Fluorescent RNA functionalized hydrogel kit, preparation method and method for detecting colorectal cancer miRNA-126

Abstract

The invention relates to a fluorescent RNA (Ribonucleic Acid) functionalized hydrogel kit, a preparation method and a method for detecting colorectal cancer miRNA-126, solves the technical problem of how to detect miRNA-126 in blood or tissue samples and is suitable for portable detection, and fluorescent RNA functionalized hydrogel is prepared from methacrylic acid hyaluronic acid, an acryloyl-modified fluorescent RNA aptamer and N, N-dimethylformamide. The fluorescent RNA aptamer is obtained by crosslinking the fluorescent RNA aptamer and N, N '-methylene bisacrylamide, and the covalent crosslinking between the fluorescent RNA aptamer and the hydrogel can prevent the RNA aptamer from being released into the external environment, so that the stability and the anti-interference capability of the hydrogel kit are improved.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more specifically, to a fluorescent RNA-functionalized hydrogel kit, its preparation method, and a method for detecting colorectal cancer miRNA-126. Background Technology

[0002] Colorectal cancer is the third most common cancer worldwide, with high morbidity and mortality rates, and remains a significant public health concern. Clinically, colonoscopy is widely recognized as the "gold standard" for diagnosing colorectal cancer. However, colonoscopy is invasive, complex, and time-consuming, and carries risks of bleeding and perforation; long-term monitoring via colonoscopy also faces challenges in patient compliance. Therefore, the demand for non-invasive diagnostic methods in colorectal cancer screening is growing.

[0003] Due to their non-invasive collection and low cost, cancer-related biomarkers are well-suited for widespread application in clinical practice. Minimal RNAs (miRNAs) play a crucial role in gene expression and immune-mediated diseases. Studies have shown that miRNA-126 can inhibit the growth, invasion, metastasis, and epithelial-mesenchymal transition of colon cancer cells, indicating that miRNA-126 is a potential cancer-related biomarker. Therefore, quantifying the expression level of miRNA-126 in blood or tissue samples can provide crucial evidence to support the early detection or monitoring of colorectal cancer.

[0004] Therefore, how to detect miRNA-12 in blood or tissue samples is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This application aims to solve the technical problem of how to detect miRNA-126 in blood or tissue samples, and provides a fluorescent RNA functionalized hydrogel kit, preparation method, and method for detecting miRNA-126 in colorectal cancer.

[0006] Fluorescent RNA aptamers, also known as fluorescent "light-up" aptamers, can activate the fluorescence of non-fluorescent dyes by folding into specific structures. Fluorescent RNA aptamers are powerful tools for RNA visualization and detection, offering advantages such as high quantum yield, rapid fluorescence activation, small size, and good resistance to photobleaching. More importantly, these fluorescent RNA sensors exhibit low background noise and a high signal-to-noise ratio, making them promising for broad applications in the field of fluorescence visualization.

[0007] Hydrophilic hydrogels have advantages in terms of load-bearing capacity and mechanical stability due to their porous polymer network structure, and can uniformly immobilize fluorescent RNA in the pores.

[0008] A first aspect of this disclosure provides a fluorescent RNA-functionalized hydrogel kit comprising a fluorescent RNA-functionalized hydrogel and a non-fluorescent dye.

[0009] Preferably, the fluorescent RNA-functionalized hydrogel comprises a solvent, methacrylamide, bisacrylamide, a photoinitiator, and fluorescent RNA, wherein the methacrylamide, bisacrylamide, photoinitiator, and fluorescent RNA are cross-linked.

[0010] Preferably, the non-fluorescent dye is HBC 530.

[0011] Preferably, the fluorescent RNA is acryloyl-modified fluorescent RNA.

[0012] A second aspect of this disclosure provides a method for detecting colorectal cancer miRNA-126 using a fluorescent RNA-functionalized hydrogel kit, comprising the following steps: Step S1, preparation of miRNA-126 detection solution: The sample to be tested is prepared in double-distilled water without RNase to form the test sample; the test sample is then mixed with a non-fluorescent dye to form the miRNA-126 detection solution. Step S2: The fluorescent RNA functionalized hydrogel is co-incubated with the miRNA-126 detection solution; Step S3: A fluorescence microscope is used to collect fluorescence signals and identify miRNA-126.

