Dual Signal Amplification Using Gold Nanoparticles-Enhanced Zinc

Oct 10, 2016 - Using Au nanoparticles (NPs)-decorated, water-soluble, ZnSe-COOH nanoflakes (NFs), an ultrasensitive photoelectrochemical (PEC) biosens...
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Dual Signal Amplification Using Gold Nanoparticles-Enhanced Zinc Selenide Nanoflakes and P19 Protein for Ultrasensitive Photoelectrochemical Biosensing of MicroRNA in Cell Wenwen Tu,† Huijuan Cao,† Long Zhang, Jianchun Bao, Xuhui Liu, and Zhihui Dai* Jiangsu Key Laboratory of Biofunctional Materials and Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, People’s Republic of China S Supporting Information *

ABSTRACT: Using Au nanoparticles (NPs)-decorated, watersoluble, ZnSe-COOH nanoflakes (NFs), an ultrasensitive photoelectrochemical (PEC) biosensing strategy based on the dual signal amplification was proposed. As a result of the localized surface plasmon resonance (SPR) of Au NPs, the ultraviolet−visible absorption spectrum of Au NPs overlapped with emission spectrum of ZnSe-COOH NFs, which generated efficient resonant energy transfer (RET) between ZnSe-COOH NFs and Au NPs. The RET improved photoelectric conversion efficiency of ZnSe-COOH NFs and significantly amplified PEC signal. Taking advantage of the specificity and high affinity of p19 protein for 21−23 bp double-stranded RNA, p19 protein was introduced. P19 protein could generate remarkable steric hindrance, which blocked interfacial electron transfer and impeded the access of the ascorbic acid to electrode surface for scavenging holes. This led to the dramatic decrease of photocurrent intensity and the amplification of PEC signal change versus concentration change of target. Using microRNA (miRNA)-122a as a model analyte, an ultrasensitive signal-off PEC biosensor for miRNA detection was developed under 405 nm irradiation at −0.30 V. Owing to RET and remarkable steric hindrance of p19 protein as dual signal amplification, the proposed strategy exhibited a wide linear range from 350 fM to 5 nM, with a low detection limit of 153 fM. It has been successfully applied to analyze the level of miRNA-122a in HeLa cell, which would have promising prospects for early diagnosis of tumor.

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spectrum of Au NPs overlapped with the emission spectrum of ZnSe-COOH NFs, which generated resonant energy transfer (RET) between ZnSe-COOH NFs and Au NPs.20−25 The RET wonderfully improved photoelectric conversion efficiency of the ZnSe-COOH NFs, which was advantageous for developing plasmonic-enhanced PEC biosensing platform. MicroRNAs (miRNAs) with a length of 17−24 nucleotides are a new class of biomarkers that perform a critical role in malignancies.26−28 The current research of miRNAs determination relies on Northern blot,29,30 reverse transcriptase− polymerase chain reaction (RT-PCR),31,32 and microarrays.33,34 Northern blot is time-consuming and needs large amounts of samples.35 RT-PCR requires short primers and reverse transcription, which increases experimental cost and design complexity.36 Microarray suffers from poor reproducibility, inferior selectivity, and inaccuracy.37 In this work, an innovative PEC biosensing platform has been proposed based on ZnSeCOOH NFs/Au NPs to develop a sensitive, fast, simple, specific, low-cost, and convenient method for miRNAs

