MiRNA colorimetric-photo-thermal bimodal aptamer lateral chromatography test strip based on photo-thermal effect of AuPt nanorods as well as preparation method and application of miRNA colorimetric-photo-thermal bimodal aptamer lateral chromatography test strip

Abstract

The invention provides a miRNA (micro Ribonucleic Acid) colorimetric-photo-thermal bimodal aptamer lateral chromatography test strip based on a photo-thermal effect of an AuPt nanorod as well as a preparation method and application of the miRNA colorimetric-photo-thermal bimodal aptamer lateral chromatography test strip. According to the test strip, an AuPt nanorod is used as a bifunctional signal carrier, a recognition probe is constructed through a sulfhydrylation nucleic acid aptamer, a hybridization chain reaction (HCR) signal amplification system is combined, a capture probe is fixed in a nitrocellulose membrane detection area, and specific recognition and signal amplification of a target miRNA-21 are achieved. During detection, a sample is laterally migrated and HCR cascade hybridization is triggered, so that a large number of AuPt nanorods are enriched on a detection line, and a macroscopic colorimetric strip is generated; meanwhile, after 808 nm laser irradiation, a temperature rise signal is recorded through infrared thermal imaging, and photo-thermal quantitative analysis is achieved. The method has the advantages of high sensitivity, dual-mode mutual test, simple operation, on-site rapid detection and the like, is suitable for rapid screening of miRNA-21, and can be expanded to detection of other miRNA markers by replacing aptamers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection and nanobiosensing technology, specifically relating to a miRNA colorimetric-photothermal dual-modal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods, its preparation method, and its application. Background Technology

[0002] MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules, 18-25 nucleotides in length, that regulate gene expression at the post-transcriptional level and are widely involved in important biological processes such as cell proliferation, differentiation, and apoptosis. Numerous studies have shown that the expression levels of various miRNAs are closely related to the occurrence, development, and metastasis of tumors. For example, miRNA-21 has been confirmed to be abnormally highly expressed in various solid tumors such as breast cancer, lung cancer, and colorectal cancer, making it a highly promising biomarker for early tumor diagnosis, prognostic assessment, and efficacy monitoring. Therefore, establishing a rapid, sensitive, and specific miRNA detection method is of great significance for achieving early disease screening and precision medicine.

[0003] Currently, commonly used miRNA detection methods in clinical practice mainly include quantitative real-time polymerase chain reaction (qRT-PCR), gene chips, and high-throughput sequencing. Although these methods have high sensitivity and specificity, they usually rely on complex instruments and equipment, professional operators, and long testing cycles. Furthermore, sample pretreatment is cumbersome and costly, making it difficult to promote their application in primary healthcare institutions, community screening, or point-of-care testing (POCT) scenarios.

[0004] Lateral chromatography test strips, with their advantages of ease of operation, speed, intuitiveness, low cost, and no need for complex instruments, have become one of the mainstream platforms in the POCT field, widely used in pregnancy testing, infectious disease screening, and food safety monitoring. Traditional lateral chromatography techniques often use colloidal gold or latex microspheres as markers to achieve qualitative or semi-quantitative detection through colorimetric signals. However, when dealing with low-abundance, small-molecule targets such as miRNAs, the sensitivity of traditional colorimetric methods is often insufficient, making it difficult to reliably detect low-concentration samples. Furthermore, the single colorimetric signal is easily affected by the reader's subjective visual differences and ambient light interference, which can easily lead to false positives or false negatives in cases of weak positivity.

[0005] To improve detection sensitivity, researchers have attempted improvements in both signal labeling and signal amplification strategies. Regarding labeling, noble metal nanomaterials, especially gold nanorods, have attracted attention due to their unique localized surface plasmon resonance effect and significant photothermal conversion capabilities. By irradiating with near-infrared lasers, gold nanorods can efficiently convert light energy into heat energy, generating a temperature rise signal that can be accurately measured by infrared thermal imagers, thus providing a possibility for constructing photothermal sensing models. However, single gold nanorods suffer from limited photothermal stability, easy surface oxidation, and tendency to aggregate in complex biological media, affecting the stability and repeatability of their signal output. Constructing a core-shell structure with gold and other metals (such as platinum) promises to enhance the chemical stability and photothermal properties of the material while maintaining strong plasmon resonance characteristics.