[0013] Preferably, the concentration of miRNA-126 in the sample is calculated based on the fluorescence signal.

[0014] A third aspect of this disclosure provides a method for preparing a fluorescent RNA-functionalized hydrogel kit, wherein the fluorescent RNA-functionalized hydrogel is prepared by the following method: A photoinitiator and bisacrylamide were added to a dissolving solution to dissolve the photoinitiator. Subsequently, methacrylated hyaluronic acid and acryloyl-modified fluorescent RNA were added to the above solution to obtain a mixed solution. The mixed solution was then dropped into the wells of an immunofluorescence slide and exposed to achieve cross-linking.

[0015] A fourth aspect of this disclosure provides a fluorescent RNA-functionalized hydrogel comprising a solution, methacrylamide, bisacrylamide, a photoinitiator, and fluorescent RNA, wherein the methacrylamide, bisacrylamide, photoinitiator, and fluorescent RNA are cross-linked.

[0016] The beneficial effects of this disclosure are that the kit can effectively detect miRNA-126 qualitatively and quantitatively; the linear range is 0.1~10000 nM, and the limit of detection is 23.8 pM. Furthermore, it allows for visual detection.

[0017] It exhibits good stability and anti-interference characteristics. It also boasts advantages such as high signal-to-noise ratio, simplicity, and versatility.

[0018] It can be used to identify biomarkers for colorectal cancer, providing a basis for early screening of colorectal cancer.

[0019] It is fast and quick to detect, making it suitable for point-of-care testing (POCT). It enables portable testing applications.

[0020] Further features and aspects of this disclosure will be clearly described in the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0021] Figure 1 This is the detection result of Example 2, a standard curve graph of different concentrations of miRNA-126 and the gray value of fluorescence signal; Figure 2 This is a comparison graph of fluorescence intensity of the detection results of Example 1 and Comparative Examples 1, 2, and 3; Figure 3 This is a comparison chart of fluorescence intensity results from Example 3; Figure 4 This is a comparison chart of fluorescence response values ​​from the detection results of Example 4. Detailed Implementation

[0022] The application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] The specific embodiments described below are merely preferred embodiments of this application, and the scope of protection of this application is not limited thereto. Those skilled in the art can make modifications or variations based on the principles, concepts, and spirit of this application, and the resulting technical solutions should all be covered within the scope of protection of this application.

[0024] Example 1

[0025] The kit includes a fluorescent RNA-functionalized hydrogel and a non-fluorescent dye. The fluorescent RNA-functionalized hydrogel includes a solvent, methacrylamide, bisacrylamide, a photoinitiator, and fluorescent RNA. The methacrylamide, bisacrylamide, photoinitiator, and fluorescent RNA are cross-linked. The mass ratio of bisacrylamide to methacrylamide can be 1:4.

[0026] Fluorescent RNA-functionalized hydrogels were prepared using the following method: Step (1), Preparation of methacrylamide hyaluronic acid: 2 g of hyaluronic acid (molecular weight: 20-40 kDa) was dissolved in 100 mL of ultrapure water and stirred at 4°C for 7 hours until completely dissolved. Then, 1.6 mL of methacrylic anhydride was added, and the pH was adjusted to 8-9 with 2.5 M NaOH. o Stir at C for 12 hours. Add anhydrous ethanol (more than three times the volume of the mixture) to the above solution and collect the flocculent product. Redissolve the collected product in 100 mL of ultrapure water. o Stir at C for 7 hours. The resulting solution is then... o Dialysis was performed at C for 48 hours. The dialysate was collected and freeze-dried for 3 days.

[0027] Step (2), Preparation of fluorescent RNA functionalized hydrogel: Add 10 g of photoinitiator I-2959 and 10 mg of bisacrylamide to 800 μL of HEPES buffer, and incubate the mixture at 37°C. o Incubate at C for 10 minutes to completely dissolve the photoinitiator I-2959. Then, add 40 mg of methacrylamide hyaluronic acid and 50 μL of 10 μM acryloyl-modified fluorescent RNA to the solution. Add 5 μL of the resulting mixture to the wells of an immunofluorescence slide. Finally, expose to a blue lamp at 470 nm for 40 min to achieve cross-linking, thus obtaining a fluorescent RNA-functionalized hydrogel.

