Surface-Initiated-Reversible-Addition–Fragmentation-Chain-Transfer

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Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Surface-Initiated-Reversible-Addition−Fragmentation-ChainTransfer Polymerization for Electrochemical DNA Biosensing Qiong Hu,†,‡ Dongxue Han,†,‡ Shiyu Gan,*,† Yu Bao,† and Li Niu*,†,‡ †

Center for Advanced Analytical Science, School of Chemistry and Chemical Engineering, MOE Key Laboratory for Water Quality and Conservation of the Pearl River Delta, Guangzhou University, Guangzhou 510006, PR China ‡ School of Civil Engineering, Guangzhou University, Guangzhou 510006, PR China

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ABSTRACT: Sensitive detection of biomolecules is integral for biomarker screening and early diagnosis. Herein, surface-initiated reversible-addition−fragmentation-chain-transfer (SI-RAFT) polymerization is exploited as a novel amplification strategy for highly sensitive electrochemical biosensing of DNA. Briefly, thiol-terminated peptide nucleic acid (PNA) probes are first self-assembled onto a gold electrode for the specific capture of target-DNA fragments; the carboxyl-groupcontaining dithiobenzoate 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD) is then tethered to the hybridized PNA− DNA heteroduplexes by means of the well-established carboxylate− Zr4+−phosphate chemistry and serves as the chain-transfer agent (CTAs) for subsequent SI-RAFT polymerization, which is thermally initiated in the presence of 2,2′-azobis[2-(2-imidazolin2-yl)propane] dihydrochloride (VA-044) as the free-radical initiator and ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through SI-RAFT polymerization, one target-DNA fragment can be labeled by a large number of electroactive Fc tags, giving rise to significant amplification of the electrochemical signal. The SI-RAFT-polymerization-based strategy does not involve the use of natural enzymes or complex nanomaterials, offering the benefits of low cost and easy operation. Under optimal conditions, the electrochemical signal is linearly related to the logarithm of the concentration of target DNA over the range from 10 aM to 10 pM (R2 = 0.997), with a detection limit down to 3.2 aM, which is much lower than those of other amplification-by-polymerization-based methods. By virtue of its easy operation, low cost, and high efficiency, the SI-RAFTpolymerization-based amplification strategy is believed to have great application prospects in the sensitive detection of biomolecules.

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without compromising the detection sensitivity and costeffectiveness. Inspired by the amplification-by-polymerization-based approach,14−17 we have very recently reported the use of surfaceinitiated electrochemically mediated atom-transfer radical polymerization (SI-eATRP) as an amplification strategy for the electrochemical biosensing of DNA18,19 and protein-kinase activity.20 The formation of polymers as a result of SI-eATRP brings a large number of electroactive tags to the electrode surface, which can therefore greatly improve the detection sensitivity. Moreover, the SI-eATRP-based strategy features several merits: (1) it is operationally simple, low-cost, and highly efficient; (2) the degree of amplification can be well controlled by the applied potential; (3) the coupling of polymer chains to binding sites based on de novo polymerization (i.e., “grafting from”) can be greatly accelerated when compared with that of the direct use of ready-made polymers (i.e., “grafting to”), because the loss in conformational entropy

n the early stage of disease development, the levels of diagnostically relevant biomarkers (e.g., circulating tumor DNAs, ctDNAs) are generally very low.1,2 For this reason, methods capable of sensitively and selectively detecting biomolecules of interest are of cardinal importance in biomarker screening and early diagnosis. Among various bioanalytical methods, electrochemical biosensing not only combines the high sensitivity of electroanalytical techniques with the high selectivity of molecular-recognition processes but also offers the benefits of low cost, fast response, and good portability,3−5 thus holding great potential in bioanalytical applications. To improve the detection sensitivity, however, most of the reported electrochemical biosensors rely on the use of either natural enzymes (e.g., alkaline phosphatase, ALP)6,7 or complex nanomaterials (e.g., surface-functionalized metal nanoparticles, NPs)8−10 for signal amplification, which limits their application scope because of the poor stability (e.g., temperature and pH sensitivity) and high cost of natural enzymes11 and the laborious and lengthy operations involved in the synthesis and postfunctionalization of nanomaterials.12,13 Therefore, the challenge in electrochemical biosensing lies, to a large extent, in simplifying the amplification strategy © XXXX American Chemical Society

