Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc

Jan 6, 2017 - Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just ...
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Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA−RNA Hybrids Chiew San Fang,† Kwang-sun Kim,† Byeongjun Yu,‡ Sangyong Jon,‡ Moon-Soo Kim,§ and Haesik Yang*,† †

Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, Korea Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea § Department of Chemistry, Western Kentucky University, Bowling Green, Kentucky 42101, United States ‡

S Supporting Information *

ABSTRACT: Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA− RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA−RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium−tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA−DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.

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sequence similarity within miRNA families.46,47 In the absence of target amplification, highly sensitive and specific detection is generally obtained using double biospecific binding in a sandwich-type assay format. However, the short length of the target miRNAs makes it difficult to achieve strong and specific double hybridization by using two single-stranded (ss) DNA or RNA probes (capture and detection probes),48 because each hybridization requires at least 15-base pairing. To overcome this issue, novel approaches have been developed, including (i) “double hybridization to a target miRNA”, based on the hybridization of a target with the short DNA capture and detection probes modified with a high-affinity DNA analogue such as a locked nucleic acid (LNA);49 (ii) “double hybridization to a long capture probe”, based on target-enabled hybridization of a long capture probe with a detection probe (not with a target miRNA);50−52 (iii) “hybridization followed by antibody or antibody-like binding”, based on specific binding of a protein probe to a probe−target hybrid;53−57 and (iv) “hybridization along with labeling/modification”, based on enzymatic (or chemical) labeling/modification of a target

on-protein-coding microRNAs (miRNAs) that are involved in post-transcriptional gene regulation have emerged as important clinical biomarkers for disease diagnosis, prognosis, and therapy.1−3 Abundant studies have revealed that changes in miRNA expression profiles are associated with cancers, heart diseases, neurological disorders, etc.1−6 Favorably, unlike normal RNA molecules, circulating miRNAs are highly stable in body fluids such as serum and blood,7−12 which significantly lowers the possibility of false negative results that frequently occur in RNA detection. Current standard methods for identifying and quantifying miRNAs are based on Northern blot,13 cloning,14−18 microarray,19−24 and reverse transcriptase polymerase chain reaction (RT-PCR).25−29 However, these methods are complicated, time-consuming, and/or costly.29−34 In recent years, various miRNA detection methods have been developed to overcome these drawbacks.35−41 In particular, electrochemical methods have increasingly gained interest because of their capability to be integrated within small instruments and their high sensitivities, which are suitable characteristics for point-of-care testing.32,42−46 When it comes to miRNA detection, both high sensitivity and high specificity are crucial because of (i) the short length of miRNA sequences (19−23 nucleotides), (ii) the low abundance of miRNAs (0.2 fM to 20 pM), and (iii) the © XXXX American Chemical Society

