Magnetically-induced self-assembled nanoprobe for detecting KRAS gene as well as preparation method and application of magnetically-induced self-assembled nanoprobe

Abstract

The invention provides a magnetic induction self-assembly type nanoprobe for detecting a KRAS gene as well as a preparation method and application of the magnetic induction self-assembly type nanoprobe, and belongs to the technical field of electrochemical biosensing. The magnetic induction self-assembly type nano probe for detecting the KRAS gene comprises an MGCE and an alpha-Fe2O3 / Fe3O4 (at) Au-ssDNA / BSA / KRAS compound, wherein the alpha-Fe2O3 / Fe3O4 (at) Au-ssDNA / BSA / KRAS compound is magnetically self-assembled to the surface of the MGCE; the magnetic induction self-assembly type nanoprobe is used as a working electrode to construct an electrochemical biosensor, the linear range of detectable concentration of the electrochemical biosensor is 10 pM to 1 mu M, the detection limit is as low as 1.93 pM, the quantification limit is 5.84 pM, and the electrochemical biosensor has the advantages of high sensitivity and wide detection range and has good application in KRAS gene detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical biosensing, and particularly relates to a magnetic induction self-assembled nanoprobe for detecting the KRAS gene, a preparation method thereof, and an application thereof. Background Art

[0002] Early detection of tumors is of great significance in the fields of tumor prevention, diagnosis, and disease monitoring. Detecting tumor-related or specific biomarkers is a very effective approach. Among them, Kirsten rat sarcoma viral oncogene homolog (KRAS gene) belongs to the guanosine triphosphatase (GTPase) family and has an important impact on cell growth, survival, and differentiation. The KRAS gene is one of the most frequently mutated oncogenes in various malignant tumors such as lung cancer, colon cancer, and pancreatic cancer, and this gene exists in 1 out of every 7 cases of human cancers. Therefore, developing a sensitive and efficient method for detecting the expression level of the KRAS gene in tumor tissues is of great significance.

[0003] There are many methods for detecting the KRAS gene. For example, surface-enhanced Raman scattering (SERS), fluorescence imaging, surface plasmon resonance (SPR) technology, and colorimetric methods, etc., but they all have different degrees of deficiencies, such as high cost, complex equipment, narrow application range, etc. The electrochemical biosensor detection method has the advantages of high sensitivity, low cost, fast detection speed, excellent stability, etc. However, in the existing technology, the electrochemical biosensor is rarely used for KRAS gene detection, and it still has the disadvantages of insufficient sensitivity and narrow application range. Therefore, it is necessary to develop a magnetic induction self-assembled nanoprobe for detecting the KRAS gene and assemble it into an electrochemical biosensor for detecting the KRAS gene. Summary of the Invention

[0004] In view of some deficiencies in the existing technology, the present invention provides a magnetic induction self-assembled nanoprobe for detecting the KRAS gene, a preparation method thereof, and an application thereof; the magnetic induction self-assembled nanoprobe for detecting the KRAS gene includes MGCE, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex; the present invention also constructs an electrochemical biosensor by using the above magnetic induction self-assembled nanoprobe as a working electrode. The linear range of the concentration that the electrochemical biosensor can detect is 10 pM–1 μM, the detection limit is as low as 1.93 pM, and the quantification limit is 5.84 pM. It has the advantages of high sensitivity and wide detection range, and has good application in detecting the KRAS gene.

[0005] To achieve the above technical objectives, the present invention adopts the following technical means:

[0006] The present invention first provides a magnetically induced self-assembled nanosensor for detecting KRAS gene, and the nanosensor includes MGCE, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex;

[0007] The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex includes: α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite, thiol ssDNA single strand assembled on the surface of α-Fe 2 O 3 / Fe 3 O 4 @Au nanorod surface through Au-S bond, blocker BSA specifically adsorbed on the material surface, and KRAS gene of the sample to be detected.

[0008] Preferably, the nucleotide sequence of the ssDNA single strand is:

[0009] 5′-SH-TCTTGCCTACGCCACCAGCTCCAACTAC-3′ (SEQ ID No.1); the ssDNA single strand is a DNA containing a thiol (-SH) and a specific nucleic acid sequence, and is used for specifically recognizing and binding to the KRAS gene;

[0010] The nucleotide sequence of the KRAS gene is:

[0011] 5′-GTAGTTGGAGCTGGTGGCGTAGGCAAGA-3′ (SEQ ID No.2);

[0012] The KRAS gene specifically binds to the ssDNA single strand according to the principle of base complementary pairing, and the sensitive detection of the KRAS gene is realized through the change of the electrical signal.

[0013] The present invention also provides a construction method of the above magnetically induced self-assembled nanosensor for detecting KRAS gene, and the construction method includes:

[0014] (1) Mix the thiol ssDNA single strand solution with α-Fe 2 O3 / Fe 3 O 4 @Au nanocomposite to obtain a mixed solution, incubate the mixed solution, and after the incubation, form α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex;

[0015] (2) Disperse the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex into the BSA solution for reaction to block non-specific binding sites, and obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex;

[0016] Disperse the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex with the sample to be detected, the KRAS gene, for incubation, and after the incubation, obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex;

[0017] (3) Resuspend the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex to obtain a resuspended solution, then drop the resuspended solution onto the surface of the MGCE to complete the magnetic induction self-assembly process, and after the self-assembly, dry it to obtain the magnetic induction self-assembled nanosensor for detecting the KRAS gene.

[0018] Preferably, in step (1), the final concentration of the α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite is 3-18 mg / mL; the final concentration of the thiol-modified ssDNA single strand is 3-18 μM;

[0019] The conditions for the incubation are: incubate at 35-38 °C for 2-4 h.

[0020] Preferably, the α-Fe 2 O3 / Fe 3 O 4 The final concentration of the α-FeO / Fe@Au nanocomposite is 15 mg / mL; the final concentration of the thiol-modified single-stranded ssDNA is 4 μM;

[0021] The incubation conditions are: incubation at 37 °C for 3 h.