[0028] Acrylyl-modified fluorescent RNA was obtained from commercially available products.

[0029] The method for detecting colorectal cancer miRNA-126 using the above kit includes the following steps: Step S1, preparation of miRNA-126 detection solution: The purchased lyophilized miRNA-126 powder was dissolved in double-distilled water without RNase to form a test sample containing miRNA-126. 10 μL of the 1 nM test sample was mixed with 5 μL of 10 mM non-fluorescent dye HBC530 to form the miRNA-126 detection solution.

[0030] Step S2: The fluorescent RNA-functionalized hydrogel is co-incubated with the miRNA-126 detection solution, as follows: Add 3 μL of miRNA-126 detection solution to the well of a glass slide and co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. oIncubate in a humid environment at C for 40 min. miRNA-126 pairs complementaryly with the corresponding sequence of fluorescent RNA, specifically recognizing and capturing miRNA-126. After miRNA-126 binds to fluorescent RNA, the conformation of fluorescent RNA changes, forming a new structure. The non-fluorescent dye HBC 530 in the detection solution binds to the fluorescent RNA and emits light.

[0031] Step S3: Acquire fluorescence signals using a fluorescence microscope. The fluorescence signal was observed under a fluorescence microscope (10x objective) and obtained. The average fluorescence intensity of the green channel was analyzed using ImageJ software to quantify miRNA-126 and obtain the concentration of miRNA-126 in the sample.

[0032] Example 2

[0033] Quantitative analysis was performed on different concentrations of miRNA-126.

[0034] The first step is to prepare methacrylated hyaluronic acid: 2 g of hyaluronic acid (molecular weight: 20-40 kDa) was dissolved in 100 mL of ultrapure water and stirred at 4°C for 7 hours until completely dissolved. Then, 1.6 mL of methacrylic anhydride was added, and the pH was adjusted to 8-9 with 2.5 M NaOH. o Stir at C for 12 hours. Add anhydrous ethanol (more than three times the volume of the mixture) to the above solution and collect the flocculent product. Redissolve the collected product in 100 mL of ultrapure water. o Stir at C for 7 hours. The resulting solution is then... o Dialysis was performed at C for 48 hours. The dialysate was collected and freeze-dried for 3 days.

[0035] The second step is to prepare a fluorescent RNA-functionalized hydrogel: Add 10 g of photoinitiator I-2959 and 10 mg of bisacrylamide to 800 μL of HEPES buffer, and incubate the mixture at 37°C. o Incubate at C for 10 minutes to completely dissolve photoinitiator I-2959. Then, add 40 mg of methacrylamide hyaluronic acid and 50 μL of 10 μM acryloyl-modified fluorescent RNA to the solution. Add 5 μL of the resulting mixture to the wells of an immunofluorescence slide. Finally, expose for 40 min to achieve cross-linking, thereby obtaining a fluorescent RNA-functionalized hydrogel.

[0036] The third step is to prepare detection solutions of different concentrations of miRNA-126: Different concentrations (0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) of miRNA-126 were prepared in double-distilled water without RNase and mixed with 5 μL of 10 mM non-fluorescent dye HBC 530 to form miRNA-126 detection solutions of different concentrations.

[0037] The fourth step involved co-incubating the fluorescent RNA-functionalized hydrogel with six different concentrations of miRNA-126 detection solutions, as follows: Add 3 μL of miRNA-126 detection solution to the well of a glass slide and co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0038] Step S3: Acquire fluorescence signals using a fluorescence microscope. Fluorescence signals were observed under a fluorescence microscope (10x objective) and obtained. The average fluorescence intensity of the green channel was analyzed using ImageJ software to quantify miRNA-126 at different concentrations in the detection solution, thus obtaining the concentration of miRNA-126. Figure 1 The standard curves for different concentrations of miRNA-126 and the gray values ​​of fluorescence signals are shown. The linear regression equation between the logarithm of miRNA-126 concentration and the gray value of fluorescence signal intensity is as follows: Gray value =3.938lg(miRNA-126 / nM)+23.497( R 2 =0.997), the detection limit is 23.8 pM ( ). S / N =3).

[0039] The following feasibility analysis is conducted using a comparative method with a blank control.

