Skin interstitial fluid miRNA multicolor visualization semi-quantitative detection method

Abstract

The application provides a skin interstitial fluid miRNA multicolor visualization semi-quantitative detection method, which comprises the following steps: incubating a skin interstitial fluid sample with magnetic nano probes of surface immobilized hairpin probes H1, opening H1 and triggering a subsequent hybridization chain reaction of hairpin probes H2 and H3 in the presence of target miRNA, and constructing a biotin-rich DNA complex on the surface of the magnetic beads; performing enzyme labeling by using an alkaline phosphatase-streptavidin coupling compound, and catalyzing ascorbic acid phosphate to generate ascorbic acid potassium iodate, so that the color of the solution changes, and high-sensitivity detection of the target miRNA is realized. The multicolor visualization detection method can produce near-full-spectrum color changes by combining the gold nano double-cone local surface plasmon resonance property change, and is beneficial to improving the portability of the sensor and the accuracy of the visual analysis.

Description

Technical Field

[0001] This invention relates to the field of nanobiosensing technology, and in particular to a multi-color visualization semi-quantitative detection method for miRNA in interstitial fluid of the skin. Background Technology

[0002] Point-of-care testing (POCT) diagnostic technologies typically involve the analysis of biomarkers in bodily fluids and have garnered significant attention in personalized medicine and health management. Currently, blood-based bodily fluids (such as serum and plasma) are most commonly used due to their rich content of biomarkers reflecting health status. However, blood collection requires specialized procedures and can cause tissue damage, potentially leading to pain or infection. While bodily fluids such as tears and sweat can serve as alternative samples for POCT, the significant fluctuations in biomarker concentrations limit their application in the diagnosis of certain diseases.

[0003] In recent years, a novel body fluid—interstitial fluid (ISF)—has shown application potential in the field of point-of-care testing (POCT). ISF is a biofluid filling the extracellular space, acting as a medium for substance exchange between cells and capillaries. Its biomarker composition is similar to that of blood, making it an important source of clinical information for disease diagnosis and treatment. Furthermore, ISF contains unique biomarkers not found in plasma, allowing for a more precise reflection of the physiological state of local tissue cells. Therefore, developing efficient methods for detecting ISF biomarkers is of great significance to the development of biodiagnostic technologies.

[0004] Although ISF appears readily available, its hydrogel properties make collection challenging. A crucial step before detecting ISF biomarkers is sample collection from the skin. An ideal ISF extraction method should be minimally invasive, painless, and easy to operate. Currently, various ISF sampling methods exist (such as suction blistering and core implantation), but these methods suffer from complexity requiring professional personnel and potential discomfort or infection. In recent years, microneedle patches have gained attention for their non-invasive and efficient extraction of ISF and in-situ detection. Hydrogel microneedle patches made from biocompatible polymers (such as polyvinyl alcohol, hyaluronic acid, and gelatin) can directly penetrate the skin to extract ISF without auxiliary equipment. During sampling, ISF biomarkers react with reagents within the hydrogel microneedles to generate detection signals, leading to the development of various POCT biosensors based on colorimetric, fluorescence, and electrochemical methods. However, the ISF is immobilized within the hydrogel network and can only react with the built-in reagents, limiting its application. Although biomarkers can be partially recovered through centrifugation, a significant amount of biomarkers remain. Therefore, it is crucial to develop novel sampling methods that can rapidly extract sufficient amounts of ISF while retaining complete biomarker information. Summary of the Invention

[0005] In view of the above situation, the main objective of this invention is to propose a multi-color visualization semi-quantitative detection method for miRNA in interstitial fluid of the skin to solve the above-mentioned technical problems.

[0006] This invention proposes a multi-color visualization semi-quantitative detection method for miRNA in skin interstitial fluid, the method comprising the following steps:

[0007] Step 1: Press the microneedle patch onto the sterilized skin surface to form micron-sized pores; use a vacuum negative pressure device to apply negative pressure to the skin surface with micron-sized pores, so that the interdermal fluid in the dermis diffuses to the skin surface and is collected to obtain the interdermal fluid.