hotoelectrochemical (PEC) sensing has received increasing attention due to its desirable analytical performance for future bioassay.1−5 Metal oxide semiconductors as traditional photoelectrochemically active materials have been intensively investigated.6,7 Nevertheless, only absorption in ultraviolet range hinders its application in PEC bioanalysis.8,9 Cadmium and plumbum-containing quantum dots (QDs) can be excited under visible light irradiation.10,11 However, the heavy metals elements are highly toxic and are a menace to cells, limiting their utilization in biological systems.12,13 In addition, the photoelectric conversion efficiencies of cadmium- and plumbum-containing QDs were low.14 ZnSe-COOH nanoflakes (NFs) are good candidates for photoelectronic devices, owing to their low toxicity, outstanding photostability, excellent water solubility, satisfactory biocompatibility, and ease of preparation.15 However, ZnSe-COOH NFs have not been utilized in PEC sensing up to now due to its generation of slight photocurrent under visible light irradiation. In this work, after Au nanoparticles (NPs) grafted to the surface of ZnSe-COOH NFs, the photocurrent of ZnSe-COOH NFs/Au NPs significantly enhanced compared with that of ZnSe-COOH NFs alone. As a result of the localized surface plasmon resonance (SPR) of Au NPs,7,16−19 the ultraviolet absorption © 2016 American Chemical Society

Received: June 21, 2016 Accepted: October 10, 2016 Published: October 10, 2016 10459

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were bought from China Sinopharm Chemical Company. Except the above reagents, all reagents were analytical grade in this work. In this work, the supporting electrolyte was 0.1 M tris(hydroxymethyl)aminomethane (Tris)-HCl buffered saline and its pH value was 7.4 unless indicated otherwise. The washing solution was phosphate buffered saline (PBS; 10 mM, pH 7.4, CNaCl = 0.3 M). Double-distilled water (DDW) was used in all assays. P19 protein was purchased from New England Biolabs. Diethylpyrocarbonate (DEPC)-treated deionized water was obtained from TaKaRa Biotechnology Co., Ltd. The DNA was obtained from Shanghai Sangon Biological Engineering Technology and Services Co. with HPLC purification. The synthetic miRNAs and oligonucleotide probes were purchased from GenePharma Company (Shanghai, China). Their base sequences were listed as follows: help DNA, 5′-SH-GGGGGG-3′; probe RNA, 5′-p AAC ACC AUU GUC ACA CUC CAU ACC CCC C-3′; target miRNA-122a, 5′-p UGG AGU GUG ACA AUG GUG UUUG-3′; MiRNA21, 5′-p UAU UGC ACA UUA CUA AGU UGC A-3′; MiRNA-32, 5′-p UAU UGC ACA UUA CUA AGU UGC A-3′. Instruments. The PEC determinations were carried out on a PEC workstation (Zahner, Germany) with a three-electrode system at room temperature under 405 nm excitation at −0.30 V, except where otherwise indicated. The modified indium tin oxide (ITO) electrode (sheet resistance 20−25 Ω/square, the modified area was 0.25 cm2) was the working electrode. The platinum wire was the counter electrode and Ag/AgCl electrode was the reference electrode. The supporting electrolyte for PEC measurements was 0.1 M Tris-HCl buffered saline in the presence of 25 mM ascorbic acid. High purity nitrogen inlet to the solutions which bubbled at least 15 min before each experiment, and a nitrogen atmosphere was maintained over the test solutions throughout the PEC detection process. H7650 type transmission electron microscope (TEM, Hitachi, Japan) was operated at an accelerating voltage of 80 kV to analyze the morphology and size of the nanocomposites. A Cary 60 UV−vis spectrometer (Agilent, U.S.A.) was used to record ultraviolet−visible (UV−vis) absorption spectra of the nanomaterials. A LS 50B fluorophotometer (PerkinElmer) was utilized to collect fluorescence spectrum. Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) was performed on an Agilent 7500ce ICP-MS (U.S.A.). Electrochemical impedance spectroscopic measurements were operated on an Autolab potentiostat/galvanostat PGSTAT302N system (Metrohm, Netherlands) in K3[Fe(CN)6] (5 mM)/K4[Fe(CN)6] (5 mM) solution containing 0.1 M KCl. The impedance spectra were recorded in the frequency range of 10−1−105 Hz. The amplitude of the applied sine wave potential in each case was 10 mV. Fourier transform infrared (FTIR) spectra were detected on Tensor 27 (Bruker, Germany) at room temperature. Synthesis of Oil-Soluble ZnSe NFs. ZnSe NFs was synthesized as follows. Briefly, 0.0682 g ZnCl2 and 10 mL of ODE were mixed at room temperature in a 100 mL three-neck flask. Then, the reactor was heated to 250 °C at a rate of 5 °C/ min and maintained at that temperature for 30 min. Subsequently, the Se precursor solution that obtained by dissolving 0.0555 g of SeO2 in 5 mL of terpineol under ultrasonication until the solution turned clear was quickly injected into the reaction system. After reacted at 250 °C for 1 h, the reactor was naturally cooled down to room temperature. The ZnSe NFs products were purified by precipitation with ethanol, and followed by centrifugation at 12000 rpm for 3 min.

