Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical

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Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay Ting Hou, Ningning Xu, Wenxiao Wang, Lei Ge, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02523 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Analytical Chemistry

Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay

Ting Hou, Ningning Xu, Wenxiao Wang, Lei Ge*, Feng Li* College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, P. R. China * Corresponding authors. Tel/Fax: 86-532-86080855 E-mails: [email protected] (F. Li), [email protected] (L. Ge)

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ABSTRACT In conventional photoelectrochemical (PEC) analysis, photoactive materials are usually immobilized on electrode surfaces, and such immobilization procedures are tedious and time consuming, and it is also difficult to prepare electrodes with good reproducibility. To circumvent such limitations, we propose here a truly immobilization-free diffusivity-mediated PEC bionsensing strategy for microRNA assay, using methylene blue (MB) in solution as the photoactive probe, and non-modified indium tin oxide (ITO) glass as the working electrode. The hybridization between the target microRNA and the MB-labelled single-stranded DNA probe (MB-DNA) triggers the digestion of MB-DNA by T7 exonuclease (T7 Exo), thus to generate MB-labelled mononucleotide, and then the released target microRNA initiates the subsequent cycling processes and generates a large amount of MB-labelled mononucleotides. Due to the diffusivity difference between MB-DNAs and MB-labelled mononucleotides, significantly increased photocurrent signal is observed for MB-labelled mononucleotides as compared to that of MB-DNAs. Therefore, via this “signal-on” mode and the T7 Exo-facilitated signal amplification, a facile and highly sensitive immobilization-free PEC microRNA assay is readily realized, with a detection limit down to 27 aM. Moreover, this strategy exhibits excellent specificity and is successfully applied in detecting microRNA spiked in serum samples. Since all the reactions take place in homogenous solutions and no electrode modification is needed, this PEC biosensing strategy exhibits the advantages of simplicity, rapidness and good reproducibility. More significantly, it provides a novel concept to design truly immobilization-free PEC biosensing systems, and shows potential to be applied in bioanalysis and biochemical research.

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INTRODUCTION Photoelectrochemical (PEC) sensing, as a newly emerged analytical approach, has undergone tremendous development and been applied in assaying all sorts of analytes, including nucleic acids, proteins, enzymes, small molecules, metal ions and so on.1-3 Because of the total separation of the excitation source of light and the readout signal of current, PEC assays exhibit excellent sensitivity comparable to that of electrochemiluminescence (ECL) and generally higher than that of the traditional electrochemical methods.4 Moreover, since electronic detection devices, rather than sophisticated and expensive optical equipment, are adopted, PEC instrumentation demonstrates additional advantages of simplicity, low cost and miniaturizbility.5 In PEC analysis, the photo-induced formation of electron-hole pairs in a photoactive material is utilized to initiate oxidation-reduction reactions.5,6 To ensure sufficient charge transfer, photoactive materials and target-recognition probes are usually immobilized on the electrode surfaces through physical and/or chemical interactions.7,8 However, the immobilization procedures are usually tedious and time consuming, and it is also difficult to prepare modified electrodes with little variation. Moreover, in the cases where target-recognition probes, such as nucleic acids, peptides, proteins, etc. are fastened to the electrode surfaces, the processing conditions need to be carefully optimized to ensure desired density and orientation of the surface-bound probes, and the binding efficiency between the targets and the probes may be impaired, due to the restriction of the probes’ configurational freedom and possible geometry changes caused by the steric hindrance effect of the electrode surface.9,10 Therefore, to circumvent the intrinsic drawbacks of such immobilization-based assays, it is highly desirable to develop immobilization-free PEC sensing strategies by avoiding the attachment of either photoactive materials or target-recognition probes on electrode surfaces. Recently, our research group has investigated the possibility to realize immobilization-free PEC

