O2

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Paper Supported Self-Powered System based on Glucose/O2 Biofuel Cell for Visual MicroRNA-21 Sensing Yanhu Wang, Lina Zhang, Kang Cui, Shenguang Ge, Peini Zhao, and Jinghua Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20034 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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ACS Applied Materials & Interfaces

Paper Supported Self-Powered System based on Glucose/O2 Biofuel Cell for Visual MicroRNA-21 Sensing Yanhu Wang,† Lina Zhang,‡ Kang Cui,†* Shenguang Ge,§ Peini Zhao,†* Jinghua Yu†

†School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China ‡ Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, China §Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan 250022, P.R. China

*Corresponding author: Kang Cui, Peini Zhao E-mail: [email protected], [email protected] Telephone: +86-531-82767161

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ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The exploitation of self-powered device that get rid of the power source restriction represents the development tendency of sensing system. Herein, a paper supported glucose/O2 biofuel cell (BFC) based self-powered sensing platform for visual analysis was developed. The BFC device utilized gold nanoparticles modified paper fibers as the electrode to wire glucose oxidase (GOx) and bilirubin oxidase for the fabrication of bioanode and biocathode. To implement an assay protocol, a target responsive cargo release system based on mesoporous silica nanocarrier controlled by microRNA-21 (miRNA-21) was designed. During the BFC operation, undesired H2O2, the side product of glucose oxidation which would be deleterious for GOx, was generated leading to inevitable degeneration of BFC performance. Based on the H2O2 mediated iodide oxidation reaction to form iodine that further modulated the starch chromogenic reaction, undesired H2O2 could be effective removed resulted in remarkably improved BFC performance as well as provided a means for visual signal readout. Thanks to the dual output signals (maximum power output density or length of blue bar), enhanced analysis reliability and sensitive detection of miRNA-21 over a range of 5 fM-100 pM were achieved. Moreover, this study demonstrates a proof of concept in visualized BFC based self-powered system for sensing application, and provides a blueprint to advance future sensors and analysis devices powered by BFC in a wide variety of in-vitro applications.

Keywords: biofuel cell, self-powered, distance-based visualized, paper, miRNA-21.

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Introduction MicroRNAs (miRNAs), as a small noncoding sequence with 18 to 25 nucleotides in length, play a vital role in a various of biological processes such as gene expression, transcription and biological progress including cell proliferation, differentiation, apoptosis, and hematopoiesis.1-4 And the abnormal expression of miRNAs would lead to the formation, invasion, and metastasis of cancer.5, 6 Based on this, miRNAs has been regarded as a promising class of biomarkers for early diagnosis.2 Moreover, the exactly tracing miRNAs would become an attractive strategy to enable the advancement of personal early diagnosis.7 To meet the urgent demands, various techniques have been established, such as photoelectrochemical,8 electrochemiluminescence9 and fluorescence10 et al. And satisfactory results have been demonstrated. Nevertheless, the need of external stimulation leading to additional energy consumption still remains a challenge prior to popularization. The recently emerging biofuel cells (BFCs) based self-powered sensors that worked independently and sustainably, have attracted considerable attention.11-15 Generally, a BFC usually uses enzymes as the catalysts to convert chemical energy from renewable fuels (i.e., glucose, fructose, lactose, methanol, ethanol, and lactate) into electrical energy.13,

16

Glucose oxidase

(GOx), possess deeply buried strongly bound redox centres in protein shell revealed excellent electron transfer vehicle, has been demonstrated to be one of the most commonly biocatalyst used in BFC for glucose oxidation device without the need of mediators.17-19 However, it should be pointed out that unfavourable H2O2 was generated during the reaction between glucose and oxygen under GOx catalysis, which would lead to inevitable degradation BFC performance.20 Therefore, it is necessary to eliminate the generated harmful H2O2 to keep the catalytic activity of anodic GOx. To address this challenge, innovative strategies have been developed. For example, a multienzyme bioanode was fabricated through co-immobilization of GOx and catalase by Kwon’s group. And the generated harmful H2O2 by the oxidation of glucose was decomposed under the catalysis of adjacent catalase, and improved BFC performance was achieved.20 Shim et al. developed a horseradish peroxidase based biocathode that fed by H2O2, the byproduct of glucose oxidation.21 Then Stoica et al. and Cosnier et al proposed an efficient biocathode fed by glucose and O2 through the co-immobilization of GOx and HRP, which exhibited markedly stability and 3



