A biomarker detection method based on photoinduced electrodynamics to drive reactive oxygen channels and its application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a biomarker detection method and application based on photoexcitation electrically driven reactive oxygen channels. The present invention first provides a detection system for photoexcitation electrically driven reactive oxygen channels, which mainly includes a reaction module, a photoexcitation module, an electric drive module and an reactive oxygen channel reaction reagent. The reactive oxygen channel reaction reagent includes a donor bead complex containing a photosensitizer and an acceptor bead complex containing polyphenols, and the donor bead complex includes a metal organic framework containing a photosensitizer coupled with a first reactant; the acceptor bead complex includes melanin-like nanoparticles containing polyphenols coupled with a second reactant; the first reactant and the second reactant are configured to connect or identify a target substance in a biological sample to be detected to form a donor bead-acceptor bead connector. Using the detection device provided by the present invention to perform biological sample detection, a "wash-free and separation-free" homogeneous biomarker analysis can be achieved, with high sensitivity and strong anti-interference ability.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a biomarker detection method and application based on photoinduced electroactive oxygen channels. Background Art

[0002] At present, in the biomedical field, analyzing the information of molecular markers such as nucleic acids and proteins in tumor tissues has become an effective method for perioperative diagnosis of tumors. For example, the detection of microRNA (miRNA). In addition, blood protein biomarkers such as carcinoembryonic antigen (CEA) are also considered to be biomarkers for breast cancer and other cancers. Although researchers at home and abroad have developed a variety of sensing methods based on optical / electrical / chemical modes to detect biomarkers, they have not yet been widely recognized in preclinical and clinical applications. The main reasons for this major challenge include: (1) After potential cancer-related miRNA or protein is released into the extracellular blood circulation, due to the dilution effect, the degree of up-regulation and down-regulation becomes insignificant. In addition, the degradation effect of enzymes in plasma will further reduce the recognition of small changes. (2) Biomarker miRNA or protein of non-specific origin can also be expressed in inflammatory diseases and various organs, resulting in a high false positive rate in the test results. Statistics show that the concentration of disease-related biomarkers in the interstitial fluid of the local tumor microenvironment is 1000-1500 times higher than that in the blood (Adv. Mater. 2024, 36, 2307; Mol. On-col. 2021, 15, 429.). Therefore, analyzing biomarkers in interstitial fluid can effectively overcome the disadvantages of blood sample testing.

[0003] In addition, miRNAs related to tumor microenvironment regulation are used as indicators for monitoring tumor progression and metastasis, and miRNAs related to microenvironment immunity and drug resistance can be used as molecular indicators for tumor companion diagnosis and prognosis. Therefore, analyzing biomarkers in tumor tissue can effectively alleviate the various disadvantages of blood sample testing. However, most electrochemical biosensors based on tumor tissue biomarker detection cannot operate in a sample-in-answer-out (SIAO) mode, that is, they need to go through multiple signal conversion or processing steps in the middle, including washing, target labeling, adding reagents to process samples, and amplifying and transducing signals, etc., in order to obtain the final result. If untreated clinical samples are encountered, the currently commonly used electrochemical biosensors cannot even directly detect and analyze the biomarkers to be tested in untreated clinical samples.

[0004] At present, there have been reports at home and abroad on methods of constructing nanosensors based on heterogeneous interface photoelectric reactions for detecting miRNA (Anal. Chem. 2023, 95, 12097-12103). However, the electron-hole recombination time in the photoelectric materials used in these methods is much lower than the time for electron transfer to the electrode or the time for the electrode electron to transfer to the material, resulting in carrier recombination, reducing the photocurrent signal, and thus reducing the detection sensitivity. In addition, heterogeneous reactions require complex solid modification steps for the electrodes, and repeated washing is required to remove unbound substances, accumulating detection errors.

[0005] Photochemiluminescence technology - a new fourth-generation chemiluminescence technology, because it is a homogeneous reaction platform, no separation system is required during the detection process, it has the advantages of no-wash, low background, high sensitivity, good repeatability, simple operation and short time consumption, and has attracted the attention of more and more IVD companies. Photochemiluminescence technology, also known as luminescent oxygen channeling immunoassay (LOCI) technology, was first proposed in 1994. There are also reports on the use of this technology to detect biomarkers, but there are also some problems. There are also reports that photoinduced chemiluminescence oxygen channel immunoassay is used for protein analysis as a common homogeneous wash-free detection technology (ACSOmega2022,7,2344-2355; Anal.Chem.2019,91,5777-5785.), but because of the complex construction of its photosensitive microspheres and luminescent microspheres, the luminescent substance is easily photobleached, and the dissipative consumption of the chemiluminescent substance in the luminescent microspheres leads to unstable chemiluminescent signal output. For another example, the publication number is CN117368483A (20240109), and the invention name is "A method and kit for detecting kidney injury molecule-1 based on homogeneous photochemiluminescence immunoassay technology". This patent uses double antibody sandwich immunoassay and photochemiluminescence characteristics to detect antibodies. This patented 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 large instruments to implement, so it is costly.

[0006] In summary, it is necessary to propose a new method and strategy to make up for or alleviate the shortcomings of existing technologies. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a method and related applications for detecting biomarkers using light-initiated electrochemical-driven reactive oxygen channel. Light-initiated electrochemical-driven reactive oxygen channel assay (LEOCA) is a homogeneous detection method of light-excited synchronous electron transfer. Its technical principle is that after laser irradiation of the donor-acceptor composite reaction system, the donor produces singlet oxygen, and the acceptor captures the singlet oxygen to produce an oxidation product, which then undergoes a cascade redox cycle at the electrode interface and generates an amplified current signal; this process is a photochemical oxidation-electrochemical reduction cascade electrochemical signal amplification process. The specific technical scheme of the present invention is as follows.

