Boosting performance of self-powered biosensing device with high-energy enzyme biofuel cells and cruciform DNA

Journal Pre-proof Boosting Performance of Self-Powered Biosensing Device with High-Energy Enzyme Biofuel Cells and Cruciform DNA Fu-Ting Wang, Yi-Han Wang, Jing Xu, Ke-Jing Huang, Zhen-hua Liu, Yun-fei Lu, Shu-yu Wang, Zi-wei Han PII:

S2211-2855(19)31017-1

DOI:

https://doi.org/10.1016/j.nanoen.2019.104310

Reference:

NANOEN 104310

To appear in:

Nano Energy

Received Date: 1 September 2019 Revised Date:

4 November 2019

Accepted Date: 17 November 2019

Please cite this article as: F.-T. Wang, Y.-H. Wang, J. Xu, K.-J. Huang, Z.-h. Liu, Y.-f. Lu, S.-y. Wang, Z.-w. Han, Boosting Performance of Self-Powered Biosensing Device with High-Energy Enzyme Biofuel Cells and Cruciform DNA, Nano Energy, https://doi.org/10.1016/j.nanoen.2019.104310. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.

Boosting Performance of Self-Powered Biosensing Device with High-Energy Enzyme Biofuel Cells and Cruciform DNA Fu-Ting Wang, Yi-Han Wang, Jing Xu, Ke-Jing Huang*, Zhen-hua Liu, Yun-fei Lu, Shu-yu Wang, Zi-wei Han College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China

*Corresponding author. E-mail address: [email protected] (K.J. Huang);

1

Abstract A self-powered microRNA (miRNA) biosensing device is fabricated with high-energy enzymatic biofuel cell (EBFC) and cruciform DNA (cDNA). Sulfur-selenium co-doped graphene/gold nanoparticles (S-Se-GR/AuNPs) is synthesized and used as supporting substrate of biocathode and bioanode. The glucose oxidase (GOD) molecules are bonded to carboxyl-functionalized AuNPs through the condensation reaction between the amino groups in enzyme and carboxyl groups on the AuNPs to form the bioanode. The ultra-thin porous carbon shell/AuNPs-complementary strand of cDNA (UPCS/AuNPs-cDNA) is meticulously designed. The potassium ferricyanides are then inserted in mesoporous UPCS/AuNPs as the biocathode electron acceptor, and the cruciform DNA bioconjugate is synthesized as signal amplifier. When target miRNA is added, the hybridization reaction happens between miRNA and capture probe DNA on the biocathode to open the capture probe DNA chain. Cruciform DNA bioconjugate is immobilized onto the biocathode through base pairing with the capture DNA on the biocathode, which can release electron acceptor [Fe(CN)6]3-, resulting in the dramatically increase of the open circuit voltage of the EBFCs. The self-powered biosensor responds linearly in the miRNA level range of 0.5-10000 fM with a detection limit of 1.5×10-16 mol L-1. Detection of miRNA in spiked serum samples is also realized with the self-powered biosensor, demonstrating its great potential in the clinical applications. Keywords: Enzymatic biofuel cell; Self-powered biosensors; MicroRNA; Cruciform DNA; Signal amplification

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1. Introduction As a kind of fuel cells, enzymatic biofuel cells (EBFCs) [1,2] have rapid developed recently due to their unique merits of simple device requirements. Even more crucial, EBFC could provides sustainable renewable energy under moderate condition [3-5]. Electrochemical self-powered sensing device based on EBFC [6,7] has been the focused area of frontier research due to the fascinating characteristics of subminiature sizes, simple fabrication process, low cost and good anti-interference ability, and thus are widely applied in biomolecular assays [8,9], cytosensing [10,11], immunoassays [12,13] and drug release fields [14]. Among them, the design effective approaches of self-powered sensing assay is primarily including inhibition effect, substrate effect and enzyme activity [15]. However, the biorecognition of biofuel by enzymes or oligonucleotide probe on electrodes may require complicated experimental steps, leading to the low enzyme loading level and therefore obviously damages the EBFCs’ performance. Thus, it is very important to fabricate new sensing platforms of EBFC for improving the enzyme loading quantities [16]. Immobilizing enzymes on the conductive substrates with large surface area is a simple but effective method to improve the enzyme loading quantities [17]. Carbon materials have become a research focus due to their effectiveness. When graphene (GR) or carbon nanomaterials are applied as enzymes scaffolds, EBFCs usually show attractive characteristics [18-20]. Recently, it has been reported that carbon materials doped with heteroatoms can markedly enhance their electrochemical activities, and greatly facilitate the electron transfer rates. For example, Gai et al. [21] prepared a

3

heteroatom doping carbon nitride nanosheets and used for microbial fuel cells. It greatly enhanced the electron transfer rate and provided abundant space for enzymes loading. Au nanoparticles (AuNPs) are mostly recommended owing to the fact that they can greatly increase the current response of the modified electrode with a good conductive ability and immobilization of biomolecular by Au-S bond, and have been widely used to construct electrochemical biosensors [22, 23]. The abnormal expression of miRNA in tissues by the injury of cells or tissues in serum may develop into a oncogene [24]. Therefore, a number of methods have been used to detect miRNAs in biological samples, including Northern blot analysis [25], magnetic relaxation switch [26], micro-arrays [27,28], surface-enhanced Raman spectroscopy [29]and high-throughput sequencing techniques [30]. However, these methods usually are expensive, depending on complicated configuration and sophisticated instruments, and showing low sensitivity. Electrochemical methods are simple and sensitive, and have been widely used for miRNAs detection [31,32]. Recently, in order to further improve the sensitivity of the electrochemical biosensor, various amplification strategies are used in the construction of biosensors, such as duplex specific nuclease-catalyzed reaction [33], ligase chain reaction [34] and catalytic hairpin assembly [35]. Attracted by the fascinating qualities of EBFC [36], in this work, an EBFC based electrochemical self-powered biosensor for sensitive detection of miRNAs is developed

by

using

sulfur-selenium

co-doped

graphene/gold

nanoparticles

(S-Se-GR/AuNPs) as supporting substrate, which can provide sufficient space for the

4

enzyme loading and facilitate the electron transfer. A cruciform DNA bioconjugate is prepared to improve space effect of the ordinary single-strand DNA and enhance accessibility of the biosensor due to its special stereo structure [37]. This assay combines the advantages of self-powered systems, S-Se-GR/AuNPs and cruciform DNA bioconjugate, and therefore shows a low detection limit and a wide linear range for target miRNA, which shows promising as one powerful analytical technique for biomarkers diagnostics.

