Magnetic self-assembly type electrochemical biosensor for detecting PIK3CA gene as well as construction method and application of magnetic self-assembly type electrochemical biosensor

Abstract

The invention provides a magnetic self-assembly type electrochemical biosensor for detecting a PIK3CA gene as well as a construction method and application of the magnetic self-assembly type electrochemical biosensor, and belongs to the technical field of electrochemical biosensing. The magnetic self-assembly type electrochemical biosensor takes an alpha-Fe2O3 / Fe3O4 (at) Au-ssDNA / BSA (Bovine Serum Albumin) nanoprobe as a working electrode, and the nanoprobe is obtained by assembling a sulfydryl ssDNA single chain capable of specifically capturing a PIK3CA gene on an alpha-Fe2O3 / Fe3O4 (at) Au nanorod; the linear range of the magnetic self-assembly type electrochemical biosensor is 1 pM to 1 mu M, the detection limit is as low as 0.18 pM, the quantification limit is 0.56 pM, and the magnetic self-assembly type electrochemical biosensor has good application in detection of PIK3CA genes.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical biosensing, and particularly relates to a magnetic self-assembled electrochemical biosensor for detecting the PIK3CA gene, a construction method thereof, and an application thereof. Background Art

[0002] The detection of tumor markers is of great significance in the fields of tumor prevention, diagnosis, and disease monitoring. The PIK3CA gene is involved in the regulation of life processes such as the proliferation, differentiation, and apoptosis of tumor cells, and is an important target for the development of anti-tumor drugs. Research shows that among patients with hormone receptor-positive (HR+) and human epidermal growth factor receptor 2 (HER2-) negative breast cancer, 40% have mutations in the PIK3CA gene. This mutation can lead to enhanced intracellular signal transduction, abnormal proliferation of normal cells, and ultimately the formation of malignant tumors. At the same time, it is also one of the main culprits affecting the endocrine therapy effect and prognosis of breast cancer patients. Therefore, it is of great significance to develop a sensitive and efficient method for detecting the expression level of the PIK3CA gene in tumor tissues.

[0003] At present, there are many methods for detecting the PIK3CA gene, such as PCR, local surface plasmon resonance (LSPR), colorimetry, etc. However, the above methods all have different degrees of deficiencies, such as high cost, complex equipment, narrow application range, etc. Using an electrochemical biosensor for PIK3CA gene detection has the advantages of high sensitivity, low cost, fast detection speed, excellent stability, etc., and electrochemical biosensors are widely used in the detection of target genes. However, existing electrochemical biosensors are generally gradually modified on a GCE, which not only requires an additional immobilization procedure but also is inconvenient to operate. Therefore, it is necessary to develop a new magnetic self-assembled electrochemical biosensor for detecting the PIK3CA gene. Summary of the Invention

[0004] Aiming at the deficiencies existing in the prior art, the present invention provides a magnetic self-assembled electrochemical biosensor for detecting the PIK3CA gene, a construction method thereof, and an application thereof; the magnetic self-assembled electrochemical biosensor uses an α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanoprobe as a working electrode, and the nanoprobe is obtained by assembling a thiol ssDNA single strand capable of specifically capturing the PIK3CA gene on α-Fe 2 O 3 / Fe 3 O 4It is obtained on @Au nanorods; the linear range of the magnetic self-assembly type electrochemical biosensor is 1 pM to 1 μM, the detection limit is as low as 0.18 pM, and the quantification limit is 0.56 pM, which has good application in detecting the PIK3CA gene.

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

[0006] The present invention first provides a nanoprobe for detecting the PIK3CA gene, and the nanoprobe includes MGCE and a complex magnetically self-assembled on the surface of MGCE; the complex includes α-Fe 2 O 3 / Fe 3 O 4 @Au nanorods, a thiol ssDNA single strand assembled on α-Fe 2 O 3 / Fe 3 O 4 @Au nanorods through Au-S bonds, a blocker BSA specifically adsorbed on the surface of α-Fe 2 O 3 / Fe 3 O 4 @Au nanorods, and the sample to be detected, the PIK3CA gene.

[0007] Preferably, the nucleotide sequence of the ssDNA single strand is: 5′-SH-AGT GAT TTT AGA GAG-3′ (SEQ ID No.1), which is a DNA containing a thiol (-SH) and a specific nucleic acid sequence for specifically recognizing and binding to the PIK3CA gene;

[0008] The nucleotide sequence of the PIK3CA gene is: 5′-CTC TCT AAA ATC ACT-3′ (SEQ ID No.2).

[0009] The PIK3CA gene specifically binds to the ssDNA single strand through the base complementary pairing principle, and the sensitive detection of the PIK3CA gene is achieved through the change of the electrical signal.

[0010] The present invention also provides a preparation method of the above nanoprobe for detecting the PIK3CA gene, and the method includes:

[0011] (1) Mix a solution of a thiol-modified ssDNA single strand with α-Fe 2 O 3 / Fe 3 O 4 @Au nanorods to obtain a mixture, incubate the mixture, and after the incubation ends, form α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex;

[0012] (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, obtaining α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex;

[0013] Incubate the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex with the sample to be detected, the PIK3CA gene. After the incubation, obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex;

[0014] (3) Resuspend the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex to obtain a resuspension, then drop the resuspension onto the surface of the MGCE to complete the magnetic-induced self-assembly process. After the self-assembly, dry it to obtain the nanosensor for detecting the PIK3CA gene.