[0008] Step 2: Wash the streptavidin-modified magnetic beads with washing buffer to obtain washed magnetic beads; perform high-temperature heating and annealing on biotin-modified hairpin probe H1 to obtain pretreated hairpin probe H1; incubate the washed magnetic beads and pretreated hairpin probe H1, wash them with washing buffer after incubation, and disperse them in dispersion buffer to obtain magnetic nanoprobes.

[0009] Step 3: Add the interstitial fluid of the skin to the magnetic nanoprobe for incubation. After incubation, wash with washing buffer to obtain the washed magnetic nanoprobe. Add biotin-modified hairpin probe H2 and biotin-modified hairpin probe H3 to the washed magnetic nanoprobe for hybridization chain reaction. After the reaction, wash with washing buffer to obtain the reacted magnetic nanoprobe.

[0010] Step 4: Add the alkaline phosphatase-streptavidin coupling compound to the reacted magnetic nanoprobe for incubation, and wash with washing buffer to obtain alkaline phosphatase-labeled magnetic bead complex.

[0011] Step 5: Add ascorbate phosphate prepared with glycine buffer to the alkaline phosphatase-labeled magnetic bead complex for reaction. After the reaction is completed, collect the supernatant by magnetic separation. Mix the supernatant with glycine buffer, potassium iodate solution and gold nanoparticle biconical solution and incubate. Observe the color change of the solution and perform qualitative and semi-quantitative analysis of miRNA to obtain the detection results.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1. Compared with existing ISF extraction technologies, the ISF sampling technology used in this invention can extract up to 89.25 μL of sample in 20 minutes, and the biomarker information in the sample is completely preserved. The sampling process causes little damage to the skin, and the extracted sample volume can meet the analytical testing requirements.

[0014] 2. Existing ISF biomarker detection technologies typically only have single or two color changes, making it difficult to achieve visually visualized quantification. The multi-color visualization detection method proposed in this invention combines the changes in the local surface plasmon resonance properties of gold nanoparticles with bipyramidal surfaces, which can generate near-full spectrum color changes, thus improving the portability of the sensor and the accuracy of visualization analysis.

[0015] 3. The multicolor visualization semi-quantitative detection method for miRNA in interstitial fluid described in this invention, by combining hybridization chain reaction and enzyme-induced signal amplification technology, can achieve the detection of miRNA-21 as low as 38.13 fM, which shows comparable or higher sensitivity compared to other visualization detection methods.

[0016] 4. Traditional miRNA analysis and detection require complex separation and extraction steps, which are complicated and require professional personnel. However, the ISF sample extracted in this invention does not have the interference of the matrix in the blood, so it can be analyzed and detected without additional processing.

[0017] 5. Traditional ISF extraction and biomarker detection require special equipment and raw materials, while the equipment and raw materials designed in this invention are commercial products (stainless steel microneedle patches and vacuum cupping device). At the same time, traditional Chinese medicine cupping therapy has high acceptance and can be used for extraction in home settings.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by means of embodiments of the invention. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the principle of a multi-color visualization semi-quantitative detection method for miRNA in skin interstitial fluid proposed in this invention;

[0020] Figure 2 Characterization of the commercially available microneedles, vacuum negative pressure device, and microneedle dimensions used in this invention;

[0021] Figure 3 These are images showing the microneedle transdermal test and recovery process used in this invention.

[0022] Figure 4 This is a typical extraction process of the ISF extraction method described in this invention;

[0023] Figure 5 This is an ISF sample image extracted by the ISF extraction method described in this invention;

[0024] Figure 6 The spectra and transmission electron microscopy (TEM) characterization images of gold nanobipyramidal nanoparticles exhibiting different colors under the action of iodine.

