Magnetic self-assembly OPN protein detection nanoprobe as well as construction method and application thereof

Abstract

The invention provides a magnetic self-assembly OPN protein detection nanoprobe as well as a construction method and application thereof, and belongs to the technical field of biological analysis. An alpha-Fe2O3 / Fe3O4 (at) Au-Report / MCH electrode modifier and a CRISPR / Cas13a system are mixed and then act to obtain a magnetic alpha-Fe2O3 / Fe3O4 (at) Au-MCH nano-composite, and the nano-composite is assembled to the surface of MGCE by utilizing the magnetism of the nano-composite to form the magnetic self-assembly OPN protein detection nano-probe. The magnetic self-assembly OPN protein detection nanoprobe can be used as a working electrode of an electrochemical RNA aptamer sensor for detecting the content of OPN protein; the electrochemical RNA aptamer sensor has a relatively low detection limit (0.33 pg / mL), a relatively wide linear range (1 pg / mL to 1 ng / mL) and relatively high sensitivity, and has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioanalysis, and in particular relates to a magnetic self-assembled OPN protein detection nanoprobe and a construction method and application thereof. Background Art

[0002] Human osteopontin (OPN) is a glycosylated phosphoprotein that was first discovered in normal human tissues and is expressed in a variety of tissues in the body. OPN participates in normal physiological activities of the human body, not only regulating the metastasis, invasion and proliferation of tumor cells, but also playing a role in physiological processes such as inflammatory response and angiogenesis. OPN directly or indirectly activates intracellular signaling pathways by binding to receptors, mediates the interaction between cells and cells, and between cells and extracellular matrix, and thus participates in regulating cell survival. However, further studies have found that OPN is also involved in the pathological process of tumors, especially in tissues of malignant diseases such as hepatocellular carcinoma, lung cancer, colorectal cancer, and breast cancer. Its expression is highly elevated in human cancers and acts as a tumor promoter. There is already a lot of evidence that abnormal expression of OPN is closely related to tumor occurrence, progression, and especially metastasis of various tumor types. Therefore, with the increasing burden of cancer worldwide, it is necessary to strengthen the early detection of OPN.

[0003] Common tumor marker detection methods include radioimmunoassay, chemiluminescence immunoassay, and enzyme-linked immunosorbent assay. However, the above methods have the disadvantages of short service life of the kit, risk of radioactive contamination, short luminescence process, high instrument failure rate, poor reagent stability, low detection accuracy, low experimental flexibility, and low sensitivity. As an emerging biosensor, immunosensor is favored for its high specificity, sensitivity and stability in identifying substances. It integrates traditional immunoassay and biosensor technology, and combines many advantages of both. It not only reduces analysis time, improves sensitivity and test accuracy, but also makes the determination process simple and easy to automate, and has broad application prospects. Therefore, it is necessary to develop a sensor for early detection of OPN. Summary of the invention

[0004] In view of some deficiencies in the prior art, the present invention provides a magnetic self-assembled OPN protein detection nanoprobe and a construction method and application thereof; the present invention mixes an α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier and a CRISPR / Cas13a system to obtain a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite, and assembles the nanocomposite onto the surface of MGCE by its own magnetism to form a magnetic self-assembled OPN protein detection nanoprobe; the magnetic self-assembled OPN protein detection nanoprobe can be used as a working electrode of an electrochemical RNA aptamer sensor to detect the OPN protein content; the electrochemical RNA aptamer sensor has a lower detection limit (0.33pg / mL), a wider linear range (1pg / mL to 1ng / mL) and a higher sensitivity, and has good practicality.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0006] The present invention first provides a magnetic self-assembled OPN protein detection nanoprobe, which comprises: MGCE, a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite magnetically self-assembled on the surface of MGCE; the magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite is obtained by mixing an α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier and a CRISPR / Cas13a system;

[0007] The α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier includes an α-Fe2O3 / Fe3O4@Au nanocomposite material, a reporter assembled on the surface of the α-Fe2O3 / Fe3O4@Au nanocomposite material through Au-S bonds, and a blocker MCH specifically adsorbed on the surface of the material;

[0008] The CRISPR / Cas13a system is formed by mixed hybridization of Cas13a / crRNA and dsRNA / OPN, wherein Cas13a / crRNA is obtained by combining Cas13a enzyme and crRNA, dsRNA is formed by annealing single-stranded Aptamer and single-stranded Activator, and dsRNA / OPN is formed by combining dsRNA and OPN protein.

[0009] Preferably, the nucleotide sequence of the Reporter is as shown in SEQ ID No.1;

[0010] The nucleotide sequence of the crRNA is shown in SEQ ID No.2;

[0011] The nucleotide sequences of the single-chain Aptamer and the single-chain Activator are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. The present invention also provides a method for preparing the above-mentioned magnetic self-assembled OPN protein detection nanoprobe, the preparation method comprising:

[0012] (1) A quantitative reporter solution is mixed with an α-Fe2O3 / Fe3O4@Au nanocomposite to form a suspension and then incubated for reaction. After the reaction is completed, the supernatant is removed by magnetic separation to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter;

[0013] Solid α-Fe2O3 / Fe3O4@Au-Reporter was dispersed into a certain amount of MCH solution for reaction to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation again to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification.

[0014] (2) reacting the Cas13a enzyme solution and the crRNA solution in 10×Reaction Buffer (for Cas13a) to form a Cas13a / crRNA binary complex; mixing and incubating the Cas13a / crRNA binary complex with the dsRNA / OPN solution to obtain a Cas13a / crRNA / Activator ternary complex, activating the cleavage activity of the Cas13a enzyme, and obtaining the CRISPR / Cas13a system;

[0015] (3) The CRISPR / Cas13a system is mixed with a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier to perform a cleavage reaction, and magnetic separation is performed after the reaction to obtain a solid magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite;

[0016] (4) using DEPC water to resuspend the solid magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite to obtain a nanocomposite resuspension; then dropping the obtained nanocomposite resuspension onto the surface of MGCE to complete the magnetically induced self-assembly process, and drying after the self-assembly is completed to obtain the magnetic self-assembled OPN protein detection nanoprobe.