efficient for oxygen reduction.22, 23 Different from those methods for consuming undesired H2O2, Kokoh et al. and Zou et al. developed abiotic nanomaterials based anode for glucose electrooxidation that avoided H2O2 generation.24, 25 And satisfactory technological advances have been achieved based on those approaches. On the other hand, further progress in the arena of signal translation has been hindered by the sophisticated readout system.26 Meanwhile, the exploration of equipment-free read-out system provide a promising alternative approach to advance analytical chemistry.27 H2O2, as a cosubstrate for peroxidases, has been demonstrated to be effective co-reactant in colorimetric reaction, that enables to diminish the benefits of a test being instrument free.27, 28 Hereon, we advocate to design an equipment-free readout system for the BFC based self-powered device in which take advantage of consumption undesired H2O2 to overcome the dependence on sophisticated equipment readout system as well as promote the BFC performance. Given the advantages of cheapness, ease-of-operation, lightweight, and compatibility with biological samples, paper is one of the most promising alternatives to conventional substrates.29, 30 And researchers have been successful in employing paper based fuel cells to realize the energy requirements of portable tests.12,

31-33

Herein, a paper supported glucose/O2 BFC based

self-powered device equipped with a colorimetric distance readout system is fabricated for miRNA-21 sensing. The presented BFC consists of rationally wiring GOx and bilirubin oxidase (BOD) on gold nanoparticles modified paper fibers as electrode for the construction of bioanode and biocathode. Meanwhile, efficient removing of bioanode generated unwelcome H2O2 by the catalytic oxidation of iodide to yield iodine further reacted with starch that leads to a sensitive blue color presenting, not only resulted in enhanced power output, but also provided a new simple visible display strategy without complicated signal conversion procedures. Meanwhile, the double model (colorimetric and electrochemical) would ensure the quantification reliability as well as offer more choices for sensing. Benefitting from the feature of BFC device, we believe that the as-prepared device could be expanded for wearable noninvasive health monitoring.

Experimental Section Device Fabrication All layouts for the paper-based BFC device were engineered through Adobe illustrator CS4 4


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and then presented on a piece of paper sheet (Whatman #1 filter paper) through a wax printer. As shown in Scheme 1A, the designed paper-based device contains five individual patterned papers: a solution-inlet layer (layer a), a solution-transfer layer (layer b), an electrolyte layer (layers c), an electrode layer (layer d), and an reaction layer (layer e). After printing, the resulting papers were transferred into an oven heated at 150 °C for 90 s to form the hydrophobic patterns. Subsequently, screen-printed carbon electrodes were printed onto hydrophilic pattern (Scheme 1B, 12-14). Materials preparation Synthesis of mesoporous silica nanomaterial (MSN): A reported literature with modifications was referred for the preparation of MSN.34 Briefly, 1.0 g N-cetyltrimethylammonium bromide (CTAB) was first dispersed into 480 mL ultrapure water to form a uniform solution. Then, 0.28 g NaOH was added into the above solution at room temperature. After 20 min stirring, 5.0 g tetraethyl orthosilicate (TEOS) was added and the mixture was heated to 80 °C under vigorously agitation. This process was maintained for 2 h to obtain white precipitates. To remove the CTAB in the mesopores, the obtained products were washed successively by a mixture of methanol and concentrated HCl (v/v = 10:1) at 65 °C for 48 h. Finally, the wet products were centrifuged at 8000 rpm for 10 min, then thoroughly rinsed with ethanol and dried in an oven under vacuum at 60 °C overnight. Amine functionalized MSN were prepared through refluxing 200 mg as-prepared MSN with 4 mL (3-aminopropyl)triethoxysilane (APTES) in 80 mL anhydrous toluene at 120 ºC under N2 for 20 h. Afterward, the resulted product was collected by centrifugation, washed thoroughly with toluene, and dried in vacuum. Preparation of miRNA-21 probe (Cu2+@MSN-DNA cap): Aforementioned amine functionalized MSN