[0025] Figure 7 The color changes, spectra, and working curves of gold nanobipyramidal nanoparticles under different target miRNA concentrations are shown in the present invention. Detailed Implementation

[0026] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0027] These and other aspects of the embodiments of the present invention will become clear from the following description and accompanying drawings. In these descriptions and drawings, some specific embodiments of the present invention are specifically disclosed to illustrate some ways of implementing the principles of the embodiments of the present invention; however, it should be understood that the scope of the embodiments of the present invention is not limited thereto.

[0028] Example 1:

[0029] Please see Figure 1 This embodiment provides a multi-color visualization semi-quantitative detection method for miRNA in skin interstitial fluid, the method comprising the following steps:

[0030] Step 1: Disinfect the skin surface with 75% alcohol; press a commercial stainless steel microneedle patch onto the skin surface for about 30 seconds to form micron-level pores. The stainless steel microneedle patch is 3×2cm in size and has a 14×10 microneedle array. Each microneedle is cylindrical, and the length of the microneedle can be adjusted within the range of 0~3mm. The diameter of the microneedle is 285μm; remove the microneedle patch, invert the vacuum cupping device onto the skin surface where micron-level pores have been formed, and use the suction device to remove the air from the cup, so that the vacuum cup adheres to the skin and maintains negative pressure for 20 minutes; move the piston on the top of the vacuum cup to balance the internal and external pressure to remove the vacuum cup, and use a pipette to collect the interdermal fluid that has diffused to the skin surface;

[0031] Step 2: Take 890 μL of streptavidin-modified magnetic beads with a concentration of 1 mg / mL, and wash them three times with washing buffer (containing 1 mM EDTA, 1 M NaCl, 0.05% Tween-20 and 10 mM Tris-HCl, pH=7.5) to obtain washed magnetic beads; take 85 μL of biotin-modified hairpin probe H1 with a concentration of 3 μM, heat at 90℃ for 10 min and then anneal to obtain pretreated hairpin probe H1; add the pretreated hairpin probe H1 to the washed magnetic beads, incubate at 36.5℃ for 1 h, and then wash three times with washing buffer to remove unreacted pretreated hairpin probe H1; finally, disperse the magnetic beads in dispersion buffer (containing 100 mM NaCl and 20 mM MgCl2, pH=8.0) to obtain magnetic nanoprobes;

[0032] Step 3: Take 50 μL of magnetic nanoprobe and add 4.5 μL of interstitial fluid containing the target miRNA (e.g., miRNA-21). Incubate at 36.5℃ for 1 h, then wash three times with washing buffer to obtain the washed magnetic nanoprobe. Add 4.5 μL of 5 μM biotin-modified hairpin probe H2 and 4.5 μL of 5 μM biotin-modified hairpin probe H3 to the washed magnetic nanoprobe, and perform a hybridization chain reaction at 36.5℃. After reacting for 1.5 h, wash three times with washing buffer to remove unreacted biotin-modified hairpin probe H2 and unreacted biotin-modified hairpin probe H3 to obtain the reacted magnetic nanoprobe.

[0033] Step 4: Add 4.5 μL of alkaline phosphatase-streptavidin coupling compound (ALP-SA) at a concentration of 20 ng / mL to the reacted magnetic nanoprobes, incubate at 36.5 °C for 30 min, and then wash three times with washing buffer to obtain alkaline phosphatase-labeled magnetic bead complexes.

[0034] Step 5: Add 50 μL of 1 mM ascorbate phosphate (prepared from 5 mM glycine buffer at pH 9.5) to the alkaline phosphatase-labeled magnetic bead complex and react at 36.5 °C for 20 min. Then collect the supernatant by magnetic separation. Mix the supernatant with 50 μL of 0.2 M glycine buffer at pH 3.2, 50 μL of 4 mM potassium iodate solution, and 80 μL of gold nanoparticle bipyramidal (AuNBPs) solution and incubate at 36.5 °C for 30 min. Perform qualitative and semi-quantitative analysis by observing the color change of the solution to obtain the detection results.

