: ecco come: in danaro
pagano meno che possono, il rimanente somministrano in piviali
logori, in pianete fruste, in dalmatiche usate e manipole rammendate
e stole rattoppate, candelieri dorati a mecca, residenze ristuccate,
madonne ricucite, cristi tenuti su con la colla, santi ritinti.
Don Liborio non stava a guardarla tanto pel sottile, e o non faceva
contrasto, ovvero ne faceva tanto da migliorare il mercato senza
però lasciarselo fuggire di mano, quindi anche egli punto da scrupolo
(o che credete, che po' poi non avesse coscienza anche don
Liborio?) di commettere simonia, portatesi tutte queste ciarpe in
Corsica, negoziava coi preti dei paesi poveri come san Quintino, che
sonava a messa co' tegoli, e dava in elemosina, vale a dire in salario
delle messe, o candelieri, o pianete, o vasi di fiori sbiaditi a prezzo
esorbitante, di rado aggiungendo danari, od aggiungendovene a
spizzico: a questo modo si procurava certificati della puntuale
celebrazione delle messe, che spediti in Francia, accettavansi per
buoni, bastando ai committenti francesi avere tanto in mano da
ninnare la coscienza perchè si addormentasse. Tuttavia la
confessione non fu mai più concessa a don Liborio: gli permisero la
predicazione, però che assai si mostrasse prestante in simile
faccenda; invero copiosa era la messe che racoglieva nel suo
apostolato, massime nel propagare il domma della Immacolata
Concezione [28].
 Capitolo IX.
AMORE TERRENO.

E se la Parca ti proceda amica, ella non può fare a meno, che

pigliando un tosone di oro per filare la tua vita, non ci mescoli dentro
alquanto di lana scura: così ordinarono i fati; mentre se all'opposto,
per elezione, o per destino, la Parca scelga per te il vello nero, tutti
bui si succederanno i tuoi giorni, e pieni di amarezza: fra le tenebre
del tuo sepolcro e quelle della tua vita, aspetta e vedrai che non ci
corre differenza alcuna.
I casi avversi chiama la gente Sventura, come se fossero una cosa
sola, e di qualità pari; invece la Sventura è molteplice, simile in tutto
ai serpenti di Laocoonte; o se pure ella ha un corpo solo, mirala (Dio
ti preservi da provarla!) i suoi capi sono infiniti! Cotesta idra spietata
ti lacera il corpo, e nello stesso punto lo spirito; nel punto stesso
t'investe la facoltà del pensare e quella del sentire; il sepolcro non ti
possiede ancora, ed ogni giorno senti la morte.... Ch'è mai l'uomo fra
gli artigli della Sventura?
Eppure, guarda cotesta onda mostruosa dell'Oceano! Anco il mare
conosce le sue catene di monti, pari a quelle della Imalaia, delle
Cordigliere e delle Alpi, quantunque esse sieno mobili, ed ei le faccia
e le disfaccia senza posa.... Osserva.... osserva.... vedi quel punto
nero che apparisce e scomparisce?.... Non vedi nulla! Ecco, guarda
con questo telescopio e fa' presto, chè il punto nero sta per
iscomparire per sempre. Lo vedi!.... Ebbene, l'onda mostruosa investì
uno dei Leviatan del mare, i quali col ferro e col fuoco portano la
schiavitù e la morte; lo travolse giù nell'abisso, lo trabalzò fino al
cielo, e quivi lo disperse ai venti insieme con le sue spume; tutto
scomparve, alberi, vele e la bandiera che nei giorni sereni, ventilata
dalle brezze dell'Oceano, pareva che collo zufolare delle pieghe
dicesse: anche l'Oceano mi riconosce signora!....
Cotesto punto nero è un uomo, che combatte contro l'Oceano. Con
quali forze? Non ci pensa. Per quanto tempo?..... È già sparito, ma
ha combattuto. Vissero e vivono anime non degne di trovarsi
abiettate dentro un corpo di creta, che non piegano alla forca
caudina del fato, e Bruto sputò la sua anima in faccia alla Sventura
esclamando: «Vergognati della tua onnipotenza». E così Spartaco
schiavo, e Catilina patrizio: entrambi caduti supini sul campo di
battaglia, entrambi laceri di ferite nel petto, e con gli occhi aperti,
trucemente fissi nel cielo, e co' ferri stretti nel pugno sfidavano ad
un punto e maledivano i fati. La paura consacrò la fama di costoro
agli Dei infernali, e la esecrazione mantiene sopra essi e la rinnova
contro quelli che gli somigliano, perchè dura la medesima paura.
Furono virtuosi? Io non lo so; so bene quest'altro, che chi li spense
sarebbe stato in ogni caso più malvagio di loro.
Impertanto possono combattersi i fati; vincersi no. Taluna di quelle
povere creature che si chiamano re ebbe la presunzione di farsi
sudditi i fati, e Carlo di Angiò al primo urto di sventura
superbamente vantava: «Buono studio vince rea fortuna.» Quando
poi sentì trafiggersi da strali più fitti, che non appaiono atomi dentro
il raggio del sole, curvò la testa supplicando: «Sire Dio, fa' che la mia
caduta sia a piccoli passi!»
Concetto degno di re, non già di uomo, imperciocchè dimostri
com'egli non intendesse perseverare nei supremi contrasti, bensì
accomodarsi agli eventi a patto gli fornissero un nido dove riparare.
Pure di non essere portato via, gli bastava durare sbattuto, come la
pianta marina abbarbicata sul fianco dello scoglio vive vita di
tremito.
Ai giorni nostri l'uomo, pauroso di rimanere sbranato di un tratto
dalle granfie del leone, preferisce disfarsi lentamente in polvere sotto
la roditura del tarlo; non vi state a confondere; se Napoleone I
avesse provato appetito della bella morte, l'avrebbe trovata: io non
dirò che la sua scelta fosse coraggiosamente codarda [29], ma egli è
certo che volle vivere per giustificare lo abuso delle facoltà
concessegli da Dio.
Perduto il trono, intese conservare la fama: e convertita Sant'Elena
in pulpito, si mise a predicare concetti che non ebbe mai; o se pure
egli li accolse nella mente, e' fu per disperderli. Vincitore, oppresse la
umanità; vinto, la ingannò. Oh! non badate al tiranno caduto, che
favella di libertà; le sue parole hanno per fine di costituire il
fondamento di un altro trono. Dall'isola di Sant'Elena, Napoleone I
legò al mondo Napoleone III, nella medesima guisa che Augusto
legava ai Romani Tiberio.
Il poeta della Francia ha pianto sulla demolizione della colonna di
piazza Vendôme, doveva piangere quando fu eretta. Tutti i popoli di
Europa conservano memorie di avere sbranato, e di essere stati
sbranati: se le tigri e i leoni conoscessero le arti, avrebbero anch'essi
le loro colonne traiane, napoleoniche e nelsoniane.
Le arti cortigiane possono lamentare la dispersione dei trofei di
