An IL5 / Bi2MoO6 / MXene photoelectric active composite material, a low-background photoelectrochemical aptamer sensor, its preparation method and application

Abstract

This invention relates to an IL5 / Bi2MoO6 / MXene photoactive composite material, a low-background photoelectrochemical aptamer sensor, its preparation method, and its applications. The sensor is constructed by sequentially modifying chitosan, glutaraldehyde, complementary DNA strands, and a gold nanoparticle-labeled aptamer. A fixed capture probe and APT-Au synergistically achieve dual signal quenching, constructing an extremely low detection baseline. When the target analyte MUC1 is present, its specific binding to the aptamer causes APT-Au to detach, dissolving the dual quenching and significantly restoring the photocurrent, thereby achieving quantitative detection. This invention solves the problems of low efficiency, heterojunction instability, and high baseline in single quenching modes of existing photoelectric materials, achieving highly sensitive, highly specific, rapid, and low-cost detection of the breast cancer biomarker MUC1 in human serum samples, providing a novel, industrially feasible technical solution for large-scale early screening of breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection and photoelectrochemical sensing technology, and particularly relates to an IL5 / Bi2MoO6 / MXene photoactive composite material, a low-background photoelectrochemical aptamer sensor, its preparation method, and its application in the detection of the breast cancer marker MUC1. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide. According to global cancer data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, breast cancer accounts for 25% of all cancers in women worldwide, ranking first among malignant tumors in women.

[0003] Studies have confirmed that early screening, early diagnosis, and early intervention for breast cancer are core means to improve the 5-year survival rate and reduce the mortality rate, and are also the top priority of current breast cancer prevention and control efforts. However, the mainstream clinical methods for breast cancer detection, including breast magnetic resonance imaging (MRI), mammography, and histopathological biopsy, all have unavoidable limitations: breast MRI is expensive and time-consuming, making it difficult to use for large-scale initial screening; mammography has a low detection rate for lesions in dense breast tissue and carries the risk of ionizing radiation; histopathological biopsy, as a diagnostic standard, is an invasive procedure that can cause physical and psychological trauma to patients, and the long post-sampling pathological testing cycle makes rapid screening impossible. These shortcomings make it difficult for existing technologies to be widely applied in large-scale early breast cancer screening in primary healthcare institutions.

[0004] With the development of precision medicine and molecular diagnostic technology, in vitro detection technology based on tumor-specific biomarkers has become a core research direction for early breast cancer screening. Mucin1 (MUC1) is a high-molecular-weight glycoprotein located on the cell membrane surface. It exhibits specific abnormally high expression in breast cancer tissue, and its expression level is significantly positively correlated with the pathological grade, clinical stage, and invasive and metastatic ability of breast cancer. It has been confirmed by numerous studies at home and abroad as a core specific biomarker for early diagnosis and prognostic assessment of breast cancer. Quantitative detection technology based on MUC1 has also become a research hotspot in the field of early breast cancer diagnosis.

[0005] Currently, various technical approaches have been developed for the detection of MUC1, including electrochemiluminescence (ECL) immunoassay, fluorescence immunoassay, and photoelectrochemical (PEC) sensing. Among them, photoelectrochemical sensing technology has shown great application potential and industrialization value in the field of trace biomarker detection due to its core advantages such as simple detection equipment, convenient operation, strong resistance to background interference, high detection sensitivity, and miniaturization integration. It has become the mainstream research and development direction for MUC1 detection technology.

[0006] The core performance of photoelectrochemical sensors is determined by photoelectroactive materials, whose core function is to achieve efficient conversion of light to electrical signals, while providing active binding sites for photoelectrochemical reactions and bio-recognition elements.

[0007] Bi₂MoO₆ (bismuth molybdate) is a typical n-type semiconductor material with a band gap of 2.5-2.8 eV. It boasts advantages such as simple synthesis, good chemical stability, low biotoxicity, and abundant active sites, and has been widely used in photocatalysis, environmental pollutant degradation, and photoelectrochemical sensing. However, pure-phase Bi₂MoO₆ materials have intrinsic defects: firstly, the high recombination rate of photogenerated carriers, with photogenerated electron-hole pairs readily recombinating within the material, leads to low photoelectric conversion efficiency; secondly, the small specific surface area limits the number of biorecognition active sites available. These defects make it difficult for pure-phase Bi₂MoO₆ to meet the high-sensitivity detection requirements of photoelectrochemical sensing applications, necessitating modification and optimization to improve its photoelectric performance.

[0008] Existing modification methods for Bi₂MoO₆ mainly include elemental doping, defect engineering, and semiconductor heterostructure construction. Among these, constructing heterojunctions can create a built-in electric field through band matching between materials, achieving efficient separation and migration of photogenerated carriers, which is one of the most effective modification strategies for improving semiconductor photoelectric conversion efficiency. For n-type Bi₂MoO₆ materials, n-type heterojunctions have become the mainstream modification direction due to their advantages such as a wide variety of selectable materials, simple preparation processes, and good stability and batch repeatability of composite materials. Furthermore, the charge transport efficiency of heterojunctions can be further improved by introducing highly conductive carbon materials.

[0009] MXenes are a class of two-dimensional transition metal carbide / nitride / carbonitride materials that exhibit typical n-type semiconductor characteristics in band matching and interfacial charge behavior. They also possess tunable optical properties, ultra-high specific surface area, excellent metallic conductivity, and good hydrophilicity, making them ideal materials for constructing n-type heterojunctions with Bi2MoO6.

[0010] However, the Bi2MoO6 / MXene heterojunctions constructed in the existing technology generally suffer from weak interfacial bonding, poor material dispersion, and structural instability during long-term use. These problems directly lead to the degradation of sensor photoelectric performance, and a decrease in detection repeatability and stability, becoming a key bottleneck restricting the industrial application of this composite material.

[0011] Ionic liquids (ILs), as a class of room-temperature molten zwitterionic compounds, possess excellent ionic conductivity, chemical stability, and anti-interference capabilities. Existing research has confirmed that when ionic liquids are combined with semiconductor photoactive materials, they can significantly improve the conductivity of composite materials and suppress photogenerated carrier recombination. At the same time, they can regulate the microstructure of semiconductor materials through interfacial interactions, thereby improving the stability and dispersion of heterojunction interfaces. However, there are currently no mature technical solutions for using ionic liquids to regulate the microenvironment of Bi2MoO6 / MXene heterojunction interfaces and simultaneously address the defects in photoelectric properties and structural stability of materials.

[0012] Besides photoactive materials, the choice of detection mode is another crucial factor determining the detection performance of photoelectrochemical sensors. Currently, the mainstream detection modes of photoelectrochemical sensors are divided into two categories: signal-off and signal-on. While signal-off detection modes are simple to design and easy to construct, they suffer from drawbacks such as high background interference, low detection sensitivity, and a tendency to produce false positives, making them unsuitable for trace detection of tumor markers. In contrast, signal-on detection modes offer core advantages such as high detection sensitivity, low background signal, strong specificity, and better suitability for detecting low-concentration target analytes, making them the mainstream research direction for the photoelectrochemical detection of tumor markers. However, the high sensitivity of signal-on detection modes is highly dependent on an extremely low initial detection baseline. The photoactive materials themselves generate intrinsic photocurrent under illumination, which severely compresses the signal amplification space for target analyte detection, directly leading to a decrease in the detection signal-to-noise ratio and an increase in the detection limit, making ultra-trace detection impossible. Therefore, it is essential to efficiently quench the initial photocurrent signal of the sensor to construct an extremely low detection baseline.