2. Experimental 2.1 Reagents and apparatus Polyazacyclopropane

(PEI),

ethylenediamine

tetraacetic

acid

(EDTA),

glucose-oxidase (GOD), potassium chloride (KCl), sodium chloride (NaCl), 6-mercaptol-1-hexyl alcohol (MCH), graphite powder, selenium powder, chlorauric acid (HAuCl4·3H2O), polyethylene glycol octyl phenyl ether (Triton-100) and poly diallyldimethylammonium chloride (PDDA) were purchased from Aladdin Reagent Co. Ltd. Absolute ethyl alcohol, flower of sulfur, sodium citrate Qiongzhi powder, Tris-Boric acid and Tris-HCl were obtained in China Pharmaceutical Group Chemical Reagent Co. Ltd. All oligonucleotides were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and the sequences are showed in Table 1.

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Table 1 Sequences of the oligonucleotides. Oligonucleotides

Sequences 5’-SH-(CH2)6-TTTTTCAACATCAGTCTGATAAGCTAGTGGCTTCA

Capture probe DNA AAGATGTTGAATT-3’ miRNA-21

5’-UAG CUU AUC AGA CUG AUG UUG A-3’

A DNA

5’-SH-(CH2)6-GGCAAGCTAATGGTGAGCACGGCAGG-3’

B DNA

5’-SH-(CH2)6-CCTGCCGTGCTCACCGAATGCTAGGG-3’ 5’-SH-(CH2)6-CCCTAGCATTCGGACTATGGCATGAGTTTGAAGC

C DNA CAC-3’ D DNA

5’-CTCATGCCATAGTCCATTAGCTTGCCAATTCAACAT-3’

miRNA-141

5’-UAA CAC UGU CUG GUA AAG AUG G-3’

miRNA-155

5’-UUA AUG CUA AUC GUG AUA GGG GU-3’

miRNA-199a

5’-ACA GUA GUC UGC ACA UUG GUU A-3’

smRNA

5’-UAG CUU AUC AGA AUG AUG UUG A-3’

tmRNA

5’-UAG CUU GUC AGA AUG AUG AUG A-3’

NC

5’-CGU AGC GAU UCU ACA GGU AAU C-3’

smRNA: single-base mismatch RNA; tmRNA: three-base mismatch RNA; NC: non-complementary; the underlined portions represent the mutation bases in target RNA. The morphology of the materials was observed on a S-4800 scanning electron microscope (SEM, Hitachi, Japan) and Tecnai G2 F20 transmission electron microscope (TEM, FEI, USA). X-ray Photoelectron Spectroscopy (XPS) was recorded on K−ALPHA 0.5EV X-ray Photoelectron Spectrometer (Thermo Fisher Scientific, USA). The X-ray powder diffraction pattern (XRD) was detected by Smartlab 9 multifunctional X-ray powder diffractometer (science, Japan). Measurement of specific surface area (BET) was by ASAP2460 automatic specific

6

surface area aperture analyzer (McMeretek, China). Raman spectral (Raman) was recorded by an inV ia Raman spectrometer (renishaw, UK). EIS was detected by one RST5200F electrochemical workstation (Zhengzhou Shi Rui Si Instrument Technology Co. Ltd., China) within a frequency range of 0.1 to 104 Hz in a 0.1 M PBS (pH 7.4) solution containing 5.0 mM [Fe(CN)6]3-/[Fe(CN)6]4- and 0.1 mM KCl. Cyclic voltammetry (CV) and open circuit voltage (EOCV) were detected by a RST5200F electrochemical workstation. Three-electrode system was used with the fabricated anode or cathode as the working electrode, an Ag/AgCl as the reference electrode and a platinum electrode as the auxiliary electrode.

2.2 Preparation of electrode supporting substrate S-GR, Se-GR, and S-Se-GR were prepared as follows [38]. 5 mg S-GR, Se-GR and S-Se-GR were respectively dissolved in 2.5 mg mL-1 PEI solution, and then ultrasonicated for 0.5 h to obtain S-GR, Se-GR and S-Se-GR suspension. Centrifugation was used to remove the excessive PEI solution. The collected product was mixed with 50 mL AuNPs and then incubated for 120 min. The excessive AuNPs were removed by centrifugation at 8000 rpm for 10 min. The mixture then was added into the solution containing 1 mg mL-1 EDC and 1 mg mL-1 NHS, and incubated for 1 h. The obtained activated S-GR/AuNPs, Se-GR/AuNPs and S-Se-GR/AuNPs were then respectively dispersed in PBS (0.1mol L-1, pH 7.4) to form a homogeneous suspension of 1 mg mL-1.

