Electrochemical aptamer sensor for detecting human epididymis protein 4 in ovarian cancer and its detection method

Abstract

This invention provides an electrochemical aptasensor for detecting human epididymis protein 4 (HE4) in ovarian cancer. Using PDDA@Ni3(HITP)2 / AuNPs as the sensing interface and NH2-MIL-53(Al)@KB / AuNPs / Tb / SP as the tracer marker, the aptasensor's electrical signal is synergistically amplified, enabling ultrasensitive detection of HE4. This method provides a new diagnostic approach for early diagnosis of ovarian cancer. Compared with traditional HE4 detection methods, the present method offers advantages such as high sensitivity, strong specificity, rapid detection, ease of operation, low-cost equipment and materials, and no pollution.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection, and in particular to an electrochemical aptamer sensor for detecting ovarian cancer human epididymis protein 4 and a detection method thereof. Background Art

[0002] Ovarian cancer is a common malignant tumor that poses a serious threat to the female reproductive system. Often referred to as a "silent killer," ovarian cancer is difficult to treat with curative intent because its symptoms are often vague and it is often diagnosed in the late stages. Therefore, improving the early diagnosis of ovarian cancer and conducting a comprehensive preoperative evaluation are crucial for treatment and prognosis. Human epididymis protein 4 (HE4) is a novel ovarian cancer tumor marker approved by the US Food and Drug Administration (FDA) as a key component of the Risk of Malignancy Algorithm (ROMA) for determining preoperative risk of ovarian cancer and monitoring advanced disease. Therefore, the development of rapid, simple, sensitive, and specific HE4 assays with low detection limits is crucial.

[0003] Currently, HE4 analysis techniques primarily focus on electrochemical immunosensors, enzyme-linked immunosorbent assays (ELISAs), chemiluminescence immunoassays (CLEIAs), fluorescence assays, electrochemiluminescence (ECL), and electrochemiluminescence immunoassays (ECLIA). While widely used, some of these methods also have limitations. For example, ELISAs often exhibit false positives, while fluorescence assays have low sensitivity and are susceptible to interferences. Therefore, as the clinical utility of serum HE4 increases, the development of sensitive, economical, and rapid detection methods, as well as accurate quantitative analysis, is crucial. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides an electrochemical aptamer biosensor for detecting ovarian cancer human epididymis protein 4 (HE4).

[0005] Unless otherwise specified, all parts described in the present invention are parts by weight and all percentages described are mass percentages.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] An electrochemical aptamer biosensor for detecting human epididymis protein 4 (HE4) in ovarian cancer is characterized in that the method for constructing the electrochemical aptamer biosensor for detecting human epididymis protein 4 in ovarian cancer is as follows: first, a PDDA@Ni3(HITP)2 solution is dripped onto the surface of a clean glassy carbon electrode and dried at room temperature; then, the dried electrode is immersed in 1% HAuCl4 and electrochemically deposited at a potential of -0.2 V to obtain a gold nanoparticle (AuNPs) layer; TAP is dripped onto the obtained gold nanoparticle (AuNPs) layer and incubated at room temperature for 12-13 hours; then, a 1% bovine serum albumin (BSA) solution is dripped onto the electrode and incubated at room temperature for 40-50 minutes; then, human epididymis protein 4 (HE4) is dripped onto the electrode and incubated at room temperature for 1-2 hours; finally, a tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP is dripped onto the electrode and incubated at room temperature for 2-3 hours, thereby obtaining an electrochemical aptamer biosensor for detecting HE4.

[0008] The preparation method of the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP is as follows: adding a signal probe (SP) to the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion, stirring in an ice bath for 12-13 hours, centrifuging and washing, and then redispersing the precipitate in ultrapure water to obtain the obtained product.

[0009] The preparation method of the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion is as follows: Tb is added to the NH2-MIL-53(Al)@KB / AuNPs dispersion, stirred at room temperature for 12-13 hours, centrifuged and washed, and then the precipitate is dispersed in ultrapure water to obtain the obtained product.

[0010] The preparation method of the NH2-MIL-53(Al)@KB / AuNPs dispersion is as follows: AuNPs are added to the NH2-MIL-53(Al)@KB dispersion, stirred for 6-7 hours under ice bath conditions, centrifuged and washed, and then the precipitate is dispersed in ultrapure water to obtain the obtained product.

[0011] The preparation method of the NH2-MIL-53(Al)@KB dispersion is as follows: dissolving NH2-MIL-53(Al) powder in ultrapure water, adding KB to the dispersion, ultrasonicating until uniform dispersion, and gently stirring for 6-7 hours to obtain the dispersion.

[0012] The preparation method of the NH2-MIL-53(Al) powder is as follows: aluminum chloride hexahydrate (AlCl3·6H2O) is dissolved in ultrapure water, 2-aminoterephthalic acid (NH2-H2BDC) is added under magnetic stirring, and stirring is continued for 30-35 minutes. After that, a deionized water solution containing urea is added dropwise and stirred for 30-35 minutes. The solution is then transferred to a reactor and reacted in an oven at 150-160°C for 5-6 hours. After cooling to room temperature, the mixture is centrifuged at 8000-10000 rpm and washed with ultrapure water. The product is then dispersed in a mixed solvent of DMF and methanol, stirred at room temperature for 1 day, and finally centrifuged, washed, and dried to obtain a light yellow powder NH2-MIL-53(Al) nanomaterial.