was

dispersed

into

10

1-ethyl-3-(3-dimethylaminopropyl)

mL

PBS

(0.01

carbodiimide

M,

pH

7.4)

hydrochloride

containing (EDC),

200 115

mg mg

N-Hydroxysuccinimide (NHS) under stirring at 6 °C for 24 h. Then the pellet was obtained via centrifugation and redispersed into 1.0 mL of PBS (0.01 M, pH 7.4) prior to use. After that, 100 μL 100 μM anchor-DNA (S1 and S2) was added into above mixture and shaken at 37 °C for 5 h. Unbonded anchor-DNA were removed through centrifugation. Anchor-DNA modified MSN was dispersed into 10 mL ultrapure water containing 100 mg CuSO4, and stirred at 6 °C for 12 h. The as-prepared products were collected by centrifugation (12 000 rpm, 20 min) and washed several 5



times with deionized water, then reacted with obtained G-quadruplex structure through the hybridization between S3 and S4 at 37 °C. Based on the hybridization reaction between the anchor-DNA and formed G-quadruplex structure, the DNA hybrid was capped onto the surfaces of the MSN and the G-quadruplex structures prevented the leakage of the Cu2+ from the mesopores. The final Cu2+@MSN-DNA cap were collected by centrifugation, washed with ultrapure water several times, and finally dispersed in 5 mL PBS for further use.

Fabrication of Bioanode and Biocathode The bare paper fibers were wholly covered with an Au NPs layers via a in situ growth method to improve the conductivity as well as the biocompatibility of the bare paper electrode according to previous work.12 After that, 5 mg·mL-1 of GOx and BOD solution (in 0.01 M PBS pH 7.4, 20 μL) were dropped on to Au NPs modified paper bioanode or biocathode electrode (PAE/PCE-Au) and maintained for 2 h respectively. Finally, physically absorbed excess GOx and BOD were rinsed with PBS (0.01 M, pH 7.4).

Verification of the Distance-Based Colorimetric Assay To investigate the feasibility of the distance-based readout system, a simple preliminary experiment was carried out. First, a mixture PBS solution (pH 7.4) with KI (50 mM) and 5 % starch was sprayed onto the straight channel to ensure droplets uniformly diffusion. After drying the reagent, the paper-based device was folded and assembled as shown in Scheme S2D, and various concentrations of H2O2 were spotted onto the inlet and incubated at ambient temperature for 1 min. The introduced H2O2 induces the oxidation of iodide to yield a colored readout signal (blue bar) in the presence of starch which can be directly acquired by naked eye. And the length of blue bar is related to the concentration of H2O2.

Biofuel Cell Operation To assess the practical applicability for miRNA-21 quantification, the glucose/O2 BFC based self-powered device was assembled using the as-prepared PAE-Au-GOx bioanode and PCE-Au-BOD biocathode. Prior miRNA-21 assaying, the Cu2+@MSN-DNA cap was preloaded within the reaction zone (10 and 11 in Scheme S1A). And 20 µL solution containing variation 6


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concentration of miRNA-21 was dropped in to the reaction zone 10 (Scheme S1A) and kept for 30 min allowing miRNA-21 binding with anti-miRNA-21 (S3) to unlock the gate, thereby causing the release of Cu2+ from Cu2+@MSN-DNA cap (Scheme S3). For comparison, 20 µL PBS was dropped in to the reaction zone 11 (Scheme S1A) and kept for 30 min, and layer d and e were stacked and assembled as shown in Scheme S2C folding along pre-defined creases and clamped, followed by a 2 h incubation at room temperature. During this procedure, the released Cu2+ permeates through the upper paper and bounded to the surface of GOx. For BFC operation, layer e was first cut off, and layers from a to d were folded as Scheme S2D, then glued together tightly (Scheme S2E). Finally, 100 µL 50 mM glucose in PBS was dropped into the inlet on layer a. The solution can wick vertically into the solution transfer layer, further wetting the electrolyte layer, finally reaching electrode layer to initiate redox reaction on bioanode and biocathode. Power output related to amount of miRNA-21 was recorded. Meanwhile, the generated H2O2 from the oxidation of glucose in bioanode migrated to the straight channel to oxidize iodide further reacted with starch resulted in a blue visualization reaction. Thus, based on both the colorimetric and electrochemical signal output, the amount of target analyte could be quantified.

