Cancer-Specific MicroRNA Analysis with a Nonenzymatic Nucleic Acid

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Biological and Medical Applications of Materials and Interfaces

Cancer-specific MicroRNA Analysis with A Non-enzymatic Nucleic Acid Circuit Dan Zhu, Bang Lu, Yu Zhu, Zihao Ma, Yaqi Wei, Shao Su, Lihua Wang, Shiping Song, Ying Zhu, Lianhui Wang, and Jie Chao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01653 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Applied Materials & Interfaces

Cancer-specific MicroRNA Analysis with A Non-enzymatic Nucleic Acid Circuit Dan Zhu,a Bang Lu,a Yu Zhu,a Zihao Ma,a Yaqi Wei,a Shao Su,a Lihua Wang,b Shiping Song,b Ying Zhu,b Lianhui Wang, a Jie Chaoa,* a Key

Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for

Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications, Nanjing

210023, China b

Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility,

CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China

ABSTRACT Sensitive detection of gastric cancer-related biomarkers in human serum provides a promising means for early cancer diagnosis. Herein, we report the design of nucleic acid circuit for gastric cancer-related microRNA-27a (miRNA-27a) detection based on dual toehold-mediated circular strand displacement amplification (CSDA). In the presence of miRNA -27a, the hybridization between miRNA-27a and probe DNA (pDNA) on magnetic beads (MBs) through toehold 1 leads to the release of fluorescent DNA (f-DNA) and the exposure of a new toehold 2 on linker DNA (l-DNA). After hybridization with catalytic DNA (c-DNA), CSDA is initiated and target miRNA-27a is released to participate in the next cyclic reaction, therefore greatly enhanced fluorescence signal is produced. The efficient magnetic separation makes the sensitive detection of miRNA-27a be accomplished within 45 min. With the efficient CSDA, the detection limit of the system (0.8 pM) is ~100 folds lower than that of the system based on strand displacement without CSDA (79.3 pM). Furthermore, the system also showed good stability and sensitivity to discriminate single-base mismatch, which allows the

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detection of miRNA-27a in human serum samples. This study provides a novel platform and approach for rapid quantitative determination of miRNA, which has great potential in clinical diagnosis and disease treatment. Keywords: DNA circuit; circular strand displacement amplification (CSDA); microRNA (miRNA) detection; magnetic separation; enzyme-free


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INTRODUCTION Gastric cancer is one of the most common cancers with the third highest fatality rate.1 The occurrence and progression of gastric cancer is in multiple stages and affected by multiple factors.2 Recent studies have shown that the generation and growth of tumors is closely related to non-coding RNA, which can regulate the expression of genes and proteins during the physiological processes and participate in various pathological processes in the human body.3 For instance, Xu regarded miRNA-27a, an endogenous and non-coding microRNA (miRNA) with 21 nucleotides, as the biomarker for gastric cancer and atrophic gastritis, which interacts with H. pylori in the carcinogenic process.4 Hua found that miRNA-23a/27a/24-2 cluster could suppress the expression of SOCS6 and acted as the oncogenes in gastric cancer.5 Rogoveanu believed that the abnormal expression of miRNA-27a is closely related to the development of gastric cancer and multiple physiological processes.6 Therefore, developing methods for quantitative determination of miRNA in human serum is vitally important to the diagnosis and identification of cancers and related diseases.7-10 Although polymerase chain reaction (PCR), DNA microarray and northern blotting has been extensively used as the common methods for miRNA detection, it still encounters the problem of timeconsuming processes and low sensitivity.11,12 Developing novel methods for sequencespecific diagnosis with fast response, high sensitivity and selectivity is still desperately needed.13-17 In recent years, enzyme-free nucleic acid based amplification circuits has received increased attention, such as rolling circle amplification (RCA),18, 19 hybridization chain reaction (HCR)20, 21 and catalyzed hairpin assembly (CHA).22 The specific nucleic acid sequences are easy to be synthesized and precisely modified, which enables the strategies with design flexibility and enhanced stability.23-26 Compared to the strategy without cyclic amplification, methods based on these cyclic nucleic acid circuits exhibit enhanced capability for signal amplification, therefore have been applied in the detection of gene, protein, metal ions and even cells.27-30 For instance, Huang