[0017] Preferably, in step (1), the preparation steps of the α-Fe2O3 / Fe3O4@Au nanocomposite include:

[0018] S1. A mixed aqueous solution of FeCl3·6H2O and NaH2PO4·2H2O is subjected to a hydrothermal reaction, and after the reaction, the mixture is centrifuged and washed to obtain α-Fe2O3 nanosheets; the α-Fe2O3 nanosheets are then mixed with glucose and calcined at high temperature, and after the calcination, the mixture is ground to obtain magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheets;

[0019] S2. preparing a polyethyleneimine (PEI) solution, and then adding a magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheet powder thereto, placing in a high temperature water bath under magnetic stirring, and after the reaction is completed, centrifuging, washing and drying to obtain an α-Fe2O3 / Fe3O4-PEI nanocomposite material;

[0020] The α-Fe2O3 / Fe3O4-PEI nanocomposite was ultrasonically dispersed in ultrapure water to form a suspension, and then the reaction environment was built into a low-temperature state. Ice HAuCl4 solution was added thereto and ultrasonically stirred. Subsequently, trisodium citrate solution and sodium borohydride solution were sequentially added thereto for reduction reaction. After mechanical stirring, the suspension was centrifuged, washed and dried to obtain a magnetic α-Fe2O3 / Fe3O4@Au nanocomposite material.

[0021] Preferably, in step S1, the aqueous solution of FeCl3 and NaH2PO4 has a FeCl3 concentration of 12mM to 38mM and a NaH2PO4 concentration of 3mM to 12mM; the hydrothermal reaction is carried out at 160°C to 240°C for 6h to 30h; the mass ratio of the α-Fe2O3 nanosheets to glucose is 1:0 to 1:16; the high temperature calcination is carried out at 400°C to 800°C for 1h to 8h;

[0022] In step S2, the mass ratio of the magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheets, PEI, HAuCl4 and trisodium citrate is (1-15):(50-350):(1-20):(10-400);

[0023] The conditions of the water bath reaction are: reacting at 90° C. for 1 h to 10 h while maintaining magnetic stirring; the conditions of the reduction reaction are: reacting at 0° C. for 15 to 40 min; and the dosage of the sodium borohydride solution is 0.075% (W / V).

[0024] Preferably, in step (1), the concentration of the α-Fe2O3 / Fe3O4@Au nanocomposite in the suspension is 5 mg / mL to 20 mg / mL;

[0025] The dosage ratio of the Reporter solution to the MCH solution is 40 μL:60 μL, wherein the concentration of the Reporter is 50 nM to 200 nM; the concentration of the MCH solution is 1 mM;

[0026] The incubation reaction conditions are: incubation at 30-37°C for 3-4h;

[0027] After adding the MCH solution, react at 25-37°C for 0.5-2h.

[0028] Preferably, the concentration of the α-Fe2O3 / Fe3O4@Au nanocomposite in the suspension is 10 mg / mL; and the concentration of the Reporter is 140 nM.

[0029] Preferably, in step (2), the dosage ratio of the Cas13a enzyme solution, the crRNA solution and the 10×Reaction Buffer (forCas13a) is 2 μL: 10 μL: 2 μL, wherein the molar concentration ratio of the Cas13a enzyme solution and the crRNA solution is 20: 1 to 1: 1;

[0030] The conditions for reacting the Cas13a enzyme solution and the crRNA solution in 10×Reaction Buffer (for Cas13a) are: reacting at 25-37° C. for 40-60 min;

[0031] The dosage ratio of the Cas13a / crRNA binary complex and the dsRNA / OPN solution is 14 μL:30 μL;

[0032] The conditions for mixed hybridization of the Cas13a / crRNA binary complex and the dsRNA / OPN solution are: incubation at 37° C. for 25 to 60 minutes.

[0033] Preferably, the molar concentration ratio of Cas13a and crRNA is 10:1;

[0034] Preferably, the preparation step of the dsRNA / OPN solution comprises: annealing a single-stranded Aptamer and a single-stranded Activator to obtain dsRNA, and then reacting the dsRNA with OPN protein to obtain a dsRNA / OPN solution after the reaction is completed;

[0035] During the preparation process:

[0036] The molar concentration and dosage of the single-chain Aptamer and the single-chain Activator are the same; the annealing conditions are: constant temperature at 95°C for 5 minutes and then cooled to room temperature; the dosage ratio of the dsRNA and OPN protein is 10μL:20μL, and the concentration of dsRNA is 5μM~20μM; the reaction conditions of the dsRNA and OPN protein are: reaction at 20~40°C for 30~90min.

[0037] Preferably, the concentration of the dsRNA is 10 μM.

[0038] Preferably, in step (3), the dosage ratio of the CRISPR / Cas13a system to the solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier is 44 μL: 0.24-0.68 mg;

[0039] The cleavage reaction time is 10 to 100 minutes.

[0040] Preferably, the dosage ratio of CRISPR / Cas13a system to solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier is 44 μL:0.4 mg;

[0041] The cleavage reaction time is 30 min.

[0042] The present invention also provides the use of the magnetic self-assembled OPN protein detection nanoprobe in preparing an electrochemical RNA aptamer sensor for detecting OPN protein.

[0043] The present invention also provides an electrochemical RNA aptamer sensor for detecting OPN protein. The electrochemical RNA aptamer sensor uses the magnetic self-assembled OPN protein detection nanoprobe as a working electrode, an Ag / AgCl electrode and a Pt electrode as a reference electrode and an auxiliary electrode, respectively, and a [Fe(CN)6] 3- / 4- The solution was used as an electrolyte, and the OPN protein content was converted into a current response through an electrochemical workstation to detect the OPN protein.

[0044] Preferably, in the electrolyte, the concentration of KCl is 0.1 M, [Fe(CN)6] 3- / 4- The concentration of the solution is 5 mM;

[0045] When the electrochemical RNA aptamer sensor is used for detection, the electrochemical impedance spectroscopy (EIS) is measured in the range of 0.1 Hz to 10 kHz with a signal amplitude of 0.1 V.

[0046] Compared with the prior art, the beneficial effects of the present invention are:

[0047] (1) The effective fixation of the signal conversion element of the electrochemical sensor on the electrode surface is a key step affecting the performance of the electrochemical RNA aptamer sensor. The present invention prepares a magnetically controllable magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheet with a large specific surface area and high surface energy. The magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheet exhibits an appropriate saturation magnetization intensity, which allows the signal nanoprobe to be self-assembled and fixed on the MGCE surface under the induction of its magnetic force. In addition, the sheet-like structure of the nanosheet can effectively reduce the resistance of the spatial site during the electron transfer process, promote the contact between the active substance and the electrolyte, and amplify the current signal. In addition, the larger specific surface area can anchor more gold nanoparticles (AuNPs), thereby promoting the fixation of the signal nanoprobe on the α-Fe2O3 / Fe3O4@Au nanocomposite material through the Au-S bond, laying a favorable foundation for the subsequent preparation of signal transmission tools. The α-Fe2O3 / Fe3O4@Au nanocomposite of the present invention coats Au nanoparticles on the surface of α-Fe2O3 / Fe3O4 while retaining its magnetic properties and still achieving magnetic self-assembly. The MGCE magnetic core used in the present invention can quickly remove the α-Fe2O3 / Fe3O4@Au, so that the electrode can be recycled and regenerated simply and quickly; it has low requirements on the required equipment, is simple to operate, and is easy to carry.