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analysis. For example, we have developed a biphasic PEC sensing strategy11 to detect acetylcholinesterase activity and inhibition, in which the enzymatic product-mediated formation of CdS quantum dots (QDs) is carried out in homogenous solution, and PEC responses are measured upon dropping the in situ formed photoactive CdS QDs onto the electrode. Another ultrasensitive immobilization-free PEC biosensor12 has also been fabricated, in which the cascaded quadratic signal ampliﬁcation reactions take place in solution, and the insoluble catalytic products produce an insulating barrier on CdS QDs-TiO2 modified electrode to decrease the photocurrent. In these PEC sensing approaches, the target recognition and the subsequent catalytic reactions occur in homogeneous solution, and high sensitivity and excellent selectivity for biosensing have been successfully achieved. However, the immobilization of pre-synthesized or in-situ formed photoactive semiconductor and/or QDs on electrodes is still inevitable. Therefore, to further simplify the experimental protocols, it is of great interest and signiﬁcance to realize truly immobilization-free PEC sensing by eliminating the procedures of depositing photoactive substances on electrode surfaces. In the past few years, efforts have been made to develop immobilization-free electrochemical detection strategies.13 Ever since the publication of Hsing and coworkers’ pioneering work14 of DNA sensing based on solution-phase electrochemical molecular beacon, all sorts of immobilization-free electrochemical methods have been proposed and successfully applied in the assay of different types of targets, such as metal ions, small molecules, microRNAs, and enzyme activities.15-25 In these assays, through the ingenious utilization of the diffusivity difference between free electrochemical indicators (for example, methylene blue or ferrocene) and those incorporated with long DNA strands (either by labelling14,16,18,21-23,25 or by intercalating into DNA double strands15 or DNA G-quadruplexes17,19), tedious and time-consuming electrode modification processes are avoided, and thus facile and convenient homogenous electrochemical assays are readily realized. In addition, both enzyme-mediated and enzyme-free signal amplification strategies have also been developed to

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significantly improve the assay sensitivity. For instance, exonuclease14,21,26,27 and nicking endonuclease22 have been used to achieve target recycling amplification, and recently our group have also proposed an enzyme-free and label-free homogenous electrochemical strategy based on hybridization chain reaction for highly sensitive microRNA assay.19 As we can see, the aforementioned diffusivity-mediated homogenous electrochemical strategies provide a novel and practical route to immobilization-free detection, and it is rational to deduce that the idea of biosensing based on diffusivity difference between free signaling molecules and those associated with DNA structures can thus be extended to other fields like PEC sensing. In PEC processes, photoactive materials are essential for the charge carrier separation and transfer upon photo illumination, and semiconductors, QDs, carbon-based materials such as polymeric carbon nitride and carbon nanotubes, nanocomposites, small organic molecules, metal complexes and so on have all been used as photoactive probes.5,8,28-30 Among them, the most commonly used semiconductors, QDs, carbon nitride and nanocomposites exhibit the unique features of chemical stability, high carrier mobility and long carrier lifetimes, and have been widely utilized in PEC sensing, photocatalytic reactions as well as PEC devices. However, in order to take full advantage of their merits, such photoactive probes need to be immobilized on electrode substrates, and thus it is difficult to adopt them in immobilization-free PEC sensing, in which photoactive probes are in solution phase. Whereas, small photoactive organic molecules, such as organic dye molecules, are better candidates. Methylene blue (MB) is a photoactive dye molecule, and a few examples of MB-based photo-electrodes for PEC sensing have been reported.31-33 For instance, PEC detection of ascorbic acid (AA) via MB immobilized in α-zirconium phosphate has been realized33, and wavelength-resolved ratiometric PEC strategies based on the incorporation of MB and CdS QDs have been developed for highly sensitive detection of copper ion31 and microRNA32. However, in these methods, the photoactive MB was still immobilized on electrodes either by drop casting31,33 or via DNA-facilitated tethering32, and thus was

