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12 arrays

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  • 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
  • 6. 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
  • 7. fluorescence detection of Cy5-labeled Streptavidin using a 4X4 photodiode array IC biochip. Excitation by a 12 mW He±Ne laser (632.8 nm).
  • 8. Single detectors vs. Vectors and arrays Single Vector Array 9
  • 9. 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)
  • 10. DNA Arrays (Gene chips)
  • 11. Example of a DNA Array (note green, yellow red colors; also note that only part of the total array is depicted)
  • 12. 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
  • 13. 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
  • 14. • 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.
  • 15. 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
  • 16. • DNA microarrays can be used to measure changes in gene expression levels, to detect single nucleotide polymorphisms (SNPs) , to genotype or resequence mutant genomes.
  • 17. 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.
  • 18. 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
  • 19. 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
  • 20. • Step 3- Make single-stranded DNA from the mRNA using “color coded” nucleotides.
  • 21. 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)
  • 22. 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.
  • 23. Control cDNA Experimental cDNA
  • 24. Step 5: Extract a small amount in a pipette.
  • 25. Step 6: Insert into first well.
  • 26. Step 7: Extract more cDNA and … … insert into second well, etc.
  • 27. Step 8: Continue until all wells are filled.
  • 28. Step 9: Allow to hybridize, then wash away all single-stranded DNA.
  • 29. 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)
  • 30. Step 10: Scan with a laser set to detect the color & process results on computer.
  • 31. 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
  • 32. PROTEIN MICROARRAY
  • 33. 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.
  • 34. 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
  • 35. Protein Array Fabrication  Protein substrates     Polyacrylamide or agarose gels Glass Nanowells Proteins deposited on chip surface by robots Benfey & Protopapas, 2005
  • 36. 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
  • 37. 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
  • 38. Expression Array  Probes (antibody) on surface recognize target proteins.  Identification of expressed proteins from samples.  Typical quantification method for large # of expressed proteins.
  • 39. Interaction Array  Probes (proteins, peptides, lipids) on surface interact with target proteins.  Identification of protein interactions.  High throughput discovery of interactions .
  • 40. Functional Array  Probes (proteins) on surface react with target molecules .  Reaction products are detected.  Main goal of proteomics.
  • 41. Sample Preparation  Labeled  Fluorescent Dye     Cy3/Cy5 via Lysines Photochemical Radioisotope May interfere
  • 42.  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
  • 43. Detection & Quantification  Scanner    Reference spots   Detects dye Adjusts for background Labeled known concentrations Computational Analysis
  • 44. 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

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