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Catalytic hairpin assembly-mediated surface charge density on the electrode for sensitive potentiometric detection of microRNA-21 in IgA-nephropathy
Biochemical Engineering Journal 140 (2018) 9–16
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Catalytic hairpin assembly-mediated surface charge density on the electrode for sensitive potentiometric detection of microRNA-21 in IgA-nephropathy ⁎
Qian Zhoua,b, , Dianping Tangb, a b
T
⁎
Institute of Environmental and Analytical Science, School of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, Henan, PR China Key Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou, 350116, PR China
H I GH L IG H T S
potentiometric DNA biosensor was developed for microRNA-21 detection. • ACatalytic hairpin assembly is utilized to increase the surface charge density. • Target recycling is used for the signal amplification. •
A R T I C LE I N FO
A B S T R A C T
Keywords: Potentiometric biosensor Catalytic hairpin assembly MicroRNA-21 Target recycling Surface charge density IgA-nephropathy
Methods based on electrochemical biosensors have been developed for detection of microRNA-21 (miRNA-21), but most involve complicated labeling or stripping procedures and are unsuitable for routine use in diagnosis/ treatment of IgA-nephropathy. Herein we report on the proof-of-concept of simple and sensitive potentiometric DNA biosensor for specific detection of miRNA-21 coupling and catalytic hairpin assembly (CHA) with target recycling. This system consists of two hairpin DNA probes and one single-stranded DNA (capture probe). To decrease the background signal, the thiolated capture probe is covalently conjugated onto a cleaned gold electrode through typical Au-S bond. Upon target miRNA-21 introduction, the analyte opens hairpins DNA in turn to propagate the CHA reaction between two alternating hairpins accompanying the release of target miRNA21 for recycling, thereby resulting in formation of numerous nicked double-helixes. Meanwhile, the newly produced CHA product hybridizes with the immobilized capture DNA probe on the electrode, thus causing the change in the surface charge density for potentiometric measurement. Under optimum conditions, CHA-based DNA biosensor presents good potentiometric responses for determination of target miRNA-21 at a level as low as 43 fM. An intermediate precision of < 10% is accomplished with the batch-to-batch identification. The specificity and stability of CHA-based biosensor are satisfactory. Importantly, CHA-based potentiometric biosensor offers promise for the label-free, rapid, simple, cost-effective analysis of biological samples.
1. Introduction IgA-nephropathy, the most common glomerulonephritis in China, is characterized by deposition of IgA antibody in the glomerulus [1]. MicroRNA-21 is significantly increased in both glomerular and tubularinterstitial tissues of patients with IgA-nephropathy [2]. MicroRNA (miRNA; a class of endogenously expressed 18–25 nucleotides with small noncoding single-strand RNA molecules) is crucially important in regulating the post-transcriptional gene expression in a broad range of the animals, plants, and viruses [3]. Emerging evidences reveal that miRNAs can be identified as the biomarkers for the diagnostic and
prognostic purposes since their expression levels are associated with different pathological conditions including cancers, e.g., hepatocellular carcinoma, lung cancer, breast cancer and gastric carcinoma [4]. Recent research has highlighted the potential of miRNAs to serve as physiological indicators of disease process among clinically depressed patients [5]. As an important robust oncogenic circulating miRNA, miRNA-21 has been also reported to be a key for various cancers and infectious diseases via a variety of regulatory functions on the inflammation, osteogenesis and osteoclastogenesis [6,7]. Therefore, ongoing effort has been done in the world for miRNA-21 detection due to its low abundances, small size, and high degree of sequence similarity
⁎ Corresponding authors at: Key Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou, 350116, PR China. E-mail addresses: [email protected] (Q. Zhou), [email protected], [email protected] (D. Tang).
https://doi.org/10.1016/j.bej.2018.09.004 Received 27 June 2018; Received in revised form 12 August 2018; Accepted 4 September 2018 Available online 06 September 2018 1369-703X/ © 2018 Elsevier B.V. All rights reserved.
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presenting obstacles [8]. Recently, different schemes and strategies have been developed for the detection of miRNA-21. Tian et al. reported an electrochemical biosensor for miRNA-21 detection through the superlattices assembly of gold nanoparticles [9]. Yin et al. designed a signal-on fluorescence biosensor for the microRNA-21 detection based on the strand displacement reaction and Mg2+-dependent DNAzyme cleavage [10]. Peng et al. developed ultrasensitive colorimetric detection of microRNA-21 based on the duplex-specific nuclease-assisted target recycling and horseradish peroxidase cascading signal amplification [11]. Zhang et al. constructed an enzyme-free isothermal target-recycled amplification strategy for the direct detection of miRNA-21, combining with polyacrylamide gel electrophoresis [12]. Despite some advances in this field, there is still the requirement to explore new strategies for improvement of the sensitivity and simplification of the assays. To the best of our knowledge, there is no reports focusing on the potentiometric methods for the detection of miRNA-21 until now. Potentiometric assay, one of most widely used straightforward methods, has extensively applied in the bioanalytical fields since no external excitement (e.g., current or voltage) is required during the measurement [13]. Typically, the signal is recorded on the basis of the formed electrical double layer from the biological bindings and the variation of electrical potential close to the surface [14]. Zhang et al. used soluble molecularly imprinted polymer to construct a potentiometric sensor for the determination of bisphenol AF [15]. Kajisa et al. prepared biocompatible poly(catecholamine)-film electrode for potentiometric cell sensing [16]. For the development of high-efficient potentiometric detection system, however, signal amplification and noise reduction are very crucial. Jakhar and Pundir employed the urease nanoparticles for the construction of an improved potentiometric urea biosensor [17]. Li et al. fabricated a potentiometric competitive immunoassay for the determination of aflatoxin B1 in food by using antibody-labeled gold nanoparticles [18]. Lv et al. utilized polyion oligonucleotide-decorated gold nanoparticles with tunable surface charge density for the amplified signal output of the potentiometric immunosensor [19]. Since DNA molecules can form negatively charged polyion complexes, the oligonucleotides can be utilized to control the intensity of surface charges on the substrate. Herein, we design a novel potentiometric detection protocol for target miRNA-21 on the basis of catalytic hairpin assembly (Scheme 1). Initially, a short-stranded DNA capture probe is immobilized on a cleaned gold disk electrode, and then reacts with target miRNA-21, thereby exposing a new sticky, which hybridizes with the sticky end of
Table 1 The sequences of the oligonucleotides used in this work. Name
Sequences (5'→3')
Hairpin DNA1
5'-TCA ACA TCA GTC TGA TAA GCT ACC ATG TGT AGA TAG CTT ATC AGA CTC TAC TCA-3' 5'-TAA GCT ATC TAC ACA TGG TAG CTT ATC AGA CTC CAT GTG TAG A-3' 5'-UAG CUU AUC AGA CUG AUG UUG A-3' 5'-UAG CUU AUC GGA CUG AUG UUG A-3'
Hairpin DNA2 Target miRNA-21 Single-base mismatch miRNA-21 Two-base mismatch miRNA-21 Three-base mismatch miRNA-21 miRNA-15 miRNA-16 miRNA-141 miRNA-143
5'-UAG CUU UUC GGA CUG AUG UUG A-3' 5'-UAG CUU UUC GAA CUG AUG UUG A-3' 5'-UAG 5'-UAG 5'-UAA 5'-UGA
CAG CAC AUA AUG GUU UGU G-3' CAG CAC GUA AAU AUU GGC G-3' CAC UGU CUG GUA AAG AUG G-3' GAU GAA GCA CUG UAG CUC A-3'
hairpin DNA1 and hairpin DNA2. In this regard, target miRNA-21 triggers a chain reaction of hybridization events between two hairpin probes to form a nicked long double-helix. Thanks to the increasing negative charges on the surface, the electrode potential changes relative to background signal. By monitoring the shift in the potential, we can quantitatively evaluate the level of target miRNA-21. The aim of this work is to develop a simple and portable potentiometric detection system for miRNA-21 with high sensitivity. 2. Experimental section 2.1. Materials and reagents All the oligonucleotides including HPLC-purified miRNA-21, capture probe, hairpin DNA1 and hairpin DNA2 were synthesized by Shanghai Sangon Biotechnol. Co., Ltd. (Shanghai, China). The sequences for detailed DNA and RNA are listed in Table 1, which were designed consulting to the literature [20]. 6-Mercapto-1-hexanol (MCH; 99%) and salmon sperm DNA were obtained from Sigma-Aldrich (St. Louis, MO, USA). DNA/RNS stock solutions were obtained through dissolving the corresponding oligonucleotides into Tris-HCl buffer (100 mM, pH 7.4). Each oligonucleotide was heated to 90 °C for 5 min, and slowly cooled down to room temperature before use. Ultrapure water at 18.2 MΩ cm was obtained from a Millipore water purification system (Millipore, Inc., Bedford, MA). Tris(hydroxymethyl)
Scheme 1. Schematic illustration of catalytic hairpin assembly (CHA)-mediated surface charge density on the capture DNA-modified gold electrode for the sensitive potentiometric detection of microRNA-21 (miRNA-21) by coupling with target recycling amplification. 10
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the possible error resulting from different additions of samples and deduct the responses induced by nonspecific adsorption, the response for each sensor was recorded until equilibrium was reached (shift less than 1.0 mV min−1). The control tests with normal samples were performed accordingly. The electric potential of DNA sensor toward target miRNA-21 was calculated referring to the equation: ΔE = En – E0 (where E0 and En represents the steady-state electrical potentials of DNA biosensor in the absence and presence of target analyte, respectively).
aminomethane (Tris) was achieved from Shanghai Chem. Re. Inc. (Shanghai, China). All other chemicals used in this study were of analytical grade and used as received. Phosphate buffer solution (PBS) with different pH values was prepared by using 10 mM phosphate-buffered saline and 0.1 M KCl (used as the supporting electrolyte). 2.2. Cell culture and cell lysis Human podocytes and human tubule epithelial (HK2) cells were cultured in RPMI-1640 and DMEM/F12 medium that contained 10% fetal bovine serum and penicillin-streptomycin (100 IU mL−1). To monitor the effect of IgA-human mesangial cell (HMC) medium, different preparations of IgA-HMC medium were diluted 8-fold with medium containing 0.5% fetal bovine serum. Initially, the cells were maintained at 37 °C in a humidified atmosphere (95% air + 5% CO2). The cancer cell density was determined by using hemocytometer prior to experiments. Cells were collected in the exponential phase of growth. Isolation of total RNAs were performed from target cell lines using TRIzol® reagent when the cells reached 80% confluency. Briefly, an aliquot containing 1.0 × 106 cells was dispersed into a 1.5-mL eppendorf tube, washed twice with ice-cold PBS (0.1 M, pH 7.4), and resuspended into 200-μL ice-cold CHARPS lysis buffer containing 10 mM Tris-HCl, pH 7.5, 1.0 mM MgCl2, 1.0 mM EGTA, 0.1 mM PMSF, 0.5% CHAPS and 10% glycerol. The mixture was incubated for 30 min on ice and centrifuged at 16,000g at 4 °C for 20 min. The supernatant was collected as cell extract for analysis or flash frozen in liquid nitrogen at −80 °C.
