Application of CRISPR / Cas12a-nanoprotease dual-mode aptamer sensing method in KIM-1 detection

Abstract

The application provides application of a CRISPR / Cas12a-nanoplasma bimodal aptamer sensing method in KIM-1 detection. The aptamer used in the method is an oligonucleotide aptamer with high specificity and high affinity for KIM-1, and simultaneously serves as a target recognition element and an activation switch of a CRISPR system; a CRISPR / Cas12a system is used as a signal amplifier, and the activity of the CRISPR / Cas12a system is controlled by the binding event of the aptamer and the target; FeNi MOF@AgNPs-MBs reporting substrate is used as a cutting unit and a bimodal signal source, and the cutting product is used to generate measurable fluorescence and colorimetric signals. The method provided by the application has high detection sensitivity and good accuracy, and the detection time is shortened from 4 hours required by ELISA to less than 2 hours, and the analysis efficiency is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and medical detection technology, specifically relating to the application of a dual-modal aptamer sensing method based on CRISPR / Cas12a and MOF nanozymes in the detection of molecule-1 of kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a common and serious complication in critically ill patients, and its high mortality rate is a major concern. Currently, urine volume (UV) and serum creatinine (sCr) levels are the gold standard for clinical diagnosis of AKI, but they are not sensitive to early kidney injury and are difficult to use for early warning. Studies have shown that kidney injury molecule-1 (KIM-1) in urine can sensitively and non-invasively reflect the process of kidney injury and recovery, and is considered a highly promising biomarker for early AKI. Therefore, developing an efficient and accurate method for detecting urinary KIM-1 is of great significance for the early clinical diagnosis and intervention of AKI.

[0003] Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for detecting disease biomarkers. Although ELISA technology is mature, antibody preparation is costly, the detection process is cumbersome and time-consuming, and cross-reactivity may occur, affecting the specificity of the detection. Furthermore, existing methods are mostly single-signal outputs (such as colorimetry or fluorescence), which are easily affected by instrument errors, operational fluctuations, or complex sample matrices (such as urine), leading to reduced reliability and accuracy of the detection results. Banks et al. (Analyst 2014; 139: 5362) first developed a green electrochemical sensor based on waste fish scale-derived hydroxyapatite (HAp) for the direct detection of the key AKI biomarker KIM-1. This work successfully prepared HAp using black carp scales via a two-step calcination method and used it as a modifying material to construct a carbon paste electrode (CMCPE). Direct electrochemical detection of KIM-1 protein was achieved. The sensing performance is highly dependent on the crystal structure of HAp, with HAp calcined at 900°C exhibiting the best electron transfer capability due to its optimal lattice parameters. This sensor demonstrated good analytical performance for KIM-1 detection in buffer solution, with a linear range of 0.01–0.20 µg / mL. -1 The detection limit is 0.017 µg / mL. -1This method has been successfully applied to the spiked recovery validation of human urine samples. However, this direct chemical detection method relies on the direct oxidation of KIM-1 on the electrode, which is a non-specific recognition mechanism. It is highly susceptible to interference from other electroactive substances in the urine sample (such as uric acid and ascorbic acid), resulting in poor specificity and low accuracy. Furthermore, it depends on the electrochemical activity of KIM-1 itself; if other biomarkers need to be detected, the electrochemical behavior of the biomarker on a specific electrode material needs to be re-explored, thus lacking universality.

[0004] Cheng (Nano Research 2023; 17: 4329-4337) developed a fully printed microarray based on antibody recognition and integrated with a photonic crystal, utilizing its fluorescence enhancement properties to achieve instantaneous and rapid quantitative detection of KIM-1 in urine. This sensor can complete detection within 10 minutes, with a detection limit as low as 8.75 pg·mL⁻¹ and an accuracy of 94.2%. Its high sensitivity and stability were validated in clinical urine samples. This photonic crystal microarray-based immunoassay method still relies on antibody recognition. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an application of a dual-modal aptamer sensing method based on CRISPR / Cas12a and nanozymes in the detection of KIM-1 in kidney injury, achieving efficient and accurate detection of KIM-1 protein in the urine of AKI patients.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of a KIM-1 nucleic acid aptamer in the detection of kidney injury molecule-1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] A nucleic acid aptamer for KIM-1 is used in the preparation of KIM-1 detection reagents.

[0009] A KIM-1 detection method includes a nucleic acid aptamer, Cas12a / crRNA, and FeNi MOF@AgNPs-MBs; the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.1, specifically: 5'-AGC AGC ACA GAG GTC AGA TGAATA CAG ATG CAC CCG GGG TCC CT ATG CGT GCT ACC GTG AA-3'.

[0010] Furthermore, the nucleotide sequence of the crRNA is shown in SEQ ID NO.2, specifically: 5'-UAA UUU CUA CUAAGU GUA GAU GCA UCU GUA UUC AUC UGA CCU C-3'; FeNi MOF@AgNPs-MBs contain ssDNA with a 5' end modified with -SH-(CH2)6 and a 3' end modified with biotin, the ssDNA sequence of which is shown in SEQ ID NO.3, specifically: 5'-SH-(CH2)6-TTAATT AAT TAA TTA ATT AAT TAA TTA ATT AAT AAT TAA TTA ATT AAT TAATTA ATT AAT AAT AAT TAA TTA ATT AAT AAT AAT AAT AAT AAT AAT AAT AAT A-Biotin-3'.

[0011] Furthermore, the preparation method of the FeNi MOF@AgNPs-MBs includes the following steps:

[0012] (1) First, FeNi MOF was synthesized. FeCl3·6H2O, NiCl2·6H2O and 2-aminoterephthalic acid (NH2-BDC) were dissolved in N,N-dimethylformamide (DMF). 2 mL of 0.4 M NaOH solution was added dropwise, and the mixture was stirred and mixed. After high-temperature reaction, FeNi MOF was obtained. (2) FeNi MOF and AgNO3 were dissolved in methanol for the first stirring reaction. After centrifugation and washing, the mixture was resuspended in water and NaBH4 was added. The second stirring reaction was carried out under light-protected conditions to synthesize FeNi MOF@AgNPs.

[0013] (3) After incubating ssDNA with -SH-(CH2)6 at the 5' end and biotin at the 3' end with TCEP, FeNi MOF@AgNPs were added and the mixture was shaken to obtain FeNi MOF@AgNPs-ssDNA.

[0014] (4) FeNi MOF@AgNPs-ssDNA was obtained by mixing FeNi MOF@AgNPs-MBs with streptavidin-coated magnetic beads (MBs) and reacting them with a vortex reaction.

[0015] As described above, the FeNi MOF@AgNPs-MBs preferably exhibits the following characteristics: in step (1), the stirring and mixing rate is 100-500 rpm; the high-temperature reaction time is 6-12 hours; the reaction temperature is 80-120℃; in step (2), the first stirring time is 1-5 hours; the second stirring time is 10-50 min; in step (3), the incubation time is 0.5-2 hours; the mixing and shaking reaction rate is 100-300 rpm; the mixing and shaking time is 10-24 hours; and in step (4), the mixing and shaking reaction rate is 100-300 rpm; the mixing and shaking time is 0.5-2 hours.

