Ultrasensitive and Facile Detection of MicroRNA via a Portable

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Ultrasensitive and Facile Detection of MicroRNA via a Portable Pressure Meter Lu Shi,† Jing Lei,† Bei Zhang,† Baoxin Li,† Chaoyong James Yang,‡ and Yan Jin*,† †

Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710119, China ‡ State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China S Supporting Information *

ABSTRACT: The upregulation of microRNA (miRNA) is highly related with some kinds of tumor, such as breast, prostate, lung, and pancreatic cancers. Therefore, for an important tumor biomarker, the point-of-care testing (POCT) of miRNA is of significant importance and is in great demand for disease diagnosis and clinical prognoses. Herein, a POCT assay for miRNA detection was developed via a portable pressure meter. Two hairpin DNA probes, H1 and H2, were ingeniously designed and functionalized with magnetic beads (MBs) and platinum nanoparticles (PtNPs), respectively, to form MBs-H1 and PtNPs-H2 complexes. In the presence of target microRNA 21 (miR-21), the cyclic strand displacement reaction (SDR) between MBs-H1 and PtNPs-H2 was triggered to continuously form the MBs-H1/PtNPs-H2 duplex. Owing to the amplification of cyclic SDR, numerous PtNPs were enriched onto the surface of MBs to catalytically decompose H2O2 for the generation of much O2. The gas pressure value has a linear relationship with the logarithmic value of miR-21 concentration in the range of 10 fM to 10 pM. The limit of detection is 7.6 fM, which is more sensitive than that in a number of previous reports. Hairpin DNA probes and magnetic separation highly ensured the specificity and reliability. Single-base mutation was easily discriminated, and the detection of miR-21 in the serum sample achieved satisfactory result. Therefore, it offers a reliable POCT strategy for the detection of miRNA, which is of great theoretical and practical importance for POCT clinical diagnostics. KEYWORDS: point-of-care testing, microRNA, portable pressure meter, gas pressure, strand displacement reaction

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INTRODUCTION MicroRNAs (miRNAs) are single-stranded, small noncoding endogenous genes (18−22 nt) that play crucial roles in mediating many important biological processes.1,2 Increasing evidence has indicated that miRNA dysregulation and expression are directly correlated with various diseases, especially human cancers, heart diseases, and diabetes.3 Therefore, miRNAs are regarded as significant biomarkers for cancer diagnosis and prognosis.4,5 There are many malignancies that show high microRNA 21 (miR-21) expression levels, such as breast cancer, pancreatic cancer, brain tumor, leukemia, colorectal cancer, and so on.6 Thus, effective detection of miR-21 is important for biomedical research and clinical diagnosis as well as therapeutic targets in cancer treatment. In the past decades, many approaches have been developed for miRNA detection, including Northern blotting,7,8 quantitative reverse transcription polymerase chain reaction,9,10 oligonucleotide microarrays,11 electrochemical methods,12,13 fluorescence methods,14 nanoparticle amplification methods, and so on.15,16 These methods provide useful analytical platforms for miRNA detection. However, these strategies have some limitations, such © XXXX American Chemical Society

as requirement of high-precision thermal cycling equipment, complex operation, especially expensive and cumbersome instruments, which limited their application at home. Thus, it is highly necessary to develop an ultrasensitive, facile, costeffective, and portable method for the miRNA assay. Owing to the demand for clinical application, researchers aimed at exploring feasible and reliable point-of-care testing (POCT) methods. Jiang et al. developed the POCT strategy for the detection of miRNA using a personal glucometer.17 In addition, Li et al. recently developed a new POCT method for miRNA detection using a pH test paper.18 The pH-response strategy is easy and friendly, but it cannot realize the qualitative detection without UV−vis spectrometry, which is similar to the limitation of the colorimetric detection of miRNA.19,20 Despite some progress, the POCT of miRNA is still rare. It is in great demand to develop a more facile, highly sensitive, and cost-effective POCT method for quantitative detection. Received: February 13, 2018 Accepted: March 27, 2018

