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22 January 2007 Specific immobilization of human immunoglobulin G on gold-coated silicon microcantilever array
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Abstract
We demonstrate a procedure for immobilizing human immunoglobulin G (IgG) on an array of gold-coated silicon microcantilevers. The procedure employed protein A for the specific immobilization of human IgG on the gold surface. Protein A bound specifically to the gold-coated upper surface of the silicon microcantilever and had no interaction with the silicon surface. It binds to the constant Fc regions of human IgG keeping the antigen binding sites on the variable Fab region free to bind to antigens. Fluorescent microscopy was done to analyze qualitatively the biomolecular binding of human IgG using FITC labeled goat anti-human IgG. The immobilization densities of protein A and human IgG were 112±19 ng/cm2 and 629±23ng/cm2, as determined employing horse radish peroxidase (HRP) labeled biomolecules by 3, 3', 4, 4'-tetramethyl benzidine (TMB) substrate assay. The uniformness of the biomolecular coatings was further determined by atomic force microscopy (AFM). Surface plasmon resonance (SPR) was used to cross-validate the immobilization density of functional human IgG molecules immobilized on the gold surface w.r.t. that obtained by TMB substrate assay.
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Sandeep Kumar Vashist, Rupinder Tewari, Ram Prakash Bajpai, Lalit Mohan Bharadwaj, and Roberto Raiteri "Specific immobilization of human immunoglobulin G on gold-coated silicon microcantilever array", Proc. SPIE 6465, Microfluidics, BioMEMS, and Medical Microsystems V, 64650G (22 January 2007); doi: 10.1117/12.695439; https://doi.org/10.1117/12.695439
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