Hydrogel-based protein microchips: manufacturing, properties, and applications.
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.
Rubina
A Yu
AY
Russian Academy of Sciences, Moscow, Russia.
Dementieva
E I
EI
Stomakhin
A A
AA
Darii
E L
EL
Pan'kov
S V
SV
Barsky
V E
VE
Ivanov
S M
SM
Konovalova
E V
EV
Mirzabekov
A D
AD
D004867:Equipment Design; D019544:Equipment Failure Analysis; D020100:Hydrogels; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000145:classification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
D020100:Hydrogels; D011506:Proteins