[0008] A detection device for light-excited electrically driven active oxygen channels, the detection device comprising a reaction module, a light-excitation module, an electrically driven module and an active oxygen channel reaction reagent;

[0009] The reaction module comprises a reaction pool (the reaction pool may be optionally open at the top), and the reaction pool is configured to place (inside) the biological sample to be detected and the active oxygen channel reaction reagent;

[0010] The optical excitation module includes at least one laser emitter;

[0011] The electric drive module includes at least one set of electrodes and an electric signal reader;

[0012] The active oxygen channel reaction reagent includes a donor bead complex containing a photosensitizer and an acceptor bead complex containing polyphenols, wherein the donor bead complex includes a metal organic framework containing a photosensitizer coupled with a first reactant; the acceptor bead complex includes a melanin-like nanoparticle containing polyphenols coupled with a second reactant; the first reactant and the second reactant are configured to connect or identify a target substance in a biological sample to be detected to form a donor bead-acceptor bead connector;

[0013] During detection (when the target is present), the laser emitted by the at least one laser emitter is irradiated onto the donor bead-acceptor bead connector, causing the donor bead complex to generate singlet oxygen that diffuses to the acceptor bead, and the polyphenol in the acceptor bead complex captures the singlet oxygen and is oxidized to generate benzoquinone;

[0014] The oxidized acceptor beads containing benzoquinone collide with the electrode surface, and a reduction voltage is applied to the electrode to trigger the electrochemical reduction of benzoquinone to polyphenol, resulting in a cascade redox cycle and generating an amplified current signal (this process is a photochemical oxidation-electrochemical reduction cascade electrochemical signal amplification process);

[0015] The electric signal reader is configured to read the current signal.

[0016] Further, the donor bead composite includes one or more metal organic frameworks selected from zirconium-based metal organic frameworks, gadolinium-based metal organic frameworks and iron-based metal organic frameworks.

[0017] Further, the receptor bead complex comprises one or more photosensitizers selected from fluoroboron dipyrrole, metal phthalocyanine and porphyrin and their derivatives.

[0018] Furthermore, the particle size range of the donor beads is 60-200 nm; the particle size range of the acceptor beads is 10-20 nm.

[0019] Furthermore, the configuration ratio of the donor bead complex to the acceptor bead complex in the active oxygen channel reactor is 1:10.

[0020] Furthermore, the distance between the donor bead and the acceptor bead connector is less than 200 nm.

[0021] Furthermore, the biological sample to be detected is selected from interstitial fluid.

[0022] Furthermore, the biological sample to be detected can also be selected from at least one of blood, urine, body fluid and a combination thereof.

[0023] Furthermore, the target substance in the biological sample to be detected includes nucleic acid or protein; when the target substance is nucleic acid, the first reactant and the second reactant are the first hairpin nucleic acid and the second hairpin nucleic acid; when the target substance is protein, the first reactant and the second reactant are the capture antibody and the detection antibody.

[0024] Furthermore, the electric drive module is arranged at the bottom of the reaction pool and is configured to provide voltage and read electrical signals; the light excitation module is arranged on the top and / or side of the reaction pool.

[0025] Further, in a preferred embodiment of the present invention, the melanin-like nanoparticles are polydopamine, but this is not limiting.

[0026] Melanin-like nanoparticles are nanometer-sized products of monomers containing benzene ring structures and polyphenol groups after oxidative polymerization in aqueous solution, and can also be selected from isomelanin, poly-L-dopa, polygallic acid or polycaffeic acid.

[0027] A method for detecting biomarkers using the above-mentioned detection device for light-excited electrically driven reactive oxygen channels, wherein the biomarkers include nucleic acids or proteins, and the detection steps are as follows:

[0028] S01: adding a biological sample to be tested into the reaction pool of the reaction module, when the biological sample to be tested contains a target nucleic acid, the target nucleic acid can recognize and respond to the first hairpin nucleic acid and trigger the exposed single-stranded region in the first hairpin nucleic acid to induce the hybridization of the second hairpin nucleic acid chain, and obtain a donor bead-acceptor bead connector of the target nucleic acid through hybridization self-assembly of the hairpin nucleic acid; or, when the sample to be tested contains a target antigen, the target antigen can recognize and bind to the capture antibody and the detection antibody to form a sandwich immune structure, and obtain a donor bead-acceptor bead connector of the target antigen, and the distance between the donor bead-acceptor bead connector is less than 200nm;

[0029] S02: Turning on the laser emitter of the light excitation module, irradiating the laser emitted by the laser emitter onto the donor bead-acceptor bead connector, wherein the donor bead complex generates singlet oxygen, the singlet oxygen diffuses in the biological sample to be tested, and the polyphenols in the acceptor bead complex capture the singlet oxygen and are oxidized to generate benzoquinone;

[0030] S03: The electrodes and the electric signal reader of the electric drive module are turned on at the same time, the acceptor beads that generate benzoquinone collide on the surface of the electrode, and a reduction voltage is applied to the electrode to trigger the electrochemical reduction of benzoquinone to polyphenol, a cascade redox cycle occurs and an amplified current signal is generated (this process is a photochemical oxidation-electrochemical reduction cascade electrochemical signal amplification process), and the electric signal reader reads the current signal (the current signal can be the current intensity);

[0031] S04: establishing a standard curve in advance according to the concentration and current intensity value of the target nucleic acid or the target protein standard;

[0032] S05: Substituting the current intensity signal read by the electrical signal reader into the standard curve to calculate the content of the target nucleic acid or the target antigen in the biological sample to be tested.