[0013] Existing signal quenching methods for signal-on sensors mainly rely on the steric hindrance effect generated by the capture probe fixed at the sensing interface to hinder the migration of electron donors in the electrolyte to the electrode surface, thereby suppressing photocurrent. However, this single steric hindrance signal quenching method has limited efficiency in suppressing photocurrent and cannot achieve an extremely low detection baseline, making it difficult to meet the needs of ultra-trace detection of MUC1 in early breast cancer screening.

[0014] Gold nanoparticles (AuNPs) are a type of nanomaterial with excellent biocompatibility and high surface activity. They exhibit a strong localized surface plasmon resonance effect, which can competitively absorb photon energy from excitation sources and efficiently capture photogenerated electrons, thereby intensifying the recombination of photogenerated carriers and achieving efficient quenching of photoelectric signals. This provides a feasible path for constructing a dual signal quenching strategy. However, in current technologies, there is no mature technical solution that combines the steric hindrance effect with the AuNPs plasmon resonance effect to construct a dual signal quenching system and achieve extremely low background and ultra-high sensitivity detection of MUC1. Summary of the Invention

[0015] The technical problem solved by this invention: Addressing the shortcomings of existing technologies in early breast cancer detection and MUC1 photoelectrochemical (PEC) sensing, this invention aims to solve the following core technical issues:

[0016] 1. This invention addresses the problems of high photogenerated carrier recombination rate, small specific surface area, and low photoelectric conversion efficiency of pure-phase Bi2MoO6 semiconductor materials, which make it difficult to meet the high-sensitivity detection requirements of PEC sensors. At the same time, it overcomes the industry pain points of weak interfacial bonding, poor material dispersion, and insufficient structural stability of existing Bi2MoO6 / MXene heterojunctions, which lead to the degradation of sensor photoelectric performance and reduced detection repeatability.

[0017] 2. To address the technical shortcomings of existing signal-on PEC sensors, which rely on a single spatial steric hindrance effect to achieve signal quenching, resulting in limited photocurrent suppression efficiency, high detection baseline, and consequently low detection signal-to-noise ratio and high detection limit, thus failing to achieve ultra-trace detection of the breast cancer biomarker MUC1.

[0018] 3. It addresses the problems of traditional breast cancer clinical detection methods (MRI, mammography, pathological biopsy) being expensive, time-consuming, and invasive, making it difficult to achieve large-scale early screening; at the same time, it makes up for the shortcomings of existing MUC1 detection technology, which cannot simultaneously achieve a wide linear detection range, low detection limit, high stability and strong anti-interference ability, and cannot be adapted to the accurate quantitative detection of complex matrix in clinical human serum.

[0019] In view of the technical problems existing in the prior art, this invention constructs a low-background "Signal-On" photoelectrochemical aptamer sensor based on ionic liquid-modulated Bi2MoO6 / MXene heterojunction interface microenvironment and a dual signal quenching strategy. The defects of heterojunction photoelectric performance and structural stability are solved simultaneously by ionic liquid modification, and dual signal efficient quenching is achieved through cDNA steric hindrance and AuNPs plasmonic resonance effect to construct an extremely low detection baseline. Finally, it realizes highly sensitive, highly specific and wide linear range quantitative detection of MUC1 in human serum samples, filling the gap of the prior art and providing a low-cost, rapid and convenient new detection technology solution for early large-scale screening of breast cancer.

[0020] It should be noted that, in this invention, unless otherwise specified, the specific meaning of "comprising" in relation to composition definition and description includes both open-ended meanings such as "comprising," "including," etc., and closed-ended meanings such as "composed of," etc., and similar meanings.

[0021] To solve the aforementioned technical problems, the present invention adopts the following solution:

[0022] [First technical solution]

[0023] A method for preparing an IL5 / Bi2MoO6 / MXene photoelectric active composite material includes the following steps:

[0024] S1: Preparation of MXene nanomaterials

[0025] In a fume hood, Ti3AlC2 powder was placed in a reaction vessel, and HF solution was slowly added dropwise until the powder was completely submerged. The mixture was gently stirred and etched at room temperature. After the reaction was completed, the mixture was separated, the precipitate was washed, and the precipitate was dried and ground to obtain black MXene nanomaterial powder.

[0026] Preparation of S2: IL5 / Bi2MoO6 / MXene photoelectric active composite material

[0027] S21 was weighed out according to a mass ratio of 9-27:4, Bi(NO3)3·5H2O and Na2MoO4·2H2O were dissolved in ethylene glycol and ultrasonically dispersed until completely dissolved to obtain bismuth source solution and molybdenum source solution, respectively.

[0028] S22 Mix the obtained bismuth source solution and molybdenum source solution, add anhydrous ethanol, stir continuously, then add the MXene nanomaterial powder prepared in step S1 and 1-aminopropyl-3-methylimidazolium bromide (IL5) to the mixture, and continue stirring to obtain a homogeneous precursor mixture.

[0029] S23 The precursor mixture was transferred to a hydrothermal reactor and the reaction was sealed. After the reaction was completed, it was naturally cooled to room temperature, centrifuged to obtain the precipitate, washed, dried and ground to obtain the IL5 / Bi2MoO6 / MXene composite material.

[0030] Furthermore, in step S1, the HF solution has a mass fraction of 45%; the stirring etching time is 10-14 hours.

[0031] After the reaction is complete, the mixture is centrifuged at a speed of 3000-8000 r / min.

[0032] The washing precipitate was obtained by washing the precipitate sequentially with ultrapure water and anhydrous ethanol until the pH of the washing solution was neutral.

[0033] The precipitate was dried in an oven at 50-70℃ and then ground to obtain black MXene nanomaterial powder.

[0034] Furthermore, in step S22, the stirring time after adding anhydrous ethanol is 15-25 min, and the stirring time is 10-20 min; the mass ratio of MXene nanomaterial powder to 1-aminopropyl-3-methylimidazolium bromide (IL5) is 2:1; the amount of 1-aminopropyl-3-methylimidazolium bromide added is 1-2% of the total mass of Bi(NO3)3·5H2O and Na2MoO4·2H2O.

[0035] In step S23, the sealing reaction is carried out by heating at 140-180°C for 8-16 hours, and the precipitate is dried by placing it in an oven at 50-70°C.

[0036] In step S1 of the present invention, the reaction vessel is a polytetrafluoroethylene (PTFE) vessel;

[0037] In step S21 of the present invention, the operation of dissolving Bi(NO3)3·5H2O and Na2MoO4·2H2O in ethylene glycol does not have particular limitations on the amount of ethylene glycol used or the ultrasonic dispersion time, as long as it is suitable for the two substances to be completely dissolved.

[0038] In step S22, the addition of anhydrous ethanol further mixes the bismuth source solution and the molybdenum source solution evenly and helps to control the reaction rate. In some embodiments of the present invention, the amount added is generally selected to be an anhydrous ethanol to ethylene glycol volume ratio of 1-2:1.

[0039] In step S23, the hydrothermal reactor is a hydrothermal reactor with a polytetrafluoroethylene liner, and the precipitate is washed 3-6 times sequentially with ultrapure water and anhydrous ethanol.

[0040] [Second Technical Solution]

[0041] An IL5 / Bi2MoO6 / MXene photoelectric active composite material was prepared according to the above-described preparation method.

[0042] [Third technical solution]

[0043] A low-background photoelectrochemical aptamer sensor, wherein the working electrode is fabricated using the aforementioned IL5 / Bi2MoO6 / MXene photoelectroactive composite material.

[0044] [Fourth technical solution]

[0045] A method for fabricating a low-background photoelectrochemical aptamer sensor includes the following steps:

[0046] Step 1: Fabrication of the substrate working electrode:

[0047] The IL5 / Bi2MoO6 / MXene photoelectric active composite material was dispersed in ultrapure water to prepare a uniform dispersion.

