Article pubs.acs.org/ac

Highly Sensitive and Selective MicroRNA Detection Based on DNABio-Bar-Code and Enzyme-Assisted Strand Cycle Exponential Signal Amplification Haifeng Dong,† Xiangdan Meng,† Wenhao Dai,† Yu Cao,† Huiting Lu,§ Shufeng Zhou,‡ and Xueji Zhang*,† †

Research Center for Bioengineering and Sensing Technology, University of Science & Technology Beijing, Beijing 100083, P.R. China § Department of Environmental Science and Engineering, School of Chemistry and Environment, Beijing University of Aeronautics & Astronautics, Beijing 100083, P.R. China ‡ Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, Florida 33612, United States ABSTRACT: Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNPDNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

M

real-time polymerase chain reaction (PCR) and microarraybased detection of miRNA16 are widely used for miRNA detection. However, these strategies also suffer from inefficient drawbacks; including low limit of detection (LOD), timeconsumption, expensive equipment and complex operation,17 which limit their biological and biomedical application. Various reliable and sensitive strategies are continuously explored for miRNA detection.18−20 In 2001, Chad A. Mirkin first reported a PCR-free high sensitive and specific detection strategy, termed as DNA-bio-bar-code amplification (BCA) assay, for protein analysis.21 Afterward, various BCA based assays were exploited for DNA and protein analysis.22−27 To our best knowledge, the BCA based miRNA detection has not been explored much owing to unique properties of miRNA. For the miRNA detection, new techniques should be introduced into the BCA system to improve the affinity of

icroRNA (miRNA), a class of endogenous noncode small molecules (18−22nt),1 plays a vital regulated role in gene expression or reverse transcription.2 It is associated with a wide range of biological processes, such as cell proliferation, apoptosis and death.3 The dysregulation of miRNA expressions are related to various diseases, especially human cancers, neurological diseases, viral infections and diabetes.4−7 The significant regulated role in biological and pathological processes make miRNA useful for clinical disease diagnosis, gene therapy and discovery of new anticancer drugs.8 For example, high miRNA-21 expression levels were observed in various malignancies including breast cancer,9 pancreatic cancer,10 brain tumor,11 leukemia,12 colorectal cancer,13 etc. Thus, miRNA-21 expression profiles are emerging as promising biomarkers for diagnosis and prognosis the onset of disease states, and providing an attractive pathway in gene therapy for genetic disorders and potential drug targets.14 However, the detection of miRNA is challenging due to the low abundance, short size, high sequence homology among miRNA family members and susceptibility to degradation.15 Northern blotting, © XXXX American Chemical Society

Received: January 5, 2015 Accepted: April 1, 2015

A

DOI: 10.1021/acs.analchem.5b00029 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Scheme 1. Schematic Illustration of miRNA Detection Based on DNA-Bio-Bar-Code and Enzyme-Assisted Target Cycle Exponential Signal Amplification

Table 1. miRNA and DNA Oligonucleotides Sequence Used in the Experimenta

a

Red letters, LNA; green letters, mismatched bases; underlined blue letters, the recognition sites of the nicking enzyme Nb·BbvCI; orange letters, the hybridization region between probes on Au-NP and the reporter probe.

forming a hairpin structure.31 It can rapidly and specifically detect a given nucleic acid sequence in homogeneous solution. However, the 1:1 hybridization ratio of traditional MB with its target strand limits its sensitivity and expanded application.32 To overcome this, several promising nicking enzyme-assisted signal amplification systems have been reported,33−37 which rely on the cleavage of MB by nicking the enzyme in the presence of a target. The sequence-independent cleavage leads to improved sensitivity without any target sequences restriction,

probes to form a stable sandwich structure with short size miRNA. Furthermore, wide dynamic linear range and high sensitivity remain great challenges because of expression variety3 and low abundance of miRNA.8 Locked nucleic acid (LNA) is a class of bicyclic high-affinity RNA analogues.28,29 It shows at least a 10-fold higher efficiency than traditional DNA probes,30 attracting intense attention in miRNA detection. As a typical homogeneous solution detection strategy, molecular beacon (MB) typically consists of an oligonucleotide sequence complementary to the target and self-complementary ends B