[0033] Furthermore, the sequence of the first hairpin nucleic acid includes that shown in SEQ ID NO.1 or SEQ ID NO.3; the sequence of the second hairpin nucleic acid includes that shown in SEQ ID NO.2 or SEQ ID NO.4; and the target antigen includes carcinoembryonic antigen.

[0034] A method for detecting biomarkers by using light-excited electrically driven reactive oxygen channels, wherein the biomarkers are nucleic acids, comprises the following steps:

[0035] S01: preparing a donor bead complex containing a photosensitizer, wherein the donor bead complex is a metal organic framework containing a photosensitizer coupled to a first hairpin nucleic acid;

[0036] S02: preparing an acceptor bead complex containing polyphenols, wherein the acceptor bead complex is a melanin-like nanoparticle coupled with a second hairpin nucleic acid;

[0037] 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 nucleic acid, the target nucleic acid can recognize and respond to the first hairpin nucleic acid and trigger the exposed single-stranded region in the first hairpin nucleic acid to induce hybridization of the second hairpin nucleic acid chain, and a donor bead-acceptor bead connector is formed through hybridization self-assembly of the hairpin nucleic acids, and the distance between the donor bead-acceptor bead connector is less than 200 nm;

[0038] S04: adding the donor bead-acceptor bead connector to the electrode interface, applying a voltage of -0.8 to -0.1 V, and irradiating the donor bead-acceptor bead connector with a laser, wherein the donor beads in the donor bead-acceptor bead connector generate singlet oxygen after laser irradiation, and the singlet oxygen diffuses in the biological sample to be tested, and the polyphenols in the acceptor beads are oxidized to generate benzoquinone after capturing the singlet oxygen, and applying a reduction voltage at the electrode interface to trigger the electrochemical reduction of benzoquinone to polyphenols, a cascade redox cycle occurs and an amplified current signal is generated, and the current signal is read;

[0039] S05: Establishing a standard curve in advance according to the concentration and current intensity value of the target nucleic acid standard;

[0040] S06: Substituting the read current signal into the standard curve to calculate the content of the target nucleic acid in the biological sample to be tested.

[0041] Further, the donor bead composite includes one or more metal organic frameworks selected from zirconium-based metal organic frameworks, gadolinium-based metal organic frameworks and iron-based metal organic frameworks.

[0042] Furthermore, the metal organic framework is covalently coupled to the first hairpin nucleic acid via an amide bond; and the melanin-like nanoparticles are covalently coupled to the second hairpin nucleic acid via a Michael addition reaction.

[0043] Furthermore, the particle size range of the donor beads is 60-200 nm; the particle size range of the acceptor beads is 10-20 nm.

[0044] Further, the receptor bead complex comprises one or more photosensitizers selected from fluoroboron dipyrrole, metal phthalocyanine and porphyrin and their derivatives.

[0045] Furthermore, the donor bead complex coupled with the first hairpin nucleic acid and the acceptor bead complex coupled with the second hairpin nucleic acid were mixed at a ratio of 1:10.

[0046] Furthermore, the sequence of the first hairpin nucleic acid includes that shown in SEQ ID NO.1 or SEQ ID NO.3; the sequence of the second hairpin nucleic acid includes that shown in SEQ ID NO.2 or SEQ ID NO.4.

[0047] Furthermore, the photosensitizer contained in the donor bead complex is tetracarboxybenzoporphyrin, and the applied laser wavelength is 630-660 nm.

[0048] Further, in some embodiments, the donor beads are obtained by reacting tetracarboxyphenylporphyrin and zirconium oxychloride octahydrate in benzoic acid and N,N-dimethylformamide, and the mass ratio of the tetracarboxyphenylporphyrin to the zirconium oxychloride octahydrate is 1 / 6-1 / 2.

[0049] Furthermore, in some embodiments, the receptor beads are prepared by first dispersing dopamine in ethanol, adding tris(hydroxymethyl)aminomethane salt and stirring the mixture to react, mixing the reaction solution with anhydrous ethanol and then centrifuging the mixture, ultrasonicating the centrifugal precipitate in a sodium hydroxide solution, adjusting the pH to neutral, and freeze-drying the mixture to finally obtain melanin nanoparticle receptor beads.

[0050] Furthermore, the first hairpin nucleic acid is coupled to the donor beads, and the second hairpin nucleic acid is coupled to the acceptor beads in a coupling buffer, which includes 10-20 mmol / L tris(hydroxymethyl)aminomethane hydrochloride, 110-140 mmol / L sodium chloride, 1-5 mmol / L potassium chloride, 0.1-1.0 mmol / L calcium chloride, 0.1-1.0 mmol / L magnesium chloride and deionized water, and has a neutral pH value.

