Electrochemically Mediated Surface-Initiated de Novo Growth of

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Electrochemically Mediated Surface-Initiated de novo Growth of Polymers for Amplified Electrochemical Detection of DNA Qiong Hu, Qiangwei Wang, Gengzhi Sun, Jinming Kong, and Xueji Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02039 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 16, 2017

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Analytical Chemistry

Electrochemically Mediated Surface-Initiated de novo Growth of Polymers for Amplified Electrochemical Detection of DNA Qiong Hu,ǁ,† Qiangwei Wang,ǁ,† Gengzhi Sun,ǂ Jinming Kong,*,† and Xueji Zhang*,‡ †

School of Environmental and Biological Engineering, Nanjing University of Science and

Technology, Nanjing 210094, P. R. China. E-mail: [email protected]. Tel.: +86-25-84303109. Fax: +86-25-84303109. ǂ

Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 211816, P. R. China. ‡

Chemistry Department, College of Arts and Sciences, University of South Florida, East Fowler Ave, Tampa, Florida 33620-4202, United States. E-mail: [email protected].

ǁ

Q.H. and Q.W. contributed equally to this work.

Corresponding authors: Jinming Kong, Xueji Zhang

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ABSTRACT: The development of convenient and efficient strategies without involving any complex nanomaterials or enzymes for signal amplification is of great importance in bioanalytical applications. In this work, we report the use of electrochemically mediated surface-initiated atom transfer radical polymerization (SI-eATRP) as a novel amplification strategy based on the de novo growth of polymers (dnGOPs) for the electrochemical detection of DNA. Specifically, the capture of target DNA (tDNA) by the immobilized peptide nucleic acid (PNA) probes provides a high density of phosphate groups for the subsequent attachment of ATRP initiators onto the electrode surface by means of the phosphate-Zr4+-carboxylate chemistry, followed by the de novo growth of electroactive polymer via the SI-eATRP. De novo growth of long polymeric chains enables the labeling of numerous electroactive probes, which in turn greatly improves the electrochemical response. Moreover, it circumvents the slow kinetics and poor coupling efficiency encountered when nanomaterials or preformed polymers are used, and features sufficient flexibility and simplicity in controlling the degree of signal amplification. Under optimal conditions, it allows a highly sensitive and selective detection of tDNA within a broad linear range from 0.1 fM to 0.1 nM (R2 = 0.996), with the detection limit down to 0.072 fM. Compared with the unamplified method, more than 1.2 × 106-fold sensitivity improvement in DNA detection can be achieved. By virtue of its simplicity, high efficiency and cost-effectiveness, the proposed dnGOPs-based signal amplification strategy holds great potential in bioanalytical applications for the sensitive detection of biological molecules.


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With benefits from the molecular level understanding of disease states, their diagnosis and prognosis on the basis of the detection of biological molecules, e.g., nucleic acids and proteins, have been intensively investigated.1 As we known, during the early stage of disease development, however, the biomarkers of interest are generally at an extremely low level. For this reason, clinical and field-portable diagnostic devices require the detection of biological molecules with high sensitivity and selectivity, without a compromise of the simplicity, rapidity, or cost-effectiveness. During the past decade, many efforts have been made worldwide to improve the sensitivity of bioanalytical methods.2–5 Intuitively, this can be realized by a supplemental amplification of the molecular recognition events, e.g., nucleic acid hybridizations and antigen-antibody interactions. The versatility of this approach is exemplified by the broad use of enzymes,2,6,7 nanomaterials,3,8 and polymeric materials,1,9,10 etc., in bioanalytical applications. Among them, the signal amplification based on the use of polymeric materials, which can either introduce directly numerous signaling probes or provide a large number of functional sites for the further modification, is particularly effective. With this in mind, various polymeric materials-based amplification strategies have been reported. The first set of these strategies is based on the direct use of preformed polymeric materials. For example, Leclerc and co-workers have reported the use of ferrocene-conjugated cationic polythiophene as the electroactive probe for the amplified electrochemical detection of DNA, where the positively-charged polythiophene backbone was designed to electrostatically interact with the captured, negatively-charged DNA.11 Similar examples include the use of polysaccharides-mediated in situ deposition of metal nanoparticles for the ultrasensitive detection of DNA.10,12 The other set involves the de novo growth of polymers (dnGOPs). Recently, He and co-workers have reported the use of de novo polymer growth as a signal amplification strategy in visual detection of DNA, in which DNA hybridization and ligation reactions enabled the attachment of initiators onto solid substrates, leading to a change of substrate opacity as a result of the subsequent dynamic growth of polymer brushes.13,14 However, the involvement of initiator-modified single-stranded DNA (ssDNA) as the secondary detection probe and the subsequent enzymatic ligation renders a trade off between cost-effectiveness and sensitivity. Atom transfer radical polymerization (ATRP), known as a reversible deactivation controlled/“living” radical polymerization (CRP) technique and proceeds through a concerted atom transfer mechanism via an inner-sphere electron-transfer (ISET) process,15–17 was firstly reported by Matyjaszewski and co-workers in 1995.18 Apart from its broad use in polymer synthesis, it has also ACS Paragon Plus Environment


