Detection of Nucleic Acids in Complex Samples via Magnetic

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Technical Note

Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-assisted Catalyzed Hairpin Assembly and “DD-A” FRET Hongmei Fang, Nuli Xie, Min Ou, Jin Huang, Wenshan Li, Qing Wang, Jianbo Liu, Xiaohai Yang, and Kemin Wang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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Analytical Chemistry

Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-assisted Catalyzed Hairpin Assembly and “DD-A” FRET Hongmei Fang,† Nuli Xie,† Min Ou, Jin Huang, Wenshan Li, Qing Wang, Jianbo Liu, Xiaohai Yang* and Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China * To whom correspondence should be addressed. Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected], [email protected].

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Nucleic acids, as one kind of significant biomarkers, have attracted tremendous attentions and exhibited immense values in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and donor donoracceptor fluorescence resonance energy transfer (“DD-A” FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labelled probe as FRET donor) and H2 (TAMRA-labelled probe as FRET acceptor). Firstly, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMBs complexes were separated and resuspended in the reaction buffer containing H1 and H2. As a result, numerous H1-H2 duplexes were formed during CHA process, inducing an obvious FRET signal. In contrast, CHA could not be trigger and the FRET signal was weak while target was absent. With the aid of magnetic separation and “DD-A” FRET, it was demonstrated to effectively eliminate errors from background interference. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application.


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INTRODUCTION Nucleic acids are a type of biological macromolecules that play important roles in diseases diagnosis and cancer therapy.1,2 Some of nucleic acids with short oligonucleotide sequences have drawn considerable attention in recent years,3,4 such as microRNA (miRNA), piwi-interacting RNA (piRNA) and small nucleolar RNA (snoRNA). Most of them have been used as diseases markers or therapeutic agents because their abnormal expression levels often portend the occurrence of diseases.5-8 Therefore, the detection of these biomarkers is of great value in clinical diagnosis and cancer therapy. However, it suffers from the problems of detection sensitivity and signal accuracy since rather low abundance of the targets exists in complex samples including cells,9 exosomes10,11 or body fluids.12,13 Signal amplification, which can be classified into enzyme-assisted and enzyme-free approaches, has become a popular and potent strategy to solve the problem of detection sensitivity in the past decades. Compared to enzyme-assisted approach, enzyme-free signal amplification possesses unique advantages in the field of in-vitro detection, due to its simple, robust, low-cost features,14,15 as well as the exemption of complicated enzyme reaction. Typical enzyme-free amplification mainly includes hybridization chain reaction (HCR),16,17 catalyzed hairpin assembly (CHA)18 and entropy-driven catalysis (EDC),19-21 etc. The principle of CHA signal amplification is to utilize toehold-mediated strand displacement mechanism to continuously assemble DNA duplexes and translate the input of nucleic acids targets into an amplified signal with low detection limit.22-24 However, the detection of nucleic acids often meets the problem of signal accuracy when applied in complex environment where coexisting multiple components tend to have considerable interference on the amplified signals. The experiment results have a high risk to be false positive signals. Thus, it is crucial to eliminate the risk in order to acquiring credible


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signals. The procedure of enriching and separating targets in advance is very helpful to decrease background and avoid system interference, especially while detecting in complex samples.25 Over the past years, many effective technologies have been reported, including magnetic separation,26,27 capillary electrophoresis28 and paper-based chromatography,29-31 etc. Among them, magnetic microbead (MMB)-based technologies have been widely used owing to their user-friendliness and excellent handleability. It has been proven to effectively separate and detect cells,32 nucleic acids33-35 and protein,36 via modifying recognition molecules onto the surface of MMBs. Alternatively, another approach to improve the detection accuracy is ratiometric signal outputs. For example, fluorescence resonance energy transfer (FRET) is a frequently-used method, because FRET can provide built-in corrections for environmental impacts through recording the intensity ratio of two fluorescent emissions at the same time instead of a single fluorescent intensity, effectively eliminating systematic fluctuation and decreasing the risk of false positives.37,38 Recently, our group proposed an enhanced donor donor-acceptor (“DD-A”) FRET mode, which was found to show higher efficiency.39 To be specific, two adjacent donors were used to excite one acceptor and transfer energy via resonance between molecules. With these strategies, it is capable to greatly enhance signal accuracy while quantifying RNA in complex environment.40-41 Herein, a novel assay that combined magnetic separation and “DD-A” FRET-based CHA signal amplification was developed to detect nucleic acids in complex samples. The working principle was illustrated in Figure 1. Firstly, DNA or RNA targets were recognized by the Captures immobilized on MMBs, so that the hairpin structures of Captures were opened to expose the sequences that could trigger CHA (Step 1). Next, with the aid of magnetic separation, MMBs were separated and the targets were enriched from complex samples (Step 2).