[0016] More preferably, in step (1), the stirring rate is 200 rpm; the high-temperature reaction time is 8 hours; and the reaction temperature is 100 ℃. In step (2), the first stirring time is 3 hours; and the second stirring time is 30 min. In step (3), the incubation time is 1 hour; the mixing and shaking reaction rate is 150 rpm; and the mixing and shaking time is 16 hours. In step (4), the mixing and shaking reaction rate is 150 rpm; and the mixing and shaking time is 1 hour.

[0017] In the FeNi MOF@AgNPs-MBs described above, preferably, in step (1), FeCl3·6H2O, NiCl2·6H2O, 2-aminoterephthalic acid (NH2-BDC), and N,N-dimethylformamide (DMF) are mixed in a molar concentration-volume ratio of 1:1:1:20 to 5:1:1:20, wherein the first three are molar concentrations in mmol / L, and the last is in volume in mL. In step (2), FeNi MOF, AgNO3, and methanol are mixed in a mass-volume ratio of 30:1:4 to 15:5:2, wherein the first two are mass units in mg, and the last is in volume in mL; the concentration range of NaBH4 is 3.3 mg / mL to 10 mg / mL. In step (3), ssDNA, TCEP and FeNi MOF@AgNPs are administered at a molar concentration and mass ratio of 1:100:1 to 10:100:1, where the first two are in mmol / L and the last is in mg.

[0018] More preferably, in step (1), FeCl3·6H2O, NiCl2·6H2O, 2-aminoterephthalic acid (NH2-BDC), and N,N-dimethylformamide (DMF) are disposed in a molar concentration to volume ratio of 3:1:1:20. In step (2), FeNiMOF, AgNO3, and methanol are disposed in a mass to volume ratio of 30:3:4; the concentration of NaBH4 is 3.3 mg / mL. In step (3), ssDNA, TCEP, and FeNi MOF@AgNPs are disposed in a molar concentration to mass ratio of 10:100:1.

[0019] A method for detecting KIM-1 includes the following steps:

[0020] S1. Mix the test solution with the nucleic acid aptamer and incubate; wherein the sequence of the nucleic acid aptamer is shown in SEQ ID NO.1;

[0021] S2. Add the Cas12a / crRNA mixture that has been mixed and incubated at room temperature; wherein the sequence of the crRNA is shown in SEQ ID NO.2;

[0022] S3. The mixture was incubated for a period of time at 37°C for the first time; the cleavage substrate FeNi MOF@AgNPs-MBs was added, and the mixture was incubated for a second time at 37°C; the supernatant was collected after magnetic separation; wherein, FeNi MOF@AgNPs-MBs contains ssDNA modified with -SH-(CH2)6 at the 5' end and biotin modified at the 3' end, and the ssDNA sequence is shown in SEQ ID NO.3;

[0023] S4. Add H2O2, 3,3',5,5'-tetramethylbenzidine (TMB), and sodium acetate solution to the supernatant, mix and incubate at 50°C for 15 min, and then perform UV detection; or add NaOH solution to the supernatant for fluorescence detection.

[0024] S5. Mix the KIM-1 standard solution with the nucleic acid aptamer and perform steps S1-S4. At the same time, perform the above experiment with the blank solution to obtain the blank value. Plot the logarithm of the KIM-1 concentration on the x-axis and the difference between the colorimetric or fluorescence reading of the KIM-1 standard solution and the blank value on the y-axis to perform linear fitting and obtain the linear equation, i.e., the standard curve, for the colorimetric or fluorescence mode.

[0025] S6. Subtract the blank value from the fluorescence reading and colorimetric value of the test solution obtained in step S4, and substitute them into the corresponding standard curve to obtain the concentration of KIM-1 to be tested.

[0026] In the KIM-1 detection method described above, preferably, the solvent in the test solution, KIM-1 standard solution, and nucleic acid aptamer solution is buffer A. Buffer A has the following formulation: containing 10.0 mM Tris-HCl, 10.0 mM NaCl, and 5.0 mM MgCl2 at a final concentration, with a pH of 7.3. In step S3, the solvent in the FeNi MOF@AgNPs-MBs solution is buffer B. Buffer B has the following formulation: containing 10.0 mM Tris-HCl, 10.0 mM NaCl, 5.0 mM MgCl2, and 0.01% Tween 20 at a final concentration, with a pH of 7.3.

[0027] As described above, in the KIM-1 detection method, preferably, in step S1, the mixing and incubation time is 30 min; in step S2, the mixing and incubation time is 10 min; in step S3, the first mixing and incubation time is 30 min; and the second mixing and incubation time is 30 min.

[0028] In the KIM-1 detection method described above, preferably, in step S1, the concentration of the nucleic acid aptamer is 5 nM, and the volume ratio of the aptamer to the test solution is 1:1; in step S2, the concentration of Cas12a and CrRNA is 50 nM, and the volume ratio of the aptamer to the test solution is 1:1; in step S3, the volume ratio of FeNi MOF@AgNPs-MBs to the aptamer to the test solution is 1:1; in step S4, the volume ratio of the supernatant, H2O2, 3,3',5,5'-tetramethylbenzidine (TMB), and sodium acetate solution is 1:1:1:37; and the concentration of NaOH is 15 mM, and the volume ratio of the NaOH to the supernatant is 2:5.

[0029] In the KIM-1 detection method described above, preferably, in step S4, the maximum absorption wavelength for colorimetric detection is 652 nm; the excitation wavelength for fluorescence detection is 332 nm, and the emission wavelength is 425 nm.

[0030] The beneficial effects of this invention are as follows:

[0031] The present invention provides an aptamer Apt20-Truc1 that can bind to KIM-1 with high specificity and high affinity, the sequence of which is shown in SEQ ID NO.1 and has an affinity of 23.4 pM.

[0032] This invention provides a synergistic detection system for the efficient and accurate detection of KIM-1. It employs a nucleic acid aptamer as both a target recognition element and an activation switch for the CRISPR system; the CRISPR / Cas12a system acts as a signal amplifier, its activity controlled by the binding event between the aptamer and the target; the FeNi MOF@AgNPs-MBs reporter substrate serves as the cleaved unit and a dual-modal signal source, with its cleavage products used to generate measurable fluorescence and colorimetric signals.

[0033] This invention provides a dual-modal aptamer sensing method based on CRISPR / Cas12a and MOF nanozymes for the detection of KIM-1. The linear range is 0.1 ng / mL to 100 ng / mL (colorimetric and fluorescence modes), the detection limit in colorimetric mode is 58.7 pg / mL, and the detection limit in fluorescence mode is 34.4 pg / mL, both with good selectivity.

[0034] Verification with actual samples showed that the detection method provided by this invention is highly consistent with the ELISA method (average relative deviations of -2.7% for colorimetric mode and 3.2% for fluorescence mode), demonstrating good accuracy. More significantly, the method of this invention reduces the detection time from 4 hours required by ELISA to less than 2 hours, resulting in a substantial improvement in analytical efficiency. Attached Figure Description

[0035] Figure 1 Images of the first round of screening and agarose gel electrophoresis of KIM-1 nucleic acid aptamers;

[0036] Figure 2 Capillary electrophoresis images of the second to fourth rounds of screening for KIM-1 nucleic acid aptamers;

[0037] Figure 3 The results show the affinity and specificity assays for six aptamer candidate sequences.

[0038] Figure 4 A schematic diagram of the secondary structure of Apt20 and its truncated sequence;

[0039] Figure 5 TEM characterization results for FeNi MOF and FeNi MOF@AgNPs;

[0040] Figure 6 The results are FT-IR characterizations of NH2-BDC, FeNi MOF, and FeNi MOF@AgNPs.

