Multi-miRNA detection hydrogel particle based on color coding and preparation method thereof

Abstract

The invention discloses a preparation method of multi-miRNA (micro Ribonucleic Acid) detection hydrogel particles based on color coding, which comprises the following steps: firstly, obtaining hydrogel particles coded in different colors, and then modifying miRNA detection aptamers for capturing miRNA. And mixing the obtained hydrogel particles modified by the aptamer with a miRNA solution, and carrying out miRNA detection. The hydrogel has weakest non-specific binding, so that the hydrogel is beneficial to detection of a target object in a complex biological sample. Color coding of different markers is achieved for the first time, and the method is used for mixed detection of multiple types of tumor markers miRNA.

Description

Technical Field

[0001] The invention belongs to the technical field of miRNA detection, and particularly relates to a multi-miRNA detection hydrogel particle based on color coding and a preparation method thereof. Background Art

[0002] In recent years, miRNA has attracted widespread attention as a tumor marker. Changes in the expression levels of tumor-related miRNAs are associated with tumor burden and malignant progression. In the early stages of liver cancer, the levels of miRNA-21, miRNA-122, and miRNA-223 are elevated. Many studies have shown that the detection of multiple miRNAs is important for early diagnosis of tumors, differential diagnosis of benign and malignant tumors, monitoring of treatment effects, and prognosis evaluation. However, traditional detection methods such as PCR are time-consuming and labor-intensive, and can only detect a single tumor marker at a time, while gene chips require expensive and bulky analytical equipment and cannot perform rapid screening tests on a large number of samples outside the laboratory. Microarray technology provides high sensitivity and multiplexing capabilities, but its use in the clinic is limited by complexity and low detection volume. Therefore, it is of great practical significance to establish an accurate, diverse, rapid, and low-cost tumor marker detection method. Summary of the invention

[0003] The main purpose of the present invention is to provide a technology that can detect miRNA in batches, efficiently and quickly. By capturing miRNA with hydrogel modified with nucleic acid aptamers, color-coded hydrogel particles are used to detect different miRNAs. In addition, the hydrogel has the weakest non-specific binding, which makes it beneficial for detecting targets in complex biological samples. MiRNA-122, miRNA-21, miRNA-223 and miRNA-27a are selected as targets and combined with nucleic acid aptamers on the surface of hydrogels using the principle of complementary pairing. Provide a reference for the rapid, accurate and portable detection of multiple tumor markers.

[0004] In order to achieve the above object, the present invention provides the following technical solution: a method for preparing multi-miRNA detection hydrogel particles based on color coding, comprising the following steps:

[0005] (1) Preparing hydrogel particles of different colors: adding dyes of different colors to a sodium alginate solution, mixing evenly, and setting the solution aside;

[0006] (2) solidification: adding the sodium alginate solution obtained in step (1) dropwise into a saturated calcium chloride solution to obtain hydrogel particles coded in different colors;

[0007] (3) Aptamer modification: combining the hydrogel particles obtained in step (2) with different miRNA detection aptamers to obtain detection aptamer-modified hydrogel particles;

[0008] (4) miRNA detection: The aptamer-modified hydrogel particles obtained in step (3) are mixed with the miRNA solution to perform miRNA detection.

[0009] As a preferred embodiment, in step (1), 1 mL of a sodium alginate solution with a concentration of 2-5% is mixed with 2 μL of a dye (green, purple, yellow, blue, black, pink) respectively.

[0010] As a preferred embodiment, in step (2), the sodium alginate solution is sprayed into the calcium chloride solution using a syringe to generate smaller hydrogel particles.

[0011] As a preferred embodiment, in step (3), the prepared hydrogel particles are dispersed in 1 mL of water, and 2 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide solid (NHS) are added respectively, and stirred in an ice-water bath (300r, 120min) to activate the carboxyl group. Then 3 μL of miRNA detection aptamer solution is added, and the ice-water bath stirring is continued (300r, 120min). Centrifuge and collect the hydrogel particles modified with the detection aptamer. In addition, all miRNA detection aptamers are modified with Acrydite (acrylamide) at the 5' end and NH2 (amino) at the 3' end.