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Subsequently, MMBs were resuspended and incubated in the buffer containing H1 (double FAM-labelled probe as FRET donor) and H2 (TAMRA-labelled probe as FRET acceptor) to trigger CHA. The resulting solution would generate a large number of H1/H2 duplexes in which the donors and acceptor were close in proximity, inducing an obvious FRET signal (Step 3). In contrast, CHA could not be triggered due to the blocked hairpin stem-loop structures and FRET signal was weak while the target was absent. As a result, the influence of coexisting components in complex samples was effectively eliminated. Moreover, it was demonstrated that signal sensitivity and accuracy were obviously improved by magnetic separation, CHA signal amplification and ratiometric signal outputs. EXPERIMENTAL SECTION Materials. SYBR Gold and streptavidin-modified MMBs (diameter 2.8 µm, 10 mg/mL, Catalog nos. 11205D) were purchased from Invitrogen (Shanghai, China). 6×DNA loading buffer and the ribonuclease (RNase) inhibitor were obtained from TaKaRa Biomedical Technology Co. Ltd (Beijing, China). All the oligonucleotide sequences were synthesized by Sangon Biotechnology Inc (Shanghai, China) and purified through high performance liquid chromatography.

They

were

quantified

by

BioSpec-nano

micro-volume

UV-Vis

spectrophotometer (Shimadzu, Japan) before use. All the sequences of oligonucleotide were listed in Table S1. Capture probe was dissolved into Binding & Washing buffer solution (B&W buffer, pH=7.5, containing 5 mM Tris-HCl, 0.5 mM EDTA and 1 M NaCl) and other probes were dissolved into sodium phosphate–sodium chloride buffer solution (SPSC buffer, containing 50 mM Na2HPO4 and 400 mM NaCl, pH=7.5 and adjusting pH with 5 M HCl). All the hairpin probes were formed by heating to 95 °C for 6 min and then slowly cooled to room temperature before use. All other reagents were of analytical grade and used as received. All solutions were


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prepared using ultrapure water (≥ 18.2 MΩ cm, Millipore), and then treated with 0.1% diethylpyrocarbonate and autoclaved to protect RNA from RNase degradation. Gel electrophoresis (E1). 24 µL SPSC buffer containing 1 µM H1, 1 µM H2, 100 nM Trigger, 100 nM Capture and 100 nM DNA-21, was incubated for 3 hours on a thermostatic metal shaker bath (37 °C and 300 rpm; Bioer, Hangzhou, China). Next, 10 µL of them was mixed with 2 µL of 100× SYBR Gold and 2 µL of 6× loading buffer. And then 10 µL of them ran at 99 V for 50 min in 2% agarose gel prepared by 1× TBE (pH=8.0, containing 89.0 mM Tris-HCl, 89.0 mM H3BO3 and 2.0 mM EDTA·2Na). The gel was photographed using the Azure C600 multifunctional imaging system (Azure Biosystems, USA). Fluorescence measurement. All fluorescence spectra were measured using a Hitachi F-7000 fluorescence spectrometer equipped with an aqueous thermostat (37 °C, Amersham Biosciences, Sweden). The excitation wavelength was set at 488 nm, the emission spectra were collected from 510 to 650 nm, the scanning speed was 1200 nm/second and the response time was 2 seconds. The excitation and emission slits were all 5 nm band-pass with a 700 V PMT voltage and a 0.2×1 cm2 quartz cuvette. The ratio of fluorescence intensity at 520 nm and 580 nm was calculated to evaluate the performance. The feasibility of “DD-A” FRET-based CHA (E2). 150 µL SPSC buffer containing 100 nM H1, 100 nM H2, 10 nM Capture and 10 nM DNA-21, was incubated for 3 hours on a thermostatic metal shaker bath (37 °C, 300 rpm). And then the fluorescence spectra were measured. The control experiment without DNA-21 was performed under the same conditions. Optimization of the ratio of donor probe and acceptor probe (E3). 150 µL SPSC buffer containing a certain ratio of H1 and H2, 10 nM Capture and 10 nM DNA-21. The concentration ratios of H1 and H2 were respectively 300 nM: 100 nM, 200 nM: 100 nM, 100 nM: 100 nM, 100