DOI: 10.1021/acs.analchem.5b00029 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

the thiol group-DNA for 1 h at room temperature. Afterward, 1 mL of the AuNPs (1.6 nM) was transferred to solution containing TECP-treated DNA and incubated at room temperature for 24 h. Thirty μL Tris acetate buffer (500 mM, pH 8.2) and 40 μL NaCl/NaPO3 solution was then dropwise added into the vial, with slight shaking to obtain AuNPs-DNA.41 The solution was kept at room temperature for another day before hybridization. Oligonucleotide Functionalization of MNPs. Thirty microliters of MNPs (0.5 mg/mL) were reacted with 30 μL of MNPs probe (100 μM), and 4 mg of EDC was then added to the solution (pH 5.4) for 15 min, followed by adding 1 mg of NHS into the solution and reacting for 8 h in phosphatebuffered saline (PBS) (10 mM, pH 7.4). The resulting MNPsDNA were washed with PBS (10 mM, pH 7.4) three times and stored in 120 μL of PBS (10 mM, pH 7.4) before use. MiRNA-21 Fluorescence Detection. Fifty microliters of AuNPs-DNA (two-component DNA modified), 8 μL of MNPs-DNA (single-component DNA modified), 2.6 μL of 25 μM reported probe (RP), and miRNA-21 (different concentration) were added in NaOH pretreated vial containing 50 μL PBS (10 mM, pH 7.4, 137 mM NaCl), keeping the mixture to hybridize for 1 day at room temperature or 37 °C for 2 h. The resulting sandwich structure was collected by a powerful magnetic field and washed by PBS (10 mM, pH 7.4) three times to remove the unreacted miRNA and AuNPs. Then the solution was heated to 65 °C for 10 min to release the RP, collecting the RP from the supernatant obtained by magnetic separation. Two microliters of MB probe (diluted in annealing buffer containing 10 mM Tris, 50 mM NaCl and 1 mM EDTA, pH 8.0) were added into a 100 μL PCR-tube and incubated for 2 min at 95 °C and cooled down to temperature before using to make the probe perfectly fold into a hairpin structure.37 The 2 μL of RP, 1 μL of Nb·BbvCI (NEB), 5 μL of CutSmart buffer, and 40 μL of PBS (10 mM, pH 7.0, 137 mM NaCl) were added in the solution mentioned above for 1 h at 50 °C. Afterward, PBS buffer (10 mM, pH 7.4, 137 mM NaCl) was added into the solution to an eventual volume of 200 μL. Fluorescence intensity was recorded at 520 nm with an excitation wavelength of 490 nm. MiRNA Analysis in Cell Lysates and Cell Medium. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. The amount of the cells was counted with a hemocytometer. HeLa cells harvested were first washed with PBS (10 mM, pH 7.4) two times. The resulting cells were then recycled three times in 37 °C and liquid nitrogen for 3 min. After this process, 40 μL of chloroform and 200 μL of PBS (10 mM, pH 7.4) were added into the solution containing the cells lysates with violent shaking for 15 s, and then kept at room temperature for 3 min. The solution was then centrifuged at 12000 rpm for 15 min to collect the supernatant. Then, 100 μL isopropanol was added into the supernatant, followed by thorough mixing and keeping the solution at room temperature for 10 min. The mixture was centrifuged at 13000 rpm for 10 min to precipitate the RNA. Finally, the resulting RNA was redispersed in 30 μL of DEPC-treated water. All other detection steps involving miRNA detection in the resulting RNA were the same as mentioned above. For detection miRNA in cell medium, HeLa cells were first cultured in DMEM at 37 °C for 36 h, and the cell media were