[0006] In terms of signal amplification strategies, enzyme-free nucleic acid amplification technologies, such as hybridization chain reactions (HCR), have been introduced into lateral chromatography systems to enhance signals due to their advantages such as high efficiency, specificity, and lack of need for precise temperature control equipment. HCR triggers the cascade hybridization of hairpin probes by the target analyte, forming a long-chain double helix structure that can enrich a large number of signal markers in situ, thereby significantly amplifying the detection signal. However, how to efficiently and controllably integrate the HCR reaction into the fluid environment of lateral chromatography and achieve synergistic effects with novel nanolabeled probes remains a technical challenge.

[0007] In summary, there is a lack of existing miRNA detection methods that can simultaneously meet the requirements of high sensitivity, high specificity, ease of operation, rapid on-site detection, and reliable result interpretation. Specifically, the following technical problems urgently need to be solved: (1) The sensitivity of traditional lateral chromatography colorimetric methods for detecting miRNA is insufficient; (2) The reliability of single detection modes (such as colorimetry only or photothermal only) is limited and easily affected by interference; (3) The signal intensity and stability of existing nanolabels need to be improved; (4) The compatibility of efficient signal amplification strategies with lateral chromatography platforms needs to be optimized.

[0008] Therefore, the present invention aims to provide an innovative solution that overcomes the shortcomings of the prior art through material design, mechanism innovation and system integration. Summary of the Invention

[0009] The core of this invention lies in constructing a colorimetric-photothermal dual-modal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods, and achieving highly sensitive, reliable, rapid and convenient detection of target miRNAs by introducing a hybridization chain reaction signal amplification mechanism.

[0010] On one hand, this invention provides a miRNA colorimetric-photothermal dual-modal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods, employing the following technical solution: A miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods includes a base plate (2) and a sample pad (1), a conjugation pad (3), a nitrocellulose membrane (6) and an absorbent pad (7) arranged sequentially on the base plate. The binding pad is loaded with AuPt nanorod photothermal colorimetric probes, which are AuPt nanorods with surface-modified thiolized hairpin probes that can specifically bind to target miRNAs. The nitrocellulose membrane has a detection line (4) and a control line (5). The detection line has a capture probe that is used to capture the target miRNA and trigger a hybridization chain reaction.

[0011] Preferably, the AuPt nanorods are platinum-coated gold nanorods with longitudinal localized surface plasmon resonance peaks located at 700~900 nm, major axis dimensions of 40~120 nm, and minor axis dimensions of 10~40 nm. The thiolized hairpin probes include a first hairpin probe H1 and a second hairpin probe H2. H1 and H2 can undergo a hybridization chain reaction triggered by the target miRNA to form a long nucleic acid structure. H1 is modified with a thiol group at its 5' or 3' end, and H2 is modified with a thiol group at its 5' or 3' end.

[0012] Preferably, the capture probe is a DNA tetrahedral structure probe, which is formed by the self-assembly of four single-stranded DNA molecules, wherein at least one single-stranded DNA molecule is modified with a thiol group or biotin at its end for immobilization on a nitrocellulose membrane.

[0013] Preferably, when the target miRNA is miRNA-21, the first hairpin probe H1 has the nucleotide sequence shown in SEQ ID NO: 1, the second hairpin probe H2 has the nucleotide sequence shown in SEQ ID NO: 2; and / or, the four single-stranded DNAs used to form the DNA tetrahedral structure probe have the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0014] Preferably, a quality control probe is fixed on the quality control line. The quality control probe is an oligonucleotide sequence that is partially complementary or reverse complementary to the nucleic acid probe modified on the surface of the AuPt nanorod photothermal colorimetric probe.

[0015] Preferably, the sample pad is soaked in a pretreatment solution containing 10-50 mmol / L Tris-HCl, 1-3×SSC, 0.5-1.5% BSA, 0.1-0.5% Tween-20, 1-5% sucrose and 0.5-5 mmol / L MgCl2, with a pH of 7.4-8.0.