[0013] The preparation method of the gold nanoparticles (AuNPs) is as follows: 1% HAuCl4 solution is added to ultrapure water and boiled, then 1% trisodium citrate solution is added and the mixture is boiled for 15-20 minutes. After cooling, the mixture is restored to its original volume with ultrapure water to obtain a transparent wine-red solution, which is the AuNPs.

[0014] The preparation method of the PDDA@Ni3(HITP)2 dispersion is as follows: Ni3(HITP)2 powder is dissolved in ultrapure water, PDDA is added, and the mixture is stirred at room temperature for 4-5 hours. After centrifugation and washing, the precipitate is dispersed in ultrapure water to obtain a PDDA@Ni3(HITP)2 base material dispersion.

[0015] The preparation method of the Ni3(HITP)2 powder is as follows: nickel chloride hexahydrate (NiCl2·6H2O) is added to water, HATP·6HCl is added to water, and the two solutions are mixed to form a uniform solution; concentrated ammonia water is then added to the mixture, stirred at 60-65°C for 1-2 hours, and then stirred at room temperature for another 2-3 hours, washed by centrifugation with water 2-3 times, and dried in a vacuum oven at 25-28°C for 12-15 hours to obtain a black powder Ni3(HITP)2 nanomaterial.

[0016] An electrochemical aptamer biosensor for detecting human epididymis protein 4 (HE4) in ovarian cancer is characterized in that the method for constructing the electrochemical aptamer biosensor for detecting human epididymis protein 4 in ovarian cancer comprises the following steps:

[0017] (1) Preparation of tracer markers;

[0018] 1) NH2-MIL-53(Al): Dissolve 3 mmol of aluminum chloride hexahydrate (AlCl3·6H2O) in 15 mL of ultrapure water. Add 3 mmol of 2-aminoterephthalic acid (NH2-H2BDC) under magnetic stirring. Stir for 30 minutes. Then, add 15 mL of deionized water containing 6 mmol of urea dropwise to the mixture and stir for 30 minutes. The resulting solution was then transferred to a reactor and reacted in an oven at 150°C for 5 hours. After cooling to room temperature, the mixture was centrifuged at 8000 rpm and washed several times with ultrapure water. The product was then dispersed in 20 mL of DMF and 20 mL of methanol, stirred at room temperature for 1 day, washed by centrifugation, and dried to obtain a pale yellow NH2-MIL-53(Al) nanomaterial.

[0019] 2) NH2-MIL-53(Al)@KB: Dissolve 2 mg of the NH2-MIL-53(Al) powder prepared in step 1) in 2 mL of ultrapure water. Add 2 mg of KB to the dispersion and sonicate until uniformly dispersed. Stir gently for 6 h to obtain an NH2-MIL-53(Al)@KB dispersion.

[0020] 3) Gold nanoparticles (AuNPs): Add 1 mL of 1% HAuCl4 solution to 100 mL of ultrapure water and bring to a boil. Then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, return the volume to the original volume with ultrapure water to obtain a transparent wine-red solution, which is the AuNPs. Store at 4°C.

[0021] 4) NH2-MIL-53(Al)@KB / AuNPs: 1 mL of AuNPs prepared in step 3) was added to the NH2-MIL-53(Al)@KB dispersion prepared in step 2). The mixture was stirred on ice for 6 h. After centrifugation and washing, the precipitate was dispersed in 2 mL of ultrapure water to obtain the NH2-MIL-53(Al)@KB / AuNPs dispersion.

[0022] 5) NH2-MIL-53(Al)@KB / AuNPs / Tb: Add 2 mg of Tb to the NH2-MIL-53(Al)@KB / AuNPs dispersion prepared in step 4), stir at room temperature for 12 h, centrifuge and wash, and then disperse the precipitate in 2 mL of ultrapure water to obtain the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion;

[0023] 6) NH2-MIL-53(Al)@KB / AuNPs / Tb / SP: Add 200 μL of 2 μM signal probe (SP) to 1 mL of the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion prepared in step 5). Stir on ice for 12 h. After centrifugation and washing, redisperse the precipitate in 1 mL of ultrapure water to obtain the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP.

[0024] (2) Preparation of substrate materials;

[0025] 1) Ni₃(HITP)₂: Weigh 0.0808 g of nickel chloride hexahydrate (NiCl₂·6H₂O) and add it to 5 mL of water. Add 0.1218 g of HATP·6HCl to 35 mL of water, then mix the two solutions to form a homogeneous solution. Add 1.125 mL of concentrated ammonia to the mixture, stir at 60°C for 1 hour, then at room temperature for another 2 hours. Wash the mixture three times by centrifugation and dry it in a vacuum oven at 25°C for 12 hours to obtain a black Ni₃(HITP)₂ nanomaterial.

