Enzyme-Free Photoelectrochemical Biosensor Based on the Co

Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/ascecg

Enzyme-Free Photoelectrochemical Biosensor Based on the CoSensitization Effect Coupled with Dual Cascade Toehold-Mediated Strand Displacement Amplification for the Sensitive Detection of MicroRNA-21 Yanxin Chu,†,§,∥ Rong Wu,§,∥ Gao-Chao Fan,‡,§ An-Ping Deng,*,† and Jun-Jie Zhu*,§

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†

The Key Lab of Health Chemistry & Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering & Materials Science, Soochow University, Suzhou 215123, P.R. China ‡ Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China § State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210023, P.R. China S Supporting Information *

ABSTRACT: An ultrasensitive photoelectrochemical (PEC) biosensor was developed based on cosensitization of biocompatible CuInS2/ZnS quantum dots (ZCIS QDs) and N-doped carbon dots (N-CDs) coupled with dual cascade toehold-mediated strand displacement amplification (dual cascade TSDA) for microRNA-21 (miRNA-21) detection. On the one hand, the TiO2/Au hybrid structure was used to immobilize double stranded DNA (thiolated capture strand and carboxylated signal strand), which could capture glutathione stabilized ZCIS QDs and N-CDs. The original TiO2/Au/ZCIS/N-CDs structure formed a cascade band gap arrangement, which provided a good band position for effective charge carrier separation, thus improving PEC performance and resulting in an evident decrease in photocurrent signal after the release of signal strands (SIG). On the other hand, the sensitivity of the biosensor was further enhanced by enzyme-free dual cascade TSDA, which was initiated by the target miRNA-21, like a molecular machine, and consumed the substrates and fuels, repeatedly used the target miRNA-21, and released a large number of reporter strands (RS). Subsequently, the released RS replaced SIG to prevent ZCIS QDs and N-CDs from sensitizing the electrode, which remarkably suppressed the photocurrent signal. The introduction of TSDA could produce high amplification capacity and specificity for the target miRNA-21 with advantages of simple primer design and mild reaction conditions. Impressively, with the cascade band gap arrangement for enhanced PEC performance and enzyme-free dual cascade TSDA for amplification capacity and specificity, the PEC biosensor exhibited excellent application in miRNA-21 analysis with a linear range from 1 pM to 100 nM and a low detection limit of 0.31 pM. This PEC biosensor retained good specificity, stability, and reproducibility and provided an effective method for PEC biosensor construction for microRNA. Moreover, the designed PEC biosensor was environmentally friendly, green manufactured, and self-powered and therefore compatible with the purpose of sustainable chemistry. KEYWORDS: photoelectrochemistry, biosensing, microRNA-21, toehold-mediated strand displacement, cosensitization

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INTRODUCTION

Nevertheless, these methods possess intrinsic drawbacks, including low sensitivity, poor specificity, high cost, and sophisticated instrumentation. Therefore, considering that miRNA concentration is at a very low level in biological samples, a simple, inexpensive, fast, and sensitive platform is urgently needed for clinical sample sensing for miRNAs.

MicroRNAs (miRNAs) are single-stranded, short-chain RNAs that function as transcriptional regulators factors for gene expression. Disorders of miRNAs are connected with human diseases such as diabetes, angiocardiopathy, and cancer. Therefore, analysis of miRNA expression is important to the early diagnosis of cancers, pathogenesis studies, and timely treatment of diseases.1−5 Recently, several conventional assays have been developed for miRNA analysis, including Northern blot, microarrays, and real-time polymerase chain reaction.6 © XXXX American Chemical Society

Received: April 25, 2018 Revised: July 23, 2018 Published: August 10, 2018 A

DOI: 10.1021/acssuschemeng.8b01857 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Scheme 1. (A) Schematic Illustration of Dual Cascade TSDA Initiated by miRNA-21; (B) Construction Process of the Designed PEC Biosensor for miRNA-21 Detection