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been intensively studied for surface functionalization because of the broad availability of monomers (e.g., acrylonitrile, (meth)acrylates, (meth)acrylamides, and styrenes), good control over the thickness, and the high orthogonality.15,19–22 In particular, the advent of aqueous ATRP at room temperature further speeds up its application in biologically relevant fields.9,23–26 Among the family of ATRP reactions, the electrochemically mediated ATRP (i.e., eATRP) stands out by virtue of its ability to precisely control over the initiation, cessation, and rejuvenation of a polymerization process, well-controlled reaction rate, and high tolerance to O2.16,21,22 Nevertheless, it has never been reported as a strategy for the dnGOPs for the amplified detection of biological molecules, to the best of our knowledge. In this work, we demonstrate the potential of exploiting electrochemically mediated surface-initiated ATRP (SI-eATRP) as a convenient and efficient signal amplification strategy based on the dnGOPs for the amplified detection of biological molecules, using the electrochemical detection of DNA as a model in our proof-of-concept experiment. To this end, thiolated peptide nucleic acid (PNA) probes are firstly immobilized onto gold electrode via the self-assembly of a well-defined monolayer for the capture of target DNA (tDNA). After hybridization, ATRP initiators, α-bromophenylacetic acid (BPAA), are attached to the hybridized PNA/DNA heteroduplexes by means of the phosphate-Zr4+-carboxylate chemistry, followed by the de novo growth of electroactive polymeric chains, with ferrocenylmethyl methacrylate (FMMA) as the monomer, via the SI-eATRP, where the activator, CuI/Me6TREN (Me6TREN = tris(2-dimethylaminoethyl)amine), is in situ electrochemically generated at a constant applied potential. De novo growth of long polymeric chains leads to the labeling of numerous FMMA, which in turn greatly improves the resulting electrochemical response. Moreover, it can circumvent the slow kinetics and poor coupling efficiency encountered when nanomaterials or preformed polymers, due to their bulky size, are used, and is featured by sufficient flexibility and simplicity in controlling the degree of signal amplification as well. Compared with the unamplified method, a tremendous improvement in detection sensitivity can be achieved. By virtue of its simplicity, high efficiency and cost-effectiveness, the dnGOPs-based signal amplification strategy is believed to hold great potential in bioanalytical applications for the sensitive detection of biological molecules. Moreover, the high compatibility of the dnGOPs by electrical addressing with microfabrication technology makes it an ideal solution for the high-throughput detection on microelectrode arrays.


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EXPERIMENTAL SECTION Materials and Reagents. Thiolated peptide nucleic acid (PNA) was custom-made by Panagene Inc. (South Korea). Oligonucleotides, including target DNA (tDNA), single-base mismatched DNA (SBM), three bases mismatched DNA (TBM) and control DNA (Control), were all synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The sequences of PNA and oligonucleotides are listed below (with mismatch(es) underlined): PNA: 5’-HS-(CH2)11-AAC CAT ACA ACC TAC TAC CTC A-3’; tDNA: 5’-TGA GGT AGT AGG TTG TAT GGT T-3’; SBM: 5’-TGA GGT AGT AGG TTG TGT GGT T-3’; TBM: 5’-TGA GGT ATT AGA TTG TGT GGT T-3’; Control: 5’-ACT TAC CTT TGC TCA TTG ACG A-3’. Ferrocenylmethyl methacrylate (FMMA), tris(2-dimethylaminoethyl)amine

(Me6TREN),

zirconium dichloride oxide octahydrate (ZrOCl2), α-bromophenylacetic acid (BPAA), and 6-mercapto-1-hexanol (MCH) were purchased from Sigma-Aldrich (St. Louis, MO). Copper(II) bromide (CuBr2), potassium bromide (KBr), potassium hexafluorophosphate (KPF6), and lithium perchlorate trihydrate (LiClO4) were all obtained from J&K Scientific Ltd. (Shanghai, China). Normal human serum (NHS) was purchased from Shanghai YiJi Industrial Co., Ltd. (Shanghai, China). N,N-Dimethylformamide (DMF) and other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were of at least analytical pure and used as supplied. Ultrapure water (≥ 18.25 MΩ) purified by Millipore Milli-Q water purification system was used in all experiments. 0.1 M Na2HPO4/NaH2PO4 (PBS, pH 7.4) was prepared and used as the stocking solution and washing buffer for oligonucleotides. Oligonucleotide samples used in this work were prepared by serial dilution with 0.1 M PBS. Electrochemical characterization of the newly formed electroactive polymers with cyclic voltammetry (CV) at different scan rates, together with square wave voltammetry (SWV) measurements, was carried out in 1.0 M LiClO4 for its ability to stabilize the oxidized form of ferrocene (i.e., ferrocenium cation).27,28 10 mM CuIIBr/Me6TREN (1:1.1 in molarity) was prepared by dissolving CuBr2 and Me6TREN into DMF. The eATRP cocktail (pH 7.0) was freshly prepared by successively adding 1.8 mL of DMF, 0.1 mL of 10 mM CuIIBr/Me6TREN, 0.1 mL of 10 mM FMMA (dissolved in DMF), 1.0 mL of 1.0 M KBr, and 7.0 mL of 0.1 M KPF6 into the electrochemical cell; the final concentrations of CuIIBr/Me6TREN and FMMA were both at ACS Paragon Plus Environment