showing promising potential in sensitive detection of oligonucleotides. Herein, a highly sensitive and specific miRNA detection strategy was designed by using an exponential signal amplification system based on a BCA system with LNA modified probe and Nb·BbvCI nicking enzyme-assisted strand cycle. As shown in Scheme 1, sandwiching target miRNA with two-component oligonucleotide functionalized gold nanoprobes (AuNPs-DNA) (Scheme 1) and the single-component oligonucleotide functionalized magnetic nanoprobes (MNPsDNA) (Scheme 1) make the AuNPs-DNA separated from solution by a magnetic field. The release of reported probe (RP) hybridized with bio-bar-code DNA immobilized on AuNPs under high temperature can trigger a Nb·BbvCI nicking enzyme-assisted strand cycle, which produces an amplified signal for sensitive miRNA detection. We demonstrate that the exponential signal amplification system renders the proposed assay with high sensitivity, while the sandwich hybridization structure allows the assay to detect the target with high selectivity.38,39 The proposed strategy also shows good performance of analysis miRNA in cell lysates.



EXPERIMENTAL SECTION Materials and Reagents. The Nb·BbvCI nicking enzyme and its CutSmart buffer were purchased from the New England Biolabs. MNPs (20 mg/mL) modified with carboxyl (−COOH) group were provided by Zhengzhou Innosep Biosciences Co., Ltd. (China). 1-(3-(Dimethylamino)propyl)3-ethylcarbodiimidehydrochloride (EDC), disodium ethylenediaminetetraacetate dehydrate (EDTA), N-hydroxysuccinimide (NHS), and tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich (USA). Ultrapure water obtained from a Millipore water purification system (⩾18 MΩ, Milli-Q, Millipore) was used in all runs. Oligonucleotides used in our experiment were purchased from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai). All the RNA sequences were obtained by GenePharma (Shanghai) and diluted in diethy pyrocarbonate (DEPC) water to a concentration of 20 μM for storage. Their sequences were presented in Table 1. Instruments. The morphologies of AuNPs and sandwich structure were examined by transmission electron microscopy (TEM). The UV−visible (UV−vis) spectra measurement was recorded by a UV-1800 spectrometer (Shimadzu, Japan). All the fluorescence experiments were carried out by an F-4500 fluorescence spectrometer (HITACHI, Japan). A Verity 96Well Thermal Cycler-PCR machine (Applied Biosystems, USA) was used for temperature control involved in the enzyme reaction. Synthesis and Oligonucleotide Functionalization of AuNPs. The distillation flask and stir bars were soaked by aqua regia solution and rinsed thoroughly with H2O before use. Two mL of HAuCl4 solution (1 wt %) was added to 48 mL of pure water under stirring, and then heated until boiling. Then 5 mL of sodium citrate solution (1 wt %) was rapidly added to the boiling solution until colorless solution changed to red wine, maintaining 10 min.40 The glass vials were soaked in 12 M NaOH for 1 day and then rinsed with copious amounts of Milllipore water; 12.67 μL of 100 μM bio-bar-code DNA strand and 2.6 μL of 5 μM AuNPs probe (100:1)24 were added into the NaOH pretreated glass vial. Then 1.67 μL acetate buffer (500 mM, pH 5.2) and 2.5 μL TCEP (10 mM) were added to the glass vial to activate C

DOI: 10.1021/acs.analchem.5b00029 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

experiments have been performed to verify the feasibility of enzyme-assisted strand cycle signal amplification. As shown in Figure 2A, the MB displays a weak fluorescence background

collected by centrifugation. One microliter of the culture medium from different amount of original HeLa cells was added in PBS (10 mM, pH 7.4, 137 m M NaCl) solution containing 50 μL of AuNPs-DNA, 8 μL of MNPs-DNA, and 2.6 μL of 25 μM RP in NaOH pretreated vial to a volume of 100 μL, keeping the mixture to hybridize for 37 °C for 2 h. All the other detection steps were the same as mentioned above.



