Porous hydrogel microneedle for miRNA (micro Ribonucleic Acid) detection as well as preparation method and detection method of porous hydrogel microneedle
Through the design of porous hydrogel microneedle and osmotic pressure-driven technology, the trauma risk and large sample collection problems of traditional miRNA detection methods are solved, painless, fast and ultra-sensitive miRNA detection is achieved, and the diagnostic process is simplified. It is an important means of early cancer detection.
Patent Information
- Application Number
- CN202411452002.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2024-12-31
AI Technical Summary
Traditional miRNA detection methods have problems with trauma risk and large sample collection and long time, making it difficult to achieve sensitive diagnosis of early cancer.
Using porous hydrogel microneedles, prepared by polyethylene glycol and lithium chloride modified hydrogel, the pyramid-shaped needle tip and high porosity structure are designed, combined with the osmotic pressure driving force, and the miRNA-21, miRNA-221 and miRNA- 155 capture efficiency and ultra-sensitive detection through special probes and Au nanoparticles.
Painless, fast and ultra-sensitive miRNA detection is achieved, the diagnostic process is simplified, the number and time of sample collection is reduced, and the rich interstitial fluid samples are provided, providing an important means for early diagnosis of cancer.
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Figure CN119214647A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of functional microneedle biological detection, and more specifically to a porous hydrogel microneedle for miRNA detection and a preparation and detection method thereof. Background Art
[0002] miRNA is a class of non-protein-coding endogenous single-stranded RNA consisting of about 22 nucleotides. In recent years, more and more studies have shown that in addition to regulating various life activities, the abnormal expression of miRNA is closely related to the occurrence, development, deterioration and metastasis of various cancers. However, the traditional way of obtaining miRNA is through blood, which not only causes trauma to patients, but also requires corresponding treatment and care for the wound surface. With the development of microneedle technology, microneedles have shown broad application prospects in the field of disease diagnosis and monitoring. Microneedles are composed of micro needles of 25 to 2500 μm. The micron-sized needle tip can quickly recover after being inserted into the skin, and will not significantly damage the skin structure. It is widely used in the detection of biomarkers in the interstitial fluid (ISF) of skin tissue. Therefore, sensitive detection of miRNA based on microneedles is of vital significance for the early diagnosis of cancer.
[0003] Chinese patent "Application No.: CN202211116354.3" reports a multi-parameter SERS-active microneedle for in-situ detection of inflammatory environments, which achieves rapid detection of inflammatory sites through the surface enhanced Raman scattering effect of metal nanoparticles. Chinese patent "Application No.: CN202311009753.4" reports a microfluidic-microneedle detection device and method, which connects the microneedle extraction chamber with the microfluidic channel to achieve rapid detection. Summary of the invention
[0004] Purpose of the invention: The present invention provides a porous hydrogel microneedle for miRNA detection and a preparation and detection method thereof, which improves the capture efficiency of miRNA-21, miRNA-221 and miRNA-155 in ISF by microneedles through osmotic pressure driving, and ultimately achieves ultra-sensitive diagnosis of miRNA.
[0005] Technical solution: The porous hydrogel microneedle used in the present invention for miRNA detection and its preparation and detection method are achieved by the following methods:
[0006] The microneedle has a porous bionic structure and osmotic pressure driving force, a porosity of 30-35%, a microneedle height of 1000-1200 μm, a pyramid-shaped needle tip, and a bottom diameter of 350-400 μm; the hydrogel microneedle is realized by modifying the hydrogel with polyethylene glycol and lithium chloride.
[0007] The present invention also provides the hydrogel microneedle preparation method comprising the following steps: dissolving polyvinyl alcohol (12wt%), polyvinyl pyrrolidone (6wt%), crosslinking agent glutaraldehyde (0.003wt%) and polyethylene glycol (0.1-0.2wt%) in deionized water, heating the resulting mixture to 90°C and stirring for 3h. Then pouring it into a PDMS mold, vacuuming and removing bubbles and drying it at room temperature for 24h. Finally, the microneedle is peeled off from the mold and immersed in a solution of 20-35wt% lithium chloride for 2h to obtain an osmotic pressure-driven microneedle, the microneedle height is 1000-1200μm, the needle tip is designed to be pyramid-shaped, the bottom diameter is 350-400μm, and the porosity is 30-35%.