[0048] The dispersion was uniformly drop-coated onto the pretreated ITO conductive surface and allowed to air dry at room temperature to obtain the IL5 / Bi2MoO6 / MXene / ITO substrate working electrode.

[0049] Step 2: Amine functionalization modification:

[0050] Chitosan acetic acid solution was added dropwise to the electrode modification area and incubated in a constant temperature environment of 35-39℃ for 0.5-1.5h to introduce active amino groups onto the electrode surface.

[0051] Step 3: Aldehyde activation:

[0052] Add glutaraldehyde solution at the same position as in step 2, and incubate in a constant temperature environment of 35-39℃ for 0.5-1.5h to activate the aldehyde group on the electrode surface through the aldehyde-amine crosslinking reaction;

[0053] Step 4: Capture probe fixation:

[0054] Immobilize the capture probe on the electrode surface and incubate it in an environment of 2-8℃ for 10-14 hours;

[0055] Step 5: Non-specific site blocking:

[0056] Add PBS buffer solution containing BSA and incubate at a constant temperature of 35-39℃ for 40-80 min to block unreacted active sites on the electrode surface.

[0057] Step 6: Aptamer hybridization and second signal quenching:

[0058] An APT-Au complex solution was dropped onto the electrode surface and incubated in a constant temperature environment of 35-39℃ for 60-80 min to finally obtain the low background photoelectrochemical aptamer sensor.

[0059] Furthermore, in step 1, the concentration of the dispersion is 0.5-2 mg / mL;

[0060] In step 2, the chitosan acetic acid solution has a mass fraction of 0.5%-2%.

[0061] In step 3, the glutaraldehyde solution has a mass fraction of 3%-7%.

[0062] In step 4, the concentration of the capture probe solution is 10-20 μM;

[0063] In step 5, the concentration of BSA in the PBS buffer solution is 0.5%-2%.

[0064] In the preparation of the sensor of the present invention, the amount of substances added in steps 1-6 above varies according to the area of ​​the pretreated ITO conductive glass. In the present invention, the effective modified area of ​​the electrode is generally limited to 0.15-0.25 cm², and the typical amount added is 15-25 μL.

[0065] Furthermore, the capture probe is a single-stranded DNA that can hybridize complementaryly with an aptamer;

[0066] The APT-Au complex solution was prepared according to the following method:

[0067] First, gold nanoparticles (AuNPs) were prepared by sodium citrate reduction and stored in a refrigerator at 2-8°C in the dark for later use.

[0068] Then, a 10-20 μM APT solution and an AuNPs solution are mixed evenly and incubated at a constant temperature of 5-39°C for 0.5-1.5 h, wherein the molar ratio of APT to AuNPs is 1:1, to obtain the APT-Au complex solution.

[0069] In this invention, the low-background photoelectrochemical aptamer sensor is constructed in a clean environment at room temperature. After each reaction step, the electrode surface is rinsed with PBS buffer solution at pH 7.0-8.0 to remove unbound substances.

[0070] In the preparation method of the low background photoelectrochemical aptamer sensor of the present invention, the capture probe is fixed on the electrode surface by covalent bonding of aldehyde group and amino group, and the first photocurrent signal quenching is achieved by utilizing the steric hindrance effect formed by the capture probe.

[0071] The purpose of sealing the unreacted active sites on the electrode surface is to eliminate non-specific adsorption interference.

[0072] The complementary cDNA sequence is: 5'-NH2-(CH2)6-TTTTTTCGCTTGCGCATG-3';

[0073] MUC1 aptamer (APT) sequence: 5'-GCAGTTGATCCTTCATGCGCAAGCG-(CH2)6-SH-3';

[0074] In this method, APT-Au is immobilized on the electrode surface through base complementary pairing between cDNA and APT, and the second photocurrent signal quenching is achieved by utilizing the local surface plasmon resonance effect of AuNPs.

[0075] [Fifth technical solution]

[0076] The application of a low-background photoelectrochemical aptamer sensor in the detection of the breast cancer biomarker MUC1 includes the following steps:

[0077] The characteristic polypeptide sequence of MUC1 is: APDTRPAPG.

[0078] (1) Establishment of the detection system:

[0079] A standard three-electrode system was used, with the prepared aptamer sensor as the working electrode, a platinum wire electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode. The supporting electrolyte was 100 mL of PBS buffer solution with pH = 7.0-8.0 containing 0.1-0.5 mol / L L-ascorbic acid.

[0080] The detection was performed using the current-time mode of an electrochemical workstation. The excitation source was an LED lamp with a wavelength of 400-450nm and a power of 80-120W. The intermittent illumination mode was used, with the on / off state switching every 10 seconds. All detections were performed at room temperature.

[0081] (2) Quantitative detection process:

[0082] 15-25 μL of MUC1 standard solution or human serum sample of different concentrations was dropped onto the surface of the prepared low-background photoelectrochemical aptamer sensor electrode. The electrode was then incubated at a constant temperature of 35-39℃ for 60-80 min. MUC1 specifically binds to APT, causing APT-Au to detach from the electrode surface, thus eliminating the double quenching effect. After incubation, the electrode was rinsed with PBS buffer solution and then placed in the detection system for photocurrent testing. Stable photocurrent response values ​​were recorded.

[0083] (3) Plotting the standard curve and calculating the results:

[0084] A standard curve was plotted with the logarithm of the concentration of the MUC1 standard solution on the x-axis and the corresponding photocurrent response value on the y-axis, and a linear regression equation was obtained by fitting the curve. The photocurrent response value of the sample to be tested was substituted into the linear regression equation to calculate the concentration of MUC1 in the sample.

[0085] The photoelectrochemical aptamer sensor based on dual signal quenching designed in this invention has a unique reaction mechanism:

[0086] This invention employs a one-pot hydrothermal method to synthesize IL5 / Bi2MoO6 / MXene composite materials. In this process, the ionic liquid acts as an interface modifier and structure directing agent, with its cations adsorbing onto the surface and edges of MXene nanosheets through electrostatic and coordination interactions. Simultaneously, its functional groups can also interact with the Bi2MoO6 precursor.

[0087] This effect effectively regulates the nucleation and growth process of Bi2MoO6 on the MXene surface, promotes a tight interfacial bond between the two, and thus forms a heterojunction composite material with good dispersion and stable structure, avoiding the agglomeration and interfacial separation problems caused by simple physical mixing.

[0088] This invention constructs a Bi2MoO6 / MXene nn-type homo-heterojunction and utilizes the built-in electric field formed by band matching of the materials to achieve efficient separation and migration of photogenerated electron-hole pairs, fundamentally solving the inherent defect of high carrier recombination rate in pure-phase Bi2MoO6. At the same time, by regulating the microenvironment of the heterojunction interface with ionic liquid, it not only further improves the conductivity of the composite material and suppresses photogenerated carrier recombination, but also significantly improves the dispersion and structural stability of the heterojunction, avoiding the degradation of photoelectric performance of the sensor during long-term use.

[0089] The sensor preparation and single-sample detection process of this invention can be completed within 2 hours. It has a low operating threshold and does not require professional pathology personnel. The detection process only requires the collection of peripheral venous serum from the patient. It is non-invasive, has no risk of ionizing radiation, and is easily accepted by the examinee. It can meet the clinical rapid detection needs of tertiary hospitals and is also suitable for large-scale early breast cancer screening in primary medical institutions.

[0090] The dual-signal quenching strategy of the photoelectrochemical aptamer sensor designed in this invention constructs an extremely low detection baseline, significantly improving detection sensitivity and signal-to-noise ratio. The underlying theory is as follows:

[0091] This invention overcomes the limitations of existing single-signal quenching technologies and pioneers the construction of a dual signal quenching system combining "cDNA spatial steric hindrance effect + AuNPs localized surface plasmon resonance effect":

[0092] The first layer of inhibition is achieved by using the steric hindrance formed by immobilized cDNA to prevent the migration of electron donors in the electrolyte to the electrode surface.

