Sensitive detection of HIV gene by coupling exonuclease III-assisted target recycling and guanine nanowire amplification

Sensors and Actuators B 238 (2017) 1017–1023

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Sensitive detection of HIV gene by coupling exonuclease III-assisted target recycling and guanine nanowire amplification Yan Li Huang, Zhong Feng Gao, Hong Qun Luo ∗ , Nian Bing Li ∗ 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

a r t i c l e

i n f o

Article history: Received 26 January 2016 Received in revised form 22 July 2016 Accepted 26 July 2016 Available online 26 July 2016 Keywords: HIV gene Guanine nanowire amplification Exonuclease III-assisted target recycling

a b s t r a c t In this work, a simple, label-free electrochemical biosensor was constructed with the combination of exonuclease III (Exo III)-assisted target recycling and guanine nanowire amplification for the detection of HIV gene. The presence of target DNA leads to target recycling process, which could obtain a digestion product named as help DNA with the assistance of Exo III. The released help DNA can hybridize with the G-quadruplex sequence-locked hairpin probe. Then, the active G-quadruplex sequence could form Gquadruplex structure in the presence of K+ , which could trigger the formation of guanine nanowire with the help of Mg2+ . The hemin/G-quadruplex repeat units could effectively catalyze the H2 O2 -mediated oxidation of 3,3 ,5,5 -tetramethyl benzidine dihydrochloride (TMB·2HCl) accompanied by a change in color of the reaction solution and an increased electrochemical current signal in the presence of hemin. The developed amplification strategy provides a sensitive and selective approach for the detection of target DNA with a detection limit of 3.6 pM. Furthermore, the proposed DNA sensor has a promising potential for biosensing in real settings. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Sensors have been widely used in various fields for the detection and analysis. Most commonly used sensors can be classified as mass sensor, optical sensor, piezoelectric sensor, and biosensor etc. based primarily on the transducing mechanism [1]. Some of the sensors are accurate and have many advantages. For example, Mishra et al. constructed the SPR sensor utilizing ITO as a plasmonic material has the characteristics of low response time and probe reusability [2,3]. Modafferi et al. reported a resistive sensor (RS) that is sample and high sensitive for monitoring low level concentrations of ammonia [4]. Among those sensors, the biosensors for highly sensitive detection of specific DNA sequences have become imperative in the field of diseases, clinical diagnosis, and clinical therapy [5–8]. Over the past decades, various DNA biosensors have been developed, including fluorescence imaging [9], optics [10], electrochemistry [11,12], electrogenerated chemiluminescence (ECL) [13], long period grating (LPG) [14], and surface plasmon resonance (SPR) techniques [15,16]. However, most of the sensors are expensive, large size and require sophisticated instruments and operations. Thus, DNA electrochemical biosensor is particularly attractive because of the

∗ Corresponding authors. E-mail addresses: [email protected] (H.Q. Luo), [email protected] (N.B. Li). http://dx.doi.org/10.1016/j.snb.2016.07.144 0925-4005/© 2016 Elsevier B.V. All rights reserved.

advantages of fast response, simple instrumentation, high specificity and sensitivity [17]. Much effort has been devoted to improve the sensitivity and selectivity of DNA electrochemical biosensor for target DNA detection. Gold nanoparticles [18], polymerase chain reaction (PCR) [19], hybridization chain reaction (HCR) [20], and enzyme catalytic reaction [21] have been used for signal amplification. Thereinto, exonuclease was especially attractive for the detection of target DNA [22,23]. Exonuclease III (Exo III) is a kind of exonuclease which is effective on 3 -terminus of duplex DNAs with a blunt or recessed 3 -terminus and has no activity on single-stranded DNA or duplex DNAs with a protruding 3 -terminus [24,25]. Thus, Exo III can be used for the digestion of HP1 -target structure and the recycling of target in electrochemical sensor. Hemin/G-quadruplex, a type of artificial DNAzyme, is also a fascinating catalytic tool for signal amplification which is widely used in the construction of electrochemical biosensor for DNA detection [26,27]. The Pu-18 c-myc template sequence d(AGGGTGGGGAGGGTGGGG) could form parallel-stranded Gquadruplex structure with K+ [28]. In the presence of Mg2+ , G-quadruplex DNA could be specifically recognized and precipitated to form higher-order superpolymer structure [29–31]. Recently, our group first reported a potential use of guanine nanowire amplification (GWA) in DNA detection [32]. However, it is imperative to further improve the analytical performance of the

