WO2023245854A1 - 检测小分子物质的试剂组合、试剂盒、检测***及检测方法 - Google Patents

检测小分子物质的试剂组合、试剂盒、检测***及检测方法 Download PDF

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WO2023245854A1
WO2023245854A1 PCT/CN2022/114296 CN2022114296W WO2023245854A1 WO 2023245854 A1 WO2023245854 A1 WO 2023245854A1 CN 2022114296 W CN2022114296 W CN 2022114296W WO 2023245854 A1 WO2023245854 A1 WO 2023245854A1
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nucleic acid
iso
small molecule
acid molecule
fluorescence
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PCT/CN2022/114296
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French (fr)
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曹丹
成舜
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南京浦光生物科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
    • G01N33/541—Double or second antibody, i.e. precipitating antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • This application belongs to the field of chemiluminescence detection technology, and specifically relates to a reagent combination, a kit, a detection system and a detection method for detecting small molecule substances.
  • CLIA Chemiluminescence immunoassay
  • the antibody When there are no small molecule substances in the blood sample, the antibody cannot specifically bind to the small molecule substances in the blood. At this time, the luminescent group generally does not have luminescent activity and will not produce strong fluorescence in addition to the background fluorescence. strength.
  • the antibody can specifically bind to the small molecule substance, and the luminescent group emits fluorescence under the action of the luminescent substrate. Therefore, the presence of small molecule substances in the blood can be determined by using a chemiluminescence detection instrument to detect the presence of fluorescence in the solution to be tested.
  • the above method uses a single fluorescent group to emit fluorescence, and determines whether there is a small molecule substance based on whether the single fluorescent group emits fluorescence. Since a single fluorescent group will emit background fluorescence in the absence of small molecule substances, this background fluorescence will affect the acquisition of fluorescence, thus causing large errors when testing the content of small molecule substances.
  • the purpose of some embodiments of the present application is to provide a reagent combination, kit, detection system and detection method for detecting small molecule substances, which can avoid the background fluorescence of a single fluorescent group in the existing chemiluminescence detection method to affect the detection results. cause impact.
  • Some of the embodiments mentioned herein may be from the same embodiment or from different embodiments.
  • some embodiments of the present application provide a reagent combination for detecting small molecule substances.
  • the reagent combination at least includes: a first conjugate, a second conjugate, a third conjugate and an oxidation inhibiting reagent.
  • the first conjugate is formed by coupling at least a first nucleic acid molecule and an antibody.
  • the first nucleic acid molecule contains a first hybridization region and a second hybridization region.
  • the first hybridization region and the second hybridization region are not directly adjacent, but are separated by 2 or 5 nucleotides.
  • the complementarity determining region (CDR) of an antibody can specifically bind to small molecules.
  • the nucleic acid molecules are single-stranded DNA. In this application, when describing each single-stranded DNA, the connection mode from left to right is from the 5' end to the 3' end.
  • the second conjugate is composed of at least a substrate protein conjugate, a second nucleic acid molecule and a second fluorescent group coupled in sequence.
  • the substrate protein conjugate is composed of at least a small molecule substrate coupled to a scaffold protein.
  • the small molecule substrate is the same substance as the small molecule substance to be measured.
  • substances bound to scaffold proteins are called small molecule substrates, and substances free in the solution to be tested are called small molecule substances.
  • the second nucleic acid molecule has a third hybridization region and a fourth hybridization region.
  • the third hybridization region is directly connected to the fourth hybridization region, with no intervening nucleotides in between.
  • the third hybridization zone is complementary to the second hybridization zone.
  • the small molecule substrate has only one epitope, which can specifically bind to the CDR of the antibody, and the binding is reversible and can be reversed by small molecules free in the solution to be tested. . That is, the small molecule substance in the free state and the small molecule substrate in the bound state can competitively bind to the antigenic epitope of the antibody.
  • the CDR of each antibody can only bind to one small molecule substrate or one small molecule substance at the same time.
  • the third conjugate is formed by coupling at least a first fluorescent group and a third nucleic acid molecule.
  • the third nucleic acid molecule contains a fifth hybridization region and a sixth hybridization region.
  • the fifth hybridization region is directly connected to the sixth hybridization region, with no intervening nucleotides in between.
  • the fifth hybridization zone is complementary to the fourth hybridization zone, and the sixth hybridization zone is complementary to the first hybridization zone.
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are all single-stranded DNA.
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule can be assembled into a stem-loop structure.
  • the structure of the neck is ⁇ -shaped, and the paired regions present a double helix structure.
  • the first fluorescent group can be oxidized by the oxidizing agent. and emits first fluorescence in the absence of antioxidants, and the first fluorescence serves as excitation light to excite the second fluorescence based on the fluorescence resonance energy transfer effect under the condition that the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are paired with each other.
  • the group emits a second fluorescence, so that the content of the small molecule substance can be obtained based on the intensity of the second fluorescence.
  • the intensity of the second fluorescence is not obtained at all, it means that the content of the small molecule substance is too high, and all the antibodies are bound to the free small molecule substance, rather than to the small molecule substrate in the substrate protein conjugate.
  • the detection limit is exceeded. At this time, the content of small molecule substances cannot be obtained based on the fluorescence intensity.
