Microbial Biosensors 1298057903 Phpapp02
Microbial Biosensors 1298057903 Phpapp02
⦿ Health Care
⚫ Measurement of Metabolites
⚫ Market Potential
⚫ Diabetes
⚫ Insulin Therapy
⚫ Artificial Pancreas
⦿ Military Application
⦿ Environmental Monitoring
⚫ Air and Water Monitoring
in : Biosensors (www.egtoget.com) 2
The ability to determine the occurrence of food
contamination due to foodborne pathogens at
every stage of food production, processing, and
distribution is crucial to improving the safety of
our food supply.
There are more than 250 known food borne
diseases caused by bacterial and viral infections in
the United States.
Annually, these foodborne diseases result in an
estimated 76 million illnesses, 325,000
hospitalizations, 5,000 deaths, and 6 billions
dollars in unneeded expenditure. Bacterial
contamination accounts for 91% of total
foodborne diseases.
Salmonella sp., Escherichia coli, Listeria
monocytogenes, Staphylococcus aureus,
Campylobacter jejuni, Campylobacter coli
and Bacillus cereus were found to be main source
of bacterial contaminations in our food supply.
3
5
6
⦿ A microbial biosensor consists of a transducer in conjunction
with immobilised viable or non-viable microbial cells. Non-
viable cells obtained after permeabilisation or whole cells
containing periplasmic enzymes have mostly been used as
an economical substitute for enzymes. Viable cells make use
of the respiratory and metabolic functions of the cell, the
analyte to be monitored being either a substrate or an
inhibitor of these processes.
Bioluminescence-based microbial biosensors have also been
developed using genetically engineered
microorganisms constructed by fusing the lux gene
with an inducible gene promoter for toxicity and
bioavailability testing (7).
7
Advantage
s• able to metabolise a wide range of chemical
compounds
• have a great capacity to adapt to adverse
conditions and to develop the ability to degrade
new molecules with time.
• are also amenable for genetic modifications
through mutation or through recombinant DNA
technology and serve as an economical source of
intracellular enzymes.
8
یکیژولویب رصانع عاونا
9
ه د ش صال خ ی ا ه میزنآ
10
یاهلولس
لماک
⦿ ه دنز ری غ م ه و ه دنز مرف ر د مل ه لل م اک ی ا هل وللس
ت ی م ه ا هدنز یاهلولس .دنریگیم رارق هدافتس ا د ر و م
دنراد اهروسنسویب تخا لس رد یا هل ظ ح ل م ل لباق ار یفلت خ م .
ک لین اگرا ی ا هبیک رت ه دنز ی ا هب و ر کی م ر جن م یز ا و ه ری غ
ای یز ا و ه تر وص ب .د ن کی م زی ل وب ات م دیس کا ی د ،
ک اینومآ هللمج زا ی تلولصحم دلیلوتهب ل دب م زا ه د افتسا اب هک
د وشی م ...و ا ه دی س ا ،ن ب رک ین ا م ز ه دنز
ا ر ت س ب وس ه م ی ا ه ل و ل س .د ن و شی م ر وتین و م ف ل ت خ م ی ا ه
ه ب ذ ج تلیفر ظ هل ک دلن وشی م ه د افتلس ا ت یالعفص لخاش
ن اونع بملس یناگراورکیم طلسوت ی د ر ا و م رد .دوش هتفرگ رلظن رد
ی لسفنتی لکیلوباتم
ی کیژولویبن ژیسکا زاین هبساحم هل م ج زا .
11
ی ا هتی د و د حم
ل م ا ک یاهلولس
12
هدافتسا للکشم نلیا رب هلبلغ یاهشور زا یلکی .1
ی ا هل ولس و هک تل ل سا رل لی ذپ ذ وفن ی ا هش ور زا
ی ا هل ل ح) یللی ای میش ( ،د ا م جن ا) یلل کی زیف نیرت جی ( کین اگ ر ا
ار .دوشیم هدافتس ا (نی یاپاپ ای موزوزیل )یمیزنآ
نئولوت هللم ج زا کلیناگرا ی اه ل ل ح هدافتلسا ش و ر ییایمیش رامیت نی،
ا .دشابیم لوناتوب و لوناتا ،مرفورلک ی ز ا س ا د ج هلیلسوب یلکچوک یا هذفنم
داجیا ثلعاب عیرس ذوفن ثلعاب هلک دوشیم لوللس حطلس زا الهدیپیل ییاشغ
ک چ و ک یاهذفن م نی ا زا تلوص حم ای ارتسبوس یریگولج یلولس ن و ر د
ی ا ه ملیزنآ جورخ زا و ه د ش ل ولس گر م ثل ع اب دلنی ا رف
نلی ا م ا جن ا دلن چ ر ه .د وشی م ورد ی ا ه میزنآ زا علبنم کلی
ناونعب الما دوشیم
ه د ا س ی ا ه د ر ک ل م ع یارب.دنکیم لللمع یلولس
اهروسنسویب
. دنریگیم رارق هدافتسا د ر و م
13
ت سا هد ش ی سررب اهشنکاو ن یا ش ه ا ک ی ارب ی یاهشور . 2.