[0035] It should be noted that, in Figure 1In the diagram, H1-bio represents the biotin-modified hairpin probe H1, H2-bio represents the biotin-modified hairpin probe H2, and H3-bio represents the biotin-modified hairpin probe H3. All biotin-modified hairpin probes H1, H2, and H3 have stem-loop structures. When miRNA-21 is added, the sequence of miRNA-21 binds complementaryly to the biotin-modified hairpin probe H1 and opens the stem-loop structure. The exposed end of H1 binds complementaryly to the sequence of H2 and opens the stem-loop structure of H2. The exposed end of H2 binds complementaryly to the sequence of H3 and opens the stem-loop structure of H3. The exposed end of H3 binds complementaryly to the sequence of H2 and opens the stem-loop structure of H2, triggering the next round of stem-loop opening, forming a long-chain double-stranded polymer, and achieving signal amplification. Figure 1 In the diagram, A represents the sampling process of interstitial fluid in the skin according to the present invention. Figure 1 B in the diagram is a schematic diagram of multicolor visualization sensing detection of miRNA in extracted interstitial fluid of the skin.

[0036] Example 2:

[0037] This embodiment provides a multi-color visualization semi-quantitative detection method for miRNA in skin interstitial fluid, the method comprising the following steps:

[0038] Step 1: Disinfect the skin surface with 75% alcohol; press a commercial stainless steel microneedle patch onto the skin surface for about 30 seconds to form micron-level pores. The stainless steel microneedle patch is 3×2cm in size and has a 14×10 microneedle array. Each microneedle is cylindrical, and the length of the microneedle can be adjusted within the range of 0~3mm. The diameter of the microneedle is 285μm; remove the microneedle patch, invert the vacuum cupping device onto the skin surface where micron-level pores have been formed, and use the suction device to remove the air from the cup, so that the vacuum cup adheres to the skin and maintains negative pressure for 20 minutes; move the piston at the top of the vacuum cup to balance the internal and external pressure to remove the vacuum cup, and use a pipette to collect the interdermal fluid that has diffused to the skin surface;

[0039] Step 2: Take 900 μL of streptavidin-modified magnetic beads with a concentration of 1 mg / mL, and wash them three times with washing buffer (containing 1 mM EDTA, 1 M NaCl, 0.05% Tween-20 and 10 mM Tris-HCl, pH=7.5) to obtain washed magnetic beads; take 90 μL of biotin-modified hairpin probe H1 with a concentration of 3 μM, heat at 90℃ for 10 min and then anneal to obtain pretreated hairpin probe H1; add the pretreated hairpin probe H1 to the washed magnetic beads, incubate at 37℃ for 1 h, and then wash three times with washing buffer to remove unreacted pretreated hairpin probe H1; finally, disperse the magnetic beads in dispersion buffer (containing 100 mM NaCl and 20 mM MgCl2, pH=8.0) to obtain magnetic nanoprobes;

[0040] Step 3: Take 50 μL of magnetic nanoprobe and add 5 μL of interstitial fluid containing the target miRNA (e.g., miRNA-21). Incubate at 37°C for 1 h, then wash three times with washing buffer to obtain the washed magnetic nanoprobe. Add 5 μL of 5 μM biotin-modified hairpin probe H2 and 5 μL of 5 μM biotin-modified hairpin probe H3 to the washed magnetic nanoprobe, and perform a hybridization chain reaction at 37°C. After reacting for 1.5 h, wash three times with washing buffer to remove unreacted biotin-modified hairpin probe H2 and unreacted biotin-modified hairpin probe H3 to obtain the reacted magnetic nanoprobe.

[0041] Step 4: Add 5 μL of alkaline phosphatase-streptavidin coupling compound (ALP-SA) at a concentration of 20 ng / mL to the reacted magnetic nanoprobes, incubate at 37 °C for 30 min, and then wash three times with washing buffer to obtain alkaline phosphatase-labeled magnetic bead complexes.

