In-vitro immunodetection method based on optical excitation electrically-driven active oxygen channel

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an in-vitro immunodetection method based on an optically-excited electrically-driven active oxygen channel. The method provided by the invention is used for detecting the target protein in a biological sample, and comprises the following steps: preparing a donor bead compound containing a photosensitizer and a receptor bead compound containing polyphenol, putting the donor bead compound and the receptor bead compound into a biological sample solution to be detected, and when the biological sample to be detected contains the target protein, detecting the target protein in the biological sample to be detected. The target protein recognizes and is combined with the capture antibody and the detection antibody to form a sandwich immune structure, and a donor bead-acceptor bead connector of the target protein is obtained for detection. The method is accurate in detection result and high in sensitivity.

Description

[0001] Divisional application This application is a divisional application of a Chinese patent application with the application number 2024109894766, the application date of July 22, 2024, and the invention title of "A method for detecting biomarkers based on photoexcitation-driven electroactive oxygen channels and its application". Technical field

[0002] The present invention belongs to the field of bioengineering technology, and specifically relates to an in vitro immunoassay method based on photoexcitation-driven electroactive oxygen channels. Background art

[0003] In recent years, the incidence of cancer in China has been continuously rising, seriously threatening the health of residents. Facing the severe cancer prevention and control situation, early diagnosis and early treatment have become the key links to reduce the cancer mortality rate. At present, the detection of protein tumor markers has been widely used in the early screening of tumors. Currently, common protein tumor markers include prostate-specific antigen (PSA) related to prostate cancer, carcinoembryonic antigen (CEA) related to digestive tract tumors, and alpha-fetoprotein (AFP) related to hepatocellular carcinoma, etc.

[0004] Photoinduced chemiluminescence technology - a new type of fourth-generation chemiluminescence technology. Since it is a homogeneous reaction platform and does not require a separation system during the detection process, it has the advantages of being wash-free and having a low background, and has attracted more and more attention from in vitro diagnostic (IVD) enterprises. Photoinduced chemiluminescence technology is also known as Luminescent oxygenchanneling immunoassay (LOCI) technology, which was first proposed in 1994. At present, there are also reports on using this technology to detect biomarkers, but there are still some problems. There are also reports that photoinduced chemiluminescence oxygen channel immunoassay, as a common homogeneous wash-free detection technology, has been used for protein analysis (ACS Omega 2022, 7, 2344 - 2355; Anal. Chem. 2019, 91, 5777 - 5785.). However, due to the complex construction of its photosensitive microspheres and luminescent microspheres, the luminescent substances are easily photo-bleached, and the chemiluminescent substances in the luminescent microspheres are dissipated and consumed, resulting in unstable output of chemiluminescent signals. Another example is the patent with the publication number CN117368483A (January 9, 2024), and the invention title of "A method for detecting kidney injury molecule-1 and a kit based on homogeneous photoinduced chemiluminescence immunoassay technology". This patent uses a double-antibody sandwich immunoassay method and the characteristics of photoinduced chemiluminescence to detect antibodies. This patent method is based on the principle of light input - light output, and there may be problems of mutual interference between light signals. In addition, this method requires a large instrument with a complex optical path system to achieve, so the cost is high.

[0005] Electrochemical sensing is a new generation of IVD technology, with technical advantages in terms of miniaturization of equipment, low raw material costs, rapid response, and high sensitivity. Abroad, companies such as Roche and Atlas Genetics aim at "sample in, result out" for sensing. Through the integration of methods such as microfluidic channel technology and droplet manipulation technology, they avoid the loss of targets caused by repeated separation and purification, and shield the interference of electroactive molecules. They have successively developed electrochemical sensing technologies based on microelectrode systems and electron transfer materials. However, the design of the pre-sensing processing chip is complex, and there are problems such as baseline drift interference and frequent electrode calibration in the sensing mode based on interfacial cumulative electrical signals. Related products have not entered China yet. Designing and developing an anti-interference electrochemical sensor based on cascaded electron transfer is a major scientific problem that urgently needs to be solved for the development of IVD reagent materials accessible at the grass-roots level.

