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Electrochemical scanning of DNA point mutations via MutS protein-mediated mismatch recognition
Biosensors and Bioelectronics 24 (2009) 1955–1961
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Electrochemical scanning of DNA point mutations via MutS protein-mediated mismatch recognition Huan Chen, Xiang-Jun Liu, Ya-Li Liu, Jian-Hui Jiang ∗ , Guo-Li Shen, Ru-Qin Yu ∗ State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Yuelu District, Changsha 410082, PR China
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Article history: Received 2 July 2008 Received in revised form 11 September 2008 Accepted 30 September 2008 Available online 15 October 2008 Keywords: Mediated mismatch recognition MutS protein Gene mutation Methylene blue Single nucleotide polymorphisms
a b s t r a c t MutS protein is an important part of the DNA repair system which can specifically recognize and bind all possible single-base mismatches as well as 1–4 base insertion or deletion loops with varying affinities independent of other proteins or cofactors. In this paper, a new approach for electrochemical gene mutation detection based on the utilization of MutS protein for the mutation recognition and spontaneously intercalated methylene blue (MB) markers for electrochemical signal generation is described. This method involves the immobilization of MutS protein onto the gold electrode, the hybridization of target DNA to form homoduplex or heteroduplex DNA, the application of MutS protein for the mutation recognition, and finally the intercalation of MB. The background is very low because MutS protein binds DNA containing mispaired and unpaired bases but does not bind equally well to DNA without mismatches or single-stranded DNA. The proposed approach has been successfully implemented for the identification of single-base mutation in −28 site of the -thalassemia gene with a detection limit of 5.6 × 10−13 M, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation and occur once every 100–300 bases in genome (Sachidanandam et al., 2001). A large number of bacterial drug resistances, human pathogenic and genetic diseases are associated with SNPs of particular genes (Wabuyele et al., 2003). Rapid and sensitive mutation detection methods are urgently demanded for the medical diagnosis of the diseases. Conventional approaches for SNP detection involved DNA direct sequencing (Kwok et al., 1994; Zakeri et al., 1998), single-strand conformation polymorphism (Hayashi, 1991; Inazuka et al., 1997) and denaturing gradient gel electrophoresis (Fischer and Lerman, 1983; Henco et al., 1994; Gelfi et al., 1996) analysis. These methods are often time-consuming and of relatively high cost. Other approaches include the allelespecific DNA microarray (Wang et al., 1998; Hacia, 1998), the allele-specific TagMan assay (Livak et al., 1995), the templatedirected dye terminator incorporation assay (Chen and Kwok, 1997) and the ligase detection reaction (Tobe et al., 1996; Landegren et al., 1988; Barany, 1991). These methods offer advantages of high
∗ Corresponding authors. Tel.: +86 731 8821961; fax: +86 731 8821916. E-mail addresses: [email protected] (J.-H. Jiang), [email protected] (R.-Q. Yu). 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.09.029
fidelity and sensitivity, but they could only screen individuals for known polymorphisms. SNP detection encompasses another important area of scanning DNA sequences for previously unknown polymorphisms. MutS protein is a mismatch binding protein that can specifically recognizes and binds to heteroduplex DNA with up to 3 mismatched bases in a row. Recently, the specificity of the biological interaction of MutS with mismatched DNA has been exploited as a promising tool for detection of mismatched DNA (Lishanski et al., 1994; Ellis et al., 1994; Gotoh et al., 1997; Geschwind et al., 1996; Nelson, 1995). Wagner et al. (1995) reported the utilizing of MutS protein for SNP detection based on a nitrocellulose membrane-based chemiluminescence mode. Su et al. (2004) combined the MutS-based mismatch recognition with quartz crystal microbalance measurements for the detection of gene mutations. Behrensdorf et al. (2002) and Bi et al. (2003) separately developed a DNA chip and a MutS protein chip for rapid screening of SNPs. The specific binding of dye-labeled MutS protein with surface-bound DNA or dye-labeled DNA with surface-bound MutS protein is revealed by the fluorescence images. Recent years, much attention has been focused on electrochemical methods for the MutS–DNA binding. General electrochemical methods, cyclic voltammetry, electrochemical quartz crystal microbalance and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS
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probe (Cho et al., 2006). Palecˇek et al. (2004) reported an electrochemical method for the determination of unlabeled MutS protein and detection of point mutations in DNA (Masaˇrík et al., 2007) An impedance study of MutS binding to a DNA mismatch has been reported (Li et al., 2004). An electrochemical assay for gene mutation was reported using a MutS protein-modified electrode at which the binding of mismatched duplex inhibited the diffusion current of ferricyanide (Han et al., 2002, 2006). However, these electrochemical methods are often inadequate to generate a highly sensitive signal. In this study, we proposed a novel and simple approach for the scanning of unknown gene mutations based on specific interaction between immobilized MutS protein and mismatched DNA heteroduplex with intercalated electroactive indicators for immediate voltammetric readout. The MutS protein was immobilized on the Au electrode surface via adsorption on a self-assembled layer of Au colloids through a 1,6-hexanedithiol (HDT) monolayer. This immobilization protocol allowed for stable and high loading of MutS protein on the electrode surface and a facilitated electrochemistry of the redox indicators, thus offering the possibility of sensitivity enhancement. The occurrence of mutation in target genes resulted in the formation of heteroduplex, which could specifically bind to the immobilized MutS protein on the electrode. The adsorption of heteroduplex on the electrode surface could then be probed electrochemically using an external redox indicator MB. MB is an electroactive molecule that can be selectively intercalated in dsDNA but exhibits weak adsorption on proteins (Jin et al., 2007; Kelley and Barton, 1997; Xiao et al., 2005; Boon and Barton, 2003; Boon et al., 2003). Therefore, the electrochemical current of MB can be used as the indicator for the amount of dsDNA adsorbed on the electrode surface, provided the target sequence is fixed. The developed biosensor is based on selective binding of mismatched dsDNA with a specified sequence on surface-immobilized MutS protein. The amount of dsDNA adsorbed on the electrode reflects the concentration of mismatched DNA in the sample, thus can be probed by DPV current of MB selectively intercalated in adsorbed dsDNA. This design allows a label-free detection of mismatch, which to our knowledge has not been reported previously. The role of Au colloids in the detection is to facilitate the immobilization of MutS protein with high loading and maximal activity as well as improve the conductivity of the electrode surface. MB is used for probing the binding of MutS protein to mismatch. The results obtained revealed that the developed approach allowed a highly sensitive, rapid and label-free detection of mutations, either known or unknown, in target genes.