  • the concentration of the small molecule substance in the solution to be tested is low, the binding degree of the small molecule substance to the antibody is weak, and the binding degree of the small molecule substrate to the antibody is strong, then the intensity of the second fluorescence is stronger. powerful. Therefore, the concentration of small molecule substances in the solution to be measured is inversely proportional to the intensity of the second fluorescence.
  • the functional relationship between the intensity of the second fluorescence and the content of the small molecule substance can be obtained in advance, thereby establishing a one-to-one numerical relationship between the two. Therefore, the measurement of the content of the small molecule substance can be converted into It is a numerical measurement of fluorescence intensity.
  • Oxidation inhibiting agents include antioxidants.
  • the antioxidant can inhibit the first fluorescent group from being oxidized and emitting the first fluorescence.
  • the first fluorescent group in various embodiments of the present application emits light in an oxidative manner, rather than fluorescing when exposed to excitation light. Therefore, it is necessary to minimize the oxidation of the first fluorescent group by oxidizing substances in the solution to be measured.
  • Background fluorescence Background fluorescence is a type of noise, which will affect the true value of fluorescence measurement, thereby affecting the accuracy of the measurement results of small molecule content.
  • Antioxidants and oxidants cannot exist in the sample to be tested at the same time, so as to prevent the oxidative luminescence effect of the oxidants on the first fluorescent group from being neutralized by the antioxidants. Therefore, antioxidants need to be removed before adding oxidizing agents.
  • the following basic conditions are required for the fluorescence resonance energy transfer effect between the first fluorescent group and the second fluorescent group: the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are to be Under the condition that there are no small molecule substances in the test solution, the intensity of the second fluorescence produced is maximum.
  • the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are partially complementary and paired under the condition that there is a lower concentration of small molecule substances in the solution to be tested. At this time, the second fluorescence will also be generated.
  • Complementary pairing will form a neck-ring structure such that the distance between the first fluorophore and the second fluorophore is smaller than the limiting distance at which fluorescence resonance energy transfer can occur, for example, in the range of 70 angstroms to 99 angstroms, or 7 nm to 10 nm In the range.
  • the first fluorescent group may be an acridinium ester
  • the second fluorescent group may be a quantum dot
  • the maximum emission wavelength of the first fluorescent group is 430 nm.
  • the maximum absorption wavelength of the second fluorescent group may be in the range of 420 nm to 520 nm, for example, it may be 470 nm.
  • the maximum emission wavelength of the second fluorescent group may be in the range of 595 nm to 615 nm, such as 605 nm.
  • the quantum dots are core-shell structure quantum dots, and the core layer material is selected from one or more of CdSe, CdS, CdTe, CdSeTe, CdZnS, ZnTe, CdSeS, PbS and PbTe, wherein
  • the shell material is selected from one or more of ZnS, ZnSe, ZnSeS, PbS and PbSeS.
  • the particle size range of the quantum dots may be 3 nm to 5 nm, or may be 4.1 nm to 4.2 nm.
  • the sugar ring at the 3' end of the first nucleic acid molecule is covalently connected to the amino group of the antibody through the first coupling agent.
  • the sugar ring at the 5' end of the first nucleic acid molecule is not modified.
  • the sugar ring at the 3' end of the first nucleic acid molecule is modified with an NH2C7 modification group, and the NH2C7 modification group is covalently connected to the amino group of the antibody through the first coupling agent.
  • the first coupling agent is disuccinimide suberate sodium salt.
  • the 3' end of the second nucleic acid molecule is connected to a second fluorescent group.
  • the sugar ring at the 3' end of the second nucleic acid molecule is modified with a thiol group
  • the surface of the second fluorescent group is modified with an amino group
  • the thiol group is covalently connected to the amino group on the surface of the second fluorescent group through a third coupling agent.
  • the third coupling agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester.
  • the sugar ring at the 5' end of the second nucleic acid molecule is covalently connected to the amino group of the scaffold protein of the substrate protein conjugate through a second coupling agent.
  • the sugar ring at the 5' end of the second nucleic acid molecule is modified with an NH2C6 modification group, and the NH2C6 modification group is covalently connected to the amino group of the scaffold protein through a second coupling agent.
  • the second coupling agent is disuccinimide suberate sodium salt.
  • the 5' end of the third nucleic acid molecule is covalently linked to the first fluorescent group.
  • the sugar ring at the 5' end of the third nucleic acid molecule is modified with an NH2C6 modification group, and the NH2C6 modification group is covalently connected to the first fluorescent group.
  • the first hybridization region in the first nucleic acid molecule, is located upstream of the second hybridization region in order from the 5' end to the 3' end.
  • the third hybridization region is located upstream of the fourth hybridization region.
  • the fifth hybridization region is located upstream of the sixth hybridization region.
  • the first nucleic acid molecule has 55 nucleotides
  • the first hybridization region covers the 3rd to 10th base positions of the first nucleic acid molecule starting from the 5' end
  • the second hybridization region covers The 13th to 19th base positions from the 5' end of the first nucleic acid molecule.
