Label-free magnetic self-assembly nano-probe, construction method thereof and application of label-free magnetic self-assembly nano-probe in detection of PrPC protein

Abstract

The invention provides a label-free magnetic self-assembly nanoprobe, a construction method thereof and application of the label-free magnetic self-assembly nanoprobe in detection of PrPC protein. The label-free magnetic self-assembly nanoprobe disclosed by the invention comprises a magnetic Fe3O4 / Fe2O3 (at) Au heteroplasmon nanorod array and a sulfhydrylation aptamer nucleic acid chain which is combined with the magnetic Fe3O4 / Fe2O3 (at) Au heteroplasmon nanorod array through an Au-S bond; a biosensor prepared from the label-free magnetic self-assembly nanoprobe is good in reproducibility and stability; the RSD value of multiple times of detection (intraday / day) is 1.55%; the storage time of multiple groups of sensors is 0-15 days, and the change of response signal values does not exceed 4%; the biosensor has a relatively low detection limit (1pg / mL), a relatively wide linear range (1 * 10 < pg / mL >-1 * 10 < 5 > pg / mL) and relatively good sensitivity, has very good application in detection of PrPC, and has very good practicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalysis and sensor preparation, and specifically relates to a label-free magnetic self-assembled nanoprobe and a construction method thereof and a method for detecting PrP C Application of protein. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, and the oral cavity is the most common site of head and neck tumors. More than 90% of oral cancers originate from squamous epithelial tissue, and oral squamous cell carcinoma (OSCC) accounts for more than 40% of head and neck malignancies. Oral squamous cell carcinoma is a common and aggressive tumor, and its early diagnosis and effective treatment have always been the focus of medical research. The 5-year survival rate of oral squamous cell carcinoma after early detection is more than 90%, and the 5-year survival rate in the last 50 years is 90%. The detection rate of advanced OSCC is often only 30%, and the 5-year survival rate in the late stage is less than 50%. The high mortality rate of patients is mainly due to delayed diagnosis, therefore, suggesting that early diagnosis of OSCC is still one of the most effective strategies to improve treatment and improve prognosis.

[0003] Tumor markers refer to a class of substances that undergo abnormal changes during the occurrence, proliferation, metastasis or recurrence of tumor cells due to the expression of related genes in tumor cells or their collective response to tumors. C ) is encoded by the human prion protein gene (PRNP), and immunohistochemical techniques have been used to demonstrate that PrP C It is overexpressed in OSCC tissues, and the research results show that it may be an important marker for the diagnosis and treatment of OSCC patients, and has great potential as a new biological tumorigenic agent and cancer treatment target.

[0004] A variety of methods have been applied to the detection of tumor markers, including enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and atomic absorption spectroscopy (AAS). Although these methods have good sensitivity and selectivity, they require highly sophisticated instruments and experienced operators, and the detection cost is high, which limits their application in practice. In recent years, the application of electrochemistry in the field of sensing detection has attracted more and more attention. As an effective detection method, electrochemical sensors have the advantages of low price, simple and fast operation, high sensitivity, and real-time detection of complex biological systems; magnetic nanomaterials have been widely used in the biomedical field due to their excellent magnetic properties and good biocompatibility. The introduction of nanomaterials into electrochemical sensors is conducive to improving their electron transfer efficiency, thereby realizing sensitive detection of biological samples. Therefore, it is necessary to develop a method for detecting cellular prion protein (PrP C ) electrochemical biosensor. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a label-free magnetic self-assembled nanoprobe and a construction method thereof and a method for detecting PrP C Application of protein; The label-free magnetic self-assembled nanoprobe of the present invention comprises a magnetic Fe3O4 / Fe2O3@Au heterogeneous nanorod array and a thiol-aptamer nucleic acid chain bonded to the nanorod array via an Au-S bond; The biosensor prepared by the label-free magnetic self-assembled nanoprobe has good reproducibility and stability: the RSD value of multiple detections (intra-day / inter-day) is 1.55%; the response signal value of multiple sets of sensors is stored for 0 to 15 days, and the change is no more than 4%; The biosensor has a low detection limit (1pg / mL), a wide linear range (1×10 pg / mL~1×10 5 pg / mL) and good sensitivity in detecting PrP C It has good application and is very practical.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0007] The present invention first provides a label-free magnetic self-assembled nanoprobe, which comprises a magnetic Fe3O4 / Fe2O3@Au nanocomposite and a thiol-modified aptamer nucleic acid bonded to the nanocomposite via an Au-S bond;

[0008] The thiolated aptamer nucleic acid is: 5'-SH-(CH2)6-TGCTGGAGGATATCGGTGTTTATGGTGTCTGTCTTCCTCC AGC-3'.