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method for DNA detection. The amplification strategy, such as combining endonuclease-assisted target recycling with rolling circle amplification (RCA) [33], combining enzymatic recycling with gold nanoparticles (AuNPs) [34], can further amplify the electrochemical signal and could detect target DNA sensitively and selectively. But until now, research that combines exonuclease-assisted target recycling and GWA methods has not been reported. In this paper, we proposed an electrochemical biosensor for the sensitive detection of human immunodeficiency virus (HIV) gene by coupling Exo III-assisted target recycling and guanine nanowire amplification. The designed electrochemical biosensor is simple, label-free, and signal-on, which leads to a lower detection limit. The proposed DNA electrochemical biosensor holds great potential for the development of ultrasensitive platform in bioanalysis and disease diagnostics. 2. Experimental

containing 5 mM [Fe(CN)6 ]4− /3− (pH 7.4). CV was performed at a scan rate of 100 mV s−1 , and EIS was recorded with the frequency ranging from 0.1 Hz to 10 kHz at an amplitude of 5 mV. Amperometric detection was performed at 150 mV and the electrocatalytic current was measured at 100 s.

2.3. Gold electrode pretreatment The bare gold electrode was initially immersed in piranha solution (H2 SO4 and 30% H2 O2, 3:1 by volume) for 5 min. The electrode was polished to a mirror-like surface with 0.05 ␮m alumina powder and then washed by sonication in ultrapure water, ethanol and ultrapure water for 3 min each. The gold electrode was electrochemically cleaned in 0.5 M H2 SO4 solution by the potential range from −0.2 to 1.6 V with a scan rate of 100 mV s−1 until a stable cyclic voltammogram was established. Finally, the prepared electrode was washed with ultrapure water and dried with nitrogen.

2.1. Materials The HPLC-purified oligonucleotides were purchased from Sangon Biotech Co., Ltd., Shanghai, China, and the sequences are listed below: Hairpin probe 1 (HP1 ): 5 -TTTCGAGGGTGGGTGAATTACGACCC ACCCTCGAAAATCTCTAGCAGT-3 Hairpin probe 2 (HP2 ): 5 -AGGGTGGGGAGGGTGGGGCCTTCACCCACCCTCGAAA(CH2 )6 -HS-3 c-myc: 5 -AGGGTGGGGAGGGTGGGG-3 Target HIV DNA: 5 -ACTGCTAGAGATTTTCCA CAT-3 Single-base mismatched DNA: 5 -ACAGCTAGAGATTTTCCA CAT-3 Four-base mismatched DNA: 5 -ACAGCCACAGGTTTTCCACAT-3 Unmatched DNA: 5 -CAGTAGCTGTCGGCGATAAGC-3 Exo III was purchased from Takara Biotechnology Co., Ltd., Dalian, China. Hemin, Dithiothreitol (DTT), and 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES) were purchased from Sangon Biotech Co., Ltd., Shanghai, China. 3,3 ,5,5 -Tetramethyl benzidine dihydrochloride (TMB·2HCl) were purchased form Xiya Chemical Industry Co., Ltd., Shandong, China. Dimethylsulfoxide (DMSO) and hydrogen peroxide (H2 O2 , 30%) were purchased from Chengdu Kelong Chemical Reagents Co., Ltd., Chengdu, China. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All the reagents mentioned were analytical reagent grade and were used without further purification. All solutions were prepared with ultrapure water (18.2 M cm). The oligonucleotide HP2 was dissolved in the immobilization buffer (20 mM pH 7.4 Tris-HCl buffer containing 200 mM NaCl and 1 mM TCEP). Hybridization buffer was 20 mM Tris-HCl buffer containing 200 mM NaCl (pH 7.4). The oligonucleotide c-myc was prepared in 10 mM Tris-HCl buffer (pH 7.4) containing 0.5 M KCl and 0.12 M Mg2+ . 2.2. Apparatusere All electrochemical measurements were performed on a CHI660D electrochemical workstation (Shanghai CHI Instruments Co., China). The three electrode system contained Ag/AgCl (sat.KCl) reference electrode, platinum wire as the counter electrode and modified gold electrode (␸ = 2 mm, Tianjin Aidahengsheng Technology Co., Ltd., China) as the working electrode. The biosensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 0.1 M phosphate buffer (PB)