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DOI: 10.1021/acsami.8b02551 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces Table 1. Sequences of the Oligonucleotides Used in the Experiments name

sequence

DNA H1 DNA H2 MiRNA-21 MiRNA-210 MiRNA-214 SM miR-21 TM miR-21

biotin-5′-TTTTTTTTTTTCAACATCAGTCTGATAAGCTACCATGTGTAGATAGCTTATCAGACT-3′ SH-5′-TTTTTTTTTTTAAGCTATCTACACATGGTAGCTTATCAGACTCCATGTGTAGA-3′ 5′-UAG CUU AUC AGA CUG AUG UUG A-3′ 5′-CUG UGC GUG UGA CAG CGG CUG A-3′ 5′-ACA GCA GGC ACA GAC AGG CAG U-3′ 5′-UAG CUU AUA AGA CUG AUG UUG A-3′ 5′-UAG CUU AUA ACC CUG AUG UUG A-3′

Scheme 1. Schematic Illustration of Gas Pressure-Based POCT Assay for Sensitive Detection of MicroRNA

Healthcare Lifesciences. RNA-free water and RNAase inhibitor were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Total RNA Extractor (Trizol), diethylpyrocarbonate-treated water, and citrate buffered saline (SSC) buffer (20×) solution were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Healthy human serum was donated by an affiliated hospital of Shaanxi Normal University. All metal salts were purchased from Xi’an chemical reagent Co., Ltd. (Xi’an, China). All other reagents were of analytical reagent grade. Deionized water obtained from a Millipore filtration system was used all throughout the experiment. Apparatus. Gel electrophoresis was performed on a vertical electrophoresis system (Bio-Rad Laboratories Inc.). Transmission electron microscopy (TEM) images were recorded using Tecnai G2 F20 Field Transmittance Electron Microscopy (FEI, Japan). The pressure meter was purchased from PASSTECH (Xiamen, China). The Molecular Imager system was purchased from Shanghai Peiqing Science & Technology Co. Ltd. (Shanghai, China). Synthesis of Platinum Nanoparticles (PtNPs). PtNPs were prepared according to the published literature.26 Initially, a 12.3 μL aliquot of 2.56 M H2PtCl6 was added to 14.1 mL of an aqueous solution containing 2.68 mM sodium citrate under vigorous stirring. Afterward, 1.75 mL of freshly prepared sodium borohydride (NaBH4) (7.3 mg) solution was added dropwise, and the solution was kept stirring for another 30 min. Ultimately, the synthesized PtNPs were centrifuged with 12 000 rpm to remove redundant electrolyte and stored at 4 °C before use. Oligonucleotide Functionalization of PtNPs. The aforementioned PtNPs and thiol-labeled H2 were incubated at a PtNPs-H2 concentration ratio of 1:10 for 24 h. The PtNPs-H2 was obtained by further centrifugation, washed with SSC buffer, and stored at 4 °C for further use.

Herein, we develop a facile and reliable POCT strategy for ultrasensitive detection of miRNAs by using a cheap and portable gas pressure meter as readout. Gas pressure monitoring is very common in daily life. However, the application of gas pressure measurements in bioanalysis and biosensing is rare.21−25 Yang et al. have creatively developed an immunoassay for highly sensitive detection of biomarkers by using a portable pressure meter.21 Our group previously developed a facile and reliable POCT assay for the detection of telomerase activity based on change in gas pressure.24 The digital pressure meter makes it easy to achieve facile, rapid, and reproducible detection. To further improve analytical performance and explore more extensive application, signal amplification technique was introduced to achieve more diverse biosensing. In this assay, strand displacement reaction (SDR) was utilized to enrich the catalyst of H2O2 decomposition reaction. In the presence of the target miR-21, the SDR between magnetic beads (MBs)-H1 and platinum nanoparticles (PtNPs)H2 was triggered to the cyclic form MBs-H1/PtNPs-H2 complex. Owing to the catalytic decomposition of H2O2 by PtNPs, miR-21 can be quantitatively and sensitively detected by a portable pressure meter. To our knowledge, this is the first time that the pressure meter was used for ultrasensitive detection of miRNA.

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EXPERIMENTAL SECTION

Materials and Reagents. All oligonucleotides listed in Table 1 were synthesized and purified by Sangon Biotech Co. (Shanghai, China). Chloroplatinic acid (H2PtCl6) was obtained from Aladdin (Shanghai, China). Streptavidin magnetic beads (MBs) were purchased from Ge B


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ACS Applied Materials & Interfaces