[0006] In summary, it is necessary to propose an in vitro immunoassay method based on a photoexcitation electro-driven reactive oxygen species channel to supplement the deficiencies of existing technologies. Summary of the Invention

[0007] The purpose of the present invention is to provide an in vitro immunoassay method based on a photoexcitation electro-driven reactive oxygen species channel to partially solve or alleviate the above deficiencies in the existing technology. The present invention specifically adopts the following technical solutions.

[0008] A method for detecting a biomarker using a photoexcitation electro-driven reactive oxygen species channel, wherein the biomarker is a protein, and the specific steps are as follows: S01: Prepare a donor bead complex containing a photosensitizer, where the donor bead complex is a conjugate of a metal-organic framework containing a photosensitizer and a capture antibody; S02: Prepare a receptor bead complex containing polyphenols, where the receptor bead complex is a conjugate of melanin-like nanoparticles and a detection antibody; S03: Put the donor bead complex and the receptor bead complex into a biological sample solution to be tested. When the biological sample to be tested contains a target protein, the target protein recognizes and binds to the capture antibody and the detection antibody to form a sandwich immune structure, obtaining a donor bead-receptor bead conjugate of the target protein, and the distance of the donor bead-receptor bead conjugate is less than 200 nm; the biological sample to be detected is selected from at least one of interstitial fluid, blood, urine, body fluid, and their combinations; S04: Add the donor bead-receptor bead linker to the electrode interface and irradiate it with a laser. The donor bead complex in the donor bead-receptor bead linker generates singlet oxygen upon laser irradiation. The singlet oxygen diffuses in the biological sample to be measured. After the polyphenol in the receptor bead complex captures the singlet oxygen, it is oxidized to produce benzoquinone. Apply a reduction voltage at the electrode interface to trigger the electrochemical reduction of benzoquinone to polyphenol, resulting in a cascaded redox cycle and generating an amplified current signal. Read the current signal. S05: Establish a standard curve in advance based on the concentration of the target protein standard and the current intensity value. S06: Substitute the read current signal into the standard curve to calculate the content of the target protein in the biological sample to be measured.

[0009] Furthermore, the metal-organic framework is coupled to the capture antibody through an amide bond.

[0010] Furthermore, the melanin-like nanoparticles are coupled to the detection antibody through a Michael addition reaction or through a Schiff base reaction.

[0011] Furthermore, the photosensitizer includes one or more selected from fluoroboron dipyrrole, metal phthalocyanine, and porphyrin.

[0012] As a preference, the photosensitizer is tetracarboxyphenyl porphyrin; correspondingly, the applied laser wavelength is 630 - 660 nm.

[0013] Furthermore, the metal-organic framework includes one or more selected from zirconium-based metal-organic frameworks, gadolinium-based metal-organic frameworks, and iron-based metal-organic frameworks.

[0014] In some embodiments, the donor bead is obtained by reacting tetracarboxyphenyl porphyrin and zirconium oxychloride octahydrate in benzoic acid and N,N-dimethylformamide, and the mass ratio of tetracarboxyphenyl porphyrin to zirconium oxychloride octahydrate is 1 / 6 - 1 / 2.

[0015] In some embodiments, for the receptor bead, first disperse dopamine in ethanol, add tris(hydroxymethyl)aminomethane salt and stir well for reaction. Take the reaction solution, mix it with absolute ethanol and then centrifuge. Take the centrifuged precipitate, ultrasonicate it in sodium hydroxide solution, adjust the pH to neutral, and finally obtain the melanin-like nanoparticle receptor bead by freeze-drying.

[0016] Furthermore, the particle size range of the donor bead is 60 - 200 nm.

[0017] Furthermore, the particle size range of the receptor bead is 10 - 20 nm.