[0041] Figure 7 XRD characterization results of FeNi MOF and FeNi MOF@AgNPs;

[0042] Figure 8 XPS characterization results for FeNi MOF@AgNPs;

[0043] Figure 9 The results represent the activity characterization of FeNi MOF@AgNPs-type peroxidases.

[0044] Figure 10 The results are the enzyme reaction kinetics determination results for FeNi MOF@AgNPs;

[0045] Figure 11 EPR characterization results for FeNi MOF and FeNi MOF@AgNPs;

[0046] Figure 12 The fluorescence characterization results of FeNi MOF and FeNi MOF@AgNPs are shown.

[0047] Figure 13 VSM characterization results for MBs and FeNi MOF@AgNPs-MBs;

[0048] Figure 14 Characterization results of Zeta potentials for FeNi MOF, FeNi MOF@AgNPs, and FeNi MOF@AgNPs-MBs;

[0049] Figure 15 The result is a linear result for the colorimetric mode;

[0050] Figure 16 Linear results for fluorescence mode;

[0051] Figure 17 Selectivity results were detected in both colorimetric and fluorescence modes. Detailed Implementation

[0052] To address the key technical bottlenecks in the practical detection of KIM-1 using existing CRISPR-based sensing technologies: firstly, CRISPR systems struggle to directly respond to protein targets, resulting in a disconnect between recognition and signal amplification; secondly, traditional signal output methods are limited in their accuracy in complex biological samples; and thirdly, there is a lack of an efficient platform integrating recognition, amplification, and multimodal detection. Therefore, this invention provides a dual-modal aptamer sensing method based on CRISPR / Cas12a and nanozymes. By simultaneously using the KIM-1 aptamer as both the recognition element and the CRISPR system activation chain, protein target signal transduction is achieved. Furthermore, the trans-cleavage activity of CRISPR is utilized to cleave the magnetically coupled ssDNA-FeNi MOF@AgNPs complex substrate. After magnetic separation, the fluorescence signal of MOF@AgNPs and its peroxidase-catalyzed colorimetric reaction are detected to achieve dual-modal signal output, ultimately enabling efficient and accurate detection of KIM-1 in urine samples. The present invention aims to develop a bimodal aptamer sensing method based on CRISPR / Cas12a and nanozymes for efficient and accurate detection of KIM-1 protein in the urine of AKI patients.

[0053] This invention designs a cascade reaction system coupling target recognition and signal amplification: First, capillary electrophoresis combined with a urine matrix is ​​used to screen for nucleic acid aptamers that specifically bind to KIM-1. These aptamers serve as recognition elements and simultaneously as activation switches for the CRISPR / Cas12a system. The aptamers trigger the trans-cleavage activity of the Cas12a / crRNA complex. When the target KIM-1 is present, its specific binding to the aptamer reduces the trans-cleavage activity of the Cas12a / crRNA complex. Activated Cas12a cleaves the magnetically bead-coupled ssDNA-FeNiMOF@AgNPs complex substrate (FeNi MOF@AgNPs-MBs). After magnetic separation, the released FeNi MOF@AgNPs nanozyme simultaneously generates a fluorescence signal and catalyzes TMB color development, thereby achieving precise dual-modal detection of KIM-1.

[0054] This invention constructs a detection system based on the synergistic effects of nucleic acid aptamers, CRISPR system, and nanozymes by screening nucleic acid aptamers that bind to KIM-1 with high affinity and high specificity, and by preparing a dual-modal signal source FeNi MOF@AgNPs.

[0055] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0056] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art. Unless otherwise specified, all reagents used in the examples are analytical grade or higher and can be commercially available products. For example, the fluorescently labeled ssDNA40N and nucleic acid sequences described in Examples 1-3 and 3-5 below were purchased from Sangon Biotech Co., Ltd.; the high-throughput sequencing of the conventional PCR products in Example 1 below was performed by Sangon Biotech Co., Ltd.; fused silica capillaries were purchased from Hebei Handan Development Zone Aotech Biotechnology Co., Ltd.; KIM-1 and neutrophil gelatinase-associated lipotransferase (NGAL) were purchased from Nanjing Oukai Biotechnology Co., Ltd. (Nanjing, China); recombinant human urinary globulin (UMOD) protein and liver-type fatty acid-binding protein (L-FABP) were purchased from Shanghai Yubo Biotechnology Co., Ltd. (Shanghai, China); human serum albumin (HSA) and human immunoglobulin (IgG) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA); tissue metalloproteinase inhibitor 2... TIMP-2 and insulin-like growth factor binding protein 7 (IGFBP-7) were purchased from Beijing Biopharmaceutical Co., Ltd. (Beijing, China); FeCl3·6H2O, NiCl2·6H2O, 2-aminoterephthalic acid (NH2-BDC), N,N-dimethylformamide (DMF), AgNO3, NaBH4, PBS buffer, 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) were purchased from Innovent (Beijing, China); CRISPR / Cas12a and 10×NEB buffer were purchased from New England Biolabs (NEB) (Massachusetts, USA).

[0057] Example 1

[0058] (1) Preparation of raw random deoxy oligonucleotide library (ssDNA40N) stock solution: Purchased crystalline fluorescently labeled random deoxy oligonucleotide library (ssDNA40N) (SEQ ID NO.4: 5'-FAM-AGCAGCACAGAGGTCAGATG-(N40)-CCTATGCGTGCTACCGTGAA-3'; N40 indicates that the middle 40 bases are random), centrifuged at 3000 rpm for 5 min, added pure water to prepare 10 μM raw ssDNA40N stock solution, cooled on ice after 5 min in a 94℃ metal bath, and stored at -20℃.

[0059] The original ssDNA40N stock solution was diluted with PBS to obtain a 400 nM ssDNA40N sample solution.

[0060] 3.0 μL of raw KIM-1 (8 μM) sample solution and 3 μL of 400 nM ssDNA40N sample solution were mixed and incubated together in a metal bath at 37 ℃ for 40 min. After incubation, the mixture contained unbound free ssDNA and KIM-1 bound ssDNA-KIM-1 complex, which were separated and analyzed by capillary electrophoresis.

[0061] The capillary used in capillary electrophoresis was a fused silica capillary with a total length of 50.2 cm, an effective length of 40 cm, and an inner diameter of 75 μm. The mixture was injected into a Beckman P / ACE MDQ capillary electrophoresis apparatus for electrophoresis. The electrophoresis buffer was a borate-borax solution, prepared by mixing 50 mM borax solution and 20 mM boric acid solution at a volume ratio of 3:2, with a pH of 8.7. The injection conditions were 0.5 psi for 8 s. The electrophoresis analysis conditions were 10 min for 10 min, 20 kV for 20 kV, and 25 °C for 25 °C. The outlet end of the capillary was the negative electrode, and the inlet end was the positive electrode. During electrophoresis, free ssDNA and the ssDNA-KIM-1 complex migrate at different rates in the capillary, passing sequentially through the detection window at the capillary tip. Peaks of both free ssDNA and the ssDNA-KIM-1 complex can be detected and separated, and the ssDNA-KIM-1 complex can be directly collected. Laser-induced fluorescence (CE-LIF) detection was used during the online capillary reaction, with excitation wavelength 488 nm and emission wavelength 520 nm. The LIF detector results are as follows: Figure 1 As shown in the left figure.