[0009] The present invention also provides a method for preparing the label-free magnetic self-assembled nanoprobe, the preparation method comprising:

[0010] (1) mixing and incubating the Fe3O4 / Fe2O3@Au nanocomposite suspension with the thiolated aptamer nucleic acid solution, and then performing magnetic separation and washing after the incubation to obtain Fe3O4 / Fe2O3@Au / Apt;

[0011] (2) adding bovine serum albumin (BSA) to Fe3O4 / Fe2O3@Au / Apt, mixing by ultrasound until uniform, obtaining a suspension, and then incubating the suspension at room temperature, and after the incubation, performing magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt / BSA;

[0012] (3) Resuspending Fe3O4 / Fe2O3@Au / Apt / BSA to obtain a resuspension, dropping the resuspension onto the surface of a clean magnetic glassy carbon electrode (MGCE), inducing self-assembly by magnetic force, and drying at room temperature to obtain the label-free magnetic self-assembled nanoprobe.

[0013] Preferably, in step (1), the method for preparing the Fe3O4 / Fe2O3@Au nanocomposite comprises:

[0014] S1. Preparation of Fe3O4 / Fe2O3 heterostructure nanorods:

[0015] FeCl3·6H2O and CO(NH2)2 are dissolved in pure water, mixed evenly and reacted in a water bath, washed, separated and dried after the reaction, ground the dried product and evenly mixed with urea, and then calcined to obtain Fe3O4 / Fe2O3 heterogeneous nanorods;

[0016] S2. Preparation of Fe3O4 / Fe2O3@Au nanocomposites:

[0017] The Fe3O4 / Fe2O3 heterogeneous nanorod powder is dispersed in a polyethyleneimine (PEI) solution, and then heated in a water bath for reaction. After the reaction is completed, it is washed and magnetically separated to obtain a Fe3O4 / α-Fe2O3@PEI intermediate.

[0018] The Fe3O4 / α-Fe2O3@PEI intermediate is dispersed in ultrapure water, a chloroauric acid solution is added thereto and mixed evenly, and then a sodium borohydride solution is added thereto for mixed reaction. After the reaction is completed, the Fe3O4 / α-Fe2O3@Au nanocomposite is obtained by washing and magnetic separation.

[0019] Preferably, in step S1, the dosage ratio of FeCl3·6H2O to CO(NH2)2 is 10g:4g; the water bath heating condition is: water bath heating at 75-95°C for 2-4h; the dosage ratio of the dried product to urea is 1g:6g; the calcination condition temperature range is 350-450°C, and the duration is 1-5h;

[0020] In step S2, the dosage ratio of Fe3O4 / Fe2O3 heterogeneous nanorod powder and polyethyleneimine solution is 50mg:150mL; the concentration of the polyethyleneimine solution is 0.5-2g / mL; the water bath heating condition is: heating reaction at 80-90°C for 1-3h;

[0021] The dosage ratio of the Fe3O4 / α-Fe2O3@PEI intermediate, chloroauric acid solution and sodium borohydride solution is 15 mg:1 mL:9 mL, wherein the concentration of the chloroauric acid solution is 20-40 mg / mL, and the concentration of the sodium borohydride solution is 0.05-0.1 wt%.

[0022] Preferably, in step (1), the dosage ratio of the Fe3O4 / Fe2O3@Au nanocomposite suspension to the thiolated aptamer nucleic acid solution is 15 μL:15 μL; wherein the concentration of the Fe3O4 / Fe2O3@Au nanocomposite suspension is 10-30 mg / mL, and the concentration of the thiolated aptamer nucleic acid solution is 5-10 μM.

[0023] Preferably, in step (1), the mixed incubation condition has a temperature range of 30 to 70° C. and a duration of 10 to 50 min.

[0024] Preferably, in step (2), the dosage ratio of Fe3O4 / Fe2O3@Au / Apt to bovine serum albumin solution is 0.5 mg:30 μL; and the concentration range of the bovine serum albumin solution is 0.1 to 0.5 wt%.

[0025] Preferably, in step (2), the mixed incubation condition has a temperature range of 15 to 30° C. and a duration of 60 to 120 min.

[0026] Preferably, in step (3), the resuspension solution is ultrapure water, and the amount used ranges from 10 to 35 μL.

[0027] The present invention also provides the above-mentioned label-free magnetic self-assembled nanoprobe for preparing and detecting PrP C Application of proteins in electrochemical biosensors.

[0028] The present invention also provides a method for detecting PrP CThe electrochemical biosensor for protein, wherein the sensor uses the label-free magnetic self-assembled nanoprobe as the working electrode, silver / silver chloride as the reference electrode, a platinum wire electrode as the counter electrode, and [Fe(CN)6] 3- / 4- The solution was used as the electrolyte and the PrP was detected using an electrochemical workstation. C protein.

[0029] Preferably, PrP C The solution was dropped onto the surface of the label-free magnetic self-assembled nanoprobe, incubated for a period of time, and detected in the range of 0.1 to 0.6V.

[0030] Preferably, the PrP C The concentration of the solution is 1×10 pg / mL~1×10 5 pg / mL, the dosage is 10μL;

[0031] The incubation conditions are: incubation at 40° C. for 30 min.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The label-free magnetic self-assembled nanoprobe of the present invention is an efficient, rapid, economical, highly specific and practical nano tumor marker detection probe, which fills the gaps in existing detection technology. The magnetic nanomaterial used in the label-free magnetic self-assembled nanoprobe of the present invention has the advantages of small volume and large specific surface area, which can more effectively bind to biomolecules, and modify the surface thereof to improve its dispersion stability, and can be subsequently separated under the action of a magnetic field.