7

2.3 Preparation of bioanode 50 mL activated S-GR/AuNPs, Se-GR/AuNPs and S-Se-GR/AuNPs suspensions were respectively added into 100 µL GOD solution (4 mg mL-1) and incubated for 12 h to immobilize GOD to form GOD/S-GR/AuNPs, GOD/Se-GR/AuNPs and GOD/S-Se-GR/AuNPs. 50 µL above solution was then dropped onto a carbon paper electrode (CP:1 cm×1 cm) and dried at 37 °C to form GOD/S-GR/AuNPs/CP, GOD/Se-GR/AuNPs/CP and GOD/S-Se-GR/AuNPs/CP.

2.4 Loading of [Fe(CN)6]3- into cruciform DNA bioconjugate UPCS/AuNPs Four DNAs (A, B, C, D) was mixed in an equimolar ratio and then heated at 95 °C for 10 min to assemble 1 µM cDNA solution. UPCS/AuNPs was prepared based on the reported protocol after some modification [39]. 6 mL UPCS/AuNPs hybrid (1 mg mL-1) was added into 2 mL potassium ferricyanide (1.0 mol L-1) and incubated at room temperature overnight to form [Fe(CN)6]3-/UPCS/AuNPs. Then, 100 µL of 1 µM cDNA was incubated with [Fe(CN)6]3-/UPCS/AuNPs at 4 °C for 12 h. The obtained cDNA/[Fe(CN)6]3-/UPCS/AuNPs was then dipped in 40 µL MCH (1 mM) for 30 min to eliminate the non-specificity adsorption of electrode surface, and then the residual [Fe(CN)6]3-, cDNA, and MCH were removed by centrifugation. The mixture was then added into 1 mL PBS to obtain cruciform DNA bioconjugate.

2.5 Preparation of biocathode 50 µL UPCS/AuNPs (1 mg mL-1) was applied on CP electrode and thereafter

8

dried at 37 °C for 120 min. Subsequently, it was immersed in a solution containing 10 mg mL-1 EDC and 10 mg mL-1 NHS for 30 min. After rinsed with ultrapure water to remove excess EDC and NHS, the activated electrodes were coated with 75 µL capture probe DNA (1 µM) and then stored at 4 °C for 12 h to form the capture probe DNA/UPCS/AuNPs/CP.

After

washed

with

ultrapure

water,

the

DNA/UPCS/AuNPs/CP was incubated in 20 µL MCH (1 mM) for 30 min to obtain the biocathode. The electrode was incubated with 40 µL miRNA at 37 °C and incubated for 70 min. 50 µL cruciform DNA bioconjugate was applied on above electrode and kept at 37 °C for 50 min for the electrochemical measurements.

2.6 Preparation of self-powered miRNA biosensor A membrane-less EBFC was fabricated with the prepared cathodes and anodes. The supporting electrolyte for CV was 10 mL of 0.1 M PBS. 50 µL cruciform DNA bioconjugate was applied on the electrode and the open circuit voltage (E0OCV) of the EBFC was measured. The E0OCV of the EBFC was detected in the solutions of miRNA-21 or the serum sample with 50 µL of cruciform DNA bioconjugate.

3. Results and discussion 3.1 Assembly of self-powered miRNA biosensor Scheme 1 is the mechanism of the fabrication process of self-powered biosensor. S-Se-GR/AuNPs is firstly prepared and used for supporting substrate of biocathode and bioanode. GOD is then immobilized on the surface of bioanode by Au

9

nanoparticles to obtain the GOD/S-Se-GR/AuNPs/CP. Potassium ferricyanide anions are entrapped in positively charged porous UHCS/AuNPs. [Fe(CN)6]3-/UPCS/AuNPs is then bonded to cDNA by the Au–S bond to form the cruciform DNA bioconjugate. When miRNA-21 is added, cruciform DNA bioconjugate is immobilized onto the biocathode through base pairing with the capture DNA. The electron from the anode transfers to cathode, resulting in the decrease of potassium ferricyanide anions. Therefore, EOCV value dramatically increases owing to the strong catalytic capacity of the anode on glucose oxidation. The obtained EOCV value is positively correlated with the level of miRNA, and then sensitive determination of miRNA is realized.

Scheme 1. Schematic illustration of the fabrication of the anode and cathode (A), cDNA (B), cruciform DNA bioconjugate (C) and the self-powered biosensor (D).

10

3.2 Characterization of materials As shown in Figure 1, the morphologies of S-Se-GR and S-Se-GR/AuNPs are characterized by SEM, TEM and HRTEM. S-Se-GR shows typical layered structure and the folds caused by the lattice defects and impurity doping (Figure 1A and 1B) [40]. Figure 1C shows the element mapping diagrams of C, S, and Se elements. S and Se elements are evenly decorated onto the surface of S-Se-GR, indicating the successful doping of S and Se elements in GR. The SEM and TEM images of S-Se-GR/AuNPs show that the AuNPs are distributed evenly on the S-Se-GR (Figure 1D and 1E). The fingerprint structure of S-Se-GR and the lattice fringes of AuNPs are displayed in Figure 1F. The stripe spacing is 0.24 nm, which corresponds to Au (111) crystal planes. The morphology of CP modified by S-Se-GR/AuNPs and GOD is characterized by SEM. As displayed in Figure 2, the S-Se-GR/AuNPs/CP (Figure 2A) is compared with bare CP (Figure 2B), and it shows S-Se-GR/AuNPs is uniformly distributed on the surface of CP electrode. As displayed in Figure 2C, CP is uniformly covered

with

AuNPs.

Figure

2D

shows

a

uniform

GOD/S-Se-GR/AuNPs/CP due to the functionalization of GOD.

11

biofilm

forms

on

Figure 1. SEM (A), TEM (B) and element mapping images (C) of S-Se-GR; SEM (D), TEM (E) and HRTEM images (F) of S-Se-GR/AuNPs.

Figure 2. SEM of S-Se-GR/AuNPs/CP (A) and bare CP (B); HRSEM of S-Se-GR/AuNPs/CP (C); SEM of GOD/S-Se-GR/AuNPs/CP (D).