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nM: 200 nM and 100 nM: 300 nM. The experiments were performed according to the conditions of E2. Modification of MMBs (E4). The modification was carried out according to the product manual of MMBs. First, 200 µg of the purchased MMBs suspension was washed 3 times with 50 µL B&W buffer. After magnetic separation, MMBs were resuspended in 80 µL B&W buffer containing 1 µM Capture and then incubated for 0.5 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm). After magnetic separation, the supernatant was saved to measure the absorbance. MMBs were washed 3 times with 50 µL B&W buffer and resuspended in 40 µL SPSC buffer for the detection. Optimization of CHA amplification (Step 3, E5). The experiments consisted of the test experiment with DNA-21 and the control experiment without DNA-21. Firstly, the amount of immobilized Capture was optimized. The amounts of immobilized Capture probe were respectively 5, 10, 15 and 20 pmol. 150 µL SPSC buffer containing 100 nM H1, 200 nM H2, a certain amount of immobilized Capture and 10 nM DNA-21 was incubated for 3 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm). After magnetic separation, the supernatant was saved for the fluorescence measurement. Secondly, CHA reaction time was optimized under 1, 2, 3, 4, 5 and 6 hours, respectively. 10 pmol immobilized Capture was chosen and other conditions of the experiments were the same as the above. Optimization of capture and enrichment (Step 1-2, E6). Firstly, the capture temperature was optimized. 3 mL SPSC buffer containing 2 nM DNA-21 and 10 pmol immobilized Capture was respectively incubated at 25, 30, 37 and 45 °C for 6 hours in a thermostatic shaker (300 rpm, Yiheng, Shanghai). After magnetic separation, MMBs were washed 3 times with 500 µL B&W buffer and resuspended in 150 µL SPSC buffer containing 100 nM H1 and 200 nM H2, and then


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incubated for 4 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm). After magnetic separation, the supernatant was retained for the fluorescence measurement. Secondly, the capture time was optimized. 3 mL SPSC buffer containing 2 nM DNA-21 and 10 pmol immobilized Capture was respectively incubated for 2, 4, 6, 8 and 10 hours in a thermostatic shaker (37 °C, 300 rpm). After magnetic separation, MMBs were washed 3 times with 500 µL B&W buffer and resuspended in 150 µL SPSC buffer containing 100 nM H1 and 200 nM H2, and then incubated for 4 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm). After magnetic separation, the supernatant was retained to for the fluorescence measurement. DNA-21 detection procedures (E7). 3 mL SPSC buffer containing different concentrations of DNA-21 and 10 pmol immobilized Capture was incubated for 6 hours in a thermostatic shaker (37 °C, 300 rpm). After magnetic separation, MMBs were washed 3 times with 500 µL B&W buffer and resuspended in 150 µL SPSC buffer containing 100 nM H1 and 200 nM H2, and then incubated for 4 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm). After magnetic separation, the supernatant was used for the fluorescence measurement. DNA-21, single-base mismatched DNA (sDNA-21), three-base mismatched DNA (tDNA-21) and random DNA (Random DNA) were chosen to test the selectivity of DNA-21 detection. The detection was performed according to the above procedures. MiRNA-21 detection procedures (E8). The procedures of miRNA-21 detection was similar to that of DNA-21 detection. MiRNA-21, single-base mismatched RNA (smiRNA-21), threebase mismatched RNA (tmiRNA-21), random RNA (Random RNA) and homologous miRNAs (miRNA-429, miRNA-141 and let-7a) were chosen to evaluate the selectivity of miRNA-21 detection. Because miRNAs are susceptibility to degradation, so the procedures were carefully performed to avoid RNA degradation in the whole experiment.