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0.1 mM. Apparatus. All electrochemical experiments, including CV, SWV and linear sweep voltammetry (LSV), were performed at room temperature on a RST5200F electrochemical workstation (Suzhou Risetest Electronic Co. Ltd., China) with a conventional three-electrode cell set-up, comprising a modified gold electrode (Φ = 2.0 mm) or a bare glassy carbon electrode (GCE, Φ = 3.0 mm) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the counter electrode, respectively. Electrochemical impedance spectroscopy (EIS) was performed on Autolab/PGSTAT302N (Eco Chemie, Netherlands), within the frequency of 0.1 Hz to 100 KHz in 5.0 mM [Fe(CN)6]3-/4-, containing 0.1 M KNO3, at open circuit potential of 0.18 V, and the amplitude of the applied sine wave potential was set to 5.0 mV. Unless otherwise stated, all potentials mentioned in this work are referred to SCE. The scanning electron microscopy (SEM) image was collected on a Quanta FEG 250 field emission scanning electron microscopy (FEI, USA). Electrochemical Detection of DNA. Prior to use, the gold electrode was sequentially polished with 0.3 and 0.05 µm alumina slurries until a mirror-like surface was obtained, followed by ultrasonic cleaning with absolute ethanol and ultrapure water.28 Next, it was immersed in freshly prepared “Piranha” solution (30% H2O2 and 98% H2SO4, 1:3 v/v, Caution! it is strongly corrosive and must be handled with extreme care) for 15 min. Then, it was subjected to CV treatment in 0.5 M H2SO4 within the potential of -0.3 V and 1.5 V, with a scan rate of 0.1 V s-1, until a reproducible cyclic voltammogram was achieved.28 Finally, the electrode was ultrasonically cleaned with ultrapure water, and dried with N2 prior to use. For the immobilization of capture probes, 5.0 µL of 0.5 µM PNA solution was added onto the gold electrode, which was incubated subsequently at 37 °C for 1.5 h. After a rinse with ultrapure water, the PNA coated electrode was further immersed in 2.0 mM MCH (dissolved in 60% ethanol) for 30 min to block the nonspecific binding of DNA,28 and washed with 60% ethanol and ultrapure water. DNA hybridization was performed by pipetting 10 µL of oligonucleotides onto the obtained electrode, followed by an incubation at 37 °C for 1.5 h. Afterwards, the resulting electrode was moderately rinsed with 0.1 M PBS (pH 7.4) to remove the unhybridized oligonucleotides. After an incubation of 15 min at 37 oC in 5.0 mM ZrOCl2 (dissolved in 10% ethanol), the electrode was moderately rinsed with 10% ethanol and then immersed in 1.0 mM BPAA (freshly prepared with 15% ethanol) at 37 oC for 30 min for the attachment of ATRP initiators, BPAA, to the hybridized ACS Paragon Plus Environment

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PNA/DNA heteroduplexes via the phosphate-Zr4+-carboxylate chemistry. After that, the electrode was moderately rinsed with 15% ethanol and ultrapure water to remove the unbound initiators. The labeling of FMMA via the eATRP is conducted by immersing the as-prepared electrode in the eATRP cocktail, followed by the electrochemical reduction of CuIIBr/Me6TREN into CuI/Me6TREN under potentiostatic conditions (vide infra). Subsequently, the electrode was subjected to LSV treatment (initial potential: 0 V; final potential: 0.2 V; scan rate: 1.0 V s-1) and rinsed with DMF and ultrapure water to remove any physically adsorbed Cu0 and FMMA. Finally, the obtained electrode was immersed in 1.0 M LiClO4, and then SWV was performed by scanning the potential from 0 V to 0.6 V (increase potential: 4.0 mV s-1; potential amplitude: 25 mV, quiet time: 30 s) to record the oxidation current of the coupled FMMA.

RESULTS AND DISCUSSION Principle of the dnGOPs-based Electrochemical Detection of DNA. The principle of exploiting SI-eATRP as a signal amplification strategy based on the dnGOPs for the electrochemical detection of DNA is illustrated in Scheme 1. The PNA, of which the backbone is composed of neutrally-charged N-(2-aminoethyl)glycine unit instead of the negatively-charged deoxyribose phosphate as observed in DNA,29 is used as the capture probe due to its excellent specificity in discriminating even a single base mismatch.30 The hybridization of PNA with tDNA results in the enrichment of a high density of phosphate groups on the electrode surface, onto which BPAA can thus be attached via the phosphate-Zr4+-carboxylate chemistry. Before we discuss the mechanism of SI-eATRP, it is worth to point out that, in copper-amine complexes, the preferred coordination numbers of the open-shell 3d9 CuII center and the closed-shell 3d10 CuI center are five and four, respectively.31,32 Considering that the Me6TREN is a tetradentate ligand, one Br- ion or solvent molecule (e.g., H2O) is required to fill up the coordination sphere of CuII, while the ligand alone can complete the coordination sphere of CuI.33 Consequently, in the presence of Me6TREN and Br-, CuII and CuI are in the form of CuIIBr/Me6TREN and CuI/Me6TREN, respectively.33 According to the previous reports,15,32 the 3d9 CuII center is in a five-coordinate trigonal bipyramidal (Tbp) geometry with a Br- ion at the fifth site, whereas the 3d10 CuI adopts a four-coordinate tetrahedral geometry. During the eATRP, a constant potential is applied to generate the CuI/Me6TREN activators electrochemically through one-electron reduction of the air-stable CuIIBr/Me6TREN deactivators in the close proximity of electrode surface to trigger the ACS Paragon Plus Environment