[0022] Preferably, in step (1), the α-Fe 2 O 3 / Fe 3 O 4 The preparation steps of the @Au nanocomposite include:

[0023] S1. Hydrothermally react the mixed aqueous solution of ferric chloride hexahydrate and cetyltrimethylammonium bromide (CTAB), and after the reaction, centrifuge and wash to obtain β-FeOOH nanorods;

[0024] Mix the β-FeOOH nanorods evenly with the reducing agent maltose and then calcine at high temperature. After the calcination, grind to obtain magnetic α-Fe 2 O 3 / Fe 3 O 4 heterogeneous nanorods;

[0025] S2. Mix the magnetic α-Fe 2 O 3 / Fe 3 O 4 heterogeneous nanorods thoroughly in an aqueous solution of polyethyleneimine (PEI), perform ultrasonic treatment and then heat the reaction. After the reaction, centrifuge, wash, and dry to obtain the solid powder α-Fe 2 O 3 / Fe 3 O 4 @PEI;

[0026] Disperse α-Fe 2 O 3 / Fe 3 O 4 -PEI ultrasonically in ultrapure water to form an α-Fe 2 O 3 / Fe 3 O 4 @PEI suspension, add ice-cold HAuCl 4 solution and then stir ultrasonically to obtain an α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 suspension, and then add sodium citrate solution dropwise to obtain α-Fe2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / citrate trisodium suspension;

[0027] Add dropwise NaBH 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / citrate trisodium suspension, and stir mechanically until the color turns purple-black, then perform magnetic separation, washing, drying, and grinding to obtain α-Fe 4 O 2 O 3 / Fe 3 O 4 @Au nanocomposite.

[0028] Preferably, in step S1, in the mixed aqueous solution of ferric chloride hexahydrate and CTAB, the dosage ratio of ferric chloride hexahydrate to cetyltrimethylammonium bromide is 2 mmol: 3.75 mmol; the conditions of the hydrothermal reaction are: hydrothermal reaction at 80-120 °C for 2 h in a programmable temperature-controlled furnace; the mass ratio of the β-FeOOH nanorods to the reducing agent maltose is 1: (1-10); the conditions of the high-temperature calcination are: high-temperature calcination at 200-700 °C for 2 h;

[0029] In step S2, the mass ratio of the magnetic α-Fe 2 O 3 / Fe 3 O 4 heterogeneous nanorods to PEI is (20-35): 1, and the concentration range of the polyethyleneimine PEI solution is 0.005-0.02 g / mL; the conditions of the heating reaction are: heating reaction at 90 °C for 2 h;

[0030] The α-Fe 2 O 3 / Fe 3 O 4 @PEI suspension and the HAuCl 4 solution have a volume ratio of (140-160): 1, and the concentration range of the HAuCl 4 solution is 15-25 mg / mL;

[0031] The citrate trisodium solution and the α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4The volume ratio of the suspension is 1:50, and the concentration range of the trisodium citrate solution is 35-40 mM;

[0032] The NaBH 4 solution and α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / trisodium citrate suspension has a volume ratio of 1:(15-19), where the concentration range of the NaBH 4 solution is 0.06-0.09 wt%.

[0033] Preferably, in step S1, the mass ratio of the β-FeOOH nanorods to the reducing agent maltose is 1:10;

[0034] In step S2, the mass ratio of the magnetic α-Fe 2 O 3 / Fe 3 O 4 heterostructure nanorods to PEI is 30:1, and the concentration range of the polyethyleneimine PEI solution is 0.01 g / mL;

[0035] The α-Fe 2 O 3 / Fe 3 O 4 @PEI suspension and the HAuCl 4 solution have a volume ratio of 150:1, and the concentration range of the HAuCl 4 solution is 20 mg / mL;

[0036] The concentration range of the trisodium citrate solution is 38 mM;

[0037] The NaBH 4 solution and α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / trisodium citrate suspension has a volume ratio of 1:17, where the concentration range of the NaBH 4 solution is 0.075 wt%.

[0038] Preferably, in step (2), the dosage ratio of the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex to the BSA solution is 15 mg:1 mL;

[0039] The concentration of the BSA solution is 0.20 - 0.30 wt%.

[0040] Preferably, the concentration of the BSA solution is 0.25%.

[0041] Preferably, in step (2), the dosage ratio of α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex to the KRAS gene is 15 mg: 1 mL;

[0042] The concentration of the KRAS gene is 10 pM - 1 μM.

[0043] The present invention also provides the application of the above magnetically induced self-assembled nanosensor for detecting the KRAS gene in the preparation of an electrochemical biosensor for detecting the KRAS gene.

[0044] The present invention also provides an electrochemical biosensor for detecting the KRAS gene. The electrochemical biosensor uses the above magnetically induced self-assembled nanosensor for detecting the KRAS gene as the working electrode, the Ag / AgCl electrode and the Pt electrode as the reference electrode and the auxiliary electrode respectively, and a [Fe(CN) 6 3- / 4- solution containing KCl as the electrolyte. The content of KRAS is converted into a current response through an electrochemical workstation to detect the KRAS gene.

[0045] Preferably, the electrolyte includes: 5 mM K 4 Fe(CN) 6 、5 mM K 3 Fe(CN) 6 、0.1 M KCl;

[0046] When the electrochemical biosensor is detecting, the EIS parameters are: the frequency range is 0.01 Hz - 100 kHz, and the signal amplitude is 1 mV - 10 mV;

[0047] The CV parameters are: the scanning voltage is -0.5 V - 0.5 V, and the scanning speed is 10 mV / s - 100 mV / s.