detection. The attachment of Au NPs to ZnSe-COOH NFs offered strong localized SPR for electrical field amplification effect.8,21 RET restrained electron−hole pairs recombination and improved photoinduced electron transport,21 thus, significantly promoting photovoltaic conversion efficiency of ZnSe-COOH NFs and amplifying PEC signal. In order to further promote the sensitivity and selectivity of PEC detection, p19 protein was introduced. P19 protein from carnation Italian ring spot virus binds with high affinity to 21− 23 bp double-stranded RNA (dsRNA) and sequenceindependent manner.38,39 P19 protein as huge-volume and insulated layer blocked interfacial electron transfer and generated remarkable steric hindrance for electron donor to photoinduced holes. After P19 protein combined with dsRNA, the photocurrent intensity dramatically decreased compared with that of dsRNA alone, which amplified photocurrent signal change versus concentration change of target. Using miRNA122a as a model analyte, an ultrasensitive signal-off PEC biosensor was designed via dual signal amplification (Scheme 1). RET significantly improved initial photocurrent intensity or Scheme 1. Schematic Illustration of the PEC Biosensing Platform Based on SPR of Au NPs Enhanced RET and Remarkable Steric Hindrance of p19 Protein as Dual Signal Amplification

blank signal of the signal-off biosensor and p19 protein amplified photocurrent signal change versus concentration change of target, which was the dual signal amplification. The developed strategy exhibited excellent analytical performance and was successfully applied to detect the content of miRNA122a in HeLa cell, which would have promising perspective for clinical diagnoses of cancer.

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EXPERIMENTAL SECTION Materials and Reagents. Trichloroethyl phosphate (TCEP), 1,4-dithiothreitol (DTT), disodium ethylenediaminetetraacetic acid (EDTA), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide (EDC), 1-octadecene (ODE, > 90%), Nhydroxysuccinimide (NHS), and tris(hydroxymethyl)-aminomethane (Tris) were obtained from Sigma-Aldrich. Ascorbic acid (AA) and chloroauric acid (HAuCl4·4H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. Zinc chloride (ZnCl2, 98%) and selenium dioxide (SeO2, 98%) were obtained from Shanghai MeiXing and XinBao Chemical Reagent Co. Ltd., respectively. The terpineol (95%), dihydrolipoic acid (DHLA, 98%), absolute alcohol, and heptane 10460

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photocurrent measurements. The excitation wavelength and the applied potential for PEC analysis of miRNA was 405 nm and −0.30 V, respectively. Cellular extracts were prepared according to the previous researches.42,43 After the PEC biosensor was incubated with the obtained cellular extracts (20 μL) for 1 h at 37 °C, 0.01 M PBS was utilized to rinse the surface of the biosensor. Subsequently, the resulting PEC biosensor was incubated with p19 protein for 1 h at 37 °C and washed with PBS. Finally it was inserted into the above supporting electrolyte to detect miRNA-122a in cellular extracts.