2.4. Formation of the biosensor The oligonucleotide HP1, HP2 , and c-myc were heated at 95 ◦ C for 5 min and naturally cooled down to room temperature before use. The HP2 probe contains c-myc region at the 5 terminus and a thiol group at the 3 terminus. After dried with nitrogen, the cleaned electrode was first immersed in 20 ␮L of 200 ␮M DTT solution (dissolved in 20 mM Tris-HCl containing 200 mM NaCl) for 30 min. The HP2 was dissolved in the immobilization buffer containing 1 mM TCEP and the mixture was incubated in the dark at 4 ◦ C for 1 h to reduce disulfide bonds. Next, the electrode was immersed in 20 ␮L of 0.5 ␮M HP2 solution for 30 min to get a monolayer DNA. Meanwhile, 1 ␮M HP1 , 30 U Exo Ш and different concentrations of the target DNA in the hybridization buffer at a final volume of 20 ␮L were incubated at 37 ◦ C for 60 min. Then, the mixture was heated to 70 ◦ C for 20 min to inactivate the Exo Ш. The modified electrode was immersed into the mixture above for 2 h at 37 ◦ C. To form the G-quadruplex complex, the resulting electrode was immersed in 50 ␮L of 0.5 M K+ solution. The electrode was gently rinsed and incubated with 1.5 ␮M c-myc solution to form G-wire structure. Finally, the electrode was incubated in 25 ␮L of 0.2 mM hemin for 0.5 h at 37 ◦ C before biocatalytic reaction in 5 mM TMB·2HCl – 2 mM H2 O2 solution (prepared with 20 mM pH 6.5 HEPES buffer containing 500 mM NaCl and 50 mM KCl). The electrode was thoroughly rinsed with the hybridization buffer after each step in this work.

3. Results and discussion 3.1. Principle of the strategy The electrochemical DNA sensor architecture and its working principle are shown in Scheme 1. In our probe design, the hairpin probe (HP1 ) is 3 -protruding termini at the stem part which cannot be digested by Exo Ш. However, in the presence of target HIV DNA, it can hybridize with the 3 -protruding terminus of HP1 to form a stem with blunt terminus, which can be cleaved by Exo Ш to release the target DNA. With each digestion cycle, a digestion product (named help DNA) was obtained, which could hybridize with the capture probe (HP2 ). Then, the released c-myc region from the HP2 can form a parallel G-quadruplex structure with the help of K+ . After the addition of c-myc, the c-myc region from HP2 could trigger the formation of guanine nanowire within 10 min in the presence of Mg2+ [32,35]. With the help of hemin, the hemin/G-quadruplex repeat units which compose of HRP-mimicking DNAzyme could trigger the electrochemical H2 O2 -mediated oxidation of TMB·2HCl.

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Scheme 1. Schematic illustration of the proposed biosensor fabrication process.

Fig. 1. CV (A) and EIS (B) of the bare GE (a), DTT/GE (b), HP2 /DTT/GE (c), c incubated with 1 ␮M HP1 containing 100 nM target and 30 U Exo Ш (d), and d incubated with 1.5 ␮M c-myc (e). Inset: the Randles circuit model for impedance analysis: Rs is the solution resistance, Cdl is the double-layer capacitance, Ret is the charge transfer resistance, and Zw is the Warburg impedance.