Received: July 30, 2018 Accepted: September 14, 2018

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DOI: 10.1021/acs.analchem.8b03416 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry of the polymers can significantly suppress the diffusion of random-coil chains through the monolayer to the binding sites;20,21 and (4) the high compatibility of the SI-eATRP process with microfabrication technology via potential control makes it applicable to high-throughput detection on microelectrode arrays.18,22 Nevertheless, the inherent drawbacks of the eATRP may limit its potential applications in some cases. Specifically, the involvement of toxic transition-metal catalysts raises concerns for biomedical applications; moreover, the nonspecific binding of positively charged metal cations (e.g., Cu2+) to biomolecules (e.g., nucleic acids), as well as the electrostatic deposition of transition metals under negative potential, may interfere with the following electrochemical measurements.23 To bypass these drawbacks and thus broaden the application scope of the amplification-by-polymerizationbased approach, therefore, it is highly desirable to perform surface-initiated polymerization in a transition-metal-free manner. Reversible-addition−fragmentation-chain-transfer (RAFT) polymerization, first reported by the CSIRO group in 1998,24 has become one of the most versatile and effective reversible-deactivation-radical-polymerization (RDRP) techniques for the synthesis of complex polymers (e.g., hyperbranched polymers) with controlled molecular weights, narrow molecular weight distributions, and high end-group fidelity.24−29 Unlike other controlled or “living” radical-polymerization techniques such as ATRP30 and nitroxide-mediated polymerization (NMP),31 RAFT polymerization is based on degenerative chain transfer, which is mediated by chaintransfer agents (CTAs) such as dithioesters, dithiobenzoates, dithiocarbamates, and trithiocarbonates,26,32−34 and it is compatible with a wide range of vinyl monomers (e.g., (meth)acrylates, (meth)acrylamides, and acrylonitrile) and reaction conditions.25,35−37 In particular, it eliminates the use of toxic transition-metal catalysts. With these in mind, He and co-workers have reported the use of SI-RAFT polymerization as an amplification strategy for the visual biosensing of DNA, with a detection limit of ∼1.0 fM being achieved.23 Although a significant improvement in the detection sensitivity can be observed, the combined use of a CTA-modified oligonucleotide fragment as the auxiliary detection probe and T4 DNA ligase for the ligation of the capture probe and the detection probe undoubtedly renders a sacrifice of operational simplicity and cost-effectiveness. Furthermore, the visualization-based approach inherently suffers from the drawbacks of limited detection sensitivity and poor accuracy.38 By exploiting SI-RAFT polymerization as a novel amplification strategy, we describe herein an electrochemical biosensor for the highly sensitive detection of DNA. The sequential preparation of the SI-RAFT-polymerization-based electrochemical DNA biosensor is illustrated in Scheme 1. In consideration of its superb specificity in differentiating even a single-base mismatch,39,40 peptide nucleic acid (PNA), a synthetic nucleic acid analogue with the negatively charged deoxyribose phosphate backbone of DNA being replaced by a neutral N-(2-aminoethyl)glycine component,39,41 serves as the recognition element for the specific capture of target-DNA fragments. After hybridization, the dithiobenzoates, 4-cyano-4(phenylcarbonothioylthio)pentanoic acid (CPAD), are tethered to the PNA−DNA duplexes by means of well-established carboxylate−Zr4+−phosphate chemistry. In the presence of 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) as the azo free-radical initiator, SI-RAFT polymer-

Scheme 1. Schematic Illustration of the Preparation of the SI-RAFT-Polymerization-Based Electrochemical DNA Biosensor

