LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUSLY DETECTING MULTIPLE MICRORNAS BASED ON DUPLEX-SPECIFIC NUCLEASE (DSN) CYCLIC AMPLIFICATION TECHNOLOGY

Abstract

A liquid chromatography method for simultaneously detecting multiple microRNAs based on a duplex-specific nuclease (DSN) cyclic amplification technology comprises the following steps: designing a fluorophore-modified single-stranded DNA probe according to a target microRNA to be detected and loading the probe onto a surface of a streptavidin-coated magnetic bead (MB) to serve as a detection probe; adding a target microRNA sample to be detected and DSN to the detection probe, fully mixing the same, and incubating the mixture; after the incubation, completely removing the magnetic bead and the unreacted DNA probe to obtain a separated solution; and injecting the separated solution into a high-performance liquid chromatography system for separation and quantification.

Claims

1. A liquid chromatography method for simultaneously detecting multiple microRNAs based on a duplex-specific nuclease (DSN) cyclic amplification technology, comprising the following steps: (1) designing a fluorophore-modified single-stranded DNA probe according to a target microRNA to be detected and loading the probe onto a surface of a streptavidin-coated magnetic bead (MB) to serve as a detection probe; (2) adding a target microRNA sample to be detected and DSN to the detection probe, fully mixing the same, and incubating the mixture; (3) after the incubation, completely remove the magnetic bead and the unreacted DNA probe to obtain a separated solution; and (4) injecting the separated solution into a high-performance liquid chromatography system for separation and quantification.

2. The liquid chromatography method for simultaneously detecting multiple miRNAs based on a DSN cyclic amplification technology according to claim 1, wherein in step (1), a ratio of a molar amount of a streptavidin binding site coated on the magnetic bead and a molar amount of the DNA probe is (3-5):1.

3. The liquid chromatography method for simultaneously detecting multiple microRNAs based on a DSN cyclic amplification technology according to claim 1, wherein in step (1), the loading process is performed in a 2×B&W buffer solution and the buffer solution is prepared from Tris-HCl, EDTA, and NaCl.

4. The liquid chromatography method for simultaneously detecting multiple microRNAs based on a DSN cyclic amplification technology according to claim 1, wherein in step (2), the target microRNA to be detected is selected from a combination of two, three or more of different target miRNAs; and the target miRNA is a miRNA with 18-25 nucleotides.

5. The liquid chromatography method for simultaneously detecting multiple microRNAs based on a DSN cyclic amplification technology according to claim 1, wherein in step (2), the target miRNA is selected from the group consisting of miRNA-122, miRNA-155, and miRNA-21, and the corresponding single-stranded DNA probe in step (1) is selected from the group consisting of P122, P155, and P21.

6. The liquid chromatography method for simultaneously detecting multiple miRNAs based on a DSN cyclic amplification technology according to claim 1, wherein in step (2), the incubation is performed at 36-38° C. for 140-160 min.

7. The liquid chromatography method for simultaneously detecting multiple microRNAs based on a DSN cyclic amplification technology according to claim 1, wherein in step (3), the magnetic bead and the unreacted DNA probe are completely removed using a permanent magnet to reduce a background interference.

8. The liquid chromatography method for simultaneously detecting multiple microRNAs based on a DSN cyclic amplification technology according to claim 1, wherein in step (4), the high-performance liquid chromatography system uses a C18 reverse phase chromatographic column and a gradient elution mode.

9. The liquid chromatography method for simultaneously detecting multiple microRNAs based on a DSN cyclic amplification technology according to claim 8, wherein the gradient elution mode is that a proportion of methanol is changed from 10% to 60% in 20 min; and a mobile phase consists of an organic phase and an aqueous phase containing TEAA.

