Construction method and application of photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi2MoO6

Abstract

The invention provides a construction method and application of a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi2MoO6 (Bi-Bi2MoO6), and relates to the technical field of chemical biosensors. The preparation method comprises the following steps: firstly, preparing a bifunctional sensing material Bi-Bi2MoO6 with photoelectric activity and peroxidase-like property through a one-step ethylene glycol reduction method; then, carrying out sandwich immune reaction and glucolase catalytic reaction of a genotoxic marker phosphorylated histone (gamma H2AX) in a microwell plate to generate an immune reaction product containing a signal factor H2O2; then, H2O2 is used as an electron acceptor of the Bi-Bi2MoO6-based anode photoelectrochemical sensor to promote the current polarity of the ITO / Bi < 2 + x > MoO6 electrode to be reversed (the anode is switched into the cathode), so that zero-background, high-sensitivity and high-selectivity detection of gamma H2AX is realized; meanwhile, H2O2 and Bi-Bi2MoO6 + TMB form a peroxidase catalysis system, so that visual, rapid, simple and convenient colorimetric detection of the target protein gamma H2AX is realized. The photoelectrochemical / colorimetric dual-mode immunosensor designed by the invention has high sensitivity and selectivity, is simple to operate, quick in response and good in stability, and has relatively strong application potential in actual detection of phosphorylated histone.

Description

Technical Field

[0001] This invention relates to the field of chemical and biological sensor technology, and in particular to a method for constructing and applying a bifunctional Bi-doped Bi2MoO6 photoelectrochemical / colorimetric dual-mode immunosensor. Background Technology

[0002] Cancer is one of the leading causes of death worldwide. The International Agency for Research on Cancer's "Global Cancer Report 2020" indicates that over 19.3 million new cancer cases were diagnosed globally in 2020, with my country accounting for 4.57 million, ranking first globally. 70-90% of these cancers are caused by environmental factors, such as toxic chemicals, air, water, and food pollution. Carcinogens are generally classified into genotoxic carcinogens (GCs) and non-genotoxic carcinogens (Non-GCs). Most carcinogens are GCs, which can directly or indirectly damage cellular DNA, leading to gene mutations or in vivo mutagenesis, and ultimately tumors or cancer. A key characteristic is that they can pose a threat to human genetic material even at low concentrations. Therefore, the screening and assessment of genotoxic substances are crucial for the ecological environment and human health.

[0003] Phosphorylated histone (γH2AX) is a product of phosphorylation at the S-139 position of the C-terminus of histone H2AX after DNA damage caused by genotoxic substances, especially DNA double-strand breaks (DSBs). The number of DSBs is directly proportional to the concentration of γH2AX; therefore, γH2AX is a biomarker indicating genotoxicity. Currently, most γH2AX detection technologies developed domestically and internationally rely on large-scale instruments and equipment, involve cumbersome and time-consuming experimental procedures, have low reliability of detection results, or are difficult to implement for rapid on-site detection. Therefore, there is an urgent need to develop a sensitive, accurate, reliable, and rapid on-site detection technology for the precise quantitative analysis of intracellular γH2AX, thereby providing important research value and technical support for early disease prevention and screening for genotoxic substances.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bidoped Bi2MoO6, so as to at least solve one of the technical problems existing in the prior art.

[0006] The second objective of this invention is to provide a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on duplex Bi-doped Bi2MoO6 and its application in the detection of phosphorylated histones, a biomarker of genotoxicity.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] In a first aspect, the present invention provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on Bi-doped Bi₂MoO₆, comprising the following steps:

[0009] (1) Preparation of Bi-Bi2MoO6: A mixed solution of Bi(NO3)3·5H2O, Na2MoO4·2H2O and polyvinylpyrrolidone was stirred, heated, centrifuged, washed and dried sequentially to obtain a yellow powder of Bi-Bi2MoO6. The Bi-Bi2MoO6 powder was dispersed in deionized water to obtain a Bi-Bi2MoO6 dispersion.

[0010] The preferred amount of Bi(NO3)3·5H2O is 2-6 mmol; the preferred amount of Na2MoO4·2H2O is 1-3 mmol. The preferred amount of polyvinylpyrrolidone is 1-2 g.

