Overview of microarrays

Microarrays consist of spots of biological solutions generated on a rectangular grid. Microarray technology includes applications for functional genomics, pharmacogenomics, SNP genotyping, proteomics and cell signalling. Microarrays can be fabricated by dispensing the presynthesized biological materials (cDNA, PCR products, peptides/proteins, etc.) or by actually producing the biomaterial directly on the substrate by in situ synthesis.

Microarrays can contain several thousand samples in the area of a few square centimeters. The manufacturing of these arrays by means of deposition of pre-synthesized biomaterials from separate tubes may be impractical due to the separate synthesis, purification and logistic requirements. In situ synthesis has the advantage of only requiring very small volumes of the different synthesis reagents regardless of the number of different end products within the microarray. Synthetically derived surface-bound biomaterials reduce the limitations related to logistic and handling problems. The synthesized materials can be modified with cross-linking reagents, dyes or biotin, and nucleotide analogues. Ink-jet is ideal for dispensing the fluids involved in the synthesis as it is a non-contact technology and it does not cross-contaminate the fluids.

MicroFab has been working on the microarray production by in situ synthesis of both DNA and peptides. The two projects were sponsored by NIH SBIR grants (Phase I for DNA and Phase I & II for peptides).

DNA microarrays

DNA microarray analysis has received significant attention as a technology with the promise to further advance biotechnology and our understanding of basic molecular pathways of the cell. The development of high-density DNA arrays has arisen, as a consequence of the worldwide genome sequencing efforts. Genome sequencing projects have provided critical tools for the next generation of studies in genomics. The appeal and demand for high-density DNA arrays has been driven by researchers who desire to efficiently scan thousands of genes for expression activity in cellular and tissue systems within a single experiment. DNA microarray technology includes such applications as mutation detection, functional genomics, pharmacogenomics, SNP genotyping, gene expression studies, proteomics and cell signaling.

MicroFab Technologies, Inc. was a recipient of an NIH Phase I SBIR for the in situ synthesis of oligonucleotide microarrays for gene expression studies. Our ink-jet technology was used to deliver small volumes of synthesis reagents to a substrate. Protogene Laboratories also utilized our ink-jet devices to synthesize onto glass oligonucleotide arrays of up to 80-mer in length, which was marketed as Flex Chip technology. These FlexChips were readily customizable for applications including gene mapping, SNP genotyping, gene expression monitoring, and other pharmacogenomics uses. MicroFab Technologies optimized and provided ink-jet devices to Protogene to be incorporated into their in situ DNA synthesis equipment. This system was comprised of a computer controlled X and Y stage to position the substrate under the ink-jet nozzles. A separate ink-jet device was provided for each of the four nucleotide monomers with a fifth device for delivery of the activating reagent for synthetic coupling. The glass substrate and ink-jet devices where contained within an anhydrous chamber during synthesis.

Left - Overview of the process steps during in situ ink-jet DNA synthesis. Right bottom - MicroFab Technologies ink-jet devices mounted on the Protogene Laboratories ink-jet DNA synthesizer. Right top - Reservoirs with the four phosphoramidite solutions suplying the ink-jet devices.

The solvent used for the monomer and activator reagents can be a mixture of acetonitrile and adiponitrile or propylene carbonate. The key to this system was the use of a patterned substrate to segregate fluids by differences in surface tension (see figure below). The micro-droplets of reagents are deposited on the narrow pitch functionalized binding sites created by thousands of wetting features on the glass slide.

Micro-patterned substrate concept utilized by Protogene Laboratories for their in situ dna synthesis on glass substrates [Image courtesy of Protogene Laboratories].

Peptide microarrays

Peptide research is important to life science and drug industry in many ways, since peptides play critical functional roles in cells and tissues. By using a combinatorial approach the synthetic peptides are grouped in very large numbers in a single unit, a peptide library in a microarray format, thus allowing a massively-accelerated sample processing. A flexible peptide microarray that can be analyzed in an automated fashion is a desired tool in the drug discovery process to reduce screening time. Some of the applications of the peptide microarray are:

• Defining minimum protein-protein interaction domains - epitope mapping
• Key residues identification - identify the residue involved in binding
• General drug screening - interaction with the drugs to determine drug leads
• Protein conformation probe - evaluation of the binding for multiple binding domains
• Interaction between protein and other molecules - protein-DNA, protein-polysaccharide, protein-cell, protein-metal interactions

When using the in situ synthesis the peptide are produced at the desired location in the microarray, one residue at a time. This methodology has its main advantage in the limited number of fluids involved in the process: the 20 (natural) amino acids and the fluids required for washing, blocking (capping), and deprotecting. The synthesis starts with a derivatized substrate on which the amino acids are dispensed (can have different amino acids at each location). After deposition and a coupling time for the reaction to complete the substrate undergoes capping where the linking sites on the subtrates are blocked for future reactions. The coupled amino acids are deprotected at all locations and then the next amino acids in the peptide chains are deposited at each location. Washing steps are also done globally to remove the residual reagents.

At MicroFab we have built a synthesis system based on Fmoc chemistry. The amino acid dispense subsystem is presented in the figure to the right. The camera in the picture determines the initial offset of the dispensers that will be used in the control program to make sure all the amino acids that need to be dispensed at one location of the microarray will completely overlap.

Peptide synthesized at MicroFab were verified by Bromophenol staining (detects the free amine at the end of the synthesis), HPLC and MS analysis of the cleaved peptides, and antibody attachment and staininig on the peptide directly within the microarray. Some examples are included below.

Bromophenol Blue staining of Enkephalin synthesized on cellulose support.

Evaluation of Enkephalin synthesis by HPLC analysis. Elution time for Enkephalin is 9.85 minutes.

Evaluation of Enkephalin synthesis by antibody attachment and Western blot. The dark spot next to 18 indicate the synthesis location where antibody attachment was performed.

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