[0012] The aptamer sequence for detecting miRNA-21 is: 5'-GATATATTTTATCAACATCAGTCTGATAAGCTA-3'; the aptamer sequence for detecting miRNA-122 is: 5'-GATATATTTACCTCACACTGTTACCACAAAC-3'; the aptamer sequence for detecting miRNA-233 is: 5'-GATATATTTGACAAACTGACTGGGGTATTT-3'; the aptamer sequence for detecting miRNA-27a is: 5'-GATATATTTTGTGTAGATTCACAGTGG-3'.

[0013] As a preferred embodiment, in step (4), the hydrogel particles are dispersed in 50 μL of TET containing 700 mM NaCl. 5 μL of miRNA is added to 95 μL of TE buffer containing 700 mM NaCl. Then, the above two solutions are mixed in a metal bath for 90 minutes (55°C, 1500 rpm) to capture the target miRNA. Subsequently, 245 μL of hybridization buffer containing 5 μL of biotin-modified universal aptamer (sequence 5'-TAAAATATATAAAAAAAAAAAA-3') is added and heated at 21.5°C for 30 minutes. Then, 1 μL of 2 g / mL streptavidin-R-phycoerythrin conjugate (SA-PE) is added, and the reaction is continued at 21.5°C for 30 minutes at a speed of 1500 r / min. This is mainly because the biotin-modified universal aptamer can bind to the hydrogel particles that have captured the target miRNA, and then based on the biotin-streptavidin reaction, the surface of the hydrogel particles that have captured the target miRNA is coated with R-phycoerythrin fluorescence. By taking fluorescent pictures of the hydrogel particles and measuring the fluorescence intensity, the content of the target miRNA can be calculated.

[0014] The beneficial effects of the present invention are as follows: the preparation method of the multi-miRNA detection hydrogel particles based on color coding realizes the color coding of different markers for the first time, and is used for mixed detection of various types of tumor marker miRNAs.

[0015] The present invention selects miRNA-122, miRNA-21, miRNA-223 and miRNA-27a as targets, and uses the principle of complementary base pairing to bind miRNA to the detection aptamer on the surface of the hydrogel. In addition, the universal aptamer modified based on biotin can be complementary paired with some bases on the miRNA detection aptamer, and streptavidin-coupling and phycoerythrin-R-phycoerythrin conjugate (SA-PE) is added. Based on the specific reaction of biotin-streptavidin, the intensity of the R-phycoerythrin fluorescence signal on the hydrogel particles is detected, thereby calculating the content of the target miRNA. The present invention realizes the color coding of different markers for the first time, which is used for mixed detection of various types of tumor marker miRNA, and the obtained hydrogel particles have high detection specificity and can be produced in large quantities and with high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a physical picture of hydrogel particles with different color codes described in Example 1 of the present application;

[0017] Figure 2 This is a fluorescence spectrum of the hydrogel particles described in Example 2 of the present application for detecting miR-21 at different concentrations:

[0018] a is the fluorescence spectrum; b is the quantitative analysis of fluorescence intensity;

[0019] Figure 3 This is a fluorescence picture of the hydrogel particles described in Example 2 of the present application used for detecting miR-21 at different concentrations;

[0020] Figure 4 This is a fluorescence spectrum of the hydrogel particles described in Example 3 of the present application for detecting miR-122 at different concentrations:

[0021] a is the fluorescence spectrum; b is the quantitative analysis of fluorescence intensity;

[0022] Figure 5 This is a fluorescence picture of the hydrogel particles described in Example 3 of the present application used for detecting miR-122 at different concentrations;

[0023] Figure 6 This is a fluorescence spectrum of the hydrogel particles described in Example 4 of the present application for detecting miR-233 at different concentrations:

[0024] a is the fluorescence spectrum; b is the quantitative analysis of fluorescence intensity;

[0025] Figure 7 This is a fluorescence picture of the hydrogel particles described in Example 4 of the present application used for detecting miR-233 at different concentrations;

[0026] Figure 8 This is a fluorescence spectrum of the hydrogel particles described in Example 5 of the present application for detecting miR-27a at different concentrations:

[0027] a is the fluorescence spectrum; b is the quantitative analysis of fluorescence intensity;

[0028] Fig. 9 This is a fluorescence picture of the hydrogel particles described in Example 5 of the present application used for detecting miR-27a at different concentrations;

[0029] Fig.10 This is a fluorescence picture of the hydrogel particles described in Example 6 of the present application used for detecting miR-21 and miR-122 at the same concentration;

[0030] Fig.11 This is a fluorescence image of the hydrogel particles described in Example 7 of the present application used to detect miR-21, miR-122 and miR-233 at the same concentration. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with examples. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0032] Example 1

[0033] A method for preparing color-coded multi-miRNA detection hydrogel particles comprises the following steps:

[0034] Dyes of different colors (green, purple, yellow, blue, black, pink) are added to a 2-5% sodium alginate solution and evenly aspirated with a syringe. Then, the sodium alginate solution is sprayed into the calcium chloride solution using a syringe to generate hydrogel particles with different color codes. Figure 1 shown.