[0011] The heating conditions are as follows: the mixed solution is placed in a high-temperature oven, preferably heated at 160°C for 12-18 hours, specifically 15 hours.

[0012] Preparation of the Bi-Bi2MoO6 dispersion: preferably 0.5-3 mL is used for every 5 mg of Bi-Bi2MoO6, specifically 1 mL.

[0013] (2) Preparation of glucose oxidase and Au-labeled secondary antibody: GOx and Ab2 were injected into the Au NPs solution and shaken at 4°C. Further, BSA blocking buffer was added to the suspension and incubated to block non-specific adsorption sites. Finally, the product was collected by high-speed centrifugation and redispersed in PBS solution for later use.

[0014] Ab2 is a detection antibody for γH2AX.

[0015] The glucose oxidase and Au-labeled secondary antibody are GOx-AuNPs-Ab2.

[0016] The concentration of GOx is preferably 0.1-1 mg / mL, and the volume used is preferably 150-250 μL.

[0017] The concentration of Ab2 is preferably 0.1-1 mg / mL, and the volume used is preferably 40-60 μL.

[0018] The preferred concentration of the AuNPs solution is 10 mg / mL, and the preferred volume is 1 mL.

[0019] The oscillation time is preferably 1-3 hours.

[0020] The BSA concentration is 1% w / v, the volume used is 100 μL, and the incubation time is preferably 8-12 h.

[0021] The PBS solution had a concentration of 0.1 mmol / L and a pH of 7.4.

[0022] (3) Target recognition and generation of signal molecule H2O2: Glutaraldehyde solution was added to a 96-well microplate and incubated with shaking. The solution was then poured out and washed three times with PBS. Next, Ab1 was added to the wells, incubated with shaking, and then washed three times with PBS. BSA solution was then added, and incubated with shaking to block non-specific adsorption sites. After rinsing, γH2AX standard solution was added to the wells, and the process was repeated with shaking incubation, pouring out, and washing with PBS. Then GO was added... x -AuNPs-Ab2 solution was incubated to form sandwich-shaped immune complexes. After rinsing, glucose solution was added to the wells for incubation, catalyzing the generation of H2O2.

[0023] Ab1 is a capture antibody for γH2AX.

[0024] The concentration of the glutaraldehyde solution is preferably 1-6 mmol / L, the volume used is preferably 50-150 μL, the shaking time is preferably 3-5 h, and the shaking temperature is room temperature.

[0025] The Ab1 concentration is preferably 5-10 μg / mL, the volume is preferably 50-150 μL, the shaking incubation time is preferably 3-5 h, and the shaking incubation temperature is preferably 30-50℃.

[0026] The γH2AX, GO x The preferred volume of AuNPs-Ab2 and BSA added is 30-60 μL, the preferred incubation temperature is 30-50℃, and the preferred incubation time is 40-70 min.

[0027] The concentration of the γH2AX standard solution is from 0.0001 ng / mL to 500 ng / mL.

[0028] The glucose concentration is preferably 4-6 mmol / L, the volume is preferably 250-350 μL, the incubation time is preferably 10-60 min, and the incubation temperature is preferably 20-50℃.

[0029] (4) Construction of photoelectrochemical channels: In this invention, an indium tin oxide (ITO) electrode was used as the electrode substrate. The substrate was thoroughly ultrasonicated three times with deionized water and ethanol, and then dried. Further, a Bi-Bi₂MoO₆ dispersion was dropwise added to the pretreated ITO electrode surface and dried, allowing the material to be physically adsorbed onto the electrode surface, resulting in an ITO / Bi-Bi₂MoO₆ electrode. Further, an immunoreaction product containing H₂O₂ was dropwise added to the above electrode surface and incubated. Finally, the above ITO / Bi-Bi₂MoO₆ electrode was used as the working electrode, a platinum electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The electrode was placed in an electrolytic cell containing PBS electrolyte. A three-electrode detection mode was used, with the working electrode surface irradiated by a light source. The photocurrent signal of the signal molecules was detected using time-current technology on an electrochemical workstation (CHI660E).

[0030] The concentration of the Bi-Bi2MoO6 dispersion is preferably 3-6 mg / mL, and the volume used is preferably 5-25 μL.

[0031] The volume of the H2O2-containing immunoreaction product is preferably 50-200 μL, and the incubation time on the electrode surface is 20-60 min.