[0062] Figure 1 The bottom spectral line in the left figure is the ssDNA40N spectral line, which produces a signal peak at 7.8 min. Figure 1 The second line from the bottom is the spectrum of the mixture of 3.0 μL KIM-1 (8 μM) sample solution and 3.0 μL 400 nM ssDNA40N sample solution incubated together. The signal peak at 7.8 min coincides with the elution time of the ssDNA40N peak in the spectrum below. Compared with the spectrum below, the ssDNA40N signal peak in the second line from the bottom is lower, and a new signal peak is generated at 3.0 min. This is because some ssDNA40N reacts with KIM-1 to form the ssDNA40N-KIM-1 complex. The signal peak at 3.0 min is determined to be the ssDNA40N-KIM-1 complex peak.

[0063] The above-mentioned complex region was subjected to conventional PCR amplification. Symmetrical PCR was performed using the following amplification system: 264 µL ddH2O, 132 µL 15 µM upstream primer, 132 µL 15 µM downstream primer, and 550 µL PCR Mix enzyme (upstream primer: SEQ ID NO.5: 5'-AGCAGCACAGAGGTCAGATG-3'-; downstream primer: SEQ ID NO.6: 5'-TTC ACG GTA GCA CGC ATAGG-3'). Conventional PCR amplification conditions were: 94 ℃, 1 min; 94 ℃, 30 s; 59 ℃, 30 s; 72 ℃, 30 s; 28 cycles were performed. DNase / RNase-free deionized water and 2×Taq PCR Mastermix were purchased from Tiangen Biotech Co., Ltd. The agarose gel electrophoresis conditions were as follows: 30 mL of 0.5×TBE buffer and 0.6 g of agarose were used to prepare a 2% agarose gel. Electrophoresis was performed on a Bio-Rad electrophoresis apparatus at 90 V for 30 min. Agarose, GeneGreen nucleic acid dye, 6×DNA Loading Buffer, and 50bp DNA Ladder were purchased from Tiangen Biotech Co., Ltd. The results are as follows: Figure 1 As shown in the figure on the right.

[0064] Figure 1 The right side shows the gel electrophoresis results of the PCR amplification products. Figure 1 The rightmost band is the DNA marker, with lengths from bottom to top of 50bp, 100bp, 150bp, 200bp, 250bp, 300bp...500bp. The length of the ssDNA40N-KIM-1 complex band matches the 80bp position in the DNA marker, thus identifying it as the amplification product of the target length.

[0065] (2) Alcohol precipitation and gel extraction: Collect the PCR product in two 1.5 mL centrifuge tubes (400 μL each), add 900 μL of pre-cooled anhydrous ethanol, centrifuge at 4 ℃ and 13000 rmp for 15 min, and discard the supernatant; add 250 μL of 70% pre-cooled anhydrous ethanol, mix well, centrifuge at 4 ℃ and 13000 rmp for 5 min, discard the supernatant, repeat this operation, dry the bottom precipitate, and redissolve it in 20 μL ddH2O. The ethanol precipitation product was subjected to agarose gel electrophoresis. The appropriate length of band was cut, crushed, and 500 μL of ddH2O was added. The mixture was vortexed, followed by 500 μL of Tris-saturated phenol. The mixture was then vortexed and centrifuged at 4 °C and 13000 rpm for 15 min. The supernatant was then added to chloroform:isoamyl alcohol (24:1), vortexed, and centrifuged at 4 °C and 13000 rpm for 5 min. The supernatant was then added to 20 μL of 3 M sodium acetate and 900 μL of pre-cooled anhydrous ethanol and incubated overnight at -20 °C. The mixture was then centrifuged at 4 °C and 13000 rpm for 15 min. The precipitate was dried and reconstituted in 20 μL of ddH2O for the next step of asymmetric PCR.

[0066] Asymmetric PCR: The procedure is the same as for symmetric PCR, except that the upstream primer is replaced with a fluorescent primer (SEQ ID NO.5: 5'-FAM-AGCAGCACAGAGGTCAGATG-3'), and the downstream primer (SEQ ID NO.6: 5'-TTC ACG GTA GCA CGC ATAGG-3') is replaced with the same volume of ddH2O. 28 rounds of amplification were performed. Subsequent alcohol precipitation and gel extraction were performed as above, and the resulting product became the secondary library for the next round of screening.

[0067] (3) Take 2 μL of the original KIM-1 (8 μM) sample solution, 2 μL of the second-round secondary library sample solution, and 2 μL of healthy human urine matrix, respectively, mix them, and incubate them together in a metal bath at 37 ℃ for 40 min. After incubation, the mixture contains unbound free ssDNA and KIM-1 bound ssDNA-KIM-1 complexes. These are separated and analyzed by capillary electrophoresis. The results are as follows: Figure 2 As shown on the left. The ssDNA40N-KIM-1 complex was collected and subjected to symmetrical PCR, alcohol precipitation and gel extraction, as well as asymmetric PCR, alcohol precipitation and gel extraction to obtain the secondary library for the third round of screening.

[0068] Repeat the same steps as in the second round, perform capillary electrophoresis separation and analysis, and the results are as follows. Figure 2As shown in the figure. The ssDNA-KIM-1 complex was collected and subjected to symmetrical PCR, alcohol precipitation and gel extraction, and asymmetric PCR, alcohol precipitation and gel extraction to obtain the fourth round of screening secondary library.

[0069] Two μL of 20 μM UMOD protein sample solution, two μL of fourth-round secondary library sample solution, and two μL of healthy human urine matrix were mixed and incubated together in a 37 ℃ metal bath for 40 min. After incubation, the mixture contained unbound free ssDNA and UMOD-bound ssDNA-UMOD complexes, which were then separated and analyzed by capillary electrophoresis. The results are shown below. Figure 2 As shown on the right. Free ssDNA was collected, symmetrical PCR was performed, and the symmetrical PCR products were then subjected to high-throughput sequencing.

[0070] Figure 2 The bottom spectral line in the left figure is the second-round secondary library spectral line; Figure 2 The second line from the bottom in the left figure is the spectrum of a mixture of 2 μL of the original KIM-1 (8 μM) sample solution, 2 μL of the second round of secondary library sample solution, and 2 μL of healthy human urine matrix. Compared with the lower line, the ssDNA40N signal peak in the second line from the bottom is reduced, and a new signal peak is generated at 3.0 min. This is because some of the secondary library reacts with KIM-1 to form the ssDNA-KIM-1 complex.

[0071] Figure 2 The spectral lines at the bottom of the middle image represent the third round of secondary library spectral lines; Figure 2 The second line from the bottom in the middle figure is the spectral line of a mixture of 2 μL of the original KIM-1 (8 μM) sample solution, 2 μL of the third round of secondary library sample solution, and 2 μL of healthy human urine matrix. Compared with the line below, a new signal peak was generated at 3.0 min, which is due to the reaction of some secondary library with KIM-1 to form the ssDNA-KIM-1 complex.

[0072] Figure 2 The bottom spectral line in the right figure is the fourth round secondary library spectral line; Figure 2 The second line from the bottom in the right figure is the spectrum of a mixture of 2 μL 20 μM UMOD protein sample solution, 2 μL fourth-round secondary library sample solution, and 2 μL healthy human urine matrix incubated together. Compared with the spectrum below, the ssDNA40N signal peak in the second line from the bottom is reduced, and a new signal peak is generated at 3.0 min. This is because some of the secondary library reacts with UMDO to generate an ssDNA-UMDO complex. The free ssDNA is collected, and after symmetrical PCR, high-throughput sequencing is performed.