[0015] Preferably, in step (1), the final concentration of the α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomplex is 3 - 18 mg / mL; the final concentration of the thiol-modified ssDNA single strand is 1 - 3 μM;

[0016] The conditions for the incubation are: incubate at 30 - 38 °C for 2 - 4 h.

[0017] Preferably, the final concentration of the α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomplex is 16 mg / mL; the final concentration of the thiol-modified ssDNA single strand is 2 μM;

[0018] The incubation conditions are: incubation at 37 °C for 3 h.

[0019] Preferably, in step (1), the α-Fe 2 O 3 / Fe 3 O 4 @Au nanorod preparation steps include:

[0020] S1. Hydrothermally react a mixed aqueous solution of ferric chloride hexahydrate and cetyltrimethylammonium bromide (CTAB), and after the reaction, centrifuge and wash to obtain β-FeOOH nanorods;

[0021] 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;

[0022] S2. Thoroughly mix the magnetic α-Fe 2 O 3 / Fe 3 O 4 heterogeneous nanorods with an aqueous solution of polyethyleneimine (PEI), ultrasonically treat and then heat react. After the reaction, centrifuge, wash, and dry to obtain a solid powder of α-Fe 2 O 3 / Fe 3 O 4 @PEI;

[0023] 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 an ice-cold HAuCl 4 solution and then ultrasonically stir to obtain an α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 suspension, and then dropwise add a sodium citrate solution to obtain an α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / sodium citrate suspension;

[0024] To α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 A NaBH 4 solution was added dropwise to the suspension of / citrate trisodium, and mechanically stirred until the color changed to purple-black, then magnetically separated, washed, dried, and ground to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite.

[0025] 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 4 mmol:7.5 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:(1-20); the conditions of the high-temperature calcination are: high-temperature calcination at 200-700 °C for 2 h;

[0026] In step S2, the mass ratio of the magnetic α-Fe 2 O 3 / Fe 3 O 4 heterostructure 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;

[0027] The volume ratio of the α-Fe 2 O 3 / Fe 3 O 4 @PEI suspension to the HAuCl 4 solution is (140-160):1, and the concentration range of the HAuCl 4 solution is 15-25 mg / mL;

[0028] The volume ratio of the citrate trisodium solution to the α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 suspension is 1:50, and the concentration range of the citrate trisodium solution is 35-40 mM;

[0029] The NaBH 4 solution and the α-Fe 2 O 3 / Fe3 O 4 @PEI / HAuCl 4 / The volume ratio of the sodium citrate suspension is 1:(15 - 19), where NaBH 4 The solution concentration ranges from 0.06 to 0.09 wt%.

[0030] Preferably, in step S1, the mass ratio of the β-FeOOH nanorods to the reducing agent maltose is 1:10;

[0031] In step S2, the magnetic α-Fe 2 O 3 / Fe 3 O 4 The mass ratio of the heterostructure nanorods, PEI is 30:1, and the concentration range of the polyethyleneimine PEI solution is 0.01 g / mL;

[0032] The α-Fe 2 O 3 / Fe 3 O 4 @PEI suspension and the HAuCl 4 The volume ratio of the solution is 150:1, and the concentration range of the HAuCl 4 solution is 20 mg / mL;

[0033] The concentration range of the sodium citrate solution is 38 mM;

[0034] The NaBH 4 solution and the α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / The volume ratio of the sodium citrate suspension is 1:17, where NaBH 4 The solution concentration ranges from 0.075 wt%.

[0035] Preferably, in step (2), the dosage ratio of the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex and the BSA solution is 16 mg:1 mL;

[0036] The concentration of the BSA solution is 0.20 - 0.30 wt%.

[0037] Preferably, the concentration of the BSA solution is 0.25%.

[0038] Preferably, in step (2), the α-Fe 2 O3 / Fe 3 O 4 The dosage ratio of the Au-ssDNA / BSA complex to the PIK3CA gene is 16 mg: 1 mL;

[0039] The concentration of the PIK3CA gene is 1 pM to 1 μM.

[0040] The present invention also provides the use of the above-mentioned nanoprobe for detecting the PIK3CA gene in the preparation of a magnetic self-assembled electrochemical biosensor for detecting the PIK3CA gene.

[0041] The present invention also provides a magnetic self-assembled electrochemical biosensor for detecting the PIK3CA gene. The electrochemical biosensor uses the above-mentioned nanoprobe as the working electrode, an Ag / AgCl electrode and a Pt electrode as the reference electrode and the auxiliary electrode respectively, and a [Fe(CN) 6 3- / 4- solution as the electrolyte, and converts the PIK3CA content into a current response through an electrochemical workstation to detect the PIK3CA gene.