[0042] Step 5: Add 50 μL of 1 mM ascorbate phosphate (prepared from 5 mM glycine buffer at pH 9.5) to the alkaline phosphatase-labeled magnetic bead complex and react at 37 °C for 20 min. Then collect the supernatant by magnetic separation. Mix the supernatant with 50 μL of 0.2 M glycine buffer at pH 3.2, 50 μL of 4 mM potassium iodate solution, and 80 μL of gold nanoparticle bipyramidal (AuNBPs) solution and incubate at 37 °C for 30 min. Perform qualitative and semi-quantitative analysis by observing the color change of the solution to obtain the detection results.

[0043] Example 3:

[0044] This embodiment provides a multi-color visualization semi-quantitative detection method for miRNA in skin interstitial fluid, the method comprising the following steps:

[0045] Step 1: Disinfect the skin surface with 75% alcohol; press a commercial stainless steel microneedle patch onto the skin surface for about 30 seconds to form micron-level pores. The stainless steel microneedle patch is 3×2cm in size and has a 14×10 microneedle array. Each microneedle is cylindrical, and the length of the microneedle can be adjusted within the range of 0~3mm. The diameter of the microneedle is 285μm; remove the microneedle patch, invert the vacuum cupping device onto the skin surface where micron-level pores have been formed, and use the suction device to remove the air from the cup, so that the vacuum cup adheres to the skin and maintains negative pressure for 20 minutes; move the piston at the top of the vacuum cup to balance the internal and external pressure to remove the vacuum cup, and use a pipette to collect the interdermal fluid that has diffused to the skin surface;

[0046] Step 2: Take 910 μL of streptavidin-modified magnetic beads with a concentration of 1 mg / mL, and wash them three times with washing buffer (containing 1 mM EDTA, 1 M NaCl, 0.05% Tween-20 and 10 mM Tris-HCl, pH=7.5) to obtain washed magnetic beads; take 95 μL of biotin-modified hairpin probe H1 with a concentration of 3 μM, heat at 90℃ for 10 min and then anneal to obtain pretreated hairpin probe H1; add the pretreated hairpin probe H1 to the washed magnetic beads, incubate at 37.5℃ for 1 h, and then wash three times with washing buffer to remove unreacted pretreated hairpin probe H1; finally, disperse the magnetic beads in dispersion buffer (containing 100 mM NaCl and 20 mM MgCl2, pH=8.0) to obtain magnetic nanoprobes;

[0047] Step 3: Take 50 μL of magnetic nanoprobe and add 5.5 μL of interstitial fluid containing the target miRNA (e.g., miRNA-21). Incubate at 37.5℃ for 1 h, then wash three times with washing buffer to obtain the washed magnetic nanoprobe. Add 5.5 μL of 5 μM biotin-modified hairpin probe H2 and 5.5 μL of 5 μM biotin-modified hairpin probe H3 to the washed magnetic nanoprobe, and perform a hybridization chain reaction at 37.5℃. After reacting for 1.5 h, wash three times with washing buffer to remove unreacted biotin-modified hairpin probe H2 and unreacted biotin-modified hairpin probe H3 to obtain the reacted magnetic nanoprobe.

[0048] Step 4: Add 5.5 μL of alkaline phosphatase-streptavidin coupler (ALP-SA) at a concentration of 20 ng / mL to the reacted magnetic nanoprobes, incubate at 37.5 °C for 30 min, and then wash three times with washing buffer to obtain alkaline phosphatase-labeled magnetic bead complexes.

[0049] Step 5: Add 50 μL of 1 mM ascorbate phosphate (prepared from 5 mM glycine buffer at pH 9.5) to the alkaline phosphatase-labeled magnetic bead complex and react at 37.5 °C for 20 min. Then collect the supernatant by magnetic separation. Mix the supernatant with 50 μL of 0.2 M glycine buffer at pH 3.2, 50 μL of 4 mM potassium iodate solution, and 80 μL of gold nanoparticle bipyramidal (AuNBPs) solution and incubate at 37.5 °C for 30 min. Perform qualitative and semi-quantitative analysis by observing the color change of the solution to obtain the detection results.