[0093] The second stage involves AuNPs competitively absorbing photons of the excitation light, efficiently capturing photogenerated electrons, and intensifying carrier recombination to achieve deep quenching of the photocurrent.

[0094] The aforementioned dual quenching strategy can reduce the initial photocurrent baseline of the sensor to less than 10% of that in the unquenched state, greatly expanding the signal amplification space for target detection and making the sensor's detection limit for MUC1 extremely low, thus successfully achieving ultra-trace detection of MUC1.

[0095] The working principle by which this invention achieves excellent detection results is as follows:

[0096] After the sensor was constructed but before contacting the target material MUC1, cDNA and APT-Au were immobilized on the electrode surface. At this time, both photocurrent generation and conduction pathways were doubly suppressed:

[0097] 1. Electron donor transport is hindered: Ascorbic acid (AA) in the electrolyte, acting as an electron donor, needs to diffuse to the photoelectric material on the electrode surface to provide electrons to complete the photoelectrochemical reaction. The dense monolayer formed by cDNA constitutes a physical barrier, severely hindering the transport of AA.

[0098] 2. The photoelectric conversion process is suppressed: The excitation light is largely absorbed by AuNPs, reducing the photon flux reaching the Bi2MoO6 / MXene material; simultaneously, the photogenerated electrons generated by photoexcitation in Bi2MoO6 are easily captured and annihilated by neighboring AuNPs, failing to be effectively transferred to the external circuit. These two effects together result in an extremely low background signal (initial photocurrent I0) in the detection system.

[0099] When the target compound MUC1 is introduced and specifically binds to the aptamer APT, the APT-Au complex dissociates from the electrode surface and enters the solution because the binding force of MUC1-APT is much stronger than that of APT-cDNA. The changes that occur at this point are:

[0100] 1. Elimination of steric hindrance: Due to its flexibility and negative charge, the single strand of cDNA becomes loose after losing its hybridization target, which weakens the physical barrier it forms and greatly reduces the resistance to the transmission of AA to the electrode surface.

[0101] 2. Quenching center removal: The removal of AuNPs eliminates their competitive absorption of photons and their ability to trap photogenerated electrons. The optoelectronic material can then receive more photons, and the generated photogenerated electrons can flow more efficiently to the electrodes.

[0102] Therefore, the photocurrent signal recovers significantly from an extremely low I0 to a relatively high I0 signal change value ΔI = I - I0, which is proportional to the concentration of MUC1. By measuring ΔI, quantitative analysis of MUC1 can be achieved. This principle of converting the presence or absence of a target analyte into a significant "switching" change in the photocurrent signal is the fundamental reason why this invention achieves high-sensitivity, low-background detection.

[0103] This invention provides an IL5 / Bi2MoO6 / MXene photoactive composite material, a low-background photoelectrochemical aptamer sensor, and its preparation method and application in the detection of the breast cancer biomarker MUC1 have the following beneficial effects:

[0104] 1. The photoelectric active material designed in this invention exhibits a significant performance leap, breaking through the core bottleneck of low photoelectric conversion efficiency in semiconductors. It not only further enhances the conductivity of the composite material and suppresses photogenerated carrier recombination, but also significantly improves the dispersion and structural stability of the heterojunction, avoiding photoelectric performance degradation during long-term sensor use. Testing shows that the photocurrent response value of the IL5 / Bi2MoO6 / MXene composite material is more than 5 times that of pure-phase Bi2MoO6 and more than 2 times that of the unmodified Bi2MoO6 / MXene composite material, achieving a qualitative leap in photoelectric conversion efficiency.

[0105] 2. The dual signal quenching strategy of the low-background photoelectrochemical aptamer sensor designed in this invention constructs an extremely low detection baseline, significantly improving detection sensitivity and signal-to-noise ratio, and achieving deep quenching of photocurrent. This dual quenching strategy can reduce the initial photocurrent baseline of the sensor to less than 10% of the unquenched state, greatly expanding the signal amplification space for target detection. This results in a detection limit of only 10 pM for MUC1, far superior to most existing MUC1 photoelectrochemical sensors based on a single quenching mode, successfully achieving ultra-trace detection of MUC1.

[0106] 3. The low-background photoelectrochemical aptamer sensor designed in this invention exhibits comprehensive and excellent detection performance, fully meeting the needs of practical clinical applications. The sensor prepared in this invention achieves a linear detection range of 20 pM-10 μM for MUC1, completely covering the concentration range of MUC1 in clinical breast cancer diagnosis, and can simultaneously meet the dual detection requirements of early breast cancer screening and disease progression monitoring. Based on the specific recognition between the aptamer and the target, the sensor has extremely strong anti-interference capabilities, achieving accurate quantitative detection even in the complex biological matrix of human serum, with a stable spiked recovery rate between 95% and 105%. Simultaneously, the sensor demonstrates excellent batch repeatability and long-term storage stability, fully complying with the technical standards of clinical in vitro diagnostic reagents.

[0107] 4. The low-background photoelectrochemical aptamer sensor designed in this invention has low detection cost and is easy to operate, enabling large-scale widespread application. All raw materials used in this invention are commercially available conventional reagents, and the core composite material is synthesized using a one-pot hydrothermal method. The process is simple and controllable, requiring no expensive large-scale precision equipment, and possesses extremely high industrial application value and socio-economic benefits.

[0108] 5. The low-background photoelectrochemical aptamer sensor designed in this invention has strong technical versatility and broad application potential. The core technology system of this invention includes a method for modifying heterojunction interfaces using ionic liquids and a low-baseline sensing interface construction strategy with dual signal quenching. It can not only be used for the detection of the breast cancer biomarker MUC1, but also, by changing the aptamer sequence, can be rapidly extended to the photoelectrochemical detection of other tumor biomarkers, pathogenic microorganisms, environmental pollutants, and other targets. This provides a universal technical platform for the development of highly sensitive photoelectrochemical sensors and possesses extremely strong technical extensibility. Attached Figure Description

[0109] Figure 1 : This is a schematic diagram illustrating the process of constructing a low-background photoelectrochemical aptamer sensor and its application in the detection of the breast cancer biomarker MUC1 in this invention;

[0110] Figure 2 The microstructure and structure of the materials prepared in Example 1 and Comparative Example 1 of this invention are as follows: (A) Bi2MoO6, (B) Mxene, (C) Bi2MoO6 / Mxene prepared in Comparative Example 1, (D) SEM image of Bi2MoO6 / IL5 / Mxene, (E) Elemental mapping image of Bi2MoO6 / IL5 / Mxene, and (F) TEM image of Bi2MoO6 / IL5 / Mxene.

