A new assay format for electrochemical immunosensors: polyelectrolyte-based separation on membrane carriers combined with detection of peroxidase activity by pH-sensitive field-effect transistor.
A new rapid immunotechnique combining separation of reactants by filtration through a porous membrane and potentiometric detection of the bound enzyme label by a pH-sensitive field-effect transistor is proposed. The complexes to be detected are formed by the method described earlier in (Anal. Chem. 71 (1999) 3538), including a homogeneous binding of immunoreactants and a polyanion carrier (polymethacrylate) followed by heterogeneous separation on a membrane incorporating an immobilized polycation (poly-N-vinyl-4-ethylpyridinium). The proposed technique for a sensitive detection of peroxidase label is based on the measurement of pH changes in the optimised substrate solution containing o-phenylenediamine, hydrogen peroxide and ascorbic acid. The antigens studied were herbicide atrazine and hormone testosterone. Their specific detection is realised via competitive binding of free and peroxidase-labelled antigens by antibodies integrating with a (staphylococcal protein A-polyanion) conjugate. The total analysis time is 20-25 min. The range of quantitative detection is 0.2-100 ng ml(-1) for atrazine and 5-300 ng ml(-1) for testosterone. Data scatter of replicate tests varies from 3 to 10%. Application of protein A-polyanion conjugate allows to use the proposed protocol for different antigens without additional treatment of specific antisera.
Institute of Biochemistry and Physiology of Micro-organisms, Russian Academy of Sciences, Nauki Prospect 5, 142290 Pushchino, Russia.
Yu V
YV
Plekhanova
A N
AN
Reshetilov
E V
EV
Yazynina
A V
AV
Zherdev
B B
BB
Dzantiev
D000936:Antigen-Antibody Complex / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D001280:Atrazine / Q000032:analysis* / Q000276:immunology; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004573:Electrolytes / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D005240:Feasibility Studies; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques / Q000295:instrumentation* / Q000379:methods; D008567:Membranes, Artificial; D009195:Peroxidase / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011109:Polymethacrylic Acids / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology; D013739:Testosterone / Q000032:analysis* / Q000276:immunology; D014173:Transistors, Electronic; D014462:Ultrafiltration / Q000295:instrumentation* / Q000379:methods
D000936:Antigen-Antibody Complex; D004573:Electrolytes; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011109:Polymethacrylic Acids; D013205:Staphylococcal Protein A; C030613:polymethacrylic acid; D013739:Testosterone; D009195:Peroxidase; D001280:Atrazine