Biomimetic Nanochannel-ionchannel Hybrid for Ultrasensitive and

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Biomimetic Nanochannel-ionchannel Hybrid for Ultrasensitive and Label-Free Detection of MicroRNA in Cells Xiao-Ping Zhao, Fei-Fei Liu, Wen-Chao Hu, Muhammad Rizwan Younis, Chen Wang, and Xing-Hua Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05536 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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Analytical Chemistry

Biomimetic Nanochannel-ionchannel Hybrid for Ultrasensitive and Label-Free Detection of MicroRNA in Cells Xiao-Ping Zhao,† Fei-Fei Liu,† Wen-Chao Hu,† Muhammad Rizwan Younis,‡ Chen Wang,*† Xing-Hua Xia*‡

†Key

Laboratory of Drug Quality Control and Pharmacovigilance (China

Pharmaceutical University), Ministry of Education; Key Laboratory of Biomedical Functional Materials, School of Science, China Pharmaceutical University, Nanjing, 211198, China ‡State

Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing 210093, China *To whom correspondence should be addressed. E-mail: [email protected]; [email protected]

ABSTRACT： A biomimetic nanochannel-ionchannel hybrid coupled with electrochemical detector was developed for label-free and ultrasensitive detection of microRNA (miRNA) in cells. Probe single stranded DNA (ssDNA) was first immobilized on the outer surface of the nanochannel-ionchannel hybrid membrane, which can hybridize with the target miRNA in cells. Due to the unique mass transfer property of the hybrid, the DNA-miRNA hybridization kinetics can be sensitively monitored in real-time using the electrochemical technique. More importantly, due to the super small size of the ionchannels, the DNA probe immobilization and hybridization process can be carried out on the outer surface of the ionchannel side, which can effectively avoid the blockage and damage of channels, and thus considerably enhance the reproducibility and accuracy of the method. Using this strategy, the miRNA ranging from 0.1 fM to 0.1 μM can be facilely detected with a low detection limit of 15.4 aM, which is much lower than most reported work. The present strategy provides a sensitive and label-free miRNA detection platform, which will be of great significance in

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biomedical research and clinical diagnosis. Keywords: microRNA, nanochannel-ionchannel hybrid, outer surface, hybridization kinetics, electrochemical detector

INTRODUCTION MicroRNAs (miRNA) play crucial role in the biological processes,1-3 which are directly correlated to many diseases such as heart diseases, human cancers, and diabetes.4 Accordingly, miRNA is considered as a biomarker in the cancer diagnosis and prognosis.5 Developing highly sensitive methods for miRNA detection is of great clinical significance. In recent years, there have been many methods proposed for miRNA detection such as Northern blotting,6 fluorescence method,7 electrochemical assay,8-11 surface plasmon resonance,12,13 and surface-enhanced raman scattering (SERS).14,15 These methods have provided powerful analytical platforms for miRNA detection. As an example, a photoelectrochemical method has been developed for miRNA detection.8 Using Au nanoparticles enhanced resonant energy transfer principle and dual signal amplification, a low miRNA detection limit of 153 fM was achieved. In another report, an electrochemiluminescence assay strategy was used.11 The detection limit for target miRNA-155 could be achieved as low as 36 aM. In those approaches, a large amount of sample is usually required, which is very hard in the cases of some scarce and expensive samples. It still remains challenging to develop miniaturized, sensitive and specific biosensors for miRNA detection. To date, many novel biosensing platforms based on nanopore/channel have been constructed due to the highly specific and sensitive response.16-31 There are two types of fundamentals concerned. The first one is to measure the transient ion current change when single molecules pass through the nanochannel. For example, Long’s group has proposed a type of single nanometer-sized nanopipettes for developing new sensors.16-21 Based on the change of resistance-pulse signals when the target passes through the nanopores, a series of molecules could be successfully detected. In the second principle, special probe molecules are firstly immobilized on the walls of nanochannels. After molecular recognition, the changes of the current-voltage (I-V)