Figure 1. (a) Gas pressure change under different conditions. The concentrations of miR-21, DNA H1, DNA H2, and MBs are 10 pM, 100 nM, 100 nM, and 0.5 mg mL−1, respectively. (b) PAGE analysis. Cell Culture. The MCF-7, HepG2, and A549 cells were cultured in Dulbecco’s modified Eagle’s medium medium, and the HL-7702 cells were cultured in 1640 medium supplemented with 10% (v/v) fetal bovine serum and 100 U mL−1 of penicillin−streptomycin. Then, the cells were maintained at 37 °C under a humidified atmosphere (95% air and 5% CO2). The cells were then collected and resuspended after trypsinization and washing with fresh medium. Total RNA Extraction from Cell Lyases. The cellular extracts were prepared by using the Trizol Reagent from Sangon Biotech Co. (Shanghai, China) according to the manufacturer’s protocol. We selected human lung adenocarcinoma cells (A549), human breast adenocarcinoma cells (MCF-7), and human hepatocellular liver carcinoma cells (HepG2) as the miRNA expression level. Normal human liver cells (HL-7702) were chosen as the control. The tumor cell lines were processed with cell counting before extraction. Briefly, cells were collected and lysed with an appropriate amount of Trizol reagent, followed by the addition of chloroform and centrifugation. The RNA extracts in the aqueous phase were separated, followed by the addition of isopropyl alcohol and washed with ethyl alcohol. Finally, the extracted RNA solution was diluted and stored at −80 °C for further use. Polyacrylamide Gel Electrophoresis (PAGE). The samples for gel electrophoresis assays were prepared as follows: (1) miR-21 was used as sample 1; (2) DNA H1 was used as sample 2; (3) DNA H2 was used as sample 3; (4) the mixture of DNA H1 and miR-21 was incubated and used as sample 4; (5) the mixture of DNA H1 and DNA H2 was incubated and used as sample 5; (6) the mixture of DNA H1 and miR-21 was incubated and used as sample 6; (7) the mixture of miR-21, DNA H1, and DNA H2 was incubated and used as sample 7. All samples were prepared at 30 °C for 40 min. A 12.5% native polyacrylamide gel loaded with 2 μL samples was run for 30 min at 200 V in 1× Tris−borate− ethylenediaminetetraacetic acid. Then, the gels were visualized with Ag staining and imaged with a molecular imaging system. Detection of Gas Pressure. The gas pressure was measured by a portable gas pressure meter. Gas pressure value is digitally displayed. First, the MBs were magnetically separated and washed three times. Second, MBs were resuspended into 100 μL of 30% H2O2 solution. Then, the wells of a 96-well plate were sealed by a rubber seal immediately. Finally, the needle of the digital pressure meter was inserted into the rubber-sealed wells of a 96-well plate one by one. The value of the gas pressure was directly read within 3 s. The gas pressure was read immediately.

H1) via streptavidin and biotin interaction. The 5′-thiolated DNA H2 was covalently coupled to the surface of PtNPs via Pt− S self-assembly to form the PtNPs-H2 complex. In the presence of target miR-21, the SDR reaction between MBs-H1 and PtNPsH2 will be triggered. First, the miR-21 unfolds DNA H1 by matching with the stem of DNA H1 (green portion). Then, the exposed portion of DNA H1 (blue and red portion) will hybridize with PtNPs-H2. The long and stable H1/H2 duplex displaced miR-21 to open another DNA H1. Owing to the targettriggered cyclic SDR, a number of H1/PtNPs-H2 complexes continuously formed on the surface of MBs. Then, the MBs were magnetically separated and washed three times with SSC buffer to remove free PtNPs. Finally, the PtNP-enriched MBs were incubated with 100 μL of 30% H2O2 solution to decompose H2O2, leading to the generation of much of oxygen. The gas pressure was facilely and directly detected by inserting the needle of a digital pressure meter into the rubber-sealed wells of a 96well plate. Therefore, the concentration of miRNA could be sensitively and portably detected by monitoring the change of gas pressure. To check the feasibility and reliability of the proposed assay, miR-21 was selected as the model target. The gas pressure, as we had expected, has reached ∼123.8 kPa when 10 pM miR-21 was coincubated with MBs-H1 and PtNPs-H2 (Figure 1a). That is, it is possible to utilize this POCT strategy for miR-21 detection. To further test the feasibility and reliability, several negative control experiments were performed. In general, H2O2 very slowly decomposes into water and oxygen. First, the self-decomposition of H2O2 was studied to avoid false-positive error. As shown in Figure 1a, under the current experimental conditions, the selfdecomposition of H2O2 is very weak. The gas pressure maintained around 7 kPa. Thus, the H2O2 itself generates such negligible O2 that it can be deducted as low background signal. Second, the influence of SSC buffer on the decomposition of H2O2 was studied. The gas pressure of 30% H2O2 in the 2× SSC buffer is ∼7 kPa, which is similar to the gas pressure that was generated by the self-decomposition of H2O2. It indicated that the SSC buffer has no influence on H2O2 decomposition. Third, the influence of MBs on the output of gas pressure was studied because magnetic nanoparticles possess some functions of mimic peroxidases according to the previous report.27 It will cause falsepositive result if MBs could promote the rate of H2O 2