  • the second nucleic acid molecule has 53 nucleotides
  • the third hybridization region covers 37 to 43 base positions of the second nucleic acid molecule starting from the 5' end
  • the fourth hybridization region covers the second nucleic acid molecule starting from the 5' end.
  • the third nucleic acid molecule has 22 nucleotides.
  • the fifth hybridization region covers the 3rd to 10th base positions of the third nucleic acid molecule starting from the 5' end.
  • the sixth hybridization region covers the third nucleic acid molecule starting from the 5' end. The 11th to 18th base positions.
  • the complementary pairing between the six DNA sequences causes the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule to hybridize in pairs to form a neck-ring structure, and the shape of the neck is ⁇ -shaped.
  • the first hybridization region is GCTGAGT from the 5' end to the 3' end
  • the sixth hybridization region is AACTCAGC from the 5' end to the 3' end.
  • the second hybridization region is CAACGAC from the 5' end to the 3' end
  • the third hybridization region is GTCGTTG from the 5' end to the 3' end.
  • the fourth hybridization region is GCTGAAT from the 5' end to the 3' end
  • the fifth hybridization region is ATCTCAGC from the 5' end to the 3' end.
  • Each hybridization zone is single-stranded DNA, not double-stranded DNA. The two DNA sequences within the same single-stranded DNA do not pair with each other, but pair with the DNA sequences of other single-stranded DNA.
  • the full-length sequence of the first nucleic acid molecule is shown in SEQ ID No: 1
  • the full-length sequence of the second nucleic acid molecule is shown in SEQ ID No: 2
  • the full-length sequence of the third nucleic acid molecule is shown in SEQ ID No: 1.
  • the sequence is shown as SEQ ID No: 3.
  • G in the first nucleic acid molecule, the second nucleic acid molecule and/or the third nucleic acid molecule can be replaced by iso G, and C can be replaced by iso C.
  • iso G and iso C are unnatural base pairs. Using non-natural base pairs for pairing can effectively avoid mismatching between the first nucleic acid molecule, the second nucleic acid molecule, and/or the third nucleic acid molecule and the natural nucleic acid in the test solution, thereby preventing mismatching from affecting the formation of the neck-loop structure. This avoids measurement errors caused by mismatching.
  • the bonding method of iso G and iso C is:
  • G in the first hybridization region and the sixth hybridization region is replaced by iso G, and C is replaced by iso C.
  • the first hybridization region is iso G iso CT iso GA iso GTT from the 5' end to the 3' end
  • the sixth hybridization region is AA iso CT iso CA iso G iso C from the 5' end to the 3' end.
  • G in the second hybridization region and the third hybridization region is replaced by iso G, and C is replaced by iso C.
  • the second hybridization region is iso CAA iso C iso GA iso C from the 5' end to the 3' end
  • the third hybridization region is iso GT iso C iso GTT iso G from the 5' end to the 3' end.
  • G in the fourth hybridization region and the fifth hybridization region is replaced by iso G, and C is replaced by iso C.
  • the fourth hybridization region is iso G iso CT iso GA iso GAT from the 5' end to the 3' end
  • the fifth hybridization region is AT iso CT iso CA iso G iso C from the 5' end to the 3' end.
  • the full-length sequence of the first nucleic acid molecule is:
  • the full-length sequence of the first nucleic acid molecule may be a sequence shown in SEQ ID No: 1 in which at least part or all of G is replaced by iso G and at least part or all of C is replaced by iso C the sequence formed.
  • the full-length sequence of the second nucleic acid molecule is:
  • the full-length sequence of the second nucleic acid molecule is the sequence shown in SEQ ID No: 2, in which at least part or all of G is replaced by iso G and at least part or all of C is replaced by iso C. sequence formed.
  • the full-length sequence of the third nucleic acid molecule is:
  • the full-length sequence of the third nucleic acid molecule is the sequence shown in SEQ ID No: 3, in which at least part or all of G is replaced by iso G and at least part or all of C is replaced by iso C. sequence formed.
  • the oxidation-inhibiting agent further includes a carrier molecule with a surface-bound antioxidant. Because the first fluorescent group emits oxidative light instead of stimulated light emission, once oxidative substances are present, the first fluorescent group may emit fluorescence.
  • the function of antioxidants is mainly to prevent the first fluorescent group from being oxidized by these substances with oxidizing ability, thereby generating background fluorescence that may affect the measurement results. These substances with oxidizing ability can come from the solution to be tested or blood samples.
  • the antioxidant is selected from any one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols, and glutathione.
  • the oxidizing agent includes an alkaline solution of hydrogen peroxide. Oxidizing agents can also be called chemiluminescent substrates of the first fluorescent group because these substrates themselves are oxidizing.
  • the carrier molecule may be graphene oxide.
  • Part of the carboxyl groups on graphene oxide is combined with the hydroxyl groups on the antioxidant through a sulfoxide condensation agent, and part of the carboxyl groups on graphene oxide is through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt
  • the acid salt combines with the amino group on the antioxidant, so that the antioxidant attaches to the graphene oxide. Because antioxidants and oxidizing agents cannot be added to the solution to be tested at the same time, otherwise the oxidizing agent will not be able to oxidize and emit light from the first fluorescent group, so the antioxidant needs to be removed from the solution to be tested before adding the oxidizing agent. Combining antioxidants on graphene oxide carriers is more conducive to antioxidant removal.
