CN114414642A

CN114414642A - Electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein

Info

Publication number: CN114414642A
Application number: CN202210016811.5A
Authority: CN
Inventors: 余秋波; 文韬
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2022-01-07
Filing date: 2022-01-07
Publication date: 2022-04-29
Anticipated expiration: 2042-01-07
Also published as: CN114414642B

Abstract

The invention belongs to the technical field of electrochemical sensors, and discloses an electrochemical sensor for detecting human papillomavirus type 16 E6 oncoprotein, and preparation of the electrochemical sensor for detecting human papillomavirus type 16 E6 oncoprotein The method includes: synthesizing materials; establishing an electrochemical biosensor; using a direct method and an indirect method to determine the target, and drawing a standard curve; analyzing the repeatability, stability and specificity of the sensor; wherein, the synthetic material includes: a synthetic dendrimer Ternary nanoparticles PdBP NSs; synthesis of mesoporous silica MSNs; synthesis of signal tags. The electrochemical sensor for the detection of human papillomavirus type 16 E6 oncoprotein provided by the invention can realize quantitative detection, is simple to operate, does not require additional professional technicians, has low cost, wide linear range and low detection limit, and can It can effectively solve the problems existing in the existing methods for detecting E6 oncoprotein, such as relatively complicated operations, expensive equipment, and the need for professional technicians.

Description

Electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein

Technical Field

The invention belongs to the technical field of electrochemical sensors, and particularly relates to an electrochemical sensor for detecting human papilloma virus 16 type E6 oncoprotein.

Background

Currently, cervical cancer is the fourth most common cancer that threatens the health of a woman. In 2018, over 30 ten thousand women died from cervical cancer. Most patients are caused by persistent infection with high risk type HPV, of which HPV16 accounts for about 50%. Studies have shown that detection of over-expressed E6 oncoprotein can distinguish between transient infections and premalignant or cancerous females. Thus, the HPV 16E 6 oncoprotein is considered to be a biomarker for cervical cancer. Immunocytochemistry, immunogold agglutination, and the Onco E6 assay are currently methods for detecting E6 oncoprotein. However, they also have certain disadvantages, such as relatively complicated operation, expensive equipment and the need for skilled personnel. The electrochemical immunosensor is an alternative technology, and has the advantages of simple operation, high cost benefit, high sensitivity and low detection limit, so that the method becomes a potential strategy for detecting the biomarkers. Therefore, there is a need to design a new electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein.

Through the above analysis, the problems and defects of the prior art are as follows: the existing method for detecting the E6 oncoprotein has relatively complex operation and expensive equipment, and needs professional technicians.

The difficulty in solving the above problems and defects is: the method is only required to be simple, the low cost is not enough to solve the practical problem, and the designed new method also needs to have a wider linear range and a lower detection limit. In addition, protease is a biological macromolecule, the activity of which is greatly influenced by pH and temperature, some proteases require metal ions as an activator, and the preparation process of the protease is complex and expensive. The significance of solving the problems and the defects is as follows: in contrast, nanoparticles may be a good choice. Many nano materials with excellent catalytic performance can be synthesized under the conditions of low temperature and normal pressure. The nano material has stable performance, is easy to store and is not easy to degrade.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an electrochemical sensor for detecting human papilloma virus 16 type E6 oncoprotein and a preparation method thereof.

The invention is realized in such a way that a preparation method of an electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein comprises the following steps:

step one, synthesizing materials;

step two, establishing an electrochemical biosensor;

step three, measuring the target by using a direct method and an indirect method respectively, and drawing a standard curve;

and step four, analyzing the repeatability, stability and specificity of the sensor.

The cervical cancer can be rapidly and sensitively quantitatively detected by the human papilloma virus 16 type E6 oncoprotein, and is a common cancer of women. Human papillomavirus type 16E 6 oncoprotein is a reliable biomarker for cervical cancer. Although there are other methods for detecting E6 oncoprotein, electrochemical methods have advantages such as low cost, convenience and rapidity. In this work, a novel dual-signal electrochemical immunosensor based on a high-efficiency catalyst and a signal label is developed for rapidly and sensitively detecting the human papillomavirus type 16E 6 oncoprotein. Here, in order to achieve rapid detection, the palladium-boron-phosphorus dendritic ternary nanospheres (PdBP NSs) not only serve as a catalyst catalyzing H2O2, but also serve as a carrier material capturing antibodies. In addition, in order to realize sensitive detection, the mesoporous silica nanocomposite carrying methylene blue and coated with chitosan (MBSi-Chi) is synthesized to be used as a signal label, and an electrochemical signal can be generated. Under optimal conditions, the linear range of the unlabeled immunosensor is from 100 fg/mL to 4ng/mL, the detection limit is 72.8fg/mL (S/N ═ 3), the linear range of the sandwich immunosensor is from 50fg/mL to 4ng/mL, and the detection limit is 34.1fg/mL (S/N ═ 3). The prepared dual-signal immunosensor has ideal specificity, stability and repeatability, which means potential application in clinical laboratories.