sangue; la umanità se ne rallegra. Il cantore che lusinga gl'istinti
feroci del popolo non riceverà mai il premio dello amplesso di Dio:
bene l'amore sarà una corda della sua lira, non già un sentimento
del suo cuore. Fin qui i francesi delirarono ubbriacarsi di sangue, più
che di vino, ed oggi, non si potendo inebriare col sangue altrui,
bevono il proprio.
E l'uomo ragnatelo, che fu Napoleone III, il quale prima ridusse la
Francia in condizione d'insetto, e poi la risucchiò; adesso torna,
pieno di speranza, a ordire la sua tela per riagguantare la mosca
morta.... Almeno Belzebub era il demonio delle mosche vive! Anco
co' denti fradici si mangia, anco con la viltà si campa, anco allo
strepito delle maledizioni assuefannosi le orecchie, e si dorme:
uomini siffatti prima di ogni altra cosa vogliono vivere, ed a ragione;
curano la materia, perchè sono e sentono essere totalmente ed
unicamente materia.
 *
Con lo amore si cammina a gran giornate, e poichè il conte Ludovico
ed Eponina si amavano senza incontrare ostacoli, potete immaginare
voi se la macchina scivolasse a tutto vapore. Però bisogna dire che lo
amore di questa non fosse uguale in tutto e per tutto allo amore di
quello; la differenza chi sapeva cercarla la trovava. Era l'amore di
Eponina amore di conquista e trionfale; amore, che nato appena,
squassato l'arco gridò: «Valgo, e voglio regnare solo»: amore, che di
ogni fiore fece ghirlanda ed anco, pur troppo, di ogni pruno siepe;
amore, di quelli che alternano il nudrimento con desiderii terreni e
con aspirazioni divine: simili alla rondine, la quale rasenta la terra
per terminare la sua curva in mezzo dei cieli, essi pigliano per volare
le ali in presto così dalle passioni come dallo ingegno e dai talenti;
che la rondine anco quando rade la terra vola, e lo amore posandosi
sulla materia alia impaziente a levarsi più in alto: però Eponina se
avesse voluto spegnere il suo amore avrebbe potuto; certo le
sarebbe stato mestieri pigliarsi il cuore e adoperarlo a modo di pietra
per ischiacciargli il capo, ma lo avrebbe potuto: Ludovico all'opposto,
quando pure avesse voluto, non avrebbe potuto per propria virtù;
ma, in forza d'impulsi esterni, avrebbe potuto, anco senza volerlo.
Natura da paternostro, la quale non si ripromette resistere alle
tentazioni, ma si raccomanda quotidianamente a Dio per non essere
indotto in tentazione.
La madre Isabella invece di temperare gli ardori della figliola, gettava
legna sul fuoco, e poi ci soffiava dentro: se l'avessi a dire proprio
come la penso, io per me credo, che mutatis mutandis (per valermi
dello stile dei notari) ella fosse invaghita del contino Anafesti, poco
meno di Eponina. O come mai? Ordinariamente la va così;
garbavano alla Isabella i modi del contino, spruzzati in pelle in pelle
di nobilesca albagia, il suo fare amabilmente contegnoso, la grazia
della persona, lo incesso, la parola, il volto, e tutto, perfino il
balbutire, vizio col quale i gentiluomini di razza manifestano la
propria virtù. Isabella, a fine di conto, popolana nacque, e venne
educata da pari sua: però tu che leggi, se sei popolano, devi
confessare che grande è la potenza dei titoli sopra i cervelli
popoleschi..... e sul tuo.
Quando un popolano pesta le mani ed i piedi gridando uguaglianza,
per ordinario non gli do retta, imperciocchè io pensi che uguaglianza
gli appetisca sì, ma a patto di diventare co' marchesi marchese.
Allorchè tu presenti al popolano un conte, quantunque spiantato, tu,
il più delle volte, lo miri, confuso per non saperlo onorare
abbastanza, facendogli di berretta, e profondendogli inchini: caso
mai il popolano od abbia, o si immagini avere l'amicizia di un titolato,
tu lo udrai ricordare a tutto pasto il suo amico barone, o conte, o
marchese, od anco cavaliere scusso. Là dove il popolo è condannato
a starsi terra terra, come la porcellana, urla uguaglianza; se avvenga
poi ch'ei si alzi un sommesso, lo proverai superbo come tutti i servi
diventati padroni. E tu che mi leggi, ricorda come un popolano, anzi
plebeo, erpicato un dì nei Consigli della Corona, a mo' di zucca sopra
la pergola, immaginasse la vendita dei titoli di nobiltà, e ne
prescrivesse la tariffa: egli pose a prezzo l'onore, nella stessa guisa
che la Curia romana ci aveva messo il paradiso con la vendita delle
indulgenze: così mentre la nuova nobilea niente acquista che turpe
non sia, la vecchia perde il pochissimo lustro che le avanza.
Una volta l'antica nobiltà era in parte rispettata, e col manto orrevole
di fodera di vaio spelacchiata, tanto la sua figura la faceva; adesso la
nuova, infagottata nei mantelli, col soppanno di pelle di gatto di
fresco scorticato, pone parecchia buona gente in sospetto della
propria pelle. Un dì i nobili vecchi disprezzavano i nuovi, e non a
torto: oggi i vecchi ed i nuovi si disprezzano vicendevolmente, e a
ragione. Una volta i nobili vecchi mandavano fuori a correre il palio
titoli e servitù, i nobili nuovi ci hanno aggiunta una puledra che si
chiama Rapina. Affermano che il Giusti (il gran cantore toscano, che
dal bellico in giù fu moderato e dal bellico in su rivoluzionario, fiera
divina [30]), quando cantò di un pirata in cappamagna, pigliasse la
mira sopra un tale dei tali, per me credo ch'egli intendesse
bersagliare tutta la classe dei pubblicani.
Napoleone I, magno conoscitore dei peccati umani, che forse poteva
curare da Dio ed invece volle approfittarsene da tiranno, fomentò il
guazzabuglio fra la nobilea viziosamente spiantata e la nobilea
colpevolmente arricchita; e travasando fanciulle plebee con grosse
doti sulle famiglie feudali, diceva che a cotesta maniera bisognava
letamare l'antica nobiltà sterilita.
Certo, non può negarsi, e' ci ha di quelli i quali si mostrano e sono
alieni davvero da siffatte distinzioni artificiali, ma se tu la squattrini
pel sottile, troverai che a ciò li conduce non mica amore di
uguaglianza, bensì studio di non vedersi menomata la legittima
disuguaglianza da essi ottenuta per opere eccelse o di mano o
d'ingegno, nè vada confusa con la turpe disuguaglianza venduta a
tariffa che del vile anco è fregio [31].
Eccetto questo caso che, raro sempre, ogni dì più si stema, titoli e
croci non furono mai tanto agognati quanto in questi tempi di fior di
democratici, e dai repubblicani larghi di cintura più che più, i.........
informino.