Received: November 21, 2016 Accepted: December 9, 2016

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miRNAs [miRNA-21 (R), miRNA-125b, miRNA-155, SBM miRNA (R′), and TBM miRNA] were sourced from GE Dharmacon (Lafayette, CO). All DNA and miRNA sequences are provided in Table S1. JAZ (J, ZNF346, CSBEP026691MO) was purchased from Cusabio (Wuhan, China). Nonstick RNase-free microfuge tubes and RNase-free tips were purchased from Ambion (Waltham, MA), Axygen (Union City, CA), and Thermo Scientific (San Diego, CA). Indium−tin oxide (ITO) electrodes were obtained from Samsung Corning (Daegu, Korea). A polymer used to form the polymeric self-assembled monolayer (pSAM) was made of poly(ethylene glycol) methyl ether methacrylate, 3(trimethoxysilyl)propyl methacrylate, and acrylosuccinimide as reported previously.70,71 The polymer is referred to as poly(TMSMA-r-PEGMA-r-NAS). 37.5:1 acrylamide/bis solution was purchased from Bio-Rad (Hercules, CA). Amicon Ultra centrifugal filters (10 and 30 K) were obtained from Millipore (Cork, Ireland). RNase-free water was prepared by treating double-distilled water with 0.1% DEPC for at least 12 h at 22 ± 2 °C. The water was then autoclaved at 110 °C for 20 min to eliminate residual DEPC. RNase-free water was used to prepare the following buffers. Tris buffer containing Zn2+ (TZ buffer, pH 8.5) consisted of 100 mM tris, 90 mM KCl, 0.1 mM ZnCl2, and 1 mM MgCl2. HEPES buffer (pH 7.5) consisted of 100 mM HEPES, 90 mM KCl, 0.1 mM ZnCl2, and 1 mM MgCl2. Tris buffer containing EDTA (TE buffer, pH 6.5) consisted of 10 mM tris and 1 mM EDTA. JAZ-binding buffer (pH 7.3) consisted of 20 mM HEPES, 20 mM KCl, 1 mM MgCl2, 10 μM ZnCl2, 5 mM dithiothreitol, 10% glycerol, and 0.1% Tween-20. Blocking buffer was TZ buffer containing 0.1% casein blocking buffer 10×. Washing buffer (pH 7.3) consisted of 50 mM tris, 1 mM NaCl, and 0.05% Tween-20. All of the buffers were stored in the refrigerator at 4 °C and regularly replaced by newly prepared buffers. Human serum was diluted 10-fold with TE buffer prior to spiking the target miRNA-21. Preparation of ALP-Conjugated JAZ. ALP-conjugated JAZ was prepared according to the reported procedure.62 To maintain the JAZ structure during the conjugation, Zn2+containing HEPES buffer was used. One milliliter of HEPES containing 55 μg/mL JAZ and 50 μL of HEPES buffer containing 0.5 mg/mL sulfo-SMCC were mixed and incubated for 30 min at 22 ± 2 °C. Sulfo-SMCC-conjugated JAZ was filtered by centrifugation using the 10 K filter for 20 min at 12 000 rpm to remove excess sulfo-SMCC. Subsequently, the filtrate was dissolved in 1 mL of HEPES buffer. One milliliter of HEPES buffer containing 200 μg/mL ALP and 10 μL of HEPES buffer containing 2 mg/mL SATP were mixed, and the mixture was then incubated for 30 min at 22 ± 2 °C. The resulting solution was mixed with 20 μL of deacetylation solution (HEPES buffer) containing 0.012 g/mL EDTA and 0.044 g/mL hydroxylamine hydrochloride for 2 h at 22 ± 2 °C, and the mixture was then filtered by centrifugation using the 30 K filter for 20 min at 12 000 rpm to remove excess hydroxylamine. The filtrate was dissolved in 1 mL of HEPES buffer. The solution containing sulfo-SMCC-conjugated JAZ and the solution containing SATP-conjugated ALP were mixed at a molar ratio of 1:1 for 2 h at 22 ± 2 °C. To filter the ALPconjugated JAZ, the final mixture was filtered by centrifugation using the 30 K filter for 20 min at 12 000 rpm. Finally, the filtrate was dissolved in 1 mL of TZ buffer. Preparation of Sensing Electrodes and Detection of MiRNA. ITO electrodes (1 cm × 2 cm) were pretreated by

miRNA or a probe−target hybrid before or after hybridization.3,58,59 Although many approaches allow sensitive and selective miRNA detection, a further development in detection scheme is required to obtain simple and multiplex point-of-care testing. In the third method mentioned above, an antibody-like protein probe can bind to either an RNA−RNA or a DNA− RNA hybrid formed between a capture probe and a target miRNA. However, to date, only the specific binding of antibody-like probes to RNA−RNA hybrids has been employed for miRNA detection.54−57 In this case, chemically unstable and readily secondary-structure forming ssRNAs are required as the capture probes. It is well-known that zinc finger proteins bind sequencespecifically to double-stranded (ds) DNAs,60 and this sequencespecific binding has been applied successfully to dsDNA detection.61,62 Interestingly, some zinc finger proteins such as Just Another Zinc finger protein (JAZ, ZNF346) and Specificity Protein 1 (SP1) can recognize and bind to DNA−RNA hybrids.63−65 JAZs bind nonsequence-specifically and preferably to A-form dsRNAs over B-form dsDNAs, and they do not bind to ssDNAs and ssRNAs.64−66 Because DNA−RNA hybrids resemble A-form dsRNAs, JAZs also bind to DNA− RNA hybrids with high affinity.64,65 Unlike JAZs, SP1s bind to dsDNAs as well as DNA−RNA hybrids.64 Therefore, JAZs could be more useful than SP1s as affinity probes for the selective binding to DNA−RNA hybrids in the presence of ssRNAs, dsDNAs, and DNA−DNA hybrids. When JAZs are used as detection probes for DNA−RNA hybrids, ssDNAs that are more stable than ssRNAs can be used as capture probes. Herein, we report an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA−RNA hybrid formed between a ssDNA capture probe and a target miRNA-21. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical−chemical−chemical (ECC) redox cycling involving an ALP product (hydroquinone, HQ). 67−69 Detection specificity was investigated using DNA−DNA hybrid, noncomplementary ssDNA, noncomplementary miRNAs (miRNA125b and miRNA-155), single-base mismatched (SBM) miRNA, and three-base mismatched (TBM) miRNA. Detection limits for miRNA-21 spiked in buffer and diluted human serum were determined.