The composition and structural characteristics of the samples were tested by EDX, XRD, XPS and Raman spectrum. The elements in S−GR samples are detected by EDS technique (Figure 3A). The inset exhibits the atoms and weight contents of S−GR. It displays the sample consists of C, O and S elements. The S element contents

12

of 8.79% (wt%) in the S−GR sample indicates the successful doped of S. Figure 3B shows Se-GR samples are composed of C, O, Se elements and Figure 3C displays the S-Se-GR sample contains C, O, S, Se elements. The Se element contents in the Se−GR sample is 31.92% (wt%) and the S and Se element contents in the S-Se-GR sample are 10.91% (wt%) and 17.67% (wt%), respectively. This confirms the successful doping of the hetero atoms in the three samples. The XRD patterns of the S−GR, Se−GR, S−Se−GR and S−Se−GR/AuNPs are shown in Figure 3D. Obviously, the S−GR, Se−GR and S−Se−GR show two distinct diffraction peaks. The peak at 25.92° is corresponding to graphitic (002) crystal planes and that at 42.26° is corresponding to graphitic (100) crystal planes [41]. The peaks at 37.57°, 43.65°, 64.18° and 77.10° belong to the (111), (200), (220) and (311) crystal planes of Au in the sample [42], respectively. The XPS was also carried out to study the chemical compositions of samples. Figure 3E shows C, S, Se and O elements in the sample. The atom contents of C, O, S and Se in the S-Se-GR sample are 68.87%, 2.85%, 11.08% and 17.20%, respectively, which are well consistent with the EDX results.

13

Figure 3. EDX pattern of S-GR (A), Se-GR (B), S-Se-GR (C); XRD pattern (D), XPS survey spectra (E) and Raman spectra (F) of S-GR, Se-GR, S-Se-GR and S-Se-GR/AuNPs.

The Raman spectrum of the samples is presented in Figure 3F. The D and G peaks of carbon are obviously observed. ID/IG has been widely applied to study defect degree in graphitized carbon samples [43]. The ID/IG intensity of the S-GR, Se-GR and S-Se-GR samples are 0.838, 0.841, and 0.839, respectively. Furthermore, the ID/IG 14

intensity increases with the doping process of S and Se, indicating the increase of the defect density of S-Se-GR. AuNPs obviously increase the scattering signal of S−Se−GR/AuNPs because AuNPs can capture and focus the lights. The XPS spectrum of S−Se−GR/AuNPs is displayed in Figure 4. The characteristic peaks of C 1s, S 2p, Se 3d and Au 4f are clearly observed, further confirming the AuNPs have been successfully assembled on S−Se−GR surface.

Figure 4. XPS survey spectra of C 1s spectra (A), S 2p spectra (B), Se 3d spectra (C) and Au 4f spectra (D) of S-Se-GR/AuNPs.

3.3 Electrochemical performance of biosensor As

shown

in

Figure

5,

the

CV

curves

of

GOD/GR/AuNPs/CP,

GOD/S-GR/AuNPs/CP, GOD/Se-GR/AuNPs/CP and GOD/S-Se-GR/AuNPs/CP are investigated. All electrodes present two obvious GOD redox bands at about -0.49 V

15

(Figure 5A-D), indicating redox of GOD can favourably occur on the electrode surface. The peak-to-peak separation of various scan rates is lower than 20 mV, suggesting the redox process is nearly reversible. The apparent surface coverage (Γ*cat) of GOD on S-Se-GR/AuNPs/CP electrode is estimated as 3.25×10-10 mol cm-2 by using the equation ip=n2F2νAГcat*/4RT (Figure 5D). This value is about 4 times larger than that of GR/AuNPs/CP, 3.5 times larger than that of S-GR/AuNPs/CP and 1.0 times larger than that of Se-GR/AuNPs/CP. The results verifies that the S-Se-GR/AuNPs/CP has the largest surface coverage of GOD. The conductive characteristics of different bioanodes are studied by EIS (Figure 6A).The semicircle size of S−Se−GR/AuNPs shows the smallest compared with the other electrodes, indicating good conductivity. Each preparation process of biocathode was studied by EIS. The CNLS fitting method on the basis of the electrical equivalent circuit was used to analyze impedance spectra, as shown in the inset of Figure 6A and Figure 6B (the detail the values are exhibited in Table S1 and S2). In Figure 6B, the semicircle of the bare electrode decreases (curve a) after modified by S−Se−GR/AuNPs (curve b), indicating its good conductivity. The semicircle markedly increases after the further loading of capture DNA (curve c) due to the negative charge DNA. Similarly, the semicircle further increases when the MCH is loaded (curve d). The semicircle significantly increases after the modification of miRNA (curve e) and cruciform DNA bioconjugate (curve f) due to the negative charge miRNA and the steric hindrance of the cruciform DNA bioconjugate.

16

Figure 5. (A) CVs of GOD/GR/AuNPs/CP in N2 saturated PBS (pH 7.4), ν: (a) 80, (b) 100, (c) 120, (d) 150, (e) 170, (f) 200, (g) 250, (h) 300, (i) 350 mV s-1; (B) CVs of GOD/S-GR/AuNPs/CP in N2 saturated PBS (pH 7.4), ν: (a) 80, (b) 100, (c) 120, (d) 150, (e) 170, (f) 200, (g) 250, (h) 300, (i) 350, (j) 400 mV s-1; (C) CVs of GOD/Se-GR/AuNPs/CP in N2 saturated PBS (pH 7.4), ν: (a) 120, (b) 150, (c) 170, (d) 200, (e) 250, (f) 300, (g) 350, (h) 400, (i) 450, (j) 500, (k) 600 mV s-1; (D) CVs of GOD/S-Se-GR/AuNPs/CP in N2 saturated PBS (pH 7.4), ν: (a) 100, (b) 120, (c) 150, (d) 170, (e) 180, (f) 200, (g) 250, (h) 300, (i) 350, (j) 400, (k) 450, (l) 500, (m) 550, (n) 600 mV s-1.

