A method for detecting LDL based on CRISPR-Cas12a and NrGO-Hemin-Mn3O4 NPs

Abstract

A method for detecting LDL based on CRISPR-Cas12a and NrGO-Hemin-Mn3O4NPs electrochemical aptamer sensor. Au NPs are modified on the surface of SPE by electrodeposition technology, and ssDNA / NrGO-Hemin-Mn3O4NPs probe is loaded on the surface of Au NPs / SPE through Au-SH bond. The specific recognition of LDL aptamer to LDL makes the activation chain cDNA separate and be recognized by Cas12a-crRNA to form a ternary complex with cutting ability. Through the trans cleavage of the ternary complex to its substrate ssDNA, the probe is separated from the electrode surface, and the electrical signal changes. The DPV of the electrochemical workstation is used for scanning, and the current response peak is recorded to draw the working curve, realizing the high sensitive detection of LDL with the minimum detection limit of 0.057 nM.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a method for detecting LDL based on the CRISPR-Cas12a system and nanocomposite materials. Background Technology

[0002] Low-density lipoprotein (LDL) is a lipoprotein particle that carries cholesterol into peripheral tissue cells and can serve as an important biomarker for atherosclerotic cardiovascular disease (ASCVD). Commonly used LDL detection techniques include ultracentrifugation, enzyme-linked immunosorbent assay (ELISA), Friedewald formula calculation, and homogeneous assay—surfactant scavenging method. Patent publication CN 112697565A discloses a method for detecting serum LDL using gel electrophoresis, achieving accurate separation and quantitative analysis of serum LDL through four steps: sample collection, staining, gel tube preparation, and electrophoresis. Patent publication CN 115754312A discloses an LDL detection kit that uses latex of different particle sizes conjugated with monoclonal or polyclonal antibodies to detect LDL. These methods are costly and complex, therefore, there is a need to establish a rapid, sensitive, and easy-to-use LDL detection method. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a self-signal amplification function of trans-cleavage of single-stranded ssDNA by a system composed of nitrogen-doped reduced graphene oxide-heme-manganese tetroxide nanocomposite material (NrGO-Hemin-Mn3O4NPs), combined with aptamers and clustered regularly spaced short palindromic repeat sequences and their associated proteins (CRISPR-Cas12a), to create a highly sensitive electrochemical sensor for detecting LDL based on aptamer-mediated CRISPR-Cas12a. The lowest detection limit of this method is 0.057 nM.

[0004] The detection principle of this invention is as follows: Using the LDL aptamer (LDLapt) as both the specific recognition element and the co-triggered element, a complementary strand of LDLapt (cDNA) and a partially complementary CRISPR promoter sequence (crRNA) are designed and synthesized. After incubation with Cas12a protein, a Cas12a-crRNA binary complex is obtained. LDLapt and cDNA partially pair complementaryly to form a double-stranded LDLapt-cDNA. In the presence of LDL, the higher affinity between LDL and LDLapt causes LDLapt to preferentially bind to LDL, releasing cDNA. When the Cas12a-crRNA binary complex specifically recognizes cDNA, a Cas12a-crRNA-cDNA ternary complex is formed, activating the trans-cleavage activity of Cas12a on the substrate ssDNA, thereby constructing a CRISPR-Cas12a signal amplification system capable of cleaving ssDNA. NrGO-Hemin-Mn3O4 NPs were used as electroactive materials, combined with thiolized ssDNA (ssDNA-SH), to form an ssDNA / NrGO-Hemin-Mn3O4 NPs signal probe through π-π adsorption. Gold nanoparticles (Au NPs) were deposited on the surface of a screen-printed electrode (SPE) using electrodeposition technology, and the ssDNA / NrGO-Hemin-Mn3O4 NPs signal probe was immobilized on the electrode surface through Au-SH bonds, forming an electrochemical sensing interface. An activated CRISPR-Cas12a system was introduced into the electrochemical sensing interface. Due to the trans-cleavage capability of the CRISPR-Cas12a system for the substrate ssDNA, some signal probes detached from the electrode surface, resulting in a weakened electrical signal. The current response of the LDL sensing interface was detected using differential pulse voltammetry (DPV). The current value was negatively correlated with the concentration of LDL, thus enabling the detection of LDL.