[0048] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0049] (1) The present invention provides a magnetically induced self-assembled nanosensor for detecting the KRAS gene. Compared with other detection technologies, the detection method of the present invention has the advantages of small device volume, low cost, good detection performance, fast information reading, etc. During the preparation process of the nanosensor, α-Fe 2 O​3 / Fe 3 O 4 @Au rod-shaped nanocomposites are used in a signal amplification strategy, effectively expanding the detection range, improving the sensitivity and specificity of detection, and achieving a more convenient and straightforward accurate detection of the KRAS gene; the α-Fe 2 O 3 / Fe 3 O 4 @Au rod-shaped nanocomposites are magnetic and can be adsorbed onto the surface of the MGCE through magnetic induction self-assembly technology; this requires low experimental equipment requirements, is simple and easy to operate, and is portable. In addition, in the present invention, a thiol ssDNA single strand is connected to the surface of the α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposites through Au-S bonds, with simple and quick operation, less consumables, and no need for other materials.

[0050] (2) The present invention constructs a magnetic induction self-assembled nanosensor for detecting the KRAS gene into an electrochemical biosensor. The electrochemical sensor realizes a high-affinity binding effect between the ssDNA probe and the KRAS gene based on the principle of base complementary pairing, significantly enhancing the sensitivity, specificity, and stability of detecting the KRAS gene; the electrochemical sensor requires a small amount of probe for each detection, has simple equipment and easy operation, and low cost; the reaction conditions are mild, and it is easy for operators to conduct experiments.

[0051] (3) The linear range of the detectable concentration of the electrochemical biosensor of the present invention is 10 pM to 1 μM, the detection limit is as low as 1.93 pM, and the quantification limit is 5.84 pM. It has a low detection limit, a wide linear range, and good sensitivity, and can better detect the KRAS gene. Description of the Drawings

[0052] Figure 1 For the transmission electron microscope photograph of the α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposites, where the scale bar in the figure is 20 nm;

[0053] Figure 2 For the cyclic voltammogram of the construction process of the magnetic induction self-assembled nanosensor for detecting the KRAS gene.

[0054] Figure 3 For the alternating current impedance spectrum of the construction process of the magnetic induction self-assembled nanosensor for detecting the KRAS gene.

[0055] Figure 4Peak currents for the magnetic induction self-assembled nanoprobes for detecting the KRAS gene to detect single-base mismatch (SBM, 1 μM), double-base mismatch (DBM, 1 μM), blank group (TE-Buffer), and KRAS (1 μM) solution.

[0056] Figure 5 Linear relationship diagram for the magnetic induction self-assembled nanoprobes for detecting the KRAS gene using ssDNA single strands as recognition probes to detect different concentrations of KRAS. Detailed implementation manners

[0057] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. Obviously, the described embodiments are only a part of the embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative efforts, such as embodiments that only change the use without changing the basic principles involved in the claims, fall within the protection scope of the present invention.

[0058] Raw materials used in the following examples:

[0059] Tris(2-carboxyethyl)phosphine, abbreviated as TCEP; bovine serum albumin, abbreviated as BSA; TCEP and BSA were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Kirsten rat sarcoma viral oncogene homolog (KRAS gene) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0060] The nucleotide sequence information involved is:

[0061] The nucleotide sequence of the ssDNA single strand is: 5′-SH-TCTTGCCTACGCCACCAGCTCCAACTAC-3′ (SEQ ID No.1), where SH represents a mercapto group;

[0062] The nucleotide sequence of the KRAS gene is: 5′-GTAGTTGGAGCTGGTGGCGTAGGCAAGA-3′ (SEQ ID No.2);

[0063] The nucleotide sequence of single-base mismatch (SBM) is: 5′-GTAGTTGCAGCTGGTGGCGTAGGCAAGA-3′ (SEQ ID No.3);

[0064] The nucleotide sequence of double-base mismatch (DBM) is: 5′-GTAGTTGCAGCTGGTGGCGTTGGCAAGA-3′ (SEQ ID No.4).

[0065] Example 1: Magnetic α-Fe 2 O 3 / Fe3 O 4 Preparation of @Au Nanocomposite

[0066] (1) Weigh 2 mmol of ferric chloride hexahydrate and 3.75 mmol of CTAB. After dissolving them uniformly in water, use the hydrothermal method to hydrothermally react at 120 °C for 2 h to prepare β-FeOOH nanorods. Mix the β-FeOOH nanorods and the reducing agent maltose evenly at a mass ratio of 1:10 and calcine them at 400 °C for 2 h. After the calcination, magnetic α-Fe 2 O 3 / Fe 3 O 4 heterogeneous nanorods are obtained.

[0067] (2) Add 150 mL of double-distilled water to 1.5 g of polyethyleneimine (PEI) to dissolve it, obtaining a polyethyleneimine (PEI) solution. Then add 50 mg of ground α-Fe 2 O 3 / Fe 3 O 4 and mix well. After ultrasonic treatment for 30 min, heat and react at 90 °C for 2 h. After the reaction, centrifuge the solution, discard the supernatant, and collect the solid. First, wash the obtained solid precipitate once with absolute ethanol, and then wash it twice with double-distilled water. After the washing, dry the obtained precipitate in a vacuum oven at 60 °C and grind it into powder to obtain α-Fe 2 O 3 / Fe 3 O 4 @PEI for standby.

[0068] (3) Ultrasonically disperse 15 mg of α-Fe 2 O 3 / Fe 3 O 4 @PEI in 150 mL of double-distilled water. Then add 1 mL of a solution with a concentration of 20 mg / mL of HAuCl 4 solution, and ultrasonically treat it in an ice bath for 30 min. After the ultrasonic treatment, add 3 mL of a solution with a concentration of 38 mM of trisodium citrate solution and stir for 1 min to obtain α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / trisodium citrate suspension;

[0069] (4) Under mechanical stirring conditions, slowly drip 9 mL of a freshly prepared solution with a concentration of 0.075 wt% of NaBH 4 solution into α-Fe 2 O 3 / Fe 3O 4 @PEI / HAuCl 4 in the @PEI / HAuCl / sodium citrate suspension, mechanical stirring was maintained for 15 min until the color of the suspension gradually changed from wine red to purple black. After collecting the solid by magnetic separation, it was washed with ethanol and water respectively, and dried in vacuum at 60 °C and ground to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite.

