Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins

Author’s Accepted Manuscript Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins Zhong Feng Gao, Yan Li Huang, Wang Ren, Hong Qun Luo, Nian Bing Li www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(15)30619-9 http://dx.doi.org/10.1016/j.bios.2015.11.070 BIOS8200

To appear in: Biosensors and Bioelectronic Received date: 9 September 2015 Revised date: 20 November 2015 Accepted date: 23 November 2015 Cite this article as: Zhong Feng Gao, Yan Li Huang, Wang Ren, Hong Qun Luo and Nian Bing Li, Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.11.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Guanine nanowire based amplification strategy: enzyme-free biosensing of nucleic acids and proteins Zhong Feng Gao a, Yan Li Huang a, Wang Ren a,b, Hong Qun Luo a,*, Nian Bing Li a,* a

Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China

b

College of Chemistry and Pharmaceutical Engineering, Sichuan University of Science and Engineering, Zigong, 643000, PR China

*

Corresponding Author

Tel: +86 23 68253237; fax: +86 23 68253237; *Hong Qun Luo: E-mail: [email protected]; *Nian Bing Li: E-mail: [email protected].

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Abstract Sensitive and specific detection of nucleic acids and proteins plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids and proteins (thrombin) at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10 min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform.

Keywords: DNA nanotechnology; Electrochemistry; Guanine nanowire; Signal amplification; Biosensors

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1. Introduction Over the last decade, DNA nanotechnology has opened up new avenues towards highly selective and ultrasensitive detection of biomolecules and proteins (Gerasimova and Kolpashchikov, 2014; Lei and Ju, 2012; Yang et al., 2014; Zhang et al., 2013). Methods that use polymerase chain reaction (Alcantara et al., 2012; Botes et al., 2013; El-Sagheer and Brown, 2009; Lai et al., 2006), recombinase polymerase amplification (Santiago-Felipe et al., 2014), helicase-dependent amplification (Andresen et al., 2009; Jeong et al., 2009), loop-mediated isothermal amplification (Han, 2013; Hsieh et al., 2014; Liang et al., 2012; Notomi et al., 2000), rolling circle amplification (Bi et al., 2013; Deng et al., 2014; Hamblin et al., 2012; Russell et al., 2014), and hybridization chain reaction (Choi et al., 2014; Ge et al., 2014; Liu et al., 2013; Xuan and Hsing, 2014) have been widely used to improve the limits of detection. Although these methods have adequate sensitivity, they often require expensive thermal cycler, professional operation, and suffer from time-consuming derivatization (Wang et al., 2014; Xu et al., 2014). Developing new amplification strategies which afford faster reaction process with high selectivity and sensitivity is imperative for biosensing. Guanine nanowire (G-wire), first reported by Sen’s laboratory, has attracted considerable attention due to its peculiar characteristics (Fahlman and Sen, 1998; Hessari et al., 2014; Ilc et al., 2013; Sen and Gilbert, 1992). In the presence of specific metal cations, the G-quartets spontaneously assemble into long and stiff G-wire with a rapid process (Borovok et al., 2008; Davis, 2004; Keniry, 2001; Marsh and Henderson, 1994; Marsh et al., 1995; Miyoshi et al., 2007; Shi et al., 2013). The G-wire structure possesses various merits, 3

including mechanically stable, heat resistant, and in-sensitive to DNase I treatment (Kotlyar et al., 2005; Protozanova and Macgregor, 1996; Yatsunyk et al., 2013). Recently reports suggest a potential use of G-wire in molecular electronics and optical devices (Calzolari et al., 2002; Changenet-Barret et al., 2010; Hua et al., 2012; Liu et al., 2010). We attempted to expand this concept by constructing a simple, label-free, and enzyme-free electrochemical DNA sensor using guanine nanowire-based amplification strategy. In this paper, we propose to use guanine nanowire as a new signal amplification method for the electrochemical detection of nucleic acids and proteins under isothermal conditions. The guanine nanowire-based amplification method, termed GWA, is a nonenzymatic reaction strategy that leads to a better detection limit, enhanced electrochemical signal strength, and anti-interference capability.

