Basic principle of optical immunosensor using fluorescence anisotropy

An immunosensor, utilizing immobilized antibody, is a promising sensing device for its high specificity and broad spectrum for detecting object. But physical adsorption is often an obstacle for its luck of enzymatic reactions. Fluorescence anisotropy immunosensor is a method which is, in principle, free from interference of physical adsorption. In this study 3 reagents, FITC, DNS-Cl and PAS, were employed for fluorescent labeling of antibody and lifetime of fluorophore was investigated to achieve optimum sensitivity. PAS, fluorophore with longest lifetime of up to 100 ns, showed the highest sensitivity which is in accordance with the correlation time of rotational relaxation of antibody, suggesting the importance of fluorescence lifetime being comparable with the correlation time of anisotropy decay. Immobilization procedure of antibody was also investigated to reduce interference of physical adsorption. Antibody immobilized on hydrophobic surface showed substantial anisotropy change by adsorption of non-antigenic protein but antibody on hydrophilic substrate showed no detectable anisotropy change. Further improvement of experimental condition will lead to application for microanalysis or implantable sensor. For practical use of this method, fluorescence measurement with higher S/N ratio is still to be attained.

[1]  L. Stryer,et al.  Segmental flexibility in an antibody molecule. , 1970, Journal of molecular biology.

[2]  H. Tanaka,et al.  Fluorescence study of N-(3-pyrene)maleimide conjugated to rabbit skeletal F-actin and plasmodium actin polymers. , 1976, Biochimica et biophysica acta.

[3]  A. Ellington,et al.  Adapting selected nucleic acid ligands (aptamers) to biosensors. , 1998, Analytical chemistry.

[4]  J. Steinkamp,et al.  Analysis of fluorescence lifetime and quenching of FITC-conjugated antibodies on cells by phase-sensitive flow cytometry. , 1996, Cytometry.

[5]  E Tamiya,et al.  Fluorescence polarization immunoassay employing immobilized antibody. , 1991, Biosensors & bioelectronics.

[6]  S. Avrameas,et al.  Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. , 1969, Immunochemistry.

[7]  A. Andreoni,et al.  Fluorochromes with long-lived fluorescence as potential labels for pulsed laser immunocytofluorometry: photophysical characterization of pyrene derivatives. , 1994, Journal of biochemical and biophysical methods.

[8]  V. Schumaker,et al.  Segmental flexibility of immunoglobulin G antibody molecules in solution: a new interpretation. , 1981, Biochemistry.

[9]  C. Kolvenbach,et al.  Ionic-strength- and pH-dependent conformational states of human plasma fibronectin. , 1991, Biochemistry.

[10]  K. Maxwell,et al.  Use of laser nephelometry in the measurement of serum proteins. , 1976, Clinical chemistry.

[11]  G. Shulman.  Comparison of specific protein assays in biological fluids by radial immunodiffusion and laser nephelometer. , 1979, Clinical biochemistry.