[0048] (2) The present invention fixes the α-Fe2O3 / Fe3O4@Au nanocomposite and the thiol-modified Reporter via Au-S bonds, which is quick and simple to operate, has a strong covalent bond, and does not require the use of other materials. In addition, the α-Fe2O3 / Fe3O4@Au nanocomposite can utilize the conductivity of Au nanoparticles to amplify the current signal and reduce the interference of the background signal. The MB modified at the end of the Reporter also has the function of transducing signals. In this way, the OPN protein can be better detected, with a lower detection limit (0.33pg / mL), a wider linear range (1pg / mL to 1ng / mL), and better sensitivity.

[0049] (3) The protein detection nanoprobe of the present invention introduces a Cas13a / crRNA binary complex and a dsRNA / OPN mixture in the presence of an Activator. The Cas13a protein is a multifunctional protein containing a double-stranded RNA binding domain and a nuclease domain. The nuclease domain has RNA endonuclease activity and can non-specifically cut RNA. CrRNA is a processed RNA molecule containing a sequence complementary to the target RNA and can specifically recognize the target RNA. Multiple crRNAs can be clustered to improve targeting efficiency. When the crRNA-bound Cas13a undergoes base pairing with the target RNA, the nuclease domain of Cas13a is activated to non-specifically cut the surrounding RNA molecules. This is called the "collateral cleavage" effect. Once activated, Cas13a will continue to perform RNA internal cutting until there is no RNA that can be cut around. Therefore, after being activated by the Activator, the Cas13a / crRNA binary complex will non-specifically cut the Reporter, which will cause the number of modifiers on the surface of the matrix material to change, thereby causing the signal transduced by MGCE to change, thereby quantitatively detecting the target substance. In addition, the method of directly transferring the binary complex formed by Cas13a and crRNA to the probe surface during the preparation process has the advantages of being safer, faster, having lower off-target effects, and having higher editing efficiency.

[0050] (4) The aptamer sensor prepared by the present invention does not require excessive processing of the test sample, and human serum samples can be used for detection; the detection process is fast and convenient, and the test results can be obtained in a short time. The amount of probe required for each test is extremely small and can be prepared in batches at low cost; the reaction conditions are mild, the process is easy to control, and no special training is required for operators, which has good clinical development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Transmission electron microscope images of magnetic α-Fe2O3 / Fe3O4 heterostructure nanosheets (A) and α-Fe2O3 / Fe3O4@Au nanocomposites (B).

[0052] Figure 2 CV curves of the construction of magnetic self-assembled OPN protein detection nanoprobe and each modification stage during the detection process: bare MGCE (a), MGCE / α-Fe2O3 / Fe3O4@Au (b), MGCE / α-Fe2O3 / Fe3O4@Au-Reporter / MCH (c), and MGCE / α-Fe2O3 / Fe3O4@Au-MCH (d).

[0053] Figure 3The magnetic self-assembled OPN protein detection nanoprobe was used to detect the DPV current response of nucleolin (NCL), heterogeneous nuclear ribonucleoprotein A1 (HN-PNP-A1), human papillomavirus (HPV), alpha-fetoprotein (AFP), microtubule-associated protein (Tau), target protein (OPN) and their mixed protein group (Mix).

[0054] Figure 4 DPV curve (A) and linear relationship diagram (B) of the electrochemical RNA aptamer sensor based on magnetic self-assembled OPN protein detection nanoprobe for detecting different OPN protein concentrations. DETAILED DESCRIPTION

[0055] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0056] In the following examples, the technical means used are conventional means well known to those skilled in the art. Various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified; the reagents, materials, etc. involved are all obtained from commercial sources unless otherwise specified.

[0057] The Cas13a enzyme involved in the following examples was purchased from Shenzhen Yizhi Biotechnology Co., Ltd., crRNA was purchased from Guangzhou Bolais Biotechnology Co., Ltd., and other nucleotide sequences were purchased from Sangon Biotechnology Co., Ltd., and the nucleotide sequences involved were:

[0058] Reporter:SH-(CH2)6-UUU UUU UUU UUU UUU UCC C-MB(SEQ ID No.1);

[0059] crRNA:GAU UUA GAC UAC CCC AAA AAC GAA GGG GAC UAA AAC UAG CAU GAU UAACCG UAU CGG CAU UGA (SEQ ID No. 2);

[0060] Aptamer:CGG CCA CAG AAU GAA AAA CCU CAU CGA UGU UGC AUA GUU G(SEQ IDNo.3);

[0061] Activator:UCA AUG CCG AUA CGG UUA AUC AUG CUA (SEQ ID No. 4);

[0062] In the above nucleotide sequence, the single-stranded Aptamer can bind to the single-stranded Activator through the principle of base complementary pairing to form a double-stranded RNA (dsRNA). In addition, the Activator can bind to the Cas13a / crRNA binary complex under the guidance of crRNA to form a Cas13a / crRNA / Activator ternary complex, and finally obtain the CRISPR / Cas13a system; Aptamer can bind to the target protein (OPN protein) through high specificity and affinity, so the addition of OPN protein can force dsRNA to disassemble to form Aptamer / OPN complex and free Activator.

[0063] Example 1: Preparation of magnetic α-Fe2O3 / Fe3O4@Au nanocomposites

[0064] (1) Weigh 0.054 g NaH2PO4 and 0.54 g FeCl3·6H2O and dissolve them in 80 mL deionized water. Mix them by magnetic stirring to obtain a mixed solution. Place 80 mL of the mixed solution in a hydrothermal reactor and heat it at 220°C for 24 h. After the reaction, wash the mixed solution with ethanol and deionized water alternately and centrifuge it several times until the supernatant is clear. Dry it in an oven to obtain the precursor α-Fe2O3 nanosheets.

[0065] (2) Using glucose as a reducing agent and α-Fe2O3 nanosheets as a precursor, magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheets were prepared by a high-temperature calcination reduction method. The specific process is as follows:

[0066] The precursor α-Fe2O3 nanosheets and glucose were placed in a crucible at a mass ratio of 1:12 and mixed evenly, and then placed in a programmable temperature-controlled furnace and calcined at 600°C for 4 hours. After calcination, they were ground to obtain magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheets.

