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.

[1]  G. S. Wilson,et al.  Biosensors : fundamentals and applications , 1987 .

[2]  T. R. Roberts,et al.  Pesticide chemistry and bioscience, the food-environment challenge , 1999 .

[3]  C P Price,et al.  Immunosensors: technology and opportunities in laboratory medicine. , 1996, Clinical chemistry.

[4]  V. A. Kabanov,et al.  Basic Properties of Soluble Interpolyelectrolyte Complexes Applied to Bioengineering and Cell Transformations , 1994 .

[5]  Bruce D. Hammock,et al.  Immunochemical techniques for environmental analysis I. Immunosensors , 1995 .

[6]  Donald G. Buerk,et al.  Biosensors: Theory and Applications , 1995 .

[7]  Robert J. Huber,et al.  Chemically sensitive field-effect transistors. , 1976, Biomedical engineering.

[8]  P Bergveld,et al.  A new approach to immunoFET operation. , 1990, Biosensors & bioelectronics.

[9]  K. Solomon.  Integrating Environmental Fate and Effects Information: The Keys to Ecotoxicological Risk Assessment for Pesticides , 1999 .

[10]  B. Hammock,et al.  Microplate immunoassay technique using polyelectrolyte carriers: Kinetic studies and application to detection of the herbicide atrazine , 1999 .

[11]  H. Nygren,et al.  Kinetics of antigen-antibody reactions at solid-liquid interfaces. , 1988, Journal of immunological methods.

[12]  N. Gupta,et al.  A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes , 1992, Steroids.

[13]  D Neumeier,et al.  Biotinylated steroid derivatives as ligands for biospecific interaction analysis with monoclonal antibodies using immunosensor devices. , 2000, Analytical biochemistry.

[14]  K. Rajkowski,et al.  A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyclonal anti-testosterone immunoglobulins. , 1989, Clinica chimica acta; international journal of clinical chemistry.

[15]  N A Lee,et al.  Environmental monitoring of pesticides by immunoanalytical techniques: validation, current status, and future perspectives. , 2001, Journal of AOAC International.

[16]  J. Sigoillot,et al.  Isolation and characterization of surfactant degrading bacteria in a marine environment , 1990 .

[17]  S. Aust,et al.  Roles of efficient substrates in enhancement of peroxidase-catalyzed oxidations. , 1997, Biochemistry.

[18]  C. McNeil,et al.  Separation-free electrochemical immunosensor for rapid determination of atrazine. , 1998, Biosensors & bioelectronics.

[19]  P Atanasov,et al.  Immunosensors: electrochemical sensing and other engineering approaches. , 1998, Biosensors & bioelectronics.

[20]  E. Gorovits,et al.  An investigation on the catalytic mechanism of enhanced chemiluminescence: immunochemical applications of this reaction. , 1989, Journal of bioluminescence and chemiluminescence.

[21]  D. Chan,et al.  Immunosensors--principles and applications to clinical chemistry. , 2001, Clinica chimica acta; international journal of clinical chemistry.

[22]  B. Geurts,et al.  Diffusion limited immunochemical sensing. , 1989, Bulletin of mathematical biology.

[23]  J. Rishpon,et al.  A rapid and sensitive heterogeneous immunoelectrochemical assay using disposable electrodes. , 1992, Journal of immunoassay.

[24]  H. Hara,et al.  Availability and development of an enzyme immunomicrosensor based on an ISFET for human immunoglobulins , 1990 .

[25]  Anatoly V. Zherdev,et al.  Immunosensor for the determination of the herbicide simazine based on an ion-selective field-effect transistor , 2000 .

[26]  M. Nakamura,et al.  An automated ELISA system using a pipette tip as a solid phase and a pH-sensitive field effect transistor as a detector. , 1995, Journal of immunological methods.

[27]  Mikhail A. Tsyganov,et al.  pH track of expanding bacterial populations , 1992 .

[28]  Anatoly V. Zherdev,et al.  Development and comparative study of different immunoenzyme techniques for pesticides detection , 1996 .

[29]  P. Woias,et al.  Protein detection with a novel ISFET-based zeta potential analyzer. , 1999, Biosensors & bioelectronics.

[30]  A. Barbaro,et al.  Immunoenzymatic assay using CHEMFET devices , 1991 .

[31]  G. Harsányi,et al.  Sensors in Biomedical Applications: Fundamentals, Technology and Applications , 2000 .

[32]  B. Hammock,et al.  Immunoassay techniques for detection of the herbicide simazine based on use of oppositely charged water-soluble polyelectrolytes. , 1999, Analytical chemistry.

[33]  Izumi Kubo,et al.  Immuno-FET sensor , 1989 .