[0018] Furthermore, the melanoid nanoparticles are nanoscale products formed by the oxidative polymerization of monomers containing benzene ring structures and polyphenol groups in an aqueous solution.

[0019] As a preference, the melanoid nanoparticles include allomelanin, polydopamine, polypyrogallol or polycaffeic acid.

[0020] Furthermore, the configuration ratio of the donor bead complex to the receptor bead complex is 1:10.

[0021] As a preference, the target protein includes carcinoembryonic antigen.

[0022] As a preference, the voltage applied in S04 is -0.8 to -0.1 V.

[0023] Beneficial technical effects: The present invention provides a method for detecting protein markers by using a method of photoexcitation-driven electroactive oxygen channel. The donor bead complex and the receptor bead complex included in the method of the present invention are nanoparticles free in the detection system. Therefore, the method of the present invention adopts a strategy of avoiding electrode modification by avoiding immobilization, and realizes "washing-free and separation-free" homogeneous electrochemistry through uniform mass transfer collision, and can be repeatedly detected. In addition, it can effectively overcome the defect that photoelectrochemistry requires repeated washing and separation steps to remove unbound substances that cause background signals, and has high detection sensitivity. In the current prior art, it is often necessary to immobilize optoelectronic nanomaterials on the electrode. The non-uniformity of the modified electrode and the uncontrollable packing density of the modified probe seriously limit the repeatability of electrochemical detection. In addition, the electron-hole recombination time in the optoelectronic materials used in these methods is much lower than the time for electrons to transfer to the electrode or for the electrode electrons to transfer to the material, resulting in carrier recombination, reducing the photocurrent signal, and thus reducing the detection sensitivity.

[0024] Furthermore, the strategy adopted by the method of the present invention is to convert light energy into electrical energy, that is, light input - electrical output. Specifically, a negative potential is applied for reduction to avoid interference from electroactive substances in the physiological matrix. In the transient active oxygen channel - electron transfer cascade process under collision constraint, the oxidation event on the surface of the receptor bead amplifies the current signal through the quinone reduction potential, thereby obtaining an electrical signal, so the sensitivity is high.

[0025] Specifically, when the method of the present invention detects the target protein in a biological sample, a sandwich immunization result of double antibodies binding to the antigen to be detected is adopted to achieve amplification of the target signal. The experimental results of the present invention show that this method can detect the target protein in the concentration range of 10 pg mL -1 - 50 ng mL -1 of the concentration range. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 Schematic diagram for simulating the method of the present invention for detecting nucleic acids by using a photoexcited electro-driven reactive oxygen species channel; Figure 2 Schematic diagram for simulating the method of the present invention for detecting proteins by using a photoexcited electro-driven reactive oxygen species channel; Figure 3 Transmission electron microscope (TEM) photograph of the donor beads prepared by the method of the present invention (scale bar 50 nm); Figure 4 Transmission electron microscope (TEM) photograph of the acceptor beads prepared by the method of the present invention (scale bar 200 nm); Figure 5 Electron paramagnetic resonance (ESR) spectrum of singlet oxygen generated by photoexcitation of the donor beads in the method of the present invention; Figure 6 Detection time-current curve of miRNA detected by the photoexcited electro-driven reactive oxygen species channel method in one embodiment of the present invention; Figure 7 Schematic diagram of the detection linear range of miRNA detected by the photoexcited electro-driven reactive oxygen species channel method in one embodiment of the present invention; Figure 8 Anti-interference performance evaluation diagram of the photoexcited electro-driven reactive oxygen species channel method in one embodiment of the present invention; Figure 9 Detection time-current curve of CEA detected by the photoexcited electro-driven reactive oxygen species channel method in one embodiment of the present invention; Figure 10 Schematic diagram of the detection linear range of CEA detected by the photoexcited electro-driven reactive oxygen species channel method in one embodiment of the present invention; Figure 11 Schematic diagram of the spiked recovery results of the simulated actual samples in one embodiment of the present invention; Figure 12 Schematic diagram of the detection system structure of the photoexcited electro-driven reactive oxygen species channel provided by the present invention; Figure 13 Schematic diagram of the working principle of the detection system of the photoexcited electro-driven reactive oxygen species channel provided by the present invention. Detailed implementation manners

