In vitro immunoassay method based on photoexcitation-driven electrodriven active oxygen channel

Abstract

The present application belongs to the technical field of bioengineering, and particularly relates to an in-vitro immune detection method based on photoexcitation electric driving active oxygen channel. The method provided by the present application is used for detecting target proteins in biological samples, and comprises the following steps: preparing a donor bead complex containing a photosensitizer and a receptor bead complex containing polyphenol; and placing the donor bead complex and the receptor bead complex into a solution of a biological sample to be detected. When the biological sample to be detected contains target proteins, the target proteins recognize and combine the capture antibody and the detection antibody to form a sandwich immune structure, so that a donor bead-receptor bead connector of the target proteins is obtained for detection. The detection result of the method is accurate and has high sensitivity.

Description

[0001] Divisional application This application is a divisional application of Chinese Patent Application No. 2024109894766, filed on July 22, 2024, entitled "A method and application for detecting biomarkers based on photoexcited electrically driven reactive oxygen species channels". Technical Field

[0002] This invention belongs to the field of bioengineering technology, specifically relating to an in vitro immunoassay method based on photoexcited electrically driven reactive oxygen species channels. Background Technology

[0003] In recent years, the incidence of cancer in my country has continued to rise, seriously threatening the health of residents. Faced with the severe situation of cancer prevention and control, early diagnosis and early treatment have become key links in reducing cancer mortality. At present, protein tumor marker detection is widely used in early tumor screening. Common protein tumor markers include prostate-specific antigen (PSA) associated with prostate cancer, carcinoembryonic antigen (CEA) associated with gastrointestinal tumors, and alpha-fetoprotein (AFP) associated with hepatocellular carcinoma.

[0004] Photo-induced chemiluminescence (PET) technology—a novel fourth-generation chemiluminescence technology—has attracted increasing attention from in vitro diagnostic (IVD) companies due to its homogeneous reaction platform, eliminating the need for a separation system during detection and offering advantages such as wash-free processing and low background. Also known as luminescent oxygen channeling immunoassay (LOCI), PET was first proposed in 1994. While there are reports of its use for biomarker detection, some issues remain. PET has been reported as a common homogeneous wash-free detection technique for protein analysis (ACS Omega 2022, 7, 2344-2355; Anal. Chem. 2019, 91, 5777-5785). However, its complex structure of photosensitive and luminescent microspheres, the luminescent material's susceptibility to photobleaching, and the dissipative consumption of chemiluminescent material within the luminescent microspheres lead to unstable chemiluminescence signal output. For example, patent CN117368483A (20240109), entitled "A Method and Kit for Detecting Kidney Injury Molecular-1 Based on Homogeneous Photochemiluminescence Immunoassay," utilizes a double-antibody sandwich immunoassay and photochemiluminescence properties to detect antibodies. This patented method is based on the light input-light output principle, which may lead to interference between light signals. Furthermore, this method requires large instruments with complex optical paths, resulting in high costs.

[0005] Electrochemical sensing is a next-generation IVD technology with advantages such as miniaturized equipment, low raw material costs, rapid response, and high sensitivity. Internationally, companies like Roche and Atlas Genetics, focusing on "sample in, result out" sensing, have integrated microfluidic channel technology and droplet manipulation techniques to avoid target loss caused by repeated separation and purification, and to shield against interference from electroactive molecules. They have successively developed electrochemical sensing technologies based on microelectrode systems and electron transfer materials. However, the design of sensor pretreatment chips is complex, and sensing modes based on interface-accumulated electrical signals suffer from baseline drift interference and frequent electrode calibration issues. Related products have not yet entered the Chinese market. Designing and developing interference-resistant electrochemical sensors based on cascaded electron transfer is a crucial scientific problem urgently needing to be solved in the development of readily available IVD reagent materials.

[0006] In summary, it is necessary to propose an in vitro immunoassay method based on photoexcited electrically driven reactive oxygen species channels to supplement the shortcomings of current technology. Summary of the Invention

[0007] The purpose of this invention is to provide an in vitro immunoassay method based on photoexcited electrically driven reactive oxygen species channels, which partially solves or alleviates the above-mentioned deficiencies in the prior art. The specific technical solution adopted by this invention is as follows.