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EXPERIMENTAL SECTION Chemicals and Solutions. Ru(NH3)62+, Ru(NH3)63+, hydroxylamine hydrochloride, tris(2-carboxyethyl)phosphine (TCEP) hydrochloride, methanol, H2O2, NH4OH, MgCl2, ZnCl2, KCl, HQ, ethylenediaminetetraacetic acid (EDTA), 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), diethyl pyrocarbonate (DEPC), tris(hydroxymethyl)aminomethane (tris), glycerol, casein blocking buffer 10×, ALP, and human serum were obtained from Sigma-Aldrich. Hydroquinone diphosphate (HQDP) was obtained from DropSens (Llanera, Spain). Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) was obtained from Thermo Scientific (Waltham, MA). N-Succinimidyl-Sacetylthiopropionate (SATP) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Amine-terminated ssDNA capture probe, its complementary ssDNA (D), noncomplementary ssDNA, fluorescein amidite (FAM)-terminated ssDNA (F-D), and FAM-terminated single-base mismatched ssDNA (F-D′) were obtained from Genotech (Daejeon, Korea). All B

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Analytical Chemistry dipping them in a mixed solution of H2O, H2O2 (30%), and NH4OH (30%) at a volume ratio of 5:1:1 for 1 h at 70 °C.72 The ITO electrodes were washed with methanol. To obtain a pSAM of poly(TMSMA-r-PEGMA-r-NAS) on ITO electrodes, the pretreated ITO electrodes were dipped in methanol containing 0.4% poly(TMSMA-r-PEGMA-r-NAS) for 1 h at 20 °C, followed by washing with methanol. The modified ITO electrodes were cured in an oven for 10 min at 100 °C. To immobilize an amine-terminated ssDNA capture probe onto the pSAM/ITO electrodes, 70 μL of HEPES buffer containing 10 nM amine-terminated ssDNA was dropped onto the ITO electrodes and then kept at 20 °C for 3 h. Next, the sensing ssDNA/pSAM/ITO electrodes were blocked by dropping 70 μL of blocking buffer containing casein and incubated for 30 min at 20 °C. To perform the hybridization, 70 μL of TE buffer (or 10-fold diluted human serum with TE buffer) containing different concentrations of miRNA-21 was dropped onto the casein-treated sensing electrodes, which were subsequently incubated at 20 °C for 1 h. The final layer was prepared by dropping 70 μL of binding buffer containing 1 μg/mL ALPconjugated JAZ, and the electrodes were then kept at 20 °C for 30 min. Finally, the electrodes were washed with washing buffer and then kept in contact with a 70 μL drop of TZ buffer (pH 8.5). The resulting electrodes were then stored in a refrigerator at 4 °C until use. Teflon electrochemical cells were assembled with a sensing ITO electrode, an Ag/AgCl (3 M NaCl) reference electrode, and a platinum counter electrode. The exposed geometric area of each ITO electrode was ca. 0.28 cm2. For the enzymatic reaction, the cells were stored for 10 min at 37 °C in 1.2 mL of TZ buffer containing 1.0 mM Ru(NH3)63+, 1.0 mM HQDP, and 2.0 mM TCEP. Electrochemical measurements were carried out using a CHI 1040C potentiostat (CH Instruments, Austin, TX). EMSA and Hybridization Test. FAM-terminated ssDNA and FAM-terminated single-base mismatched ssDNA were used as fluorescent capture probes for the EMSA and hybridization test. To investigate the selective binding of JAZ to a DNA−RNA hybrid, ssDNA and miRNA (or ssDNA) were hybridized at a molar ratio of 1:1.5 in tris-EDTA buffer (pH 8.0) for 1 h at 4 °C prior to JAZ binding. JAZ was then allowed to bind to the DNA−RNA (or DNA−DNA) hybrid at a molar ratio of 1:100 in JAZ-binding buffer for 1 h at 4 °C. All samples were loaded with 4 μL of 20% glycerol to a final volume of 20 μL prior to electrophoresis through a 12% (w/v) polyacrylamide gel (37.5:1 acrylamide:bis(acrylamide)) with 0.5× trisborate buffer (pH 7.6) free of EDTA at 150 V for 4 h at 22 ± 2 °C. In the hybridization test, the hybridizations between ssDNA and miRNA (or ssDNA) were performed at a molar ratio of 1:1.5 in tris-EDTA buffer (pH 8.0) for 1 h at 25 °C. Five different samples were loaded with 4 μL of 20% glycerol to a final volume of 20 μL and then electrophoresed through 7% (w/v) polyacrylamide gel (37.5:1 acrylamide:bis(acrylamide)) with 0.5× tris-borate-EDTA buffer (pH 7.6) at 150 V for 3 h at 22 ± 2 °C. The EMSA and hybridization tests were visualized and imaged with the ChemiDoc MP imaging system and Image Lab Software (Bio-Rad).