[0050] To verify the effectiveness of this invention, the microneedle patch described in this invention is a currently commercially available disposable skin puncture needle, such as... Figure 2 As shown in Figure A, the microneedle patch has a size of 3×2cm and a 14×10 microneedle array. Each microneedle is conical in shape and has a diameter of 285μm (e.g., ...). Figure 2 As shown in C), the length of the microneedles can be adjusted within the range of 0~3mm to adapt to skin of different thicknesses; the vacuum negative pressure device used in this invention is the vacuum cupping device used in traditional Chinese medicine cupping therapy (such as...). Figure 2 As shown in B), it has the advantages of simple equipment, easy operation, and high social acceptance.

[0051] It should be noted that, Figure 2 In the diagram, A represents a schematic representation of the commercially available microneedles used in this invention. The product technical requirements number for these commercially available microneedles is: Su Lian Xie Bei 20240387. Figure 2 B in the diagram is a schematic diagram of the vacuum negative pressure device used in this invention. Figure 2 C in the figure represents the microneedle size characterization.

[0052] Because microneedle patches are made of stainless steel, they possess sufficient mechanical strength to easily pierce the epidermis and stratum corneum, creating hundreds of micropores. Furthermore, microneedle patches cause minimal damage to the skin, and the resulting micron-sized pores disappear naturally within 2 minutes (e.g., Figure 3 (As shown); After the microneedle patch creates micron-sized pores on the skin surface, a vacuum cupping device generates negative pressure on the skin surface. Driven by this negative pressure, the interdermal fluid in the dermis diffuses from the dermis along these micron-sized pores to the skin surface. Finally, collecting the diffused interdermal fluid achieves efficient extraction (e.g., Figure 4 (As shown).

[0053] Please refer to Table 1. According to the extraction technique of the present invention, 15 healthy volunteers were recruited for interdermal fluid extraction. It was found that the volume of extracted interdermal fluid varied from 15 to 89.25 μL depending on age, extraction site, sex, weight, etc., with the maximum extraction volume being 89.25 μL (e.g., ...). Figure 5 (As shown).

[0054] Table 1. Volunteer Information

[0055]

[0056] According to the principle of this invention, when the target miRNA21 is present in the extracted interstitial fluid sample, the sample can open the biotin-modified hairpin probe H1, exposing the initiation sequence, thereby catalyzing a hybridization chain reaction between the biotin-modified hairpin probes H2 and H3. Since the biotin-modified hairpin probes H2 and H3 are labeled with biotin, alkaline phosphatase (ALP) can be modified onto magnetic beads through the interaction between biotin and avidin. ALP catalyzes the production of ascorbic acid from ascorbate phosphate, which in turn reduces potassium iodate to iodine. Iodine possesses oxidizing and coordinating properties, enabling rapid etching of gold nanobipyramidal nanoparticles (AuNBPs), altering their aspect ratio and producing rich color variations. Therefore, the iodine produced by the reaction of different concentrations of ascorbic acid with potassium iodate can produce different degrees of etching effects on AuNBPs, resulting in color differences (e.g., ...). Figure 6 (As shown).

[0057] Based on this, the amount of ALP modified on the surface of magnetic beads varies with different concentrations of miRNA21, which ultimately results in different degrees of etching of AuNBPs, producing obvious color differences and enabling qualitative and quantitative analysis of the target analytes.

[0058] It should be noted that, Figure 6 In the image, A represents the transmission electron microscopy (TEM) characterization of AuNBPs during the etching process when 0 mM ascorbic acid was added. Figure 6 The upper right corner of A in the figure is the corresponding color after etching with AuNBPs solution; Figure 6 B in the image represents the TEM characterization of AuNBPs during the etching process when 0.25 mM ascorbic acid was added. Figure 6 C in the figure represents the TEM characterization of AuNBPs during the etching process when 0.5 mM ascorbic acid was added. Figure 6 D in the image represents the TEM characterization of AuNBPs during the etching process when 0.75 mM ascorbic acid was added. Figure 6E in the figure represents the TEM characterization of AuNBPs during the etching process when 1 mM ascorbic acid was added. Figure 6 F in the figure represents the corresponding spectrum of AuNBPs during the etching process.

