Bio-chips, also known as lab-on-a-chip devices, can provide portable, low-cost, and low-power platforms for integrating sensors and other components. DNA microarrays allow high-throughput screening by placing probes for thousands of genes on a single chip. mRNA is extracted from experimental and control samples, converted to fluorescent cDNA, and hybridized on the chip. The resulting colors indicate gene expression levels. Protein microarrays similarly attach thousands of proteins to a chip and use probes to study protein interactions, expression profiles, and biochemical functions through detection of reaction products. Technical challenges include maintaining protein activity and structure during immobilization and detection.

1. Bio-chips (Lab-on-a-chip)
1

2. System architectures

3. 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.)

4. 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 SENSOR
AND 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
4

5. System architectures
• Chips – flat platforms, sensors below or above the chip
T. Vo-Dinh et al. , Sensors and Actuators, B 74 (2001) 2-11
5

7. 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
7

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

9. Single detectors vs. Vectors and arrays
Single
Vector
Array
9

10. MICROARRAYS
It is a 2D array on a solid substrate (usually a glass slide or silicon thinfilm cell) that assays large number of biological material using highthroughput 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)

11. DNA Arrays (Gene chips)

12. Example of a DNA Array
(note green, yellow red colors;
also note that only part of the total
array is depicted)

13. Example of a DNA Array
(note green, yellow red colors;
also note that only part of the total
array is depicted)
http://www.biomed.miami.edu/arrays/images/agilent_array.jpg
41,000+ unique human genes
and transcripts represented, all
with public domain annotations

14. 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 hybridi
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

15. • 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.

16. 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.
Affymetrix
Agilent Technologies
Applied
CombiMatrix
Eppendorf
GE Healthcare
Genetix
Greiner Bio-One
Illumina, Inc.
Kreatech
Micronit Microfluidics
Nanogen, Inc.
NimbleGen
Ocimum Biosolutions
Roche Diagnostics
SCHOTT Nexterion
STMicroelectronics

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

18. 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.

19. DNA Array: Single-stranded copy DNA Oligonucleotides for
each gene in a different well.
cDNA
gene 1
cDNA
gene 2
cDNA
gene 3
cDNA
gene 4
cDNA
gene 5

20. 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

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

22. Extract mRNA from Control Cells
Make single-stranded cDNA
using green nucleotides (e.g.
Quantum dots)
cDNA = complementary DNA (DNA synthesized from RNA)
Extract mRNA from
Experimental/pathological Cells
Make single-stranded cDNA
using red nucleotides (e.g.
Quantum dots)

23. 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.

24. Control cDNA
Experimental cDNA

25. Step 5: Extract a small
amount in a pipette.

26. Step 6: Insert into first
well.

27. Step 7: Extract
more cDNA and …
… insert into
second well, etc.

28. Step 8: Continue until all wells are
filled.

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

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

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

32. 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 in
control
cells
Mostly in
control
cells
Mostly in
Only in
Same in
experimental experimental
both cells
cells
cells

34. PROTEIN MICROARRAY

35. Part1
Protein 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.

36. Protein Array VS DNA Microarray
Target:
Binding:
Stability:
Surface:
Printing:
Amplification:
Proteins
(Big, 3D)
3D affinity
Low
Glass
Arrayer
Cloning
DNA
(Small, 2D)
2D seq
High
Glass
Arrayer
PCR

37. Protein Array Fabrication

Protein substrates




Polyacrylamide or
agarose gels
Glass
Nanowells
Proteins deposited
on chip surface by
robots
Benfey & Protopapas, 2005

38. Protein Attachment




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
Benfey & Protopapas, 2005

39. Protein Interactions


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 function
Antigen–
antibody
Protein–
protein
Aptamers
Enzyme–
substrate
Receptor–
ligand
Benfey & Protopapas, 2005

40. Expression Array

Probes (antibody) on surface recognize
target proteins.

Identification of expressed proteins from
samples.

Typical quantification method for large # of
expressed proteins.

45. Interaction Array
 Probes (proteins, peptides, lipids) on
surface interact with target proteins.
 Identification of protein interactions.
 High throughput discovery of interactions .

46. Functional Array
 Probes (proteins) on surface react with
target molecules .
 Reaction products are detected.
 Main goal of proteomics.

50. Sample Preparation

Labeled

Fluorescent Dye




Cy3/Cy5 via Lysines
Photochemical
Radioisotope
May interfere

51. 
Unlabeled

Antibody Sandwich


Surface Plasmon
resonance




2nd antibody with label
incubated on top of sample
Measure electromagnetic
waves
Angle changes in the order
of 0.1° with 1 nm film
adsorption
Needs special equipment
Don’t affect protein
structure

52. Detection & Quantification

Scanner



Reference spots


Detects dye
Adjusts for
background
Labeled known
concentrations
Computational
Analysis

57. 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