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

Journal of Chemistry, Vol. 44 (3), P. 377 - 381, 2006<br /> <br /> <br /> RAPID ELECTROCHEMICAL DETECTION OF SINGLE AND<br /> DOUBLE NUCLEOTIDE MISMATCHES (MUTATIONS) OF<br /> SEQUENCES FROM HIV SAMPLES<br /> Received 15 April 2005<br /> Tran Dai Lam , Bui Tien Trinh2, Nguyen Duc Chien2<br /> 1<br /> <br /> 1<br /> Faculty of Chemical Technology, Hanoi University of Technology (HUT)<br /> 2<br /> International Training Institute for Materials Science, HUT<br /> <br /> SUMMARY<br /> For the first time, the detection of DNA sequences, related to HIV (found in AIDS patients),<br /> using Methylene Blue (MB) as the redox indicator was performed. The voltametric signals have<br /> been investigated on bared chitosan (CS) modified; probe immobilized (DNA single strand) and<br /> target-hybridized (DNA double strand) electrodes sequentially by means of Cyclic Voltammetry<br /> (CV) and Square Wave Voltammetry (SWV). The response of hybridization between the probe and<br /> perfectly complementary, double-base mismatch, single-base mismatch of analyzed targets were<br /> clearly identified. The response of CS modified electrode with non-complementary target<br /> sequence and the response of CS-free electrode (adsorption only) with complementary target<br /> were used as control references to confirm whether the detected signals correspond trully to the<br /> hybridization and respond selectively to the targets.<br /> <br /> I - INTRODUCTION has been used in the most studies [3, 4]. In our<br /> previous report [5], we have presented the<br /> In recent years, there has been considerable application of functionalized poly(5-hydroxy-<br /> interest in the development of DNA sensors due 1,4-naphthoquinone-co-5-hyroxy-3-acetic acid-<br /> to their numerous applications such as the 1,4-naphthoquinone) for DNA biosensor (the<br /> analysis of unknown or mutant genes, diagnosis carboxylic group is serving as a linker for DNA<br /> of infectious agents in various environments and probe immobilization, quinone group as a<br /> detection of analytes (drugs, pollutants, etc. transducer for DNA probe-DNA target<br /> Traditional methods for DNA detection, based hybridization. In present study, we report the<br /> on the radioisotopic and fluorescent detection, construction of DNA sensors based on CS<br /> are labor and time consuming, and are, thus, not modified electrode with MB as redox indicator.<br /> well suited for routine and rapid medical The detection of HIV sequences is successfully<br /> analyses, particularly for point-of-care tasks [1, performed and the discrimination against the 1-<br /> 2]. Among some new approaches for DNA base and 2-base mismatch was effectively<br /> detection, electrochemical detection has many achieved.<br /> advantages such as reduction of the assay time,<br /> simple protocol and therefore can be used for II - EXPERIMENTAL<br /> on-site monitoring. In this context,<br /> oligonucleotides (ODN, a short fragment of 1. Chemicals<br /> DNA) immobilization at an electrode surface CS used was medical grade (MW = 200.000,<br /> 377<br /> determined by viscometry measurements; DA = with diamond paste of decreasing particle size.<br /> 70%, determined by IR analysis and Voltammetric measurements were<br /> potentiometric titration [6]). N'-(3-dimethyl- performed on AUTOLAB PGSTAT 12<br /> aminopropyl)-N-ethylcarbodiimide Electrochemical Analyser (EcoChemie, the<br /> hydrochloride (EDC) was provided by Sigma. Netherlands) under the control of GPES version<br /> N-methyl-imidazole (MIA) was from Acros. 4.9.005. The parameters for CV: scan rate: 50<br /> PBS buffer (0.137 M NaCl, 0.0027 M KCl, mV/s; potential range: -0.5 V - +0.2 V vs.SCE.<br /> 0.0081 M Na2HPO4, 0.00147 M KH2PO4, pH The parameters for SWV: frequency: 12.5 Hz;<br /> 7.4) was from Sigma. Aqueous solutions were start potential: -0.6 V; end potential: +0.4 V;<br /> made with bi-distilled water. All other reagents step 8 mV; amplitude: 25 mV. Prior to SWV<br /> used were of analytical grade. CH3COOH, measurements, the electrodes were held for 120<br /> methylene Blue (China), were of analytical s at the starting potential for conditioning.