[0005] This invention is carried out according to the following steps:

[0006] Step 1: Construction of the CRISPR-Cas12a signal amplification system

[0007] (1) Synthesis of Cas12a-crRNA binary complex: crRNA was annealed at high temperature and then mixed with Cas12a. After incubation in a water bath, the Cas12a-crRNA binary complex was obtained.

[0008] (2) Synthesis of LDLapt-cDNA probe: cDNA and LDLapt were mixed and incubated in a water bath. After annealing, the mixture was centrifuged and the unbound cDNA was discarded to obtain the LDLapt-cDNA probe.

[0009] (3) Synthesis of Cas12a-crRNA-cDNA ternary complex: LDL was added to the LDLapt-cDNA probe. The specific binding between LDL and LDLapt caused the cDNA to detach. The supernatant containing cDNA was collected by centrifugation and added to the Cas12a-crRNA binary complex. After mixing and incubation, the Cas12a-crRNA-cDNA ternary complex with cleavage ability was obtained.

[0010] Step 2: Preparation of ssDNA / NrGO-Hemin-Mn3O4NPs signal probe

[0011] (1) Preparation of nitrogen-doped reduced graphene oxide (NrGO): Graphene oxide (GO) was added to pure water and ultrasonically crushed to obtain a GO solution. Urea was added to the GO solution, and after stirring, it was transferred to a reaction vessel and subjected to hydrothermal reaction under high temperature and high pressure to obtain a NrGO solution.

[0012] (2) Preparation of nitrogen-doped reduced graphene oxide-hemin (NrGO-Hemin): Hemin was dissolved in ammonia and then added to NrGO solution. After mixing, the solution was stirred in a water bath to obtain NrGO-Hemin solution.

[0013] (3) Preparation of manganese tetroxide (Mn3O4): Manganese chloride tetrahydrate (MnCl2·4H2O) and polyvinylpyrrolidone (PVP) were mixed and stirred in a water bath, and then NaOH solution was added. After stirring in a water bath, Mn3O4 solution was obtained.

[0014] (4) Preparation of nitrogen-doped reduced graphene oxide-heme chloride-manganese tetroxide (NrGO-Hemin-Mn3O4NPs): Mn3O4 solution and NrGO-Hemin solution were mixed, PVP was added and stirred, and the supernatant was removed by centrifugation to obtain NrGO-Hemin-Mn3O4NPs solution.

[0015] (5) Preparation of ssDNA / NrGO-Hemin-Mn3O4 NPs signal probe: ssDNA and NrGO-Hemin-Mn3O4 NPs solution were ultrasonically mixed and incubated and then centrifuged to obtain ssDNA / NrGO-Hemin-Mn3O4 NPs signal probe.

[0016] Step 3: Electrode modification and sensing interface construction

[0017] (1) Preparation of Au NPs / SPE: The screen-printed electrode (SPE) was activated by cyclic voltammetry (CV) in a dilute H2SO4 solution; the activated SPE was placed in a chloroauric acid (HAuCl4) solution and constant potential deposition was performed using it to obtain the AuNPs / SPE electrode.

[0018] (2) Preparation of ssDNA / NrGO-Hemin-Mn3O4 NPs / Au NPs / SPE sensing interface: ssDNA / NrGO-Hemin-Mn3O4 NPs solution was added dropwise and incubated on the AuNPs / SPE surface. The active sites were then blocked with bovine serum albumin (BSA) solution to obtain the ssDNA / NrGO-Hemin-Mn3O4NPs / AuNPs / SPE electrochemical sensing interface.

[0019] Step 4: Plotting the LDL working curve

[0020] (1) The Cas12a-crRNA-cDNA ternary complex obtained in step 1 was added to the ssDNA / NrGO-Hemin-Mn3O4 NPs / AuNPs / SPE electrochemical sensing interface obtained in step 3. After incubation, the electrode surface was rinsed with phosphate buffer solution (PBS) to obtain the working electrode. The obtained working electrode was placed in PBS and scanned using DPV technology of an electrochemical workstation. At the same time, a blank group was set up and the response current value of the sensor was recorded.

[0021] (2) Detect LDL at different concentrations, record the peak current, plot the LDL working curve based on the relationship between the peak response current and LDL concentration at different concentrations, and calculate the minimum detection limit of this method.