17

Figure 6. (A) EIS of GR/AuNPs/CP (a) , S-GR/AuNPs/CP (b), Se-GR/AuNPs/CP (c), and S-Se-GR/AuNPs/CP (d); (B) EIS of CP electrode (a), S-Se-GR/AuNPs/CP (b), capture DNA/S-Se-GR/AuNPs/CP (c), MCH/capture DNA/S-Se-GR/AuNPs/CP (d), miRNA/MCH/capture

DNA/S-Se-GR/AuNPs/CP

(e)

and

cruciform

DNA

bioconjugate/miRNA/MCH/capture DNA/S-Se-GR/AuNPs/CP (f). Inset shows the electrical equivalent circuit used for fitting impedance spectra. Rs: solution resistance; Rct: charge-transfer resistance; Cdl: double layer capacitance; ZW: Warburg impedance resulting from the diffusion of ions.

From Figure 7A, the CV curves of the anode with or without glucose are recorded.

Clearly,

two

redox

peaks

appear

at

about

-0.49

V

on

GOD/S−Se−GR/AuNPs electrode in the absence of glucose, which indicates the bioanode is sensitive to glucose. The result is related to the theory of the O2 mediates GOD for glucose oxidation. The observed oxidation peaks gradualy increases when glucose is added because the oxidation of glucose requires consumption of O2 (C6H12O6+O2+H2O → C6H12O7+H2O2), resulting in decrease of reduction peak signal [44]. In order to determine the existence of miRNA can immobilize the cDNA on the electrode surface and trigger recycling release of potassium ferricyanide ions, the effect of different concentrations of miRNA on cathode signal are studied by CV (Figure 7B). Two smaller redox peaks appear in the absence of target miRNA. The 18

current signal inch by inch increases with the increase of miRNA concentration from 0 to 1000 fM, indicating the higher concentration of miRNA-21 can immobilize more cDNAs on the cathode. Moreover, the results confirm the cDNA contains [Fe(CN)6]3-.

Figure 7. (A) CVs of the bioanode in PBS (pH 7.4) without glucose (a) and with 5 mM glucose (b); (B) CVs responses of [Fe(CN)6]3- at the cathode in the presence of miRNA with different concentrations (from a to g: 0, 1, 5, 10, 50, 100 and 1000 fM).

Agarose gel electrophoresis was also used to confirm the immobilization of cDNA (Figure 8). The bands in lanes 2, 3, 4, 5 are single strand A, B, C and D, respectively. The band of the sixth path is annealing product of A and B. Band of the seventh path is annealing product of A, B and C. The results suggest the cDNA is difficult to form by two or three DNAs. The slowest band is observed on lane 8 when four DNA chains are mixed, confirming the successful assembly of cDNA.

19

Figure 8. Agarose gel electrophoresis results of the products and sequences of cDNA. Lane 1: 20 bp DNA ladder marker; Lanes 2, 3, 4, 5: single strand A, B, C, D, respectively; Lane 6: annealing products of strand A and strand B; Lane 7: annealing products of strand A, strand B and strand C; Lane 8: cDNA.

The reaction time of miRNA and capture DNA was optimized (Figure 9A). Electrochemical response signal gradually increases with the hybrid time increasing, and it then begins to level off after 80 min, indicating the saturated binding reaches between the miRNA and capture probe. Thus, 80 min is used in the subsequent experiments. Obviously, the current signal depends on the loaded [Fe(CN)6]3- and the immobilized cDNA. The effect of hybrid reaction time between cDNA and capture DNA was studied, and the results were showed in Figure 9B. Obviously, the electrochemical response signal gradually increases with the incubation time enhancing, and then the signal begins to level off after 50 min. So the time of 50 min was used. Similarly, the cathode current increases with the increasing of [Fe(CN)6]3-

20

concentration (Figure 9C), and the signal begins to decrease when more than 200 mM L-1 [Fe(CN)6]3- is used, suggesting the saturated binding reaches between the [Fe(CN)6]3- and UHCS/AuNPs. Therefore, 200 mM L-1 of [Fe(CN)6]3- was selected.

Figure 9. The effect of the various conditions on the peak currents: (A) the hybridization reaction time between the target miRNA and the capture probe; (B) the hybridization reaction time between the cruciform DNA bioconjugate and the terminus of capture probe, (C) the [Fe(CN)6]3- concentration, (D) the cDNA concentration. The concentration of miRNA was 1.0 pM.

The effect of cDNA concentration was studied. The results in Figure 9D indicates that the peak current gradually increases with the increase of cDNA concentration, and the maximum value is obtained at 1 µM. Therefore, 1 µM of cDNA was chosen. 21