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MiRNA-21 detection in serum (E9). First, the inactivated fetal bovine serum (FBS, 56 °C and 30 minutes) was treated with 0.2 U/µL RNase inhibitors. Next, 50 µL of different concentrations of miRNA-21 was added to 450 µL FBS and filtrated using a 30K ultrafiltration centrifuge tube. The filtrate containing different concentrations of miRNA-21 was saved. And then 300 µL of the filtrate was added to 2.7 mL SPSC buffer containing 10 pmol immobilized Capture, obtaining 3 mL of the sample. Other experiments were performed as same as DNA-21 detection procedures. MiRNA-21 detection in cell culture medium (E10). HepG-2 cells (human hepatocellular liver carcinoma), HeLa cells (human cervical cancer) and SMMC-7721 cells (human hepatocellular carcinoma) were purchased from cell bank of the Chinese Academy of Science (Shanghai, China). First, the cells were washed 3 times by 4 mL D-Hank’S, mixed with 20 mL fresh RPMI 1640 medium (supplemented with 10% FBS and 100 U/mL penicillin-streptomycin) and incubated at 37 °C for 24 hours under a 5% CO2 atmosphere. The cell media were collected and centrifuged to remove the residual cells at 24,000 rpm for 30 minutes by a centrifugation (CS150NX, Hitachi, Japan). The cell media were divided into two equal parts for the detection using this method and qRT-PCR (Micoread, Beijing, China). For the detection using this method, the cell media were treated with RNase inhibitor and filtrated using a 30K ultrafiltration centrifuge tube, obtaining 3 mL of the filtrate. Other experiments were performed according to DNA-21 detection procedures. Note: unless noted otherwise, all the experiments were repeated 3 times at least in this work. RESULTS AND DISCUSSION In this work, the Captures immobilized on MMBs were designed to separate and concentrate targets from complex samples and expose the sequences to initiate CHA, instead of direct target-


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initiated CHA. This optimization could be beneficial to eliminate the interference of coexisting components and decrease background of CHA. In addition, hairpin structures would restrain non-target binding, improving the selectivity of our method. Firstly, CHA reaction (Step 3) was validated by gel electrophoresis (detailed procedures are described in E1). As shown in Figure 2, both lane 1-3 had only one band which was located at the same position, in the absence of CHA trigger sequence. Similarly, only one band showed in lane 5 because the hairpin structure of Captures could not be opened to initiate CHA without synthetic DNA-21. While the Trigger or DNA-21 was present, new band of H1/H2 duplexes emerged and original band disappeared in lane 4 and 6. It demonstrated that CHA reaction successfully occurred with low background and numerous H1/H2 duplexes were generated. To confirm the feasibility, a fluorescent assay (detailed procedures are described in E2) based on “DD-A” FRET was performed. In this work, “DD-A” model was finally used because of its higher FRET efficiency than traditional donor-acceptor (D-A) as well as donor-acceptor acceptor (D-AA) and donor donor-acceptor acceptor (DD-AA) FRET model, according to our previous paper.39 H1 was labeled with two FAM as FRET donor and H2 was labeled with TAMRA as FRET acceptor. The fluorescence spectra between 510 - 650 nm were collected under the excitation of 488 nm, and the intensity ratio (FA/FD) of green fluorescence at 520 nm and red fluorescence at 580 nm was calculated. We denoted the ratio of (FA/FD) 1/ (FA/FD) 0 as effective amplification factor (R), where (FA/FD) 1 and (FA/FD) 0 were the intensity ratios with and without target, respectively. As shown in Figure 3, in the presence of DNA-21, the intensity of FAM sharply decreased while the intensity of TARMA increased, indicating an obvious FRET signal. As a result, the value of FA/FD changed from low to high. This result demonstrated that the concentration of DNA-21 could be well transformed into “DD-A” FRET signal.