[0035] Example 2

[0036] The above-mentioned hydrogel particles with different color codes were dispersed in 1 mL of deionized water, and 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added respectively. The reaction was carried out in an ice-water bath for 120 min to activate the carboxyl groups on the surface of the hydrogel particles. Then, 3 μL of detection aptamer was added (the sequence of miRNA-21 detection aptamer was 5'-GATATATTTTATCAACATCAGTCTGATAAGCTA-3'; the sequence of miRNA-122 detection aptamer was 5'-GATATATTTACCTCACACTGTTACCACAAAC-3'; the sequence of miRNA-233 detection aptamer was 5'-GATATATTTGACAAACTGACTGGGGTATTT-3'; the sequence of miRNA-27a detection aptamer was 5'-GATATATTTTGTGTAGATTCACAGTGG-3'), and the reaction was carried out in an ice water bath for 120 minutes to obtain hydrogel particles modified with different miRNA detection aptamers.

[0037] The results are as follows Figure 2 As shown in the figure, after modification with different miRNA detection aptamers, the surface charge of the hydrogel particles decreased, which was mainly due to the negative charge carried on the surface of the nucleic acid aptamer, indicating that the nucleic acid aptamer has been successfully modified on the surface of the hydrogel particles.

[0038] Example 3

[0039] The following experiments were performed in a clean bench to avoid inactivation of miRNA. 50 μL of hydrogel particles were dispersed in 50 μL of TET containing 700 mM NaCl. 0.5, 1.0, 3.0, and 5.0 μL of miRNA-21 were added to 95 μL of TET buffer containing 700 mM NaCl, respectively.

[0040] Then, the above two solutions were mixed in a metal bath for 90 min (55° C., 1500 r / min) to capture the target miRNA.

[0041] Subsequently, 245 μL of hybridization buffer containing 5 μL of biotin-modified universal aptamer (the universal aptamer sequence is 5'-TAAAATATATAAAAAAAAAAAA-3') was added and heated at 21.5°C for 30 minutes. Then 1 μL of 2g / mL streptavidin-R-phycoerythrin conjugate (SA-PE) was added and the reaction continued for 30 minutes at 21.5°C and the speed was 1500r / min. After the reaction, it was wrapped in tin foil to avoid light and stored in a refrigerator at 4°C. The fluorescence intensity changes of the hydrogel particles were measured using a fluorescence spectrometer, and the fluorescence images of the hydrogel particles were taken using an inverted fluorescence microscope.

[0042] The results are as follows Figure 3 and 4 As shown, as the concentration of miRNA-21 increases, the fluorescence signal on the surface of the hydrogel particles increases, indicating that the hydrogel particles can specifically bind to miRNA-21, and the intensity of the fluorescence signal is proportional to its concentration.

[0043] Example 4

[0044] The following experiments were performed in a clean bench to avoid miRNA inactivation. 50 μL of hydrogel particles were dispersed in 50 μL of TET containing 700 mM NaCl. 0.5, 1.0, 3.0, and 5.0 μL of miRNA-122 were added to 95 μL TET buffer containing 700 mM NaCl. Then, the two solutions were mixed in a metal bath for 90 minutes (55°C, 1500 r / min) to capture the target miRNA. Subsequently, 245 μL of hybridization buffer containing 5 μL of biotin-modified universal aptamer (sequence 5'-TAAAATATATAAAAAAAAAAAA-3') was added and heated at 21.5°C for 30 minutes. Then, 1 μL of 2 g / mL streptavidin-R-phycoerythrin conjugate (SA-PE) was added and the reaction continued at 21.5°C for 30 minutes at a speed of 1500 r / min. After the reaction, it was wrapped in tin foil to avoid light and stored in a refrigerator at 4°C. The fluorescence intensity changes of the hydrogel particles were measured using a fluorescence spectrometer, and the fluorescence images of the hydrogel particles were taken using an inverted fluorescence microscope. Figure 5 and 6 As shown, as the concentration of miRNA-122 increases, the fluorescence signal on the surface of the hydrogel particles increases, indicating that the hydrogel particles can specifically bind to miRNA-122, and the intensity of the fluorescence signal is proportional to its concentration.

