Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids |
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Authors: | Kagan Kerman Masato Saito Eiichi Tamiya |
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Institution: | (1) School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi City Ishikawa, 923-1292, Japan;(2) Present address: Department of Chemistry, The University of Western Ontario, 1151 Richmond Street, London, ON, N6A 5B7, Canada;(3) Present address: Department of Applied Physics, Graduate School of Engineering, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan |
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Abstract: | Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination
with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms
(SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids
and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive
indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface.
When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached
to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal
from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very
effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic
digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring
the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the
target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism
(GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect
SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and
pharmaceutical applications.
Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant
structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively
choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Electrochemical DNA biosensor Chitosan nanoparticles Peptide nucleic acids Single-nucleotide polymorphism detection Nuclease S1 |
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