Quantitative analysis method for disease-related miRNA based on single-particle signal amplification and enzyme-free assistance CRISPR / Cas12a

Abstract

The application of a CRISPR (clustered regularly spaced short palindromic repeat) detection platform in miRNA detection is increasingly increased, and particularly, high sensitivity and wide application are achieved by combining an enzyme-based nucleic acid pre-amplification strategy. Although a huge success is achieved, the introduction of an additional amplification enzyme may cause negative interference between primer residues, cost increase and non-specific amplification, sometimes a false positive result is caused, and even a CRISPR system is inactivated. In order to solve the problems, an enzyme-free amplification CRISPR / Cas12a miRNA detection method is designed, and a hybridization chain reaction (HCR) amplification and single particle analysis strategy is combined. The HCR amplification utilizes the self-assembly characteristic of nucleic acid and avoids the use of any enzyme, so that efficient amplification is realized under mild conditions. In addition, single particle analysis by inductively coupled plasma mass spectrometry (Sp-ICPMS) can provide direct signal amplification by analyzing a single nanoparticle that retains a large number of metal atoms. In the invention, the target miRNAs can start an HCR amplification loop and can be converted into double-stranded DNA (dsDNA) with PAM regions, and the PAM regions can be recognized by a CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / Cas) system. The activated Cas12a can effectively destroy a cross-linking system of the orthogonal nano particles, so that various nano aggregates with different detectable Sp-ICPMS strengths are generated. Finally, the limit of detection (LOD) of the fM level and excellent selectivity are realized. A desired result is also obtained by a serum labeling recovery test. It is reported for the first time that the HCR-CRISPR / Cas12a single particle analysis strategy is used for miRNA detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical chemistry, and relates to the field of analytical chemistry sensors based on Single Particle Inductively Coupled Plasma Mass Spectrometry (Sp-ICP-MS), in particular to a sensitive analytical method for disease-related miRNA based on the CRISPR / Cas12a shear enzyme and hybrid chain reaction assisted single particle inductively coupled plasma mass spectrometry. BACKGROUND

[0002] miRNAs are a class of non-coding RNA nucleic acids with 20-22 nt in length. miRNAs can be involved in gene expression, cell development and various disease development, and other important physiological processes. Studies have shown that some miRNAs are closely related to the occurrence, metastasis and apoptosis of tumors, can feedback the relevant information of the disease, and are important biomarkers. Therefore, the development of high-sensitivity miRNA detection methods has important significance for life science research, disease diagnosis and personal medical effect monitoring. Various signal amplification strategies, such as PCR, RPA, and LAMP, are also used as auxiliary methods for signal amplification and widely used in miRNA quantitative analysis. Although they have achieved great success, the inevitable signal leakage and the interference of various proteases often also increase the complexity of the detection system and lead to inaccurate detection results. This paper proposes a nucleic acid quantitative method for sensitive detection of miRNA and combines the CRISPR system with the hybrid chain reaction (HCR) auxiliary, while also realizing a potential enzyme-free single-particle biological analysis method for on-site detection. Single-particle inductively coupled plasma mass spectrometry (Sp-ICP-MS) has good resolution and high sensitivity detection capability without the advantage of mass spectrometry overlap. AuNPs aggregates composed of linker and two sets of nucleic acid-labeled gold nanoparticles (ArmA / ArmB-AuNPs) as signal output means. When the target miRNA exists, the presence of the target miRNA in the system will mediate the occurrence of the HCR reaction. First, the target miRNA and the hairpin DNA H1 in the system are combined by the base complementary principle, and the hairpin DNA H1 is opened and reacts with hairpin DNA H2 and H3 in sequence, and releases the DNA duplex containing the PAM sequence of the activatable nuclease Cas12a-crRNA. When the trans-cleavage ability of Cas12a is activated, the linker in the AuNPs aggregate unit will be cut, and AuNPs aggregates of different sizes will be obtained. After the AuNPs aggregates obtained are sufficiently diluted, they can be used for downstream Sp-ICP-MS analysis. This method successfully achieves a detection limit (LOD) of femtomolar level, and through serum spiking recovery analysis, the method can provide effective analysis results. SUMMARY

[0003] The application aims to provide a high-efficiency CRISPR / Cas12a-based enzyme-free amplification miRNA single-nanoparticle bioanalysis method and its application in quantitative detection of disease-related miRNA; the principle of the application is that the method first converts target miRNA into a DNA duplex based on HCR reaction. The DNA duplex contains a Cas12a-crRNA complex that can be activated downstream. Through the principle of base complementarity, crRNA will recognize and bind the PAM sequence region in the duplex, and release the anti-single-stranded DNA (ssDNA) transcleavage ability. The cross-linked DNA (linker DNA) mixed in the solution will be randomly cut, thereby changing the state of gold nanoparticle aggregate formation. With the increase of the concentration of target miRNA in the solution, the specific morphology of gold nanoparticles will gradually decrease from large to small. By performing homogeneous analysis on gold nanoparticles containing different concentrations of target miRNA solution through the intensity mode of single-nanoparticle ICP-MS (SP-ICP-MS), the sensitive detection of miRNA can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0004] Figure 1 It is a mechanism diagram of the analysis method of the application;

