阅读说明：本技术 一种用于microRNA定量分析的纸基检测系统及其制备方法和检测方法 (Paper-based detection system for quantitative analysis of microRNA, and preparation method and detection method thereof ) 是由 李勇 吴云华 于 2019-09-25 设计创作，主要内容包括：本发明涉及生物医学分析领域,尤其涉及一种用于microRNA定量分析的纸基检测系统及其制备方法和检测方法,所述纸基检测系统整合了靶标microRNA分子的特异性识别、滚环扩增和生物发光共振能量转移传感过程,仅利用智能手机就可在滤纸上实现microRNA的高灵敏度定量分析。此外,本发明所提供的纸基检测系统可在室温条件下长期保存,且所有检测反应可在滤纸上完成,无需繁琐的溶液配制和昂贵的专业化大型仪器。因此,具有易于运输、便携化程度高、使用简便、成本低廉、可直接向医疗条件落后地区或基层医疗单位推广的优点。(The invention relates to the field of biomedical analysis, in particular to a paper-based detection system for quantitative analysis of microRNA, a preparation method and a detection method thereof. In addition, the paper-based detection system provided by the invention can be stored for a long time at room temperature, and all detection reactions can be completed on filter paper without complicated solution preparation and expensive specialized large-scale instruments. Therefore, the portable medical device has the advantages of easy transportation, high portability, simple and convenient use, low cost and capability of being directly popularized to regions with laggard medical conditions or basic medical units.)

1. The paper-based detection system for quantitative analysis of microRNA is characterized in that: the system at least comprises a rolling circle amplification filter paper piece part and a bioluminescence resonance energy transfer sensing filter paper piece part, and is used for realizing the specific recognition, rolling circle amplification and bioluminescence resonance energy transfer sensing processes of target molecules.

2. A method of making the paper-based detection system of claim 1, characterized in that: the preparation method of the paper-based detection system comprises the following steps:

(1) manufacturing and sealing filter paper sheets: cutting the filter paper into circular paper sheets with the diameter of 6mm, and sealing the paper sheets by using 50-100 mg/mL bovine serum albumin and 0.2-1% (v/v) Tween 20 solution;

(2) preparing a rolling circle amplification filter paper sheet: preparing a rolling circle amplification reaction solution, wherein the reaction solution comprises the following components: 20-100 mM Tris-acetate with pH of 7.0-8.0, 10-20 units phi29DNA polymerase, 0.1-0.5 mM dNTPs, 10-100 nM circular DNA probe, 1-5 mM dithiothreitol, 10-20 mM magnesium acetate, 50-100 mM potassium acetate, 20-60 units RNase inhibitor; dripping the solution onto a closed filter paper sheet, rapidly cooling with liquid nitrogen, and freeze-drying in a freeze dryer to obtain a rolling ring amplification filter paper sheet;

(3) preparing a bioluminescence resonance energy transfer sensing filter paper sheet: preparing 20-50 mM Tris-Cl with pH 7.0-8.0, 50-150 mM NaCl, 1-5 mM MgCl₂，0.05～0.2mM ZnCl₂10-100 nM donor protein, 50-500 nM acceptor protein, 0.1-1 mg/mL BSA, 0.5-2 mM tris (2-carbonylethyl) phosphate, 0.1-0.5. mu.M single-stranded DNA fragment, 1-5% (w/v) mannitol; and dripping the solution onto the sealed filter paper sheet, and drying to obtain the bioluminescence resonance energy transfer sensing filter paper sheet.

3. The method of making a paper-based detection system according to claim 2, characterized in that: the energy donor protein and the energy receptor protein can specifically identify a rolling circle amplification product of a target microRNA, and the sequences of the energy donor protein and the energy receptor protein are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the single-stranded DNA fragment sequence can be specifically hybridized with a rolling circle amplification product of microRNA, and the sequence is shown as SEQ ID NO. 3.

4. The method of making a paper-based detection system according to claim 2, characterized in that: the circular DNA probe is designed according to a sequence of let-7a, and is prepared by a chemical synthesis technology; the probe sequence is shown in SEQ ID NO. 4.

5. The method of making a paper-based detection system according to claim 2, characterized in that: and respectively placing the rolling ring amplification filter paper sheet and the bioluminescence resonance energy transfer sensing filter paper sheet in a cell culture plate, covering a layer of paper towel on the surface, placing the paper towel in a closed container containing a drying agent, and storing the paper towel in a dark place at room temperature.

6. A method of testing the paper-based testing system of claim 1, comprising the steps of:

(1) paper-based rolling circle amplification: dripping a microRNA sample onto a rolling circle amplification filter paper sheet, and incubating for 2-4 hours at 30-37 ℃ to obtain a first reaction system;

(2) paper-based bioluminescence resonance energy transfer sensing: adding bioluminescence resonance energy transfer sensing filter paper into the first reaction system, and continuously incubating at room temperature for 10-30 min to obtain a second reaction system;

(3) signal reading: and adding a luciferase substrate into the second reaction system to obtain a detection system, putting the detection system into a cassette, and finally reading the detection result through an intelligent equipment terminal.