Further, the composite material in the first step comprises:

(1) synthesizing dendritic ternary nano particles PdBP NSs;

(2) synthesizing mesoporous silica MSN;

(3) a signal tag is synthesized.

Further, the synthesis process of the dendritic ternary nanoparticles in the step (1) comprises the following steps:

to 5mL of a 9mg/mL DODAC solution was added 500. mu.L of 0.337M NH₄F. 500 μ L of 0.101M H₃BO₃And 800. mu.L of 8.5M Na₂PdCl₄(ii) a After magnetic stirring at 400rpm for 5min, 400. mu.L of 10 wt% NH was added to the mixed solution₃·H₂Continuously stirring until the solution becomes colorless;

500 μ L of 2.5mg NaH was added₂PO₂Heating the obtained mixed solution in a silicon oil bath at 95 ℃ for 20min, dropwise adding 500 mu L DMAB, and continuing to heat for 30 min; centrifuging at 12000rpm for 10min, removing supernatant, collecting precipitate, and cleaning with ultrapure water, anhydrous ethanol and ultrapure water respectively under the same centrifugation condition; and finally, obtaining PdBP powder through freeze drying treatment.

Further, the synthesis process of the mesoporous silica in the step (2) comprises:

adding 25mg CTAB and 7mg NaOH into 12mL of ultrapure water, moving the solution into a flask, and heating the flask to 80 ℃ in a silicon oil bath for 1 h; dripping 125 mu L of TEOS into the flask, and keeping heating for 2 h; centrifuging the obtained solution at 10000rpm for 10min, removing supernatant, collecting precipitate, and washing with methanol for 3 times under the same centrifugation condition; dispersing the precipitate obtained after washing in absolute ethyl alcohol;

adding 4mL of the extract into the absolute ethyl alcohol dispersion, and heating in an oil bath at 90 ℃ for 45 min; centrifuging at 10000rpm for 10min, removing supernatant, collecting precipitate, and cleaning with ultrapure water, anhydrous ethanol and ultrapure water respectively under the same centrifugation condition; the extraction step was repeated once and the washed precipitate was dried under vacuum at 37 ℃ for 20 h.

Further, the extract consisted of 7.2mL of absolute ethanol and 0.8mL of concentrated hydrochloric acid.

Further, the synthesizing process of the signal tag in the step (3) includes:

mixing 1mL of 10mM methylene blue solution MB with 1mL of 5mg/mL MSN dispersion, performing ultrasonic treatment for 30min, and magnetically stirring at 400rpm at room temperature for 4 h; adding 10 mu L of glacial acetic acid and 4mg of chitosan Chi into the solution, and violently stirring at 1500rpm at room temperature for 12 h;

centrifuging at 10000rpm for 10min, removing supernatant, collecting precipitate, cleaning with ultrapure water for 3 times under the same centrifugation condition, and collecting precipitate MBSi-Chi; vacuum drying the collected MBSi-Chi at 37 ℃ for 20h, and dispersing the MBSi-Chi in ultrapure water to obtain 1mg/mL dispersion liquid;

adding 400 μ L of MBSi-Chi, 1.2mL of E6 antibody and 466 μ L of 5% glutaraldehyde solution into a small beaker, and magnetically stirring for 12h in a refrigerator at 4 ℃; adding 40 mu L of 0.5% BSA into the solution, and continuing stirring for 6 h; centrifuging at 10000rpm for 10min, removing supernatant, collecting precipitate, and centrifuging with Na under the same conditions₂CO₃/NaHCO₃The buffer was washed 3 times and the resulting signal tag, i.e., the MBsi-Chi complex, was redispersed in 500. mu.L of ultrapure water and stored at 4 ℃.