2. Material and methods 2.1. Materials and instruments All oligonucleotides were synthesized and purified by Sangon Inc., Shanghai, China, and the sequences are listed in Table S1 in supporting information. The TMB substrate (TMB, 3,3’,5,5’-tetramethylbenzidine) was obtained from Sangon Inc., Shanghai, China, in the format of a ready-to-use reagent (H2O2 included). All other chemicals were analytical grade and used without further purification. A CHI660d electrochemical station (Chenhua Instruments Co., Shanghai, China) was used for electrochemical experiments with a conventional three-electrode cell, which contains a platinum wire as the counter electrode, an Ag/AgCl (3 M KCl) reference electrode, and a gold electrode as the working electrode (2.0 mm diameter, CHI Co., Ltd., Shanghai, China). Gold electrodes were cleaned following the reported protocol (Gao et al., 2014). Electrochemical impedance spectroscopy was recorded at a frequency ranging from 10 4

kHz to 100 mHz by 5 mV amplitude. Cyclic voltammetry was recorded at a scan rate of 0.1 V/s. Amperometric detection was carried out at 150 mV and the electrocatalytic current was measured at 100 s after the HRP-mimicking reaction reached the steady state. AFM imaging was performed in tapping-mode in an atmosphere of dry helium on a Digital Instruments Nanoscope IIIa (Digital Instruments, Santa Barbara, CA). For practical applications, human serum, which obtained from healthy volunteers, was diluted 200-fold for the detection. All measurements were conducted at room temperature and repeated at least three times to obtain statistically meaningful results. 2.2. Direct growth of guanine nanowire The cleaned gold electrode was first immersed in 200 M DTT solution for 10min. Then, the gold surface was incubated with 1 M thiolated c-myc probe (prepared with 10 mM pH 8.0 Tris-EDTA buffer containing 100 mM NaCl and 10 mM tris(2-carboxyethyl) phosphine hydrochloride) for 30 min. The obtained gold electrode was incubated with 1.5 M c-myc probe solution (prepared with 10 mM Tris-HCl buffer, 0.5 M KCl, 0.12 M Mg2+, pH 7.4). It is worth noting that the immersion procedure between the sensor and c-myc probe solution should be performed as fast as possible after the addition of Mg2+. Before amperometric measurement, the resulting electrode was treated with 0.2 mM hemin (dissolved by 10 mM HEPES buffer, 50 mM KCl, 1% DMSO, pH 8.0) for 30 min to induce G-wire/hemin complexes. 2.3. For DNA detection The DTT-modified sensor was immersed in 1 M hairpin DNA solution for 30 min. After incubation with a series of concentrations of target DNA (dissolved in 10 mM Tris-HCl buffer, 100 mM NaCl, 5mM MgCl2, pH 7.4), the resulting electrode was gently rinsed and incubated

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with 1.5 M c-myc probe to form G-wire. After that, the electrode was kept in 0.2 mM hemin for 30 min before biocatalytic reaction in TMB solution. 2.4. For thrombin detection The DTT-modified sensor was immersed in 1 M thiolated TBA solution for 30 min and different concentrations of TB (dissolved in 10 mM Tris-HCl buffer, 150 mM NaCl, 50 mM KCl, 5 mM MgCl2, pH 7.4) for 1 h. Then, 1.5 M L-DNA was captured on the sensor after 1 h incubation due to the TBA region. And the c-myc region triggers the GWA procedure for further electrochemical detection.

3. Results and discussion 3.1. Direct growth of guanine nanowire To demonstrate the detection principle, one question to be answered first was whether the G-wire could be constructed. We have previously reported the telomeric oligonucleotide d(AGGGTGGGG), namely c-myc (detailed sequence see Table S1), which adopted a head to tail parallel-stranded G-quadruplex structure in the presence of K+ (Shi et al., 2013). After the addition of Mg2+, high-order G-wire superstructure formed, which consists of G-quadruplex repeat units. This is because Mg2+ facilitates the π−πstacking of G-quadruplexes via phosphate charge neutralization to form an axial extension filamentous polymer (Chen, 1997). Fig. 1A illustrates the details for direct growth of G-wire on gold substrate. Initially, thiolated c-myc (HS-c-myc) DNA probe was anchored onto the dithiothreitol (DTT) modified electrode surface. G-quadruplex can be formed on the electrode surface with the help of K+. In the presence of Mg2+ and free c-myc probes, G-quadruplexes can spontaneously self-assemble into long, linear G-wire within 10 min (Fig. S1), which could form hemin/G-quadruplex repeat units after incubation