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curve could be monitored, based on which the analyte can be sensitively measured. As an example, Ali et al. presented a selective sensing system based on single synthetic conical nanochannels for the sequence-specific DNA detection.25 The peptide nucleic acid (PNA) was first modified in the nanochannels. Upon PNA/DNA hybridization, the I-V curves changed by the varied surface charge, based on which the specific detection of single-stranded DNA could be easily achieved. These methods open up new avenues in developing highly-sensitive sensors based on nanopore/nanochannel. As a special nanostructure with array nanochannels, porous anodic alumina (PAA) membrane has drawn more and more attraction for constructing nanochannel-based biosensors due to its perfect chemical and mechanical stability. Moreover, the high channel densities in PAA leads to amplified ionic current response by several orders of magnitude compared to single nanochannel, and accordingly could considerably enhance the detection sensitivity.32-40 Presently, using PAA coupling with electrochemical method, a nanofluidic platform was established for label-free detection of DNA and insulin.34 However, up to now most interest has been focused on the usage of the inner nanochannels, and the barrier layer in PAA was usually removed. Recently, the existence of ion channels with super small size in PAA barrier layer has been confirmed.41-44 The novel nanochannel-ionchannel hybrid features highly asymmetric geometry and charge distribution, and accordingly unique ionic current rectification (ICR) could be highly anticipated. It has been proved that significant ICR phenomenon could occur in nanochannels with charged outer surface but neutral inner wall.45 Therefore, minor change in the outer surface charge may result in an obvious ionic current response, making the detection simple, direct and sensitive. In this work, the nanochannel-ionchannel hybrid was fabricated for probe immobilization and target miRNA recognition. First, the probe single stranded DNA (ssDNA) was immobilized on the outer surface of ionchannels through chemical coupling with (3-aminopropyl) trimethoxysilane (APTMS) modified on PAA beforehand. When the target miRNA-10b (miR-10b) was added, miR-10b could be



specially recognized by ssDNA probe through hybridization. Owing to the different charge density on the outer surface as well as varied effective ionchannel size after target miRNA recognition, the mass transport property of the nanochannel-ionchannel hybrid changes accordingly. Using a home-made electrochemical cell (as illustrated in Scheme 1B), the I-V property of the hybrid can be detected in real-time, which enables in situ detection of miRNA with a sensitive and label-free format (Scheme 1C). The as-prepared method could be regenerated by simply immersing the hybrid membrane in RNase H solution, allowing for renewable use for miRNA detection. Using the present method, ultralow detection limit of miR-10b with 15.4 aM can be successfully detected with excellent repeatability and specificity.

Scheme 1. (A) Illustration of surface modification and miRNA detection on the nanochannel-ionchannel hybrid. (B) Schematic diagram of the setup for miRNA detection. (C) I-V properties of nanochannel-ionchannel hybrid under different conditions. The scan rate is 100 mV/s. Pink line: pure hybrid; Red curve: APTMS immobilized hybrid; Green line: probe ssDNA immobilized hybrid; Blue curve: miR-10b hybridized hybrid.

EXPERIMENTAL SECTION


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Materials and Reagents. Potassium hydroxide, Tin (П) chloride (SnCl2), hydrogen peroxide (30% H2O2), phosphoric acid (H3PO4) were from Sinopharm Chemical Reagent

Co.,

Ltd.

(3-aminopropyl)