ization is thermally triggered under mild conditions using ferrocenylmethyl methacrylate (FcMMA) as the monomer and VA-044 as the surface-tethered CTA. Through SI-RAFT polymerization, one target-DNA fragment can be labeled by a large number of electroactive Fc tags, leading to significant amplification of the electrochemical signal, thereby greatly improving the detection sensitivity. Clearly, the SI-RAFTpolymerization-based strategy does not involve the use of either natural enzymes or complex nanomaterials, thus offering the benefits of low cost and easy operation. Superior to the SIeATRP-based strategy, it eliminates the use of toxic transitionmetal catalysts and thus is compatible with biomedical applications, and the following electrochemical measurements are free of potential interference from nonspecifically bound or electrostatically deposited transition metals. Under optimal conditions, the as-prepared electrochemical biosensor allows the highly sensitive detection of target DNA down to 3.2 aM with a linear range of 10 aM to 10 pM. Compared with the visualization-based approach, a significant improvement in detection sensitivity has been achieved. Results also indicate that the as-prepared electrochemical DNA biosensor holds great potential in genotyping of single-nucleotide polymorphisms (SNPs). In addition, its potential application for DNA biosensing in complex biological matrices has been validated using 10% normal human serum samples. Owing to its easy operation, low cost, and high efficiency, the SI-RAFTpolymerization-based amplification strategy is highly applicable to the sensitive detection of biomolecules.

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EXPERIMENTAL SECTION Materials and Reagents. A thiol-terminated PNA probe (5′-HS-(CH2)11-AAC CAT ACA ACC TAC TAC CTC A-3′) was custom-made by Chengdu CP Biochem Company, Ltd. (Chengdu, China). All oligonucleotide fragments, including target single-stranded DNA (ssDNA; 5′-TGA GGT AGT AGG TTG TAT GGT T-3′, tDNA), single-base-mismatched ssDNA (5′-TGA GGT AGT AGG TTG TGT GGT T-3′, SBM), three-base-mismatched ssDNA (5′-TGA GGT ATT AGA TTG TGT GGT T-3′, TBM), and control ssDNA (5′ACT TAC CTT TGC TCA TTG ACG A-3′, cDNA), were synthesized and purified (HPLC) by Sangon Biotech B

DOI: 10.1021/acs.analchem.8b03416 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Scheme 2. Principle of the SI-RAFT-Polymerization-Based Amplification Strategy

Company, Ltd. (Shanghai, China). 6-Mercapto-1-hexanol (MCH) and FcMMA were supplied by Sigma-Aldrich (St. Louis, MO). Lithium perchlorate trihydrate (LiClO4), VA-044, and zirconium dichloride oxide octahydrate (ZrOCl2) were purchased from J&K Scientific Ltd. (Shanghai, China). Normal human serum (NHS) and CPAD were collected from Shanghai YiJi Industrial Company, Ltd. (Shanghai, China) and Aladdin Bio-Chem Technology Company, Ltd. (Shanghai, China), respectively. N,N-Dimethylformamide (DMF), absolute ethanol (EtOH), potassium nitrate (KNO3), and other chemicals were commercially supplied by Sinopharm Chemical Reagent Company, Ltd. (Shanghai, China). All chemicals were of analytical purity or better and used as supplied. The ultrapure water (resistivity ≥ 18.25 MΩ cm) used in the work was purified by a Millipore Milli-Q purification system (Burlington, MA). The SI-RAFT-polymerization solution (freshly prepared with 15% DMF) was a mixture of VA-044 and FcMMA; the final concentration of FcMMA was 0.1 mM. Apparatus. Square-wave voltammetry (SWV) and cyclic voltammetry (CV) were performed using a CHI 760D electrochemical workstation (Shanghai, China). Electrochemical-impedance spectroscopy (EIS) was performed at an opencircuit potential (OCP) of 0.18 V in 0.1 M KNO3 containing 5.0 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1 in stoichiometric ratio) as the redox probe on an Autolab/PGSTAT302N potentiostat/galvanostat (Eco Chemie, Utrecht, Netherlands), with a sine-wave-potential amplitude of 5 mV and a frequency range of 0.1 MHz to 0.1 Hz. All electrochemical experiments were carried out at room temperature (∼25 °C) with a gold working electrode (ϕ = 2.0 mm), a platinum-wire auxiliary electrode, and a saturated-calomel reference electrode (SCE). Unless otherwise specified, all potentials mentioned in this work are referred to SCE. Scanning electron microscopy (SEM) was performed at an accelerating voltage of 30 kV using a Quanta 250F field-emission scanning electron microscope (FEI, Hillsboro, OR). SI-RAFT-Polymerization-Based Electrochemical Biosensing of DNA. The surface of the gold electrode was polished to a mirrorlike finish prior to use with 0.3 and 0.05 μm alumina slurries and ultrasonically cleaned in EtOH and