Figure 1. Schematic representation of miRNA detection based on selective JAZ binding to a DNA−RNA hybrid.

forming the DNA−RNA hybrid. An ALP-conjugated JAZ binds selectively but nonsequence-specifically to the hybrid. However, the JAZ does not bind to ssDNA, ssRNA, dsDNA, and DNA− DNA hybrids.64−66 This unique binding behavior offers selective miRNA detection even in the presence of DNA− DNA hybrids and nonspecifically adsorbed ssRNA, ssDNA, and dsDNA. High signal amplification is required to obtain ultrasensitive miRNA detection. Because enzymatic amplification alone is not sufficient to fulfill this requirement, multiple signal amplification was employed in this study. During incubation in TZ buffer containing HQDP, Ru(NH3)63+, and TCEP, the ALPconjugated JAZ that is specifically bound to the DNA−RNA hybrid rapidly converts HQDP into HQ, which reacts with Ru(NH3)63+, generating benzoquinone (BQ) and Ru(NH3)62+. The generated BQ reacts with TCEP, producing HQ and TCEPO (the oxidized form of TCEP). The regenerated HQ reacts again with Ru(NH3)63+. As a result, chemical−chemical (CC) redox cycling occurs during the incubation.67−69 This CC redox cycling induces an increase in Ru(NH3)62+ concentration at the vicinity of the sensing electrode. When Ru(NH3)62+ is electrochemically oxidized, the ECC redox cycling involving ITO electrode, Ru(NH3)63+/Ru(NH3)62+, BQ/HQ, and TCEP is induced.67−69 This ECC redox cycling occurs continuously in a steady state as long as the ALP product, HQ, and TCEP are present at the vicinity of the electrode, which allows high electrochemical signal (charge) to be obtained. Consequently, a triple amplification based on (i) enzymatic amplification by ALP, (ii) CC redox cycling, and (iii) ECC redox cycling offers high signal levels. During this process, the main contributors to high signal amplification are the enzymatic amplification and the ECC redox cycling. Signal and Background Levels. In this study, Tris buffer containing Zn2+ (TZ buffer) was used for the entire detection process to maintain the binding activity of JAZ. Although divalent cation Zn2+ activates ALP,73 Zn2+ may inversely affect ECC redox cycling. To investigate this, signal and background levels for the ECC redox cycling in TZ buffer were compared using cyclic voltammetry and chronocoulometry (Figure 2). The cyclic voltammogram obtained in TZ buffer containing Ru(NH3)62+ (curve i of Figure 2a) shows a near-reversible electrochemical behavior even at a low electrocatalytic ITO electrode, because Ru(NH3)62+ undergoes a fast outer-sphere electron-transfer reaction. However, the cyclic voltammogram obtained in TZ buffer containing HQ (curve ii of Figure 2a)

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RESULTS AND DISCUSSION Detection Principle. Figure 1 shows a schematic representation of the electrochemical miRNA detection using selective JAZ binding to DNA−RNA hybrid. MiRNA in sample hybridizes with the ssDNA capture probe on sensing electrode, C