[0059] like Figure 7 A and Figure 7 As shown in Figure B, AuNBPs exhibit different color changes and absorption spectra at different concentrations of miRNA21 (0.25, 0.5, 10, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100 pM).

[0060] Statistical analysis revealed a linear relationship between the concentration of miRNA21 in the extracted interstitial fluid samples (0.25–100 pM) and the absorption peak shift of AuNBPs (e.g., ...). Figure 7 (As shown in C), its linear equation is:

[0061] y = 2.65x + 6.92 (R) 2 =0.993);

[0062] Where y represents the absorption peak shift of AuNBPs, x represents the concentration of miRNA21 in the extracted interstitial fluid sample, and R 2 This represents the correlation coefficient.

[0063] It should be noted that, Figure 7 In the diagram, A represents the color change of AuNBPs as a function of miRNA-21 concentration. Figure 7 In the diagram, B represents the absorption spectra of AuNBPs at different miRNA-21 concentrations. Figure 7 In the figure, C represents the linear relationship between the maximum absorption wavelength shift and the concentration of miRNA-21.

[0064] It should be understood that although the steps in the flowcharts of the various embodiments of the present invention are shown sequentially according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the various embodiments may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these sub-steps or stages is not necessarily sequential, but can be performed alternately or in turn with other steps or at least a portion of the sub-steps or stages of other steps.

[0065] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for detecting non-diagnostic miRNAs in interstitial fluid using multicolor visualization and semi-quantitative methods, characterized in that, The method includes the following steps: Step 1: Press the microneedle patch onto the sterilized skin surface to form micron-sized pores; use a vacuum negative pressure device to apply negative pressure to the skin surface with micron-sized pores, so that the interdermal fluid in the dermis diffuses to the skin surface and is collected to obtain the interdermal fluid. Step 2: Wash the streptavidin-modified magnetic beads with washing buffer to obtain washed magnetic beads; Biotin-modified hairpin probe H1 was subjected to high-temperature heating and annealing to obtain pretreated hairpin probe H1; washed magnetic beads were incubated with pretreated hairpin probe H1, and after incubation, they were washed with washing buffer and dispersed in dispersion buffer to obtain magnetic nanoprobes. Step 3: Add the interstitial fluid of the skin to the magnetic nanoprobe for incubation. After incubation, wash with washing buffer to obtain the washed magnetic nanoprobe. Biotin-modified hairpin probes H2 and H3 were added to the washed magnetic nanoprobes for a hybridization chain reaction. After the reaction was completed, the nanoprobes were washed with washing buffer to obtain the reacted magnetic nanoprobes. Step 4: Add the alkaline phosphatase-streptavidin coupling compound to the reacted magnetic nanoprobe for incubation, and wash with washing buffer to obtain alkaline phosphatase-labeled magnetic bead complex. Step 5: Add ascorbate phosphate prepared with glycine buffer to the alkaline phosphatase-labeled magnetic bead complex for reaction. After the reaction is completed, collect the supernatant by magnetic separation. Mix the supernatant with glycine buffer, potassium iodate solution and gold nanoparticle biconical solution and incubate. Observe the color change of the solution and perform qualitative and semi-quantitative analysis of miRNA to obtain the detection results.

2. The non-diagnostic detection method for multi-color visualization semi-quantitative non-diagnostic miRNA in interstitial fluid according to claim 1, characterized in that, In step 1, the microneedle patch is made of stainless steel, the size of the microneedle patch is 3×2cm, the microneedles in the microneedle patch are distributed in a 14×10 array, the individual microneedles are cylindrical, the length of the microneedles is adjustable in the range of 0~3mm, the diameter of the microneedles is 285μm, and the negative pressure is applied for 20min.