Under the optimized condition, miRNA-21 was detected with self-powered biosensor. As shown in Figure 10A, the EOCV of the EBFC biosensor is only 0.2 V without miRNA-21 due to there is hardly no [Fe(CN)6]3-. When [Fe(CN)6]3- is liberated from cruciform DNA bioconjugate in the presence of miRNA-21, a large EOCV is observed. The EOCV value increasingly enhances with the increase of the target miRNA level (Figure 10B). The relationships between miRNA concentration and EOCV was determined. A linear range was obtained from 0.5 to 10000fM (Figure 10C) with an equation of EOCV = 0.50 log c + 0.07 (R=0.994). The limit of detection was 0.15 fM (S/N=3), which was similar to or lower than some reported methods (Table 2). To evaluate the selectivity of the self-powered biosensor, three miRNAs (miRNA-155, miRNA-141 and miRNA-199a) and base mismatched sequences (smRNA, tmRNA and NC) were used as the interfering substances (1 pM). Figure 10D shows the ∆EOCV values of these interferes are much lower than that of target miRNA, demonstrating the excellent selectivity of self-powered biosensor. To study the reproducibility of the self-powered biosensor, five biosensors were fabricated and then used to detect 1 pM target miRNA. A RSD of 7.4% was obtained, suggesting good reproducibility. The cycle stability of bioanode and biocathode was evaluated. The CV profiles of bioanode and biocathode kept almost unchanged when they undergone 100 cycles of successive potential scan in PBS (pH 7.4), indicating good cycle stability. The long-term stability of biosensor was also studied. The self-powered biosensors was fabricated and kept at 4°C for 10 days. Only 94.6%

22

current signal was kept, demonstrating its excellent long-term stability. A recovery experiment was carried out to study the application potential of self-powered biosensor in real samples. The human serum sample (obtained from affiliated hospital of Xinyang normal University) containing miRNA-21 were measured. Samples 1 and 2 are from two healthy persons. Samples 3 and 4 are from two cancer patients. As shown in Table 3, the recoveries (92.0-106.2%) and RSDs (3.52-7.04%) suggested that the self-powered biosensor has great potential to be applied for miRNA bioassay in clinical diagnosis.

Figure 10. (A) EOCV of self-powered biosensor in various levels of miRNA (from a to i: 0, 0.5, 1, 5, 10, 50, 100, 1000, 10000 fM); (B) The variation of EOCV as a function of miRNA concentration; (C) The linear relationship between EOCV and the logarithm of miRNA-21 concentration from 0.5 to 10000 fM; (D) EOCV changes of the self-powered biosensor resulted from various detection substances.

23

Table 2. Analytical performances of different assays for miRNA detection. Technique Colorimetry Chronocoulom etry SERS Fluorescence LSPR DPV Fluorescence Fluorescence Electrophoresis EBFC

Strategy pH-responsive isothermal amplified system Gold-loaded nanoporous superparamagnetic nanocubes DNA-mediated gold-silver nanomushroom Catalyzed hairpin assembly Label-free nanoprobe Enzyme-free sensing Target-catalyzing signal amplification Target recycling amplification Separation-assisted double cycling signal amplification Nanomaterials and cruciform DNA hybrids

Linear range

LOD

Ref.

20 fM-20 nM

9.3 fM

45

0.1 pM-1 µM

0.1 pM

46

1 pM-10 nM

10 fM

47

0.5 nM-50 nM 10 pM-1 µM 50 fM-0.5 nM 10 fM-500 fM 10 pM-2 nM

72 pM 5 pM 18 fM 3 fM 4.2 pM

48 35 49 50 51

20 fM-20 pM

8 fM

52

0.5 fM-10 pM

0.15 fM

This work

SERS: surface-enhanced Raman scattering; LSPR: localized surface plasmon resonance; DPV: differential pulse voltammetry.

Table 3. Measurement of miRNA-21 in human serum samples. Serum samples

miRNA-21 Concentration (fM) Detected Added Found (n=3)

1

Not detected

2

Not detected

3

0.37

4

0.54

RSD (%)

Recovery (%)

0.5 1.0 10 50 0.5 1.0 0.5

0.52 0.92 10.62 50.41 0.86 1.41 1.03

4.88 4.21 7.04 4.28 3.58 3.99 3.52

104 92 106.20 100.82 98.85 103.16 99

1.0

1.51

5.84

98.05

4. Conclusion In summary, a high-energy EBFC based self-powered electrochemical biosensor is developed for sensitive determination of miRNA with cruciform DNA bioconjugate

24

for signal amplification. S-Se-GR/AuNPs is prepared as electrode supporting substrate not only provides big specific surface area and excellent conductivity, but also assists enlarging enzymes loading. DMM is used for signal readout and can reduce the cost of detection, because it is cheaper than other electrochemical detection devices.

This

assay

combines

the

advantages

of

self-powered

systems,

S-Se-GR/AuNPs and cruciform DNA bioconjugate. Therefore a low detection limit (1.5×10-16 mol L-1) and a large linear range (0.5-10000 fM) are obtained for target miRNA detection with good selectivity. The self-powered biosensor is successfully applied in serum samples analysis, which shows the great potential for the clinical applications.

Acknowledgments 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.

References [1] C. Xu, C. F. Pan, Y. Liu, Z. L. Wang, Nano Energy. 1 (2012) 259-272. [2] T. Zhang, H. Chai, F. Y. Meng, Z. Z. Guo, Y. Jiang, P. Miao, ACS Appl. Mater. Interfaces. 10 (2018) 36796-36804. [3] S. Cosnier, Gross. A. J, Goff. A. Le, M. Holzinger, J. Power Sources. 325 (2016) 252-263.