Results and Discussion Materials characterization Through a self-catalysis reduction process, an interconnected Au NPs layers were grown on microfibers of cellulose paper. Figure 1 presents the morphologies of the scanning electron microscopy (SEM) image evolution of the paper fibers with modification. The bare paper fibers exhibited a 3D network structure which would be favorable for solution infiltration and oxygen permeation. Compared with bare fibers (Figure 1A), it could be clearly seen that the Au NPs tightly arranged on the cellulose microfibers for PAE/PCE-Au (Figure 1B). Furthermore, energy dispersive X-ray (EDX) mapping analysis (Figure 1C) revealed a homogeneous distribution of Au, C and O elements, demonstrating the fiber was fully covered by Au element. X-ray diffraction (XRD) pattern was further used to describe the crystal structure evolution of paper electrode (Figure 1D). The XRD peaks at 22.88° collected from bare paper fiber was arose from the (002) planes of cellulose (JCPDS 03-0289) (curve a). The diffraction peaks apart from bare paper peaks could be well matched those of the cubic phase of Au NPs (JCPDS 04-0784) (curve b), providing 7



a solid evidence for the growth of Au NPs layers on cellulose fibers.35 All these results indicated a successful preparation of the PAE/PCE-Au. Figure 1 E showed the typical SEM images of enzymes loaded on the PAE/PCE-Au, with the similar morphology of PAE/PCE-Au. The amplificatory SEM imaging (Figure 1F) revealed that it was hard to discriminate individual enzymes, but the enzyme agglomerates homogenously distributed through the nanofibres could be seen, hinting the successful immobilization of enzymes. In addition, electrochemical impedance spectra were carried out to evaluate the successful immobilization of enzymes on PAE/PCE-Au (Figure S1). There was an obvious increase of electron-transfer resistance indicating an inhibition of the electron transfer between the redox-probe and electrode, which was ascribed to the anchor of enzymes. This was consistent with the observation from SEM images (Figure 1E and F).

Figure 1 SEM micrographs of bare paper nanofibre network before (A) and after (B) Au NPs modification; EDX mapping images (C) of the PAE/PCE-Au; XRD patterns (D) of the bare paper nanofibre network before (a) and after (b) Au NPs modification; SEM image of enzyme fixed on PAE/PCE-Au with low (E) and high (F) magnifications.

The SEM image in Figure 2A demonstrated that the as-prepared MSN exhibited a spherical morphology with diameter around 80 nm. TEM image in Figure 2B also revealed a homogeneous distribution of the as-prepared MSN with consistent size distribution observed from SEM image. From high resolution TEM (HRTEM) image in Figure 2B inset, large amount of ordered 8


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nanopores (about 2 nm in diameter) can easily be distinguished. The large amount of pores would be favorable for cargo loading. Figure 2C shows the zeta potential evolution of MSN after different modification. The zeta-potential was switched from -13.43 to +15.7 mV after amination treatment. After anchor-DNA immobilization, the zeta-potential further decreased. The decreased zeta-potential was indicative of more negative charged molecules fixed. After Cu2+ loading, the value further increased to -8.6 mV, further suggesting the effective loading of Cu2+ onto the MSN. Finally, the zeta-potential value of Cu2+@MSN-DNA cap drastically decreased to -53.5 mV, indicating the successful immobilization of DNA hybrid. In addition, the pore volume and pore diameter

of

bare

MSN

and

Cu2+@MSN-DNA

cap

were

investigated

by

the

Barrett-Joyner-Halenda (BJH) pore distribution (Figure 2D) and N2 adsorption desorption isotherm (Figure 2E). It could be noted that both the pore volume and pore diameter decreased for Cu2+@MSN-DNA cap compared with bare MSN agreed with previous report well,36,