[0042] Preferably, the electrolyte includes: 5 mM K 4 Fe(CN) 6 、5 mM K 3 Fe(CN) 6 、0.1 M KCl;

[0043] When the electrochemical biosensor is detecting, the EIS parameters are: the frequency range is 0.01 Hz to 100 kHz, and the signal amplitude is 1 mV to 10 mV;

[0044] The CV parameters are: the scanning voltage is -0.5 V to 0.5 V, and the scanning speed is 10 mV / s to 100 mV / s.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] (1) The present invention provides a nanoprobe for detecting the PIK3CA 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 nanoprobe, α-Fe 2 O 3 / Fe 3 O 4 @Au rod-shaped nanocomposites are used for the signal amplification strategy, effectively expanding the detection range, improving the detection sensitivity and specificity, and realizing more convenient, simple and accurate detection of the PIK3CA gene; the α-Fe 2 O 3 / Fe​3 O 4 @Au rod-shaped nanocomposites have magnetism and can be adsorbed onto the surface of MGCE through magnetic induction self-assembly technology; this requires low experimental equipment, is simple and easy to operate, and is portable. In addition, in the present invention, single-stranded mercapto-ssDNA is linked to α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite surface, which is simple and fast to operate, consumes few materials, and does not require other materials.

[0047] (2) The present invention constructs a magnetically induced self-assembled nanosensor for detecting the PIK3CA gene into an electrochemical biosensor. The electrochemical sensor realizes the high-affinity binding of the ssDNA probe and the PIK3CA gene based on the principle of base complementary pairing, significantly enhancing the sensitivity, specificity, and stability of detecting the PIK3CA gene; the electrochemical sensor requires a small amount of probe for each detection, the equipment is simple and easy to operate, and the cost is low; the reaction conditions are mild, and it is easy for operators to conduct experiments.

[0048] (3) The linear range of the detectable concentration of the electrochemical biosensor of the present invention is 1 pM to 1 μM, the detection limit is as low as 0.18 pM, and the quantification limit is 0.56 pM. It has a low detection limit, a wide linear range, and good sensitivity, and can better detect the PIK3CA gene. Description of the Drawings

[0049] Figure 1 is the transmission electron microscope photograph of α-Fe 2 O 3 / Fe 3 O 4 @Au nanorods, where the scale bar in the figure is 50 nm;

[0050] Figure 2 is the cyclic voltammogram of the electrochemical process of constructing the nanosensor;

[0051] Figure 3 is the electrochemical impedance spectroscopy of the process of constructing the nanosensor;

[0052] Figure 4 are the peak currents of the nanosensor for detecting single-base mismatch (SBM, 1 μM), double-base mismatch (DBM, 1 μM), non-complementary sequence (NC), and PIK3CA (1 μM) solutions.

[0053] Figure 5 is the linear relationship diagram of the nanosensor detecting different concentrations of PIK3CA using single-stranded ssDNA as the recognition probe. Detailed Embodiments

[0054] 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. All other embodiments obtained by those skilled in the art without creative work, 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.

[0055] The raw materials used in the following examples are:

[0056] 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.; the PIK3CA gene was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0057] The nucleotide sequence information involved is:

[0058] The nucleotide sequence of the ssDNA single strand is: 5′-SH-AGT GAT TTT AGAGAG-3′ (SEQ ID No.1), where SH represents a mercapto group;

[0059] The nucleotide sequence of the PIK3CA gene is: 5′-CTC TCT AAA ATC ACT-3′ (SEQ ID No.2);

[0060] The nucleotide sequence of the single-base mismatch (SBM) is: 5′-CTC TCT AAA G TC ACT-3′ (SEQ ID No.3);

[0061] The nucleotide sequence of the double-base mismatch (DBM) is: 5′-CTC TCT G AA ATC G CT-3′ (SEQ ID No.4);

[0062] The nucleotide sequence of the non-complementary sequence (NC) is: 5′-GAC TGC GAG GTC GCT-3’ (SEQ ID No.5). Example 1: Preparation of α-Fe 2 O 3 / Fe 3 O 4 @Au nanorods

[0063] (1) Weigh 4 mmol of ferric chloride hexahydrate and 7.5 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.

[0064] (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.

[0065] (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 an α-Fe 2 O 3 / Fe 3 O 4 @PEI / HAuCl 4 / trisodium citrate suspension;

[0066] (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 3 O 4 @PEI / HAuCl 4In the 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, dried in vacuum at 60 °C, and ground to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite.

[0067] Figure 1 For α-Fe 2 O 3 / Fe 3 O 4 @Au nanorod transmission electron microscopy photograph; it can be seen from the electron microscopy photograph that gold nanoparticles with an average diameter of 6.2 nm are coated on the surface of the α-Fe 2 O 3 / Fe 3 O 4 heterogeneous nanorod surface.

[0068] Example 2: Preparation of a nanoprobe for detecting the PIK3CA gene

[0069] In this example, a nanoprobe for detecting the PIK3CA 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 PIK3CA gene. The specific steps are as follows:

[0070] S1. Preparation of a nanoprobe for detecting the PIK3CA gene:

[0071] (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, and after vortexing and mixing, it was activated at 30 °C for 1 h to eliminate disulfide bonds and set aside;

[0072] To 30 μL of the activated thiol-ssDNA single-strand solution with a concentration of 2 μM, 0.48 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite was added, and it 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.