<br /> grade.<br /> 3. ODN quantification<br /> 2. Electrochemical cell and electrodes<br /> All sequences of ODN were synthesized by<br /> All electrochemical measurements were Alpha DNA, Quebec, Canada. Five sets of ODN<br /> carried out in three-electrode cell configuration (5' 3') were used (sequences and calculated<br /> with a Pt counter electrode, saturated calomel molar absorption coefficient are given in table 1).<br /> reference electrode (SCE). The working pGEM is served as the probe; c-HIV, 1M-HIV,<br /> electrodes were glassy carbon disks (Tokai, 3 2M-HIV, A20 as complementary, 1-base<br /> mm). The working electrodes were polished mismatch, 2-base mismatch, non complementary<br /> prior to use on a cloth (Struers) sequentially (completely unmatched) targets, respectively.<br /> Table 1: Sequences and calculated molar absorption coefficient of probe and targets<br /> 260<br /> Code (103. M-1.cm-1) Sequences (5' 3')<br /> pGEM 226.0 CTC TCG CAC CCA TCT CTC TCC TTC TAG<br /> c-HIV 216.8 A GAA GGA GAG AGA TGG GTG C<br /> 1M-HIV 217.5 A GAA GGA GAG AAA TGG GTG C<br /> 2M-HIV 219.4 A GAA GAA GAG AAA TGG GTG C<br /> A20 243.4 A AAA AAA AAA AAA AAA AAA A<br /> <br /> 0.5<br /> Concentration was determined from<br /> absorbance measurements at 260 nm on Agilent 1<br /> 0.4<br /> 8453 UV-Vis spectrophotometer and calculated<br /> molar absorption coefficient.<br /> Absorption (a.u)<br /> <br /> <br /> <br /> <br /> 0.3 2<br /> 4. DNA immobilization-hybridization 3<br /> procedure<br /> 0.2<br /> <br /> DNA immobilization is based on reaction<br /> between amine group on CS polymer and 0.1<br /> <br /> phosphate group of ODN probe sequence. EDC<br /> of 1.5.10-2 M in MIA solution was used to 0.0<br /> 250 260 270 280 290 300 310 320<br /> activate the amine group, via the formation of<br /> Wavelength (nm)<br /> intermediate labile ester. Hybridization<br /> experiments were carried out in 1x PBS solution Figure 1: UV-vis spectra of (1) pGEM;<br /> (pH 7) containing the targets at room (2) c-HIV; (3) A20<br /> 378<br />  temperature. Hybridization time was 30 min. can see that CS films present the NH2 groups,<br /> which can act as reactive groups for the covalent<br /> III - RESULTS AND DISCUSSION immobilization of phosphate-terminated DNA<br /> via phosphoramidate linkage.<br /> 1. Film characterization b) Electroactivity in MB<br /> a) IR analysis The film electroactivity was investigated in<br /> The IR spectra (figure not shown) showed 1x PBS buffer containing 100 µM of MB.<br /> the following characteristic peaks with their Reduction of MB proceeds by the participation<br /> attributions ( /cm-1) at 3429 ( O-H, N-H in of two electrons and one proton under the<br /> NH2), 2880 ( C-H), 1650 ( C-O), amide I), experimental conditions to form Leuco-MB blue<br /> 1595 ( N-H), amide II). From the IR data, one according the following equation:<br /> <br /> N H<br /> N<br /> + 2e- + H+<br /> (H3C)2N S N(CH3)2 S<br /> (H3C)2N N(CH3)2<br /> Methylene Blue Leucomethylene Blue<br /> <br /> Cv's showed a quasi-reversible wave pair at anodic and cathodic waves may be a result from<br /> -0.25V/-0.2V vs.SCE (45±5 mV separation kinetic complexity in the MB leucoMB<br /> between anodic Ea and cathodic Ec peak transformation. The CV's and SWV's were very<br /> potentials) (Fig. 2, left). The asymmetry of the stable upon cycling.<br /> -6<br /> 3.0x10<br /> -7 1.6x10<br /> <br /> -6<br /> -7 1.4x10<br /> 2.0x10<br /> -6<br /> 1.2x10<br /> -7<br /> 1.0x10<br /> -6<br /> 1.0x10<br /> Current (A)<br /> <br /> <br /> <br /> <br /> Current (A)<br /> <br /> <br /> <br /> <br /> 0.0<br /> -7<br /> 8.0x10<br /> -7<br /> -1.0x10<br /> -7<br /> 6.0x10<br /> -7<br /> -2.0x10 -7<br /> 4.0x10<br /> <br /> -7<br /> -3.0x10 -7<br /> 2.0x10<br /> <br /> -7<br /> -4.0x10 0.0<br /> -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3<br /> Potential (V vs SCE) Potential (V vs SCE)<br /> <br /> Figure 2: CV's (left) and SWV's (right) of CS modified electrodes in MB<br /> <br /> 2. Electrochemical detection of hybridization were recorded sequentially before and after each<br /> step (immobilization and hybridization) in MB<br /> The main objective of all DNA detection is<br /> solution under the same experimental<br /> to define DNA recognition interface (between conditions.