trimethoxysilane

(APTMS),

1-ethyl-3-

(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC, ≥98%), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC) and N-hydroxy succinimide (NHS, 98%) were from Sigma Aldrich (Shanghai, China). Potassium chloride (KCl), acetone, oxalic acid dehydrate, chromic acid (H2CrO4) were from Shanghai Ling Feng Chemical Reagent Co., Ltd. RNA-free water, RNase H and trypsinization were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Tris (2,2-Bipyridine) ruthenium dichloride (Ru(bpy)3Cl2) was purchased from Meryer Chemical Technology Co., Ltd (Shanghai, China). Total RNA Extractor (Trizol), and diethylpyrocarbonate-treated water (DEPC water), were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Corning Co. (Manassas). DMEM culture medium was from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). The HeLa cell (human cervical cancer cells) was obtained from Shanghai Institutes for Biological Sciences (China). Silica gel films were from Shanghai Zhang's silicone rubber products Co., Ltd. In all the above experiments, deionized water with a resistivity of 18.2 MΩ/cm was used. All oligonucleotides as follow were synthesized and purified by Sangon Biotech Co. (Shanghai, China). Single straned DNA (ssDNA): 5’-COOH-CACAAATTCGGTTCTACAGGGTA-3’; miR-10b:5’-UACCCUGUAGAACCGAAUUUGUG-3’ miRNA-21(miR-21):5’-UAGCUUAUCAGACUGUGUUGA-3’ miRNA-141(miR-141):5’-UAACACUGUCUGGUAAAGAUGG-3’ miRNA-122 (miR-122):5’-UGGAGUUGACAAUGGUGUUUG-3’ Instrumentation. The fabricated nanochannel-ionchannel hybrid was characterized by a scanning electron microscope (SEM, S-4800, Japan). The immobilization of



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APTMS and ssDNA was characterized using an X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Fisher Scientific, USA) and Fourier-transform infrared (FTIR) spectroscopy (Nicolet 6700 model 912A0637). The electrochemical experiments were conducted in 1 mM KCl solution using an electrochemical workstation (CHI660E, Chenhua, China). The anode and cathode are two Ag/AgCl electrodes. Cell Culture. HeLa cells were cultured in culture-flasks with DMEM medium respectively containing penicillin (100 μgmL-1), streptomycin (100 μgmL-1), and 10% FBS at 37 °C under 5% CO2 in the cell incubator. The cells were resuspended after trypsinization. Total RNA Extraction from Cell Lyases. HeLa cells were extracted using the Trizol Reagent.46 Before extraction, the cells were processed with cell counting. Firstly, the cells were collected and lysed using Trizol reagent, followed by the addition of chloroform and centrifugation. Finally, the miRNA extracts were separated and washed with isopropyl alcohol and then diluted. The extracted miRNA solution at -80 °C before use. Fabrication of Nanochannel-Ionchannel Hybrid. PAA was fabricated using electrochemical anodizationmethod.44 Briefly, a high purity aluminum (Al) substrate was first degreased in acetone, then rinsed with water and etched in 1.0 M KOH. The first anodization process was conducted in 0.3 M oxalic acid at 50 V voltage for 0.5 h. After the first anodization, the formed irregular oxide layer needs to be removed using a mixture solution of phosphoric acid (5 wt.%) and chromic acid (1.8 wt.%) at 60 °C for 40 min. Subsequently, the second anodization was conducted under the same condition as the first anodization. The aluminum substrate on the formed hybrid was removed by immersing in saturated SnCl2 solution. Finally, the fabricated PAA was then put into boiled hydrogen peroxide (30% H2O2) at 100 °C for 30 minutes to generate abundant -OH groups on the channel surface. Surface

Modification

of

Nanochannel-Ionchannel

Hybrid.

For

grafting

amino-groups on PAA, 2 mL APTMS (1% in acetone) was dropped on the ionchanel side of PAA at 4 °C for 12 h. Then the PAA was washed using acetone and deionized


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water, baked at 120 °C for 1 h for better crosslink. After that, the ionchannel side was added with 2 mL mixed ssDNA solutions (1 μM ssDNA, 0.9 mg/mL EDC and 0.8 mg/mL NHS) at 4 °C overnight. The PAA hybrid was then washed with distilled water and incubated with 1 mg/mL BSA solution for 1 h to prevent possible nonspecific adsorption. Each modification step was followed by complete rinsing by deionized water. For fluorescence labeling of PAA, the APTMS modified outer surface of PAA membrane was treated by FITC (20 µgmL-1) in 100 mM phosphate buffer saline (PBS buffer, pH 7.4) for 1 h. The unbounded dye was washed off by PBS buffer. Then the prepared membrane was characterized by the laser scanning confocal microscopy (LSCM). Eletrochemical Measurement. The I-V measurement was performed using CHI 660E electrochemical workstation. Briefly, the PAA hybrid clamped between two silica gel slabs was placed between two half cells (2 mL). To achieve the miRNA detection, 1800 mL aqueous electrolyte (1 mM KCl) was filled in both halves of the cell. Then, different concentrations of miR-10b (20 μL) solutions (prepared by DEPC water) were added on the outer surface of the ionchannel side. The final concentration of miRNA in the cell is 0.1 μM-0.1 fM, which is determined by follow equation: 20 𝐶= × 𝐶0 1800 + V where 𝐶 was the final concentration of miRNA, V was the total volume of added miRNA, 𝐶0 was the concentration of added miRNA (10 μM-10 fM). Finally, the miRNA was incubated for 20 min at room temperature. The I-V curves of the hybrid were measured using two Ag/AgCl electrodes inserting into each half-cell solution.