[0070] Figure 1 For α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite transmission electron microscope photograph; it can be seen from the electron microscope photograph that gold nanoparticles with an average diameter of 7.1 nm are coated on the surface of the α-Fe 2 O 3 / Fe 3 O 4 heterogeneous nanorod surface.

[0071] Example 2: Preparation of a magnetically induced self-assembled nanoprobe for detecting the KRAS gene

[0072] In this example, a magnetically induced self-assembled nanoprobe for detecting the KRAS gene was first prepared and assembled into an electrochemical biosensor. The electrochemical properties of the nanocomposites at each stage during the preparation of the nanoprobe were investigated to evaluate the feasibility of detecting the KRAS gene. The specific steps are as follows:

[0073] S1. Preparation of a magnetically induced self-assembled nanoprobe for detecting the KRAS gene:

[0074] (1) A certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) was added to the ssDNA dry powder, sealed and vortexed to mix evenly to prepare a mother liquor with a concentration of 100 μM. 10 mM TCEP was added at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100. After vortexing and mixing, it was activated at 30 °C for 1 h to eliminate disulfide bonds and set aside;

[0075] To the activated 30 μL of 4 μM thiol-ssDNA single-strand solution, 0.45 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite was added, and it was continuously shaken and incubated in a constant temperature shaker at 37 °C for 3 h. After the incubation ended, α-Fe 2 O 3 / Fe 3 O4 @Au-ssDNA complex

[0076] (2) Disperse the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex into 30 μL of 0.25% BSA solution, incubate at 37 °C for 1 h to block non-specific binding sites, and after incubation, obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex. Then add all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomplex and 30 μL of 1 μM KRAS gene, incubate with shaking at 37 °C for 20 min, and after incubation, centrifuge and wash to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex.

[0077] (3) Resuspend the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex in PBS solution and wash twice, then resuspend in 30 μL of ultrapure water to obtain a suspension with a concentration of 15 mg / mL; Drop 9 μL of the suspension onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dries, a uniform magnetic nanoparticle composite film is formed on the electrode surface to obtain the magnetic induction self-assembled nanosensor for detecting KRAS gene.

[0078] Example 3: Preparation of a magnetic induction self-assembled nanosensor for detecting KRAS gene

[0079] (1) Add a certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) to the ssDNA dry powder, seal and vortex to mix evenly to prepare a mother liquor with a concentration of 100 μM. Add 10 mM TCEP at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100, vortex and mix evenly, and activate at 30 °C for 1 h to eliminate disulfide bonds for standby;

[0080] Add 0.15 mg of α-Fe 2 O3 / Fe 3 O 4 @Au nanocomposite was continuously incubated with shaking in a constant temperature shaker at 37 °C for 3 h. After the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was obtained.

[0081] (2) All the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complexes were dispersed into 30 μL of 0.25% BSA solution and incubated at 37 °C for 1 h to block non-specific binding sites. After the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complexes were obtained. Then all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomposites were incubated with 30 μL of 1 μM KRAS gene at 37 °C with shaking for 20 min. After the incubation, centrifugation and washing were carried out to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complexes.

[0082] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complexes were resuspended in PBS solution and washed twice, and then resuspended in 30 μL of ultrapure water to obtain a suspension with a concentration of 5 mg / mL; 9 μL of the suspension was dropped onto the surface of MGCE pretreated with 3 mm alumina powder. After the liquid was dried, a uniform magnetic nanocomposite film was formed on the electrode surface to obtain the magnetic induction self-assembled nanosensor for detecting KRAS gene.

[0083] Example 4: Preparation of a magnetic induction self-assembled nanosensor for detecting KRAS gene

[0084] (1) Add a certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) to the ssDNA dry powder, seal and vortex to mix evenly to prepare a mother liquor with a concentration of 100 μM. Add 10 mM TCEP at a molar ratio of 1:100, C(ssDNA):C(TCEP)=1:100, vortex to mix evenly and activate at 30 °C for 1 h to eliminate disulfide bonds for standby;

[0085] Add 0.30 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite to the activated 30 μL of 3 μM thiol ssDNA single-strand solution, continuously oscillate and incubate in a constant-temperature shaker at 37 °C for 3 h. After the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex is obtained.

[0086] (2) Disperse all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex into 30 μL of 0.25% BSA solution, incubate at 37 °C for 1 h to block non-specific binding sites. After the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex is obtained. Then disperse all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomposite with 30 μL of 1 μM KRAS gene and oscillate and incubate at 37 °C for 20 min. After the incubation, centrifuge and wash to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex.

[0087] (3) Disperse α-Fe 2 O 3 / Fe 3 O 4The @Au-ssDNA / BSA / KRAS complex was resuspended in PBS solution and washed twice, and then resuspended in 30 μL of ultrapure water to obtain a suspension with a concentration of 10 mg / mL. 9 μL of the suspension was dropped onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dried, a uniform magnetic rice composite film was formed on the electrode surface, and the magnetic induction self-assembled nanosensor for detecting the KRAS gene was obtained.

[0088] Example 5: Preparation of a magnetic induction self-assembled nanosensor for detecting the KRAS gene

[0089] (1) A certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) was added to the ssDNA dry powder, and the mixture was sealed and vortexed to prepare a stock solution with a concentration of 100 μM. 10 mM TCEP was added at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100. After vortexing and mixing, the mixture was activated at 30 °C for 1 h to eliminate disulfide bonds and set aside.

[0090] To the activated 30 μL of 3 μM thiol-ssDNA single-stranded solution, 0.54 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite was added, and the mixture was continuously shaken and incubated in a constant temperature shaker at 37 °C for 3 h. After the incubation, the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was obtained.