Further, the direct method in the third step comprises:

using 0.3 μm and 50nm Al respectively₂O₃Polishing the electrode into a mirror surface by using the powder, respectively carrying out ultrasonic treatment on the electrode for 5min in the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;

dripping 10 mu L of electrode modification material dendritic ternary nano particles PdBP NSs on the surface of the electrode, and drying at 37 ℃;

thirdly, dripping 10 mu L E6 antibody on the surface of the dried electrode, and incubating for 2h at 37 ℃;

fourthly, washing the electrode with ultrapure water, drying the electrode at room temperature, dripping 6 mu L of 0.5 percent BSA, and incubating for 30min at room temperature;

fifthly, rinsing the electrode with ultrapure water, drying at room temperature, then dropwise adding 8 mu L E6 protein, and incubating for 2h at 37 ℃;

sixthly, washing the incubated electrode with a washing buffer solution, and then drying the electrode in nitrogen;

seventhly, the electrodes are placed in 5mL of 0.1M PBS solution for characterization, and 20 mu L of 3.0M H solution is added every 40s₂O₂Measuring the current value by an ampere current method;

and drawing a working curve according to the linear relation between the obtained current change value and the concentration of the target protein.

Further, the indirect method in step three comprises:

using 0.3 μm and 50nm Al respectively₂O₃Polishing the electrode into a mirror surface by using powder, respectively carrying out ultrasonic treatment on the electrode for 5min in the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;

sixthly, washing the electrode with ultrapure water, drying at room temperature, dripping 10 mu L of signal label, namely MBsi-Chi compound, and incubating for 2h at 37 ℃;

seventhly, washing the incubated electrode with a cleaning buffer solution, and then drying the electrode in nitrogen;

placing the electrode in 5mL of 0.1M PBS solution for characterization, and measuring the current value of the electrode by a differential pulse voltammetry;

and ninthly, drawing a working curve according to the linear relation between the obtained current change value and the concentration of the target protein.

Further, the PBS solution included 0.1MNa₂HPO₄，0.1M KH₂PO₄And 0.1M KCl.

The invention also aims to provide the electrochemical sensor for detecting the human papillomavirus type 16E 6 oncoprotein, which is prepared by the preparation method of the electrochemical sensor for detecting the human papillomavirus type 16E 6 oncoprotein.

By combining all the technical schemes, the invention has the advantages and positive effects that: the electrochemical sensor for detecting the human papillomavirus type 16E 6 oncoprotein, provided by the invention, can realize quantitative detection, is simple to operate, does not need additional professional technicians, is low in cost, wide in linear range and low in detection limit, and can effectively solve the problems that the existing method for detecting the E6 oncoprotein is relatively complex in operation, expensive in equipment, needs professional technicians and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a flow chart of a preparation method of an electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein according to an embodiment of the present invention.

FIG. 2 is a flow chart of an electrode construction provided by an embodiment of the present invention; a is a signal label (the synthesis process of the MBSi-Chi compound) diagram provided by the embodiment of the invention; and B is a schematic diagram of the electrode construction process provided by the embodiment of the invention.

Fig. 3-5 are schematic diagrams of experimental results provided by embodiments of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In view of the problems in the prior art, the present invention provides an electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein, which is described in detail below with reference to the accompanying drawings.

As shown in fig. 1, a method for preparing an electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein according to an embodiment of the present invention includes the following steps:

s101, synthesizing materials;

s102, establishing an electrochemical biosensor;

s103, measuring the target by using a direct method and an indirect method respectively, and drawing a standard curve;

and S104, analyzing the repeatability, stability and specificity of the sensor.

The technical solution of the present invention is further described below with reference to specific examples.

1. Composite material

(1) The synthesis process of the dendritic ternary nanoparticles (PdBP NSs) comprises the following steps: to 5mL DODAC solution (9mg/mL) was added 500. mu.L NH₄F(0.337M)、500μL H₃BO₃(0.101M) and 800. mu.L Na₂PdCl₄(8.5M). After magnetic stirring at 400rpm for 5min, 400. mu.L of 10 wt% NH was added to the mixed solution₃·H₂O was continuously stirred until the solution became colorless. Then, 500. mu.L of NaH was added₂PO₂(2.5 mg). The mixed solution obtained above is placed in a silicon oil bath and heated at 95 ℃ for 20min, and then 500. mu.L DMAB is added dropwise and the heating is continued for 30 min. Centrifuging at 12000rpm for 10min, removing supernatant, collecting precipitate, and washing with ultrapure water, anhydrous ethanol and ultrapure water under the same centrifugation condition. And finally, obtaining PdBP powder through freeze drying treatment.