RESULTS AND DISCUSSION Characterization of Functionalized Nanoprobe. The as-prepared AuNPs and the sandwich structure were characterized by TEM. As shown in Figure 1A, the generated AuNPs Figure 2. (A) Fluorescence response of PBS (10 mM, pH 7.4, 137 mM NaCl) solution containing (a) 2 μL of MB (10 μM), (b) 2 μL of MB (10 μM), and 2 μL of RP (0.4 μM), and (c) 2 μL of MB (10 μM), 2 μL of RP (0.4 μM), and 2 μL of Nb·BbvCI (10000 U) in 1 h. (B) The corresponding real-time kinetic response of these three solutions. The statistical significance (P < 0.05) was determined by the two-tailed Student t test (*P < 0.05, **P < 0.01, ***P < 0.001), as compared with the control at the same time point.

signal of about 44.4 (curve a), upon addition of RP, the fluorescence intensity sharply increase to 92.7, indicating the RP can efficiently recognize the MB and separate the fluorescent dye from the quencher (curve b). The introduction of Nb·BbvCI can further cause the increase of the fluorescence intensity, leading to a 3.2-fold higher intensity than MB (curve c). It demonstrates that the hybridization RP with MB induces the exposing of the active site for Nb·BbvCI recognition, which further efficiently triggers the strand cycle for signal amplification. The corresponding real-time kinetic study of these solutions mentioned above have also been investigated (Figure 2B). The fluorescence intensity increased sharply until the reaction time increased to 60 min and achieved to a plateau after 60 min, which indicated the hybridization reaction had been finished at 60 min. Optimization of Experimental Conditions. The choice of the temperature for RP release is significant. It should give RP the ability to be efficiently released from the sandwich structure to obtain the high signal-to-noise ratio. As shown in Figure 3A, the fluorescence intensity increased along with the increase of release temperature up to 65 °C, and further

Figure 1. Characterization of AuNPs and MNPs functionalized with DNA: (A) TEM of AuNPs, (B) sandwich structure, (C) UV−visible spectra of Au and AuNPs-DNA, and (D) MNPs and MNPs-DNA.

have a uniform spherical crystallite with an average diameter of 18 nm. The UV−vis spectra were used to characterize the functionalization of the AuNPs-DNA42 and MNPs-DNA probe. The as-prepared AuNPs shows an obvious absorbance peak at 523 nm and after functionalizing with DNA, a 4 nm red shift associated with the change of localized surface plasmon resonance effect was observed in the AuNPs-DNA nanoprobe, which indicated DNA was successfully modified on AuNPs (Figure 1C).43 The comparison of the UV−vis spectrum of MNPs and MNPs-DNA is shown in Figure 1D; a strong characteristic absorbance peak of DNA located at 258 nm was observed in MNPs-DNA in comparison with MNPs, revealing the DNA was efficiently modified on the MNPs surface.44 In the presence of the target, the AuNPs-DNA and MNPs-DNA probe can hybridize with part of the target to form a sandwich structure. As shown in Figure 1B, the TEM image showed that each 57 nm MNP was surrounded by several 18 nm AuNPs to form a core−shell analogous structure. The free AuNPs-DNA observed resulted from the separation of the sandwich during the TEM operation process. Enzyme-Assisted Strand Cycle Signal Amplification. Normally, the DNA BCA assay can detect a target oligonucleotide sequence down to fM level; some of them achieve aM level. It is competitive for DNA detection but still does not work well for miRNA analysis due to the low abundant level of miRNA. Keeping this issue in mind, we introduce a novel enzyme-assisted strand cycle signal amplification into DNA-BCA based assay to further improve the sensitivity for miRNA detection. Fluorescence intensity

Figure 3. Influence of (A) RP release temperature, (B) nicking enzyme reaction temperature, (C) nicking enzyme reaction time, and (D) amount of Nb·BbvCI to the proposed miRNA detection strategy. D