[0045] Example 5

[0046] The following experiments were performed in a clean bench to avoid miRNA inactivation. 50 μL of hydrogel particles were dispersed in 50 μL of TET containing 700 mM NaCl. 0.5, 1.0, 3.0, and 5.0 μL of miRNA-223 were added to 95 μL TET buffer containing 700 mM NaCl. Then, the above two solutions were mixed in a metal bath for 90 minutes (55°C, 1500 r / min) to capture the target miRNA. Subsequently, 245 μL of hybridization buffer containing 5 μL of biotin-modified universal aptamer (sequence 5'-TAAAATATATAAAAAAAAAAAA-3') was added and heated at 21.5°C for 30 minutes. Then, 1 μL of 2 g / mL streptavidin-R-phycoerythrin conjugate (SA-PE) was added and the reaction continued at 21.5°C for 30 minutes at a speed of 1500 r / min. After the reaction, it was wrapped in tin foil to avoid light and stored in a refrigerator at 4°C. The fluorescence intensity changes of the hydrogel particles were measured using a fluorescence spectrometer, and the fluorescence images of the hydrogel particles were taken using an inverted fluorescence microscope. Figure 7 and 8As shown, as the concentration of miRNA-233 increases, the fluorescence signal on the surface of the hydrogel particles increases, indicating that the hydrogel particles can specifically bind to miRNA-233, and the intensity of the fluorescence signal is proportional to its concentration.

[0047] Example 6

[0048] The following experiments were performed in a clean bench to avoid miRNA inactivation. 50 μL of hydrogel particles were dispersed in 50 μL of TET containing 700 mM NaCl. 0.5, 1.0, 3.0, and 5.0 μL of miRNA-27a were added to 95 μL TET buffer containing 700 mM NaCl. Then, the above two solutions were mixed in a metal bath for 90 minutes (55°C, 1500 r / min) to capture the target miRNA. Subsequently, 245 μL of hybridization buffer containing 5 μL of biotin-modified universal aptamer (sequence 5'-TAAAATATATAAAAAAAAAAAA-3') was added and heated at 21.5°C for 30 minutes. Then, 1 μL of 2 g / mL streptavidin-R-phycoerythrin conjugate (SA-PE) was added and the reaction continued at 21.5°C for 30 minutes at a speed of 1500 r / min. After the reaction, it was wrapped in tin foil to avoid light and stored in a refrigerator at 4°C. The fluorescence intensity changes of the hydrogel particles were measured using a fluorescence spectrometer, and the fluorescence images of the hydrogel particles were taken using an inverted fluorescence microscope. Fig. 9 and 10 As shown, as the concentration of miRNA-27a increases, the fluorescence signal on the surface of the hydrogel particles increases, indicating that the hydrogel particles can specifically bind to miRNA-27a, and the intensity of the fluorescence signal is proportional to its concentration.

[0049] Example 7

[0050] The following experiments were performed in a clean bench to avoid miRNA inactivation. 50 μL of mixed hydrogel particles (half red and half blue) were dispersed in 50 μL TET containing 700 mM NaCl. 1.0 μL miRNA-122 and 1.0 μL miRNA-21 were added to 95 μL TET buffer containing 700 mM NaCl. Then, the above two solutions were mixed in a metal bath for 90 minutes (55°C, 1500 r / min) to capture the target miRNA. Subsequently, 245 μL hybridization buffer containing 5 μL biotin-modified universal aptamer (sequence 5'-TAAAATATATAAAAAAAAAAAA-3') was added and heated at 21.5°C for 30 minutes. Then, 1 μL of 2g / mL streptavidin-R-phycoerythrin conjugate (SA-PE) was added and the reaction continued at 21.5°C for 30 minutes at a speed of 1500 r / min. After the reaction, the particles were wrapped in tin foil to avoid light and stored in a refrigerator at 4°C. Fluorescence images of the hydrogel particles were taken using an inverted fluorescence microscope. Fig.11 As shown, the hydrogel particles we prepared can be used for the mixed detection of miRNA-122 and miRNA-21 at the same time.