*
I nostri amanti non si erano promessi con parole di legittimare
l'affetto onde si sentivano presi davanti il prete od il notaro, perchè
nell'amore quando è di quello buono, ciò che parla meno sono le
parole: con gli occhi, col sorriso, col tremito, con gli effluvi della
persona se lo dicevano e promettevano sempre. O chi avrebbe
voluto contrastarlo? Ed anco volendo, o chi lo avrebbe potuto? La
signora contessa, madre di Ludovico, lo amava troppo per pensare
nè manco per sogno a far cosa che gli tornasse molesta, cotesti non
sono tiri da mamme amorose, massime se di figli unici; certo ella
aveva preso lingua e le sarebbe stato caro di concimare con più
letame plebeo, che non avrebbe potuto Eponina, la sua casa
sfruttata, ma poi fiat voluntas tua. E quanto a babbo Marcello, non ci
si pensava neppure; di tante cortesie lo colmava, tanto volentieri con
lui si tratteneva, che si giudicava sicuro dovesse parergli toccare il
cielo col dito accasando la sua cara figliuola con Ludovico. O non ci è
un arnese che ci prenunzia il tempo cattivo? Sicuramente che ci è, e
parecchi lo serbano in casa sotto forma di cappuccino, il quale
quando la stagione mette al vento o al piovoso, si incappuccia, e se
al buono, scapucciasi. Ora domando io o perchè non potrebbe essere
corredata del suo barometro anche l'anima? O che difficoltà! Per me
non ce ne vedo alcuna. Ma chi lo ha visto? Come è egli fatto? Chi lo
fabbrica? Oh! se non si vede si sente. Quanto al fabbricante mi
prevarrò dell'arguzia subalpina del ministro Galvagno: Rispondo che
non rispondo. Lepidezza di cui rimase sbigottito quel desso che la
profferì, e parve prodigiosa tanto là nelle parti del Piemonte, che il
Municipio di Torino deliberò conservarla nell'acquavite, allato ai feti
mostruosi, dentro il Museo di Storia naturale.
Fatto sta, che mal sonno aveva dormito Eponina, ed Isabella peggio:
entrambe si erano alzate di pessimo umore: fin lì avevano trascurato
le squisite mondizie della persona, loro cura e delizia. Eponina
trascurò il pappagallo, che indarno ripeteva indiavolato: Eponina!
Eponina! Isabella pestò la zampetta al suo Cialì: la prima erasi
versato addosso la tazza del caffè, l'altra aveva rotto una caraffa di
cristallo. Tutto insomma presagiva un giorno uzziaco. Con sospiro
affannoso le donne aspettavano la posta del mattino, dacchè
Ludovico, quantunque passasse la serata a veglia in casa Marcello,
pure prima di coricarsi scrivesse una epistola erotica, breve o lunga,
conforme gli frullava, e la faceva impostare, ovvero usciva ad
impostarla egli medesimo, onde la fanciulla dell'animo suo la
ricevesse la mattina per tempo: ghiribizzi d'amore.
Queste lettere specificano a parte a parte.... Rassicurati lettore; io
non vo' dirti davvero che cosa e come dicessero; ho fatto per
metterti paura: tu pure ne avrai scritte, rammentale, ed immagina
che quelle del conte non saranno state più argute nè più sceme delle
tue: piacevano a chi le dettava, piacevano a cui le riceveva; contenti
loro, contenti tutti....
— Eccolo! Eccolo! — esclamò Eponina dalla finestra dove si era
affacciata. — Dio mio! fanno la leva dei gottosi per fornire di fattorini
la posta.... è uno scandalo.... ne vo' scrivere al Barbavara.... ed
occorrendo anco al ministro.
Credo inutile dire che il fattorino non era neppure di leva pel servizio
militare, mancandogli giusto otto mesi a compiere venti anni, e lesto
in gamba così da dare tre punti a Mercurio, e le linguaccie dicevano
che la prestezza non era la sola qualità da lui posseduta in comune
con Mercurio.
Il pacchetto è consegnato alla portinaia; questa, punta dalla
padrona, lo porta su di volo: Eponina in capo di scala glielo strappa
di mano e riscontrando foglio per foglio mormora:
— Giornali.... anco giornali.... maledetti quanti giornali vivono al
mondo! (e per questa volta dalla maledizione non rimase escluso
veruno, nemmeno la Novità del Sonzogno, che la Eponina come
patriotta preferiva a qualunque altro giornale di mode parigino)....
Lettere per papà.... una per te, mammà.... per me nulla, o Dio! Nulla
per me.
E la povera giovane sarebbe stramazzata sul pavimento, dove pronta
al soccorso non l'avesse accolta nelle sue braccia la madre: se non
che di corto ripigliava animo come sicura di non potere essere
dimenticata, nè s'ingannò, che scorso un quarto d'ora appena, la
cameriera discreta accostandole le labbra all'orecchio ci susurrò:
— Gaspero l'aspetta di là, in sala, per consegnarle una lettera del
padrone nelle sue proprie mani.
— Che novità son queste! Ditegli che venga qua.
— Gliel'ho già detto, e mi ha risposto avere ordine di parlare a lei
sola.
Eponina ansando va a pigliare la lettera; sul punto di aprirla nota
come Gaspero, dopo fatto un profondo inchino, accennasse a
svignarsela, onde ella imperiosamente gli comandò: — Non vi
movete. E Gaspero di cui il mestiere era obbedire si fermò, perchè
tra l'ordine del padrone un po' stantio, e quello della Eponina fresco
fresco nella cronologia della obbedienza, prevaleva l'ultimo. Eponina
con una ondata di virtù visiva lesse di un tratto:

«Amor mio!
«Che io ti ami non importa dirtelo; chè tu conosci quanto me, forse
meglio di me, che sono cosa interamente tua; quando pure volessi
non potrei dimenticarti, e tu sai se io lo voglia; eppure una terribile
necessità mi stringe la gola sforzandomi a lasciarti. Io mi conserverò
intero all'amor mio, perchè il mio amore è il mio cuore; ma sarei
peggio, che tristo se pretendessi, od anco ti consigliassi a respingere
gli omaggi che ti verranno fatti da altri certo non più devoti, ma più
fortunati di me, Eponina di una cosa ti supplico, ed è non credere
verbo di quanto ti verrà fatto udire a carico dell'onor del tuo
Ludovico. Per le ossa del padre mio, per la vita della madre mia, per
l'amor nostro, io ti giuro che la colpa altrui mi precipita in questa
desolazione. Mentre tu leggerai questa lettera, Milano avrà avuto da
me l'ultimo addio».

— Ho capito — disse imperturbata Eponina e fissando di un tratto gli

occhi dentro gli occhi di Gaspero gli domandò:
— E quando parte il vostro padrone?
Il servo preso così a soqquadro rispose:
— E' non me l'ha anche detto.
— Dunque si trova in casa?
— Oh! no signora, egli è partito.
Allora Eponina, abbrancato con incredibile violenza Gaspero pel
petto, gridò:
— Guai a te se mentisci! Chè dalle tue bugie può uscirne un
precipizio, che i tuoi occhi non basterebbero per piangere; dove si
trova in questo momento Ludovico?
Il servo, conquiso dagli sguardi di Eponina, terribili di amore e di
furore, come persona costretta dal fascino, rispose: — In coscienza
io non lo so, uscì di casa stanotte, e non lo abbiamo visto più. La
signora contessa mi ha ordinato piangendo di fare i bauli per un
viaggio lungo e di portarli a Venezia; la mia partenza è stabilita a
stasera per l'ultima corsa della ferrovia.
— Prendi e bevi — disse Eponina porgendogli una moneta, e l'altro
corrucciato respingendola soggiunse:
— Nè prendo, nè bevo.... palesando il segreto del mio padrone ho
commesso errore, ed ora vuole ella col suo danaro convertirmelo in
colpa?
— Hai ragione, scusa, va'.
Eponina tornata alla madre la mette a parte del successo, e a lei,
che si confessava povera di consiglio, risolutamente favella:
— Madre mia, qui il tempo stringe, e come vedi lo indugio piglia
vizio, io non voglio nè posso essere di altri che di Ludovico; nel mio
amore sta la mia vita; divisa da lui, o ammattisco, o mi ammazzo: le
parole non montano; andiamo a trovare papà, e facciamo in modo
ch'egli acconsenta subito al mio matrimonio con Ludovico; ottenuto il
consenso paterno, lascia a me il pensiero di scovar lui; ci uniremo e
poi partiremo insieme, dacchè io intenda partecipare come moglie
alle sue ree del pari che alle sue prospere fortune.
I gesti e i detti di Eponina soggiogavano, e poi la madre conosceva a
prova l'arduo volere di lei, sicchè estimando ogni opposizione vana,
si piegò ad accompagnarla nello scrittoio del padre.
Ivi rinvennero Marcello, il quale seduto davanti al banco si reggeva la
testa con le mani, in atto di leggere un foglio; da più di un'ora ci
teneva gli occhi su, e una volta in fondo, tornava da capo; al
comparire della moglie e della figlia, tolse via con precipitazione il
foglio, che si ripose in tasca, nel mentre che co' cenni invitava le
donne ad assettarsi: il suo volto era torbido, e taceva; sarebbe
toccato a Isabella incominciare, ma sì, tentava rinvenire il bandolo
della matassa, e non ci riusciva; allora, come sempre, risoluta
Eponina prese a favellare: brevi le sue parole e quasi incise sopra
metallo; la voce stessa rendeva il suono che esce dalle vibrazioni di
una corda metallica.... ma ahimè! non corda di metallo, bensì la più
delicata fra le fibre del suo cuore mandava fuori quel suono infelice.
La udì Marcello, con sembiante di mano in mano più triste; quando
ella ebbe finito il suo discorso, il padre esitò, gli balenarono gli occhi,
aperse le labbra per parlare, ma, appena ne fu uscito un suono
inarticolato, le richiuse; tuttavia gli occhi di Eponina, fitti sopra
Marcello, scottavano; non ci era verso da sottrarsi alla risposta,
ond'egli all'ultimo cupamente sentenziò:
— Prima di saperti moglie a costui io vorrei vederti morta.
— Perchè?
— Tanto ti basti, Eponina, e non costringermi ad affliggerti e ad
affliggermi di più; a questo pensa, che il padre piglia cura della
felicità e della fama della sua figliuola per lo meno quanta ce ne può
pigliare la figliuola stessa.
— E tu, padre, rammenta che io ho coscienza, che io ho volere, e
non posso commettere al giudizio altrui ciò che è sostanza dell'anima
mia; riconosco in te l'autorità di chiarirmi e di consigliarmi, ma volere
e pensare spettano a me.
— E pure bisogna che per questa volta sia così. La causa che mi fa
dire venne confidata all'onore di tuo padre; pretenderesti tu che io
mancassi al mio onore?
— Bisogna.... bisogna... — mormorava Eponina.
E il padre di rincalzo: — E il suo celarsi e il fuggire dello sciagurato
non ti dice nulla, figlia mia?
— Nulla, però che io sappia come noi altri cristiani adoriamo un
innocente, che fu lacerato ed infamato con la morte degli schiavi.
— Eponina! — esclamò il padre, ed aperse le braccia: la figliuola vi si
gettò dentro, ma non piansero, non aggiunsero parola. Tutto quello
che potevano dirsi, si erano detto.
 *
Da parecchi giorni Ludovico Anafesti si trova a Vienna sotto nome
mentito. Senza amicizie, mal pratico del paese, ignaro della lingua
tedesca, non bene fornito a danari, erasi ridotto a stare nel primo
albergo che gli avevano proposto, e quivi viveva di pessima voglia
struggendosi nell'angoscia; pur troppo lo premeva il male e lo
spaventava il peggio. Dalla madre fin qui veruna lettera: mandava
Gaspero due o tre volte il giorno alla posta, e quando il servo dopo
breve ora tornando gli annunziava da lontano: Niente! il capo gli
cascava giù peso sul petto come se glielo avessero empito di
piombo, a mo' che fece il plebeo Settimuleio con quello del suo
amico Caio Gracco. Per ordinario il giovine taceva; però di tratto in
tratto lo spasimo che lo lacerava gli faceva forza a lamentarsi con
parole tronche, nelle quali ricorrevano spesso i nomi di Eponina e
della madre: imprecava al destino che lo aveva forzato ad
abbandonare questi unici amori dell'anima sua: aggiungeva poi non
so che di generosità sprecata..... di condizione insopportabile.....
dando in certa guisa a divedere che l'immane sacrifizio incominciava
a pesargli.
— Senza colpa nè peccato ho perso tutto, — egli diceva — casa,
nome, patria, madre ed Eponina, che a quest'ora, o non pensa a me,
o con orrore ci pensa.... Oh! se tu sapessi quanto patisco per te....
tu mi saresti accanto a temperarmi il fiele che bevo.... Io non sono
avvezzo al dolore, e questo è troppo, ed incomincia adesso.
Gaspero da dieci minuti gli stava impalato davanti, aspettando la
occasione opportuna per favellargli, la quale parendogli ora venuta
incominciò:
— Il padrone della locanda, signor Bruksteiner, persona garbata, mi
ha messo a parte essere uso di questa locanda regolare il conto co'
forestieri che si fermano una volta in capo a dieci giorni; al quale
effetto....
E gli porgeva la nota: Ludovico ci getta gli occhi sopra, e vede che
ella sommava niente meno che a cinquanta fiorini, ond'è che
rendendola a Gaspero, lo avvertì languidamente:
— Gaspero, paga e poi procura subito di trovarmi un albergo di cui il
padrone sia meno garbato, ma più discreto, chè, andando avanti di
questo passo, in poco più di un mese mi troverei al verde.
— O la signora contessa non le diede le gioie? Forse a Vienna le
gioie costano come ghiaie?
— O Gaspero, tu ti hai a rendere capace come nella vendita delle
gioie, quando si scapita un terzo si scapita poco; che se caschi in
mano al giudeo, il quale di questi commerci si è imposto, e noi lo
sopportiamo tiranno, fa' conto che s'ei non le giudicherà ghiaiottoli,
la batterà di lì: e poi io tengo sacri questi ornamenti materni, e
sebbene comprenda che un giorno o l'altro mi toccherà a venderli,
pure io sentirei rimorso ad affrettare la necessità di disfarmene.
Gaspero, provvisto di tanti bei marenghi d'oro, andava alla volta del
locandiere garbato, nè stette molto a tornarsene tutto raggiante
verso il suo padrone, nel cavo della mano manca stringeva tuttavia i
marenghi di Ludovico, e con la destra agitava un borsellino di
moneta; era fuori di sè dall'allegrezza (perchè se il vino letifica il
cuore dell'uomo, a mille doppi lo esalta il danaro, col quale si compra
e vino e pane e carne e ogni altra cosa) e con voce che aveva preso
l'argentino del metallo ragguagliava il padrone, come il sig.
Bruksteiner, proprietario della locanda l'Aquila Imperiale, avesse in
quella stessa mattina ricevuta la somma di franchi duemila da
consegnarsi al signor Giulio Bonatti; ond'è, che prelevatine duecento
in saldo del conto, gliene contava milleottocento, dei quali, pregava
gli facesse un bocconcino di riscontro, per sua regola.
— Ma io non li aspettava....
— In coscienza, signor Ludovico, che sia benedetto, le pare questa
una buona ragione per rifiutarli?
— Non ho detto questo: temo ci sia equivoco e non vorrei....
— In quanto a questo, dorma fra due guanciali; il signor Bruksteiner
ha scritto sopra i suoi registri, e l'ho visto io con questi occhi
veggenti: Ricevuto da M. Hans Kreutzer franchi duemila in oro da
passarsi al signor Giulio Bonatti, n. 8. Tante cose ci tocca a pigliare,
che non aspettiamo e che non vorremmo pigliare, che la sarebbe
bella respingere i quattrini, che pigliamo più che volentieri. Dia retta
a me, li pigli addirittura, chè per me, mi pare di vederlo, li manda la
sua signora madre.
— No, Gaspero, non vengono da mammà; me lo dice il cuore; non
mica perchè non volesse, ma perchè temo, poveretta! che non
possa.
— O chi vuole che, a questi lumi di luna, mandi a spasso duemila
franchi, se non è la madre?
— Sta' zitto; pochi amori vincono quello della madre, pure ve ne ha
uno che vince anco lui.