37

which

might be attributed to modification. Furthermore, to investigate the stability of the Cu2+@MSN-DNA cap, the change of the current intensity of Cu2+ was monitored using chronoamperometry in a traditional three electrode system where glassy carbon electrode worked as working electrode, Ag/AgCl as reference electrode and a Pt as counter electrode with a bias voltage of -0.4 V (Figure 2F). No obvious signal change could be seen until the introduction of miRNA-21 (curve a). For comparison, only a slight current increase appeared for bare PBS which was attributed to the addition of target miRNA-21 induced solution perturbation (curve b). All those results suggested the pores of MSN are fully blocked and nearly no leakage of Cu2+ occurs.

Figure 2 SEM (A) and TEM (B) images of as-prepared MSN, inset in (B) is the HRTEM image of MSN; Zeta potentials evolution (C) of MSN with different modification; Pore size distributions 9



(D) and nitrogen adsorption-desorption isotherms (E) of bare MSN (a) and Cu2+@MSN-DNA cap; (F) Chronoamperometric response of Cu2+@MSN-DNA cap solution (curve a) and bare PBS solution (curve b) after the addition of miRNA-21.

Electrochemical Study of Glucose Oxidation and Oxygen Reduction in Bioanode and Biocathode The amperometric response of the as fabricated PAE-Au-GOx to successive addition of glucose solution was recorded at 0 V (vs Ag/AgCl) in 0.01 M PBS (pH 7.4). As illustrated in Figure 3A, a steady and prompt anodic current response was observed with the addition of glucose (curve a). The increase of the current response is due to the redox-active mediator FAD in glucose oxidation catalyzed by GOx.38 And the current responses increased with the continue addition of glucose (curve a). While, there was no amperometric response recorded for the PAE-Au (curve b), demonstrating that the generated catalytic current was attributed to the presence of GOx. Figure 3B shows the cyclic voltammetry (CV) behavior of PAE-Au-GOx to the bioelectrocatalyzed performance toward glucose with a scan rate of 50 mV·s-1. In the absence of glucose, a redox reaction peak of FAD/FADH2 within GOx was observed at -0.48 V vs. Ag/AgCl (curve a in Figure 3B).39 While, no biocatalytic current response toward glucose could be observed. With the introduction of 50 mM glucose, a remarkable anodic current increase arose, which was due to the efficient oxidation of glucose. And the decrease in the cathodic current was attributed to the reduction of H2O2,40 also demonstrated the oxidation of glucose catalyzed by GOx. Figure 3C showed the polarization curve of the PAE-Au (curve a) and PAE-Au-GOx (curve b) in the presence of 5 mM glucose. Both the anodic current response of PAE-Au and PAE-Au-GOx increased with the increasing of applied potential from -0.6 to 0.8 V. Finally, the anodic current for PAE-Au-GOx reached a moderately high current density of about 18.5 μA/cm2 at 0.1 V, while no obvious catalytic current was observed for the PAE-Au in the range of 0.15-0.8 V, which indicated a lack of reactivity of the PAE-Au with respect to the direct oxidation of glucose.19 Moreover, the current density increased with glucose concentration increasing and achieved higher current density of 105 μA cm−2 (0.1 V) with 50 mM glucose (curve c). To investigate the inhibition effect of Cu2+ toward the catalytic performance of GOx, the polarization curve of PAE-Au-GOx upon the addition of Cu2+ was recorded. The anodic catalytic current response 10


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decreased after the addition of 10 μM Cu2+ (curve d), which obviously indicating the efficient inhibition of Cu2+ toward GOx enzyme.41 The inhibition was ascribed to the coordination of the Cu2+ to the reduced form of the flavin cofactor (FADH2) in the active site of GOx that further restrained FADH2 from reducing O2.42

Figure 3 (A) Chronoamperometric response of PAE-Au-GOx (a) and PAE-Au (b) bioanode with successive addition of 50 mM glucose; (B) CV curves of PAE-Au-GOx bioanode without (a) and with (b) 50 mM glucose introduction; (C) Polarisation curves of PAE-Au bioanode (a) and PAE-Au-GOx bioanode (b-d) in the in the presence of 5 mM glucose (a and b), 50 mM glucose (c) and mixture of 10 μM Cu2+ and 50 mM glucose (d).