7. The detection method according to claim 6, characterized in that: the reading of the detection result by the intelligent equipment terminal is realized by shooting the detection result by the intelligent equipment terminal and analyzing the bioluminescence resonance energy transfer signals of each sample in the picture by image processing software.

Technical Field

The invention relates to the field of biomedical analysis, in particular to a paper-based detection system for quantitative analysis of microRNA (ribonucleic acid), and a preparation method and a detection method thereof.

Background

The microRNA which is abnormally expressed in a human body not only directly participates in the generation and development processes of major diseases such as tumors and the like, but also is an important marker for early diagnosis of the diseases, selection of clinical treatment schemes and evaluation of drug efficacy. Therefore, the accurate quantitative analysis of the microRNA is of great significance to the research of pathogenesis of diseases and clinical treatment.

Currently, the main technologies applied to microRNA detection in the market include: real-time fluorescent quantitative polymerase chain reaction (qRT-PCR), gene chip, RNA sequencing, nano-analysis, molecular beacons, isothermal amplification, and the like. Although the technology can well realize the quantitative analysis of microRNA, the problems that the detection reagent needs to be stored and transported at low temperature, the experiment operation is complicated, and large-scale analytical instruments and equipment and professional technicians need to be matched still exist. The problems lead to high cost of the corresponding method, and seriously restrict the popularization of the diagnosis method based on the microRNA in regions with laggard medical conditions or basic medical units.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a paper-based detection system for quantitative analysis of microRNA, which integrates the rolling circle amplification and bioluminescence resonance energy transfer sensing processes of target microRNA molecules, and can realize high-sensitivity quantitative analysis of microRNA on filter paper only by using a smart phone; in addition, the paper-based detection system provided by the invention can be stored for a long time at room temperature, and all detection reactions can be completed on filter paper without complicated solution preparation and expensive specialized large-scale instruments. Therefore, the portable medical device has the advantages of easy transportation, high portability, simple and convenient use, low cost and capability of being directly popularized to regions with laggard medical conditions or basic medical units.

The technical scheme provided by the invention is a paper-based detection system for microRNA quantitative analysis, which at least comprises a rolling circle amplification filter paper sheet part and a bioluminescence resonance energy transfer sensing filter paper sheet part, and is used for realizing the processes of specific identification of target molecules, rolling circle amplification and bioluminescence resonance energy transfer sensing.

A method of making a paper-based detection system, the method of making the paper-based detection system comprising the steps of:

(1) manufacturing and sealing filter paper sheets: cutting the filter paper into circular paper sheets with the diameter of 6mm, and sealing the paper sheets by using 50-100 mg/mL bovine serum albumin and 0.2-1% (v/v) Tween 20 solution;

(2) preparing a rolling circle amplification filter paper sheet: preparing a rolling circle amplification reaction solution, wherein the reaction solution comprises the following components: 20-100 mM Tris-acetic acid with pH of 7.0-8.0, 10-20 units phi29DNA polymerase, 0.1-0.5 mM dNTPs, 10-100 nM circular DNA probe, 1-5 mM dithiothreitol, 10-20 mM magnesium acetate, 50-100 mM potassium acetate, 20-60 units RNase inhibitor; dripping the solution onto a closed filter paper sheet, rapidly cooling with liquid nitrogen, and freeze-drying in a freeze dryer to obtain a rolling ring amplification filter paper sheet;

(3) preparing a bioluminescence resonance energy transfer sensing filter paper sheet: preparing 20-50 mM Tris-Cl with pH 7.0-8.0, 50-150 mM NaCl, 1-5 mM MgCl₂，0.05～0.2mM ZnCl₂10-100 nM donor protein, 50-500 nM acceptor protein, 0.1-1 mg/mL BSA, 0.5-2 mM tris (2-carbonylethyl) phosphate, 0.1-0.5. mu.M single-stranded DNA fragment, 1-5% (w/v) mannitol; and dripping the solution onto the sealed filter paper sheet, and drying to obtain the bioluminescence resonance energy transfer sensing filter paper sheet.

Moreover, the energy donor protein and the energy acceptor protein can specifically identify a rolling circle amplification product of a target microRNA, and the sequences of the energy donor protein and the energy acceptor protein are respectively shown as SEQ ID No.1 and SEQ ID No. 2; the single-stranded DNA fragment sequence can be specifically hybridized with a rolling circle amplification product of microRNA, and the sequence is shown as SEQ ID NO. 3.

The circular DNA probe is designed according to the sequence of let-7a, and is prepared by a chemical synthesis technology; the probe sequence is shown in SEQ ID NO. 4.

Moreover, the preservation method of the paper-based detection system comprises the following steps: and respectively placing the rolling ring amplification filter paper sheet and the bioluminescence resonance energy transfer sensing filter paper sheet in a cell culture plate, covering a layer of paper towel on the surface, placing the paper towel in a closed container containing a drying agent, and storing the paper towel in a dark place at room temperature.