[0073] Example 2

[0074] The affinity and specificity of the following 6 aptamer candidate sequences were determined using CE.

[0075] Apt3 (SEQ ID NO.7): 5'-FAM-AGC AGC ACA GAG GTC AGA TGA ATA CAG ATG CACCCG GGG GAT GTT GTG CTC AAG CGT CTC CCT ATG CGT GCT ACC GTG AA-3'; Apt4 (SEQ ID NO.8): 5'-FAM- AGC AGC ACA GAG GTC AGA TGC CGC AGG CAGCTG CCA TTA GTC TCT ATC CGT GAC GGT ATG CCT ATG CGT GCT ACC GTG AA-3'; Apt7 (SEQ ID NO.9): 5'-FAM- AGC AGC ACA GAG GTC AGA TGT TTG TTT GTG AGAGGG GGG TAT GGC CTC AAA GCA TGG AAG CCT ATG CGT GCT ACC GTG AA-3'; Apt20 (SEQ ID NO.10): 5'-FAM- AGC AGC ACA GAG GTC AGA TGA ATA CAG ATGCAC CCG GGG GAT GCT GTG CTC AAG CGT CTC CCT ATG CGT GCT ACC GTG AA-3'; Apt53 (SEQ ID NO. 11): 5'-FAM- AGC AGC ACA GAG GTC AGA TGT GTC TGC GCGCGT CAT CCG AAC ACT TAC ATC GCT GCG ATG CCT ATG CGT GCT ACC GTG AA-3'; Apt266 (SEQ ID NO. 12): 5'-FAM- AGC AGC ACA GAG GTC AGA TGC CGC AGG CAGCTG CCA TTA GTC TCT ATC CGT GAC GGT ATA CCT ATG CGT GCT ACC GTG AA-3'.

[0076] (1) Affinity: Different concentrations of aptamer sequences (15.6, 31.2, 62.5, 125, 250, 500 nM) were mixed with 4.0 μM KIM-1 and incubated at 37 ℃ for 40 min before CE analysis. Based on the change in peak area of ​​the KIM-1-Apt complex with KIM-1 concentration, nonlinear fitting was performed using the following formula:

[0077]

[0078] Where I0 represents the Apt peak area of ​​the blank sample, I represents the Apt peak area of ​​a sample at a certain concentration, constant represents a constant, and K represents a constant. D The value represents affinity, and [target] is the KIM-1 concentration. The left side of the formula represents the binding ratio of the aptamer Apt or the peak area ratio of the KIM-1-Apt complex, and the ordinate is obtained through nonlinear fitting to determine constant and K. D Value. Result as follows Figure 3 As shown above.

[0079] Figure 3 The above shows the affinity of the six aptamer candidate sequences: Apt3 is 220 nM, Apt4 is 181 nM, Apt7 is 754 nM, Apt20 is 125 nM, Apt53 is 145 nM, and Apt266 is 278 nM.

[0080] (2) Specificity: 4.0 μM UMOD, human serum albumin (HSA), liver-type fatty acid-binding protein (L-FABP), human immunoglobulin (IgG), urine matrix, and PBS buffer were mixed with 400 nM of six aptamer candidate sequences and incubated at 37 ℃ for 40 min, followed by CE analysis. The selectivity of different aptamer candidate sequences was obtained based on the change in the peak area of ​​free ssDNA. The results are as follows: Figure 3 As shown below.

[0081] Figure 3 The following are the specificities of the six aptamer candidate sequences: Apt3, Apt20, and Apt53 showed good selectivity; Apt4, Apt7, and Apt266 showed varying degrees of cross-reactivity with other proteins.

[0082] Example 3

[0083] Aptamer sequence truncation: (1) The Apt20 sequence, which has excellent affinity and specificity, was optimized and truncated. First, the binding sites of Apt20 and KIM-1 were analyzed by molecular docking (http: / / hdock.phys.hust.edu.cn / ). Based on the interaction results between Apt20 and KIM-1 in the molecular docking results, redundant sequences were truncated without changing the secondary structure of the aptamer (http: / / www.unafold.org / mfold / applications / dna-folding-form.php) to obtain Apt20-Truc1, Apt20-Truc1a and Apt20-Truc2. The results of their secondary structures are as follows. Figure 4 As shown. Wherein:

[0084] Apt20-Truc1 (SEQ ID NO.1): 5'-AGC AGC ACA GAG GTC AGA TGA ATA CAG ATGCAC CCG GGG TCC CT ATG CGT GCT ACC GTG AA-3';

[0085] Apt20-Truc1a (SEQ ID NO.13): 5'-AGC AGC ACA GAG GTC AGA TGA ATA CAG ATGCAC CC GG ATG CGT GCT A-3';

[0086] Apt20-Truc2 (SEQ ID NO. 14): 5'-CAG CAC AGA GGT CAG ATG AAT ACA GAT GCACCC GGG GTC CCT ATG CGT GCT A-3'.

[0087] The affinities of Apt20, Apt20-Truc1, Apt20-Truc1a, and Apt20-Truc2 with KIM-1 were determined using the surface plasmon resonance (SPR) method, and the affinities were 11.7 nM, 23.4 pM, 58.7 pM, and 1.9 nM, respectively. The optimal affinity sequence, Apt20-Truc1, was selected for establishing the sensing method.

[0088] Example 4

[0089] (1) FeNi MOF was first synthesized via a hydrothermal method: 0.810 g FeCl3·6H2O, 0.238 g NiCl2·6H2O, and 0.181 g 2-aminoterephthalic acid (NH2-BDC) were dissolved in 20 mL N,N-dimethylformamide (DMF), and 2 mL 0.4 M NaOH solution was added dropwise. After stirring for 10 min, the resulting yellow viscous liquid was sealed in a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 100 °C for 8 h. After the autoclave cooled naturally, the generated FeNi MOF was collected by centrifugation and washed three times each with DMF and methanol. After drying at 50 °C for 12 h, FeNi MOF was obtained for later use.

[0090] Synthesis of FeNi MOF@AgNPs: 150 mg FeNi MOF and 15 mg AgNO3 were dissolved in methanol and stirred in the dark for 3 h. The product was collected by centrifugation, and excess reactants were washed away with deionized water. The product was resuspended in 20 mL of deionized water, and 0.67 mL of 3.3 mg / mL NaBH4 was added. The mixture was stirred in the dark for 30 min. Excess reactants were washed away with deionized water, and the product was dried at 50 °C for 12 h for later use.

[0091] The preparation of FeNi MOF@AgNPs was confirmed using a series of characterization methods. First, the morphology of FeNi MOF and FeNi MOF@AgNPs was characterized using scanning electron microscopy (TEM). The results are as follows: Figure 5 As shown, FeNi MOF possesses a highly uniform bipyramidal structure; in the morphology of FeNi MOF@AgNPs, silver nanoparticles are observed to be firmly loaded on the outer surface of FeNi MOF. Because the cluster size of the silver nanoparticles is small, their loading does not alter the bipyramidal structure of FeNi MOF.