[0091] (2) All of the above-obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was dispersed into 30 μL of 0.25% BSA solution and incubated at 37 °C for 1 h to block non-specific binding sites. After the incubation, the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex was obtained. Then, all of the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomposite was incubated with 30 μL of 1 μM KRAS gene at 37 °C with shaking for 20 min. After the incubation, centrifugation and washing were performed to obtain α-Fe 2 O 3 / Fe 3 O4 @Au-ssDNA / BSA / KRAS complex

[0092] (3) Add α-Fe 2 O 3 / Fe 3 O 4 The @Au-ssDNA / BSA / KRAS complex was resuspended in PBS solution and washed twice, and then resuspended in 30 μL of ultrapure water to obtain a resuspension with a concentration of 18 mg / mL; 9 μL of the resuspension was dropped onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dried, a uniform magnetic rice composite film was formed on the electrode surface to obtain the magnetic induction self-assembled nanosensor for detecting the KRAS gene.

[0093] Example 6: Preparation of a magnetic induction self-assembled nanosensor for detecting the KRAS gene

[0094] (1) Add a certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) to the ssDNA dry powder, seal and vortex to mix evenly to prepare a mother liquor with a concentration of 100 μM. Add 10 mM TCEP at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100, vortex and mix evenly, and activate at 30 °C for 1 h to eliminate disulfide bonds for standby;

[0095] Add 0.45 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite to the activated 30 μL of 5 μM thiol-ssDNA single-strand solution, and continuously oscillate and incubate in a constant temperature shaker at 37 °C for 3 h. After the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was obtained.

[0096] (2) Disperse all the above α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex into 30 μL of 0.25% BSA solution, incubate at 37 °C for 1 h to block non-specific binding sites. After the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex was obtained. Then all the obtained α-Fe 2 O 3 / Fe 3 O4 The Au-ssDNA / BSA nanocomplex was incubated with 30 μL of 1 μM KRAS gene at 37 °C with shaking for 20 min. After incubation, centrifugation and washing were performed to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex.

[0097] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex was resuspended in PBS solution and washed twice, and then resuspended in 30 μL of ultrapure water to obtain a suspension with a concentration of 15 mg / mL. 9 μL of the suspension was dropped onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dried, a uniform magnetic nanocomposite film was formed on the electrode surface to obtain the magnetic induction self-assembled nanosensor for detecting the KRAS gene.

[0098] Example 7: Preparation of a magnetic induction self-assembled nanosensor for detecting the KRAS gene

[0099] (1) A certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) was added to the ssDNA dry powder, and the mixture was sealed and vortexed to prepare a mother liquor with a concentration of 100 μM. 10 mM TCEP was added at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100. After vortexing, it was activated at 30 °C for 1 h to eliminate disulfide bonds and reserved;

[0100] To the activated 30 μL of 4 μM thiol-ssDNA single-strand solution, 0.45 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomplex was added, and it was continuously incubated with shaking in a constant temperature shaker at 37 °C for 3 h. After incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was obtained.

[0101] (2) All of the above-obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was dispersed into 30 μL of 0.25% BSA solution and incubated at 37 °C for 1 h to block non-specific binding sites. After incubation, α-Fe2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex. Then all the resulting α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanoparticles were incubated with 30 μL of 1 μM KRAS gene at 37 °C with shaking for 10 min. After incubation, centrifugation and washing were performed to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex.

[0102] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex was resuspended in PBS solution and washed twice, and then resuspended in 30 μL of ultrapure water to obtain a suspension with a concentration of 15 mg / mL; 9 μL of the suspension was dropped onto the surface of MGCE pretreated with 3 mm alumina powder. After the liquid dried, a uniform magnetic nanoparticle composite film was formed on the electrode surface to obtain the magnetic induction self-assembled nanosensor for detecting KRAS gene.

[0103] Example 8: Preparation of Magnetic Induction Self-Assembled Nanosensor for Detecting KRAS Gene

[0104] (1) A certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) was added to the ssDNA dry powder, and the mixture was sealed and vortexed to prepare a stock solution with a concentration of 100 μM. 10 mM TCEP was added at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100. After vortexing, the mixture was activated at 30 °C for 1 h to eliminate disulfide bonds and reserved for use;

[0105] To the activated 30 μL of 4 μM thiol-ssDNA single-strand solution, 0.45 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanoparticles were added, and the mixture was continuously incubated with shaking in a constant temperature shaker at 37 °C for 3 h. After incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was obtained.

[0106] (2) Disperse all of the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex into 30 μL of 0.25% BSA solution, and incubate at 37 °C for 1 h to block non-specific binding sites. After incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex is obtained. Then, disperse all of the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomplex with 30 μL of 1 μM KRAS gene, and incubate with shaking at 37 °C for 30 min. After incubation, centrifuge and wash to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex.

[0107] (3) Resuspend the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex in PBS solution and wash twice, then resuspend in 30 μL of ultrapure water to obtain a suspension with a concentration of 15 mg / mL; Drop 9 μL of the suspension onto the surface of MGCE pretreated with 3 mm alumina powder. After the liquid dries, a uniform magnetic rice composite film is formed on the electrode surface to obtain the magnetic induction self-assembled nanosensor for detecting KRAS gene.

[0108] Example 9: Preparation of a magnetic induction self-assembled nanosensor for detecting KRAS gene

[0109] (1) Add a certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) to the ssDNA dry powder, seal and vortex to mix evenly to prepare a mother liquor with a concentration of 100 μM. Add 10 mM TCEP at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100, vortex and mix evenly, then activate at 30 °C for 1 h to eliminate disulfide bonds, and set aside;

[0110] Add 0.45 mg of α-Fe 2 O 3 / Fe 3 O 4@Au nanocomposites were continuously incubated with shaking in a constant temperature shaker at 37 °C for 3 h. After incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA composites were obtained.

[0111] (2) All of the above-obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA composites were dispersed into 30 μL of 0.25% BSA solution and incubated at 37 °C for 1 h to block non-specific binding sites. After incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA composites were obtained. Then, all of the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomposites were incubated with shaking at 37 °C for 40 min with 30 μL of 1 μM KRAS gene. After incubation, centrifugation and washing were performed to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS composites.