[0067] (3) Dissolve 1.5 g of polyethyleneimine (PEI) in 150 mL of distilled water and stir magnetically for 0.5 h to obtain a PEI aqueous solution; add 50 mg of magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheets to the PEI aqueous solution and disperse them by ultrasonication for 0.5 h to obtain a mixed solution. Then transfer the mixed solution obtained above to a round-bottom flask and stir magnetically for 2 h in a 90°C water bath. After the reaction is completed, centrifuge, wash, and dry to obtain an α-Fe2O3 / Fe3O4-PEI nanocomposite.

[0068] (4) Weigh 15 mg of magnetic α-Fe2O3 / Fe3O4-PEI nanocomposite, ultrasonically disperse it in 150 mL of double distilled water to form a suspension, and then control the temperature of the entire reaction environment to ≤0°C. Subsequently, add 1 mL of 48.6 mM HAuCl4 solution to the suspension, ultrasonically stir for 30 min, and after stirring, slowly add 3 mL of trisodium citrate solution (38.8 mM) and 9 mL of freshly prepared NaBH4 (0.075%) solution to the reaction solution in sequence, and keep mechanical stirring for 1 min and 15 min respectively, until the solution turns purple-black, and centrifuge, wash and dry to obtain the magnetic α-Fe2O3 / Fe3O4@Au nanocomposite.

[0069] Figure 1 The transmission electron microscope photos of magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheets and magnetic α-Fe2O3 / Fe3O4@Au nanocomposites show that the morphology of α-Fe2O3 / Fe3O4 is a uniform circular sheet structure with an average diameter and thickness of 230nm and 130nm respectively. Au nanoparticles are evenly distributed on the surface of magnetic α-Fe2O3 / Fe3O4 heterogeneous nanosheets, and the average diameter of Au nanoparticles is about 6.65nm.

[0070] Example 2: Construction of magnetic self-assembly OPN protein detection nanoprobe

[0071] (1) Reporter was prepared into a 50 μM stock solution with DEPC water, and then Reporter and TCEP (10 mM) were mixed at a molar ratio of 1:100 and reacted at 37°C for 1 h to remove disulfide bonds. After the reaction, the Reporter solution was diluted with DEPC water to a concentration of 140 nM for later use.

[0072] (2) 40 μL of 280 nM Reporter solution was mixed with 0.4 mg of α-Fe2O3 / Fe3O4@Au nanocomposite to form a suspension (10 mg / mL), which was incubated at 37°C for 3 h. After the incubation, the Reporter solution that was not fixed to the matrix material was removed by magnetic separation, and then it was dispersed in 60 μL of blocking agent MCH solution and reacted at room temperature for 30 min to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification.

[0073] (3) 2 μL of 100 nM Cas13a enzyme solution and 10 μL of 10 nM crRNA solution were assembled in 2 μL of 10× Reaction buffer and incubated at 37° C. for 40 min to form a Cas13a / crRNA binary complex for later use.

[0074] Equal volumes of 20 μM Aptamer and 20 μM Activator were mixed, and the water temperature was raised to 95°C and kept constant for 5 min. It was slowly cooled to room temperature to complete the annealing process to form dsRNA (10 μM). 10 μL dsRNA (10 μM) was mixed with 20 μL OPN protein (100 pg / mL) and incubated at 37°C for 30 min. After the incubation, a dsRNA / OPN mixture was obtained.

[0075] 14 μL of Cas13a / crRNA binary complex and 30 μL of dsRNA / OPN mixture were mixed and reacted at 37°C for 30 min. dsRNA was disassembled under the action of OPN protein to obtain Aptamer / OPN complex and free Activator. The free Activator combined with Cas13a / crRNA under the guidance of crRNA to form Cas13a / crRNA / Activator ternary complex, activating the enzymatic activity of Cas13a and obtaining CRISPR / Cas13a system.

[0076] (4) 0.4 mg of α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification was mixed with 44 μL of CRISPR / Cas13a system obtained in the above reaction and reacted. The CRISPR / Cas13a-mediated cleavage reaction was carried out at 37°C for 30 min to cleave the single-stranded reporter immobilized on the α-Fe2O3 / Fe3O4@Au surface. After the reaction, the reaction solution was removed by magnetic separation to obtain a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite.

[0077] The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomplex was washed with DEPC water and then dispersed with 40 μL of DEPC water. 9 μL of the heavy suspension (10 mg / mL) was added dropwise to the clean MGCE surface to realize the magnetic self-assembly process. After the assembly was completed, it was dried at 37°C to obtain a magnetic self-assembled OPN protein detection nanoprobe. The prepared magnetic self-assembled OPN protein detection nanoprobe was tested by DPV in the range of -0.5V to 0V, and the peak current was measured to be 134.26 μA. Example 3: Construction of magnetic self-assembled OPN protein detection nanoprobe

[0078] (1) Reporter was prepared into a 50 μM stock solution with DEPC water, and then Reporter and TCEP (10 mM) were mixed at a molar ratio of 1:100 and reacted at 37°C for 1 h to remove disulfide bonds. After the reaction, Reporter solution was diluted with DEPC water to a concentration of 120 nM for later use.

[0079] (2) 40 μL of 120 nM Reporter solution was mixed with 0.32 mg of α-Fe2O3 / Fe3O4@Au nanocomplex to form a suspension (8 mg / mL), and the mixture was incubated at 37°C for 3 h. After the incubation, the Reporter solution not fixed to the matrix material was removed by magnetic separation. Then, 60 μL of blocking agent MCH was added and reacted at room temperature for 30 min to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification.

[0080] (3) Assemble 2 μL of 100 nM Cas13a enzyme solution and 10 μL of 5 nM crRNA solution in 2 μL of 10× Reaction buffer and incubate at 37° C. for 40 min to form a Cas13a / crRNA binary complex for later use.

[0081] Equal volumes of 20 μM Aptamer and 20 μM Activator were mixed, and the water temperature was raised to 95°C and kept constant for 5 min. It was slowly cooled to room temperature to complete the annealing process to form dsRNA (10 μM). 10 μL dsRNA (8 μM) was mixed with 20 μL OPN protein (100 pg / mL) and incubated at 37°C for 30 min. After the incubation, a dsRNA / OPN mixture was obtained.

[0082] 14 μL of Cas13a / crRNA binary complex and 30 μL of dsRNA / OPN mixture were mixed and reacted at 37°C for 30 min. dsRNA was disassembled under the action of OPN protein to obtain Aptamer / OPN complex and free Activator. The free Activator combined with Cas13a / crRNA under the guidance of crRNA to form Cas13a / crRNA / Activator ternary complex, activating the enzymatic activity of Cas13a and obtaining CRISPR / Cas13a system.