demonstrated a CHA-based DNA circuit for miRNA-21 detection with a fluorescence anisotropy method, which led to the limit of detection of 47 pM.31 Compared to the system without CHA, the sensitivity was improved ~4.3 folds. Wang reported a CHAHCR based circuit for signal amplified detection of miRNA-21.32 Their results suggested that the detection limit of the system based on coupled CHA-HCR was 100~200 times lower than that of systems based on single CHA or HCR. Despite the progress made, it always takes a relatively long time to carry out the circular circuit from 1 h to even 12 h.32-35 Designing efficient nucleic acid circuits with fast response holds great promise for rapid and sensitive diagnosis of biomolecules. In this work, we propose a non-enzymatic DNA circuit for gastric cancer-related miRNA-27a based on circular strand displacement amplification (CSDA) and magnetic separation. Nanomaterials have great potential in in vitro and in vivo analysis for its excellent intrinsic property.36-38 Magnetic beads (MBs) are herein employed as the platform for DNA circuit reaction, which is efficient for rapid separation and collection of signal molecules due to the large surface area.39-41 With the assistance of efficient magnetic separation, the DNA circuit can be accomplished within 45 min. The designed efficient approach also shows high selectivity and sensitivity for miRNA-27a detection. The detection limit of the system is 0.80 pM, which is ~100 folds lower than that of the strand displacement-based strategy without CSDA. EXPERIMENTAL SECTION Materials. DNA oligonucleotides and miRNA sequences synthesized and purified by Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China) are listed in Table S1. Magnetic separator and MBs modified with streptavidin (10 mg/mL, 1 μm in diameter) were purchased from Thermos Fisher Scientific (Waltham, the U.S.A.). Bovine serum albumin (BSA) were purchased from Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Tris (hydroxymethyl)


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aminomethane (Tris), sodium chloride (NaCl), EDTA and magnesium chloride hexahydrate (MgCl2) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Diethylpyrocarbonate (DEPC) treated deionized water was obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Buffer solutions used in this work were listed as follows: buffer A: 10 mM Tris–HCl, 1 mM EDTA and 2 M NaCl, pH 7.4; buffer B: 5 mM Tris–HCl, 0.5 mM EDTA and 1 M NaCl, pH 7.4; buffer C: 50 mM Tris–HCl, 140 mM NaCl, 1 mM MgCl2 and 0.1% BSA, pH 7.4; buffer D: 50 mM Tris–HCl, 140 mM NaCl, 1 mM MgCl2, pH 7.4; All buffer solutions were prepared with DEPC treated deionized water to avoid the degradation of RNA. Apparatus. Fluorescence

signals

were

measured

on

an

RF-5301PC

fluorescence

spectrophotometer (Shimadzu, Japan). The UV-Vis spectra were characterized by UV3600 Fourier transform infrared spectroscopy (FT-IR) (Shimadzu, Japan). Solution was incubated with an Eppendorf F1.5 thermomixer (Germany). The pH analysis of buffers was monitored on a PB-10 pH meter (Sartorius, Germany). The image of gel was taken using GBOX-F3-E (Gene Company Limited, the U.S.A). Immobilization of DNA probes on MBs. Firstly, 12 μL MBs was diluted and washed by buffer A for three times to remove passivating agent and preservative on the surface of MBs. Then, 50 μL buffer B containing 0.5 μM biotin modified p-DNA was added to MBs for 30 min incubation at 37 °C with slight shaking. After magnetic separation, the probe DNA (p-DNA) modified MBs was washed with buffer C for three times and then incubated in buffer C for 10 min to block active sites on the surface. The as-prepared MBs was suspended in buffer D again, followed by the addition of 0.5 μM fluorescent DNA (f-DNA) and 0.5 μM linker DNA (l-DNA). After 50 min incubation at 37 °C, the f-DNA/l-DNA/pDNA/MB probe was washed by buffer D for three times and dispersed in buffer D for further use.