[0034] The present invention adopts Fe3O4 / Fe2O3@Au nanocomposite to construct a label-free magnetic self-assembled nanoprobe. Its unique signal amplification strategy can reduce the interference of background signals, does not require the use of electrically active redox markers, and does not require pretreatment. The detection process is fast and convenient, and the detection results can be obtained in a few minutes.

[0035] The present invention designs and synthesizes thiolated aptamer nucleic acids, which have the advantages of rapid in vitro synthesis, high stability, strong affinity, short preparation cycle, good specific recognition ability, high repeatability, low price, easy modification, and easy in vitro mass preparation. In addition, the thiolated aptamer nucleic acid has a small and flexible structure, which can bind to smaller targets and hidden domain structures that some antibodies cannot bind to.

[0036] The label-free magnetic self-assembled nanoprobe prepared by the present invention comprises a magnetic Fe3O4 / Fe2O3@Au nanocomposite and a thiolated aptamer nucleic acid bonded to the nanocomposite via an Au-S bond; the thiolated aptamer nucleic acid is firmly fixed on the surface of the magnetic Fe3O4 / Fe2O3@Au nanocomposite via the Au-S bond and is evenly distributed, so that the aptamer nucleic acid can better detect PrP. C The detection has a lower detection limit (1pg / mL) and a wider linear range (1×10pg / mL~1×10 5 pg / mL) and good sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the transmission microscopy characterization image of Fe3O4 / Fe2O3@Au nanocomposite.

[0038] Figure 2 PrP C Cyclic voltammetry characterization of each stage in the construction process of the protein label-free magnetic self-assembled probe MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA; among them, from a to b, bare electrode (MGCE), MGCE / Fe3O4 / Fe2O3, MGCE / Fe3O4 / Fe2O3@Au, MGCE / Fe3O4 / Fe2O3@Au / Apt, MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA, MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA, MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA / PrP C .

[0039] Figure 3 PrP C Protein label-free magnetic self-assembled probes for detecting PrP C , AFP, NCL, HNRNPA, OPN, and their mixtures.

[0040] Figure 4 PrP C Concentration and linear range that can be detected by the protein label-free magnetic self-assembled probe. DETAILED DESCRIPTION

[0041] In order to make the purpose, features and advantages of the present invention clearer and easier to understand, the present invention will be further described below in conjunction with specific embodiments and drawings, so that relevant technical personnel can better understand the technical solution of the present invention. Obviously, the described embodiments are only part of the present invention, and any other embodiments obtained without creative work, such as embodiments that only change the purpose without affecting the basic principles involved in the claims, should be deemed to be within the scope of protection of the present invention. The source of the reagents used, the trade name, and if it is necessary to list their components, are all indicated when they first appear, and the same reagents used thereafter are not otherwise specified, and are the same as the first stated content; the reagents, materials, etc. involved are all obtained through commercial channels unless otherwise specified.

[0042] In the following examples, tri(2-carboxyethyl)phosphine (TCEP) and bovine serum albumin (BSA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; PrP C Proteins were purchased from Sangon Biotechnology Co., Ltd.; ribonucleoprotein (HNRNPA) and osteopontin (OPN) were purchased from Jinan Protein Technology Co., Ltd.; alpha-fetoprotein (AFP) and nucleolar protein (NCL) were purchased from Sewell Biotechnology Co., Ltd.; the thiol-modified aptamer nucleic acid sequence was: 5'-SH-(CH2)6-TGCTGGAGGATATCGGTGTTTATGGTGTCTGTCTTCCTCC AGC-3', which was synthesized by Sangon Biotechnology Co., Ltd.

[0043] Example 1: PrP C Construction of label-free magnetic self-assembly probes for proteins

[0044] S1. Preparation of Fe3O4 / Fe2O3@Au nanocomposites:

[0045] S1.1 Preparation of magnetic Fe3O4 / Fe2O3 heterostructure nanorods:

[0046] Dissolve 10.812g hexahydrate and ferric chloride FeCl3·6H2O and 4.8048g urea CO(NH2)2 in 50mL pure water, mix well to obtain suspension, heat the suspension in a water bath at 85℃ for 4h under magnetic stirring, and obtain an orange-red suspension after the reaction. Wash the obtained suspension by centrifugation, dry and obtain a precipitate. Mix 0.1g precipitate with 0.6g urea, calcine at 400℃ for 2h, and obtain magnetic Fe3O4 / Fe2O3 heterogeneous nanorods after calcination.

[0047] S1.2 Preparation of Fe3O4 / Fe2O3@Au nanocomposites:

[0048] 50 mg of Fe3O4 / α-Fe2O3 heterogeneous nanorod powder was dispersed in 150 mL of 1 g / mL polyethyleneimine (PEI) solution to form a suspension. The suspension was heated in a water bath at 90°C for 2 h under magnetic stirring. After the reaction, it was washed and magnetically separated to obtain a red-black solid, namely, Fe3O4 / α-Fe2O3@PEI intermediate.