[0073] (2) Disperse 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 25 °C for 30 min to block non-specific binding sites. After incubation, obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex. Then, centrifuge and wash all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomplex with 30 μL of 1 μM PIK3CA gene at 37 °C with shaking for 20 min. After incubation, centrifuge and wash to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex.

[0074] (3) Resuspend the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex in PBS solution and wash twice, then resuspend in 30 μL of ultrapure water to obtain a suspension with a concentration of 16 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 nanocomplex film is formed on the electrode surface to obtain the nanosensor for detecting the PIK3CA gene.

[0075] S2. Application of the nanosensor in detecting the PIK3CA gene:

[0076] (1) Respectively, place the 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 / PIK3CA was used as the working electrode, and the Ag / AgCl electrode and the Pt electrode were used as the reference electrode and the auxiliary electrode respectively. They were placed in the electrolyte solution (5 mM K 4 Fe(CN) 6 、5 mM K 3 Fe(CN) 6 、0.1 M KCl), and connected to an electrochemical workstation (purchased from Shanghai Chenhua Instrument Co., Ltd., CHI660E) to obtain an electrochemical biosensor.

[0077] Among them, for MGCE / α-Fe 2 O 3 / Fe 3 O 4 : 0.48 mg of α-Fe 2 O 3 / Fe 3 O 4 was added to 30 μL of ultrapure water to prepare a suspension with a concentration of 16 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.

[0078] For MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au: 0.48 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au was added to 30 μL of ultrapure water to prepare a suspension with a concentration of 16 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.

[0079] For MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA: 0.48 mg of α-Fe 2 O 3 / Fe 3 O 4@Au was continuously incubated in a constant temperature shaker at 37 °C for 3 h, and α-Fe was obtained after the incubation ended. 2 O 3 / Fe 3 O 4 @Au-ssDNA complex. The supernatant was removed by magnetic separation, and the solid was resuspended in 30 μL of ultrapure water to obtain a resuspension with a concentration of 16 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.

[0080] MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA: 0.48 mg of α-Fe was added to 30 μL of 2 μM mercapto-ssDNA single-strand solution. 2 O 3 / Fe 3 O 4 @Au, and it was continuously incubated in a constant temperature shaker at 37 °C for 3 h. After the incubation ended, α-Fe was obtained. 2 O 3 / Fe 3 O 4 @Au-ssDNA complex. The supernatant was removed by magnetic separation, and the solid was dispersed into 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 16 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. MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA: Prepared from step S1.

[0081] (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.

[0082] Among them, the CV parameters were -0.1 to 0.7 V, and the scanning rate was 100 mV / s.

[0083] The EIS frequency range is 0.1 Hz to 10 kHz, the signal amplitude is 5 mV, the DPV voltage is 0 to 0.6 V, the vibration amplitude is 0.05 V, the pulse width is 0.06 s, the sampling width is 0.02 s, and the pulse period is 0.5 s.

[0084] The detection results are as Figure 2 and Figure 3 shown.

[0085] It can be seen from Figure 2 that, taking the bare MGCE electrode (curve a) as a reference, after modification with α-Fe 2 O 3 / Fe 3 O 4 the current signal of the electrode (curve b) decreases significantly; however, after modification with α-Fe 2 O 3 / Fe 3 O 4 @Au (curve c), the current signal increases significantly, successfully amplifying the electrical signal. This is because of the excellent conductivity of Au nanoparticles, which accelerates the electron transfer of the modified electrode and thus improves the sensitivity of the electrochemical sensor. ssDNA is single-stranded DNA containing thiol modification. TCEP is used to reduce the disulfide bond of ssDNA to thiol, which then serves as a probe to combine with the Au nanoparticles on the surface of the α-Fe 2 O 3 / Fe 3 O 4 @Au material to obtain MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA (curve d). Compared with curve c, the redox current decreases. On the one hand, since the single-stranded ssDNA contains a phosphate backbone, it is negatively charged, resulting in mutual repulsion with the redox probe [Fe(CN) 6 3- / 4- which is also negatively charged in the electrolyte; on the other hand, due to the steric hindrance effect of DNA, it is difficult for [Fe(CN) 6 3- / 4- to achieve electron transfer on the electrode surface, resulting in the attenuation of the electrochemical signal. BSA is added to the successfully prepared α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA to block non-specific sites, obtaining 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 decreases. After incubating the probe molecule with the PIK3CA gene, ssDNA specifically recognizes the PIK3CA gene through base complementary pairing, and MGCE / α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA (curve f), compared with curve e, the redox current decreases, proving that the constructed probe molecule can detect the PIK3CA gene. The decrease in current is also due to the phosphate backbone and steric hindrance effect of the PIK3CA gene.

[0086] From Figure 3 it can be seen that the semicircle diameter of the material electrode after gold coating (curve c) is smaller than that of the material electrode before gold coating (curve b), proving that the resistance of the modified electrode is smaller after the material is gold coated. The electrode modified with ssDNA (curve d) has a larger radius than curve c, proving that the ssDNA single-strand recognition probe is 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 the electron transfer resistance. BSA can achieve the effect of 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 / PIK3CA (curve f) electrode is larger, indicating that the PIK3CA gene is successfully bound to the ssDNA single-strand recognition probe.

