Advanced Technologies

in Biological Research

µ-arrays

Carina Huber-Gries
Biochips and microarrays
Biochips are parallelized, miniaturized and automatized biochemical analysis
methods, often performed as microarrays.

Microarrays are arrays of biomolecular probes immobilized onto solid

supports for highly parallel miniaturized analysis systems

▪ miniaturization
▪ functional integration → „lab-on-a-chip“
▪ parallelism → thousands of reactions at the same time
▪ automation

Spot Ø 50 – 150 µm
Spot volume pl to nl

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Biochips and microarrays
Solid supports – chip platforms Glass or plastic slides

PCR assay in tube for virus detection

Liu, Q et al., Clin. Chem., 53: 188-194, 2007

http://www.illumina.com

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Biochips and microarrays: probe arraying

High density arrays:

▪ in situ synthesis
Low density arrays:
▪ contact printing
▪ non-contact printing
▪ µ-contact printing (micropatterning)

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Biochips and microarrays: probe arraying

in situ synthesis for high density DNA arrays → photolithographic techniques

photolithography

▪ high-density oligonucleotide probe DNA

microarrays (approximately 25 nucleotides)

▪ photolithography: use light to create a pattern

▪ light-directed combinatorial chemical synthesis

▪ solid support, selectively synthesized probes

Affymetrix GeneChip oligonucleotide microarray
directly on the surface

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Biochips and microarrays: probe arraying
in situ synthesis for high density DNA arrays → photolithographic techniques
Chemical synthesis cycle
▪ linker molecules (OH groups) and protecting
groups on the free end are removed by light

▪ UV light is directed through a

photolithographic mask to deprotect and
activate selected sites with OH groups

▪ coupling with incoming nucleotides that

attach to the activated sites

▪ change of exposure sites to the coordinates

on the array where next nucleotide will be
attached

▪ process is repeated, 25 nucleotides possible

Affymetrix GeneChip oligonucleotide microarray

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Biochips and microarrays: probe arraying
in situ synthesis for high density DNA arrays → photolithographic techniques
application of DNA microarrays
▪ cDNA of the control sample (healthy tissue) is labelled in green
▪ experimental sample (diseased tissue) is labelled in red
▪ no mutation: red and green samples will bind equally on
complementary sequences on chip (normal sequence)
▪ mutation: no binding of red sample to “normal sequence” and
binding of red sample to complementary sequence of diseased
DNA.
Not significant: ▪ the ratio of the two signals
not present in cells at a given gene position
reflects the relative
present in both cell types
abundances of the
Significant: corresponding mRNAs in
only in healty cells the different samples.
only in diseased cells
Proceedings of the Nature Research Society, 2, 02010 (2018)

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Biochips and microarrays: probe arraying
Deposition of pre-synthezised elements
low density arrays
▪ larger molecules: oligos, antibodies
▪ for low-density arrays
▪ contact printing
▪ non contact printing
▪ µ-contact printing

Faraday Discuss., 2019, 219, 9

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Biochips and microarrays: probe arraying
Deposition of pre-synthesized elements
contact printing

www.arrayit.com

☺ high throughput
 pin determines spot volume
and size https://www.youtube.com/watch?v=XxLcVYNi2Fo

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Biochips and microarrays: probe arraying
Deposition of pre-synthesized elements
non-contact printing
▪ uses a piezo crystal for drop generation
▪ expansion when voltage is applied – droplet releases from nozzle

Lab Chip, 2015, 15, 2538–2558

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Biochips and microarrays: probe arraying
Deposition of pre-synthesized elements
non-contact printing
☺ independent of substrate material characteristics
☺ low risk of contamination
 optimum voltage applied to piezo crystal
depends on composition of solution
 satellites

Phys. Fluids 18, 072102 (2006) https://www.youtube.com/watch?v=LoyYge-gPp0

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Biochips and microarrays: probe arraying
Deposition of pre-synthesized elements → non-contact printing
the effect of the spotting buffer
▪ different buffer compositions (PBS vs. CBS buffer)
▪ effect of additives (e.g. triton X-100)

effects of different additives (e.g. triton X-100) and concentrations of additives on

spot quality and protein immobilization (rabbit IgG immobilized on solid supports)

