Functional fullerene-molybdenum disulfide fabricated electrochemical DNA biosensor for Sul1 detection using enzyme-assisted target recycling and a new signal marker for cascade amplification

Journal Pre-proof Functional fullerene-molybdenum disulfide fabricated electrochemical DNA biosensor for Sul1 detection using enzyme-assisted target recycling and a new signal marker for cascade amplification Huan You, Zhaode Mu, Min Zhao, Jing Zhou, Yonghua Yuan, Lijuan Bai

PII:

S0925-4005(19)31682-X

DOI:

https://doi.org/10.1016/j.snb.2019.127483

Reference:

SNB 127483

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

21 March 2019

Revised Date:

25 November 2019

Accepted Date:

26 November 2019

Please cite this article as: You H, Mu Z, Zhao M, Zhou J, Yuan Y, Bai L, Functional fullerene-molybdenum disulfide fabricated electrochemical DNA biosensor for Sul1 detection using enzyme-assisted target recycling and a new signal marker for cascade amplification, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127483

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Functional fullerene-molybdenum disulfide fabricated electrochemical DNA biosensor for Sul1 detection using enzyme-assisted target recycling and a new signal marker for cascade amplification Huan Youa1, Zhaode Mua1, Min Zhaob, Jing Zhoua, Yonghua Yuana, Lijuan Baia Engineering Research Center for Pharmacodynamic Evaluation of Chongqing, College of

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a

Pharmacy, Chongqing Medical University, Chongqing 400016, PR China b

Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of

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Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China

Polyamidoamine (PAMAM) functionalized fullerene (C60) and molybdenum disulfide

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

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Highlights

(MoS2) nanohybrid was first synthesized and employed as sensing platform.

Exonuclease III-assisted target recycling and MoS2 doped polyaniline (MoS2-PANI)

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nanocomposite were used for signal amplification.

The proposed biosensor successfully detected the specific DNA sequence with

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wide linear range and low detection limit.

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This method showed high specificity and sensitivity for PCR products of Sul1 gene obtained from Salmonella typhimurium.

* Corresponding author. Tel: +86-023-68485161; Fax: +86-023-68485161. E-mail address: [email protected] (L. Bai) 1

These two authors contributed equally to this work. 1

Abstract: This work describes a DNA biosensor for sensitive detection of the specific Sul1 gene of Salmonella typhimurium. First of all, polyamidoamine (PAMAM) functionalized fullerene (C60) and molybdenum disulfide (MoS2) nanohybrid is synthesized and employed as sensing platform. Secondly, MoS2 doped polyaniline (PANI) nanocomposite is designed as

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redox nanoprobe to enhance electrochemical signal. Moreover, exonuclease III (exo III)-assisted target recycling strategy impels a large amount of single-stranded capture probe to hybridize with signal marker, resulting in a significantly enhanced response signal. With

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the cascade amplification strategy, the proposed biosensor shows high sensitivity for target DNA detection with a wide detection linear range from 40 fM to 40 nM. More importantly, it

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has been applied for assay of Sul1 from Salmonella typhimurium with high specificity and

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sensitivity, which provides a methodological guidance for detection of pathogenic bacteria. Keywords: Electrochemical DNA biosensor; Exonuclease III-assisted target recycling;

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1. Introduction

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Polyaniline; Fullerene; Molybdenum disulfide

Salmonella typhimurium is a kind of food-borne bacterium that threatens public health

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[1]. Data from the United States Center for Disease Control and Prevention reveal that about 1.4 million cases of Salmonella infections occur annually [2,3], which are most related to the Salmonella-infected animal-derived foodstuffs, such as eggs, poultry and beef, as well as vegetables and fruits [4]. Worse still, the emergence of various drug resistance genes, such as sulphonamide resistance protein gene (Sul1), makes the treatment of bacterial infections more

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and more difficult. Therefore, rapid and efficient detection of Sul1 is of great importance for guiding clinical medication more reasonably and scientifically. At present, polymerization chain reaction (PCR) is the main method for detecting Sul1 of Salmonella with relatively high sensitivity [5-7]. However, it has some shortcomings, such as expensive equipment, complicated and time-consuming operation. Up to now, biosensing techniques based on the hybridization of DNA probes have attracted more and more attention in different research

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fields [8-10]. Owing to the advantages of low cost, simple operation process, as well as high sensitivity and stability, electrochemical DNA biosensors have been widely applied in pathogen detection [11,12]. Accordingly, a new type of electrochemical DNA biosensor based

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on nanomaterials and biological amplification technology is developed for detection of Sul1

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gene in this work.