3.2. Electrochemical characterization of the biosensor The cyclic voltammetry of ferricyanide was chosen as a marker to characterize the fabrication process and investigate the changes in the electrode behavior. As shown in Fig. 1A, the peak current of the DTT/Au electrode (curve b) decreases compared to that of the bare electrode (curve a). This is attributed to the fact that DTT could be chemisorbed onto the gold electrode surface via Au S bonds, which could provide enough free space for the subsequent assembly of DNA probe. Then, insert-assembly of the thiolated HP2 resulted in an obvious electrochemical response (curve c), which could be explained by that HP2 could insert into the loosely molecular layer of DTT and hinder the interfacial electron transfer. After hybridizing with the help DNA, the electrochemical signal decreased (curve d). And it further decreased observably after the

addition of c-myc (curve e), due to the fact that the c-myc adopted a head to tail parallel-stranded G-quadruplex structure to form high-order G-wire superstructure. Electrochemical impedance spectroscopy is an effective technique to investigate the modification process of the electrode. The obtained EIS results were fitted to the Randles circuit (Inset, Fig. 1B). As observed in Fig. 1B, the bare gold electrode (GE) shows a tiny semicircle domain (curve a), indicating a rapid electron transfer process of [Fe(CN)6 ]3−/4− . The Ret increased substantially with the incubation of DTT (curve b). After the thiolated HP2 was selfassembled onto the DTT/GE, the Ret increased significantly (curve c), because the increasing oligonucleotides on the electrode hindered the electron-transfer. The Ret kept increasing (curve d) after hybridizing with the help DNA. With the formation of guanine nanowire, a further increased Ret value is observed (curve e).

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the formation of G-quadruplex at 5 terminus of HP2 could strongly bind hemin to form the catalytic DNAzyme that catalyzed the H2 O2 mediated oxidation of TMB·2HCl. It was found that great current increment (7.26 nA) was obtained after the addition of c-myc (curve c in Fig. 2). This is because the c-myc region from HP2 could trigger the formation of guanine nanowire within 10 min in the presence of Mg2+ , resulting in a great increased signal (recorded as I). Compared to the traditional single G-quadruplex signal transmission unit, the signal is greatly improved via the signal amplification of G-wire. 3.4. Optimization of the experimental conditions

Fig. 2. Amperometric responses of (a) 0 nM target after guanine nanowire amplification, 100 nM target before (b) and after (c) guanine nanowire amplification.

3.3. Amplification effect of G-wire In order to investigate the signal amplification effect of G-wire, we used amperometric curves to measure the current from the electrocatalytic reaction. As shown in Fig. 2, in the absence of target DNA, only a weak signal (recorded as I0 ) is observed (curve a in Fig. 2), because the help DNA cannot be released and the G-wire structure can not be formed on the gold electrode. Compared curve a with curve b in Fig. 2, the current increment is 3.64 nA after the addition of 100 nM target DNA. This is attributed to the fact that

In order to maximize the response signal of the proposed sensor, various conditions were optimized. The amounts of Exo Ш were connected with the degree of the respond signal amplification. In Fig. 3A, as the amount of Exo Ш was increased to 30 U, the electrochemical response reached to the maximum and remained a steady value. Thus, the amount of Exo Ш was set at 30 U for subsequent experiments. The incubation time of Exo Ш with HP1 and target DNA complex had great effect on Exo Ш-assisted target recycling process. As shown in Fig. 3B, with increasing incubation time, the electrochemical response sharply increased and tended to a steady value after 60 min. In this case, we selected 60 min as the optimal incubation time. The immobilization time and concentration of HP2 were important parameters that influenced the reaction. Fig. 3C shows that the electrochemical signal increased with increasing immobilization time and reached maximum at 30 min, and then it decreased

Fig. 3. Optimization of experimental conditions: (A) Effect of Exo III concentration. (B) Effect of the incubation time of target recycling and Exo Ш digestion. (C) Effect of the incubation time of capture probe HP2 . (D) Effect of the concentration of capture probe HP2. Concentrations: 1 ␮M HP1 , 100 nM target DNA, 1.5 ␮M c-myc, 0.2 mM hemin. Error bars represent the standard deviation of three independent experiments.