DOI: 10.1021/acs.analchem.5b00029 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

exponential amplification assay.37 The high sensitivity can be explained that each target is responding to hundreds of RP, and the RP can further trigger numerous endonuclease-assisted strand cycles to produce exponential amplified fluorescence signal for detection. Specificity is another vital factor to evaluate a biosensor. The specificity of this strategy for miRNA detection was verified by measuring the fluorescence intensity response to three types of miRNA sequences including complementary target (CM, 300 fM), single-base mismatched strand (SM, 2 nM) and threebases mismatched strand (TM, 2 nM) in the same condition. As shown in Figure 5, the fluorescence intensity showed

increasing of the temperature caused the slight decrease of the intensity, which may have resulted from the introduction of some AuNPs-DNA dissociated from the sandwich structure at high temperature quenching the fluorescent dye. Thus, 65 °C was selected in the following experiments. The reaction temperature, the reaction time and the amount of Nb·BbvCI involved in the enzyme-assisted strand cycle also influenced the performance of the proposed assay. Figure 3B revealed the fluorescence intensity increased while the reaction temperature of enzyme-assisted strand cycle was raised up to 49 °C, and flatted in the range from 49 to 55 °C. However, the fluorescence intensity decreased sharply after 55 °C, which was attributed to the optimal reaction temperature of Nb·BbvCI and release efficiency of RP from the RP/MB duplex structure (Scheme 1). The effect of reaction time is shown in Figure 3C. The largest fluorescence intensity was obtained at 60 min and the reaction time of 60 min was regarded as the optimized condition. As shown in Figure 3D, the fluorescence intensity increased with the amount of Nb·BbvCI (10000 U) in the range from 0 to 1 μL and reached to a plateau at 1 and 1 μL Nb·BbvCI was chosen as the optimal volume of nicking enzyme. Assay Performance for MiRNA Detection. Under optimized conditions, this strategy for miRNA detection was evaluated. To avoid the influence of background to the fluorescence response, the fluorescence intensity was normalized as relative fluorescence intensity change F/F0 to investigate the proposed assay PL response to miRNA, where F and F0 are FAM intensities of MB at 523 nm in the presence and absence of the target, respectively. As shown in Figure 4A, the

Figure 5. Fluorescent intensity of this strategy after incubation with control, complementary target (CM, 0.3 pM), single-base mismatch strand (SM, 2 nM), three-base mismatch strand (TM, 2 nM), and miRNA-1 (2 nM), miRNA-16 (2 nM), miRNA-24 (2 nM), miRNA26a (2 nM), as well as pre-miRNA-21 (2 nM). The statistical significance (P < 0.05) was determined by the two-tailed Student t test (*P < 0.05, **P < 0.01, ***P < 0.001), as compared with the control.

negligible changing even after addition of SM and TM at a concentration of 2 nM, while the CM exhibited a strong fluorescent intensity at the concentration of 300 fM (Figure 5A), which was 2.7-fold and 2.8-fold higher than the intensity of the SM and TM, respectively (Figure 5B). The results clearly demonstrated that the proposed assay had excellent discrimination capability for a sequence with base-mismatch, which may be attributed to the unique sandwich structure in the DNA-BCA.21 Furthermore, the capability of the proposed assay for distinguishing the premature and mature miRNA-21 as well as the common interference from other miRNAs46 were also investigated. As shown in Figure 5C, the miRNA-1, miRNA-16, miRNA-24, miRNA-26a, and pre-miRNA-21 presented slight fluorescence intensity change compared to the fluorescence intensity change of CM at the same concentration (2 nM), which was approximately 2.5-flod higher than the intensity of the other miRNAs and pre-miRNA-21 detected with this strategy (Figure 5D). These results indicated that this proposed method had high specificity and promising application for miRNA detection in real samples. MiRNA Detection in Cell Lysates. The feasibility of this strategy for miRNA detection in a real sample was also investigated by analyzing the miRNA extracted from HeLa cells. As shown in Figure 6A, the fluorescence intensity increased