[0087] In summary, the magnetically induced self-assembled nanosensor for detecting the PIK3CA gene described in the present invention can be used to detect the PIK3CA gene.

[0088] S3. Investigation of the detection limit and detection range of the electrochemical sensor:

[0089] In this step, the electrochemical biosensor assembled above is used to detect different concentrations of PIK3CA, and a standard curve is plotted to investigate the detection limit, quantification limit, and detection range of the electrochemical biosensor. The specific steps are as follows:

[0090] α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA was incubated with PIK3CA 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 PIK3CA concentration. The results are shown as follows Figure 5 As can be seen from the figure, when α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomposite was used as a probe, the detection limit for detecting PIK3CA was 0.18 pM (LOD = 3σ / slope), the quantification limit was 0.56 pM (LOQ = 10σ / slope), and the linear range was 1 pM - 1 μM. Therefore, the linear quantification of PIK3CA concentration in the dynamic range of 1 pM - 1 μM was achieved by using this electrochemical biosensor, which had high sensitivity and specificity.

[0091] Example 3: Preparation of a nanoprobe for detecting the PIK3CA gene

[0092] (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, and after vortexing and mixing, it was activated at 30 °C for 1 h to eliminate disulfide bonds and set aside;

[0093] 0.24 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite was added to 30 μL of the activated thiol-ssDNA single-strand solution with a concentration of 2 μM, and the mixture 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.

[0094] (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, α-Fe2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA complex. Then all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanoparticles were incubated with 30 μL of 1 μM PIK3CA gene at 37 °C with shaking for 20 min. After incubation, centrifugation and washing were carried out to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex.

[0095] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA 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 8 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 to obtain the nano-probe for detecting PIK3CA gene.

[0096] The magneto-induced self-assembled nano-probe for detecting PIK3CA gene prepared was used as the working electrode, the Ag / AgCl electrode and the Pt electrode were used as the reference and auxiliary electrodes respectively, and the [Fe(CN) 6 3- / 4- solution containing KCl was used as the electrolyte to construct an electrochemical sensor. The differential pulse voltammetry (DPV) was used to optimize the experimental conditions and evaluate the analytical performance of the sensor.

[0097] The results of the performance analysis and evaluation showed that the biosensor had strong selectivity for PIK3CA. Although the detection was not as good as that in Example 2, trace amounts of PIK3CA gene could still be detected. In addition, the sensitivity of the sensor was insufficient, which was manifested by the too low current response during the construction of the sensor monitored by CV and EIS.

[0098] Example 4: Preparation of nano-probe for detecting PIK3CA gene

[0099] ​(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, and set aside;

[0100] Add 0.30 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite to 30 μL of the activated thiol-ssDNA single-strand solution with a concentration of 2 μM, 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 is obtained.

[0101] (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, 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 PIK3CA gene and incubate at 37 °C with shaking for 20 min. After the incubation, centrifuge and wash to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex.

[0102] (3) For α-Fe 2 O 3 / Fe 3 O 4The @Au-ssDNA / BSA / PIK3CA 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 10 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 nano-probe for detecting the PIK3CA gene.

[0103] The magnetically induced self-assembled nano-probe for detecting the PIK3CA gene prepared was used as the working electrode, the Ag / AgCl electrode and the Pt electrode were used as the reference electrode and the auxiliary electrode respectively, and the [Fe(CN) 6 3- / 4- solution containing KCl was used as the electrolyte to construct an electrochemical sensor. The differential pulse voltammetry (DPV) was used to optimize the experimental conditions and evaluate the analytical performance of the sensor.

[0104] The results of the performance analysis and evaluation showed that the biosensor had a certain selectivity for PIK3CA. Although it was not as good as that in Example 2, trace amounts of the PIK3CA gene could still be detected, which was manifested by an obvious current response only to the PIK3CA gene group. In addition, its sensitivity was insufficient, which was manifested by a slightly lower current response degree than the experimental expected effect during the construction of the sensor monitored by CV and EIS.

[0105] Example 5: Preparation of the nano-probe for detecting the PIK3CA gene

[0106] (1) A certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) was added to the ssDNA dry powder, and it was 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;

[0107] To 30 μL of the activated thiol-ssDNA single-strand solution with a concentration of 2 μM, 0.60 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nano-complex was added, and it was continuously oscillated and incubated in a constant temperature shaker at 37 °C for 3 h. After the incubation ended, the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complex was obtained.

[0108] (2) All of the above-obtained α-Fe 2 O 3 / Fe​3 O 4 Disperse the @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 all the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomplex is incubated with 30 μL of 1 μM PIK3CA gene at 37 °C with shaking for 20 min. After incubation, centrifuge and wash to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex.

[0109] (3) Resuspend the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex in PBS solution and wash twice, then resuspend in 30 μL of ultrapure water to obtain a suspension with a concentration of 20 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 complex film is formed on the electrode surface to obtain the nanosensor for detecting the PIK3CA gene.