[0022] Step 5: Detection of LDL in actual serum samples

[0023] (1) Add the actual serum sample to be tested to the LDLapt-cDNA probe, centrifuge and take the supernatant containing cDNA, add it to the Cas12a-crRNA binary complex to obtain the Cas12a-crRNA-cDNA ternary complex containing the serum sample.

[0024] (2) The above ternary complex was dropped onto the ssDNA / NrGO-Hemin-Mn3O4 NPs / Au NPs / SPE electrochemical sensing interface in step 3, incubated for a period of time, rinsed and then immersed in PBS. The DPV technology of the electrochemical workstation was used for scanning, and a blank group was set up to record the current response peak.

[0025] (3) Based on the working curve obtained in step 4, the concentration of LDL in the actual serum sample to be tested is calculated.

[0026] Furthermore, in step 1, the concentrations of Cas12a, crRNA, LDLapt, and cDNA are all 5.0 μM;

[0027] Furthermore, the annealing temperature in step 1 is 95°C, and the annealing time is 10 hours.

[0028] Furthermore, in step 2, the hydrothermal method for preparing NrGO has a reaction temperature of 180℃ and a reaction time of 12h.

[0029] Furthermore, the concentration of ssDNA used in step 2 is 10.0 μM;

[0030] Furthermore, in step 3, the concentration of the dilute H2SO4 solution is 0.5 mol / L, and the CV scan voltage is -1.2V to 1.2V;

[0031] Furthermore, in step 3, the concentration of the HAuCl4 solution is 0.01%, the constant voltage is -0.5V, and the scanning time is 2min;

[0032] Furthermore, in step 3, the amount of ssDNA / NrGO-Hemin-Mn3O4 NPs and Cas12a-crRNA-cDNA dropped onto the electrode surface is 2.5 μL each;

[0033] Furthermore, in steps 4 and 5, the DPV scan has a voltage range of 0.2V to 1.0V.

[0034] As a preferred option:

[0035] In steps 4 and 5, the CRISPR-Cas12a system was cut at an electrode surface at a temperature of 40°C for 40 min; the Cas12a-crRNA concentration was 6.0 μM; the PBS buffer pH was 6.0; and the ssDNA / NrGO-Hemin-Mn3O4NPs probe concentration was 1.0 mg / mL. The sensor response current showed the largest difference from the blank control response current.

[0036] Step 1 involves constructing a signal amplification system based on the CRISPR-Cas12a system, capable of cleaving its substrate ssDNA. Step 2 synthesizes NrGO-Hemin-Mn3O4 NPs with high electroactivity and a large specific surface area, which bind to ssDNA via π-π binding to form an ssDNA / NrGO-Hemin-Mn3O4 NPs nano-signal probe. Step 3 forms an ssDNA / NrGO-Hemin-Mn3O4 NPs / AuNPs / SPE sensing interface. The construction of this biosensing interface in step 3 is a crucial step for LDL detection in steps 4 and 5. The LDL working curve from step 4 provides the calculation basis for determining the LDL concentration in actual serum samples in step 5. It is evident that steps 1-5 are mutually supportive and work together to enable the detection of LDL using an electrochemical aptamer sensor based on the CRISPR-Cas12a system and NrGO-Hemin-Mn3O4 NPs.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] 1. The CRISPR-Cas12a signal amplification system constructed in this patent utilizes its ability to cleave substrate ssDNA to amplify the signal and improve the sensitivity of the sensor in detecting LDL.

[0039] 2. The synthesized NrGO-Hemin-Mn3O4NPs nanocomposite material possesses a unique morphology, large specific surface area, high electroactivity, and excellent electron transfer rate. Using LDLapt as the specific recognition element and co-triggering element for sensing improves the sensor's specificity in detecting LDL.

[0040] 3. An ssDNA / NrGO-Hemin-Mn3O4 NPs signal probe was immobilized on the surface of an SPE. Activated CRISPR-Cas12a was introduced into the electrochemical system. The cleavage of the substrate ssDNA by CRISPR-Cas12a caused the signal probe to detach from the electrode surface. Sensitive detection of LDL was achieved by detecting the change in the redox current of Hemin in the signal probe. The detection limit of this method was 0.057 nM. Attached Figure Description

[0041] Figure 1 Schematic diagram of an electrochemical aptamer sensor based on CRISPR-Cas12a and NrGO-Hemin-Mn3O4NPs for detecting LDL.