[0028] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0030] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0031] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0032] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is only for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0033] Glossary of terms: The light-excited electro-driven active oxygen channel (LEOCA) described in the present invention is a homogeneous detection method for light-excited synchronous electron transfer. Specifically, after a donor-acceptor composite reaction system is irradiated with a laser, the donor generates singlet oxygen, and the acceptor captures the singlet oxygen to generate an oxidation product, and then undergoes a cascade chemical oxidation-electrochemical reduction process at the electrode interface to generate an electrochemical signal, which can also be simply referred to as the LEOCA signal.

[0034] The cascade redox cycle described in the present invention refers to the singlet oxygen generated by photoexciting the donor bead complex diffusing to the surface of the acceptor bead complex in the presence of the target substance and oxidizing the polyphenol groups in the acceptor bead to form benzoquinone. The benzoquinone that collides and contacts on the electrode surface is reduced to polyphenol again at a negative potential, generating an electrochemical signal. The product benzoquinone produced by the former chemical oxidation event is the substrate for the latter electrochemical reduction event, triggering the subsequent electrochemical reduction reaction, so it is called the cascade redox cycle reaction.

[0035] The donor bead-acceptor bead linker described in the present invention refers to a structure formed by the first reactant on the donor bead complex and the second reactant on the acceptor bead complex connecting with the target substance in the biological sample to be detected, specifically referring to the structure of the first reactant-target substance-second reactant between the donor bead and the acceptor bead after connection.

[0036] Example 1 This example provides an example of a method for detecting biomarkers using a photoexcited electro-driven reactive oxygen channel. The biomarker detected in this example is nucleic acid.

[0037] (1) Use the first hairpin nucleic acid conjugated with a metal-organic framework containing a porphyrin-based photosensitizer as a nano donor bead, and the second hairpin nucleic acid conjugated with a polydopamine particle containing polyphenol as a nano acceptor bead. The target nucleic acid recognizes and responds to the first hairpin nucleic acid strand to trigger the naked single-stranded region, thereby inducing the hybridization of the second hairpin nucleic acid strand and finally forming a catalytic hairpin self-assembly complex.

[0038] (2) Irradiate the detection system with excitation light. The singlet oxygen ( 1 O 2 ) generated by the donor bead in the complex diffuses to the acceptor bead. The polyphenol in the acceptor bead captures 1 O 2 and can be oxidized to produce benzoquinone, and undergoes a cascade chemical oxidation-electrochemical reduction process during the instantaneous collision at the electrode interface, generating an electrochemical signal. It should be noted that due to 1 O 2 having high activity and a short half-life, only when the distance between the donor bead and the acceptor bead is close enough (forming a catalytic hairpin self-assembly complex), the 1 O 2 released by the donor bead can effectively reach the acceptor bead, thereby generating an electrochemical signal.

[0039] (3) When there is no target in the system, that is, no catalytic hairpin self-assembly complex is formed, the distance between the donor bead and the acceptor bead is greater than 200 nm, and the 1 O 2 generated by the free donor bead dissipates rapidly in the system, and almost no electrochemical signal is generated.

[0040] In summary, through catalytic hairpin self-assembly between nucleic acids, high-energy 1 O 2 transfer is achieved between the two types of donor and acceptor beads, inducing the process of photoexcitation-driven electrochemically active oxygen channels. The detection principle diagram is as Figure 1 shown.

[0041] In this example, the nucleic acid sample to be tested is selected from tissue interstitial fluid.

[0042] Example 2 This example provides another method example for detecting biomarkers using photoexcitation-driven electrochemically active oxygen channels. The biomarker detected in this example is protein.