2. Experimental methods 2.1. Oligonucleotides and reagents
water was obtained through a Nanopure Infinity ultrapure water system (Barnstead/thermolyne Corp., Dubuque, IA) and had an electric resistance >18.3 MW. 2.2. Buffer and solutions The hybridization buffer and incubation buffer between MutS protein and dsDNA were a 10 mM Tris–HCl (pH 7.5) buffer solution containing 300 mM NaCl and 20 mM MgCl2 . PBS buffer (pH 7.4, 0.067 M Na2 HPO4 and 0.067 M KH2 PO4 ) containing 100 mM NaCl was used for the DPV analysis. 2.3. Apparatus The apparatus used for impedance and cyclic voltammetry measurement was CHI760B electrochemical workstation (Shanghai Chenhua Instruments, Shanghai). All electrochemical measurements were conducted in 1/15 M PBS (containing 0.1 M NaCl, pH 7.4). Impedance measurements were performed in the frequency range from 0.1 to 10,000 Hz. Differential pulse voltammetry (DPV) responses reported were given as the background-subtracted currents. Electrochemical experiments were performed in an electrochemical cell containing a three-electrode system. A gold electrode (2 mm diameter) was used as the working electrode with a saturated calomel electrode (SCE) as the reference electrode and a platinum electrode as the auxiliary electrode. All measurements were performed in a 10-mL cell and the temperature was kept at room temperature. All potentials measured were with respect to the SCE. 2.4. Modification of electrode with MutS protein Prior to surface modification, the gold electrode was treated with “piranha” solution, and washed thoroughly with a copious amount of doubly distilled water. The electrode was polished to a mirror with 0.05 mm alumina, and then sonicated for 2 min in alcohol and distilled water, respectively. Then, the electrochemical pretreatments were performed by cycling from a potential of −0.3 to +1.5 V versus SCE reference electrode in a 0.1 M H2 SO4 solution for 12 min. The bare gold electrode was placed in a freshly made 1,6hexanedithiol solution in dry ethanol. After adsorption for 30 min, the electrode was thoroughly washed with ethanol and water to remove physically adsorbed 1,6-hexanedithiol and dried at room temperature. Subsequently, the electrode was immersed in the colloidal Au solution overnight. After sufficiently rinsing, the electrode was incubated in MutS protein solution (0.05 mg mL−1 ) for 1 h at 37 ◦ C.
Deoxyoligonucleotides including DNA target 1:, 5 -gggag ggc agg agc cag ggc tgg gca ta g aag tca ggg cag agc cat cta ttg ctt
2.5. Electrochemical measurement procedure
aca ttt gc ttctg-3 , DNA target 2: 5 -gggag ggc agg agc cag ggc tgg gca ta a aag tca ggg cag agc cat cta ttg ctt aca ttt gc ttctg-3 , and detection probe: 5 -cagaa gc aaa tgt aag caa tag atg gct ctg ccc tga ctt t ta tgc cca gcc ctg gct cct gcc ctccc-3 ) were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Tris was from Roche. Trisodium citrate, NaCl, KCl and MgCl2 were obtained from Amresco (Solon, OH). The size of gold nanoparticles was verified as 20 nm by TEM (Hitachi TEM 800, Japan). The concentration of colloid Au is about 1.3 × 1011 particles/mL in citrate buffer. 1,6-Hexanedithiol (HDT) was obtained from Sigma–Aldrich. HAuCl4 ·3H2 O and methylene blue (MB) are obtained from Aldrich. HAuCl4 ·3H2 O, trisodium citrate and other chemicals were all of analytical grade. Ultrapure
In a typical experiment, appropriate amount of DNA target 1 or 2 was added to the hybridization buffer containing 100 nM detection probe, followed by heating at 77 ◦ C for 60 min. After hybridization, the MutS protein-modified electrode was immersed in the hybridization solution and incubated for 60 min at 37 ◦ C, followed by washing with detection buffer. Then, the gold electrode was immersed in 20 M MB solution for 5 min. Finally, the electrode was introduced into the electrochemical cell containing 10 mL of PBS buffer for DPV analyses. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV.
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3. Results and discussion 3.1. Schematic representation of principle of mutation detection The analytical principle of mutation detection the MutS protein based are shown in Scheme 1. A self-assembled monolayer of disulfides on gold electrode was used as the base interface for the assembly of the Au colloids. This enabled the formation of highly ordered and well-organized structure of gold nanoparticles on the electrode surface. Due to large surface free energy and surface area, nanoparticles show much stronger affinity in adsorbing proteins, improving the surface loading of protein. Also, nanoparticle layer can enhance the conductivity of the biosensor interface and maximally maintain the activity of the immobilized protein, thus improving the detection sensitivity. Therefore, use of nanoparticle layer for protein immobilization is helpful for sensitivity enhancement in biosensors (Rembaum and Dreyer, 1980; Martin and Micher, 1998; Crumbliss et al., 1992; Wang et al., 2004; Tang et al., 2008; Liu et al., 2003; Grabar et al., 1995). The colloidal Au layer then provided a platform for strong adsorption of MutS protein as the recognition element. This immobilization protocol not only allowed for high loading of MutS protein, but also preserved the activity of the proteins. MutS protein is a mismatch binding protein that can specifically recognizes and binds to heteroduplex DNA with up to 3 mismatched bases. The occurrence of mutation in target genes resulted in the formation of heteroduplex, which could specifically bind to the immobilized MutS protein on the electrode. As a result, a certain redox indicator as MB that could selectively intercalate in the duplexes was utilized for probe the adsorption of heteroduplex on the electrode surface, where the DPV current of MB could reveal the amount of DNA heteroduplex arising from gene mutations. 3.2. Characterization of the electrode processes by impedance spectroscopy
Fig. 1. Electrochemical impedance spectra of electrode processes: (a) bare Au electrode, (b) 1,6-hexanedithiol/gold electrode and (c) Au-colloid/1,6hexanedithiol/gold electrode, (d) MutS protein/Au-colloid/1,6-hexanedithiol/gold electrode,(e) electrode incubated with full-matched dsDNA and (f) single-base mismatch of dsDNA immobilized electrode in the frequency range from 0.1 Hz to 10 kHz. All the measurements were performed in 0.067 MPBS (containing 0.1 M KCl, 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], pH 7.4).