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surface-initiated polymerization. As depicted in Scheme 1, the mechanism of the SI-eATRP can be described as follows: 1) the CuIIBr/Me6TREN deactivators are electrochemically reduced into the CuI/Me6TREN activators, with which the reaction of the surface-attached BPAA initiators produces the initiating radicals and the higher oxidation state deactivators (a portion of the deactivators returns to the electrode surface to be reduced again); 2) these radical species propagate to form polymeric chains by reacting with the methacrylic monomers, i.e., FMMA, or are reverted back to the dormant species (~Pn−X) by reacting with the deactivators; and 3) by repeating such sequential reactions, long electroactive polymers can eventually be obtained.16,33 Electrogeneration of the CuI/Me6TREN activator is carried out at a constant potential (Eapp), the ∆E = Eapp – E°’ (E°’ represents the apparent formal potential of the CuII/CuI couple in the presence of the ligand and is estimated from E1/2 = (Eox + Ered)/232,33) can be chosen to control the CuIIBr/Me6TREN to CuI/Me6TREN ratio at the electrode surface, according to the Nernst equation:

= °’ + ln

[ /] [ /]

(1)

where L = Me6TREN, and R and F are the universal gas constant and the Faraday constant, respectively. In the absence of mass transport restrictions, the concentration of CuI/Me6TREN can be dictated by the Eapp, allowing a good control over the polymerization rate. In the case of the eATRP with methyl acrylate (MA) as the monomer, for example, an accelerated polymerization was observed by Magenau et al. at more negative potentials, whereas more positive potentials led to a slower polymerization.16 Also, they have succeeded to precisely control over the initiation, cessation, and rejuvenation of the polymerization, simply by varying the Eapp. Moreover, as a result of the electrostatic interaction, the in situ generated CuI/Me6TREN activators will locate and enrich at the electrode surface, thereby significantly accelerating the growth rate of polymeric chains. With this in mind, Zhou and co-workers have successfully grafted gradient brushes from substrate surface by changing the tilt angle between the working electrode and the substrate, which was based on the gradient distribution of the CuI/Me6TREN activator.22 De novo growth of polymeric chains not only circumvents the slow kinetics and poor coupling efficiency encountered when bulky nanomaterials or preformed polymers are used, but also provides sufficient flexibility and simplicity in controlling the degree of signal amplification. Due to the labeling of numerous electroactive probes, a tremendous improvement in detection sensitivity can therefore be achieved. Electrochemical Characterizations. To compare the stabilizing effects of the Me6TREN ligand toward CuII and CuI cations, the redox properties of the CuII/CuI couple in the absence and ACS Paragon Plus Environment

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presence of the ligand were investigated with CV, using a bare GCE as the working electrode and 0.5 M KNO3 (N2 saturated) as the supporting electrolyte. As shown in Figure 1A, the E°’ of the CuII/CuI couple is about -0.03 V vs. SCE (Figure 1A,b); for the [CuII/Me6TREN]/[CuI/Me6TREN] couple, it shifts to -0.54 V vs. SCE (Figure 1A,c). Therefore, the complexation of Cu2+ and Cu+ by the ligand leads to a negative shift of the E°’ of the CuII/CuI couple by about 0.51 V, revealing that the Me6TREN ligand preferentially stabilizes the higher oxidation state of the metal.32,33 The relative stability of the CuII/Me6TREN and CuI/Me6TREN complexes (i.e., the ratio of their stability constants, βII and βI) can be calculated from the reduction peak potential, according to Equation 2: ⊖ ⊖ [ /]/[ /] = [ / ] +

ln

(2)

⊖ ⊖ II I where [ /]/[ /] and [ / ] represent the reduction peak potentials of the Cu /Cu

couple in the presence and absence of the ligand, respectively. As calculated on the basis of the shift of the reduction peak potential (-0.63 V vs. SCE for the [CuII/Me6TREN]/[CuI/Me6TREN] couple, and -0.08 V vs. SCE for the CuII/CuI couple), the stability constant of the CuII/Me6TREN complex is about 109.3-fold of that of the CuI/Me6TREN complex.32 For CuII/Me6TREN, the ligand and the 3d9 CuII center is in a t25 configuration, which favors the coordination of the CuII center with all the four spatially-confined N atoms simultaneously. However, the formation of the CuI/Me6TREN complex is less favored because (1) the 3d10 CuI center has poorer electron-withdrawing character due to its lower charge and (2) the t26 configuration of the CuI center disfavors the formation of coordination bonds with all the four N atoms of the ligand. As a result, the CuII/Me6TREN complex is more stable than the CuI/Me6TREN complex. Prior to the SI-eATRP, cyclic voltammogram of the PNA/MCH/tDNA/Zr4+/BPAA-modified electrode in the FMMA-free eATRP cocktail was acquired to ascertain the potential range appropriate for accurate control of the electrochemical generation of the CuI/Me6TREN activator. As shown in Figure S1, the cathodic scan results in a reduction peak at the potential of -0.55 V vs. SCE, which can be assigned to the conversion of the CuIIBr/Me6TREN deactivator into the CuI/Me6TREN activator.33 According to Equation 1, a higher concentration of the CuI/Me6TREN activator can be obtained at a more negative potential; however, this generally results in a deterioration of the polymerization control presumably due to the loss of C-Br functional groups.33 On the other hand, more positive potentials provide a better control of polymer growth at the expense of growth rate. A leverage of the polymerization control and the growth rate, in the