The biocatalytic performance of the assembled PCE-Au-BOD toward oxygen reduction reaction (ORR) was examined. Figure 4A shows CV of the as-prepared biocathode in the N2-saturated or air-saturated 0.01 M PBS (pH 7.4) at room temperature. No observable redox response under anaerobic condition for the PCE-Au-BOD (curve a). However, in the air-saturated conditions, the resulting biocathode demonstrated outstanding bioelectrocatalytic activity for ORR at 0.58 V (VS Ag/AgCl) (curve b), similar to the redox potential of the Cu ion at the type Ι Cu site of BOD.43 Neither copper site in BOD nor oxygen reduction current was observed without loading of BOD on PCE-Au (curve c). All those results demonstrated that the as-prepared biocathode could be used an efficient biocatalyst for O2 reduction. Meanwhile, Figure 4B showed the polarization curves of the biocathode. No obvious catalytic current were observed under N2-saturated conditions (curve a). While, biocatalytic O2 reduction of was observed at +0.48 V (vs Ag/AgCl) in air-saturated conditions (curve b), which is close to the thermodynamic equilibrium potential of E0O2/H2O (0.61 V at pH 7.0).44 And the electrocatalytic current reached 280 µA·cm-2 near +0.35 V (vs Ag/AgCl). The formation of Au NPs layers is favorable for the direct electron communication between BOD and paper electrode. 11



Figure 4 CV responses (A) of PCE-Au-BOD biocathode (a and b) and bare PCE-Au (c) in 0.1 M PBS (pH 7.4) under N2-saturated (a) and ambient air (b and c) atmosphere; polarization curves (B) of the PCE-Au-BOD biocathode in 0.1 M PBS (pH 7.4) under N2-saturated (a) and ambient air (b) atmosphere.

Performances of the as-assembled BFC

Figure 5 Dependence of the power density on the cell voltage (A) in 0.1 M PBS (pH 7.4) under ambient air atmosphere containing 50 mM glucose under different conditions: (a) removing of undesired H2O2, (b) without removing of undesired H2O2, and (c) removing of undesired H2O2 and introduction of 10 μM Cu2+ to bioanode; (B) stability of the power output of the BFC operating by the continuous addition of glucose at 0 h (a) and 2.5 h (b).

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Scheme 1 Working principle of the as-fabricated BFC based self-powered platform without (A) and with (B) the presence of Cu2+.

The compartment-less glucose/O2 BFC was assembled as indicated in Scheme 1. The power output of the assembled glucose/O2 BFCs on the cell voltage in 0.01 M PBS (pH 7.4) containing 50 mM glucose is shown in Figure 5A. The BFC device yielded a maximum power output density of 132 µW·cm-2 at 0.61 V, and gains an open circuit potential of 0.77 V (curve a), which exhibited superior or comparable performance than previous reported work, even though the experimental conditions are difference different (Table S1) While, without the removing of H2O2 generated from bioanode, both the maximum power output and open circuit potential decreased (curve b). The result suggested that it was necessary to remove the undesired H2O2 keep the BFC performance. Besides, to understand the inhibition effect of Cu2+ toward BFC, the maximum power output decreased from 132 µW·cm-2 to 103 µW·cm-2, and the open circuit potential shifted from 0.76 V to 0.72 V with the addition of 10 μM Cu2+ to bioanode (curve c), which demonstrated the efficient inhibition of Cu2+ toward the GOx. Due to the low capacity of the electrolyte reservoir, the assembled BFC could continuous operate for 2.0 h (curve a in Figure 5B). Interestingly, the power output of this device can be fully recovered by rejecting additional glucose solution (curve b). The result clear indicated that the power output was mainly influenced by the biofuel depletion.45 Based on this, it would be possible to make continuously power output by providing enough fuels. And the working procedure of the biofuel cell could be summarized as follows: glucose + 𝑂2 +𝐺𝑂𝑥(𝐹𝐴𝐷)→𝑔𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐻2𝑂2 +𝐺𝑂𝑥(𝐹𝐴𝐷𝐻2) GOx(𝐹𝐴𝐷𝐻2)→2𝐻 + +2𝑒 ― +𝐺𝑂𝑥(𝐹𝐴𝐷) 13


