A reusable and specific protein A-coated piezoelectric biosensor for flow injection immunoassay.
A hydrophilic matrix of periodate-oxidized dextran was used as a double-sided linker to covalently immobilize Staphylococcus aureus protein A (SpA) molecules onto a poly-L-lysine-modified piezoelectric crystal surface to improve their stability, activity, and binding specificity with human immunoglobulin G (IgG) in flow injection assays. The prepared sensing crystals displayed best sensitivity and reusability at a flow rate of 140 microL/min. A human IgG concentration as low as 0.3 nM can be detected by this system. Up to 19 successive assay repetitions were achieved without significant loss of sensitivity using the same crystal. The analysis of adsorption kinetics indicates that such a preparation can greatly increase the amount of available active human IgG binding sites on immobilized SpA. Hardly any response arising from unspecific binding was detected. In addition, the sensing crystal prepared by this method was found to retain activity better than one prepared via direct deposition when stored in either wet or dry states. Finally, the prepared SpA-coated crystals were applied to the affinity immobilization of polyclonal goat anti-Schistosoma japonicum glutathione-S-transferase (GST) and were able to subsequently detect GST and its genetically engineered mutant either in a purified form or in the crude cell lysate.
Lu
H C
HC
Department of Chemical Engineering, Chang Gung University, Taoyuan, Taiwan 333, ROC.
Chen
H M
HM
Lin
Y S
YS
Lin
J W
JW
D000818:Animals; D000909:Antibodies, Helminth; D015374:Biosensing Techniques; D001709:Biotechnology; D017743:Equipment Reuse; D004926:Escherichia coli / Q000235:genetics; D017022:Flow Injection Analysis / Q000379:methods*; D005982:Glutathione Transferase / Q000032:analysis / Q000235:genetics / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis; D007163:Immunosorbent Techniques; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D013205:Staphylococcal Protein A
D000909:Antibodies, Helminth; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D005982:Glutathione Transferase