*
Compiacetevi ripassare le Alpi e tornar meco a Milano. Eponina è
sparita. Dopo le novissime parole favellate col padre suo, ella parve
tranquillarsi: alla madre, che blanda industriavasi consolarla, rispose:
— Non fa caso; vedi, io non mi sgomento; ho fede nella innocenza
del mio sposo Ludovico; e il tempo la chiarirà.
Convenne a mensa insieme con gli altri; certo, se si dicesse che il
pranzo fu lieto, sarebbe bugia, ma nè anco fu tristo come si
presagiva: più confusi degli altri apparvero Marcello ed Isabella, i
quali, quantunque amassero del pari tutti i loro figliuoli, pure della
Eponina andavano orgogliosi, però ebbero caro che, terminato il
pranzo, ella proponesse di recarsi a veglia dalla signora Claudia tanto
per isvagarsi.
— Va' pure, le dissero a coro i parenti, e fa' di cacciare i tristi
pensieri, pensando che dopo il tempo cattivo ne viene il buono.
Tu ti rammenti sicuramente, mio diletto lettore, di donna Claudia? La
zia biscottina del rompicollo il quale poneva ogni sua speranza, per
rammendare gli strappi fatti nel proprio patrimonio, nella eredità di
lei? Questa signora abitava un quartiere nel medesimo palazzo dove
aveva stanza Marcello. La signora Claudia in gioventù coltivò
parecchie maniere di amori; il suo cuore era un porto capace per
tutti; nella lunga navigazione della vita aveva dovuto far getto ora di
questo ora di quell'altro amore, ma però ne aveva conservati due più
preziosi di tutti, co' quali costa costa ella si augurava riparare in
braccio alla divina provvidenza, voglio dire, l'amore dei biscottini con
la cioccolata e quello di santa madre Chiesa, la quale, come ognuno
sa, è sposa legittima di Gesù Cristo, redentore nostro. Costei era un
po' maligna, un po' linguaggia, anco un zinzino scandalosa; la
tacciavano altresì di avarizia, ma per acquistarsi la gloria del paradiso
non intendeva miserie, sparnazzava alla grande; del rimanente pulita
come una gatta, bella favellatrice e dama di tratto signorile; si
mostrava svisceratissima per la Isabella, che assai aveva usanza con
le figliuole in casa sua, e la signora Claudia accoglieva tutte con
festa, ma sua delizia era Arria. Questa ogni dì per non poche ore se
ne stava allato a lei, ed ella l'ammaestrava nell'arte del ricamo in
seta ed in oro, nel fabbricare fiori artificiali e a miniare Gesù
bambino e i santi, cose tutte nelle quali riputavasi ed era certamente
valentissima.
Arria, secondo il consueto, in cotesto dì, dopo le nove di sera tornò
alla casa paterna: interrogata perchè non fosse venuta seco
Eponina, ebbe a rispondere non averla veduta dal pranzo in fuori, nè
in casa della signora Claudia esserci punto stata: dapprima
crederono che parlasse per celia, ma e' fu uno istante, chè tosto
subentrava la dolorosa realtà.
A cui legge riuscirà più agevole immaginare la desolazione della
famiglia, che a me descriverla; però me ne passo. Le fantasie
germogliavano, si urtavano nel cervello di quella povera gente, e via
via più angosciose: più delle altre importuna ricorreva quella che
disperata avesse posto fine ai suoi giorni, onde Marcello, che se ne
chiamava in colpa, guaiva come se lo trafiggesse il male dei denti nel
cuore; anche egli voleva darsi moto, ma non avendo balìa di reggersi
in piedi stramazzava, nè gli altri, compresi interamente dal proprio
affanno, badavano a lui; correvano privi di consiglio; i parenti e gli
amici convenuti a casa durarono tutta la notte nella ricerca piena di
agonia, e non venne lor fatto rinvenire nulla, come accade sempre
quando la mente si volta tutta ad un punto che non è il vero. Chi
può ridire le ansie di cotesta notte? Chi lo spasimo dei genitori? Chi
le smanie di tutti? La mattina si radunarono in casa Marcello:
tampoco se si fossero incontrati altrove si sarebbero riconosciuti,
tanto apparivano nelle sembianze mutati. Rovistata da cima in fondo
la camera di Eponina, non occorsero in iscritto, ovvero in indizio altro
qualunque, capace di fornire lume: giunse la posta, e con la posta,
bontà di Dio! una lettera, la quale, sebbene sconfortante, di fronte
allo sgomento che li travagliava, parve sollievo.

La lettera di Eponina diceva così:

«Io corro sopra le traccie dello sposo che la mia anima si è eletto per
istarmi con lui e partecipare le sue fortune. Per me lo stimo, anzi lo
so innocente di qualunque colpa, che altri, o illuso o perfido, possa
apporgli: e fosse anche reo, la parte della donna è quella di portare
coraggiosamente la croce del marito. A Maria bastò l'anima per
accompagnare Gesù al patibolo e per consolarne l'agonia: ora nel
patire, tutte le donne hanno da sentirsi Marie: che, se ella era madre
io sono sposa; e questo amore o supera quello o lo ragguaglia. Non
porto invano il nome di Eponina. Ad ogni modo chi accusa e
condanna deve provare la colpa; e trattandosi d'indurmi a pestare il
capo di persona a me congiunta coi vincoli più solenni, che
conoscano le creature umane, io non devo, nè posso starmene al
giudizio altrui: se lo facessi, sarebbe viltà, se altri lo pretendesse,
commetterebbe ingiustizia. Considerati i nostri tempi in confronto
agli antichi, oggi il padre che impone alla figlia di spegnere il suo
amore già consentito e benedetto da lui, solo per cieca obbedienza
alla autorità paterna, è più tiranno del padre romano, al quale si
concedeva la vendita dei figli sanguinolenti.»

Il povero Marcello nel sentirsi trattare da tiranno levò le mani al cielo

e diede in un sospiro desolato, tuttavia giova osservare che qualche
volta anco la buona gente, senza accorgersene, passa il segno
reputando che la intenzione benevola temperi la rigidezza del
comando.