10. The liquid chromatography method for simultaneously detecting multiple microRNAs based on a DSN cyclic amplification technology according to claim 1, wherein processes of the method are all performed in a dark place.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a mechanism diagram of a high- performance liquid chromatography analysis method for simultaneously detecting miRNA-122, miRNA-155, and miRNA-21 based on a DSN cyclic amplification technology;

[0029] FIG. 2A shows chromatograms of DNA probes, the chromatogram of the DNA probes with different base sequences of the same length (25 nt);

[0030] FIG. 2B shows chromatograms of DNA probes, the chromatogram of the DNA probes with the same base sequence (cytosine C and guanine G) of different lengths;

[0031] FIG. 2C shows chromatograms of DNA probes, the chromatogram of the DNA probes with the same base sequence (thymine T) of different lengths;

[0032] FIG. 3 is a graph of fluorescence signals verifying feasibility of simultaneously detecting miRNA-122, miRNA-155, and miRNA-21,

[0033] fluorescence signals of (a) blank, (b) 0.4 U DSN, (c) 100 pM miRNA-122+100 pM miRNA-155+100 pM miRNA-21, (d) 100 pM miRNA-122+0.4 U DSN, (e) 100 pM miRNA-155+0.4 U DSN, (f) 100 pM miRNA-21+0.4 U DSN, and (g) 100 pM miRNA-122+100 pM miRNA-155+100 pM miRNA-21+0.4 U DSN, experimental conditions: 100 nM of DNA probes, 25 mM of Mg.sup.2+, pH 8.0 and incubation at 40° C. for 180 min, and error bars represent standard deviation of three independent experiments;

[0034] FIG. 4A shows optimization of experimental conditions for 100 pM of target miRNA-122, miRNA-155 and miRNA-21, and 100 nM of DNA probes: a concentration of DSN from 0.1 U to 0.5 U;

[0035] FIG. 4B shows optimization of experimental conditions for 100 pM of target miRNA-122, miRNA-155 and miRNA-21, and 100 nM of DNA probes: a pH value of a buffer solution from 7 to 9;

[0036] FIG. 4C shows optimization of experimental conditions for 100 pM of target miRNA-122, miRNA-155 and miRNA-21, and 100 nM of DNA probes: a concentration of mg.sup.2+ from 15 mM to 35 mM;

[0037] FIG. 4D shows optimization of experimental conditions for 100 pM of target miRNA-122, miRNA-155 and miRNA-21, and 100 nM of DNA probes: chromatograms of probes corresponding to different concentrations of mg.sup.2+;

[0038] FIG. 4E shows optimization of experimental conditions for 100 pM of target miRNA-122, miRNA-155 and miRNA-21, and 100 nM of DNA probes: an incubation temperature from 30° C. to 50° C.;

[0039] FIG. 4F shows optimization of experimental conditions for 100 pM of target miRNA-122, miRNA-155 and miRNA-21, and 100 nM of DNA probes: incubation time from 60 min to 210 min;

[0040] FIG. 5A show calibration curves for three miRNAs: miRNA-122; experimental conditions: 100 nM of DNA probes, 25 mM of Mg.sup.2+, 0.4 U DSN, pH 8.0 and incubation at 40° C. for 180 min; and error bars represent standard deviation of three independent experiments;

[0041] FIG. 5B show calibration curves for three miRNAs: miRNA-155; experimental conditions: 100 nM of DNA probes, 25 mM of Mg.sup.2+, 0.4 U DSN, pH 8.0 and incubation at 40° C. for 180 min; and error bars represent standard deviation of three independent experiments;

[0042] FIG. 5C show calibration curves for three miRNAs: miRNA-21; experimental conditions: 100 nM of DNA probes, 25 mM of Mg.sup.2+, 0.4 U DSN, pH 8.0 and incubation at 40° C. for 180 min; and error bars represent standard deviation of three independent experiments;

[0043] FIG. 6 is a graph for selectively detection of miRNA-122, miRNA-155, and miRNA-21: peak areas for M1 (single base mismatch), M2 (double base mismatch) and NM (complete mismatch), interfering miRNA at a concentration of 10 nM, and each miRNA at a concentration of 100 pM; experimental conditions: 100 nM of DNA probes, 25 mM of Mg.sup.2+, 0.4 U DSN, pH 8.0 and incubation at 40° C. for 180 min; and error bars represent standard deviation of three independent experiments; and