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not in solution phase. Whereas, another organic dye molecule, toluidine blue (TB), has been adopted in immobilization-free PEC assay of AA,34 in which TB in photo excited state (TB*) oxidized AA to produce leuco-TB and oxidized AA, and the subsequent conversion of leuco-TB back to TB resulted in electrochemical current signals. Since MB has similar molecular structure as TB (Figure S1 in Supporting Information), we can infer that it is reasonable to use MB as the photoactive species in solution phase to carry out immobilization-free PEC assays. Herein, inspired by the ideas of homogenous electrochemical sensing and photoactive organic dye-based PEC analysis, we proposed a facile and highly sensitive diffusivity-mediated immobilization-free PEC biosensing strategy using MB in solution phase as the photoactive probe and bare indium tin oxide (ITO) glass as the working electrode. In this strategy, microRNAs (miRNAs) are chosen as the proof-of-concept analyte, which are small noncoding RNAs containing around 19 to 23 nucleotides, and have been regarded as biomarkers for diseases (e.g. cancers) and potential targets in disease diagnosis and therapy.35-37 An MB-labelled single-stranded DNA probe (MB-DNA) is designed, whose sequences are complementary to part of the target miRNA. In the absence of the target miRNA, MB-DNA can resist the digestion by T7 exonuclease (T7 Exo) and remains its single-stranded conformation. Whereas, in the presence of the target miRNA, MB-DNA is digested by T7 Exo upon its hybridization with the target miRNA, thus generating MB-labelled mononucleotide and releasing the target miRNA to initiate the subsequent cycling process and produce a large amount of MB-labelled mononucleotides. Due to the diffusivity difference, MB-labelled mononucleotides can easily migrate to the surface of the electrode, but MB-DNAs are kept away from the electrode because of the repulsion between the both negatively charged MB-DNA and the ITO electrode surface. Moreover, upon visible light illumination, MB can transform to leuco-MB, and the subsequent current generation occurs in the presence of strong reducing agents like

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AA,31,33 as illustrated below: hν

ν MB  → MB*

MB* + AA → leuco - MB + oxidized AA leuco - MB → MB + e -

As a result, upon visible light illumination and in the presence of strong reducing agent (AA), significantly increased photocurrent signal is observed for MB-labelled mononucleotides as compared to that of MB-DNAs. Therefore, highly sensitive detection of miRNA is readily realized via this “signal-on” and signal amplification approach. Since all the reactions take place in homogeneous solution and no electrode modification is necessary, the as-proposed diffusivity-mediated PEC strategy is truly immobilization free and exhibits the unique merits of simplicity, rapidness and good repeatability. EXPERIMENTAL SECTION Reagents and Materials. T7 Exo and 10× NEBuffer 4 (500 mM potassium acetate, 200 mM Tris acetate, 100 mM magnesium acetate, 10 mM dithiothreitol, pH 7.9 at 25 °C) were purchased from New England Biolabs, Ltd. (Beijing, China). HPLC-purified and freeze-dried DNA oligonucleotides and miRNAs were ordered from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China), with their sequences listed in Table S1 (in Supporting Information). Ascorbic acid (AA) and diethyl pyrocarbonate (DEPC) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Gel red was purchased from Solebo Biotechnology Co., Ltd. (Beijing，China). Human serum was obtained from Sigma-Aldrich (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane (Tris), Na2HPO4, NaH2PO4, NaCl, KCl, and MgCl2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were of analytical grade and used without further treatment. Indium tin oxide (ITO) glass was purchased from Shenzhen Nanbo Display Technology Co. LTD. (Shenzhen, China). DNA oligonucleotides and miRNA were diluted with 10 mM Tris-HCl buffer (pH 7.0) to give the stock solutions.

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Prior to use, DNA stock solutions were heated up to 95 °C, maintained at this temperature for 10 min, and then slowly cooled down to room temperature. Ultrapure water (resistivity of 18.2 MΩ cm at 25 °C) was provided by a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA). DEPC-treated ultrapure water was used throughout the experiments. Apparatus. PEC measurements were carried out using a Zahner PEC measurement system (ZAHNER-elektrik GmbH & Co. KG, Germany), with a RTR02 light (627 nm) as the accessory light source. All PEC experiments were carried out at room temperature using a conventional three-electrode system: an ITO electrode with an active area of 1.0 cm2 as the working electrode, an Ag/AgCl as the reference electrode, and a platinum wire as the counter electrode. Native polyacrylamide gel electrophoresis (PAGE) was performed on a Bio-Rad electrophoresis system (Bio-Rad Laboratories, Inc., USA), and the gel was imaged using a Gel Doc XR+ Imaging System (Bio-Rad Laboratories, Inc., USA). ITO Electrode Pretreatment. ITO glass was cut into 1.0 cm × 4.0 cm slices, and the obtained ITO electrodes were pretreated as follows: firstly, the ITO electrodes were cleaned by ultrasonic treatment in 1 M NaOH in water/ethanol mixture (1:1, v/v), acetone, and ultrapure water for 30 min, respectively; next, the ITO electrodes were immersed in 1 mM NaOH solution for 5 h at room temperature, followed by sonication in ultrapure water for 10 min, and then blown dry with nitrogen gas. Upon these treatments, negatively charged ITO electrode surface was obtained and ready for use in the PEC measurements. PEC Detection of miRNA. The miRNA assay was performed as follows: first, 30 µL MB-DNA (10 µM) and 20 µL target miRNA with different concentrations were added to 37 µL Tris-HCl buffer (10 mM Tris, 100 mM NaCl, 20 mM KCl, and 10 mM MgCl2, pH 7.4), and the mixture was incubated at 37 °C for 2 h. Next, 3 µL T7 Exo (2 U/µL) and 10 µL 1× NEBuffer 4 (20 mM Tris-HAc, 50 mM KAc, 10 mM MgAc2, 1 mM DTT, pH 7.9 at 25 °C) was added to the above reaction solution to bring the volume to 100 µL, and then