[0051] A method for detecting a biomarker by using light-induced electrical activation of an active oxygen channel, wherein the biomarker is a protein, comprises the following steps:

[0052] S01: preparing a donor bead complex containing a photosensitizer, wherein the donor bead complex is a metal organic framework containing a photosensitizer coupled to a capture antibody;

[0053] S02: preparing an acceptor bead complex containing polyphenols, wherein the acceptor bead complex is a melanin-like nanoparticle coupled with a detection antibody;

[0054] S03: placing the donor bead complex and the acceptor bead complex into a biological sample solution to be tested. When the biological sample to be tested contains a target antigen, the target antigen can recognize and bind to the capture antibody and the detection antibody to form a sandwich immune structure, forming a donor bead-acceptor bead connector, and the distance between the donor bead-acceptor bead connector is less than 200 nm;

[0055] S04: adding the donor bead-acceptor bead connector to the electrode interface, applying a voltage of -0.8 to -0.1 V, and irradiating the donor bead-acceptor bead connector with a laser, wherein the donor beads in the donor bead-acceptor bead connector generate singlet oxygen after laser irradiation, and the singlet oxygen diffuses in the biological sample to be tested, and the polyphenols in the acceptor beads are oxidized to generate benzoquinone after capturing the singlet oxygen, and applying a reduction voltage at the electrode interface to trigger the electrochemical reduction of benzoquinone to polyphenols, a cascade redox cycle occurs and an amplified current signal is generated, and the current signal is read;

[0056] S05: establishing a standard curve in advance according to the concentration and current intensity value of the target antigen standard;

[0057] S06: Substituting the read current signal into the standard curve to calculate the content of the target antigen in the biological sample to be tested.

[0058] Furthermore, the metal organic framework is coupled to the capture antibody via an amide bond; and the melanin-like nanoparticles are coupled to the detection antibody via Michael addition or Schiff base reaction.

[0059] Furthermore, the target antigen includes carcinoembryonic antigen.

[0060] Application of the above method in the preparation of a photoinduced electrically driven reactive oxygen channel biomarker detection reagent or kit.

[0061] Beneficial technical effects

[0062] The present invention provides a method for detecting biomarkers by using a photo-induced electrochemical method to drive active oxygen channels. The donor bead complex and the acceptor bead complex contained in the active oxygen channel reaction reagent provided by the present invention are free nanoparticles in the detection system. Therefore, the method of the present invention adopts a non-solidification strategy to avoid electrode modification, and realizes "wash-free and separation-free" homogeneous electrochemistry through uniform mass transfer collision, which can be detected repeatedly. In addition, it can also effectively overcome the defect that photoelectrochemistry requires repeated washing and separation steps to remove unbound substances that cause background signals, and the detection sensitivity is high. At present, the prior art often requires that photoelectric nanomaterials be immobilized on electrodes, and the unevenness of the modified electrodes and the uncontrollable stacking 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 lower than the time for electrons to transfer to electrodes or electrons from electrodes to materials, resulting in carrier recombination, reducing photocurrent signals, and thus reducing detection sensitivity.

[0063] 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, negative potential reduction is applied to avoid interference from electroactive substances in the physiological matrix; in the collision-constrained transient reactive oxygen 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, so the sensitivity is high.

[0064] In the detection of specific nucleic acids, the present invention adopts a catalytic hairpin self-assembly strategy to achieve target cyclic amplification, and then combines the cascade redox cycle electron transfer signal amplification strategy of the receptor beads to achieve dual synergistic electrical signal amplification and improve the sensitivity of detection. In addition, there is no need for complex RNA extraction, purification, and multi-step separation and washing processes. In the specific detection of proteins, the present invention uses a sandwich immune result of double antibodies combined with the antigen to be tested to achieve target signal amplification.

[0065] Finally, the electrochemical workstation equipment provided by the present invention has the advantages of miniaturization, low cost, convenient operation, and providing efficient and fast detection results, and has a good industrialization prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0067] Figure 1 A schematic diagram of a method for simulating the present invention of detecting nucleic acids by using light-induced electrical activation of reactive oxygen species channels;

[0068] Figure 2 A schematic diagram of a method for simulating the present invention of detecting proteins by using light-induced electrical activation of reactive oxygen species channels;

[0069] Figure 3 Transmission electron microscope (TEM) photograph of donor beads prepared by the method of the present invention (scale bar 50 nm);

[0070] Figure 4 This is a transmission electron microscope (TEM) photograph of the acceptor beads prepared by the method of the present invention (scale bar 200 nm);

[0071] Figure 5 The electron paramagnetic resonance (ESR) spectrum of singlet oxygen generated by photoexcitation of donor beads in the method of the present invention;

[0072] Figure 6 This is a detection time-current curve diagram of detecting miRNA using a photoinduced electroactive oxygen channel method in one embodiment of the present invention;

[0073] Figure 7 This is a schematic diagram of the linear range of detecting miRNA using the photoinduced electroactive oxygen channel method in one embodiment of the present invention;

[0074] Figure 8 This is a diagram for evaluating the anti-interference performance of the photoelectrification-driven active oxygen channel method in one of the embodiments of the present invention;

[0075] Fig. 9 This is a detection time-current curve diagram of detecting CEA using a photoinduced electroactive oxygen channel method in one embodiment of the present invention;

[0076] Fig.10 This is a schematic diagram of the detection linear range of CEA detection using the photoinduced electroactive oxygen channel method in one embodiment of the present invention;

[0077] Fig.11 This is a graph showing the recovery results of simulating actual sample spike addition in one of the embodiments of the present invention;

[0078] Fig.12 A schematic diagram of the structure of a detection system for photoinduced electroactive oxygen channels provided by the present invention;

[0079] Fig.13 Schematic diagram of the working principle of the detection system for light-induced electro-driven active oxygen channels provided by the present invention. DETAILED DESCRIPTION

[0080] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0081] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0082] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0083] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0084] As used in this specification, the term "about" typically means + / - 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.