[0032] The pH of the electrolyte PBS is 5-9.

[0033] (5) Construction of colorimetric channels: The immunoreaction product was added dropwise to an acetate buffer containing Bi-Bi2MoO6 and TMB. The mixture was incubated at room temperature, and the UV absorption curve of the sample was measured at a wavelength of 550–800 nm to obtain the maximum absorption wavelength at 652 nm.

[0034] The volume of the immunoreaction product is preferably 50-200 μL.

[0035] The volume of the Bi-Bi2MoO6 is preferably 40-60 μL, and the concentration used is preferably 4-6 mg / mL.

[0036] The volume of the TMB is preferably 40-60 μL, and the concentration used is preferably 8-14 mM.

[0037] The concentration of the acetic acid buffer is preferably 0.1-0.5 mmol / L, the volume used is preferably 1-2 mL, and the pH is 4.0-7.0.

[0038] The preferred incubation time for the mixture is 10-40 min, and the preferred incubation temperature is 20-50℃.

[0039] Secondly, the present invention provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on duplex Bi-doped Bi2MoO6 and its application in the detection of phosphorylated histones, a biomarker of genotoxicity.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) Compared with undoped Bi2MoO6, Bi-doped Bi2MoO6 not only has a better PEC response, but also has stronger peroxidase catalytic activity.

[0042] (2) By utilizing the dual-functional properties of Bi-Bi2MoO6 sensing material, not only was the construction and sensing of dual-mode sensors realized, but the preparation steps of dual-mode immunosensors were also simplified, which greatly improved the signal response and detection sensitivity.

[0043] (3) The accuracy and reliability of the detection results are greatly improved by combining photoelectrochemical and colorimetric dual-mode immunosensors.

[0044] (4) The Bi-Bi2MoO6-based dual-mode immunosensor can achieve the detection of the genotoxic biomarker phosphorylated histone γH2AX in a simple, rapid, visual, highly sensitive and selective manner. Attached Figure Description

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is a schematic diagram illustrating a method for constructing a bifunctional Bi-doped Bi2MoO6 photoelectrochemical / colorimetric dual-mode immunosensor.

[0047] Figure 2 The images show the colorimetric response signal (A) and standard curve (B) and photoelectrochemical response signal (C) and standard curve (D) of the dual-mode immunosensor constructed in Example 1 of this invention for detecting γH2AX standard solutions of different concentrations. The concentrations of the γH2AX standard solutions were: 0.0001, 0.0002, 0.0005, 0.001, 0.01, 0.05, 1.00, 2.00, 100, and 500 ng / mL.

[0048] Figure 3The dual-mode immunosensor constructed in Example 1 of this invention exhibits selectivity for γH2AX in both photoelectrochemical (A) and colorimetric (B) detection channels in the presence of other H2A family proteins. Implementation

[0049] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0050] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzyme-catalyzed reaction techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, experimental procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0051] This invention was supported by the National Natural Science Foundation of China (No: 22304168). The method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi2MoO6 provided by this invention has been experimentally verified and can be applied in the detection of genotoxicity biomarkers.

[0052] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Example 1

[0054] This invention provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆, the implementation method of which is as follows: Figure 1 As shown.

[0055] The specific steps are as follows:

[0056] (1) Preparation of Bi-Bi2MoO6: 5 mmol of Bi(NO3)3·5H2O, 2 mmol of Na2MoO4·2H2O, and 1.5 g of polyvinylpyrrolidone were dissolved in 80 mL of ethylene glycol. The mixed solution was sonicated for 30 min, then transferred to a 100 mL reactor and heated at 160 °C for 15 h. After naturally cooling to room temperature, the solution was collected by centrifugation, washed three times with deionized water, and dried in a 60 °C oven to obtain a yellow powder of Bi-Bi2MoO6. 5 mg of the Bi-Bi2MoO6 powder was dispersed in 1 mL of deionized water to obtain a 5 mg / mL Bi-Bi2MoO6 dispersion.

