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Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering

Nature Biotechnology volume 22, pages 1133–1138 (2004)Cite this article

Abstract

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.

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Figure 1: Structure and SERRS spectra of substrates and component dyes.
Figure 2: SERRS spectra with time following lipase-catalyzed cleavage of substrate 1.
Figure 3: Change in SERRS response at 1,420 cm−1 before and after addition of enzyme.
Figure 4: Variation in rates of enzyme hydrolysis monitored by SERRS.
Figure 5: Dilution of Pseudomonas cepacia lipase monitored for each enantiomer.

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Acknowledgements

The authors wish to thank the BBSRC for funding this work.

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Author notes

  1. Barry D Moore and Duncan Graham: These authors contributed equally to the work.

Authors and Affiliations

  1. Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, G1 1XL, UK

    Barry D Moore, Lorna Stevenson, Chris Cassidy & Duncan Graham

  2. School of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, EH9 3JJ, UK

    Alan Watt, Sabine Flitsch & Nicolas J Turner

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  1. Barry D Moore
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  2. Lorna Stevenson
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Correspondence to Barry D Moore or Duncan Graham.

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Supplementary information

Supplementary Fig. 1

Rate of colloid Aggregation (PDF 63 kb)

Supplementary Fig. 2

SERRS monitoring of complete reaction profile (PDF 45 kb)

Supplementary Methods (PDF 260 kb)

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Moore, B., Stevenson, L., Watt, A. et al. Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering. Nat Biotechnol 22, 1133–1138 (2004). https://doi.org/10.1038/nbt1003

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