(1) (2)


𝑂2 +4𝐻 + + 4𝑒 ―

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𝐵𝑂𝐷

(3)

2𝐻2𝑂

𝐻2𝑂2 + 𝐼 ― →𝐼2 + 𝐻2𝑂

(4)

𝑛𝐼2 +6𝑛(𝐶6𝐻10𝑂5)→2𝑛(𝐶18𝐻30𝑂3𝐼)(𝑏𝑙𝑢𝑒)

(5)

Feasibility Verification of Distance-Readout Strategy

Figure 6 (A) Pictures of KI solution and mixture of KI and H2O2; (B) photograph of starch solution, mixture of starch and KI and mixture of starch, KI and H2O2; response of the distance based readout strategy toward different amount of H2O2 (C) and Cu2+ (D).

To demonstrate the hypothesis of that H2O2 presence would oxidize the iodide to iodine, we mixed the KI with H2O2. A brilliant yellow color was observed (Figure 6A), indicating the oxidization of iodide. For the classical starch chromogenic reaction, KI was first mixed with starch, no color change occurred (middle of Figure 6B). While once H2O2 was added, a deep blue occurred (right of Figure 6B), confirming the generation of iodine. To verify the feasibility of the distance readout strategy, different amount of H2O2 were added into inlet on layer a. In this reaction, the H2O2 travels along the channel by capillary action, and the iodide was oxidized by H2O2, and subsequently reacted with the pre-embedded starch generating a blue bar (Figure 6C), whose length was in proportion to the concentration of H2O2. For the miRNA-21 sensing, the introduction of miRNA-21 would lead to the release of Cu2+. To verify the leakage of Cu2+ would inhibit the biocatalysis activity toward GOx, further influencing the H2O2 generation, various 14


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amount of Cu2+ was added into the bioanode for signal gathering (6 in Scheme S1A), for comparison, no Cu2+ was dropped in the bioanode for calibration (4 in Scheme S1A). It is observed that with the Cu2+ concentration increased, the length of distance bar gradually decreased, while almost no discernible length changes were observed in the control one (Figure 6D), confirmed the efficient inhibition effect of Cu2+ toward GOx.

Quantification for miRNA-21 For miRNA-21 analysis, target miRNA-21 with various concentrations was added into the reaction zone for quantification (10 in Scheme 1A). In such process, miRNA-21 worked as a key to unlock the Cu2+@MSN-DNA cap via competing against anchor-DNA (S1) to fully hybridize with anti-miRNA-21 (S3) triggering the release of Cu2+ (Scheme S3). The released Cu2+ then bonded to the surface of GOx resulted in the GOx biocatalytic performance decrease (Scheme 1B). And the maximum power output decreased with the addition of miRNA-21 (Figure 7A). Figure 7B displays the calibration curve, the maximum power output density linearly decreased with the increasing of miRNA-21, and an excellent relationship was acquired (R2= 0.9835) in the range of 5 fM to 100 pM (average of eleven measurements). The linear regression equation was Pmax= 134-23.3lgc, (Pmax is the maximum power output density, c is the concentration of miRNA-21), with a low detection limit of 2.7 fM (at a signal-to-noise ratio of 3). Figure 7C and D show the reduction in length of blue stripe upon the increasing of miRNA-21. These results demonstrated the practical application and its potential to be used for on-site assay in the future. It is worth noting that the linear range and detection limit of our method present comparable to those of previously reported miRNA-21 sensors (Table 1).