[0083] (4) 0.32 mg of α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification was mixed with 44 μL CRISPR / Cas13a system obtained in the above reaction and reacted. The CRISPR / Cas13a-mediated cleavage reaction was carried out at 37°C for 20 min to cleave the single-stranded reporter fixed on the α-Fe2O3 / Fe3O4@Au surface. After the reaction, the reaction solution was removed by magnetic separation to obtain a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite.

[0084] The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite was washed with DEPC water and then dispersed with 40 μL DEPC water. 9 μL of the resuspension (8 mg / mL) was added to the clean MGCE surface to realize the magnetic self-assembly process. After the assembly was completed, it was dried at 37°C to obtain a magnetic self-assembly OPN protein detection nanoprobe. The prepared magnetic self-assembly OPN protein detection nanoprobe was tested by DPV in the range of -0.5V to 0V, and the peak current was measured to be 130.68 μA.

[0085] Example 4: Construction of magnetic self-assembly OPN protein detection nanoprobe

[0086] (1) Reporter was prepared into a 50 μM stock solution with DEPC water, and then Reporter and TCEP (10 mM) were mixed at a molar ratio of 1:100 and reacted at 37°C for 1 h to remove disulfide bonds. After the reaction, the Reporter solution was diluted with DEPC water to a concentration of 100 nM for later use.

[0087] (2) 40 μL of 100 nM Reporter solution was mixed with 0.24 mg of α-Fe2O3 / Fe3O4@Au nanocomplex to form a suspension (6 mg / mL), and the mixture was incubated at 37°C for 3 h. After the incubation, the Reporter solution not fixed to the matrix material was removed by magnetic separation. Then, 60 μL of blocking agent MCH was added and reacted at room temperature for 30 min to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification.

[0088] (3) 2 μL of 100 nM Cas13a enzyme solution and 10 μL of 2.5 nM crRNA solution were assembled in 2 μL of 10× Reaction buffer and incubated at 37° C. for 40 min to form a Cas13a / crRNA binary complex for later use.

[0089] Equal volumes of 20 μM Aptamer and 20 μM Activator were mixed, and the water temperature was raised to 95°C and kept constant for 5 min. It was slowly cooled to room temperature to complete the annealing process to form dsRNA (10 μM). 10 μL dsRNA (6 μM) was mixed with 20 μL OPN protein (100 pg / mL) and incubated at 37°C for 30 min. After the incubation, a dsRNA / OPN mixture was obtained.

[0090] 14 μL of Cas13a / crRNA binary complex and 30 μL of dsRNA / OPN mixture were mixed and reacted at 37°C for 30 min. dsRNA was disassembled under the action of OPN protein to obtain Aptamer / OPN complex and free Activator. The free Activator combined with Cas13a / crRNA under the guidance of crRNA to form Cas13a / crRNA / Activator ternary complex, activating the enzymatic activity of Cas13a and obtaining CRISPR / Cas13a system.

[0091] (4) 0.24 mg of α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification was mixed with 44 μL CRISPR / Cas13a system obtained in the above reaction and reacted. The CRISPR / Cas13a-mediated cleavage reaction was carried out at 37°C for 40 min to cleave the single-stranded reporter fixed on the α-Fe2O3 / Fe3O4@Au surface. After the reaction, the reaction solution was removed by magnetic separation to obtain a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite.

[0092] The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite was washed with DEPC water and then dispersed with 40μL DEPC water. 9μL of the resuspension (6mg / mL) was added to the clean MGCE surface to realize the magnetic self-assembly process. After the assembly, it was dried at 37°C to obtain the magnetic self-assembly OPN protein detection nanoprobe. The prepared magnetic self-assembly OPN protein detection nanoprobe was tested by DPV in the range of -0.5V to 0V, and the peak current was measured to be 131.45μA.

[0093] Example 5: Construction of magnetic self-assembly OPN protein detection nanoprobe

[0094] (1) Prepare Reporter with DEPC water to make a 50 μM stock solution, then mix Reporter and TCEP (10 mM) at a molar ratio of 1:100 and react at 37°C for 1 h to remove disulfide bonds. After the reaction, dilute with DEPC water to a 160 nM Reporter solution for later use.

[0095] (2) 40 μL of 160 nM Reporter solution was mixed with 0.48 mg of α-Fe2O3 / Fe3O4@Au nanocomposite to form a suspension (12 mg / mL), and the mixture was incubated at 37°C for 3 h. After the incubation, the Reporter solution not fixed to the matrix material was removed by magnetic separation. Then, 60 μL of blocking agent MCH was added and reacted at room temperature for 30 min to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification.

[0096] (3) 2 μL of 100 nM Cas13a enzyme solution and 10 μL of 20 nM crRNA solution were assembled in 2 μL of 10× Reaction buffer and incubated at 37° C. for 40 min to form a Cas13a / crRNA binary complex for later use.

[0097] Equal volumes of 20 μM Aptamer and 20 μM Activator were mixed, and the water temperature was raised to 95°C and kept constant for 5 min. It was slowly cooled to room temperature to complete the annealing process to form dsRNA (10 μM). 10 μL dsRNA (12 μM) was mixed with 20 μL OPN protein (100 pg / mL) and incubated at 37°C for 30 min. After the incubation, a dsRNA / OPN mixture was obtained.

[0098] 14 μL of Cas13a / crRNA binary complex and 30 μL of dsRNA / OPN mixture were mixed and reacted at 37°C for 30 min. dsRNA was disassembled under the action of OPN protein to obtain Aptamer / OPN complex and free Activator. The free Activator combined with Cas13a / crRNA under the guidance of crRNA to form Cas13a / crRNA / Activator ternary complex, activating the enzymatic activity of Cas13a and obtaining CRISPR / Cas13a system.

[0099] (4) 0.48 mg of α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification was mixed with 44 μL CRISPR / Cas13a system obtained in the above reaction and reacted. The CRISPR / Cas13a-mediated cleavage reaction was carried out at 37°C for 50 min to cleave the single-stranded reporter fixed on the α-Fe2O3 / Fe3O4@Au surface. After the reaction, the reaction solution was removed by magnetic separation to obtain a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite.

[0100] The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite was washed with DEPC water and then dispersed with 40μL DEPC water. 9μL of the resuspension (12mg / mL) was added to the clean MGCE surface to realize the magnetic self-assembly process. After the assembly was completed, it was dried at 37°C to obtain the magnetic self-assembly OPN protein detection nanoprobe. The prepared magnetic self-assembly OPN protein detection nanoprobe was tested by DPV in the range of -0.5V to 0V, and the peak current was measured to be 129.33μA.