[0044] FIG. 7 shows chromatograms of an original sample and a serum sample containing a certain amount of miRNAs and the established method is used to detect the chromatograms of the original sample and the spiked serum sample; and experimental conditions: 100 nM of DNA probes, 25 mM of Mg.sup.2+, 0.4 U DSN, pH 8.0 and incubation at 40° C. for 180 min.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The method of the present disclosure will be described in more detail below with reference to the accompanying drawings and specific examples.

[0046] A liquid chromatography method for detecting multiple microRNAs based on a DSN cyclic amplification technology comprises the following steps:

[0047] Firstly adding 40 .Math.L of streptavidin-coated magnetic beads (MBs) with a particle size of 300 nm into a 1.5-mL brown polypropylene centrifuge tube, performing magnetic separation to remove a solvent, and retaining the MBs in the tube; after washing 3 times with a 1xB&W buffer solution, redispersing the MBs in 90 .Math.L of a 2xB&W buffer solution, and adding 5 .Math.L of 10 .Math.M of DNA probes of different lengths (in the example, P122 is 37 nt, P155 is 35 nt, and P21 is 58 nt); gently vortexing at a room temperature for 15 min to ensure adequate binding of biotin on the probes and streptavidin from the MBs performing magnetic separation to obtain a supernatant and fluorescence detection at an excitation wavelength of 495 nm and an emission wavelength of 518 nm; estimating the coupling efficiency between the probes and the MBs according to fluorescence data; wherein it is estimated that there are about 1.16x10.sup.4 DNA probes (the sum of P122, P155, and P21 coupled to each MB accounts for about 12% of the total capacity of the MBs), and thus, there is a sufficient space for target miRNA hybridization and DSN cleavage; and finally, washing and dispersing the obtained long and short DNA probes and MB conjugates in a hybridization buffer solution, and storing the same at 4° C. for a standby application.

[0048] Before the target miRNAs are detected, all experimental conditions are optimized, including conjugation time, the dose of DSN, concentration of Mg.sup.2+, pH of buffer solution, and incubation temperature and time. The optimization results are shown in FIG. 4.

[0049] It can be seen from FIG. 4A that the peak area of the fluorescence signal increases significantly when the dose of DSN is changed from 0.10 U to 0.50 U, while the peak area of the fluorescence signal of P122, P155, and P21 all tends to be stable when DSN > 0.40 U. To improve detection sensitivity and keep costs low maximally, 0.4 U is used as the optimal dose of DSN, which is sufficient to detect miRNAs as low as a femtomolar level. The pH value will strongly affect the cleavage rate of the DNA probes and thus affect measured amplification capacity. To maximally improve the amplification capacity, the pH value of the sample solution is adjusted from 7 to 9. It is reported that the DSN has a working range of 3.5 to 8.5 with the maximum activity at a pH of about 6.6 and the DSN is deactivated when pH value < 3.0 or > 9.0. In addition, the pH value of the solution also has a significant effect on the fluorescence of the fluorescein labels. In an acidic medium, the fluorescein is typically protonated. Instead, the fluorescein is deprotonated in an alkaline medium. Both protonated and deprotonated states change the conformation of the fluorescein, and thus the fluorescence efficiency is reduced. According to the observation of our experiment (FIG. 4B), pH 8.0 is found to be the best choice for the method. The presence of Mg.sup.2+ is of great significance in promoting the cleavage of the DSN and the efficient hybridization of the miRNA/DNA heteroduplexes. Therefore, the concentration of Mg.sup.2+ plays a key role in detecting miRNAs. The present disclosure evaluates the effect of the parameter and the results are shown in FIG. 4C and D. It can be seen that the peak area increases with the increase of the content of Mg.sup.2+, but the peak area decreases significantly when the concentration of Mg.sup.2+ is above 25 mM. The possible reason is that the DSN activity is reduced at high ionic strength. In view of these results, 25 mM Mg.sup.2+ is selected as the optimum concentration for the measurement. Another important variable is the incubation temperature of the reaction. The incubation temperature directly affects the hybridization efficiency and the cleavage activity of the DSN. In practical use, a melting temperature should be 10-15° C. higher than a hybridization temperature to achieve high hybridization efficiency while ensuring good selectivity. Under the current experimental conditions, the melting temperatures of miRNA-122, miRNA-155, and miRNA-21 are estimated to be about 53° C., 54° C., and 51° C., separately. Therefore, theoretically, the experiment should be performed below 45° C. The cleavage by the DSN is good at a temperature above 45° C. and has an optimum temperature of 60° C. As shown in FIG. 4E, the peak areas of the three miRNAs reach a maximum at 40° C. Therefore, 40° C. is used as the incubation temperature for the measurement. A series of incubation time from 60 min to 210 min is used in the experiment. FIG. 4F shows that as the incubation time increases from 60 min to 180 min, the peak area of the fluorescence signal increases rapidly, after which the peak areas of the three miRNAs tend to be stable. These results indicate that 180 min of incubation is sufficient to cleave the DNA probes. In view of the reaction efficiency, since no significant difference is observed after a longer incubation time, 180 min is selected as the optimal time for a subsequent experiment.