[0008] The present invention also provides a method for detecting miRNA using the hydrogel microneedle, comprising the following steps:
[0009] (1) The capture probe I was first dissolved to a concentration of 50-150 μM at 0.5-1.0 OD, and then 1-2 μL of 1-2 M tris(2-carbonylethyl)phosphine hydrochloride (TCEP) solution was added and mixed thoroughly, and the reduction reaction was carried out at room temperature for 30-40 min. The needle body and needle tip of the prepared microneedle were inserted into the solution and allowed to stand for 10-20 min, then taken out, rinsed with PBS buffer, and immersed in 5-8% BSA solution at room temperature for 1-2 h to encapsulate the detection probe I.
[0010] (2) The capture probe I osmotic pressure-driven microneedle prepared in step (1) is immersed in the liquid to be tested or inserted into living tissue, and after reacting for 6 to 8 hours, it is taken out and rinsed with PBS buffer to obtain a microneedle bound to the miRNA to be tested.
[0011] (3) Dissolve 0.5-1.0OD SH-probe II to 50-150μM, add 5-7μL 1-2M tris(hydroxymethyl)acrylate buffer (TECP), mix well, and let stand at room temperature for 20-30 minutes. Add 500-600μL Au nanoparticle solution (0.1-0.2mg / mL) and reduced SH-probe II to a glass bottle, and then place it in a microwave oven for medium-high temperature heating for 3-5min. Then resuspend the mixture with Tris-EDTA (TE) buffer at pH 7.0-8.0, and then centrifuge and wash twice at 20000-30000rpm, each time for 15-20min. The obtained Au-SH probe II solution is stored at 4℃.
[0012] (4) Immerse the microneedle prepared in step (2) in the Au-SH probe II solution suspension prepared in step (3), let it stand for 1 to 2 hours, then take out the microneedle, allow the microneedle to bind to Au, and finally measure the fluorescence intensity using a fluorescence device.
[0013] Among them, the capture probe I of the present invention includes miRNA-21 capture probe, miRNA-221 capture probe and miRNA-155 capture probe.
[0014] Beneficial effects: 1. The present invention provides a porous hydrogel microneedle for miRNA detection, which can be used for ultra-sensitive detection of cancer-related miRNA. The hydrogel microneedle is modified by introducing polyethylene glycol to optimize the porous structure of the microneedle. By introducing more porous channels in a microneedle of limited volume, its surface area can be increased, and the capacity for interstitial fluid extraction can be improved; at the same time, lithium chloride is used to increase the osmotic pressure of the interstitial fluid in the hydrogel matrix, promote the diffusion of skin interstitial fluid in the application area and effectively enter the microneedle matrix. This osmotic pressure-compounded high-swelling microneedle can collect a large amount of skin interstitial fluid in a shorter time, providing a rich source of samples for a variety of analyses or biomarker studies.
[0015] 2. The present invention provides a porous hydrogel microneedle for miRNA detection, which has a height of 1000-1200 μm, a pyramid-shaped needle tip, a porosity of 30-35%, and excellent mechanical strength. Compared with traditional clinical disease diagnosis methods such as blood routine tests, computer tomography and nuclear magnetic resonance, this technology can simplify the detection process, reduce the amount of sample collection, shorten the diagnosis time, and facilitate the operation of medical staff. It can penetrate the epidermis of the skin into the dermis, which can avoid stimulation of nerve fibers, thereby achieving painless detection and reducing the pain of patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 It is a schematic diagram of the porous hydrogel microneedle for miRNA detection and its preparation method of the present invention; wherein: 1 is a microneedle, 2 is an osmotic pressure microneedle, and 3 is a probe-modified microneedle. DETAILED DESCRIPTION
[0017] The present invention provides a porous hydrogel microneedle for miRNA detection and a preparation and detection method thereof. The specific embodiments of the present invention are described below in conjunction with the accompanying drawings.
[0018] Example 1
[0019] The microneedle has a porous bionic structure and osmotic pressure driving force, a porosity of 30%, a microneedle height of 1000 μm, a needle tip designed to be pyramid-shaped, and a bottom diameter of 350 μm; the hydrogel microneedle is realized by modifying the hydrogel with polyethylene glycol and lithium chloride.