2.5. RT-PCR analysis for target miRNA-21 Total RNA sample from cells was extracted by using mirVana™ miRNA Isolation Kit. Template complementary DNA was prepared by using reverse transcriptase, and miRNA-21 expression was quantified with an ABI 7300 Sequence Detection System (Applied Biosystem, ABI, USA) by using SYBR green PCR Master Mix (Takara, Shiga, Japan). The relative expression of miRNA-21 was calculated by using the 2T−ΔΔC method based on the relative DNA oligonucleotides. All RT-PCR reactions were performed in triplicate. 2.6. Statistical analyses The signals in all experiments referred to the average responses of the reaction with the corresponding standard deviations in triplicate, unless otherwise indicated. The change in the electrical potential was collected and registered as the sensor signal relative to target miRNA-21 concentration. All measurements were performed at room temperature (25 ± 1.0 °C). Statistical analysis of the data from multiple groups was performed by one-way ANOVA followed by Student-Newman-Kuels test. Data from two groups were compared by t-test (note: p < 0.05 was considered significant).
2.3. Preparation of DNA sensor A gold disk electrode with 2.0 mm in diameter was first polished carefully with 0.3 and 0.05-μm alumina slurry, followed by successive sonication in ultrapure water and ethanol for 5 min and dried in air. Prior to modification, the pretreated gold electrode was first cleaned with hot piranha solution [a 3:1 (v/v) mixture containing concentrated H2SO4 and 30% H2O2] for 10 min at room temperature, and then continuously scanned within the applied potential range from −0.3 to 1.5 V in freshly prepared deoxygenated 0.5 M H2SO4 at 100 mV s−1 until a stable cyclic voltammogram was obtained. After being washed with ultrapure water and absolute ethanol, the cleaned gold electrode was immersed into capture DNA probe (0.5 μM), and incubated for 6 h at room temperature. During this process, the thiolated capture probe was covalently conjugated onto the gold electrode through the classical Au-S bond. The resulting gold electrode was washed as before. Following that, the modified gold electrode was incubated with 1.0-mM MCH in 10 mM Tris-HCl buffer, pH 7.4, for 60 min. Finally, the asprepared DNA sensor was suspended over pH 7.4 PBS at 4 °C when not in use.
3. Results and discussion 3.1. Design and characterization of CHA-based potentiometric DNA biosensor As a newly developed detection scheme for practical application in future, a simple, rapid and sensitive method would be advantageous. In this work, target miRNA-21 is quantitatively evaluated by a portable potentiometer. The assay mainly consists of two label-free hairpin DNA probes and one short-stranded capture probe. Each hairpin H1 has a stem of 14 base pairs enclosing an 11-base loop and an additional sticky end at the 5′ and 3′ ends, whereas hairpin H2 contains a stem of 11 base pairs enclosing 14-base loop and an additional sticky end at the 5′ end. The electrical potential is amplified on the basis of target-induced catalytic hairpin assembly between two DNA hairpins. To reduce the background signal of DNA sensor, thiolated capture probe is firstly immobilized onto the cleaned gold electrode via the Au-S bond. In the presence of target miRNA-21, the analyte initially pairs with the 5′ sticky end of hairpin H1 to open the hairpin. The newly exposed sticky end of hairpin H1 nucleates at the sticky end on hairpin H2 and opens the hairpin, which undergoes a catalytic hairpin assembly reaction. During this process, target miRNA-21 is displaced from the nicked double-helix to execute the next cycling reaction between two hairpin probes (i.e., target recycling reaction). Following that, the newly formed double-helix is captured to the electrode by the immobilized capture DNA. Due to introduction of negatively charged oligonucleotides on the electrode, the local electrical potential is changed relative to the initial signal. By monitoring the shift in the electrical potential, we can quantitatively evaluate the concentration of miRNA-21 in the sample. In contrast, the CHA reaction cannot be carried out in the absence of target miRNA-21, and hairpin probes cannot be conjugated to the electrode. To realize our design, one important concern for the development of
2.4. Catalytic hairpin assembly with miRNA-21 and potentiometric measurement Scheme 1 gives schematic illustration of potentiometric DNA sensing strategy toward miRNA-21 on capture probe-modified gold electrode coupling with target-triggered catalytic hairpin assembly for the signal amplification. The assay consisted of the following steps. Firstly, the different-level miRNA standard was added into the mixture containing 0.5 μM hairpin DNA1 and 0.5 μM hairpin DNA2, and reacted for 150 min (i.e., CHA reaction time) at 37 °C in a centrifugal tube to execute catalytic hairpin assembly. Following that, capture DNA-modified gold electrode was immersed into the resulting mixture, and reacted for another 40 min (i.e., hybridization time) at 37 °C. After being washed with pH 7.4 PBS, the resulting electrode was monitored in pH 7.4 PBS (10 mM) by using a digital ion analyzer with a two-electrode system comprising of the modified gold electrode (as the working electrode) and an Ag/AgCl reference electrode (filled with 3.5 M KCl). To avoid 11
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Fig. 1. (A) Gel electrophoresis: (M) 0.5 μM DNA marker, (a) 0.5 μM miRNA-21, (b) 0.5 μM hairpin DNA1, (c) 0.5 μM hairpin DNA2, (d) 0.5 μM hairpin DNA1 + 0.5 μM miRNA-21, (e) 0.5 μM hairpin DNA1 + 0.5 μM miRNA-21 + 0.5 μM hairpin DNA2, and (f) 0.5 μM hairpin DNA1 + 0.5 μM hairpin DNA2 (note: The incubation time was 2.5 h for all mixture reactions); (B–D) AFM images of (B) gold substrate, (C) capture DNA-modified gold substrate and (D) capture DNA-modified gold substrate after reaction with CHA product in the presence of miRNA-21.
USA) (Fig. 1B–D). Fig. 1B gives typical AFM image of the cleaned gold substrate (surface roughness: 312.1 ± 13.7 nm; Fig. S1 in the Supporting Information). As shown in Fig. 1C, the assembly of thiolated capture probes on the gold substrate caused the change of the surface roughness (surface roughness: 397.4 ± 27.3 nm; Fig. S2). When the capture probe-modified gold electrode hybridized with the products of CHA reaction, significantly, the surface of gold substrate (Fig. 1D) became rougher than that of Fig. 1C (surface roughness: 658.5 ± 34.1 nm; Fig. S3). Vaguely, we seemed to observe the structures of long nicked oligonucleotides. As control test, the surface morphology of capture DNA-modified gold substrate was studied after incubation with hairpins DNA1 and DNA2 in the absence of target miRNA21. As seen from Fig. S4, the surface roughness was 318.9 ± 17.4 nm, which was almost the same as that in Fig. S1, indicating that hairpins DNA1 and DNA2 were not nonspecifically adsorbed onto the substrate without target analyte. Therefore, we might preliminarily make a conclusion that our design is feasible.
CHA-based potentiometric DNA sensor is whether the analyte could readily cause the catalytic hairpin assembly between two hairpins. To clarify this issue, we first used gel electrophoresis to investigate two hairpin DNA probes in the absence and presence of miRNA-21 (Fig. 1A). Lanes 'a–c' shows gel electrophoresis images of miRNA-21, hairpin H1 and hairpin H2, respectively. When mixture of miRNA-21 with hairpin H1, the spot at lane 'd' slightly migrated relative to lane 'b', which might be ascribed to the fact that the base number of the formed miRNA21/ hairpin DNA1 structure was more than that of hairpin DNA1 alone. Upon reaction of miRNA-21 with hairpins H1/H2, favorably, the migration of the formed complexes at lane 'e' was obviously slower than those of hairpins H1/H2. Importantly, the mixture of hairpin H1 with hairpin H2 in the absence of target miRNA-21 did not cause their selfhybridization reaction (lane 'f'). The results revealed that catalytic hairpin assembly between two hairpins could be implemented through target miRNA-21. Logically, another question arises to whether the newly formed nicked double-helix could be captured on the electrode by the immobilized capture probe. To this end, gold substrates before and after modification with the oligonucleotides were characterized by using atomic force microscopy (AFM; Nanoscope IIID Instruments, Bruker,
3.2. Evaluation of feasibility and control experiments Electrochemical impedance spectroscopy (EIS) is a widely used tool 12
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Fig. 2. (A) Nyquist diagrams for (a) gold electrode, (b) capture DNA-modified gold electrode, (c,d) sensor 'b' after reaction with 0.5 μM hairpin H1 and 0.5 μM hairpin H2 in the (c) presence and (d) absence of 0.1 nM miRNA-21, and (e) sensor 'b' after reaction with 0.5 μM hairpin H1 and 0.1 nM miRNA-21 in 10 mM pH 7.4 PBS containing 5.0 mM Fe(CN)64−/3− with the range from 10-2 to 105 Hz at an alternate voltage of 5 mV (inset: equivalent circuit); (B) electrode potentials of (a) capture DNA-modified gold electrode, (b) sensor 'b' + 0.1 nM miRNA-21, (c) sensor 'b' + 0.5 μM hairpin H1, (d) sensor 'b' + 0.5 μM hairpin H2, (e) sensor 'b' + 0.5 μM hairpin H1 + 0.5 μM hairpin H2, (f) sensor 'b' + 0.1 nM miRNA-21 + 0.5 μM hairpin H1 and (g) sensor 'b' + 0.1 nM miRNA-21 + 0.5 μM hairpin H1 + 0.5 μM hairpin H2 in pH 7.4 PBS (10 mM) (note: Sensor 'b' stands for capture DNA-modified gold electrode).