[0111] Figure 3: Infrared spectra of materials (A) Ti3AlC2 (a) and Mxene (b), (B) Bi2MoO6 (a) and Mxene (b), and materials Bi2MoO6 / Mxene (c) and Bi2MoO6 / IL5 / Mxene (d) in Example 1 and Comparative Example 1 of the present invention;

[0112] Figure 4 : XRD spectra of materials (A) Ti3AlC2(a) and Mxene(b), (B) Bi2MoO6(a) and Mxene(b), and materials Bi2MoO6 / Mxene(c) and Bi2MoO6 / IL5 / Mxene(d) in Example 1 and Comparative Example 1 of the present invention;

[0113] Figure 5 (A) The photocurrent response of the sensor prepared in Example 1 of the present invention to different concentrations of MUC1, MUC1 concentrations (from a to i): 10 μM, 5 μM, 2 μM, 0.1 μM, 10 nM, 1 nM, 0.1 nM, 50 pM, 20 pM; (B) Calibration curves of the corresponding concentrations of MUC1;

[0114] Figure 6 (A) Photocurrent response of the sensor prepared in Example 1 of the present invention in 0.1 μM MUC1 (a), 10 μM GLU (b), AA (c), UA (d), DA (e), CREA (f), Cys (g), BSA (h), 1 μM CEA (i), CA125 (j); (B) Photocurrent response of 0.1 μM MUC1 (a) with 10 μM interference substances GLU (b), AA (c), UA (d), DA (e), CREA (f), Cys (g), BSA (h) and 1 μM interference substances CEA (i), CA125 (j); (C) Schematic diagram of the repeatability of detecting 0.1 μM MUC1;

[0115] Figure 7 : This is a graph showing the stability test results of the sensor prepared in Example 1 of the present invention; Detailed Implementation

[0116] The present invention will be further described below with reference to specific embodiments and accompanying drawings:

[0117] Regarding the core reagents and raw materials in the embodiments of this invention:

[0118] Sodium hydroxide (NaOH), acetone, ethanol, titanium aluminum carbide (Ti3AlC2), hydrofluoric acid (HF), bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), sodium molybdate dihydrate (Na2MoO4·2H2O), ethylene glycol (EG), 1-aminopropyl-3-methylimidazolium bromide (IL5), tetrachloroauric acid (HAuCl4), sodium citrate, chitosan (CS), glutaraldehyde (GLD), bovine serum albumin (BSA), sodium dihydrogen phosphate, disodium hydrogen phosphate, L-ascorbic acid (AA), glacial acetic acid; MUC1 protein, MUC1 aptamer (APT), and complementary strand cDNA were all purchased from commercial biotechnology companies.

[0119] Among them: complementary cDNA sequence: 5'-NH2-(CH2)6-TTTTTTCGCTTGCGCATG-3';

[0120] MUC1 aptamer (APT) sequence: 5'-GCAGTTGATCCTTCATGCGCAAGCG-(CH2)6-SH-3';

[0121] MUC1 characteristic polypeptide sequence: APDTRPAPG

[0122] Example 1 of preparation of IL5 / Bi2MoO6 / MXene photoelectric active composite material

[0123] S1: Preparation of MXene nanomaterials

[0124] In a fume hood, Ti3AlC2 powder was placed in a polytetrafluoroethylene container, and 45% HF solution was slowly added dropwise until the powder was completely submerged. The mixture was then gently stirred and etched at room temperature for 12 hours.

[0125] After the reaction was complete, the mixture was centrifuged at 6000 r / min.

[0126] The precipitate was washed successively with ultrapure water and anhydrous ethanol until the pH of the washing solution was neutral.

[0127] The precipitate was dried in a 60°C oven and then ground to obtain black MXene powder.

[0128] Preparation of S2: IL5 / Bi2MoO6 / MXene photoelectric active composite material

[0129] Weigh (0.9g) Bi(NO3)3·5H2O and (0.2g) Na2MoO4·2H2O according to a mass ratio of 18:4, dissolve them in 10mL of ethylene glycol respectively, and sonicate for 15min until completely dissolved to obtain bismuth source solution and molybdenum source solution;

[0130] The two were then mixed, 20 mL of anhydrous ethanol was added, and the mixture was stirred for 20 min. Then, 0.04 g of MXene powder and 0.02 g of 1-aminopropyl-3-methylimidazolium bromide (IL5) were added to the mixture. The precursor mixture was then transferred to a polytetrafluoroethylene-lined hydrothermal reactor, sealed, and heated at 160 °C for 12 h.

[0131] After the reaction was complete, the mixture was allowed to cool to room temperature, centrifuged to obtain the precipitate, and washed 3-6 times successively with ultrapure water and anhydrous ethanol.

[0132] The precipitate was dried in a 60℃ oven and then ground to obtain the IL5 / Bi2MoO6 / MXene composite material.

[0133] Example 2 of preparation of IL5 / Bi2MoO6 / MXene photoelectric active composite material

[0134] S1: Preparation of MXene nanomaterials

[0135] In a fume hood, Ti3AlC2 powder was placed in a polytetrafluoroethylene container, and 45% HF solution was slowly added dropwise until the powder was completely submerged. The mixture was then gently stirred and etched at room temperature for 14 hours.

[0136] After the reaction was complete, the mixture was centrifuged at 3000 r / min.

[0137] The precipitate was washed successively with ultrapure water and anhydrous ethanol until the pH of the washing solution was neutral.

[0138] The precipitate was dried in a 50°C oven and then ground to obtain black MXene powder.

[0139] Preparation of S2: IL5 / Bi2MoO6 / MXene photoelectric active composite material

[0140] Weigh (0.45g) Bi(NO3)3·5H2O and (0.2g) Na2MoO4·2H2O according to a mass ratio of 9:4, dissolve them in 5mL of ethylene glycol respectively, and sonicate for 10min until completely dissolved to obtain bismuth source solution and molybdenum source solution;

[0141] The two were then mixed, 20 mL of anhydrous ethanol was added, and the mixture was stirred for 15 min. Then, 0.13 g of MXene powder and 0.065 g of 1-aminopropyl-3-methylimidazolium bromide (IL5) were added to the mixture. The precursor mixture was then transferred to a polytetrafluoroethylene-lined hydrothermal reactor, sealed, and heated at 140 °C for 16 h.

[0142] After the reaction was complete, the mixture was allowed to cool to room temperature, centrifuged to obtain the precipitate, and washed 3-6 times successively with ultrapure water and anhydrous ethanol.

[0143] The precipitate was dried in a 50°C oven and then ground to obtain the IL5 / Bi2MoO6 / MXene composite material.

[0144] Example 3 of preparation of IL5 / Bi2MoO6 / MXene photoelectric active composite material

[0145] S1: Preparation of MXene nanomaterials

[0146] In a fume hood, Ti3AlC2 powder was placed in a polytetrafluoroethylene container, and 45% HF solution was slowly added dropwise until the powder was completely submerged. The powder was then gently etched at room temperature with stirring for 10 hours.

[0147] After the reaction was complete, the mixture was centrifuged at 8000 r / min.

[0148] The precipitate was washed successively with ultrapure water and anhydrous ethanol until the pH of the washing solution was neutral.

[0149] The precipitate was dried in a 70°C oven and then ground to obtain black MXene powder.

[0150] Preparation of S2: IL5 / Bi2MoO6 / MXene photoelectric active composite material

[0151] Weigh (1.35g) Bi(NO3)3·5H2O and (0.2g) Na2Mo4·2H2O according to a mass ratio of 27:4, dissolve them in 15mL of ethylene glycol respectively, and sonicate for 20min until completely dissolved to obtain bismuth source solution and molybdenum source solution;

[0152] The two were then mixed, 30 mL of anhydrous ethanol was added, and the mixture was stirred for 25 min. Then, 0.62 g of MXene powder and 0.31 g of 1-aminopropyl-3-methylimidazolium bromide (IL5) were added to the mixture. The precursor mixture was then transferred to a hydrothermal reactor lined with polytetrafluoroethylene, sealed, and heated at 180 °C for 8 h.

[0153] After the reaction was complete, the mixture was allowed to cool to room temperature, centrifuged to obtain the precipitate, and washed 3-6 times successively with ultrapure water and anhydrous ethanol.

[0154] The precipitate was dried in a 70°C oven and then ground to obtain the IL5 / Bi2MoO6 / MXene composite material.

[0155] Comparative Example 1: Preparation of Bi2MoO6 / MXene composite material:

[0156] The preparation method of the Bi2MoO6 / MXene composite material in Comparative Example 1 of the present invention is completely consistent with the steps of Example 1 above, except that IL5 is not added.