[0043] (1) A metal-organic framework conjugated with a capture antibody and containing a porphyrin-based photosensitizer is used as a nano-donor bead, and a polydopamine particle conjugated with a detection antibody and containing polyphenols is used as a nano-acceptor bead. The capture antibody and the detection antibody are recognized and responded by the target antigen to form a sandwich immune structure, and finally a donor bead-acceptor bead complex is formed.

[0044] (2) The detection system is irradiated with excitation light. The singlet oxygen ( 1 O 2 ) generated by the donor beads in the complex diffuses to the acceptor beads. The polyphenols in the acceptor beads capture 1 O 2 and are oxidized to produce benzoquinone, which undergoes a cascade chemical oxidation-electrochemical reduction process at the electrode interface instantaneously upon collision, generating an electrochemical signal. It should be noted that due to 1 O 2 having high activity and a short half-life, only when the distance between the donor beads and the acceptor beads is close enough (the capture antibody-target antigen-detection antibody binding), the 1 O 2 released by the donor beads can effectively reach the acceptor beads, thus generating an electrochemical signal.

[0045] (3) When the target antigen is absent, the capture antibody and the detection antibody in the detection system cannot bind, the distance between the donor beads and the acceptor beads is greater than 200 nm, and the 1 O 2 generated by the free donor beads dissipates rapidly in the system, and almost no electrochemical signal is generated.

[0046] In summary, through the binding of antigen and antibody between the two types of donor and acceptor beads, high-energy 1 O 2 transfer is achieved, inducing the process of photoexcitation-driven electrochemically active oxygen channels. The detection principle diagram is as Figure 2 shown.

[0047] In this example, the nucleic acid sample to be tested is selected from tissue interstitial fluid.

[0048] Example 3 This example provides an example of a preparation method for various materials in a detection system.

[0049] (1)Preparation of donor beads Add 50 - 150 mg of tetracarboxyphenyl porphyrin, 300 mg of zirconium oxychloride octahydrate, 2.2 - 3.3 g of benzoic acid, and 80 - 140 mL of N,N - dimethylformamide into a flask, ultrasonically dissolve for 5 min, and stir at 90 °C for 5 hours; after the reaction is completed, centrifuge at 15000 rpm for 10 - 20 min, and then wash 3 times with N,N - dimethylformamide. Finally, collect the precipitate, which is the zirconium - based metal - organic framework nano - donor beads, and disperse them in absolute ethanol for storage.

[0050] It can be understood that the preparation of the zirconium - based metal - organic framework nano - donor beads in this example is only an example, and gadolinium - based metal - organic frameworks or iron - based metal - organic frameworks can also be selected.

[0051] (2)Preparation of receptor beads Add 55 mg of dopamine hydrochloride into a flask, ultrasonically disperse it in a mixture of 100 mL of deionized water and 20 mL of absolute ethanol, add 120 mg of tris - (hydroxymethyl) aminomethane salt to the reaction mixture, and stir at room temperature for 72 hours; mix the reaction stock solution with absolute ethanol, centrifuge at 16000 rpm for 10 - 20 min, discard the supernatant to obtain the precipitate, and repeat three times; dissolve 20 mg of the precipitate in 10 mL of 0.1 M sodium hydroxide solution, ultrasonically treat at an output power of 600 W for 5 - 10 min, and then adjust the pH value to 7.0 using hydrochloric acid; freeze - dry the obtained polydopamine receptor beads and disperse them in water for storage.

[0052] It can be understood that the preparation of the polydopamine receptor beads in this example is only an example, and eumelanin, poly - L - dopa, poly - gallic acid, poly - caffeic acid, etc. can also be selected.

[0053] Perform morphological characterization (TEM) on the donor beads and receptor beads, as Figure 3 、 Figure 4 shown. Qualified donor beads are spherical nanostructures with a size range of 100 - 150 nm, and qualified receptor beads are spherical nanostructures with a size range of 10 - 20 nm. Further analyze the donor beads using electron spin resonance (ESR), and the spectrum is as Figure 5 shown. After the ESR spin - trapping agent TEMP acts on the donor beads under light, an ESR signal is generated, indicating that the donor beads that can generate singlet oxygen by photo - excitation are successfully synthesized by this method.