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the mixture solution was incubated at 37 °C for another 45 min. Finally, 100 µL of phosphate buffer (PB, 0.1 M Na2HPO4, 0.1 M NaH2PO4, pH 7.0) containing 0.2 M AA was added to the above reaction solution, and then the PEC measurement was carried out. For PEC miRNA detection in human serum samples, standard addition method was adopted by adding standard miRNA solution with different concentrations into Tris-HCl buffer containing 100-fold diluted human serum, and the other procedures were the same as those mentioned above. Nondenaturing Polyacrylamide Gel Electrophoresis (PAGE). To avoid the interference of methylene blue on PAGE results, non-labelled DNA, with the same sequence as that of MB-DNA but without MB labelling, was used in the gel electrophoresis experiments. Briefly, different reaction solutions were mixed with 6× loading buffer and loaded on the 12% nondenaturing polyacrylamide gel. Then gel electrophoresis was carried out in 1× TBE buffer (pH 7.9, 9 mM Tris-HCl, 9 mM boric acid, 0.2 mM EDTA) at 110 V for 55 min at room temperature. After being strained by Gel-Red dye solution for 30 min, the gel was imagined using the Gel Doc XR+ Imaging System. RESULTS AND DISCUSSION Principle of Immobilization-Free PEC miRNA Assay. The proposed mechanism of the immobilization-free PEC strategy for miRNA assay is schematically illustrated in Scheme 1. A single-stranded DNA modified with MB at the 5′-terminus, denoted as MB-DNA, was designed and adopted as the signal probe, which hybridizes with part of the target miRNA sequences from the 3′-end. In the absence of the target miRNA, as illustrated in Scheme 1(a), MB-DNA retains its single-stranded conformation, and thus can resist the digestion by T7 Exo, a sequence-independent nuclease that catalyzes the stepwise removal of mononucleotides from the recessed or blunt 5′-termini of double-stranded DNA or RNA-DNA duplex.38-40 Due to the repulsion between the both negatively charged single-stranded MB-DNA

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and the ITO electrode surface, it is rather difficult for MB-DNA to reach the ITO electrode surface. Upon visible light (λ = 627 nm) illumination, MB can be excited to MB*, and the subsequent oxidation of AA (acting as the electron donor) by MB* to produce leuco-MB and oxidized AA takes place. However, for MB-DNA, the electrons generated via the regeneration of MB from leuco-MB are unable to transfer to the ITO electrode due to the low diffusivity of MB-DNA toward the electrode surface. Therefore, a rather small PEC signal is observed.