[0112] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS composites were resuspended in PBS solution and washed twice, and then resuspended in 30 μL of ultrapure water to obtain a suspension with a concentration of 15 mg / mL; 9 μL of the suspension was dropped onto the surface of MGCE pretreated with 3 mm alumina powder. After the liquid dried, a uniform magnetic nanoparticle composite film was formed on the electrode surface to obtain the magnetic induction self-assembled nanosensor for detecting KRAS gene.

[0113] Example 10: Preparation of a Magnetic Induction Self-Assembled Nanosensor for Detecting KRAS Gene

[0114] (1) Add a certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) to the dry ssDNA powder, seal and vortex to mix evenly to prepare a mother liquor with a concentration of 100 μM. Add 10 mM TCEP at a molar ratio of 1:100, C(ssDNA):C(TCEP) = 1:100, vortex to mix evenly and activate at 30 °C for 1 h to eliminate disulfide bonds, and set aside;

[0115] Add 0.45 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite to the activated 30 μL of 4 μM thiol-ssDNA single-stranded solution, and continuously oscillate and incubate in a constant temperature shaker at 37 °C for 3 h. After the incubation, obtain the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex.

[0116] (2) Disperse all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex into 30 μL of 0.25% BSA solution, and incubate at 37 °C for 1 h to block non-specific binding sites. After the incubation, obtain the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex. Then disperse all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomposite with 30 μL of 1 μM KRAS gene and oscillate and incubate at 37 °C for 50 min. After the incubation, centrifuge and wash to obtain the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS complex.

[0117] (3) For α-Fe 2 O 3 / Fe 3 O 4The @Au-ssDNA / BSA / KRAS complex was resuspended in PBS solution and washed twice, and then resuspended in 30 μL ultrapure water to obtain a resuspension with a concentration of 15 mg / mL; 9 μL of the resuspension was dropped onto the MGCE surface pretreated with 3 mm alumina powder, and after the liquid dried, a uniform magnetic rice complex film was formed on the electrode surface to obtain the magnetically induced self-assembled nanoprobe for detecting the KRAS gene.

[0118] Example 11: Investigation of the electrochemical performance of magnetically induced self-assembled nanoprobes for detecting KRAS gene

[0119] In this example, the magnetically induced self-assembled nanoprobe for detecting the KRAS gene prepared in Example 2 and the intermediate products obtained in each stage were assembled into an electrochemical biosensor, and the electrochemical performance of the nanoprobe during the preparation process was investigated to evaluate its feasibility of detecting the KRAS gene. The specific steps are as follows:

[0120] (1) Bare MGCE, MGCE / α-Fe 2 O 3 / Fe 3 O 4 、MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au、MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA、MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA and MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS was used as the working electrode, Ag / AgCl electrode and Pt electrode were used as the reference and auxiliary electrodes respectively, and the electrolyte (5mMK 4 Fe(CN) 6 , 5mM K 3 Fe(CN) 6 , 0.1 M KCl), connected to an electrochemical workstation (purchased from Shanghai Chenhua Instrument Co., Ltd., CHI660E), to obtain an electrochemical biosensor.

[0121] Among them, MGCE / α-Fe 2 O 3 / Fe3 O 4 : 0.45 mg α-Fe 2 O 3 / Fe 3 O 4 Add 30 μL of ultrapure water to prepare a suspension with a concentration of 15 mg / mL. Drop 9 μL of the suspension onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dries, a uniform magnetic rice composite film is formed on the electrode surface.

[0122] MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au: 0.45 mg α-Fe 2 O 3 / Fe 3 O 4 Add 30 μL of ultrapure water to @Au to prepare a suspension with a concentration of 15 mg / mL. Drop 9 μL of the suspension onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dries, a uniform magnetic rice composite film is formed on the electrode surface.

[0123] MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA: Add 0.45 mg α-Fe to 30 μL of a 4 μM mercapto-ssDNA single-strand solution 2 O 3 / Fe 3 O 4 @Au, and continuously oscillate and incubate in a constant-temperature shaker at 37 °C for 3 h. After the incubation, an α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex is obtained. Magnetically separate and remove the supernatant, resuspend the solid in 30 μL of ultrapure water to obtain a resuspended solution with a concentration of 15 mg / mL; drop 9 μL of the resuspended solution onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dries, a uniform magnetic rice composite film is formed on the electrode surface.

[0124] MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA: Add 0.45 mg α-Fe to 30 μL of a 4 μM mercapto-ssDNA single-strand solution 2 O 3 / Fe 3 O4 @Au was continuously oscillated and incubated in a constant temperature shaker at 37 °C for 3 h. After the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was obtained. The supernatant was removed by magnetic separation, and the solid was dispersed in 30 μL of 0.25% BSA solution and incubated at 37 °C for 1 h to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA. The supernatant was removed by magnetic separation and resuspended in 30 μL of ultrapure water to obtain a resuspension with a concentration of 15 mg / mL; 9 μL of the resuspension was dropped onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dried, a uniform magnetic nanocomposite film was formed on the electrode surface. MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS: Prepared from step S1.

[0125] (2) The above electrochemical biosensors were measured using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) respectively to monitor the step-by-step construction process of the sensors; differential pulse voltammetry (DPV) was used to optimize the experimental conditions and evaluate the analytical performance of the electrochemical biosensors.

[0126] Among them, the CV parameters were -0.1 to 0.7 V and the scanning rate was 100 mV / s.

[0127] The EIS frequency range was 0.1 Hz to 10 kHz, the signal amplitude was 5 mV, the DPV voltage was 0 to 0.6 V, the vibration amplitude was 0.05 V, the pulse width was 0.06 s, the sampling width was 0.02 s, and the pulse period was 0.5 s.

[0128] The detection results are as Figure 2 and 3 shown.