[0157] Example 1 of the construction of a low-background photoelectrochemical aptamer sensor

[0158] The sensor construction example 1 in this invention is carried out using the IL5 / Bi2MoO6 / MXene photoactive composite material prepared in Example 1 above.

[0159] The pretreatment method for the ITO conductive glass in the sensor construction example of this invention is as follows:

[0160] ITO conductive glass was placed in 1 mol / L sodium hydroxide solution, ultrapure water, acetone and anhydrous ethanol in sequence, and ultrasonically cleaned for 15 min each. After removal, it was dried with high-purity nitrogen gas for later use.

[0161] When using it, identify the conductive surface of ITO with an ohmmeter, and stick insulating conductive adhesive with a circular hole in the middle on the conductive surface. The effective modification area of ​​the electrode is limited to 0.15-0.25 cm².

[0162] In practical applications, the effective area of ​​ITO conductive glass can be adapted to the specific needs of the test. The amount of material added in each step can be selected accordingly based on the size of the area. For the area mentioned above, the amount is generally selected as 15-25μL.

[0163] In the construction example of this invention, the effective area of ​​the ITO conductive glass is set to 0.2 cm².

[0164] Regarding the preparation of AuNPs nanoparticles and APT-Au composites in the sensor construction examples of the present invention

[0165] The procedure for preparing AuNPs by sodium citrate reduction is as follows:

[0166] Add 50 mL of ultrapure water to a three-necked flask, reflux and heat to boiling, and slowly add 0.5 mL of 1% (w / w) HAuCl4 aqueous solution to the boiling water while stirring continuously;

[0167] After the solution boils again, quickly add 0.5 mL of 1% sodium citrate aqueous solution and stir continuously under reflux for 30 min.

[0168] After heating is stopped, the solution is allowed to cool naturally to room temperature to obtain a wine-red AuNPs solution, which is then stored in a refrigerator at 6°C away from light for later use.

[0169] Preparation of APT-Au complex:

[0170] A 20 μM APT solution and AuNPs solution were mixed thoroughly at a volume ratio of 1:1.2, with the molar ratio of APT solution to AuNPs being 1:1. The mixture was then incubated at a constant temperature of 37 °C for 1 h to obtain an APT-Au complex solution, which was prepared and used immediately.

[0171] The sensor of this invention was constructed in a clean environment at room temperature. After each reaction step, the electrode surface was rinsed with PBS buffer solution at pH 7.0-8.0 to remove unbound substances. The specific steps are as follows:

[0172] Step 1: Substrate electrode fabrication:

[0173] The IL5 / Bi2MoO6 / MXene photoactive composite material prepared in Example 1 was dispersed in ultrapure water to prepare a homogeneous dispersion of 1 mg / mL.

[0174] Take 20 μL of dispersion and uniformly drop it onto the pretreated ITO conductive surface, and let it air dry at room temperature to obtain the IL5 / Bi2MoO6 / MXene / ITO working electrode;

[0175] Step 2: Amine functionalization modification:

[0176] 20 μL of 1% chitosan acetic acid solution was added dropwise to the electrode-modified area and incubated at 37°C for 1 h to introduce active amino groups onto the electrode surface.

[0177] Step 3: Aldehyde activation:

[0178] Add 20 μL of 6% glutaraldehyde solution at the same position and incubate at a constant temperature of 37°C for 1 h to activate the aldehyde group on the electrode surface through the aldehyde-amine crosslinking reaction.

[0179] Step 4: Capture probe cDNA fixation (first signal quenching):

[0180] 20 μL of 15 μM cDNA solution was dropped onto the electrode surface and incubated at 6 °C for 12 h. The cDNA was immobilized on the electrode surface by the covalent binding of aldehyde and amino groups. The steric hindrance effect formed by the cDNA was used to achieve the first photocurrent signal quenching.

[0181] Step 5: Blocking non-specific sites;

[0182] Add 20 μL of PBS buffer solution containing 1% BSA and incubate at 37°C for 50 min to block unreacted active sites on the electrode surface and eliminate nonspecific adsorption interference.

[0183] Step 6: Aptamer hybridization and second signal quenching:

[0184] 20 μL of APT-Au complex solution was dropped onto the electrode surface and incubated at 37 °C for 70 min. APT-Au was immobilized on the electrode surface through base complementarity pairing between cDNA and APT. The second photocurrent signal quenching was achieved by utilizing the local surface plasmon resonance effect of AuNPs, and finally the APT-Au-CDNA / IL5 / Bi2MoO6 / MXene / ITO photoelectrochemical aptamer sensor was prepared.

[0185] Example 2 of constructing a low-background photoelectrochemical aptamer sensor

[0186] In Construction Example 2 of the present invention, the operation process is the same as that in Construction Example 1, except that the concentrations in each step are changed:

[0187] In step 1, the dispersion concentration was 0.5 mg / mL; in step 2, the chitosan-acetic acid solution had a mass fraction of 0.5%, and the incubation temperature was 35℃ for 1.5 h; in step 3, the glutaraldehyde solution had a mass fraction of 3%, and the solution was incubated at a constant temperature of 35℃ for 1.5 h; in step 4, the cDNA solution had a concentration of 10 μM, and the solution was incubated at 2℃ for 10 h; in step 5, the PBS buffer solution containing 0.5% BSA was incubated at a constant temperature of 35℃ for 80 min; and in step 6, the APT-Au complex solution was added dropwise, and the solution was incubated at a constant temperature of 35℃ for 80 min.

[0188] Example 3: Construction of a low-background photoelectrochemical aptamer sensor

[0189] In Construction Example 3 of the present invention, the operation process is the same as that in Construction Example 1, except that the concentrations in each step are changed:

[0190] In step 1, the dispersion concentration was 2 mg / mL; in step 2, the chitosan-acetic acid solution had a mass fraction of 2%, and the incubation temperature was 39℃ for 0.5 h; in step 3, the glutaraldehyde solution had a mass fraction of 7%, and the solution was incubated at a constant temperature of 39℃ for 0.5 h; in step 4, the cDNA solution had a concentration of 20 μM, and the solution was incubated at 8℃ for 14 h; in step 5, the PBS buffer solution containing 2% BSA was incubated at a constant temperature of 39℃ for 40 min; and in step 6, the APT-Au complex solution was added dropwise, and the solution was incubated at a constant temperature of 39℃ for 60 min.

[0191] Regarding the PEC quantitative detection method of MUC1 in this invention

[0192] (1) Establishment of the detection system:

[0193] A standard three-electrode system was used, with the aptamer sensor prepared in Example 1 above as the working electrode, the platinum wire electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode. The supporting electrolyte was 100 mL of PBS buffer solution with pH = 7.0-8.0 containing 0.1-0.5 mol / L L-ascorbic acid (electron donor).

[0194] The detection was performed using the current-time (it) mode of an electrochemical workstation. The excitation source was an LED lamp with a wavelength of 450 nm and a power of 120 W. The intermittent illumination mode was used, and the on / off state was switched every 10 seconds. All detections were performed at room temperature.

[0195] Quantitative detection process:

[0196] 20 μL of MUC1 standard solution or human serum sample of different concentrations was added to the surface of the prepared sensor electrode and incubated at 37°C for 70 min. MUC1 specifically binds to APT, causing APT-Au to detach from the electrode surface and the double quenching effect disappears. After incubation, the electrode was rinsed with PBS buffer solution and placed in the detection system for photocurrent testing, and stable photocurrent response values ​​were recorded.

[0197] (2) Plotting the standard curve and calculating the results:

[0198] A standard curve was plotted with the logarithm of the concentration of the MUC1 standard solution on the x-axis and the corresponding photocurrent response value on the y-axis. A linear regression equation was then fitted to the curve. The photocurrent response value of the sample was substituted into the linear regression equation to calculate the concentration of MUC1 in the sample. Relevant detection results are as follows: Figure 5 As shown.