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RESULTS AND DISCUSSION Proof of Principle. Scheme 1 illustrates the principle of the POCT strategy for detection of miRNA. The 5′ biotinylated DNA H1 was combined with streptavidin-modified MBs (MBsC


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Figure 2. Influences of experimental parameters on analytical performance: (a) effect of the concentration of MBs, (b) effect of reaction time between MBs and DNA H1, (c) effect of the different kinds of buffer, and (d) effect of the concentration of SSC buffer.

Characterization of PtNPs. TEM was employed to analyze surface morphologies and lateral dimensions of the synthesized PtNPs. As displayed in Figures S2a and S2b, the TEM images showed that it has a satisfactory dispersion and the size of PtNP is around 4 nm. The catalytic decomposition of H2O2 by PtNPs is directly related to the sensitivity of this method. Thus, we tested the catalytic ability of PtNPs. As shown in Figure S2c, as expected, the pressure value gradually increased with increasing concentration of H2O2 and reaction time. Figure S2d shows that there is a good linear relationship between the value of gas pressure and the concentration of the PtNPs. All these results indicated that the catalytic ability of PtNPs is optimal. Assessment of Stability. In this work, the stability of pressure measurements depends on the gas leakage. The wells of the 96-well plate were tightly sealed with rubber seal. To further prevent gas leakage induced by high pressure, the four sides of the 96-well plate were fixed with clamps. Herein, the stability of pressure measurements was investigated. The concentrations of H2O2 and PtNPs were fixed. Then, the pressure was measured by a portable pressure meter after 3 and 6 h. As shown in Table S1, the pressure fell by 3.3% within 3 h, suggesting that the gas leakage has no effect on pressure measurements. Moreover, the reproducibility of pressure measurements was investigated. The H2O2/PtNPs mixture was added to the 48 wells of a 96-well plate. Then, the pressure of each well was measured by a portable pressure meter after 10 min. As shown in Figure S3, the pressure of each well was in the range of 87.8 ± 3.2% kPa, suggesting that the reproducibility of pressure measurements was satisfactory. Therefore, the stability of pressure measurements is sufficient to ensure reliability and accuracy. Optimization of Experimental Conditions. Several parameters could affect the reliability and sensitivity of gas pressure measurement. To achieve optimal outcome, the influence of these parameters was systematically investigated. First, the influence of MBs on the detection of gas pressure was

decomposition. As shown in Figure S1a, it is clear that the gas pressure is very low when 0.5 mg mL−1 MBs are incubated with 100 μL of 30% H2O2 solution. The MBs cannot decompose H2O2 even when prolonging the reaction time. It is suggested that MBs with the concentration of 0.5 mg mL−1 will not cause nonspecific gas pressure output. In addition, the effect of the concentration of MBs on the decomposition of H2O2 was also investigated. As shown in Figure S1b, MB has almost no effect on the decomposition of H2O2 as the concentration of MBs increased to 1.25 mg mL−1. Finally, the influence of selfhybridization between DNA H1 and PtNPs-H2 was studied. It was discovered that the gas pressure is slightly higher than that of 100 μL of 30% H2O2 solution when 100 μL of 30% H2O2 solution was incubated with MBs-H1 and PtNPs-H2. The gas pressure reached ∼16 kPa. The outcome may be attributed to the nonspecific adsorption of PtNPs onto the surface of MBs or nonspecific self-hybridization between DNA H1 and H2. In this work, hairpin DNA probes H1 and H2 were carefully designed to avoid nonspecific self-hybridization. The long and stable stems of DNA H1 and H2 ensure that the SDR only can be triggered by the target miR-21. To verify this, polyacrylamide gel electrophoresis (PAGE) assay was performed. As shown in Figure 1b, only the target miR-21 can trigger the SDR (line 7). As anticipated, DNA H1 did not hybridize with DNA H2 in the absence of miR-21 (line 4), and no new DNA band appeared. Thus, nonspecific self-hybridization has little effect on the output of gas pressure. By optimizing magnetic separation and washing, the gas pressure of 100 μL of 30% H2O2 solution containing MBs-H1 and PtNPs-H2 stayed around 16 kPa, which is the highest background signal. Therefore, the gas pressure of 100 μL of 30% H2O2 solution containing MBs-H1 and PtNPs-H2 was regarded as blank signal all throughout the experiment. On the basis of the above discussion, we concluded that it is a feasible and reliable POCT strategy for the detection of miR-21 via a portable gas pressure meter. D