[0026] 2) PDDA@Ni3(HITP)2: Dissolve 1.5 mg of the Ni3(HITP)2 powder prepared in step 1) in 1 mL of ultrapure water. Add 200 μL of PDDA and stir at room temperature for 4 h. After centrifugation and washing, disperse the precipitate in 1 mL of ultrapure water to obtain a PDDA@Ni3(HITP)2 base material dispersion.

[0027] (3) Construction of chemogenic aptamer sensor for HE4 detection:

[0028] 1) Treat the HE4 aptamer (Apt), capture probe (CP), and signal probe (SP) with 20 mM Tris-HCl (pH 7.4) buffer at room temperature and store until use;

[0029] 2) Take 200 μL of the Apt and CP prepared in step 1) and mix them, heat them at 95°C for 5 minutes, and then incubate them at 37°C for 2 hours to obtain a triple-helix aptamer probe (TAP).

[0030] 3) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 minutes, then rinse with ultrapure water for later use;

[0031] 4) The electrodes obtained in step 3) were polished to a mirror surface using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The electrodes were then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dried for later use.

[0032] 5) Electrochemically activate the electrode obtained in step 4) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0033] 6) Add 10 μL of the substrate material PDDA@Ni3(HITP)2 solution onto the surface of the glassy carbon electrode cleaned in step 5) and dry at room temperature;

[0034] 7) Immersing the electrode prepared in step 6) in 1% HAuCl4 and electrochemically depositing it at a potential of -0.2 V for 30 s to obtain a gold nanoparticle (AuNPs) layer;

[0035] 8) Add 10 μL of TAP prepared in step 2) onto the electrode prepared in step 7) and incubate at room temperature for 12 h.

[0036] 9) Add 10 μL of 1% bovine serum albumin (BSA) solution to the electrode obtained in step 8) and incubate at room temperature for 40 minutes.

[0037] 10) Add 10 μL of different concentrations of target human epididymis protein 4 (HE4) to the electrode obtained in step 9) and incubate at room temperature for 1.5 h.

[0038] 11) 10 μL of the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP was added dropwise to the electrode prepared in step 10) and incubated at room temperature for 2 h to obtain an electrochemical aptasensor for HE4 detection.

[0039] This study prepared a composite material consisting of an aluminum-based metal-organic framework (NH2-MIL-53(Al)) supporting Ketjen black (KB)-modified electroactive material, toluidine blue (Tb), coupled with gold nanoparticles (AuNPs). The nanoparticles were then loaded with a large number of signal probes (SP) through the bonding between the metal particles and amino groups, ultimately creating the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP. KB is a highly efficient, high-purity conductive carbon black used in lithium batteries, renowned for its unique morphology and ultrahigh conductivity. In recent years, it has also been widely used in electrochemical sensors to improve conductivity. However, due to its small nanoparticle size, it is not very stable. Metal-organic frameworks (MOFs) are widely used in various fields due to their advantages such as adsorption, high porosity, and large specific surface area. However, MOFs generally exhibit poor electrical conductivity. NH2-MIL-53(Al) was used to modify KB to form a composite material (NH2-MIL-53(Al)@KB). This synergistic effect improves electron transfer efficiency and stability, resulting in enhanced electrocatalytic performance. Although inherently electrochemically inactive, it can support small molecules such as Tb, resulting in a significant and stable signal response. Subsequently, AuNPs were assembled to serve as active binding sites for SP, increasing the SP loading capacity. Alternatively, MOFs offer these advantages while also exhibiting excellent electrical conductivity. Ni3(HITP)2, used in this work, is one such material. PDDA was used to improve its properties, and a layer of AuNPs was electrodeposited on the surface as a sensing interface, enabling the incorporation of a large number of triple-helical aptamer probes (TAPs) for further signal amplification. Generally speaking, the inherent flexibility of oligonucleotides is highly sensitive to their working environment, making their conformational structure unstable and leading to weak affinity and selectivity. However, TAP, based on the Watson-Crick and Hoogsteen base pairing mechanisms, has a unique structural feature. It consists of two aptamer sequences flanked by loops and a three-stranded oligonucleotide. This stable conformational structure achieves stronger binding affinity and stability. Therefore, in this invention, PDDA@Ni3(HITP)2 / AuNPs is used as the sensing interface and NH2-MIL-53(Al)@KB / AuNPs / Tb / SP is used as the tracer marker. The synergistic amplification of the electrical signal of the aptamer sensor achieves ultrasensitive detection of HE4, providing a new diagnostic approach for the early diagnosis of ovarian cancer patients.

[0040] The present invention also provides a method for detecting HE4 using the electrochemical aptasensor.

[0041] The method of detecting HE4 using an electrochemical aptamer sensor of the present invention comprises the following steps:

[0042] 1) The sensor electrode was placed in a 0.1 M PBS (pH = 7.4) characterization solution and quantitatively detected using the DPV technique in the range of -0.7 V to 0.1 V;

[0043] 2) Based on the linear relationship between the DPV response obtained in step 1) and the logarithmic value of HE4 concentration, a working curve is drawn;

[0044] 3) The sample to be tested is detected by the sensor, and the obtained current value is calculated using the working curve prepared in step 2) to obtain the HE4 concentration of the sample to be tested.