carbon dot (N-CDs),26 Cu2S nanowire arrays/CDs.27 The efficiently enhanced PEC performance can be subsequently achieved, which benefits from excitation wavelength-dependent photoluminescence behavior, broad absorption range, and high electrical conductivity of CDs.28 Moreover, large amounts of functional groups, such as carboxyl and hydroxyl groups, make CDs disperse well in water and conjugate easily with nanoparticles.29 In addition, the N-doping method is used to further extend visible light absorption. Compared with most heavy metal semiconductor quantum dots, N-CDs are environmentally friendly and promising in PEC analysis. Moreover, a self-powered PEC matrix was achieved without external potential application due to the high photocurrent conversion efficiency of TiO2/Au/ZCIS/N-CDs cosensitized structure, and it conforms to the aim of sustainable chemistry with respect to energy conservation. Signal amplification is another key factor in artificial biosensors, including hybridization chain reaction (HCR),30 rolling circle amplification (RCA),31 cyclic enzymatic amplification method (CEA),32 and toehold-mediated strand displacement amplification (TSDA).33 Among them, CEA and RCA are limited by the intrinsic properties of the nucleases used because most of the them are not applicable to targets. HCR amplification also has several deficiencies, such as high background signal or environmental sensitivity, which hinder more extensive application. However, TSDA, driven by the configurational entropy of the released molecule, is superior because it circumvents many problems including addition of auxiliary substances, mediation of enzymes, sophisticated construction of primer, and accurate control of reaction temperature. It allows for the target molecules to catalyze the release of the output strand (OS), which in turn functions as a catalyst for other reactions. Thus, the target is reused, and signal amplification is achieved. Toehold domain exchange is conducted by a hybridization reaction and forms a

As a clean, sustainable, and environmentally friendly energy, solar energy has attracted worldwide attention, which is the key input for the photoelectrochemical (PEC) process. Based on the incorporation of PEC method and biosensing technique, PEC detection as a newly developed analytical technology, potentially possesses superiorities of simple equipment, selfpowering ability, and low cost, which are consistent with the aims of sustainable chemistry. Moreover, benefiting from the complete separation of input (light source) and output (photocurrent signal), high sensitivity and low background can be obtained.7,8 Of course, the enhanced PEC intensity is a key factor in promoting the performance of PEC biosensors, which mainly depends on the photocurrent response efficiency of semiconductor materials.9 A cosensitization structure with cascade boundary is a promising strategy, which can promote the harvest of light, the separation of charge, and the photocurrent output.10 For the PEC bioanalysis, semiconductor materials such as TiO2 and ZnO have been used in previous research, but few works focused on cosensitization strategies such as ZnO/CdS/CdTe, TiO2−NTs/CdS:Mn/ CdTe, and TiO2/CdSeTe/CdS:Mn structures.11−13 In recent years, inorganic semiconductor quantum dots (QDs) have been the focus of considerable attention in PEC applications, which possess ideal characteristics, such as convenient synthesis, high absorption coefficients, and size and composition-tunable photoluminescence from the visible to the near-infrared.11,14−21 Among them, the environmental friendly I (B)-III (A)-VI (A) QDs such as CuInSe2 and CuInS2 possess a narrow bandgap, which match with the solar spectrum well and are ideal sensitizers.22,23 Recently, a noninjection and high yield rate colloidal synthesis technology for monodisperse CIS QDs was developed.24 Carbon quantum dots (CDs) have also been used to form cosensitized structures for photocatalytic and photoelectric systems such as Bi2O3 inverse opal/CDs,25 TiO2 nanowire arrays/nitrogen-doped B