زا ی د ا د ع ت ن دش جر ا خ ث ع اب ل ول ل س یری ذپ ذ وفن
ل م ع نلیا هلک ه د ش ملک یللوکلوم نز و الب الهروتکافوک ل ول س کی
ه چ رگ .د ه دی م ش ه اک ار هت س ا و خ ان ی ا هشنک ا و ار زوتکل
یلول س ل خ ا د زادیزوتکلگاتب ل م ا ش ر م خ م ل م ا ک رد لول س ن ا م ه
و لوناتا هب co2هکیالح رد دنکیم لیدبت
زوتکالگ و زکولگ هلب طلقف ار زوتکلریذپذوفن طلیارش رگید یکی .دنکیم
لیدبت اهروتکافوک نتفر تس د زا تلعب یاهشور زا هدافتسا اهمیزنآ ندرک
العفریغ یاهشور زا هدافتسا ه د ر م یاهلوللس زا هلکینامز هدوب
ش ل ه اک ر د ر ل گ ی د یلکیزیف ر د هتس ا و خ ان ی ا هشنک ا و
ش ور .د وش هتس اوخان یکیلوبات م ی ا ه ه ا ر ندرک هکول ب هدنز
یاهلولس
ای
ت سا ال قتنا متسیس .
14
د ا و م ثبیتت
ی کیژ ول ویب
دن وی یی ای میش ی ا ه ش ور ⦿
پ یسنال ووک
ت ل اصتا
یض ر ع
یزادنا هلت
15
دن ویپ
یسن ل ا و وک
⦿ میزنآ ثبیتت یارب جیار کینکت کی یسنال ووک دنویپ
لولس ثبیلتت یارب هلک تلسا اله یداب یلتنآ و ا ه نی ا یس اس ا
ت ل کش م زا ی ک ی .تس ی ن یب س ان م ش ور ی ا ه ه و ر گ
ض ر ع م رد اهلولس هلک تلسنیا کلینکت شنک ا و ت خس طی ارش و
ی وق ه دن ه د ش ن ک ا و ل م اع یاهلولس هلب ندیلسر بیلسآ ثلعاب
هلک هلتفرگ رارق یلول س ن و ر د یاهمیزن آ روطنیمه و ه د ش
هدنز
دنکیم ادیپش ه ا ک.
16
یکیزیف یاهشور
17
یزادنا هلت
یزتنس یاهرمیلپ
لکال لینیو یلپ و ناتروا یلپ س ا س ا رب یاهلژوردیه -دیمآ لیرکآ یلپ بیسآ ال متحا تابیکرت نیا
یاهتیدودحم نیرتمهم زا یکی.دشابیم
ت سا هدنز ی اهلولس هبن دیسر .
یعیبط یاهرمیلپ
اهرمیلپ
- ن انیگاراک -ت انیژآل ل م ا ش اهلولس ن تخادنا م ا د هبی اربهدافتسا د ر و م ی ارب
ن یا هک دشابیم ...ن ازوتیک -ن یئاپب و ذ هطقن ابزراگآ
تدنتسه دیفم رایسبهدنز ی اهلولس ت ثبی.
18
رد یبورکیم یاهروسنسویب یاهدربراک
اذغ
19
ریش تیفیک لرتنک
21
اهنیماتیو نییعت
22
یبورکیم یاهروسنسویب یاهدربراک
طیحم رد
23
Biosnsors for detection
of:
L. monocytogenes
24
Salmonella
25
26
Salmonella
27
Salmonella
28
Magnetoelastic biosensor for the detection of
Salmonella typhimurium in food products
29
Salmonella(2
)
⦿ Existing biosensor technologies such as surface
wave devices, optical and thickness-shear mode
acoustic
resonators offer sensitivity, but are limited by their need
for direct physical contacts. For example quartz crystal
microbalance (QCM) needs direct physical contacts (i.e.
electrical wiring) to obtain sensor response which prohibit
their usage in the sealed containers. Magnetoelastic
sensors don’t need direct electrical contacts for the
actuation and data acquisition; this makes magnetoelastic
sensors suitable for the remote detection of pathogens.