[0005] Figure 2 It is a characterization diagram of ArmA / ArmB-DNA labeled gold nanoparticles in the analysis method of the application;

[0006] Figure 3 It is a linear diagram of the feasibility of single-particle analysis of gold nanoparticle aggregates used in the analysis method of the application;

[0007] Figure 4 It is an electrophoresis characterization diagram and cleavage verification of the hybridization chain reaction used in the analysis method of the application;

[0008] Figure 5 It is a linear diagram of target miRNA and real-time data of Sp-ICP-MS intensity mode in the analysis method of the application;

[0009] Figure 6 It is a specificity analysis diagram of Sp-ICP-MS mode for target miRNA in the analysis method of the application;

[0010] Figure 7 It is an application diagram of Sp-ICP-MS mode for serum standard addition recovery in the analysis method of the application. DETAILED DESCRIPTION

[0011] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. The water used in the following examples is ultrapure water treated by an ultrapure water purification system.

[0012] The present application is carried out according to the following preferred specific steps.

[0013] A. Preparation of 25 nm AuNPs

[0014] A1. Take 100 uL of 10% chloroauric acid (w / v) into 60 mL of ultrapure water, put a magnetic stirrer into a three-necked flask, reflux boil in a heating jacket and keep boiling for 20 min;

[0015] A2. Quickly add 1.2 mL of 1% sodium citrate reducing agent solution, keep refluxing for 30 min under stirring boil;

[0016] Turn off the temperature control system of the heating jacket, keep stirring with the stirrer, and when the solution slowly cools to room temperature, transfer the solution to a black 100 mL storage bottle, and adjust the volume to 100 mL.

[0017] B. Functionalization of gold nanoparticles (ArmA / ArmB-DNA-AuNPs)

[0018] B1. Take 1 mL of AuNPs, wash with 0.01 M of boric acid buffer solution for three times (4°C, 11000 rpm, 10 min / time), adjust the pH to about 8.0-8.5;

[0019] B2. Reduce 1.9 nmol of thiol-modified ArmA-DNA and ArmB-DNA with 10-fold molar excess of TCEP-HCl at 37°C for 30 min;

[0020] B3. After the reaction, add the TCEP-treated ArmA / ArmB-DNA to 1 mL of pre-centrifuged washed AuNPs solution (1.60 nmol L-1) using the freeze-labeling method. Mix well by vortex, and store in a -20°C refrigerator overnight in the dark;

[0021] B4. Take the obtained ArmA / ArmB-DNA-AuNPs out of the refrigerator, thaw at room temperature. Centrifuge wash 3 times (10 min, 4°C, 11000 rpm) with PBS wash buffer (10 mM PB; 150 mM NaCl; 0.01% Tween-20; pH 7.4) to remove excess Arm-DNA, and finally store at 4°C for later use.

[0022] C. Hybridization chain reaction (HCR)

[0023] C1. Dissolve H1, u-H2 and u-H3 in Tris-HCl buffer solution (20 mM Tris-HCl, 800 mM NaCl, 5 mL KCl);

[0024] C2. Put 1 uM H1, 1.5 uM u-H2 and u-H3 into a polymerase chain reaction thermal cycler, respectively, and incubate at 95°C for 5 minutes to start the annealing reaction, and gradually cool to 4°C at a rate of 1°C / min;

[0025] C3. Then mix the above obtained hairpin DNA at a final concentration of 250 nM H1, 500 nM u-H2 and 500 nM u-H3;

[0026] C4. Then add different concentrations of miR-21 to the above mixed solution, and incubate at 37°C for 30 minutes to perform HCR reaction.

[0027] D. mi-21 detection based on HCR amplification and Sp-ICP-MS construction

[0028] D1. Mix the same concentration of Cas12a and crRNA in 1x NEBuffer 2.1 buffer, and incubate at 37°C for 15 minutes to form Cas12a-crRNA ribonucleoprotein complex particles (RNPs);

[0029] D2. Add 20 uL HCR product and 10 uL linker DNA (2.5 uM) to the pre-assembled Cas12a-crRNA RNPs;

[0030] D3. Add the same concentration of labeled gold nanoparticles tag (800 pmol L-1 of Arm-A / B-AuNPs) respectively;

[0031] D4. Incubate at 37°C for 30 minutes to perform trans-splicing and gold nanoparticle cross-linking reaction, and the obtained different state of gold nanoparticle aggregates are used for subsequent analysis after sufficient dilution.