  • two fluorophores are used to generate the second fluorescence for measurement, the first fluorophore emits oxidation light, the second fluorophore emits light excitation, and the first fluorophore
  • the first fluorescence emitted by the group can excite the second fluorescent group to emit second fluorescence based on fluorescence resonance energy transfer.
  • the second fluorescence is collected, it can be determined whether there is a small molecule substance in the solution to be measured or the concentration of the small molecule substance can be obtained.
  • the intensity of the second fluorescence reaches the maximum value, it means that the free small molecule substance will not Competitively binds the antibody to the bound small molecule substrate, so the solution to be tested does not contain small molecule substances at all.
  • the content of small molecule substances is inversely proportional to the intensity of the second fluorescence, the content of small molecule substances can also be obtained based on the intensity of the second fluorescence.
  • This detection method eliminates the influence of the background fluorescence of the first fluorescent group on the measurement results and improves the sensitivity and accuracy of the detection.
  • the two fluorescent groups in various embodiments of the present application can emit fluorescence signals without setting up additional excitation light sources, which also reduces the complexity of the detection system and enables mobile detection.
  • the first fluorescent group in each embodiment of the present application emits flash light, the detection time is very short, and the detection results can be obtained quickly.
  • Some embodiments of the present application also provide a method for detecting small molecule substances, which includes the following steps:
  • the first conjugate is formed by coupling at least a first nucleic acid molecule and an antibody.
  • the first nucleic acid molecule contains a first hybridization region and a second hybridization region, and the antibody can bind to the small Specific binding of molecular substances.
  • the second conjugate is composed of at least a substrate protein conjugate, a second nucleic acid molecule and a second fluorescent group coupled in sequence.
  • the second nucleic acid molecule has a third hybridization region and a fourth hybridization region.
  • the third hybridization region is connected to the third hybridization region.
  • the two hybrid regions are complementary.
  • Substrate protein conjugates are small molecule substrates that specifically bind to the complementarity determining regions of antibodies.
  • the third conjugate is formed by coupling at least a first fluorescent group and a third nucleic acid molecule.
  • the third nucleic acid molecule contains a fifth hybridization region and a sixth hybridization region, the fifth hybridization region is complementary to the fourth hybridization region, and the sixth hybridization region is complementary to the first hybridization region.
  • the antibody forms an immune complex with the substrate protein conjugate by binding to the small molecule substance, and the first nucleic acid molecule , the second nucleic acid molecule and the third nucleic acid molecule form a neck ring structure, and the first fluorescent group and the second fluorescent group are located on the same side of the neck ring structure, so fluorescence resonance energy transfer can occur between the two fluorescent molecules, thus , the second fluorescent group is excited to emit second fluorescence.
  • the content of the small molecule substance is obtained based on the intensity of the second fluorescence and the one-to-one correspondence between the fluorescence intensity and the content of the small molecule substance.
  • a filter When collecting the intensity of the second fluorescence, in order to avoid the interference of the first fluorescence on the second fluorescence, a filter can be used to filter out the fluorescence generated after the first fluorescent group is oxidized, and only allow the second fluorescence to pass through the filter. slice, thereby collecting the second fluorescence emitted by the second fluorescent group, and obtaining the content of the small molecule substance based on the intensity of the second fluorescence.
  • the solution to be tested can be derived from a blood sample.
  • the composition of blood is relatively complex and contains a variety of oxidative substances. If the first fluorescent group is oxidized by these oxidative substances, background fluorescence will be produced, which will affect the accuracy of the measurement results. Therefore, in this application, in order to reduce the background For the influence of fluorescence on the measurement results, the first fluorescence emitted by the first fluorophore is not used as the detection fluorescence signal, but the second fluorescence emitted by the second fluorophore is used as the detection fluorescence signal.
  • the working concentration of the first conjugate in the sample to be tested may be 1 nM to 20 nM.
  • the working concentration of the second conjugate can be from 1 nM to 20 nM.
  • the working concentration of the third conjugate can be 0.05 nM to 0.2 nM.
  • Working concentrations of oxidation inhibitory reagents can range from 15 ⁇ g/ml to 25 ⁇ g/ml.
  • the solution to be tested is derived from blood samples such as whole blood samples, serum samples, or plasma samples.
  • the mixing time may be from 5 minutes to 10 minutes.
  • the mixing temperature may be 36 to 37 degrees.
  • the volume of the oxidizing agent may be 200 ⁇ L
  • the oxidizing agent is an alkaline hydrogen peroxide solution
  • the pH value is 8.0.
  • the alkaline hydrogen peroxide solution is prepared by dissolving hydrogen peroxide in TBS buffer, where the final concentration of hydrogen peroxide is 0.1M and the final concentration of TBS is 10mM.
  • the small molecule substance includes triiodothyronine, tetraiodothyronine, or progesterone.
  • the antibodies in the first conjugate form an immune response with the small molecule substrate.
  • the immune complex brings the complementary sequences of the three single-stranded DNAs closer together, pairing them complementary and achieving assembly between the DNAs, thereby forming a neck-loop structure.