with 0.1 nM miRNA-21, 0.5 μM hairpin H1 or 0.5 μM hairpin H2, respectively. As shown from columns 'b-d', almost no significant changes in the potentials were observed when DNA sensors reacted with miRNA-21 (column 'b'), hairpin H1 (column 'c') and hairpin H2 (column 'd') alone. Moreover, coexistence of hairpins H1/H2 did not cause the obvious change in the potential (column 'e' vs. column 'a'). More favorably, the potential was heavily changed relative to column 'a' when 0.1 nM miRNA-21, 0.5 μM hairpin H1 and 0.5 μM hairpin H2 were simultaneous present in the incubation solution (column 'g'). For comparison, we also studied the potential of the modified electrode after hybridization with the aforementioned product (column 'f'). Obviously, the change in the potential relative to column 'a' was less than that of column 'g'. The reason could be explained as follows: (i) the base number of the product obtained by CHA reaction was more than that of only hairpin DNA1, (ii) miRNA-21 could be repeatedly utilized for reaction with hairpin DNA1 in the presence of hairpin DNA2, thus resulting in formation of numerous CHA products. These results indicated that miRNA-21 and hairpins H1/H2 could be nonspecifically adsorbed to capture probe-modified electrode, and the change in the potential derived from the interaction of target miRNA-21 with hairpins H1/H2 (i.e., the CHA product). Therefore, our strategy could be utilized for the quantitative monitoring of miRNA-21.
to investigate the interfacial characteristics of the modified electrode. Generally speaking, the experimental data for EIS can be fitted to a Randles equivalent circuit (Fig. 2A, inset) comprising of electrolyte resistance (Rs), the lipid bilayer capacitance (Cdl), charge transfer resistance (Ret) and Warburg element (Zw). The complex impedance can be presented as the sum of the real, Zre and imaginary, Zim, components that originate mainly from the resistance and capacitance of the cell. The two components, Rs and Zw, represent bulk properties of the electrolyte solution and diffusion of the applied redox probe in solution, respectively. Therefore, they are not affected by chemical transformations occurring at the electrode interface. The other two components of the circuit, Cdl and Ret, depend on the dielectric and insulating features at the electrode/electrolyte interface. In EIS, the semicircle diameter of EIS equals the electron transfer resistance, Ret. This resistance controls the electron transfer kinetics of the redox-probe at electrode interface. Its value varies when different substances are adsorbed onto the electrode surface. Fig. 2A shows the EIS results of differently modified electrodes in 10 mM pH 7.4 PBS containing 5.0 mM Fe(CN)64−/3−. The cleaned gold electrode gave a similarly straight-line resistance (Nyquist 'a'). Upon assembly of capture DNA probe, the resistance increased to 682 Ω (Nyquist 'b'), indicating that the negatively charged oligonucleotide backbone inhibited the electron transfer of Fe(CN)64−/3− from the solution to the base electrode. When capture probe-modified gold electrode reacted with the CHA product, significantly, the resistance heavily increased (Nyquist 'c'), which was ascribed to the repulsive interaction between negative charges. One question to be produced was whether the strong resistance originated from the nonspecific adsorption toward the CHA product. As seen from Nyquist 'd', the resistance was almost the same as that of capture DNA probe-modified electrode (Nyquist 'b') in the absence of target miRNA-21. Logically, the product after hairpin DNA1 reacted with miRNA-21 could be also hybridized with the immobilized capture DNA on the electrode. In this regard, the impedimetric spectroscopy of capture DNA-modified electrode after reaction with miRNA-21 and hairpin DNA1 was investigated (Nyquist 'e'), and resistance was slightly lower than that of Nyquist 'c' in the simultaneous presence of hairpins DNA1 and DNA2. The reason might be attributed to the fact that the structure of CHA product was similar with that after reaction of hairpin DNA1 with target miRNA-21, shown in Scheme 1. The change in the resistance indicated that this system could be utilized for the detection of miRNA-21 on the basis of the CHA amplification strategy. Next, we also investigated the electrical potentials of the differently modified electrodes in pH 7.4 PBS (10 mM) by using a digital ion analyzer with a two-electrode system comprising of the modified gold electrode (as the working electrode) and an Ag/AgCl reference electrode (filled with 3.5 M KCl) (Fig. 2B). Column 'a' gives the potential of the newly prepared DNA sensor modified with capture probe (E0 = −10.2 mV). As control tests, the as-prepared DNA sensor was incubated
3.3. Optimization of experimental conditions As mentioned above, the shift in the potential stemmed from the reaction of DNA sensor with the CHA product. So, the amount of CHA product would directly affect the sensitivity of potentiometric sensor. By using 0.1 nM miRNA as an example, we investigated the effect of CHA reaction time between miRNA-21 and hairpins H1/H2 on the potential of DNA sensor by changing the CHA reaction time at a fixed hybridization reaction time of 60 min to ensure adequate reaction of CHA product with capture DNA-modified electrode. As shown in Fig. 3A, the change in the potential increased with the increasing CHA reaction time, and tended to level off after 150 min. A long incubation time did not cause the significant change of potentiometric sensor. Hence, 150 min was used as the CHA reaction time. Similarly, the reaction time of CHA product with DNA sensor also affected the potential response of the potentiometric sensor. A short reaction time was unfavorable for the conjugation of CHA product onto the electrode, thereby resulting in a low sensitivity. Experimental results indicated that the maximum change in the potential could be acquired after 40 min (Fig. 3B) at this condition of 150-min CHA reaction time. To save the assay time, the potential was registered after the reaction was executed for 40 min as the sensor signal relative to the concentration of target miRNA-21 throughout the text.
13
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Fig. 3. Dependence of electrode potential (ΔE = En−E0) of CHA-based potentiometric DNA sensor on (A) CHA reaction time and (B) hybridization time between capture DNA-modified gold electrode and the CHA product (0.1 nM miRNA-21 used in this case).
3.4. Calibration plots of CHA-based potentiometric DNA biosensor toward miRNA-21
Table 2 Comparsion of CHA-based potentiometric immunoassay with other detection schemes for target miRNA-21 on the analytical properties including linear range, LOD and amplification strategy.
Under the above-optimized conditions, target miRNA-21 standards with different concentrations were monitored on capture DNA-modified gold electrode coupling with catalytic hairpin assembly between two hairpins for the signal amplification. As indicated from Fig. 4A, the shifts in the potential relative to the background signal increased with the increment of miRNA-21 concentration, and exhibited a sigmoidal 'S' response relationship between the electrode potential and the logarithm of miRNA-21 level. Moreover, a good linear relationship between the electrode potential (mV) and the logarithm of miRNA-21 concentration (nM) could be obtained within the dynamic working range from 0.1 pM to 10 nM. The regression equation could be fit as y (mV) = 5.9139 × logC[miRNA-21] + 55.392 (nM, R2 = 0.9968, n = 6). The limit of detection (LOD) calculated from the slope of the calibration graph was estimated to 43 fM. Apparently, analytical properties of our strategy were comparable with those of other detection schemes (Table 2). Such a high sensitivity should be ascribed to the catalytic hairpin assembly and target recycling amplification.
Method
Materials/ amplification
Linear range
LOD
Ref.
Amperometry
Enzyme amplification Enzyme and nanogold DNAzyme and nanogold Target recycling CdTe QDs Nanogold
1.0–5000 pM
0.4 pM
[21]
0.1–70 pM
0.06 pM
[22]
0.01–500 pM
6.0 fM
[23]
50–500 aM 100 aM–0.1 nM 10–200 nM
8.0 aM 33 aM 221 pM
[24] [25] [26]
50 pM–5.0 nM
19 pM
[27]
0.1 pM–10 nM
43 fM
This work
Chronoamperometry Voltammetry Chronoamperometry Stripping voltammetry Resonance light scattering Gluometer Potentiometry
Mesoporous silica CHA/target recycling
Table 3 Reproducibility and precision of CHA-based potentiometric immunoassay toward low-middle-high miRNA-21 concentrations by using the same-batch or different-batch DNA biosensors.
3.5. Reproducibility, specificity and stability of CHA-based potentiometric biosensor
Conc.
The reproducibility of CHA-based potentiometric sensor was studied by analyzing three low-middle-high miRNA-21 standards within the linear range with the same-batch or various-batch DNA sensors. The results are summarized in Table 3. Obviously, the relative standard deviations (RSDs) for sensor-to-sensor reproducibility were higher than those of the intra-assays. Inspiringly, all the RSDs were less than 10%, indicating a good reproducibility and precision for the batch
1.0 pM 1.0 nM 5.0 nM
Intra-assay (Conc.)