[0040] Comparative Example 1 The first step is to prepare methacrylated hyaluronic acid: 2 g of hyaluronic acid (molecular weight: 20-40 kDa) was dissolved in 100 mL of ultrapure water and stirred at 4°C for 7 hours until completely dissolved. Then, 1.6 mL of methacrylic anhydride was added, and the pH was adjusted to 8-9 with 2.5 M NaOH. o Stir at C for 12 hours. Add anhydrous ethanol (more than three times the volume of the mixture) to the above solution and collect the flocculent product. Redissolve the collected product in 100 mL of ultrapure water. o Stir at C for 7 hours. The resulting solution is then... oDialysis was performed at C for 48 hours. The dialysate was collected and freeze-dried for 3 days.

[0041] The second step is to prepare a hydrogel that does not contain fluorescent RNA: 10 g of photoinitiator I-2959 and 10 mg of bisacrylamide were added to 800 μL of HEPES buffer. The mixture was incubated at 37 °C for 10 min to allow photoinitiator I-2959 to completely dissolve. Subsequently, 40 mg of methacrylamide hyaluronic acid was added to the above solution to form a mixed solution. 5 μL of the mixed solution was dropped into the wells of an immunofluorescence slide. Finally, the slide was exposed for 40 min to achieve cross-linking, thereby obtaining a hydrogel without fluorescent RNA.

[0042] The third step is to prepare the miRNA-126 detection solution: Take 10 μL of 1 nM miRNA-126 solution (prepared in double-distilled water without RNase) and mix it with 5 μL of 10 mM non-fluorescent dye HBC 530 to form the miRNA-126 detection solution.

[0043] Step 4: Co-incubate the hydrogel without fluorescent RNA with the miRNA-126 detection solution: Add 3 μL of miRNA-126 detection solution to the well of the glass slide and co-incubate with the hydrogel without fluorescent RNA at 45°C. o Incubate in a humid environment at C for 40 min.

[0044] Fifth step: fluorescence signal is acquired using a fluorescence microscope; no fluorescence signal is detected.

[0045] Comparative Example 2 The first step is to prepare methacrylated hyaluronic acid: 2 g of hyaluronic acid (molecular weight: 20-40 kDa) was dissolved in 100 mL of ultrapure water and stirred at 4°C for 7 hours until completely dissolved. Then, 1.6 mL of methacrylic anhydride was added, and the pH was adjusted to 8-9 with 2.5 M NaOH. o Stir at C for 12 hours. Add anhydrous ethanol (more than three times the volume of the mixture) to the above solution and collect the flocculent product. Redissolve the collected product in 100 mL of ultrapure water. o Stir at C for 7 hours. The resulting solution is then... o Dialysis was performed at C for 48 hours. The dialysate was collected and freeze-dried for 3 days.

[0046] The second step is to prepare a fluorescent RNA-functionalized hydrogel: Add 10 g of photoinitiator I-2959 and 10 mg of bisacrylamide to 800 μL of HEPES buffer, and incubate the mixture at 37°C. oIncubate at C for 10 minutes to completely dissolve photoinitiator I-2959. Then, add 40 mg of methacrylamide hyaluronic acid and 50 μL of 10 μM acryloyl-modified fluorescent RNA to the solution. Add 5 μL of the resulting mixture to the wells of an immunofluorescence slide. Finally, expose for 40 min to achieve cross-linking, thereby obtaining a fluorescent RNA-functionalized hydrogel.

[0047] The third step is to prepare a detection solution that does not contain miRNA-126: prepare 5 μL of 10 mM non-fluorescent dye HBC 530 to form a detection solution that does not contain miRNA-126.

[0048] Step 4: Co-incubate the fluorescent RNA-functionalized hydrogel with the detection solution without miRNA-126: Add 3 μL of the detection solution without miRNA-126 to the well of the glass slide and co-incubate with the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0049] The fifth step involves using a fluorescence microscope to collect fluorescence signals, which are weak.