[0008] A method for detecting biomarkers, wherein the biomarkers are proteins, using photoexcitation of electrically driven reactive oxygen species channels, comprises the following steps: S01: Prepare 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: Prepare a receptor bead complex containing polyphenols, wherein the receptor bead complex is a conjugate of melanin-like nanoparticles and detection antibodies; S03: The donor bead complex and the acceptor bead complex are placed in the biological sample solution to be tested. When the biological sample to be tested contains the target protein, the target protein recognizes and binds to the capture antibody and the detection antibody to form a sandwich immune structure, thereby obtaining the donor bead-acceptor bead conjugate of the target protein. The distance between the donor bead-acceptor bead conjugates 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 combinations thereof. S04: The donor bead-acceptor bead connector is added to the electrode interface and irradiated with a 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. The polyphenol in the acceptor bead complex captures the singlet oxygen and is oxidized to produce benzoquinone. A reduction voltage is applied to the electrode interface to trigger the electrochemical reduction of benzoquinone to polyphenol, resulting in a cascade redox cycle and generating an amplified current signal. The current signal is read. S05: Establish a standard curve in advance based on the concentration and current intensity value of the target protein standard; S06: Input the read current signal into the standard curve to calculate the content of the target protein in the biological sample to be tested.

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

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

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

[0012] As a preferred embodiment, the photosensitizer is tetracarboxyphenylporphyrin; 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 beads are obtained by reacting tetracarboxyphenylporphyrin and zirconium oxychloride octahydrate in benzoic acid and N,N-dimethylformamide, wherein the mass ratio of tetracarboxyphenylporphyrin to zirconium oxychloride octahydrate is 1 / 6 to 1 / 2.

[0015] In some embodiments, the receptor beads are obtained by first dispersing dopamine in ethanol, adding tris(hydroxymethyl)aminomethane salt and stirring the mixture thoroughly, mixing the reaction solution with anhydrous ethanol and centrifuging, taking the centrifuged precipitate and sonicating it in sodium hydroxide solution, adjusting the pH to neutral, and freeze-drying it to finally obtain melanin-like nanoparticle receptor beads.

[0016] Furthermore, the donor beads have a particle size range of 60-200 nm.

[0017] Furthermore, the particle size of the acceptor beads ranges from 10 to 20 nm.

[0018] Furthermore, the melanin-like 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 preferred embodiment, the melanin-like nanoparticles include isomelanin, poly-L-DOPA, polygallic acid, or polycaffeic acid.

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

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

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

[0023] Beneficial technical effects: This invention provides a method for detecting protein biomarkers using photoexcited electro-driven reactive oxygen species (ROS) channels. The donor and acceptor bead complexes in this method are free nanoparticles in the detection system. Therefore, this method employs a non-immobilization strategy to avoid electrode modification, achieving homogeneous electrochemistry without washing or separation through uniform mass transfer collisions, enabling repeatable detection. Furthermore, it effectively overcomes the drawback of photoelectrochemistry requiring repeated washing and separation steps to remove unbound substances that cause background signals, resulting in high detection sensitivity. Current technologies often require immobilizing photoelectric nanomaterials on electrodes. The inhomogeneity of the modified electrodes and the uncontrollable packing density of the modified probes severely limit the repeatability of electrochemical detection. In addition, the electron-hole recombination time in the photoelectric materials used in these methods is much shorter than the time for electron transfer to the electrode or for electron transfer from the electrode to the material, leading to carrier recombination, reducing the photocurrent signal, and thus reducing detection sensitivity.

[0024] Furthermore, the strategy employed in this invention is to convert light energy into electrical energy, i.e., light input-electrical output. Specifically, a negative potential reduction is applied to avoid interference from electroactive substances in the physiological matrix; during the collision-constrained transient reactive oxygen species channel-electron transfer cascade process, the oxidation event on the surface of the acceptor bead amplifies the current signal through the quinone reduction potential, thereby obtaining an electrical signal, thus resulting in high sensitivity.