The obtained crude product was washed by absolute ethanol and heptane for 5 times to remove byproducts. Finally, the product was dried and stored in vacuum, which was used for characterization and analysis. Surface Modification of ZnSe NFs and Synthesis of Au NPs. A mixture of as-prepared ZnSe NFs sample (0.05 mmol), cyclohexane (15 mL), ethanol (15 mL), and DHLA (0.15 g) was stirred at room temperature for 48 h. The product was then isolated by centrifugation at 12000 rpm for 10 min, washed with deionized water, ZnSe-COOH NFs was obtained, and then dried at vacuum for further use. The Au NPs were synthesized according to the previous report with a minor modification.40 Briefly, all glassware was cleaned in aqua regia (HCl/HNO3 = 3:1), rinsed with H2O, and then oven-dried prior to use. A total of 400 μL of cysteamine solution (213 mM) was injected into 40 mL of HAuCl4 solution (1.42 mM) under stirring for 20 min at room temperature, and then 2 mL of NaBH4 solution (10 mM) was added under stirring, which resulted in a change in the solution color from pale yellow to wine-red. After the color changed, the solution was further stirred for 15 min followed by cooling to room temperature. Finally, the Au NPs colloidal solution obtained was placed in a refrigerator for further use. Fabrication of the PEC Biosensor. A piece of bulked ITO (sheet resistance 20−25 Ω/square) was incised to small pieces of rectangular ITO, and then the nonconductive rubberized fabric with hollow-carved geometrical area of 0.25 cm2 (0.5 cm × 0.5 cm) was pasted on the small piece of rectangular ITO to obtain the modified electrode (ITO electrode; Figure S-1A in Supporting Information). The ITO electrode was sonicated in acetone and NaOH (1 M) in 1:1 (V/V) ethanol/water and then in water for 15 min, respectively. A ZnSe-COOH NFs solution (20 μL, 1.5 mg·mL−1) was dropped on the electrode to form ZnSe-COOH modified ITO electrode (ITO/ZnSeCOOH). PBS (20 μL) containing NHS (0.005 M) and EDC (0.01 M) was placed on the above electrode and then maintained 60 min, rinsing twice with PBS. Before drying, Au NPs solution (20 μL) was dropped onto the electrode surface. The Au NPs grafted on the ZnSe-COOH NFs via amidation. After rinsing, 20 μL of 1 μM help DNA was applied to the ZnSe-COOH NFs/Au NPs modified electrode with stay overnight at 4 °C. Subsequently, in order to get rid of unlinked help DNA, 0.01 M PBS was used to swill the as-prepared modified electrode. The resulting modified electrode was blocked with monoethanolamine (MEA, 1 mM) and incubated for 1 h at 4 °C. MEA could block the nonspecific sites and reduce the nonspecific adsorption. After rinsing that modified electrode above, 20 μL of 1 μM probe RNA was introduced for 2 h incubation at 37 °C, and then it was rinsed to obtain the PEC biosensor. Synthesis of depositing Au NPs directly on the surface of ZnSe-COOH NFs was according to the reported method.41 The other related experimental procedures for the modification of the electrode were as same as the above process. Detection Procedure. MiRNA hybridization was performed by incubating the PEC biosensor with 20 μL of target miRNA solution containing various concentrations of miRNA122a for 1 h at 37 °C. In order to get rid of unhybridized target miRNA, 0.01 M PBS was used to swill the as-prepared modified electrode, and then incubated with p19 protein for 1 h at 37 °C. After the PEC biosensor was washed with PBS again (Figure S1B in Supporting Information), it was inserted into 0.1 M N2saturated Tris-HCl saline containing 25 mM AA to carry out

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RESULTS AND DISCUSSION Characterization. The TEM image of Au NPs displayed that the morphology was spherical and the average diameter was 13 nm (Figure 1A). Figure 1B revealed a few layer sheets,

Figure 1. TEM images of (A) Au NPs, (B) ZnSe-COOH NFs, and (C) ZnSe-COOH NFs/Au NPs.