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against deionized water for 24 h to remove unreacted reactant. The prepared N-CDs were stored at 4 °C for future use. Preparation of the ITO/TiO2/Au Electrode. First, the ITO electrode was cleaned by sequential ultrasonic and solvent treatment for removing possible organics and enhancing the hydrophilia.11 Afterward, the electrode was dried at 80 °C for 12 h. Subsequently, TiO2 (20 μL, 1 mg/mL) was spread dropwise on the ITO electrode to form a thin film with an area of 0.25 cm2 and then calcined for 30 min at 450 °C in a muffle furnace. Then, the TiO2 modified electrode was immersed in HAuCl4 (1 wt %, pH 4.5) for 10 min followed by deionized water cleaning and drying in air atmosphere. Subsequently, the electrode was calcined at a temperature of 300 °C for 120 min.41 Finally, the color of the prepared ITO/TiO2/Au electrode was fuchsia. Enzyme-Free DNA Strand Displacement Circulation Procedure. The three-strand DNA partial complementary duplex linker strand (L1) was synthesized by mixing equal volume of Template Strand 1 (TS1, 1 μM), Supporting Strand 1 (SS1, 1 μM), and Output Strand (OS, 1 μM) in PBS (0.01 M) with heat treatment at 95 °C for 5 min and then slowly cooled to 25 °C. The probe (L2) was prepared with the same method (with Template Strand2 (TS2, 1 μM), Supporting Strand2 (SS2, 1 μM), Report Strand (RS, 1 μM) in PBS (0.01 M)). Fuel strand 1 (FS1) and Fuel strand 2 (FS2) probe solutions were mixed with prepared linker strands to obtain L1 and L2 sensing solutions, respectively. Subsequently, 20 μL of miRNA-21 with various concentrations (11 pM−11 μM) was added into 100 μL of L1 sensing solution (the mixture of L1 and FS1) and incubated at 25 °C for 1 h. Afterward, 100 μL of L2 sensing solution (the mixture of L2 and FS2) was introduced into the above mixture and incubated for another 1 h. Electrophoresis analysis was carried out to test the correctness of the designed TSDA. Native Polyacrylamide Gel Electrophoresis (PAGE). Then, sample (10 μL) and 6× loading dye (2 μL) were mixed and analyzed using 8% native PAGE at 150 V for approximately 60 min in 1× TBE buffer. The gels were stained with ethidium bromide (EB) for 15 min, followed by recorded through a Bio-Rad gel imaging system (U.S.A.). Fabrication of the Biosensor. Typically, 100 μL of thiolated capture strand (CS, 2 μM) was activated with 2.5 μL of TCEP (10 mM) for 1 h, and then 100 μL of carboxylated signal strand (SIG, 2 μM) was incubated with CS for another 1 h to form a duplex strand probe (CS/SIG). Then, 15 μL of CS/SIG was added and incubated with ITO/TiO2/Au electrode at 4 °C in a moisture atmosphere overnight. The DNA-modified electrode was blocked with MCH (1 mM, 15 μL) for 1 h. Subsequently, 15 μL of sensing solution incubated with miRNA-21 with various concentrations was incubated with the electrode for 3 h at 25 °C. After that, the electrode was immersed in the EDC/NHS (10 mg/mL, 2:1, v/v) for 30 min to activate the carboxylic group of SIG. Then, 15 μL of ZCIS QDs was incubated with the electrode for 12 h at 4 °C. Finally, 400 μL of NCDs was activated by EDC/NHS (10 mg/mL, 2:1 v/v, 10 μL) for 30 min at 25 °C, and 15 μL of activated N-CDs was incubated with the electrode for 12 h at 4 °C. The electrode was rinsed with washing buffer after each step. PEC Measurement. A xenon lamp provided an excitation light with a spectral range of 200 to 2500 nm. The electrolyte was ascorbic acid (AA, in PBS pH = 7.4, 0.1 M), which served as an electron donor during detection process. Before measurement, the electrolyte was deaerated with pure nitrogen for 15 min.

duplex strand to make the nonterminal toehold inactive, which prevents nonspecific strand displacement.34,35 Due to the outstanding amplification capacity and specificity, it has been applied in detecting small molecules (e.g., ATP) and biomacromolecules (e.g., miRNAs and DNA).36−38 The integration of catalytic TSD and a PEC biosensor with a cosensitized structure would certainly be desirable and could potentially improve the detection limits of the target molecules. However, few works focused on this superior strategy coupled with PEC sensing are reported until now. In order to remain consistent with the development trend of nontoxic materials and green technology, we present a novel, ultrasensitive PEC assay based on the sensitization effect of a TiO2/Au/ZCIS/N-CDs hybrid structure coupled with signal amplification of an enzyme-free dual cascade TSDA for miRNA-21 detection. As shown in Scheme 1, the TiO2 nanoparticles were used as a substrate of cosensitization by forming a mesoporous film on an indium tin oxide (ITO) electrode. Then, Au nanoparticles were deposited to form the TiO2/Au structure to immobilize the thiolated capture strand (CS). Without a target, glutathione-stabilized CuInS2/ZnS quantum dots (ZCIS QDs) and N-CDs were successfully bonded to the carboxylated signal strand (SIG) by coupling reaction between carboxyl and amino groups, which enhanced the photocurrent signal. Conversely, with the introduction of miRNA-21, a DNA-fueled molecular machine carried out the enzyme-free dual cascade TSDA, which was started by hybridization of the target microRNA-21 (miRNA-21) with the terminal toehold domain on the linker strands (L1 and L2), consumed the fuel strands (FS1 and FS2), reused the target miRNA-21, and consequently released abundant reporter strand (RS) on the L2. Subsequently, the released RS could displace the carboxylated SIG, which prevents cosensitization effect of ZCIS QDs and N-CDs and dramatically suppresses the PEC signal. This work extended the application of a DNA-fueled molecular machine (viz., dual cascade TSDA) in the PEC field, and the designed PEC biosensor exhibited good sensitivity and selectivity for miRNA21 detection.