Electrophoresis experiment. Polyacrylamide gel electrophoresis (PAGE) analysis was conducted in the absence of magnetic beads to characterize the hybridization process. The gel was run in 10% acrylamide solution with 1 × TBE buffer at 75 V constant voltage for 1.5 h at room temperature. Then, the gel was stained with Gel-Red for 10 min to indicate the position of DNA and photographed. DNA circuits for miRNA-27a detection. The prepared f-DNA/l-DNA/p-DNA/MB probe was then incubated with buffer D containing different concentrations of target miRNA-27a and 0.5 μM catalytic DNA (cDNA) to run the DNA circuit at 37 °C (final volume of 50 μL). After 45 min incubation, the supernatant was collected after magnetic separation for fluorescence detection. For target miRNA-27a detection without CSDA, the f-DNA/l-DNA/p-DNA/MB probe was incubated with 50 μL buffer D containing different concentrations of target miRNA27a at 37 °C. After 45 min incubation, the supernatant was collected for fluorescence measurement. Specificity experiment. Different kinds of miRNA targets (10 nM) including miRNA-27a, single-base mismatched miRNA-27a (SM-miRNA-27a), miRNA-21 and a non-complementary miRNA with random sequence (Random miRNA) was diluted in buffer D was mixed with f-DNA/l-DNA/p-DNA/MB probe and 0.5 μM c-DNA (final volume of 50 μL), respectively. After 45 min incubation at 37 °C, the supernatant was collected after magnetic separation for fluorescence detection. MiRNA detection in the human serum. The detection of miRNA-27a in the human serum was performed by the standard addition method. The human serum was diluted 100 times by adding 10 μL serum into


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990 μL buffer D. Then, 50 pM /100 pM /1000 pM miRNA-27a targets dissolved in the diluted human serum was added into f-DNA/l-DNA/p-DNA/MB probe and 0.5 μM cDNA (final volume of 50 μL). After 45 min incubation at 37 °C, the supernatant was collected after magnetic separation for fluorescence detection. The fluorescence was finally converted to molar concentration of miRNA-27a by comparing with the standard linear calibration curve to calculate the recovery. RESULTS AND DISCUSSION Detection principle of the CSDA-based DNA circuit. Firstly, magnetic probes were prepared by firstly assembling probe DNA (p-DNA) onto streptavidin modified MBs by streptavidin-biotin conjunction, followed by the hybridization of FAM-labeled fluorescent DNA (f-DNA) and linker DNA (l-DNA) with toehold 1 exposed in the terminal of magnetic probes. In the conventional strand displacement assay (system without CSDA in Figure 1), l-DNA could hybridize with miRNA-27a through toehold 1 recognition and f-DNA was released after strand displacement. After the magnetic separation, the fluorescence can be collected and detected by fluorescence spectrometer. Since the strand displacement was carried out in an equivalent ratio (1:1) between released f-DNA and target miRNA-27a, low fluorescence was observed. To provide amplified fluorescent signals, a CSDA-based DNA circuit was introduced, whose principle and process was displayed in Figure 1. After miRNA-27a being combined onto magnetic probes through toehold 1 hybridization, the addition of a new catalytic DNA (c-DNA) led to a new round of displacement through toehold 2 recognition. Therefore, miRNA-27a was released into solution and then participate in the next circle to release more f-DNA from the magnetic probes. After the repeated cycling reaction, large amounts of f-DNA could be collected and an enhanced fluorescence signal could be observed.



