Format

Send to

Choose Destination
ACS Nano. 2016 Aug 23;10(8):7476-84. doi: 10.1021/acsnano.6b02062. Epub 2016 Aug 5.

Label-Free Electrical Detection of Enzymatic Reactions in Nanochannels.

Author information

1
Department of Mechanical Engineering, Boston University , Boston, Massachusetts 02215, United States.
2
Department of Mechanical Engineering, Ajou University , Suwon 443-749, South Korea.
3
Department of Pharmaceutical Chemistry, University of California , San Francisco, California 94158, United States.
4
Department of Mechanical Engineering, Stanford University , Stanford, California 94305, United States.

Abstract

We report label-free electrical detection of enzymatic reactions using 2-D nanofluidic channels and investigate reaction kinetics of enzymatic reactions on immobilized substrates in nanoscale-confined spaces. Trypsin proteolysis is chosen for demonstration of the detection scheme. When trypsin cleaves poly-l-lysine coated on the surface of silica nanochannels, the resulting change of surface charge density can be detected by monitoring the ionic conductance of the nanochannels. Our results show that detection of such surface enzymatic reactions is faster than detection of surface binding reactions in nanochannels for low-concentration analytes. Furthermore, the nanochannel sensor has a sensitivity down to 5 ng/mL, which statistically corresponds to a single enzyme per nanochannel. Our results also suggest that enzyme kinetics in nanochannels is fundamentally different from that in bulk solutions or plain surfaces. Such enzymatic reactions form two clear self-propagating reaction fronts inside the nanochannels, and the reaction fronts follow square-root time dependences at high enzyme concentrations due to significant nonspecific adsorption. However, at low enzyme concentrations when nonspecific adsorption is negligible, the reaction fronts propagate linearly with time, and the corresponding propagation speed is related to the channel geometry, enzyme concentration, catalytic reaction constant, diffusion coefficient, and substrate surface density. Optimization of this nanochannel sensor could lead to a quick-response, highly sensitive, and label-free sensor for enzyme assay and kinetic studies.

KEYWORDS:

electrical detection; enzyme kinetics; immobilized substrates; nanochannels; surface enzymatic reactions; trypsin proteolysis

PMID:
27472431
DOI:
10.1021/acsnano.6b02062
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center