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EXPERIMENTAL SECTION

Preparation of ZCIS QDs. 1. Preparation of CIS QDs. Watersoluble, glutathione stabilized CIS QDs were synthesized according to the method reported by Chen et al.39 Briefly, CuCl2 (0.01 mmol), InCl3 (0.04 mmol), sodium citrate (0.16 mmol), glutathione (0.02 mmol), and deionized water (20 mL) were added in a three-neck flask. Then, 0.062 mmol Na2S was injected into the mixture with stirring. Subsequently, the reaction mixture was heated to 95 °C and maintained for 40 min. Eventually, the glutathione-stabilized Cu−In− S quantum dots were obtained. 2. Preparation of ZnS Shell-Coated CIS QDs. Precursor solutions were obtained by mixing 0.368 g of glutathione, 0.061 g of thiourea, 0.176 g of Zn(OAc)2·2H2O, and 20 mL of deionized water (the pH value was adjusted to the range of 6.0 by 1.0 M NaOH solution and stored at 4 °C for use), and 400 μL of stock solution was injected into the as-prepared CIS reaction solution and maintained for another 45 min at the same temperature to obtain the ZnS shell over the CIS core. The resulting product ZCIS QDs is precipitated by ethanol, purified by ultrafiltration, and placed in a refrigerator at 4 °C for further use. Synthesis of N-CDs. For the synthesis of N-CDs, a typical hydrothermal reaction proceeded.40 Then, citric acid (1.314 g), urea (1.502 g), and deionized water (30 mL) were mixed and stirred to obtain a clarified mixture. The mixture was sealed in a Teflon-lined, stainless-steel autoclave at 160 °C for 8 h. The product was dialyzed

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RESULTS AND DISCUSSION Characterization of TiO2/Au Structure. Figure 1A shows the ITO/TiO2 electrode morphology. The mesoporous TiO2 film was composed of TiO2 nanoparticles with size of a 22−28 nm. When the Au nanoparticles (Au NPs) were deposited on the TiO2 film (Figure 1B), a number of smaller size nanoparticles (approximately 6−10 nm) were observed. Moreover, the color change of the electrode surface from white to fuchsia further confirmed the deposition of Au NPs. As shown in Figure 1C, Au NPs appeared on the bare ITO C


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fringes in HRTEM image (Figure S1B). The optical properties were investigated by photoluminescence spectra and UV−vis absorption. Figure 3A indicates the fluorescence emission

Figure 3. PL (A) spectra and UV−vis absorption (B) spectra of NCDs.

spectra of N-CDs varying by excitation wavelength, which verifies that the fluorescent emission of N-CDs was excitationdependent. Moreover, with excitation wavelength ranging from 350 to 450 nm, the emission peak of N-CDs varied from 450 to 504 nm, which may be due to the quantum effect caused by unevenly sized CDs and different surface traps on the NCDs.43 As shown in Figure 3B, the UV−vis spectrum indicates that N-CDs had characteristic absorption of 340 nm. The photograph images in Figure S2B further demonstrate that NCDs had good dispersity in aqueous solutions and exhibited strong blue color under 365 nm ultraviolet light. Electrophoresis Analysis. As shown in Figure 4A, to confirm the DNA amplification circuit, PAGE was carried out.

Figure 1. SEM images of (A) ITO/TiO2, (B) ITO/TiO2/Au, and (C) ITO/Au electrodes. The embedded images in (A) and (B) are corresponding photograph images. (D) EDS characterization of ITO/ TiO2/Au electrode.

electrode, which was similar to the deposition on TiO2 film. Figure 1D shows the EDS of the ITO/TiO2/Au electrode, which also confirmed the existence of Au and Ti elements. Characterization of ZCIS QDs. To verify the successful synthesis of ZCIS QDs, HRTEM was carried out. As shown in Figure S1A, the lattice fringes of ZCIS QDs could be clearly seen, and the mean size was 2−3 nm. Compared with the fluorescence emission spectrum of CIS QDs, as shown in Figure 2A, ZCIS QDs exhibited a stronger fluorescence signal