illustrating the nanoflakes shape of ZnSe-COOH. The singlesheet nature shown in Figure 1C identified the better dispersion of ZnSe-COOH NFs in water after the introduction of Au NPs. The Au NPs mainly grafted on the edge of ZnSeCOOH NFs (Figure 1C), owing to that Au NPs attached covalently to the surface of ZnSe-COOH by amidation, which might be beneficial for photoinduced electron transfer and RET between Au NPs and ZnSe-COOH NFs, leading to plasmonicenhanced PEC performance of ZnSe-COOH NFs/Au NPs. In addition, from the ICP-MS analyses, the content of Au NPs on ZnSe-COOH NFs were 369 ng by the addition of 20 μL of Au NPs colloid solution (Figure S-2 in Supporting Information). To verify the successful preparation of ZnSe-COOH NFs and Au NPs, and illustrate RET, UV−vis absorption, and fluorescence spectra were performed (Figure 2). ZnSe-COOH NFs showed a broad absorption peak at 368 nm (Figure 2A, curve a) and a strong PL emission peak at about 390 nm (Figure 2B, curve b), overlapping well with broad plasmon absorption spectrum of Au NPs (Figure 2B), which played an important role in RET. This phenomenon suggested the formation of ZnSe-COOH NFs and Au NPs and implied that it 10461

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redox probe accessing the layer. After Au NPs was coated onto the ZnSe-COOH NFs surface, the impedance spectrum of ZnSe-COOH NFs/Au NPs modified electrode (curve b) showed a lower Ret compared to that of ZnSe-COOH NFs modified electrode (curve a), which could be ascribed to that the excellent electron transfer property of Au NPs facilitated Fe(CN)63−/4− to reach the surface of the modified electrode. This result indicated that Au NPs were successfully immobilized on ZnSe-COOH NFs surface through the reaction between −COOH and -NH2 groups. After immobilization of help DNA, Ret increased obviously (curve c), which was attributed to that the negatively charged phosphate backbone of DNA repelled the access of negatively charged redox probe to the surface of the modified electrode. However, Ret declined when the modified electrode above was blocked by MEA (curve d). The electrostatic interaction between the negatively charged probe and the positively charged MEA10 might promote the transport of Fe(CN)63−/4− to the surface of the modified electrode. Subsequently, Ret stepwise promoted when help DNA was hybridized with probe RNA (curve e) and miRNA-122a (curve f), implying an efficient hybridization reaction occurred on the modified electrode surface. The increased Ret might be attributed to that negative charge of RNA phosphate backbone introducing by hybridization reaction hindered the access of the negatively charged probe ([Fe(CN)6 ] 3− / 4− ) to the electrode surface. After the introduction of p19 protein, Ret increased dramatically (curve g), suggesting the successful assembly of p19 protein in dsRNA. This phenomenon was agreed with the fact that p19 protein layer was huge-volume and insulated, which could block the interfacial electron transfer and generate remarkable steric hindrance for impeding the transfer of [Fe(CN)6]3−/4− to the surface of the modified electrode. This variation of Ret confirmed the formation of biosensing interface for miRNA detection. Moreover, the IR spectra further characterized the immobilization of components of the PEC biosensor (Figure S4 in Supporting Information), the emergence of the amide I (1636 cm−1) and amide II (1564 cm−1) bands verified the covalent assembly of Au NPs on the surface of ZnSe-COOH NFs by the amidation reaction on the biosensing interface. PEC Behaviors of the Modified Electrodes. As the hydrophilic and biocompatible material, ZnSe-COOH NFs were used as the photoelectrochemically active species to produce PEC signal under 405 nm excitation at −0.30 V. A cathodic photocurrent of 814 nA was observed at ZnSe-COOH NFs modified ITO electrode in N2-saturated Tris-HCl saline containing 25 mM AA (Figure 4A, curve a). After Au NPs covalently bond to ZnSe-COOH NFs through amidation reaction, ZnSe-COOH NFs/Au NPs modified electrode exhibited a significantly enhanced PEC response (photocurrent of 1130 nA; Figure 4A, curve b), which was 1.4-fold of the photocurrent obtained at ZnSe-COOH NFs modified electrode. The much improved PEC response might mainly ascribe to SPR of Au NPs enhanced RET between ZnSeCOOH NFs and Au NPs.11 RET promoted charge separation and restrained electron−hole pairs recombination in ZnSeCOOH NFs/Au NPs,21 which significantly promoted photoelectric conversion efficiency of ZnSe-COOH NFs in the visible light region, leading to the amplification of the PEC signal (Scheme 1). While Au NPs was deposited directly on the surface of ZnSe-COOH, a cathodic photocurrent of 940 nA was observed (Figure S-5 in Supporting Information, curve b), which was smaller than that of ZnSe-COOH NFs/Au NPs