[0045] Compared with the prior art, the preparation method and application of the electrochemical aptasensor for detecting HE4 of the present invention have the following outstanding features:

[0046] This paper prepares a composite material of an aluminum-based metal-organic framework (NH2-MIL-53(Al)) loaded with Ketjen black (KB)-modified electroactive material toluidine blue (Tb) coupled with gold nanoparticles (AuNPs). Then, a large number of signal probes (SP) are loaded through the bonding between the metal particles and the amino groups, and finally the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP is prepared. In terms of material selection, NH2-MIL-53(Al) is a mesoporous molecular sieve with diamond-shaped pores. Due to its outstanding molecular loading capacity (187.8 m 2 This novel nanostructured aptamer has attracted considerable attention due to its high surface area (<0.05 nm), large pore size (4.8 nm), and broad acid-base adaptability (pH 4.00-8.00). This work, for the first time, combines NH2-MIL-53(Al) with KB to form a composite material (NH2-MIL-53(Al)@KB). This significantly improves the signal response and stability of either NH2-MIL-53(Al) or KB combined with Tb, achieving signal amplification and thus enhancing sensor sensitivity. Subsequently, AuNPs were assembled as active binding sites for the signal probe (SP), increasing the loading capacity. Furthermore, in this work, a PDDA@Ni3(HITP)2 composite was synthesized by stirring Ni3(HITP)2 and PDDA as a sensing interface, capturing a large number of triple-helix aptamer probes (TAPs) for further signal amplification. Through these methods, the prepared electrochemical aptamer sensor was successfully applied for the ultrasensitive detection of HE4. Compared with the traditional HE4 detection method, the advantages of the present invention are high sensitivity, strong specificity, rapid detection, convenient operation, low equipment and material prices, and no pollution, thereby providing a new analytical method for the detection of HE4.

[0047] The beneficial effects of the present invention are:

[0048] 1) A composite material composed of Ketjen Black (KB) supported by an aluminum-based metal-organic framework (NH2-MIL-53(Al)) can synergistically improve the efficiency and stability of electron transfer and possess better electrocatalytic ability. The electroactive substance Toluidine Blue (Tb) is then modified to obtain a significant and stable signal response, thereby improving sensor performance, achieving signal amplification, and increasing detection sensitivity.

[0049] 2) Based on the preparation of Ni3(HITP)2 substrate materials, part of the sensor construction process can be characterized by electrochemical means within a certain voltage range.

[0050] 3) HE4 aptamers were used for the first time in the detection of HE4 for target identification. The triple-helix aptamer probe has high specificity and can improve the selectivity and stability of the sensor, thus providing a new research direction and analytical method for the detection of trace HE4.

[0051] 4) All the materials involved can be synthesized under laboratory conditions, which are simple to operate, the raw materials are inexpensive, low in toxicity, and environmentally friendly. In addition, the amount used each time is extremely small, which reduces the experimental cost.

[0052] 5) The entire detection and analysis method has clear and simple steps, high sensitivity, and rapid signal response.

[0053] 6) The electrochemical aptamer sensor prepared by this method can provide a new method for the detection of HE4; the electrochemical aptamer sensor prepared by this invention can also be applied to other biological sample determinations, food, drug and environmental monitoring, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Cyclic voltammetry (A) and impedance voltammetry (B) of different modified electrodes in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a voltage range of -0.2 to 0.6 V and a scan rate of 100 mV / s.

[0055] Figure 2 Figure 1 is the detection result of different concentrations of HE4 by the sensor of the present invention, wherein Figure A is the cyclic voltammogram of the sensor scanning 0, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM HER-2 in 0.1 M PBS (pH 7.0); Figure B is the calibration curve of the sensor current response value and the logarithmic value of different HE4 concentrations.

[0056] Figure 3 This is the sensor stability test result, which is the stability test graph obtained after the sensor incubated with 0.1 nM HE4 was scanned continuously for 50 cycles;

[0057] Figure 4 The figure shows the reproducibility of five different glassy carbon electrodes incubated with 0.001 nM HE4 and scanned under the same conditions.

[0058] Figure 5 This is the specificity detection graph of the HE4 aptamer sensor, where the interfering substances are ultrapure water, neuron-specific enolase (NSE, 1 nM), human epidermal growth factor receptor 2 (HER-2, 1 nM), human serum albumin (HSA, 1 nM), cytokeratin 19 fragment (CYFRA 21-1, 1 nM), prostate-specific antigen (PSA, 1 nM), and carcinoembryonic antigen (CEA, 1 nM). DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0060] The aptamers involved in the examples of the present invention were synthesized by Shanghai Sangon Co., Ltd., and the specific sequences are as follows:

[0061] Sequence of HE4-binding aptamer chain (Apt):

[0062] 5'-NH2-(CH2)6-CTTCTCTTTATCGTACGACAGTCATCCTACACTCTCTTC-3'