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Figure 3. (a) Gas pressure of the different concentrations of miR-21. (b) Relation between gas pressure and miR-21 concentration. Inset: linear correlation between gas pressure value and logarithmic of miR-21 concentration. Error bars are standard deviations of three repetitive experiments.

recorded by the portable pressure meter increased obviously when the concentration of miR-21 ranged from 10 fM to 10 pM. As can be seen in the inset of Figure 3b, there is a good linear relationship between the gas pressure signal and the logarithmic value of the miR-21 concentration. The coefficient of determination (R2) was 0.9984, the linear regression equation is P (Pa) = 26 311.9 lg C (pM) + 100 724.2, and the detection limit for miR-21 was calculated to be 7.6 fM (S/N = 3). As shown in Table S2, this POCT assay exhibited higher sensitivity compared to that in some previous reports. Selectivity of the Pressure Sensor for miR-21 Detection. It is very challenging for miRNA assays to discriminate differences between different miRNA members. To investigate the selectivity of this POCT strategy, several different miRNAs were employed, including miR-210, miR-214, single-base mismatched miR-21 (SM miR-21), and three-base mismatched miR-21 (TM miR-21). The concentration of miRNAs was set as 10 pM. As displayed in Figure 4, only in

optimized, including the concentration of MBs and reaction time. As shown in Figures 2a and S4a, the gas pressure increased with the increase of MB concentration and reached a plateau when the concentration of MBs increased to 0.5 mg mL−1. The blank signal slowly increased as the concentration of MBs increased. The higher concentration of MBs may catalyze H2O2 because of the functions of mimic peroxidase. On the contrary, the lower concentration of MBs did not offer enough binding site for DNA H1, which is not conducive to the next SDR. Therefore, 0.5 mg mL−1 was chosen for further investigation. Meanwhile, the reaction time between streptavidin-coated MBs and biotinylated DNA H1 also was explored. As shown in Figures 2b and S4b, the gas pressure value increased to the maximum when the reaction time was 20 min. Thus, the optimal reaction time between MBs and biotinylated DNA H1 was 20 min. Second, the influence of reaction medium on analytical performance was investigated. The reaction buffer affects the stability of the hairpin structure, the efficiency of DNA hybridization, and SDR. Thus, the four most frequently used buffers were investigated to choose the optimal buffer, including Tris−(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer. As shown in Figures 2c and S4c, the optimal buffer is the SSC buffer. Then, the concentration of SSC buffer was optimized by setting the concentrations as 0.5×, 1×, 1.5×, 2×, and 2.5×. As shown in Figures 2d and S4d, when the concentration of SSC buffer is 2×, the gas pressure value reached the highest. Therefore, 2× SSC buffer was used as the optimal buffer all throughout the experiment. Finally, the factors influencing SDR were investigated, such as the ratio of DNA H1 and DNA H2 concentration, the reaction time, reaction temperature, and so on. According to the results in Figure S5a,b, the change in gas pressure reached the maximum when the H1/H2 ratio was 1: 1. Thus, the optimal concentration of DNA H1 and DNA H2 is 100 nM. The efficiency of SDR depended on the reaction time. It was found from Figure S5c,d that the changes in gas pressure rose gradually as the reaction time increased. A plateau was observed when the reaction time was more than 40 min. Therefore, 40 min was designated as the SDR reaction time. Moreover, temperature affects the efficiency of SDR and the stability of the hairpin DNA probes. By monitoring the change in gas pressure, it was discovered that the optimal temperature was 30 °C (Figure S5e,f). Sensitivity of miR-21 Detection. Under optimized conditions, the sensitivity of the detection of miR-21 by this strategy was studied. Figure 3a depicts that the gas pressure

Figure 4. Response of gas pressure for the detection of different miRNAs by the POCT assay. The concentration of miRNAs was 10 pM. Error bars represent the standard deviations for three replicates.

the presence of the target miR-21, significant gas pressure was detected. The responses of miR-210 and miR-214 were close to the blank signal. Compared with miR-21, SM miR-21, and TM miR-21 contain one and three mutation bases, respectively. The pressure outputs of SM miR-21 and TM miR-21 are similar to the blank signal as well. This revealed that single-base mutation was easily discriminated by this POCT strategy. In this work, two E


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Figure 5. Detection of miR-21 in different cell lines: (a) MCF-7 cell, (b) HepG2 cells, (c) A549 cells, and (d) HL-7702 cells. Error bars represent the standard deviations for three replicates.