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subsequent experiments the SI-eATRP is carried out at -0.55 V, at which the CuI/Me6TREN to CuIIBr/Me6TREN ratio at the electrode surface is about 25.3 according to Equation 1. We further verified the feasibility of exploiting SI-eATRP as a signal amplification strategy based on the dnGOPs for the electrochemical detection of DNA. As can be seen from Figure 1B, an intense oxidation current at the potential of ~ 0.304 V (vs. SCE) can be clearly observed for the PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified electrode (Figure 1B,a). The peak potential, corresponding to the electrochemical oxidation of ferrocene into ferrocenium cation, falls into the typical redox potential range of ferrocene.27,28 On the other hand, only a very weak oxidation current can be detected if PNA (Figure 1B,b) or tDNA (Figure 1B,c) was not added during the modification of the electrode; likewise, in the absence of BPAA (Figure 1B,d), CuIIBr/Me6TREN (Figure 1B,e), or FMMA (Figure 1B,f), almost no oxidation current, except a faint background charging current,28 can be observed. Such a high signal-to-noise (S/N) ratio is essential for the sensitive detection of tDNA. Moreover, it is clear that the intense oxidation current can only be observed for the PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified electrode, excluding the possibility that the electrochemical signal is due to the nonspecific adsorption of FMMA. These results demonstrate that the dnGOPs-based signal amplification strategy can be applied to the electrochemical

detection

of

DNA.

The

surface

morphology

of

the

PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified electrode was characterized with SEM. As shown in Figure S2, a high density of hill-like eminences can be observed after the SI-eATRP, and they are perpendicularly distributed as is observed for the PNA/DNA heteroduplexes,10 confirming the formation of polymers. Further, the newly formed electroactive polymers were characterized with CV at different scan rates. As shown in Figure 1C, the anodic and cathodic peak currents vary linearly along with the scan rate from 0.01 to 1.0 V s-1, indicating that the redox process is not diffusion-dependent, and thus confirms that the electroactive polymers are covalently attached to the electrode surface.27,28,34 Of note, the poor reversibility of the redox process is likely due to the complicated monolayer, impeding the interfacial electron transfer.35.36 In addition, the sequential modification of the electrode was also tracked with EIS, owing to its high sensitivity to the subtle changes occurring at solid-liquid interfaces.37 In EIS, the semicircle, observed at high frequency region, corresponds to the charge-transfer resistance (Rct).37 Figure 1D shows the Nyquist plots of the same electrode at different stages of the modification process. In the equivalent circuit model (see inset in Figure 1D), Rs and CPE represent the solution resistance and ACS Paragon Plus Environment

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the constant-phase element, respectively. As shown in Figure 1D, the bare electrode shows the smallest Rct (~ 128 Ω; curve a). After immobilization of PNA probes, an increase in the Rct of ~137% is observed as a result of the formation of well-defined self-assembled monolayer (SAM) (~ 303 Ω; curve b).38 Subsequently, the blocking of the nonspecific binding sites on the electrode surface with MCH leads to a further increase in the Rct (~ 490 Ω; curve c). This is because that the more densely-packed monolayer further blocks the transfer of charge. The capture of tDNA by PNA probes results in a large increase in the Rct (~ 984 Ω; curve d), indicative of the formation of a high density of PNA/DNA heteroduplexes.37 Interestingly, the complexation of Zr4+ ions with the phosphate groups of the captured tDNA is also accompanied by a large increase in the Rct (~ 1.30 kΩ; curve e). This observation can be tentatively interpreted as follows: 1) the electrostatic interaction between the positively-charged Zr4+ ions and the negatively-charged [Fe(CN)6]3-/4- leads to the adsorption of [Fe(CN)6]3-/4- to the PNA/DNA heteroduplexes; and 2) the pre-adsorbed [Fe(CN)6]3-/4- are electrostatically repellent to the transfer of charge. The attachment of initiators causes a further increase in the Rct (~ 3.03 kΩ; curve f), owing to the expulsion of [Fe(CN)6]3-/4from the electrode surface as a result of a drastic decrease of surface hydrophilicity. Finally, a significant increase in the Rct is observed after the SI-eATRP (~ 7.01 kΩ; curve g), indicative of the de novo growth of numerous highly hydrophobic polymeric chains. The progressive increase of Rct demonstrates that the modification process proceeds smoothly. Effects of pH and eATRP Time. pH is an important factor that may drastically affect the stability of the CuIIBr/Me6TREN and CuI/Me6TREN complexes and thus the growth of polymeric chains. Both the CuIIBr/Me6TREN and CuI/Me6TREN complexes and the Me6TREN ligand can be involved in proton transfer reactions.33 An acidic environment is believed to favor the protonation of the ligand, decreasing its ability to stabilize the metal cations, leading to the dissociation of the CuIIBr/Me6TREN and CuI/Me6TREN complexes and the disproportionation of CuI.33 Considering that the OH− is a much stronger ligand than Br- ion, a higher pH, on the other hand, can result in the formation of the CuIIOH/Me6TREN complex.33 In such case, the presence of OH- tends to suppress the polymerization efficiency, since either the activation or deactivation reaction will be hampered. Therefore, the SI-eATRP was carried out at pH 7.0. The growth of polymeric chains correlates with the coupling of electroactive probes onto the electrode surface. As a CRP technique, eATRP offers the benefit of tuning the degree of polymerization. That is, longer polymerization time results in the enrichment of more electroactive ACS Paragon Plus Environment