ultrapure water. Then, it was soaked in piranha solution, a 3:1 (v/v) mixture of 98% H2SO4 and 30% H2O2 (Caution! It is highly corrosive!), for 15 min and rinsed with ultrapure water. Afterward, it was electrochemically pretreated in a 0.5 M H2SO4 solution by cycling the applied potential between −0.3 and 1.5 V, with a scan rate of 0.1 V s−1, until a stable voltammogram was observed.19 Lastly, it was soaked in NaBH4 solution for 15 min, ultrasonically cleaned in ultrapure water, and dried with N2. The immobilization of the thiol-terminated PNA probes through gold−sulfur self-assembly was fulfilled by the addition of 5.0 μL of 0.5 μM PNA aqueous solution onto the electrode surface and then incubation for 1.0 h at 37 °C, followed by a thorough rinse with ultrapure water. The electrode surface was then soaked in 3.0 mM MCH solution (prepared with 60% EtOH) for 0.5 h at 37 °C to block the residual binding sites18 and sequentially rinsed with EtOH and ultrapure water. After that, 10 μL of oligonucleotide sample (prepared with 0.1 M NaH2PO4/Na2HPO4 in PBS, pH 7.4) was added onto the electrode surface, which was subsequently incubated for 1.5 h at 37 °C and gently rinsed with 0.1 M PBS (pH 7.4). Afterward, the electrode surface was soaked in 5.0 mM ZrOCl2 solution (prepared with 15% EtOH) for 15 min at 37 °C and gently rinsed with ultrapure water. After a soak in 0.5 mM CPAD solution (prepared with 15% EtOH) for 0.5 h at 37 °C and a successive rinse with 15% EtOH and ultrapure water, the electrode surface was soaked in the SI-RAFTpolymerization solution (400 μL); this was followed by incubation for 1.5 h at 47 °C. After that, the electrode surface was sequentially rinsed with EtOH and ultrapure water to remove the physically adsorbed species (e.g., FcMMA). Lastly, the resulting electrode was dipped into 0.5 M LiClO4 solution, and then SWV (increase potential: 4.0 mV, potential amplitude: 25 mV, frequency: 15 Hz, quiet time: 15 s) was applied to record the electrochemical signal.

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RESULTS AND DISCUSSION Principle of the SI-RAFT-Polymerization-Based Amplification Strategy. Typically, the RAFT process involves a reversible addition−fragmentation equilibrium in which transfer of the thiocarbonylthio moiety, SC(Z)S−, of the CTA between the dormant chains and the propagating radicals C

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Analytical Chemistry serves to maintain the living character of the polymerization process.24,28 Because of the inevitable termination process, a continuous supply of initiating radicals is essential in RAFT polymerization.26 Accordingly, VA-044 is used herein as the thermal radical initiator by virtue of its good water solubility and, in particular, low decomposition temperature (the 10 h half-life decomposition temperature in water is 44 °C).26 As shown in Scheme 2, the principle of the SI-RAFT-polymerization-based amplification strategy can be described as follows:24−26,36 (I) By cleavage of two C−N bonds, the azo initiators (i.e., VA-044) thermally decompose to produce N2 and alkyl radicals (I·). (II) These alkyl radicals attack the methacrylic monomers (i.e., FcMMA) to form oligomeric radicals (Pn·). (III) The Pn· radicals react with the surfacetethered CTAs (1; i.e., CPAD) to produce the radical intermediates (2), which can fragment either back into the surface-tethered CTAs and the Pn· radicals or into the surfacetethered reinitiating radicals (3) and the thiocarbonylthiogroup-capped dormant chains (4). (IV) By repeatedly reacting with the FcMMA monomers, these reinitiating radicals propagate to form the polymeric radicals (Pm·), which react with the thiocarbonylthio-group-capped dormant chains to produce the intermediate radical species (5), which can fragment either back into the P m · radicals and the thiocarbonylthio-group-capped dormant chains or into the thiocarbonylthio-group-capped polymer chains and the Pn· radicals (the Pn· radicals can also propagate by repeatedly reacting with the FcMMA monomers). As a result of the continuous reversible addition−fragmentation (step IV), long thiocarbonylthio-group-capped polymer chains can be de novo grafted from each of the CTA-tethered sites. Therefore, SIRAFT polymerization can bring a large number of electroactive Fc tags to each of the captured target-DNA fragments, thereby greatly improving the electrochemical signal and thus the detection sensitivity. Feasibility of the Electrochemical DNA Biosensing. To probe the feasibility of exploiting SI-RAFT polymerization as an amplification strategy for the electrochemical biosensing of DNA, the square-wave voltammograms of differently prepared electrodes were collected for a comparison. As can be seen from Figure 1A, during the step-by-step modification of the electrode surface, if the PNA probe (curve a), target DNA (curve b), Zr4+ (curve c), CPAD (curve d), VA-044 (curve e), or FcMMA (curve f) has not been added, no clear oxidation peak can be observed; on the other hand, a strong oxidation current with a peak potential at ∼0.3 V can be clearly observed when all the above components have been added during the sequential modification of the electrode surface (curve g). The peak potential, of which the oxidation current is derived from the electrochemical oxidation of the Fc tags of the electroactive polymers into the ferrocenium cations,18,42 falls into the typical oxidation-potential range of the Fc tag.19,20,42 The fact that no clear oxidation current can be observed if only PNA probe (curve a), target DNA (curve b), Zr4+ (curve c), CPAD (curve d), or VA-044 (curve e) is not added (in each case, FcMMA is added) indicates that the nonspecific adsorption of FcMMA monomers onto the electrode surface is negligible. These results clearly show that SI-RAFT polymerization can indeed be exploited as an amplification strategy for the electrochemical biosensing of DNA. SEM Characterization. The surface morphologies of the electrodes obtained before and after SI-RAFT polymerization