25

[4] K. Stolarczyk, M. Kizling, D. Majdecka, K. Żelechowska, J. F. Biernat, J. Rogalski, R. Bilewicz, J. Power Sources. 249 (2014) 263-269. [5] Y. Chen, P. P. Gai, J. R. Zhang, J. J. Zhu, J. Mater. Chem. A. 3 (2015) 11511-11516. [6] Y. H. Wang, L. N. Zhang, K. Cui, S. G. Ge, P. N. Zhao, J. H.Yu, ACS Appl. Mater. Interfaces. 11 (2019) 5114-5122. [7] C. C. Gu, P. P. Gai, L. Han, Q. Y. Liu, F. Li, Chem. Commun. 54 (2018) 5438-5441. [8] D. Majdecka, S. Draminska, D. Janusek, P. Krysinski, R. Bilewicz, Biosens. Bioelectron. 102 (2018) 383-388. [9] T. Hou, W. Li, X. J. Liu, F. Li, Anal. Chem. 87 (2015) 11368-11374. [10] P. P. Gai, R. B. Song, C. Zhu, Y. S. Ji, W. J. Wang, J .R. Zhang, J. J. Zhu, Chem. Commun. 51 (2015) 16763-16766. [11] P. P. Gai, Y. S. Ji, W. J. Wang, R. B. Song, C. Zhu, Y. Chen, J. R. Zhang, J. J. Zhu, Nano Energy. 19 (2016) 541-549. [12] J. S. Cheng, Y. J. Han, L. Deng, S. J. Guo, Anal. Chem. 86 (2014) 11782-11788. [13] Y. H. Wang, L. Ge, P. P. Wang, M. Yan, J. H. Yu, S. G. Ge, Chem. Commun. 50 (2014) 1947-1949. [14] M. Gamella, N. Guz, S. Mailloux, J. M. Pingarrón, E. Katz, ACS Appl. Mater. Interfaces. 6 (2014) 13349-13354. [15] M. Zhou, Y. Du, C. G. Chen, B. L. Li, S. J. Dong, E. K. Wang, J. Am. Chem. Soc. 132 (2010) 2172-2174. [16] M. Acunzo, G. Romano, D. Wernicke, Adv. Biolog Regul. 57 (2015) 1-9. [17] L. L. Wang, H. H. Shao, W. J. Wang, J. R. Zhang, J. J. Zhu, Nano. Energy. 44 (2018) 95-102.

26

[18] X. G. Duan, K. O'Donnell, H. Q. Sun, Y. X. Wang, S. B. Wang, Small. 11 (2015) 3036–3044. [19] A. Y. Chen, S. Y. Ma, Y. Zhuo, Y. Q. Chai, R. Yuan, Anal. Chem. 88 (2016) 3203-3210. [20] W. Z. Lv, L. Li, M. C. Xu, J. X. Hong, X. X. Tang, L. G. Xu, Y. H. Wu, R. Zhu, R. F. Chen, W. Huang, Adv. Mater. (2019) 1900682. [21] P. P. Gai, R. B. Song, C. Zhu, Y. S. Ji, Y. Chan, J. R. Zhang, J. J. Zhu, Chem. Commun. 51 (2015) 14735-14738. [22] H. L. Shuai, K. J. Huang, X. Wu, J. Mater. Chem. B 5 (2017) 5362-5372. [23] L. Kuang, S. P. Cao, L. Zhang, Q.H. Li, Z. C. Liu, R.P. Liang, J. D. Qiu, Biosens. Bioelectron. 85 (2016) 798-806. [24] Z. X. Chen, Y. J. Peng, Y. Cao, H. Wang, J. R. Zhang, H. Y. Chen, J. J. Zhu, Nano. Lett. 18 (2018) 3759-3765. [25] S. W. Kim, Z. H. Li, P. S. Moore, Y. Chang, B. John, Nucleic Acids. Res. 38 (2010) e98-e98. [26] W. J. Lu, Y. P. Chen, Z. Liu, W. B. Tang, Q. Feng, J, S, Sun, X. Y. Jiang, ACS Nano. 10 (2016) 6685-6692. [27] E. Clancy, M. Burke, V. Arabkari, T. Barry, H. Kelly, R. M. Dwyer, M. J. Kerin, T. J. Smith, Anal. Bioanal. Chem. 409 (2017) 3497-3505. [28] Z. X. Wang, H. B. Bian, J. R. Wang, Z. X. Cheng, K. M. Wang, W. De, J. Surg. Oncol. 104 (2011) 847-851. [29] F. Davis, S. P. J. Higson, Biosens. Bioelectron. 22 (2007) 1224−1235. [30] J. H. Schulte, T. Marschall, M. Martin, P. Rosenstiel, P. Mestdagh, S. Schlierf, T. Thor, J. Vandesompele, A. Eggert, S. Schreiber, S. Rahmann, A. Schramm, Deep Nucleic Acids Res. 38 (2010) 5919–5928.

27

[31] Y. H. Yuan, Y. D. Wu, B. Z. Chi, R. P. Liang, J. D. Qiu, Biosens. Bioelectron. 97 (2017) 325-331. [32] L. Chen, L. P. Song, Y. C. Zhang, P. Wang, Z. D. Xiao, Y. G. Gou, F. F. Chao, ACS Appl. Mater. Interfaces. 8 (2016) 11255. [33] L. Ge, W. X. Wang, F. Li, Anal. Chem. 89 (2017) 11560-11261. [34] L. Zhang, J. H. Wang, J. X. Zhang, Y. Q. Liu, L. Z. Wu, J. J. Shen, Y. Zhang, Y. L. Hu, Q. L. Fan, W. Huang, ACS sens. 2 (2017) 1435-1440. [35] W. Shen, H. M. Deng, Z. Q. Gao, J. Am. Chem. Soc. 134 (2012) 14678-14681. [36] X. Hun, G. L. Xie, X. L. Luo, Chem. Commun. 51 (2015) 7100-7103. [37] K. J. Huang, Y. J. Liu, Q. F. Zhai, J. Mater. Chem. B 3 (2015) 8180–8187. [38] Y. X. Chen, K. J. Huang, K. X. Niu, Biosens. Bioelectron. 99 (2018) 612–624. [39] W. Yang, W. Yang, F. Ding, L. Sang, Z. P. Ma, G. J. Shao, Carbon. 111 (2017) 419-427. [40] X. G. Meng, C. Yu, X. D. Song, H. L. Jin, S. Wang, L. L. Huang, J. S. Qiu, Z. Q. Lin, Angew. Chem. Int. Edit. 57 (2018) 4682-4686. [41] L. L. Xing, G. G. Zhao, K. J. Huang, X. Wu, Dalton Transactions 47 (2018) 2256-2265. [42] D. Zhang, Y. Guo, Z. Zhao. Appl Catal B: Environ. 226 (2018) 1-9. [43] M. Wooten, S. Karra, M. G. Zhang, W. Gorski, Anal. Chem. 86 (2013) 752−757. [44] M. Christwardana, K. J. Kim, Y. Kwon, Sci. Rep. 6 (2016) 30128. [45] C. Feng, X. X. Mao, H. Shi, B. Bo, X. X. Chen, T. S. Chen, X. L. Zhu, G. X. Li, Anal. Chem. 89 (2017) 6631-6636. [46] M. K. Masud, M. N. Islam, M. H. Haque, S. Tanake, Chem. Commun. 53 (2017) 8231-8234.