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probes. During the eATRP, CV was conducted periodically to real-time monitor the dynamic coupling of electroactive probes. As seen from Figure S3, in the presence of tDNA (10 µL, 0.1 nM), the oxidation current increases drastically with the eATRP time during the first 20 min, after which no significant increase can be observed, presumably due to the radical-radical termination and/or the significantly increased steric hindrance associated with the dynamic growth of polymeric chains.19,39 The fast reaction kinetics indicate that the dnGOPs based on the SI-eATRP is highly efficient. In the following experiments, the eATRP time was set to 30 min, on the basis of a leverage of the eATRP time and the amount of covalently coupled electroactive probes. Analytical Performance. Under optimal conditions, the analytical performance of the dnGOPs-based electrochemical detection of DNA was studied with varying tDNA concentrations. As shown in Figure 2A, the SWV curves show a well-defined oxidation peak at the potential of 0.304 V vs. SCE, and the oxidation current increases linearly along with tDNA concentration over 7 orders of magnitude from 0.1 fM to 0.1 nM. A “signal-on” method like this is highly tolerant to false-positive results. As shown in Figure 2B, there is a good linear relationship between the oxidation current and the logarithm of tDNA concentration. The linear regression equation is I (µA) = 9.65 + 1.92 lg [CtDNA/pM] (R2 = 0.996). The limit of detection (LOD) is calculated to be 0.072 fM, on the basis of 3σ/slope, where σ is the standard deviation of the control. Given that the sample volume for the electrochemical detection is 10 µL, the minimum amount of tDNA that can be accurately detected is calculated to be 0.72 zmol. The LOD is lower than many other polymeric materials-based methods, as summarized in Table 1.9,11–14,40–42 When compared with our previous work in which the electroactive probe, aminoferrocene, was directly labelled to the free 5’-terminal phosphate group, only in a stoichiometric ratio of 1:1, of the captured tDNA,28 more than 1.2 × 106-fold sensitivity improvement in DNA detection has been achieved (0.072 fM vs. 93 pM). The improved sensitivity is partially due to the excellent sensitivity of SWV. Compared with those strategies based on the direct use of preformed polymeric materials, the approach of dnGOPs can eliminate the need for ex-situ synthesis and purification of the polymeric materials. More importantly, it can circumvent the slow kinetics and poor coupling efficiency encountered when nanomaterials or preformed polymers, due to their bulky size, are used. Taking advantage of the high efficiency of the dnGOPs based on the SI-eATRP, a high density of electroactive probes can be covalently coupled to the electrode surface, leading to a tremendous improvement in detection sensitivity. Considering that the electroactive polymers are grown from an initiator-attached ACS Paragon Plus Environment

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substrate surface, this “grafting-from” or “in-situ” synthesis by electrical addressing is also highly compatible with microfabrication technology. As a proof-of-concept experiment, a gold microelectrode array was designed for the electrochemical detection of tDNA. Similar to the macroelectrode, an intense oxidation current at the potential of ~ 0.304 V vs. SCE can be clearly observed for the PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified microelectrode array (Figure S4,a), while only a very weak oxidation current can be detected if tDNA (Figure S4,b) was not added during the modification of the microelectrode array. Consequently, the dnGOPs-based signal amplification strategy holds great potential for the high-throughput detection of biological molecules on microelectrode arrays by virtue of its simplicity and high efficiency. The reproducibility of the dnGOPs-based electrochemical detection of DNA was evaluated by the intra- and inter-assays. The coefficients of variation of the intra-assay and inter-assay, derived from seven repetitive measurements at a tDNA concentration of 0.1 nM, are 4.2% and 4.9%, respectively. The results indicate that the demonstrated method has high reproducibility. In addition, the storage stability of the PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified electrode was also tested by a long-term storage experiment. We found that, up to 94.7% of the electrochemical response can be retained even after three weeks, when it was stored in a moisture-saturated environment at 4 °C. Thus, the PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified electrode possesses satisfactory stability. Selectivity and Anti-interference Ability. To evaluate the selectivity, the SWV responses from three non-complementary oligonucleotides, including SBM, TBM, and Control, were compared with that from the tDNA. As shown in Figure 3A, the SWV response from SBM is only about 24.6% of that from tDNA, indicating that the dnGOPs-based electrochemical detection of DNA has high selectivity toward even a single base mismatch. The SWV response from TBM is about 86.5% lower off than that of the tDNA, while no significant difference relative to the background signal can be observed for the Control. The notable difference results from the hybridization efficiency of different oligonucleotides with the PNA probe, since the presence of mismatched base(s) makes it thermodynamically unfavourable for the formation of PNA/DNA heteroduplexes. These results demonstrate that the dnGOPs-based electrochemical detection of DNA is highly selective, showing great potential toward genotyping of single nucleotide polymorphisms (SNPs). The high selectivity can be ascribed to the excellent specificity of PNA probes.30 The potential interference from complex serum samples on the analytical performance of the ACS Paragon Plus Environment


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dnGOPs-based electrochemical detection of DNA has also been investigated. The tDNA samples were prepared by spiking tDNA into 5% NHS, and the SWV response from 5% NHS sample was compared with that from 0.1 M PBS (pH 7.4). As shown in Figure 3B, the SWV responses from 5% NHS samples are approximately 97.6%, 91.3% and 82.3% of that from the PBS buffer for 0.1 nM, 0.1 pM and 0.1 fM tDNA, respectively. As a result, the dnGOPs-based electrochemical detection of DNA holds great potential in clinical applications, owing to its good analytical performance in the presence of complex serum samples.