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in the presence of HQDP and the charge value measured at 100 s in the presence of HQ were 998 and 20 μC, respectively. Accordingly, the signal-to-background ratio under this condition was 50. To obtain ultrasensitive miRNA detection, it is crucial to minimize nonspecific adsorption of ALP-conjugated JAZ on the sensing electrode. In this study, a pSAM of poly(TMSMA-rPEGMA-r-NAS) was used to obtain low nonspecific adsorption. Poly(TMSMA-r-PEGMA-r-NAS) consisted of an anchor part for anchoring onto the ITO electrode, a bioreactive moiety for immobilizing the amine-terminated ssDNA capture probe, and a bioinert moiety containing poly(ethylene glycol) for repelling unwanted nucleic acids/proteins.70,71 However, only the pSAM was not sufficient to obtain low nonspecific adsorption of ALP-conjugated JAZ. To further reduce the nonspecific adsorption, the sensing electrodes were treated and blocked with casein. Curve i of Figure 3 was obtained with a

Figure 2. (a) Cyclic voltammograms recorded (at a scan rate of 20 mV) using bare ITO electrodes in TZ buffer containing (i) 1.0 mM Ru(NH3)62+, (ii) 1.0 mM HQ, (iii) 2.0 mM TCEP, and (iv) 1.0 mM Ru(NH3)62+, 1.0 mM HQ, and 2.0 mM TCEP. (b) Chronocoulograms recorded at 0.05 V at bare ITO electrodes in TZ buffer containing (i) 1.0 mM Ru(NH3)63+ and 2.0 mM TCEP, (ii) 1.0 mM Ru(NH3)63+, 2.0 mM TCEP, and 1.0 mM HQDP, and (iii) 1.0 mM Ru(NH3)63+, 2.0 mM TCEP, and 1.0 mM HQ.

Figure 3. Chronocoulograms recorded at 0.05 V using (i, ii, iv) caseintreated and (iii) casein-untreated sensing electrodes with (i, ii, iii) none and (iv) 100 nM complementary ssDNA (i) without and (ii, iii, iv) with treatment with ALP-conjugated JAZ.

shows a near-irreversible electrochemical behavior. Accordingly, the direct oxidation of HQ at the applied potential of 0.05 V in chronocoulometry was slow. The direct oxidation of TCEP was negligible within the tested potential range (curve iii of Figure 2a). Importantly, the anodic currents in the cyclic voltammogram obtained in TZ buffer containing Ru(NH3)62+, HQ, and TCEP (curve iv of Figure 2a) were much higher than those obtained in TZ buffer containing only Ru(NH3)62+ (curve i of Figure 2a), indicating that the ECC redox cycling process described in Figure 1 is fast. The overall results indicate that the effect of Zn2+ in the ECC redox cycling was not significant. Chronocoulograms shown in Figure 2b were obtained at 0.05 V, an applied potential at which Ru(NH3)63+ oxidation is fast while Ru(NH3)63+ reduction/oxidation and TCEP oxidation are slow.67 In TZ buffer containing Ru(NH3)63+ and TCEP, the chronocoulometric signal was low (curve i of Figure 2b). This result shows that the direct reaction between Ru(NH3)63+ and TCEP is slow, although the difference between their formal potentials is large. In the presence of HQDP (the ALP substrate), the chronocoulometric signal (curve ii of Figure 2b) was slightly higher than that obtained in its absence (curve i of Figure 2b). However, in the presence of HQ (the ALP product), the chronocoulometric signal was substantially increased (curve iii of Figure 2b). In fact, the charge obtained in the presence of HQDP corresponds to background level, whereas the charge yielded in the presence of HQ corresponds to signal level. Accordingly, the chronocoulometric results indicate that the signal level is very high as compared to the background level. The linear increase of the charge with time indicates that the ECC redox cycling occurred in a steady state with no variations in rate. The charge value measured at 100 s