RSD (%)
1
2
3
1.02 0.92 5.1
0.98 0.98 4.8
1.06 1.09 4.9
3.92 8.65 3.10
Inter-assay
RSD (%)
1
2
3
1.09 0.99 4.9
1.12 1.21 5.2
0.93 1.12 5.4
9.76 9.99 4.87
Fig. 4. (A) Electrode potential responses (ΔE = En−E0) of CHA-based DNA biosensor toward miRNA-21 standards with various concentrations in 10 mM PBS (pH 7.4), (B) the specificity of this system against miRNA-15 (10 nM), miRNA-16 (10 nM), miRNA-21 (0.1 nM), miRNA-141 (10 nM), miRNA-143 (10 nM) and the mismatched oligonucleotides (10 nM) including single-base mismatch miRNA-21 (M-1), two-base mismatch miRNA-21 (M-2) and three-base mismatch miRNA-21 (M-3); and (C) comparison of miRNA-21 copies in cancer cell lysates including human podocyte cell, human tubule epithelial (HK2) cell and HeLa cell obtained by using CHA-based DNA biosensor and the referenced RT-PCR kit. 14
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Acknowledgements
preparation of the potentiometric sensors. Next, the specificity of CHA-based potentiometric DNA sensor was investigated by challenging other miRNAs, such as miRNA-15, miRNA16, miRNA-141 and miRNA-143, and the mismatched oligonucleotides including single-base mismatch miRNA-21 (M-1), two-base mismatch miRNA-21 (M-2) and three-base mismatch miRNA-21 (M-3) (note: No specific reason for the site selection of the mismatched bases). Fig. 4B displays the electrochemical responses of this system in the presence and absence of target miRNA-21. As seen from Fig. 4B, the strong shifts in the electrode potential relative to background signal could be observed in the only presence of target miRNA-21. In contrast, the similar results with blank sample were achieved toward these non-target analytes alone. Furthermore, their coexistence did not cause the significant change in the electrode potentials. Unfavorably, the mismatched oligonucleotides (< three mismatched bases) would interfere the signal of potentiometric sensor to some extent. On a whole, our designed potentiometric sensor could specifically distinguish from other miRNA family members. Further, the long-time stability of the prepared DNA sensor was monitored over six-month period by storing DNA sensors at 4 °C when not in use. During this period, DNA sensors with the same batch were used for the detection of 0.1 nM miRNA-21 intermittently (every 15 days). The electrode potentials could preserve 98.3%, 96.2%, 95.4%, 94.8%, 92.1% and 90.7% of the initial signal at 1st, 2nd, 3rd, 4th, 5th and 6th month, respectively, suggesting good stability.
Support by the National Natural Science Foundation of China (21675029 & 21475025), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R11) is gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.bej.2018.09.004. References [1] G. D’Amico, The commonest glomerulonephritis in the world: IgA nephropathy, Q. J. Med. 64 (1987) 709–727. [2] H. Bao, S. Hu, C. Zhang, S. Shi, W. Qin, C. Zeng, K. Zen, Z. Liu, Inhibition of miRNA21 prevents fibrogenic activation in podocytes and tubular cells in IgA nephropathy, Biochem. Biophys. Res. Commun. 444 (2014) 455–460. [3] K. Phiwpan, X. Zhou, MicroRNAs in regulatory T cells, Cancer Lett. 423 (2018) 80–85. [4] C. Croce, Causes and consequences of miRNA dysregulation in cancer, Nat. Rev. Genet. 10 (2009) 704–714. [5] H. Yuan, D. Mischoulon, M. Fava, M. Otto, Circulating microRNAs as biomarkers for depression: many candidates, few finalists, J. Affect. Disord. 233 (2018) 68–78. [6] J. Chan, A. Krichevsky, K. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells, Cancer Res. 65 (2005) 6029–6033. [7] I. Negoi, S. Hostiuc, M. Sartelli, R. Negoi, M. Beuran, MicroRNA-21 as a prognostic biomarker in patients with pancreatic cancer–a systematic review and metal-analysis, Am. J. Surgery 215 (2017) 515–524. [8] H. Yang, A. Hui, G. Pampalakis, L. Soleymani, F. Liu, E. Sargent, S. Kelley, Direct, electronic microRNA detection for the rapid determination of differential expression profiles, Angew. Chem. Int. Ed. 48 (2009) 8461–8464. [9] L. Tian, K. Qian, J. Qi, Q. Liu, C. Yao, W. Song, Y. Wang, Gold nanoparticles superlattices assembly for electrochemical biosensor detection of microRNA-21, Biosens. Bioelectron. 99 (2018) 564–570. [10] H. Yin, B. Li, Y. Zhou, H. Wang, M. Wang, S. Ai, Signal-on fluorescence biosensor for microRNA-21 detection based on DNA strand displacement reaction and Mg2+dependent DNAzyme cleavage, Biosens. Bioelectron. 96 (2017) 106–112. [11] W. Peng, Q. Zhao, J. Piao, M. Zhao, Y. Huang, B. Zhang, W. Gao, D. Zhou, G. Shu, X. Gong, J. Chang, Ultra-sensitive detection of microRNA-21 based on duplexspecific nuclease-assisted target recycling and horseradish peroxidase cascading signal amplification, Sens. Actuators B Chem. 263 (2018) 289–297. [12] J. Zhang, W. Zhang, Y. Gu, Enzyme-free isothermal target-recycled amplification combined with PAGE for direct detection of microRNA-21, Anal. Biochem. 550 (2018) 117–122. [13] X. Pei, B. Zhang, J. Tang, B. Liu, W. Lai, D. Tang, Sandwich-type immunosensors and immunoassays exploiting nanostructure labels: a review, Anal. Chim. Acta 758 (2013) 1–18. [14] J. Shu, D. Tang, Current advantages in quantum-dots-based photoelectrochemical immunoassays, Chem. Asian J. 12 (2017) 2780–2789. [15] H. Zhang, R. Yao, N. Wang, R. Liang, W. Qin, Soluble molecularly imprinted polymer-based potentiometric sensor for determination of biophenol AF, Anal. Chem. 90 (2018) 657–662. [16] T. Kajisa, Y. Yanagimoto, A. Saito, T. Sakata, Biocompatible poly(catecholamine)film electrode for potentiometric cell sensing, ACS Sens. 2 (2018) 476–483. [17] S. Jakhar, C. Pundir, Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor, Biosens. Bioelectron. 100 (2018) 242–250. [18] Q. Li, S. Lv, M. Lu, Z. Lin, D. Tang, Potentiometric competitive immunoassay for determination of aflatoxin B1 in food by using antibody-labeled gold nanoparticles, Microchim. Acta 183 (2016) 2815–2822. [19] S. Lv, Z. Lin, K. Zhang, M. Lu, D. Tang, Polyion oligonucleotide-decorated gold nanoparticles with tunable surface charge density for amplified signal output of potentiometric immunosensor, Anal. Chim. Acta 964 (2017) 67–73. [20] Q. Li, F. Zeng, N. Lyu, J. Liang, Highly sensitive and specific electrochemical biosensor for microRNA-21 detection by coupling catalytic hairpin assembly with rolling circle amplification, Analyst 143 (2018) 2304–2309. [21] Y. Zhou, Z. Zhang, Z. Xu, H. Yin, S. Ai, MicroRNA-21 detection based on molecular switching by amperometry, New J. Chem. 36 (2012) 1985–1991. [22] H. Yin, Y. Zhou, H. Zhang, X. Meng, S. Ai, Electochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system, Biosens. Bioelectron. 33 (2012) 247–253. [23] X. Meng, Y. Zhou, Q. Liang, X. Qu, Q. Yang, H. Yin, S. Ai, Electrochemical determination of microRNA-21 based on bio bar code and hemin/G-quadruplet DNAzyme, Analyst 138 (2013) 3409–3415. [24] X. Zhang, D. Wu, Z. Liu, S. Cai, Y. Zhao, M. Chen, Y. Xia, C. Li, J. Zhang, J. Chen, An ultrasensitive label-free electrochemical biosensor for microRNA-21 detection based on a 2’-O-methyl modified DNAzyme and duplex-specific nuclease assisted target cycling, Chem. Commun. 50 (2014) 12375–12377.
3.6. Monitoring of real samples and evaluation of method accuracy As a newly developed detection method, the accuracy of this method was evaluated by assaying the real samples. Initially, the cell lysates were extracted from three tumor cells including human podocyte cell, human tubule epithelial (HK2) cell and HeLa cell with high miRNA-21 expression level. Thereafter, these samples were detected by using CHA-based potentiometric sensor. The high-level miRNA-21 cell lysates were calculated according to the dilution ratio. These results (copies per cell) were compared with those obtained from the referenced RT-PCR method (Fig. 4C). The RSD values between two methods were 3.6–8.2%. In addition, the accuracy of this system was also evaluated by using standard addition method. Three miRNA-21 standards including 0.01, 0.5 and 5.0 nM were initially spiked in the lysis buffer, and then detected by using our strategy. The levels of miRNA-21 in these spiked samples was 0.089, 0.52 and 5.68 nM, respectively. The recoveries were 89–112.6%. These result indicated that this system could be utilized as an optional scheme for detection of target miRNA21 in the complex samples with good accuracy.
4. Conclusions In summary, this study successfully demonstrates a new approach toward the development of advanced potentiometric DNA sensor for the detection of miRNA-21 by combination with catalytic hairpin assembly for the amplification of detectable signal. Although the oligonucleotides before and after formation of double-stranded DNA can cause the change in the potential to some extent, the signal is relatively weak. Introduction of catalytic hairpin assembly with target recycling is used to amplify the electrical signal. Compared with conventional electrochemical detection strategies, e.g., voltammetric or impedimetric assays, CHA-based potentiometric sensing protocol exhibits the following advantages, e.g., label free, simply operation, low cost and short assay time. Nevertheless, only one limitation of our system is that CHA reaction time is relatively long (∼2.5 h). Maybe, it can be improved by controlling the reaction temperature. Therefore, future work should be focus on the improvement of reaction conditions.
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scattering method based on aggregation of gold nanoparticles for selective detection of microRNA-21, RSC Adv. 6 (2016) 83078–83083. [27] K. Deng, Y. Zhang, X. Tong, Sensitive electrochemical detection of microRNA-21 based on propylamine-functionalized mesoporous silica with glucometer readout, Anal. Bioanal. Chem. 410 (2018) 1863–1871.