[0025] Specifically, the method of this invention employs a sandwich immunoassay with double antibodies binding to the antigen to be tested when detecting target proteins in biological samples, thereby amplifying the target signal. Experimental results of this invention show that this method can detect target proteins in 10 pg mL... -1 - 50ng mL -1 The target protein was detected within a certain concentration range. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0027] Figure 1 A schematic diagram illustrating the method of detecting nucleic acids using photoexcited electrically driven reactive oxygen species channels as described in this invention; Figure 2 A schematic diagram illustrating the method of detecting proteins using photoexcited electrically driven reactive oxygen species channels as described in this invention; Figure 3 Transmission electron microscope (TEM) image of the donor bead prepared by the method of the present invention (scale bar 50 nm). Figure 4 Transmission electron microscope (TEM) image of the acceptor bead prepared by the method of the present invention (scale bar 200 nm). Figure 5 This is the electron paramagnetic resonance (ESR) spectrum of singlet oxygen generated by pearlescent excitation of the donor in the method of the present invention; Figure 6 This is a detection time-current curve of miRNA detection using photoexcited electro-driven reactive oxygen species method in one embodiment of the present invention; Figure 7 This is a schematic diagram of the detection linear range of miRNA using the photoexcited electro-driven reactive oxygen species method in one embodiment of the present invention; Figure 8 This is an evaluation diagram of the anti-interference performance of the photoexcited electrically driven active oxygen channel method in one embodiment of the present invention; Figure 9 This is a detection time-current curve of CEA detected using the photoexcited electrically driven reactive oxygen species channel method in one embodiment of the present invention; Figure 10 This is a schematic diagram of the detection linear range of CEA using the photoexcited electro-driven reactive oxygen species channel method in one embodiment of the present invention; Figure 11 This is a diagram showing the simulated actual sample spike recovery results in one embodiment of the present invention; Figure 12 This is a schematic diagram of the detection system for photoexcited electrically driven reactive oxygen species channels provided by the present invention; Figure 13 This is a schematic diagram illustrating the working principle of the photoexcited electrically driven reactive oxygen species detection system provided by the present invention. Detailed Implementation

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

[0029] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0030] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

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

[0032] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges 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 the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0033] Definition of the noun: The photoexcited electrodriven reactive oxygen species channel (LEOCA) described in this invention is a homogeneous detection method for photoexcited synchronous electron transfer. Specifically, after laser irradiation of the donor-acceptor composite reaction system, the donor generates singlet oxygen, and the acceptor captures the singlet oxygen to produce oxidation products. Subsequently, a cascade chemical oxidation-electrochemical reduction process occurs at the electrode interface to generate an electrochemical signal, which can also be simply referred to as the LEOCA signal.

[0034] The cascaded redox cycle described in this invention refers to the process where, in the presence of the target substance, singlet oxygen generated by photoexcitation of the donor bead complex diffuses to the surface of the acceptor bead complex and oxidizes the polyphenolic groups in the acceptor beads to generate benzoquinone. The benzoquinone, which collides and contacts at the electrode surface, is then reduced to polyphenol at a negative potential, generating an electrochemical signal. The benzoquinone product of the former chemical oxidation event is the substrate of the latter electrochemical reduction event, triggering subsequent electrochemical reduction reactions; therefore, it is called a cascaded redox cycle reaction.

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

[0036] Example 1 This embodiment provides an example of a method for detecting biomarkers using photo-excited electrically driven reactive oxygen species channels. The biomarker detected in this embodiment is nucleic acid.

[0037] (1) A metal-organic framework containing a porphyrin photosensitizer is coupled with a first hairpin nucleic acid as a nano donor bead, and a polydopamine particle containing polyphenols is coupled with a second hairpin nucleic acid as a nano acceptor bead. The target nucleic acid recognizes and responds to the first hairpin nucleic acid chain, triggering the exposed single-strand region, thereby inducing the hybridization of the second hairpin nucleic acid chain and finally forming a catalytic hairpin self-assembly complex.

[0038] (2) After being irradiated with excitation light, the singlet oxygen generated by the donor beads in the complex ( 1 O2 diffuses into the receptor beads, where polyphenols are captured. 1 O2 can oxidize benzoquinone to form benzoquinone, which undergoes a cascade of chemical oxidation-electrochemical reduction processes upon instantaneous collision at the electrode interface, generating an electrochemical signal. It is important to note that, due to... 1 O2 has high activity and a short half-life; the release of oxygen from the donor bead is only possible when the donor and acceptor beads are sufficiently close (forming a catalytic hairpin self-assembly complex). 1 Only when O2 reaches the acceptor bead can an electrochemical signal be generated.

[0039] (3) When there is no target in the system, i.e., 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 free donor bead produces 1 O2 dissipates rapidly in the system, producing almost no electrochemical signal.