[0054] (3)Preparation of hairpin nucleic acid The hairpin nucleic acid was chemically synthesized and purified by HPLC, then dissolved in annealing buffer to prepare a solution, and placed in a 95°C PCR instrument for denaturation for 5 min, and then the temperature was gradually decreased to 25°C at a rate of 0.1°C per second to form a stable catalytic hairpin structure.

[0055] Annealing buffer: The solvent is deionized water, and the components and contents of its solution are: Tris hydrochloride, 20 mmol / L; sodium chloride, 140 mmol / L; potassium chloride, 5 mmol / L; pH is 7.4.

[0056] It can be understood that in this embodiment, the capture antibody and the detection antibody can also be prepared according to the conventional technical means in the art.

[0057] (4)Preparation of the first hairpin nucleic acid donor bead complex / the second hairpin nucleic acid acceptor bead complex The zirconium-based metal-organic framework donor beads and the first hairpin nucleic acid were mixed and incubated at 37°C in coupling buffer for 2 hours to obtain the first hairpin nucleic acid donor bead complex. The polydopamine receptor beads and the second hairpin nucleic acid were mixed and incubated at 37°C in coupling buffer for 4 hours to obtain the second hairpin nucleic acid receptor bead complex.

[0058] Coupling buffer: The solvent is deionized water, and the components and contents of its solution are: Tris hydrochloride, 20 mmol / L; sodium chloride, 140 mmol / L; potassium chloride, 5 mmol / L; calcium chloride, 1 mmol / L; magnesium chloride, 1 mmol / L; pH is 7.4.

[0059] It can be understood that in this embodiment, the capture antibody donor bead complex and the detection antibody receptor bead complex can also be prepared according to a similar method and other conventional technical means in the art.

[0060] Example 4 This embodiment provides an actual nucleic acid sample detection example and an investigation of the performance of the detection method.

[0061] (1)The zirconium-based metal-organic framework donor beads and the first hairpin nucleic acid were mixed and incubated at 37°C in Tris-HCl buffer for 2 hours; the polydopamine receptor beads and the second hairpin nucleic acid were mixed and incubated at 37°C in Tris-HCl buffer for 4 hours; the donor and receptor beads were mixed evenly as the detection system. In this embodiment, the particle size of the donor beads is 100 nm, and the particle size of the receptor beads is 10 nm. The mixing ratio of the donor beads to the receptor beads is 1:10.

[0062] (2)A sample containing the tumor tissue lysate or tumor interstitial fluid to be tested, which contains the miRNA to be tested, was added to the detection system, and it was mixed evenly and incubated at 37°C for 30 - 120 min to form a test dispersion.

[0063] (3) Add the dispersion dropwise to a screen-printed carbon electrode, and use an electrochemical workstation to detect the strength of the electrical signal. In a homogeneous environment, under the excitation condition of a 630 nm laser (the photosensitizer selected in this example is tetracarboxyphenyl porphyrin), singlet oxygen transfer occurs between the donor and acceptor beads. A voltage of -0.2 V is applied to the electrode for reduction to generate an electrochemical signal. A standard curve is established based on the concentration of the miRNA standard and the current intensity value. Substitute the current intensity of the sample to be tested into the standard curve to calculate the content of miRNA in the sample to be detected.

[0064] It can be understood that when different photosensitizers are selected, the applied laser wavelength will be different.

[0065] Table 1 Sequence Information It can be understood that the above sequences are only examples, not limitations.

[0066] The results of detecting miRNA (H1-H2) in the embodiments of the present invention are as Figure 6 shown. As the concentration of the target miRNA (H1-H2) increases (10 pM - 1 nM), the electrochemical current intensity also increases. As Figure 7 shown, when the concentration of miRNA (H1-H2) is in the range of 10 pM - 1 nM, the current intensity has a good linear relationship with the miRNA concentration (R 2 = 0.998), and the detection limit is 3.4 pM. This indicates that this detection system can accurately detect miRNA in the sample to be tested.