[0032] E. Single nanoparticle ICP-MS analysis

[0033] E1. Before introducing ICP-MS for single particle mode analysis, all samples need to be diluted sufficiently to avoid excessive counting of each pulse signal reading period, while collecting intensity data and pulse frequency data of AuNP probes;

[0034] E2. Single particle mode analysis was performed on the sample by setting the dwell time to 200 us and the scan time to 20 s.

[0035] F. Sp-ICP-MS parameter settings and cleaning

[0036] F1. The liquid suction pump tube of the Sp-ICP-MS was inserted into a solution containing 1% nitric acid in water;

[0037] F2. The solution was signal collected by setting the frequency counting mode, the collection time was 20 s, and the speed was 200 us / time.

[0038] G. Standard addition recovery analysis

[0039] G1. The collected healthy human serum was diluted 10 times;

[0040] G2. Different contents of miR-21 (0, 100, 500 pM) were added respectively;

[0041] G3. After the obtained sample was mixed thoroughly, centrifugal treatment was performed (15000 rpm, 15 minutes);

[0042] G4. The sample supernatant was taken, and single nanoparticle analysis was performed using the above method.

[0043] Further description will be made in combination with the drawings of the specification.

[0044] As shown in the specification, Figure 1 the present application is a kind of based on HCR-CRISPR / Cas12a without amplification enzyme involved in the miRNA single nanoparticle counting biological analysis method.

[0045] Example 1, characterization of gold nanoparticle probe;

[0046] In order to reveal the success of gold nanoparticle probe labeling in Sp-ICP-MS experiment, first of all, the labeling of gold nanoparticles with DNA was characterized, and ultraviolet visible spectrophotometry, dynamic light scattering method and Zeta potential were used for verification. Figure 2 (a) The ultraviolet analysis result shown can see that the gold nanoparticles labeled with ArmA / ArmB-DNA have obvious red shift compared with bare gold nanoparticles without labeling; through Figure 2 (b) Zeta potential results also show that the ArmA / ArmB-DNA-AuNPs have more negative charge than the original nanoparticles due to the negative charge of DNA, which also proves the successful labeling of DNA on gold nanoparticles; at the same time,Figure 2 The results of the particle size analysis by dynamic light scattering of (c) also show that the ArmA / ArmB-DNA sequences are successfully labeled on the surface of the gold nanoparticles.

[0047] Example 2, Investigation of the feasibility of single particle analysis of gold nanoparticle aggregates;

[0048] To demonstrate that the proposed gold nanoparticle aggregates can be detected by the single particle analysis mode, different concentrations of linker DNA were added to the solution containing the same concentration of ArmA / ArmB-DNA-AuNPs. After the reaction and sufficient dilution, the Sp-ICP-MS single nanoparticle analysis mode was used for analysis. As shown in Figure 3 As shown in (a), (b), (c), and (d), the resulting gold nanoparticle aggregates can be sensitively detected by the Sp-ICP-MS single nanoparticle counting mode, and the intensity signal and linker DNA concentration have good probe linearity (R2=0.98).

[0049] Example 3, Characterization of miRNA-induced hybridization chain reaction (HCR);

[0050] To demonstrate that the hybridization chain reaction (HCR) is induced by the target miRNA, electrophoretic analysis was performed to characterize the hairpin DNAs (H1, H2, H3) used and the target miRNA. Figure 4 As shown in (a), the nucleic acid sequences used in the present application can successfully induce HCR reaction by the target miRNA. It is also proved that the hairpin sequence H1 used can be used to identify the target miRNA, and the three hairpin DNAs do not affect each other in the absence of the target miRNA. And the formation of nanoparticle aggregates by the designed HCR-mediated CRISPR / Cas12a nanoparticle aggregate splitting method was also investigated. As shown in Figure 4 As shown in (b)-(c), we observed the different nanoparticle aggregation conditions caused by different concentrations of target miRNA in the detection system by TEM. It can be clearly seen that after the addition of the target miRNA, the detection system can successfully cleave the linker DNA and result in a dispersed nanoparticle solution. In the absence of the target miRNA, the linker DNA successfully connects the ArmA / ArmB-DNA-AuNPs label, and finally forms obvious nanoparticle aggregates. The above results all test the ability of the proposed HCR-mediated CRISPR / Cas12a nanoparticle aggregate splitting method to detect the target miRNA.