RESULTS AND DISCUSSION Characterization of miR-10b Functionalized Hybrid. SEM images were used to characterize the morphology of the fabricated nanochannel-ionchannel hybrid. It can be observed that there are many hexagons with the size of about 100 nm in the barrier layer (Figure 1A). The thickness of barrier layer of PAA is about ~70 nm (Figure S1). To estimate the size of the ionchannel, a fluorescent probe molecule of Ru(bpy)3Cl2



with size of 1.3 nm was tested on the present hybrid device. Firstly, Ru(bpy)3Cl2 was added in the nanochannel side. After the transmembrane potential (2 V) was applied for 60 min, the fluorescence of electrolyte in the ionchannel side was measured (Figure S2). The result indicates that there are no Ru(bpy)3Cl2 molecules that transfer from the nanochannel side to ionchannel side due to size exclusion effect. Thus the diameter of ionchannel could be estimated to less than 1.3 nm. The diameter of nanochannel is about 50 nm (Figure 1B). Regular cylinder array nanochannels which are parallel to each other could be clearly seen in Figure 1C. The thickness of the whole membrane is estimated to be about 50 μm from Figure 1D. To show the presence of APTMS and probe ssDNA in the hybrid, XPS spectra was used. An obvious peak of Si 2p in APTMS (red curve in Figure 1E) exhibits the successful incorporation of APTMS within hybrid. However, no Si 2p peak is observed in the case of the pure hybrid (black curve). Similarly, no P 2p peak (black curve in Figure 1F) is observed in pure hybrid, while a clear signal of P 2p peak (red curve) occurs for ssDNA modified hybrid, which is attributed to the P element in the probe ssDNA. The whole XPS spectra for the pure and modified hybrid are shown in Figure S3 in the Supporting Information. In addition, in order to prove the carboxylic modified ssDNA covalently coupled with the amine-groups APTMS, FTIR was further used. By using pure hybrid as the reference, the absorption of amide I and amide II vibrations can be monitored (Figure 1G). As seen, the amide I band (1630 cm-1) corresponding to the C=O stretching vibration of the peptide linkage is observed. The amide II band (1523 cm-1) is due to the C-N stretching. These results demonstrated the efficient immobilization of APTMS and ssDNA on the ionchannel side has been achieved. To verify the modification only occurs on PAA outer surface, the fluorescein isothiocyanate (FITC) was used to be bounded selectively with APTMS-modifed PAA. As shown in Figure S4, it is clear that only the barrier layer of PAA membrane exhibits bright green fluorescence, demonstrating the precisely modification of FITC on the outer surface of the PAA membrane.


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Figure 1. (A-D): SEM images of the as-prepared nanochannel-ionchannel hybrid (A: top, B: bottom, C: cross section, D: the whole cross section). (E) XPS spectra of the pure hybrid (black curve) and APTMS modified hybrid (red curve). (F) XPS spectra of pure hybrid (black curve) and ssDNA modified hybrid (red curve). (G) FTIR spectrum of ssDNA-modified hybrid.