[0057] (2) Preparation of glucose oxidase and Au-labeled secondary antibody: 200 μL of 0.5 mg / mL glucose oxidase (GOx) and 50 μL of 0.5 mg / mL γH2AX secondary antibody (Ab2) were injected into 1 mL of 10 mg / mL AuNPs solution and shaken on a shaker at 4 °C for 80 min. Then, 100 μL of 1% w / v BSA blocking buffer was added to the suspension and incubated at 4 °C for 12 h to block non-specific adsorption sites. Finally, the obtained GOx-AuNPs-Ab2 conjugate was centrifuged at 12000 rpm at 4 °C for 10 min and redispersed in 1.0 mL of 0.1 mmol / L PBS solution (pH 7.4) for further use.

[0058] (3) Target recognition and generation of the signal molecule H2O2: First, 100 μL of 25% w / v 5.0 mmol / L glutaraldehyde solution was added to each well of a 96-well microplate and shaken at room temperature for 4 h. Then, the solution was poured out and washed three times with PBS (pH 7.4). Next, 10 μg / mL γH2AX primary antibody (Ab1) was added to each well at a volume of 100 μL and shaken at 37 °C for 4 h. Then, the plate was washed three times with PBS, followed by the addition of 100 μL of 1% w / v BSA solution and incubation at 4 °C for 60 min. After rinsing, 50 μL of γH2AX standard solutions of different concentrations were added to each well and incubated at 37 °C with shaking for 60 min. Further, 50 μL of GO was added. x -AuNPs-Ab2 solution was incubated with shaking at 37°C for 60 min to form a sandwich-shaped immune complex. After washing with PBS, 300 μL of 5 mmol / L glucose solution was added to the wells and incubated at 37°C for 15 min. During this process, the glucase modified on the γH2AX secondary antibody catalyzed the glucose substrate to generate an immunoreaction product containing H2O2, wherein the concentration of H2O2 was quantitatively correlated with the concentration of γH2AX.

[0059] (4) Construction of photoelectrochemical channels: In this invention, an indium tin oxide (ITO, 1cm × 2cm) electrode was used as the electrode substrate. The electrode was thoroughly sonicated three times with deionized water and ethanol, and then dried at 60°C. Next, 20 μL of a 5 mg / mL Bi-Bi₂MoO₆ dispersion was added dropwise to the pretreated ITO electrode surface, and the electrode was dried at 60°C for 30 min to allow the material to be physically adsorbed onto the electrode surface, thus obtaining an ITO / Bi-Bi₂MoO₆ electrode. Further, 100 μL of an immunoreaction product containing H₂O₂ was added dropwise to the above electrode surface and incubated for 30 min. Finally, the ITO / Bi-Bi2MoO6 electrode was used as the working electrode, the platinum electrode as the counter electrode, and the Ag / AgCl electrode as the reference electrode. The electrode was placed in an electrolytic cell containing 30 mL of PBS (0.1 mol / L, pH 7.4) electrolyte. The surface of the working electrode was irradiated with a 100W 430 nm LED light source. The photocurrent signal of the signal molecule was detected using time-current technology on an electrochemical workstation (CHI660E).

[0060] (5) Construction of the colorimetric channel: 100 μL of the immunoreaction product was added to 1 mL of 0.1 M, pH 5.5 acetate buffer containing 50 μL of 5 mg / mL Bi-Bi2MoO6 and 50 μL of 10 mM TMB. The mixture was incubated at room temperature for 30 min, and the UV absorption curve of the sample was measured at wavelengths of 550–800 nm to obtain the maximum absorption wavelength at 652 nm.

[0061] (6) Establishment of standard curves: γH₂AX standard solutions of different concentrations were added to steps (3), (4), and (5) to obtain molecular detection solutions with different gradient concentrations of sample-related concentration signals. These solutions were then detected using an electrochemical workstation and a UV spectrophotometer. A linear fit was performed with the logarithm of the γH₂AX concentration as the abscissa and the photocurrent signal as the ordinate to establish a standard curve for the photoelectrochemical detection channel. Similarly, a linear fit was performed with the logarithm of the γH₂AX concentration as the abscissa and the absorbance as the ordinate to establish a standard curve for the colorimetric detection channel.

[0062] The concentration of the γH2AX standard solution is from 0.0001 ng / mL to 500 ng / mL, for example, it can be 0.0001, 0.0002, 0.0005, 0.001, 0.01, 0.05, 1.00, 2.00, 100 and 500 ng / mL.