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The probes H1 and H2 were of great importance in this work. They not only served as FRET donor probe and acceptor probe respectively, but also were essential components of CHA. Sufficient amounts of H2 should be added into reaction solution, which guarantee cyclic replacement of the Captures from Capture/DNA-21/H1 complexes. But excess H2 could also spontaneously hybridize with H1, generating high background. Thus, the concentration ratio of H1 and H2 should be optimized (detailed procedures are described in E3). Figure S1 indicated that R value maximized when the concentration ratio of H1 and H2 was 1:2. Therefore, the concentration ratio 1: 2 was chosen for the following studies. To separate and concentrate targets from complex samples, the Captures were immobilized onto streptavidin-modified MMBs surface by the affinity interaction between streptavidin and biotin (detailed procedures are described in E4). The surface coverage of immobilized Captures was estimated (the details were described in S1) using equation (1): Surface coverage = (Cbefore–Cafter) × V × NA / S

(1)

Where Cbefore and Cafter were the molar concentrations of Capture in solution before and after modification onto the MMBs. V was the volume of Capture solution. NA is Avogadro constant. S was the total superficial area of MMBs. According to this equation, the concentrations of Capture in solution before and after modification were measured by the characteristic absorption peak of DNA at 260 nm. It was respectively calculated as 1.1 µM and 0.6 µM. 50 µg MMBs were incubated with 20 µL Capture solution, so it displayed that 10 pmol Capture were immobilized onto MMBs. Because the known concentration of MMBs was about 7×104 bead/µg with a average diameter of 2.8 µm, so the total superficial area of 50 µg MMBs was 8.6 × 107 µm2. Therefore, the surface coverage of immobilized Captures was calculated to be 7 × 1010 mm-2.


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Importantly, Capture was a crucial component of CHA amplification (Step 3). The amount of immobilized Capture could directly affect detection performance. Appropriate amounts of immobilized Captures were beneficial to CHA reaction, but redundant amount of Capture would give rise to the waste of materials. Therefore, the optimization on the amount of immobilized Captures was necessary (detailed procedures are described in E5). As shown in Figure S2 (A), given that all the surface coverages were same, as the amount of immobilized Captures increased, FRET ratio increased and reached a plateau when the amount of immobilized Captures was 10 pmol or more. Thus, 10 pmol of immobilized Captures was chosen for the following detection. In addition, CHA time also affected the formation yield of H1-H2 duplexes. Figure S2 (B) indicated that FRET signal increased with more incubation time and reached a plateau at 4 hours, suggesting 4 hours were the best reaction time for CHA amplification. Due to quick magnetic responsiveness, MMBs have been widely used for separation and enrichment of targets in complex samples. With the assistance of MMBs, this strategy could further eliminate the interference of coexisting components and amplify target signal. Two significant factors of affecting the enrichment efficiency were the incubation time and temperature. As shown in Figure S3 (A) (detailed procedures are described in E6), FRET ratio maximized with an unchanging background when the temperature was 37 °C. Figure S3 (B) displayed that FRET signal gradually increased with more enriching time and reached maximum at 6 hours, but then declined when the time was 8 h. The decline might be owing to the saturation of the enrichment reaction and the hydrolysis of a few probe.42 Consequently, it was chosen to incubate 6 hours at 37 °C when using MMBs for targets enrichment. Under these optimal conditions, we used this system to detect synthetic DNA-21 that was the mimic of miRNA-21 (detailed procedures are described in E7). A series of fluorescence spectra