[0085] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted 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 independent numerical values ​​within this range. For example, the range The description of 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., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0086] Glossary:

[0087] The photoexcited electrically driven reactive oxygen channel (LEOCA) described in the present invention is a homogeneous detection method for photoexcited synchronous electron transfer, which specifically refers to that after laser irradiation of the donor-acceptor composite reaction system, the donor produces singlet oxygen, and the acceptor captures the singlet oxygen to produce oxidation products, which then undergo a cascade chemical oxidation-electrochemical reduction process at the electrode interface to generate an electrochemical signal, which can also be referred to as a LEOCA signal.

[0088] The cascade redox cycle described in the present invention refers to the singlet oxygen generated by the photoexcited donor bead complex in the presence of the target, which diffuses to the surface of the acceptor bead complex and oxidizes the polyphenol groups in the acceptor bead to generate benzoquinone. The benzoquinone that collides and contacts the electrode surface is reduced to generate polyphenol at a negative potential, generating an electrochemical signal. The product benzoquinone generated by the former chemical oxidation event is the substrate of the latter electrochemical reduction event, triggering the subsequent electrochemical reduction reaction, and is therefore called a cascade redox cycle reaction.

[0089] The donor bead-acceptor bead connector described in the present invention refers to a structure formed by connecting the first reactant on the donor bead complex and the second reactant on the acceptor bead complex with the target substance in the biological sample to be detected, and specifically refers to the first reactant-target substance-second reactant structure between the donor bead and the acceptor bead after connection.

[0090] Example 1

[0091] This embodiment provides an example of a method for detecting a biomarker by using light-induced electrical driving of reactive oxygen species channels. The biomarker detected in this embodiment is nucleic acid.

[0092] (1) A first hairpin nucleic acid coupled to a metal organic framework containing a porphyrin-based photosensitizer is used as a nanodonor bead, and a second hairpin nucleic acid coupled to polydopamine particles containing polyphenols is used as a nanoacceptor bead. The target nucleic acid recognizes and responds to the first hairpin nucleic acid chain to trigger the exposed single-stranded region, thereby inducing the hybridization of the second hairpin nucleic acid chain to ultimately form a catalytic hairpin self-assembly complex.

[0093] (2) After the excitation light is irradiated to the detection system, the singlet oxygen ( 1 O2) diffuses to the acceptor beads, and the polyphenols in the acceptor beads are captured 1 O2 can be oxidized to produce benzoquinone, which undergoes a cascade chemical oxidation-electrochemical reduction process at the instant of instantaneous collision at the electrode interface to generate an electrochemical signal. 1 O2 has high activity and short half-life. Only when the distance between the donor beads and the acceptor beads is close enough (forming a catalytic hairpin self-assembly complex), the O2 released by the donor beads will 1 O2 can effectively reach the acceptor beads and generate electrochemical signals.

[0094] (3) When there is no target in the system, that is, no catalytic hairpin self-assembly complex is formed, the distance between the donor and acceptor beads is greater than 200 nm, and the free donor beads produce 1 O2 dissipates rapidly in the system and generates almost no electrochemical signal.

[0095] In summary, the two donor and acceptor beads achieve high energy by self-assembly of hairpins catalyzed by nucleic acid 1 The transfer of O2 induces the process of photoinduced electrical activation of active oxygen channels. The detection principle diagram is shown in Figure 1 shown.

[0096] The nucleic acid sample to be tested in this embodiment is selected from interstitial fluid.

[0097] Example 2

[0098] This embodiment provides another example of a method for detecting a biomarker by using light-excited electrically driven reactive oxygen species channels. The biomarker detected in this embodiment is a protein.

[0099] (1) A capture antibody coupled to a metal organic framework containing a porphyrin-based photosensitizer is used as a nanodonor bead, and a detection antibody coupled to polydopamine particles containing polyphenols is used as a nanoacceptor bead. The target antigen recognizes and responds to the capture antibody and the detection antibody to form a sandwich immune structure, and finally forms a donor bead-acceptor bead complex.

[0100] (2) After the excitation light is irradiated to the detection system, the singlet oxygen ( 1 O2) diffuses to the acceptor beads, and the polyphenols in the acceptor beads are captured 1 O2 is then oxidized to produce benzoquinone, which undergoes a cascade chemical oxidation-electrochemical reduction process at the instant of instantaneous collision at the electrode interface, generating an electrochemical signal. 1 O2 has high activity and short half-life. Only when the distance between the donor beads and the acceptor beads is close enough (capture antibody-target antigen-detection antibody binding), the donor beads will release 1 O2 can effectively reach the acceptor beads and generate electrochemical signals.

[0101] (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 beads and the acceptor beads is greater than 200 nm, and the free donor beads produce 1 O2 dissipates rapidly in the system and generates almost no electrochemical signal.

[0102] In summary, the two donor and acceptor beads achieve high energy through the combination of antigen and antibody. 1 The transfer of O2 induces the process of photoinduced electrical activation of active oxygen channels. The detection principle diagram is shown in Figure 2 shown.

[0103] The nucleic acid sample to be tested in this embodiment is selected from interstitial fluid.

[0104] Example 3

[0105] This embodiment provides an example of a method for preparing various materials of a detection system.