[0063] like Figure 2The figures show the photoelectrochemical response signal (A) and standard curve (B) and the colorimetric response signal (C) and standard curve (D) of the dual-mode immunosensor constructed in Example 1 of this invention for detecting γH2AX. Based on S / N = 3 and the standard curve, the detection limit of the photoelectrochemical channel was calculated to be 0.32 fg / mL, and the detection limit of the colorimetric detection channel was calculated to be 70.8 fg / mL.

[0064] (7) γH2AX sample detection: Replace the γH2AX standard solution in step (3) with the actual sample, and proceed according to steps (1)-(5) to obtain the photocurrent response value and the maximum absorption wavelength at 652nm. Substitute these values ​​into the standard curve in (6) to obtain the concentration of γH2AX in the sample to be tested.

[0065] Example 2

[0066] This embodiment provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆, which differs from Embodiment 1 in that:

[0067] Dissolve 3 mmol of Bi(NO3)3·5H2O, 1 mmol of Na2MoO4·2H2O and 1.5 g of polyvinylpyrrolidone in 80 mL of ethylene glycol;

[0068] Other preparation conditions were the same as in Example 1.

[0069] Example 3

[0070] This embodiment provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆, which differs from Embodiment 1 in that:

[0071] Dissolve 6 mmol of Bi(NO3)3·5H2O, 2 mmol of Na2MoO4·2H2O and 1.5 g of polyvinylpyrrolidone in 80 mL of ethylene glycol;

[0072] Other preparation conditions were the same as in Example 1.

[0073] Example 4

[0074] This embodiment provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆, which differs from Embodiment 1 in that:

[0075] Dissolve 2 mmol of Bi(NO3)3·5H2O, 1 mmol of Na2MoO4·2H2O and 1.5 g of polyvinylpyrrolidone in 80 mL of ethylene glycol;

[0076] Other preparation conditions were the same as in Example 1.

[0077] Example 5

[0078] This embodiment provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆, which differs from Embodiment 1 in that:

[0079] Dissolve 4 mmol of Bi(NO3)3·5H2O, 2 mmol of Na2MoO4·2H2O and 1.5 g of polyvinylpyrrolidone in 80 mL of ethylene glycol;

[0080] Other preparation conditions were the same as in Example 1.

[0081] Example 6

[0082] This embodiment provides a method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆, which differs from Embodiment 1 in that:

[0083] Dissolve 6 mmol of Bi(NO3)3·5H2O, 3 mmol of Na2MoO4·2H2O and 1.5 g of polyvinylpyrrolidone in 80 mL of ethylene glycol;

[0084] Other preparation conditions were the same as in Example 1.

[0085] Example 7

[0086] The selectivity of a bifunctional Bi-doped Bi₂MoO₆-based photoelectrochemical / colorimetric dual-mode immunosensor for γH₂AX includes the following steps:

[0087] The photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆ prepared in Example 1 was used to detect interfering H₂A family proteins, including H₂A.X, H₂A, H₂A.1, and H₂A.2. The concentration of γH₂AX was 50 pg / mL, and the concentration of the other H₂A family protein standard solutions was 500 pg / mL. The detection results are as follows: Figure 3 As shown in photoelectrochemical (A) and colorimetric (B), the photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi2MoO6 provided by the present invention exhibits good selectivity for γH2AX.

[0088] Application Example 1

[0089] The photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆ described in Example 1 was used to detect γH₂AX in HepG2 cells, including the following steps:

[0090] (1) Cell Culture and Lysis: HepG2 cells were cultured in α-MEM complete medium containing 10% fetal bovine serum (v / v), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. When the cell density was greater than 80%, the cells were passaged at a ratio of 1:3, with the medium changed every 2-3 days during passage. When the cell density exceeded 80%, the cells were digested with trypsin-EDTA solution and lysed at a ratio of 5 × 10⁶ cells / mL. 6 Cells were plated at a density of 10 cm² in 10 cm culture dishes and allowed to adhere overnight before being treated with the specified compound. HepG2 cells were lysed using commercially available RIPA lysis buffer, centrifuged at high speed, and the cell lysis samples were collected.

