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Bio-chips (Lab-on-a-chip)

System architectures

White lines correspond to metal electrodes that connect to individual nanowire devices. The position of the microfluidic channel used to deliver sample is highlighted in blue and has a total size of 6 mm × 500 μm, length × width. The image field is 4.4 × 3.5 mm.

(B) Optical image of one row of addressable device elements from the region highlighted by the red-dashed box in A. The red arrow highlights the position of a device. The image field is 500 × 400 μm.

C) Scanning electron microscopy image of one silicon nanowire device. The electrode contacts are visible at the upper right and lower left regions of the image. (Scale bar: 500 nm.)

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Bio-chips• Portable, • low cost in high volumes, • low power, • can be integrated with other components

Chii-Wann Lin et al, DEVELOPMENT OF MICROMACHINED ELECTROCHEMICAL SENSORAND PORTABLE METER SYSTEM, a Proceedings of the 20th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Vol. 20, No 4,1998

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System architectures• Chips – flat platforms, sensors below or above the chip

T. Vo-Dinh et al. , Sensors and Actuators, B 74 (2001) 2-11

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Schematic diagram of an integrated DNA biochip system

Vo-Dinh T, Alarie JP, Isola N, Landis D, Wintenberg AL, Ericson, MN (1999) Anal Chem 71 :358–363

fluorescence detection of Cy5-labeled Streptavidin using a 4X4 photodiode array IC biochip. Excitation by a 12 mW He±Ne laser (632.8 nm).

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Single detectors vs. Vectors and arrays

Single

Vector Array

MICROARRAYS

It is a 2D array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large number of biological material using high-throughput screening methods. Types of microarrays include:

• DNA microarrays, • oligonucleotide microarrays

• MMChips, for surveillance of microRNA populations• Protein microarrays• Tissue microarrays• Cellular microarrays (also called transfection microarrays)

• Chemical compound microarrays• Antibody microarrays• Carbohydrate arrays (glycoarrays)

DNA Arrays (Gene chips)

Example of a DNA Array(note green, yellow red colors;

also note that only part of the totalarray is depicted)

http://www.biomed.miami.edu/arrays/images/agilent_array.jpg

Example of a DNA Array(note green, yellow red colors;

also note that only part of the totalarray is depicted)

41,000+ unique human genes and transcripts represented, all with public domain annotations

an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called probes, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target)

the probes are attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip

• Probe-target hybridization is usually detected and quantified by detection of fluorophore-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. Since an array can contain tens of thousands of probes, a microarray experiment can accomplish many genetic tests in parallel.

Colloquially known as an Affy chip when an Affymetrix chip is used. Other microarray platforms, such as Illumina, use microscopic beads, instead of the large solid support.

AffymetrixAgilent TechnologiesAppliedCombiMatrixEppendorf GE Healthcare Genetix Greiner Bio-One Illumina, Inc. Kreatech Micronit Microfluidics Nanogen, Inc. NimbleGen Ocimum Biosolutions Roche DiagnosticsSCHOTT Nexterion STMicroelectronics

• DNA microarrays can be used to measure changes in gene expression levels, to detect single nucleotide polymorphisms (SNPs) , to genotype or resequence mutant genomes.

Step 1: Create a DNA array (gene “chip”) by placing single-stranded DNA/ Oligonucleotides for each gene to be assayed into a separate “well” on the chip.

cDNAgene 1

cDNAgene 2

cDNAgene 3

cDNAgene 4

cDNAgene 5

DNA Array: Single-stranded copy DNA Oligonucleotides for each gene in a different well.

Step 2: Extract mRNA from biological tissues subjected to an experimental treatment and from the same tissue subjected to a control treatment. Or from normal and from pathological

tissue

• Step 3- Make single-stranded DNA from the mRNA using “color coded” nucleotides.

Extract mRNA from Control Cells

Extract mRNA from Experimental/pathological Cells

Make single-stranded cDNA

using green nucleotides (e.g. Quantum dots)

Make single-stranded cDNA

using red nucleotides (e.g. Quantum dots)

cDNA = complementary DNA (DNA synthesized from RNA)

Step 4: After making many DNA copies of the RNA, extract an equal amount of cDNA from the controls & experimentals and place it into a

container.