The current investigation mainly focuses on the
development of a biosensor that addresses these
limitations through remote and real-time sensing of
pathogens in food products. The Langmuir Blodgett (LB)
technique was used for antibody immobilization on the
magnetoelastic sensors(2).
30
Salmonella
Fig. Schematic drawing illustrating the wireless nature of the magnetoelastic biosensors and the
basic principle for detecting bacterial cells. The fundamental resonant frequency of the
biosensor is f0 without antigen binding, which shifts (decreases) to fanalyte due to the increased
mass of antigen binding to antibody immobilized on the sensor surface(2).
31
Bacterial binding measurements
32
33
⦿ When the test temperature and humidity are constant, the resonant
frequency change of the magnetoelastic sensor depends only on
the mass change (Δm) on its surface. If the mass increase is small
compared to the mass of the sensor the shift in the resonant
frequency is given by:
34
Sensor surface bacterial densities of 0.105, 0.075 and 0.105
cells/µm2 were observed on the samples which were exposed to S. typhimurium suspensions in water, fat-free milk, and
apple juice respectively at the highest bacterial
concentrations(2).
46
E. Coli (2)
36
E. coli O157:H7 is most well known to both microbiologists
and general public. This organism was first recognized in 1982,
and has the ability to cause life threatening complications
hemorrhagic colitis and hemolytic uremic syndrome (HUS) in
humans(6). Illness due to E. coli O157:H7 infection is often
misdiagnosed and generally needs invasive and expensive medical
tests before it is correctly diagnosed. Primary sources for
exposure to E. coli O157:H7 are consumption of ground beef,
sprouts lettuce, salami, unpasteurized milk, and juice
contaminated by pathogens. Since the loss caused by E. coli
O157:H7 is enormous in terms of medical cost and product
recall, it is extremely urgent to develop some rapid and sensitive
methods to detect the bacteria in food or water supplies(6).
37
⦿ As to the selectivity, DNA is an ideal target for specific detection
of pathogenic bacteria. The DNA probe, which is an
oligonucleotide sequence immobilized on a fixed support and able
to hybridize the complementary strand present in solution, is a
powerful molecular tool for monitoring and detecting specific
microorganisms in the environment or in food. It is well known
that QCM is a very sensitive mass-measuring sensor, and the
crystal resonance frequency decreases with a mass increase on the
Au electrode surface.
38
Nanoparticles are a new class of materials, which has been adopted for
improving the detection limit and sensitivity of the DNA biosensors. In
order to form a more effective and sensitive system, we adopted the gold
nanoparticles in the construction of E. coli O157: H7 DNA biosensor for
the goal of signal amplification, because (i) colloidal Au has a good
biocompatibility; (ii) gold nanoparticle has a larger surface area, and
helps to immobilize more biofunctional molecules onto the surface of the
sensor; (iii) avidin-conjugated Au nanoparticle could be used as “mass
enhancer” to amplify the frequency change depending on its relatively
large mass compared to DNA target.
39
The whole detection course of this QCM DNA sensor mainly included
two parts: sensor fabrication and detection. During the process of sensor
fabrication, how to immobilize more ssDNA probes was pivotal for the
following bacteria DNA detection, so two important routes for DNA
probes immobilization were introduced to this part, which were:
• Self-assembled monolayers (SAMs).
• Au-thiol binding.
A stepwise decrease of Δf was observed in each fabrication step(6).
51
41
E.
coli
D etection of E .coli
in foods
⦿ One of the many applications of surface plasmon
resonance (SPR) technology is the detection of E.
coli O 157:H7. Surface plasmon resonance is a
quantum optical-electrical phenomenon based on
the fact that energy carried by photons of light
can be transferred to electrons in a metal. The
wavelength of light at which coupling occurs is
characteristic of the particular metal and the
environment of the metal surface illuminated. This
transfer can be observed by measuring the
amount of light reflected by the metal surface (1,
in, 2).