A paper-based detection method for quantitative analysis of microRNA comprises the following steps:

(1) paper-based rolling circle amplification: dripping a microRNA sample onto a rolling circle amplification filter paper sheet, and incubating for 2-4 hours at 30-37 ℃ to obtain a first reaction system;

(2) paper-based bioluminescence resonance energy transfer sensing: adding bioluminescence resonance energy transfer sensing filter paper into the first reaction system, and continuously incubating at room temperature for 10-30 min to obtain a second reaction system;

(3) signal reading: and adding a luciferase substrate into the second reaction system to obtain a detection system, putting the detection system into a cassette, and finally reading the detection result through an intelligent equipment terminal.

And the reading of the detection result by the intelligent equipment terminal is realized by shooting the detection result by the intelligent equipment terminal and analyzing the bioluminescence resonance energy transfer signal of each sample in the picture by image processing software.

Compared with the prior art, the invention has the beneficial effects that: (1) after the paper-based detection system is stored for 3 months at room temperature, the analysis performance can not be obviously attenuated, so that low-temperature storage is not needed, and the difficulty and the cost of storage and transportation are obviously reduced; (2) the detection process can be completed only by dripping the microRNA sample on the filter paper sheet, complex solution preparation and harsh experimental condition control are not needed, and the obtained result can be read by a smart phone, so that the method has the advantages of high portability degree, easiness in operation and no limitation of personnel and experimental instruments.

Drawings

FIG. 1 is a schematic view of the detection principle of the paper-based detection system provided by the present invention;

FIG. 2 is a graph showing the results of electrophoresis of the energy donor protein and the energy acceptor protein prepared in example 1 of the present invention;

FIG. 3 is a graph showing the linear response of the bioluminescence resonance energy transfer signal to the change in the concentration of let-7a in example 2 of the present invention;

FIG. 4 is a graph showing the linear response of the bioluminescence resonance energy transfer signal to the change in the concentration of let-7a in example 3 of the present invention.

Detailed Description

The invention aims to protect a paper-based detection method and a paper-based detection system for microRNA quantitative analysis and a preparation method of the paper-based detection system.

A paper-based detection system for a microRNA quantitative analysis detection method at least comprises a rolling circle amplification filter paper piece part and a bioluminescence resonance energy transfer sensing filter paper piece part, and is used for realizing the specific identification, rolling circle amplification and bioluminescence resonance energy transfer sensing processes of target molecules.

A method of making a paper-based detection system, the method of making the paper-based detection system comprising the steps of:

(1) manufacturing and sealing filter paper sheets: cutting the filter paper into circular paper sheets with the diameter of 6mm, and using 50-100 parts

Sealing the paper sheets by mg/mLBSA and 0.2-1% (v/v) Tween 20 solution;

(2) preparing a rolling circle amplification filter paper sheet: preparing a rolling circle amplification reaction solution, wherein the reaction solution comprises the following components: 20-100 mM Tris-acetic acid (pH 7.0-8.0), 10-20 units phi29DNA polymerase, 0.1-0.5 mM dNTPs, 10-100 nM circular DNA probe, 1-5 mM dithiothreitol, 10-20 mM magnesium acetate, 50-100 mM potassium acetate, 20-60 units RNase inhibitor; dripping the solution onto a closed filter paper sheet, rapidly cooling with liquid nitrogen, and freeze-drying in a freeze dryer to obtain a rolling ring amplification filter paper sheet;

(3) preparing a bioluminescence resonance energy transfer sensing filter paper sheet: the composition contains 20-50 mM Tris-Cl (pH 7.0-8.0), 50-150 mM NaCl, 1-5 mM MgCl₂，0.05～0.2mM ZnCl₂10 to 100nM donor protein, 50 to 500nM acceptor protein, 0.1 to 1mg/mL BSA, 0.5 to 2mM tris (2-carbonylethyl) phosphate (TCEP), 0.1 to 0.5. mu.M single-stranded DNA fragment, 1 to 5% (w/v) mannitol; dripping the solution onto a closed filter paper sheet, and drying to obtain a bioluminescence resonance energy transfer sensing filter paper sheet; the energy donor protein and the energy receptor protein can specifically identify a rolling circle amplification product of a target microRNA, and the sequences of the energy donor protein and the energy receptor protein are respectively shown as SEQ ID No.1 and SEQ ID No. 2; the single-stranded DNA fragment sequence can be specifically hybridized with a rolling circle amplification product of microRNA, and the sequence is shown as SEQ ID NO. 3.

A paper-based detection method for quantitative analysis of microRNA comprises the following steps:

(1) paper-based rolling circle amplification: dripping a microRNA sample onto a rolling circle amplification filter paper sheet, and incubating for 2-4 hours at 30-37 ℃ to obtain a first reaction system;

(2) paper-based bioluminescence resonance energy transfer sensing: adding bioluminescence resonance energy transfer sensing filter paper into the first reaction system, and continuously incubating at room temperature for 10-30 min to obtain a second reaction system;

(3) signal reading: and adding a luciferase substrate into the second reaction system to obtain a detection system, putting the detection system into a cassette, and finally reading the detection result through an intelligent equipment terminal.

The present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to the examples.