[0063] Sequence of capture probe (CP): 5'-NH2-(CH2)6-AAAGAAGAGAAG-3'

[0064] Sequence of signal probe (SP): 5'-NH2-(CH2)6-TTCTCTTCTTT-3'

[0065] Equipment used and technical parameters:

[0066] Instrumentation: Differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were performed using a Metrohm Autolab BV electrochemical workstation (Modular Instruments, Switzerland). Electrochemical detection was performed using a three-electrode system: a modified glassy carbon electrode (4 mm diameter) as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. pH was monitored using a pH meter (S210 SevenCompact, Mettler-Toledo, Shanghai, China). Electrochemical three-electrode DPV was performed in 0.1 M PBS solution (pH 7.4) with a voltage range of -0.7 V to 0.1 V ( vs.SCE), CV scans were performed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution at 100 mV / s, and the voltage range was set from -0.6 V to 0.2 V ( vs. SCE). Example 1

[0067] The preparation of the electrochemical aptamer biosensor for detecting human epididymis protein 4 (HE4) in ovarian cancer according to the present invention is carried out according to the following steps:

[0068] (1) Preparation of tracer markers;

[0069] 1) NH2-MIL-53(Al): Dissolve 3 mmol of aluminum chloride hexahydrate (AlCl3·6H2O) in 15 mL of ultrapure water. Add 3 mmol of 2-aminoterephthalic acid (NH2-H2BDC) under magnetic stirring. Stir for 30 minutes. Then, add 15 mL of deionized water containing 6 mmol of urea dropwise to the mixture and stir for 30 minutes. The resulting solution was then transferred to a reactor and reacted in an oven at 150°C for 5 hours. After cooling to room temperature, the mixture was centrifuged at 8000 rpm and washed several times with ultrapure water. The product was then dispersed in 20 mL of DMF and 20 mL of methanol, stirred at room temperature for 1 day, washed by centrifugation, and dried to obtain a pale yellow NH2-MIL-53(Al) nanomaterial.

[0070] 2) NH2-MIL-53(Al)@KB: Dissolve 2 mg of the NH2-MIL-53(Al) powder prepared in step 1) in 2 mL of ultrapure water. Add 2 mg of KB to the dispersion and sonicate until uniformly dispersed. Stir gently for 6 h to obtain an NH2-MIL-53(Al)@KB dispersion.

[0071] 3) Gold nanoparticles (AuNPs): Add 1 mL of 1% HAuCl4 solution to 100 mL of ultrapure water and bring to a boil. Then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, return the volume to the original volume with ultrapure water to obtain a transparent wine-red solution, which is the AuNPs. Store at 4°C.

[0072] 4) NH2-MIL-53(Al)@KB / AuNPs: 1 mL of AuNPs prepared in step 3) was added to the NH2-MIL-53(Al)@KB dispersion prepared in step 2). The mixture was stirred on ice for 6 h. After centrifugation and washing, the precipitate was dispersed in 2 mL of ultrapure water to obtain the NH2-MIL-53(Al)@KB / AuNPs dispersion.

[0073] 5) NH2-MIL-53(Al)@KB / AuNPs / Tb: Add 2 mg of Tb to the NH2-MIL-53(Al)@KB / AuNPs dispersion prepared in step 4), stir at room temperature for 12 h, centrifuge and wash, and then disperse the precipitate in 2 mL of ultrapure water to obtain the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion;

[0074] 6) NH2-MIL-53(Al)@KB / AuNPs / Tb / SP: Add 200 μL of 2 μM signal probe (SP) to 1 mL of the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion prepared in step 5). Stir on ice for 12 h. After centrifugation and washing, redisperse the precipitate in 1 mL of ultrapure water to obtain the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP.

[0075] (2) Preparation of substrate materials;

[0076] 1) Ni₃(HITP)₂: Weigh 0.0808 g of nickel chloride hexahydrate (NiCl₂·6H₂O) and add it to 5 mL of water. Add 0.1218 g of HATP·6HCl to 35 mL of water, then mix the two solutions to form a homogeneous solution. Add 1.125 mL of concentrated ammonia to the mixture, stir at 60°C for 1 hour, then at room temperature for another 2 hours. Wash the mixture three times by centrifugation and dry it in a vacuum oven at 25°C for 12 hours to obtain a black Ni₃(HITP)₂ nanomaterial.

[0077] 2) PDDA@Ni3(HITP)2: Dissolve 1.5 mg of the Ni3(HITP)2 powder prepared in step 1) in 1 mL of ultrapure water. Add 200 μL of PDDA and stir at room temperature for 4 h. After centrifugation and washing, disperse the precipitate in 1 mL of ultrapure water to obtain a PDDA@Ni3(HITP)2 base material dispersion.

[0078] (3) Preparation of electrochemical aptasensor for HE4 detection:

[0079] 1) Treat the HE4 aptamer (Apt), capture probe (CP), and signal probe (SP) with 20 mM Tris-HCl (pH 7.4) buffer at room temperature and store until use;

[0080] 2) Take 200 μL of the Apt and CP prepared in step 1) and mix them, heat them at 95°C for 5 minutes, and then incubate them at 37°C for 2 hours to obtain a triple-helix aptamer probe (TAP).