[0020] A porous hydrogel microneedle for miRNA detection and its preparation and detection method are as follows:
[0021] (1) Preparation of osmotic pressure-driven microneedles
[0022] Polyvinyl alcohol (10 wt%), polyvinyl pyrrolidone (5 wt%), crosslinking agent glutaraldehyde (0.002 wt%) and polyethylene glycol (0.1 wt%) were dissolved in deionized water, and the resulting mixture was heated to 90 ° C and stirred for 3 h. It was then cast into a PDMS mold, and dried at room temperature for 24 h after vacuum removal of bubbles. Finally, the microneedles were peeled off from the mold and immersed in a 20 wt% lithium chloride solution for 2 h. The microneedle height was 1000 μm, the needle tip was designed to be pyramid-shaped, the bottom diameter was 350 μm, and the porosity was 30%.
[0023] (2) Osmotic pressure microneedle fixed capture probe I
[0024] The miRNA-21 capture probe was first dissolved to 100 μM at a concentration of 0.5 OD, and then 1 μL of 1 M tris(2-carbonylethyl)phosphine hydrochloride (TCEP) solution was added and mixed thoroughly, and the reduction reaction was carried out at room temperature for 30 minutes. The needle body and needle tip of the microneedle prepared in step (1) were inserted into the solution and left to stand for 10 minutes, then taken out, rinsed with PBS buffer, and immersed in 5% BSA solution at room temperature for 1 hour to encapsulate the detection probe I.
[0025] (3) The capture probe modified microneedle prepared in step (2) was immersed in a solution containing 2 mL of 1 μM miRNA-21, incubated at 37° C. for 2 h, and then the microneedle was repeatedly washed with PBS solution to remove the unfixed miRNA-21.
[0026] (4) Dissolve 0.5OD SH-probe II to 50μM, add 5μL 1M tris(hydroxymethyl)acrylate buffer (TECP), mix well, and let stand at room temperature for 20min; add 500μL Au nanoparticle solution (0.1mg / mL) and reduced SH-probe II into a glass bottle, and then place it in a microwave oven for medium-high temperature heating for 3min; then resuspend the mixture with Tris-EDTA (TE) buffer at pH 8.0, and then centrifuge and wash twice at 20000rpm, each time for 15min; the obtained Au-SH probe II solution is stored at 4°C.
[0027] (5) Subsequently, the microneedles prepared in step (3) were incubated with the Au-SH probe II solution at 37°C for 2 h. The microneedles were then washed several times with PBS solution to remove excess Au-SH probe II. After the incubation, the microneedles were dried and placed under the objective lens of a Raman / fluorescence spectrometer equipped with a confocal system, and a 532nm laser excitation light source was used to collect fluorescence signals near 680nm. By this method, ultrasensitive detection of 4.3pM concentration of miRNA-221 can be achieved.
[0028] Example 2
[0029] The microneedle has a porous bionic structure and osmotic pressure driving force, with a porosity of 35%, a microneedle height of 1100 μm, a pyramid-shaped needle tip, and a bottom diameter of 380 μm; the hydrogel microneedle is realized by modifying the hydrogel with polyethylene glycol and lithium chloride.
[0030] A porous hydrogel microneedle for miRNA detection and its preparation and detection method are as follows:
[0031] (1) Preparation of osmotic pressure-driven microneedles
[0032] Polyvinyl alcohol (12 wt%), polyvinyl pyrrolidone (6 wt%), crosslinking agent glutaraldehyde (0.003 wt%) and polyethylene glycol (0.15 wt%) were dissolved in deionized water, and the resulting mixture was heated to 80 ° C and stirred for 2 h. It was then cast into a PDMS mold, and dried at room temperature for 20 h after vacuum removal of bubbles. Finally, the microneedles were peeled off from the mold and immersed in a 25 wt% lithium chloride solution for 3 h to obtain osmotic pressure-driven microneedles with a microneedle height of 1100 μm, a pyramid-shaped needle tip, a bottom diameter of 380 μm, and a porosity of 35%.