[0049] Take 15 mg of Fe3O4 / α-Fe2O3@PEI intermediate powder and disperse it in 150 mL of ultrapure water. Add 1 mL of 20 mg / mL chloroauric acid and drop 9 mL of 0.075 wt% sodium borohydride solution. After the solution turns from red to black, centrifuge the suspension obtained by the reaction to obtain a precipitate, wash and dry to obtain a magnetic Fe3O4 / Fe2O3@Au nanocomposite.

[0050] Figure 1 This is a transmission electron microscope photograph of the Fe3O4 / Fe2O3@Au nanocomposite. It can be seen from the figure that the Fe3O4 / Fe2O3@Au nanocomposite is obtained by uniformly dispersing gold nanoparticles on the surface of the Fe3O4 / Fe2O3 magnetic heterostructure with a nanorod structure. The length of the Fe3O4 / Fe2O3 magnetic heterostructure nanorod is 299.4nm and the diameter is 35.7nm; the diameter of the gold nanoparticle is about 10nm.

[0051] S2.PrP C Construction of label-free magnetic self-assembly probe for proteins:

[0052] S21. The Fe3O4 / Fe2O3@Au nanocomposite obtained in step S1 is dispersed in ultrapure water to prepare a Fe3O4 / Fe2O3@Au nanocomposite suspension with a concentration of 20 mg / mL. The Fe3O4 / Fe2O3@Au nanocomposite suspension is mixed with an equal amount of 7.5 μM thiolated aptamer nucleic acid solution and incubated at 4°C for 12 h. After the incubation, the mixture is subjected to magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt.

[0053] S22. Add 30 μL of 0.25 wt% BSA solution to 0.5 mg Fe3O4 / Fe2O3@Au / Apt, mix by ultrasound until uniform, and then incubate the suspension at room temperature in the dark for 1 h. After the incubation, perform magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt / BSA.

[0054] S23. Resuspend Fe3O4 / Fe2O3@Au / Apt / BSA with 30 μL ultrapure water to obtain a resuspension, and drop 9 μL of the resuspension onto the surface of a clean magnetic glassy carbon electrode (MGCE), induce self-assembly by magnetic force, and dry at room temperature to obtain the label-free magnetic self-assembled nanoprobe, recorded as MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA.

[0055] S3.PrP C Protein label-free magnetic self-assembled probe for detecting PrP C Application of protein:

[0056] 9 μL of 100 pg / mL PrP C The protein solution was added dropwise to the MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA surface prepared in step S2 and incubated at 40°C for 30 min to allow it to specifically bind to the thiolated aptamer nucleic acid. C MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode for protein solution.

[0057] The test was carried out using an electrochemical workstation and a three-electrode system to drop PrP C The MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode of the protein solution was used as the working electrode, the silver / silver chloride electrode was used as the reference electrode, and the platinum wire electrode was used as the counter electrode. 5 mM [Fe(CN)6] 3- / 4- The solution was used as an electrolyte, and differential pulse voltammetry (DPV) was performed in the range of 0.1 to 0.6 V using an electrochemical workstation.

[0058] After testing, the DPV current signal of the label-free magnetic self-assembled nanoprobe was 51.72 μA. Based on the "off-type" detection principle, the lower the current signal response value, the more PrP C Proteins are recognized by label-free magnetic self-assembled probes.

[0059] Example 2: PrP C Construction of label-free magnetic self-assembly probes for proteins

[0060] S1. The preparation method of Fe3O4 / Fe2O3@Au nanocomposite is the same as that in Example 1.

[0061] S2.PrP C Construction of label-free magnetic self-assembly probe for proteins:

[0062] S21. The Fe3O4 / Fe2O3@Au nanocomposite obtained in step S1 is dispersed in ultrapure water to prepare a Fe3O4 / Fe2O3@Au nanocomposite suspension with a concentration of 15 mg / mL. The Fe3O4 / Fe2O3@Au nanocomposite suspension is mixed with an equal amount of 5 μM thiolated aptamer nucleic acid solution and incubated at 4°C for 12 h. After the incubation, the mixture is subjected to magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt.

[0063] S22. Add 30 μL of 0.1 wt% BSA solution to 0.5 mg Fe3O4 / Fe2O3@Au / Apt, mix by ultrasound until uniform, and then incubate the suspension at room temperature in the dark for 0.5 h. After the incubation, perform magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt / BSA.

[0064] S23. Resuspend Fe3O4 / Fe2O3@Au / Apt / BSA with 10 μL ultrapure water to obtain a resuspension, and drop 9 μL of the resuspension onto the surface of a clean magnetic glassy carbon electrode (MGCE), induce self-assembly by magnetic force, and dry at room temperature to obtain the label-free magnetic self-assembled nanoprobe, recorded as MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA.