(2) The synthesis process of the Mesoporous Silica (MSN) comprises the following steps: containing 25mg CTAB, 7mg NaOH was added to 12mL of ultrapure water, transferred to the flask, and the flask was heated to 80 ℃ in a silicon oil bath for 1 hour. Then, 125 μ L of TEOS was added dropwise to the flask, and heating was maintained for 2 hours. The resulting solution was centrifuged at 10000rpm for 10min, the supernatant was removed and the precipitate was collected and washed 3 times with methanol under the same centrifugation conditions. Subsequently, the precipitate obtained after washing was dispersed in anhydrous ethanol. The extract consisted of 7.2mL of absolute ethanol and 0.8mL of concentrated HCl. 4ml of the extract was added to the above absolute ethanol dispersion, and the mixture was oil-bathed at 90 ℃ for 45 min. Then, centrifugation was carried out at 10000rpm for 10min, the supernatant was removed and the precipitate was collected, and the mixture was washed once with ultrapure water, absolute ethanol and ultrapure water under the same centrifugation conditions. The previous extraction step was repeated once. Finally, the precipitate obtained after washing was dried under vacuum at 37 ℃ for 20 hours.

(3) The process of synthesizing the signal tag: first, 1mL of methylene blue solution (MB, 10mM) was mixed with 1mL of MSN dispersion (5mg/mL), sonicated for 30min, and then magnetically stirred at room temperature for 4h (400 rpm). Next, 10. mu.L of glacial acetic acid and 4mg of chitosan (Chi) were added to the above solution, and vigorously stirred at room temperature for 12 hours (1500 rpm). Centrifuging at 10000rpm for 10min, removing supernatant, collecting precipitate, washing with ultrapure water under the same centrifugation condition for 3 times, and collecting precipitate (MBSi-Chi).Thirdly, the collected MBSi-Chi is dried in vacuum at 37 ℃ for 20 h. Then, MBSi-Chi was dispersed in ultrapure water to obtain a dispersion (1 mg/mL). mu.L of MBSi-Chi, 1.2mL of E6 antibody and 466. mu.L of 5% glutaraldehyde solution were added to a small beaker and magnetically stirred in a refrigerator at 4 ℃ for 12 h. Then, 40. mu.L of 0.5% BSA was added to the above solution, and stirring was continued for 6 hours. Centrifuging at 10000rpm for 10min, removing supernatant, collecting precipitate, and centrifuging with Na under the same conditions₂CO₃/NaHCO₃The buffer was washed 3 times and the resulting signal tag (MBsi-Chi complex) was redispersed in 500. mu.L of ultrapure water and stored at 4 ℃.

2. Establishing an electrochemical biosensor, measuring a target, and drawing a standard curve.

(1) The direct method comprises the following steps:

using 0.3 μm and 50nm Al respectively₂O₃Polishing the electrode into a mirror surface by using powder, then respectively carrying out ultrasonic treatment on the electrode for 5min according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;

dripping 10 mu L of electrode modification material dendritic ternary nano particles (PdBP NSs) on the surface of the electrode, and drying at 37 ℃;

seventhly, placing the electrode in 5mL containing 0.1MNa₂HPO₄，0.1M KH₂PO₄Characterization was performed in 0.1M KCl in 0.1M PBS and 20. mu.L of 3.0M H was added every 40s₂O₂Measuring the current value by an ampere current method;

(2) An indirect method:

sixthly, washing the electrode with ultrapure water, drying at room temperature, dripping 10 mu L of signal label (MBsi-Chi compound), and incubating for 2h at 37 ℃;

eighthly, placing the electrode in 5mL containing 0.1MNa₂HPO₄，0.1M KH₂PO₄Performing characterization in 0.1M PBS solution of 0.1M KCl, and measuring the current value by differential pulse voltammetry;

The electrode construction flow chart is shown in FIG. 2; wherein, A of FIG. 2 represents the synthesis process of a signal tag, i.e., MBSi-Chi complex; b of fig. 2 represents an electrode construction process.

The technical effects of the present invention will be described in detail with reference to experiments.

