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Figure 10.7. (A) Aldehyde group attachment to the substrate; (B) immobilization of a protein at an array site and subsequent quenching of the unreacted site with BSA. (Reproduced with permission from

http://cgr.harvard.edu/macbeath/research/protein_microarrays.)

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Figure 10.8. (A) Preparation of BSA-NHS slides; (B) immobilization of proteins and subsequent quenching with glycine. (Reproduced with permission from http://cgr.harvard.edu/macbeath/ research/protein_microarrays.)

that can react with surface amines on the proteins (step (2) in (A)). The unre-acted sites on the substrate are then quenched with glycene (step (2) in (B)).

A number of other approaches have been utilized to produce protein microarrays. They include the use of layers of aluminum or gold, hydrophilic polymers, and polyacrylamide gels for immobilization of capture agents. The polyacrylamide gel pad method has already been discussed in Section 10.2. Each method requires appropriate chemistry to orient each molecule in the same direction and to create the necessary hydrophilic environment for proteins.

An approach being pursued by Zyomy of Hayward, California utilizes photolithography to etch miniature wells on the surface of a silicon chip. The proteins or antibodies are immobilized in the flow chambers on the chip to maintain an aqueous solution environment.

Another approach used by Large Scale Biology of Vacaville, California utilizes hundreds of thin plastic rods, each doped with a particular antibody and bundled together in a sheaf. Chips in the form of micrometer-thin slices are then produced from this sheaf by cutting them transversely.

Recently, Lahiri and co-workers (Fang et al., 2002) have reported the fabrication of a membrane-protein microarray. They utilized surface modification with g-aminopropylsilane (GAPS) and produced microspots on GAPS by printing vesicular solutions of G-protein-coupled receptors (GPCRs) using a quill pin printer.

Capture Agents. Immobilized antibodies have been frequently used as capture agents. Protein microarrays have been an array of different antibodies that are monoclonal, polyclonal, antibody fragments, or synthetic polypeptide ligands. Other capture agents used are aptamers (single-stranded nucleic acids that complex with proteins) and oligonucleotides that bind specifically to proteins. Light-sensitive "photoaptamers" are being used by SomaLogic of Boulder, Colorado. These photoaptamers capture proteins and covalently cross-link with them when exposed to UV light. The oligonucleotides and aptamers offer the advantage that the same technology used to print the microarrays for m-RNA expression can be used.

Amplification. The technique of rolling circle amplification (RCA) (Lizardi et al., 1998) has been used to increase the sensitivity for multiplexed arrays on antibody microarray (Schweitzer et al., 2000). An adaptation of RCA is used where the 5' end of an oligonucleotide primer is attached to an antibody. Consequently, the presence of a circular DNA, DNA polymerase, and nucleotides allows rolling circle replication to generate a concatamer of circle DNA sequence copies attached to the antibody. The concatamer, which are tandem arrays of monomeric DNA molecules with complementary ends, can then be detected by hybridization with fluorescently labeled complementary oligonu-cleotide probes. This method of amplification has been reported to yield a 100-to 1000-fold improvement over detection using simply fluorescently labeled antibodies or streptavidin (Schweitzer, 2000).

Detection. Fluorescence detection is the widely used method. However, this necessitates labeling proteins with a fluorochrome. The risk is that the binding with the fluorochrome may alter the ability of the protein to bind (interact) with the immobilized capture agent. Another method is based on the use of the surface plasmon resonance technique (SPR) discussed in Chapter 9. The microarrays for this detection are fabricated by immobilizing the test proteins or antibodies on a metal (gold)-coated glass chip. Biacore is marketing SPR-based protein chips. Ciphergen of Fremont, California utilizes laser evapora-

Fluorophore Glass Slide

Figure 10.9. Schematic of the specific binding between an immobilized antibody and the fluorescently labeled protein. (Reproduced with permission from http://cgr.harvard.edu/macbeath/research/ protein_microarrays.)

tion of the captured protein spot into a benchtop time-of-flight mass spectrometer to analyze the protein.

Protein Microarray in Action. As an illustration of the use of protein microarray, Figure 10.9 shows the schematic of three different types of antibodies immobilized on a glass slide. One type of antibody selectively binds with the fluorescently labeled protein that can then be detected. This approach was used to probe protein-protein interactions between three pairs: (i) protein G and IgG, (ii) p50 and 1KB a, and (iii) FRB domain of FKBPI2, the last pair requiring a small molecule, rapamycin, to enhance interaction. The results are shown in Figure 10.10.

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