[0042] Figure 2 Scanning electron microscope (SEM) image of NrGO-Hemin-Mn3O4 NPs;

[0043] Figure 3 X-ray diffraction (XRD) spectrum of NrGO-Hemin-Mn3O4 NPs;

[0044] Figure 4 DPV curves of LDL at different concentrations were detected using an electrochemical aptamer sensor based on the CRISPR-Cas12a system and NrGO-Hemin-Mn3O4NPs. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0046] A method for detecting LDL based on a CRISPR-Cas12a system and an electrochemical aptamer sensor using NrGO-Hemin-Mn3O4 NPs is described below. Figure 1 First, the SPE was activated, and then Au NPs were electrodeposited onto the SPE to enhance conductivity. Second, the prepared ssDNA / NrGO-Hemin-Mn3O4 NPs signal probe was immobilized on Au NPs / SPE, forming an ssDNA / NrGO-Hemin-Mn3O4 NPs / AuNPs / SPE electrochemical sensing interface. NrGO exhibits excellent biocompatibility, and Hemin is an electroactive substance that can serve as an electronic medium to provide signals for the sensor. The addition of Hemin also prevents NrGO aggregation; the addition of Mn3O4 nanoparticles enhances the conductivity of the nanocomposite material. Third, the specific binding of LDL to LDLapt causes cDNA to detach from the cDNA-LDLapt. The detached cDNA can activate the CRISPR-Cas12a system, achieving trans-cleavage of the substrate ssDNA, resulting in the partial detachment of the NrGO-Hemin-Mn3O4 NPs nanomaterial from the electrode surface, thus weakening the electrical signal. By comparing the differences between current signals, quantitative analysis of LDL can be achieved.

[0047] The specific implementation steps are as follows:

[0048] Step 1: Construction of the CRISPR-Cas12a signal amplification system

[0049] (1) 40.0 μL of 5.0 μM crRNA was annealed in a water bath at 95 °C for 10 minutes and then annealed for 10 h. The resulting crRNA was then mixed with an equal volume of Cas12a and incubated in a water bath at 37 °C for 60 minutes to obtain the Cas12a-crRNA binary complex.

[0050] (2) Mix 80.0 μL of 5.0 μM cDNA with LDLapt and incubate in a water bath at 95 °C for 10 minutes, anneal for 10 h, and then centrifuge at 12000 rpm to discard the unbound cDNA and obtain the LDLapt-cDNA probe.

[0051] (3) Add 40.0 μL of LDL at different concentrations to 40.0 μL of 5.0 μM LDLapt-cDNA probe. The specific binding between LDL and LDLapt causes cDNA to detach. After centrifugation at 12000 rpm, take the supernatant containing cDNA and add it to the Cas12a-crRNA binary complex. After mixing, incubate at room temperature for 1 h to obtain the Cas12a-crRNA-cDNA ternary complex with the ability to cleave ssDNA.

[0052] Step 2: Preparation of ssDNA / NrGO-Hemin-Mn3O4 NPs signal probe

[0053] (1) Weigh 30.0 mg of GO and transfer it to a beaker containing 30.0 mL of ultrapure water. Sonicate the solution for 2 hours to obtain a GO solution with a concentration of 1.0 mg / mL. Add 900.0 mg of urea to the solution and stir at room temperature for 30 minutes. Then transfer the solution to a high-pressure reactor made of polytetrafluoroethylene and hydrothermally react at 180°C for 12 hours. Centrifuge and wash to remove excess free urea to obtain NrGO solution.

[0054] (2) Weigh 30.0 mg of Hemin, add 60.0 μL of NH3·H2O, then add 30.0 mL of ultrapure water, and stir to obtain a 1.0 mg / mL Hemin solution. Mix the Hemin solution and NrGO solution in a 1:1 ratio, then add 10.0 μL of hydrazine hydrate solution, vortex for 10 min, and then place the mixed solution in a water bath at 60 °C for 4 h. After centrifugation and washing, the NrGO-Hemin solution can be obtained.