[0016] On the other hand, the present invention also provides a method for preparing a miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods, using the following technical solution: A method for preparing a miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods includes the following steps: (1) Preparation of AuPt nanorod photothermal colorimetric probe: AuPt nanorods were incubated and coupled with a thiolized hairpin probe activated by a reducing agent that could specifically bind to the target miRNA, and the probe was obtained after purification; (2) Preparation of test strip assembly: Spray the binding pad with the probe solution obtained in step (1) and dry it; soak the sample pad in the pretreatment solution and dry it; print the detection line and the control line on the nitrocellulose membrane, the detection line containing the capture probe and the control line containing the control probe; (3) Assembly: The treated sample pad, the bonding pad, the printed nitrocellulose membrane and the absorbent pad are sequentially overlapped and pasted on the base plate to obtain the test strip.

[0017] This invention also provides a method for detecting miRNA using a colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods, characterized by comprising the following steps: (1) Add the sample solution to be tested onto the sample pad of the test strip and carry out the chromatography reaction; (2) Qualitative or semi-quantitative analysis is performed by visually inspecting the test line for colorimetric bands. (3) Irradiate the detection line and control line area with a laser with a wavelength of 808 nm, record the temperature change with an infrared thermal imager, calculate the temperature difference ΔT, and perform quantitative analysis of the target miRNA according to the standard curve.

[0018] Preferably, the laser power is 0.8~1.5 W, the irradiation time is 20~60 s, and the irradiation distance is 0.5~3 cm; the concentration range of the target miRNA in the sample solution is 5 fmol / L ~ 500 pmol / L.

[0019] The present invention also provides an application of a lateral chromatography test strip for preparing a kit for detecting miRNA-21; including changing the sequences of the thiolized hairpin probe and the capture probe, making the test strip usable for detecting miRNA-155 or miRNA-125b.

[0020] In summary, the beneficial effects of the present invention are as follows: This invention significantly improves the detection performance of lateral chromatography for miRNA by integrating AuPt nanorod photothermal probes, a hybridization chain reaction (HCR) signal amplification system, and a dual-modal readout mechanism. AuPt nanorods possess both strong near-infrared absorption and high photothermal conversion efficiency. The HCR reaction enables target-triggered nucleic acid cascade assembly and efficient enrichment of the signal nanorods, achieving fmol-level detection sensitivity, which is 1-2 orders of magnitude higher than traditional colorimetric methods. Simultaneously, the colorimetric and photothermal dual-signal output modes mutually validate each other, effectively avoiding missed detections of low-abundance or weakly positive samples, and greatly improving the reliability of the detection results.

[0021] This invention retains the core advantages of lateral chromatography: simple and rapid operation, and no need for complex instruments. The detection process requires only three steps: sample addition, chromatography, and photothermal reading, making it suitable for point-of-care testing scenarios such as primary healthcare and on-site screening. Furthermore, based on the designability of nucleic acid aptamer sequences, this platform can be easily extended to the detection of various disease-related biomarkers such as miRNA-155 and miRNA-125b by changing the probe sequence, demonstrating good versatility and application prospects. Attached Figure Description

[0022] Figure 1 Transmission electron microscopy (TEM) characterization image of the AuPt nanorods prepared in Example 1; Figure 2 High-resolution transmission electron microscopy (HRTEM) characterization image of the AuPt nanorods prepared in Example 1; Figure 3 A schematic diagram illustrating the preparation of a DNA-capturing tetrahedral probe (TDN-21); Figure 4 This is a schematic diagram of a colorimetric-photothermal dual-modal aptamer lateral chromatography apparatus; Figure 5 The prepared AuPt nanorods were prepared at 1 W / cm 2 Photothermal images under near-infrared light illumination; Figure 6 In Figure a, the image shows the photothermal signal captured by an infrared thermal imager when the concentration of miRNA-21 is 500 pmol / L. Figure 6 In Figure b, the image shows the photothermal signal captured by an infrared thermal imager when the concentration of miRNA-21 is 5 fmol / L. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments.