miRNA concentration of zero without treatment with ALPconjugated JAZ, whereas curve ii of Figure 3 was obtained after treatment with ALP-conjugated JAZ. The difference between the two chronocoulometric signals is due to the nonspecific adsorption of ALP-conjugated JAZ. Although slight nonspecific adsorption still existed, it was low enough to obtain ultrasensitive miRNA detection. In the absence of casein treatment, nonspecific adsorption was much higher (curve iii of Figure 3). The modified ITO electrodes obtained after each construction step for the preparation of sensing electrodes were checked and compared using cyclic voltammograms of Fe(CN)63− in TZ buffer (Figure S1). The peak separation between the cathodic and anodic peak potentials increased after every construction step, indicating that the electron transfer rate between Fe(CN)63− and ITO became lower and that each construction was well conducted on the ITO electrode. Specificity and Selectivity Test. To avoid false positive results, the detection should be able to discriminate between DNA−RNA hybrid and DNA−DNA hybrid. When 100 nM complementary ssDNA, instead of target miRNA-21, was allowed to hybridize with the ssDNA capture probe, the chronocoulometric signal (curve iv of Figure 3; Figure 4) was similar to that obtained with a miRNA-21 concentration of zero (curve ii of Figure 3; Figure 4). However, when 1 nM miRNA21 was allowed to hybridize with the capture probe, the chronocoulometric signal (Figure S2; Figure 4) was much larger than that obtained with a miRNA-21 concentration of D

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Figure 4. Histograms representing the charges measured at 100 s in the chronocoulograms obtained using TZ buffer containing different nucleic acids (from the left: zero miRNA-21; 100 nM complementary ssDNA; 1 nM miRNA-21; 1 nM miRNA-155; 1 nM miRNA-125b; 1 nM TBM miRNA; 1 nM SBM miRNA).

zero (curve ii of Figure 3; Figure 4). The different electrochemical behavior implies that the ALP-conjugated JAZ binds much more strongly to DNA−RNA hybrid than to DNA−DNA hybrid. Furthermore, the chronocoulometric signal for a sample solution containing only miRNA-21 was similar to the signals for sample solutions containing both miRNA-21 and excess noncomplementary ssDNA (Figure S3). This result clearly shows that the detection method can selectively quantify miRNA even in the presence of a huge excess of ssDNA. To reconfirm the selective binding of JAZ to DNA−RNA hybrid over DNA−DNA hybrid, EMSA for DNA−DNA and DNA−RNA hybrids was carried out (Figure 5a). A clear band shift was observed in the presence of JAZ in the case of DNA−RNA hybrid (lane iv of Figure 5a), whereas no band shift was observed in the case of DNA−DNA hybrid (lane ii of Figure 5a). The EMSA results are in good agreement with the electrochemical results and the fact that JAZ has a very low binding affinity toward DNA−DNA hybrids as compared to DNA−RNA hybrids.65 Because of sequence similarity within miRNA families, a highly specific and selective detection method that allows discrimination between target miRNA and mismatched miRNAs is required. The specificity of the detection method was investigated using noncomplementary miRNAs (miRNA125b and miRNA-155), SBM miRNA, and TBM miRNA. As shown in Figure 4 and Figure S2, the chronocoulometric signals obtained for noncomplementary miRNA-125b and miRNA-155 were similar to the signal obtained without miRNA-21. The chronocoulometric signals obtained for SBM miRNA and TBM miRNA were slightly higher than those obtained without miRNAs. Nevertheless, the increase in chronocoulometric signal was negligible as compared to that for target miRNA21 (Figure 4). These results suggest that the developed method is specific enough to discriminate between miRNA-21 and single-base mismatched miRNAs. The stability of single-base mismatched DNA−RNA hybrids depends on the X·Y type of the mismatched base pair (X, DNA base; Y, RNA base). The X·Y mismatch type between ssDNA capture probe and SBM miRNA was C·A. Interestingly, in the

Figure 5. (a) EMSA results for (i) F-D (FAM-terminated ssDNA) + D (complementary ssDNA), (ii) F-D + D + J (JAZ), (iii) F-D + R (miRNA-21), and (iv) F-D + R + J. (b) Hybridization test results for (i) F-D, (ii) F-D + D, (iii) F-D + R, (iv) F-D + R′ (single-base mismatched miRNA), and (v) F-D′ (FAM-terminated single-base mismatched ssDNA) + R.

gel electrophoresis experiment, the hybridization between FAM-terminated ssDNA and SBM miRNA (lane iv of Figure 5b) was too weak to be observed, whereas the hybridization between FAM-terminated ssDNA and complementary ssDNA (lane ii of Figure 5b) and the hybridization between FAMterminated ssDNA and miRNA-21 (lane iii of Figure 5b) were very strong. Furthermore, when the X·Y mismatch type is T·G, the hybridization between FAM-terminated single-base mismatched ssDNA and miRNA-21 was strong enough to be E