[0055] (3) Add 445.0 mg MnCl2·4H2O and 500.0 mg PVP to 55.0 mL of ultrapure water, stir at room temperature, and heat to 90 °C. Add 200.0 mg NaOH solid to 5.0 mL of ultrapure water, stir to dissolve, pour into MnCl2·4H2O, and stir at 90 °C for 4 h. Centrifuge and wash the resulting brownish-red solution three times to obtain Mn3O4 solution. Add 10.0 mL of Mn3O4 solution to 10.0 mL of NrGO-Hemin solution, add 10.0 mg PVP, stir for 16 h, centrifuge, wash, and dry to obtain NrGO-Hemin-Mn3O4NPs.

[0056] NrGO-Hemin-Mn3O4NPs were characterized using a Hitachi SU8020 scanning electron microscope (SEM). Figure 2 As shown. Figure 2 Image A is a SEM scan of NrGO, showing the wrinkled membrane structure. Figure 2 B is a SEM scan of NrGO-Hemin, which shows that the wrinkles on the material surface are deepened, which is due to the presence of Hemin. Figure 2 C is the SEM scan image of NrGO-Hemin-Mn3O4NPs, showing small particles attached to the surface of the wrinkled film, indicating that Mn3O4 particles were successfully attached to the NrGO-Hemin material.

[0057] X-ray diffraction was performed on different stages of the preparation process of NrGO-Hemin-Mn3O4NPs, such as... Figure 3 As shown in the figure. From curve a, it can be seen that the X-ray diffraction peaks of Mn3O4 are located at 2θ = 18.00°, 28.88°, 32.31°, 36.08°, 44.44°, 58.51°, 59.84° and 65.39°, which belong to the (101), (112), (103), (211), (220), (321), (224) and (323) reflection crystal planes, which correspond to the tetragonal spinel structure of Mn3O4. The characteristic diffraction peak at 2θ = 20.00° is the diffraction peak of carbon element in the non-metallic material NrGO-Hemin (curve b); while the characteristic diffraction peak at 2θ = 20.00° in curve c is present in the diffraction peak planes of crystals (101), (112), (103), (211), (220), (321), (224), and (323), indicating that NrGO-Hemin-Mn3O4NPs were successfully prepared.

[0058] (4) Mix 50.0 μL of 10.0 μM ssDNA-SH and 50.0 μL of 1.0 mg / mL NrGO-Hemin-Mn3O4NPs by sonication, incubate at room temperature for 10 h, centrifuge at 12000 rpm for 20 min, discard unbound ssDNA, and obtain ssDNA / NrGO-Hemin-Mn3O4NPs signal probe.

[0059] Step 3: Electrode modification and construction of biosensing interface

[0060] (1) The bare SPE was activated in 0.5 mol / L dilute sulfuric acid solution and CV scan was performed at -1.2 V to 1.2 V for 20 cycles at 0.5 V / s. After activation, the SPE was placed in a small beaker containing 0.01% HAuCl4 solution and placed on a magnetic stirrer. The SPE was scanned at a constant voltage of -0.5 V for 2 min using it scan. The SPE was washed three times with ultrapure water and dried to obtain Au NPs / SPE.

[0061] (2) Take 2.5 μL of ssDNA / NrGO-Hemin-Mn3O4NPs solution with a concentration of 1.0 mg / mL and drop it onto the surface of SPE / AuNPs electrode. Incubate at a constant temperature of 25℃ for 30 minutes, wash and dry, then block the active site with 4.0 μL of 0.5% BSA and incubate for 30 minutes to obtain the working electrode ssDNA / NrGO-Hemin-Mn3O4NPs / Au NPs / SPE.

[0062] Step 4: Plotting the LDL working curve

[0063] (1) Equal volumes of 0.005 nM to 1000.0 nM LDL standard solutions were added to 5.0 μM LDLapt-cDNA solutions, incubated at 37 °C for 30 min, and the supernatant was collected by centrifugation to obtain cDNA of different concentrations. The cDNA solutions of different concentrations were added to the Cas12a-crRNA binary complex and incubated for 1 h to form a Cas12a-crRNA-cDNA ternary complex with trans-cleavage capability. 2.5 μL of the Cas12a-crRNA-cDNA ternary complex obtained from different concentrations of cDNA was dropped onto the sensing interface of ssDNA / NrGO-Hemin-Mn3O4NPs / AuNPs / SPE, and incubated at 40 °C for 40 min to obtain the working electrode. A Cas12a-crRNA binary complex without cDNA was set as a blank group.