Figure 4. (A) Fluorescent response to the different concentrations of miRNA-21: (a) Control, (b−i) 30 zM to 0.3 pM. (B) The linear correlation between the fluorescence intensity and the logarithm of miRNA-21 concentration in the range from3 aM to 0.3 pM. The statistical significance (P < 0.05) was determined by the two-tailed Student t test (*P < 0.05, **P < 0.01, ***P < 0.001), as compared with the control.

fluorescence intensity increased with the increase of miRNA21 concentration and the relative fluorescence intensity change F/F0 showed a good linear relationship with the logarithm of miRNA-21 concentration in the range from 3 aM to 0.3 pM (Figure 4B). The regression equation between fluorescence intensity change F/F0 and miRNA concentration (M) is F /F0 (au) = 3.7906 + 0.1329lgC(M ), R2 = 0.9967

where R2 is the correlation coefficient, and the statistical significance (P < 0.05) was determined by the two-tailed Student t test. The LOD was calculated by using three times the standard deviation of the control according to previous report,45 which was estimated down to 52.5 zM. It is superior to those of DNABCA based DNA or protein assay,21−27 or some other E

DOI: 10.1021/acs.analchem.5b00029 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 6. (A) Fluorescence spectrum response to the different HeLa cell extractive: (a) Control, (b) 3 × 103, (c) 3 × 104, (d) 3 × 105, (e) 3 × 106, (f) 3 × 107. (B) The linear correlation between the fluorescence intensity and the logarithm of the number of HeLa cells in the range of 3 × 103−3 × 107. (C) Fluorescence intensity plots to the culture medium from (a) 3 × 103, (b) 3 × 104, and (c) 3 × 105 HeLa cells. The statistical significance (P < 0.05) was determined by the two-tailed Student t test (*P < 0.05, **P < 0.01, ***P < 0.001), as compared with the control.



with the increment of HeLa cells amount, which exhibited a good linear relationship with the HeLa amount in the range from 3 × 103 to 3 × 107 (Figure 6B). Meanwhile, the detection for miRNA-21 in culture medium from different amount of HeLa cells was presented as Figure 6C. Upon increasing the amount of HeLa cells, the fluorescence intensity change F/F0 ratio also increased, indicating that the feasibility of the proposed approach in detection of miRNA in culture medium. These results suggested the proposed strategy can sensitively detect miRNA extracted from 3000 HeLa cells, which indicated it was powerful for miRNA detection in real samples and had promising potential in clinical application.