[0050] The method used for optimizing all the examples of the present disclosure is as follows: 44 .Math.L of 100 nM of P122, P155, and P21 are added to a centrifuge tube, followed by 0.4 U of DSN and 5 .Math.L of target miRNAs. Then a short shaking treatment is performed for about 2 s to mix the reaction mixture fully. After incubation at 40° C. for 180 min, MBs are separated with a permanent magnet along with unreacted DNA probes. Finally, the obtained supernatant is injected into a high-performance liquid chromatography system for separation and quantification.

[0051] The high-performance liquid chromatography is performed using a Shimadzu LC-20A system equipped with a Shimadzu RP-20A fluorescence detector. Data acquisition and processing are completed using an LCsolution data analysis software (free version). A clarity reverse phase chromatographic column from Phenomenex (50×4.6 mm (internal diameter) and 3 .Math.m particle size) is used for separating miRNAs. A column temperature is maintained at 35° C. A gradient elution mode is used. The parameters of the fluorescence detector are set as an excitation wavelength of 495 nm and an emission wavelength of 518 nm.

[0052] The 1xB&W buffer solution required for an experiment at a pH of 7.5 is prepared from 5.0 mM of Tris-HCl, 0.5 mM of EDTA, and 1.0 M of NaCl. The 2xB&W buffer solution required for an experiment at a pH of 7.5 is prepared from 10.0 mM of Tris-HCl, 1.0 mM of EDTA, and 2.0 M of NaCl. The hybridization buffer solution required from an experiment at a pH of 8 is prepared from 50 mM of Tris-HCl and 25 mM MgCl.sub.2.

[0053] A stationary phase of the chromatographic column in the experiment is octadecyl (C18). The C18 column is a typical reverse phase (RP) column and commonly used to retain and separate hydrophobic compounds. However, oligonucleotides are strongly polar and difficult to retain in any RP columns. Therefore, 100 mM of TEAA is added as an ion-pairing reagent in a mobile phase to make the retention time of the DNA probes on the column longer.

[0054] In order to prevent exposure to light that could adversely affect the fluorescent properties of fluorophores, all the steps related to the fluorophores are performed in aluminium foil-wrapped centrifuge tubes.

[0055] The gradient elution mode used in the experiment is that a proportion of methanol is changed from 10% to 60% in 20 min, and a flow rate is 1 mL/min.

[0056] The mobile phase in the experiment consists of an organic phase of methanol and an aqueous phase of 100 mM of a TEAA aqueous solution and 5% acetonitrile.