[25] H. Liu, X. Bei, Q. Xia, Y. Fu, S. Zhang, M. Liu, K. Fan, M. Zhang, Y. Yang, Enzymefree electrochemical detection of microRNA-21 using immobilized hairpin probes and a target-triggered hybridization chain reaction amplification strategy, Microchim. Acta 183 (2016) 297–304. [26] M. Ren, S. Wang, C. Cai, C. Chen, X. Chen, A simple and sensitive resonance light
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Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Catalytic hairpin assembly-mediated surface charge density on the electrode for sensitive potentiometric detection of microRNA-21 in IgA-nephropathy ⁎
Qian Zhoua,b, , Dianping Tangb, a b
T
⁎
Institute of Environmental and Analytical Science, School of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, Henan, PR China Key Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou, 350116, PR China
H I GH L IG H T S
potentiometric DNA biosensor was developed for microRNA-21 detection. • ACatalytic hairpin assembly is utilized to increase the surface charge density. • Target recycling is used for the signal amplification. •
A R T I C LE I N FO
A B S T R A C T
Keywords: Potentiometric biosensor Catalytic hairpin assembly MicroRNA-21 Target recycling Surface charge density IgA-nephropathy
Methods based on electrochemical biosensors have been developed for detection of microRNA-21 (miRNA-21), but most involve complicated labeling or stripping procedures and are unsuitable for routine use in diagnosis/ treatment of IgA-nephropathy. Herein we report on the proof-of-concept of simple and sensitive potentiometric DNA biosensor for specific detection of miRNA-21 coupling and catalytic hairpin assembly (CHA) with target recycling. This system consists of two hairpin DNA probes and one single-stranded DNA (capture probe). To decrease the background signal, the thiolated capture probe is covalently conjugated onto a cleaned gold electrode through typical Au-S bond. Upon target miRNA-21 introduction, the analyte opens hairpins DNA in turn to propagate the CHA reaction between two alternating hairpins accompanying the release of target miRNA21 for recycling, thereby resulting in formation of numerous nicked double-helixes. Meanwhile, the newly produced CHA product hybridizes with the immobilized capture DNA probe on the electrode, thus causing the change in the surface charge density for potentiometric measurement. Under optimum conditions, CHA-based DNA biosensor presents good potentiometric responses for determination of target miRNA-21 at a level as low as 43 fM. An intermediate precision of < 10% is accomplished with the batch-to-batch identification. The specificity and stability of CHA-based biosensor are satisfactory. Importantly, CHA-based potentiometric biosensor offers promise for the label-free, rapid, simple, cost-effective analysis of biological samples.
1. Introduction IgA-nephropathy, the most common glomerulonephritis in China, is characterized by deposition of IgA antibody in the glomerulus [1]. MicroRNA-21 is significantly increased in both glomerular and tubularinterstitial tissues of patients with IgA-nephropathy [2]. MicroRNA (miRNA; a class of endogenously expressed 18–25 nucleotides with small noncoding single-strand RNA molecules) is crucially important in regulating the post-transcriptional gene expression in a broad range of the animals, plants, and viruses [3]. Emerging evidences reveal that miRNAs can be identified as the biomarkers for the diagnostic and
prognostic purposes since their expression levels are associated with different pathological conditions including cancers, e.g., hepatocellular carcinoma, lung cancer, breast cancer and gastric carcinoma [4]. Recent research has highlighted the potential of miRNAs to serve as physiological indicators of disease process among clinically depressed patients [5]. As an important robust oncogenic circulating miRNA, miRNA-21 has been also reported to be a key for various cancers and infectious diseases via a variety of regulatory functions on the inflammation, osteogenesis and osteoclastogenesis [6,7]. Therefore, ongoing effort has been done in the world for miRNA-21 detection due to its low abundances, small size, and high degree of sequence similarity
⁎ Corresponding authors at: Key Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou, 350116, PR China. E-mail addresses: [email protected] (Q. Zhou), [email protected], [email protected] (D. Tang).
https://doi.org/10.1016/j.bej.2018.09.004 Received 27 June 2018; Received in revised form 12 August 2018; Accepted 4 September 2018 Available online 06 September 2018 1369-703X/ © 2018 Elsevier B.V. All rights reserved.
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presenting obstacles [8]. Recently, different schemes and strategies have been developed for the detection of miRNA-21. Tian et al. reported an electrochemical biosensor for miRNA-21 detection through the superlattices assembly of gold nanoparticles [9]. Yin et al. designed a signal-on fluorescence biosensor for the microRNA-21 detection based on the strand displacement reaction and Mg2+-dependent DNAzyme cleavage [10]. Peng et al. developed ultrasensitive colorimetric detection of microRNA-21 based on the duplex-specific nuclease-assisted target recycling and horseradish peroxidase cascading signal amplification [11]. Zhang et al. constructed an enzyme-free isothermal target-recycled amplification strategy for the direct detection of miRNA-21, combining with polyacrylamide gel electrophoresis [12]. Despite some advances in this field, there is still the requirement to explore new strategies for improvement of the sensitivity and simplification of the assays. To the best of our knowledge, there is no reports focusing on the potentiometric methods for the detection of miRNA-21 until now. Potentiometric assay, one of most widely used straightforward methods, has extensively applied in the bioanalytical fields since no external excitement (e.g., current or voltage) is required during the measurement [13]. Typically, the signal is recorded on the basis of the formed electrical double layer from the biological bindings and the variation of electrical potential close to the surface [14]. Zhang et al. used soluble molecularly imprinted polymer to construct a potentiometric sensor for the determination of bisphenol AF [15]. Kajisa et al. prepared biocompatible poly(catecholamine)-film electrode for potentiometric cell sensing [16]. For the development of high-efficient potentiometric detection system, however, signal amplification and noise reduction are very crucial. Jakhar and Pundir employed the urease nanoparticles for the construction of an improved potentiometric urea biosensor [17]. Li et al. fabricated a potentiometric competitive immunoassay for the determination of aflatoxin B1 in food by using antibody-labeled gold nanoparticles [18]. Lv et al. utilized polyion oligonucleotide-decorated gold nanoparticles with tunable surface charge density for the amplified signal output of the potentiometric immunosensor [19]. Since DNA molecules can form negatively charged polyion complexes, the oligonucleotides can be utilized to control the intensity of surface charges on the substrate. Herein, we design a novel potentiometric detection protocol for target miRNA-21 on the basis of catalytic hairpin assembly (Scheme 1). Initially, a short-stranded DNA capture probe is immobilized on a cleaned gold disk electrode, and then reacts with target miRNA-21, thereby exposing a new sticky, which hybridizes with the sticky end of
Table 1 The sequences of the oligonucleotides used in this work. Name
Sequences (5'→3')
Hairpin DNA1
5'-TCA ACA TCA GTC TGA TAA GCT ACC ATG TGT AGA TAG CTT ATC AGA CTC TAC TCA-3' 5'-TAA GCT ATC TAC ACA TGG TAG CTT ATC AGA CTC CAT GTG TAG A-3' 5'-UAG CUU AUC AGA CUG AUG UUG A-3' 5'-UAG CUU AUC GGA CUG AUG UUG A-3'
Hairpin DNA2 Target miRNA-21 Single-base mismatch miRNA-21 Two-base mismatch miRNA-21 Three-base mismatch miRNA-21 miRNA-15 miRNA-16 miRNA-141 miRNA-143
5'-UAG CUU UUC GGA CUG AUG UUG A-3' 5'-UAG CUU UUC GAA CUG AUG UUG A-3' 5'-UAG 5'-UAG 5'-UAA 5'-UGA
CAG CAC AUA AUG GUU UGU G-3' CAG CAC GUA AAU AUU GGC G-3' CAC UGU CUG GUA AAG AUG G-3' GAU GAA GCA CUG UAG CUC A-3'
hairpin DNA1 and hairpin DNA2. In this regard, target miRNA-21 triggers a chain reaction of hybridization events between two hairpin probes to form a nicked long double-helix. Thanks to the increasing negative charges on the surface, the electrode potential changes relative to background signal. By monitoring the shift in the potential, we can quantitatively evaluate the level of target miRNA-21. The aim of this work is to develop a simple and portable potentiometric detection system for miRNA-21 with high sensitivity. 2. Experimental section 2.1. Materials and reagents All the oligonucleotides including HPLC-purified miRNA-21, capture probe, hairpin DNA1 and hairpin DNA2 were synthesized by Shanghai Sangon Biotechnol. Co., Ltd. (Shanghai, China). The sequences for detailed DNA and RNA are listed in Table 1, which were designed consulting to the literature [20]. 6-Mercapto-1-hexanol (MCH; 99%) and salmon sperm DNA were obtained from Sigma-Aldrich (St. Louis, MO, USA). DNA/RNS stock solutions were obtained through dissolving the corresponding oligonucleotides into Tris-HCl buffer (100 mM, pH 7.4). Each oligonucleotide was heated to 90 °C for 5 min, and slowly cooled down to room temperature before use. Ultrapure water at 18.2 MΩ cm was obtained from a Millipore water purification system (Millipore, Inc., Bedford, MA). Tris(hydroxymethyl)
Scheme 1. Schematic illustration of catalytic hairpin assembly (CHA)-mediated surface charge density on the capture DNA-modified gold electrode for the sensitive potentiometric detection of microRNA-21 (miRNA-21) by coupling with target recycling amplification. 10
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the possible error resulting from different additions of samples and deduct the responses induced by nonspecific adsorption, the response for each sensor was recorded until equilibrium was reached (shift less than 1.0 mV min−1). The control tests with normal samples were performed accordingly. The electric potential of DNA sensor toward target miRNA-21 was calculated referring to the equation: ΔE = En – E0 (where E0 and En represents the steady-state electrical potentials of DNA biosensor in the absence and presence of target analyte, respectively).
aminomethane (Tris) was achieved from Shanghai Chem. Re. Inc. (Shanghai, China). All other chemicals used in this study were of analytical grade and used as received. Phosphate buffer solution (PBS) with different pH values was prepared by using 10 mM phosphate-buffered saline and 0.1 M KCl (used as the supporting electrolyte). 2.2. Cell culture and cell lysis Human podocytes and human tubule epithelial (HK2) cells were cultured in RPMI-1640 and DMEM/F12 medium that contained 10% fetal bovine serum and penicillin-streptomycin (100 IU mL−1). To monitor the effect of IgA-human mesangial cell (HMC) medium, different preparations of IgA-HMC medium were diluted 8-fold with medium containing 0.5% fetal bovine serum. Initially, the cells were maintained at 37 °C in a humidified atmosphere (95% air + 5% CO2). The cancer cell density was determined by using hemocytometer prior to experiments. Cells were collected in the exponential phase of growth. Isolation of total RNAs were performed from target cell lines using TRIzol® reagent when the cells reached 80% confluency. Briefly, an aliquot containing 1.0 × 106 cells was dispersed into a 1.5-mL eppendorf tube, washed twice with ice-cold PBS (0.1 M, pH 7.4), and resuspended into 200-μL ice-cold CHARPS lysis buffer containing 10 mM Tris-HCl, pH 7.5, 1.0 mM MgCl2, 1.0 mM EGTA, 0.1 mM PMSF, 0.5% CHAPS and 10% glycerol. The mixture was incubated for 30 min on ice and centrifuged at 16,000g at 4 °C for 20 min. The supernatant was collected as cell extract for analysis or flash frozen in liquid nitrogen at −80 °C.