[0199] The invention will be further illustrated by referring to the accompanying drawings:

[0200] Figure 1 This is a schematic diagram illustrating the process of constructing a low-background photoelectrochemical aptamer sensor and its application in the detection of the breast cancer biomarker MUC1.

[0201] like Figure 1 As shown, it fully demonstrates the technical route of the present invention, which is divided into three stages:

[0202] The first stage: Bi2MoO6 / IL5 / MXene photoelectric active material preparation stage, layered MXene was prepared by etching Ti3AlC2 with HF, and IL5 was used as an interface modifier to synthesize nn-type heterojunction composite material through hydrothermal reaction;

[0203] The second stage: sensor construction and dual signal quenching stage. The composite material is modified on the ITO electrode, and the cDNA capture probe is fixed by CS / GLD crosslinking. After the non-specific sites are blocked by BSA, it is hybridized with APT-Au. The primary photocurrent quenching is achieved through the steric hindrance effect of cDNA, and the secondary photocurrent quenching is achieved through the surface plasmon resonance effect of AuNPs, thus constructing a low background sensing interface.

[0204] The third stage: the quantitative detection stage of MUC1. The target substance MUC1 specifically binds to APT-Au, causing it to detach from the electrode surface. The double quenching effect is eliminated, and the photocurrent signal increases with the increase of MUC1 concentration, thus realizing quantitative detection.

[0205] Figure 2 shows the microstructure and structure of the materials prepared in Example 1 and Comparative Example 1 of the present invention: (A) Bi2MoO6, (B) Mxene, (C) Bi2MoO6 / Mxene prepared in the comparative example, (D) SEM image of Bi2MoO6 / IL5 / Mxene, (E) elemental mapping image of Bi2MoO6 / IL5 / Mxene, and (F) TEM image of Bi2MoO6 / IL5 / Mxene;

[0206] from Figure 2 As can be seen from Figure 2A, the pure phase Bi2MoO6 exhibits a nanocluster structure; Figure 2B shows that the etched MXene has a typical accordion-like layered structure with no obvious lamellar stacking, confirming the successful synthesis of MXene.

[0207] Figure 2C shows that the Bi2MoO6 particles in the composite material without IL5 introduction are severely agglomerated, and the composite interface with MXene is not uniform.

[0208] Figure 2D shows that after IL5 modification, the interface between Bi2MoO6 and MXene is tight and uniform, and the original crystal form is not destroyed. Figure 2E confirms that Bi, Mo, O, Ti, Br and N elements are uniformly distributed in the composite material, and IL5 modification is successful. Figure 2F shows that the characteristic lattice fringes of Bi2MoO6 and MXene can be observed at the same time, confirming that the heterojunction interface is successfully constructed and the material has good crystallinity.

[0209] Figure 3 Infrared spectra of materials (A) Ti3AlC2 (a) and Mxene (b), (B) Bi2MoO6 (a) and Mxene (b), and materials Bi2MoO6 / Mxene (c) and Bi2MoO6 / IL5 / Mxene (d) in Comparative Example 1 of the present invention;

[0210] As shown in Figure 3, Example 1 of this invention has successfully synthesized the IL5 / Bi2MoO6 / MXene photoelectric active composite material:

[0211] In Figure 3A, Ti3AlC2 is etched by HF at a depth of 420 cm⁻¹. -1 The characteristic peaks of the Ti-Al bond completely disappeared at 570 cm⁻¹. -1 The presence of Ti-C bond characteristic peaks confirms the successful preparation of MXene;

[0212] The spectrum of Bi2MoO6 / IL5 / MXene in Figure 3B shows characteristic functional group peaks of Bi2MoO6, MXene, and IL5 simultaneously, confirming the successful synthesis of the ternary composite material.

[0213] Figure 4 The XRD spectra of materials (A) Ti3AlC2(a) and Mxene(b), (B) Bi2MoO6(a) and Mxene(b), and materials Bi2MoO6 / Mxene(c) and Bi2MoO6 / IL5 / Mxene(d) in Example 1 and Comparative Example 1 of the present invention are shown.

[0214] Figure 4 is used to characterize the crystal structure of the material: In Figure 4A, the characteristic diffraction peak of Ti3AlC2 at 39° (104) disappears after etching, and the peak at 20° (004) undergoes a low-angle shift, confirming that Ti3AlC2 has been successfully transformed into MXene;

[0215] The diffraction peaks of Bi2MoO6 / IL5 / MXene in Figure 4B are completely matched with the standard characteristic peaks of Bi2MoO6, while retaining the characteristic peaks of MXene, confirming that the heterojunction was successfully constructed and that the IL5 modification did not destroy the main crystal structure of the material.

[0216] Figure 5 The sensor (A) prepared in Example 1 of the present invention responds to the photocurrent of different concentrations of MUC1, with MUC1 concentrations (from a to i): 10 μM, 5 μM, 2 μM, 0.1 μM, 10 nM, 1 nM, 0.1 nM, 50 pM, 20 pM; (B) Calibration curves of the corresponding concentrations of MUC1;

[0217] Figure 5 is used to characterize the quantitative detection performance of the sensor: Figure 5A shows that the photocurrent intensity of the sensor increases significantly with increasing MUC1 concentration;

[0218] Figure 5B shows the linear regression equation as J = 0.9561 × Log C MUC1 +4.667, correlation coefficient R²=0.9973, linear response range of 20 pM-10 μM, and detection limit as low as 10 pM, confirm that this sensor has the advantages of wide linear range and high sensitivity.

[0219] Figure 6 (A) Photocurrent response of the sensor prepared in Example 1 of the present invention in 0.1 μM MUC1 (a), 10 μM GLU (b), AA (c), UA (d), DA (e), CREA (f), Cys (g), BSA (h), 1 μM CEA (i), CA125 (j); (B) Photocurrent response of 0.1 μM MUC1 (a) with 10 μM interference material GLU (b), AA (c), UA (d), DA (e), CREA (f), Cys (g), BSA (h) and 1 μM interference material CEA (i), CA125 (j); (C) Schematic diagram of the repeatability of detecting 0.1 μM MUC1.

[0220] Figure 6. Used to characterize sensor specificity and anti-interference capability:

[0221] Figure 6A shows that the photocurrent response of conventional interfering agents at 100 times the concentration and tumor marker interfering agents at 10 times the concentration is much lower than that of MUC1, confirming that the sensor has excellent specificity for MUC1.

[0222] Figure 6B shows that the detection photocurrent of the mixture of MUC1 and high concentration of interfering substances is not significantly different from that of MUC1 alone, confirming that the sensor has excellent anti-interference ability and is suitable for the detection of complex biological samples.

[0223] Figure 6C is a comparison of the photocurrent response of five parallelly fabricated sensors to 0.1 μM MUC1. The relative standard deviation (RSD) is 3.25%, confirming that the sensor fabrication process is stable and has good repeatability.

[0224] Figure 7 The graph shows the stability test results of the sensor prepared in Example 1 of this invention.

[0225] In the stability test, the sensor prepared in Example 1 of this invention was stored at 4°C in the dark for 14 days, and its photocurrent response value was measured again. Figure 7 As can be seen from the data, after the sensor prepared in Example 1 of this invention was stored at 4°C in the dark for 14 days, the photocurrent response value remained at more than 90% of the initial value, indicating that it has good stability.

[0226] This invention solves the problems of low efficiency, instability of heterojunctions, and high baseline of single quenching mode in existing optoelectronic materials, and realizes highly sensitive, highly specific, rapid and low-cost detection of the breast cancer marker MUC1 in human serum samples, providing a new industrializable technical solution for large-scale early screening of breast cancer.