Figure 4. Gel electrophoresis analysis image of programmable DNA amplification. (A) Lane 1: TS1; Lane 2: TS1:SS1:OS linker strand (L1); Lane 3: the mixture of L1 and miRNA-21; Lane 4: the mixture of L1 and FS1; Lane 5: the mixture of L1, miRNA-21, and FS1; Lane 6: TS1:OS:miRNA-21 complex (I1); Lane 7: TS1:FS1 complex; Lane 8: the mixture of L1 and FS1 (annealed); Lane 9: the mixture of L1, miRNA-21, and FS1 (annealed). (B) Lane 1: TS2; Lane 2: TS2:SS2:RS linker strand (L2); Lane 3: the mixture of L2 and OS; Lane 4: the mixture of L2 and FS2; Lane 5: the mixture of L2, OS, and FS2; Lane 6: TS2:RS:OS complex (I2); Lane 7: TS2:FS2 complex; Lane 8: the mixture of L2 and FS2 (annealed); and Lane 9: the mixture of L2, OS, and FS2 (annealed).

Figure 2. (A) PL spectra and (B) UV−vis absorption spectra of CIS QDs (black line) and CuInS2/ZnS core/shell QDs (red line).

from 500 to 700 nm and had 30 nm of blue-shifted fluorescence emission peak owing to the passivation originated by ZnS shell. The ultraviolet spectra show that CIS QDs and ZCIS QDs had a similar wide absorption range, and their absorption edges were below 600 nm without an obvious absorption peak (Figure 2B). The photograph images show that ZCIS QDs exhibited a strong orange fluorescence signal under ultraviolet light, which was different from CIS QDs (Figure S2A). For CIS QDs, the surface defect plays an important role in carrier capture, which reduces the charge separation efficiency. Wrapping QDs to form core/shell QDs with a passivation layer is an effective way to reduce surface trapping states for enhanced charge separation efficiency.24,42 Therefore, compared with the TiO2/Au/CIS electrode (I = 22 μA), the enhanced photocurrent intensity was obtained (I = 45 μA) for the TiO2/Au/ZCIS electrode (Figure S3). Characterization of N-Doped Carbon Dots. The carbon dots possessed a uniform size of 3−5 nm with clear lattice

For the first circuit (TSDA1), Lane 2, a band of L1, was observed with a much lower migration rate than the single strand TS1 in Lane 1; the band of complex (TS1:FS1) was in Lane 7, and the migration rate was higher than L1. When L1 was incubated with FS1 (Lane 4), a weak band was observed at the same position as that of the TS1:FS1 complex (Lane 7). Compared with Lane 7, the weak band in Lane 4 indicated a low level of spontaneous interactions between L1 and FS1. After L1 was incubated with miRNA-21 at 25 °C for 1 h (Lane 3), the band of TS1:OS:miRNA-21 intermediate complex (I1, Lane 6) appeared, and the band of L1 disappeared. After target miRNA-21 was incubated with L1 and FS1 at 25 °C for 1 h (Lane 5), the band of TS1:FS1 complex was obviously observed, and the band of L1 had almost disappeared. Compared with annealing reagents during the reaction (Lane 8 and Lane 9), the bright and narrow band in Lane 5 D


