Electrochemical biosensor modified with dsDNA monolayer for restriction enzyme activity determination

Joanna Zajda , Łukasz Górski , Elżbieta Malinowska

Abstract

A simple and cost effective method for the determination of restriction endonuclease activity is presented. dsDNA immobilized at a gold electrode surface is used as the enzymatic substrate, and an external cationic redox probe is employed in voltammetric measurements for analytical signal generation. The assessment of enzyme activity is based on a decrease of a current signal derived from reduction of methylene blue which is present in the sample solution. For this reason, the covalent attachment of the label molecule is not required which significantly reduces costs of the analysis and simplifies the entire determination procedure. The influence of buffer components on utilized dsDNA/MCH monolayer stability and integrity is also verified. Electrochemical impedance spectroscopy measurements reveal that due to pinhole formation during enzyme activity measurement the presence of any surfactants should be avoided. Additionally, it is shown that the sensitivity of the electrochemical biosensor can be tuned by changing the restriction site location along the DNA length. Under optimal conditions the proposed biosensor exhibits a linear response toward PvuII activity within a range from 0.25 to 1.50 U/μL.
Author Joanna Zajda ZMB
Joanna Zajda,,
- Department Of Microbioanalytics
, Łukasz Górski ZMB
Łukasz Górski,,
- Department Of Microbioanalytics
, Elżbieta Malinowska ZMB
Elżbieta Malinowska,,
- Department Of Microbioanalytics
Journal seriesBioelectrochemistry, ISSN 1567-5394
Issue year2016
Vol109
Pages63-69
Publication size in sheets0.5
Keywords in EnglishDNA-based biosensor; Electrochemical sensor; Restriction endonuclease activity determination
DOIDOI:10.1016/j.bioelechem.2016.01.009
URL http://www.sciencedirect.com.doi.eczyt.bg.pw.edu.pl/science/article/pii/S1567539416300093
Languageen angielski
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Score (nominal)35
ScoreMinisterial score = 35.0, 28-11-2017, ArticleFromJournal
Ministerial score (2013-2016) = 35.0, 28-11-2017, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 3.346 (2) - 2016=3.413 (5)
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