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with hemin. The hemin/G-quadruplex was found to reveal horseradish peroxidise mimicking (HRP-mimicking) activities in the presence of the H2O2 and 3, 3’,5,5’-tetramethylbenzidine (TMB) (Wen et al., 2012; Yu et al., 2015; Zhou et al., 2014). H2O2 was reduced when an external voltage was applied in the presence of an electroactive cosubstrate, TMB, generating quantitative electrochemical reduction current signals (Ge et al., 2014; Pei et al., 2010; Wen et al., 2012; Zhou et al., 2014). According to these reports, an external voltage (+150 mV vs Ag/AgCl) is required for amperometric detection in this work. (Here Fig. 1) 3.2. Feasibility tests of guanine nanowire based amplification strategy Amperometry, a direct way to characterize the HRP-mimicking catalyzed electrochemical process, was utilized to verify the feasibility of designed amplification process (Fig. 1B). Compared to the current generated by bare gold electrode (curve a), the current of c-myc modified sensor for TMB catalysis barely changed in the absence of K+ (curve b). This is because G-quadruplex hardly formed without K+ and no catalytic reaction occurred on the sensor surface. With the addition of K+, G-quadruplex was formed and a current increase was observed (curve c) due to the two-electron oxidation and reduction reactions of TMB (Wen et al., 2012). Significantly, over 300% amperometric signals increased was obtained after G-wire formed in the presence of K+ and Mg2+ (curve d), which indicate that the current response might reflect the sensitivity for further detection of analytes. Similar signal amplification phenomenon was observed by cyclic voltammetry (CV) measurements (Fig. 1C). It can be seen that curve a displays a couple of typical redox peaks of Fe(CN)63−/4−, indicating a fast charge transfer reaction of the redox probe on the bare gold electrode surface. With the successive formation of G-quadruplex on the gold surface, the difference of peak position was gradually increased,

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whereas the intensity of peak current was decreased (curve b). The CV response was further decreased after direct growth of negatively charged G-wire (curve c), which is attributed to the fact that immobilized G-wire provides a barrier for the electron transfer of Fe(CN)63−/4− at the electrode surface. Subsequently, the sensor was characterized by electrochemical impedance spectroscopy (EIS) measurements (Fig. S2), illustrating that the obtained results were in accordance with those of the CV tests. In addition, the surface coverage of G-wire on the gold electrode surface was 98.9% calculated by EIS using our reported method (Gao et al., 2013), which was much higher than that of G-quadruplexes (96.5%). The obtained EIS results were fitted to the Randles circuit: Rs (Q (RctW)) (inset, Fig. S2), where Rs means the solution resistance, Q represents the constant phase element, Rct is the charge transfer resistance, and W is the Warburg impedance. To further confirm these findings, atomic force microscopy (AFM) has been employed to directly characterize the G-wire structure. Fig.1D shows a typical AFM image of c-myc in the absence of Mg2+. A significant image of c-myc was not observed, indicating that high-order nanowire structure does not occur in the absence of Mg2+. In contrast, the AFM image revealed that c-myc folded into a high-order linear nanowire in the presence of Mg2+ (Fig. 1E). Next, we used EIS method to investigate whether G-wire could form from other topologies or structures of G-rich DNA oligomers. The c-myc modified sensor was immersed with other reported G-quadruplexes in Mg2+ solution. We found that other oligomers hardly change EIS signal under the same experimental conditions (Fig. S3). Thus, it provides an efficient tool for the discrimination of parallel-stranded G-quadruplex from other topologies DNA. 3.3. Detection of nucleic acid We first evaluated this strategy for the detection of nucleic acid. As shown in Fig. 2A, a hairpin DNA probe was first immobilized on gold surface. The hairpin probe consists of two main regions, one of which hybridizes with the target DNA and the other is the peroxidase-like