[0051] Example 8

[0052] The following experiments were performed in a clean bench to avoid miRNA inactivation. 50 μL of mixed hydrogel particles (half red and half blue) were dispersed in 50 μL of TET containing 700 mM NaCl. 1.0 μL miRNA-122, 1.0 μL miRNA-21, and 1.0 μL miRNA-123 were added to 95 μL TET buffer containing 700 mM NaCl. Then, the above two solutions were mixed in a metal bath for 90 minutes (55°C, 1500 r / min) to capture the target miRNA. Subsequently, 245 μL hybridization buffer containing 5 μL biotin-modified universal aptamer (sequence 5'-TAAAATATATAAAAAAAAAAAA-3') was added and heated at 21.5°C for 30 minutes. Then add 1 μL of 2g / mL streptavidin-R-phycoerythrin conjugate (SA-PE), and continue the reaction at 21.5°C for 30 minutes at a speed of 1500r / min. After the reaction is completed, wrap it with tin foil to avoid light and store it in a refrigerator at 4°C. Use an inverted fluorescence microscope to take a fluorescent picture of the hydrogel particles. The hydrogel particles prepared by the present invention can be used for mixed detection of miRNA-122, miRNA-21 and miRNA123 at the same time.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing color-coded multi-miRNA detection hydrogel particles, characterized in that: The following steps are involved: (1) Preparing hydrogel particles of different colors: adding dyes of different colors to a sodium alginate solution, mixing evenly, and setting the solution aside; (2) solidification: adding the solution obtained in step (1) dropwise into a saturated calcium chloride solution to obtain hydrogel particles with different color codes; (3) Aptamer modification: combining the hydrogel particles obtained in step (2) with the miRNA detection aptamer to obtain aptamer-modified hydrogel particles; In step (3), the prepared hydrogel particles are dispersed in water, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide solids are added respectively, and stirred in an ice-water bath to activate the carboxyl groups, and then the miRNA detection aptamer solution is added, and the ice-water bath is continued to stir, centrifuged, and the aptamer-modified hydrogel particles are collected; The miRNA detection aptamer is modified with Acrydite (acrylamide group) at the 5' end and NH2 (amino group) at the 3' end.

2. The method for preparing color-coded multi-miRNA detection hydrogel particles according to claim 1, characterized in that: In step (1), a sodium alginate solution with a concentration of 2-5% is mixed with 2 μL of dye.

3. The method for preparing color-coded multi-miRNA detection hydrogel particles according to claim 1, characterized in that: In step (2), the sodium alginate solution is sprayed into the calcium chloride solution to generate small hydrogel particles.

4. The method for preparing color-coded multi-miRNA detection hydrogel particles according to claim 1, characterized in that: The aptamer sequence for miRNA-21 detection is: 5'-GATATATTTTATCAACATCAGTCTGATAAGCTA-3'; the aptamer sequence for miRNA-122 detection is: 5'-GATATATTTACCTCACACTGTTACCACAAAC-3'; the aptamer sequence for miRNA-233 detection is: 5'-GATATATTTGACAAACTGACTGGGGTATTT-3'; The miRNA-27a detection aptamer sequence is: 5'-GATATATTTTGTGTAGATTCACAGTGG-3'.

5. The method for preparing color-coded multi-miRNA detection hydrogel particles according to claim 1, characterized in that: The method also includes (4) miRNA detection: mixing the aptamer-modified hydrogel particles obtained in step (3) with the miRNA solution to perform miRNA detection.

6. The method for preparing color-coded multi-miRNA detection hydrogel particles according to claim 5, characterized in that: In step (4), the hydrogel particles are dispersed in TET containing NaCl, and the miRNA is added to the TE buffer containing NaCl; then, the above two solutions are mixed in a metal bath to capture the target miRNA; a hybridization buffer containing a biotin-modified universal aptamer with a sequence of 5'-TAAAATATATAAAAAAAAAAAA-3' is added, and heated at 21.5°C; Then, streptavidin-R-phycoerythrin conjugate was added, and the reaction was continued at 21.5°C to calculate the content of the target miRNA.

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