[0227] The present invention has been described above by way of example with reference to the embodiments and accompanying drawings. Obviously, the implementation of the present invention is not limited to the above-described manner. Any improvements made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A method for preparing an IL5 / Bi2MoO6 / MXene photoelectric active composite material, characterized in that, Includes the following steps: S1: Preparation of MXene nanomaterials In a fume hood, Ti3AlC2 powder was placed in a reaction vessel, and HF solution was slowly added dropwise until the powder was completely submerged. The mixture was gently stirred and etched at room temperature. After the reaction was completed, the mixture was separated, the precipitate was washed, and the precipitate was dried and ground to obtain black MXene nanomaterial powder. Preparation of S2: IL5 / Bi2MoO6 / MXene photoelectric active composite material S21 was weighed out according to a mass ratio of 9-27:4, Bi(NO3)3·5H2O and Na2MoO4·2H2O were dissolved in ethylene glycol and ultrasonically dispersed until completely dissolved to obtain bismuth source solution and molybdenum source solution, respectively. S22 Mix the obtained bismuth source solution and molybdenum source solution, add anhydrous ethanol, stir continuously, then add the MXene nanomaterial powder prepared in step S1 and 1-aminopropyl-3-methylimidazolium bromide (IL5) to the mixture, and continue stirring to obtain a homogeneous precursor mixture. S23 The precursor mixture was transferred to a hydrothermal reactor and the reaction was sealed. After the reaction was completed, it was naturally cooled to room temperature, centrifuged to obtain the precipitate, washed, dried and ground to obtain the IL5 / Bi2MoO6 / MXene composite material.

2. The method for preparing the IL5 / Bi2MoO6 / MXene photoactive composite material according to claim 1, characterized in that: In step S1, the HF solution has a mass fraction of 45%; the stirring etching time is 10-14 hours. After the reaction is complete, the mixture is centrifuged at a speed of 3000-8000 r / min. The washing precipitate was obtained by washing the precipitate sequentially with ultrapure water and anhydrous ethanol until the pH of the washing solution was neutral. The precipitate was dried in an oven at 50-70℃ and then ground to obtain black MXene nanomaterial powder.

3. The method for preparing the IL5 / Bi2MoO6 / MXene photoactive composite material according to claim 1, characterized in that: In step S22, the stirring time after adding anhydrous ethanol is 15-25 min, and the stirring time is further 10-20 min; the mass ratio of MXene nanomaterial powder to 1-aminopropyl-3-methylimidazolium bromide (IL5) is 2:1; the amount of 1-aminopropyl-3-methylimidazolium bromide added is 1-2% of the total mass of Bi(NO3)3·5H2O and Na2MoO4·2H2O. In step S23, the sealing reaction is carried out by heating at 140-180°C for 8-16 hours, and the precipitate is dried by placing it in an oven at 50-70°C.

4. An IL5 / Bi2MoO6 / MXene photoelectric active composite material, characterized in that: It is prepared according to the preparation method described in any one of claims 1-3.

5. A low-background photoelectrochemical aptamer sensor, characterized in that: The sensor described herein utilizes the IL5 / Bi2MoO6 / MXene photoactive composite material described in claim 4 to fabricate the working electrode.

6. A method for fabricating a low-background photoelectrochemical aptamer sensor, characterized in that, It includes the following steps: Step 1: Fabrication of the substrate working electrode: The IL5 / Bi2MoO6 / MXene photoelectric active composite material was dispersed in ultrapure water to prepare a uniform dispersion. The dispersion was uniformly drop-coated onto the pretreated ITO conductive surface and allowed to air dry at room temperature to obtain the IL5 / Bi2MoO6 / MXene / ITO substrate working electrode. Step 2: Amine functionalization modification: Chitosan acetic acid solution was added dropwise to the electrode modification area and incubated in a constant temperature environment of 35-39℃ for 0.5-1.5h to introduce active amino groups onto the electrode surface. Step 3: Aldehyde activation: Add glutaraldehyde solution at the same position as in step 2, and incubate in a constant temperature environment of 35-39℃ for 0.5-1.5h to activate the aldehyde group on the electrode surface through the aldehyde-amine crosslinking reaction; Step 4: Capture probe fixation: Immobilize the capture probe on the electrode surface and incubate it in an environment of 2-8℃ for 10-14 hours; Step 5: Non-specific site blocking: Add PBS buffer solution containing BSA and incubate at a constant temperature of 35-39℃ for 40-80 min to block unreacted active sites on the electrode surface. Step 6: Aptamer hybridization and second signal quenching: An APT-Au complex solution was dropped onto the electrode surface and incubated in a constant temperature environment of 35-39℃ for 60-80 min to finally obtain the low background photoelectrochemical aptamer sensor.

7. The method for preparing a low-background photoelectrochemical aptamer sensor according to claim 6, characterized in that: In step 1, the concentration of the dispersion is 0.5-2 mg / mL; In step 2, the chitosan acetic acid solution has a mass fraction of 0.5%-2%. In step 3, the glutaraldehyde solution has a mass fraction of 3%-7%. In step 4, the concentration of the capture probe solution is 10-20 μM; In step 5, the concentration of BSA in the PBS buffer solution is 0.5%-2%; In the preparation of the sensor of the present invention, the amount of substances added in steps 1-6 varies according to the area of ​​the pretreated ITO conductive glass, and the general amount added is 15-25 μL.

8. The method for preparing a low-background photoelectrochemical aptamer sensor according to claim 6, characterized in that: The capture probe is a single-stranded DNA that can hybridize complementaryly with an aptamer; The APT-Au complex solution was prepared according to the following method: First, gold nanoparticles (AuNPs) were prepared by sodium citrate reduction and stored in a refrigerator at 2-8°C in the dark for later use. Then, a 10-20 μM APT solution and an AuNPs solution are mixed evenly and incubated at a constant temperature of 5-39°C for 0.5-1.5 h, wherein the molar ratio of APT to AuNPs is 1:1, to obtain the APT-Au complex solution.

9. The application of the low-background photoelectrochemical aptamer sensor of claim 5 in the detection of the breast cancer biomarker MUC1.

10. The application of the low-background photoelectrochemical aptamer sensor according to claim 9 in the detection of the breast cancer biomarker MUC1, characterized in that, Includes the following steps: (1) Establishment of the detection system: A standard three-electrode system was used, with the prepared aptamer sensor as the working electrode, a platinum wire electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode. The supporting electrolyte was 100 mL of PBS buffer solution with pH = 7.0-8.0 containing 0.1-0.5 mol / L L-ascorbic acid. The detection was performed using the current-time mode of an electrochemical workstation. The excitation source was an LED lamp with a wavelength of 400-450nm and a power of 80-120W. The intermittent illumination mode was used, with the on / off state switching every 10 seconds. All detections were performed at room temperature. (2) Quantitative detection process: 15-25 μL of MUC1 standard solution or human serum sample of different concentrations was dropped onto the surface of the prepared low-background photoelectrochemical aptamer sensor electrode. The electrode was then incubated at a constant temperature of 35-39℃ for 60-80 min. MUC1 specifically binds to APT, causing APT-Au to detach from the electrode surface, thus eliminating the double quenching effect. After incubation, the electrode was rinsed with PBS buffer solution and placed in the detection system for photocurrent testing. Stable photocurrent response values ​​were recorded. (3) Plotting the standard curve and calculating the results: A standard curve was plotted with the logarithm of the concentration of the MUC1 standard solution on the x-axis and the corresponding photocurrent response value on the y-axis, and a linear regression equation was obtained by fitting the curve. The photocurrent response value of the sample to be tested was substituted into the linear regression equation to calculate the concentration of MUC1 in the sample.

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