[0065] S3.PrP C Protein label-free magnetic self-assembled probe for detecting PrP C Application of protein:

[0066] 9 μL of 100 pg / mL PrP C The protein solution was added dropwise to the MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA surface prepared in step S2 and incubated at 30°C for 20 min to allow it to specifically bind to the thiolated aptamer nucleic acid. C MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode for protein solution.

[0067] PrP was carried out according to the method described in step S3 of Example 1 C Protein detection: After testing, the DPV current signal of the label-free magnetic self-assembled nanoprobe is 61.21 μA, which shows that compared with the results of Example 1, the label-free magnetic self-assembled nanoprobe was constructed under non-optimal construction conditions in this example.

[0068] Example 3: PrP C Construction of label-free magnetic self-assembly probes for proteins

[0069] S1. The preparation method of Fe3O4 / Fe2O3@Au nanocomposite is the same as that in Example 1.

[0070] S2.PrP C Construction of label-free magnetic self-assembly probe for proteins:

[0071] S21. The Fe3O4 / Fe2O3@Au nanocomposite obtained in step S1 is dispersed in ultrapure water to prepare a Fe3O4 / Fe2O3@Au nanocomposite suspension with a concentration of 30 mg / mL. The Fe3O4 / Fe2O3@Au nanocomposite suspension is mixed with an equal amount of a 7 μM thiolated aptamer nucleic acid solution and incubated at 4°C for 12 h. After the incubation, the mixture is subjected to magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt.

[0072] S22. Add 30 μL of 0.3 wt% BSA solution to 0.5 mg Fe3O4 / Fe2O3@Au / Apt, mix by ultrasound until uniform, and then incubate the suspension at room temperature in the dark for 1.5 h. After the incubation, perform magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt / BSA.

[0073] S23. Resuspend Fe3O4 / Fe2O3@Au / Apt / BSA with 20 μL ultrapure water to obtain a resuspension, and drop 9 μL of the resuspension onto the surface of a clean magnetic glassy carbon electrode (MGCE), induce self-assembly by magnetic force, and dry at room temperature to obtain the label-free magnetic self-assembled nanoprobe, recorded as MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA.

[0074] S3.PrP C Protein label-free magnetic self-assembled probe for detecting PrP C Application of protein:

[0075] 9 μL of 100 pg / mL PrP C The protein solution was added dropwise to the MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA surface prepared in step S2 and incubated at 50°C for 10 min to allow it to specifically bind to the thiolated aptamer nucleic acid. C MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode for protein solution.

[0076] PrP was carried out according to the method described in step S3 of Example 1 C Protein detection: After testing, the DPV current signal of the label-free magnetic self-assembled nanoprobe is 63.97 μA.

[0077] Example 4: PrPC Construction of label-free magnetic self-assembly probes for proteins

[0078] S1. The preparation method of Fe3O4 / Fe2O3@Au nanocomposite is the same as that in Example 1.

[0079] S2.PrP C Construction of label-free magnetic self-assembly probe for proteins:

[0080] S21. The Fe3O4 / Fe2O3@Au nanocomposite obtained in step S1 is dispersed in ultrapure water to prepare a Fe3O4 / Fe2O3@Au nanocomposite suspension with a concentration of 35 mg / mL. The Fe3O4 / Fe2O3@Au nanocomposite suspension is mixed with an equal amount of 8 μM thiolated aptamer nucleic acid solution and incubated at 4°C for 12 h. After the incubation, the mixture is subjected to magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt.

[0081] S22. Add 30 μL of 0.5 wt% BSA solution to 0.5 mg Fe3O4 / Fe2O3@Au / Apt, mix by ultrasound until uniform, and then incubate the suspension at room temperature in the dark for 2 h. After the incubation, wash by magnetic separation to obtain Fe3O4 / Fe2O3@Au / Apt / BSA.

[0082] S23. Resuspend Fe3O4 / Fe2O3@Au / Apt / BSA with 25 μL ultrapure water to obtain a resuspension, and drop 9 μL of the resuspension onto the surface of a clean magnetic glassy carbon electrode (MGCE), induce self-assembly by magnetic force, and dry at room temperature to obtain the label-free magnetic self-assembled nanoprobe, recorded as MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA.

[0083] S3.PrP C Protein label-free magnetic self-assembled probe for detecting PrP C Application of protein:

[0084] 9 μL of 100 pg / mL PrP C The protein solution was added dropwise to the MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA surface prepared in step S2 and incubated at 60°C for 40 min to allow it to specifically bind to the thiolated aptamer nucleic acid. C MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode for protein solution.

[0085] PrP was carried out according to the method described in step S3 of Example 1 CProtein detection: After testing, the DPV current signal of the label-free magnetic self-assembled nanoprobe is 95.75 μA.

[0086] Example 5: PrP C Construction of label-free magnetic self-assembly probes for proteins

[0087] S1. The preparation method of Fe3O4 / Fe2O3@Au nanocomposite is the same as that in Example 1.