In addition to above characterizations, the surface modification process of nanochannel-ionchannel hybrid was also confirmed by measuring the transmembrane ionic current response using 1 mM KCl (pH 7.4) in the two halves of cells. The I-V curves of hybrid before and after modification were recorded in Figure 2. After the nanochannel-ionchannel hybrid is prepared by two-step anodic oxidation, ionized hydroxyl groups (-O-) exposes on the PAA surface, offering a negatively charged surface at pH 7.4.41 The I-V curve of the bare hybrid membrane is indicated as the black curve in Figure 2A. It was seen that the non-linear I-V curve was observed under different potential bias on the hybrid. This asymmetric mass transfer behavior is caused by the super-asymmetric geometry of the nanochannel-ionchannel hybrid,



which was called as the ionic current rectification (ICR).24 When the ionchannel side was covalently coupled with positively charged APTMS, the surface charge on the outer surface of ionchannel changes from negative to positive, and the I-V curve with reversed rectification direction could be expected (red curve in Figure 2A). The change in the I-V properties during the modification process indicates that APTMS has been successfully modified onto the surface of the ionchannel side. Afterwards, probe ssDNA with carboxyl group was easily bound with APTMS on the ionchannel side by using a mixture solution of EDC and NHS, through which ssDNA was immobilized on the ionchannel side. Owing to the negatively charged phosphate backbone of ssDNA, the outer surface of ionchannel was negatively charged again. Therefore, the direction of ICR upon introduction of ssDNA recovered to the same as the bare hybrid (blue curve in Figure 2A). Then BSA was used to block the unmodified sites on PAA. The negatively charged BSA increases the surface charge density, resulting in a more obvious ICR (pink curve in Figure 2A). In the presence of miR-10b, miR-10b was recognized by ssDNA probe, and the miRNA-ssDNA hybridization occurs. The negatively charge density increased dramatically, leading to a remarkable ICR property (green curve in Figure 2A). To make sure the charge change after miRNA-ssDNA hybridization, the zeta potential was determined by Malver Zetasizer. As shown in Figure S5 in the revised version, the measured zeta potentials of ssDNA is -12.3 mV, after hybridization with miR-10b, the zeta potentials changed to -33.6 mV, indicating an increased negative charge density after miRNA-ssDNA hybridization. Thus the cations flow from the outer surface to the inner channels preferentially, resulting in an increased change in ICR upon miRNA-ssDNA hybridization. The rectification ratios (fR) is a parameter that reflects the extent of permselectivity of the asymmetrical nanochannels.25 It is directly related to the surface charge. Based on the data in Figure 2A, the rectification ratio fR of the nanochannel-ionchannel hybrid could be calculated. The changed rectification ratios triggered by different surface charge property were clearly shown in Figure 2B. The relative rectification ratio for miR-10b recognized hybrid was much larger than that of ssDNA and BSA modified hybrid. All the above results prove the successful surface


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modification on PAA surface.

Figure 2. (A) I-V curves of the pure hybrid (black curve) and different modification process (APTMS: red curve, ssDNA: blue curve, BSA: pink curve, miR-10b: green curve). (B) The rectification ratio before and after modifications of APTMS, ssDNA, BSA and miR-10b.

Optimization of Hybridization Time. In this miRNA sensing system, the effect of hybridization time was investigated. In order to achieve the optimum hybridization time, 0.1 μM of miR-10b solution was dropped onto the outer surface of the ionchannel side of the hybrid and hybridized for different times (10, 15, 20, 25, 30, 40, 50, 60, 70 min) at room temperature. The I-V properties after hybridizing with the target miR-10b were investigated (Figure 3A). It is found that the I-V characteristics increased continuously during the first 20 min, then decreased after that. The inset shows the enlarged part of the overlapping curves in Figure 3A (from + 0.8 V to + 1.0 V). To clearly show the change of the ionic current responses after hybridizing with miR-10b, the current values at + 1.0 V were highlighted in Figure 3B. It is clear that the current reaches the maximum at 20 min. After this period time, the current response decreases obviously. This phenomenon was also found in other previous study.47,48 It is concluded that this decreased response is possibly caused by the degradation by present RNases. The results suggest the hybridization reaction could be completed within 20 minutes on the nanochannel-ionchannel hybrid. Therefore 20 minutes of hybridization time was chosen in the following work.