[0106] (1) Preparation of donor beads

[0107] Add 50-150 mg of tetracarboxylphenylporphyrin, 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 under ultrasonication for 5 minutes, and stir at 90°C for 5 hours; after the reaction is completed, centrifuge at 15000 rpm for 10-20 minutes, and then wash with N,N-dimethylformamide 3 times. Finally, collect the precipitate, which is the zirconium-based metal organic framework nanodonor beads, and disperse it in anhydrous ethanol for storage.

[0108] It is understandable that the preparation of the zirconium-based metal-organic framework nano-donor beads in this embodiment is only an example, and a gadolinium-based metal-organic framework or an iron-based metal-organic framework may also be used.

[0109] (2) Preparation of acceptor beads

[0110] 55 mg of dopamine hydrochloride was added to a flask, and the mixture was dispersed in a mixture of 100 mL of deionized water and 20 mL of anhydrous ethanol by ultrasonic treatment. 120 mg of tris(hydroxymethyl)aminomethane) salt was added to the reaction mixture, and the mixture was stirred at room temperature for 72 hours. The reaction stock solution was mixed with anhydrous ethanol, and the mixture was centrifuged at 16000 rpm for 10-20 minutes, and the supernatant was discarded to obtain a precipitate, which was repeated three times. 20 mg of the precipitate was dissolved in 10 mL of 0.1 M sodium hydroxide solution, and the mixture was ultrasonically treated at an output power of 600 W for 5-10 minutes, and then the pH value was adjusted to 7.0 using hydrochloric acid. The polydopamine receptor beads obtained by freeze drying were dispersed in water for storage.

[0111] It is to be understood that the preparation of the polydopamine receptor beads in this embodiment is only an example, and isomelanin, poly-L-dopa, polygallic acid or polycaffeic acid etc. may also be used.

[0112] The donor and acceptor beads were characterized by TEM. Figure 3 , Figure 4 As shown in FIG. 1 , 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, and the spectrum is shown in FIG. Figure 5 As shown, the ESR spin trap TEMP reacts with the donor beads under light to generate ESR signals, indicating that the donor beads that generate singlet oxygen by photoexcitation are successfully synthesized by this method.

[0113] (3) Preparation of hairpin nucleic acid

[0114] The hairpin nucleic acid is chemically synthesized and purified by HPLC, then dissolved in an annealing buffer to prepare a solution, and placed in a 95°C PCR instrument for denaturation for 5 minutes, and then the temperature is gradually reduced to 25°C at a rate of 0.1°C per second to form a stable catalytic hairpin structure.

[0115] Annealing buffer: The solvent is deionized water, and the solution components and contents are: tris(hydroxymethyl)aminomethane hydrochloride, 20 mmol / L; sodium chloride, 140 mmol / L; potassium chloride, 5 mmol / L; pH is 7.4.

[0116] It is understandable that in this embodiment, the capture antibody and the detection antibody can also be prepared according to conventional techniques in the art.

[0117] (4) Preparation of the first hairpin nucleic acid donor bead complex / the second hairpin nucleic acid acceptor bead complex

[0118] The zirconium-based metal organic framework donor beads and the first hairpin nucleic acid were mixed and incubated in a coupling buffer at 37° C. for 2 hours to obtain the first hairpin nucleic acid donor bead complex. The polydopamine acceptor beads and the second hairpin nucleic acid were mixed and incubated in a coupling buffer at 37° C. for 4 hours to obtain the second hairpin nucleic acid acceptor bead complex.

[0119] Coupling buffer: The solvent is deionized water, and the solution components and contents are: tris(hydroxymethyl)aminomethane hydrochloride, 20mmol / L; sodium chloride, 140mmol / L; potassium chloride, 5mmol / L; calcium chloride, 1mmol / L; magnesium chloride, 1mmol / L; pH is 7.4.

[0120] It is understandable that the capture antibody donor bead complex and the detection antibody acceptor bead complex can also be prepared in this embodiment according to similar methods and other conventional techniques in the art.

[0121] Example 4

[0122] This embodiment provides an actual nucleic acid sample detection example and a performance evaluation of the detection method.

[0123] (1) The zirconium-based metal organic framework donor beads and the first hairpin nucleic acid were mixed and incubated in a Tris-HCl buffer at 37°C for 2 hours; the polydopamine acceptor beads and the second hairpin nucleic acid were mixed and incubated in a Tris-HCl buffer at 37°C for 4 hours; the donor and acceptor beads were mixed to form a detection system. In this embodiment, the particle size of the donor beads is 100 nm, and the particle size of the acceptor beads is 10 nm. The mixing ratio of the donor beads to the acceptor beads is 1:10.

[0124] (2) Add a sample of tumor tissue lysate or tumor interstitial fluid containing the miRNA to be tested to the detection system, mix and incubate at 37° C. for 30-120 min to form a dispersion to be tested.

[0125] 3) The dispersion is added dropwise to a screen-printed carbon electrode, and the intensity of the electrical signal is detected using an electrochemical workstation. In a homogeneous environment, under 630 nm laser excitation conditions (the photosensitizer selected in this embodiment is tetracarboxyphenylporphyrin), singlet oxygen is transferred between the donor and acceptor beads, and a -0.2 V voltage is applied to the electrode for reduction to generate an electrochemical signal. A standard curve is established based on the concentration and current intensity values ​​of the miRNA standard, and the current intensity of the sample to be tested is substituted into the standard curve to calculate the content of miRNA in the sample to be tested.