[0129] Figure 2 is the cyclic voltammogram of the construction process of a magnetic induction self-assembled nanoprobe for detecting the KRAS gene. It can be seen from the figure that based on the bare MGCE electrode (curve a), after modification with α-Fe 2 O 3 / Fe 3 O 4 the current signal of the modified electrode (curve b) decreased significantly; however, after modification with α-Fe 2 O 3 / Fe 3 O4 The current signal of the Au-modified electrode (curve c) increased significantly, successfully amplifying the current signal. This is because Au nanoparticles have good electrical conductivity, and after modification, they can accelerate the mutual transfer of electrons, thereby improving the sensitivity of the electrochemical sensor. ssDNA is DNA containing thiol modification. TCEP is used to reduce the disulfide bond of ssDNA to thiol, and then it is used as a probe to combine with α-Fe 2 O 3 / Fe 3 O 4 @Au material surface to obtain MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA (curve d). Compared with curve c, the redox current decreased. On the one hand, since the ssDNA single strand contains a phosphate backbone, it is negatively charged, resulting in mutual repulsion with the redox probe [Fe(CN) 6 3- / 4- in the electrolyte that also carries a negative charge; on the other hand, due to the steric hindrance effect of DNA, it makes [Fe(CN) 6 3- / 4- difficult to achieve electron transfer on the electrode surface, resulting in the attenuation of the electrochemical signal. In the successfully prepared α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA, BSA is added to block non-specific sites to obtain MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA (curve e). Compared with curve d, due to the steric hindrance effect of the protein, the redox current further decreased. When the probe molecule is incubated with the KRAS gene, ssDNA specifically recognizes the KRAS gene through base complementary pairing to obtain MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS (curve f). Compared with curve e, the redox current decreased, proving that the constructed magnetic induction self-assembled nanosensor for detecting the KRAS gene can detect the KRAS gene, and the current decrease is also due to the phosphate backbone and steric hindrance effect of the KRAS gene.

[0130] Figure 3 ​​AC impedance spectrogram of the construction process of a magnetically induced self-assembled nanoprobe for detecting the KRAS gene. It can be seen from the figure that the semicircle diameter of MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au (curve c) is smaller than that of MGCE / α-Fe 2 O 3 / Fe 3 O 4 (curve b), indicating that the electron transfer resistance of MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au is small. The semicircle diameter of MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA (curve d) is larger than that of curve c, proving that the ssDNA single-strand recognition probe has been successfully modified onto α-Fe 2 O 3 / Fe 3 O 4 @Au. Compared with curve d, with the addition of BSA, the semicircle diameter of MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA (curve e) continues to increase, indicating an increase in electron transfer resistance. BSA can play a role in blocking non-specific sites. Compared with curve e, the semicircle diameter of the MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / KRAS (curve f) electrode is larger, indicating that the KRAS gene has successfully bound to the ssDNA single-strand recognition probe. The above results prove that the construction scheme of the magnetically induced self-assembled nanoprobe for detecting the KRAS gene as an electrochemical sensor for detecting the KRAS gene is feasible.

[0131] In summary, the magnetically induced self-assembled nanoprobe for detecting the KRAS gene described in the present invention can be used to detect the KRAS gene.

[0132] Example 12: Investigation of the selectivity of the electrochemical biosensor

[0133] In this example, first, taking the magnetically induced self-assembled nanoprobe for detecting the KRAS gene prepared in Example 2 as an example, it was assembled into an electrochemical biosensor according to the method described in Example 11. The peak currents of single-base mismatch (SBM, 1 μM), double-base mismatch (DBM, 1 μM), blank group (TE-Buffer), and KRAS (1 μM) solution were detected respectively. The detection results are as Figure 4 shown.

[0134] As Figure 4 can be seen, the peak currents of the magnetically induced self-assembled nanoprobe for detecting the KRAS gene prepared in Example 2 when detecting single-base mismatch (SBM, 1 μM), double-base mismatch (DBM, 1 μM), and blank group (TE-Buffer) are significantly different from the current values of the KRAS group, indicating that it can specifically recognize the KRAS gene, and other similar genes will not interfere with its recognition and detection, and will not have a significant impact on the detection results, thus proving its excellent selectivity. Therefore, it has strong selectivity.

[0135] In this example, the above method was also used to investigate the selectivity of the magnetically induced self-assembled nanoprobes for detecting the KRAS gene prepared in Examples 3 to 10. The investigation results are shown in Table 1

[0136] Table 1. Selectivity investigation of magnetically induced self-assembled nanoprobes for detecting the KRAS gene prepared under different conditions

[0137]

[0138] As can be seen from Table 1, although Examples 3 to 10 have methods with relatively strong selectivity, they are still not as good as Example 2.

[0139] In this example, the performance of the magnetically induced self-assembled nanoprobes for detecting the KRAS gene prepared in Examples 3 to 10 was also investigated. The investigation results are shown in Table 2.

[0140] Table 2. Partial performance analysis of magnetically induced self-assembled nanoprobes for detecting the KRAS gene prepared under different conditions

[0141]

[0142] In summary, compared with Example 1, Examples 3 to 10 do not have sufficient performance advantages. Therefore, the reaction parameters in Example 1 are the optimal reaction parameters.

[0143] Example 13: Investigation of the detection limit and detection range of the electrochemical biosensor

[0144] In this example, first, taking the magnetically induced self-assembled nanoprobe for detecting the KRAS gene prepared in Example 2 as an example, it was assembled into an electrochemical biosensor according to the method described in Example 11. Then, different concentrations of KRAS were detected, and a standard curve was plotted to investigate the detection limit, quantification limit, and detection range of the electrochemical biosensor. The specific steps are as follows:

[0145] α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA was incubated with KRAS at concentrations of 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM respectively, and the obtained current values were analyzed and fitted with the logarithm of the KRAS concentration.

[0146] The investigation results are as Figure 5 shown. It can be seen from the figure that the current response is linearly negatively correlated with the logarithm of the KRAS concentration. The standard curve is I = -5.83lg C Kras +119.84 (R 2 = 0.997). Its detection limit is 1.93 pM (LOD = 3.3σ / slope), the quantification limit is 5.84 pM (LOQ = 10σ / slope), and the linear range is 10 pM to 1 μM. Its LOD and detection range are comparable to the prior art, but the sensor constructed in this example has high biocompatibility, and the construction process is simple and the operation is easy. In terms of detection, the advantage of magnetic self-assembly enables it to firmly bind to the MGCE without additional operation control, facilitating detection.