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Fig. 4. (A) Amperometric curves for the sensor tested with different target concentrations. From a to k: 0, 10 pM, 25 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 25 nM, 100 nM. (B) The linear relationship of the current change versus logarithm value of the target DNA concentration. Error bars represent the standard deviation of three independent experiments.

Table 1 Comparison of the proposed method with some reported genosensors. Signal amplification strategy

Linear range (nmol L−1 )

Detection limit (nmol L−1 )

Reference

Carbon nanotube WS2 nanosheet T7 exonuclease and graphene oxide Labeled dual hairpin DNA probe Super sandwich Gold nanoparticles and HCRa Exonuclease III and gold nanoparticle Graphene oxide and DNAzyme Polydopamine nanospheres and exonuclease III Exonuclease III and guanine nanowire

0.67–8.4 1–20 0.05–2 0.15–900 5–1000 0–0.5 0.1–10 0.1–3 0.078–5 0.01–100

0.14 0.5 0.0386 50 1.7 0.050 0.033 0.034 0.005 0.0036

[37] [38] [39] [40] [41] [42] [43] [26] [44] This work

a

HCR: hybridization chain reaction.

after 30 min. Thus, 30 min was selected to be the optimum immobilization time for all the assays in this work. Fig. 3D shows the effect of concentration of HP2 . The respond current reached maximum when the concentration of HP2 probe was 0.5 ␮M and decreased at higher concentrations. Therefore, we set the concentration of HP2 as 0.5 ␮M throughout this work. However, longer immobilization time and high concentration of HP2 would affect the subsequent DNA assembly due to the steric and electrostatic hindrances arising from the more tightly packed DNA monolayer. 3.5. Sensitivity of the sensor The sensitivity of the aptasensor was investigated with different concentrations of target DNA under optimum conditions. As shown in Fig. 4A, the electrochemical response increased with increasing target DNA concentration. The current change (I, where I was calculated by the actual current I subtracted by the blank response I0 ) exhibited a linear relationship with the logarithm value of target DNA concentration within the range from 10 pM to 100 nM. The linear regression equation was expressed as I = 17.4191 + 1.5180 lgC (unit of I is nA, the unit of C is M) with a correlation coefficient of 0.9903 (Fig. 4B). The sensitivity of the proposed sensor is defined as the slope of the calibration curve. The limit of detection (LOD) of target DNA concentration is 3.6 pM (3␴/slope, where ␴ is the standard deviation of black measurement) [36]. In addition, the limit of quantification (LOQ) is 11.8 pM, which is established by using the expression 10␴/slope. The proposed biosensor is sensitive based on this signal amplification method. A comparison of this method with some reported genosensors with the detection limit and the signal amplification strategy is

Fig. 5. Selectivity of the proposed biosensor. Amperometric values of the biosensor for (a) target DNA (100 nM), (b) single-based mismatched DNA (100 nM), (c) fourbased mismatched DNA (100 nM), and (d) unmatched DNA (100 nM) detection. Error bars represent the standard deviation of three independent experiments.

shown in Table 1. The detection limit of our proposed sensor is considerably satisfactory, which is lower than those of some reported genosensors. Therefore, the proposed sensor has good analytical performance for the detection of specific DNA sequences. 3.6. Selectivity and repeatability of the sensor To evaluate the selectivity of the biosensor, four types of DNA sequences including full matched target HIV DNA, single-base mismatched DNA, four-base mismatched DNA, and unmatched DNA were detected under the same and optimum conditions. Fig. 5 shows the response current change (I) of the proposed sensor

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Table 2 Detection of target DNA added in human serum with the proposed biosensor (n = 4). Samples