3. The non-diagnostic detection method for multi-color visualization semi-quantitative non-diagnostic miRNA in interstitial fluid according to claim 2, characterized in that, In step 2, when the magnetic beads are washed, the volume of the streptavidin-modified magnetic beads is 890-910 μL, the concentration of the streptavidin-modified magnetic beads is 1 mg / mL, and the washing is performed 3 times. In the process of obtaining the pretreated hairpin probe H1, the volume of the biotin-modified hairpin probe H1 was 85~95μL, the concentration of the biotin-modified hairpin probe H1 was 3μM, the heating temperature was 90℃, and the heating time was 10min. In the process of obtaining magnetic nanoprobes, the incubation temperature was 36.5~37.5℃ and the incubation time was 1h.

4. The non-diagnostic detection method for multi-color visualization semi-quantitative non-diagnostic miRNA in interstitial fluid according to claim 3, characterized in that, The washing buffer contains EDTA, NaCl, Tween-20 and Tris-HCl, and the pH of the washing buffer is 7.5; the dispersion buffer contains NaCl and MgCl2, and the pH of the dispersion buffer is 8.

5. The non-diagnostic detection method for multi-color visualization semi-quantitative non-diagnostic miRNA in interstitial fluid according to claim 4, characterized in that, In the washing buffer, the concentration of EDTA is 1 mM, the concentration of NaCl is 1 M, the concentration of Tween-20 is 0.05%, and the concentration of Tris-HCl is 10 mM. In the dispersion buffer, the concentration of NaCl is 100 mM and the concentration of MgCl2 is 20 mM.

6. The method for detecting non-diagnostic miRNAs in interstitial fluid using multicolor visualization semi-quantitative methods according to claim 5, characterized in that... In step 3, when the washed magnetic nanoprobes are obtained, the volume of the interstitial fluid is 4.5~5.5 μL, the volume of the magnetic nanoprobes is 50 μL, the incubation temperature is 36.5~37.5℃, the incubation time is 1 h, and the washing is performed 3 times. In the process of obtaining the reacted magnetic nanoprobes, the volume of biotin-modified hairpin probe H2 was 4.5~5.5 μL, the concentration of biotin-modified hairpin probe H2 was 5 μM, the volume of biotin-modified hairpin probe H3 was 4.5~5.5 μL, the concentration of biotin-modified hairpin probe H3 was 5 μM, the reaction temperature of the hybridization chain reaction was 36.5~37.5℃, the reaction time was 1.5 h, and the number of washings was 3.

7. The non-diagnostic detection method for multi-color visualization semi-quantitative non-diagnostic miRNA in interstitial fluid according to claim 6, characterized in that, In step 4, when obtaining the alkaline phosphatase-labeled magnetic bead complex, the volume of the alkaline phosphatase-streptavidin coupler is 4.5~5.5 μL, the concentration of the alkaline phosphatase-streptavidin coupler is 20 ng / mL, the incubation temperature is 36.5~37.5℃, the incubation time is 30 min, and the number of washes is 3.

8. The method for detecting non-diagnostic miRNAs in interstitial fluid using multicolor visualization semi-quantitative methods according to claim 7, characterized in that... In the process of obtaining the supernatant in step 5, the concentration of glycine buffer is 5mM, the pH of glycine buffer is 9.5, the volume of ascorbate phosphate is 50μL, the concentration of ascorbate phosphate is 1mM, the reaction temperature is 36.5~37.5℃, and the reaction time is 20min. During the process of obtaining the test results, the volume of glycine buffer was 50 μL, the concentration of glycine buffer was 0.2 M, the pH of glycine buffer was 3.2, the volume of potassium iodate solution was 50 μL, the concentration of potassium iodate solution was 4 mM, the volume of gold nanoparticle bipyramidal solution was 80 μL, the incubation temperature was 36.5~37.5℃, and the incubation time was 30 min.

Images