2.5. RT-PCR analysis for target miRNA-21 Total RNA sample from cells was extracted by using mirVana™ miRNA Isolation Kit. Template complementary DNA was prepared by using reverse transcriptase, and miRNA-21 expression was quantified with an ABI 7300 Sequence Detection System (Applied Biosystem, ABI, USA) by using SYBR green PCR Master Mix (Takara, Shiga, Japan). The relative expression of miRNA-21 was calculated by using the 2T−ΔΔC method based on the relative DNA oligonucleotides. All RT-PCR reactions were performed in triplicate. 2.6. Statistical analyses The signals in all experiments referred to the average responses of the reaction with the corresponding standard deviations in triplicate, unless otherwise indicated. The change in the electrical potential was collected and registered as the sensor signal relative to target miRNA-21 concentration. All measurements were performed at room temperature (25 ± 1.0 °C). Statistical analysis of the data from multiple groups was performed by one-way ANOVA followed by Student-Newman-Kuels test. Data from two groups were compared by t-test (note: p < 0.05 was considered significant).
2.3. Preparation of DNA sensor A gold disk electrode with 2.0 mm in diameter was first polished carefully with 0.3 and 0.05-μm alumina slurry, followed by successive sonication in ultrapure water and ethanol for 5 min and dried in air. Prior to modification, the pretreated gold electrode was first cleaned with hot piranha solution [a 3:1 (v/v) mixture containing concentrated H2SO4 and 30% H2O2] for 10 min at room temperature, and then continuously scanned within the applied potential range from −0.3 to 1.5 V in freshly prepared deoxygenated 0.5 M H2SO4 at 100 mV s−1 until a stable cyclic voltammogram was obtained. After being washed with ultrapure water and absolute ethanol, the cleaned gold electrode was immersed into capture DNA probe (0.5 μM), and incubated for 6 h at room temperature. During this process, the thiolated capture probe was covalently conjugated onto the gold electrode through the classical Au-S bond. The resulting gold electrode was washed as before. Following that, the modified gold electrode was incubated with 1.0-mM MCH in 10 mM Tris-HCl buffer, pH 7.4, for 60 min. Finally, the asprepared DNA sensor was suspended over pH 7.4 PBS at 4 °C when not in use.
3. Results and discussion 3.1. Design and characterization of CHA-based potentiometric DNA biosensor As a newly developed detection scheme for practical application in future, a simple, rapid and sensitive method would be advantageous. In this work, target miRNA-21 is quantitatively evaluated by a portable potentiometer. The assay mainly consists of two label-free hairpin DNA probes and one short-stranded capture probe. Each hairpin H1 has a stem of 14 base pairs enclosing an 11-base loop and an additional sticky end at the 5′ and 3′ ends, whereas hairpin H2 contains a stem of 11 base pairs enclosing 14-base loop and an additional sticky end at the 5′ end. The electrical potential is amplified on the basis of target-induced catalytic hairpin assembly between two DNA hairpins. To reduce the background signal of DNA sensor, thiolated capture probe is firstly immobilized onto the cleaned gold electrode via the Au-S bond. In the presence of target miRNA-21, the analyte initially pairs with the 5′ sticky end of hairpin H1 to open the hairpin. The newly exposed sticky end of hairpin H1 nucleates at the sticky end on hairpin H2 and opens the hairpin, which undergoes a catalytic hairpin assembly reaction. During this process, target miRNA-21 is displaced from the nicked double-helix to execute the next cycling reaction between two hairpin probes (i.e., target recycling reaction). Following that, the newly formed double-helix is captured to the electrode by the immobilized capture DNA. Due to introduction of negatively charged oligonucleotides on the electrode, the local electrical potential is changed relative to the initial signal. By monitoring the shift in the electrical potential, we can quantitatively evaluate the concentration of miRNA-21 in the sample. In contrast, the CHA reaction cannot be carried out in the absence of target miRNA-21, and hairpin probes cannot be conjugated to the electrode. To realize our design, one important concern for the development of
2.4. Catalytic hairpin assembly with miRNA-21 and potentiometric measurement Scheme 1 gives schematic illustration of potentiometric DNA sensing strategy toward miRNA-21 on capture probe-modified gold electrode coupling with target-triggered catalytic hairpin assembly for the signal amplification. The assay consisted of the following steps. Firstly, the different-level miRNA standard was added into the mixture containing 0.5 μM hairpin DNA1 and 0.5 μM hairpin DNA2, and reacted for 150 min (i.e., CHA reaction time) at 37 °C in a centrifugal tube to execute catalytic hairpin assembly. Following that, capture DNA-modified gold electrode was immersed into the resulting mixture, and reacted for another 40 min (i.e., hybridization time) at 37 °C. After being washed with pH 7.4 PBS, the resulting electrode was monitored in pH 7.4 PBS (10 mM) by using a digital ion analyzer with a two-electrode system comprising of the modified gold electrode (as the working electrode) and an Ag/AgCl reference electrode (filled with 3.5 M KCl). To avoid 11
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Fig. 1. (A) Gel electrophoresis: (M) 0.5 μM DNA marker, (a) 0.5 μM miRNA-21, (b) 0.5 μM hairpin DNA1, (c) 0.5 μM hairpin DNA2, (d) 0.5 μM hairpin DNA1 + 0.5 μM miRNA-21, (e) 0.5 μM hairpin DNA1 + 0.5 μM miRNA-21 + 0.5 μM hairpin DNA2, and (f) 0.5 μM hairpin DNA1 + 0.5 μM hairpin DNA2 (note: The incubation time was 2.5 h for all mixture reactions); (B–D) AFM images of (B) gold substrate, (C) capture DNA-modified gold substrate and (D) capture DNA-modified gold substrate after reaction with CHA product in the presence of miRNA-21.
USA) (Fig. 1B–D). Fig. 1B gives typical AFM image of the cleaned gold substrate (surface roughness: 312.1 ± 13.7 nm; Fig. S1 in the Supporting Information). As shown in Fig. 1C, the assembly of thiolated capture probes on the gold substrate caused the change of the surface roughness (surface roughness: 397.4 ± 27.3 nm; Fig. S2). When the capture probe-modified gold electrode hybridized with the products of CHA reaction, significantly, the surface of gold substrate (Fig. 1D) became rougher than that of Fig. 1C (surface roughness: 658.5 ± 34.1 nm; Fig. S3). Vaguely, we seemed to observe the structures of long nicked oligonucleotides. As control test, the surface morphology of capture DNA-modified gold substrate was studied after incubation with hairpins DNA1 and DNA2 in the absence of target miRNA21. As seen from Fig. S4, the surface roughness was 318.9 ± 17.4 nm, which was almost the same as that in Fig. S1, indicating that hairpins DNA1 and DNA2 were not nonspecifically adsorbed onto the substrate without target analyte. Therefore, we might preliminarily make a conclusion that our design is feasible.
CHA-based potentiometric DNA sensor is whether the analyte could readily cause the catalytic hairpin assembly between two hairpins. To clarify this issue, we first used gel electrophoresis to investigate two hairpin DNA probes in the absence and presence of miRNA-21 (Fig. 1A). Lanes 'a–c' shows gel electrophoresis images of miRNA-21, hairpin H1 and hairpin H2, respectively. When mixture of miRNA-21 with hairpin H1, the spot at lane 'd' slightly migrated relative to lane 'b', which might be ascribed to the fact that the base number of the formed miRNA21/ hairpin DNA1 structure was more than that of hairpin DNA1 alone. Upon reaction of miRNA-21 with hairpins H1/H2, favorably, the migration of the formed complexes at lane 'e' was obviously slower than those of hairpins H1/H2. Importantly, the mixture of hairpin H1 with hairpin H2 in the absence of target miRNA-21 did not cause their selfhybridization reaction (lane 'f'). The results revealed that catalytic hairpin assembly between two hairpins could be implemented through target miRNA-21. Logically, another question arises to whether the newly formed nicked double-helix could be captured on the electrode by the immobilized capture probe. To this end, gold substrates before and after modification with the oligonucleotides were characterized by using atomic force microscopy (AFM; Nanoscope IIID Instruments, Bruker,
3.2. Evaluation of feasibility and control experiments Electrochemical impedance spectroscopy (EIS) is a widely used tool 12
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Fig. 2. (A) Nyquist diagrams for (a) gold electrode, (b) capture DNA-modified gold electrode, (c,d) sensor 'b' after reaction with 0.5 μM hairpin H1 and 0.5 μM hairpin H2 in the (c) presence and (d) absence of 0.1 nM miRNA-21, and (e) sensor 'b' after reaction with 0.5 μM hairpin H1 and 0.1 nM miRNA-21 in 10 mM pH 7.4 PBS containing 5.0 mM Fe(CN)64−/3− with the range from 10-2 to 105 Hz at an alternate voltage of 5 mV (inset: equivalent circuit); (B) electrode potentials of (a) capture DNA-modified gold electrode, (b) sensor 'b' + 0.1 nM miRNA-21, (c) sensor 'b' + 0.5 μM hairpin H1, (d) sensor 'b' + 0.5 μM hairpin H2, (e) sensor 'b' + 0.5 μM hairpin H1 + 0.5 μM hairpin H2, (f) sensor 'b' + 0.1 nM miRNA-21 + 0.5 μM hairpin H1 and (g) sensor 'b' + 0.1 nM miRNA-21 + 0.5 μM hairpin H1 + 0.5 μM hairpin H2 in pH 7.4 PBS (10 mM) (note: Sensor 'b' stands for capture DNA-modified gold electrode).