[0051] Example 4, investigate the linearity of the method for detecting the target miRNA (miR-21);

[0052] The linearity of Sp-ICP-MS single nanoparticle counting analysis for miR-21 is shown in FIG. 2 (a) - (d). Figure 5

[0053] According to the concentration of miR-21 and the response signal of Sp-ICP-MS single nanoparticle mode intensity, the single nanoparticle intensity signal response trend of different concentrations of miR-21 was established, and then logarithmic operation and linear processing were performed. The linear relationship of 250 fM - 10 nM was obtained in the single particle counting mode (the detection limit is 220 fM, R2=0.97), and the time resolution data graph of Sp-ICP-MS (FIG. 2 (b) - (d) ) shows the real-time detection results in the single particle mode. The method can stably and smoothly detect miR-21 and distinguish from the background signal. Figure 5

[0054] Example 5, investigate the specific recognition ability of the method for the target miR-21;

[0055] In order to reveal the specificity of the method for miR-21, the interference of other disease-related miRNAs on the analysis method was analyzed. 50 nM of miR-200, miR-125, miR-342, miR-29, miR-191, miR-34 and miR-9 were added to the sample containing 5 nM AFB1, and the sample solution containing the target miR-21 and the above interference miRNA was detected by Sp-ICP-MS counting mode in parallel. By Figure 6 It can be seen that the interference miRNAs do not interfere with the detection of miR-21, which shows that the method can well distinguish the target miR-21 among different miRNAs.

[0056] Example 6, investigate the serum spiking recovery analysis ability of the method;

[0057] 1. Healthy human serum source;

[0058] The healthy human serum sample used in the application of the analysis method of the application is from Chengdu Seventh People's Hospital.

[0059] 2. Serum spiking recovery analysis results;

[0060] Using the above analysis steps, after the reaction, the sample was analyzed by Sp-ICP-MS single nanoparticle mode. The analysis results are shown in FIG. 4. Figure 7 Figure 7 ​​​The recovery analysis results of the analysis method are shown, and it can be seen that the established analysis method can obtain a recovery of 103%-110%. The above analysis results prove that the analysis method has the ability of analyzing and detecting actual samples and resisting complex matrix.

Claims

1. A method for analyzing cancer-related miRNAs based on hybridization chain reaction (HCR)-initiated CRISPR / Cas12a combined with single-particle inductively coupled plasma mass spectrometry, characterized in that: The analytical method comprises the use of HCR reaction to identify target miRNA-21 based on inductively coupled plasma mass spectrometry in single nanoparticle mode (SpICPMS), and the conversion and amplification of miRNA-21 into a long-chain DNA double strand that can activate the Cas12a cleavage enzyme. Sensitive detection of cancer-related miRNA-21 biomarkers is achieved with the assistance of trans-cleavage released from Cas12a. The nucleic acid sequence in the analytical method includes hairpin DNA1 for synthesizing specific miRNA-21 recognition and hairpin DNA2 and 3 for initiating subsequent HCR amplification. The signal output unit in the analytical method consists of two 25-nanometer-sized gold nanoparticles modified with different Arm (A and B) DNAs, and cross-linked DNA. In the presence of target miRNA-21, HCR is initiated, and the generated double-stranded DNA product activates Cas12a-crRNA and releases trans-cleavage. The cross-linked DNA in the system is cleaved, thereby altering the cross-linking of the gold nanoparticles. The size and intensity of the gold nanoparticle cross-links in the solution are analyzed using SpICPMS detection mode.

2. The HCR reaction system according to claim 1, characterized in that: The target miRNA was transformed by using hairpin DNA1, which specifically recognizes miRNA-21, and enzymatic-free isothermal HCR amplification was initiated with the assistance of hairpin DNA2 and 3. The three hairpin DNAs used can exist stably in the system in the absence of the target. In the presence of the target, miRNA-21 will open hairpin DNA1, and the resulting miRNA-DNA complex will initiate downstream hairpin DNA2 and 3 to obtain the HCR amplification product.

3. The signal output unit based on the gold nanoparticle crosslinking system according to claim 1, characterized in that: Two Arm (A) and B) DNA sequences with thiol (-SH) modified at the 5' end were first modified onto the surface of gold nanoparticles using a freezing method. After washing away excess Arm (A) and B DNA by centrifugation (centrifugation conditions: 110,000 rpm), the DNA was redissolved in an equal volume of PBS buffer solution (10 mM PB, 150 mM NaCl, 0.01% Tween-21, pH=7.5) to obtain Arm-A-AuNPs and Arm-B-AuNPs.

4. The HCR-mediated Cas12a-assisted Sp-ICPMS detection method according to claim 1, characterized in that: When the HCR amplification product is recognized by the pre-incubated Cas12a-crRNA complex, trans-cleavage is released, and the cross-linked DNA used to link Arm-A-AuNPs and Arm-B-AuNPs will be cleaved; depending on the miRNA-21 content, the degree of cross-linked DNA cleavage varies, thus yielding nanoparticle cross-links of different sizes. After the obtained solution was fully diluted, the size of single nanoparticles was analyzed using SpICPMS in single-particle mode. The measurement time was 20 s and the scan rate was 200 µs / s.

Images