42
The following Fig. shows how a change in the chemical
environment 100 nm above the thin metal layer results in a shift in
the wavelength of light, which is absorbed rather than reflected.
The most common practical implementation of SPR is to use a
metal-coated optical prism, but other practical implementations
have been demonstrated including metal-coated diffraction
gratings, optical fibres and planar waveguides. The SPR biosensor
has potential for use in rapid, real-time detection and identification
of bacteria, and to study the interaction of organisms with
different antisera or other molecular species (Fratamico et al.,
1998). The lower detection limit of the BIAcore system (a
commercial example of an SPR system) is approximately 10 pg
of analyte
mm-2.
43
E.
coli
44
L . monocytogenes
45
SPR biosensor technology is one of the most promising detection methods
currently in use for rapid pathogenic identification. It is sensitive to changes
in the thickness or refractive indices of biomaterials at the interface between
a thin gold film and an ambient medium, & is thus capable of characterizing
biomolecular interactions in real time without the need for labeling.
57
As SPR sensors are capable of detecting changes in a refractive index of several
hundred nanometers over the sensor surface, bacterial cell particles specifically
bound to an antibody can induce a large SPR angle shift, as compared with
biomolecules covering an identical surface area(4). The direct detection of
microbial cells is often difficult, owing principally to their large size. In order to
assay whole L.monocytogenes cells with an SPR biosensor, it is first necessary to
find a high-affinity antibody [monoclonal anti- L.monocytogenes mouse antibody]
for the cell and to optimize the detection surface.
In this study, a specific monoclonal antibody against L.monocytogenes was
screened using an SPR biosensor. Monoclonal antibodies were bound to protein
L, after which the L.monocytogenes cells were subjected to an affinity assay.
Protein L was immoblized on a carboxymethyl dextran surface via an amine
coupling method, and utilized repeatedly by regeneration. The monoclonal
antibody, ‘A 18’, was selected and employed for the high sensitivity detection of
L.monocytogenes. specific antibodies were screened via direct binding to cells
using an SPR biosensor. It was determined that the screened antibody evidenced a
high degree of sensitivity for the direct detection of L.monocytogenes.
47
⦿ In a study that was done in year 2007 by Joung Hyou- Arm et
al., they selected a specific monoclonal antibody (mouse IgG1)
against
L. monocytogenes , and also attempted to detect it directly. The
direct detection of microbial cells is often difficult, owing to
principally large size. In order to assay whole L. monocytogenes
cells with a SPR biosensor, it is first necessary to find a high-
affinity antibody for the cell & to optimize the detection surface
(4).
48
References
□ Maria N. Velasco-Garcia; Toby Mottram (2003). Biosensor Technology
addressing Agricultural Problems. Biosystems Engineering , 84 (1), 1–12
e Guntupalli, R., Lakshmanan, Ramji S., Johnson, Michael L., Hu, J., Huang,
Tung-Shi., Barbaree, James M., Vodyanoy, Vitaly J., Chin, Bryan A. (2007).
Magnetoelastic biosensor for the detection of Salmonella typhimurium in food
products. Sens. & Instrumen. Food Qual. 1:3–10 DOI
10.1007/s11694-006-9003-8
0 SKLADAL, P., SYMERSKA, Y., POHANKA, M., SAFAR, B., MACELA, A.
(2005). ELECTROCHEMICAL IMMUNOSENSOR FOR DETECTION OF
FRANCISELLA TULARENSIS. Detection Technologies, Implementation
Strategies and Commercial Opportunities, 221–232.
o Joung, H., et al. (2007). Screening of a Specific Monoclonal Antibody against
and Detection of Listeria monocytogenes Whole Cells Using a Surface Plasmon
Resonance Biosensor. Biotechnology and Bioprocess Engineering, 12 : 80-85.
d Nayak, M., Kotian, A., Marathe, S., Chakravortty, D. (2009). Detection of
microorganisms using biosensors—A smarter way towards detection techniques.
Biosensors and Bioelectronics 25, 661–667.
i LiJiang, W., QingShan, W., ChunSheng, W., ZhaoYing, H., Jian, J. & Ping, W.
(2008). The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-
enhanced piezoelectric biosensor. Chinese Science Bulletin, vol. 53 | no. 8 |
1175-1184.
B D’Souza, S.F.(2001). Microbial biosensors. Biosensors & Bioelectronics 16, 337–
353.
49
Any
Questions???
50
The End
Thanks
51