[0092] Infrared spectroscopy (FT-IR) characterization was performed, such as... Figure 6 As shown, the C=O stretching vibration absorption peak in the NH2-BDC ligand is located at 1691 cm⁻¹. -1 At this location, after coordination with the metal, the infrared spectrum of FeNi MOF and FeNi MOF@AgNPs shifts to 1691 cm⁻¹. -1 The -OH absorption peak in the NH2-BDC ligand is located at 2975 cm⁻¹. -1 However, this absorption peak disappears in the infrared spectra of FeNi MOF and FeNi MOF@AgNPs, indicating that it has formed coordination with the metal; in addition, the symmetric and asymmetric stretching vibrations of -NH2 in the NH2-BDC ligand are located at 3394 and 3508 cm⁻¹, respectively. -1However, in FeNi MOF and FeNi MOF@AgNPs, the peak changes to 3431 cm⁻¹ due to the formation of an internal hydrogen bond between the amino group and the electron-donating oxygen of the carboxyl group. -1 A broad absorption peak at that location.

[0093] The crystal phases of FeNi MOF and FeNi MOF@AgNPs were characterized by X-ray diffraction (XRD). Figure 7 As shown, characteristic peaks related to the MOF structure can be found in the range of 5° to 40°, and the crystallinity of FeNi MOF does not show significant loss after loading AgNPs. In addition, the XRD pattern of FeNi MOF@AgNPs shows diffraction peaks at 2θ values ​​of 38.1°, 44.3° and 64.4°, which correspond to the (111), (200) and (220) crystal planes of silver, respectively, indicating that silver nanoparticles have been successfully loaded.

[0094] The elemental composition of FeNi MOF@AgNPs was obtained by X-ray photoelectron spectroscopy (XPS), such as... Figure 8 As shown. In the XPS spectra of FeNiMOF@AgNPs ( Figure 8 (Top left image) In addition to C1s, O1s, Fe2p, and Ni2p, Ag3d was also observed, indicating that AgNPs were successfully loaded onto FeNi MOF. High-resolution Ni2p XPS image ( Figure 8 The spectrum (top right) shows the fitted Ni 2p1 / 2 (875.1 eV) and Ni 2p3 / 2 (855.6 eV) peaks, along with two satellite bands. Their splitting energies indicate that nickel exists in a divalent form. Furthermore, the weaker peak intensities suggest a low nickel content. High-resolution Fe 2p XPS spectrum ( Figure 8 The lower left figure shows two broad peaks at 711.5 and 725.1 eV, corresponding to Fe 2p3 / 2 and Fe 2p1 / 2, respectively. Similarly, the high-resolution XPS spectrum of Ag3d (…) Figure 8 In the lower right figure, the peaks at 368.2 and 374.3 eV correspond to Ag3d5 / 2 and Ag3d3 / 2, respectively.

[0095] Example 5

[0096] Characterization of peroxidase activity and fluorescence properties of FeNi MOF@AgNPs

[0097] (1) The catalytic performance of FeNi MOF@AgNPs nanozymes was evaluated using TMB as a chromogenic substrate. 12.5 μL of 1.0 mg / mL FeNi MOF@AgNPs was added to 12.5 μL of 0.2 M H2O2, 12.5 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB), and 462.5 μL of sodium acetate solution (0.1 M, pH 4.0). The mixture was incubated at 50 °C for 15 min and then detected by UV in the wavelength range of 500-750 nm.

[0098] like Figure 9 As shown, FeNi MOF@AgNPs showed almost no absorbance after incubation with H2O2. However, by mixing FeNi MOF@AgNPs with TMB, a low absorbance was observed at 652 nm, the characteristic absorption peak of oxTMB, indicating low oxidase-like activity of FeNi MOF@AgNPs. In contrast, mixing FeNi MOF@AgNPs with TMB and H2O2 exhibited stronger absorbance, suggesting that FeNi MOF@AgNPs can act as a peroxidase to catalyze the reaction with H2O2. Furthermore, the catalytic activity of FeNi MOF was further compared under the same conditions; FeNi MOF could also oxidize colorless TMB to blue oxTMB. However, the catalytic performance of FeNi MOF@AgNPs was superior to that of FeNi MOF.

[0099] (2) Determination of enzyme reaction kinetics of FeNi MOF@AgNPs nanozymes.

[0100] The concentration of TMB was varied under a fixed hydrogen peroxide concentration (0.2 M), or the concentration of H2O2 was varied under a fixed TMB concentration (20.0 mM). The main steps are as follows: 12.5 μL of FeNi MOF@AgNPs suspension (1 mg / mL), 12.5 μL of H2O2 at different concentrations (0.1, 0.5, 1.0, 3.0, 5.0, 10.0, 50.0 mM, 0.1, 0.2 M) and 12.5 μL of TMB (20.0 mM) were mixed, and 462.5 μL of sodium acetate solution (0.1 M, pH 4.0) was added and incubated at 50 ℃ for 10 min. Another experimental group used 12.5 μL of H₂O₂ (0.2 M) and 12.5 μL of TMB at different concentrations (0.1, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 20.0, 30.0 mM), keeping all other experimental conditions unchanged. Absorbance was measured at the maximum absorption wavelength of 652 nm. The absorbance at 652 nm was measured using a Meptopek P3 single-beam spectrophotometer. The absorbance values ​​were converted to TMB oxide concentration using the Lambert-Beer law, where the molar absorptivity of TMB oxide at 652 nm is 39,000 M. -1 ·cm -1 The microcuvette has an optical path length of 1 cm. The concentration change per unit time during the first 10 minutes is calculated as the initial reaction rate (V). By plotting the initial rate against the substrate concentration, the Michaelis equation curve [V = V] can be obtained. max [S] / (K m + [S])], where V is the initial reaction rate, Vmax is the maximum reaction rate, [S] is the substrate concentration, and K m This is the Michaelis constant. Ultimately, the Michaelis constant (K) can be calculated. m ) and maximum reaction rate (V max The experimental results are as follows: Figure 10 As shown, the prepared FeNi MOF@AgNPs exhibit good peroxidase-like activity.

[0101] (3) To elucidate the catalytic mechanism of FeNi MOF@AgNPs, electron paramagnetic resonance (EPR) measurements were performed using 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as a spin trapping agent to capture •OH and •O2⁻. The experimental results are as follows: Figure 11As shown in the spectrum, distinct characteristic signal peaks with intensity ratios of 1:2:2:1 and 1:1:1:1 are observed, confirming the successful capture of DMPO-•OH and DMPO-•O2⁻ adducts. Furthermore, the signal intensities induced by FeNi MOF@AgNPs during free radical capture are stronger than those induced by FeNi MOF itself. These results indicate that the introduction of AgNPs significantly enhances the peroxidase mimicry activity of FeNi MOF by strengthening its ability to generate •OH and •O2⁻ free radicals. This enhancement may stem from a synergistic effect between AgNPs and MOF.

[0102] (4) Fluorescence detection: 50 μL of FeNi MOF@AgNPs suspension (1 mg / mL) was added to 20 μL of 15 mM NaOH solution. Fluorescence detection was performed using a PerkinElmer Engight microplate reader. The excitation wavelength was 332 nm, and the emission wavelength scanning range was 375-600 nm. The experimental results are as follows: Figure 12 As shown, experimental results indicate that FeNi MOF itself exhibits weak fluorescence. However, after loading AgNPs onto its surface to form FeNi MOF@AgNPs, the localized surface plasmon resonance effect of AgNPs effectively enhances the fluorescence emission of the system, resulting in significant fluorescence. The fluorescence is further enhanced upon the addition of sodium hydroxide, which may be due to the deprotonation of the organic ligands in the MOF by OH⁻.