[0081] 3) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 minutes, then rinse with ultrapure water for later use;

[0082] 4) The electrodes obtained in step 3) were polished to a mirror surface using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The electrodes were then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dried for later use.

[0083] 5) Electrochemically activate the electrode obtained in step 4) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0084] 6) Add 10 μL of the substrate material PDDA@Ni3(HITP)2 solution onto the surface of the glassy carbon electrode cleaned in step 5) and dry at room temperature;

[0085] 7) Immersing the electrode prepared in step 6) in 1% HAuCl4 and electrochemically depositing it at a potential of -0.2 V for 30 s to obtain a gold nanoparticle (AuNPs) layer;

[0086] 8) Add 10 μL of TAP prepared in step 2) onto the electrode prepared in step 7) and incubate at room temperature for 12 h.

[0087] 9) Add 10 μL of 1% bovine serum albumin (BSA) solution to the electrode obtained in step 8) and incubate at room temperature for 40 minutes.

[0088] 10) Add 10 μL of different concentrations of target human epididymis protein 4 (HE4) to the electrode obtained in step 9) and incubate at room temperature for 1.5 h.

[0089] 11) 10 μL of the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP was added dropwise to the electrode prepared in step 10) and incubated at room temperature for 2 h to obtain an electrochemical aptasensor for HE4 detection.

[0090] Example 2

[0091] Detection of HE4 using electrochemical aptasensor

[0092] HE4 was detected using the electrochemical aptasensor constructed in Example 1, following the steps below:

[0093] 1. Draw a working curve

[0094] 1) The modified electrodes from step 5) to step 10) in step (3) of Example 1 were placed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV and EIS characterization. The current response signals were measured, and the results were as follows: Figure 1 As shown in Figure A: (a) Bare glassy carbon electrode; (b) Addition of DDA@Ni3(HITP)2 composite material; (c) Electrochemical deposition of AuNPs; (d) Addition of TAP; (e) BSA blocking; (f) Addition of HE4; (g): Addition of tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP. The impedance response signal is measured, and the results are shown in Figure 1. Figure 1 Figure B shows: (a) bare glassy carbon electrode; (b) dropwise addition of DDA@Ni3(HITP)2 composite material; (c) electrochemical deposition of AuNPs; (d) dropwise addition of TAP; (e) BSA blocking; (f) dropwise addition of HE4; (g) dropwise addition of tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP

[0095] 2) 10 μL of target HE4 at different concentrations was added to the electrode of the aptasensor prepared in Example 1, and the current changes were measured. Figure 2 As shown in A: The concentrations of a→i are: 0, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 nM.

[0096] 3) Based on the linear relationship between the obtained current change value and the logarithmic value of HE4 concentration, draw a working curve (such as Figure 2 The results showed that the current response value and the logarithm of HE2 concentration showed a good linear relationship in the range of 1 fM-10 nM, with a linear correlation coefficient of 0.9985 and a detection limit of 0.41 fM.

[0097] 2. Sensor stability test: After the sensor prepared in Example 1 was subjected to 50 consecutive CV measurements under optimal conditions, its current intensity only decreased by 8.8% (e.g. Figure 3 ), indicating that the sensor has good stability.

[0098] 3. Sensor reproducibility test: The sensor prepared in Example 1 by incubating the same concentration of HE4 (10 pM) with five different glassy carbon electrodes was subjected to DPV measurement (e.g. Figure 4 The relative standard deviation (RSD) was 2.66%, indicating that the sensor had good reproducibility.

[0099] 4. Sensor specificity test: In order to study the specificity of the proposed adaptive sensor, the following interfering substances that may be present in serum were used: neuron-specific enolase (NSE, 1 nM), human epidermal growth factor receptor 2 (HER-2, 1 nM), human serum albumin (HSA, 1 nM), cytokeratin 19 fragment (CYFRA 21-1, 1 nM), prostate-specific antigen (PSA, 1 nM) and carcinoembryonic antigen (CEA, 1 nM). The current response values (such as Figure 5 ), indicating that the sensor has good specificity.