  • Some embodiments of the present application also provide a small molecule substance detection kit, which includes: a first storage tube, a second storage tube, a third storage tube, a fourth storage tube and a fifth storage tube.
  • the first storage tube stores at least the conjugate of the first nucleic acid molecule and the antibody.
  • the second storage tube stores at least the substrate protein conjugate, the second nucleic acid molecule and the conjugate of the second fluorescent group.
  • Antibodies and substrate protein conjugates can form immune complexes with small molecule substrates in the absence of small molecule substances or the presence of a small amount of small molecule substances. On the contrary, if there is an excess of small molecule substances, then the small molecule substances will competitively bind to the antibodies originally bound to the small molecule substrate, and immune complexes will not be formed, thus preventing the assembly of single-stranded DNA.
  • the third storage tube stores at least the conjugate of the first fluorescent group and the third nucleic acid molecule.
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are assembled into a neck-loop structure based on the proximity effect under the conditions of immune complex formation.
  • the spatial conformation of the neck-loop structure makes the first fluorescent group and the second fluorescent group Spontaneously arranged on the same side of the neck ring structure, and the spatial distance between the two is smaller than the limiting distance where fluorescence resonance energy transfer can occur, for example, in the range of 70 angstroms to 99 angstroms, which is between two fluorophores Provides conditions for fluorescence resonance energy transfer to occur.
  • the fourth storage tube stores at least an antioxidant capable of inhibiting oxidation of the first fluorescent group.
  • the antioxidant can be modified on the carrier molecule to become an oxidation inhibiting agent.
  • the carrier molecule may be selected from graphene oxide.
  • the fifth storage tube stores at least the oxidant.
  • the oxidizing agent is capable of oxidizing the first fluorescent group to emit first fluorescence. Oxidants and antioxidants are not present in the sample to be tested at the same time. Before adding the oxidizing agent, the antioxidant and carrier molecules need to be removed from the sample to be tested. Because the antioxidant is modified on the carrier molecule, and the carrier molecule graphene oxide exhibits a lamellar network structure and is easily removed from the sample to be tested, the antioxidant can be removed by removing the carrier molecule.
  • graphene oxide can also adsorb the free third nucleic acid molecule, thereby adsorbing the conjugate of the first fluorescent group and the third nucleic acid molecule, so that the antioxidant inhibits the first fluorescent group in the free state from being improperly oxidized, thus Suppresses inappropriate emission of background fluorescence from the first fluorophore.
  • the first nucleic acid molecule contains a first hybridization region and a second hybridization region; the second nucleic acid molecule has a third hybridization region and a fourth hybridization region; and the third nucleic acid molecule contains a fifth hybridization region and a sixth hybridization region.
  • the first hybridization region, the second hybridization region, the third hybridization region, the fourth hybridization region, the fifth hybridization region and the sixth hybridization region contain at least part or all of the bases iso G and iso C. Since iso G is used to replace the natural base G and iso C is used to replace the natural base C, the possibility of mismatching is reduced.
  • the detection principle of the small molecule substance detection kit is as follows.
  • the antibody and the substrate protein conjugate form an immune complex in whole or in part.
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are close to each other, hybridize and self-assemble into a neck-ring structure.
  • Six DNA sequences (i.e., hybridization regions) in three nucleic acid molecules (i.e., single-stranded DNA) pair up in pairs to form the neck of the neck-loop structure, and the remaining non-DNA sequences form the ring of the neck-loop structure.
  • the two DNA sequences inside each single-stranded DNA cannot pair with each other, but can only pair with the complementary sequence of another single-stranded DNA.
  • the spatial conformation of the neck ring structure is such that the first fluorescent group and the second fluorescent group are located on the same side of the neck ring structure, and the distance between them can ensure that fluorescence resonance energy transfer occurs between them.
  • the oxidizing agent can oxidize the first fluorescent group and cause it to emit the first fluorescence.
  • the first fluorescence excites the second fluorescent group to emit the second fluorescence based on the fluorescence resonance energy transfer effect, so that according to the second The intensity of fluorescence obtains the content of small molecule substances.
  • the immune complex and the neck ring structure cannot be formed. Even if the antioxidant is removed and the subsequent oxidizing agent is added to cause the first fluorophore to emit the first fluorescence, the first If the distance between the fluorophore and the second fluorophore is greater than the limit distance at which fluorescence resonance energy transfer can occur, the first fluorophore cannot excite the second fluorophore to emit the second fluorescence. The inability to obtain the intensity of the second fluorescence indicates that the solution to be tested contains an excess of small molecular substances.
  • the second fluorescent group can emit the second fluorescence under the excitation of the excitation light. If there is no excitation of the first fluorescence, then the second fluorescent group will not emit for measuring the content of small molecule substances.
  • the second fluorescence therefore, the second fluorescence group as the detection group will basically not emit background fluorescence, which ensures the reliability of using the second fluorescence as the detection fluorescence.
  • Some embodiments of the present application provide a small molecule substance detection system, which includes: a reaction container, a micro-syringe pump, an optical filter, a light signal detection module and a calculation module.
  • the reaction vessel has a holding chamber capable of holding the solution to be tested.