[0103] Characterization of the peroxidase activity and fluorescence properties of FeNi MOF@AgNPs showed that it can be used as a good signal transduction and signal catalyst in the CRISPR / Cas12a system, thereby achieving efficient and accurate detection of targets.

[0104] Example 6

[0105] Preparation of FeNi MOF@AgNPs-MBs: The ssDNA used was modified with -SH at the 5' end and biotin at the 3' end (its sequence is (SEQ ID NO.3): 5'-SH-(CH2)6-TTAATT AAT TAA TTA ATT AAT TAA TTA ATTAAT AAT TAA TTA ATT AAT TAA TTA ATT AAT TAA TTA ATT AAT TAA TTA ATT AATAAT TAA TTA ATTA-Biotin-3'). FeNi MOF@AgNPs-ssDNA was prepared based on the chemical bonding between the thiol group and AgNPs. 187.0 μL of 10.0 μM ssDNA was mixed with 3 μL of 100 mM tris(2-carboxyethyl)phosphine (TCEP) and incubated at room temperature for 1 h. Add 1.0 mg FeNi MOF@AgNPs to the above solution and mix with 810 μL of buffer A (10.0 mM Tris-HCl, 10.0 mM NaCl, 5.0 mM MgCl2, pH 7.3) for 16 h. Collect the product by centrifugation and wash away excess reactants with buffer A.

[0106] FeNi MOF@AgNPs-MBs were prepared based on the interaction between biotin and streptavidin. 1.0 mL of streptavidin-coated magnetic beads (MBs) resuspended in buffer B (10.0 mM Tris-HCl, 10.0 mM NaCl, 5.0 mM MgCl2, 0.01% Tween 20, pH 7.3) at 1 mg / mL was added to solution (1). The mixture was shaken for 1 h, and the product was collected by magnetic separation. Excess reactants were washed away with buffer B. The product was resuspended in 0.6 mL of buffer B to obtain FeNi MOF@AgNPs-MBs, which were stored at 4 °C for later use.

[0107] The magnetic changes during the synthesis of FeNi MOF@AgNPs-MBs were detected using a vibrating sample magnetometer (VSM). The experiment is as follows: Figure 13 As shown, the magnetic response of FeNi MOF@AgNPs-MBs is related to the content of Fe3O4 MBs. Figure 13The hysteresis regression curves of Fe3O4 MBs and FeNi MOF@AgNPs-MBs both exhibit an origin-symmetric "S" shape, indicating paramagnetism. The saturation magnetization of Fe3O4 MBs and FeNiMOF@AgNPs-MBs are 53.6 and 46.2 emu / g, respectively. Although the saturation magnetization gradually decreases due to the coupling between ssDNA and FeNi MOF@AgNPs, the retained magnetism is sufficient to achieve rapid separation under the influence of an external magnetic field.

[0108] The zeta potentials of FeNi MOF, FeNi MOF@AgNPs, and FeNiMOF@AgNPs-MBs were determined using a nano-force-Zeta potential analyzer (DLS). The experimental results are as follows: Figure 14 As shown, the Zeta potentials of FeNi MOF, FeNi MOF@AgNPs, and FeNi MOF@AgNPs-MBs are -16.7, -15.5, and -21.1 mV, respectively. The negative potential of FeNi MOF itself may originate from the ionization of ligands (such as carboxyl groups) in its metal-organic framework. After composite with AgNPs, the potential slightly increases to -15.5 mV, which may be due to the relatively neutral AgNPs partially shielding the negative charge of the MOF backbone, or due to the rearrangement of surface charge distribution caused by interfacial interactions during complex formation. Finally, when ssDNA-modified magnetic beads (MBs) are fixed to the FeNiMOF@AgNPs surface via Ag-S bonds, the Zeta potential shifts significantly negatively to -21.1 mV, confirming the successful ssDNA ligation, as each phosphate group in the DNA backbone carries a high density of negative charge, thus significantly enhancing the negative charge of the entire composite surface.

[0109] The above results indicate that the FeNi MOF@AgNPs-MBs substrate chain was successfully prepared by CRISPR cleaving.

[0110] Example 7

[0111] A synergistic detection system was constructed based on the obtained nucleic acid aptamers, the constructed CRISPR cleavage substrate chain FeNi MOF@AgNPs-MBs, and the CRISPR / Cas12a system. The CrRNA sequence is (SEQ ID NO.2): 5'-UAA UUU CUA CUA AGU GUA GAU G CAU CUG UAU UCA UCU GAC CUC-3'. Different concentrations of KIM-1 (0, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0 ng / mL) were mixed with an equal volume (10 μL) of 10.0 nM aptamer and incubated at 37 °C for 30 min. Then, 20 μL of a mixture of Cas12a / crRNA (Cas12a / crRNA refers to a mixture of Cas12a and crRNA at equal concentrations of 50 nM) that had been incubated at room temperature for 10 min was added, and the mixture was incubated at 37 °C for 30 min. Finally, 20 μL of the cleavage substrate FeNi MOF@AgNPs-MBs was added, and the mixture was incubated at 37 °C for 30 min. After magnetic separation, the supernatant was collected for colorimetric and fluorescence detection.

[0112] (1) Take 12.5 μL of supernatant, add 12.5 μL of 0.2 M H2O2, 12.5 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB), and 462.5 μL of sodium acetate solution (0.1 M, pH 4.0), mix and incubate at 50 ℃ for 15 min, then perform colorimetric detection. The detection wavelength range is 550-750 nm, and the absorbance value is read at 652 nm. Plot the logarithm of KIM-1 concentration on the x-axis and the difference between the colorimetric reading of the KIM-1 standard solution and the blank value on the y-axis for linear fitting. The linear equation of the colorimetric mode, i.e., the standard curve, is Y = 0.144X + 0.155 (R² + π / 4)². 2 =0.994), each experiment was performed in triplicate, the linear range was 0.1 ng / mL-100 ng / mL, and the detection limit was 58.7 pg / mL.

[0113] (2) Take 50 μL of supernatant and add 20 μL of 15 mM NaOH solution for fluorescence detection. The excitation wavelength is 332 nm, the emission wavelength range is 350-600 nm, and the fluorescence intensity is read at 425 nm. A linear fit is performed with the logarithm of KIM-1 concentration as the x-axis and the difference between the fluorescence reading of the KIM-1 standard solution and the blank value as the y-axis to obtain the linear equation of the colorimetric mode, i.e., the standard curve: Y = 159536.7X + 193541.0 (R² + π / 4)². 2=0.995), each experiment was performed in triplicate, the linear range was 0.1 ng / mL-100 ng / mL, and the detection limit was 33.4 pg / mL.

[0114] Selectivity assays were performed using the established detection method: Equal volumes (10 μL, 50 ng / mL) of different proteins (HSA, UMOD, NGAL, TIMP-2, IGFBP-7) were mixed with 10.0 nM nucleic acid aptamers and incubated at 37 °C for 30 min; Cas12a / crRNA (20 μL, 50 nM) mixture, which had been mixed and incubated at room temperature for 10 min, was added and incubated at 37 °C for 30 min; 20 μL of cleavage substrate FeNi MOF@AgNPs-MBs was added and incubated at 37 °C for 30 min; the supernatant was collected after magnetic separation.