[0126] It is understood that when different photosensitizers are selected, the applied laser wavelength will be different.

[0127] Table 1 Sequence information

[0128]

[0129] It will be understood that the above sequence is merely an example and not a limitation.

[0130] The results of the miRNA (H1-H2) detection in the present embodiment are as follows Figure 6 As shown in Figure 2, as the concentration of target miRNA (H1-H2) increases (10pM-1nM), the electrochemical current intensity also increases. Figure 7 As shown in Figure 2, when the concentration of miRNA (H1-H2) was in the range of 10pM-1nM, the current intensity showed a good linear relationship with the miRNA concentration (R 2 =0.998), and the detection limit was 3.4 pM. This indicates that the detection system can accurately detect miRNA in the sample to be tested.

[0131] Furthermore, interfering substances, including ascorbic acid, dopamine, uric acid, glucose, norepinephrine, cysteine, etc., were added to the detection system. 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.

[0132] Example 5

[0133] This embodiment provides an actual protein sample detection example and a performance evaluation of the detection method.

[0134] (1) The zirconium-based metal organic framework donor beads and the capture antibody were mixed and incubated in a coupling buffer at 37°C for 2 hours; the polydopamine acceptor beads and the detection antibody were mixed and incubated in a Tris-HCl buffer at 37°C for 4 hours; the donor and acceptor beads were mixed to form a detection system. In this embodiment, the particle size of the donor beads is 100 nm, and the particle size of the acceptor beads is 10 nm. The mixing ratio of the donor beads to the acceptor beads is 1:10.

[0135] (2) Add a sample containing serum containing the target antigen to be detected into the detection system, mix and incubate at 37° C. for 30-120 min to form a dispersion to be detected.

[0136] 3) The dispersion is added dropwise to a screen-printed carbon electrode, and the intensity of the electrical signal is detected using an electrochemical workstation. In a homogeneous environment, under 630 nm laser excitation conditions (the photosensitizer selected in this embodiment is tetracarboxyphenylporphyrin), singlet oxygen is transferred between the donor and acceptor beads, and a -0.2 V voltage is applied to the electrode for reduction to generate an electrochemical signal. A standard curve is established based on the concentration and current intensity value of the target antigen standard, and the current intensity of the sample to be tested is substituted into the standard curve to calculate the content of the antigen in the sample to be tested.

[0137] The results of the target antigen detection in the embodiment of the present invention are as follows Fig. 9 As shown, with the increase of target CEA concentration (10pg mL -1 -50ng mL -1 ), the higher the electrochemical current intensity. Fig.10 As shown, when the CEA concentration was 10 pg mL -1 -50ng mL -1 When the current intensity is within the range, there is a good linear relationship between the current intensity and the CEA concentration (R 2 =0.993), and the detection limit was 2.6 pg mL -1 . This shows that the detection system can accurately detect the target protein in the sample to be tested.

[0138] The results of target antigen spike recovery in the simulated serum samples detected in this example are as follows Fig.11 As shown, the spiked recovery of CEA antigen was 99.3% to 108.1%, and the RSD was 0.1% to 1.05%, indicating that the detection system has good clinical application prospects.

[0139] Example 6

[0140] This embodiment provides an example of a detection device for photoinduced electrodynamic driving of reactive oxygen species channels.

[0141] A detection device for light-excited electrically driven active oxygen channels, the detection device comprising a reaction module, a light-excitation module, an electrically driven module and an active oxygen channel reaction reagent;

[0142] The reaction module comprises a reaction pool, and the reaction pool is configured to place the biological sample to be detected and the active oxygen channel reaction reagent;

[0143] The optical excitation module includes at least one laser emitter;

[0144] The electric drive module includes at least one set of electrodes and an electric signal reader;

[0145] The active oxygen channel reaction reagent includes a donor bead complex containing a photosensitizer and an acceptor bead complex containing polyphenols, wherein the donor bead complex includes a metal organic framework containing a photosensitizer coupled with a first reactant; the acceptor bead complex includes a melanin-like nanoparticle containing polyphenols coupled with a second reactant; the first reactant and the second reactant are configured to connect or identify a target substance in a biological sample to be detected to form a donor bead-acceptor bead connector;

[0146] During detection, the laser emitted by the at least one laser emitter is irradiated onto the donor bead-acceptor bead connector, causing the donor bead complex to generate singlet oxygen, and the polyphenol in the acceptor bead complex captures the singlet oxygen and is oxidized to generate benzoquinone;

[0147] The oxidized acceptor beads containing benzoquinone collide with the electrode surface, and a reduction voltage is applied to the electrode to trigger the electrochemical reduction of benzoquinone to polyphenol, completing a cascade redox cycle and generating an amplified current signal;

[0148] The electrical signal reader is configured to read the electrochemical signal.

[0149] The structural diagram of the detection device and the schematic diagram of the working principle of this embodiment are shown in Fig.12 , Fig.13 .exist Fig.12 In one of the embodiments shown, the laser emitter can be arranged on one side of the reaction pool; Fig.12 In another embodiment shown, the laser emitter can be arranged on the other side of the reaction pool; or, the laser emitter can also be arranged on the top of the reaction pool. It is understood that in order to increase the efficiency of light excitation, multiple laser emitters can be arranged. It is understood that the reaction pool can be Fig.12 The structure of the reaction container shown in the figure may also be any other carrier or system that can ensure the completion of the liquid reaction.