Figure 1. Schematic description of the detection principle of target miRNA-27a with and without circular strand displacement amplification (CSDA). Inset is the working mechanism for the CSDA-based DNA circuit. The feasibility and characterization of CSDA-based DNA circuit. From the transmission electron microscope (TEM) characterization (Figure S1), the magnetic beads is nearly spherical with the average diameter of 1 μm. The images before and after DNA immobilization suggested that the magnetic beads has a good dispersion in the solution, which is suitable for the in-solution detection. In order to confirm the feasibility of the CSDA-based DNA circuit, miRNA-27a was chosen as the analytical target because of their importance in the carcinogenesis and diagnosis of gastric cancer. As shown in Figure 2A, in the absence of target miRNA-27a, fluorescence in supernatant was low in the systems without CSDA (curve a). Moreover, the fluorescence almost unchanged even in the presence of 500 nM c-DNA (curve b), which demonstrated that DNA circuit could not work to release f-DNA without target miRNA-27a. After 10 nM target miRNA-27a was added (curve c), the relative fluorescence intensity was increased ~1.5 folds than that of the control group. When


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both target 10 nM miRNA-27a and c-DNA existed in the system (curve d), the fluorescence had a significant improvement. As shown in Figure 2B, the relative fluorescence intensity of DNA circuit (system d) was ~4.2 times higher than control group (system a) and ~2.2 times as much as the system without CSDA (system c). To verify the catalytic process of DNA circuit, native polyacrylamide gel electrophoresis (PAGE) was further performed (Figure 2C). The slow mobility of DNA nanostructure in Lane 3 clearly demonstrated the formation of f-DNA/l-DNA/p-DNA probe. After adding 10 nM target miRNA-27a (Lane 6), the intensity of f-DNA/l-DNA/p-DNA band was almost constant and the released f-DNA was nearly invisible due to the extremely low concentrations of miRNA-27a/f-DNA/l-DNA complex and f-DNA. As a comparison in Lane 7, the presence of 10 nM miRNA-27a and 500 nM c-DNA could efficiently facilitate the DNA strand displacement between DNA probes and release large amounts of f-DNA, which further confirmed the operation of DNA circuit was initiated by miRNA-27a and c-DNA.

Figure 2. (A) Fluorescence spectra and (B) relative fluorescence intensity of the system with and without CSDA in the absence and presence of 10 nM target miRNA-



27a. The concentration of c-DNA in system b and d was 500 nM, respectively. (C) Native polyacrylamide gel electrophoresis (PAGE) analysis of DNA circuit. Lane M: 20 bp marker; Lane 1: f-DNA; Lane 2: p-DNA + l-DNA; Lane 2: p-DNA + l-DNA; Lane 3: p-DNA + l-DNA+ f-DNA; Lane 4: l-DNA+ c-DNA; lane 5: p-DNA + l-DNA+ miRNA-27a; Lane 6: p-DNA + l-DNA+ f-DNA + miRNA-27a; Lane 7: p-DNA + lDNA+ f-DNA + miRNA-27a + c-DNA. [p-DNA] = [l-DNA] = [f-DNA] = 1 μM; [miRNA-27a] = 10 nM; [c-DNA] = 500 nM. The mechanism study of CSDA-based DNA circuit. Since CSDA-based DNA circuit was operated through a multi-step strand displacement, the factors that might influence the displacement process are investigated. Considering that the short length of miRNA-27a, too short toehold 1 might hinder the hybridization between miRNA-27a and l-DNA, while long toehold 1 might cause the instability of l-DNA/f-DNA hybrid and high background noise. Therefore, a set of magnetic probes with different length of toehold 1 domain from 3 to 11 bases were designed to compare the signal/noise (S/N) ratio (Figure 3A). The result in Figure 3B suggested that the S/N ratio firstly increased and then decreased when the length of toehold 1 changed from 3 to 11 bases by tuning the hybridization bases of f-DNA. The highest S/N was obtained when the base number of toehold 1 is 5 and 7 (system b and c in Figure 3B). Therefore, 7 bases were selected as the optimized length of toehold 1 due to the relatively high fluorescence signals and S/N ratio. The concentration of cDNA was then investigated because excessive c-DNA might cause the non-specific release of f-DNA while few c-DNA was not sufficient to initiate the DNA circuit. As shown in Figure 3C, the approximately constant low fluorescence signals in the presence of different concentrations of c-DNA further indicated the advanced antiinterference performance of DNA circuit due to the blocking of toehold 2 site. When the concentration of c-DNA increased from 0 to 1000 nM, the fluorescence signal was increased and the S/N reached the plateau when the c-DNA was higher than 500 nM. Therefore, 500 nM was selected for the concentration of c-DNA in the following