[0033] (2) Osmotic pressure microneedle fixed capture probe I
[0034] The miRNA-221 capture probe was first dissolved to 120 μM at a concentration of 1.0 OD, and then 2 μL of 2M tris(2-carbonylethyl)phosphine hydrochloride (TCEP) solution was added and mixed thoroughly, and the reduction reaction was carried out at room temperature for 40 min. The needle body and needle tip of the microneedle prepared in step (1) were inserted into the solution and allowed to stand for 15 min, then taken out, rinsed with PBS buffer, and immersed in 6% BSA solution at room temperature for 2 h to encapsulate the detection probe I.
[0035] (3) Immersing the capture probe modified microneedle prepared in step (2) in a solution containing a specific concentration of miRNA-221, incubating at 37° C. for 2 h, and then repeatedly washing the microneedle with PBS solution to remove unfixed miRNA-221.
[0036] (4) Dissolve 0.8OD SH-probe II to 100μM, add 6μL 1.5M tris(hydroxymethyl)acrylate buffer (TECP), mix well, and let stand at room temperature for 25min; add 550μL Au nanoparticle solution (0.15mg / mL) and reduced SH-probe II into a glass bottle, and then place it in a microwave oven for medium-high temperature heating for 4min; then resuspend the mixture with Tris-EDTA (TE) buffer at pH 7.5, and then centrifuge and wash twice at 25000rpm, each time for 18min; the obtained Au-SH probe II solution is stored at 4°C.
[0037] (5) Subsequently, the microneedles prepared in step (3) were incubated with the Au-SH probe II solution at 37°C for 2 h. The microneedles were then washed several times with PBS solution to remove excess Au-SH probe II. After the incubation, the microneedles were dried and placed under the objective lens of a Raman / fluorescence spectrometer equipped with a confocal system, and a 532nm laser excitation light source was used to collect fluorescence signals near 680nm. By this method, ultrasensitive detection of 3.3pM concentration of miRNA-221 can be achieved.
[0038] Example 3
[0039] The microneedle has a porous bionic structure and osmotic pressure driving force, a porosity of 32%, a microneedle height of 1200 μm, a needle tip designed to be pyramid-shaped, and a bottom diameter of 400 μm; the hydrogel microneedle is realized by modifying the hydrogel with polyethylene glycol and lithium chloride.
[0040] A porous hydrogel microneedle for miRNA detection and its preparation and detection method are as follows:
[0041] (1) Preparation of osmotic pressure-driven microneedles
[0042] Polyvinyl alcohol (16wt%), polyvinyl pyrrolidone (8wt%), crosslinking agent glutaraldehyde (0.004wt%) and polyethylene glycol (0.2wt%) were dissolved in deionized water, and the resulting mixture was heated to 90°C and stirred for 3h. It was then cast into a PDMS mold, and dried at room temperature for 24h after vacuum removal of bubbles. Finally, the microneedles were peeled off from the mold and immersed in a 35wt% lithium chloride solution for 3h to obtain osmotic pressure-driven microneedles with a microneedle height of 1200μm, a bottom diameter of 400μm, and a porosity of 35%.
[0043] (2) Osmotic pressure microneedle fixed capture probe I
[0044] The miRNA-155 capture probe was first dissolved to 150 μM at a concentration of 0.8 OD, and then 1.5 μL of 1.5 M tris(2-carbonylethyl)phosphine hydrochloride (TCEP) solution was added and mixed thoroughly, and the reduction reaction was carried out at room temperature for 35 min. The needle body and needle tip of the microneedle prepared in step (1) were inserted into the solution and left to stand for 20 min before being taken out, rinsed with PBS buffer, and immersed in 8% BSA solution at room temperature for 1.5 h to encapsulate the detection probe I.
[0045] (3) The capture probe modified microneedle prepared in step (2) was immersed in a solution containing a specific concentration of miRNA-155, incubated at 37° C. for 1.5 h, and then the microneedle was repeatedly washed with PBS solution to remove the unfixed miRNA-155.
[0046] (4) Dissolve 1.0OD SH-probe II to 150μM, add 7μL 2M tris(hydroxymethyl)acrylate buffer (TECP), mix well, and let stand at room temperature for 30min; add 600μL Au nanoparticle solution (0.2mg / mL) and reduced SH-probe II into a glass bottle, and then place it in a microwave oven for medium-high temperature heating for 5min; then resuspend the mixture with Tris-EDTA (TE) buffer at pH 7.0, and then centrifuge and wash twice at 30000rpm, each time for 20min; the obtained Au-SH probe II solution is stored at 4℃.