HDT self-assembled electrode. This observation is consistent with those reported previously (Wang et al., 2004; Liu et al., 2003). The impedance increment obtained were 1200 after the immobilization with MutS protein, indicating that the immobilization protocol could efficiently load the protein on the sensor surface. The reaction of the MutS protein-immobilized electrode with single-base mismatched dsDNA caused an impedance increment of 1100 , but no impedance increment was observed with full-matched dsDNA, implying that the specific recognition was achieved on the electrode interface. 3.3. Optimization of the hybridization buffer and assay time
Electrochemical impedance spectroscopy is one of the most powerful and sensitive techniques for investigating the features of surface-modified electrodes. According to Fig. 1, the impedance increase is related to the adsorption of an electrically insulated material on the electrode surface. Following the experimental procedure as described above, the impedance values were recorded after each step was finished. The modification of HDT produced an impedance increment of 3500 , suggesting the self-assembled monolayer of HDT on the electrode surface. The subsequent interaction of the electrode with Au colloids reduced the impedance, evidencing the formation of highly ordered Au colloid layer that improved the conductance of the electrode. Commonly, there is an electronic coupling between Au nanoparticles and the gold electrode through the tunneling effect (Lyon et al., 1998). Thus, Au colloid assembled electrode shows smaller impedance than the
The salt concentration in hybridization buffers was one of the most important factors that had great effects on the hybridization efficiency. The positively charged ions could reduce the repulsion between the negatively charged phosphate backbones in DNA structure. Fig. 2A displays the current responses as a function of NaCl concentrations in hybridization buffer. The response increases with NaCl concentration increased from 0.05 to 0.3 M and reaches a plateau at 0.3 M. Fig. 2B depicts the signal responses as a function of MgCl2 concentrations in hybridization buffer. The current response to 1.5 pM target 1 increases with MgCl2 concentration increased from 0 to 40 mM and reaches a plateau at 20 mM. Therefore, 0.3 M NaCl and 20 mM MgCl2 were employed in the hybridization buffer throughout the experiments. Obviously, the amount of dsDNA binding to MutS protein on the electrode is dependent upon the interaction time between MutS protein and mismatched dsDNA. One can expect that sufficient reaction time could guarantee the maximized amount of dsDNA binding on the electrode, which in turn improves the sensitivity in mutation detection. As shown in Fig. 3, the current response to dsDNA hybrid of 10 pM target 1 with detection probe increase as the reaction time is increased up to 25 min, and the current response tends to level off in a prolonged reaction time. So, the MutS–dsDNA binding time as 25 min was adopted throughout this work. 3.4. Determination of point mutations
Scheme 1. Schematic illustration of the label-free electrochemical sensor for the MutS protein-mediated mismatch recognition.
The MutS protein-modified electrode constituted a specific platform for the detection of point mutation. As shown in Fig. 4A,
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Fig. 2. The current responses detected by the MutS protein-modified electrode with different concentration of NaCl and MgCl2 in hybridization buffer. The concentrations of probe and target 1 were 100 nM and 1.5 pM, respectively. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV.
Fig. 3. Effects of the MutS–dsDNA binding time on the peak currents. The concentrations of probe and target 1 were 100 nM and 10 pM, respectively. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV. Other experimental conditions were kept unchanged in the optimized buffer.
the DPV responses of the electrode to 1.6 nM DNA target 2 was very small, ca. 10.8 nA. This is attributed to the weak interaction of MutS protein with DNA homoduplex with AT perfect match. In contrast, the DPV responses of the electrode to 1.6 nM DNA target 1 that has a single-base mismatch with the detection probe gives an appreciable current peak with a peak current of 82.3 nA, indicating that the interaction between MutS and heteroduplex with GT mismatch is much stronger than that between MutS and homoduplex with AT perfect match. The results also evidenced that the MutS protein-modified electrode offered an effective approach for the discrimination of point mutations in the target genes. As shown in Fig. 4B, the DPV currents increased as the concentration of DNA target 1 with single-base mismatch increased in the range from 1.5 pM to 1.6 nM, and the current tends to be leveled off at higher concentrations. No detectable peak current was observed when there was no DNA target (targets 1 and 2) in the sample solution, implying that the interaction of MutS protein with single-stranded detection probe is too weak to be detected. In the inset, a linear correlation between the DPV peak currents to the logarithmic concentrations of target DNA 1 was obtained in the range from 1.5 pM to 1.6 nM with a regression equation ip = 19.4 log Ctarget 1 + 215.0. The error bar means S.D. across five repetitive experiments to prove the good reproducibility of the biosensor. The limit of detection of 0.6 pM was readily achieved in terms of the rule of three times of standard deviation over the blank measurement.
Fig. 4. (A) The current responses of 1.6 nM target 2 (a) and 1.6 nM target 1 (b) reacted with 100 nM probe. (B). DPV analysis for DNA target 1 with different concentration: (a) 0 nM, (b) 1.5 pM, (c) 6.1 pM, (d) 24.4 pM, (e) 100 pM, (f) 400 pM, and (g) 1.0 nM. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV. Other experimental conditions were kept unchanged as the optimized ones.
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Fig. 5. (A) Schematic illustration of the principle of genomic samples detection. In this figure (A) shows the principle of two-step detection for wild-type sample, (B) shows the principle of two-step detection for heterozygous mutant sample and (C) shows the principle of two-step detection for homozygous mutant sample. (B) The DPV analysis for four genomic samples. Curves a show the current responses for real samples treated after step 1. Curves b show the current responses for real samples treated after step 2.
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The proposed approach was implemented for the identification of single-base mutation in −28 site of the -thalassemia gene in genomic samples with a preliminary polymerase chain amplification (PCR). Genomic DNA was extracted from blood samples according to the common method. The forward and the reverse primers are 5 -GTA CGG CTG TCA TCA CTT AGA CCT CA-3 and 5 -TGC AGC TTG TCA CAG TGC AGC TCA CT-3 , respectively. PCR amplification was performed in a 50 L system with 200 M deoxyribonucleotide triphosphate (dNTPs), 0.2 M forward and reverse primers (10 pmol for each primer), as well as ca. 300 ng of genomic DNA extracted from the samples. Amplification was achieved by thermal cycling for 30 cycles at 98 ◦ C for 15 s, 59 ◦ C for 20 s, 72 ◦ C for 15 s and a final extension at 72 ◦ C for 8 min. PCR products of 602 bp were obtained after purified using a gel extraction kit (Ambiogen) and then quantified by UV–vis spectrometer. The resulting samples were diluted using the assay buffer to appropriate concentration for mutation detection. The identification of the samples included two steps in order to discriminate between the wild-type, heterozygous mutant and homozygous mutant. In the first step, the sample of 50 L was heated to 95 ◦ C followed by slow cooling to room temperature to allow the formation of possible heteroduplexes. The sample solution was then detected using the MutS protein-modified electrode. In the first step, when the samples contained both the wild-type and the mutant genes, i.e., the case of heterozygous mutant, the treatment allowed the formation of possible heteroduplexes and a positive response was expected on the MutS protein-modified electrode. For samples from wild-type and homozygous mutant, only homoduplexes would obtain via the first step, then a negative response was expected on the MutS protein-modified electrode. In the second step, 1 L detection probe was added in the sample solution of 50 L (The final concentration was 100 nM for the detection probe). Then, the sample was heated to 95 ◦ C followed by slow cooling to room temperature to allow the formation of possible heteroduplexes. The sample solution was then detected using the MutS protein-modified electrode. In the second step, after sample was denatured at 95 ◦ C followed by annealing at lowed temperature, DNA duplex would form between all DNA single strands present in the solution. Thus, in cases where the sample contained mutant gene, possible heteroduplex with single-base mismatch would be obtained, which was expected to give positive response on the MutS protein-modified electrode. In contrast, for samples without mutant gene, no heteroduplex would form in the solution and a negative response was expected. Combined the results obtained in two steps, the genotype of the samples could be determined. Fig. 5A was the schematic representation of the principle of genomic samples detection. Fig. 5A shows the principle of the twostep detection for samples of wild-type, heterozygous mutant and homozygous mutant, respectively. Fig. 5B shows the DPV currents of four genomic samples for the identification of single-base mutation in −28 site of the -thalassemia gene. Curves a in Fig. 5B depict the DPV currents obtained after the first step and curves b represent that after the second step. Combined the results obtained in two steps, the genotype of the four samples were determined to be wild-type (sample 1), heterozygous mutant (sample 2 and 3) and homozygous mutant (sample 4).