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experiments. As an important factor affecting the DNA hybridization and dissociation,42 temperature was optimized from 25 to 45 °C. As shown in Figure 3D, the fluorescence signal and noise both get stronger as the temperature increases. The stronger noise in the absence of miRNA-27a under higher temperature might be caused by the nonspecific thermally induced dissociation of f-DNA. The maximum S/N was achieved at 37 °C, where a strong fluorescence signal could also be observed. Therefore, 37 °C was chosen as the temperature for the CSDA reaction. The kinetics study in Figure S1 suggested that the CSDA reaction reached equilibrium within 45 min, while system without CSDA took a longer time more than 1 h. The faster kinetics allowed the efficient and simple detection for miRNA-27a.

Figure 3. (A) Design of toeholds 1 with a set of base numbers (a-e: 3, 5, 7, 9 and 11) on l-DNA. (B) The corresponding fluorescence signals in the presence and absence of 10 nM miRNA-27a and the signal/noise (S/N) ratio of the system a-e in Figure 3A. (C) Effects of the concentration of c-DNA (0, 250, 500, 750 and 1000 nM) of the DNA circuit in the presence or absence of 10 nM miRNA-27a and the corresponding S/N.



(D) The fluorescence signals in the presence and absence of 10 nM miRNA-27a at different reaction temperature (25, 30, 35, 37, 40 and 45 °C) and the corresponding S/N. Analytical performance for target miRNA-27a detection. The quantitative detection performance of miRNA-27a was then investigated under the optimized experimental conditions. After incubating with a series concentration of miRNA-27a from 0 to 10 nM, fluorescence spectra of the system with and without CSDA-based DNA circuit was recorded. We defined ΔF520 nm = Fm-F0, where Fm was the measured data of fluorescence intensity at 520 nm, F0 was the control fluorescence intensity at 520 nm measured without miRNA-27a. As shown in Figure 4A and 4B, the ΔF520 nm were enhanced with the increase concentration of miRNA-27a from 0 to 10 nM in both systems. It is remarkable that the enhancement of fluorescence in the system with CSDA-based DNA circuit (Figure 4C) was almost 2-4 folds higher than that in the system only with one-step strand displacement (Figure 4A). The calibration curves in Figure 4D suggested that the ΔF520

nm

of system with CSDA-based DNA circuit

exhibited a good linear relationship with the logarithm of the concentration of miRNA27a (lg CmiRNA-27a) from 1 pM to 1 nM, while the linear detection range of system without CSDA-based DNA circuit was 100 pM to 2.5 nM (Figure 4B). The limit of detection (LOD) of the system with the DNA circuit was calculated as 0.8 pM, which was ~100 folds lower than that of the system without DNA circuit (LOD: 79.3 pM). The improved sensitivity and low detection limit was attributed to the circling reaction of miRNA-27a fueled by c-DNA. We also compared the detection performance of our assay with other reported assays, the parameters listed in Table 1 further suggested that our assay possess relatively lower detection limit and faster response towards miRNA detection.


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Figure 4. (A, C) Fluorescence spectra of the magnetic probe (A) with and (C) without CSDA-based DNA circuit incubated with different miRNA-27a concentrations from 0 to 10 nM. (B, D) The Calibration plots of ΔF520 nm versus the logarithm concentration of miRNA-27a (lg CmiRNA-27a) of both systems (B) with and (D) without CSDA-based DNA circuit. The corresponding linear regression equation was y = 14.54 lg CmiRNA-27a + 2.644 (R2 = 0.9744) and y= 14.07 lg CmiRNA-27a – 24.06 (R2 = 0.9549), respectively. Table 1. Comparison of the reported fluorescent platforms for miRNAs detection.

