Multiplex screening using enzyme inhibition, fluorescence detection and chemometrics

Fluorescence enzyme inhibition assays have been established for a number of years as valuable methods of analysis in clinical chemistry and other fields. Those in common use are normally single analyte assays. However, in many cases (e.g. drug screening) dual or multiplex assays would be much more valuable, with the advantages of increased information content, saving in time and costs, and the elimination of some sources of sampling variance. This project has investigated single and dual screening assays of enzyme inhibitors, namely 3-nitrophenylboronic acid (3-NPBA), phenylethyl /3-0- thiogalactopyranoside (PETG) and sodium vanadate (VI), using flow injection fluorescence spectroscopy and chemometric methods to resolve strongly overlapping fluorescence spectra. The single and dual screening assays have been based on flow injection analysis methodology, with immobilised enzymes on solid phase reactors to investigate the enzyme inhibitors. The assays were rapid, allowing around 15-25 measurements to be made per hour. The inhibitions of alkaline protease, alkaline phosphatase and /3-galactosidase with their inhibitors at flg/ml levels were achieved. An alternative approach to these dual assays has been investigated by the use of multivariate techniques. Such techniques allow accurate and reliable results to be obtained even from spectra that contain extremely overlapping signals. Moreover, preliminary investigation of three fluorophores which gave strongly overlapping spectra, using flow injection fluorescence spectroscopy and partial least squares (PLS-1) model has been successful. By combination of this flow injection fluorescence spectroscopy with the use of chemometrics, many applications can be envisaged in biochemical, clinical, and pharmaceutical industries. With the findings of this research the system described here can be developed for use in high throughput screening of candidate drug molecules and many screening processes throughout the different industries.