[0088] S2.PrP C Construction of label-free magnetic self-assembly probe for proteins:

[0089] S21. The Fe3O4 / Fe2O3@Au nanocomposite obtained in step S1 is dispersed in ultrapure water to prepare a Fe3O4 / Fe2O3@Au nanocomposite suspension with a concentration of 40 mg / mL. The Fe3O4 / Fe2O3@Au nanocomposite suspension is mixed with an equal amount of 9 μM thiolated aptamer nucleic acid solution and incubated at 4°C for 12 h. After the incubation, the mixture is subjected to magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt.

[0090] S22. Add 30 μL of 0.4 wt% BSA solution to 0.5 mg Fe3O4 / Fe2O3@Au / Apt, mix by ultrasound until uniform, and then incubate the suspension at room temperature in the dark for 2.5 h. After the incubation, perform magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt / BSA.

[0091] S23. Resuspend Fe3O4 / Fe2O3@Au / Apt / BSA with 35 μL ultrapure water to obtain a resuspension, and drop 9 μL of the resuspension onto the surface of a clean magnetic glassy carbon electrode (MGCE), induce self-assembly by magnetic force, and dry at room temperature to obtain the label-free magnetic self-assembled nanoprobe, recorded as MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA.

[0092] S3.PrP C Protein label-free magnetic self-assembled probe for detecting PrP C Application of protein:

[0093] 9 μL of 100 pg / mL PrP C The protein solution was added dropwise to the MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA surface prepared in step S2 and incubated at 60°C for 40 min to allow it to specifically bind to the thiolated aptamer nucleic acid. C MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode for protein solution.

[0094] PrP was carried out according to the method described in step S3 of Example 1 C Protein detection: After testing, the DPV current signal of the label-free magnetic self-assembled nanoprobe is 80.12 μA.

[0095] Example 6: PrP C Construction of label-free magnetic self-assembly probes for proteins

[0096] S1. The preparation method of Fe3O4 / Fe2O3@Au nanocomposite is the same as that in Example 1.

[0097] S2.PrP C Construction of label-free magnetic self-assembly probe for proteins:

[0098] S21. The Fe3O4 / Fe2O3@Au nanocomposite obtained in step S1 is dispersed in ultrapure water to prepare a Fe3O4 / Fe2O3@Au nanocomposite suspension with a concentration of 20 mg / mL. The Fe3O4 / Fe2O3@Au nanocomposite suspension is mixed with an equal amount of 10 μM thiolated aptamer nucleic acid solution and incubated at 4°C for 12 h. After the incubation, the mixture is subjected to magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt.

[0099] S22. Add 30 μL of 0.35 wt% BSA solution to 0.5 mg Fe3O4 / Fe2O3@Au / Apt, mix by ultrasound until uniform, and then incubate the suspension at room temperature in the dark for 3 h. After the incubation, wash by magnetic separation to obtain Fe3O4 / Fe2O3@Au / Apt / BSA.

[0100] S23. Resuspend Fe3O4 / Fe2O3@Au / Apt / BSA to obtain a resuspension, drop 9 μL of the resuspension onto the surface of a clean magnetic glassy carbon electrode (MGCE), induce self-assembly by magnetic force, and dry at room temperature to obtain the label-free magnetic self-assembled nanoprobe, recorded as MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA.

[0101] S3.PrP C Protein label-free magnetic self-assembled probe for detecting PrP C Application of protein:

[0102] 9 μL of 100 ng / mL PrP C The protein solution was added dropwise to the MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA surface prepared in step S2 and incubated at 80°C for 35 min to allow it to specifically bind to the thiolated aptamer nucleic acid. CMGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode for protein solution.

[0103] PrP was carried out according to the method described in step S3 of Example 1 C Protein detection: After testing, the DPV current signal of the label-free magnetic self-assembled nanoprobe is 63.57 μA.

[0104] Example 7. PrP C Performance evaluation of protein label-free magnetic self-assembled probes

[0105] In this example, the label-free magnetic self-assembly probe prepared in Example 1 was used as an example, and the current response of the electrode at each stage in the preparation process of the label-free magnetic self-assembly probe was measured by cyclic voltammetry to track the construction process of the label-free magnetic self-assembly probe and verify the design strategy.

[0106] The electrodes in each stage from a to b are bare electrode (MGCE), MGCE / Fe3O4 / Fe2O3, MGCE / Fe3O4 / Fe2O3@Au, MGCE / Fe3O4 / Fe2O3@Au / Apt, MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA, MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA / PrP C ;

[0107] a. Bare electrode (MGCE): clean magnetic glassy carbon electrode;

[0108] b. The preparation process of the MGCE / Fe3O4 / Fe2O3 modified electrode is as follows: ultrasonically disperse the Fe3O4 / Fe2O3 magnetic heterogeneous nanorods in step S11 of Example 1 in ultrapure water to prepare a Fe3O4 / Fe2O3 heterogeneous nanorod suspension with a concentration of 15 mg / mL; take 9 μL of the Fe3O4 / Fe2O3 nanorod suspension and add it dropwise to the surface of a clean magnetic glassy carbon electrode (MGCE), dry it at room temperature, and obtain a MGCE / Fe3O4 / Fe2O3 modified electrode for use.