[0115] Colorimetric and fluorescence measurements were performed using the methods described above. The experimental results are as follows: Figure 17 As shown in the figure. Experimental results demonstrate that the constructed detection method exhibits good selectivity in both colorimetric and fluorescence detection modes.

[0116] Example 8

[0117] Ten urine samples from AKI patients were centrifuged at 4 ℃ and 2500 g for 15 min, and the supernatant was collected and frozen at -80 ℃ for later use.

[0118] The detection method established in this invention was applied to the detection of 10 actual samples from AKI patients. The detection method was the same as in Example 7.

[0119] The KIM-1 content in 10 samples was also detected using an ELISA kit purchased from Shanghai Sangon Biotech, and the average relative deviation between the two methods was calculated; the experimental results are shown in Table 1.

[0120] Table 1. Results of KIM-1 determination in actual samples using the method of this invention and the ELISA method.

[0121]

[0122] The detection method provided by this invention yields results highly consistent with the ELISA method (average relative deviations are 3.2% for fluorescence and -2.7% for UV), demonstrating good accuracy. More significantly, the method of this invention reduces the detection time from 4 hours required by ELISA to less than 2 hours, resulting in a substantial improvement in analytical efficiency.

[0123] The CRISPR / Cas12a-nanozyme bimodal aptamer sensor constructed in this invention is the first to combine CRISPR / Cas12a, nanozyme, and aptamer to achieve the integration of "molecular recognition + signal amplification + bimodal output". Existing methods are mostly based on a single material, such as linear scanning voltammetry using carbon paste electrodes modified with hydroxyapatite (HAp) extracted from fish scales (see Zhang, Y., Zhang, W., Zhang, Q., Li, K., Liu, W., Liu, Y., & Banks, CE(2014). Analyst, 139(21), 5362-5366.) or a single detection mode, such as upconversion nanoparticle-enhanced luminescent lateral chromatography (see Arai, MS, Kim, H., Pascavis, M., Cha, B., Brambilla, G., Cho, YK, Lee, H. (2024). ACS Applied Materials & Interfaces, 16(29), 38243-38251). Furthermore, none of these methods have self-verification capabilities. This invention, however, achieves self-verification of results through dual-modal signal colorimetry + fluorescence, reducing the risk of false positives or false negatives. Compared with the prior art, the method of the present invention not only has high detection sensitivity, but also has a unique dual-signal output mode, which realizes internal self-verification of detection results and significantly improves the reliability and accuracy of the method.

Claims

1. The application of a KIM-1 nucleic acid aptamer in the preparation of a KIM-1 detection kit, characterized in that, The nucleotide sequence of its nucleic acid aptamer is shown in SEQ ID NO.

1. The detection kit also includes Cas12a / crRNA and FeNiMOF@AgNPs-MBs.

2. The application according to claim 1, characterized in that, The method for detecting KIM-1 includes the following steps: S1. Mix the test solution with the nucleic acid aptamer and incubate at 37°C; S2. Add the Cas12a / crRNA mixture that has been mixed and incubated at room temperature; the sequence of the crRNA is shown in SEQ ID NO.2; S3. The mixture was incubated for a period of time at 37°C for the first time; the cleavage substrate FeNi MOF@AgNPs-MBs was added, and the mixture was incubated for a second time at 37°C; the supernatant was collected after magnetic separation; wherein, FeNi MOF@AgNPs-MBs contains ssDNA modified with -SH-(CH2)6 at the 5' end and biotin modified at the 3' end, and the ssDNA sequence is shown in SEQ ID NO.3; S4. Add H2O2, 3,3',5,5'-tetramethylbenzidine and sodium acetate solution to the supernatant, mix and incubate at 50 °C for 15 min, and then perform ultraviolet detection. Alternatively, NaOH solution can be added to the supernatant for fluorescence detection; S5. Mix KIM-1 standard solutions of different concentrations with nucleic acid aptamers and perform steps S1-S4 respectively. At the same time, perform the above experiment with blank solution to obtain blank value. Plot the logarithm of KIM-1 concentration as the abscissa and the difference between the colorimetric or fluorescence reading of KIM-1 standard solution and the blank value as the ordinate to perform linear fitting and obtain the linear equation of colorimetric mode or fluorescence mode, i.e., standard curve. S6. Subtract the blank value from the fluorescence reading or colorimetric value of the test solution obtained in step S4 and substitute it into the corresponding standard curve to obtain the concentration of KIM-1 to be tested.

3. The application according to claim 2, characterized in that, In step S1, the mixing and incubation time is 10-50 min; in step S2, the mixing and incubation time is 10 min; in step S3, the first mixing and incubation time is 10-50 min; and the second mixing and incubation time is 10-50 min.

4. The application according to claim 2, characterized in that, In step S1, the concentration of the nucleic acid aptamer is 10 nM, and the volume ratio of the aptamer to the test solution is 1:1; in step S2, the concentration of Cas12a and CrRNA is 50 nM, and the volume ratio of the aptamer to the test solution is 1:1; in step S3, the volume ratio of FeNi MOF@AgNPs-MBs to the aptamer to the test solution is 1:1; in step S4, the volume ratio of the supernatant, H2O2, 3,3',5,5'-tetramethylbenzidine, and sodium acetate solution is 1:1:1:37; the concentration of NaOH is 15 mM, and the volume ratio of NaOH to the supernatant is 2:

5. In step S4, the maximum absorption wavelength for colorimetric detection is 652 nm; the excitation wavelength for fluorescence detection is 332 nm, and the emission wavelength is 425 nm.

5. A KIM-1 detection kit, characterized in that, It includes nucleic acid aptamers, Cas12a / crRNA, and FeNiMOF@AgNPs-MBs; the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.

1.

6. The detection kit according to claim 5, characterized in that, The nucleotide sequence of the crRNA is shown in SEQ ID NO.

2. The FeNi MOF@AgNPs-MBs contain ssDNA with 5' end modified with -SH-(CH2)6 and 3' end modified with biotin. The ssDNA sequence is shown in SEQ ID NO.

3.

7. The detection kit according to claim 5, characterized in that, The preparation method of the FeNi MOF@AgNPs-MBs includes the following steps: (1) First, FeNi MOF was synthesized. FeCl3·6H2O, NiCl2·6H2O and 2-aminoterephthalic acid were dissolved in N,N-dimethylformamide. 2 mL of 0.4 M NaOH solution was added dropwise, stirred and mixed, and reacted at 80~120℃ to obtain FeNiMOF. (2) FeNi MOF and AgNO3 were dissolved in methanol for the first stirring reaction. After centrifugation and washing, the mixture was resuspended in water and then NaBH4 was added. The second stirring reaction was carried out under light-protected conditions to synthesize FeNi MOF@AgNPs. (3) After incubating ssDNA with -SH-(CH2)6 at the 5' end and biotin at the 3' end with TCEP, FeNiMOF@AgNPs were added and the mixture was shaken to obtain FeNi MOF@AgNPs-ssDNA. (4) FeNi MOF@AgNPs-ssDNA was obtained by mixing FeNi MOF@AgNPs-ssDNA with streptavidin-coated magnetic beads and reacting with oscillation.

8. The detection kit according to claim 7, characterized in that, The reaction time in step (1) is 6 to 12 hours; In step (2), the first stirring time is 1-5 hours; the second stirring time is 10-50 minutes. In step (3), the incubation time is 0.5 to 2 hours; the mixing and shaking time is 10 to 24 hours. The mixing and oscillation time in step (4) is 0.5 to 2 hours.

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