4. Conclusion A novel electrochemical biosensing approach for point mutation detection was developed based on the specific recognition of MutS protein to DNA heteroduplexes with single-base mis-
match. This approach was the first demonstration that combined the MutS-based mismatch recognition with the implementation of redox intercalator of DNA duplex for label-free detection of gene mutations. The results obtained showed that the MutS proteinmodified electrode offered a viable platform for the discrimination between DNA heteroduplexes and homoduplexes, and the detection limit for single mismatched DNA could be achieved as low as 0.6 pM. The method had the advantages of simplicity, label-free detection, high sensitivity, rapidness and the capacity to identify unknown mutations, implying that the proposed approach might hold great promise for rapid scan of point mutations in target genes. Acknowledgements This work was supported by “973” National Key Basic Research Program (2007CB310500), and National Nature Science Foundation of China (20872027, U0632005, 20775023). References Barany, F., 1991. Proc. Natl. Acad. Sci. U.S.A. 88, 189–193. Behrensdorf, H.A., Pignot, M., Windhab, N., Kappel, A., 2002. Nucleic Acids Res. 30, e64. Bi, L.J., Zhou, Y.F., Zhang, X.E., Deng, J.Y., Zhang, Z.P., Xie, B., Zhang, C.G., 2003. Anal. Chem. 75, 4113–4119. Boon, E.M., Barton, J.K., 2003. Bioconjugate Chem. 14, 1140–1147. Boon, E.M., Jackson, N.M., Wightman, M.D., Kelley, S.O., Hill, M.G., Barton, J.K., 2003. J. Phys. Chem. B 107, 11805–11812. Chen, X., Kwok, Y.P., 1997. Nucleic Acids Res. 25, 347–353. Cho, M., Lee, S., Han, S.Y., Park, J.Y., Rahman, M.A., Shim, Y., Ban, C., 2006. Nucleic Acids Res. 34, e75. Crumbliss, A.L., Ferine, S.C., Stonehunerer, J., Tubergen, K.R., Zhao, J.G., Henkens, R.W., O’Daly, J.P., 1992. Biotechnol. Bioeng. 40, 483–490. Ellis, L.A., Taylor, G.R., Banks, R., Baumberg, S., 1994. Nucleic Acids Res. 22, 2710–2711. Fischer, S.G., Lerman, L.S., 1983. Proc. Natl. Acad. Sci. U.S.A. 80, 1579–1583. Gelfi, C., Cremonesi, L., Ferrari, M., Righetti, P.G., 1996. BioTechniques 21, 926–932. Geschwind, D.H., Rhee, R., Nelson, S.F., 1996. Genet. Anal. 13, 105–111. Gotoh, M., Hasebe, M., Ohira, T., Hasegawa, Y., Shinohara, Y., Sota, H., Nakao, J., Tosu, M., 1997. Genet. Anal. Biomol. Eng. 14, 47–50. Grabar, K.C., Reeman, R.G., Hommer, M.B., Natan, M.J., 1995. Anal. Chem. 67, 735–743. Hacia, J.G., 1998. Genome Res. 8, 1245–1258. Han, A., Shibata, T., Takarada, T., Maeda, M., 2002. Nucleic Acids Res. Suppl. 2, 287–288. Han, A., Takarada, T., Shibata, T., Nakayama, M., Maeda, M., 2006. Anal. Sci. 22, 663–666. Hayashi, K., 1991. PCR Methods Appl. 1, 34–38. Henco, K., Harders, J., Wiese, U., Riesner, D., 1994. Methods Mol. Biol. 31, 211–228. Inazuka, M., Wenz, H.M., Sakabe, M., Tahira, T., Hayashi, K., 1997. Genome Res. 7, 1094–1103. Jin, Y., Yao, X., Liu, Q., Li, J., 2007. Biosens. Bioelectron. 22, 1126–1130. Kelley, S.O., Barton, J.K., 1997. Bioconjugate Chem. 8, 31–37. Kwok, P.Y., Carlson, C., Yager, T.D., Ankener, W., Nickerson, D.A., 1994. Genomics 23, 138–144. Landegren, U., Kaiser, R., Sanders, J., Hood, L., 1988. Science 241, 1077–1080. Li, C.Z., Long, Y.T., Lee, J.S., Kraatz, H.B., 2004. Chem. Commun. 10, 574–575. Lishanski, A., Ostrander, E.A., Rine, J., 1994. Proc. Natl. Acad. Sci. U.S.A. 91, 2674– 2678. Liu, Y.J., Yin, F., Long, Y.M., Zhang, Z.H., Yao, S.Z., 2003. J. Colloid Interface Sci. 258, 75–81. Livak, K.J., Marmaro, J., Todd, A.J., 1995. Nat. Genet. 9, 341–342. Lyon, L.A., Musick, M.D., Natan, M.J., 1998. Anal. Chem. 70, 5177–5183. Martin, C.R., Micher, D.T., 1998. Anal. Chem. 5, 322A. Masaˇrík, M., Cahová, K., Kizek, R., Palecˇek, E., Fojta, M., 2007. Anal. Bioanal. Chem. 388, 259–270. Nelson, S.F., 1995. Electrophoresis 16, 279–285. Palecˇek, E., Masaˇrík, M., Kizek, R., Kuhlmeier, D., Hassmann, J., Schulein, J., 2004. Anal. Chem. 76, 5930–5936. Rembaum, A., Dreyer, W.J., 1980. Science 208, 364. Sachidanandam, R., Weissman, D., Schmidt, S.C., Kakol, J.M., Stein, L.D., Marth, G., Sherry, S., Mullikin, J.C., Mortimore, B.J., Willey, D.L., 2001. Nature 409, 928– 933. Su, X.D., Robelek, R., Wu, Y.J., Wang, G.Y., Knoll, W., 2004. Anal. Chem. 76, 489–494. Tang, L., Zeng, G.M., Shen, G.L., Li, Y.P., Zhang, Y., Huang, D.L., 2008. Environ. Sci. Technol. 42, 1207. Tobe, V.O., Taylor, S.L., Nickerson, D.A., 1996. Nucleic Acids Res. 24, 3728–3732. Wabuyele, M.B., Farquar, H., Stryjewski, W., Hammer, R.P., Soper, S.A., Cheng, Y.W., Barany, F., 2003. J. Am. Chem. Soc. 125, 6937–6945.