[0067] Further, interference substances, including ascorbic acid, dopamine, uric acid, glucose, norepinephrine, cysteine, etc., are added to the detection system. As Figure 8 shown, when using this detection system to detect 10 pM miRNA (H1-H2), the output of the current intensity basically does not change, around 1.2 μA, indicating that this detection system has good anti-interference performance in a complex physiological matrix.

[0068] Example 5 This example provides an example of detecting an actual protein sample and an investigation of the performance of the detection method.

[0069] (1) Mix and incubate the zirconium-based metal-organic framework donor beads with the capture antibody in a coupling buffer at 37 °C for 2 hours; mix and incubate the polydopamine receptor beads with the detection antibody in a Tris-HCl buffer at 37 °C for 4 hours; mix the donor and receptor beads as the detection system. In this example, the particle size of the donor beads is 100 nm, and the particle size of the receptor beads is 10 nm. The mixing ratio of the donor beads to the receptor beads is 1:10.

[0070] (2) Add a sample containing serum, which contains the target antigen to be detected, to the detection system, and mix and incubate at 37 °C for 30 - 120 min to form a test dispersion.

[0071] (3) Drop the dispersion onto a screen-printed carbon electrode, and use an electrochemical workstation to detect the electro-signal intensity. In a homogeneous environment, under the excitation of a 630 nm laser (the photosensitizer selected in this example is tetracarboxyphenyl porphyrin), singlet oxygen transfer occurs between the donor and receptor beads. The electrode applies a voltage of -0.2 V for reduction to generate an electrochemical signal. A standard curve is established based on the concentration of the target antigen standard and the current intensity value. Substitute the current intensity of the test sample into the standard curve to calculate the content of the antigen in the test sample.

[0072] (4) The results of detecting the target antigen in the examples of the present invention are as Figure 9 shown. As the concentration of the target CEA increases (10 pg mL -1 - 50 ng mL -1 ), the electrochemical current intensity is higher. As Figure 10 shown, when the CEA concentration is in the range of 10 pg mL -1 - 50 ngmL -1 interval, the current intensity has a good linear relationship with the CEA concentration (R 2 = 0.993), and the detection limit is 2.6 pg mL -1 . It shows that this detection system can accurately detect the target protein in the test sample.

[0073] The results of the spiked recovery of the target antigen in the simulated serum samples detected in this example are as Figure 11 shown. The spiked recovery rate of the CEA antigen is 99.3% - 108.1%, and the RSD is 0.1% - 1.05%, indicating that this detection system has good pre-clinical application.

[0074] Example 6 This example provides an example of a detection device for a photoexcitation electro-driven reactive oxygen species channel.

[0075] A detection device for a photoexcitation electro-driven reactive oxygen species channel, the detection device includes a reaction module, a photoexcitation module, an electro-driven module, and a reactive oxygen species channel reaction reagent; The reaction module includes a reaction cell, which is arranged to place the biological sample to be detected and the reactive oxygen species channel reaction reagent; The photoexcitation module includes at least one laser emitter; The electric drive module includes at least one set of electrodes and an electrical signal reader; The reactive oxygen species channel reaction reagent includes a donor bead complex containing a photosensitizer and an acceptor bead complex containing polyphenols. The donor bead complex includes a metal-organic framework containing a photosensitizer coupled with a first reactant; the acceptor bead complex includes a melanoid nanoparticle containing polyphenols coupled with a second reactant; the first reactant and the second reactant are arranged to connect or identify a target substance in the biological sample to be detected to form a donor bead-acceptor bead linker; During detection, the laser emitted by the at least one laser emitter irradiates the donor bead-acceptor bead linker, causing the donor bead complex to generate singlet oxygen. After the polyphenols in the acceptor bead complex capture the singlet oxygen, they are oxidized to produce benzoquinone; The oxidized acceptor bead containing benzoquinone collides on the surface of the electrode, and a reduction voltage is applied to the electrode to trigger the electrochemical reduction of benzoquinone to polyphenols on the electrode, completing a cascade redox cycle and generating an amplified current signal; The electrical signal reader is arranged to read the electrochemical signal.

