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A Strategy for Development of Electrochemical DNA Bio Sensors

A Strategy for Development of Electrochemical DNA Bio Sensors

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12/26/2012

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Title: A Strategy for Development of Electrochemical DNABiosensor based on Site-specific DNA Cleavage of RestrictionEndonucleaseAuthors: Jinghua Chen, Jing zhang, Huanghao Yang, FengfuFu, Guonan ChenPII: S0956-5663(10)00301-5DOI:doi:10.1016/j.bios.2010.05.033Reference: BIOS 3807To appear in:
Biosensors and Bioelectronics
Received date: 12-3-2010Revised date: 7-5-2010Accepted date: 24-5-2010Please cite this article as: Chen, J., zhang, J., Yang, H., Fu, F., Chen, G., AStrategy for Development of Electrochemical DNA Biosensor based on Site-specificDNA Cleavage of Restriction Endonuclease,
Biosensors and Bioelectronics
(2008),doi:10.1016/j.bios.2010.05.033This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.Themanuscriptwillundergocopyediting,typesetting,andreviewoftheresultingproobefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
 
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A Strategy for Development ofElectrochemical DNA
1
Biosensor based on Site-specific DNA Cleavage of 
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Restriction Endonuclease
34
Jinghua Chen
a,b
, Jing zhang
c
, Huanghao Yang
a
*, Fengfu Fu
a
, Guonan Chen
a*
5
a.Ministry of Education Key Laboratory of Analysis and DetectionTechnology for 
6
Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection
7
Technology for Food Safety,Department of Chemistry, Fuzhou University,
8
Fuzhou, 350002, China
9
 b.Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical
10
University, Fuzhou 350004, China.
11
c.Pharmaceutical department of Fujian College of Medical Occupation and
12
Technology, Fuzhou 350101, China
1314
Abstract
15
A new strategy for development ofelectrochemical DNA biosensor based on
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site-specific DNA cleavage of restriction endonuclease and using quantum dots as
17
reporter was reported in this paper. The biosenser was fabricated by immobilizing a
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capture hairpin probe, thiolated single strand DNA labeled with biotin group, on a gold
19
electrode. BfuCI nuclease, which is able to specifically cleave only double strand DNA
20
 but not single strand DNA, was used to reduce background current and improve the
21
sensitivity. We demonstrated that the capture hairpin probe can be cleaved by BfuCI
22 
*
Cooresponding author, e-mail:gnchen@fzu.edu.cn(G. Chen);hhyang@fzu.edu.cn(H. Yang); Tel.: +86 591 87893315; fax: +86 591 83713866
 
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nucleasein the absence of target DNA, but can not be cleaved in the presence of target
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DNA. The difference before and after enzymatic cleavage was then monitored by
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electrochemical method after the quantum dots were dissolved from the hybrids. Our 
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resuts suggested that the usage of BfuCI nuclease obviously improved the sensitivity
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and selectivity of the biosensor. We successfully applied this method to the
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sequence-selective discrimination between perfectly matched and mismatched target
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DNA including a single-base mismatched target DNA, and detected as low as 3.3×10
-14
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M of complementarytarget DNA.Furthermore, our above strategy was also verified
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with fluorescent method by designing a fluorescent molecular beacon (MB),which
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combinedthecapture hairpin probe and a pair of fluorophore(TAMRA) and quencher 
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(DABCYL). The fluorescent results is consistent with that of electroanalysis, further 
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indicated that the proposed new strategy indeed works as our expected.
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Keywords:
Electrochemical DNA Biosensor, Site-specific DNA Cleavage, Restriction
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Endonuclease, Cymbidium mosaic virus
3637
1. Introduction
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Methods for the sequence-specific DNA detection have attracted significant
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attention due to possible applications in fields ranging from virus detection to the
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diagnosis of genetic diseases[Heller et al., 2002; Balakin et al., 1998]. Consequently,
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various techniques have been employed for the detection of DNA hybridization, such
42
as chromosome analysis[Jorge et al., 1996], fluorescence in situ hybridization(Jilani
43
et al., 2008)and real-time quantitative reverse transcription PCR(Rong et al., 2002).
44

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