Claims

1. An electrochemical aptamer biosensor for detecting human epididymis protein 4 (HE4) in ovarian cancer, characterized in that: The electrochemical aptasensor for detecting human epididymis protein 4 (HE4) in ovarian cancer was constructed as follows: PDDA@Ni3(HITP)2 solution was first added to the surface of a clean glassy carbon electrode and dried at room temperature. The dried electrode was then immersed in a 1% chloroauric acid (HAuCl4) solution and electrochemically deposited at a potential of -0.2 V to obtain a layer of gold nanoparticles (AuNPs). A triple-helix aptamer probe (TAP) was added to the obtained AuNPs layer and incubated at room temperature for 12-13 hours. A 1% bovine serum albumin (BSA) solution was then added to the electrode and incubated at room temperature for 40-50 minutes. Human epididymis protein 4 (HE4) was then added to the electrode and incubated at room temperature for 1-2 hours. Finally, the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP was added to the electrode and incubated at room temperature for 2-3 hours to obtain the electrochemical aptasensor for HE4 detection. The preparation method of the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP is as follows: adding a signal probe (SP) to the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion, stirring in an ice bath for 12-13 hours, centrifuging and washing, and then redispersing the precipitate in ultrapure water to obtain the obtained product; The NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion is prepared by adding toluidine blue (Tb) to the NH2-MIL-53(Al)@KB / AuNPs dispersion, stirring at room temperature for 12-13 hours, centrifuging and washing, and then dispersing the precipitate in ultrapure water to obtain the NH2-MIL-53(Al)@KB / AuNPs dispersion. The preparation method of the NH2-MIL-53(Al)@KB / AuNPs dispersion is as follows: adding gold nanoparticles (AuNPs) to the NH2-MIL-53(Al)@KB dispersion, stirring for 6-7 hours under ice bath conditions, centrifuging and washing, and then dispersing the precipitate in ultrapure water to obtain the NH2-MIL-53(Al)@KB dispersion; The preparation method of the NH2-MIL-53(Al)@KB dispersion is as follows: dissolving NH2-MIL-53(Al) powder in ultrapure water, adding Ketjen black (KB) to the dispersion, ultrasonicating until uniform dispersion, and gently stirring for 6-7 hours to obtain the dispersion; The preparation method of the NH2-MIL-53(Al) powder is as follows: aluminum chloride hexahydrate (AlCl3·6H2O) is dissolved in ultrapure water, 2-aminoterephthalic acid (NH2-H2BDC) is added under magnetic stirring, and stirring is continued for 30-35 minutes. After that, a deionized water solution containing urea is added dropwise and stirred for 30-35 minutes. The solution is then transferred to a reactor and reacted in an oven at 150-160°C for 5-6 hours. After cooling to room temperature, the mixture is centrifuged at 8000-10000 rpm and washed with ultrapure water. The product is then dispersed in a mixed solvent of N,N-dimethylformamide (DMF) and methanol, stirred at room temperature for 1 day, and finally centrifuged, washed, and dried to obtain a light yellow powder NH2-MIL-53(Al) nanomaterial.

2. The electrochemical aptamer biosensor according to claim 1, wherein The preparation method of the gold nanoparticles (AuNPs) is as follows: 1% chloroauric acid (HAuCl4) solution is added to ultrapure water and boiled, and then 1% trisodium citrate solution is added and boiled for 15-20 minutes. After cooling, the volume is restored to the original volume with ultrapure water to obtain a transparent wine-red solution, which is the AuNPs.

3. The electrochemical aptamer biosensor according to claim 1, wherein The preparation method of the PDDA@Ni3(HITP)2 dispersion is as follows: Ni3(HITP)2 powder is dissolved in ultrapure water, PDDA is added, and the mixture is stirred at room temperature for 4-5 hours. After centrifugation and washing, the precipitate is dispersed in ultrapure water to obtain a PDDA@Ni3(HITP)2 base material dispersion.

4. The electrochemical aptamer biosensor according to claim 3, wherein The preparation method of the Ni3(HITP)2 powder comprises the following steps: adding nickel chloride hexahydrate to water, adding HATP·6HCl to water, and then mixing the two solutions to form a uniform solution; then adding concentrated ammonia water to the mixture, stirring at 60-65°C for 1-2 hours, stirring at room temperature for another 2-3 hours, washing by centrifugation with water 2-3 times, and drying in a vacuum oven at 25-28°C for 12-15 hours to obtain a black powder Ni3(HITP)2 nanomaterial.