[0076] The schematic structural diagram and working principle diagram of the detection device in this embodiment are shown in Figure 12 、 Figure 13 。In Figure 12 In one of the embodiments shown, the laser emitter can be arranged on one side of the reaction cell; in Figure 12 In another embodiment shown, the laser emitter can be arranged on the other side of the reaction cell; or, the laser emitter can also be arranged on the top of the reaction cell. It can be understood that, in order to increase the efficiency of photoexcitation, multiple laser emitters can also be arranged. It can be understood that the reaction cell can be the structure of the reaction container shown in Figure 12 , or any other carrier or system that can ensure the completion of liquid reactions.

[0077] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. All of these fall within the protection scope of the present invention.

Claims

1. A method for detecting biomarkers by using photoinduced electrokinetic driving of reactive oxygen species channels, characterized in that: The biomarker is a protein, and the specific steps are as follows: S01: preparing a donor bead complex containing a photosensitizer, wherein the donor bead complex is a conjugate of a metal organic framework containing a photosensitizer and a capture antibody; S02: preparing an acceptor bead complex containing polyphenols, wherein the acceptor bead complex is a conjugate of melanin-like nanoparticles and a detection antibody; S03: placing the donor bead complex and the acceptor bead complex into a biological sample solution to be tested, and when the biological sample to be tested contains a target protein, the target protein recognizes and binds to the capture antibody and the detection antibody to form a sandwich immune structure, thereby obtaining a donor bead-acceptor bead connector of the target protein, wherein the distance between the donor bead-acceptor bead connector is less than 200 nm; the biological sample to be tested is selected from at least one of interstitial fluid, blood, urine, body fluid and a combination thereof; S04: adding the donor bead-acceptor bead connector to the electrode interface and irradiating it with laser, the donor bead complex in the donor bead-acceptor bead connector generates singlet oxygen after laser irradiation, the singlet oxygen diffuses in the biological sample to be tested, and the polyphenol in the acceptor bead complex captures the singlet oxygen and is oxidized to generate benzoquinone; applying a reduction voltage at the electrode interface to trigger the electrochemical reduction of benzoquinone to polyphenol, a cascade redox cycle occurs and an amplified current signal is generated, and the current signal is read; S05: establishing a standard curve in advance according to the concentration and current intensity value of the target protein standard; S06: Substituting the read current signal into the standard curve to calculate the content of the target protein in the biological sample to be tested.

2. The method according to claim 1, characterized in that The metal organic framework is coupled to the capture antibody via an amide bond.

3. The method according to claim 1, characterized in that The melanin-like nanoparticles are coupled to the detection antibody via a Michael addition reaction, or via a Schiff base reaction.

4. The method according to claim 1, characterized in that The photosensitizer includes one or more selected from fluoroborane dipyrrole, metal phthalocyanine and porphyrin.

5. The method according to claim 1, characterized in that The metal organic framework includes one or more selected from a zirconium-based metal organic framework, a gadolinium-based metal organic framework and an iron-based metal organic framework.

6. The method according to claim 1, wherein: The particle size of the donor beads ranges from 60-200 nm.

7. The method according to claim 1, characterized in that The particle size of the acceptor beads ranges from 10 to 20 nm.

8. The method according to claim 1, characterized in that The melanin-like nanoparticles are nanoscale products formed by oxidative polymerization of monomers containing a benzene ring structure and a polyphenol group in an aqueous solution.

9. The method according to claim 8, characterized in that The melanin-like nanoparticles include isomelanin, poly-L-dopa, polygallic acid or polycaffeic acid.

10. The method according to claim 1, characterized in that The configuration ratio of the donor bead complex to the acceptor bead complex is 1:10.

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