Nanomaterials with unique structures and excellent electronic properties are utilized to

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improve the analytical performances of electrochemical biosensors [13-16]. The transition

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metal oxides of molybdenum disulfide (MoS2) [17-20] and the zero-dimensional carbon material of fullerene (C60) that possess large specific surface area and high conductivity have

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been widely employed as electrode materials [21,22]. However, poor solubility and unstable electrochemical property in aqueous media limit their applications. Accordingly, the

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amino-terminated polyamidoamine (PAMAM) with highly branched structure is adopted to modify C60 and MoS2, improving their water solubility and stability. Then PAMAM functionalized C60 and MoS2 nanohybrid (P-MoS2-C60) with better hydrophilicity is employed as sensing platform, which not only facilitates electron transfer, but also increases the loading amount of biomolecules. Moreover, nuclease-based target recycling strategies can surmount

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the limitation of 1:1 recognition ratio between target and signal probe in traditional nucleic acid hybridization system, for that one target can act on multiple signal probes, greatly enhancing the signal responses [23-25]. So far, nucleases such as nicking endonuclease [26,27], DNase I [28,29], Rnase H [30], exonuclease III (exo III) [31,32], and lambda exonuclease [33,34] have been applied in biosensors, showing high specificity and satisfactory catalytic activity.

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In addition, polyaniline (PANI) is one of the attractive conducting polymers because of its easy synthesis, chemical stability and good electric conductivity [35-37]. Accordingly, MoS2-doped PANI nanocomposite (MoS2-PANI) is designed and synthesized in this work for

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the first time, which not only possesses large surface area and abundant amino groups for

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further modification, but also displays excellent conductivity and redox activity. Then amino groups on the surface of MoS2-PANI could effectively load gold nanoparticles (AuNPs) and

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signal probes (SP) to obtain a new signal marker, greatly improving the electrochemical

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signal.

To achieve the cascade signal amplification, P-MoS2-C60 nanohybrid with large specific

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surface area and excellent conductivity is served as sensing platform, possessing abundant amino groups for electrochemical deposition of gold nanoparticles (dpAu). Then the partially

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complementary double-strand DNA (pdsDNA) of capture probe (CP) and assistant probe (AP) is assembled on the modified electrode. In the presence of target DNA and exo III, pdsDNA would hybridize with target DNA to form the completely complementary double-strand DNA (cdsDNA), providing a condition for exo III to digest the AP from 3' to 5' terminal. Consequently, target DNA is released to participate in the next hybridization process,

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resulting in a large number of CP liberated from the cyclic cleavage process. After the hybridization between single-stranded CP and signal marker, a significantly enhanced electrochemical signal of MoS2-PANI can be easily read out. The preparation procedure of signal marker, stepwise fabrication process of the biosensor, as well as the DNA hybridization principle of cdsDNA in the proposed sensing system, are depicted in Scheme 1.

2.1. Reagents and materials Aniline, tris-(2-carboxyethyl) phosphine hydrochloride

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2. Experimental

(TCEP),

amino-terminated

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polyamidoamine (PAMAM) dendrimer (ethylenediamine core, G 5.0), MoS2 powder (< 2 μm) and gold chloride (HAuCl4) were obtained from Sigma-Aldrich Chemical Co. (USA). C60

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powder was purchased from Nanjing Xianfeng Nano Co., Ltd. (Nanjing, China). Beef extract,

culture

collection

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peptone, agar and Salmonella typhimurium ATCC14028 strain were obtained from food safety Guangdong

huankai

Microbial

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and

Tech.

Co.,

Ltd.

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Tris(hydroxymethyl)aminomethane hydrochloride (Tris–HCl) and the FastStart Universal SYBR Green Master (Rox) (contains 2× qPCR Mix and ddH2O) were supplied by Roche

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(Switzerland). The TRIzol Kit and RevertAid First Strand cDNA Synthesis Kit K1622 were