[0057] The method of the present disclosure simultaneously detects multiple miRNAs. In the example: miRNA-122 has the limit of detection of 0.39 fM, miRNA-155 of 0.30 fM, and miRNA-21 of 0.26 fM with a linear range all of 1.0 fM to 10 pM. The established method is successfully used to detect miRNA-122, miRNA-155, and miRNA-21 in serum samples of patients with lupus erythematosus, cervical cancer, and ovarian cancer, and healthy people.

[0058] The method is used to detect miRNA-122, miRNA-155, and miRNA-21 in serum samples of healthy people, and patients with lupus erythematosus, cervical cancer, and ovarian cancer. Specific examples are as follows:

Example 1 Detection of miRNA-122, miRNA-155, and miRNA-21 in Serum Samples of Healthy People

[0059] The detection results of miRNA-122, miRNA-155, and miRNA-21 in serum samples of healthy people were shown in Table 1. Concentrations of miRNA-122, miRNA-155, and miRNA-21 were detected to be 0.063 pM, 0.057 pM, and 0.046 pM separately in serum sample 1 (healthy volunteers), and 0.067 pM, 0.053 pM, and 0.048 pM separately in serum sample 2 (healthy volunteers). To evaluate the effect of matrices, different concentrations of miRNA-122, miRNA-155, and miRNA-21 were spiked into sample 1 to obtain a good relative recovery of 101.7%-104.7% and a relative standard deviation (RSD) of 2.4 %-4.7%. These results were consistent with those of qRT-PCR. The results clearly demonstrated that the provided method had good practicability for analyzing multiple miRNAs in practical samples.

TABLE-US-00001 Detection of miRNA-122, miRNA-155, and miRNA-21 in serum samples of healthy people miRNA-122 (pM) miRNA-155 (pM) miRNA-21 (pM) Sample Spiked The method (RSD%) Relative recovery (%) Spiked The method (RSD%) Relative recovery (%) Spiked The method (RSD%) Relative recovey (%) Sample 1 Blank 0.063 (2.7) - Blank 0.057 (4.5) - Blank 0.046 (3.6) - Serum of Blank+ 0.50 0.584 (4.7) 104.1 Blank+0. 50 0.570 (3.1) 102.4 Blank+0. 50 0.571 (2.4) 104.7 healthy people Blank+ 5.00 5.151 (2.6) 101.7 Blank+5. 00 5.076 (2.7) 100.4 Blank+5. 00 5.131 (2.6) 101.6 Sample 2 Serum of healthy people Blank 0.067 (1.4) - Blank 0.053 (2.7) - Blank 0.048 (2.1) - .sup.aRelative recovery=(total concentration-blank concentration)/spiked concentration

Example 2 Detection of miRNA-155 and miRNA-21 in Serum Samples of Patient with Lupus Erythematosus

[0060] The detection results of miRNA-155 and miRNA-21 in serum samples of a patient with lupus erythematosus were shown in Table 2. 0.399 pM of miRNA-155 and 0.034 pM of miRNA-21 were separately detected in the serum samples of the patient with lupus erythematosus. Compared with healthy people, miRNA-155 was significantly overexpressed in the patient with lupus erythematosus. These results were consistent with those of qRT-PCR. The results clearly demonstrated that the provided method had good practicability for analyzing multiple miRNAs in practical samples.

TABLE-US-00002 Detection of miRNA-155 and miRNA-21 in serum samples of patient with lupus erythematosus miRNA-155 (pM) miRNA-21 (pM) Sample Spiked The method (RSD%, n=3) Relative recovery (%) Spiked The method (RSD%, n=3) Relative recovery (%) Serum of patient with lupus erythematosus Blank 0.399 (1.2) - Blank 0.034 (1.7) - .sup.aRelative recovery=(total concentration-blank concentration)/spiked concentration

Example 3 Detection of miRNA-155 and miRNA-21 in Serum Samples of Patient with Ovarian Cancer

[0061] The detection results of miRNA-155 and miRNA-21 in serum samples of a patient with ovarian cancer were shown in Table 3. 0.090 pM of miRNA-155 and 0.137 pM of miRNA-21 were separately detected in the serum sample of the patient with ovarian cancer. Compared with healthy people, miRNA-21 was significantly overexpressed in the patient with ovarian cancer. These results were consistent with those of qRT-PCR. The results clearly demonstrated that the provided method had good practicability for analyzing multiple miRNAs in practical samples.

