Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering.
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.
Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK.
Barry D
BD
Moore
Lorna
L
Stevenson
Alan
A
Watt
Sabine
S
Flitsch
Nicolas J
NJ
Turner
Chris
C
Cassidy
Duncan
D
Graham
D020099:Coated Materials, Biocompatible / Q000032:analysis / Q000737:chemistry*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry; D008049:Lipase / Q000032:analysis* / Q000737:chemistry*; D008422:Materials Testing; D008832:Microchemistry / Q000379:methods*; D043942:Nanotubes / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012834:Silver / Q000737:chemistry*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013237:Stereoisomerism
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D012834:Silver; D008049:Lipase