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provided by Thermo Fisher Scientific Co., Ltd (Shanghai, China). The exonuclease III (exo III) and NEB buffer were purchased from New England Biolabs Co., Ltd. (Beijing, China). Ultrapure water with a resistivity of 18.2 MΩ.cm was used throughout the experiment. Sangon Biotech Co., Ltd. (Shanghai, China) provided all the oligonucleotides which were purified by HPLC. The specific gene fragment of Sul1 selected as target DNA in this work was based on GeneBank database through BLAST algorithm. The sequences of 5

oligonucleotides were listed in Table S1 (see in Supplementary Material). 20 mM Tris–HCl buffer containing 5 mM KCl, 140 mM NaCl and 1 mM MgCl2 was used to dissolve the oligonucleotides. The stepwise fabrication process of the biosensor was monitored in 0.1 M phosphate buffer (PB, pH 7.0) containing 10 mM NaH2PO4, 10 mM Na2HPO4 and 2 mM MgCl2 in the whole experiment. 2.2. Apparatus and characterization

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The electrochemical measurements were carried out on a CHI 660E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd, China) with a conventional three-electrode system: a platinum wire auxiliary electrode, a saturated calomel reference

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electrode (SCE) and a modified GCE as the working electrode. Cyclic voltammetry (CV) and

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electrochemical impedance spectroscopy (EIS) measurements were performed in 5 mM K4Fe(CN)6/K3Fe(CN)6 solution containing 0.1 M KCl. Differential pulse voltammetry (DPV)

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measurements were carried out in 0.1 M PB (pH 7.0) from -0.4 to 0.3 V, with the parameters

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of 50 mV pulse amplitude and 50 ms pulse width. The scanning electron microscope of SU8010 (SEM, Japan) was used to characterize these different nanomaterials. The elements of

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P-MoS2-C60 were characterized by energy dispersive spectroscopy (EDS, Oxford X-MaxN, Britain). The PCR amplification was carried out using a SLAN real-time PCR instrument

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from Shanghai Hongshi Medical Technology Co., Ltd (Shanghai, China). The UV-Vis absorption spectroscopy was measured by a Thermo Fisher Scientific NanoDrop2000 (Shanghai, China). 2.3. Preparation of the nanocomposites 2.3.1. Synthesis of P-MoS2-C60 nanohybrid

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P-MoS2-C60 nanohybrid was synthesized via a facile phase transfer [38]. Briefly, 5 mg MoS2 powder was dissolved in 5 mL anhydrous ethanol and sonicated for 12 h. Then, 5 mL ultrapure water and 5 mL C60-toluene solution (1 mg/mL) were added into the above solution for 30 min under stirring. Next, 40 μL PAMAM was dropped into the mixture solution. After stirring for 36 h at room temperature, the precipitate was centrifuged and washed for several times, and then it was re-dispersed in 5 mL ultrapure water.

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2.3.2. Preparation of the pdsDNA

The pdsDNA solution was prepared in advance through the chain hybridization annealing reaction. The mixture solution of 500 μL sulfydryl-labeled CP (2 μM) and 550 μL

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AP (2 μM) was heated at 90 °C for 5 min, then it was cooled down to 65 °C and incubated for

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10 min. The pdsDNA complex was obtained and stored at 4 °C. Prior to use, 10 mM TCEP was added into the pdsDNA solution to reduce the formation of disulfide bonds.

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2.3.3. Preparation of the signal marker

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MoS2-PANI nanocomposite was prepared according to our previous researches [39,40] with appropriate modification. Firstly, the mixture of 100 µL aniline, 10 mL H2SO4 (0.5 M)

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and 5 mL MoS2-ethanol solution (1 mg/mL) was stirred for 30 min. Then 10 mL K2S2O8 (0.15 M) was dropped into the above solution at 0 °C and reacted for 6 h under continuous

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stirring. After centrifugation, the synthesized MoS2-PANI nanocomposite was re-dissolved in 10 mL ultrapure water and stored at 4 °C for further use. Subsequently, 5 mL MoS2-PANI solution was added into 20 mL AuNPs colloidal and incubated at 4 °C for 30 min under continuous stirring. The obtained green precipitate (MoS2-PANI-Au) was washed and centrifuged for several times. Then 200 μL amino-labeled

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SP (2 μM) and the above precipitate were re-suspended in 5 mL ultrapure water and incubated for 12 h at 4 °C to obtain the signal marker (MoS2-PANI-Au-SP) through strong interaction between the amino group of SP and AuNPs. Finally, the signal marker was centrifuged and re-dispersed in 5 mL ultrapure water. For comparison, PANI-Au-SP nanocomposite was prepared by the same procedure without the addition of MoS2. 2.4. Fabrication of the DNA biosensor

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Before modification, bare glassy carbon electrode (GCE) was soaked into piranha solution (30% H2O2/98% H2SO4 = 1:3, v/v) for 30~60 min to clear from the impurities. After that, 0.3 and 0.05 µm alumina powders were used to polish the surface of GCE, respectively.