*
Dei fratelli di Eponina fin qui non toccammo del minore; ma siccome
egli sa essere una delle dramatis personae ed anco delle più
importanti, così si strugge fra le quinte e si arrabatta per uscirne
fuori a recitare la sua parte: per me non lo tengo, esca pure, ma
prima di entrare in iscena mi permetta che io gli serva da Cicerone,
affinchè i lettori apprendano a conoscerlo per di dentro e per di
fuori. Curio nelle forme del corpo comparisce affatto diverso dai suoi
fratelli; e come questo possa succedere laddove ogni sospetto di
contrabbando sociale viene meno, domandatelo a cui lo può sapere,
e non a me, che non ne so nulla. Egli era pertanto di statura
mezzana, tarchiato e forte a meraviglia; neri gli occhi ed i capelli; la
crescente lanugine sopra le guancie pur nera; acceso in volto, che ad
ogni lieve commozione gli divampava; le labbra tremule come gli
occhi, sempre in procinto di mandare baleni; svelto, veloce al corso,
agile ai salti, cacciatore perpetuo, tiratore unico: sciabole e spade
più frequenti in sua mano, che penna o libri: anco di musica egli
sapeva, ma impaziente ad osservare la misura, lo scartavano sempre
dalla orchestra: sonava il flauto o la tromba proprio per le Muse e
per sè, imperciocchè non ci fosse verso che alcuno si fermasse per
ascoltarlo. Per confessione di quanti lo conoscevano, egli superava i
suoi fratelli in bontà e in ingegno: tuttavia non passava giorno che
qualcheduno non conciasse pel dì delle feste, e ciò perchè essendo
pronto di parole, e più di mano, mutava subito le controversie in
contesa: siccome poi la esperienza gli aveva insegnato come le
sassate di colta sieno quelle che contano, così di rado si trovava
secondo a menare le mani; però, appena vinto od anche offeso
l'avversario, sboglientiva subito e tu lo vedevi affannarglisi attorno
amoroso per consolarlo o medicarlo: nè per repulse si ristava, nè per
ingiurie e nè anco per battiture; a patto però che non fossero
troppe, nè troppo sode. Poichè in tutte le guerre si portano due
sacca, cioè quella del dare e l'altra del riscotere, egli ne riscoteva e
spesso: allora con la faccia grondante sangue ei non voleva che
alcuno lo curasse, se prima non avesse lavato e fasciato lo
avversario e gli avesse chiesto ed ottenuto il perdono, sicchè nei
presenti talora, più che altro, mosse il riso, e, strano a dirsi, a lui
fruttarono più amici i pugni dei baci. Se taluno dei conoscenti cadeva
gravemente infermo, egli, finchè durava il pericolo, lo vegliava la
notte; e se moriva, egli lì a lavarlo, a vestirlo, a deporlo nella cassa,
ad accompagnarlo alla fossa. Nel donare piuttosto eccessivo che
largo: sovente anche nella crudissima stagione tornò alla madre in
giubba nera, scarpe, calze e cappello, ma senza calzoni, però che
nello androne di casa se li fosse levati per vestirne un tapino che
moriva di freddo: quanto a danari le sue mani simili a vagli; per la
quale cosa la madre, intantochè lo riforniva di quattrini, lo
rimproverava dicendo: «Ma, Curio mio a questo modo tu darai fondo
ad una nave di sughero.»
Rispetto a scienze, s'egli avesse potuto imparare passeggiando,
come certamente fece Alessandro Magno sotto Aristotele, maestro
dei peripatetici [32], sarebbe riuscito più cosa di lui; ma fossero pure
le sedie sulle quali assettavasi imbottite e coperte di velluto, ei le
avrebbe provate intollerabili come pettini da lino: di percezione
rapidissima, chiappava la scienza a volo, o non la chiappava più;
apprendere per lui era come un buttare la moneta all'aria giocando
ad arme o testa; e tuttavolta, quantunque la fantasia gli bollisse
sempre come una caldaia a vapore, le scienze di calcolo gli
talentavano sopra tutte le altre: lo ingegno possiede le sue
contradizioni come il cuore.
Dicitore parco e preciso; e tanto più preciso quanto più gli
sconvolgeva la mente la procella della passione; vero vulcano di
Ecla, il quale ha fuoco dentro e in vetta la neve. Rispetto allo amore
per la Libertà basti dirne tanto che alla madre, la quale egli adorava
come cosa santa, certo dì che lo interrogava chi più amasse nel
mondo, rispose: la Libertà; ed insistendo ella: anche più di tua
madre? Egli esitò, si fece pallido, poi ridivenuto vermiglio
risolutamente confermò: più della madre.
Ed ora che ho fatto conoscere il mio Curio, spieghi l'ale e voli.
Quando questo giovane si fu persuaso che Eponina e Ludovico non
si erano abbandonati alla disperazione, si mise a ricercare
sottilmente dove si fossero ridotti, però che a lui paresse chiaro che i
due amanti avessero dovuto fuggire di conserva; ma siccome egli
s'ingannava nel suo supposto, così non gli venne fatto scoprire
traccia della sorella; al contrario di Ludovico, e poichè simile esito lo
confermò nel suo concetto, decise di mettersi in via per iscovarlo.
Chiesta ed ottenuta licenza, la quale tanto più volentieri gli
concessero, quanto che se la sarebbe presa da sè, dove
gliel'avessero negata, provvisto di denaro e di preghiere a procedere
prudentemente, egli partiva pel suo viaggio di scoperta.
E adesso vediamo la prudenza di Curio.
Fino a Venezia egli andò a posta sicura; a Venezia ebbe a trattenersi
alquanto per rinvenire l'orma smarrita, la quale in breve ritrovata,
subito corse a Vienna; appena giunto, eccolo a rovistare caffè, teatri
e locande, ma gli venne meno il tempo e le gambe; cadde svenuto
come quello che da più dì non aveva mangiato nè dormito: per
ventura questo accidente lo colse in una locanda dove facilmente
potè sopperire al suo bisogno: ricreato di forze, la mattina si ripose
in giro per tempissimo: oggi la fortuna gli arride più propizia; sullo
entrare nello albergo L'Aquila Imperiale, sbircia Gaspero, di cui gli
occhi s'incontrano per lo appunto co' suoi. Gaspero, nel presagio
della mala parata, s'industria sguizzare, volta la persona di scancio e
prende a camminare di traverso, a mo' dei granchi, ma Curio dietro;
insieme essi vanno su per le scale, insieme per le anticamere,
insieme pei corridoi, dove l'uno lascia l'orma l'altro mette il piede,
finchè Gaspero giunto all'uscio della stanza del suo padrone, quivi si
ferma e sta. Sopraggiunge Curio, che gli domanda:
— Dov'è il tuo padrone?
— Non sono obbligato a risponderle.
— Levati di costì e lasciami passare.
— Io non mi muovo e non la lascio passare.
— No?
L'uscio della camera schiantato dagli arpioni si apre strepitosamente,
e ruzzolano in un fascio sul tappeto insieme attaccati Gaspero e
Curio.
Ludovico, comecchè desto, stavasene supino a letto, senza neanche
il refrigerio del poeta Berni, il quale in quel medesimo atto per
passare la mattana contava i travicelli del soffitto, perchè il palco
della camera appariva stoiato e dipinto con uno stormo di amori, i
quali tiravano a segno sopra l'ospite come per avvezzarlo con la
minaccia dei loro strali agli altri più pungenti che l'oste gli
apparecchiava coi suoi conti; al rumore del tracollo egli saltò giù da
letto e, dopo rampognato acerbamente Gaspero, gli ordinava
uscisse, chiudesse la porta in fondo del corridore e colà si piantasse
di sentinella, impedendo la entrata a chi venisse a disturbarli.
Appena Gaspero aveva avuto tempo di eseguire i comandamenti di
Ludovico, che Curio, sempre in virtù della racomandatagli prudenza,
salta al collo del conte, lo scaraventa sul letto, e stringendogli la gola
da fargli schizzare gli occhi dalla fronte, digrignando i denti, urla:
— Scellerato, che hai tu fatto della mia sorella? Dov'è Eponina?
A Ludovico non riusciva articolare parola; appena poteva mandare
fuori un rantolo; sforzandosi sgusciargli di sotto, ma era niente, ogni
conato per liberare la strozza dalla fiera tanaglia gli tornava in
peggio, però che l'altro stringesse più forte. Ormai la faccia di
Ludovico era divenuta tra rossa e pavonazza (avrei potuto dire con
una parola sola infaonata, ma correva rischio di buscarmi di pedante
e non essere capito da veruno) gli balenavano gli occhi esterrefatti, e
Curio procedeva a strangolarlo, con la devozione con la quale il prete
novizio celebra la sua prima messa. Se la fortuna qui non ci mette le
mani, la vita di Ludovico è giunta al Laus Deo.
E la fortuna ce la mise. Ti ricordi aver letto (e se non lo hai letto
vallo a leggere), nella Iliade di Pallade-Minerva, che agguanta pei
capelli il piè-veloce Achille in procinto di avventarsi contro
Agamennone, re dei re, dopo averlo salutato di cuore di cervo e di
muso di cane? [33] Così per lo appunto accadde a Curio che,
sentendosi strappare i capelli dalla nuca, si voltò addietro e vide...
che vide egli mai? Vide Eponina in carne ed ossa, la quale sapendo il
fratel suo non nato da regio sangue non si permise adoperare i titoli
dati dal figliuol di Peleo al divo Atride; bensì di bestia e di insensato il
dabben Curio ne ebbe quanto ne volle....
— Ecco le solite fole da romanzieri! — esclama la signora Verdiana,
penitente di don Formicola, curato di San Satiro. — O come la
scapestrata Eponina era piovuta là dentro? Chi ce l'aveva portata? —
La non s'inquieti, signora Verdiana, e senta me. Veruno ci aveva
portato Eponina, perchè ci si era condotta da sè ed ecco come: la
povera giovane invece di recarsi a veglia dalla signora Claudia, toltasi
seco quanta più moneta poteva ed in buon dato gioie, doni dei suoi
parenti e di amici ammiratori della virtù di lei, si recò a casa di certa
amica del cuore, o se ella vuole, d'ingegno scapestrato come il suo,
e questa l'aiutò a travestirsi ad accertarle il viaggio ed a partire.
La medesima sera Eponina lasciò Milano col traino stesso sul quale
partiva Ludovico; con lui, senza che ei se ne accorgesse, scese a
Venezia, con lui continuò il viaggio e giunse a Vienna; colà fece in
modo di aver la stanza contigua a quella di Ludovico, divisa solo da
sottile parete dove era una porta di cui ella volle la chiave; e siccome
favellava stupendamente il tedesco e pagava alla grande, così non è
a dire se le facessero festa, e ai suoi voleri più che volentieri
soddisfacessero. Raccomandò al cameriere di affermare vuota la
camera abitata da lei, e la raccomandazione accompagnò con un
marengo: il cameriere, uso a dire tante bugiarderie gratis, lascio
considerare a voi se ci s'inducesse pagato! La servì a pennello, molto
più che piace a tutti gratificarsi la bellezza, ed Eponina era
bellissima; chiusa nella sua cameretta, ella gustò dolcezze ineffabili,
quali solo può immaginare il cuore di donna innamorata... ed il suo,
signora Verdiana; però che Eponina udisse sovente rammentare il
proprio nome, tra lacrime e sospiri del giovane amato, e se non
giunse ad avere contezza piena del caso che glielo svelse dal fianco,
almeno si confermò nella fiducia della sua innocenza; per la
medesima via, conosciute le angustie di lui, le sovvenne mettendosi
d'accordo col padrone della locanda, l'onesto Bruksteiner, il quale
trovandoci il suo conto la servì a braccia quadre ed ebbe per giunta
un sorriso in pagamento, che, se avesse potuto, egli avria messo nel
barattolo delle ciliege per conservarlo nello spirito.
Per le quali ragioni, ella vede bene, signora Verdiana, che la
presenza della Eponina giusto nel punto d'impedire uno sciaratto, si
spiega naturalmente senza miracoli: e caso mai ci fosse mestieri
miracolo, o che la opposizione dovrebbe muovere proprio da lei? Da
lei che crede come articolo di fede che sant'Antonio da Padova si
trovasse nel punto stesso a Padova e a Lisbona!