Figure 1. (A) Square-wave voltammograms of the electrodes prepared without the addition of (a) PNA probe, (b) target DNA, (c) Zr4+, (d) CPAD, (e) VA-044, or (f) FcMMA and that of the (g) PNA−MCH−tDNA−Zr4+−CPAD−FcMMA-modified electrode. (B) SEM image of the PNA−MCH−tDNA−Zr4+−CPAD−FcMMAmodified electrode. The scale bar is 3.0 μm. (C) Cyclic voltammograms of the PNA−MCH−tDNA−Zr4+−CPAD−FcMMA-modified electrode in 0.5 M LiClO4 solution at different scan rates (along the arrow, the scan rates are 15, 25, 50, 75, 100, 150, 250, 500, 750, and 1000 mV s−1, respectively). The inset depicts the plots of the cathodic (red line) and the anodic (black line) peak currents versus the scan rate. (D) Nyquist plots of the (a) bare electrode and of the (b) PNA-, (c) PNA−MCH-, (d) PNA−MCH−tDNA-, (e) PNA−MCH− tDNA−Zr4+ -, (f) PNA−MCH−tDNA−Zr4+ −CPAD-, and (g) PNA−MCH−tDNA−Zr4+−CPAD−FcMMA-modified electrodes. In these experiments, the concentrations of tDNA and VA-044 were 10 pM and 2.0 μM, respectively.

were analyzed by scanning electron microscopy. As can be seen from Figure S1, the surface of the PNA−MCH−tDNA−Zr4+− CPAD-modified electrode is quite smooth, with no particles being observed. On the other hand, a high density of polymer particles are evenly distributed on the surface of the PNA− MCH−tDNA−Zr 4+ −CPAD−FcMMA-modified electrode (Figure 1B). These results indicate that SI-RAFT polymerization can lead to the formation of plenty of polymer particles on the electrode surface. Further, the elemental mappings of the electrodes obtained before (Figure S2) and after (Figure S3) SI-RAFT polymerization were tracked by energydispersive X-ray spectroscopy (EDS). Before we discuss the results of EDS analysis, it is worth pointing out that the absence of characteristic elements makes it impossible to distinguish the presence of the PNA probe (composed of S, C, N, O, and H elements), MCH (composed of S, C, O, and H elements), or CPAD (composed of S, C, N, O, and H elements) from the elemental mapping. Accordingly, we only analyzed the other three components, target DNA (composed of P, C, N, O, and H elements), Zr4+, and FcMMA (composed of Fe, C, O, and H elements), on the basis of their characteristic elements. As shown in Figure S3, the traces of P (Figure S3B), Zr (Figure S3C), and Fe (Figure S3D) elements, which are characteristic of the target DNA, the Zr4+ ion, and FcMMA, respectively, represent the presence of these three components on the electrode surface. For the PNA− MCH−tDNA−Zr4+−CPAD-modified electrode (Figure S2), however, no trace of Fe element (Figure S2C) can be observed. Therefore, it is reasonable to conclude that the D