[0091] (2) Sample detection: γH2AX standard was added to the cell lysate samples to achieve final concentrations of 40 pg / mL, 80 pg / mL, 120 pg / mL, and 240 pg / mL, respectively. The photocurrent signal and absorbance were measured according to the steps in Example 1, and the concentration of γH2AX in the sample was obtained by substituting them into the standard curve. Each sample was measured 5 times, and the average value was taken to calculate the recovery rate and RSD. As shown in Table 1, the average recovery rate of the photoelectrochemical sensing mode was 94.2-114.7%, and the average recovery rate of the colorimetric sensing mode was 91.95-109.1%, with an RSD of less than 4.2%, indicating that the dual-mode immunosensor has good accuracy, reliability, and practicality.

[0092] Table 1

[0093]

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to fall outside the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fabricating a bifunctional Bi-doped Bi₂MoO₆ photoelectrochemical / colorimetric dual-mode immunosensor, characterized in that, Includes the following steps: (1) Preparation of Bi-Bi2MoO6: Bi(NO3)3·5H2O, Na2MoO4·2H2O and polyvinylpyrrolidone were added to an ethylene glycol solution. The mixed solution was subjected to ultrasonication, heating, centrifugation, washing and drying in sequence to obtain a yellow powder of Bi-Bi2MoO6. The Bi-Bi2MoO6 powder was dispersed in deionized water to obtain a Bi-Bi2MoO6 dispersion. (2) Preparation of glucose oxidase GOx and Au-labeled secondary antibody Ab2: GOx and Ab2 were injected into Au NPs solution and shaken. Then, BSA blocking buffer was added to the suspension and incubated to block non-specific adsorption sites. Finally, the product was collected by centrifugation and redispersed in PBS solution for later use. The glucose oxidase and Au-labeled secondary antibody are GOx-AuNPs-Ab2. Ab2 is a detection antibody for the target protein γH2AX. (3) Target recognition and generation of signal molecule H2O2: Glutaraldehyde solution was added to a 96-well microplate and shaken. The solution was then poured out and washed three times with PBS. Next, Ab1 was added to the wells, shaken, and washed three times with PBS, followed by blocking non-specific adsorption sites with BSA solution. After washing, different concentrations of γH2AX standards were added to the wells, and incubation, pouring, and washing were performed sequentially. Then GO was added. x The AuNPs-Ab2 solution was incubated, poured out, and washed sequentially to form a sandwich-shaped immune complex. Finally, glucose solution was added to the wells for incubation, catalyzing the production of the signaling molecule H2O2. Ab1 is a recognition antibody for the target protein γH2AX. The pH of the PBS is preferably 7-8. (4) Construction of photoelectrochemical channels: In this invention, an indium tin oxide (ITO) electrode was used as the electrode substrate, and was thoroughly ultrasonicated three times with deionized water and ethanol, respectively, and then dried. Further, a Bi-Bi₂MoO₆ dispersion was dropwise added to the surface of the pretreated ITO electrode and dried, allowing the material to be physically adsorbed onto the electrode surface, resulting in an ITO / Bi-Bi₂MoO₆ electrode. Further, an immunoreaction product containing H₂O₂ was dropwise added to the surface of the above electrode. After drying, the above ITO / Bi-Bi₂MoO₆ electrode was used as the working electrode, a platinum electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The electrode was placed in an electrolytic cell containing PBS electrolyte, using a three-electrode mode. The working electrode surface was irradiated with a light source, and the photocurrent response of the signal molecules was detected using time-current technology on an electrochemical workstation. (5) Construction of colorimetric channels: The immunoreaction product was added dropwise to an acetate buffer containing Bi-Bi2MoO6 and TMB. The mixture was incubated at room temperature, and the UV absorption curve of the sample was measured at a wavelength of 550–800 nm to obtain the maximum absorption wavelength at 652 nm.

2. The method for fabricating a bifunctional Bi-doped Bi₂MoO₆ photoelectrochemical / colorimetric dual-mode immunosensor according to claim 1, characterized in that, In step (1), the preferred amount of Bi(NO3)3·5H2O is 2-6 mmol; the preferred amount of Na2MoO4·2H2O is 1-3 mmol; and the preferred amount of polyvinylpyrrolidone is 1-2 g. The heating conditions are as follows: the mixed solution is placed in a high-temperature oven, preferably heated at 160℃ for 12-18 h, specifically 15 h; the preparation of the Bi-Bi2MoO6 dispersion: 0.5-3 mL is preferred for every 5 mg of Bi-Bi2MoO6, specifically 1 mL.