Figure 2. (A) UV−vis absorption (a) and fluorescence (b) spectra of ZnSe-COOH NFs; (B) UV−vis absorption spectrum of Au NPs.

was favorable to develop ultrasensitive PEC biosensing platform via RET. The biocompatibility of a photoelectrochemically active material is positively related to its hydrophilicity, which can be evaluated by the contact-angle measurement. The contact angles of the bare ITO electrode, ZnSe NFs and ZnSe-COOH NFs films were measured to be 34.21°, 93.30° and 40.02°, respectively (Figure S-3 in Supporting Information). The smaller contact angle of ZnSe-COOH NFs films than that of ZnSe NFs films indicated their better hydrophilicity, which was contributed to more hydrophilic groups introduced by carboxy group on ZnSe-COOH NFs. The good biocompatibility and hydrophilicity of ZnSe-COOH NFs could greatly improve loading capacity of biomolecules, thus, it was advantageous for the construction of highly sensitive PEC biosensor. The stepwise assembly process of PEC biosensing interface was monitored by electrochemical impedance spectroscopy (Figure 3). The semicircle diameter equals the electron transfer resistance (Ret), which reflects the restricted diffusion of the

Figure 3. Nyquist diagrams of (a) ZnSe-COOH NFs, (b) ZnSeCOOH NFs/Au NPs, (c) ZnSe-COOH NFs/Au NPs/help DNA, (d) ZnSe-COOH NFs/Au NPs/help DNA/MEA, (e) ZnSe-COOH NFs/ Au NPs/help DNA/MEA/probe RNA, (f) ZnSe-COOH NFs/Au NPs/help DNA/MEA/probe RNA/miRNA-122a (100 pM), and (g) ZnSe-COOH NFs/Au NPs/help DNA/MEA/probe RNA/miRNA122a/p19 protein-modified ITO electrodes in 0.1 M KCl solution containing 5 mM K3Fe(CN)6/K4Fe(CN)6 (1:1). Inset: the electrical equivalent circuit applied to fit the impedance spectra; Rs, Zw, Ret, and Cdl represent Ohmic resistance of the electrolyte, Warburg impedance, electron transfer resistance, and the double layer capacitance, respectively. 10462

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response was observed, and 96.2% of its initial photocurrent response was maintained after 30 days, which indicated that the change of PEC signal was not resulted from the instability of the electrode in such a long time or other surrounding factors. These phenomena illustrated the decrease of the PEC signal was closely related to target miRNA. Therefore, using miRNA122a as a model analyte, an ultrasensitive PEC biosensing platform was designed to realize miRNA detection based on SPR of Au NPs enhanced RET and the remarkable steric hindrance of p19 protein as dual signal amplification. SPR of Au NPs enhanced RET significantly improved initial photocurrent intensity or blank signal of the signal-off PEC biosensor and p19 protein amplified photocurrent signal change versus concentration change of target, which was the dual signal amplification. PEC Biosensing for MiRNA. Under the optimized conditions (Figures S-7 and S-8 in Supporting Information), the proposed PEC biosensing platform was applied to detection of miRNA-122a. The time-photocurrent curves of this PEC biosensor toward miRNA exhibited that the intensity of PEC signal declined stepwise with increasing concentrations of miRNA-122a (Figure 5A). The linear curve was established by

Figure 4. (A) Photocurrent responses of (a) the ZnSe-COOH NFs modified electrode, (b) after Au NPs coating, (c) after incubation with 20 μL of 1 μM help DNA, (d) after MEA blocking, (e) after hybridization with 20 μL of 1 μM probe RNA, (f) after hybridization with 20 μL of 500 pM target RNA, (g) after p19 protein immobilization. (B) PEC responses of ZnSe-COOH NFs (a) and ZnSe-COOH NFs/Au NPs (b) modified ITO electrodes.