TABLE-US-00003 Detection of miRNA-155 and miRNA-21 in serum samples of patient with ovarian cancer miRNA-155 (pM) miRNA-21 (pM) Sample Spiked The method (RSD%, n=3) Relative recovery (%) Spiked The method (RSD%, n=3) Relative recovery (%) Serum of patient with ovarian cancer Blank 0.090 (3.7) - Blank 0.137 (3.7) -

Example 4 Detection of miRNA-122, miRNA-155, and miRNA-21 in Serum Samples of Patients with Cervical Cancer

[0062] The detection results of miRNA-122, miRNA-155, and miRNA-21 in serum samples of a patient with cervical cancer were shown in Table 4. Concentrations of miRNA-122 were detected to be 0.070 pM and 0.090 pM separately in sample 3 and 4 (patients with cervical cancer), concentrations of miRNA-155 were detected to be 0.209 pM and 0.224 pM separately, and concentrations of miRNA-21 were detected to be 0.115 pM and 0.117 pM separately. These results indicated that miRNA-155 and miRNA-21 were up-regulated in the patients with cancer compared with healthy people. These results were consistent with those of qRT-PCR. The results clearly demonstrated that the provided method had good practicability for analyzing multiple miRNAs in practical samples.

TABLE-US-00004 Detection of miRNA-122, miRNA-155, and miRNA-21 in serum samples of patients with cervical cancer miRNA-122 (pM) miRNA-155 (pM) miRNA-21 (pM) Sample Spiked The method (RSD%) Relative recovery (%) Spiked The method (RSD%) Relative recovery (%) Spiked The method (RSD%) Relative recovery (%) Sample 3 Blank 0.070(4.5) - Blank 0.209(3.5) - Blank 0.115(3.9) - Serum of patient with cervical cancer Blank+0.50 0.578(3.9) 102.9 Blank+0.50 0.765(4.1) 102.4 Blank+0.50 0.621(3.3) 101.2 Blank+5.00 5.283(1.6) 104.4 Blank+5.00 5.120(3.1) 98.21 Blank+5.00 5.221(1.1) 102.2 Sample 4 Serum of patient with cervical cancer Blank 0.090(3.7) - Blank 0.224(2.4) - Blank 0.117(2.7) - .sup.aRelative recovery=(total concentration-blank concentration)/spiked concentration