with 0.1 nM miRNA-21, 0.5 μM hairpin H1 or 0.5 μM hairpin H2, respectively. As shown from columns 'b-d', almost no significant changes in the potentials were observed when DNA sensors reacted with miRNA-21 (column 'b'), hairpin H1 (column 'c') and hairpin H2 (column 'd') alone. Moreover, coexistence of hairpins H1/H2 did not cause the obvious change in the potential (column 'e' vs. column 'a'). More favorably, the potential was heavily changed relative to column 'a' when 0.1 nM miRNA-21, 0.5 μM hairpin H1 and 0.5 μM hairpin H2 were simultaneous present in the incubation solution (column 'g'). For comparison, we also studied the potential of the modified electrode after hybridization with the aforementioned product (column 'f'). Obviously, the change in the potential relative to column 'a' was less than that of column 'g'. The reason could be explained as follows: (i) the base number of the product obtained by CHA reaction was more than that of only hairpin DNA1, (ii) miRNA-21 could be repeatedly utilized for reaction with hairpin DNA1 in the presence of hairpin DNA2, thus resulting in formation of numerous CHA products. These results indicated that miRNA-21 and hairpins H1/H2 could be nonspecifically adsorbed to capture probe-modified electrode, and the change in the potential derived from the interaction of target miRNA-21 with hairpins H1/H2 (i.e., the CHA product). Therefore, our strategy could be utilized for the quantitative monitoring of miRNA-21.
to investigate the interfacial characteristics of the modified electrode. Generally speaking, the experimental data for EIS can be fitted to a Randles equivalent circuit (Fig. 2A, inset) comprising of electrolyte resistance (Rs), the lipid bilayer capacitance (Cdl), charge transfer resistance (Ret) and Warburg element (Zw). The complex impedance can be presented as the sum of the real, Zre and imaginary, Zim, components that originate mainly from the resistance and capacitance of the cell. The two components, Rs and Zw, represent bulk properties of the electrolyte solution and diffusion of the applied redox probe in solution, respectively. Therefore, they are not affected by chemical transformations occurring at the electrode interface. The other two components of the circuit, Cdl and Ret, depend on the dielectric and insulating features at the electrode/electrolyte interface. In EIS, the semicircle diameter of EIS equals the electron transfer resistance, Ret. This resistance controls the electron transfer kinetics of the redox-probe at electrode interface. Its value varies when different substances are adsorbed onto the electrode surface. Fig. 2A shows the EIS results of differently modified electrodes in 10 mM pH 7.4 PBS containing 5.0 mM Fe(CN)64−/3−. The cleaned gold electrode gave a similarly straight-line resistance (Nyquist 'a'). Upon assembly of capture DNA probe, the resistance increased to 682 Ω (Nyquist 'b'), indicating that the negatively charged oligonucleotide backbone inhibited the electron transfer of Fe(CN)64−/3− from the solution to the base electrode. When capture probe-modified gold electrode reacted with the CHA product, significantly, the resistance heavily increased (Nyquist 'c'), which was ascribed to the repulsive interaction between negative charges. One question to be produced was whether the strong resistance originated from the nonspecific adsorption toward the CHA product. As seen from Nyquist 'd', the resistance was almost the same as that of capture DNA probe-modified electrode (Nyquist 'b') in the absence of target miRNA-21. Logically, the product after hairpin DNA1 reacted with miRNA-21 could be also hybridized with the immobilized capture DNA on the electrode. In this regard, the impedimetric spectroscopy of capture DNA-modified electrode after reaction with miRNA-21 and hairpin DNA1 was investigated (Nyquist 'e'), and resistance was slightly lower than that of Nyquist 'c' in the simultaneous presence of hairpins DNA1 and DNA2. The reason might be attributed to the fact that the structure of CHA product was similar with that after reaction of hairpin DNA1 with target miRNA-21, shown in Scheme 1. The change in the resistance indicated that this system could be utilized for the detection of miRNA-21 on the basis of the CHA amplification strategy. Next, we also investigated the electrical potentials of the differently modified electrodes in pH 7.4 PBS (10 mM) by using a digital ion analyzer with a two-electrode system comprising of the modified gold electrode (as the working electrode) and an Ag/AgCl reference electrode (filled with 3.5 M KCl) (Fig. 2B). Column 'a' gives the potential of the newly prepared DNA sensor modified with capture probe (E0 = −10.2 mV). As control tests, the as-prepared DNA sensor was incubated
3.3. Optimization of experimental conditions As mentioned above, the shift in the potential stemmed from the reaction of DNA sensor with the CHA product. So, the amount of CHA product would directly affect the sensitivity of potentiometric sensor. By using 0.1 nM miRNA as an example, we investigated the effect of CHA reaction time between miRNA-21 and hairpins H1/H2 on the potential of DNA sensor by changing the CHA reaction time at a fixed hybridization reaction time of 60 min to ensure adequate reaction of CHA product with capture DNA-modified electrode. As shown in Fig. 3A, the change in the potential increased with the increasing CHA reaction time, and tended to level off after 150 min. A long incubation time did not cause the significant change of potentiometric sensor. Hence, 150 min was used as the CHA reaction time. Similarly, the reaction time of CHA product with DNA sensor also affected the potential response of the potentiometric sensor. A short reaction time was unfavorable for the conjugation of CHA product onto the electrode, thereby resulting in a low sensitivity. Experimental results indicated that the maximum change in the potential could be acquired after 40 min (Fig. 3B) at this condition of 150-min CHA reaction time. To save the assay time, the potential was registered after the reaction was executed for 40 min as the sensor signal relative to the concentration of target miRNA-21 throughout the text.
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Fig. 3. Dependence of electrode potential (ΔE = En−E0) of CHA-based potentiometric DNA sensor on (A) CHA reaction time and (B) hybridization time between capture DNA-modified gold electrode and the CHA product (0.1 nM miRNA-21 used in this case).
3.4. Calibration plots of CHA-based potentiometric DNA biosensor toward miRNA-21
Table 2 Comparsion of CHA-based potentiometric immunoassay with other detection schemes for target miRNA-21 on the analytical properties including linear range, LOD and amplification strategy.
Under the above-optimized conditions, target miRNA-21 standards with different concentrations were monitored on capture DNA-modified gold electrode coupling with catalytic hairpin assembly between two hairpins for the signal amplification. As indicated from Fig. 4A, the shifts in the potential relative to the background signal increased with the increment of miRNA-21 concentration, and exhibited a sigmoidal 'S' response relationship between the electrode potential and the logarithm of miRNA-21 level. Moreover, a good linear relationship between the electrode potential (mV) and the logarithm of miRNA-21 concentration (nM) could be obtained within the dynamic working range from 0.1 pM to 10 nM. The regression equation could be fit as y (mV) = 5.9139 × logC[miRNA-21] + 55.392 (nM, R2 = 0.9968, n = 6). The limit of detection (LOD) calculated from the slope of the calibration graph was estimated to 43 fM. Apparently, analytical properties of our strategy were comparable with those of other detection schemes (Table 2). Such a high sensitivity should be ascribed to the catalytic hairpin assembly and target recycling amplification.
Method
Materials/ amplification
Linear range
LOD
Ref.
Amperometry
Enzyme amplification Enzyme and nanogold DNAzyme and nanogold Target recycling CdTe QDs Nanogold
1.0–5000 pM
0.4 pM
[21]
0.1–70 pM
0.06 pM
[22]
0.01–500 pM
6.0 fM
[23]
50–500 aM 100 aM–0.1 nM 10–200 nM
8.0 aM 33 aM 221 pM
[24] [25] [26]
50 pM–5.0 nM
19 pM
[27]
0.1 pM–10 nM
43 fM
This work
Chronoamperometry Voltammetry Chronoamperometry Stripping voltammetry Resonance light scattering Gluometer Potentiometry
Mesoporous silica CHA/target recycling
Table 3 Reproducibility and precision of CHA-based potentiometric immunoassay toward low-middle-high miRNA-21 concentrations by using the same-batch or different-batch DNA biosensors.
3.5. Reproducibility, specificity and stability of CHA-based potentiometric biosensor
Conc.
The reproducibility of CHA-based potentiometric sensor was studied by analyzing three low-middle-high miRNA-21 standards within the linear range with the same-batch or various-batch DNA sensors. The results are summarized in Table 3. Obviously, the relative standard deviations (RSDs) for sensor-to-sensor reproducibility were higher than those of the intra-assays. Inspiringly, all the RSDs were less than 10%, indicating a good reproducibility and precision for the batch
1.0 pM 1.0 nM 5.0 nM
Intra-assay (Conc.)
