Tan Eu Vian, Lowe Christopher R
Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK.
Anal Chem. 2009 Sep 15;81(18):7579-89. doi: 10.1021/ac9008989.
Optical sensors are widely utilized in drug discovery to analyze biomolecular interactions in vitro. Aside from additional time and cost demands, other issues associated with labeled screening methods include signal interference that can arise from the label per se and/or the screened compounds. This report describes an enzyme inhibition-based holographic sensor as a potential label-free detection system, using acetylcholinesterase (acetylcholine acetylhydrolase; EC 3.1.1.7; abbreviated herein AChE) as the model enzyme. pH-responsive reflection holograms, incorporated into "smart" hydrogel films bearing ionizable monomers, were used to monitor the pH change resulting from acetic acid produced by the hydrolysis of the substrate acetylcholine. The enzyme was immobilized on the sensor by an entrapment and in situ cross-linking method; no chemical modification and/or prelabeling of the enzyme (or the substrate) was required. The fully reversible sensor exhibited good operational and storage stability, allowing relatively short assay times and repeated use of a single sensor. Apparent inhibition parameters for several drug inhibitors of the enzyme were determined. The feasibility of adapting these sensors into an array format for prospective high-throughput screening, without compromising their intrinsic optical and functional properties, was also demonstrated.
