Rapid electrochemical detection of single and double nucleotide mismatches (mutations) of sequences from HIV samples

For the first time, the detection of DNA sequences, related to HIV (found in AIDS patients), using Methylene Blue (MB) as the redox indicator was performed. The voltametric signals have been investigated on bared chitosan (CS) modified; probe immobilized (DNA single strand) and target-hybridized (DNA double strand) electrodes sequentially by means of Cyclic Voltammetry (CV) and Square Wave Voltammetry (SWV). The response of hybridization between the probe and perfectly complementary, double-base mismatch, single-base mismatch of analyzed targets were clearly identified. The response of CS modified electrode with non-complementary target sequence and the response of CS-free electrode (adsorption only) with complementary target were used as control references to confirm whether the detected signals correspond trully to the hybridization and respond selectively to the targets.
Nội dung trích xuất từ tài liệu:
Rapid electrochemical detection of single and double nucleotide mismatches (mutations) of sequences from HIV samplesJournal of Chemistry, Vol. 44 (3), P. 377 - 381, 2006 RAPID ELECTROCHEMICAL DETECTION OF SINGLE AND DOUBLE NUCLEOTIDE MISMATCHES (MUTATIONS) OF SEQUENCES FROM HIV SAMPLES Received 15 April 2005 Tran Dai Lam , Bui Tien Trinh2, Nguyen Duc Chien2 1 1 Faculty of Chemical Technology, Hanoi University of Technology (HUT) 2 International Training Institute for Materials Science, HUT SUMMARY For the first time, the detection of DNA sequences, related to HIV (found in AIDS patients), using Methylene Blue (MB) as the redox indicator was performed. The voltametric signals have been investigated on bared chitosan (CS) modified; probe immobilized (DNA single strand) and target-hybridized (DNA double strand) electrodes sequentially by means of Cyclic Voltammetry (CV) and Square Wave Voltammetry (SWV). The response of hybridization between the probe and perfectly complementary, double-base mismatch, single-base mismatch of analyzed targets were clearly identified. The response of CS modified electrode with non-complementary target sequence and the response of CS-free electrode (adsorption only) with complementary target were used as control references to confirm whether the detected signals correspond trully to the hybridization and respond selectively to the targets. I - INTRODUCTION has been used in the most studies [3, 4]. In our previous report [5], we have presented the In recent years, there has been considerable application of functionalized poly(5-hydroxy-interest in the development of DNA sensors due 1,4-naphthoquinone-co-5-hyroxy-3-acetic acid-to their numerous applications such as the 1,4-naphthoquinone) for DNA biosensor (theanalysis of unknown or mutant genes, diagnosis carboxylic group is serving as a linker for DNAof infectious agents in various environments and probe immobilization, quinone group as adetection of analytes (drugs, pollutants, etc. transducer for DNA probe-DNA targetTraditional methods for DNA detection, based hybridization. In present study, we report theon the radioisotopic and fluorescent detection, construction of DNA sensors based on CSare labor and time consuming, and are, thus, not modified electrode with MB as redox indicator.well suited for routine and rapid medical The detection of HIV sequences is successfullyanalyses, particularly for point-of-care tasks [1, performed and the discrimination against the 1-2]. Among some new approaches for DNA base and 2-base mismatch was effectivelydetection, electrochemical detection has many achieved.advantages such as reduction of the assay time,simple protocol and therefore can be used for II - EXPERIMENTALon-site monitoring. In this context,oligonucleotides (ODN, a short fragment of 1. ChemicalsDNA) immobilization at an electrode surface CS used was medical grade (MW = 200.000, 377determined by viscometry measurements; DA = with diamond paste of decreasing particle size.70%, determined by IR analysis and Voltammetric measurements werepotentiometric titration [6]). N-(3-dimethyl- performed on AUTOLAB PGSTAT 12aminopropyl)-N-ethylcarbodiimide Electrochemical Analyser (EcoChemie, thehydrochloride (EDC) was provided by Sigma. Netherlands) under the control of GPES versionN-methyl-imidazole (MIA) was from Acros. 4.9.005. The parameters for CV: scan rate: 50PBS buffer (0.137 M NaCl, 0.0027 M KCl, mV/s; potential range: -0.5 V - +0.2 V vs.SCE.0.0081 M Na2HPO4, 0.00147 M KH2PO4, pH The parameters for SWV: frequency: 12.5 Hz;7.4) was from Sigma. Aqueous solutions were start potential: -0.6 V; end potential: +0.4 V;made with bi-distilled water. All other reagents step 8 mV; amplitude: 25 mV. Prior to SWVused were of analytical grade. CH3COOH, measurements, the electrodes were held for 120methylene Blue (China), were of analytical s at the starting potential for conditioning.grade. 3. ODN quantification2. Electrochemical cell and electrodes All sequences of ODN were synthesized by All electrochemical measurements were Alpha DNA, Quebec, Canada. Five sets of ODNcarried out in three-electrode cell configuration (5 3) were used (sequences and calculatedwith a Pt counter electrode, saturated calomel molar absorption coefficient are given in table 1).reference electrode (SCE). The working pGEM is served as the probe; c-HIV, 1M-HIV,electrodes were glassy carbon disks (Tokai, 3 2M-HIV, A20 as complementary, 1-basemm). The working electrodes were polished mismatch, 2-base mismatch, non complementaryprior to use on a cloth (Struers) sequentially (completely unmatched) targets, respectively. Table 1: Sequences and calculated molar absorption coefficient of probe and targets 260 Code (103. M-1.cm-1) Sequences (5 3) pGEM 226.0 CTC TCG CAC CCA TCT CTC TCC TTC TAG c-HIV 216.8 A GAA GGA GAG AGA TGG GTG C ...