28

[47] J. Su, D. F. Wang, L. Nörbel, J. L. Shen, Z. H. Zhao, Y. Z. Dou, T. H. Peng, J. Y. Shi, S. Mathur, C. H. Fan, S. P. Song, Anal. Chem. 89 (2017) 2531-2538. [48] Y. Liu, T. Shen, J. Li, H. Gong, C. Y. Chen, X. M. Chen, C. Q. Cai, ACS sens. 2 (2017) 1430-1434. [49] J. J. Jiang, X. Y. Lin, G. W. Diao, ACS Appl. Mater. Interfaces. 9 (2017) 36688-36694. [50] Y. K. Xia, L. L. Wang, J. Li, X. Q. Chen, J. M. Lan, A. Yan, Y. Lei, S. Yang, H. H. Yang, J. H. Chen, Anal. Chem. 90 (2018) 8969-8976. [51] S. H. Xu, Y. Y. Nie, L. P. Jiang, J. Wang, G. Y. Xu, W. Wang, X. L. Luo, Anal chem. 90 (2018) 4039-4045. [52] K. J. Wei, J. J. Zhao, X. S. Luo, S. H. Qiu, F. Y. He, S. T. Li, S. L. Zhao, Analyst. 143 (2018) 1468-1474.

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Author Biographies

Fu-Ting Wang is a graduate student at Xinyang Normal University. Her current research is self-powered biosensors.

Yi-Han Wang is a graduate student at Xinyang Normal University. Her current research is electrochemical biosensors.

Jing Xu received her Ph.D. degree under the supervision of Professor Changguo Chen in School of Chemistry and Chemical Engineering at Chongqing University in 2018, with the research interests of Mg-MnO2 battery. She has worked on Xinyang Normal University since 2018. Her research focuses on prepare nanostructured electrode materials for advanced rechargeable batteries and high energy enzyme biofuel cells.

Ke-Jing Huang is currently a full professor of Chemistry and Chemical Engineering, Xinyang Normal University. He obtained his PhD in Analytical Chemistry from Wuhan University, China in 2006 and became a professor in Xinyang Normal 30

University in 2014. His research focuses on biosensors and electrochemical energy storage.

Zhen-hua Liu is an undergraduate student at Xinyang Normal University. Her current research is electrochemical self-powered biosensors.

Yun-fei Lu is an undergraduate student at Xinyang Normal University. Her current research is electrochemical self-powered biosensors.

Shu-yu Wang is an undergraduate student at Xinyang Normal University. Her current research is electrochemical biosensors.

Zi-Wei Han is an undergraduate student at Xinyang Normal University. Her current research is electrochemical self-powered biosensors.

31

Supporting Information for Boosting Performance of Self-Powered Biosensing Device with High-Energy Enzyme Biofuel Cells and Cruciform DNA Fu-Ting Wang, Yi-Han Wang, Jing Xu, Ke-Jing Huang*, Zhen-hua Liu, Yun-fei Lu, Shu-yu Wang, Zi-wei Han College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China

*Corresponding author. E-mail address: [email protected] (K.J. Huang);

32

Table S1 Parameters obtained from EIS fitting. Value Element a

b

c

d

Rs(Ω)

5.250

5.339

5.682

5.290

CPE1-T(mF)

3.425×10-4

2.731×10-4

2.415×10-4

1.302×10-4

CPE1-P(mF)

0.9838

0.8697

0.7918

0.8367

Rct(Ω)

10.06

3.350

5.409

8.104

W1-R(Ω)

111.2

87.13

108.3

144.0

W1-T(Ω)

25.67

24.59

23.52

22.75

W2-R(Ω)

0.5450

0.6518

0.6684

0.6493

Table S2 Parameters obtained from EIS fitting. Value Element a

b

c

d

e

f

Rs(Ω)

8.26

8.23

8.54

8.892

8.124

8.76

CPE1-T(mF)

1.424×10-4

5.661×10-4

2.381×10-4

2.291×10-4

1.945×10-4

1.681×10-4

CPE1-P(mF)

0.8376

0.7609

0.7309

0.7930

0.7669

0.7554

Rct(Ω)

70.06

3.106

14.40

18.33

23.78

45.28

W1-R(Ω)

1.949

0.1063

1.73

144.4

32.87

206.5

W1-T(Ω)

5.795×10-3

2.721×10-3

1.093×10-2

65.99

1.779

19.91

W2-R(Ω)

0.4104

0.3527

0.2864

0.4441

0.3032

0.6289

33

Highlights

• A self-powered biosensor is fabricated based on high-energy enzymatic biofuel cell and cruciform DNA. • Sulfur-selenium co-doped graphene/gold nanoparticles is prepared and used for supporting substrate. • [Fe(CN)6]3- is entrapped in the UPCS/AuNPs by using cruciform DNA bioconjugate signal amplification. • The self-powered biosensor shows a detection limit of 0.15 fM and high specificity towards target microRNA.

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