DNA added (nM)

DNA found (nM)

Recovery (%)

RSD (%)

1 2 3

0.1 1 25

0.0995 0.98 23.50

99.5 97.8 94.1

4.12 4.83 5.27

to various DNA sequences. It can be seen that the presence of the single-base, four-base mismatched and unmatched DNA (each 100 nM) shows reduced responses when compared with the presence of the target HIV DNA (100 nM). The comparison demonstrates that the proposed electrochemical biosensor had high specificity for the detection of target HIV DNA. The repeatability of the proposed electrochemical sensor was examined. Five different electrodes were used to detect HIV target DNA (100 nM) under the same conditions. All biosensors exhibited similar electrochemical response and the relative standard deviation (RSD) was 3.82%. These results indicate the satisfactory repeatability for target DNA detection. 3.7. Practical application In order to evaluate the applicability of the developed biosensor, we performed recovery experiments in human serum samples which were obtained from healthy individuals. With standard addition method, target HIV DNA with three different concentrations (100 pM, 1 nM, and 25 nM) were added to 100-fold-diluted human serum samples. As shown in Table 2, the recoveries were 99.5%, 97.8%, and 94.1%, respectively, which exhibited a good accuracy and good potential for target DNA detection in complex biological samples. 4. Conclusion In summary, a simple Exo Ш-assisted target recycling and guanine nanowire amplification strategy has been developed for sensitive and selective detection of target DNA. The presence of target HIV DNA switches the structure of HP1 and triggers the cleavage activity of Exo Ш towards double-strand DNA. The important digestion product help DNA could hybridize with HP2 and then the c-myc region of HP2 could trigger GWA. The developed method greatly lowers the detection limit toward target HIV DNA down to 3.6 pM. The proposed biosensor shows high sensitivity and selectivity to target DNA. In addition, the developed strategy holds great potential and may pave the way for the use of DNA detection in area of bioanalysis, disease diagnostics, and clinical biomedicine. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 21273174), the Innovation Foundation of Chongqing City for Postgraduate (No. CYB14052), and the Municipal Natural Science Foundation of Chongqing City (No. CSTC–2013jjB00002). References [1] S. Singh, Sensors – an effective approach for the detection of explosives, J. Hazard. Mater. 144 (2007) 15–28. [2] S.K. Mishra, B.D. Gupta, Surface Plasmon resonance based fiber optic sensor for the detection of CrO4 2− using Ag/ITO/hydrogel layers, Anal. Methods 6 (2014) 5191–5197. [3] S.K. Mishra, D. Kumari, B.D. Gupta, Surface Plasmon resonance based fiber optic ammonia gas sensor using ITO and polyaniline, Sens. Actuator B 171 (2012) 976–983.

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Biographies Yan Li Huang is a MS candidate in School of Chemistry and Chemical Engineering, Southwest University, China. Her major research interests include electrochemistry and electroanalytical chemistry. Zhong Feng Gao is a doctor student in School of Chemistry and Chemical Engineering, Southwest University, China. His major research interests include electrochemistry and electroanalytical chemistry. Hong Qun Luo is a professor of chemistry in School of Chemistry and Chemical Engineering, Southwest University, China. She received her MS degree in environmental chemistry from Sichuan University in 1991 and PhD degree in analytical chemistry from Southwest China Normal University in 2002. During 2006–2007, she was a visiting scholar in Tohoku University, Japan. Her research is focused on molecular spectroscopy and electrochemical sensor. Nian Bing Li is a professor of chemistry in School of Chemistry and Chemical Engineering, Southwest University, China. He received his MS degree in physical chemistry in 1997 and PhD degree in material science in 2000 from Chongqing University. During 2000–2002, he was a postdoctoral research fellow in Fuzhou University, China. Since 2006–2007, he was a postdoctoral research fellow in Korea Advanced Institute of Science and Technology (KAIST), Korea. His research interests are the developments of electrochemical devices such as chemical sensors and biosensors.