[0040] In summary, the two types of donor and acceptor beads self-assembled via inter-nucleic acid catalytic hairpins, achieving high-energy... 1 The transfer of O2 induces photoexcitation of electrically driven reactive oxygen species channels. The detection principle diagram is shown below. Figure 1 As shown.

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

[0042] Example 2 This embodiment provides another example of a method for detecting biomarkers using photoexcited electrically driven reactive oxygen species channels. The biomarker detected in this embodiment is a protein.

[0043] (1) Using a capture antibody coupled with a metal-organic framework containing a porphyrin photosensitizer as a nano donor bead, and a detection antibody coupled with a polydopamine particle containing polyphenols as a nano acceptor bead, the capture antibody and the detection antibody form a sandwich immune structure in response to the target antigen recognition, and finally form a donor bead-acceptor bead complex.

[0044] (2) After being irradiated with excitation light, the singlet oxygen generated by the donor beads in the complex ( 1 O2 diffuses into the receptor beads, where polyphenols are captured. 1 O2 is subsequently oxidized to produce benzoquinone, which undergoes a cascade of chemical oxidation and electrochemical reduction processes upon instantaneous collision at the electrode interface, generating an electrochemical signal. It is important to note that, due to... 1 O2 has high activity and a short half-life; it only releases its active ingredient when the donor bead and the acceptor bead are sufficiently close (capture antibody-target antigen-detection antibody binding). 1 Only when O2 reaches the acceptor bead can an electrochemical signal be generated.

[0045] (3) When the target antigen is not present, the capture antibody and the detection antibody in the detection system cannot bind, the distance between the donor bead and the acceptor bead is greater than 200 nm, and the free donor bead produces 1 O2 dissipates rapidly in the system, producing almost no electrochemical signal.

[0046] In summary, high-energy interactions are achieved between the two types of donor and recipient beads through the binding of antigens and antibodies. 1 The transfer of O2 induces photoexcitation of electrically driven reactive oxygen species channels. The detection principle diagram is shown below. Figure 2 As shown.

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

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

[0049] (1) Preparation of donor beads Add 50-150 mg of tetracarboxyphenylporphyrin, 300 mg of zirconium oxychloride octahydrate, 2.2-3.3 g of benzoic acid, and 80-140 mL of N,N-dimethylformamide to a flask. Dissolve by sonication for 5 min and stir at 90 °C for 5 hours. After the reaction is complete, centrifuge at 15,000 rpm for 10-20 min, and then wash three times with N,N-dimethylformamide. Finally, collect the precipitate, which is the zirconium-based metal-organic framework nanobead, and disperse it in anhydrous ethanol for storage.

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

[0051] (2) Preparation of receptor beads Add 55 mg of dopamine hydrochloride to a flask, sonicate to disperse it in a mixture of 100 mL deionized water and 20 mL anhydrous ethanol, add 120 mg of tris(hydroxymethyl)aminomethane salt to the reaction mixture, and stir at room temperature for 72 hours; take the original reaction solution and mix it with anhydrous 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, sonicate at 600 W output power for 5-10 min, and then adjust the pH value to 7.0 with hydrochloric acid; freeze-dry the obtained polydopamine acceptor beads and store them dispersed in water.

[0052] It is understood that the preparation of polydopamine receptor beads in this embodiment is only an example, and isomelanin, poly-L-dopa, polygalic acid, or polycaffeic acid can also be used.

[0053] Morphological characterization (TEM) was performed on the donor and recipient beads, such as... Figure 3 , Figure 4 As shown, qualified donor beads are spherical nanostructures with a size range of 100-150 nm, and qualified acceptor beads are spherical nanostructures with a size range of 10-20 nm. Electron spin resonance (ESR) was used to further analyze the donor beads; the resulting spectra are shown below. Figure 5 As shown, the ESR signal is generated after the ESR spin trapping agent TEMP interacts with the donor bead under illumination, indicating that the donor bead that generates singlet oxygen by photoexcitation was successfully synthesized by this method.

[0054] (3) Hairpin nucleic acid preparation The hairpin nucleic acid was chemically synthesized and purified by HPLC. It was then dissolved in annealing buffer to prepare a solution and denatured in a PCR instrument at 95°C for 5 min. The temperature was then gradually reduced 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 its components and contents are as follows: tris(hydroxymethyl)aminomethane hydrochloride, 20 mmol / L; sodium chloride, 140 mmol / L; potassium chloride, 5 mmol / L; pH is 7.4.