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Analytical Chemistry

effect) because the highly hydrophobic polymers can extrude the hydrophilic redox probes away from the electrode surface. Therefore, the decrease in Rct after SI-RAFT polymerization indicates that the positive effect plays a dominant role in the charge-transfer kinetics. As observed from the SEM images, after SI-eATRP (for details, see refs 18−20), the polymers are sparsely and disorderly distributed on the electrode surface; after SI-RAFT polymerization (Figure 1B), it can be seen that the polymers are densely and evenly distributed on the electrode surface, with the amount of polymers being much higher than that obtained after SI-eATRP. Besides, for the SIRAFT-polymerization-based strategy, we have also observed that a longer polymerization time can feature a smaller Rct (after a 1.0 h polymerization, for example, the Rct is ∼0.69 kΩ; Figure S4); that is, more polymers result in a smaller Rct. Consequently, the significant decrease in Rct observed after SIRAFT polymerization is likely due to the formation of a more densely and evenly packed polymer layer on the electrode surface, which preferentially facilitates the interfacial-chargetransfer kinetics. Optimization of VA-044 Concentration. As mentioned above, an additional supply of radical initiator is required in the RAFT-polymerization process.26 However, a lower initiator concentration has been reported as being advantageous for obtaining higher livingness of the polymerization process (i.e., a higher fraction of living chains), but in such a case, a lower polymerization rate will result.26 With this in mind, the effect of VA-044 concentration on SI-RAFT polymerization was investigated. As seen from Figure S5, the oxidation-peak current (the peak potential was at ∼0.3 V) increases with increasing VA-044 concentration until 2.0 μM; afterward, it decreases with further increases of VA-044 concentration. In the following experiments, the concentration of VA-044 was therefore set to 2.0 μM. Analytical Performance. Under optimal conditions, the analytical performance of the SI-RAFT-polymerization-based electrochemical DNA biosensor was investigated. As shown in Figure 2A, the square-wave voltammograms are featured by a

particles that distributed on the electrode surface are the electroactive ferrocenyl polymers.20 Electrochemical Characterizations. Subsequently, CV was applied to characterize the electrochemistry of the PNA− MCH−tDNA−Zr4+ −CPAD−FcMMA-modified electrode. With the scan rate from 15 to 1000 mV s−1, it can be seen that both the cathodic peak current (red line) and the anodic peak current (black line) increase linearly with the scan rate (Figure 1C). Because this observation is a typical characteristic of surface-controlled redox processes, it can be concluded that the polymers have been tethered to the electrode surface.18,42 The sequential preparation of the SI-RAFT-polymerizationbased electrochemical DNA biosensor was characterized with EIS by virtue of its ability to sensitively perceive the subtle changes occurring at the electrode interface.43 Figure 1D shows the typical Nyquist plots, collected step-by-step during the sequential preparation process, of the impedance spectra of the same electrode. In the Nyquist plot, the diameter of the semicircle at the high-frequency region equals the chargetransfer resistance (Rct), representing the charge-transfer kinetics between the redox probe (i.e., [Fe(CN)6]3−/4−) and the electrode interface.44 As expected, because of the fast charge-transfer kinetics,45 the bare electrode shows a very small Rct (∼0.14 kΩ; Figure 1D, curve a). After the modification of PNA probes, an increase in Rct (∼0.24 kΩ; Figure 1D, curve b) can be observed, which can be ascribed to the steric hindrance from the PNA monolayer.18,46 Likewise, the blocking of the residual binding sites with MCH can lead to a further increase in Rct (∼1.16 kΩ; Figure 1D, curve c); this is because the formation of the MCH monolayer further hinders the charge transfer.18,46 Because the negatively charged phosphate groups are electrostatically repellent to the redox probes,47 the formation of PNA−DNA duplexes can result in an increase in Rct (∼1.55 kΩ; Figure 1D, curve d). The complexation of Zr4+ ions to the phosphate groups of the captured target-DNA fragments causes a further increase in Rct (∼2.06 kΩ; Figure 1D, curve e). This is because the negatively charged redox probes can be electrostatically adsorbed to the positively charged Zr4+ ions, but the preadsorbed redox probes are, however, repellent to the charge transfer.18−20 The tethering of the CTAs (i.e., CPAD) to the PNA−DNA duplexes also renders an increase in Rct (∼2.62 kΩ; Figure 1D, curve f), because the redox probes are ejected from the electrode surface as a result of the decrease of surface hydrophilicity.18 After SI-RAFT polymerization (∼0.22 kΩ; Figure 1D, curve g), a significant decrease in Rct indicates the presence of plenty of highly electroactive polymers, which is in good agreement with the results of the SWV measurement (Figure 1A, curve g) and SEM characterization (Figure 1B). These results evidence the successful preparation of the SI-RAFT-polymerizationbased electrochemical DNA biosensor. In our previous reports,18−20 we found that the formation of electroactive polymers as a result of SI-eATRP can cause a significant increase in Rct. However, SI-RAFT polymerization is featured by a significant decrease in Rct, and this phenomenon has also been firmly evidenced by our repeated experiments. Theoretically, the presence of electroactive polymers has a competitive effect on the interfacial-charge-transfer kinetics. On one hand, the Fc tags are electroactive and thus can act as charge-transfer mediators, thereby greatly facilitating the charge transfer between the redox probes and the electrode surface (positive effect); on the other hand, the presence of plenty of polymers can substantially lower the charge-transfer kinetics (negative