[0047] (5) Subsequently, the microneedles prepared in step (3) were incubated with the Au-SH probe II solution at 37°C for 1.5 h. The microneedles were then washed several times with PBS solution to remove excess Au-SH probe II. After the incubation, the microneedles were dried and placed under the objective lens of a Raman / fluorescence spectrometer equipped with a confocal system, and a 532nm laser excitation light source was used to collect fluorescence signals near 680nm. By this method, ultrasensitive detection of 5.5pM concentration of miRNA-155 can be achieved.
[0048] By using the preparation method of the present application, the preparation of microneedles for detecting miRNA can be achieved, and the obtained microneedles have broad application prospects in the detection of trace substances in interstitial fluid.
[0049] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above technical features.
Claims
1. A porous hydrogel microneedle for miRNA detection, characterized in that: The microneedle has a porous bionic structure and osmotic pressure driving force, a porosity of 30-35%, a microneedle height of 1000-1200 μm, a pyramid-shaped needle tip, and a bottom diameter of 350-400 μm; the hydrogel microneedle is realized by modifying the hydrogel with polyethylene glycol and lithium chloride.
2. The method for preparing the porous hydrogel microneedle according to claim 1, characterized in that: The method comprises the following steps: dissolving 10-16wt% polyvinyl alcohol, 5-8wt% polyvinyl pyrrolidone, 0.002-0.004wt% cross-linking agent glutaraldehyde and 0.1-0.2wt% polyethylene glycol in deionized water, heating the obtained mixture to 80-90°C and stirring for 2-3h; subsequently pouring the mixture into a polydimethylsiloxane mold, vacuum removing bubbles and drying at room temperature for 20-24h; finally peeling the microneedle from the mold, and immersing the microneedle in a 20-35wt% lithium chloride solution for 2-3h to obtain an osmotic pressure-driven microneedle; the microneedle has a height of 1000-1200μm, the needle tip is designed to be pyramid-shaped, the bottom diameter is 350-400μm, and the porosity is 30-35%.
3. The method for detecting miRNA using the porous hydrogel microneedle according to claim 1, characterized in that: The following steps are involved: (1) The capture probe I is first dissolved to a concentration of 50 to 150 μM at 0.5 to 1.0 OD, and then 1 to 2 μL of 1 to 2 M tris(2-carbonylethyl)phosphine hydrochloride solution is added and mixed thoroughly, and a reduction reaction is performed at room temperature for 30 to 40 minutes; the needle body and needle tip of the porous hydrogel microneedle are inserted into the solution and allowed to stand for 10 to 20 minutes, then taken out, rinsed with PBS buffer, and immersed in a 5 to 8% BSA solution at room temperature for 1 to 2 hours to encapsulate the detection probe I; (2) immersing the capture probe I osmotic pressure-driven microneedle prepared in step (1) into the liquid to be tested, taking it out after the reaction, and washing it with PBS buffer to obtain a microneedle bound to the miRNA to be tested; (3) Dissolve 0.5-1.0OD SH-probe II to 50-150μM, add 5-7μL 1-2M tris(hydroxymethyl)acrylate buffer, mix well, and let stand at room temperature for 20-30min; add 500-600μL 0.1-0.2mg / mL Au nanoparticle solution and reduced SH-probe II into a glass bottle, and then put it into a microwave oven for medium-high temperature heating for 3-5min; then resuspend the mixture with Tris-EDTA buffer of pH 7.0-8.0, and then centrifuge and wash twice at 20000-30000rpm, each time for 15-20min; the obtained Au-SH probe II solution; (4) Immerse the microneedle prepared in step (2) in the Au-SH probe II solution suspension prepared in step (3), let it stand for 1 to 2 hours, then remove the microneedle, allow the microneedle to bind to Au, and finally measure the fluorescence intensity using a fluorescence device to determine whether miRNA exists in the test fluid based on the fluorescence intensity.
4. The method for detecting miRNA according to claim 3, characterized in that: Capture probe I includes miRNA-21 capture probe, miRNA-221 capture probe or miRNA-155 capture probe.