<br /> the probe and target sequences) and afterwards<br /> effectively discriminate complementary target The first interesting observation is that the<br /> against 1 and 2-base mutant sequences. non-complementary sequence A20 (Fig. 3,<br /> The CV's (figure not shown) and SWV's control experiment 1) does not give any signal,<br /> 379<br /> while the posterior treatment on the same formation step, the subsequent steps of<br /> electrode with complementary one of c-HIV immobilization and hybridization are<br /> showed a significant decrease in current signal. maintained) (Fig. 6). No changes in current<br /> This indicates that unspecific adsorption of non- signal were observed, indicating no<br /> complementary sequence was negligible and the hybridization in this case. This proved once<br /> change in current signal is due to hybridization again unspecific adsorption of ODN is<br /> with matched sequence. Secondly, the negligible but covalent fixation of ODN probe<br /> discrimination of c-HIV against 1M-HIV and (via the reaction of functional groups on CS<br /> 2M-HIV was clearly demonstrated a less current films and ODN) was predominant and very<br /> signal upon hybridization with c-HIV was important for effective detection of<br /> recorded (Fig. 4), while no changes were hybridization. This result also confirmed above<br /> registered for 1M-HIV (Fig. 5) and 2M-HIV signal change was really come from the true<br /> (figure not shown). hybridization but not any other phenomena like<br /> Another control experiment is carried out on non-specific adsorption or signal unstability<br /> CS-free electrode (without the CS film (selectivity of the detection).<br /> <br /> -6<br /> 1.6x10<br /> -6<br /> 1 1.6x10<br /> 1: pGEM<br /> 2 1: pGEM<br /> -6 2: A20 -6<br /> 1<br /> 1.4x10 1.4x10 2: c-HIV<br /> 3: c-HIV<br /> -6<br /> 3 -6 2<br /> 1.2x10 1.2x10<br /> Current (A)<br /> <br /> <br /> <br /> <br /> -6 -6<br /> I/A 1.0x10 1.0x10<br /> <br /> -7 -7<br /> 8.0x10 8.0x10<br /> <br /> -7 -7<br /> 6.0x10 6.0x10<br /> <br /> -7 -7<br /> 4.0x10 4.0x10<br /> <br /> -7 -7<br /> 2.0x10 2.0x10<br /> <br /> 0.0 0.0<br /> -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3<br /> Potential (V vs SCE)<br /> Potential (V vs SCE)<br /> Fig. 3: SWV’s responses on CS/pGEM Fig. 4: SWV’s responses on CS/pGEM<br /> electrode (1) before treatment; (2) after treatment electrode (1) before treatment; (2) after treatment<br /> with A20; (3) second treatment with c-HIV with treatment with c-HIV<br /> -6<br /> 1.8x10<br /> -6<br /> 1.2x10 1 Bared GC<br /> 2<br /> 1: pGEM 1.6x10<br /> -6<br /> 2 pGEM adsorption<br /> -6<br /> 2: 1M-HIV 1 3 Hyb-tion with c-HIV<br /> 1.0x10 1.4x10<br /> -6<br /> <br /> <br /> <br /> <br /> -7 1.2x10<br /> -6 3<br /> 8.0x10<br /> Current (A)<br /> Current (A)<br /> <br /> <br /> <br /> <br /> -6<br /> 1.0x10<br /> -7<br /> 6.0x10<br /> -7<br /> 8.0x10<br /> <br /> 4.0x10<br /> -7<br /> 2 -7<br /> 6.0x10<br /> <br /> -7 -7<br /> 2.0x10 1 4.0x10<br /> <br /> -7<br /> 2.0x10<br /> 0.0<br /> -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3<br /> -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 Potential (V vs SCE)<br /> Potential (V vs SCE)<br /> Fig. 6: SWV’s responses on CS-free electrode<br /> Fig. 5: SWV’s responses on CS/pGEM (1) bared GC electrode; (2) after treatment with<br /> electrode (1) before; (2) after treatment with 1M- pGEM; (3) after treatment with c-HIV<br /> HIV<br /> 380<br />  IV - CONCLUSION (Vietnamese) fund for providing Agilent UV-vis<br /> spectrophotometer, VLIR-HUT reciprocal fund<br /> In this study, direct electrochemical for AUTOLAB Electrochemical system, VLIR-<br /> detection of HIV sequences has been performed HUT Project AP05\Prj3\Nr 05.<br /> by CV and SWV. 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