Figure 2. (A) Square-wave voltammograms toward different concentrations of target DNA over the range of 10 aM to 10 pM. (B) The calibration plot of the oxidation-peak current versus the logarithm of target-DNA concentration. Error bars show the SDs of five independent measurements.

well-defined oxidation peak at the potential of ∼0.3 V, with the peak current increasing positively with increasing target-DNA concentration. Therefore, it is a “signal-on” biosensor, which is highly tolerant to false-positive results.18,42 Figure 2B shows that there is good linearity between the peak current and the logarithm of target-DNA concentration over 7 orders of magnitude from 10 aM to 10 pM. The linear-regression equation is I (μA) = 3.63 + 1.24 log CtDNA (fM) (R2 = 0.997), with the detection limit being calculated to be 3.2 aM (3σb/ slope, where σb is the standard deviation (SD) of the blank E

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Table 1. Comparison of the Analytical Performance with Those of Other Amplification-by-Polymerization-Based Methods method

signal amplification

linear range

detection limit

ref

visual visual visual visual electrochemical electrochemical electrochemical

SI-RAFT polymerization SI-ATRP SI-ATRP AGET ATRPa SI-eATRP AGET ATRP SI-RAFT polymerization

1.0 fM to 1.0 μM not provided 1.0 fM to 1.0 μM 1.0 nM to 1.0 μM 0.1 fM to 0.1 nM 0.1 nM to 1.0 μM 10 aM to 10 pM

>1.0 fM 1.0 nM 1.0 fM 1.0 nM 72 aM 15 pM 3.2 aM

23 14 48 49 18 15 this work

a

AGET ATRP: activator-generated electron transfer for ATRP.

results indicate that the electrochemical detection of DNA with this biosensor is highly reproducible. The storage stability of the PNA−MCH−tDNA−Zr4+− CPAD−FcMMA-modified electrode was examined by a longterm storage test, which was carried out using two sets of identically prepared electrodes (five electrodes for each set). One set of electrodes was measured by SWV immediately after preparation and the other was stored at 4 °C in a moisturesaturated environment. We observed that up to 96.3% of the oxidation-peak current can be retained after a fortnight of storage. Therefore, the modified electrode possesses satisfactory storage stability. The anti-interference capability of the SI-RAFT-polymerization-based electrochemical DNA biosensor was studied using NHS as the biological matrix. The serum samples were prepared by spiking a certain amount of target-DNA fragment into 10% NHS, and the oxidation-peak currents corresponding to 10% NHS samples were plotted against those from 0.1 M PBS (pH 7.4). As shown in Figure 3B, the peak currents from 10% NHS samples are, respectively, 96.7, 89.4, and 82.9% of those from 0.1 M PBS (pH 7.4) for 10 pM, 10 fM, and 10 aM target DNA. Therefore, the as-prepared electrochemical DNA biosensor holds great potential in clinical applications in view of its good anti-interference capability in the presence of complex serum matrices.