  • the microinjection pump is connected to the accommodation chamber through the injection pipe, and injects the mixture of the first conjugate, the second conjugate, the third conjugate and the oxidation inhibitor reagent into the accommodation chamber through the injection pipe, so that Mix with the solution to be tested;
  • the first conjugate is composed of at least a first nucleic acid molecule and an antibody
  • the second conjugate is at least a substrate protein conjugate, a second nucleic acid molecule, and a second fluorescent group.
  • the third conjugate is formed by coupling at least a first fluorescent group and a third nucleic acid molecule, and the oxidation inhibiting reagent contains an antioxidant that inhibits the first fluorescent group from being oxidized and emitting the first fluorescence.
  • the optical filter is disposed on the exit light path of the first fluorescence and allows the transmission of the second fluorescence having the same wavelength as the maximum emission wavelength of the second fluorescence group.
  • the light signal detection module is disposed on the exit light path of the first fluorescence and is located on the downstream side of the filter, and acquires the second fluorescence transmitted through the filter.
  • the intensity value of the second fluorescence can be 0, which means that there is an excess of small molecule substances in the solution to be measured, because the second fluorescent group emits excited light rather than oxidative light. Under the condition of no excitation light, the second fluorescent group emits light.
  • the second fluorescent group itself does not emit second fluorescence, so it will not produce background fluorescence noise. Therefore, in the presence of excess small molecule substances, the intensity of the second fluorescence can reach zero, and in the absence of small molecules, the intensity of the second fluorescence can reach zero.
  • the intensity value of the second fluorescence is inversely proportional to the content of the small molecule substance. If the first fluorescence is used as the detection fluorescence, since the first fluorescence is oxidative luminescence, and there will be oxidative substances in the solution to be measured, the first fluorescence will be oxidized by these oxidative substances and emit background fluorescence noise. Therefore, using the second fluorescence as the detection fluorescence is more accurate and has smaller error than using the first fluorescence as the detection fluorescence.
  • the calculation module converts the second fluorescence into a digital signal and obtains the content of the small molecule substance in the solution to be tested according to the functional relationship between the fluorescence intensity and the content of the small molecule substance.
  • Some embodiments of the present application use a combination of immune reaction and fluorescence resonance energy transfer effect to detect whether the solution to be tested contains small molecule substances. Specifically:
  • the antibody and the substrate protein conjugate form an immune complex based on the immune reaction, so that the distance between the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule Pulling in, triggering DNA assembly and forming a neck-loop structure.
  • the neck-loop structure causes the first fluorescent group and the second fluorescent group to be located on the same side of the neck-loop structure, thereby generating a fluorescence resonance energy transfer phenomenon between the two.
  • the second fluorescent group emits second fluorescence as a fluorescence signal to be measured.
  • the content of the small molecule substance in the solution to be measured is obtained according to the preset functional relationship between the fluorescence intensity and the content of the small molecule substance and the intensity of the fluorescence signal to be measured.
  • the immune complex and the neck ring structure cannot be formed, and the second fluorescent group will not be excited by the excitation light (i.e. the first fluorescence) and emit the second fluorescence, then The inability to capture the fluorescence signal to be measured indicates that there is an excess of small molecule substances in the solution to be measured.
  • the first fluorophore emits background fluorescence under the action of certain oxidizing substances, since the neck ring structure cannot be formed, the spatial distance between the first fluorophore and the second fluorophore is larger than the fluorescence resonance.
  • the fluorescence resonance energy transfer effect will not occur between the two fluorophores, and the second fluorophore will not emit second fluorescence based on the background fluorescence. At least this enables qualitative detection of the presence of small molecule substances in the solution to be tested and quantitative detection of the content of small molecule substances in the solution to be tested.
  • Various embodiments of the present application use the second fluorescence emitted by the second fluorescent group that is less likely to generate background fluorescence noise as the fluorescence signal to be measured, instead of using the first fluorescence emitted by the first fluorescent group that usually has background fluorescence noise as the fluorescence signal to be measured. Measuring fluorescence signals can greatly reduce the impact of background fluorescence noise on detection results, improve detection sensitivity and reduce detection errors, making the measured protein content closer to the true value.
  • Figure 1 is a schematic diagram of the immune complex and neck ring structure of some embodiments of the present application.
  • Figure 2 is a schematic diagram of a detection method according to some embodiments of the present application.
  • the antibody and the small molecule substrate will form an immune complex, and there will be an immune complex between the single-stranded DNA molecules. Form a neck ring structure.
  • an oxidant hydroogen peroxide alkali solution
  • the acridinium ester is oxidized by the oxidant and emits the first fluorescence of 430nm.
  • the first fluorescence excites the quantum dots to emit the second fluorescence of 605nm.
  • Small molecule substance 1 antibody 2, substrate protein conjugate 3, first nucleic acid molecule 4, second nucleic acid molecule 5, third nucleic acid molecule 6, first fluorescent group 7, second fluorescent group 8, first hybridization Zone 9, second hybridization zone 10, third hybridization zone 11, fourth hybridization zone 12, fifth hybridization zone 13, sixth hybridization zone 14, composite 15 of graphene oxide and antioxidant (ie, oxidation inhibitor reagent), Scaffolding protein 16, immune complex 17.