*
Mentre Ludovico si stropiccia il collo e fa prova di tossire onde
assicurarsi che nella gola non ci ha nulla di guasto, Curio sempre
ardente come tizzo acceso così rampogna la sorella:
— Ahi! trista, chi mai avrebbe detto che a te basterebbe il cuore di
fuggire via da noi, che ti amavamo come la pupilla degli occhi?
Senza rispetto pei parenti, senza vergogna per te, tu ti sei messa
dietro al tuo seduttore; ahimè! a vederti mi trovo costretto a
coprirmi la faccia.
— Curio, — rispose Eponina, ficcando i suoi occhi dentro gli occhi del
fratello — tu sei giovane troppo ed inesperto delle passioni umane
per erigerti giudice delle medesime: tuttavia sappi che la seduzione è
parola vuota di senso: vivi e proverai; la donna conosce ottimamente
quello che fa, e sebbene paia talora che si governi per moto
improvviso dell'animo, va' sicuro, che ella ha pensato più di una volta
a quello che intende di fare: se poi ella s'inganna, ciò avviene perchè
i ragionieri stessi nei loro calcoli sbagliano: ad ogni modo, io non mi
sento donna da lasciarmi sedurre. Ho seguìto Ludovico, lui
inconsapevole, egli ignorava la mia partenza da casa e la mia
presenza qui; ora per la prima volta gli apparisco davanti, e se la tua
avventatezza non era, non mi avrebbe mai vista: però io voglio che
tu sappia che lo considero come sposo dell'anima mia e intendo
essere sua per la vita. E poichè questo avrei fatto anco sapendolo
colpevole, tanto più mi tengo obbligata di farlo adesso che,
quantunque al buio del suo segreto, pure lo so innocente ed
infelice... Fratello Curio... giungono questi sensi così nuovi al tuo
cuore che ti abbisognino maggiori spiegazioni?
Così avendo favellato, Eponina sorrise blanda al suo Ludovico e gli
porse la mano in segno di pace; cui egli si recò alla bocca
coprendola di baci, ma più di lacrime assai. Curio trasognato
guardava un po' l'uno, un po' l'altra; lungamente tacque e parve
meditare; all'ultimo proruppe:
— Orsù vi credo; maledico la mia furia e vi domando perdono.
Ludovico, ti ho fatto male? Lasciami guardare un po'.... ti è rimasta
una striscia rossa, ma non è nulla, sai.
— Certo.... certo.... gusto non ce l'ho avuto.... ma non pigliartene
pensiero; con un po' di gargarismo spero uscirne.
— Bene, per ora addio, tornerò a parlarti, perchè parlarti mi bisogna,
quando ti sarai rimesso in sesto.
— Accomodati come ti piace, ma per me se tu parlassi addirittura,
l'avrei caro....
— Magari! e in due parole mi sbrigo. O perchè non ti sposi Eponina e
poi senza tanti andirivieni ve ne tornate tutti e due a casa?
— Perchè non posso.
— O come non puoi? E chi ti tiene?
— Il debito di un uomo onorato, intendimi bene, Curio, m'impedisce
sposare tua sorella, che amo quanto me stesso, mi divide dalla
madre e dalla patria, a me, dopo Eponina, sopra ogni altra cosa
dilette.
— Arzigogoli! Senti una cosa, Ludovico: o tu sposi Eponina, o io ti
ammazzo.
— Ecco daccapo la bestia che ti piglia il sopravvento — disse
Eponina — guardami e considera se ci può essere donna al mondo
più dolorosa di me: il padre potrebbe consentire le mie nozze con
Ludovico, e non vuole; Ludovico le vorrebbe, e non può; ne
domando la ragione ad ambedue, ed ambedue me la negano, come
se non ci andasse di mezzo l'anima mia, ed io accetto rassegnata il
mio destino di donna, che è quello di nascere, soffrire e morire.
— Quanto al nascere ci ho già consentito e quanto a morire quasi
assicuro che a suo tempo ci acconsentirò, ma circa al soffrire io
voglio avere le braccia libere. Pertanto, Ludovico, mettiamo le
minaccie da parte, molto più perchè adesso che ci penso,
quantunque io ti abbia detto che ti ammazzerei, potrebbe darsi
benissimo che tu ammazzassi me.
Or via, ragioniamo; tu sei giovane onesto, almeno fin qui ti conobbi
tale; però credo indovinare che qualche riguardo o impegno grosso ti
faccia impedimento a palesarmi la causa che ti muove ad agire come
fai: però tu stesso devi conoscere che questo negozio così per aria
non può stare: considera se ti convenga aprirtene con qualcheduno;
già s'intende sotto sigillo di confessione e con promessa solenne di
silenzio assoluto. Tu designa persona, la quale non dubito che per la
sua onoratezza piacerà ad Eponina ed a me; tu la informerai e noi
staremo a quanto giudicherà, taciuti i motivi del suo giudizio;
insomma basterà che ci dica: Ludovico ha ragione; noi allora
piegheremo il capo alla sorte maligna, la quale pur troppo ne può
più di noi.
— E tu accetti il partito? — chiese Ludovico ad Eponina.
E questa gli rispose:
— Poichè tu non vuoi riporre la tua fiducia in me, mi adatterò.
— Ebbene datemi un'ora per pensarci su, che per me la è faccenda
gravissima: per altri d'importanza suprema; ho bisogno di
raccogliermi; lasciatemi solo.
Eponina e Curio si ritirarono; anzi per somma delicatezza uscirono
entrambi; e così ella per la prima volta vide le strade di Vienna.
Trascorse due ore e più, si ridussero da capo allo albergo, dove
rinvennero Ludovico dolente in vista, ma pure risoluto, il quale disse:
— Sta bene: voleva non farlo, anzi mi era meco stesso obbligato a
non farlo, ma Curio ha profferito una savia parola: la malignità della
sorte ne può più di noi. Qui però siamo stranieri, non conosciamo
persona in cui ci potessimo fidare: e per giunta io non conosco la
lingua del paese.
— Dunque? — interrogò Curio a cui subito era saltata la mosca al
naso.
— Dunque — riprese umile Ludovico — ho pensato, che la persona
più acconcia a ricevere ed a custodire il mio segreto sei tu, e tu il più
atto ad ottenermi fiducia da Eponina.
— Io? — replicò Curio esitando, ma poi aggiunse: — Bene, sia: a
quando?
— Subito.
— Dove?
— Qui o altrove a tuo piacimento.
— Usciamo.
— Usciamo.
Eponina desolata li vide partire, e col cuore ancora più chiuso dopo
breve spazio di tempo li vide tornare: si tenevano a braccetto per
non traballare; le faccie e i colli a terra come se li gravasse un
medesimo giogo d'ineffabile affanno. Curio non trovava bandolo per
cominciare, Eponina per chiedere; un gemito di lei tenne luogo di
domanda. Allora Curio reggendosi con la destra ad una tavola a voce
fioca favellò:
— Sorella, Ludovico ha ragione; nel rifiutare le tue nozze egli fa
prova di rettitudine e di gentilezza. La sorte maligna ci vince. Io
quanto posso ti scongiuro, sorella, di tornartene a casa: là nelle
braccia di nostra madre riparati, finchè la tempesta duri e, se non
cessasse, morite almeno consolate con la mutua pietà. Dammi,
sorella un abbraccio; dammi un bacio.... venti baci, e addio.
— Ed ora dove vai, Curio? — domanda con crescente angoscia
Eponina.
— La gioventù italiana è corsa alla chiamata del Garibaldi nel Tirolo a
combattere le ultime battaglie della patria: io vado a cercarvi la
morte.
Ed avventatosi al collo della sorella, dopo averla baciata e ribaciata,
corre via precipitoso; senonchè, giunto in fondo al corridore, ritorna
sopra i suoi passi ed affacciato all'uscio della stanza di Eponina,
esclama:
— E rammentati bene, Eponina, che io, tuo fratello, ti faccio
testimonianza come Ludovico, ricusando di palesare a te e ad altri le
accuse che lo movono a respingere le tue nozze, dimostra tale
generosità di cui non avrei mai creduto capace la creatura umana.
Ludovico, addio, e tu pure rammenta che sei andato troppo in su per
durarci un pezzo: chi la piglia troppo alta ordinariamente fa stecca.