Scheme 1. Schematic illustration of the immobilization-free diffusivity-mediated PEC strategy for miRNA assay: (a) in the absence and (b) in the presence of the target miRNA. Whereas, with the target miRNA present in the system, as illustrated in Scheme 1(b), it hybridizes with MB-DNA to form the RNA-DNA duplex, and then T7 Exo recognizes the recessed 5′-terminus of MB-DNA in the RNA-DNA duplex, initiating the hydrolysis of MB-DNA in the direction from 5′ to 3′-end. Because MB-DNA is designed to be complementary to only part of the target miRNA sequences, the dangling 5′-end of the miRNA in RNA-DNA duplex protects it from being digested by T7 Exo. As a result, upon the digestion of MB-DNA in RNA-DNA duplex, the hydrolysis product of MB-labelled mononucleotide is formed, and the intact target miRNA is then liberated. Next, the released miRNA hybridizes with another

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MB-DNA probe to initiate the subsequent cycling digestion process, generating a considerable amount of MB-labelled mononucleotides, which possess much improved diffusivity toward the negatively charged ITO electrode surface because of their smaller sizes and less negative charges than that of MB-DNA.14,21 Eventually, as the MB-labelled mononuleotides reach the ITO electrode surface, under visible light illumination, the electrons generated via the photoreaction between MB and AA readily transfer to the ITO electrode, and thus a significantly amplified photocurrent is obtained. Moreover, free guanines released from MB-DNA upon enzymatic digestion could also act as electron donors,41 and make small contribution to the signal increment. Therefore, by adopting this signal-on approach, highly sensitive and facile quantitative determination of miRNA can be realized by monitoring the photocurrent change of the reaction system. Feasibility Study of Immobilization-free PEC miRNA Assay. The feasibility of the as-proposed immobilization-free PEC strategy for miRNA assay was investigated, by carrying out PEC measurements under different conditions. As shown in Figure 1, with only MB-DNA being present in the reaction system containing AA, a photocurrent of ca. 75 nA was obtained (curve a). With the target miRNA-155 being added to the system, a slightly decreased photocurrent was observed (curve b), which could be attributed to the increased repulsion between the as-formed RNA-DNA double helix and the negatively charged ITO electrode surface than that between single-stranded MB-DNA and ITO surface, resulting in the further reduced diffusivity of MB-DNA towards the electrode. Whereas, with T7 Exo being further added to the system, a significantly increased photocurrent was observed (curve c), which could be due to the higher diffusivity of MB-labeled mononucleotides resulted from the stepwise digestion of MB-DNA in DNA-RNA duplex by T7 Exo, thus corresponding well to the proposed mechanism of the diffusivity-mediated PEC assay for miRNA detection.

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Figure 1. The photocurrents of the reaction system containing: (a) MB-DNA, (b) MB-DNA + miRNA-155, (c) MB-DNA + miRNA-155 + T7 Exo. The concentrations of MB-DNA, miRNA-155 and T7 Exo were 1.5 µM, 100 fM and 30 U mL-1, respectively. Before PEC measurements, AA with the final concentration of 0.1 M was added to all reaction systems. Moreover, to further confirm the hybridization between MB-DNA and the target miRNA, as well as the digestion of MB-DNA in DNA-RNA duplex by T7 Exo, gel electrophoresis analysis was carried out, in which MB-DNA was replaced by non-labelled DNA with the same sequence to avoid any inference from MB. As clearly demonstrated in the PAGE image (Figure 2), when both non-labelled DNA and the target miRNA were present, as compared to the bands of non-labelled DNA (Lane a) and miRNA-155 (Lane b), a new band was observed (Lane c), corresponding to the hybridization product of DNA-RNA duplex; whereas, with T7 Exo being present in the system, the band of DNA-RNA duplex disappeared, and the band of miRNA-155 reappeared (Lane d), indicating that non-labelled DNA in the DNA-RNA duplex was successfully digested by T7 Exo, and the intact miRNA-155 was released from the duplex. Therefore, the aforementioned PEC and PAGE results evidently proved the feasibility of the as-proposed PEC sensing strategy for miRNA detection.