Detection of miR-21 in Human Serum. To further verify the capability of this strategy, the detection of miRNA in diluted human serum was investigated. The feasibility of the method was confirmed by the standard addition method. Three different concentrations of miR-21 were added into diluted human serum. As demonstrated in Table 2, the recovery rate varied from 97.7 to 102.7%, suggesting that the strategy has a potential prospect for the detection of miRNAs in diluted human serum.

hairpin DNA probes were utilized to realize molecular recognition and signal amplification. Compared to linear DNA probe, hairpin DNA probe has higher specificity due to the unique stem−loop structure. Moreover, magnetic separation greatly reduced the false-positive signal by removing the unreacted PtNPs-H2. Therefore, the selectivity of this POCT assay is excellent, which can be used for reliable and highly selective detection of miRNA. Determination of miR-21 in Different Cells. The capability of the POCT assay for detecting miR-21 in complex biological samples was evaluated. MiR-21 was extracted from three human cancer cell lines, including A549, MCF-7, and HepG2 cells. According to the experimental data in Figure 5a, the gas pressure obviously increased as the number of MCF-7 cells gradually increased from 100 to 1 000 000. The outcome is in accordance with that of a previous report.28 Similar results were obtained when HepG2 and A549 cells (Figure 5b,c) were investigated. Moreover, the expression level of miR-21 in A549 cell is higher than that of MCF-7 and HepG2 cells. Therefore, the difference in the expression level of miR-21 between different tumor cells was obviously reflected by monitoring the change in gas pressure. To further verify the reliability of miR-21 detection in a complex sample, the expression level of miR-21 in human normal liver cells (HL-7702) was studied. It is clear from Figure 5d that the expression level of miR-21 in the HL-7702 cell is much lower than that in cancer cells. The gas pressure is similar to the blank signal when the number of HL-7702 cells is under 1 × 106. The results were in good agreement with those of previous reports, which also indicated similar miR-21 expression level in these cell lines.29−31 Therefore, the POCT strategy reliably detected miR-21 in a complex sample.

Table 2. Detection of miR-21 in Diluted Human Serum Samples (n = 3) samples

added (fM)

found (fM)

recovery (%)

RSD (%)

1 2 3

10 100 1000

9.77 101.72 988.53

97.7 101.7 98.9

2.7 4.0 2.3

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CONCLUSIONS In conclusion, a facile, cost-effective, ultrasensitive POCT method had been developed for the detection of miRNA by using a portable gas pressure meter as readout. By combining the cyclic SDR with highly efficient gas-generation reaction, the ultrasensitive detection of miRNA was easily achieved with a low detection limit of 7.6 fM. Hairpin DNA probes and magnetic separation help realize highly specific and reliable detection, even in complex cell lysate and human serum. The gas pressure meter is very cheap and digital, and the decomposition of H2O2 to O2 is user friendly. Thus, the pressure-based POCT assay can be rapidly, safely, and directly performed at low cost. By introducing other functional nucleic acids, the POCT strategy can be F


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extended to detect different targets. Therefore, it offers a promising way for biomedical research and clinical diagnosis.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b02551. Effect of reaction time and concentration of MBs; TEM images of PtNPs and the decomposition of H2O2 catalyzed by PtNPs; effect of the 48 different wells; effect of the prolonged reaction time; optimization of the concentration of MBs; reaction time between MBs and H1; effect of buffer; effect of the concentration of SSC buffer; effect of concentration ratio of H1 and H2; effect of reaction time of SDR and reaction temperature of SDR; comparison of different methods (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-29-81530726. Fax: 86-2981530727. ORCID

Chaoyong James Yang: 0000-0002-2374-5342 Yan Jin: 0000-0001-8051-9950 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support through grants from the National Natural Science Foundation of China (No. 21375086) and the Fundamental Research Funds for the Central Universities (GK201701002).

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REFERENCES

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Ultrasensitive and Facile Detection of MicroRNA via a Portable

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