[0024] Example Example 1 The specific steps for preparing an AuPt nanorod are as follows: S1. Preparation of the gold seed solution: Take a clean 20 mL glass bottle and add 10 mL of 0.1 mol / L hexadecyltrimethylammonium bromide (CTAB) solution. Add 30 μL of 86 mmol / L chloroauric acid (HAuCl4) solution and gently shake to mix. Under vigorous stirring (1200 rpm), quickly add 0.5 mL of freshly prepared 10 mmol / L sodium borohydride (NaBH4) solution. The solution color rapidly changes from pale yellow to brownish-yellow. Continue stirring for 2 minutes, then stop stirring and let it stand at room temperature for 30 minutes. The resulting solution is the stock solution containing gold nanoseeds with a particle size of approximately 5–10 nm.

[0025] S2. Preparation of growth medium: In a 250 mL Erlenmeyer flask, dissolve 3.6 g CTAB and 0.4 g 5-bromosalicylic acid in 100 mL of ultrapure water preheated to 55°C. Stir magnetically until the solution is completely clear and transparent. Stop heating and allow the solution to cool naturally to room temperature, then let it stand for 15 minutes before use.

[0026] S3. Growth of Gold Nanorods: Add 2 mL of 10 mmol / L silver nitrate (AgNO3) solution to the above growth solution, gently shake, and let stand for 15 minutes. Add 100 mL of 1 mmol / L HAuCl4 solution and stir magnetically for 15 minutes. Add 0.5 mL of 0.1 mol / L ascorbic acid solution and stir vigorously for 30 seconds; the solution color rapidly changes from yellow to colorless. Immediately add 0.3 mL of the gold seed solution prepared in step 1 and quickly shake to mix for 30 seconds. Place the reaction flask at room temperature and let stand overnight (approximately 12 hours). Observe that the solution color gradually turns deep purple or wine red, indicating that gold nanorods have formed.

[0027] Synthesis of S4 and AuPt core-shell nanorods: Take 10 mL of the synthesized and preliminarily centrifuged concentrated gold nanorod solution and place it in a 50 mL centrifuge tube. Add 0.5 mL of 0.1 mol / L ascorbic acid solution and gently invert to mix. Then add 99 μL of 10 mmol / L chloroplatinic acid (H₂PtCl₆·6H₂O) solution and 80 μL of 10 mmol / L hydrochloric acid solution, and gently mix. Place the reaction system in a 27°C incubator and allow it to react in the dark for 12 hours. After the reaction is complete, the solution color may change slightly, indicating that the platinum shell has been deposited on the surface of the gold nanorods. Figure 1The image shown is a transmission electron microscope (TEM) image of the AuPt nanorods, which clearly displays the morphology and size of the nanomaterial. Numerous nanoparticles with distinct rod-like structures are uniformly dispersed in the image, with their long and short axes ranging from 40-120 nm and 10-40 nm, respectively, consistent with the design goals. The high-contrast image further confirms that the nanorods are successfully coated with a platinum shell, forming a clear core-shell structure. This structure is a key basis for the material's excellent photothermal conversion efficiency and stability. Figure 2 As shown, high-resolution transmission electron microscopy (HRTEM) images clearly reveal the lattice fringes of the AuPt nanorods, visually confirming the heterostructure formed by the gold core and platinum shell, as well as the good lattice matching and interface integrity between the two.

[0028] Example 2 The specific steps for preparing the AuPt nanorod photothermal colorimetric probe for miRNA-21 colorimetric-photothermal dual-modal detection are as follows: S1. Purification and concentration of AuPt nanorods: The synthesized AuPt nanorod solution was centrifuged at 10,000 rpm for 15 minutes at 4°C. The supernatant (containing CTAB, excess reagents, and byproducts) was carefully discarded. The precipitate was resuspended in an equal volume of ultrapure water to obtain a purified and concentrated AuPt nanorod solution.