Figure 3. (A) The hybridization kinetics of ssDNA-(miR-10b). The concentration of miR-10b was 0.1 μM. The inset shows the enlarged part of the curves from +0.8 V to +1.0 V. (B) The current values +1.0 V (from Panel A) versus the hybridization time.

Detection of miR-10b. To investigate the applicability of the proposed method for miR-10b detection, miR-10b solutions with varied concentrations from 0.1 fM to 0.1 μM were tested with a 1mM KCl electrolyte solution. Using the electrochemical linear sweep voltammetry technique, the I-V characteristics under different miR-10b concentrations were achieved, and the results were shown in Figure 4A. It can be seen that the current (measured at +1.0 V) increases with the miR-10b concentrations from 0.1 fM to 0.1 μM, which is owing to the increased negative charges by recognized miR-10b on the outer surface of the ionchannel side. In addition, it has been reported that the electrochemically confined effect in nanofluidics may also contribute to the changed current response.18,20 The linear curve was established by plotting Log I (I was the current response of miR-10b recognized hybrid at different concentrations at +1.0 V) against Log C (C was the concentration of miR-10b), as shown in Figure 4B. The calibration equation was obtained as Log I = -6.472+ 0.0535 Log C with a correlation coefficient R of 0.998. The linear range was 0.1 fM to 0.1 μM with a detection limit of 15.4 aM at 3 SD/N (SD is the standard deviation of the blank, N is the slope of the calibration curve). The various current sensors for miRNA detecting were illustrated in Table S1 in the supporting information. Compared with other miRNA detection methods, the present method could reach a wider linear rang and lower detection limit without introducing signal amplification strategy. Moreover, the


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rectification ratio exhibits a dramatic increase from 6.4 to around 27.2 with the increase of miR-10b concentration (inset in Figure 4B), which also indicates that the surface charge on the outer surface increases gradually with the miR-10b concentration. The results imply that any minor change in the surface charge may lead to a significant current response change owing to highly asymmetric geometry and charge distribution of this hybrid. In this case, the detection limit of miR-10b can be considerably improved by utilizing this hybrid device.

Figure 4. (A) I-V curves of the ssDNA modified nanochannel-ionchannel hybrid after incubation with different concentrations of miR-10b (from 10-10 μM to 0.1 μM). (B) The calibration curve of Log I-Log C, I was the current of the ssDNA-modified device hybridizing with different concentrations of miR-10b at +1.0 V, C was the miR-10b concentration. The inset was the rectification ratio after incubation with different concentrations of miR-10b.

Selectivity and Reversibility of miR-10b Detection. It is well known that the miRNA can direct hybridize with their fully complementary ssDNA sequence compared to non-complementary ones.49 Thus the ssDNA was used for special binding with miR-10b. To investigate the selectivity of the constructed nanochannel-ionchannel hybrid device, several different miRNAs were employed including miR-21, miR-122, and miR-141. The concentration of other miRNAs was set as 1 μM, while miR-10b was 0.1 μM. As displayed in Figure 5A, there is almost no significant change in the current response after the ssDNA-modified nanochannel-ionchannel hybrid was treated by the above-mentioned miRNAs. In comparison, the significant change was observed after the ssDNA-modified



nanochannel-ionchannel hybrid were treated by 0.1 μM miR-10b. When their mixtures (miR-10b/miR-21, miR-10b/miR-122, and miR-10b/miR-141) were tested for the same experiments, the similar change trend as the one of miR-10b occurs, confirming the miR-10b is the only miRNA hybridization with ssDNA. The current values at + 1.0 V in Figure 5A was displayed in Figure 5B. Only in the cases of the target miR-10b and its mixtures, the significant current change could be detected and the responses of miR-21, miR-122, and miR-141 were close to the blank signal. The result demonstrates the high selectivity of the as-prepared biosensor toward miR-10b.