[0147] In summary, the magnetically induced self-assembled nanoprobe for detecting the KRAS gene of the present invention realizes linear quantification of the KRAS concentration in the dynamic range of 10 pM to 1 μM, and has high sensitivity and specificity.

[0148] To sum up, the present invention constructs a magnetically induced self-assembled nanoprobe for detecting the KRAS gene. Among them, ssDNA specifically captures the KRAS gene through base complementary pairing, and then is adsorbed onto the surface of the magnetic glassy carbon electrode (MGCE) through magnetic self-assembly to achieve sensitive detection; the present invention constructs an electrochemical biosensor with the above nanoprobe as the working electrode. The linear range of the concentration that the electrochemical biosensor can detect is 10 pM to 1 μM, the detection limit is as low as 1.93 pM, and the quantification limit is 5.84 pM. It has the advantages of high sensitivity and wide detection range, and has good application in detecting the KRAS gene.

[0149] The described embodiments are the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Without departing from the essential content of the present invention, any obvious improvements, substitutions or modifications that those skilled in the art can make all fall within the protection scope of the present invention.

Claims

1. A magnetically induced self-assembling nanoprobe for detecting KRAS gene, characterized in that: The magnetically induced self-assembled nanoprobe includes MGCE and an α-Fe2O3 / Fe3O4@Au-ssDNA / BSA / KRAS complex magnetically self-assembled on the surface of MGCE; The α-Fe2O3 / Fe3O4@Au-ssDNA / BSA / KRAS complex comprises: an α-Fe2O3 / Fe3O4@Au nanocomposite, a thiol ssDNA single strand assembled on the surface of an α-Fe2O3 / Fe3O4@Au nanorod through an Au-S bond, a blocking agent BSA specifically adsorbed on the surface of the material, and a KRAS gene of a sample to be detected.

2. The magnetically induced self-assembling nanoprobe for detecting the KRAS gene according to claim 1, characterized in that: The nucleotide sequence of the ssDNA single strand is: 5′-SH-TCTTGCCTACGCCACCAGCTCCAACTAC-3′ (SEQ ID No. 1); The nucleotide sequence of the KRAS gene is: 5'-GTAGTTGGAGCTGGTGGCGTAGGCAAGA-3' (SEQ ID No. 2).

3. The method for constructing the magnetically induced self-assembling nanoprobe for detecting the KRAS gene according to claim 1, characterized in that: The construction method comprises: (1) mixing a thiol ssDNA single-strand solution with an α-Fe2O3 / Fe3O4@Au nanocomposite to obtain a mixed solution, incubating the mixed solution, and forming an α-Fe2O3 / Fe3O4@Au-ssDNA complex after the incubation; (2) dispersing the α-Fe2O3 / Fe3O4@Au-ssDNA complex into a BSA solution for reaction to block nonspecific binding sites, thereby obtaining an α-Fe2O3 / Fe3O4@Au-ssDNA / BSA complex; The α-Fe2O3 / Fe3O4@Au-ssDNA / BSA complex is incubated with the KRAS gene of the sample to be detected, and after the incubation, the α-Fe2O3 / Fe3O4@Au-ssDNA / BSA / KRAS complex is obtained; (3) The α-Fe2O3 / Fe3O4@Au-ssDNA / BSA / KRAS complex is resuspended to obtain a resuspension, and then the resuspension is dropped onto the surface of MGCE to complete the magnetically induced self-assembly process. After the self-assembly is completed, the process is dried to obtain the magnetically induced self-assembly nanoprobe for detecting the KRAS gene.

4. The construction method according to claim 3, characterized in that: In step (1), the final concentration of the α-Fe2O3 / Fe3O4@Au nanocomplex is 3-18 mg / mL; the final concentration of the thiol-modified ssDNA single strand is 3-18 μM; The incubation conditions are: incubation at 35-38° C. for 2-4 hours.

5. The construction method according to claim 4, characterized in that: The final concentration of the α-Fe2O3 / Fe3O4@Au nanocomposite is 15 mg / mL; the final concentration of the thiol-modified ssDNA single strand is 4 μM; The incubation conditions are: incubation at 37° C. for 3 h.

6. The construction method according to claim 1, characterized in that: In step (2), the dosage ratio of α-Fe2O3 / Fe3O4@Au-ssDNA complex and BSA solution is 15 mg:1 mL; The concentration of the BSA solution is 0.20-0.30%; The dosage ratio of α-Fe2O3 / Fe3O4@Au-ssDNA / BSA complex and KRAS gene was 15 mg:1 mL; The concentration of the KRAS gene is 10 pM to 1 μM.

7. The construction method according to claim 6, characterized in that: The concentration of the BSA solution was 0.25%.

8. Use of the magnetically induced self-assembled nanoprobe for detecting the KRAS gene according to claim 1 in preparing an electrochemical biosensor for detecting the KRAS gene.

9. An electrochemical biosensor for detecting KRAS gene, characterized in that: The electrochemical biosensor uses the magnetically induced self-assembled nanoprobe for detecting the KRAS gene as the working electrode, the Ag / AgCl electrode and the Pt electrode as the reference and auxiliary electrodes, respectively, and the [Fe(CN)6] 3- / 4- The solution was used as an electrolyte, and the KRAS content was converted into a current response through an electrochemical workstation to detect the KRAS gene.

10. The electrochemical biosensor for detecting KRAS gene according to claim 9, characterized in that: The electrolyte includes: 5mM K4Fe(CN)6, 5mM K3Fe(CN)6, 0.1M KCl; When the electrochemical biosensor is used for detection, the EIS parameters are: frequency range 0.01 Hz to 100 kHz, signal amplitude 1 mV to 10 mV; The CV parameters are: scanning voltage -0.5 V to 0.5 V, scanning speed 10 m V / s to 100 m V / s.

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