5. An electrochemical aptamer biosensor for detecting human epididymis protein 4 (HE4) in ovarian cancer, characterized in that: The method for constructing an electrochemical aptamer sensor for detecting human epididymis protein 4 in ovarian cancer comprises the following steps: (1) Preparation of tracer markers; 1) NH2-MIL-53(Al): 3 mmol of aluminum chloride hexahydrate was dissolved in 15 mL of ultrapure water. 3 mmol of 2-aminoterephthalic acid was added under magnetic stirring. After continuous stirring for 30 minutes, 15 mL of deionized water solution containing 6 mmol of urea was added dropwise and stirred for 30 minutes. The resulting solution was then transferred to a reactor and reacted in an oven at 150°C for 5 hours. After cooling to room temperature, the mixture was centrifuged at 8000 rpm and washed several times with ultrapure water. The product was then dispersed in 20 mL of N,N-dimethylformamide (DMF) and 20 mL of methanol, stirred at room temperature for 1 day, and finally centrifuged, washed, and dried to obtain a light yellow NH2-MIL-53(Al) nanomaterial. 2) NH2-MIL-53(Al)@KB: Dissolve 2 mg of the NH2-MIL-53(Al) powder prepared in step 1) in 2 mL of ultrapure water. Add 2 mg of Ketjen black (KB) to the dispersion and sonicate until uniformly dispersed. Stir gently for 6 h to obtain an NH2-MIL-53(Al)@KB dispersion. 3) Gold Nanoparticles (AuNPs): Add 1 mL of 1% chloroauric acid (HAuCl4) solution to 100 mL of ultrapure water and bring to a boil. Then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, return the volume to the original volume with ultrapure water to obtain a transparent wine-red solution, which is the gold nanoparticles (AuNPs). Store at 4°C. 4) NH2-MIL-53(Al)@KB / AuNPs: 1 mL of gold nanoparticles (AuNPs) prepared in step 3) was added to the NH2-MIL-53(Al)@KB dispersion prepared in step 2). The mixture was stirred on ice for 6 h. After centrifugation and washing, the precipitate was dispersed in 2 mL of ultrapure water to obtain the NH2-MIL-53(Al)@KB / AuNPs dispersion. 5) NH2-MIL-53(Al)@KB / AuNPs / Tb: Add 2 mg of toluidine blue (Tb) to the NH2-MIL-53(Al)@KB / AuNPs dispersion prepared in step 4), stir at room temperature for 12 h, centrifuge and wash, and then disperse the precipitate in 2 mL of ultrapure water to obtain the NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion. 6) NH2-MIL-53(Al)@KB / AuNPs / Tb / SP: 200 μL of 2 μM signal probe (SP) was added to 1 mL of NH2-MIL-53(Al)@KB / AuNPs / Tb dispersion, and the mixture was stirred on ice for 12 h. After centrifugation and washing, the precipitate was redispersed in 1 mL of ultrapure water to obtain the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP. (2) Preparation of substrate materials; 1) Ni3(HITP)2: Weigh 0.0808 g of nickel chloride hexahydrate and add it to 5 mL of water. Then, add 0.1218 g of HATP·6HCl to 35 mL of water. The two solutions are then mixed to form a homogeneous solution. 1.125 mL of concentrated ammonia is then added to the mixture. The mixture is stirred at 60°C for 1 h, then at room temperature for another 2 h. The mixture is washed three times by centrifugation with water and dried in a vacuum oven at 25°C for 12 h to obtain a black Ni3(HITP)2 nanomaterial. 2) PDDA@Ni3(HITP)2: Dissolve 1.5 mg of the Ni3(HITP)2 powder prepared in step 1) in 1 mL of ultrapure water. Add 200 μL of PDDA and stir at room temperature for 4 h. After centrifugation and washing, disperse the precipitate in 1 mL of ultrapure water to obtain a PDDA@Ni3(HITP)2 substrate dispersion. (3) Construction of chemogenic aptamer sensor for HE4 detection: 1) Treat the HE4 aptamer (Apt), capture probe (CP), and signal probe (SP) with 20 mM Tris-HCl buffer (pH 7.4) at room temperature and store until ready for use. 2) Take 200 μL of the Apt and CP prepared in step 1) and mix them, heat them at 95°C for 5 minutes, and then incubate them at 37°C for 2 hours to obtain a triple-helix aptamer probe (TAP). 3) Soak the glassy carbon electrode in a piranha solution (98% H2SO4 / 30% H2O2 = 3:1 by volume) for 30 minutes, then rinse with ultrapure water for later use. 4) The electrodes obtained in step 3) were polished to a mirror surface using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The electrodes were then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dried for later use. 5) Electrochemically activate the electrode obtained in step 4) in 0.5 M H2SO4, then rinse with ultrapure water and dry; 6) Add 10 μL of the substrate material PDDA@Ni3(HITP)2 solution onto the surface of the glassy carbon electrode cleaned in step 5) and dry at room temperature; 7) Immersing the electrode prepared in step 6) in a 1% chloroauric acid (HAuCl4) solution and electrochemically depositing it at a potential of -0.2 V for 30 s to obtain a gold nanoparticle (AuNPs) layer; 8) Add 10 μL of TAP prepared in step 2) onto the electrode prepared in step 7) and incubate at room temperature for 12 h. 9) Add 10 μL of 1% bovine serum albumin (BSA) solution to the electrode obtained in step 8) and incubate at room temperature for 40 min. 10) Add 10 μL of different concentrations of target human epididymis protein 4 (HE4) to the electrode obtained in step 9) and incubate at room temperature for 1.5 h. 11) 10 μL of the tracer marker NH2-MIL-53(Al)@KB / AuNPs / Tb / SP was added dropwise to the electrode prepared in step 10) and incubated at room temperature for 2 h to obtain an electrochemical aptasensor for HE4 detection.

6. A method for detecting human epididymis protein 4 (HE4) using the electrochemical aptasensor according to any one of claims 1 to 5, characterized in that: The steps include: 1) The sensor electrode was placed in a 0.1 M PBS characterization solution (pH = 7.4) and quantitatively detected using DPV technology within the range of -0.7 V to 0.1 V; 2) Based on the linear relationship between the DPV response obtained in step 1) and the logarithmic value of HE4 concentration, a working curve is drawn; 3) The sample to be tested is detected by the sensor, and the obtained current value is calculated using the working curve prepared in step 2) to obtain the HE4 concentration of the sample to be tested.

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