Figure 5. (A) I-V curves of ssDNA-modified hybrid by treating with different miRNAs (0.1 μM miR-10b, 1 μM miR-21, 1 μM miR-122, 1 μM miR-141, 0.1 μM miR-10b/miR-21, 0.1 μM miR-10b/miR-122, 0.1 μM miR-10b/miR-141) for 20 min at room temperature, respectively. (B) The current at +1.0 V values of the functional nanodevice from Panel A. (C) Characterization of the nanodevices regeneration ability. (D) Changes in current value at +1.0 V of ssDNA-modified hybrid device upon hybridization and dehybridization with miR-10b for several cycles.


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In addition to the properties mentioned above, the regeneration ability of the ssDNA-modified nanochannel-ionchannel hybrid biosensor was also investigated. Three successive experiments were repeated on the same nanochannel-ionchannel hybrid device by adding RNase H solution on the ionchannel side for 2 h for dehybridization.49 Afterwards the same concentration of miR-10b was added for rehybridization. By repeating the experimental process, dehybridization and rehybridization were performed successively. The result was shown in Figure 5C. It is clear that when dehybridization was carried out, the current decreased from 0.32 μA to 0.08 μA. When the hybridization was conducted, the current increased to the similar value as the initial state. Reversible variation of the ionic current of nanochannel-ionchannel hybrid at +1.0 V (Figure 5D) shows that the present nanofluidic hybrid device has a reliable reusability and can be reused for multiple times.

Detection of miR-10b in HeLa Cells. The proposed hybrid device in the clinical application for practical samples was investigated by detecting the miR-10b concentration from HeLa cells. As depicted in Figure 6A and B, it was found that the relative ion current change increases generally with the increased concentrations of HeLa cell from 104-108 cells mL-1. Therefore, the different expression level of miR-10b in different cell concentrations could be obviously reflected by monitoring the change in the ionic current. The results agree well with the previous report,50 which also indicates similar miR-10b expression level in this cell lines. The experimental results demonstrate that the constructed nanochannel-ionchannel hybrid still works well in the real cell samples, showing a key potential in the application of clinical diagnostics and biomedical research.



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Figure 6. Detection of miR-10b in HeLa cells: (A) the I-V curves of the ssDNA modified nanochannel-ionchannel hybrid for different concentrations of HeLa cells from104-108 cells mL-1; (B) the current value at +1.0 V from Panel A versus Log c (c was the concentration of HeLa cells).

CONCLUSION In summary, we have developed a novel nanochannel-ionchannel hybrid device for label-free and ultrasensitive detection of miR-10b. After ssDNA was covalently immobilized on the outer surface of the ionchannel side of hybrid, the hybridization between ssDNA and miR-10b took place. The changed surface charge on the outer surface of ionchnnel side results in varied mass transfer properties. Using the electrochemical

technique,

the

ionic

current

response

through

the

nanochannel-ionchannel hybrid could be monitored in real-time, thus allows in-situ and label-free detection of miR-10b.The results show that the present nanofluidic biosensor have highly specificity and sensitivity towards miR-10b detection with the low detection limit of 15.4 aM. Moreover, it was found that the present biosensor can be regenerated through several cycles by treating with RNase H. The practicality of our present method was validated by the analyses of real cell samples, which offered a promising way for biomedical study and early clinical diagnosis.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]


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ORCID Chen Wang: 0000-0001-6544-4065 Xing-Hua Xia: 0000-0001-9831-4048 Author Contributions Xiao-Ping Zhao and Fei-Fei Liu contributed equally. All authors have given approval to the final version of the manuscript Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (2017YFA0206500) and the National Natural Science Foundation of China (21874155, 21635004, 21575163). Supporting Information Supporting Information Available: Supporting Information is available free of charge from the Analytical Chemistry home page (http://pubs.acs.org/journal/ancham). Supplementary Figure S1, SEM image of cross section of PAA; Supplementary Figure S2, fluorescence emission spectra of samples in ionchannel side and nanochannel side; Supplementary Figure S3, the whole XPS spectra for the modifed and unmodifed PAA; Supplementary Figure S4, the LSCM cross-sectional image of the APTMS-modified PAA; Supplementary Figure S5, the zeta potential of ssDNA and miRNA-ssDNA complex; Supplementary Table S1, comparison of the performance of various sensing systems for the detection of miRNAs.

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