Construction of sandwiched self-powered biosensor based on smart nanostructure and capacitor: Toward multiple signal amplification for thrombin detection

Journal Pre-proof Construction of sandwiched self-powered biosensor based on smart nanostructure and capacitor: toward multiple signal amplification for thrombin detection Yihan Wang, Futing Wang, Ziwei Han, Kejing Huang, Xuemei Wang, Zhenhua Liu, Shuyu Wang, Yunfei Lu

PII:

S0925-4005(19)31617-X

DOI:

https://doi.org/10.1016/j.snb.2019.127418

Reference:

SNB 127418

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

11 October 2019

Revised Date:

8 November 2019

Accepted Date:

13 November 2019

Please cite this article as: Wang Y, Wang F, Han Z, Huang K, Wang X, Liu Z, Wang S, Lu Y, Construction of sandwiched self-powered biosensor based on smart nanostructure and capacitor: toward multiple signal amplification for thrombin detection, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127418

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Construction of sandwiched self-powered biosensor based on smart nanostructure and capacitor: toward multiple signal amplification for thrombin detection

Yihan Wang1, 2,‡ Futing Wang1,‡ Ziwei Han1, Kejing Huang1*, Xuemei Wang2*,

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Zhenhua Liu1, Shuyu Wang1, Yunfei Lu1

College of Chemistry and Chemical Engineering, Xinyang Normal University,

State Key Laboratory of Bioelectronics (Chien-Shiung Wu Lab), School of Biological

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Xinyang 464000, China

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Science and Medical Engineering, Southeast University, Nanjing 210096, China.

*Corresponding author. Tel.: +86-376-6390611, +86-25-83792177

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E-mail address: [email protected] (K.J. Huang), [email protected] (X.M. Wang)

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‡Yihan Wang and Futing Wang contributed equally to this work.

Research highlights 

A self-powered electrochemical biosensor is fabricatedbased on capacitor signal amplification and EBFCs.

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The EBFCs paralleling with capacitor shows an increase of 18.4 times in

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sensitivity than pure EBFCs. 

The new biosensing platform shows wide linear range and low detection limit for thrombin detection.

Abstract A self-powered electrochemical sensing platform is fabricated to sensitively

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detect protein based on capacitor signal amplification and one-compartment glucose/O2 enzymatic biofuel cells (EBFCs). SnS2 nanoflowers/Au nanoparticles (AuNPs) and DNA-carbon nanotubes bioconjugate/AuNPs are prepared and modified on carbon

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paper as electrode substrates. The sandwiched structure to craft bioanodes is elaborately

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designed to dramatically enhance the open circuit voltage of the device, triggered by protein coupled with the DNA bioconjugate containing glucose oxidase and aptamer.

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The EBFCs-based aptasensor will subsequently catalyze glucose oxidation in the

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presence of the target thrombin (TB). More importantly, a commercial capacitor is also introduced into EBFCs to accumulate charge for a higher instantaneous current. This

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will result in a significantly amplified readout signal, which can be efficiently captured by digital multimeter. The open circuit voltage can be tuned with target TB

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concentration ranging from 0.02 to 5 ng/mL with a low detection limit of 7.90 pg/mL. Once the capacitor is charged by EBFCs, a sensitivity of 42.4 µA/(ng/mL) can be discharged with an increase of 18.4 times than that of pure EBFCs. Furthermore, this capacitor/EBFC hybrid device can be extended as a common platform for the quantitative determination of other proteins and has a great clinical analysis potential.

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Keywords: Self-powered electrochemical sensing; Capacitor; Enzyme biofuel cell; Signal amplification; Protein

1. Introduction Biosensors are devices sensitive to biological substances, and their concentrations

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can be converted to electrical signals for analysis and detection. Therefore, they have

been extensively used on biomolecule detection, information collection, environmental test and clinical medical analysis. However, different type of sensing devices consume

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energy at different rates, and, thus, requires an external source to power sensor. This in

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turn greatly limits the miniaturization and portability of the sensors [1]. Therefore, a adoptable equipment needs to be developed to solve this problem.

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On the other hand, enzymatic biofuel cells (EBFCs) with oxidoreductase as

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electrocatalysts can derive energy from sugars and alcohols combined with oxygen to produce electricity [2,3]. With the power of EBFCs to convert bioenergy directly to

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electricity, people have been using EBFCs as power sources for medical implants (e.g. cardiac pacemakers) [4], drug pumps [5], biosensors [6,7] and other devices [8].

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Compared with traditional electrochemical sensors, EBFCs-based sensing platforms have advantages with their self-powered energy supplies and can reduce the cost of production cost. Therefore, they have been widely applied in fields like immunoassay [9-12], biomolecular recognition [13,4], drug release [15,16], environmental monitoring [17] and others.

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Aptamer is a single-stranded nucleic acid molecule containing a specific oligonucleotide sequence [18], which can be folded with specific binding properties for high specific affinity and selectivity with their targets [19]. The aptamer has the advantages of low cost, high stability, high specificity, and has been broadly employed as the recognition component of target in biosensor [20]. As a specific serine protease, thrombin (TB) plays an important role in the

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coagulation cascade, which can cleave fibrinogen into fibrin and stimulate platelet

aggregation [21]. Low concentration of TB has a protective effect for cells, however, higher concentration can damage cells. Therefore, it is considered as a crucial

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biomarker for diseases such as cardiovascular disease, tissue repair and inflammation.

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Recently, many researchers have used EBFCs as power supply and biosensing technology to build the self-powered biosensing platform. For example, Zhu’s group

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developed N-doped hollow carbon nanosphere as electrode materials [22]. They

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developed a highly sensitive EBFC-based self-powered biosensor to detect miRNA-21 and miRNA-141 simultaneously. Li’s group prepared one EBFCs-based assay for

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antibiotic residue [7]. The self-powered sensor has the advantages of simple process, small size, low cost, and easy portability. However, EBFCs are susceptible to the

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limitations of low current and low power density. The most direct and effective way to solve the above problems is to introduce other energy sources or develop new electrode substrate materials. As a new type of environmentally friendly energy storage device, capacitors have characteristics with high power density, long cycling stability and no pollution, etc., and

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have been widely used in electric vehicles, portable electronic products, intermittent standby power supply and pulse launcher. Recently, Crooks research group has introduced capacitors into EBFC based self-powered sensors to improve the detection sensitivity [23]. In their devices, the presence of the target adenosine triggers redox reactions at the biological anode and cathode, generating a current that directly charges the capacitor in the loop, which then amplifies the signal when it connects to the

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multimeter.

In addition, enzyme loading amount and electron transfer rate are key issues for the performance of EBFCs. Nanomaterials usually have the superiorities of ultrahigh

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specific surface area, good electrical conductivity and the ability of promoting electron

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transfer, which are beneficial to the immobilization of more enzymes. The layered SnS2 nanosheets have been reported to accelerate electron transfer, where individual S-Sn-S

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layer is integrated with Van der Waals interactions to maintain excellent chemical

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stability. Li et al. prepared multi-walled carbon nanotubes-nanoflake-like SnS2 nanocomposite and applied it to modify electrode for glucose oxidase detection. The

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assay showed a low detection limit of 4 μM [24]. Lavanya and Sekar developed an electrochemical sensor based on SnO2-SnS2 nanocomposite for selective and

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simultaneous determination of depression biomarkers serotonin and tryptophan in the presence of ascorbic acid [25]. Carbon nanotubes (CNTs) as enzyme support also can boost the transfer of electrons, and have been used as electrode substrate materials for EBFCs [26]. Au nanoparticles (AuNPs) are mostly recommended owing to the fact that they can greatly increase the current response of the modified sensor with a good

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conductive ability and immobilization of biomolecular by Au-S bond [27], and have been widely used to construct aptamer sensors [28, 29]. Herein, a sandwich-structure self-powered electrochemical biosensing platform is constructed based on EBFC and DNA bioconjugates to detect TB. For the biocathode, bilirubin oxidase (BOD) is attached on the modified carbon paper (CP) with CNTs/AuNPs (Scheme 1A). As shown in Scheme 1B, SnS2/AuNPs modified CP

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electrode is used as electrode substrate to immobilize aptamer 1 (tApt1), and then the

specific site is blocked by 6-mercapto-1-hexanol (MCH). In Scheme 1C, DNACNTs/AuNPs bioconjugate is further prepared to obtain a sandwich-structure carrier.

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Typically, glucose oxidase (GOD) and aptamer 2 (tApt2) are immobilized on the

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CNTs/AuNPs scaffold. And bovine serum albumin (BSA) is applied to block active site. Subsequently, in the presence of target TB exists, the formation of sandwiched structure

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through DNA-CNTs/AuNPs bioconjugate modified GOD connected with anode

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substrate is used to catalyze the oxidation of glucose. BOD is employed as cathodic enzymes for catalyzing oxygen reduction to generate an electrical signal. In order to

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further improve the performance of EBFC, a capacitor is introduced to obtain a large instantaneous current that is readily read out by digital multimeter. The proposed

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sensing platform integrates the ingenious merits of self-powered biosensors, nucleic acid aptamers and capacitors, displaying a great potential for clinical analysis of proteins.

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Scheme 1. Schematic illustration of fabrication of the biocathode (A), the bioanode (B),

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the DNA-CNTs/AuNPs bioconjugate (C) and the self-powered biosensor (D).

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2. Experimental Section 2.1 Materials and reagents

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SnCl4·5H2O, anhydrous ethanol, L-Cysteine, and sodium citrate were purchased

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from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Nhydroxysuccinimide (NHS), immunoglobin G (IgG), hemoglobin (HB), poly (diallydimethylammonium) HAuCl4·3H2O,

1-ethyl-3-(3-dimethyl-aminopropyl)

carbodiimide

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(PDDA),

hydrochloride (EDC) and BSA were obtained from Macklin Biochemical Technology

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Co., Ltd. (Shanghai, China). TB, BOD from Myrothecium verrucaria (E.C. 1.3.3.5), GOD from Aspergillus niger (E.C. 1.1.3.4) and prostate specific antigen (PSA) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). All DNA sequences were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) [30]. The sequences are showed in Table 1. The DNA sequences were dissolved in 10 mM Tris-HCl

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solutions (0.1 M NaCl, pH 7.4). 0.1 M phosphate buffer solutions (PBS, pH 7.4) consisting of Na2HPO4 and NaH2PO4 was adopted for the supporting electrolyte. Table 1. Sequences of the oligonucleotides. Oligonucleotides

Sequence

tApt1

5’-NH2-(CH2)6-GGT TGG TGT GGT TGG-3’

tApt2

5’-NH2-(CH2)6-AGT CCG TGG TAG GGC AGG TTG

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GGG TGA CT-3’

2.2 Apparatus and measurements

The morphologies of the material were characterized by S-4800 scanning electron

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microscope (SEM, Hitachi, Japan) and Tecnai G2 F20 transmission electron

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microscope (TEM, FEI, USA). X-ray photoelectron spectroscopy (XPS) was recorded

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by a K-ALPHA X-ray photoelectron spectrometer (Thermo Fisher Scientific, USA). The zeta potential was detected by Zetasizer Nano ZS90 nanoparticle size

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potentiometer (Malvern, China). The instantaneous current was read out by a F12E+ high precision DMM (Fluke, China). Electrochemical measurements, including linear

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sweep voltammetry (LSV), and cyclic voltammetric (CV) were tested on the CHI 660E electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China) with a

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three-electrode including the Pt wire, Ag/AgCl, the modified bioanode or biocathode as auxiliary electrode, reference electrode and working electrode, respectively. The open circuit voltage (EOCV) was detected on the CHI 660E electrochemical workstation by using a two-electrode system.

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2.3 Synthesis of SnS2/AuNPs The SnS2 nanoflowers have been reported with large specific surface area and good conductivity [31]. In this work, they were prepared as follows. 0.5 g L-Cysteine and 0.7 g SnCl4·5H2O were dissolved in 80 mL distilled water. Subsequently, this mixture was added in a 100 mL autoclave and kept under 160 ℃ for 24 h. After naturally cooled down to room temperature and alternately washed with anhydrous ethanol and

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distilled water, the yellow precipitates were collected and dried under vacuum at 60 ℃ for 8 h to obtain SnS2 nanoflowers. The SnS2 nanoflowers were then dissolved in

distilled water to form 1 mg/mL SnS2 solution. Subsequently, 500 μL 1 mg/mL of SnS2

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was added into 1 mL HAuCl4·3H2O solution, and the homogeneous mixture was

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obtained by ultrasonic treatment for 10 min and then heated. When the temperature reached 96 ℃, 500 µL of sodium citrate (1%) was rapidly added and stirred for 15 min.

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Then, the precipitate was collected and then dissolved in distilled water to form 1

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mg/mL SnS2/AuNPs solution.

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2.4 Synthesis of DNA-CNTs/AuNPs bioconjugate The CNTs were pretreated according to the literature [32]. 16 mg CNTs were then

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dissolved in 8 mL PDDA (1%) (containing 0.02 mol/L NaCl) and sonicated for 0.5 h to produce positively charged CNTs/PDDA. The mixture was centrifuged and washed with distilled water. Subsequently, CNTs/PDDA and 6 mL AuNPs (containing 1 mg/mL EDC/NHS) were stirred overnight at room temperature, and then centrifuged to remove the excessive AuNPs. 400 µL GOD (5 mg/mL) and 75 µL tApt2 (1 µmol/L) were added

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into the solution, and then incubated at 4 ℃ for 12 h. Then, the nonspecific binding sites of AuNPs were blocked with 40 µL of 1 mmol/L BSA for 30 min, and washed by PBS. Finally, the precipitate was dissolved in PBS to obtain the DNA-CNTs/AuNPs bioconjugate, which was stored at 4 ℃.

2.5 Preparation of bioanodes

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50 µL of 1 mg/mL SnS2/AuNPs was dropped on CP (0.5 cm × 0.5 cm) and dried

at vacuum under 37 ℃ for 120 min. The electrodes were immersed in 1 mg/mL EDC/NHS for 0.5 h to activate the carboxyl groups of AuNPs and the excess EDC/NHS

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was washed by distilled water. Then, 75 µL of 1 µmol/L tApt1 was applied on it and

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layed aside overnight to obtain tApt1/SnS2/AuNPs modified CP electrode. Subsequently, it was incubated in 20 µL 1 mmol/L mercaptoethanol for 0.5 h, and then

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washed by water to obtain a bioanode, which was stored at 4 ℃. Thereafter, 40 μL TB

CNTs/AuNPs

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were applied on electrode, and kept at 37 ℃ for 80 min. Finally, 50 μL of DNAbioconjugate

was

coated

on

the

resulted

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TB/MCH/tApt1/AuNPs/SnS2/CP and kept at 37 ℃ for 120 min.

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2.6 Preparation of biocathodes 50 µL of 1 mg/mL CNTs/AuNPs was added onto the CP (0.5 cm × 0.5 cm) and

dried under vacuum at 37 ℃ for 2 h. CNTs/AuNPs modified CP electrode was then immersed in 1 mg/mL EDC/NHS for 0.5 h to activate the carboxyl groups of AuNPs. The excess EDC/NHS was washed by distilled water. Subsequently, 75 µL of 8 mg/mL

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BOD solution was applied on the electrode and incubated at 4 ℃ for 12 h. The obtained BOD/CNTs/AuNPs modified biocathode was stored at 4 ℃ for standby application.

2.7 Construction of self-powered biosensors and measurements A single-chamber glucose/O2 EBFC was constructed with modified anode and cathode (Figure S1), and the supporting electrolyte was 10 mL of 0.1 M PBS with 5

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mM glucose (pH 7.4). In the absence of TB, 50 µL of DNA-CNTs/AuNPs bioconjugate was applied on the electrode and the voltage was recorded as E0OCV. When TB was added, 50 µL of DNA-CNTs/AuNPs bioconjugate was applied on the electrode and

3. Results and Discussion

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kept at 37 ℃ for 2 h. Then, the voltage was measured as EOCV.

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3.1 Characterization of SnS2 and SnS2/AuNPs nanostructures

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The nanostructure and morphology of SnS2 were studied with SEM, TEM and high-resolution TEM (HRTEM). Figure 1A shows SnS2 with a uniform flower-like

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morphology with a diameter of about 500 nm. It can be obviously observed from Figure 1B that the individual SnS2 was composed of many ultrathin nano-petal structures.

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Figure 1C and 1D show SEM and TEM images of the SnS2/AuNPs. Clearly, AuNPs are evenly distributed on SnS2 nanoflowers. The HRTEM image of SnS2/AuNPs (Figure 1E) shows that the lattice fringes of AuNPs and SnS2 are 0.24, 0.28 and 0.32 nm, corresponding to the (111) planes of Au, the (101) and (100) planes of SnS2, respectively. Compared with bare CP (Figure 1F inset), the SnS2/AuNPs are uniformly

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coated upon CP electrode (Figure 1F).

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Figure 1. (A) SEM and (B) TEM of SnS2; (C) SEM, (inset, HRSEM); (D) TEM and

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(E) HRTEM of SnS2/AuNPs; (F) SEM of SnS2/AuNPs/CP (inset, SEM of bare CP).

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Figure 2A shows the X-ray powder diffractometer (XRD) patterns of SnS2 and

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SnS2/AuNPs samples. All the diffraction peaks of the SnS2 samples are consistent with the standard card of the hexagonal phase SnS2 (JCPDS: No.023-0677). No impurity

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peaks are observed, indicating high product purity. In the XRD pattern of SnS2/AuNPs, the Au peaks are located at 37.54°, 43.84°, 64.08°, and 77.43°, corresponding to the

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(111), (200), (220), and (311) planes. The pure SnS2 and SnS2/AuNPs composites show practically identical diffraction peaks, demonstrating the inexistence of phase transformation after AuNPs reduction. Raman spectroscopy was further used to confirm the successful preparation of SnS2 (Figure 2B). The characteristic peak of SnS2 is located at 311 cm-1, corresponding to the A1g mode. The compositions of the

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SnS2/AuNPs sample were studied by SEM-energy dispersive X-ray (EDX). The results in Figure 2C demonstrate that C, O, Sn, S and Au elements exist in the SnS 2/AuNPs sample. The presence of Au indicates that Au is successfully reduced. The atomic ratio of the Sn and S elements is about 1:1.9, which is basically consistent with theoretical stoichiometric value of SnS2. The results of the Brunauer-Emmett-Teller (BET) analysis of the SnS2 nanoflowers are shown in the Figure 2D. The specific surface area

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of the SnS2 nanoflowers is detected as 26.13 m2/g. Based on the Barrett Joyner-Halenda (BJH) method, the inset of Figure 2D shows the SnS2 has main pore size of 6.98 nm. Figure 3A shows the XPS spectra of SnS2 nanoflowers and SnS2/AuNPs composite.

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Four elements of Sn, S, C, and O are detected in the SnS2 sample, and the C 1s peak is

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present at 283.69 eV, which may derive from conductive adhesive. In the highresolution XPS spectrum of Sn 3d (Figure 3B), the peaks of Sn 3d 5/2 and Sn 3d3/2 are

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observed at 487.04 and 495.45 eV, respectively, which differs by 8.41 eV, indicating Sn

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exists in the form of +4 valence [33]. The S 2p (Figure S3C) shows two states of S 2p3/2 and S 2p1/2 at 161.72 and 162.91 eV, respectively. Au (Figure 3D) display two peaks at

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83.85 eV and 87.48 eV belonging to 4f7/2 and 4f5/2, respectively.

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Figure 2. XRD patterns of SnS2 and SnS2/AuNPs (A); Raman spectrum of SnS2

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nanoflowers (B); EDX pattern of SnS2/AuNPs (C); (D) N2 adsorption-desorption

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isotherm of the SnS2 nanoflowers and pore size distribution curve (inset).

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Figure 3. XPS survey spectra of SnS2 and SnS2/AuNPs (A); Sn 3d spectra (B), S 2p

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spectra (C) and Au 4f spectra (D) of SnS2/AuNPs.

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3.2 Characterization of CNTs and CNTs/AuNPs The morphology of CNTs/AuNPs was characterized by SEM and TEM. Figure 4A

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shows a smooth structure of CNTs with diameter of approximately10 nm, which is

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consistent with the TEM image (Figure 4B). The structure of CNTs/AuNPs is displayed in Figure 4C and D. Clearly, the activated AuNPs are well-distributed on CNTs, which is beneficial to the immobilization of enzymes on the CNTs. The morphology of CNTs/AuNPs modified electrode is imaged by SEM (Figure 4E). CNTs/AuNPs is uniformly grown on CP electrode. The zeta potentials of CNTs (a), functionalized CNTs (b), AuNPs (c) and CNTs/AuNPs (d) are measured (Figure 4F). The zeta potential of 15

CNTs is detected as -17.5 mV. After treated with PDDA, the zeta potential value of CNTs reaches to 24.63 mV. The zeta potential of AuNPs is -38.77 mV. When AuNPs are combined with CNTs by electrostatic force, the related zeta potential drops to -18.6 mV. This result strongly demonstrates the successful assembly of CNTs/AuNPs composites. Figure 5A shows the XRD patterns of the CNTs and CNTs/AuNPs composites. A

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peak of the CNTs at 25.14° is corresponded to the (002) plane of graphite. The Au peaks at 37.57°, 43.65°, 64.18°, and 77.10° are indexed to the (111), (200), (220), and (311) planes. The C 1s, O 1s and Au 4f peaks are observed from the XPS spectra (Figure 5B),

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demonstrating the successful preparation of CNTs/AuNPs.

Figure 4. SEM (A) and TEM (B) of CNTs; SEM (C) and TEM (D) of CNTs/AuNPs; SEM of CNTs/AuNPs/CP (F); (F) Zeta potential of CNTs (a), functionalized CNTs (b), AuNPs (c) and CNTs/AuNPs (d).

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Figure 5. XRD pattern (A) and XPS survey spectra (B) of CNTs and CNTs/AuNPs.

3.3 Electrochemical characterization

The various modification steps of bioanode and biocathode were tested by

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electrochemical impedance spectroscopy (EIS) with 1 M [Fe(CN)6]3-/4- (containing 0.1

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M KCl) as electrolyte solution between 0.1 Hz-100 kHz (Figure 6A). The inset is the equivalent circuit for the fitting the data. Compared with bare CP (curve a), the Rct value

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of the SnS2/AuNPs modified CP electrode (curve b) significantly decreases on account

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of its superior conductivity. When the negatively charged tApt1 is fixed on the electrode (curve c), the Rct value greatly increases because of the electrostatic repulsion between

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the tApt1 and [Fe(CN)6]3-/4-. After MCH is applied on the electrode (curve d), the Rct further increases. When the electrode is incubated with TB (curve e), an increased Rct

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value is observed. Further modified with DNA-CNTs/AuNPs bioconjugate (curve f) leads to a further increased Rct value. Similarly, EIS is used to study the assembly process of biocathodes. As shown in Figure 6B, bare CP shows a low Rct value (curve a), and the Rct value decreases when CNTs/AuNPs is coated on the CP electrode due to the superior conductivity of CNTs/AuNPs. However, Rct increases after incubating with

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BOD (curve c), which is due to the high spatial steric resistance of BOD. These results

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indicate that the bioanodes and biocathodes are successfully prepared.

Figure 6. (A) EIS of CP (a), SnS2/AuNPs/CP (b), tApt1/SnS2/AuNPs/CP (c), MCH/tApt1/SnS2/AuNPs/CP

(d),

TB/MCH/tApt1/SnS2/AuNPs/CP

(e),

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CNTs/AuNPs bioconjugate/TB/MCH/tApt1/SnS2/AuNPs/CP (f); (B) EIS of CP (a), CNTs/AuNPs/CP (b) , BOD/CNTs/AuNPs/CP (c). Inset of (A) is the Randles circuit

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model for the various electrodes: RS, electrolyte solution resistance; Ret, element of interfacial electron transfer resistance; ZW, Warburg impedance resulting from the

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diffusion of ions.

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Cyclic Voltammetry (CV) tests for bioanodes and biocathodes were performed at a sweep rate of 100 mV/s. As displayed in Figure 7A, when the target TB is not present

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in the solution, the DNA-CNTs/AuNPs bioconjugate is separated from the anode electrode. Even if glucose is oxidized on the bioconjugate, the released electrons by

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GOD can not be transferred to the external circuit through the anode substrate. Therefore, the redox current measured in the substrate without glucose (PBS 7.4, curve a) and the substrate containing 5 mmol/L glucose (PBS 7.4, curve b) does not significantly change. In Figure 7B, when 2 ng/mL of target TB exists, the sensor forms a pathway because tApt1 and tApt2 can specifically recognize TB to form a sandwich

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structure. When glucose does not contain in the substrate solution, two obvious redox peaks appear around -0.5 V, which is the characteristic peak of GOD. Nevertheless, in presence of 5 mmol/L glucose in substrate solution(curve b), an increased oxidation peak and decreased reduction peak occurs, which is related to O2 mediated glucose oxidation on the electrode surface. Meanwhile, LSV is also used to confirm the formation of sandwich structure. The bioanode is subjected to LSV testing (1 mV/s)

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ranging from -0.8 to 0 V (Figure 7C). When the sandwiched structure is formed, the

DNA-CNTs/AuNPs bioconjugate containing GOD is immobilized on the surface of the bioanode, leading to a higher oxidation peak current in the glucose solution (curve b)

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than that of pure buffer solution (curve a). Figure 7D shows CV of the

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BOD/CNTs/AuNPs/CP in air saturated solution (curve a) and O2 saturated solution (curve b). Under O2 saturation condition, the redox reaction is enhanced and its

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reduction potential is approximately at 520 mV, which is similar to the redox potential

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of the T1 Cu site of BOD, indicating that it is the first electron acceptor of BOD.

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Figure 7. (A) CVs of DNA-CNTs/AuNPs bioconjugate/MCH/tApt1/SnS2/AuNPs

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bioanode in PBS (pH 7.4) without glucose (a) and with 5 mmol/L glucose (b); CVs (B) and LSV (C) of DNA-CNTs/AuNPs bioconjugate/MCH/tApt1/SnS2/AuNPs bioanode

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after incubation with 2 ng/mL TB in PBS (pH 7.4) without glucose (a) and with 5 mmol/L glucose (b); (D) CVs of BOD/CNTs/AuNPs biocathode saturated with air (a)

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and O2 (b) in PBS (pH 7.4).

3.4 Optimization of experimental conditions The experimental conditions were optimized by CV. The effect of concentration

of tApt2 was studied (Figure 8A). When it increases from 0.4 µmol/L to 1 µmol/L, the bioelectrocatalytic

current

enhances,

indicating

more

DNA-CNTs/AuNPs

bioconjugates are fixed on the anode. There is no obvious change in the concentration 20

range of 1-1.6 µmol/L, indicating DNA-CNTs/AuNPs bioconjugates are saturated on the electrode. Therefore, 1 µmol/L of tApt2 is chosen. Figure 8B shows bioelectrocatalytic currents increase with the enhancing incubation time of DNACNTs/AuNPs bioconjugate, and it tends to be stable when time is larger than 2 h. So,

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the incubation time of DNA-CNTs/AuNPs bioconjugates of 2 h is used.

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Figure 8. The degree of the upward movement at -0.6 V in the CVs for the bioanode in PBS (pH 7.4) versus (A) tApt2 concentration and (B) the incubation time between TB

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and the DNA-CNTs/AuNPs bioconjugate.

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3.5 Self-powered electrochemical biosensing of TB

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Different concentrations of target TB were detected with self-powered sensors under optimized conditions. As shown in Figure 9A, as the concentration of TB increases, a large number of DNA-CNTs/AuNPs bioconjugates are immobilized on the bioanodes, resulting in the gradually enhanced EOCV value. The relationship between EOCV and TB concentration is shown in Figure 9B. It can be seen from the inset that the TB concentration exhibits a good linear ranging from 0.02 to 5 ng/mL. The linear 21

equation is EOCV = 0.08 logc + 0.94 (R2 = 0.995) with a detection limit of 7.90 pg/mL (0.22 pmol/L) (S/N=3). The self-powered sensor is compared with other methods in

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Table 2. The results show that our biosensor displays good analytical performance.

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Figure 9. (A) EOCV of the self-powered biosensor of TB with various levels(a-i: 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 ng/mL); (B) the relation of EOCV and TB levels, and inset

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shows the calibration plot; Selectivity (C) and stability (D) of the biosensor.

Table 2. Different strategies for TB detection. 22

Strategy

Linear range

LOD

References

DNA bioconjugate and EBFC

0.6 pM-0.1 nM

0.22 pM

This work

27 aM

[28]

Sandwich-type electrochemical aptasensor based on hybridization chain reaction

10 fM-0.1 nM 0.1 pM-10 nM

11.6 fM

[30]

Nano-engineered platforms

0.5 pM-3.7 nM

58 pM

Catalytic assembly and enzyme-free

0.05 nM-100 nM

23.6 pM

Aptamer based molecular machinery Self-assembly and catalyzing cleavage

5 pM-1 nM

1.7 pM

5 pM-5 nM

1.8 pM

[34] [35] [36] [37]

Target-triggering nicking enzyme signaling strategy

1 pM-30 nM

0.32 pM

[38]

Self-assembled multilayers

1 pM-160 nM

0.156 fM

[39] [40]

A sandwich-type electrochemical aptasensor

homogeneous

10 pM -1 μM 0.1 fM-1.0 nM

3.6 Specificity, reproducibility and stability

1 pM

0.03 fM

[41]

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Aptasensor based on a capacitive transducer

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Tetrahedral DNA probe-hybridization chain reaction

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Figure 9C shows the selectivity of the biosensor by using HB (0.2 g/mL), PSA (2

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ng/mL) and IgG (20 ng/mL) as interference agent, respectively. The results display that the detection signal has no significant difference compared with the blank sample.

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However, when 2 ng/mL TB is added, the detection signal greatly increases. The results indicate that the biosensor has good selectivity. Five independent experiments are

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designed to measure the EOCV response of each sensor to study the reproducibility of

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the sensor. 5.1% of RSD is obtained indicating a good reproducibility of the biosensor. In addition, the stability of EBFC is investigated under continuous reaction time (Figure 4D). After continuous operation for 2000 s, EOCV remains about 98%, demonstrating the self-powered biosensor has a good stability.

3.7 Real samples analysis 23

A recovery experiment was carried out to study its application potential of developed method. Human serum samples were obtained from the affiliated hospital of Xinyang Normal University. Firstly, the serum samples were precipitated for 2.5 h to obtain the supernatant, which was further purified through extracting, and centrifuged for three times at 8000 rpm. Then, various concentrations of target TB were added into the diluted 10-fold purified serum with PBS buffer solution. As shown in Table 3, the

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recoveries and RSDs are 96.24%-108.15% and 3.96%-8.25%, respectively, indicating that the self-powered sensor has a superior reliability in real samples determination.

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Table 3. Determination of TB in serum samples (n=3). Added

Found

(pg/mL)

(pg/mL)

1

10

10.19

2

20

3

50

4

100

RSD (%)

Recovery (%)

4.13

101.90

21.63

5.97

108.15

48.12

8.25

96.24

102.13

7.89

102.13

3.96

99.94

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Sample

200

199.87

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5

3.8 Capacitor/EBFC hybrid device

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Introducing other sources of energy into EBFCs is an important way to improve its current and power density. For further improving the analytical performance of our biosensor, the developed EBFC was combined to a commercial capacitor. As shown in Figure 10A, the circuit is assembled on the breadboard (Figure 10B) and the current value is measured by the DMM. In the capacitor/EBFC hybrid device, the capacitor is

24

charged by the EBFC, and the DMM is introduced to detect the current value. When switches a and b are closed, c is open, and the short-circuit current of GOD/BOD EBFC is measured by DMM. When switches b and c are closed, a is opened. The capacitor current before charging is measured by the DMM. When switches a and c are closed, and b is opened, the capacitor is charged by the EBFC. When the switches b and c are closed, and a is opened, the instantaneous current of the capacitor after charging

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completing can be measured by DMM.

Figure 10. (A) Circuit diagram of the GOD/BOD EBFC combined with commercial

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capacitor and (B) a photograph of a breadboard for measuring the output current of the device by using a DMM. The optical photographs of current values of uncharged capacitor (C), GOD/BOD EBFC after incubation with 2 ng/mL TB (D), and capacitor after charging (E).

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A 1000 µF commercial capacitor was charged with the constructed GOD/BOD EBFC. After charging for 70 s, it reached the highest instantaneous current. As shown in Figure 11A, when the TB concentration is 2 ng/mL, the time-current curve of 1000 µF commercial capacitor is definitely emerged after charged by self-powered biosensor. Figure 10C shows the current value of the uncharged capacitor. Figure 10D displays the current value of the self-powered biosensor when the TB concentration is 2 ng/mL.

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Figure 10E shows the current value of the capacitor after charging. As shown in Figure 11B, the discharge current linearly increases with the increased TB concentration. When the capacitor is charged by EBFCs, it can be discharged with a sensitivity of 42.4

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µA/(ng/mL) after discharging (Figure 11C), which delivers an increase of 18.4 times

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than that of the pure EBFCs (2.3 µA/(ng/mL)). The above analysis indicated that the capacitor could provide a higher instantaneous current to enhance the sensitivity of

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device after connected with the self-powered biosensor.

26

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Figure 11. (A) The time-current curve of 1000 µF commercial capacitor during the

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charging process with the sensor in 2 ng/mL TB；Relationship between current response and concentration of TB without (B) and with the amplification by a capacitor (C). The

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inset shows the calibration curve.

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4. Conclusion

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A novel ultrasensitive self-powered biosensor is developed based on GOD/BOD EBFC for TB detection. The large specific surface area of SnS2/AuNPs combined with the superior conductivity of CNTs/AuNPs modified CP are employed as bioanode and biocathode respectively, which can provide numerous binding sites for aptamer and enzyme immobilization, and accelerate electrons transferring. Meanwhile, the good biocompatibility of the electrode material supplies a suitable microenvironment for the 27

EBFCs, improving the stability of the self-powered biosensor. The sandwich-structure of the aptamer-target-aptamer can anchor the DNA-CNTs/AuNPs bioconjugate on the surface of the bioanode and significantly enhance the performance of the sensor. The assay therefore exhibits a wide linear range (0.02-5 ng/mL) and a low detection limit (7.90 pg/mL). In addition, the specific binding of aptamer and TB shows an excellent selectivity. More importantly, a capacitor/EBFC hybrid device is elaborately designed

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for the amplification of the output current to further improve detection sensitivity.

Simultaneously, the adopted DMM is cheaper than other electrochemical detection devices, resulting in the lower cost of detection. The sensing platform combines the

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advantages of self-powered biosensors, nucleic acid aptamers and capacitor. Therefore,

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great clinical analysis potential.

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it can be extended for other protein detection by simple replacing aptamers and has a

Declaration of Interest Statement

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The authors declared that they have no conflicts of interest to this work.

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Acknowledgments

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This work was supported by the National Natural Science Foundation of China (21475115), Henan Provincial Science and technology innovation team (C20150026), and Nanhu Scholars Program of XYNU.

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Yi-Han Wang is a graduate student at Xinyang Normal University. Her current research is electrochemical biosensors. Fu-Ting Wang is a graduate student at Xinyang Normal University. Her current research is electrochemical self-powered biosensors. Zi-Wei Han is an undergraduate student at Xinyang Normal University. Her current research is electrochemical self-powered biosensors.

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Ke-Jing Huang received his PhD in 2006 from Wuhan University. Presently, he is a

professor at Xinyang Normal University. His research interests include 2D nanomaterial preparation, supercapacitor electrode materials and electrochemical

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biosensors.

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Xuemei Wang is currently a full professor of Biomedical Engineering, Southeast University. She obtained her PhD in Chemistry from Nanjing University, China in 1994

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and became a lecturer in Nanjing University in 1995. She was an Alexander von

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Humboldt Fellow in the Chemistry Department, University of Saarland, Germany, before she joined the State Key Laboratory of Bioelectronics, Southeast University in

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1998. Her research focuses on bioelectronics and biosensors, biomaterials for

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multimode bioimaging and nanomedicine.

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