[0110] Use the prepared magnetic-induced self-assembled nanosensor for detecting the PIK3CA 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- solution as the electrolyte to construct an electrochemical sensor. Optimize the experimental conditions by differential pulse voltammetry (DPV) and evaluate the analytical performance of the sensor.

[0111] ​The performance analysis and evaluation results show that the biosensor has strong selectivity for PIK3CA, but it can still detect trace amounts of the PIK3CA gene, manifested as an obvious current response only to the PIK3CA genome, while the recognition ability for other interference groups is not satisfactory. The sensitivity of this biosensor is good, and during the construction of the sensor monitored by CV and EIS, the current response is obvious. For the DPV curves of the biosensor for detecting PIK3CA genes at different concentrations, the linear range is 100 pM - 1 μM, and its linear range is worse than that of Example 2. Therefore, although using this electrochemical biosensor to detect the PIK3CA gene has certain advantages, it is not the most preferred option.

[0112] Example 6: Preparation of a nanoprobe for detecting the PIK3CA gene

[0113] (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;

[0114] Add 0.75 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite to 30 μL of the activated thiol-ssDNA single-strand solution with a concentration of 2 μM, 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.

[0115] (2) Disperse all the above α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complexes into 30 μL of 0.25% BSA solution, 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 4The @Au-ssDNA / BSA nanocomposite was incubated with 30 μL of 1 μM PIK3CA gene at 37 °C with shaking for 20 min. After incubation, centrifugation and washing were carried out to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex.

[0116] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA 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 25 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 to obtain the nanosensor for detecting PIK3CA gene.

[0117] The magnetically induced self-assembled nanosensor for detecting PIK3CA gene prepared was used as the working electrode, the Ag / AgCl electrode and the Pt electrode were used as the reference and auxiliary electrodes respectively, and the [Fe(CN) 6 3- / 4- solution was used as the electrolyte to construct an electrochemical sensor. The differential pulse voltammetry (DPV) was used to optimize the experimental conditions and evaluate the analytical performance of the sensor.

[0118] The results of performance analysis and evaluation showed that the biosensor had strong selectivity for PIK3CA, but trace amounts of PIK3CA gene could still be detected, manifested by an obvious current response only to the PIK3CA gene group, while the recognition ability for other interference groups was not satisfactory. However, its sensitivity was seriously insufficient, manifested by too low current response during the construction of the sensor monitored by CV and EIS. The DPV curves for the detection of PIK3CA gene with different concentrations by this biosensor had a narrow linear range. Compared with other methods for detecting PIK3CA and Example 2, it did not have sufficient performance advantages, and the DPV response fluctuated greatly. This phenomenon might be related to the amount of composite material in the suspension dropped on the electrode surface.

[0119] Example 7: Preparation of the nanosensor for detecting PIK3CA gene

[0120] ​(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 and mix evenly, then activate at 30 °C for 1 h to eliminate disulfide bonds, and set aside;

[0121] Add 1.20 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite to 30 μL of the activated thiol ssDNA single-strand solution with a concentration of 2 μM, 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.

[0122] (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, 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 PIK3CA gene, oscillate and incubate at 37 °C for 10 min. After the incubation, centrifuge and wash to obtain the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex.

[0123] (3) Disperse the α-Fe 2 O 3 / Fe 3 O 4The @Au-ssDNA / BSA / PIK3CA 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 40 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 complex film was formed on the electrode surface, and the nanosensor for detecting the PIK3CA gene was obtained.

[0124] The magnetically induced self-assembled nanosensor for detecting the PIK3CA gene prepared above was used as the working electrode, the Ag / AgCl electrode and the Pt electrode were used as the reference electrode and the auxiliary electrode respectively, and the [Fe(CN) 6 3- / 4- solution containing KCl was used as the electrolyte to construct an electrochemical sensor. The differential pulse voltammetry (DPV) was used to optimize the experimental conditions and evaluate the analytical performance of the sensor.

[0125] The results of the performance analysis and evaluation showed that the biosensor had strong selectivity for PIK3CA, but trace amounts of the PIK3CA gene could still be detected. It was manifested that there was an obvious current response only to the PIK3CA gene group, while the recognition ability for other interference groups was not satisfactory. However, its sensitivity was seriously insufficient, manifested by the too low current response during the construction of the sensor monitored by CV and EIS, which might be due to excessive loading on the electrode surface, hindering electron transfer. The DPV curves of the biosensor for detecting PIK3CA genes with different concentrations had a narrow linear range, and compared with other methods for detecting PIK3CA and Example 2, it did not have sufficient performance advantages.

[0126] Example 8: Preparation of a nanosensor for detecting the PIK3CA gene

[0127] (1) A certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) was added to the ssDNA dry powder, and it 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 and mixing, it was activated at 30 °C for 1 h to eliminate disulfide bonds and set aside.

[0128] To 30 μL of the activated mercapto-ssDNA single-stranded solution with a concentration of 1 μM, 0.48 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite was added, and it 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

[0129] (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 PIK3CA 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 / PIK3CA complex.