[0064] (2) The working electrode obtained above was placed in a 0.2 mol / L PBS solution with a pH of 6.0. The current peak value was recorded by DPV scanning using a CHI660E electrochemical workstation. The DPV curves for different LDL concentrations are shown in the figure. Figure 4 The LDL concentration ranges from 0.005 nM to 1000.0 nM. The difference between the peak response current at this concentration and the peak response current of the LDL blank group (Y) exhibits two linear relationships with the LDL concentration (X). Within the range of 0.1 nM to 1000.0 nM, a logarithmic linear relationship is observed. The working curve is Y = 0.6659lgX + 1.5886, with a correlation coefficient R0. 2=0.9942. A linear relationship is observed within the range of 0.005 nM to 0.1 nM, with the working curve being Y = 7.9209X + 0.1535 and a correlation coefficient R0. 2 = 0.9915. According to formula C LOD =3Sb / b (Sb represents the standard deviation of the six LDL blank groups, and b represents the slope of the standard curve), the minimum detection limit is calculated to be 0.057nM.

[0065] Step 5: Detection of LDL in actual serum samples

[0066] (1) Three types of serum samples were selected: samples 1-3 were from healthy individuals, samples 4-6 were from patients with moderate ASCVD, and samples 7-9 were from patients with severe ASCVD. The concentration of LDL in each serum sample was determined using a homogeneous assay method commonly used in clinical practice—the surfactant scavenger method. Each serum sample had two measurements: one from the clinical method and the other obtained by the method.

[0067] (2) A human serum sample with a known LDL concentration was diluted tenfold and added to the LDLapt-cDNA probe solution at a 1:1 ratio as described in step 1. After centrifugation, an equal volume of the supernatant containing cDNA was added to a 6.0 μM Cas12a-crRNA binary complex to obtain Cas12a-crRNA-cDNA. 2.5 μL of the Cas12a-crRNA-cDNA solution was added to the biosensor interface obtained in step 3 and incubated at 40°C for 40 min to achieve trans-cleavage of ssDNA. Following step 4, the working electrode after cleavage was placed in 0.2 mol / L PBS buffer solution (pH = 6.0), and DPV scanning was performed, recording the current peak value.

[0068] (3) The concentration of LDL in actual serum samples was calculated using the LDL working curve obtained in step 4. The results are shown in Table 1. The relative error was in the range of 0.868% to 5.649%, and the RSD value was 1.906% to 5.235%. The results show that the electrochemical aptamer sensor can be used to detect LDL concentration in actual serum samples.

[0069] Table 1. Detection results of LDL in actual serum samples

[0070]