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ACS Sustainable Chemistry & Engineering demonstrated that the targeted TSDA1 was near completion. The complete system behaved as expected: the untargeted reaction was slow (Lane 4), and a certain quantity of target promoted the reaction to proceed rapidly to near completion. Similar to TSDA1, the PAGE characterization of the circuit shown in Figure 4B indicated the successful synthesis of L2 (Lane 1 and Lane 2), and the OS in TSDA 1 worked as a catalyst target in TSDA 2 to accelerate the TSDA 2 reaction to approach completion (Lanes 3−9). The full formation gel including control lanes were presented in Figure S4A,B. The band of released OS and RS could be observed in Lane 8, which further proved the reliable and predictable circuit behavior. Moreover, the cascade circuit behavior was analyzed (Figure S4C in Supporting Information) compared with the slow reaction rate of untargeted reaction, and a stoichiometric quantity of miRNA-21 could enable the dual cascade TSDA to proceed rapidly to near completion. PEC Mechanism of the Biosensor. The sensitivity of the DNA sensor could be evaluated by photocurrent response change when miRNA-21 with various concentrations was added into the PEC sensing platform. As illustrated in Scheme 1A, the dual cascade TSDA was composed of two linker strands (L1, L2) and two fuel strands (F1, F2). In the TSDA 1, when the target and fuel strands were added, the DNA amplification circuit was initiated by hybridization of miRNA21 to the terminal toehold domain on the L1 complex and generated a new active toehold domain (of 4 nt) as the correct association of F1 to I1. Along with a new hybridization displacement reaction between F1 and I1, OS was released and initiated the second amplification circuit. Meanwhile, the miRNA-21 also dissociated from I1 and was reused. In the TSDA 2, the OS worked as an input strand and triggered the release of RS from L2 according to the same process. Subsequently, the RS released from L2 could substitute the carboxylated SIG on the electrode and prevent ZCIS QDs and N-CDs from sensitizing the electrode. Thus, the photocurrent was remarkably suppressed. On the contrary, SS1 and SS2 were designed to block the second toehold regions to inhibit spontaneous interactions without target. Briefly, RS was unable to be released from the TSDA followed by attachment to the CS without miRNA-21, and the unreacted Linker strands and fuel strands were washed away from the electrode surface. The amplified photocurrent response in the presence of the miRNA-21 was expected to achieve high detection sensitivity. Moreover, the sensitivity was further amplified by constructing the TiO2/Au/ZCIS/N-CDs heteronanostructure. The mechanism of photogenerated electron−hole transfer was shown in Scheme 2. Wide band gap semiconductor TiO2 (3.2 eV) can only absorb ultraviolet light, leading to low light utilization efficiency. However, ZCIS has a 1.55 eV bandgap that matches the solar spectrum and can promote electron transfer from QDs to the TiO2/Au substrate. The absorption and emission of ZCIS QDs can be adjusted to the visible and near-infrared range. In addition, N-CDs were also used as a sensitizer and introduced into the PEC sensor. During light irradiation, the photogenerated electrons produced by the carbon dots were first injected into the conduction band of the ZCIS QDs. At the same time, the electrons at the valence band (VB) of ZCIS QDs were also photoexcited and jumped to the conduction band (CB). Because the CB and VB of ZCIS QDs are higher than those of TiO2, the electrons at the CB were rapidly transmitted from ZCIS QDs to the TiO2 along the direction of the N-CDs-ZCIS-TiO2-photoanode-counter elec-

Scheme 2. Transfer Mechanism of the Photo-Generated Electrons in the PEC DNA Biosensor

trode. At the same time, the photogenerated holes were transferred to the interface between the electrode, and the electrolyte and consumed AA to accelerate charge transfer and separation efficiency for enhanced PEC performance. The above explanation shows that the TiO2/Au/ZCIS/N-CDs heteronanostructure acted as a sensitized structure for enhanced PEC response and achieved remarkable PEC signal suppression after the SIG were substituted. Therefore, the combination of dual cascade TSDA and the cosensitization effect is expected to be ultrasensitive in miRNA-21 detection. Characterization of the Biosensor. As shown in Figure 5A, the UV−vis absorption spectrum first confirmed the fabrication progress. TiO2/Au showed an absorption edge at 380 nm, corresponding to the characteristic bandgap absorption of TiO2 (∼3.2 eV). In contrast, a stronger absorption of TiO2/Au/ZCIS below 380 nm and the extended absorption edge to 600 nm of the visible light region was ascribed to the characteristic band absorption of ZCIS (∼1.55 eV). After CDs modification, a sharp absorption peak at 350 nm appeared, which belonged to the characteristic peak of the CDs. The UV−vis spectrum indicates that the ZCIS QDs and CDs could be conjugated onto prepared TiO2/Au electrodes by coupling reactions between carboxylated SIG, −NH2 groups of ZCIS QDs, and −COOH groups of CDs. Electrochemical impedance spectroscopy (EIS) also provided effective evidence of the electrode modification (Figure 5B). The impedance spectra included a semicircle (representing electron-transfer-limited process) and a linear part (representing diffusion-limited process). The electron-transfer resistance (Ret) reveals the restricted diffusion of the redox probe [Fe(CN)6]3−/4− accessing the layer. The ITO/TiO2 electrode showed a relatively small Ret (curve a), indicating small electron transfer resistance. After in situ growth of Au NPs on the TiO2 film, the Ret was reduced (curve b), which was attributed to the good conductivity of Au nanoparticles. After duplex DNA, MCH, and sensing solution were incubated with the electrode (curve c), the Ret was slightly increased due to steric hindrance increase and electron transfer efficiency reduction. The Ret significantly increased after the ZCIS QDs were immobilized on the electrode because of the poor electrical conductivity of the QDs (curve d). When N-CDs were introduced into the biosensor, the Ret decreased (curve e) because of good conductivity. The EIS characterization also E


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Figure 5. UV−vis absorption spectra (A) of (a) ITO/TiO2/Au electrode, (b) after DNA and QDs incubation, (c) after N-CDs immobilization; EIS (B) and photocurrent responses (C) of (a) the ITO/TiO2 electrode, (b) after Au deposition, (c) after duplex DNA immobilization MCH blocking and sensing solution incubation (0 M), (d) after ZCIS QDs incubation, (e) after N-CDs immobilization and photocurrent response of (f) ITO/TiO2/Au/duplex strand DNA/ZCIS/N-CDs.