Control cDNA Experimental cDNA

Step 5: Extract a smallamount in a pipette.

Step 6: Insert into first well.

… insert intosecond well, etc.

Step 7: Extractmore cDNA and …

Step 8: Continue until all wells arefilled.

Step 9: Allow to hybridize, then wash away all single-stranded DNA.

Result:(1) Some wells have no color-coded cDNA (no mRNA in either type of cell)(2) Some wells have only red (i.e., expressed only in experimental cells)(3) Some wells have only green (i.e., expressed only in control cells)(4) Some wells have both red and green in various mixtures (expressed

in both experimental and control cells)

Step 10: Scan with a laser set to detect the color & process results on

computer.

Results: The colors denote the degree of expression in the

experimental versus the control cells.

Gene not expressed in control or in experimental

cells

Only incontrol

cells

Mostly incontrol

cells

Only inexperimental

cells

Mostly inexperimental

cells

Same inboth cells

PROTEIN MICROARRAYPROTEIN MICROARRAY

1. High throughput analysis of hundreds of thousands of proteins.

2. Proteins are immobilized on glass chip.

3. Various probes (protein, lipids, DNA, peptides, etc) are used.

Part1

Protein Microarray

Protein Array VS DNA Microarray

Target: Proteins DNA(Big, 3D) (Small, 2D)

Binding: 3D affinity 2D seqStability: Low HighSurface: Glass GlassPrinting: Arrayer ArrayerAmplification: Cloning PCR

Protein Array Fabrication

Protein substratesProtein substrates Polyacrylamide or Polyacrylamide or

agarose gelsagarose gels GlassGlass NanowellsNanowells

Proteins deposited Proteins deposited on chip surface by on chip surface by robotsrobots

Benfey & Protopapas, 2005

Protein Attachment

Benfey & Protopapas, 2005

Diffusion Protein suspended in

random orientation, but presumably active

Adsorption/Absorption Some proteins inactive

Covalent attachment Some proteins inactive

Affinity Orientation of protein

precisely controlled

Diffusion

Adsorption/Absorption

Covalent

Affinity

Protein Interactions

Benfey & Protopapas, 2005

Different capture molecules must be used to study different interactions

Examples Antibodies (or antigens) for

detection Proteins for protein-protein

interaction Enzyme-substrate for

biochemical functionReceptor–

ligand

Antigen–antibody

Protein–protein

Aptamers

Enzyme–substrate

Expression Array Probes (antibody) on surface recognize

target proteins.

Identification of expressed proteins from samples.

Typical quantification method for large # of expressed proteins.

Interaction Array Probes (proteins, peptides, lipids) on

surface interact with target proteins.

Identification of protein interactions.

High throughput discovery of interactions.

Functional Array Probes (proteins) on surface react with

target molecules .

Reaction products are detected.

Main goal of proteomics.

Sample PreparationSample Preparation LabeledLabeled

Fluorescent DyeFluorescent Dye Cy3/Cy5 via Cy3/Cy5 via

LysinesLysines PhotochemicalPhotochemical RadioisotopeRadioisotope May interfereMay interfere

UnlabeledUnlabeled Antibody SandwichAntibody Sandwich

22ndnd antibody with label antibody with label incubated on top of incubated on top of samplesample

Surface Plasmon Surface Plasmon resonanceresonance

Measure electromagnetic Measure electromagnetic waveswaves

Angle changes in the Angle changes in the order of 0.1° with 1 nm order of 0.1° with 1 nm film adsorptionfilm adsorption

Needs special equipmentNeeds special equipment DonDon’’t affect protein t affect protein

structurestructure

Detection & Detection & QuantificationQuantification

ScannerScanner Detects dyeDetects dye Adjusts for Adjusts for

backgroundbackground Reference spotsReference spots

Labeled known Labeled known concentrationsconcentrations

Computational Computational AnalysisAnalysis

Technical Challenges in Protein Chips

1. Poor control of immobilized protein activity.

2. Low yield immobilization.

3. High non-specific adsorption.

4. Fast denaturation of Protein.

5. Limited number of labels – low mutiplexing