control). To our knowledge, this detection limit is much lower than those of other amplification-by-polymerization-based methods, as can be seen from Table 1. Compared with the visualization-based approach,23 for example, a more than 300fold improvement in detection sensitivity has been achieved (3.2 aM vs 1.0 fM). Such a high sensitivity can be ascribed to SI-RAFT polymerization, through which a large number of Fc tags can label each of the captured target-DNA fragments. Compared with the SI-eATRP-based electrochemical DNA biosensor,18 the detection sensitivity has been improved by a factor of 22.5, which can be ascribed to the higher loading of the electroactive polymers and the higher signal-to-background ratio of the SI-RAFT-polymerization-based biosensor. Selectivity. The selectivity of the SI-RAFT-polymerizationbased electrochemical DNA biosensor was evaluated on the basis of the oxidation-peak currents corresponding to four different oligonucleotide fragments, including target DNA (tDNA), SBM, TBM, and cDNA. The concentration of each oligonucleotide fragment was 10 pM. As can be seen from Figure 3A, the peak current corresponding to the SBM is as

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CONCLUSIONS In summary, a novel electrochemical biosensor has been designed for the highly sensitive detection of DNA by exploiting SI-RAFT polymerization as an operationally simple, low-cost, and highly efficient amplification strategy. This is the first time that SI-RAFT polymerization has been used as an amplification strategy for electrochemical biosensing. Unlike conventional amplification strategies, the SI-RAFT-polymerization-based approach does not involve the use of either natural enzymes or complex nanomaterials, thus offering the benefits of low cost and easy operation. Moreover, it eliminates the use of toxic transition-metal catalysts and thus is compatible with biomedical applications, which makes it superior to the SI-eATRP-based strategy with respect to signal amplification. The as-prepared electrochemical biosensor allows the highly sensitive detection of DNA with a linear range over 7 orders of magnitude from 10 aM to 10 pM. Compared with the visualization-based approach, more than 300-fold improvement in detection sensitivity has been achieved. Results also indicate that the SI-RAFT-based electrochemical biosensor is highly selective toward DNA detection and can be used for the genotyping of SNPs. Moreover, it is highly applicable to the biosensing of DNA in complex biological matrices. By virtue of its easy operation, low cost, and high efficiency, the SI-RAFT-polymerization-based

Figure 3. (A) Oxidation-peak currents toward different oligonucleotide fragments. The concentration of each oligonucleotide fragment was 10 pM. (B) Oxidation-peak currents in the absence and presence of 10% NHS. Error bars show the SDs of five independent measurements.

low as 26.3% of that from the tDNA, indicating that this electrochemical DNA biosensor can effectively differentiate even a single-base mismatch. In addition, the peak current corresponding to the TBM is about 14.2% of that from the tDNA, whereas for the cDNA the peak current is as small as the background current. The high selectivity of this electrochemical DNA biosensor gives it great potential in genotyping SNPs, and this can be ascribed to the superb specificity of the PNA probe.39,40 Reproducibility, Storage Stability, and Anti-interference Capability. The reproducibility of the as-prepared electrochemical DNA biosensor was studied on the basis of intra-assay and interassay experiments. In the presence of 10 pM target DNA, the intra- and interassay coefficients of variation are 4.1 and 4.7%, respectively (in both cases, the values were derived from five repetitive measurements). The F

DOI: 10.1021/acs.analchem.8b03416 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

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amplification strategy is believed to hold great potential for the sensitive detection of biomolecules.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b03416. SEM image and elemental mapping of the PNA− MCH−tDNA−Zr4+−CPAD-modified electrode, elemental mapping and effect of polymerization time on the Rct of the PNA−MCH−tDNA−Zr4+−CPAD− FcMMA-modified electrode, and optimization of VA044 concentration (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-020-39366902 (S.G.). *E-mail: [email protected] (L.N.). ORCID

Qiong Hu: 0000-0001-6360-1813 Li Niu: 0000-0003-3652-2903 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21527806, 21727815, and 21505127). REFERENCES

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DOI: 10.1021/acs.analchem.8b03416 Anal. Chem. XXXX, XXX, XXX−XXX