[0056] It is understood that the capture antibody and detection antibody can also be prepared in accordance with conventional techniques in the art in this embodiment.

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

[0058] Coupling buffer: The solvent is deionized water, and its components and contents are as follows: tris(hydroxymethyl)aminomethane 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 is understood that the capture antibody donor bead complex and the detection antibody receptor bead complex can also be prepared by similar methods and other conventional techniques in the art.

[0060] Example 4 This embodiment provides a practical example of nucleic acid sample detection and a performance evaluation of the detection method.

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

[0062] (2) Add a sample containing the lysate of the tumor tissue to be tested or the interstitial fluid of the tumor to be tested to the detection system. The sample contains the miRNA to be tested. Mix and incubate at 37°C for 30-120 min to form the dispersion to be tested.

[0063] (3) The dispersion is dropped onto the screen-printed carbon electrode. The electrical signal intensity is detected by an electrochemical workstation. In a homogeneous environment, under 630 nm laser excitation (the photosensitizer used in this embodiment is tetracarboxyphenylporphyrin), singlet oxygen transfer occurs between the donor and acceptor beads. The electrode is reduced by a voltage of -0.2 V to generate an electrochemical signal. A standard curve is established based on the concentration and current intensity of the miRNA standard. The content of miRNA in the sample to be tested can be calculated by substituting the current intensity of the sample to be tested into the standard curve.

[0064] It is understandable that the applied laser wavelength will be different when different photosensitizers are used.

[0065] Table 1 Sequence Information It is understood that the above sequences are merely examples and not limitations.

[0066] The results of miRNA (H1-H2) detection in this embodiment of the invention are as follows: Figure 6 As shown, the electrochemical current intensity increases with increasing target miRNA (H1-H2) concentration (10 pM-1 nM). Figure 7 As shown, when the miRNA (H1-H2) concentration is within the range of 10 pM-1 nM, the current intensity shows a good linear relationship with the miRNA concentration (R0). 2 = 0.998), and the detection limit is 3.4 pM. This indicates that the detection system can accurately detect miRNA in the sample.

[0067] Furthermore, interfering substances are added to the detection system, including ascorbic acid, dopamine, uric acid, glucose, norepinephrine, and cysteine, etc. Figure 8 As shown, when the detection system was used to detect 10 pM miRNA (H1-H2), the current intensity output remained basically unchanged at around 1.2 μA, indicating that the detection system has good anti-interference performance in complex physiological matrices.

[0068] Example 5 This embodiment provides an example of actual protein sample detection and a performance evaluation of the detection method.

[0069] (1) Zirconium-based metal-organic framework donor beads and capture antibody were mixed and incubated in conjugation buffer at 37°C for 2 hours; polydopamine acceptor beads and detection antibody were mixed and incubated in Tris-HCl buffer at 37°C for 4 hours; the donor and acceptor beads were then mixed to form the detection system. In this embodiment, the donor beads had a particle size of 100 nm and the acceptor beads had a particle size of 10 nm. The mixing ratio of donor beads to acceptor beads was 1:10.

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

[0071] (3) The dispersion is dropped onto the screen-printed carbon electrode. The electrical signal intensity is detected by an electrochemical workstation. In a homogeneous environment, under 630 nm laser excitation (the photosensitizer used in this embodiment is tetracarboxyphenylporphyrin), singlet oxygen transfer occurs between the donor and acceptor beads. The electrode is reduced by a voltage of -0.2 V to generate an electrochemical signal. A standard curve is established based on the concentration and current intensity of the target antigen standard. The content of antigen in the sample to be tested can be calculated by substituting the current intensity of the sample to be tested into the standard curve.

[0072] (4) The results of the detection of target antigens in the embodiments of the present invention are as follows: Figure 9 As shown, with the increase of target CEA concentration (10 pg / mL), -1 - 50 ng mL -1 The higher the electrochemical current intensity, the better. Figure 10 As shown, when the CEA concentration is 10 pg / mL -1 - 50 ngmL -1 Within the specified range, the current intensity and CEA concentration exhibit a good linear relationship (R0). 2 = 0.993), detection limit is 2.6 pg mL -1 This indicates that the detection system can accurately detect the target protein in the sample.