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Electrochemical scanning of DNA point mutations via MutS protein-mediated mismatch recognition Huan Chen, Xiang-Jun Liu, Ya-Li Liu, Jian-Hui Jiang ∗ , Guo-Li Shen, Ru-Qin Yu ∗ State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Yuelu District, Changsha 410082, PR China
a r t i c l e
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Article history: Received 2 July 2008 Received in revised form 11 September 2008 Accepted 30 September 2008 Available online 15 October 2008 Keywords: Mediated mismatch recognition MutS protein Gene mutation Methylene blue Single nucleotide polymorphisms
a b s t r a c t MutS protein is an important part of the DNA repair system which can specifically recognize and bind all possible single-base mismatches as well as 1–4 base insertion or deletion loops with varying affinities independent of other proteins or cofactors. In this paper, a new approach for electrochemical gene mutation detection based on the utilization of MutS protein for the mutation recognition and spontaneously intercalated methylene blue (MB) markers for electrochemical signal generation is described. This method involves the immobilization of MutS protein onto the gold electrode, the hybridization of target DNA to form homoduplex or heteroduplex DNA, the application of MutS protein for the mutation recognition, and finally the intercalation of MB. The background is very low because MutS protein binds DNA containing mispaired and unpaired bases but does not bind equally well to DNA without mismatches or single-stranded DNA. The proposed approach has been successfully implemented for the identification of single-base mutation in −28 site of the -thalassemia gene with a detection limit of 5.6 × 10−13 M, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation and occur once every 100–300 bases in genome (Sachidanandam et al., 2001). A large number of bacterial drug resistances, human pathogenic and genetic diseases are associated with SNPs of particular genes (Wabuyele et al., 2003). Rapid and sensitive mutation detection methods are urgently demanded for the medical diagnosis of the diseases. Conventional approaches for SNP detection involved DNA direct sequencing (Kwok et al., 1994; Zakeri et al., 1998), single-strand conformation polymorphism (Hayashi, 1991; Inazuka et al., 1997) and denaturing gradient gel electrophoresis (Fischer and Lerman, 1983; Henco et al., 1994; Gelfi et al., 1996) analysis. These methods are often time-consuming and of relatively high cost. Other approaches include the allelespecific DNA microarray (Wang et al., 1998; Hacia, 1998), the allele-specific TagMan assay (Livak et al., 1995), the templatedirected dye terminator incorporation assay (Chen and Kwok, 1997) and the ligase detection reaction (Tobe et al., 1996; Landegren et al., 1988; Barany, 1991). These methods offer advantages of high
∗ Corresponding authors. Tel.: +86 731 8821961; fax: +86 731 8821916. E-mail addresses: [email protected] (J.-H. Jiang), [email protected] (R.-Q. Yu). 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.09.029
fidelity and sensitivity, but they could only screen individuals for known polymorphisms. SNP detection encompasses another important area of scanning DNA sequences for previously unknown polymorphisms. MutS protein is a mismatch binding protein that can specifically recognizes and binds to heteroduplex DNA with up to 3 mismatched bases in a row. Recently, the specificity of the biological interaction of MutS with mismatched DNA has been exploited as a promising tool for detection of mismatched DNA (Lishanski et al., 1994; Ellis et al., 1994; Gotoh et al., 1997; Geschwind et al., 1996; Nelson, 1995). Wagner et al. (1995) reported the utilizing of MutS protein for SNP detection based on a nitrocellulose membrane-based chemiluminescence mode. Su et al. (2004) combined the MutS-based mismatch recognition with quartz crystal microbalance measurements for the detection of gene mutations. Behrensdorf et al. (2002) and Bi et al. (2003) separately developed a DNA chip and a MutS protein chip for rapid screening of SNPs. The specific binding of dye-labeled MutS protein with surface-bound DNA or dye-labeled DNA with surface-bound MutS protein is revealed by the fluorescence images. Recent years, much attention has been focused on electrochemical methods for the MutS–DNA binding. General electrochemical methods, cyclic voltammetry, electrochemical quartz crystal microbalance and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS
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probe (Cho et al., 2006). Palecˇek et al. (2004) reported an electrochemical method for the determination of unlabeled MutS protein and detection of point mutations in DNA (Masaˇrík et al., 2007) An impedance study of MutS binding to a DNA mismatch has been reported (Li et al., 2004). An electrochemical assay for gene mutation was reported using a MutS protein-modified electrode at which the binding of mismatched duplex inhibited the diffusion current of ferricyanide (Han et al., 2002, 2006). However, these electrochemical methods are often inadequate to generate a highly sensitive signal. In this study, we proposed a novel and simple approach for the scanning of unknown gene mutations based on specific interaction between immobilized MutS protein and mismatched DNA heteroduplex with intercalated electroactive indicators for immediate voltammetric readout. The MutS protein was immobilized on the Au electrode surface via adsorption on a self-assembled layer of Au colloids through a 1,6-hexanedithiol (HDT) monolayer. This immobilization protocol allowed for stable and high loading of MutS protein on the electrode surface and a facilitated electrochemistry of the redox indicators, thus offering the possibility of sensitivity enhancement. The occurrence of mutation in target genes resulted in the formation of heteroduplex, which could specifically bind to the immobilized MutS protein on the electrode. The adsorption of heteroduplex on the electrode surface could then be probed electrochemically using an external redox indicator MB. MB is an electroactive molecule that can be selectively intercalated in dsDNA but exhibits weak adsorption on proteins (Jin et al., 2007; Kelley and Barton, 1997; Xiao et al., 2005; Boon and Barton, 2003; Boon et al., 2003). Therefore, the electrochemical current of MB can be used as the indicator for the amount of dsDNA adsorbed on the electrode surface, provided the target sequence is fixed. The developed biosensor is based on selective binding of mismatched dsDNA with a specified sequence on surface-immobilized MutS protein. The amount of dsDNA adsorbed on the electrode reflects the concentration of mismatched DNA in the sample, thus can be probed by DPV current of MB selectively intercalated in adsorbed dsDNA. This design allows a label-free detection of mismatch, which to our knowledge has not been reported previously. The role of Au colloids in the detection is to facilitate the immobilization of MutS protein with high loading and maximal activity as well as improve the conductivity of the electrode surface. MB is used for probing the binding of MutS protein to mismatch. The results obtained revealed that the developed approach allowed a highly sensitive, rapid and label-free detection of mutations, either known or unknown, in target genes.
2. Experimental methods 2.1. Oligonucleotides and reagents
water was obtained through a Nanopure Infinity ultrapure water system (Barnstead/thermolyne Corp., Dubuque, IA) and had an electric resistance >18.3 MW. 2.2. Buffer and solutions The hybridization buffer and incubation buffer between MutS protein and dsDNA were a 10 mM Tris–HCl (pH 7.5) buffer solution containing 300 mM NaCl and 20 mM MgCl2 . PBS buffer (pH 7.4, 0.067 M Na2 HPO4 and 0.067 M KH2 PO4 ) containing 100 mM NaCl was used for the DPV analysis. 2.3. Apparatus The apparatus used for impedance and cyclic voltammetry measurement was CHI760B electrochemical workstation (Shanghai Chenhua Instruments, Shanghai). All electrochemical measurements were conducted in 1/15 M PBS (containing 0.1 M NaCl, pH 7.4). Impedance measurements were performed in the frequency range from 0.1 to 10,000 Hz. Differential pulse voltammetry (DPV) responses reported were given as the background-subtracted currents. Electrochemical experiments were performed in an electrochemical cell containing a three-electrode system. A gold electrode (2 mm diameter) was used as the working electrode with a saturated calomel electrode (SCE) as the reference electrode and a platinum electrode as the auxiliary electrode. All measurements were performed in a 10-mL cell and the temperature was kept at room temperature. All potentials measured were with respect to the SCE. 2.4. Modification of electrode with MutS protein Prior to surface modification, the gold electrode was treated with “piranha” solution, and washed thoroughly with a copious amount of doubly distilled water. The electrode was polished to a mirror with 0.05 mm alumina, and then sonicated for 2 min in alcohol and distilled water, respectively. Then, the electrochemical pretreatments were performed by cycling from a potential of −0.3 to +1.5 V versus SCE reference electrode in a 0.1 M H2 SO4 solution for 12 min. The bare gold electrode was placed in a freshly made 1,6hexanedithiol solution in dry ethanol. After adsorption for 30 min, the electrode was thoroughly washed with ethanol and water to remove physically adsorbed 1,6-hexanedithiol and dried at room temperature. Subsequently, the electrode was immersed in the colloidal Au solution overnight. After sufficiently rinsing, the electrode was incubated in MutS protein solution (0.05 mg mL−1 ) for 1 h at 37 ◦ C.
Deoxyoligonucleotides including DNA target 1:, 5 -gggag ggc agg agc cag ggc tgg gca ta g aag tca ggg cag agc cat cta ttg ctt
2.5. Electrochemical measurement procedure
aca ttt gc ttctg-3 , DNA target 2: 5 -gggag ggc agg agc cag ggc tgg gca ta a aag tca ggg cag agc cat cta ttg ctt aca ttt gc ttctg-3 , and detection probe: 5 -cagaa gc aaa tgt aag caa tag atg gct ctg ccc tga ctt t ta tgc cca gcc ctg gct cct gcc ctccc-3 ) were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Tris was from Roche. Trisodium citrate, NaCl, KCl and MgCl2 were obtained from Amresco (Solon, OH). The size of gold nanoparticles was verified as 20 nm by TEM (Hitachi TEM 800, Japan). The concentration of colloid Au is about 1.3 × 1011 particles/mL in citrate buffer. 1,6-Hexanedithiol (HDT) was obtained from Sigma–Aldrich. HAuCl4 ·3H2 O and methylene blue (MB) are obtained from Aldrich. HAuCl4 ·3H2 O, trisodium citrate and other chemicals were all of analytical grade. Ultrapure
In a typical experiment, appropriate amount of DNA target 1 or 2 was added to the hybridization buffer containing 100 nM detection probe, followed by heating at 77 ◦ C for 60 min. After hybridization, the MutS protein-modified electrode was immersed in the hybridization solution and incubated for 60 min at 37 ◦ C, followed by washing with detection buffer. Then, the gold electrode was immersed in 20 M MB solution for 5 min. Finally, the electrode was introduced into the electrochemical cell containing 10 mL of PBS buffer for DPV analyses. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV.
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3. Results and discussion 3.1. Schematic representation of principle of mutation detection The analytical principle of mutation detection the MutS protein based are shown in Scheme 1. A self-assembled monolayer of disulfides on gold electrode was used as the base interface for the assembly of the Au colloids. This enabled the formation of highly ordered and well-organized structure of gold nanoparticles on the electrode surface. Due to large surface free energy and surface area, nanoparticles show much stronger affinity in adsorbing proteins, improving the surface loading of protein. Also, nanoparticle layer can enhance the conductivity of the biosensor interface and maximally maintain the activity of the immobilized protein, thus improving the detection sensitivity. Therefore, use of nanoparticle layer for protein immobilization is helpful for sensitivity enhancement in biosensors (Rembaum and Dreyer, 1980; Martin and Micher, 1998; Crumbliss et al., 1992; Wang et al., 2004; Tang et al., 2008; Liu et al., 2003; Grabar et al., 1995). The colloidal Au layer then provided a platform for strong adsorption of MutS protein as the recognition element. This immobilization protocol not only allowed for high loading of MutS protein, but also preserved the activity of the proteins. MutS protein is a mismatch binding protein that can specifically recognizes and binds to heteroduplex DNA with up to 3 mismatched bases. The occurrence of mutation in target genes resulted in the formation of heteroduplex, which could specifically bind to the immobilized MutS protein on the electrode. As a result, a certain redox indicator as MB that could selectively intercalate in the duplexes was utilized for probe the adsorption of heteroduplex on the electrode surface, where the DPV current of MB could reveal the amount of DNA heteroduplex arising from gene mutations. 3.2. Characterization of the electrode processes by impedance spectroscopy
Fig. 1. Electrochemical impedance spectra of electrode processes: (a) bare Au electrode, (b) 1,6-hexanedithiol/gold electrode and (c) Au-colloid/1,6hexanedithiol/gold electrode, (d) MutS protein/Au-colloid/1,6-hexanedithiol/gold electrode,(e) electrode incubated with full-matched dsDNA and (f) single-base mismatch of dsDNA immobilized electrode in the frequency range from 0.1 Hz to 10 kHz. All the measurements were performed in 0.067 MPBS (containing 0.1 M KCl, 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], pH 7.4).
HDT self-assembled electrode. This observation is consistent with those reported previously (Wang et al., 2004; Liu et al., 2003). The impedance increment obtained were 1200 after the immobilization with MutS protein, indicating that the immobilization protocol could efficiently load the protein on the sensor surface. The reaction of the MutS protein-immobilized electrode with single-base mismatched dsDNA caused an impedance increment of 1100 , but no impedance increment was observed with full-matched dsDNA, implying that the specific recognition was achieved on the electrode interface. 3.3. Optimization of the hybridization buffer and assay time
Electrochemical impedance spectroscopy is one of the most powerful and sensitive techniques for investigating the features of surface-modified electrodes. According to Fig. 1, the impedance increase is related to the adsorption of an electrically insulated material on the electrode surface. Following the experimental procedure as described above, the impedance values were recorded after each step was finished. The modification of HDT produced an impedance increment of 3500 , suggesting the self-assembled monolayer of HDT on the electrode surface. The subsequent interaction of the electrode with Au colloids reduced the impedance, evidencing the formation of highly ordered Au colloid layer that improved the conductance of the electrode. Commonly, there is an electronic coupling between Au nanoparticles and the gold electrode through the tunneling effect (Lyon et al., 1998). Thus, Au colloid assembled electrode shows smaller impedance than the
The salt concentration in hybridization buffers was one of the most important factors that had great effects on the hybridization efficiency. The positively charged ions could reduce the repulsion between the negatively charged phosphate backbones in DNA structure. Fig. 2A displays the current responses as a function of NaCl concentrations in hybridization buffer. The response increases with NaCl concentration increased from 0.05 to 0.3 M and reaches a plateau at 0.3 M. Fig. 2B depicts the signal responses as a function of MgCl2 concentrations in hybridization buffer. The current response to 1.5 pM target 1 increases with MgCl2 concentration increased from 0 to 40 mM and reaches a plateau at 20 mM. Therefore, 0.3 M NaCl and 20 mM MgCl2 were employed in the hybridization buffer throughout the experiments. Obviously, the amount of dsDNA binding to MutS protein on the electrode is dependent upon the interaction time between MutS protein and mismatched dsDNA. One can expect that sufficient reaction time could guarantee the maximized amount of dsDNA binding on the electrode, which in turn improves the sensitivity in mutation detection. As shown in Fig. 3, the current response to dsDNA hybrid of 10 pM target 1 with detection probe increase as the reaction time is increased up to 25 min, and the current response tends to level off in a prolonged reaction time. So, the MutS–dsDNA binding time as 25 min was adopted throughout this work. 3.4. Determination of point mutations
Scheme 1. Schematic illustration of the label-free electrochemical sensor for the MutS protein-mediated mismatch recognition.
The MutS protein-modified electrode constituted a specific platform for the detection of point mutation. As shown in Fig. 4A,
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Fig. 2. The current responses detected by the MutS protein-modified electrode with different concentration of NaCl and MgCl2 in hybridization buffer. The concentrations of probe and target 1 were 100 nM and 1.5 pM, respectively. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV.
Fig. 3. Effects of the MutS–dsDNA binding time on the peak currents. The concentrations of probe and target 1 were 100 nM and 10 pM, respectively. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV. Other experimental conditions were kept unchanged in the optimized buffer.
the DPV responses of the electrode to 1.6 nM DNA target 2 was very small, ca. 10.8 nA. This is attributed to the weak interaction of MutS protein with DNA homoduplex with AT perfect match. In contrast, the DPV responses of the electrode to 1.6 nM DNA target 1 that has a single-base mismatch with the detection probe gives an appreciable current peak with a peak current of 82.3 nA, indicating that the interaction between MutS and heteroduplex with GT mismatch is much stronger than that between MutS and homoduplex with AT perfect match. The results also evidenced that the MutS protein-modified electrode offered an effective approach for the discrimination of point mutations in the target genes. As shown in Fig. 4B, the DPV currents increased as the concentration of DNA target 1 with single-base mismatch increased in the range from 1.5 pM to 1.6 nM, and the current tends to be leveled off at higher concentrations. No detectable peak current was observed when there was no DNA target (targets 1 and 2) in the sample solution, implying that the interaction of MutS protein with single-stranded detection probe is too weak to be detected. In the inset, a linear correlation between the DPV peak currents to the logarithmic concentrations of target DNA 1 was obtained in the range from 1.5 pM to 1.6 nM with a regression equation ip = 19.4 log Ctarget 1 + 215.0. The error bar means S.D. across five repetitive experiments to prove the good reproducibility of the biosensor. The limit of detection of 0.6 pM was readily achieved in terms of the rule of three times of standard deviation over the blank measurement.
Fig. 4. (A) The current responses of 1.6 nM target 2 (a) and 1.6 nM target 1 (b) reacted with 100 nM probe. (B). DPV analysis for DNA target 1 with different concentration: (a) 0 nM, (b) 1.5 pM, (c) 6.1 pM, (d) 24.4 pM, (e) 100 pM, (f) 400 pM, and (g) 1.0 nM. The DPV measurements were performed by scanning from a potential of −0.4 to 0 V versus SCE reference electrode in a 0.067 M PBS (pH 7.4). Potential-scan rate: 50 mV s−1 ; initial potential: 0 mV. Other experimental conditions were kept unchanged as the optimized ones.
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Fig. 5. (A) Schematic illustration of the principle of genomic samples detection. In this figure (A) shows the principle of two-step detection for wild-type sample, (B) shows the principle of two-step detection for heterozygous mutant sample and (C) shows the principle of two-step detection for homozygous mutant sample. (B) The DPV analysis for four genomic samples. Curves a show the current responses for real samples treated after step 1. Curves b show the current responses for real samples treated after step 2.
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The proposed approach was implemented for the identification of single-base mutation in −28 site of the -thalassemia gene in genomic samples with a preliminary polymerase chain amplification (PCR). Genomic DNA was extracted from blood samples according to the common method. The forward and the reverse primers are 5 -GTA CGG CTG TCA TCA CTT AGA CCT CA-3 and 5 -TGC AGC TTG TCA CAG TGC AGC TCA CT-3 , respectively. PCR amplification was performed in a 50 L system with 200 M deoxyribonucleotide triphosphate (dNTPs), 0.2 M forward and reverse primers (10 pmol for each primer), as well as ca. 300 ng of genomic DNA extracted from the samples. Amplification was achieved by thermal cycling for 30 cycles at 98 ◦ C for 15 s, 59 ◦ C for 20 s, 72 ◦ C for 15 s and a final extension at 72 ◦ C for 8 min. PCR products of 602 bp were obtained after purified using a gel extraction kit (Ambiogen) and then quantified by UV–vis spectrometer. The resulting samples were diluted using the assay buffer to appropriate concentration for mutation detection. The identification of the samples included two steps in order to discriminate between the wild-type, heterozygous mutant and homozygous mutant. In the first step, the sample of 50 L was heated to 95 ◦ C followed by slow cooling to room temperature to allow the formation of possible heteroduplexes. The sample solution was then detected using the MutS protein-modified electrode. In the first step, when the samples contained both the wild-type and the mutant genes, i.e., the case of heterozygous mutant, the treatment allowed the formation of possible heteroduplexes and a positive response was expected on the MutS protein-modified electrode. For samples from wild-type and homozygous mutant, only homoduplexes would obtain via the first step, then a negative response was expected on the MutS protein-modified electrode. In the second step, 1 L detection probe was added in the sample solution of 50 L (The final concentration was 100 nM for the detection probe). Then, the sample was heated to 95 ◦ C followed by slow cooling to room temperature to allow the formation of possible heteroduplexes. The sample solution was then detected using the MutS protein-modified electrode. In the second step, after sample was denatured at 95 ◦ C followed by annealing at lowed temperature, DNA duplex would form between all DNA single strands present in the solution. Thus, in cases where the sample contained mutant gene, possible heteroduplex with single-base mismatch would be obtained, which was expected to give positive response on the MutS protein-modified electrode. In contrast, for samples without mutant gene, no heteroduplex would form in the solution and a negative response was expected. Combined the results obtained in two steps, the genotype of the samples could be determined. Fig. 5A was the schematic representation of the principle of genomic samples detection. Fig. 5A shows the principle of the twostep detection for samples of wild-type, heterozygous mutant and homozygous mutant, respectively. Fig. 5B shows the DPV currents of four genomic samples for the identification of single-base mutation in −28 site of the -thalassemia gene. Curves a in Fig. 5B depict the DPV currents obtained after the first step and curves b represent that after the second step. Combined the results obtained in two steps, the genotype of the four samples were determined to be wild-type (sample 1), heterozygous mutant (sample 2 and 3) and homozygous mutant (sample 4).