[0109] c. The preparation process of MGCE / Fe3O4 / Fe2O3@Au modified electrode is as follows: ultrasonically disperse the Fe3O4 / Fe2O3@Au nanocomposite in step S12 of Example 1 in ultrapure water to prepare a Fe3O4 / Fe2O3@Au nanocomposite suspension with a concentration of 15 mg / mL; take 9 μL of the Fe3O4 / Fe2O3@Au nanocomposite suspension and add it dropwise to the surface of a clean magnetic glassy carbon electrode (MGCE), dry it at room temperature, and obtain a MGCE / Fe3O4 / Fe2O3@Au modified electrode for use.

[0110] d. The preparation process of the MGCE / Fe3O4 / Fe2O3@Au / Apt modified electrode is as follows: 9 μL of the Fe3O4 / Fe2O3@Au / Apt suspension after incubation in step S21 of Example 1 is added dropwise to the surface of a clean magnetic glassy carbon electrode (MGCE), and dried at room temperature to obtain a MGCE / Fe3O4 / Fe2O3@Au / Apt modified electrode for use.

[0111] e. The preparation process of MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA modified electrode is the label-free magnetic self-assembled nanoprobe in Example 1.

[0112] f.MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA / PrP C In step S3 of Example 1, PrP is added dropwise C MGCE / Fe3O4 / Fe2O3@Au / Apt / BSA electrode for protein solution.

[0113] The test results are as follows Figure 2 As shown in the figure, it can be seen that the current response value of curve c is higher than that of curve b. This is because the Fe3O4 / Fe2O3 heterogeneous nanoparticles have a large steric hindrance, which hinders the [Fe(CN)6] 3- / 4- The probe reaches the electrode surface, causing the current to weaken. When the Fe3O4 / Fe2O3 heterostructure nanorods are coated with evenly distributed gold nanoparticles, the conductivity of the Fe3O4 / Fe2O3@Au nanocomposite is greatly enhanced, so the peak current increases. Curve d is the peak current decrease after the thiol-modified DNA aptamer is connected to the electrode surface. This is because the negative charge of the DNA phosphate backbone interacts with [Fe(CN)6] 3- / 4- The probes have electrostatic repulsion and the steric hindrance of DNA; the peak current of curve e decreases because BSA has poor conductivity and steric hindrance; curve f is the incubation of PrP C After protein, DNA aptamer and PrP C The protein undergoes a specific binding reaction and a "closed-type" detection occurs, and the electrode electrical signal response weakens.

[0114] In summary, the results of the cyclic voltammetry current response change indicate that a label-free magnetic self-assembled probe that meets the design strategy has been successfully constructed and has the ability to specifically recognize PrP C Protein capacity.

[0115] Example 8: Electrochemical performance of label-free magnetic self-assembled probes

[0116] (1) Selective inspection:

[0117] In this example, the label-free magnetic self-assembled probe constructed in Example 1 was used as the working electrode, and an interfering protein was added to its surface to construct an electrochemical sensor, so as to examine the selectivity of the electrochemical sensor.

[0118] The interfering proteins include alpha-fetoprotein (AFP), nucleolar protein (NCL), ribonucleoprotein (HNRNPA), osteopontin (OPN) and MIX (by PrP C , AFP, NCL, HNRNPA, OPN protein mixture), the concentration of the interfering protein is 10pg / mL, the addition of interfering protein and the construction of the electrochemical sensor refer to the method described in step S3 of Example 1.

[0119] from Figure 3 It can be seen that the presence of various interferents will not affect the specific detection of the sensor, which shows that the electrochemical biosensor has good selectivity and the label-free magnetic self-assembled probe of the present invention has good selectivity.

[0120] (2) Investigation of the detection range of label-free magnetic self-assembled probes:

[0121] In this step, the label-free magnetic self-assembled probe constructed in Example 1 was used as the working electrode to construct an electrochemical sensor. Different concentrations of PrP were added to the surface of the working electrode. C Protein solution (1pg / mL~1×10 6 pg / mL), and investigated the analytical performance of the electrochemical sensor. The results were as follows Figure 4 shown.

[0122] from Figure 4 As can be seen, the current value is related to PrP C The protein concentration showed a good linear correlation, and the concentration range that the label-free magnetic self-assembly probe could detect was 1×10 pg / mL~1×10 5 pg / mL, and its linear regression equation is I(μA)=-6.4965Ig C PrPC (pg / mL)+65.8156, correlation coefficient R 2 =0.997. I represents current, IgC is PrP C The inverse of protein concentration. The detection limit was calculated by linear fitting data to be 1 pg / mL (S / N=3) and the quantification limit was 10 pg / mL (S / N=10).

[0123] In summary, the label-free magnetic self-assembled nanoprobe of the present invention comprises a magnetic Fe3O4 / Fe2O3@Au heterogeneous nanorod array and a thiol-aptamer nucleic acid chain bonded to the nanorod array via an Au-S bond; the biosensor prepared by the label-free magnetic self-assembled nanoprobe has good reproducibility and stability: the RSD value of multiple detections (intra-day / inter-day) is 1.55%; the response signal value of multiple sets of sensors is stored for 0 to 15 days, and the change in the response signal value does not exceed 4%; the biosensor has a low detection limit (1 pg / mL), a wide linear range (1×10 pg / mL~1×10 5 pg / mL) and good sensitivity in detecting PrP C It has good application and is very practical.