[0073] The results of detecting target antigen spiked recovery in simulated serum samples in this embodiment are as follows: Figure 11 As shown, the recovery rate of CEA antigen spiked was 99.3% ~ 108.1%, and the RSD was 0.1% ~ 1.05%, indicating that the detection system has good potential for clinical application.

[0074] Example 6 This embodiment provides an example of a detection device for photoexcited electrically driven reactive oxygen species channels.

[0075] A detection device for photoexcited electrically driven reactive oxygen species (ROS) channels, the detection device comprising a reaction module, a photoexcitation module, an electrically driven module, and ROS channel reaction reagents; The reaction module includes a reaction chamber, which is configured to hold the biological sample to be tested and the reactive oxygen channel reaction reagent. The optical excitation 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 comprises a donor bead complex containing a photosensitizer and an acceptor bead complex containing polyphenols. The donor bead complex comprises a metal-organic framework containing a photosensitizer coupled to a first reactant. The acceptor bead complex comprises melanin-like nanoparticles containing polyphenols coupled to a second reactant. The first reactant and the second reactant are configured to connect to or recognize target substances in the biological sample to be tested to form a donor bead-acceptor bead conjugate. During detection, the laser emitted by the at least one laser emitter irradiates the donor bead-acceptor bead connector, causing the donor bead complex to generate singlet oxygen. The polyphenols in the acceptor bead complex capture the singlet oxygen and are then oxidized to produce benzoquinone. The benzoquinone-containing acceptor beads produced by oxidation collide with the electrode surface. A reduction voltage is applied to the electrode to trigger the electrochemical reduction of benzoquinone to polyphenols, completing the cascade redox cycle and generating an amplified current signal. The electrical signal reader is configured to read the electrochemical signal.

[0076] The structural schematic diagram and working principle schematic diagram of the detection device in this embodiment are shown below. Figure 12 , Figure 13 .exist Figure 12 In one embodiment shown, the laser emitter can be positioned on one side of the reaction tank; in Figure 12 In another embodiment shown, the laser emitter may be located on the opposite side of the reaction cell; alternatively, the laser emitter may be located on top of the reaction cell. It is understood that multiple laser emitters may be provided to increase the efficiency of photoexcitation. It is understood that the reaction cell may be... Figure 12 The structure of the reaction vessel shown can also be any other carrier or system that can ensure the completion of the liquid reaction.

[0077] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for detecting biomarkers using photo-excited electrically driven reactive oxygen species channels, characterized in that, The biomarker is a protein, and the method is a non-disease diagnostic method. The specific steps are as follows: S01: Prepare 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; the photosensitizer includes one or more selected from fluoroboron dipyrrole, metal phthalocyanine and porphyrin; S02: Prepare a receptor bead complex containing polyphenols, wherein the receptor bead complex is a conjugate of melanin-like nanoparticles and detection antibodies; wherein the melanin-like nanoparticles are nanoscale products formed by oxidative polymerization of monomers containing benzene ring structures and polyphenol groups in aqueous solution, including isomelanin, poly-L-DOPA, polygallic acid or polycaffeic acid. S03: The donor bead complex and the acceptor bead complex are placed in the 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, thereby obtaining a donor bead-acceptor bead conjugate of the target protein. The distance between the donor bead-acceptor bead conjugates is less than 200 nm. The biological sample to be tested is selected from at least one of interstitial fluid, blood, urine, and combinations thereof. S04: The donor bead-acceptor bead connector is added to the electrode interface and irradiated with a 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. The polyphenol in the acceptor bead complex captures the singlet oxygen and is oxidized to produce benzoquinone. A reduction voltage is applied to the electrode interface to trigger the electrochemical reduction of benzoquinone to polyphenol, resulting in a cascade redox cycle and generating an amplified current signal. The current signal is read. S05: Establish a standard curve in advance based on the concentration and current intensity value of the target protein standard; S06: Input 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 as described in claim 1, characterized in that, The metal-organic framework is coupled to the capture antibody via an amide bond.

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

4. The method as described in claim 1, characterized in that, 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.

5. The method as described in claim 1, characterized in that, The donor beads have a particle size range of 60-200 nm.

6. The method as described in claim 1, characterized in that, The size range of the acceptor beads is 10-20 nm.

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

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