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Figure 2. Polyacrylamide gel electrophoresis (PAGE) image of the nucleic acid reaction products. Lane a: non-labelled DNA only, Lane b: miRNA-155 only, Lane c: non-labelled DNA + miRNA-155, and Lane d: non-labelled DNA + miRNA-155 + T7 Exo. The concentrations of non-labelled DNA, miRNA-155 and T7 Exo were 0.5 µM, 0.5 µM and 100 U mL-1, respectively. Optimization of Experimental Conditions. To attain the best analytical performance of the as-proposed immobilization-free PEC assay, the experimental conditions were carefully optimized, including MB-DNA concentration, reaction time for the hybridization between MB-DNA and miRNA, T7 Exo concentration and its reaction time. As demonstrated in Figure S2A (in Supporting Information), with the MB-DNA concentration increased from 0.5 to 2.0 µM, the photocurrent change (∆I, the difference between the photocurrents in the presence and absence of the target miRNA) first increased and then decreased, with the maximum value obtained at 1.5 µM of MB-DNA. The initial increase of ∆I may be due to the fact that, with the elevation of the MB-DNA concentration, more MB-labelled mononucleotides were generated via the digestion of MB-DNA in DNA-RNA duplex by T7 Exo. However, with MB-DNA concentration further increased up to 2.0 µM, for the reaction system in the presence of the target miRNA, more MB-labelled mononucleotides were generated to give further elevated photocurrent, but the photocurrent of the blank also increased to a greater extent, resulting in the decrease of ∆I. Thus, 1.5 µM was chosen as the optimal MB-DNA concentration. To ensure complete hybridization between MB-DNA and the target miRNA, their

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reaction time was optimized. With the reaction time increased from 30 to 180 min, the photocurrent initially increased and then levelled off after 120 min (Figure S2B, in Supporting Information), indicating the completion of the hybridization. So 120 min was chosen as the optimum reaction time for MB-DNA and miRNA hybridization. In the as-proposed assay, T7 Exo plays an important role, thus its concentration and reaction time were also optimized. With T7 Exo concentration increased from 2.5 to 60 U mL-1, the photocurrent increased and then reached the maximum value at 30 U mL-1 (Figure S2C, in Supporting Information). Similarly, the photocurrent reached the plateau at the T7 Exo reaction time of 45 min (Figure S2D, in Supporting Information). Therefore, 30 U mL-1 and 45 min were chosen as the optimal T7 Exo concentration and reaction time, respectively. Analytical Performance of Immobilization-Free PEC miRNA Assay. Under the optimal experimental conditions, the analytical performance of the as-proposed PEC strategy for miRNA assay was investigated, by measuring the PEC responses with different amount of miRNA-155 being added to the reaction system. As the miRNA-155 concentration increased from 0 to 10 pM, the photocurrent increased accordingly (Figure 3A), and the photocurrent change (∆I, the difference between the photocurrents in the presence and absence of miRNA) also increased with the elevated miRNA-155 concentration (Figure 3B), corresponding well to the proposed mechanism that target miRNA with higher concentration would induce the formation of more MB-labelled mononucleotides via the digestion of RNA-DNA duplex by T7 Exo, leading to the elevated photocurrent. A good linear relationship was then obtained between ∆I and the logarithm (to base 10, lg) of miRNA-155 concentration ranging from 80 aM to 10 pM (Inset of Figure 3B), with a coefficient of determination of R2 = 0.9931, and a correlation equation of ∆I = 75.04 + 16.89 lg C, where ∆I is the photocurrent change (in units of nA), and C is the miRNA-155 concentration (in units of pM). The limit of detection for miRNA-155 assay was then determined to be 27 aM (based on S/N of 3), which is comparable

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or superior to those previously reported in literature, including PEC, electrochemical and electrochemiluminescent methods (Table S2, in Supporting Information). The relative standard deviation (RSD) was determined to be 2.67% for 11 replicate measurements of 100 fM miRNA-155, indicating high precision of the as-proposed method for miRNA determination. In addition, to further verify the reproducibility of PEC signals obtained on different ITO electrodes, the photocurrents obtained on five different bare ITO electrodes in the absence and presence of 100 fM miRNA-155 were measured. As clearly demonstrated in Figure S3 (in Supporting Information), there are barely any changes in the photocurrent values obtained on these five different ITO electrodes, both in the absence and presence of the target miRNA, thus indicating the excellent reproducibility of the proposed PEC biosensing approach. Therefore, the aforementioned results clearly demonstrated that highly sensitive and reliable miRNA detection can be readily realized by the diffusivity-mediated immobilization-free PEC strategy we proposed here.

