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Title: Single enzyme electroanalysis
Traditional studies of enzymatic activity rely on the combined kinetics of millions of enzyme molecules to produce a product, an experimental approach that may wash out heterogeneities that exist between individual enzymes. Evaluating these properties on an enzyme-by-enzyme basis represents an unambiguous means of elucidating heterogeneities; however, the quantification of enzymatic activity at the single-enzyme level is fundamentally limited by the maximum catalytic rate, k cat , inherent to a given enzyme. For electrochemical methods measuring current, single enzymes must turn over greater than 10 7 molecules per second to produce a measurable signal on the order of 10 −12 A. Enzymes with this capability are extremely rare in nature, with typical k cat values for biologically relevant enzymes falling between 1 and 10 000 s −1 . Thus, clever amplification strategies are necessary to electrochemically detect the vast majority of enzymes. This review details the progress toward the electroanalytical detection and evaluation of single enzyme kinetics largely focused on the nanoimpact method, a chronoamperometric detection strategy that monitors the change in the current-time profile associated with stochastic collisions of freely diffusing entities ( e.g. , enzymes) onto a microelectrode or nanoelectrode surface. We discuss the experimental setups and methods developed in the last decade toward the quantification of single molecule enzymatic rates. Special emphasis is given to the limitations of measurement science in the observation of single enzyme activity and feasible methods of signal amplification with reasonable bandwidth.  more » « less
Award ID(s):
2003587
PAR ID:
10325051
Author(s) / Creator(s):
; ; ;
Date Published:
Journal Name:
The Analyst
Volume:
146
Issue:
11
ISSN:
0003-2654
Page Range / eLocation ID:
3413 to 3421
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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