[0050] Comparative Example 3 The first step is to prepare methacrylated hyaluronic acid: 2 g of hyaluronic acid (molecular weight: 20-40 kDa) was dissolved in 100 mL of ultrapure water and stirred at 4°C for 7 hours until completely dissolved. Then, 1.6 mL of methacrylic anhydride was added, and the pH was adjusted to 8-9 with 2.5 M NaOH. o Stir at C for 12 hours. Add anhydrous ethanol (more than three times the volume of the mixture) to the above solution and collect the flocculent product. Redissolve the collected product in 100 mL of ultrapure water. o Stir at C for 7 hours. The resulting solution is then... o Dialysis was performed at C for 48 hours. The dialysate was collected and freeze-dried for 3 days.

[0051] The second step is to prepare a fluorescent RNA-functionalized hydrogel: Add 10 g of photoinitiator I-2959 and 10 mg of bisacrylamide to 800 μL of HEPES buffer, and incubate the mixture at 37°C. o Incubate at C for 10 minutes to completely dissolve photoinitiator I-2959. Then, add 40 mg of methacrylamide hyaluronic acid and 50 μL of 10 μM acryloyl-modified fluorescent RNA to the solution. Add 5 μL of the resulting mixture to the wells of an immunofluorescence slide. Finally, expose for 40 min to achieve cross-linking, thereby obtaining a fluorescent RNA-functionalized hydrogel.

[0052] The third step is to prepare a detection solution that does not contain the non-fluorescent dye HBC 530: Prepare 10 μL of 1 nM miRNA-126 solution in double-distilled water without RNase to form a detection solution free of the non-fluorescent dye HBC 530.

[0053] Step 4: Co-incubate the fluorescent RNA-functionalized hydrogel with a detection solution free of the non-fluorescent dye HBC 530: Add 3 μL of HBC 530 (free of the non-fluorescent dye) to the well of a glass slide and co-incubate with the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0054] The fifth step involves using a fluorescence microscope to collect fluorescence signals, which are weak.

[0055] Therefore, refer to Figure 2 The fluorescence signal in Comparative Example 3 is weak (fourth bar in the figure); the fluorescence signal in Comparative Example 2 is weak (second bar in the figure); the result for Comparative Example 1 is the background fluorescence signal. The first bar in the figure represents the detection result of Example 1, with a strong fluorescence signal.

[0056] Example 3

[0057] To evaluate the selectivity of the kit, the fluorescent RNA functionalized hydrogel kit prepared in Example 1 was used to detect 1 nM miRNA-126, single-base mismatched miRNA (sm-RNA), triple-base mismatched miRNA (sm-RNA), and miRNA-21, respectively.

[0058] The first step is to prepare methacrylated hyaluronic acid: 2 g of hyaluronic acid (molecular weight: 20-40 kDa) was dissolved in 100 mL of ultrapure water and stirred at 4°C for 7 hours until completely dissolved. Then, 1.6 mL of methacrylic anhydride was added, and the pH was adjusted to 8-9 with 2.5 M NaOH. o Stir at C for 12 hours. Add anhydrous ethanol (more than three times the volume of the mixture) to the above solution and collect the flocculent product. Redissolve the collected product in 100 mL of ultrapure water. o Stir at C for 7 hours. The resulting solution is then... o Dialysis was performed at C for 48 hours. The dialysate was collected and freeze-dried for 3 days.

[0059] The second step is to prepare a fluorescent RNA-functionalized hydrogel: Add 10 g of photoinitiator I-2959 and 10 mg of bisacrylamide to 800 μL of HEPES buffer, and incubate the mixture at 37°C. o Incubate at C for 10 minutes to completely dissolve photoinitiator I-2959. Then, add 40 mg of methacrylamide hyaluronic acid and 50 μL of 10 μM acryloyl-modified fluorescent RNA to the solution. Add 5 μL of the resulting mixture to the wells of an immunofluorescence slide. Finally, expose for 40 min to achieve cross-linking, thereby obtaining a fluorescent RNA-functionalized hydrogel.

[0060] The third step is to prepare detection solutions for miRNA-126, single-base mismatched miRNA (sm-RNA), triple-base mismatched miRNA (tm-RNA), and miRNA-21, respectively: A 1 nM miRNA-126 solution was prepared in double-distilled water without RNase and mixed with 5 μL of 10 mM non-fluorescent dye HBC 530 to form a miRNA-126 detection solution.

[0061] Prepare 1 nM sm-RNA in double-distilled water without RNase, and mix it with 5 μL of 10 mM non-fluorescent dye HBC530 to form an sm-RNA detection solution.