RSD (%)
1
2
3
1.02 0.92 5.1
0.98 0.98 4.8
1.06 1.09 4.9
3.92 8.65 3.10
Inter-assay
RSD (%)
1
2
3
1.09 0.99 4.9
1.12 1.21 5.2
0.93 1.12 5.4
9.76 9.99 4.87
Fig. 4. (A) Electrode potential responses (ΔE = En−E0) of CHA-based DNA biosensor toward miRNA-21 standards with various concentrations in 10 mM PBS (pH 7.4), (B) the specificity of this system against miRNA-15 (10 nM), miRNA-16 (10 nM), miRNA-21 (0.1 nM), miRNA-141 (10 nM), miRNA-143 (10 nM) and the mismatched oligonucleotides (10 nM) including single-base mismatch miRNA-21 (M-1), two-base mismatch miRNA-21 (M-2) and three-base mismatch miRNA-21 (M-3); and (C) comparison of miRNA-21 copies in cancer cell lysates including human podocyte cell, human tubule epithelial (HK2) cell and HeLa cell obtained by using CHA-based DNA biosensor and the referenced RT-PCR kit. 14
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Acknowledgements
preparation of the potentiometric sensors. Next, the specificity of CHA-based potentiometric DNA sensor was investigated by challenging other miRNAs, such as miRNA-15, miRNA16, miRNA-141 and miRNA-143, and the mismatched oligonucleotides including single-base mismatch miRNA-21 (M-1), two-base mismatch miRNA-21 (M-2) and three-base mismatch miRNA-21 (M-3) (note: No specific reason for the site selection of the mismatched bases). Fig. 4B displays the electrochemical responses of this system in the presence and absence of target miRNA-21. As seen from Fig. 4B, the strong shifts in the electrode potential relative to background signal could be observed in the only presence of target miRNA-21. In contrast, the similar results with blank sample were achieved toward these non-target analytes alone. Furthermore, their coexistence did not cause the significant change in the electrode potentials. Unfavorably, the mismatched oligonucleotides (< three mismatched bases) would interfere the signal of potentiometric sensor to some extent. On a whole, our designed potentiometric sensor could specifically distinguish from other miRNA family members. Further, the long-time stability of the prepared DNA sensor was monitored over six-month period by storing DNA sensors at 4 °C when not in use. During this period, DNA sensors with the same batch were used for the detection of 0.1 nM miRNA-21 intermittently (every 15 days). The electrode potentials could preserve 98.3%, 96.2%, 95.4%, 94.8%, 92.1% and 90.7% of the initial signal at 1st, 2nd, 3rd, 4th, 5th and 6th month, respectively, suggesting good stability.
Support by the National Natural Science Foundation of China (21675029 & 21475025), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R11) is gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.bej.2018.09.004. References [1] G. D’Amico, The commonest glomerulonephritis in the world: IgA nephropathy, Q. J. Med. 64 (1987) 709–727. [2] H. Bao, S. Hu, C. Zhang, S. Shi, W. Qin, C. Zeng, K. Zen, Z. Liu, Inhibition of miRNA21 prevents fibrogenic activation in podocytes and tubular cells in IgA nephropathy, Biochem. Biophys. Res. Commun. 444 (2014) 455–460. [3] K. Phiwpan, X. Zhou, MicroRNAs in regulatory T cells, Cancer Lett. 423 (2018) 80–85. [4] C. Croce, Causes and consequences of miRNA dysregulation in cancer, Nat. Rev. Genet. 10 (2009) 704–714. [5] H. Yuan, D. Mischoulon, M. Fava, M. Otto, Circulating microRNAs as biomarkers for depression: many candidates, few finalists, J. Affect. Disord. 233 (2018) 68–78. [6] J. Chan, A. Krichevsky, K. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells, Cancer Res. 65 (2005) 6029–6033. [7] I. Negoi, S. Hostiuc, M. Sartelli, R. Negoi, M. Beuran, MicroRNA-21 as a prognostic biomarker in patients with pancreatic cancer–a systematic review and metal-analysis, Am. J. Surgery 215 (2017) 515–524. [8] H. Yang, A. Hui, G. Pampalakis, L. Soleymani, F. Liu, E. Sargent, S. Kelley, Direct, electronic microRNA detection for the rapid determination of differential expression profiles, Angew. Chem. Int. Ed. 48 (2009) 8461–8464. [9] L. Tian, K. Qian, J. Qi, Q. Liu, C. Yao, W. Song, Y. Wang, Gold nanoparticles superlattices assembly for electrochemical biosensor detection of microRNA-21, Biosens. Bioelectron. 99 (2018) 564–570. [10] H. Yin, B. Li, Y. Zhou, H. Wang, M. Wang, S. Ai, Signal-on fluorescence biosensor for microRNA-21 detection based on DNA strand displacement reaction and Mg2+dependent DNAzyme cleavage, Biosens. Bioelectron. 96 (2017) 106–112. [11] W. Peng, Q. Zhao, J. Piao, M. Zhao, Y. Huang, B. Zhang, W. Gao, D. Zhou, G. Shu, X. Gong, J. Chang, Ultra-sensitive detection of microRNA-21 based on duplexspecific nuclease-assisted target recycling and horseradish peroxidase cascading signal amplification, Sens. Actuators B Chem. 263 (2018) 289–297. [12] J. Zhang, W. Zhang, Y. Gu, Enzyme-free isothermal target-recycled amplification combined with PAGE for direct detection of microRNA-21, Anal. Biochem. 550 (2018) 117–122. [13] X. Pei, B. Zhang, J. Tang, B. Liu, W. Lai, D. Tang, Sandwich-type immunosensors and immunoassays exploiting nanostructure labels: a review, Anal. Chim. Acta 758 (2013) 1–18. [14] J. Shu, D. Tang, Current advantages in quantum-dots-based photoelectrochemical immunoassays, Chem. Asian J. 12 (2017) 2780–2789. [15] H. Zhang, R. Yao, N. Wang, R. Liang, W. Qin, Soluble molecularly imprinted polymer-based potentiometric sensor for determination of biophenol AF, Anal. Chem. 90 (2018) 657–662. [16] T. Kajisa, Y. Yanagimoto, A. Saito, T. Sakata, Biocompatible poly(catecholamine)film electrode for potentiometric cell sensing, ACS Sens. 2 (2018) 476–483. [17] S. Jakhar, C. Pundir, Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor, Biosens. Bioelectron. 100 (2018) 242–250. [18] Q. Li, S. Lv, M. Lu, Z. Lin, D. Tang, Potentiometric competitive immunoassay for determination of aflatoxin B1 in food by using antibody-labeled gold nanoparticles, Microchim. Acta 183 (2016) 2815–2822. [19] S. Lv, Z. Lin, K. Zhang, M. Lu, D. Tang, Polyion oligonucleotide-decorated gold nanoparticles with tunable surface charge density for amplified signal output of potentiometric immunosensor, Anal. Chim. Acta 964 (2017) 67–73. [20] Q. Li, F. Zeng, N. Lyu, J. Liang, Highly sensitive and specific electrochemical biosensor for microRNA-21 detection by coupling catalytic hairpin assembly with rolling circle amplification, Analyst 143 (2018) 2304–2309. [21] Y. Zhou, Z. Zhang, Z. Xu, H. Yin, S. Ai, MicroRNA-21 detection based on molecular switching by amperometry, New J. Chem. 36 (2012) 1985–1991. [22] H. Yin, Y. Zhou, H. Zhang, X. Meng, S. Ai, Electochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system, Biosens. Bioelectron. 33 (2012) 247–253. [23] X. Meng, Y. Zhou, Q. Liang, X. Qu, Q. Yang, H. Yin, S. Ai, Electrochemical determination of microRNA-21 based on bio bar code and hemin/G-quadruplet DNAzyme, Analyst 138 (2013) 3409–3415. [24] X. Zhang, D. Wu, Z. Liu, S. Cai, Y. Zhao, M. Chen, Y. Xia, C. Li, J. Zhang, J. Chen, An ultrasensitive label-free electrochemical biosensor for microRNA-21 detection based on a 2’-O-methyl modified DNAzyme and duplex-specific nuclease assisted target cycling, Chem. Commun. 50 (2014) 12375–12377.
3.6. Monitoring of real samples and evaluation of method accuracy As a newly developed detection method, the accuracy of this method was evaluated by assaying the real samples. Initially, the cell lysates were extracted from three tumor cells including human podocyte cell, human tubule epithelial (HK2) cell and HeLa cell with high miRNA-21 expression level. Thereafter, these samples were detected by using CHA-based potentiometric sensor. The high-level miRNA-21 cell lysates were calculated according to the dilution ratio. These results (copies per cell) were compared with those obtained from the referenced RT-PCR method (Fig. 4C). The RSD values between two methods were 3.6–8.2%. In addition, the accuracy of this system was also evaluated by using standard addition method. Three miRNA-21 standards including 0.01, 0.5 and 5.0 nM were initially spiked in the lysis buffer, and then detected by using our strategy. The levels of miRNA-21 in these spiked samples was 0.089, 0.52 and 5.68 nM, respectively. The recoveries were 89–112.6%. These result indicated that this system could be utilized as an optional scheme for detection of target miRNA21 in the complex samples with good accuracy.
4. Conclusions In summary, this study successfully demonstrates a new approach toward the development of advanced potentiometric DNA sensor for the detection of miRNA-21 by combination with catalytic hairpin assembly for the amplification of detectable signal. Although the oligonucleotides before and after formation of double-stranded DNA can cause the change in the potential to some extent, the signal is relatively weak. Introduction of catalytic hairpin assembly with target recycling is used to amplify the electrical signal. Compared with conventional electrochemical detection strategies, e.g., voltammetric or impedimetric assays, CHA-based potentiometric sensing protocol exhibits the following advantages, e.g., label free, simply operation, low cost and short assay time. Nevertheless, only one limitation of our system is that CHA reaction time is relatively long (∼2.5 h). Maybe, it can be improved by controlling the reaction temperature. Therefore, future work should be focus on the improvement of reaction conditions.
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[25] H. Liu, X. Bei, Q. Xia, Y. Fu, S. Zhang, M. Liu, K. Fan, M. Zhang, Y. Yang, Enzymefree electrochemical detection of microRNA-21 using immobilized hairpin probes and a target-triggered hybridization chain reaction amplification strategy, Microchim. Acta 183 (2016) 297–304. [26] M. Ren, S. Wang, C. Cai, C. Chen, X. Chen, A simple and sensitive resonance light
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