[0130] (3) Resuspend the α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complex in PBS solution and wash twice, then resuspend in 30 μL of ultrapure water to obtain a suspension with a concentration of 16 mg / mL; Drop 5 μL of the suspension onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid dries, a uniform magnetic nanocomplex film is formed on the electrode surface to obtain the magnetic-induced self-assembled nanosensor for detecting PIK3CA gene.

[0131] Use the prepared magnetic-induced self-assembled nanosensor for detecting PIK3CA gene as the working electrode, Ag / AgCl electrode and Pt electrode as the reference and auxiliary electrodes respectively, and the solution containing KCl and [Fe(CN) 6 3- / 4- as the electrolyte to construct an electrochemical sensor. Optimize the experimental conditions by differential pulse voltammetry (DPV) and evaluate the analytical performance of the sensor.

[0132] ​The performance analysis and evaluation results show that the biosensor has strong selectivity for PIK3CA, but it can still detect trace amounts of the PIK3CA gene, manifested as an obvious current response only to the PIK3CA genome, while the recognition ability for other interference groups is not satisfactory. During the construction of the sensor monitored by CV and EIS, the current response is too low. The DPV curves of the biosensor for detecting PIK3CA genes at different concentrations have a narrow linear range and do not have sufficient performance advantages compared with other methods for detecting PIK3CA and Example 2. Moreover, the DPV response fluctuates greatly, and this phenomenon may be related to the amount of the suspension dropped on the electrode surface.

[0133] Example 9: Preparation of a Nanoprobe for Detecting the PIK3CA Gene

[0134] (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;

[0135] Add 0.48 mg of α-Fe 2 O 3 / Fe 3 O 4 @Au nanocomposite to 30 μL of the activated thiol ssDNA single-strand solution with a concentration of 1.5 μM, 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.

[0136] (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, 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 nanocomposites were incubated with 30 μL of 1 μM PIK3CA gene at 37 °C with shaking for 40 min. After incubation, centrifugation and washing were carried out to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complexes.

[0137] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA 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 16 mg / mL; 13 μL of the suspension was dropped onto the surface of 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 nanosensor for detecting PIK3CA gene.

[0138] The magnetically induced self-assembled nanosensor for detecting PIK3CA gene prepared was used as the working electrode, the Ag / AgCl electrode and the Pt electrode were used as the reference and auxiliary electrodes respectively, and the [Fe(CN) 6 3- / 4- solution was used as the electrolyte to construct an electrochemical sensor. The differential pulse voltammetry (DPV) was used to optimize the experimental conditions and evaluate the analytical performance of the sensor.

[0139] The results of performance analysis and evaluation showed that the biosensor had weak selectivity for PIK3CA. During the construction of the sensor monitored by CV and EIS, the current response was too low. The DPV response fluctuated greatly, probably because when the suspension was dropped, the amount of the suspension was large and it was easy to form an uneven magnetic nanocomposite film.

[0140] Example 10: Preparation of a nanosensor for detecting PIK3CA gene

[0141] (1) A certain amount (refer to the instruction manual) of annealing buffer (TE Buffer) was added to the ssDNA dry powder, and it 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, and after vortexing, it was activated at 30 °C for 1 h to eliminate disulfide bonds and reserved;

[0142] 0.48 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 the incubation, α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA complexes were obtained.

[0143] (2) All of 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 of the obtained α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA nanocomposites were incubated with shaking with 30 μL of 1 μM PIK3CA gene at 37 °C for 50 min. After the incubation, centrifugation and washing were carried out to obtain α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA complexes.

[0144] (3) The α-Fe 2 O 3 / Fe 3 O 4 @Au-ssDNA / BSA / PIK3CA 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 16 mg / mL; 9 μL of the suspension was dropped onto the surface of the MGCE pretreated with 3 mm alumina powder. After the liquid was dried, a uniform magnetic rice composite film was formed on the electrode surface to obtain the nanosensor for detecting PIK3CA gene.

[0145] The magnetically induced self-assembled nanosensor for detecting PIK3CA gene prepared was used as the working electrode, the Ag / AgCl electrode and the Pt electrode were used as the reference electrode and the auxiliary electrode respectively, and the [Fe(CN) 6 3- / 4- solution containing KCl was used as the electrolyte to construct an electrochemical sensor. The differential pulse voltammetry (DPV) was used to optimize the experimental conditions and evaluate the analytical performance of the sensor.​

[0146] The performance analysis and evaluation results show that the biosensor is selective for PIK3CA, but it can still detect the PIK3CA gene. However, compared with Example 2, its selectivity performance is average. In the selectivity test experiment, there are differences in the current responses between the target group and the single-base mismatch and double-mismatch groups, but the differences are not obvious. The sensitivity of this biosensor is insufficient, and during the construction of the sensor monitored by CV and EIS, the current response is low. From the DPV curves and linear fitting curves of the detection of different concentrations of PIK3CA gene by this biosensor, it can be seen that its linear range is 100 pM to 100 nM, and the detection limit (LOD) and quantification limit (LOQ) are not superior to other methods for detecting PIK3CA.

[0147] In summary, the magneto-induced self-assembled nano-probe for detecting PIK3CA gene described in the present invention can be used to detect the PIK3CA gene.