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were indicated in Figure S4 (A). The intensity of TAMRA normalized by FAM fluorescence gradually enhanced along with growing concentrations of DNA-21 from 0 to 4 nM. A curve of FRET ratio versus concentration was drawn (Figure S4 (B), solid point), with a detection limits of 33 pM (S/N=3). Furthermore, we found that FRET ratios at each concentration point after the enrichment of MMBs were distinctly higher than those without enrichment (Figure S4 (B), hollow point), revealing that the enrichment of MMBs played a positive role in improving detection sensitivity. In addition, the selectivity of this method was also studied. Three kinds of DNAs including two mismatched DNA (sDNA-21 and tDNA-21) as well as Random DNA were chosen as control group (detailed procedures are described in E7). As shown in Figure S4 (C), with the concentrations of targets increased, the R value of DNA-21 remarkably grew while almost no signal responded to other three samples. It indicated that this method had good identification capability of single base mismatch. MiRNA-21 is a typical cancer biomarker, which overexpresses in a wide variety of cancer cells.43,

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Herein, miRNA-21 was selected as the model to investigate miRNA detection

performance of this method in buffer (detailed procedures are described in E8). As show in Figure 4, gradual increasing FRET signals were observed when the concentrations of miRNA21 ranged from 0 to 4 nM, and the detection limit was calculated as low as 34 pM (S/N=3). In addition, compared to those without MMBs enrichment (Figure 4B, hollow point), the samples with MMBs enrichment (Figure 4B, solid point) showed obviously higher signal. The detection limit of miRNA-21 was comparable to that of some reported non-protease nucleic acids detection methods, which listed in Table S2. Despite tedious procedures, our method exhibits competitive or comparable sensitivity compared with previous reported silver nanoclustersbased, FRET-based, MMB-based and other non-protease methods. In addition, this MMB and


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FRET-based method can decrease irrelative interferences. Therefore, it is capable to apply to the detection of nucleic acids in complex samples. To simplify the analysis procedures, integrating with microfluidics may be one of useful strategies in the future. Distinguishing from similar miRNAs is one of major challenges for accurate miRNAs quantification, because miRNAs often have many homologous sequences. SmiRNA-21, tmiRNA-21, three homologous miRNAs (miRNA-429, miRNA-141 and let-7a) and Random RNA were chosen to evaluate the selectivity of this method for miRNA-21 detection (detailed procedures are described in E8). Figure 5 displayed an obvious R value in the presence of miRNA-21. In contrast, FRET signals were still close to the background in the presence of miRNA-141, miRNA-141 and let-7a. It demonstrated that this method can distinguish single base mismatch in RNA sequences. To explore the practical application in complex biological samples, different concentrations of miRNA-21 were added to 10% FBS to simulate biological samples (detailed procedures are described in E9). RNase inhibitors were added into the serum samples to avoid enzyme degradation, and then the samples were filtered to remove the macromolecules to eliminate the non-specific adsorption. As demonstrated in Table 1, the recovery of miRNA-21 from the serum samples was 96-105.1% and relative standard deviation was as low as 6.5, showing good consistent with the added concentrations. Therefore, this method could effectively eliminate the interference of multiple components coexisting in the serum samples. According to previous studies, miRNAs can stably exist in complex environments such as exosomes and body fluids. They are generally called extracellular miRNAs. The detection of extracellular miRNAs has huge research values for various physiological and pathological processes.45,46 Furthermore, it has also been reported that human cells in culture can export


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miRNAs into the extracellular environment.47 Herein, we utilized this method and qRT-PCR together to evaluate the relative expression levels of miRNA-21 in human cancer cell culture media (detailed procedures are described in E10). Relative signal intensity was based on the expression ratio of miRNA-21 in target cell medium versus that in HeLa cell medium. As shown in Figure 6, the results of this method were coincident with that of qRT-PCR. It was found that three kinds of cancer cell medium existed different expression levels of miRNA-21. In detail, the abundance of miRNA-21 in the HepG-2 cell medium was obviously higher than that in the HeLa cell medium. And the abundance of miRNA-21 in the HeLa cells medium was also higher than that in the SMMC-7721 cell medium. Both results were in good accordance with previous reports about the expression levels of miRNA-21 in different types of cancer cells,48,

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suggesting that this method had a great potential for biomedical application. CONCLUSIONS In summary, a novel method was successfully developed for nucleic acids detection in complex samples. Benefit from MMB-assisted CHA and “DD-A” FRET signal outputs, this method effectively amplified the detectable signal and decreased irrelative interference. In addition, this method achieved sensitive miRNA detection in serum and cell culture media, and the results were in agreement with that of qRT-PCR. However, this method contains tedious procedures that may hinder its practical applications to clinical diagnosis and biomedical application. To further simplify the analysis procedures and develop miniaturized and portable devices, one of the strategies is to integrate with microfluidics.