[0150] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A detection system for photoinduced electrokinetic driven active oxygen channels, characterized in that: The detection system includes a reaction module, a light excitation module, an electric drive module and an active oxygen channel reaction reagent; The reaction module comprises a reaction pool, and the reaction pool is configured to place the biological sample to be detected and the active 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 electric signal reader; The active oxygen channel reaction reagent includes a donor bead complex containing a photosensitizer and an acceptor bead complex containing polyphenols, and the donor bead complex includes a metal organic framework containing a photosensitizer coupled to a first reactant; The receptor bead complex includes melanin-like nanoparticles containing polyphenols coupled to a second reactant; The first reactant and the second reactant are configured to connect or identify a target substance in a biological sample to be detected to form a donor bead-acceptor bead connection body; The distance between the donor bead and the acceptor bead connector is less than 200 nm; During detection, the laser emitted by the at least one laser emitter is irradiated onto the donor bead-acceptor bead connector, so that the donor bead complex generates singlet oxygen which diffuses to the acceptor bead, and the polyphenol in the acceptor bead complex captures the singlet oxygen and is oxidized to generate benzoquinone; During the detection, the target substance in the biological sample to be detected includes nucleic acid or protein; when the target substance is nucleic acid, the first reactant is the first hairpin nucleic acid, and the second reactant is the second hairpin nucleic acid; when the target substance is protein, the first reactant is a capture antibody, and the second reactant is a detection antibody; The oxidized acceptor beads containing benzoquinone collide with the electrode surface, and a reduction voltage is applied to the electrode to trigger the electrochemical reduction of benzoquinone to polyphenol, resulting in a cascade redox cycle and generating an amplified current signal; The electric signal reader is configured to read the current signal.

2. The detection system according to claim 1, characterized in that: The donor bead composite includes one or more metal organic frameworks selected from the group consisting of a zirconium-based metal organic framework, a gadolinium-based metal organic framework, and an iron-based metal organic framework.

3. The detection system according to claim 1, characterized in that: The donor bead complex includes one or more photosensitizers selected from the group consisting of fluoroboron dipyrrole, metal phthalocyanine, and porphyrin.

4. The detection system according to claim 1, wherein: The particle size of the donor beads ranges from 60 to 200 nm; the particle size of the acceptor beads ranges from 10 to 20 nm.

5. The detection system according to claim 1, characterized in that: The configuration ratio of the donor bead complex to the acceptor bead complex in the active oxygen channel reaction reagent is 1:

10.

6. The detection system according to claim 1, characterized in that: The biological sample to be detected is selected from interstitial fluid.

7. The detection system according to claim 1, characterized in that: The electric drive module is arranged at the bottom of the reaction pool, and is configured to provide voltage and read electrical signals; the light excitation module is arranged at the top and / or side of the reaction pool.

8. A method for detecting biomarkers using the detection system for photoinduced electrically driven reactive oxygen channels according to any one of claims 1 to 7, characterized in that: The biomarkers include nucleic acids or proteins, and the detection steps are as follows: S01: adding a biological sample to be tested into the reaction pool of the reaction module, when the biological sample to be tested contains a target nucleic acid, the target nucleic acid can recognize and respond to the first hairpin nucleic acid and trigger the exposed single-stranded region in the first hairpin nucleic acid to induce the hybridization of the second hairpin nucleic acid chain, and obtain a donor bead-acceptor bead connector of the target nucleic acid through hybridization self-assembly of the hairpin nucleic acid; or, when the biological sample to be tested contains a target antigen, the target antigen can recognize and bind to the capture antibody and the detection antibody to form a sandwich immune structure, and obtain a donor bead-acceptor bead connector of the target antigen, and the distance between the donor bead-acceptor bead connector is less than 200 nm; S02: Turning on the laser emitter of the light excitation module, irradiating the laser emitted by the laser emitter onto the donor bead-acceptor bead connector, wherein the donor bead complex generates singlet oxygen, the singlet oxygen diffuses in the biological sample to be tested, and the polyphenols in the acceptor bead complex capture the singlet oxygen and are oxidized to generate benzoquinone; S03: The electrodes of the electric drive module and the electric signal reader are turned on simultaneously, the acceptor beads producing benzoquinone collide on the surface of the electrode, a reduction voltage is applied to the electrode to trigger the electrochemical reduction of benzoquinone into polyphenol, a cascade redox cycle occurs and an amplified current intensity signal is generated, and the electric signal reader reads the current intensity signal; S04: establishing a standard curve in advance according to the concentration and current intensity value of the target nucleic acid or the target antigen standard; S05: Substituting the current intensity signal read by the electrical signal reader into the standard curve to calculate the content of the target nucleic acid or the target antigen in the biological sample to be tested.

9. The method according to claim 8, characterized in that The sequence of the first hairpin nucleic acid includes that shown in SEQ ID NO.1 or SEQ ID NO.3; the sequence of the second hairpin nucleic acid includes that shown in SEQ ID NO.2 or SEQ ID NO.4; the 5' end of the nucleic acid shown in SEQ ID NO.1 is modified with an NH2 group; the 3' end of the nucleic acid shown in SEQ ID NO.2 is modified with an SH group; the 5' end of the nucleic acid shown in SEQ ID NO.3 is modified with an NH2 group; the 3' end of the nucleic acid shown in SEQ ID NO.4 is modified with an SH group; the target antigen includes a carcinoembryonic antigen.

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