CONCLUSIONS In summary, we have demonstrated the potential of exploiting the SI-eATRP as a convenient and efficient signal amplification strategy based on the dnGOPs for the amplified electrochemical detection of DNA. A “signal-on” method like this is highly tolerant to false-positive results. De novo growth of long polymeric chains leads to the labeling of numerous electroactive probes and thus greatly improves the resulting electrochemical response. The dnGOPs based on the SI-eATRP is highly efficient, which can effectively circumvent the slow kinetics and poor coupling efficiency associated with the direct use of bulky nanomaterials or preformed polymers, and is featured by sufficient flexibility and simplicity in controlling the degree of signal amplification as well. Compared with the unamplified method, a tremendous improvement in detection sensitivity has been achieved without the necessity of any complex nanomaterials or enzymes. By virtue of its simplicity, high efficiency and cost-effectiveness, the dnGOPs-based signal amplification strategy is believed to hold great potential in bioanalytical applications for the sensitive detection of biological molecules. Moreover, the high compatibility of the dnGOPs by electrical addressing with microfabrication technology makes it an ideal solution for the high-throughput detection on microelectrode arrays.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Tel.: +86-25-84303109. Fax: +86-25-84303109. *E-mail: [email protected]. Author Contributions ǁ

Q.H. and Q.W. contributed equally to this work. ACS Paragon Plus Environment

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21575066).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.xxxxxxx. Figures including (1) potential range of the SI-eATRP, (2) SEM image of the electrode after the SI-eATRP, (3) optimization of eATRP time and (4) proof-of-concept experiment on microelectrode arrays. (PDF)

REFERENCES (1) Sikes, H. D.; Hansen, R. R.; Johnson, L. M.; Jenison, R.; Birks, J. W.; Rowlen, K. L.; Bowman, C. N. Nat. Mater. 2008, 7, 52–56. (2) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010–3011. (3) Hansen, J. A.; Mukhopadhyay, R.; Hansen, J. Ø.; Gothelf, K. V. J. Am. Chem. Soc. 2006, 128, 3860–3861. (4) Xia, F.; White, R. J.; Zuo, X.; Patterson, A.; Xiao, Y.; Kang, D.; Gong, X,; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2010, 132, 14346–14348.\ (5) Wang, W. J.; Li, J. J.; Rui, K.; Gai, P. P.; Zhang, J. R.; Zhu, J. J. Anal. Chem. 2015, 87, 3019–3026. (6) Ge, Z.; Lin, M.; Wang, P.; Pei, H.; Yan, J.; Shi, J.; Huang, Q.; He, D.; Fan, C.; Zuo, X. Anal. Chem. 2014, 86, 2124–2130. (7) Yang, F.; Yang, X.; Wang, Y.; Qin, Y.; Liu, X.; Yan, X.; Zou, K.; Ning, Y,; Zhang, G. J. Anal. Chem. 2014, 86, 11905–11912. (8) Cui, H. F.; Xu, T. B.; Sun, Y. L.; Zhou, A. W.; Cui, Y. H.; Liu, W.; Luong, J. H. Anal. Chem. 2015, 87, 1358–1365. (9) Wu, Y.; Liu, S.; He, L. Anal. Chem. 2009, 81, 7015–7021. (10) Hu, Q.; Hu, W.; Kong, J.; Zhang, X. Microchim. Acta 2015, 182, 427–434. ACS Paragon Plus Environment


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(11) Le Floch, F.; Ho, H. A.; Harding-Lepage, P.; Bédard, M.; Neagu-Plesu, R.; Leclerc, M. Adv. Mater. 2005, 17, 1251–1254. (12) Kong, J.; Ferhan, A. R.; Chen, X.; Zhang, L.; Balasubramanian, N. Anal. Chem. 2008, 80, 7213–7217. (13) Lou, X.; Lewis, M. S.; Gorman, C. B.; He, L. Anal. Chem. 2005, 77, 4698–4705. (14) He, P.; Zheng, W.; Tucker, E. Z.; Gorman, C. B.; He, L. Anal. Chem. 2008, 80, 3633–3639. (15) Matyjaszewski, K.; Xia, J. Chem. Rev. 2001, 101, 2921–2990. (16) Magenau, A. J.; Strandwitz, N. C.; Gennaro, A.; Matyjaszewski, K. Science 2011, 332, 81–84. (17) De Paoli, P.; Isse, A. A.; Bortolamei, N.; Gennaro, A. Chem. Commun. 2011, 47, 3580–3582. (18) Wang, J. S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614–5615. (19) Ma, H.; Hyun, J.; Stiller, P.; Chilkoti, A. Adv. Mater. 2004, 16, 338–341. (20) Baskaran, D.; Mays, J. W.; Bratcher, M. S. Angew. Chem. Int. Ed. 2004, 43, 2138–2142. (21) Li, B.; Yu, B.; Huck, W. T.; Zhou, F.; Liu, W. Angew. Chem. 2012, 124, 5182–5185. (22) Li, B.; Yu, B.; Huck, W. T.; Liu, W.; Zhou, F. J. Am. Chem. Soc. 2013, 135, 1708–1710. (23) Gao, W.; Liu, W.; Mackay, J. A.; Zalutsky, M. R.; Toone, E. J.; Chilkoti, A. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 15231–15236. (24) Zhang, Z. B.; Yuan, S. J.; Zhu, X. L.; Neoh, K. G.; Kang, E. T. Biosens. Bioelectron. 2010, 25, 1102–1108. (25) Bortolamei, N.; Isse, A. A.; Magenau, A. J.; Gennaro, A.; Matyjaszewski, K. Angew. Chem. 2011, 123, 11593–11596. (26) Yuan, L.; Wei, W.; Liu, S. Biosens. Bioelectron. 2012, 38, 79–85. (27) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 9134–9137. (28) Hu, Q.; Deng, X.; Yu, X.; Kong, J.; Zhang, X. Biosens. Bioelectron. 2015, 65, 71–77. (29) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895–1897. (30) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. A. J. Am. Chem. Soc. 1996, 118, 7667–7670. (31) Pintauer, T.; Reinöhl, U.; Feth, M.; Bertagnolli, H.; Matyjaszewski, K. Eur. J. Inorg. Chem. 2003, 2003, 2082−2094. (32) Hu, Q.; Zhou, B.; Li, F.; Kong, J.; Zhang, X. Chem. Asian J. 2016, 11, 3040−3045. (33) Fantin, M.; Isse, A. A.; Gennaro, A.; Matyjaszewski, K. Macromolecules 2015, 48, ACS Paragon Plus Environment