(photocurrent of 1130 nA) prepared by covalent combination (Figure S-5 in Supporting Information, curve a). The Au NPs mainly grafted on the edge of ZnSe-COOH NFs via covalent combination, which might be more advantageous for photoinduced electron transfer and RET than that of the direct deposition of Au NPs on the surface of ZnSe-COOH NFs, leading to larger plasmonic-enhanced PEC response. In addition, periodically controlling the light, the PEC signal could be turned on and off. The enhanced photocurrent was relatively stable after five on−off cycles accompanying with a slight decline (Figure 4B, curve b). With the assembly of help DNA (Figure 4A, curve c) and MEA (Figure 4A, curve d), and efficient hybridization with probe RNA (Figure 4A, curve e) and target miRNA (Figure 4A, curve f), respectively, the photocurrents stepwise decreased owing to the fact that increasing steric hindrance of decorated membranes impeded the diffusion of electron donor to valence band of ZnSe-COOH NFs. After p19 protein was introduced to PEC biosensing interface, the photocurrent remarkably fell to 450 nA (Figure 4A, curve g) that was merely 63% of the PEC response (710 nA) before immobilization of p19 protein (Figure 4A, curve f). The significantly declined photocurrent was due to that immense steric hindrance of hydrophobic protein layer blocked the interfacial electron transfer and impeded the AA diffusion to the electrode surface for scavenging the holes.38,39 The introduction of p19 protein amplified PEC signal change versus concentration change of target. However, in the absence of target miRNA (Figure S-6 in Supporting Information, curve b), the PEC response (744 nA) was much larger than that of in the presence of miRNA (450 nA; Figure S-6 in Supporting Information, curve d), which was attributed that P19 protein did not bind to single-strand RNA and it only bond with high affinity to 21−23 bp doublestranded RNA.38,39 Moreover, the PEC stability of the modified electrode without target miRNA and p19 protein for 30 days was examined. No obvious decrease in the photocurrent

Figure 5. (A) PEC responses of the biosensor to different concentrations of miRNA-122a (0, 350 fM, 500 fM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 6 nM, and 7 nM from top to bottom). (B) Linear calibration.

plotting ΔI (ΔI = I − I0, I0 was the current response when miRNA-122a concentration was zero; I was the current response of miRNA-122a at different concentrations) against logC (C was different concentration of miRNA-122a; Figure 5B). The calibration equation was ΔI = 102.3 + 124.0 lg C, with a correlation coefficient R of 0.997. The linear range was 350 fM to 5 nM with a detection limit of 153 fM at 3 S/N. This linear range was wider or the detection limit was lower than those of some other methods (Table S-1 in Supporting Information).44−48 Moreover, the sensitivity was estimated to be 4.96 × 105 AM−1 cm−2. The photocurrent signal change versus concentration change of the target was much larger than that of some recent researches (Table S-2 in Supporting Information) due to the introduction of p19 protein with remarkable steric hindrance as signal amplification. This 10463

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CONCLUSION An ultrasensitive PEC biosensing platform for miRNA detection was designed based on the dual signal amplification. SPR of Au NPs enhanced RET significantly improved initial photocurrent intensity or blank signal of the signal-off PEC biosensor and p19 protein amplified photocurrent signal change versus concentration change of target, which was the dual signal amplification. The RET improved photoelectric conversion efficiency of the ZnSe-COOH NFs, which significantly amplified PEC signal. P19 protein generated remarkable steric hindrance which blocked the interfacial electron transfer and impeded the AA diffusion to the electrode surface for scavenging the holes, leading to the dramatical decrease of the photocurrent intensity. Introduced by carboxy groups, ZnSe-COOH NFs displayed better hydrophilicity that could greatly improve loading capacity of biomolecules, and thus, it was advantageous for the construction of highly sensitive PEC biosensor. After introducing MiRNA-122a, which was a model analyte, a signal-off PEC biosensor for ultrasensitive detection of miRNA was proposed. This variation of Ret confirmed the formation of miRNA biosensing interface. The developed strategy displayed excellent sensitivity, good selectivity, and wide linear concentration range toward miRNA122a detection, and it was successfully applied to analyze the content of miRNA-122a in HeLa cell. This would open an avenue for miRNAs analysis in tumor cells and provide a promising perspective for application to clinical diagnoses of cancer. In addition, the proposed method without using enzyme-based amplification or other PCR methods undoubtedly reduces the design complexity and experimental cost, which avoids the influence due to enzyme denaturation.