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deoxyribozyme c-myc. In the absence of target, the c-myc was hybridized and prevented from forming the G-quadruplex. Upon hybridization with the target, the hairpin probe was opened and released the c-myc, which could trigger the GWA event. (Here Fig. 2) To test the signal amplification ability of our proposed GWA strategy, we used amperometry to measure the current from the electrocatalytic reaction. It can be seen from Fig. 2B, we obtain a current of ∼18 nA when we detect the target DNA with GWA method, which is two times higher than the current (∼9 nA) without the GWA method. The relatively large difference of current makes our assay sensitive. The electrochemical signal of the sensor was optimized in terms of hybridization time (Fig. S4). The amperometric intensities are controlled by the concentrations of target in the sensing system (Fig. 2C). When the concentration of target DNA increases, the amperometric current are intensified. Fig. 2D depicts the resulting calibration curve, which illustrates that the current changes (I, I = I – I0, where I and I0 are the current intensity in the presence and absence of target DNA, respectively) of the catalytic process reach a saturation value at a concentration of target corresponding to 100 nM. The regression equation is I = 3.9159 logC + 42.9021 with a correlation coefficient of 0.9792. The detection limit of 15 pM was obtained in this assay (3/slope, where  is the standard deviation). Compared to the traditional single G-quadruplex signal transmission unit (Pavlov et al., 2004), the detection limit is improved by two orders of magnitude. Notably, our proposed strategy is better or competitive with many reported methods (Table S2). Next, the specificity of the described detection strategy was further investigated by exposing the DNA sensor to different nucleic acids, including perfect complementary target DNA (T), 1 nt-mismatched DNA (T1), 2 nt-mismatched DNA (T2), 3 nt-mismatched DNA (T3), and random DNA (Tr). Impressively, we

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found that our designed strategy generated much higher amperometric current for target DNA than other analogous DNA (Fig. 2E). A comparison of our proposed sensor with other sensors on the signal ratio of single base mismatched DNA to perfectly complementary target DNA is shown in Table S3. It can be seen that this sensor has better selectivity compared with part of previously reported sensors. To evaluate the practical ability of the proposed method in complex matrixes, recovery tests were conducted in diluted human serum samples. By spiking standard target DNA solution into human serum samples, the recoveries were calculated from 98.3 to 105.0% (Table S4). The high selectivity, acceptable recovery and low matrix effect might ensure the practicality of the proposed strategy. 3.4. Detection of protein To demonstrate the general applicability of our strategy, we applied the GWA strategy to the analysis of protein. Thrombin (TB) was chosen as a model molecule. As shown in Fig. 3, a thiol-modified thrombin binding aptamers (TBA) was first self assembled on the gold surface. TBA is known to be of G-rich sequence, which is easy to fold into G-quadruplex in the presence of K+. In order to decrease the background signal, a partial complementary DNA (C-DNA) was employed to hybridize with TBA. A link DNA probe (L-DNA), which contains TBA region and c-myc region, was captured on the sensor and used for triggering GWA. To prevent interstrand entanglement, C-DNA was employed to hybridize with L-DNA. After the addition of TB and L-DNA, a sandwich type aptasensor was fabricated and C-DNA was displaced as waste. The c-myc region from L-DNA could trigger the formation of guanine nanowire and amplify the electrochemical signal. The enhancement of the amperometric signals was achieved in the presence of analyte, indicating the occurrence of GWA (Fig. 4A). (Here Fig. 3)

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The sensitivity of the fabricated electrochemical DNA biosensor based on the GWA method was investigated. As shown in Fig. 4B, the amperometric current increased when the concentration of TB increased, indicating that the GWA event is highly dependent on the concentration of TB. As shown in Fig. 4C, the average increase of amperometric current (I) showed a good linear correlation with the concentrations of TB ranging from 1 pM to 50 nM. The regression equation is I = 35.2338 logC + 2.8591 with a correlation coefficient of 0.9783. The detection limit of this assay is 0.29 pM (3/slope, where  is the standard deviation).This low detection limit might be attributed to the high amplification efficiency of G-wire. The sensitivity of this assay is better than that of some published strategies, and the details are shown in Table S5. (Here Fig. 4) Next, we investigated the practicality, matrix effect, and specificity of this assay. Based on the highly specific binding of the TBA to the thrombin, the current signal responding to human serum was similar to that in blank buffer (Fig. 4D). The background signal obtained in human serum was slightly higher than that in blank buffer, which might be due to the interferences of the complex matrices. The amount of TB spiked in serum was evaluated and the recovery was obtained varying from 100.0 to 102.0% (Table S6). To investigate the specificity of this sensing system, we challenged the biosensor with other possible interferences, including bovine serum albumin (BSA), hemoglobin (HB), and lysozyme. As shown in Fig. 4E, with the addition of non-target molecules, no apparent change in the amperometric current was observed com-pared with that of the blank sample test. However, the presence of TB resulted in the dramatic increase in the amperometric current. Even when BSA, HB, and lysozyme coexisted with TB, the

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amperometric response was almost the same with that of TB, indicating the high specificity of the proposed DNA sensor for thrombin detection.