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chronocoulograms. The calculated detection limit for miRNA-21 in 10-fold diluted human serum was approximately 30 fM. The result clearly shows that it is even possible to obtain ultrasensitive miRNA detection in serum. MiRNA in serum was detected in a wide range of concentrations ranging from 30 fM to 1 nM. All results suggest that the detection method is highly sensitive and promising toward detecting miRNAs in body fluids. Table S2 represents the comparison of our method with the electrochemical methods developed for miRNA detection. This table clearly shows that our method is highly sensitive and selective. Because the detection method is based on a sandwichtype assay, it can be applied to standard assay formats such as microplate and microarray without modification in detection procedure. Importantly, the detection method can be readily extended to multiplex detection of miRNAs with only one JAZ detection probe due to its nonsequence-specific binding character.

observed (lane v of Figure 5b). These results are in good accordance with the reported stability order of the mismatched base pair,74 where the stability order of the X·Y mismatch type on microarrays is as follows: T·G ≈ T·U ≈ T·C > G·U ≈ A·C ≈ C·C ≈ C·U ≈ A·A ≈ A·G ≥ C·A > G·G ≈ G·A. Therefore, the gel electrophoresis results confirm the single-base mismatch discrimination capability (with the X·Y mismatch type of C·A) observed in the electrochemical experiments (Figure 4). miRNA-21 Detection. The method was applied to the detection of miRNA-21 in buffer and serum. Figure S4a shows chronocoulograms that were obtained for different concentrations of miRNA-21 in TE buffer after the final sensing electrodes were incubated for 10 min in TZ buffer containing Ru(NH3)63+, HQDP, and TCEP. The chronocoulometric signal increased with increasing miRNA-21 concentration. Figure S4b represents a calibration plot that was drawn using the charge data recorded at 100 s in the chronocoulogram. The calculated detection limit for miRNA-21 in TE buffer was approximately 2 fM, indicating that the detection method is ultrasensitive. Moreover, miRNA-21 was detected within a wide range of concentrations, ranging from 2 fM to 1 nM. Body fluids contain many species that may interfere with ultrasensitive miRNA detection. To investigate this possibility, chronocoulograms were obtained for different concentrations of miRNA-21 in 10-fold diluted human serum (Figure 6a). The chronocoulometric signal increased with increasing miRNA-21 concentration. Figure 6b represents a calibration plot that was drawn using the charge data recorded at 100 s in the

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CONCLUSIONS In this study, we have designed and demonstrated highly specific and ultrasensitive electrochemical detection of miRNA21 in a standard sandwich-type assay format. The selective binding of JAZ to the DNA−RNA hybrid formed between chemically stable ssDNA capture probe and target miRNA-21 was used to obtain highly specific detection. Enzymatic amplification coupled with ECC redox cycling was performed to obtain ultrasensitive detection. pSAM-modified and caseintreated ITO electrode was used to minimize nonspecific adsorption. The calculated detection limits for miRNA-21 in buffer and 10-fold diluted serum were approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. Because JAZ binds nonsequence-specifically to any DNA−RNA hybrid, the developed method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ. It could also be a promising method for point-of-care testing of miRNAs in body fluids. We believe that the present approach will open a new avenue for developing sensitive and specific miRNA profiling methods and thereby fulfill the needs in biochemical research as well as in clinical diagnosis.

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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/acs.analchem.6b04609. Supporting data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

Figure 6. (a) Chronocoulograms obtained at 0.05 V using the sensing electrodes with various concentrations of miRNA-21 spiked in 10-fold diluted human serum. (b) Calibration plot: concentration dependence of the charges at 100 s in panel a. Each experiment was carried out with three different electrodes for the assay of the same sample. The data were subtracted by the mean value obtained from seven measurements at a concentration of zero. The dashed line corresponds to 3 times the standard deviation (SD) of the charge at a concentration of zero.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (2015R1A2A2A01002695 and 2016M3A7B4910538). This material was also supported by the Ministry of Trade, Industry & Energy (MOTIE, Korea), under Industrial Technology Innovation Program (no. 10062995). F

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DOI: 10.1021/acs.analchem.6b04609 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.6b04609 Anal. Chem. XXXX, XXX, XXX−XXX