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Transcript of 12 arrays
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Bio-chips (Lab-on-a-chip)
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System architectures
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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
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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
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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)
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DNA Arrays (Gene chips)
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Example of a DNA Array(note green, yellow red colors;
also note that only part of the totalarray is depicted)
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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
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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
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• 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.
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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
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• DNA microarrays can be used to measure changes in gene expression levels, to detect single nucleotide polymorphisms (SNPs) , to genotype or resequence mutant genomes.
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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.
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cDNAgene 1
cDNAgene 2
cDNAgene 3
cDNAgene 4
cDNAgene 5
DNA Array: Single-stranded copy DNA Oligonucleotides for each gene in a different well.
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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
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• Step 3- Make single-stranded DNA from the mRNA using “color coded” nucleotides.
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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)
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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.
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Control cDNA Experimental cDNA
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Step 5: Extract a smallamount in a pipette.
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Step 6: Insert into first well.
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… insert intosecond well, etc.
Step 7: Extractmore cDNA and …
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Step 8: Continue until all wells arefilled.
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Step 9: Allow to hybridize, then wash away all single-stranded DNA.
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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)
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Step 10: Scan with a laser set to detect the color & process results on
computer.
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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
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PROTEIN MICROARRAYPROTEIN MICROARRAY
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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
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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
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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
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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
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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
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Expression Array Probes (antibody) on surface recognize
target proteins.
Identification of expressed proteins from samples.
Typical quantification method for large # of expressed proteins.
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Interaction Array Probes (proteins, peptides, lipids) on
surface interact with target proteins.
Identification of protein interactions.
High throughput discovery of interactions.
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Functional Array Probes (proteins) on surface react with
target molecules .
Reaction products are detected.
Main goal of proteomics.
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Sample PreparationSample Preparation LabeledLabeled
Fluorescent DyeFluorescent Dye Cy3/Cy5 via Cy3/Cy5 via
LysinesLysines PhotochemicalPhotochemical RadioisotopeRadioisotope May interfereMay interfere
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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
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Detection & Detection & QuantificationQuantification
ScannerScanner Detects dyeDetects dye Adjusts for Adjusts for
backgroundbackground Reference spotsReference spots
Labeled known Labeled known concentrationsconcentrations
Computational Computational AnalysisAnalysis
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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