As shown in FIGS. 3-5, the present invention designs a dual-signal immunosensor for the detection of human papillomavirus type 16E 6 oncoprotein. In one aspect, label-free immunosensors are fabricated based on palladium-boron-phosphorus nanospheres (PdBP NSs) for rapid detection. PdBP NSs are ternary nanoparticles with dendritic structures that provide a larger specific surface area, enabling them to immobilize more antibodies through stable covalent bonds between Pd and amino groups. In addition, PdBP NSs were found to exhibit high catalytic activity on H2O 2. The reasons may be as follows: 1) the B/P alloy promotes the decomposition of the oxygen-containing intermediate on the surface of Pd; 2) the dendritic structure of PdBP NSs exposes more active sites and accelerates mass/electron transfer. In summary, PdBP NSs have a large specific surface area, good conductivity and high catalytic efficiency, and are used for the first time to design an immunosensor.

On the other hand, in order to further improve the sensitivity, a sandwich type immunosensor was manufactured using PdBP NSs as a substrate material and an MBSi-Chi nanocomposite material as a signal marker for detection of human papillomavirus type 16E 6 oncoprotein. Methylene Blue (MB) is widely used in biosensors due to its electrochemical redox activity. Methylene blue is susceptible to electropolymerization, resulting in a different morphology. This phenomenon will limit its applications. Mesoporous Silica Nanoparticles (MSN) are a common support with large specific surface area and pore structure. The encapsulation of methylene blue into mesoporous silica (MBSi NPs) not only avoids its electropolymerization but also amplifies the signal response. Chitosan is a biocompatible molecule containing reactive amino groups. Glutaraldehyde is used as a linker to bind the antibody to the nanoparticles via schiff base. Finally, the MBSi-Chi nanocomposite is synthesized to be used as a signal label.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Claims

1. a preparation method for the electrochemical sensor of human papillomavirus type 16 E6 oncoprotein detection, it is characterized in that, the preparation method of the electrochemical sensor for human papillomavirus type 16 type E6 oncoprotein detection comprises The following steps:

Step 1, synthesizing materials;

Step 2, establishing an electrochemical biosensor;

Step 3, use the direct method and the indirect method respectively to determine the target, and draw a standard curve;

The fourth step is to analyze the repeatability, stability and specificity of the sensor.

2. The preparation method of an electrochemical sensor for human papillomavirus type 16 E6 oncoprotein detection as claimed in claim 1, wherein the synthetic material in the step 1 comprises:

(1) Synthesis of dendritic ternary nanoparticles PdBP NSs;

(2) Synthesis of mesoporous silica MSN;

(3) Synthesized signal label.

3. The preparation method of an electrochemical sensor for human papillomavirus type 16 E6 oncoprotein detection according to claim 2, wherein the synthesis process of the dendritic ternary nanoparticles in the step (1) comprises the following steps: :

Add 500 μL 0.337M NH ₄ F, 500 μL 0.101 M H ₃ BO ₃ and 800 μL 8.5 M Na ₂ PdCl ₄ to 5 mL 9 mg/mL DODAC solution; add 400 μL 10wt% to the mixed solution after magnetic stirring at 400 rpm for 5 min NH ₃ ·H ₂ O was continuously stirred until the solution became colorless;

Add 500 μL 2.5 mg of NaH ₂ PO ₂ , place the obtained mixed solution in a silicone oil bath at 95°C for 20 min, add 500 μL DMAB dropwise, and continue heating for 30 min; centrifuge at 12,000 rpm for 10 min, remove the supernatant to collect the precipitate, Under the same centrifugation conditions, it was washed once with ultrapure water, absolute ethanol and ultrapure water respectively; finally, PdBP powder was obtained by freeze-drying.

4. The preparation method of the electrochemical sensor for human papillomavirus type 16 E6 oncoprotein detection as claimed in claim 2, wherein the synthesis process of the mesoporous silica in the step (2) comprises:

Add 25mg CTAB and 7mg NaOH to 12mL ultrapure water, then transfer to a flask, heat the flask to 80°C in a silicone oil bath for 1h; add 125μL TEOS dropwise to the flask, keep heating for 2h; heat the resulting The solution was centrifuged at 10,000 rpm for 10 min, the supernatant was removed to collect the precipitate, and washed three times with methanol under the same centrifugal conditions; the precipitate obtained after washing was dispersed in anhydrous ethanol;

Add 0.8 mL of concentrated hydrochloric acid and 7.2 mL of absolute ethanol to the beaker and stir to obtain an extract;

4 mL of the extract was added to the above-mentioned absolute ethanol dispersion, heated in an oil bath at 90 °C for 45 min; centrifuged at 10,000 rpm for 10 min, the supernatant was removed to collect the precipitate, and ultrapure water, absolute ethanol and ultra-pure water were used under the same centrifugal conditions. The pure water was washed once respectively; the extraction step was repeated once, and the precipitate obtained after washing was vacuum-dried at 37 °C for 20 h.