developed strategy of PEC biosensing displayed excellent sensitivity and wide concentration range toward miRNA detection owing to the dual signal amplification. SPR of Au NPs enhanced RET significantly improved initial photocurrent intensity or blank signal of the signal-off PEC biosensor and p19 protein amplified photocurrent signal change versus concentration change of target, which was the dual signal amplification. Reproducibility of the Developed Method. Both the intra-assay and interassay precision of the developed method were examined. The relative standard deviations (RSD) of intra-assay were 3.2%, 2.7%, and 2.1% at 500 fM, 10 pM, and 500 pM, respectively. Whereas the interassay RSD of 4.4%, 4.2%, and 3.9% were obtained by measuring the samples of the same concentrations with six electrodes prepared independently under identical experimental conditions. These results indicated good reproducibility of the developed method. Selectivity of MiRNAs Assay. The PEC responses of the biosensor toward miRNA-32, miRNA-21, and miRNA-122a were measured to investigate the selectivity of the designed method. At the same concentration, the normalized PEC response (take the normalized PEC response of miRNA-122a as 1) of miRNA-32 and miRNA-21 were only ∼7% (Figure 6,

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ASSOCIATED CONTENT

S Supporting Information *

Figure 6. Photocurrent changes of the PEC biosensor toward (a) miRNA-32 (100 pM), (b) miRNA-21 (100 pM), and (c) miRNA122a (100 pM). (d) The relative expression of miRNA-122a in HeLa cell.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02381. Additional information as noted in text (PDF).

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column a) and ∼14% (Figure 6, column b) of the PEC signal of miRNA-122a (Figure 6, column c), suggesting negligible interference of miRNA-32 and miRNA-21. Furthermore, this level of mismatch discrimination ability was better than that of other hybridization-based miRNA biosensors.49,50 The reason might be the introduction of p19 protein which has high affinity and specificity for 21−23 bp dsRNA. These results verified that except miRNA-122a, the other two test samples did not change PEC response obviously, indicating an excellent specificity for miRNAs assay. PEC Analysis of MiRNAs in Cell. To confirm the practicability and feasibility of the developed method, the level of miRNA-122a in cell was detected. HeLa (cervical cancer cells) cell lysates were prepared. According to the linear curve (Figure 5B), once the photocurrent change was recorded after HeLa cell lysates were modified on the PEC biosensing interface, the concentration of miRNA-122a in HeLa cell lysates could be estimated. The relative expression of miRNA122a in HeLa cell lysates was given (Figure 6, column d). The average content of miRNA-122a in HeLa cell lysates was found to be 50 pM. Since the linear range of the PEC biosensor for miRNA-122a detection was wide enough (350 fM to 5 nM), the proposed strategy held great potential for assaying the level of miRNA expression in tumor cells.

AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: +86-25-85891051. E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work (W.T. and H.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by NNSFC (21475062, 21533012, 21175069), Natural Science Research of Jiangsu Higher Education Institutions (15KJB150016) and PAPD of Jiangsu Higher Education Institutions.

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DOI: 10.1021/acs.analchem.6b02381 Anal. Chem. 2016, 88, 10459−10465

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DOI: 10.1021/acs.analchem.6b02381 Anal. Chem. 2016, 88, 10459−10465