15 arrays

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

1

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.

C) Scanning electron
microscopy image of one
(B) Optical image of one row of silicon nanowire device.
addressable device elements from the The electrode contacts are
region highlighted by the red-dashed visible at the upper right
box in A. The red arrow highlights the and lower left regions of the
position of a device. The image field is image. (Scale bar: 500 nm.)
500 × 400 μm.

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 3
Engineering in Medicine and Biology Society, Vol. 20, No 4,1998

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

T. Vo-Dinh et al. , Sensors and Actuators, B 74 (2001) 2-11 4

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
6

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

Single detectors vs. Vectors and arrays

Single

Vector Array 8

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)

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

41,000+ unique human genes
Example of a DNA Array
(note green, yellow red colors; and transcripts represented, all
also note that only part of the total with public domain annotations
array is depicted)

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

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

• 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.

• 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.

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

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
Extract mRNA from Control Cells Experimental/pathological Cells

Make single-stranded cDNA Make single-stranded cDNA
using green nucleotides (e.g. using red nucleotides (e.g.
Quantum dots) 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 small
amount in a pipette.

Step 6: Insert into first
well.

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

Step 8: Continue until all wells are
filled.

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

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.

Protein Array VS DNA Microarray

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

Protein Array Fabrication
 Protein substrates
 Polyacrylamide or
agarose gels
 Glass
 Nanowells
 Proteins deposited
on chip surface by
robots

Benfey & Protopapas, 2005

Protein Attachment
 Diffusion
 Protein suspended in Diffusion
random orientation, but
presumably active Adsorption/
 Adsorption/Absorption Absorption
 Some proteins inactive

 Covalent attachment
Covalent
 Some proteins inactive

 Affinity
 Orientation of protein
Affinity
precisely controlled


Protein Interactions
 Different capture molecules Antigen–
must be used to study antibody
different interactions
 Examples Protein–
 Antibodies (or antigens) for protein
detection
 Proteins for protein-protein
Aptamers
interaction
Enzyme–
 Enzyme-substrate for
substrate
biochemical function
Receptor–
ligand

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.

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

15 arrays

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