Page 16 of 22

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6862−6875. (34) Grabowska, I.; Singleton, D. G.; Stachyra, A.; Góra-Sochacka, A.; Sirko, A.; Zagórski-Ostoja, W.; Radecka, H.; Stulz, E.; Radecki, J. Chem. Commun. 2014, 50, 4196–4199. (35) Devaraj, N. K.; Dinolfo, P. H.; Chidsey, C. E.; Collman, J. P. J. Am. Chem. Soc. 2006, 128, 1794−1795. (36) Cañete, S. J. P.; Lai, R. Y. Chem. Commun. 2010, 46, 3941–3943. (37) Dubuisson, E.; Yang, Z.; Loh, K. P. Anal. Chem. 2011, 83, 2452−2460. (38) Liu, X.; Qu, X.; Fan, H.; Ai, S.; Han, R. Electrochim. Acta 2010, 55, 6491−6495. (39) Wu, Y.; Xue, P.; Hui, K. M.; Kang, Y. Biosens. Bioelectron. 2014, 52, 180−187. (40) Okelo, G. O.; He, L. Biosens. Bioelectron. 2007, 23, 588–592. (41) Qian, H.; He, L. Sens. Actuators, B 2010, 150, 594–600. (42) Liu, S.; Su, W.; Li, Z.; Ding, X. Biosens. Bioelectron. 2015, 71, 57–61.



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FIGURE CAPTIONS: Scheme 1. Principle of the dnGOPs-based electrochemical detection of DNA. Figure 1. (A) Cyclic voltammograms (CVs) of the bare GCE (a), and in the presence of 1.0 mM CuBr2 (b) or 1.0 mM CuIIBr/Me6TREN (c). Scan rate: 0.1 V s-1. Supporting electrolyte: 0.5 M KNO3

(N2

saturated).

(B)

Square

wave

voltammograms

(SWVs)

of

the

PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified electrode (a), and of those prepared in the absence of PNA (b), tDNA (c), BPAA (d), CuIIBr/Me6TREN (e), or FMMA (f). (C) CVs of the newly formed electroactive polymers on the electrode surface at the scan rates of 10, 25, 50, 75, 100, 150, 250, 500, 750 and 1000 mV s-1. Inset: The linear relationship between anodic (black line) and cathodic (red line) peak currents and scan rate. (D) The Nyquist plots of EIS of the bare gold electrode

(a),

and

of

PNA/MCH/tDNA/Zr4+-,

the

(b) (f)

PNA-,

(c)

PNA/MCH-,

(d)

PNA/MCH/tDNA-,

PNA/MCH/tDNA/Zr4+/BPAA-,

or

(e) (g)

PNA/MCH/tDNA/Zr4+/BPAA/FMMA-modified gold electrode. The concentration of tDNA used for all the experiments was 0.1 nM. The eATRP time was 30 min. Figure 2. (A) SWVs toward different concentrations of tDNA over the range of 0.1 fM to 0.1 nM. (B) The calibration plot between the oxidation current and the logarithm of tDNA concentration. Error bars represent the standard deviations of seven independent measurements. Figure 3. (A) SWV responses toward different oligonucleotides (0.1 nM, 10 µL). (B) SWV responses in the absence or presence of 5% NHS. Error bars represent the standard deviations of seven independent measurements.


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Scheme 1

Figure 1

Figure 2



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Figure 3


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Table 1. Comparison of the analytical performance of our method with other polymeric materials-based methods. Method

Linear range

LOD

Ref.

Electrochemical

0.1 nM−1.0 µM

15 pM

9

Electrochemical

1.0 nM−1.0 µM

0.5 nM

11

Electrical

0.1 fM−0.1 pM

3.0 fM

12

Visual

Not provided

1.0 nM

13

Visual

1.0 fM−1.0 µM

> 1.0 fM

14

Visual

1.0 fM−1.0 µM

1.0 fM

40

Visual

1.0 nM−1.0 µM

1.0 nM

41

Electrochemical

0.1 nM−1.0 µM

10 pM

42

Electrochemical

0.1 fM−0.1 nM

0.072 fM

This work



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for TOC only


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Electrochemically Mediated Surface-Initiated de Novo Growth of

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