Claims

1. A method for detecting LDL using an electrochemical aptamer sensor based on CRISPR-Cas12a and NrGO-Hemin-Mn3O4NPs, comprising the following steps: Step 1: Construction of the CRISPR-Cas12a signal amplification system (1) Synthesis of Cas12a-crRNA binary complex: crRNA was mixed with Cas12a and incubated in a water bath to obtain Cas12a-crRNA binary complex. (2) Synthesis of LDLapt-cDNA probe: cDNA and LDLapt were mixed and incubated in a water bath, annealed, centrifuged, and the unbound cDNA was discarded to obtain LDLapt-cDNA probe. (3) Synthesis of Cas12a-crRNA-cDNA ternary complex: LDL was added to the LDLapt-cDNA probe. The specific binding between LDL and LDLapt caused the cDNA to detach. The supernatant containing cDNA was collected by centrifugation and added to the Cas12a-crRNA binary complex. After mixing and incubation, the Cas12a-crRNA-cDNA ternary complex with cleavage ability was obtained. Step 2: Preparation of ssDNA / NrGO-Hemin-Mn3O4NPs signal probe (1) Preparation of nitrogen-doped reduced graphene oxide (NrGO): Graphene oxide (GO) was added to pure water and ultrasonically crushed to obtain a GO solution. Urea was added to the GO solution, and after stirring, it was transferred to a reaction vessel and subjected to hydrothermal reaction under high temperature and high pressure to obtain a NrGO solution. (2) Preparation of nitrogen-doped reduced graphene oxide-heme chloride NrGO-Hemin: Hemin was dissolved in ammonia water and then added to NrGO solution. After mixing, the mixture was stirred in a water bath to obtain NrGO-Hemin solution. (3) Preparation of manganese tetroxide Mn3O4: Manganese chloride tetrahydrate MnCl2·4H2O and polyvinylpyrrolidone PVP were mixed and stirred in a water bath, and then NaOH solution was added. After stirring in a water bath, Mn3O4 solution was obtained. (4) Preparation of nitrogen-doped reduced graphene oxide-heme chloride-manganese tetroxide nanocomposite NrGO-Hemin-Mn3O4NPs: Mn3O4 solution and NrGO-Hemin solution were mixed, PVP was added and stirred, and the supernatant was removed by centrifugation to obtain NrGO-Hemin-Mn3O4NPs. (5) Preparation of ssDNA / NrGO-Hemin-Mn3O4NPs signal probe: ssDNA and NrGO-Hemin-Mn3O4NPs solution were ultrasonically mixed evenly, incubated and then centrifuged to obtain ssDNA / NrGO-Hemin-Mn3O4NPs signal probe. Step 3: Electrode modification and sensing interface construction (1) Preparation of Au NPs / SPE: The screen-printed electrode SPE was placed in a 0.5 mol / L H2SO4 solution and activated by cyclic voltammetry; the activated SPE was placed in a solution containing 0.01% chloroauric acid HAuCl4 and deposited at a constant potential of -0.5 V for 2 min using it to obtain the Au NPs / SPE electrode; (2) Preparation of ssDNA / NrGO-Hemin-Mn3O4NPs / Au NPs / SPE sensing interface: ssDNA / NrGO-Hemin-Mn3O4NPs solution was added dropwise and incubated on Au NPs / SPE, and then the active site was blocked with bovine serum albumin (BSA) solution to obtain the ssDNA / NrGO-Hemin-Mn3O4NPs / Au NPs / SPE electrochemical sensing interface; Step 4: Plotting the LDL working curve (1) The Cas12a-crRNA-cDNA ternary complex obtained in step 1 was added to the ssDNA / NrGO-Hemin-Mn3O4NPs / Au NPs / SPE electrochemical sensing interface in step 3. After incubation, the working electrode was obtained by rinsing. The working electrode was placed in phosphate buffer solution PBS and scanned using DPV technology of an electrochemical workstation to record the response current value of the sensor. At the same time, a blank group was set up to record the current response peak value. (2) Detect LDL at different concentrations, record the peak current, and plot the LDL working curve based on the difference between the peak response current at that concentration and the peak response current of the LDL blank group and the LDL concentration. Step 5: Detection of LDL in actual serum samples (1) Add the actual serum sample to be tested to the LDLapt-cDNA probe, centrifuge and take the supernatant containing cDNA, add it to the Cas12a-crRNA binary complex to obtain the Cas12a-crRNA-cDNA ternary complex containing the serum sample. The above ternary complex was dropped onto the ssDNA / NrGO-Hemin-Mn3O4NPs / Au NPs / SPE electrochemical sensing interface in step 3, incubated, rinsed, and then immersed in PBS. The DPV technology of the electrochemical workstation was used for scanning, and a blank group was set up to record the current response peak. (2) Based on the working curve obtained in step 4, the concentration of LDL in the actual serum sample to be tested is calculated.

2. The method according to claim 1, characterized in that: In step 1, the concentrations of Cas12a, crRNA, LDLapt, and cDNA were all 5.0 μM.

3. The method according to claim 1, characterized in that: The annealing temperature in step 1 is 95℃, and the annealing time is 10h.

4. The method according to claim 1, characterized in that: In step 2, the hydrothermal method for preparing NrGO is carried out at a temperature of 180℃ and a reaction time of 12h.

5. The method according to claim 1, characterized in that: The concentration of ssDNA used in step 2 is 10.0 μM.

6. The method according to claim 1, characterized in that: In step 3, the amount of ssDNA / NrGO-Hemin-Mn3O4 NPs and Cas12a-crRNA-cDNA dropped onto the electrode surface was 2.5 μL each.

7. The method according to claim 1, characterized in that: In steps 4 and 5, the DPV scan is performed with a voltage range of 0.2V to 1.0V.

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