5. the preparation method of the electrochemical sensor that is used for the detection of human papillomavirus type 16 E6 oncoprotein as claimed in claim 2, is characterized in that, the synthesis process of the signal label in described step (3) comprises:

Mix 1 mL of 10 mM methylene blue solution MB with 1 mL of 5 mg/mL MSN dispersion, sonicate for 30 min, and magnetically stir at 400 rpm for 4 h at room temperature; add 10 μL of glacial acetic acid and 4 mg of chitosan Chi to the solution, and at room temperature at 1500 rpm Stir vigorously for 12h;

Centrifuge at 10,000 rpm for 10 min, remove the supernatant to collect the precipitate, wash with ultrapure water three times under the same centrifugal conditions and collect the precipitated MBSi-Chi; after vacuum drying the collected MBSi-Chi at 37 °C for 20 h, the MBSi-Chi Disperse in ultrapure water to obtain a dispersion of 1 mg/mL;

Add 400 μL of MBSi-Chi, 1.2 mL of E6 antibody and 466 μL of 5% glutaraldehyde solution into a small beaker, stir magnetically for 12 h in a 4°C refrigerator; add 40 μL of 0.5% BSA to the solution, continue stirring for 6 h; at 10000 rpm Centrifuge for 10 min, remove the supernatant to collect the precipitate, wash three times with Na ₂ CO ₃ /NaHCO ₃ buffer under the same centrifugation conditions, and re-disperse the obtained signal tag, namely the MBSi-Chi complex in 500 μL of ultrapure water, 4 Store at ℃ until use.

6. The preparation method of an electrochemical sensor for human papillomavirus type 16 E6 oncoprotein detection as claimed in claim 1, wherein the direct method in the step 3 comprises:

(1) After polishing the electrodes into mirror surfaces with Al ₂ O ₃ powders of 0.3 μm and 50 nm, respectively, ultrasonic electrodes were ultrasonicated in the order of ultrapure water, absolute ethanol, and ultrapure water for 5 minutes each, and dried at room temperature for use;

(2) 10 μL electrode modification material dendritic ternary nanoparticle PdBP NSs was dropped on the electrode surface and dried at 37°C;

(3) Drop 10 μL of E6 antibody on the surface of the electrode after drying, and incubate at 37°C for 2 hours;

(4) Rinse the electrode with ultrapure water and dry it at room temperature, add 6 μL of 0.5% BSA dropwise, and incubate at room temperature for 30 minutes;

(5) Rinse the electrode with ultrapure water and dry it at room temperature, add 8 μL E6 protein dropwise, and incubate at 37°C for 2h;

(6) Rinse the incubated electrode with washing buffer and dry it in nitrogen;

(7) The electrode was placed in 5mL 0.1M PBS solution for characterization, 20μL, 3.0MH ₂ O ₂ was added every 40s, and its current value was measured by amperometric method;

(8) Draw a working curve according to the linear relationship between the obtained current change value and the target protein concentration.

7. The preparation method of an electrochemical sensor for human papillomavirus type 16 E6 oncoprotein detection as claimed in claim 1, wherein the indirect method in the step 3 comprises:

(1) After polishing the electrodes into mirror surfaces with Al ₂ O ₃ powders of 0.3 μm and 50 nm, respectively, ultrasonic electrodes were ultrasonicated in the order of ultrapure water, absolute ethanol and ultrapure water for 5 minutes each, and dried at room temperature for use;

(6) Rinse the electrode with ultrapure water and dry it at room temperature, add 10 μL of the signal tag, namely the MBsi-Chi complex, and incubate at 37°C for 2h;

(7) Rinse the incubated electrode with washing buffer and dry it in nitrogen;

(8) The electrode is placed in 5mL 0.1M PBS solution for characterization, and its current value is measured by differential pulse voltammetry;

(9) Draw a working curve according to the linear relationship between the obtained current change value and the target protein concentration.

8. The preparation method of an electrochemical sensor for human papillomavirus type 16 E6 oncoprotein detection according to claim 6 or 7, wherein the PBS solution comprises 0.1M Na ₂ HPO ₄ , 0.1M KH ₂ PO ₄ and 0.1M KCl.

9. A preparation method for the detection of human papillomavirus type 16 E6 oncoprotein prepared by applying the method for preparing an electrochemical sensor for detection of human papillomavirus type 16 E6 oncoprotein according to any one of claims 1 to 7 electrochemical sensor.