  • first nucleic acid molecule 4 second nucleic acid molecule 5
  • third nucleic acid molecule 6 first fluorescent group 7, second fluorescent group 8, first hybridization Zone 9, second hybridization zone 10, third hybridization zone 11, fourth hybridization zone 12, fifth hybridization zone 13, sixth hybridization zone 14, composite 15 of graphene oxide and antioxidant (ie, oxidation inhibitor reagent), Scaffolding protein 16, immune complex 17.
  • antioxidant ie, oxidation inhibitor reagent
  • Some embodiments of the present application provide a combination of reagents.
  • This reagent combination is used to detect whether there are excess small molecule substances in the solution to be tested, thereby achieving qualitative detection of small molecule substances.
  • the reagent combination can also obtain the concentration of the small molecule substance in the solution to be tested on the premise that there is a small amount of the small molecule substance in the solution to be tested, thereby achieving quantitative detection of the content of the small molecule substance.
  • the solution to be tested may be derived from blood samples or other non-blood samples.
  • the blood sample can be from a whole blood sample, a serum sample or a plasma sample.
  • the types of small molecule substances to be detected are not particularly limited.
  • the small molecule substance may be triiodothyronine, tetraiodothyronine, or progesterone, etc.
  • the molecular weight of the small molecule substance to be detected can be in the range of 100 to 5000 KD, but in some cases it is not limited to the above range.
  • the reagent combination includes: a first conjugate, a second conjugate, a third conjugate, and an oxidation inhibiting reagent.
  • the first conjugate is formed by coupling at least a first nucleic acid molecule and an antibody.
  • the sugar ring at the 3' end of the first nucleic acid molecule is modified with an NH2C7 modification group, and the NH2C7 modification group is covalently connected to the amino group of the antibody through the first coupling agent.
  • the NH2C7 modification group modifies the sugar ring at the 3’ end of the first nucleic acid molecule, not the base.
  • the first coupling agent may be disuccinimide suberate sodium salt.
  • the amino group connected to the first coupling agent in the antibody is not located on the CDR of the antibody, otherwise the antibody cannot specifically bind to small molecules.
  • the 3' end of the first nucleic acid molecule is not modified.
  • the full-length sequence of the first nucleic acid molecule is as shown in SEQ ID No: 1.
  • the specific sequence may be ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG, but is not limited to the above sequence.
  • the first nucleic acid molecule contains a first hybridization region and a second hybridization region.
  • the first hybridization region may be located upstream of the second hybridization region in order from the 5' end to the 3' end.
  • the first hybridization region may be GCTGAGT from the 5' end to the 3' end
  • the second hybridization region may be CAACGAC from the 5' end to the 3' end.
  • complementary pairing does not occur between the first hybridization zone and the second hybridization zone. In various embodiments of the present application, complementary pairing does not occur between the two DNA sequences contained in each single-stranded DNA. Only the single-stranded DNA between different reagents undergo complementary pairing to assemble into a neck-loop structure.
  • the first nucleic acid molecule has 55 nucleotides
  • the first hybridization region covers the 3rd to 10th base positions from the 5' end of the first nucleic acid molecule
  • the second hybridization region covers the first nucleic acid Base positions 13 to 19 starting from the 5' end of the molecule.
  • antibodies can specifically bind to small molecule substances.
  • Each antibody molecule can only bind to one small molecule substance, but cannot bind to multiple small molecule substances at the same time.
  • the CDR of the antibody molecule can only recognize one antigenic epitope of the small molecule substance, so the specific binding of the two can be achieved.
  • the second conjugate may be sequentially coupled with at least a substrate protein conjugate, a second nucleic acid molecule, and a second fluorescent group.
  • the substrate protein conjugate is composed of at least a small molecule substrate coupled to a scaffold protein.
  • the small molecule substrate is the same as the small molecule substance, so the small molecule substrate and the small molecule substance can competitively bind to the same antibody.
  • the concentration of small molecule substances is very high, the small molecule substrate can basically not bind to the CDR of the antibody. Therefore, when there is no or a small amount of small molecule substances in the solution to be tested, the antibody and the substrate protein conjugate can undergo an immune reaction to form an immune complex.
  • the full-length sequence of the second nucleic acid molecule is as shown in SEQ ID No: 2.
  • the specific sequence may be TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC, but is not limited to the above sequence.
  • the second nucleic acid molecule has a third hybridization region and a fourth hybridization region.
  • the third hybridization region is located upstream of the fourth hybridization region.
  • the third hybridization region is GTCGTTG from the 5' end to the 3' end
  • the fourth hybridization region is GTCTGAGAT from the 5' end to the 3' end.
  • the third hybridization zone is complementary to the second hybridization zone. There is no complementary relationship between the third hybridization region and the fourth hybridization region, therefore, the possibility of mismatching within the DNA strand is reduced.
  • the second nucleic acid molecule has 53 nucleotides.
  • the third hybridization region covers the 37th to 43rd base positions from the 5' end of the second nucleic acid molecule, and the fourth hybridization region covers the 44th to 51st base positions from the 5' end of the second nucleic acid molecule.
  • the 3' end of the second nucleic acid molecule is linked to a second fluorescent group.