FINE DEL VOLUME PRIMO.

 NOTE:

1. Ferecide filosofo fu maestro di Pittagora; e che i pidocchi non allignino

sopra gli asini e sopra i montoni lo attesta Plinio; Stis. Mundi, l. II, cap. 39.
2. Demetrio Falereo ebbe 300 statue, le quali, lui vivente, furono tutte in un
giorno atterrate.
3. Tucidide, Stor., VIII.
4. Marrone chiamano in commercio il sensale non autorizzato ad esercitare il
prossenetico, e la legge dichiara nulli i contratti, o partiti, che i mercanti
stipulano a mediazione di loro.
5. Anche nei tempi antichi costumò la confessione, imperciocchè ai sacerdoti
importasse sempre per le medesime ragioni sapere i fatti altrui; tuttavia anco
in cotesti tempi non mancarono intelletti, i quali con un po' di senno si
ribellassero a cotesto gravissimo giogo pretesco. Lisandro essendosi recato in
Samotracia per ottenere certa risposta dall'Oracolo, disse al sacerdote, il quale
gli faceva pressa dintorno affinchè gli confessasse se non tutti, almeno il più
reo peccato ch'egli avesse commesso: «Bene sta, o sacerdote, ma in virtù di
che mi comandi tu questo? Sei tu che lo vuoi, ovvero gli Dei?» «Gli Dei —
rispose il sacerdote.» «Così stando le cose — ripiglia Lisandro — ritirati da
parte, e se gli Dei me lo comanderanno, io obbedirò loro come conviene.» Ed
Antalcida, del pari confortato a rivelare le colpe commesse durante la sua vita,
rispose: «Questo volete per voi, imperciocchè gli Dei se alcuna ne commisi la
sanno.» Nè mancano altri, ma bastano questi riportati da Plutarco nel Trattato
degli Apoftegmi e detti notabili dei Lacedemoni.
6. La dominazione francese cessò non per virtù nostra, ma per infelicità altrui,
epperò senza veruna sicurezza di libertà vera. A Roma andammo, ma sarebbe
troppo più lo scapito che il guadagno se ci avessimo a stare ai patti proposti
dal Governo.
7. Nel giornale Gli Stati Uniti, che si stampa a Ginevra, n. 3, anno III,
occorrono notizie che importa propagare. G. Battista Say nel Trattato di
Economia fino dal 1819 scriveva: «Gl'imprestiti, arme più funesta assai della
polvere da cannone della quale si potranno servire lungo tempo, giusto in
virtù dello abuso che ne fanno... Così facile mezzo offre il credito pubblico alla
dissipazione dei grandi capitali, che molti pubblicisti sono condotti ad
estimarlo perniciosissimo ai popoli. Il Governo potente per facoltà di pigliare
quattrini in prestito si mescola in tutti gl'interessi politici, sicchè assumendo
imprese gigantesche, le quali mettono capo così alla gloria come al vituperio,
genera sempre lo sfinimento; egli combatte le guerre o le fa combattere; egli
compra tutto quanto può comprare, perfino il sangue e la coscienza degli
uomini; allora l'ambizione, l'orgoglio e la perversità nelle mani loro raccolgono
i capitali, i frutti della industria onesta e della buona condotta....» Il signor
Larroque ha stampato un'opera intitolata Della guerra e degli eserciti
permanenti, donde estraggo questi fatti.
Soldati di terra e di mare, eccettuate guardie nazionali, milizie, riserve,
landewers, 5,157,099.
Perdita di somme corrispondenti al guadagno del lavoro di cotesti uomini L.
3,202,985,500.
Valore improduttivo dei mobili e degl'immobili per uso della guerra L.
19,535,000,000; interesse su questa somma al 4 per cento L. 781,100,000.
Debiti cagionati dalla guerra L. 68,304,844,187; interessi sopra questa somma
L. 2,716,905,529.
Spesa annuale di guerra L. 9,818,853,968, vale a dire che consuma 19/20
della entrata generale, che somma a L. 10,116,294,065; ed in parecchi Stati le
spese di guerra superano la medesima entrata.
Tolta questa enorme spesa, venduti gl'immobili e parte dei mobili guerreschi,
ecco nel giro di pochi anni estinto il debito europeo, fondati istituti, industrie
promosse, lavoro accertato, moltiplicate le vie del guadagno, e spenti la
ignoranza, la miseria e il delitto: o diminuiti assai con continua speranza di
meglio, essendo rimosse a tutti questi guai le cause di esistere. Così si
pareggiano i bilanci, le altre le sono ciurmerie, nè manco credute da cui le
propone.
Ai posteri lasceremo eredità di debiti, e perchè mai non potranno accettarla
con benefizio d'inventario? Ma, osserverà taluno, non tutte le spese riuscirono
o disutili, o dannose, come strade, porti, acquedotti, e via discorrendo: bene
sta: scevreranno le une dalle altre, e mentre pagheranno le seconde,
lasceranno le prime a carico di cui le fece.
Tre o quattro monarchi investiti del diritto di fare la guerra adesso possono, se
li piglia la mattana, spingere 10 milioni di uomini a sgozzarsi fra loro: — così
essendo, vi par egli che possano durare le monarchie, costituzionali o no,
imperciocchè anco il re costituzionale possieda il diritto di bandire la guerra?
8. Di Teramene parla a lungo Senofonte nel libro II delle Storie: fu capitano di
mare, e negoziò la dura pace tra Sparta e Atene, onde vennero addosso a
Welcome to our website – the perfect destination for book lovers and
knowledge seekers. We believe that every book holds a new world,
offering opportunities for learning, discovery, and personal growth.
That’s why we are dedicated to bringing you a diverse collection of
books, ranging from classic literature and specialized publications to
self-development guides and children's books.

More than just a book-buying platform, we strive to be a bridge

connecting you with timeless cultural and intellectual values. With an
elegant, user-friendly interface and a smart search system, you can
quickly find the books that best suit your interests. Additionally,
our special promotions and home delivery services help you save time
and fully enjoy the joy of reading.

Join us on a journey of knowledge exploration, passion nurturing, and

personal growth every day!

ebookbell.com