(1) Dong, H. F.; Jin, S.; Ju, H. X.; Hao, K. H.; Xu, L. P.; Lu, H. T.; Zhang, X. J. Anal. Chem. 2012, 84, 8670−8674. (2) Johnson, B. N.; Mutharasan, R. Analyst 2014, 139, 1576−1588. (3) Gregory, R. I.; Yan, K. P.; Amuthan, G.; Chendrimada, T.; Doratotaj, B.; Cooch, N.; Shlekhattar, R. Nature 2004, 432, 235−240. (4) Campuzano, S.; Pedrero, M.; Pingarrón, J. M. Anal. Bioanal. Chem. 2014, 406, 27−33. (5) Ryoo, S. R.; Lee, J.; Yeo, J.; Na, H. K.; Kim, Y. K.; Jang, H.; Lee, J. H.; Han, S. W.; Lee, Y.; Kim, V. N.; Min, D. H. ACS Nano 2013, 7, 5882−5891. (6) Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem. 2008, 80, 2319−2325. (7) Bi, S.; Zhang, J. L.; Hao, S. Y.; Ding, C. F.; Zhang, S. S. Anal. Chem. 2011, 83, 3696−3702. (8) Duan, R. X.; Zuo, X. L.; Wang, S. T.; Quan, X. Y.; Chen, D. L.; Chen, Z. F.; Jiang, L.; Fan, C. H.; Xia, F. J. Am. Chem. Soc. 2013, 135, 4604−4607. (9) Volinia, S.; Calin, G. A.; Liu, C. G.; Ambs, S.; Cimmino, A.; Petrocca, F.; Visone, R.; Iorio, M.; Roldo, C.; Ferracin, M.; Prueitt, R. L.; Yanaihara, N.; Lanza, G.; Scarpa, A.; Vecchione, A.; Negrini, M.; Harris, C. C.; Croce, C. M. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 2257−2261. (10) Lee, E. J.; Gusev, Y.; Jiang, J.; Nuovo, G. J.; Lerner, M. R.; Frankel, W. L.; Morgan, D. L.; Postier, R. G.; Brackett, D. J.; Schmittgen, T. D. Int. J. Cancer 2006, 120, 1046−1054. (11) Chan, J. A.; Krichevsky, A. M.; Kosik, K. S. Cancer Res. 2005, 65, 6029−6033. (12) Loffler, D.; Brocke-Heidrich, K.; Pfeifer, G.; Stocsits, C.; Hackermuller, J.; Kretzschmar, A. K.; Burger, R.; Gramatzki, M.; Blumert, C.; Bauer, K.; Cvijic, H.; Ullmann, A. K.; Stadler, P. F.; Horn, F. Blood 2007, 110, 1330−1333. (13) Schetter, A. J.; Leung, S. Y.; Sohn, J. J.; Zanetti, K. A.; Bowman, E. D.; Yanaihara, N.; Yuen, S. T.; Chan, T. L.; Kwong, D. L.; Au, G. K. J. Am. Med. Assoc. 2008, 299, 425−436. (14) Wu, K. L.; Li, L. W.; Li, S. Y. Tumour Biol. 2014, 1−9. (15) Xi, Q.; Zhou, D. M.; Kan, Y. Y.; Ge, J.; Wu, Z. K.; Yu, R. Q.; Jiang, J. H. Anal. Chem. 2014, 86, 1361−1365. (16) Várallyay, É.; Burgyán, J.; Havelda, Z. Nat. Protoc. 2008, 3, 190− 196. (17) Zhang, Q.; Chen, F.; Xu, F.; Zhao, Y. X.; Fan, C. H. Anal. Chem. 2014, 86, 8098−8105. (18) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503− 1506. (19) Qavi, A. J.; Bailey, R. C. Angew. Chem., Int. Ed. 2010, 122, 4712− 4715. (20) Li, J.; Yao, B.; Huang, H.; Wang, Z.; Sun, C. H.; Fan, Y.; Chang, Q.; Li, S. L.; Wang, X.; Xi, J. Z. Anal. Chem. 2009, 81, 5446−5451. (21) Nam, J. M.; Park, S. J.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 3820−3821. (22) Jaffrezic-Renault, N.; Martelet, C.; Chevolot, Y.; Cloarec, J. P. Sensors 2007, 7, 589−614.



CONCLUSION In conclusion, an ultrasensitive and selective fluorescence biosensor for miRNA-21 detection based on DNA BCA and Nb·BbvCI assisted strand cycle exponential signal amplification was designed. In this strategy, each target miRNA is responding to hundreds of RP, and the RP can trigger strand cycle amplification to unfold numerous MBs and produce an exponential amplified fluorescent signal for detection. Under the optimized experimental condition, this assay exhibited linear range over 6 orders of magnitude with LOD down to zeptomolar level for miRNA detection, and excellent discrimination for single-base, three-bases mismatched miRNA and complementary target. Moreover, the feasibility of miRNA detection in HeLa lysates was demonstrated. Given the high sensitivity, selectivity and reliability for real sample analysis, this proposed strategy has promising potential application in biology research, clinical disease diagnosis or discovery of new anticancer drugs.



REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel./fax: +86 10 82376993. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The work was supported by National Natural Science Foundation of China (NSFC Grant No. 21305008, 21475008, 21275017, 21127007), China Postdoctoral Special Foundation (No. 11175039), and Ph.D. Programs Foundation of Ministry of Education of China (No.11170197), the Fundamental Research Funds for the Central Universities (No. 06199045) and the Chinese 1000 Elites program and USTB start-up fund. F

DOI: 10.1021/acs.analchem.5b00029 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry (23) Thaxton, C. S.; Hill, H. D.; Georganopoulou, D. G.; Stoeva, S. I.; Mirkin, C. A. Anal. Chem. 2005, 77, 8174−8178. (24) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932−5933. (25) Hill, H. D.; Mirkin, C. A. Nat. Protoc. 2006, 1, 324−336. (26) Nam, J. M.; Thaxton, S.; Mirkin, C. A. Science 2003, 301, 1884− 1886. (27) Hill, H. D.; Vega, R. A.; Mirkin, C. A. Anal. Chem. 2007, 79, 9218−9223. (28) Kloosterman, W. P.; Wienholds, E.; de Bruijn, E.; Kauppinen, S.; Plasterk, R. H. A. Nat. Methods 2006, 3, 27−29. (29) Castoldi, M.; Schmidt, S.; Benes, V.; Noertholm, M.; Kulozik, A. E.; Hentze, M. W.; Muckenthaler, M. U. RNA 2006, 12, 913−920. (30) Dong, H. F.; Lei, J. P.; Ding, L.; Wen, Y. Q.; Ju, H. X.; Zhang, X. J. Chem. Rev. 2013, 113, 6207−6233. (31) Hartig, J. S.; Grüne, I.; Najafi-Shoushtari, S. H.; Famulok, M. J. Am. Chem. Soc. 2004, 126, 722−723. (32) Zuo, X. L.; Xia, F.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 1816−1818. (33) Hu, Y. H.; Xu, X. Q.; Liu, Q. H.; Wang, L.; Lin, Z. Y.; Chen, G. N. Anal. Chem. 2014, 86, 8785−8790. (34) Li, L.; Wang, Q.; Feng, J.; Tong, L. L.; Tang, B. Anal. Chem. 2014, 86, 5101−5107. (35) Song, Y.; Li, W. K.; Duan, Y. F.; Li, Z. J.; Deng, L. Biosens. Bioelectron. 2014, 55, 400−404. (36) Zhou, D. M.; Du, W. F.; Xi, Q.; Ge, J.; Jiang, J. H. Anal. Chem. 2014, 86, 6763−6767. (37) Wang, G. L.; Zhang, C. Y. Anal. Chem. 2012, 84, 7037−7042. (38) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547−1562. (39) Lei, J. P.; Ju, H. X. Chem. Soc. Rev. 2012, 41, 2122−2134. (40) Xia, H. B.; Bai, S.; Hartmann, J.; Wang, D. Y. Langmuir 2009, 26, 3585−3589. (41) Liu, J. W.; Lu, Y. Nat. Protoc. 2006, 1, 246−252. (42) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79, 4215−4221. (43) Choi, J. H.; Kim, H.; Choi, J. H.; Choi, J. W.; Oh, B. K. Acs. Appl. Mater. Inter. 2013, 5, 12023−12028. (44) Zhu, X. L.; Han, K.; Li, G. X. Anal. Chem. 2006, 78, 2447−2449. (45) Wang, Y. X.; Li, J. S.; Jin, J. Y.; Wang, H.; Tang, H. X.; Yang, R. H.; Wang, K. M. Anal. Chem. 2009, 81, 9703−9709. (46) Yin, C.; Salloum, F. N.; Kukreja, R. C. Circ. Res. 2009, 104, 572−575.

G

DOI: 10.1021/acs.analchem.5b00029 Anal. Chem. XXXX, XXX, XXX−XXX

Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-as...
480KB Sizes 2 Downloads 6 Views