Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering Barry D Moore 1,3 , Lorna Stevenson 1 , Alan Watt 2 , Sabine Flitsch 2 , Nicolas J Turner 2 , Chris Cassidy 1 & Duncan Graham 1,3 Measurement of enzyme activity and selectivity at in vivo concentrations is highly desirable in a range of fields including diagnostics, functional proteomics and directed evolution. Here we demonstrate how surface-enhanced resonance Raman scattering (SERRS), measured using silver nanoparticles, can be used to detect the activity of hydrolases at ultra-low levels. This approach was made possible by designing ‘masked’ enzyme substrates that are initially completely undetected by SERRS. Turnover of the substrate by the enzyme leads to the release of a surface targeting dye, and intense SERRS signals proportional to enzyme activity are generated. The method was used to rapidly screen the relative activities and enantioselectivities of fourteen enzymes including examples of lipases, esterases and proteases. In the current format the sensitivity of the technique is sufficient to detect 500 enzyme molecules, which offers the potential to detect multiple enzyme activities simultaneously and at levels found within single cells. Detection of enzyme activity and selectivity has attracted a large amount of interest because of an increased need for functional protein analysis since the sequencing of the human genome. In response to this demand, high-throughput screening methods have been devel- oped, mainly by adapting existing techniques. These include UV-vis 1,2 and IR spectroscopy 3 , gas chromatography 4 , mass spectrometry 5,6 , capillary electrophoresis 7 , colorimetric analysis 8–10 and fluorescence 11 . The use of UV-vis and fluorescence spectroscopy permits determina- tions in the range of several thousand samples per day but often the sensitivity and selectivity are not adequate. To overcome this, intern- ally quenched fluorescent substrates have been developed to provide more sensitive enzyme detection 12 . These substrates have a fluoro- phore and quencher on the same molecule separated by a linker that is cleaved by the action of the enzyme. Initially very low fluorescence is observed but after enzyme cleavage the quencher is separated from the fluorophore and emission occurs. This allows very sensitive monitor- ing (picomole levels have been achieved) but nevertheless the approach can suffer from the background fluorescence commonly found with cellular extracts. In addition the broad nature of fluores- cence emissions provides limited potential for multiplexing whereby many enzyme reactions can be studied simultaneously. A key enabling technology in biotechnology would therefore be the development of a technique that allowed rapid and simultaneous characterization of multiple enzyme activities and specificities at a concentration and purity found in living cells. To achieve this goal the detection technique must be extremely sensitive and capable of identifying multiple components within a mixture, without separa- tion. SERRS fulfils these criteria and is thus applied in this study. SERRS requires the target species to be colored and to adsorb onto a roughened metal surface. It produces an enhanced vibrational spectrum of the target species that is characterized by multiple sharp peaks equivalent to a ‘fingerprint’ of the target species 13,14 . The metal surface may be in the form of an electrode or a vapor- deposited metal film, but a particularly convenient form is that of citrate-reduced silver nanoparticles. These provide excellent and reproducible SERRS spectra 15 . The silver nanoparticles are B40 nm in diameter and can be easily prepared as a monodispersed suspen- sion. To provide maximum enhancement they are aggregated into discrete clusters by the addition of agents that alter the effective surface charge, such as sodium chloride or poly-(L-lysine) 16 . The benefits of using SERRS and nanoparticles in terms of sensitivity and selectivity have previously been demonstrated by the detection of DNA at ultra-low concentrations 17,18 and by the multiplexed genotyping of the mutational status of the transmembrane conduc- tance regulator gene for cystic fibrosis 19,20 . The crucial step in obtaining SERRS is getting the target species to adsorb onto the surface of the silver nanoparticle. We have studied this aspect in considerable detail and have found that benzotriazole-based com- pounds exhibit a strong propensity to bind to the silver nanoparticle surface by displacing the citrate surface layer. Direct complexation onto the silver leads to generation of signals relative to species in solution that remain undetected. Published online 8 August 2004; doi:10.1038/nbt1003 1 Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK. 2 School of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JJ, UK. 3 These authors contributed equally to the work. Correspondence and requests for materials should be addressed to B.D.M. (b.d.moore@strath.ac.uk) or D.G. (duncan.graham@strath.ac.uk). NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 9 SEPTEMBER 2004 1133 ARTICLES © 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology