Analyst View Article Online

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

PAPER

Cite this: DOI: 10.1039/c5an00566c

View Journal

Simple and fast electrochemical detection of sequence-specific DNA via click chemistrymediated labeling of hairpin DNA probes with ethynylferrocene Qiong Hu,a Xianbao Deng,a Jinming Kong,*a Yuanyuan Dong,a Qianrui Liua and Xueji Zhang*a,b A universal and straightforward electrochemical biosensing strategy for the detection and identification of sequence-specific DNA via click chemistry-mediated labeling of hairpin DNA probes (hairpins) with ethynylferrocene was reported. In the target-unbound form, the immobilized hairpins were kept in the folded stem–loop configuration with their azido terminals held in close proximity of the electrode surface, making them difficult to be labeled with ethynylferrocene due to the remarkable steric hindrance of the densely packed hairpins. Upon hybridization, they were unfolded and underwent a large conformational change, thus enabling the azido terminals to become available for its subsequent conjugation with ethynylferrocene via the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC). After that, the quantitatively labeled ethynylferrocene could be exploited as the electroactive probes to monitor the DNA hybridization. As the unfolded hairpins were labeled in a stoichiometric ratio of 1 : 1, the electrochemical measurement based on differential pulse voltammetry enabled a reliable quantification of sequencespecific DNA. Under optimal conditions, the strategy could detect target single-stranded DNA (ssDNA) down to 0.296 pM with a good linear response over the range from 1 pM to 1 nM, and had excellent

Received 22nd March 2015, Accepted 13th April 2015

specificity in the genotyping of single-nucleotide polymorphisms. Furthermore, it also exhibited good

DOI: 10.1039/c5an00566c

detection reliability in serum samples and required no complicated protocols. More importantly, the simplicity of this strategy together with its compatibility with microfluidic chips makes it show great potential

www.rsc.org/analyst

in clinical applications, where simple procedures are generally preferred.

Introduction The fast screening of genetic diseases in clinical diagnosis requires the development of reliable, highly sensitive, and highly selective biosensing strategies with excellent specificity toward the genotyping of single-nucleotide polymorphisms (SNPs).1–3 Although numerous approaches have been proposed to circumvent these challenges, certain limitations still remain. To overcome these drawbacks, novel and alternative methodologies are urgently needed. Compared with the conventional optical methods, such as fluorescence spectrometry,4 ultraviolet absorption spectrophotometry,5 and colorimetry,6 electrochemical methods have received more attention by

a School of Environmental and Biological Engineering, Nanjing University of Science & Technology, Nanjing 210094, P. R. China. E-mail: [email protected]; Tel: +86-25-84303109 b Chemistry Department, College of Arts and Sciences, University of South Florida, East Fowler Ave, Tampa, Florida 33620-4202, USA. E-mail: [email protected]

This journal is © The Royal Society of Chemistry 2015

virtue of their intrinsic characteristics of high sensitivity, simple instrumentation, relatively low cost, and amenability to miniaturization.7,8 During the past few decades, molecular beacons (MBs) have been extensively researched in fields ranging from in vitro genotyping to in vivo studies within living cells, thanks to their simplicity and especially their outstanding specificity toward SNPs.9–11 MB is a hairpin-like single-stranded oligonucleotide and was always kept in the folded stem–loop configuration with its terminals held in close proximity in the targetunbound form, due to the formation of a thermostable stem.9,10,12 Generally, the complementary stem of MB determines the hairpin configuration, which is quite important both for the low background signal in the target-unbound form and for the improved selectivity of MB toward the genotyping of SNPs in comparison with linear oligonucleotide probes.9 Meanwhile, the loop portion of the hairpin is a complementary region; it can specifically recognize and hybridize with the predetermined target.9,10,12 The interaction of MBs

Analyst

View Article Online

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

Paper

with their target is of high specificity and even the single base mismatch (SBM) in the complete sequence can be effectively discriminated.9 Consequently, it has been exploited as an exclusive alternative to peptide nucleic acid (PNA) in the field of genotyping.13 Generally, electrochemical interrogation of DNA hybridization can be conveniently achieved via measuring the electrochemical signal of the quantitatively labeled electroactive probes, such as ferrocene-based derivatives3,12,14 and methylene blue.3,12,15,16 The electroactive probe is routinely modified at one terminal of the MB with its another counterpart immobilized on the electrode; thus its distance to the electrode surface will be altered significantly impelled by the formation of the rigid duplex resulting from DNA hybridization, which in turn results in a large signal change.3,12,14 For example, a highly adaptable and robust strategy for MB-based electrochemical interrogation of DNA hybridization has been established by Plaxco and co-workers.12 In this strategy, the immobilized MBs labeled with ferrocene probes were utilized as the hybridization sensing elements; the distance between the probes and the electrode would be greatly changed impelled by the hybridization-induced conformational change; the quantitative analysis of DNA hybridization therefore could be easily accomplished via monitoring the change of electrochemical response. However, the coupling of amino-labeled hairpin-like oligonucleotide with carboxy-containing ferrocene is quite uncontrollable and inefficient, since the nucleotides are also highly reactive sites available for the binding of ferrocene probes. Meanwhile, the resulting amide linkages are also chemically unstable and liable to hydrolysis. In addition, complicated protocols for the further separation and purification of the prepared MBs are also time-consuming and troublesome. Therefore, to improve the applicability of MBs in the field of DNA biosensing, further progress can still be achieved by choosing a more reliable and straightforward strategy for the labeling of electroactive probes. Click chemistry, firstly proposed by Sharpless in 2001,17 has undeniably become one of the most popular in chemistry and materials science. It is generally used to describe a class of chemistry reactions tailored to generate products quickly and reliably by conjugating small units together as nature does, with a yield close to 100% as well as a preferential and rapidly occurring irreversibility, high selectivity and orthogonality, in a modular and process-driven manner.17,18 Of all currently identified click reactions, the class of heteroatom cycloadditions, especially the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, is the most reliable and versatile category. The products of Huisgen 1,3-dipolar cycloaddition are chemically stable 1,2,3-triazoles, and the reactions can proceed fast and efficiently both in organic solvents and in aqueous solutions under mild reaction conditions facilitated by the catalysis of Cu(I).17,19 More importantly, the azido and alkynyl are widely absent in the biological systems and are inert under a wide range of conditions and do not interact with the presented water, oxygen, biological molecules, and other functionalities.17,19 Thus, the well-established Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) has great application

Analyst

Analyst

value as a chemoselective platform for the functionalization or ligation of biomaterials, including site-specific modification of proteins/viruses, protein/oligonucleotide microarrays, functionalized biomolecules and cell surfaces, etc.20 Currently, attempts have been made to integrate click chemistry into the biosensing of DNA, taking into account its simplicity and efficiency. For example, Sun and Peng have proposed a strategy that is highly specific and capable of discriminating SBM under mild conditions based on the templatedirected fluorogenic oligonucleotide ligation using the CuAAC.2 Meanwhile, Lai and co-workers have developed an electrochemical DNA biosensing approach via potentialassisted click chemistry, where the CuAAC was exploited as a simple and efficient strategy for the immobilization of methylene blue-labeled MBs.16 However, to our knowledge, there has been no report on the electrochemical detection of sequencespecific DNA based on click chemistry-mediated labeling of electroactive probes. Herein, a novel electrochemical biosensing strategy that was applicable for the simple and fast detection of sequencespecific DNA was reported for the first time based on exploiting click chemistry-mediated facile labeling of the unfolded hairpins with ethynylferrocene. In this work, the bifunctional hairpins with thiol and azido at 5′ and 3′ terminals were firstly self-assembled on the gold electrode to serve as the immobilized capture probes for the specific recognition of target ssDNA in the following step. The hairpin has five complementary bases at either terminal of its sequence, which tend to form a thermostable double-stranded stem in the targetunbound form in the presence of MgCl2, and thus can localize the azido moiety to close proximity of the electrode surface, while the loop portion can specifically recognize and hybridize with the predetermined complementary ssDNA.9,12,21 Upon hybridization, the hairpins were unfolded and underwent a large conformational change due to the formation of the rigid duplexes to enable the azido moieties to become available for their subsequent conjugation with alkynyl-containing electroactive probes, ethynylferrocene, in the presence of Cu(I) as the efficient catalyst. Finally, the quantitatively labeled electroactive probes were measured to monitor the hybridization via differential pulse voltammetry (DPV). As the unfolded hairpins were labeled in the stoichiometric ratio of 1 : 1, the electrochemical analysis of ethynylferrocene enabled a reliable quantification of sequence-specific DNA. Under optimal conditions, the strategy presented a good linearity between the oxidation currents and logarithms of ssDNA concentrations in the range from 1 pM to 1 nM, and it had excellent specificity in the genotyping of SNPs. As the labeling of electroactive probes could be easily accomplished through the efficient CuAAC, the strategy was straightforward and labour-saving, allowing for its superiority in eliminating complicated protocols. Results also showed that it held good detection reliability in serum samples. More importantly, the simplicity of this strategy together with its compatibility with microfluidic chips makes it show great potential in clinical applications, where simple procedures are generally preferred.

This journal is © The Royal Society of Chemistry 2015

View Article Online

Analyst

Paper

Experimental section

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

Chemicals and reagents The bifunctional hairpin DNA probe sequence and the DNA sequences used in this work were all purchased from Sangon Biotech Co., Ltd (Shanghai, China) and were purified by highperformance liquid chromatography (HPLC). Their sequences are listed below: hairpin DNA probe (hairpin): 5′-SH-(CH2)6-CCA CGC TTG TGG GTC AAC CCC CGT GG-N3-3′. Target complementary ssDNA (cDNA): 5′-GGG GTT GAC CCA CAA G-3′. Single-base mismatched ssDNA (SBM, mismatch underlined): 5′-GGG GTC̲ GAC CCA CAA G-3′. Three bases mismatched ssDNA (TBM, mismatches underlined): 5′-GGG GTC̲ GT̲C G̲CA CAA G-3′. Control ssDNA (Control): 5′-TTC AGC TCT ATC AAT C-3′. Ethynylferrocene and L-ascorbic acid (AA) were purchased from J&K Scientific Ltd (Shanghai, China). Copper(II) sulfate (CuSO4), 6-mercapto-1-hexanol (MCH), and lithium perchlorate (LiClO4) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS, Defined) was purchased from Shanghai YiJi Industrial Co., Ltd (Shanghai, China). Unless stated otherwise, all other chemical reagents were of analytical grade and were used without further purification. Ultrapure water obtained from a Millipore Milli-Q water purification system (≥18.25 MΩ) was used throughout all experiments. Tris-EDTA buffer (TE, 10 mM Tris-HCl, 1 mM EDTA, pH = 8) was prepared and used as the stocking solution and washing buffer for the hairpin and ssDNA. Different concentrations of ssDNA used in this work were prepared by serial dilution with TE buffer. 1 M LiClO4 was utilized as the electrolyte for voltammetric measurements allowing for its advantage in maintaining the stability of ferrocenium.12,22 Apparatus All electrochemical experiments were conducted at room temperature with a conventional three-electrode system consisted of a modified gold electrode (2 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire as the auxiliary electrode. Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were performed on a CHI 760D electrochemical workstation (Shanghai, China). Electrochemical impedance spectroscopy (EIS) measurements were performed on an Autolab potentiostat/galvanostat PGSTAT302N (Metrohm, Netherlands).

Scheme 1 Schematic illustration of electrochemical detection of sequence-specific DNA based on click chemistry-mediated labeling of electroactive probes.

Piranha solution (98% H2SO4 and 30% H2O2, 3 : 1 v/v, Caution! it is dangerous and must be handled carefully) under ultrasonication. The electrode was then electrochemically treated in freshly prepared 0.5 M H2SO4 by CV within the potential of −0.2 V and 1.5 V with a scan rate of 0.1 V s−1 until a stable cyclic voltammogram was achieved.3,8 Subsequently, it was rinsed thoroughly with absolute ethanol and ultrapure water, followed by drying with nitrogen prior to modification. Immobilization of the capture probes was achieved by incubating the pretreated electrode in TE solution containing 1 μM hairpins. After washing with TE buffer, the residual nonspecific binding sites on the electrode were blocked with 2 mM MCH solution (freshly dissolved in 70% ethanol), followed by rinsing with 70% ethanol and ultrapure water.8,12 After that, DNA hybridization was performed by incubating the obtained electrode in 10 μL of TE buffer that contained certain concentrations of ssDNA and 0.1 mM MgCl2 at 37 °C, followed by washing with TE buffer to remove the unhybridized ssDNA. Subsequently, 30 μL of freshly prepared solution of ethynylferrocene, CuSO4 and AA with the concentrations of CuSO4 and AA respectively kept at 0.1 mM and 0.2 mM (freshly dissolved in 70% ethanol) was pipetted onto the electrode and then incubated at 25 °C for the labeling of electroactive probes. Finally, the electrode was moderately rinsed with absolute ethanol and ultrapure water, and dried with nitrogen. Electrochemical measurements The as-prepared electrode was immersed in 10 mL of 1 M LiClO4 solution, and then DPV was conducted in the potential range from 0.1 V to 0.5 V to record the oxidation current of ethynylferrocene.12,23

Preparation of the modified electrode The scheme for electrochemical detection of sequence-specific DNA by exploiting click chemistry-mediated labeling of electroactive probes is illustrated in Scheme 1. Prior to use, the gold electrode was manually polished with 0.3 and 0.05 μm alumina slurries to obtain a mirror-like surface, and then it was thoroughly rinsed with ultrapure water followed by ultrasonic cleaning in absolute ethanol and ultrapure water for 2 min. After that, it was immersed for 10 min in a fresh

This journal is © The Royal Society of Chemistry 2015

Results and discussion Electrochemical characterization of the modified electrode To verify the feasibility of the strategy for the electrochemical detection of sequence-specific DNA based on click chemistrymediated labeling of hairpins with ethynylferrocene, the modified electrode was characterized by DPV. As shown in Fig. 1A, merely a feeble background current due to the charging

Analyst

View Article Online

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

Paper

Analyst

Fig. 1 Differential pulse voltammograms of the modified electrode without adding cDNA (a) or CuSO4 (b), as well as in the presence of all reagents (c) (A); the cyclic voltammograms of the labeled electroactive probes on the electrode at scan rates from 10 to 200 mV s−1 (B). (Inset) The linear relationship between anodic (black line) and cathodic (red line) peak currents and scan rates toward 50 pM cDNA in 1.0 M LiClO4; the Nyquist plots of impedance spectra of the bare gold electrode (a), hairpins (b), hairpins/MCH (c), hairpins/MCH/cDNA (d), and hairpins/MCH/cDNA/ethynylferrocene (e) modified gold electrode (C).

process would appear provided that cDNA was not added to the system (Fig. 1A,a). This was mainly because the hairpins were kept in the folded configuration in the target-unbound form and therefore the remarkable steric hindrance of the densely packed hairpins disabled the conjugation of azidomodified terminals with ethynylferrocene. Meanwhile, only a relatively low oxidation current could be observed without the catalysis of Cu(I), because of the sluggish reaction kinetics (Fig. 1A,b).17,19 However, a strong oxidation peak appeared at a potential of around 0.384 V provided that the electroactive probes had been successfully labeled to the unfolded hairpins under the catalysis of Cu(I) (Fig. 2A,c). The resulting potential value was within the typical redox potential range of ferrocene,3,12,24 and the oxidation current was a result of the electrochemical oxidation of ferrocene into ferrocenium.8,25,26 The obtained results evidenced that ethynylferrocene could be effectively labeled to the accessible azido moieties of the unfolded hairpin via the efficient CuAAC. More importantly, the oxidation current appeared only when the hairpins had been unfolded by the cDNA, and this strongly guaranteed the reliability of the electrochemical response. Thus, the strategy was quite feasible.

In addition, the modified electrode was further characterized by CV (Fig. 1B). As can be seen, the voltammograms obtained for the labeled ethynylferrocene at different scan rates from 10 to 200 mV s−1 revealed that the redox process of ferrocene/ferrocenium was quasi electrochemically reversible at low scan rates.27 However, the reversibility would get worse simultaneously with the further increase of scan rate. Additionally, the favorable linearity between the anodic and cathodic peak currents and scan rates provided further evidence that the redox probes were confined at the electrode surface.12,25,27 In other words, the strategy reported here for the facile labeling of hairpins with electroactive probes based on CuAAC proceeded fluently and efficiently. The preparation process of the modified electrode was monitored by EIS. EIS is a reliable and informative technique for monitoring the microscopic interfacial changes.28 In EIS, the diameter of the semicircle portion at high frequencies represents the electron-transfer resistance (Ret), which dominates the electron transfer kinetics of the presented redox probe at the electrode surface.29 In this work, it was conducted within the frequency range of 0.1 Hz to 100 kHz in 10 mL of 0.1 M KNO3 containing 5 mM [Fe(CN)6]3−/4− as the redox probe at an

Fig. 2 Effects of the labeling time (A), the ethynylferrocene concentration (B), and the hybridization time (C) on the analytical performance of this strategy toward 50 pM cDNA in 1.0 M LiClO4.

Analyst

This journal is © The Royal Society of Chemistry 2015

View Article Online

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

Analyst

open circuit potential of 0.18 V, with a voltage amplitude of 5 mV. Fig. 1C shows the Nyquist plots of impedance spectra at different stages of the preparation process. The bare gold electrode exhibited a small Ret due to the fast electron-transfer process (Fig. 1C,a).30 After the immobilization of hairpins, the Ret increased dramatically (Fig. 1C,b). The reason may be that the remarkable electrostatic repulsion between the densely packed negatively charged hairpins and the [Fe(CN)6]3−/4− redox probe slowed down the interfacial electron transfer.31 After MCH was utilized to block the electrode surface, the Ret increased accordingly (Fig. 1C,c). This was mainly because the more densely packed monolayer rendered a slower electrontransfer.8 Further increase in the Ret could be observed when the hybridization had been accomplished (Fig. 1C,d), suggesting an efficient DNA hybridization on the modified electrode surface.32 Finally, a further enhancement in the Ret could be witnessed after incubation within the ethynylferrocene-containing solution under the catalysis of Cu(I) (Fig. 1C,e). This could be ascribed to the steric effect of the covalently labeled electroactive probes, revealing the successful labeling of ethynylferrocene. These results showed that the preparation process proceeded successfully and further confirmed the feasibility and effectiveness of the reported strategy. Optimization of experimental conditions To improve the analytical performance of the strategy, several important experimental parameters were carefully investigated. Apart from the labeling time, the concentration of ethynylferrocene and the hybridization time were also optimized. The concentration of ssDNA can be conveniently monitored via measuring the electrochemical response of the labeled electroactive probes. Predictably, more labeling time can introduce more electroactive probes. That is to say, the analytical performance of this strategy was partially controlled by the labeling time. To improve the analytical performance, the effect of labeling time on electrochemical response was firstly investigated. As shown in Fig. 2A, the oxidation current increased drastically with the increase of labeling time until 90 min; afterwards, no significant changes in current intensity could be still observed; this might be attributed to the fact that the unfolded hairpins had already been saturated with ethynylferrocene. Therefore, 90 min was selected as the optimal labeling time, and was adopted throughout the subsequent experiments. Admittedly, the concentration of ethynylferrocene was another important parameter in optimizing the analytical performance. Thus, the effect of ethynylferrocene concentration on the electrochemical response was also investigated. It can be seen that the oxidation current was significantly enhanced with increasing the concentration of ethynylferrocene, which then tended to reach a plateau after 1.4 mg mL−1 (Fig. 2B). As a result, 1.4 mg mL−1 was accepted as the optimal concentration of ethynylferrocene and was used in the following experiments. Apart from the two parameters discussed above, the hybridization time undoubtedly also had a considerable effect on the

This journal is © The Royal Society of Chemistry 2015

Paper

analytical performance, given that more hybridization time would result in more unfolded hairpins, which was available for the subsequent labeling of electroactive probes. To achieve the best analytical performance, the effect of hybridization time was further investigated. As shown in Fig. 2C, the oxidation current increased rapidly simultaneously with the continuous increase of hybridization time until 75 min and then it tended to reach a plateau after 90 min. The obtained results indicated that the optimal hybridization time was 90 min, and it was adopted in the following experiments. Analytical performance Under optimal conditions, the relationship between the electrochemical response and the cDNA concentration was evaluated. As shown in Fig. 3, the oxidation current increased linearly along with the increase of cDNA concentration. The calibration plot showed a good linearity between the current intensities and logarithms of cDNA concentrations over a range from 1 pM to 1 nM, with a detection limit down to 0.296 pM (S/N = 3). The linear regression equation was I (μA) = 0.209 + 0.176 lg [CDNA/pM] (R2 = 0.9995), with an acceptable relative standard deviation (RSD) of 4.53% (n = 8). The sensitivity was much lower than many other hairpin-based DNA biosensing strategies (as summarized in Table 1). The improved sensitivity benefited most from the relatively low background signal of the reported strategy, by virtue of the outstanding superiority of hairpin probes and the highly selective labeling mediated by CuAAC. In addition, compared with other methods, complicated protocols for the quantitative analysis of sequence-specific DNA were unnecessary for the strategy and the response signal was quite reliable. Therefore, the analytical performance of the strategy designed for the simple and fast electrochemical detection of sequence-specific DNA was quite acceptable. Specificity and stability of this strategy To evaluate the specificity of this strategy, the electrochemical responses of three types of non-complementary ssDNA, including SBM, TBM, and Control, were compared with that of cDNA. As shown in Fig. 4, the current intensities from SBM,

Fig. 3 Differential pulse voltammograms (A), and calibration plot (B) of this strategy toward different concentrations of cDNA in the range from 1 pM to 1 nM.

Analyst

View Article Online

Paper Table 1

Analyst Comparison of the analytical performance of this strategy with other hairpin-based DNA biosensing strategies

Electroactive probe

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

Methylene blue Ferrocene Ferrocene Ferrocene Methylene blue Ethynylferrocene a

Detection method a

SWV DPV CV DPV DPV DPV

Linear range (M) −11

Detection limit (M) −6

5.0 × 10 –1.0 × 10 3.5 × 10−10–2.5 × 10−9 1.0 × 10−9–1.0 × 10−5 5.0 × 10−12–5.0 × 10−9 2.3 × 10−12–2.3 × 10−9 1.0 × 10−12–1.0 × 10−9

−11

2.51 × 10 2.75 × 10−10 1.0 × 10−9 3.5 × 10−12 1.7 × 10−12 2.96 × 10−13

Mode

Ref.

Signal-off Signal-off Signal-off Signal-off Signal-on Signal-on

3 11 12 33a 33b This strategy

SWV: square wave voltammetry.

Fig. 4 DPV responses of this strategy toward four types of 50 pM ssDNA.

TBM, and Control were approximately 28.9%, 12.4%, and 7.6% of the value obtained from cDNA, respectively. The notable difference was a result of the hybridization efficiencies of different types of ssDNA with the loops of the immobilized hairpins, considering that only the fully matched cDNA could specifically and perfectly bind to the loop portions of the hairpins and unfold them simultaneously, while the others could not.9 More importantly, the labeling of redox probes to the hairpins happened only when the latter had been successfully unfolded by the DNA hybridization, because there were no other potential sites available for the binding of ethynylferrocene due to the high selectivity of CuAAC. The obtained results showed that the novel strategy demonstrated here had excellent specificity in differentiating complementary and mismatched oligonucleotide fragments, indicating that it was highly specific and showed great potential in the genotyping of SNPs. In addition, the stability of the modified electrode was challenged by a long-term storage assay. It was immersed in 1 M LiClO4 solution and kept at 4 °C, and nearly 95.2% of the initial response was retained after a storage period of three weeks. The partially lost electrochemical response might be attributed to the relative instability of the constructed system in oxidative LiClO4 solution. The results indicated that the as-prepared electrode possessed satisfactory stability.

Analyst

Fig. 5 DPV responses of this strategy toward 50 pM cDNA in TE buffer (A), 1% serum sample (B), and 5% serum sample (C).

Detection capability in serum samples To evaluate the analytical reliability and application potential of this strategy in real samples, the interference effect of complicated fetal bovine serum (FBS) on its analytical performance was researched. The oxidation currents from 50 pM cDNA in FBS samples were compared with that obtained from 50 pM cDNA in TE buffer. The assay results from 15 repeated experiments are presented in Fig. 5. The oxidation currents from 1% (B) and 5% (C) FBS samples were approximately 93.5% and 88.6% of that from TE buffer (A), respectively. All these results showed that the strategy showed acceptable analytical reliability and application potential in clinical applications, allowing for its good detection capability in complicated systems, especially in serum samples.

Conclusions In summary, a universal, straightforward and selective electrochemical biosensing strategy for the detection and identification of sequence-specific DNA based on exploiting click chemistry-mediated facile labeling of hairpin DNA probes with ethynylferrocene was reported, where the alkynyl-containing probes could be easily labeled to the azido-terminated hairpins, which had been previously unfolded by DNA hybridization, via the efficient CuAAC. Using the hairpins as the

This journal is © The Royal Society of Chemistry 2015

View Article Online

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

Analyst

immobilized capture probes for the specific recognition of target ssDNA significantly improved the specificity, making the strategy show great potential in the genotyping of SNPs. The superiority of this system improved the sensitivity and enabled a detection limit of 0.296 pM under optimal conditions. In addition, complicated protocols for the DNA electrochemical detection and identification were unnecessary and this new approach exhibited acceptable reliability for the biosensing of target ssDNA in serum samples. Its superior analytical performance, such as simple, straightforward, labour- and time-saving, satisfactory stability and reliability and excellent specificity, implicated that it shows great potential in clinical applications. More importantly, the strategy based on click chemistry-mediated labeling of electroactive probes was quite simple, convenient and selective. Furthermore, it could be fully integrated into microfluidic chips and realized multicomponent analysis capabilities at the same time.

Acknowledgements We are grateful to Nanjing University of Science and Technology for its start-up funding and National Natural Science Foundation of China (no. 21345002) for funding this project. We appreciate the financial support received from the Priority Academic Program Development of Jiangsu Higher Education Institutions. We are grateful to the Technology Foundation for Selected Overseas Scholar in Nanjing.

Notes and references 1 (a) I. Singh, C. Wendeln, A. W. Clark, J. M. Cooper, B. J. Ravoo and G. A. Burley, J. Am. Chem. Soc., 2013, 135, 3449–3457; (b) C. Song, X. Yang, K. Wang, Q. Wang, J. Huang, J. Liu, W. Liu and P. Liu, Anal. Chim. Acta, 2014, 827, 74–79. 2 H. Sun and X. Peng, Bioconjugate Chem., 2013, 24, 1226– 1234. 3 Y. Du, B. J. Lim, B. Li, Y. S. Jiang, J. L. Sessler and A. D. Ellington, Anal. Chem., 2014, 86, 8010–8016. 4 (a) R. Hu, T. Liu, X. B. Zhang, S. Y. Huan, C. C. Wu, T. Fu and W. H. Tan, Anal. Chem., 2014, 86, 5009–5016; (b) S. Wang, Y. Zhang, Y. Ning and G. J. Zhang, Analyst, 2015, 140, 434–439. 5 M. J. Holden, R. J. Haynes, S. A. Rabb, N. Satija, K. Yang and J. R. Blasic Jr., J. Agric. Food Chem., 2009, 57, 7221– 7226. 6 N. Zhang and D. H. Appella, J. Am. Chem. Soc., 2007, 129, 8424–8425. 7 (a) T. G. Drummond, M. G. Hill and J. K. Barton, Nat. Biotechnol., 2003, 21, 1192–1199; (b) I. Willner and M. Zayats, Angew. Chem., Int. Ed., 2007, 46, 6408–6418; (c) Q. Hu, W. Hu, J. Kong and X. Zhang, Microchim. Acta, 2015, 182, 427–434.

This journal is © The Royal Society of Chemistry 2015

Paper

8 Q. Hu, X. Deng, X. Yu, J. Kong and X. Zhang, Biosens. Bioelectron., 2015, 65, 71–77. 9 S. Tyagi and F. R. Kramer, Nat. Biotechnol., 1996, 14, 303– 308. 10 L. G. Kostrikis, S. Tyagi, M. M. Mhlanga, D. D. Ho and F. R. Kramer, Science, 1998, 279, 1228–1229. 11 X. Miao, X. Guo, Z. Xiao and L. Ling, Biosens. Bioelectron., 2014, 59, 54–57. 12 C. Fan, K. W. Plaxco and A. J. Heeger, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 9134–9137. 13 C. Briones and M. Moreno, Anal. Bioanal. Chem., 2012, 402, 3071–3089. 14 C. E. Immoos, S. J. Lee and M. W. Grinstaff, ChemBioChem, 2004, 5, 1100–1103. 15 Y. Xiao, A. A. Lubin, B. R. Baker, K. W. Plaxco and A. J. Heeger, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 16677– 16680. 16 S. J. P. Cañete and R. Y. Lai, Chem. Commun., 2010, 46, 3941–3943. 17 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed., 2001, 40, 2004–2021. 18 (a) Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless and M. G. Finn, J. Am. Chem. Soc., 2003, 125, 3192–3193; (b) W. H. Binder and R. Sachsenhofer, Macromol. Rapid Commun., 2007, 28, 15–54; (c) C. R. Becer, R. Hoogenboom and U. S. Schubert, Angew. Chem., Int. Ed., 2009, 48, 4900–4908; (d) E. Lallana, E. Fernandez-Megia and R. Riguera, J. Am. Chem. Soc., 2009, 131, 5748–5750. 19 (a) C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem., 2002, 67, 3057–3064; (b) J. Lahann, Click chemistry for biotechnology and materials science, Wiley, West Sussex, 2009; (c) R. Kluger, J. Am. Chem. Soc., 2010, 132, 6611–6612. 20 (a) T. S. Seo, Z. Li, H. Ruparel and J. Ju, J. Org. Chem., 2002, 68, 609–612; (b) S. Punna, E. Kaltgrad and M. G. Finn, Bioconjugate Chem., 2005, 16, 1536–1541; (c) P. C. Lin, S. H. Ueng, M. C. Tseng, J. L. Ko, K. T. Huang, S. C. Yu, A. K. Adak, Y. J. Chen and C. C. Lin, Angew. Chem., Int. Ed., 2006, 45, 4286–4396; (d) E. Lallana, R. Riguera and E. Fernandez-Megia, Angew. Chem., Int. Ed., 2011, 50, 8794– 8804; (e) D. C. Kennedy, C. S. McKay, M. C. Legault, D. C. Danielson, J. A. Blake, A. F. Pegoraro, A. Stolow, Z. Mester and J. P. Pezacki, J. Am. Chem. Soc., 2011, 133, 17993–18001. 21 X. Wang, P. Dong, W. Yun, Y. Xu, P. He and Y. Fang, Biosens. Bioelectron., 2009, 24, 3288–3292. 22 L. Fu, D. Tang, J. Zhuang, W. Lai, X. Que and G. Chen, Biosens. Bioelectron., 2013, 47, 106–112. 23 D. Kang, X. Zuo, R. Yang, F. Xia, K. W. Plaxco and R. J. White, Anal. Chem., 2009, 81, 9109–9113. 24 C. J. Yu, Y. J. Wan, H. Yowanto, J. Li, C. L. Tao, M. D. James, C. L. Tan, G. F. Blackburn and T. J. Meade, J. Am. Chem. Soc., 2001, 123, 11155–11161. 25 H. Brisset, A. E. Navarro, N. Spinelli, C. Chaix and B. Mandrand, Biotechnol. J., 2006, 1, 95–98. 26 K. Heinze and H. Lang, Organometallics, 2013, 32, 5623– 5625.

Analyst

View Article Online

Published on 13 April 2015. Downloaded by University of Illinois at Chicago on 05/05/2015 13:58:00.

Paper

27 I. Grabowska, D. G. Singleton, A. Stachyra, A. Góra-Sochacka, A. Sirko, W. Zagórski-Ostoja, H. Radecka, E. Stulz and J. Radecki, Chem. Commun., 2014, 50, 4196–4199. 28 R. K. Shervedani and S. Pourbeyram, Biosens. Bioelectron., 2009, 24, 2199–2204. 29 L. Alfonta, I. Willner, D. J. Throckmorton and A. K. Singh, Anal. Chem., 2001, 73, 5287–5295. 30 K. Wang, Z. Sun, M. Feng, A. Liu, S. Yang, Y. Chen and X. Lin, Biosens. Bioelectron., 2011, 26, 2870–2876.

Analyst

Analyst

31 H. He, J. Xia, X. Peng, G. Chang, X. Zhang, Y. Wang, K. Nakatani, Z. Lou and S. Wang, Biosens. Bioelectron., 2013, 49, 282–289. 32 C. Zhang, J. Lou, W. Tu, J. Bao and Z. Dai, Analyst, 2015, 140, 506–511. 33 (a) G. Chatelain, M. Ripert, C. Farre, S. Ansanay-Alex and C. Chaix, Electrochim. Acta, 2012, 59, 57–63; (b) H. F. Cui, L. Cheng, J. Zhang, R. Liu, C. Zhang and H. Fan, Biosens. Bioelectron., 2014, 56, 124–128.

This journal is © The Royal Society of Chemistry 2015

Simple and fast electrochemical detection of sequence-specific DNA via click chemistry-mediated labeling of hairpin DNA probes with ethynylferrocene.

A universal and straightforward electrochemical biosensing strategy for the detection and identification of sequence-specific DNA via click chemistry-...
1MB Sizes 0 Downloads 6 Views