Amplification strategy

DSN-assisted amplification Double-Strand Displacement Rolling circle amplification (RCA), strand displacement amplification (SDA)

Linear

Detection

range

limit

1 pM-10 μM 5 nM-1 μM 51 pM-166 nM

Time

Reference

1.03 pM

2h

Ref.43

5 nM

2h

Ref.44

50 pM

6h

Ref.45



Catalytic hairpin assembly

0 nM-200

(CHA)

nM

Rolling circle amplification

6.4 pM-100

(RCA)

nM

Circular strand displacement amplification (CSDA)

1 pM-1 nM

290 pM

6.4 pM

0.81 pM

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75

Ref.46

min

Ref.47

2h 45 min

This work

The selectivity of the magnetic probe. The specificity of this proposed magnetic probe with CSDA-based DNA circuit was then evaluated by testing four different miRNAs, including miRNA-27a, single-base mismatched miRNA-27a (SM-miRNA-27a), miRNA-21, miRNA-155, miRNA-486 and a non-complementary miRNA with random sequence (Random miRNA) under the same experimental conditions. As exhibited in Figure 5, the ΔF520 nm was obviously strong in the presence of 10 nM miRNA-27a, while ~4.5 folds lower in intensity in the existence of SM-miRNA-27a. Moreover, the systems incubated with the same concentration of the miRNA-21, miRNA-155, miRNA-486 and non-complementary Random miRNA exhibited very weak fluorescence enhancement approximated to the control group. Take the probe without CSDA a comparison, the ΔF520

nm

for SM-

miRNA-27a was ~2.9 folds lower in intensity than that for target miRNA-27a (Figure S3). The results indicated that the proposed biosensor has an enhanced capability of distinguishing single base mismatches and displayed excellent selectivity for the determination of target miRNA-27a from other nonspecific miRNAs, which might have potential in the detection of biological samples.


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Figure 5. ΔF520 nm of the magnetic probe based on CSDA-based DNA circuit in the absence (control) and presence of target miRNA-27a, single-base mismatched miRNA-27a (SM-miRNA-27a), miRNA-21, miRNA-155, miRNA-486 and a noncomplementary miRNA with random sequence (Random miRNA) under the same experimental conditions. The concentration of all miRNAs was 10 nM. Detection of target miRNA-27a in serum samples. To evaluate the performance of our magnetic probe with CSDA-based DNA circuit in real biological samples, recovery test was conducted in human serum by spiking different concentrations of miRNA-27a into 100-fold diluted human serum sample. The biosensor response to the spiked samples with 100 pM and 1 nM miRNA-27a was measured to calculate the recoveries. As shown in Table 2, the recovery of miRNA-27a in human serum at 50 pM, 100 pM and 1 nM was 98.1%, 106.4% and 102.4%, respectively. Experiments revealed a favorable reproducibility for detection according to the RSD data (n = 3) in Table 2. The results suggested the proposed probe performed well in complex actual samples and had great potential for practical applications. Table 2. The recovery test of miRNA-27a in the human serum samples. Each sample was tested at least three times.