[0029] S2. Activation of thiolized hairpin probes: Hairpin probes H1-21 and H2-21 were dissolved in phosphate-buffered saline (PBS) at pH 7.4 to prepare 50 μmol / L stock solutions. 100 μL of the probe solution was taken, and 5 μL of 1 mmol / L tris(2-carboxyethyl)phosphine (TCEP) solution was added. After mixing, the solution was incubated at room temperature for 30 minutes to reduce the disulfide bonds at the probe ends and expose the active thiol groups.

[0030] Table 1. Sequences of hairpin probes H1 and H2 for miRNA-21

[0031] S3. Coupling of the probe with AuPt nanorods: Take 500 μL of purified AuPt nanorod solution, add 50 μL of TCEP-activated hairpin probe miRNA-21-H1 solution and 50 μL of activated miRNA-21-H2 solution (total volume ratio 10:1:1), and gently invert to mix. Add an appropriate amount of NaCl solution to make the final NaCl concentration in the mixture reach 100 mmol / L to promote the dense assembly of the probe on the nanorod surface. Place the mixture in a refrigerator at 4℃ and incubate in the dark for 12 hours to allow the thiol groups to form stable Au-S / Pt-S bonds with the Au / Pt surface.

[0032] S4. Purification of the conjugated probe: Centrifuge the incubated solution at 10,000 rpm for 15 minutes at 4°C, and discard the supernatant containing free, unbound probe. Resuspend the precipitate to its original volume with PBS buffer (pH 7.4) containing 0.1% BSA to obtain the purified miRNA-21-H1 / H2-AuPt nanorod photothermal colorimetric probe solution, and store it at 4°C in the dark for later use.

[0033] like Figure 3 The diagram shows the process of self-assembling a capture DNA tetrahedral probe (TDN-21) via thermal annealing. This three-dimensional structure is constructed from four terminally modified single-stranded DNAs (A, B, C, D) through complementary base pairing. As a capture probe immobilized on the detection line, it is used to specifically bind to the target miRNA-21 and trigger a subsequent hybridization chain reaction.

[0034] Example 3 The construction of the hybridization chain reaction (HCR) system and the assembly of the test strips for the colorimetric-photothermal dual-modal detection of miRNA-21 are as follows: S1. Preparation of the DNA Tetrahedral Capture Probe (TDN-21): Dissolve four single-stranded DNA strands (A, B, C, D) separately in 5 mmol / L Tris-HCl buffer (pH 8.0) containing 10 mmol / L TCEP and 20 mmol / L MgCl2 to prepare a 1 mmol / L stock solution, then dilute to a working solution of 200 μmol / L. Mix equal volumes of strands A, B, C, and D to obtain a final concentration of 50 μmol / L. Place the mixture in a PCR instrument and heat at 95°C for 10 minutes, then rapidly transfer to ice and quench to 4°C, subsequently storing at -20°C. This process allows the four strands to self-assemble into a tetrahedral structure.

[0035] Table 2 miRNA-21 and probe sequences

[0036] S2. Processing and assembly of each component of the test strip: The test strip consists of a base plate, on which a sample pad, a conjugation pad, a nitrocellulose membrane (NC membrane), and an absorbent pad are sequentially laid. These components are connected by an overlapping structure to ensure continuous liquid flow. The NC membrane has two functional bands: a detection line (T line) and a control line (C line). The detection line is used to specifically recognize the target miRNA, while the control line is used to verify the effectiveness of the test strip. Figure 4 The image shown is a schematic diagram of a photothermal lateral chromatography device for detecting miRNA.

[0037] Sample pad treatment: Immerse the glass cellulose sample pad in the pretreatment solution (containing 10 mmol / L Tris-HCl pH8.0, 1×SSC, 1% BSA, 0.3% Tween-20, 3% sucrose, 2 mmol / L MgCl2) for 20 minutes, then remove and dry in an oven at 37℃ for 1 hour.

[0038] Bonding pad treatment: The polyester fiber bonding pad is uniformly sprayed with 21-H1 / H2-AuPt probe solution (70 μmol / L) at a spraying rate of 10 μL / cm, and then dried.

