Gel-based microarrays (biochips) consisting of nanoliter and sub-nanoliter gel drops on hydrophobic substrate are a versatile technology platform for immobilization of proteins and other biopolymers. Biochips provide a highly hydrophilic environment, which stabilizes immobilized molecules and facilitates their interactions with analytes. The probes are immobilized simultaneously with gel polymerization, evenly distributed throughout individual elements, and are easily accessible because of large pores. Each element is an isolated nanotube. Applications of biochips in the studies of protein interactions with other proteins, nucleic acids, and glycans are described. In particular, biochips are compatible with MALDI-MS. Biochip-based assay of prostate-specific antigen became the first protein microarray approved for clinical use by a national regulatory agency. In this review, 3-D immobilization is compared with mainstream technologies based on surface immobilization.
Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.
A method of simultaneous analysis of staphylococcal enterotoxins using hydrogel-based microarrays (biochips) has been developed. The method allows simultaneous quantitative detection of seven enterotoxins: A, B, C1, D, E, G, and I in a single sample. The development of the method included expression and purification of recombinant toxins, production of panels of monoclonal antibodies (mAbs) against the toxins, and design and manufacturing of an experimental biochip for the screening of mAbs and selection of optimal pairs of primary and secondary antibodies for each toxin. The selected mAbs have high affinity toward their targets and no cross-reactivity with unrelated enterotoxins. Finally, a diagnostic biochip was designed for quantitative analysis of the toxins, and the analytical protocols were optimized. The sensitivity of the detection reached 0.1-0.5 ng/mL, depending on the type of enterotoxin. The evaluation of the resulting biochip using spiked food samples demonstrated that the sensitivity, specificity, and reproducibility of the proposed test system fully satisfy the requirements for traditional immunoanalytical systems. The diagnostic biochips manufactured on reflecting metal-coated surfaces shortened the time of analysis from 17 to 2 h without loss of sensitivity. The method was successfully tested on samples of food and biological media.
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