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Figure 3. (A) Photocurrents of the reaction system in the presence of miRNA-155 with different concentrations: (a) 0 fM, (b) 0.08 fM, (c) 0.1 fM, (d) 0.5 fM, (e) 1 fM, (f) 5 fM, (g) 10 fM, (h) 100 fM, (i) 1 pM and (j) 10 pM. (B) The photocurrent change ∆I versus the concentration of miRNA-155, in which ∆I = I – I0, where I0 is the photocurrent in the absence of miRNA-155, and I is the photocurrent in the presence of miRNA-155. Inset: the linear relationship between the photocurrent change ∆I and the logarithm of miRNA-155 concentration. The error bars represent the standard deviation of five repetitive measurements. Specificity of PEC miRNA Assay. To investigate the specificity of the as-proposed PEC strategy for miRNA assay, miRNA-155 was substituted by three other types of miRNAs, namely miRNA-141, miRNA-143, and miRNA-199a, respectively, all with the same concentration of 100 fM, i.e. 10 times of miRNA-155 concentration (10 fM), and the PEC responses of the reaction system were then measured. As shown in Figure 4, a large photocurrent change (∆I) was observed for miRNA-155, whereas relatively small photocurrent changes were obtained in the presence of miRNA-141, miRNA-143 or miRNA-199a. These results demonstrated excellent specificity of the as-proposed PEC sensing strategy towards the target miRNA.

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Figure 4. Comparison of the photocurrent change (∆I) of the reaction system in the presence of miRNA-155 (10 fM), miRNA-141 (100 fM), miRNA-143 (100 fM) and miRNA-199a (100 fM), respectively, where ∆I is the difference between the photocurrents with miRNA present and that in the absence of miRNA. The error bars represent the standard deviation of five repetitive measurements. Detection of miRNA in Serum Samples. Moreover, the applicability of the as-proposed PEC strategy for miRNA assay in real samples was further inspected, through the measurement of PEC responses of 100-fold diluted human serum samples. As illustrated in Table 1, no miRNA-155 was detected in the non-spiked human serum sample, and for the human serum samples spiked with different amount of miRNA-155 (1.00, 10.0 and 100 fM), the recoveries were found to be in the range of 94.9% to 104.2%, with the relative standard deviation (RSD) no larger than 3.40%. Therefore, these results demonstrate the accuracy of the as-proposed diffusivity-mediated and immobilization-free PEC assay for selective detection of target miRNA in human serum samples, suggesting its potential to be applied in real sample monitoring. Table 1. Detection results of miRNA-155 spiked in human serum samples. Sample No.

Added / fM

Mean measured (n=6) / fM

Mean recoverya

RSD

1 2 3 4

0 1.00 10.00 100.00

Not detected 0.95 ± 0.02 10.29 ± 0.35 100.42 ± 1.53

94.9% 102.9% 104.2%

2.11% 3.40% 1.52%

a

Recovery = (Cmeasured/Cadded) × 100% 17



CONCLUSIONS In summary, we have developed a truly immobilization-free diffusivity-mediated PEC biosensing strategy using the organic dye MB in solution phase as the photoactive probe and bare ITO glass as the working electrode. By taking advantage of the diffusivity difference between MB incorporated with long DNA strands and those with mononucleotides toward negatively charged ITO electrode surface, a “signal-on” PEC detection mode is readily constructed. Moreover, via the signal amplification strategy based on T7 Exo-assisted target recycling, highly sensitive detection of miRNA, the proof-of-concept analyte, has been realized, with a detection limit down to 27 aM, which is comparable or superior to those reported in literature. Moreover, this strategy demonstrates excellent selectivity to distinguish the target miRNA from other types of miRNAs, and has been successfully applied to detect the target miRNA spiked in serum samples. Due to the facts that all the reactions take place in homogenous solutions and there is no need to modify the electrode surface, the as-proposed strategy exhibits the unique features of simplicity, rapidness and good repeatability. More significantly, the strategy we proposed here provides a novel concept to design truly immobilization-free PEC biosensing systems, and shows great potential to be applied in bioanalysis and biochemical research. AUTHOR INFORMATION Corresponding Authors *Tel./Fax: 86-532-86080855. E-mail: [email protected] (F. Li), [email protected] (L. Ge) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Nos. 21675095, 21575074, and 31501570), and the Special Foundation for Distinguished Taishan Scholar of

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For TOC only

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Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical

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