[0148] Example 11: Investigation of the performance of the electrochemical biosensor

[0149] In this example, first, taking the nano-probe for detecting PIK3CA gene prepared in Example 2 as an example, it was assembled into an electrochemical biosensor according to the method described in Example 2, and the peak currents of single-base mismatch (SBM, 1 μM), double-base mismatch (DBM, 1 μM), non-complementary sequence (NC, 1 μM) blank group (TE-Buffer), and PIK3CA (1 μM) solution were detected respectively. The detection results are as Figure 4 shown.

[0150] From Figure 4 it can be seen that the peak currents of the nano-probe for detecting PIK3CA gene prepared in Example 2 when detecting single-base mismatch (SBM, 1 μM), double-base mismatch (DBM, 1 μM), and non-complementary sequence (NC, 1 μM) blank group (TE-Buffer) are significantly different from the current values of the PIK3CA group, indicating that it can specifically recognize the PIK3CA 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.

[0151] In summary, the magneto-induced self-assembled nano-probe for detecting PIK3CA gene described in the present invention realizes linear quantification of PIK3CA concentration in the dynamic range of 10 pM to 1 μM, and has high sensitivity and specificity.

[0152] In summary, the present invention constructs a magnetically induced self-assembled nanoprobe for detecting the PIK3CA gene. Among them, ssDNA specifically captures the PIK3CA gene through base complementary pairing, and then is adsorbed onto the surface of a 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 1 pM to 1 μM, the detection limit is as low as 0.18 pM, and the quantification limit is 0.56 pM. It has the advantages of high sensitivity and a wide detection range, and has good application in detecting the PIK3CA gene.

[0153] The above embodiments are the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Without departing from the essence of the present invention, any obvious improvements, substitutions or modifications that those skilled in the art can make all belong to the protection scope of the present invention.

Claims

1. A nanoprobe for detecting the PIK3CA gene, characterized in that: The nanoprobe includes MGCE and a complex magnetically self-assembled on the surface of MGCE; the complex includes α-Fe2O3 / Fe3O4@Au nanorods, thiol ssDNA single strands assembled on the α-Fe2O3 / Fe3O4@Au nanorods through Au-S bonds, a blocker BSA specifically adsorbed on the surface of the α-Fe2O3 / Fe3O4@Au nanorods, and a PIK3CA gene of a sample to be detected.

2. The nanoprobe for detecting the PIK3CA gene according to claim 1, characterized in that: The nucleotide sequence of the thiol ssDNA single strand is: 5′-SH-AGT GAT TTT AGA GAG-3′ (SEQ ID No. 1); The nucleotide sequence of the PIK3CA gene is: 5′-CTC TCT AAAATC ACT-3′ (SEQ ID No. 2).

3. The method for preparing a nanoprobe for detecting the PIK3CA gene according to any one of claims 1 or 2, characterized in that: The method comprises: (1) mixing a thiol-modified ssDNA single-strand solution with α-Fe2O3 / Fe3O4@Au nanorods 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 PIK3CA gene of the sample to be detected, and after the incubation, the α-Fe2O3 / Fe3O4@Au-ssDNA / BSA / PIK3CA complex is obtained; (3) resuspending the α-Fe2O3 / Fe3O4@Au-ssDNA / BSA / PIK3CA complex to obtain a resuspension, then dropping the resuspension onto the MGCE surface to complete the magnetically induced self-assembly process, and drying after the self-assembly to obtain the nanoprobe for detecting the PIK3CA gene.

4. The preparation 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 1-3 μM; The incubation conditions are: incubation at 30-38° C. for 2-4 hours.

5. The preparation method according to claim 4, characterized in that: The final concentration of the α-Fe2O3 / Fe3O4@Au nanocomposite is 16 mg / mL; the final concentration of the thiol-modified ssDNA single strand is 2 μM; The incubation conditions are: incubation at 37° C. for 3 h.

6. The preparation method according to claim 3, characterized in that: In step (2), the dosage ratio of α-Fe2O3 / Fe3O4@Au-ssDNA complex and BSA solution is 16 mg:1 mL; The concentration of the BSA solution is 0.20-0.30 wt %.

7. The preparation method according to claim 3, characterized in that: In step (2), the dosage ratio of α-Fe2O3 / Fe3O4@Au-ssDNA / BSA complex to PIK3CA gene is 16 mg:1 mL; The concentration of the PIK3CA gene is 1 pM to 1 μM.

8. Use of the nanoprobe for detecting the PIK3CA gene according to claim 1 in preparing a magnetic self-assembly electrochemical biosensor for detecting the PIK3CA gene.

9. A magnetic self-assembly electrochemical biosensor for detecting PIK3CA gene, characterized in that: The electrochemical biosensor uses the nanoprobe described in claim 1 as a working electrode, an Ag / AgCl electrode and a Pt electrode as a reference electrode and an auxiliary electrode, respectively, and a [Fe(CN)6] 3- / 4- The solution was used as an electrolyte, and the PIK3CA content was converted into a current response through an electrochemical workstation to detect the PIK3CA gene.

10. The magnetic self-assembly electrochemical biosensor for detecting PIK3CA 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; CV parameters are: scanning voltage -0.5V to 0.5V, scanning speed 10mV / s to 100mV / s.

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