[0101] Example 6: Construction of magnetic self-assembly nanoprobe for OPN protein detection

[0102] (1) Prepare Reporter with DEPC water to make a 50 μM stock solution, then mix Reporter and TCEP (10 mM) at a molar ratio of 1:100 and react at 37°C for 1 h to remove disulfide bonds. After the reaction, dilute with DEPC water to a 180 nM Reporter solution for later use.

[0103] (2) 40 μL of 180 nM Reporter solution was mixed with 0.56 mg of α-Fe2O3 / Fe3O4@Au nanocomposite to form a suspension (14 mg / mL), and the mixture was incubated at 37°C for 3 h. After the incubation, the Reporter solution not fixed to the matrix material was removed by magnetic separation. Then, 60 μL of blocking agent MCH was added and reacted at room temperature for 30 min to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification.

[0104] (3) 2 μL of 100 nM Cas13a enzyme solution and 10 μL of 50 nM crRNA solution were assembled in 2 μL of 10× Reaction buffer and incubated at 37° C. for 40 min to form a Cas13a / crRNA binary complex for later use.

[0105] Equal volumes of 20 μM Aptamer and 20 μM Activator were mixed, and the water temperature was raised to 95°C and kept constant for 5 min. It was slowly cooled to room temperature to complete the annealing process to form dsRNA (10 μM). 10 μL dsRNA (14 μM) was mixed with 20 μL OPN protein (100 pg / mL) and incubated at 37°C for 30 min. After the incubation, a dsRNA / OPN mixture was obtained.

[0106] 14 μL of Cas13a / crRNA binary complex and 30 μL of dsRNA / OPN mixture were mixed and reacted at 37°C for 30 min. dsRNA was disassembled under the action of OPN protein to obtain Aptamer / OPN complex and free Activator. The free Activator combined with Cas13a / crRNA under the guidance of crRNA to form Cas13a / crRNA / Activator ternary complex, activating the enzymatic activity of Cas13a and obtaining CRISPR / Cas13a system.

[0107] (4) 0.56 mg of α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification was mixed with 44 μL CRISPR / Cas13a system obtained in the above reaction and reacted. The CRISPR / Cas13a-mediated cleavage reaction was carried out at 37°C for 60 min to cleave the single-stranded reporter fixed on the α-Fe2O3 / Fe3O4@Au surface. After the reaction, the reaction solution was removed by magnetic separation to obtain a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite.

[0108] The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite was washed with DEPC water and then dispersed with 40 μL DEPC water. 9 μL of the resuspension (14 mg / mL) was added dropwise to the clean MGCE surface to realize the magnetic self-assembly process. After the assembly was completed, it was dried at 37°C to obtain the magnetic self-assembly OPN protein detection nanoprobe. The prepared magnetic self-assembly OPN protein detection nanoprobe was tested by DPV in the range of -0.5V to 0V, and the peak current was measured to be 134.26 μA.

[0109] Example 7: Construction of magnetic self-assembly nanoprobe for OPN protein detection

[0110] (1) Prepare Reporter with DEPC water to make a 50 μM stock solution, then mix Reporter and TCEP (10 mM) at a molar ratio of 1:100 and react at 37°C for 1 h to remove disulfide bonds. After the reaction, dilute with DEPC water to a 200 nM Reporter solution for later use.

[0111] (2) 40 μL of 200 nM Reporter solution was mixed with 0.64 mg of α-Fe2O3 / Fe3O4@Au nanocomplex to form a suspension (16 mg / mL), and the mixture was incubated at 37°C for 3 h. After the incubation, the Reporter solution not fixed to the matrix material was removed by magnetic separation. Then, 60 μL of blocking agent MCH was added and reacted at room temperature for 30 min to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification.

[0112] (3) 2 μL of 100 nM Cas13a enzyme solution and 10 μL of 100 nM crRNA solution were assembled in 2 μL of 10× Reaction buffer and incubated at 37° C. for 40 min to form a Cas13a / crRNA binary complex for later use.

[0113] Equal volumes of 20 μM Aptamer and 20 μM Activator were mixed, and the water temperature was raised to 95°C and kept constant for 5 min. It was then slowly cooled to room temperature to complete the annealing process to form dsRNA (10 μM). 10 μL dsRNA (16 μM) was mixed with 20 μL OPN protein (100 pg / mL) and incubated at 37°C for 30 min. After the incubation, a dsRNA / OPN mixture was obtained.

[0114] 14 μL of Cas13a / crRNA binary complex and 30 μL of dsRNA / OPN mixture were mixed and reacted at 37°C for 30 min. dsRNA was disassembled under the action of OPN protein to obtain Aptamer / OPN complex and free Activator. The free Activator combined with Cas13a / crRNA under the guidance of crRNA to form Cas13a / crRNA / Activator ternary complex, activating the enzymatic activity of Cas13a and obtaining CRISPR / Cas13a system.

[0115] (4) 0.64 mg of α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification was mixed with 44 μL CRISPR / Cas13a system obtained in the above reaction and reacted. The CRISPR / Cas13a-mediated cleavage reaction was carried out at 37°C for 70 min to cleave the single-stranded reporter fixed on the α-Fe2O3 / Fe3O4@Au surface. After the reaction, the reaction solution was removed by magnetic separation to obtain a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite.

[0116] The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite was washed with DEPC water and then dispersed with 40 μL DEPC water. 9 μL of the resuspension (16 mg / mL) was added dropwise to the clean MGCE surface to realize the magnetic self-assembly process. After the assembly was completed, it was dried at 37°C to obtain a magnetic self-assembly OPN protein detection nanoprobe. The prepared magnetic self-assembly OPN protein detection nanoprobe was tested by DPV in the range of -0.5V to 0V, and the peak current was measured to be 125.89 μA.

[0117] Example 8: Construction of electrochemical RNA aptamer sensor

[0118] In this example, bare electrode MGCE, MGCE / α-Fe2O3 / Fe3O4@Au, and MGCE / α-Fe2O3 / Fe3O4@Au-Reporter / MCH were used as controls, and CV was used to monitor the construction process of the electrochemical RNA aptamer sensor based on the magnetic self-assembled OPN protein detection nanoprobe. Among them, the preparation method of MGCE / α-Fe2O3 / Fe3O4@Au is: disperse the magnetic α-Fe2O3 / Fe3O4@Au nanocomposite in DEPC water to prepare a magnetic α-Fe2O3 / Fe3O4@Au nanocomposite suspension with a concentration of 10 mg / mL, then take 9 μL and drop it onto the surface of the cleaned MGCE, and dry it at 37°C to obtain the MGCE / α-Fe2O3 / Fe3O4@Au electrode modification. MGCE / α-Fe2O3 / Fe3O4@Au-Reporter / MCH and MGCE / α-Fe2O3 / Fe3O4@Au-MCH were prepared in Example 2.