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It was then ultrasonically rinsed for several minutes with absolute ethanol and water

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separately.

The clean surface of GCE was coated with 10 μL suspension of P-MoS2-C60

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nanohybrid and dried at room temperature. Subsequently, the electrochemical deposition of gold nanoparticles (dpAu) on the above modified electrode was performed in HAuCl4 (1%) at

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-0.2 V for 30 s. Next, 21 μL of the prepared pdsDNA (1 μM) solution was dropped onto the

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dpAu/P-MoS2-C60/GCE surface and incubated for 16 h at room temperature. The modified electrode was then soaked into 3 wt% BSA solution to block the non-specific binding sites for

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1 h. Subsequently, 20 μL of the mixture solution containing 18 μL target DNA, 1 μL exo III (2000 U/mL) and 1 μL NEB buffer was dropped onto the surface of the modified electrode and incubated at 37 °C for 2 h. Finally, 10 μL of the signal marker was added and kept for another 2 h at room temperature. After that, the enhanced electrochemical signal was recorded by DPV in 0.1 M PB (pH 7.0).

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3. Results and Discussion 3.1. Characterization of nanomaterials The surface morphologies of the as-prepared nanocomposites were investigated by SEM. As shown in Fig. 1A, structure of PAMAM-C60 was quite different from the spherical C60. The single-layered PAMAM-MoS2 with large surface area (Fig. 1B) was also obtained by phase transfer method. In Fig. 1C, the P-MoS2-C60 nanohybrid showed larger specific surface

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area than that of sole C60 and MoS2. It can be seen from Fig. 1D that the porous MoS2-PANI was successfully synthesized, which is favorable to facilitate electron transfer between PANI and electrode surface. Moreover, a large number of shiny AuNPs were decorated on the

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surface of MoS2-PANI (Fig. 1E), which could provide abundant active sites to label with SP.

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In addition, elemental compositions of P-MoS2-C60 were analyzed by EDS, and the signature

of P-MoS2-C60 nanohybrid.

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peaks for C, Mo and S were observed in Fig. 1F, further indicating the successful preparation

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3.2. Electrochemical characterization of the biosensor The stepwise fabrication process of the DNA biosensor was monitored by CV and EIS.

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In Fig. 2A, the peak currents of P-MoS2-C60 modified GCE (curve b) and dpAu/P-MoS2-C60 modified GCE (curve c) were larger than that of bare GCE (curve a), which was attributed to

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the excellent conductivity of P-MoS2-C60 and dpAu. After pdsDNA (curve d) and BSA (curve e) were assembled onto the dpAu/P-MoS2-C60/GCE, electrochemical signal responses decreased because the non-conductive substances of pdsDNA and BSA could enormously hinder the electron transfer. When the modified electrode was incubated with the mixture solution of target DNA and exo III, the signal responses increased (curve f), attributing to the

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digestion of AP through exo III-assisted target recycling. As shown in Fig. 2B, the results of EIS were consistent with that of CV measurements, revealing that the biosensor was successfully constructed for target DNA detection. 3.3. Amplification performance of the designed DNA biosensor In this work, the control experiments were performed to assess the amplification performance of the biosensor and evaluate the specific roles of AP, exo III and signal marker.

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In Fig. 3, curve b in all panels represented the DPV response of the proposed biosensor incubated with 40 nM target DNA. As shown in Fig. 3A, when target DNA was absent, the cdsDNA could not be formed and AP would not be digested by exo III. Consequently, no

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significant peak current (curve a) was acquired because there was no released CP to hybridize

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with signal marker. In the absence of pdsDNA, DPV response was negligible (Fig. 3B, curve a), owing to the fact that signal marker could not be introduced onto the electrode surface. In

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addition, no remarkable electrochemical response signal was obtained without exo III (Fig.

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3C, carve a), because cdsDNA could exist stably on the electrode surface and the signal marker could not hybridize with CP. Moreover, it can be seen from Fig. 3D that the current

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response from signal marker of MoS2-PANI-Au-SP (curve b) was much larger than that from PANI-Au-SP (curve a), indicating MoS2 with large specific surface area and high

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conductivity can facilitate electron transfer and further improve the electrochemical response signal of PANI. As a result, the cascade signal amplification strategy was successfully applied to enhance the specificity and sensitivity of the designed biosensor. 3.4. Detection performances of the biosensor 3.4.1. DPV responses and calibration plot

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To evaluate the application of the proposed biosensor for Sul1 detection, different concentrations of target DNA were investigated and the results were recorded by DPV in 0.1 M PB (pH 7.0). As shown in Fig. 4A, peak current responses raised gradually along with the increased concentrations of target DNA. The corresponding calibration plot for target DNA was shown in Fig. 4B. The peak currents were proportional to the logarithm value of target DNA concentrations with a wide range from 40 fM to 40 nM, and the detection limit was

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estimated as 29.57 fM. The regression equation was I = 28.33 + 4.14 lgc with a correlation coefficient of 0.9942. In addition, the analytical performances of the developed biosensor for Sul1 detection were compared with those of other methods reported in the literatures, and the

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results were shown in Table S2 (see in Supplementary Material). The proposed DNA

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biosensor showed a wider linear range with lower detection limit, indicating that MoS 2-PANI nanocomposite and exo III-catalyzed target recycling could efficiently enhance the

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electrochemical response signal and improve the detection performances.

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3.4.2. Specificity, reproducibility and stability of the biosensor Non-complementary DNA and single-base-mismatched DNA were chosen to evaluate

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the specificity of the biosensor. The concentrations of target DNA and interfering oligonucleotides were 4 nM and 40 nM, respectively. As shown in Fig. 5, DPV responses of

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blank, single-base-mismatched DNA and non-complementary DNA were negligible compared with that of target DNA, while the signal responses from the mixture and target DNA were almost the same. These results revealed that the designed method can be applied to detect target DNA with good selectivity.

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The reproducibility of the electrochemical biosensor was evaluated by analyzing target DNA (4 nM) with five electrodes which were prepared in the same batch. The obtained peak currents were given in Fig. S1A (see in Supplementary Material). All of the electrodes exhibited similar peak currents with a relative standard deviation (RSD) of 0.83%, demonstrating that the reproducibility of the DNA biosensor was acceptable. To assess the long-term storage stability of the proposed DNA biosensor, the five

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electrodes prepared above were stored at 4 °C for 20 days. As shown in Fig. S1B (see in Supplementary Material), the peak currents were still retained more than 92.9% of its initial responses after 20 days, indicating that the DNA biosensor displayed excellent stability.

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3.4.3. Recovery test of the biosensor

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In order to preliminarily study the analytical reliability and application of the DNA biosensor, recovery test was performed by standard addition method in human serum

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according to our previous work [41] and the results were provided in Table S3 (see in

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Supplementary Material). The recovery rates of three target DNA concentrations (0.004, 0.4, 4 nM) were 98%, 103% and 103%, with RSD of 9.8%, 3.9% and 2.8%, respectively. These

samples.

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results demonstrated that the developed biosensor provided a potential application in complex

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3.4.4. Detection of Sul1 in Salmonella typhimurium PCR products of real samples are often used to evaluate the potential application of DNA

biosensors [39,42]. Therefore, the PCR products of Sul1 separated from Salmonella typhimurium and the synthetic target DNA with different concentrations (0, 0.0004, 0.04, 40 nM) were analyzed respectively by the proposed DNA biosensor. The details of bacterial

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culture and preparation of the PCR products were provided in Supplementary Material. Fig. 6A showed the peak currents of DPV responses for different concentrations of PCR products. It can be seen from Fig. 6B that peak currents of PCR products were in accordance with that of the synthetic target DNA, indicating the possibility of this method in practical application.

4. Conclusion

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This work constructed a novel electrochemical DNA biosensor for highly sensitive detection of Sul1 based on cascade amplification strategy. Firstly, P-MoS2-C60 nanohybrid as sensing platform could increase the effective specific surface area of the electrode and

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facilitate electron transfer. Secondly, MoS2-PANI nanocomposite displayed excellent conductivity and redox activity, greatly enhancing the electrochemical response signal. In

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addition, exo III-catalyzed target recycling further improved the specificity and sensitivity of

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the biosensor. Accordingly, the proposed biosensor showed low detection limit of 29.57 fM for target DNA with a wide linear dynamic range from 40 fM to 40 nM. It also exhibited good

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specificity, acceptable reproducibility and stability. In view of these results, this method provides an innovative technology platform for pathogen detection and can be applied for

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other targets by reasonable design of the corresponding nucleic acid sequences. Declaration of interests

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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Acknowledgements 13

This work is supported by National Natural Science Foundation of China (81601856), Ba Yu Scholars Funding Scheme in Colleges and Universities of Chongqing City (2019), Funds for High Level Young Science and Technology Talent Cultivation Plan in Chongqing Medical University (2019), the Third Batch of Young Backbone Teachers Funding Program in Colleges and Universities of Chongqing City ([2016] No. 53) and Funds for Young Science

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and Technology Talent Cultivation Plan of Chongqing City (cstc2014kjrc-qnrc00004).

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Biographies Huan You is a master degree candidate at College of Pharmacy, Chongqing Medical University. Her research interests are the application of nanomaterials and the fabrication of electrochemical aptasensors. Zhaode Mu is a professor of College of Pharmacy, Chongqing Medical University. She received her PhD degree from College of Laboratory Medicine, Chongqing Medical University in 2007. She has been working on the preparation of nanocomposites and the

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application of electrochemical biosensors.

Min Zhao is a postdoctor of College of Laboratory Medicine, Chongqing Medical University. She received her PhD from College of Chemistry and Chemical Engineering, Southwest

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University in 2017. Her research subject is the fabrication of metal nanoclusters for

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pharmaceutical and biomedical analysis.

Jing Zhou received her PhD degree from Chengdu Institute of Organic Chemistry, Chinese

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Academy of Sciences in 2015. She is a lecturer of College of Pharmacy, Chongqing Medical University. Her research interest is the synthesis of novel nanomaterials for the construction

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of electrochemical devices.

Yonghua Yuan received her PhD from College of Pharmacy, Chongqing Medical University in 2007. She is an associate professor of College of Pharmacy, Chongqing Medical University.

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She has been working on the preparation of nanocomposites and the application of

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electrochemical biosensors.

Lijuan Bai is an associate professor of College of Pharmacy, Chongqing Medical University. She received her PhD degree from College of Chemistry and Chemical Engineering, Southwest University in 2014. She has been working on the construction methodology of new electrochemical biosensors and their applications in biomedical analysis and disease diagnosis.

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Figure captions Scheme 1 (A) Illustration of the preparation process of P-MoS2-C60 and signal marker. (B) Schematic representation of the stepwise fabrication procedure of the constructed electrochemical DNA biosensor for target DNA. (C) The DNA hybridization principle of cdsDNA in the proposed sensing system, which provided the recognition condition for exo III

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to cleave AP from 3' to 5' terminal.

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Fig. 1. The SEM images of (A) PAMAM-C60, (B) PAMAM-MoS2, (C) P-MoS2-C60, (D)

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MoS2-PANI and (E) MoS2-PANI-Au. (F) The EDS picture of P-MoS2-C60.

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Fig. 2. (A) CV and (B) EIS of different modified electrodes in 5 mM Fe(CN)64+/3+ solution containing 0.1 M KCl: (a) bare GCE, (b) P-MoS2-C60/GCE, (c) dpAu/P-MoS2-C60/GCE, (d)

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pdsDNA/dpAu/P-MoS2-C60/GCE, (e) BSA/pdsDNA/dpAu/P-MoS2-C60/GCE and (f) the step

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e incubated with the mixture solution of target DNA and exo III.

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Fig. 3. DPV responses of various modified electrodes. Curve a was the biosensor incubated

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(A) without target DNA, (B) in the absence of pdsDNA, (C) without exo III and (D) with PANI-Au-SP. Curve b in all panels represented the DPV responses of the biosensor incubated with 40 nM target DNA in 0.1 M PB (pH 7.0).

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Fig. 4. (A) DPV responses of the biosensor incubated with different concentrations of target

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DNA (0, 0.00004, 0.0004, 0.004, 0.04, 0.4, 4, 40 nM) in 0.1 M PB (pH 7.0). (B) The calibration plots of DPV peak currents versus the logarithm of target DNA concentrations.

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The error bars represented the standard deviation of three parallel determination results).

Fig. 5. (A) DPV responses and (B) the corresponding histograms of the biosensor incubated

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with (a) mixture of single-base-mismatched DNA (40 nM), non-complementary DNA (40 nM) and target DNA (4 nM), (b) target DNA (4 nM), (c) blank, (d) single-base-mismatched DNA (40 nM) and (e) non-complementary DNA (40 nM). The error bars represented the standard deviation of three parallel determination results.

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Fig. 6. (A) The peak currents of DPV responses for different concentrations of PCR products

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(0, 0.0004, 0.04, 40 nM). (B) The contrast histograms of peak currents from spiked PCR products and the target DNA with same concentrations. The error bars represented the

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standard deviation of three parallel determination results.

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