3. The method for fabricating a bifunctional Bi-doped Bi₂MoO₆ photoelectrochemical / colorimetric dual-mode immunosensor according to claim 1, characterized in that, In step (2), the concentration of GOx is 0.1-1 mg / mL, and the volume used is 150-250 μL; the concentration of Ab2 is 0.1-1 mg / mL, and the volume used is 40-60 μL; the concentration of AuNPs solution is preferably 10 mg / mL, and the volume used is preferably 1 mL; the shaking time is preferably 1-3 h; the concentration of BSA is 1% w / v, the volume used is 100 μL, and the incubation time is preferably 8-12 h. The concentration of PBS solution is 0.1 mmol / L, and the pH is 7.

4.

4. The method for fabricating a bifunctional Bi-doped Bi₂MoO₆ photoelectrochemical / colorimetric dual-mode immunosensor according to claim 1, characterized in that, The glutaraldehyde solution concentration in step (3) is preferably 1-6 mmol / L, the volume used is preferably 50-150 μL, the shaking incubation time is preferably 3-5 h, and the shaking temperature is room temperature; the Ab1 concentration is preferably 5-10 μg / mL, the volume is preferably 50-150 μL, the shaking incubation time is preferably 3-5 h, and the shaking incubation temperature is preferably 30-50 °C; the γH2AX standard solution, GO x The preferred volume of AuNPs-Ab2 and BSA added is 30-60 μL, the preferred incubation temperature is 30-50℃, and the preferred incubation time is 40-70 min; the preferred glucose concentration is 4-6 mmol / L, the preferred volume is 250-350 μL, the preferred incubation time is 10-60 min, and the preferred incubation temperature is 20-50℃.

5. The method for fabricating a bifunctional Bi-doped Bi₂MoO₆ photoelectrochemical / colorimetric dual-mode immunosensor according to claim 1, characterized in that, In step (3), the concentration of the γH2AX standard solution is 0.0001 ng / mL to 500 ng / mL.

6. The method for fabricating a bifunctional Bi-doped Bi₂MoO₆ photoelectrochemical / colorimetric dual-mode immunosensor according to claim 1, characterized in that, The concentration of the Bi-Bi2MoO6 dispersion in step (4) is preferably 3-6 mg / mL; the volume used is preferably 5-25 μL; the volume of the immunoreaction product containing H2O2 is preferably 50-200 μL, and the incubation time on the electrode surface is 20-60 min; the pH of the electrolyte PBS is 5-9.

7. The method for fabricating a bifunctional Bi-doped Bi₂MoO₆ photoelectrochemical / colorimetric dual-mode immunosensor according to claim 1, characterized in that, The volume of the immunoreaction product in step (5) is preferably 50-200 μL; the volume of Bi-Bi2MoO6 is preferably 40-60 μL, and the concentration is preferably 4-6 mg / mL; the volume of TMB is preferably 40-60 μL, and the concentration is preferably 8-14 mM; the concentration of the acetate buffer is preferably 0.1-0.5 mmol / L, the volume is preferably 1-2 mL, and the pH is 4.0-7.0; the incubation time of the mixture is preferably 10-40 min, and the incubation temperature is preferably 20-50℃.

8. The method for constructing a photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆ according to any one of claims 1-7, characterized in that, The detection limit for phosphorylated histones by the photoelectrochemical channel is 0.32 fg / mL, and the detection limit for the colorimetric detection channel is 70.8 fg / mL.

9. The application of the method for constructing a bifunctional Bi-doped Bi2MoO6 photoelectrochemical / colorimetric dual-mode immunosensor according to any one of claims 1-8 in the detection of phosphorylated histones, a biomarker of genotoxicity.

10. The photoelectrochemical / colorimetric dual-mode immunosensor based on bifunctional Bi-doped Bi₂MoO₆ according to any one of claims 1-9, characterized in that, With high sensitivity, good selectivity, and low cost, it can quickly, visually, and accurately detect phosphorylated histones, a biomarker of genotoxicity, enabling the screening of genotoxic substances.

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