[0062] Prepare 1 nM tm-RNA in double-distilled water without RNase, and mix it with 5 μL of 10 mM non-fluorescent dye HBC530 to form a tm-RNA detection solution.

[0063] A 1 nM miRNA-21 solution was prepared in double-distilled water without RNase and mixed with 5 μL of 10 mM non-fluorescent dye HBC 530 to form a miRNA-21 detection solution.

[0064] The fourth step involves co-incubating the fluorescent RNA-functionalized hydrogel with detection solutions for miRNA-126, smRNA, tmRNA, and miRNA-21, respectively, as follows: Add 3 μL of miRNA-126 detection solution to the well of a glass slide and co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0065] Add 3 μL of smRNA detection solution to the well of a glass slide and co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0066] Add 3 μL of tm-RNA detection solution to the well of a glass slide and co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0067] Add 3 μL of miRNA-21 detection solution to the well of a glass slide and co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0068] Step 5: Acquire fluorescence signals using a fluorescence microscope: Fluorescence images were obtained by observing under a fluorescence microscope (10x objective). The average fluorescence intensity of the green channel was analyzed using ImageJ software to quantify miRNA-126, sm-RNA, tm-RNA, and miRNA-21. Figure 3 The images show the fluorescence intensity of miRNA-126, sm-RNA, tm-RNA, and miRNA-21. The fluorescence signal recoveries of the control groups (sm-RNA, tm-RNA, and miRNA-21) were 22.3%, 12.5%, and 10.1%, respectively. Meanwhile, the signal recovery rate of the blank group was only 7.3%. These results demonstrate the excellent selectivity of the hydrogel kit for fluorescent RNA functionalization.

[0069] Example 4

[0070] To investigate the kit's resistance to interference in complex environments, buffer and normal human serum were used as test matrices, with 1 nM miRNA-126 added to the matrices.

[0071] The first step is to prepare methacrylated hyaluronic acid: 2 g of hyaluronic acid (molecular weight: 20-40 kDa) was dissolved in 100 mL of ultrapure water and stirred at 4°C for 7 hours until completely dissolved. Then, 1.6 mL of methacrylic anhydride was added, and the pH was adjusted to 8-9 with 2.5 M NaOH. o Stir at C for 12 hours. Add anhydrous ethanol (more than three times the volume of the mixture) to the above solution and collect the flocculent product. Redissolve the collected product in 100 mL of ultrapure water. o Stir at C for 7 hours. The resulting solution is then... o Dialysis was performed at C for 48 hours. The dialysate was collected and freeze-dried for 3 days.

[0072] The second step is to prepare a fluorescent RNA-functionalized hydrogel: Add 10 g of photoinitiator I-2959 and 10 mg of bisacrylamide to 800 μL of HEPES buffer, and incubate the mixture at 37°C. o Incubate at C for 10 minutes to completely dissolve photoinitiator I-2959. Then, add 40 mg of methacrylamide hyaluronic acid and 50 μL of 10 μM acryloyl-modified fluorescent RNA to the solution. Add 5 μL of the resulting mixture to the wells of an immunofluorescence slide. Finally, expose for 40 min to achieve cross-linking, thereby obtaining a fluorescent RNA-functionalized hydrogel.

[0073] The third step is to prepare the miRNA-126 detection solution: Five 1 nM miRNA-126 solutions were prepared using buffer solution and normal human serum at different dilutions (10%, 20%, 30%, 50%, and 70%). These solutions were then mixed with 5 μL and 10 mM of the non-fluorescent small molecule dye HBC-530 to form five miRNA-126 detection solutions.

[0074] Fourth, the fluorescent RNA-functionalized hydrogel was co-incubated with the five miRNA-126 detection solutions obtained in the third step, as follows: Take 3 μL of the first miRNA-126 detection solution and add it to the well of the glass slide. Co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0075] Take 3 μL of the second miRNA-126 detection solution and add it to the well of the glass slide. Co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0076] Take 3 μL of the third miRNA-126 detection solution and add it to the well of the glass slide. Co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0077] Take 3 μL of the fourth miRNA-126 detection solution and add it to the well of the glass slide. Co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C. o Incubate in a humid environment at C for 40 min.