ASSOCIATED CONTENT Supporting Information.


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The Supporting Information is available free of charge on the ACS Publications website. Sequences of oligonucleotide used in this work, the comparison of this method with other non-protease methods, optimization of the concentration ratio of H1 and H2, optimization of CHA amplification (Step 3), optimization of capture and enrichment (Step 1-2), the performance for DNA-21 detection using this method, the calculation of the total superficial area and the surface coverage of Capture probes. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; *E-mail: [email protected] ORCID Jin Huang: 0000-0002-2890-682X Qing Wang: 0000-0002-7337-0999 Jianbo Liu: 0000-0001-8282-4078 Xiaohai Yang: 0000-0001-8122-7140 Kemin Wang: 0000-0001-9390-4938 Author Contributions †

Fang, H. and Xie, N. contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This work was supported by the National Natural Science Foundation of China (21675047, 21735002 and 21521063), the Key Point Research and Invention Program of Hunan Province (2017DK2011). REFERENCES (1) Bell, J. Nature 2004, 429, 453-456. (2) Zhao, Y.; Chen, F.; Li, Q.; Wang, L.; Fan, C. Chem. Rev. 2015, 115, 12491-12545. (3) Xi, X.; Li, T.; Huang, Y.; Sun, J.; Zhu, Y.; Yang, Y.; Lu, Z. Non-Coding RNA 2017, 3, 9. (4) Graybill, R. M.; Bailey, R. C. Anal. chem.2016, 88, 431-450. (5) Kilic, T.; Erdem, A.; Ozsoz, M.; Carrara, S. Biosens. Bioelectron. 2018, 99, 525-546. (6) Castanotto, D.; Rossi, J. J.; Sarver, N. Adv Pharmacol. 1994, 25, 289-317. (7) Bartel, D. P. Cell 2004, 116, 281-297. (8) Brennecke, J.; Hipfner, D. R.; Stark, A.; Russell, R. B.; Cohen, S. M. Cell 2003, 113, 25-36. (9) Duan, R.; Zuo, X.; Wang, S.; Quan, X.; Chen, D.; Chen, Z.; Jiang, L.; Fan, C.; Xia, F. J. Am. Chem. Soc. 2013, 135, 4604-4607. (10) Siravegna, G.; Marsoni, S.; Siena, S.; Bardelli, A. Nat. Rev. Clin. oncol. 2017, 14, 531-548. (11) Wan, J. C. M.; Massie, C.; Garcia-Corbacho, J.; Mouliere, F.; Brenton, J. D.; Caldas, C.; Pacey, S.; Baird, R.; Rosenfeld, N. Nature Rev. Cancer 2017, 17, 223-238. (12) Schwarzenbach, H.; Hoon, D. S.; Pantel, K. Nature Rev. Cancer 2011, 11, 426-437. (13) Weber, J. A.; Baxter, D. H.; Zhang, S.; Huang, D. Y.; Huang, K. H.; Lee, M. J.; Galas, D. J.; Wang, K. Clin. Chem. 2010, 56, 1733-1741. (14) Chen, J.; Tang, L.; Chu, X.; Jiang, J. Analyst 2017, 142, 3048-3061. (15) Deng, R.; Zhang, K.; Li, J. Acc. Chem. Res. 2017, 50, 1059-1068. (16) Dong, J.; Cui, X.; Deng, Y.; Tang, Z. Biosens. Bioelectron. 2012, 38, 258-263.


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Figure 1. Schematic of a fluorescent method for the detection of nucleic acids in complex samples. Step 1: The hairpin probes, i.e. Captures, immobilized on magnetic microbeads (MMBs) were opened by targets, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Step 2: With the aid of magnetic separation, MMBs were separated and the targets were concentrated from complex samples. Step 3: MMBs were resuspended in the buffer containing hairpin probes H1 and H2, generating numerous H1-H2 duplexes and obvious ratiometric FRET signal. On the contrary, CHA could not happen when the target was absent.