Frontiers in Bioscience 12, 3768-3773, May 1, 2007

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Biochips and microarrays: probe arraying
Deposition of pre-synthesized elements
µ-contact printing
(Aminosilane) micropatterning

Li H., et al., Langmuir 2010, 26(8), 5603–5609

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Biochips and microarrays: probe arraying
Deposition of pre-synthesized elements
µ-contact printing
▪ stamp 'inked' with solution of molecules (e.g. proteins, thiols,…) → stamp is coated with biomolecule solution
▪ stamp is dried and pressed onto the surface to be patterned
▪ molecules are transferred directly from the stamp to the surface

https://www.youtube.com/watch?v=Acm_bH413wk&t=4s

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Biochips and microarrays: assay design and applications
▪ DNA-based
▪ diagnostic arrays
▪ genotyping
▪ gene expression

▪ Protein-based
▪ forward vs. reverse phase assays
▪ sandwich / inhibition / competitive immunoassays
▪ examples

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Biochips and microarrays: assay design and applications
▪ DNA-based
▪ diagnostic arrays
▪ genotyping
▪ gene expression

▪ Protein-based
▪ forward vs. reverse phase assays
▪ sandwich / inhibition / competitive immunoassays
▪ examples

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Biochips and microarrays: assay design and applications
Example: DNA-microarrays for gene expression studies
▪ determine what genes are active in a cell and at what levels
▪ compare the gene expression profiles of a control vs treated
▪ determine what genes have increased / decreased during an experimental condition
Scatter plots:
data analysis for DNA expression chips
▪ significance analysis, pathway analysis …
▪ intensities of experimental samples versus
normal samples
▪ quick look at the changes and overall quality
of microarray

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Biochips and microarrays: assay design and applications
▪ DNA-based
▪ diagnostic arrays
▪ genotyping
▪ gene expression

▪ Protein-based
▪ forward vs. reverse phase assays
▪ sandwich / inhibition / competitive immunoassays
▪ examples (medical diagnosis)
▪ effect of incubation time

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Biochips and microarrays: assay design and applications
Classes of protein microarrays: based on ELISA principles
Forward phase protein microarray:
analyte(s) of interest captured from the solution phase by a
capture molecule, usually an antibody
▪ sandwich immunoassay
▪ binding inhibition assay
▪ competitive immunoassay

Reverse phase microarrays:

analytes (e.g. cells lysate) immobilized on the solid phase;
analyte-specific ligand (e.g. antibody) applied in solution; bound
antibodies detected by secondary tagging and signal
amplification
screening for molecular markers (e.g. drug discovery)
K.M. Sheehan et al., Molecular & Cellular Proteomics 4:346-355, 2005

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Biochips and microarrays: assay design and applications
Sandwich immunoassay:
need of two antibodies.
first antibody captures the antigen (sample).
second labelled antibody (sometimes called tracer) is binding to the bound antigen.

the more probe is immobilized, the greater the fluorescence signal.

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Biochips and microarrays: assay design and applications
Binding inhibition assay: signal inversely proportional to unknown antigen concentration.

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Biochips and microarrays: assay design and applications
Competitive immunoassay:

Unknown antigen (analyte) competes with a fluorescence labelled antigen to bind to antibodies.

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Biochips and microarrays: assay design and applications
Example: diagnosis of sepsis: scientific question most relevant:
Sepsis yes / no?
→ protein biomarker chip different concentration range of biomarkers (mg/L vs. ng/L)
Type of bacteria that caused the sepsis?
→ DNA chip, different expected cell numbers (10 cells vs. 100 cells/mL blood)
more sensitive less sensitive

U. Sauer et al., Anal. Biochem., 2011

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Biochips and microarrays: assay design and applications
Effect of incubation time
▪ limit of detection is improved with incubation time
▪ assay is more reproducible
▪ cross reactivity is diminished

cross reactivity: antibody binds unspecifically to an

antigen which has similar three-dimensional
structures (epitopes)
Cv coefficient of variation