[0078] Take 3 μL of the fifth miRNA-126 detection solution and add it to the well of the glass slide. Co-incubate it with the fluorescent RNA in the fluorescent RNA-functionalized hydrogel at 45°C.o Incubate in a humid environment at C for 40 min.

[0079] Step 5: Acquire fluorescence signals using a fluorescence microscope: Fluorescence images were obtained by observing under a fluorescence microscope (10x objective). The average fluorescence intensity of the green channel was analyzed using ImageJ software for quantitative detection of the fluorescence signal. Figure 4 The images show the fluorescence signals of the five miRNA-126 detection solutions. In the serum-containing group, the fluorescence response values ​​corresponding to 10%, 20%, 30%, 50%, and 70% serum concentrations were 89.6%, 80.6%, 65.4%, 45.2%, and 22.5%, respectively. These results indicate that the kit exhibits excellent anti-interference capabilities.

[0080] Covalent cross-linking between fluorescent RNA aptamers and hydrogels prevents the RNA aptamers from being released into the external environment, thereby improving the stability and anti-interference ability of the hydrogel kit.

Claims

1. A hydrogel kit for fluorescent RNA functionalization, characterized in that, This includes fluorescent RNA-functionalized hydrogels and non-fluorescent dyes.

2. The hydrogel kit for fluorescent RNA functionalization according to claim 1, characterized in that, The fluorescent RNA-functionalized hydrogel comprises a solvent, methacrylamide, bisacrylamide, a photoinitiator, and fluorescent RNA, wherein the methacrylamide, bisacrylamide, photoinitiator, and fluorescent RNA are cross-linked.

3. The hydrogel kit for fluorescent RNA functionalization according to claim 1, characterized in that, The non-fluorescent dye is HBC 530.

4. The hydrogel kit for fluorescent RNA functionalization according to claim 1, characterized in that, The fluorescent RNA is an acryloyl-modified fluorescent RNA.

5. A method for detecting colorectal cancer miRNA-126 using the fluorescent RNA-functionalized hydrogel kit according to claim 1, characterized in that, Includes the following steps: Step S1, preparation of miRNA-126 detection solution: The sample to be tested is prepared in double-distilled water without RNase to form the test sample; the test sample is then mixed with a non-fluorescent dye to form the miRNA-126 detection solution. Step S2: The fluorescent RNA functionalized hydrogel is co-incubated with the miRNA-126 detection solution; Step S3: A fluorescence microscope is used to collect fluorescence signals and identify miRNA-126.

6. A method for detecting colorectal cancer miRNA-126 using a fluorescent RNA-functionalized hydrogel kit as described in claim 2, characterized in that, Includes the following steps: Step S1, preparation of miRNA-126 detection solution: The sample to be tested is prepared in double-distilled water without RNase to form the test sample; the test sample is then mixed with a non-fluorescent dye to form the miRNA-126 detection solution. Step S2: The fluorescent RNA functionalized hydrogel is co-incubated with the miRNA-126 detection solution; Step S3: A fluorescence microscope is used to collect fluorescence signals and identify miRNA-126.

7. The method for detecting colorectal cancer miRNA-126 according to claim 6, characterized in that, The concentration of miRNA-126 in the sample was calculated based on the fluorescence signal.

8. A method for preparing the fluorescent RNA functionalized hydrogel kit according to claim 2, characterized in that, Fluorescent RNA-functionalized hydrogels were prepared using the following method: A photoinitiator and bisacrylamide were added to a dissolving solution to dissolve the photoinitiator. Subsequently, methacrylated hyaluronic acid and acryl-modified fluorescent RNA were added to the above solution to obtain a mixed solution. The mixed solution was then dropped into the wells of an immunofluorescence slide. Exposure enables cross-linking.

9. A fluorescent RNA-functionalized hydrogel, characterized in that, It includes a solution, methacrylamide, bisacrylamide, a photoinitiator, and fluorescent RNA, wherein the methacrylamide, bisacrylamide, photoinitiator, and fluorescent RNA are cross-linked.

10. The hydrogel kit for fluorescent RNA functionalization according to claim 2, characterized in that, The mass ratio of bisacrylamide to methacrylamide hyaluronic acid is 1:4.

Images