Preliminary study of real-time fiber optic based protein C biosensor.
Deficiency of protein C (PC), one of the human body's key anticoagulants, can lead to massive thrombotic complications. There is a diagnostic need to perform real-time assays, in order to quickly identify and treat this disease. An immuno-optical biosensor for the diagnosing of PC deficiencies and monitoring of PC concentrations is being developed for this purpose. Monoclonal antibody against PC (anti-PC) is immobilized on the surface of a tapered quartz fiber that is enclosed in a glass tube (capacity approximately 200 microL). Following sample injection, PC within a sample binds to the anti-PC in a highly specific reaction. The system is then probed with a fluorophore-tagged secondary antibody against PC. Excitation light is applied through the fiber, and the fluorescence intensity is correlated with the PC concentration in the sample. This study presents (1) a feasibility, direct binding assay, (2) a comparison of methods to immobilize anti-PC upon the fiber (direct immobilization vs an avidin-biotin bridge), and (3) effectiveness of an elution step to regenerate the fiber. PC-deficient patients typically have a concentration range less than 2.5 microg/mL. It was found that the sensor could detect PC levels down to 0.1 microg/mL in pure buffer with minimal optimization. Avidin-biotin immobilization of the primary antibody produced enhanced signals, up to 470% of the original intensities. Preliminary fiber regeneration tests achieved nearly a 50% increase in fiber lifetime with the use of a CaCl(2) elution step. Ultimately, further development may lead to automation and the use of the system as a multi-blood factor analyzer.
Spiker
J O
JO
Department of Chemical and Biochemical Engineering and Department of Biological Sciences, University of Maryland-Baltimore County, Baltimore, Maryland 21250, USA.
Kang
K A
KA
D000818:Animals; D000911:Antibodies, Monoclonal; D001360:Avidin; D015374:Biosensing Techniques / Q000379:methods* / Q000592:standards; D001709:Biotechnology; D001710:Biotin; D002021:Buffers; D005069:Evaluation Studies as Topic; D005336:Fiber Optic Technology; D006801:Humans; D051379:Mice; D055100:Optical Fibers; D011486:Protein C / Q000032:analysis* / Q000276:immunology / Q000592:standards; D020151:Protein C Deficiency / Q000175:diagnosis
D000911:Antibodies, Monoclonal; D002021:Buffers; D011486:Protein C; D001360:Avidin; D001710:Biotin