Chemical attachment of monoclonal antibody on gold surface

Abstract

A 5 MHz quartz crystal mibrobalance (QCM) sensor was developed for detection of Vibrio harveyi, a bacteria causing luminous in shrimp. Immobilization of monoclonal antibody (MAb) against V. harveyi onto gold electrode of quartz crystal involved a three-step procedure. The procedure includes: (1) a formation of self- assembled monolayer (SAM) of carboxyl-terminated alkanethiol, (2) an activation of carboxyi groups by N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDCI), and (3) an attachment of the MAb to the activated carboxyl groups. The stepwise chemical modification of the gold-coated substrate was characterized by reflection-absorption infrared spectroscopy, water contact angle measurements and QCM. It has been demonstrated that the developed QCM-based immunosensor can be used for the detection of V. harveyi with a working range of 10³-10⁷ CFU/mL, a slight cross reactivity to V. vulnificus, and no specificity to V. paraheamolyticus. Controlling the density of MAb which is the active binding site of bacteria by the use of mixed SAMS of alkanethiols and 1%BSA as a blocking reagent can significantly improve the binding efficiency of the targeted bacteria.