Example 5 Comparison of Different Methods for Detecting miRNAs

[0063] The method of the present disclosure was compared with other methods for the detection of multiple miRNAs or methods related to HPLC reported in recent years, and the results were shown in Table 5. The method for analyzing miRNAs using fluorescence detection was highly selective but not sensitive (Wang, R.; Xu, X.; Li, X.; Zhang, N.; Jiang, W. pH-responsive ZnO nanoprobe mediated DNAzyme signal amplification strategy for sensitive detection and live cell imaging of multiple microRNAs. Sens. Actuators, B. 2019, 293, 93-99.Jie, G.; Zhao, Y.; Wang, X.; Ding, C. Multiplexed fluorescence detection of microRNAs based on novel distinguishable quantum dot signal probes by cycle amplification strategy. Sens. Actuators, B. 2017, 252, 1026-1034). The electrochemiluminescence (ECL)-based miRNA measurement methods showed high sensitivity, but had disadvantages such as synthesizing complex materials (Feng, X.; Gan, N.; Zhang, H.; Li, T.; Cao, Y.; Hu, F.; Jiang, Q. Ratiometric biosensor array for multiplexed detection of microRNAs based on electrochemiluminescence coupled with cyclic voltammetry. Biosens. Bioelectron. 2016, 75, 308-314.Peng, L.; Zhang, P.; Chai, Y.; Yuan, R. Bi-directional DNA Walking Machine and Its Application in an Enzyme-Free Electrochemiluminescence Biosensor for Sensitive Detection of MicroRNAs. Anal. Chem. 2017, 89 (9), 5036-5042). The HPLC-MS/MS-based analysis showed high selectivity but often involved tedious data analysis (Kuang, Y; Cao, J.; Xu, F.; Chen, Y. Duplex-Specific Nuclease-Mediated Amplification Strategy for Mass Spectrometry Quantification of miRNA-200c in Breast Cancer Stem Cells. Anal. Chem. 2019, 91 (14), 8820-8826. Liu, L.; Xu, Q.; Hao, S.; Chen, Y. A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe. Sci. Rep. 2017, 7 (1), 5669). Furthermore, these analyses relied on the detection of peptide chains and indirectly relied on the insertion of targeted proteomics into miRNAs for quantification. More importantly, most of these analytical methods cannot detect multiple miRNAs in a single run. Furthermore, it can be seen that the limit of detection (LOD) measured in the experiment is much lower than the LOD reported in the literature. Although Nakayama et al. identified more than a dozen human cellular miRNAs in a single untargeted nanoflow LC-MS/MS, the method still requires more sophisticated instruments and has a lower LOD than other genetics methods (Nakayama, H.; Yamauchi, Y.; Taoka, M.; Isobe, T. Direct Identification of Human Cellular MicroRNAs by Nanoflow Liquid Chromatography-High-Resolution Tandem Mass Spectrometry and Database Searching. Anal. Chem. 2015, 87 (5), 2884-2891). It can be seen that the method of the present disclosure has an advantage of better simultaneously detecting multiple miRNAs.

TABLE-US-00005 Comparison of different methods for detecting miRNAs analytical methods target sample mechanism single run/test LOD linear range fluorescence miRNA-21 cells pH-responsive ZnO nanoprobe mediated DNAzyme no 54 pM 100 pM-30 nM miRNA-373 38 pM 100 pM-20 nM miRNA-141 cells quantum dot signal probes with cycle amplification strategy no 1.5 pM 5.0 pM-50.0 miRNA-21 1.5 pM nM ECL miRNA-21 serum Ru(bpy).sub.3.sup.2+- Silica@Poly-L-lysine-Au NPs no 6.3 fM 0.02-150 pM miRNA-141 8.6 fM 0.03-150 pM miRNA-21 PBS bi-directional DNA walking machine no 1.51 fM 5 fM-500 pM miRNA-155 1.67 fM surface-enhanced Raman scattering miRNA-21 cells electromagnetic hot spots vis target-mediated nanoparticle dimerization strategy yes 1 pM 1.0 pM- 10 nM miRNA-155 1 pM LC-MS/MS hsa-let7 family hsa-miRNA-15b, 16, 21, 23a, 24, 25, 27a, 27b, 30b, 30c, 98, 106b, 125b, 365a cells nano-flow LC-high-resolution tandem MS and RNAsequence database searching yes 300 amol 300 amol to 300 fmol miRNA-21 cells quasi-targeted proteomics approach no 1 pM ( LOQ) 1 pM-100 nM miRNA-let7a miRNA-200c miRNA-125a miRNA-15b miRNA-200c cells DSN mediated amplification — 1 fM (LOQ) 1 fM-200 fM miRNA-21 cells proteomics approach via a covalently immobilized DNA peptide probe — 5 pM (LOQ) 5 pM-10 nM HPLC-fluorescence miRNA-122 serum long and short probes based DSN mediated amplification yes 0.39 fM 1.0 fM-100 pM miRNA-155 0.30 fM 1.0 fM-100 pM miRNA-21 0.26 fM 1.0 fM-100 pM