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Biochips and microarrays: detection schemes
▪ with label
▪ fluorescence
▪ absorbance / colorimetric
▪ chemiluminescence
▪ without label
▪ surface plasmon resonance (SPR)
▪ reflectance interference spectroscopy (RIfS)
▪ signal enhancement possibilities

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Biochips and microarrays: detection schemes
▪ with label
▪ fluorescence
▪ absorbance / colorimetric
▪ chemiluminescence
▪ without label
▪ surface plasmon resonance (SPR)
▪ reflectance interference spectroscopy (RIfS)
▪ signal enhancement strategies

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Biochips and microarrays: detection schemes
Signal enhancement strategies: Dendrimers for DNA microarrays

- Dendrimers containing multiple copies of fluorophores

- Plasmonic-enhanced fluorescence
- Target amplification techniques (rolling circle amplification)
- Immuno-PCR
- Absorbance based enhancement

3DNA® dendrimer by Genisphere Inc.

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Biochips and microarrays: detection schemes
Signal enhancement strategies: Dendrimers for DNA microarrays

Sensors 2006, 6, 901-914

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Biochips and microarrays: detection schemes
Signal enhancement strategies: Plasmonic enhanced fluorescence

▪ Increasing the sensitivity of fluorescence assays involves increasing the total number of detected
photons
▪ Metal enhanced fluorescence (MEF) occurs when fluorophores are within about 100 A from noble
metal nanostructures
▪ The MEF effect is thought to be a result of surface plasmons induced by the incident light or by the
excited fluorophores.
▪ Enhancements of 10-to 40-fold can be reached

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Biochips and microarrays: detection schemes
Signal enhancement strategies: Plasmonic enhanced fluorescence

C.R. Sabanayagam et al., Nucleic Acids

Res (2007) 35 (2): e13.
E.G. Matveeva, et al., Journal of Immunological Methods 302 (2005) 26–35

Surface hybridization. Intensity scans of Cy5- and Cy3-labeled target oligonucleotides hybridized to
probes arrayed onto silver island films (SIF) and glass substrates. Note the log scale on the x -axis. ( A
and B ) show the average intensity versus spotting concentration for Cy5 and Cy3, respectively, on SIF
substrates (empty circles) and glass (filled circles). ( C ) Plots of the intensity enhancement factor
versus spotting concentration from the hybridization data. Cy5 is shown in red, and Cy3 is shown in
green. SIF= silver island films
(reduction of Ag+)

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Biochips and microarrays: detection schemes
Signal enhancement strategies: Target amplification techniques (rolling circle amplification)

Scheme of immunoassays with rolling circle amplification

A. oligonucleotide primer is attached to an antibody
B. The antibody-DNA conjugate binds to its specific target molecules
C. circular DNA hybridizes to its complementary sites on the primer,
in presence of DNA polymerase and nucleotides, rolling-circle
replication occurs
D. long single DNA molecule is generated that remains attached to
the antibody
E. this DNA product is detected by hybridization of multiple
fluorescent, complementary oligonucleotide probes

Schweitzer B., et al., Nature Biotechnol., 20 (2002) 359-365 Trends in Microbiology

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Biochips and microarrays: detection schemes
Signal enhancement strategies: Immuno PCR
☺ 100-10000 fold more sensitive than ELISA  complicated multi-step protocol
☺ significant advances in the detection of very low analyte concentrations  complicated synthesis of DNA-antibody conjugate
☺ allows early identification of tumor- and disease-associated antigens  sometimes high background

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Biochips and microarrays: detection schemes
Signal enhancement strategies: Immuno PCR

Absorbance based enhancement: sliver precipitation

www.eppendorf.at

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Biochips and microarrays: learnings
Biochips are arrays of biomolecular probes immobilized onto solid
supports for highly parallel miniaturized analysis systems

• Substrates
• Biomolecules: DNA, proteins

• Immobilization principles: covalent, electrostatic, affinity, adsorption

• Arraying: contact vs. non-contact spotting vs. µCP

• Assay formats & applications

• Detection and signal enhancement

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