Figure 6. (A) Photocurrent responses of the PEC biosensor to different concentration of miRNA-21 (0, 10−3, 10−2, 10−1, 1, 10, 100, and 1000 nM). (B) Calibration curve of the PEC miRNA biosensor. The error bars display the standard deviations of five parallel tests.

Table 1. Analysis Performance Comparison methods

target microRNA

linear range (M)

LOD (fM)

assay time

refs

fluorescence fluorescence photoelectrochemistry DPV ECL electrochemistry photoelectrochemistry

let-7d miRNA-21 miRNA-122a miRNA-21 miRNA-20a miRNA-21 miRNA-21

10−11−10−10 10−12−10−8 3.5 × 10−13−5 × 10−9 2 × 10−10−3.88 × 10−7 10−13−10−8 2 × 10−11−10−7 10−12−10−7

8400 300 153 10000 100 3400 310

16 h 40 min 4h 15.5 h 18.5 h 8h 27 h

44 45 46 47 48 49 this work

Analytical Performance. On the basis of dual cascade TSDA coupled with the sensitization effect, miRNAs could be sensitively detected by the PEC biosensor (Figure 6). Under the optimized experimental conditions, along with the increase in concentration of miRNA-21, the PEC signal gradually decreased. The photocurrent signal was found to be logarithmically related to the miRNA-21 concentration varying from 1 pM to 100 nM. The linear regression equation was I = 34.64−6.31 log C (nM) with a correlation coefficient of 0.9917, and the limit of detection (LOD) of the biosensor (S/ N = 3) was 0.31 pM. In addition, the leakage of DNA amplification circuit was estimated by comparing photocurrent values of curve e (I = 53.4 μA) and curve f (I = 58 μA) in Figure 5C. 4.6 μA of photocurrent decrease was observed, which was caused by nonspecific release of RS even in the absence of target. The intra/interassay precisions were determined by five parallel measurements of miRNA-21 at concentrations of 10 pM, 100 pM, and 1 nM with the relative standard deviation (RSD) values of 4.4%/5.1%, 3.9%/5.6%, and 4.6%/4.9%, respectively, indicating a good stability and reproducibility of the PEC DNA biosensor. As shown in Table 1, the detection performance of this proposed method was

suggests the biosensor was successfully fabricated layer by layer. Moreover, the fabrication process could be also monitored by the photocurrent response change (Figure 5C). The ITO/TiO2/Au electrode showed a small photocurrent. It was because of low photocurrent conversion efficiency of narrow ultraviolet absorption of TiO2 (curve b, I = 27 μA). After Au deposition, further decreased intensity (curve b, I = 27 μA) was observed. Because of the lower Fermi level of Au, there is a transfer of photogenerated electrons from TiO2 to Au for equilibrating the Fermi level between them. Subsequently, the photocurrent decreased after the duplex strand DNA (CS/SIG), MCH, and sensing solution were incubated in sequence (current c, I = 15 μA) because of the increase in steric hindrance and the reduction in electron transfer efficiency. The photocurrent markedly increased as the ZCIS QDs modified the duplex DNA on the electrode due to the sensitization effect of ZCIS QDs (curve d, I = 42 μA). The N-CDs enhanced the photocurrent response because of its high electrical conductivity and broad band optical absorption (current e, I = 52 μA). All these results demonstrated the successful fabrication of the PEC biosensor. F


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potential interferents such as albumin, triglycerides, and total cholesterol contained in serum. Nevertheless, a good recovery and mild matrix effect are observed in 1:10 diluted serum samples, indicating that a good feasibility is achieved with the proposed method. All these results demonstrated the PEC sensor has a favorable anti-interference, and possible matrix effects have no significant influences toward target miRNA detection.