Table 1 Comparison of the elaborated biosensor with other analytical strategies. Method

Target

Liner range

Detection limit

Reference

Electrochemiluminescence

miRNA

0.1 fM-10 pM

0.03 fM

9

Fluorescence

miRNA

-

4

5.36 fM

46

100 aM-100 pM Electrochemical

miRNA

20 fM-50 pM 15



Page 16 of 22

Electrochemical

miRNA

100 fM-25 pM

25 fM

47

PEC

miRNA

1 fM-5 pM

0.35 fM

48

PEC

miRNA

1 fM-150 pM

0.74 fM

8

Electrochemical

miRNA

5 fM-100 pM

2.7 fM

This work

Figure 7 (A) the variation of power outputs of the fabricated BFC at different concentrations of target miRNA-21; (B) Plot of Pmax vs miRNA-21 concentrations, inset is linear relationship between Pmax and the logarithm of miRNA-21 concentrations; (C) and (D) quantification of miRNA-21 using a distance-based readout strategy.

To investigate the anti-interference ability of this method, other three kinds of non-target miRNA (single base mismatched, three base mismatched and non-complementary miRNA) were selected to perform contrast experiments at same concentration (1 pM) and same experiment conditions. As shown in Figure 8A and B, only in the presence of target miRNA-21 exhibited string signal response, whereas the other non-complementary miRNA had no obvious effect on the signal response compared with the blank. Such an excellent selectivity of the self-power sensor can stand interference from these co-existing species. The repeatability and reproducibility were also investigated by intra- and inter-assay. And variation coefficients for intra- and inter-assay were 3.4 % and 4.7 %, showing a satisfactory repeatability and reproducibility. Moreover, after 1 month storage at 4 °C, no obvious changes were observed which reflected the good stability of this method. 16


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Figure 8 Selectivity investigation (A and B) of the proposed method; (C) practical application of the proposed method from the blank experiment, MCF-7 and Hela cancer cells with different cell numbers.

Finally, Hela cancer cells and MCF-7 cancer cells were used to investigate the expression level of miRNA-21 in tumor cells. Prior to measuring, the tumor cell sample was accurate counted, followed by lysing to extraction miRNAs via a commercial miRNA extraction instrument.8 As shown in Figure 8C, negligible signal changes were observed for the miRNA-21 extracted from Hela cancer cells due to the low expression in Hela cells. It was found that with the increasing amount of MCF-7 cancer cells, the corresponding signal attenuation gradually. It was a direct experimental evidence to demonstrate that miRNA-21 mainly acted as a dominant breast carcinoma-specific miRNAs, thus exhibited a high expression in MCF-7 cancer cells.9 Meanwhile, to further evaluate the target miRNA-21 detection in complex samples, the presented sensing platform was carried out to monitor miRNA-21 in normal human serum samples without miRNA-21 expression based on the method of standard addition. As can be seen (Table S2), the recoveries of the proposed method are in the range from 97 % to 106.5 %, and the relative standard deviation are below 5 %, indicating that this platform holds a great promise in real biological samples.

Conclusion In summary, a paper supported enzyme glucose/O2 BFC based self-powered device equipped with a distance-based readout system was developed for miRNA-21 quantification. Thanks to the efficient removing the undesired H2O2, the byproduct of glucose oxidation, not only a visual quantitative approach can be implemented based on the classical chromogenic reaction between iodine and starch, but also enhanced BFC performance could be achieved. Such a self-powered 17



device realized a broad linear range from 5 fM to 100 pM appeared with a detection limit of 2.7 fM toward miRNA-21. Meanwhile, it should be pointed out that the present method would overcome the external power source restriction, and would be actually accomplished without any sophisticated instruments that provide a low cost, flexible and ubiquitous sensing strategy. In addition, our results exhibit rational design strategies for making efficient BFC and push the frontier of BFC device.

Acknowledgements This work was financial supported by the Major Program of Shandong Province Natural Science Foundation (ZR2017ZC0124), the program for Taishan Scholar of Shandong province (ts201712048), National Natural Science Foundation of China (21874055, 51632003), and the Key Research and Development Program of Shandong Province, China (2018GGX103037). Supports from the 111 Project of International Corporation on Advanced Cement-based Materials (No. D17001) is greatly appreciated.

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