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H1

+

−

+

+

+

+

H2

−

+

+

+

+

+

Trigger

−

−

−

+

−

−

Capture

−

−

−

−

+

+

DNA-21

−

−

−

−

−

+

Figure 2. Gel electrophoresis analysis of CHA product. 1 µM H1, 1 µM H2, 100 nM Trigger, 100 nM Capture and 100 nM DNA-21. The samples were incubated for 3 hours on a thermostatic metal shaker bath (37 °C, 300 rpm). The 2% agarose gel was run at 99 V for 50 min. (+ means in the presence of, − means in the absence of).


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500

Fluorescence (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FAM

No target Target

400 300 200 100 TAMRA

0 520

540

560

580

600

620

640

Wavelength (nm)

Figure 3. The feasibility of “DD-A” FRET-based CHA validated by the fluorescent method. 150 µL SPSC buffer containing 100 nM H1, 100 nM H2, 10 nM Capture and 10 nM DNA-21, was incubated for 3 hours on a thermostatic metal shaker bath (37 °C, 300 rpm).


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2.0

A 4 nM

FA/FD

1.5 1.0 0 nM

0.5 0.0 520

540

560

580

600

620

640

Concentration of miRNA-21 (nM) 2.0

FA/FD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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B

1.5

With enrichment Without enrichment

1.0

0.5 0

1

2

3

4

Concentration of miRNA-21 (nM)

Figure 4. (A) Fluorescence profile corresponding to different concentrations of miRNA-21 (0, 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2 and 4 nM). (B) Calibration curves of acceptor-to-donor ratio (FA/FD) vs miRNA-21 concentration using this method with (solid point) or without (hollow point) MMB enrichment. 3 mL SPSC buffer containing different concentrations of miRNA-21 and 10 pmol immobilized Capture was incubated for 6 hours in a thermostatic shaker (37 °C, 300 rpm). After magnetic separation, MMBs were resuspended in 150 µL SPSC buffer containing 100 nM H1 and 200 nM H2, then incubated for 4 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm).


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4.0

3.0 R

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.0 1.0 21 A- A-21 -21 9 N 2 R N A 1 mi miR iRN NA-4 A-14 t-7a A s tm iR le RN N m miR om nd Ra

2n 1.5 nM 1n M 0.75 M 0.5 n nM M 0 nM

Figure 5. Selectivity evaluation of this method for miRNA-21. The ordinate was effective amplification factor (R). 3 mL SPSC buffer containing different concentrations of targets and 10 pmol immobilized Capture was incubated for 6 hours in a thermostatic shaker (37 °C, 300 rpm). After magnetic separation, MMBs were resuspended in 150 µL SPSC buffer containing 100 nM H1 and 200 nM H2, then incubated for 4 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm).


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Table 1. Detection of miRNA-21 in 10% FBS. Samples

CAdded (nM)

CFound (nM)

Recovery* (%)

RSD (%)

1

0.100

0.105

105.1

3.1 (n=3)

2

0.200

0.196

98.0

4.6 (n=3)

3

0.500

0.480

96.0

6.5 (n=3)

*Recovery (%) = (CFound / CAdded) × 100 %


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Figure 6. Comparison of miRNA-21 levels detected in cell culture media using this method (blue histogram) and qRT-PCR (pink histogram). Relative signal intensity was based on the expression ratio of miRNA-21 in target cell medium versus that in HeLa cell medium. 3 mL filtrate containing 10 pmol immobilized Capture was incubated for 6 hours in a thermostatic shaker (37 °C, 300 rpm). After magnetic separation, MMBs were resuspended in 150 µL SPSC buffer containing 100 nM H1 and 200 nM H2, then incubated for 4 hours on a thermostatic metal shaker bath (37 °C, 1300 rpm).


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For TOC only:


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Detection of Nucleic Acids in Complex Samples via Magnetic

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