compared with some reported methods on miRNA detection. The LOD of 0.31 pM was lower than those of assays mentioned, including fluorescence (8.4 pM),44 DPV (10 pM),47 and electrochemistry (3.4 pM).49 Long incubation time was essential (Figure S5), and the linear range was comparable. As shown in Figure 7, miRNA-21, the single-base mismatched miRNA-21 (SM miRNA-21), and another

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CONCLUSIONS In summary, a PEC biosensor for sensitive detection of miRNA-21 was achieved under a mild condition with a TiO2/ Au/ZCIS/N-CDs cosensitization structure combined with an enzyme-free dual cascade TSDA. As a PEC matrix, the TiO2/ Au/ZCIS/N-CDs sensitization structure could remarkably enhance the photocurrent signal because of the cascade band gap arrangement, effective charge separation, and high utilization of excitation light, which further improved the detection sensitivity. The enzyme-free DNA cycle amplification achieved by dual cascade TSDA exhibited outstanding amplification capacity and specificity and had advantages in nonintroduction of auxiliary substances, nonmediation of enzymes, simple primer design, and mild reaction conditions. Moreover, the sensor is environmentally friendly and has the features of simple post-treatment, self-powering ability, and low cost, all of which are advantages for a new generation of green manufactured biosensors. On basis of the proposed strategy, the PEC sensor exhibited a low LOD of 0.31 pM and a linear range of 1 pM−100 nM for miRNA-21 detection. The developed PEC detection method can be used as an attractive candidate for tracking horizontal miRNA detection, which can provide valuable information for early clinical diagnosis.

Figure 7. ΔI (I0 nM − I1 nM) of photocurrent response of (a) miRNA21, (b) SM miRNA-21, and (c) miRNA-141 in the concentration of 1 nM.

miRNA (miRNA-141) were selected to investigate the selectivity. As expected, the PEC response (ΔI = (I0 nM − I1 nM)) for complementary target miRNA-21 was obviously the highest among three kinds of miRNA sequences at the same concentration of 1 nM. The cross-reactivities of miRNA-141 and SM miRNA-21 were 12.6% and 22.8%, respectively. This result demonstrates that the biosensor was selective and could distinguish miRNA-21 from other noncomplementary sequence and SM sequence. Stability of the PEC Biosensor. As shown in Figure S6, the long-term storage stability of the biosensor was investigated. The ITO/TiO2/Au/CAP/SIG/MCH electrodes were made and kept in a refrigerator at 4 °C. At different storage times (such as 1, 2, 3, 4, 5, 6, and 7 days), the electrodes were incubated with sensing solution at two different concentrations (0 M and 1 nM). The photocurrent response for 0 M and 1 nM decreased progressively with the storage time increased from 1 to 7 days. As a result, the electrode after storage of 7 days still retained 88% and 80% for 0 M and 1 nM, compared with the newly prepared electrodes. Good stability of the PEC sensor may be attributed to two aspects: first, duplex DNA strands can be firmly bond to the electrode surface through Au−S bonds; second, both TiO2 and Au have good chemical stability and biocompatibility. Recovery Experiment in Spiked Blood Sample. Spiked blood sample was evaluated as a complex biological environment to explore the practicability of the proposed PEC sensor. After a series of processes with methanol treatment, centrifugation, and adjusting the pH to neutral, the blood sample was stored at −20 °C for later use. In the recovery experiment, two different concentrations of analyte miRNA-21 were spiked into diluted human serum. After 5 parallel detection processes, the results are shown in Table S2. When 5-fold dilution with PBS was produced, the recovery rates vary from 76 to 84% for 1 pM and 81 to 87% for 10 pM spiked in the samples, which indicates a minor negative matrix effect. The recovery rates were improved to 85−94% and 89−93% for 1 and 10 pM spikes when the spiked serum samples were diluted 1:10 in PBS. The matrix effect is mainly caused by the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b01857. Experimental details, materials and apparatus, additional HRTEM, photograph images, PEC data, PAGE and sequences of the oligonucleotides (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (A. D.). *E-mail: [email protected] (J.J.Z.). ORCID

Yanxin Chu: 0000-0003-0008-2387 Gao-Chao Fan: 0000-0002-4645-3115 An-Ping Deng: 0000-0002-6451-9384 Jun-Jie Zhu: 0000-0002-8201-1285 Author Contributions ∥

Y.X.C. and R.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully appreciate funding from the National Natural Science Foundation of China (21603099, 31772053, and 21175097), the International Cooperation Foundation from the Ministry of Science and Technology (2016YFE0130100), G


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Enzyme-Free Photoelectrochemical Biosensor Based on the Co

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