[0039] NC membrane printing: A capture probe solution consisting of TDN-21 (50 μmol / L) and streptavidin (0.5 mg / mL) was sprayed at a rate of 1 μL / cm onto the detection line (T line) of the nitrocellulose membrane. Control probe DNA-21 (100 μmol / L) was sprayed at the same rate onto the control line (C line). The sequence of control probe DNA-21 (SEQ ID NO: 8) is TCAACATCAGGGATTTTTT, and the distance between the T and C lines was 5 mm. The membrane was dried at 37°C after printing.

[0040] Assembly: On the PVC base plate, the prepared sample pad, bonding pad, printed NC film and absorbent pad are pasted together with an overlap width of 2mm, and then cut into test strips of the required width.

[0041] Example 4 The detection process and signal reading for miRNA-21 colorimetric-photothermal dual-modal detection are detailed below: S1. Sample Detection: Dilute or reconstitute the target miRNA-21 standard and the test sample using loading buffer (10 mmol / L Tris-HCl pH 7.4, 1×SSC, 5 mmol / L MgCl2, 1% BSA, 1% Tween-20, 3% sucrose, 0.02% NaN3). Add 100 μL of sample solution vertically to the sample pad of the test strip.

[0042] S2. Chromatography and Colorimetric Interpretation: Allow the sample to undergo free chromatography at room temperature for 5 minutes. After chromatography, observe the NC membrane directly with the naked eye. If a clear gray band appears at the T line, it is considered a positive colorimetric result; the intensity of the band color can be used for semi-quantitative estimation. The C line should always show a band as a process control.

[0043] S3. Quantitative Analysis of Photothermal Signals: A portable laser with an output wavelength of 808 nm, adjusted to 1 W, was used to vertically irradiate the test strip for 30 seconds at a distance of approximately 1 cm directly above the T / C line area. Simultaneously, an infrared thermal imager was used at a distance of approximately 10 cm from the test strip to capture and record the temperature changes in the T and C line areas in real time. The average temperature difference (ΔT) between the T and C lines after laser irradiation stabilized was calculated using thermal imaging analysis software. Based on a ΔT-concentration standard curve established beforehand using different concentrations of miRNA-21 standards, the accurate concentration of miRNA-21 in the test sample was calculated. The detection limit for miRNA-21 using this method is approximately 0.94 fmol / L. Figure 5 The image shows the photothermal heating of the prepared AuPt nanorods under 808 nm near-infrared laser irradiation, which intuitively demonstrates that the material has efficient photothermal conversion performance. Its temperature rises rapidly and remains stable, which provides a key material science basis for its use as a photothermal signal carrier for quantitative detection. Figure 6 The results of the detection of high-concentration and extremely low-concentration miRNA-21 samples were compared directly, showing that the test strip had a significant T-line colorimetric band at a concentration of 500 pmol / L, and could still be clearly detected by photothermal temperature difference (ΔT) at concentrations as low as 5 fmol / L. Both of these results together verify that the dual-modal detection system described in this invention has both high sensitivity and reliable signal output capability.

[0044] Example 5 The preparation of the AuPt nanorod photothermal colorimetric probe for the colorimetric-photothermal dual-modal detection of miRNA-155 differs from Example 2 in that the hairpin probes H1-21 and H2-21 are replaced with hairpin probes H1-155 and H2-155 in step S2, and the concentration is changed from 50 μmol / L to 70 μmol / L. A purified 155-H1 / H2-AuPt nanorod photothermal colorimetric probe solution is finally obtained.

[0045] Table 3. Sequences of hairpin probes H1 and H2 for miRNA-155

[0046] Example 6 The specific steps for constructing the hybridization chain reaction (HCR) system and assembling the test strip in the colorimetric-photothermal dual-modal detection of miRNA-155 are as follows: S1. Preparation of the DNA Tetrahedral Capture Probe (TDN-21): Dissolve four single-stranded DNA strands separately in 5 mmol / L Tris-HCl buffer (pH 8.0) containing 10 mmol / L TCEP and 20 mmol / L MgCl2 to prepare a 100 μmol / L stock solution, then dilute to a 200 μmol / L working solution. Mix equal volumes of strands A, B, C, and D to obtain a final concentration of 50 μmol / L. Place the mixture in a PCR instrument and heat at 95°C for 10 minutes, then rapidly transfer to ice and quench to 4°C, subsequently storing at -20°C. This process allows the four strands to self-assemble into a tetrahedral structure.