4. Conclusion A novel electrochemical biosensing approach for point mutation detection was developed based on the specific recognition of MutS protein to DNA heteroduplexes with single-base mis-
match. This approach was the first demonstration that combined the MutS-based mismatch recognition with the implementation of redox intercalator of DNA duplex for label-free detection of gene mutations. The results obtained showed that the MutS proteinmodified electrode offered a viable platform for the discrimination between DNA heteroduplexes and homoduplexes, and the detection limit for single mismatched DNA could be achieved as low as 0.6 pM. The method had the advantages of simplicity, label-free detection, high sensitivity, rapidness and the capacity to identify unknown mutations, implying that the proposed approach might hold great promise for rapid scan of point mutations in target genes. Acknowledgements This work was supported by “973” National Key Basic Research Program (2007CB310500), and National Nature Science Foundation of China (20872027, U0632005, 20775023). References Barany, F., 1991. Proc. Natl. Acad. Sci. U.S.A. 88, 189–193. Behrensdorf, H.A., Pignot, M., Windhab, N., Kappel, A., 2002. Nucleic Acids Res. 30, e64. Bi, L.J., Zhou, Y.F., Zhang, X.E., Deng, J.Y., Zhang, Z.P., Xie, B., Zhang, C.G., 2003. Anal. Chem. 75, 4113–4119. Boon, E.M., Barton, J.K., 2003. Bioconjugate Chem. 14, 1140–1147. Boon, E.M., Jackson, N.M., Wightman, M.D., Kelley, S.O., Hill, M.G., Barton, J.K., 2003. J. Phys. Chem. B 107, 11805–11812. Chen, X., Kwok, Y.P., 1997. Nucleic Acids Res. 25, 347–353. Cho, M., Lee, S., Han, S.Y., Park, J.Y., Rahman, M.A., Shim, Y., Ban, C., 2006. Nucleic Acids Res. 34, e75. Crumbliss, A.L., Ferine, S.C., Stonehunerer, J., Tubergen, K.R., Zhao, J.G., Henkens, R.W., O’Daly, J.P., 1992. Biotechnol. Bioeng. 40, 483–490. Ellis, L.A., Taylor, G.R., Banks, R., Baumberg, S., 1994. Nucleic Acids Res. 22, 2710–2711. Fischer, S.G., Lerman, L.S., 1983. Proc. Natl. Acad. Sci. U.S.A. 80, 1579–1583. Gelfi, C., Cremonesi, L., Ferrari, M., Righetti, P.G., 1996. BioTechniques 21, 926–932. Geschwind, D.H., Rhee, R., Nelson, S.F., 1996. Genet. Anal. 13, 105–111. Gotoh, M., Hasebe, M., Ohira, T., Hasegawa, Y., Shinohara, Y., Sota, H., Nakao, J., Tosu, M., 1997. Genet. Anal. Biomol. Eng. 14, 47–50. Grabar, K.C., Reeman, R.G., Hommer, M.B., Natan, M.J., 1995. Anal. Chem. 67, 735–743. Hacia, J.G., 1998. Genome Res. 8, 1245–1258. Han, A., Shibata, T., Takarada, T., Maeda, M., 2002. Nucleic Acids Res. Suppl. 2, 287–288. Han, A., Takarada, T., Shibata, T., Nakayama, M., Maeda, M., 2006. Anal. Sci. 22, 663–666. Hayashi, K., 1991. PCR Methods Appl. 1, 34–38. Henco, K., Harders, J., Wiese, U., Riesner, D., 1994. Methods Mol. Biol. 31, 211–228. Inazuka, M., Wenz, H.M., Sakabe, M., Tahira, T., Hayashi, K., 1997. Genome Res. 7, 1094–1103. Jin, Y., Yao, X., Liu, Q., Li, J., 2007. Biosens. Bioelectron. 22, 1126–1130. Kelley, S.O., Barton, J.K., 1997. Bioconjugate Chem. 8, 31–37. Kwok, P.Y., Carlson, C., Yager, T.D., Ankener, W., Nickerson, D.A., 1994. Genomics 23, 138–144. Landegren, U., Kaiser, R., Sanders, J., Hood, L., 1988. Science 241, 1077–1080. Li, C.Z., Long, Y.T., Lee, J.S., Kraatz, H.B., 2004. Chem. Commun. 10, 574–575. Lishanski, A., Ostrander, E.A., Rine, J., 1994. Proc. Natl. Acad. Sci. U.S.A. 91, 2674– 2678. Liu, Y.J., Yin, F., Long, Y.M., Zhang, Z.H., Yao, S.Z., 2003. J. Colloid Interface Sci. 258, 75–81. Livak, K.J., Marmaro, J., Todd, A.J., 1995. Nat. Genet. 9, 341–342. Lyon, L.A., Musick, M.D., Natan, M.J., 1998. Anal. Chem. 70, 5177–5183. Martin, C.R., Micher, D.T., 1998. Anal. Chem. 5, 322A. Masaˇrík, M., Cahová, K., Kizek, R., Palecˇek, E., Fojta, M., 2007. Anal. Bioanal. Chem. 388, 259–270. Nelson, S.F., 1995. Electrophoresis 16, 279–285. Palecˇek, E., Masaˇrík, M., Kizek, R., Kuhlmeier, D., Hassmann, J., Schulein, J., 2004. Anal. Chem. 76, 5930–5936. Rembaum, A., Dreyer, W.J., 1980. Science 208, 364. Sachidanandam, R., Weissman, D., Schmidt, S.C., Kakol, J.M., Stein, L.D., Marth, G., Sherry, S., Mullikin, J.C., Mortimore, B.J., Willey, D.L., 2001. Nature 409, 928– 933. Su, X.D., Robelek, R., Wu, Y.J., Wang, G.Y., Knoll, W., 2004. Anal. Chem. 76, 489–494. Tang, L., Zeng, G.M., Shen, G.L., Li, Y.P., Zhang, Y., Huang, D.L., 2008. Environ. Sci. Technol. 42, 1207. Tobe, V.O., Taylor, S.L., Nickerson, D.A., 1996. Nucleic Acids Res. 24, 3728–3732. Wabuyele, M.B., Farquar, H., Stryjewski, W., Hammer, R.P., Soper, S.A., Cheng, Y.W., Barany, F., 2003. J. Am. Chem. Soc. 125, 6937–6945.
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