Sample

Added (pM)

Founded (pM)

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Recovery (%)

RSD (%)

98.1

0.641

106.4

0.856

102.4

1.209

53.64 1

50

47.15 46.37 100.5

2

100

117.2 101.4 1141

3

1000

958.3 973.3

CONCLUSION In summary, we demonstrated an enzyme-free amplification strategy based on magnetic separation and dual toehold-mediated DNA circuit for sensitive detection of gastric cancer-related miRNA-27a. This CSDA-based DNA circuit system enables efficient and rapid detection of miRNA-27a with improved sensitivity and performance. The detection limit is 0.8 pM, which is ~100 folds lower than that of the system without DNA circuit (LOD: 79.3 pM). Compared to other reported biosensors for miRNA detection, this assay possesses several advantages. Firstly, CSDA-based DNA circuit is conducted to provide recycled miRNA-27a and amplified fluorescence signals. The enzyme-free manner allows the robust and convenient detection of target miRNA. Secondly, MBs with large surface area provides a platform to achieve the collection of large amounts of signal molecules, which are conducive to DNA circuit reaction. Thirdly, with the assistance of magnetic separation, the detection can be accomplished within 45 min, which is convenient and efficient. This study provides a novel platform and approach for rapid quantitative determination of miRNAs, which has great potential in clinical diagnosis and disease treatment. ASSOCIATED CONTENT


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Supporting Information Supporting table S1 and supporting Figure S1-S3 is available free of charge on the ACS Publications website. DNA and RNA sequences; TEM images of magnetic beads before and after DNA immobilization; Kinetics for miRNA-27a detection of magnetic probe with and without CSDA; Selectivity study of magnetic probe without CSDA. AUTHOR INFORMATION Corresponding Author * [email protected] ACKNOWLEDGEMENTS This work was ﬁnancially supported by the National Key Research and Development Program of China (2017YFA0205302), the National Natural Science Foundation of China (21605087, 61771253), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R37), the Key Research and Development Program of Jiangsu (BE2018732), the Natural Science Key Fund for Colleges and Universities in Jiangsu Province (17KJA430011), the National Postdoctoral Program for Innovative Talents (BX201700123), the China Postdoctoral Science Foundation funded project (2018M630586), and the LU JIAXI International team program supported by the K.C. Wong Education Foundation and CAS. CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES (1) Siegel, R. L., Miller, K. D., Jemal, A. Cancer Statistics. 2018. CA-Cancer J. Cli.



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2018, 68, 7-30. (2) Hartgrink, H. H., Jansen, E. P. M., van Grieken, N. C. T., van de Velde, C. J. H. Gastric Cancer. Lancet. 2009, 374, 477-490. (3) Huang, Y., Yu, J.. Circulating MicroRNAs and Long Non-Coding RNAs in Gastric Cancer Diagnosis: An Update and Review. World J. Gastroentero. 2015, 21, 98639886. (4) Xu, Q., Chen, T., He, C., Sun, L., Liu, J., Yuan, Y. MiR-27a Rs895819 is Involved in Increased Atrophic Gastritis Risk, Improved Gastric Cancer Prognosis and Negative Interaction with Helicobacter Pylori. Sci. Rep. 2017, 7. (5) Hua, K., Chen, Y., Chen, C., Tang, Y., Huang, T., Lin, Y., Yeh, T., Huang, K., Lee, H., Hsu, M., Chi, C., Wu, C., Lin, C., Ping, Y. MicroRNA-23a/27a/24-2 Cluster Promotes Gastric Cancer Cell Proliferation Synergistically. Oncol. Lett. 2018, 16, 2319-2325. (6) Rogoveanu, I., Burada, F., Cucu, M. G., Vere, C. C., Ioana, M., Cimpeanu, R. A. Association of MicroRNA Polymorphisms with the Risk of Gastric Cancer in a Romanian Population. J. Gastrointest. Liver. 2017, 26, 231-238. (7) Pei, H., Liang, L., Yao, G., Li, J., Huang, Q., Fan, C. Reconfigurable ThreeDimensional DNA Nanostructures for the Construction of Intracellular Logic Sensors. Angew. Chem. Inter. Ed. 2012, 51, 9020-9024. (8) Wen, Y., Pei, H., Shen, Y., Xi, J., Lin, M., Lu, N., Shen, X., Li, J., Fan, C. DNA Nanostructure-Based

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Ultrasensitive

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Table of Contents (TOC)


Cancer-Specific MicroRNA Analysis with a Nonenzymatic Nucleic Acid

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