[0047] Table 2 miRNA-21 and probe sequences

[0048] S2. Processing and assembly of each component of the test strip: The test strip consists of a base plate on which a sample pad, a conjugation pad, a nitrocellulose membrane (NC membrane), and an absorbent pad are sequentially laid. These components are connected via an overlapping structure to ensure continuous liquid flow. The NC membrane has two functional bands: a detection line (T line) and a control line (C line). The detection line is used to specifically recognize the target miRNA, while the control line is used to verify the effectiveness of the test strip.

[0049] Sample pad treatment: Immerse the glass cellulose sample pad in the pretreatment solution (containing 10 mmol / L Tris-HCl pH8.0, 1×SSC, 1% BSA, 0.3% Tween-20, 3% sucrose, 2 mmol / L MgCl2) for 20 minutes, then remove and dry in an oven at 37℃ for 1 hour.

[0050] Binding pad treatment: The polyester fiber binding pad was uniformly sprayed with the miRNA-155-H1 / H2-AuPt probe solution (70 μmol / L) prepared in the previous step at a spraying rate of 10 μL / cm, and then dried.

[0051] NC membrane printing: At the detection line (T line) of the nitrocellulose membrane, a capture probe solution consisting of TDN-21 (50 μmol / L) and streptavidin (0.5 mg / mL) was sprayed at a rate of 1 μL / cm. At the control line (C line), the control probe DNA-155 (100 μmol / L) was sprayed at the same rate. The sequence of the control probe DNA-155 (SEQ ID NO: 16) is TCAACATCAGGGATTTTTT(5´-3´), and the distance between the T line and the C line is 5 mm. The membrane was dried at 37°C after printing.

[0052] Assembly: On the PVC base plate, the prepared sample pad, bonding pad, printed NC film and absorbent pad are pasted together with an overlap width of 2mm, and then cut into test strips of the required width.

[0053] Example 7 The detection process and signal reading for miRNA-155 colorimetric-photothermal dual-modal detection are detailed below: S1. Sample Detection: Dilute or reconstitute the target miRNA-155 standard or the sample to be tested with loading buffer (10 mmol / L Tris-HCl pH 7.4, 1×SSC, 5 mmol / L MgCl2, 1% BSA, 1% Tween-20, 3% sucrose, 0.02% NaN3). Take 100 μL of sample solution and drop it vertically onto the sample pad of the test strip.

[0054] S2. Chromatography and Colorimetric Interpretation: Allow the sample to undergo free chromatography at room temperature for 5 minutes. After chromatography, observe the NC membrane directly with the naked eye. If a clear gray band appears at the T line, it is considered a positive colorimetric result; the intensity of the band color can be used for semi-quantitative estimation. The C line should always show a band as a process control.

[0055] S3. Quantitative Analysis of Photothermal Signals: A portable laser with an output wavelength of 808 nm, adjusted to 1 W, was used to vertically irradiate the test strip for 30 seconds at a distance of approximately 1 cm directly above the T / C line area. Simultaneously, an infrared thermal imager was used at a distance of approximately 10 cm from the test strip to capture and record the temperature changes in the T and C line areas in real time. The average temperature difference (ΔT) between the T and C lines after laser irradiation stabilized was calculated using thermal imaging analysis software. Based on a ΔT-concentration standard curve established beforehand using different concentrations of miRNA-155 standards, the accurate concentration of miRNA-155 in the test sample was calculated. The detection limit for miRNA-155 using this method is approximately 1.15 fmol / L.

[0056] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods, characterized in that, Includes a base plate (2) and a sample pad (1), a conjugation pad (3), a nitrocellulose membrane (6) and an absorbent pad (7) arranged sequentially on the base plate (2); The binding pad (3) is loaded with an AuPt nanorod photothermal colorimetric probe, which is an AuPt nanorod with a thiolized hairpin probe that can specifically bind to the target miRNA. The nitrocellulose membrane (6) is provided with a detection line (4) and a control line (5). The detection line (4) is provided with a capture probe for capturing target miRNA and initiating a hybridization chain reaction.