4. Conclusions In summary, a novel amplification method was developed for nucleic acid and protein detection based on guanine nanowire. Significantly, our strategy allows the overall performance of DNA sensors to be improved: the amplification rate can be decreased from hours to minutes and detection limits can also be enhanced. In addition, the proposed DNA sensor worked well in complex mixtures and in serum, revealing high practical applicability. We expect the GWA strategy could be used in combination with other amplification methods, such as enzyme-assisted target recycling, nanomaterials-assisted method, et al., to reach an ultrasensitive sensing platform in future. The GWA method holds great potential to have a broad influence on a variety of diagnostic applications and may pave the way for the use of guanine nanowire as a new amplification method for biosensing in real-world settings.

Acknowledgment This work was financially supported by the National Natural Science Foundation of China (No. 21273174), the Municipal Science Foundation of Chongqing City (No. CSTC– 2013jjB00002), and the Innovation Foundation of Chongqing City for Postgraduate (CYB14052).

Appendix A. Supplementary information

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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.201X.XX.XXX.

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Figure captions: Fig. 1. (A) Schematic illustration of direction growth of guanine nanowire on gold surface. (B) Amperometric curves for the bare gold electrode in the presence of K+/Mg2+ in TMB solution (a), c-myc modified sensor (b) in the absence of K+, (c) in the presence of K+, and (d) in the presence of K+/Mg2+ in TMB solution (ready-to-use reagent, H2O2 included). (C) Cyclic voltammograms of the sensor at different stages: (a) bare gold electrode; (b) G-quadruplexes modified gold electrode; (c) G-wires modified gold electrode. The CV measurements were carried out in 100 mM phosphate buffer containing 5 mM Fe(CN)63−/4− and 0.2 M NaCl. AFM images of the c-myc solution incubated with (D) K+ and (E) K+/Mg2+. Fig. 2. (A) Schematic illustration of nucleic acid detection by guanine nanowire amplification. (B) Amperometric responses of (a) 0 nM target after GWA, 100 nM target (b) before and (c) after GWA. (C) Amperometric curves for the sensor tested with a series of target DNA concentrations. From a to h: 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 100 nM, 1000 nM, and 2000 nM. (D) Plot of the current increase versus target DNA concentration. Inset depicts the responses of the sensing system toward DNA at low concentration. (E) Selectivity investigation of the proposed method for 2 M target DNA against 2 M T1, T2, T3, and Tr. Amperometric measurements were conducted in TMB solution (ready-to-use reagent, H2O2 included). Fig. 3. Schematic illustration of thrombin detection by guanine nanowire amplification. Fig. 4. (A) Amperometric responses of (a) 0 nM TB after GWA, 50 nM TB (b) before and (c) after GWA. (B) Amperometric curves for the sensor tested with a series of thrombin concentrations. From a to i: 1 pM, 10 pM, 100 pM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, and

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500 nM. (C) Plot of the current increase versus thrombin concentration. Inset depicts the responses of the sensing system toward thrombin at low concentration. (D) Amperometric currents of 10 pM, 100 pM, and 1000 pM thrombin detection in (a) buffer solution and (b) diluted human serum solution. (E) Specificity investigation for thrombin (10 nM) detection against the interference proteins, BSA (10 nM), HB (10 nM), lysozyme (10 nM), Mixture (containing 10 nM thrombin, 10 nM BSA, 10 nM HB, and 10 nM lysozyme). Amperometric measurements were conducted in TMB solution (ready-to-use reagent, H2O2 included).

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Graphical abstract

Highlights  A new signal amplification strategy based on guanine nanowire is proposed.  Guanine nanowire based amplification strategy was used for DNA and protein assay.  The detection limits were improved by two orders of magnitude using this strategy.

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