[0119] Determination method:

[0120] The electrochemical workstation and three-electrode system were used for the test. The bare electrodes MGCE, MGCE / α-Fe2O3 / Fe3O4@Au, MGCE / α-Fe2O3 / Fe3O4@Au-Reporter / MCH, and MGCE / α-Fe2O3 / Fe3O4@Au-MCH were used as working electrodes, and the Ag / AgCl electrode and Pt electrode were used as reference and auxiliary electrodes, respectively. The reaction mixture was prepared by mixing 0.1 M KCl and 5 mM [Fe(CN)6] 3- / 4- The solution was used as the electrolyte to construct an electrochemical RNA aptamer sensor for detection. The measurement results were as follows Figure 2 shown.

[0121] from Figure 2 As can be seen from the figure, curve a is a bare electrode, and curve b is the electrode surface on which α-Fe2O3 / Fe3O4@Au nanocomplexes are added. Since the gold nanoparticles on the surface of the nanomaterial have excellent conductivity, they can significantly amplify the current signal, so the redox current increases, indicating that the MGCE / α-Fe2O3 / Fe3O4@Au modified electrode was successfully constructed. Curve c is the Reporter with thiol and methylene blue modified at both ends fixed to the α-Fe2O3 / Fe3O4@Au nanocomplexes by Au-S bonds, and MCH is used to block its nonspecific adsorption sites to construct the MGCE / α-Fe2O3 / Fe3O4@Au-Reporter / MCH modified electrode. Since the negatively charged phosphate skeleton in the ssRNA chain of Reporter binds to [Fe(CN)6] 3- / 4- Electrostatic repulsion occurs, causing [Fe(CN)6] 3- / 4- At the same time, the arrangement of Reporter on the surface of the nanocomposite increases the steric hindrance, resulting in a significant decrease in the redox current and an increase in the charge transfer resistance. Curve d is the reaction solution containing CRISPR / Cas13a, the Cas13a enzyme activity is activated, and the Reporter fixed on the α-Fe2O3 / Fe3O4@Au surface is cleaved. It can be seen that the nucleotide chain is cut into short fragments, resulting in a decrease in the steric hindrance on the electrode surface, and the [Fe(CN)6] 3- / 4- It is easier to reach the electrode surface, so the redox current increases. It can be seen that the present invention successfully constructs a magnetic self-assembly OPN protein detection nanoprobe.

[0122] Example 9: Performance evaluation of electrochemical RNA aptamer sensor

[0123] In this example, an electrochemical RNA aptamer sensor was constructed based on magnetic self-assembly OPN protein detection nanoprobes, and the interfering protein and target protein of the same concentration were detected and compared to investigate the selectivity of the electrochemical sensor. The specific steps are as follows.

[0124] 100 pg / mL protein solutions of nucleolin (NCL), heterogeneous nuclear ribonucleoprotein A1 (HN-PNP-A1), human papillomavirus (HPV), alpha-fetoprotein (AFP), microtubule-associated protein (Tau), target protein (OPN) and their mixed protein group (Mix) were prepared respectively;

[0125] According to the preparation method in Example 2, appropriate amounts of dsRNA and NCL, HN-RNP-A1, AFP, Tau, OPN and Mix protein solutions were mixed and incubated at 37° C. for 30 min to obtain dsRNA mixed solutions containing different interferors.

[0126] 14 μL of Cas13a / crRNA binary complex and 30 μL of dsRNA mixed solution containing different interfering substances were mixed and incubated at 37°C for 30 min to obtain Cas13a / crRNA / Activator ternary complex. 44 μL of Cas13a / crRNA binary complex and dsRNA mixed solution containing different interfering substances were mixed with 0.4 mg of α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier and reacted. The CRISPR / Cas13a-mediated cleavage reaction was reacted at 37°C for 30 min, and the reporter was cut indiscriminately, and then the magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomplex was obtained by magnetic separation.

[0127] The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite was washed with DEPC water and then dispersed with 40 μL of DEPC water. 9 μL of the resuspension (10 mg / mL) was added dropwise to the clean MGCE surface for magnetically induced self-assembly. The nanoprobe was dried at 37°C to obtain an electrochemical RNA aptamer sensor based on magnetic self-assembled OPN protein detection nanoprobes. The working electrode after drying was tested on an electrochemical workbench. The test results are shown in FIG. Figure 3 shown.

[0128] from Figure 3 It can be seen that compared with the blank group, the current response of the interfering protein is significantly lower than that of the target protein and the mixed group, and the current response of the mixed group (Mix) is similar to that of the target protein, indicating that the magnetic self-assembly OPN protein detection nanoprobe constructed by the present invention has good selectivity.

[0129] Example 10: Performance analysis of electrochemical RNA aptamer sensor for detecting OPN protein

[0130] In this example, 20 μL of an OPN protein solution with a concentration ranging from 0.1 pg / mL to 100 ng / mL was mixed with 10 μL of prepared dsRNA (10 μM) and incubated at 37° C. for 30 min, and then mixed with 14 μL of the Cas13a / crRNA binary complex prepared in Example 2, and incubated at 37° C. for 30 min to obtain a CRISPR / Cas13a system.

[0131] The CRISPR / Cas13a system was reacted with the α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier at 37°C for 30 min. Finally, the mixture was dropped onto the clean MGCE surface according to the treatment method in Example 2. The magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite was tested by DPV in the range of -0.5V to 0V, and the DPV peak current was measured to investigate the analytical performance of the electrochemical sensor. The results are shown in FIG. Figure 4 shown.

[0132] from Figure 4 It can be seen that the current response value is linearly correlated with the logarithm of the concentration of OPN protein, and the linear equation is: I = 7.8lg OPN + 120.1 (R 2 =0.995), the detection limit was 0.33pg / mL (LOD = 3σ / slope), and the quantification limit was 1.09pg / mL (LOQ = 10σ / slope). The results show that changes in target protein concentration can affect the trans-cleavage activity of CRISPR / Cas13a, thereby causing changes in the electrical signal on the electrode surface.

[0133] In summary, the magnetic self-assembly OPN protein detection nanoprobe of the present invention has a low detection limit and a wide linear range. When the OPN protein concentration is in the range of 1pg / mL to 10ng / mL, the current I shows a good linear correlation with the OPN protein concentration. The preparation method is simple, the selectivity is good, and sensitive detection of OPN protein can be achieved. Therefore, this detection strategy provides a reference for detecting protein macromolecules using electrochemical sensors with CRISPR / Cas13a.