2. The miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods according to claim 1, characterized in that, The AuPt nanorods are platinum-coated gold nanorods with longitudinal localized surface plasmon resonance peaks located at 700~900 nm, major axis dimensions of 40~120 nm, and minor axis dimensions of 10~40 nm. The thiolized hairpin probe includes a first hairpin probe H1 and a second hairpin probe H2. H1 and H2 can undergo a hybridization chain reaction triggered by the target miRNA to form a long nucleic acid structure. The 5' or 3' end of H1 is modified with a thiol group, and the 5' or 3' end of H2 is modified with a thiol group.

3. The miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods according to claim 1, characterized in that, The capture probe is a DNA tetrahedral structure probe, which is formed by the self-assembly of four single-stranded DNA strands, wherein at least one single-stranded DNA strand is modified with a thiol group or biotin at its end for immobilization on the nitrocellulose membrane (6).

4. The miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods according to claim 3, characterized in that, When the target miRNA is miRNA-21, the first hairpin probe H1 has the nucleotide sequence shown in SEQ ID NO: 1, the second hairpin probe H2 has the nucleotide sequence shown in SEQ ID NO: 2; and / or, the four single-stranded DNAs used to form the DNA tetrahedral structure probe have the nucleotide sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

5. The miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods according to claim 1, characterized in that, The quality control line (5) is fixed with a quality control probe, which is an oligonucleotide sequence that is partially complementary or reverse complementary to the nucleic acid probe modified on the surface of the AuPt nanorod photothermal colorimetric probe.

6. The miRNA colorimetric-photothermal bimodal aptamer lateral chromatography test strip based on the photothermal effect of AuPt nanorods according to claim 1, characterized in that, The sample pad (1) is soaked in a pretreatment solution containing 10~50 mmol / L Tris-HCl, 1~3×SSC, 0.5~1.5% BSA, 0.1~0.5% Tween-20, 1~5% sucrose and 0.5~5 mmol / L MgCl2, with a pH of 7.4~8.

0.

7. A method for preparing a lateral chromatography test strip according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of AuPt nanorod photothermal colorimetric probe: AuPt nanorods were incubated and coupled with a thiolized hairpin probe activated by a reducing agent that could specifically bind to the target miRNA, and the probe was obtained after purification; (2) Preparation of test strip assembly: Spray the conjugate pad (3) with the probe solution obtained in step (1) and dry it; soak the sample pad (1) in the pretreatment solution and dry it; print the detection line (4) and the control line (5) on the nitrocellulose membrane (6), wherein the detection line (4) contains a capture probe and the control line (5) contains a control probe; (3) Assembly: The treated sample pad (1), the binding pad (3), the printed nitrocellulose membrane (6) and the absorbent pad (7) are sequentially overlapped and pasted on the base plate (2) to obtain the test strip.

8. A method for detecting miRNA using the lateral chromatography test strip according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Add the sample solution to be tested to the sample pad (1) of the test strip and carry out the chromatography reaction; (2) Visually inspect the test line (4) for colorimetric bands to perform qualitative or semi-quantitative analysis; (3) Irradiate the detection line (4) and control line (5) region with a laser with a wavelength of 808 nm, record the temperature change using an infrared thermal imager, calculate the temperature difference ΔT, and perform quantitative analysis of the target miRNA according to the standard curve.

9. The method according to claim 8, characterized in that, The laser power is 0.8~1.5 W, the irradiation time is 20~60 s, and the irradiation distance is 0.5~3 cm; the concentration range of the target miRNA in the sample solution to be tested is 5 fmol / L~500 pmol / L.

10. An application of the lateral chromatography test strip according to any one of claims 1-6, characterized in that, A kit for detecting miRNA-21 can be prepared; the application includes making the test strip usable for detecting miRNA-155 or miRNA-125b by changing the sequences of the thiolated hairpin probe and the capture probe.

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