[0124] 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 label-free magnetic self-assembled nanoprobe, characterized in that: The label-free magnetic self-assembled nanoprobe comprises a magnetic Fe3O4 / Fe2O3@Au nanocomposite and a thiolated aptamer nucleic acid bound to the nanocomposite via an Au-S bond; The thiolated aptamer nucleic acid is: 5'-SH-(CH2)6-TGCTGGAGGATATCGGTGTTTATGGTGTCTGTCTTCCTCC AGC-3'.

2. The method for preparing the label-free magnetic self-assembled nanoprobe according to claim 1, characterized in that: The preparation method comprises: (1) mixing and incubating the Fe3O4 / Fe2O3@Au nanocomposite suspension with the thiolated aptamer nucleic acid solution, and then performing magnetic separation and washing after the incubation to obtain Fe3O4 / Fe2O3@Au / Apt; (2) adding bovine serum albumin to Fe3O4 / Fe2O3@Au / Apt, mixing by ultrasound until uniform, obtaining a suspension, and then incubating the suspension at room temperature, and after the incubation, performing magnetic separation and washing to obtain Fe3O4 / Fe2O3@Au / Apt / BSA; (3) Resuspending Fe3O4 / Fe2O3@Au / Apt / BSA to obtain a resuspension, dropping the resuspension onto the surface of a clean magnetic glassy carbon electrode, inducing self-assembly by magnetic force, and drying at room temperature to obtain the label-free magnetic self-assembled nanoprobe.

3. The preparation method according to claim 2, characterized in that: In step (1), the preparation method of the Fe3O4 / Fe2O3@Au nanocomposite comprises: S1. Preparation of Fe3O4 / Fe2O3 heterostructure nanorods: FeCl3·6H2O and CO(NH2)2 are dissolved in distilled water, mixed evenly and reacted in a water bath, washed, separated and dried after the reaction, ground the dried product and evenly mixed with urea, and then calcined to obtain Fe3O4 / Fe2O3 heterogeneous nanorods; S2. Preparation of Fe3O4 / Fe2O3@Au nanocomposites: The Fe3O4 / Fe2O3 heterogeneous nanorod powder is dispersed in a polyethyleneimine (PEI) solution, and then heated in a water bath for reaction. After the reaction is completed, it is washed and magnetically separated to obtain a Fe3O4 / α-Fe2O3@PEI intermediate. The Fe3O4 / α-Fe2O3@PEI intermediate is dispersed in ultrapure water, a chloroauric acid solution is added thereto and mixed evenly, and then a sodium borohydride solution is added thereto for mixed reaction. After the reaction is completed, the Fe3O4 / α-Fe2O3@Au nanocomposite is obtained by washing and magnetic separation.

4. The preparation method according to claim 2, characterized in that: In step (1), the dosage ratio of the Fe3O4 / Fe2O3@Au nanocomposite suspension to the thiolated aptamer nucleic acid solution is 15 μL:15 μL; wherein the concentration of the Fe3O4 / Fe2O3@Au nanocomposite suspension is 10-30 mg / mL, and the concentration of the thiolated aptamer nucleic acid solution is 5-10 μM.

5. The preparation method according to claim 2, characterized in that: In step (1), the mixed incubation condition has a temperature range of 30 to 70° C. and a duration of 10 to 50 minutes.

6. The preparation method according to claim 2, characterized in that: In step (2), the dosage ratio of Fe3O4 / Fe2O3@Au / Apt to bovine serum albumin solution is 0.5 mg:30 μL; the concentration range of the bovine serum albumin solution is 0.1-0.5 wt%; The mixed incubation condition has a temperature range of 15 to 30° C. and a duration of 60 to 120 minutes.

7. The preparation method according to claim 2, characterized in that: In step (3), the resuspension solution is ultrapure water, and the dosage range is 10 to 35 μL.

8. The label-free magnetic self-assembled nanoprobe according to claim 1 is used for the preparation and detection of PrP C Application of proteins in electrochemical biosensors.

9. A method for detecting PrP C An electrochemical biosensor for proteins, characterized in that The sensor uses the label-free magnetic self-assembled nanoprobe described in claim 1 as a working electrode, silver / silver chloride as a reference electrode, a platinum wire electrode as a counter electrode, and [Fe(CN)6] containing KCl as a 3- / 4- The solution was used as the electrolyte and the PrP was detected using an electrochemical workstation. C protein.

10. The method for detecting PrP according to claim 9. C An electrochemical biosensor for proteins, characterized in that PrP C The solution was dropped onto the surface of the label-free magnetic self-assembled nanoprobe, incubated for a period of time, and detected in the range of 0.1 to 0.6 V; The PrP C The concentration of the solution is 1×10 pg / mL~1×10 5 pg / mL, the dosage is 10μL; The incubation conditions are: incubation at 40° C. for 30 min.

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