[0134] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A magnetic self-assembled OPN protein detection nanoprobe, characterized in that: The magnetic self-assembled OPN protein detection nanoprobe includes: MGCE, a magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite magnetically self-assembled on the surface of MGCE; the magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite is obtained by mixing an α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier and a CRISPR / Cas13a system; The α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier includes an α-Fe2O3 / Fe3O4@Au nanocomposite material, a reporter assembled on the surface of the α-Fe2O3 / Fe3O4@Au nanocomposite material through Au-S bonds, and a blocker MCH specifically adsorbed on the surface of the material; The CRISPR / Cas13a system is formed by mixed hybridization of Cas13a / crRNA and dsRNA / OPN, wherein Cas13a / crRNA is obtained by the combination of Cas13a enzyme and crRNA, dsRNA is formed by annealing of single-stranded Aptamer and single-stranded Activator, and dsRNA / OPN is formed by the combination of dsRNA and OPN protein.

2. The magnetic self-assembled OPN protein detection nanoprobe according to claim 1, characterized in that: The nucleotide sequence of the reporter is shown in SEQ ID No. 1; The nucleotide sequence of the crRNA is shown in SEQ ID No. 2; The nucleotide sequences of the single-chain Aptamer and single-chain Activator are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

3. The method for preparing the magnetic self-assembled OPN protein detection nanoprobe according to claim 1 or 2, characterized in that: The preparation method comprises: (1) A quantitative reporter solution was mixed with α-Fe2O3 / Fe3O4@Au nanocomposite to form a suspension and then incubated for reaction. After the reaction, the supernatant was removed by magnetic separation to obtain solid α-Fe2O3 / Fe3O4@Au-Reporter; Solid α-Fe2O3 / Fe3O4@Au-Reporter was dispersed into a certain amount of MCH solution for reaction to block nonspecific adsorption sites. After the reaction, the supernatant was removed by magnetic separation again to obtain a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification. (2) reacting the Cas13a enzyme solution and the crRNA solution in 10× Reaction Buffer (for Cas13a) to form a Cas13a / crRNA binary complex; mixing the Cas13a / crRNA binary complex with the dsRNA / OPN solution and incubating it to obtain a Cas13a / crRNA / Activator ternary complex, activating the cleavage activity of the Cas13a enzyme, and obtaining the CRISPR / Cas13a system; (3) The CRISPR / Cas13a system was mixed with a solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modifier to undergo a cleavage reaction, and magnetic separation was performed after the reaction to obtain a solid magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite; (4) Using DEPC water to resuspend the solid magnetic α-Fe2O3 / Fe3O4@Au-MCH nanocomposite to obtain a nanocomposite resuspension; then adding the obtained nanocomposite resuspension to the MGCE surface to complete the magnetic induced self-assembly process, and drying after the self-assembly is completed to obtain the magnetic self-assembled OPN protein detection nanoprobe.

4. The method for preparing the magnetic self-assembled OPN protein detection nanoprobe according to claim 3, characterized in that: In step (1), the concentration of the α-Fe2O3 / Fe3O4@Au nanocomposite in the suspension is 5 mg / mL to 20 mg / mL; The dosage ratio of the Reporter solution and the MCH solution is 40 μL:60 μL, wherein the concentration of the Reporter solution is 50 nM to 200 nM; the concentration of the MCH solution is 1 mM; The incubation reaction conditions are: incubation at 30-37°C for 3-4h; After adding a certain amount of MCH solution, react at 25-37°C for 0.5-2h.

5. The method for preparing the magnetic self-assembled OPN protein detection nanoprobe according to claim 4, characterized in that: The concentration of the α-Fe2O3 / Fe3O4@Au nanocomposite in the suspension is 10 mg / mL; the concentration of the reporter is 140 nM.

6. The method for preparing the magnetic self-assembled OPN protein detection nanoprobe according to claim 3, characterized in that: In step (2), the dosage ratio of the Cas13a enzyme solution, crRNA solution and 10×Reaction Buffer (for Cas13a) is 2 μL:10 μL:2 μL, wherein the molar concentration ratio of the Cas13a enzyme solution and the crRNA solution is 20:1 to 1:1; The reaction conditions of the Cas13a enzyme solution and crRNA solution in 10×Reaction Buffer (for Cas13a) are as follows: reaction at 25-37°C for 40-60 minutes; The dosage ratio of the Cas13a / crRNA binary complex and the dsRNA / OPN solution is 14 μL:30 μL; the conditions for the mixed hybridization of the Cas13a / crRNA binary complex and the dsRNA / OPN solution are: incubation at 37° C. for 25 to 60 minutes; The preparation steps of the dsRNA / OPN solution include: annealing a single-stranded Aptamer and a single-stranded Activator to obtain dsRNA, and then reacting the dsRNA with OPN protein to obtain the dsRNA / OPN solution after the reaction is completed.

7. The method for preparing the magnetic self-assembled OPN protein detection nanoprobe according to claim 6, characterized in that: The molar concentration ratio of the Cas13a and crRNA is 10:1; During the preparation of dsRNA / OPN solution: The molar concentration and dosage of the single-chain Aptamer and the single-chain Activator are the same; the annealing conditions are: constant temperature at 95°C for 5 minutes, and then cooled to room temperature; the dosage ratio of the dsRNA and OPN protein is 10 μL:20 μL, and the concentration of the dsRNA is 5 μM to 20 μM; the reaction conditions of the dsRNA and OPN protein are: reaction at 20 to 40°C for 30 to 90 minutes.

8. The method for preparing the magnetic self-assembled OPN protein detection nanoprobe according to claim 3, characterized in that: In step (3), the ratio of the CRISPR / Cas13a system to the solid α-Fe2O3 / Fe3O4@Au-Reporter / MCH electrode modification is 44 μL: 0.24-0.68 mg; The cleavage reaction time is 10 to 100 minutes.

9. Use of the magnetic self-assembled OPN protein detection nanoprobe according to claim 1 or 2 in the preparation of an electrochemical RNA aptamer sensor for detecting OPN protein.

10. An electrochemical RNA aptamer sensor for detecting OPN protein, characterized in that: The electrochemical RNA aptamer sensor uses the magnetic self-assembled OPN protein detection nanoprobe according to claim 1 or 2 as a working electrode, an Ag / AgCl electrode and a Pt electrode as a reference and auxiliary electrode, respectively, and [Fe(CN)6] 3- / 4- The solution was used as an electrolyte, and the OPN protein content was converted into a current response by an electrochemical workstation to detect the OPN protein.

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