  • the sugar ring at the 3' end of the second nucleic acid molecule is modified with a thiol group
  • the surface of the second fluorescent group is modified with an amino group
  • the thiol group is covalently connected to the amino group on the surface of the second fluorescent group through a third coupling agent.
  • the third coupling agent may be 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester.
  • the sugar ring at the 5' end of the second nucleic acid molecule is modified with an NH2C6 modification group, and the NH2C6 modification group is covalently connected to the amino group of the substrate protein conjugate through a second coupling agent.
  • the second coupling agent is disuccinimide suberate sodium salt.
  • the amino group connecting the scaffold protein to the second coupling agent in the substrate protein conjugate is not located at the binding site between the scaffold protein and the small molecule substrate, otherwise the scaffold protein cannot specifically bind to the small molecule substrate.
  • the third conjugate can be formed by coupling at least a first fluorescent group and a third nucleic acid molecule.
  • the sugar ring at the 5' end of the third nucleic acid molecule is modified with an NH2C6 modification group, and the NH2C6 modification group is covalently connected to the first fluorescent group.
  • the full-length sequence of the third nucleic acid molecule is as shown in SEQ ID No: 3.
  • the specific sequence can be CGATCTCAGCAACTCAGCAGCG, but is not limited to the above sequence.
  • the third nucleic acid molecule contains a fifth hybridization region and a sixth hybridization region, and the fifth hybridization region and the sixth hybridization region are not complementary to each other.
  • the fifth hybridization region is located upstream of the sixth hybridization region in order from the 5' end to the 3' end.
  • the third nucleic acid molecule has 22 nucleotides
  • the fifth hybridization region covers the 3rd to 10th base positions of the third nucleic acid molecule starting from the 5' end
  • the sixth hybridization region covers the third nucleic acid Base positions 11 to 18 starting from the 5' end of the molecule.
  • the sequence of the fifth hybridization region from the 5' end to the 3' end can be ATCTCAGC.
  • the sequence of the sixth hybridization region from the 5' end to the 3' end can be AACTCAGC.
  • the fifth hybridization zone is complementary to the fourth hybridization zone
  • the sixth hybridization zone is complementary to the first hybridization zone
  • the third hybridization zone is complementary to the second hybridization zone.
  • the first fluorescent group emits first fluorescence under conditions capable of being oxidized by an oxidizing agent, and the first fluorescence is based on fluorescence resonance under conditions in which the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule pair with each other.
  • the energy transfer effect excites the second fluorescent group to emit second fluorescence, so as to obtain the content of the small molecule substance based on the intensity of the second fluorescence.
  • the second fluorescent group When there is no or a small amount of small molecule substances in the solution to be measured, since the distance between the first fluorescent group and the second fluorescent group satisfies the conditions for the generation of fluorescence resonance energy transfer, the second fluorescent group will undergo the third Second fluorescence, the wavelength of the second fluorescence is different from that of the first fluorescence. Therefore, using the second fluorescence as the detection fluorescence will not be affected by the background fluorescence emitted by the first fluorophore. If the second fluorescence intensity is not obtained from the solution to be tested, it means that there is an excess of small molecule substances in the solution to be tested.
  • the antibody When there is no or a small amount of small molecule substances in the solution to be tested, the antibody combines with the small molecule substances to form a stable immune complex, and three single-stranded DNAs form a neck-loop structure.
  • the immune complex and the neck ring structure can make the distance between the first fluorescent group and the second fluorescent group sufficient to produce a fluorescence resonance energy transfer effect, so that according to the difference between the intensity and intensity content of the second fluorescence emitted by the second fluorescent group
  • the standard curve determines the content of small molecule substances.
  • the first fluorophore emits fluorescence without irradiation of excitation light, but emits light under the oxidation effect of the oxidant.
  • the second fluorophore needs to be illuminated by excitation light to emit fluorescence. Without the irradiation of excitation light, the second fluorophore itself will not actively emit fluorescence, so it is not easy to generate background fluorescence. Therefore, this application The measurement results of the examples are not interfered by the background fluorescence of the second fluorophore. Compared with the method of using only one fluorescent group to emit fluorescence, the methods of various embodiments of the present application can effectively avoid the interference of the background fluorescence of fluorescent molecules, so the measurement results are more accurate.
  • first fluorescent group and the second fluorescent group need to be able to undergo fluorescence resonance energy transfer, and the following conditions need to be met: first conjugate, second conjugate, third conjugate
  • the substances are completely complementary in the absence of small molecule substances, and partially complementary in the presence of a small amount of small molecule substances. Complementary pairing can form a neck-ring structure such that the distance between the first fluorophore and the second fluorophore is less than or equal to 100 Angstroms.
  • the first fluorescence emitted by the first fluorescent group can become the excitation light of the second fluorescent group, thereby exciting the second fluorescent group to generate second fluorescence (as shown in Figures 1 and 2).
  • the first fluorescent group can be an acridinium ester.
  • Acridinium ester does not require excitation light to emit fluorescence, but achieves oxidation and luminescence in the presence of an oxidizing agent. Since there may be oxidizing substances in the solution to be tested, it can also oxidize acridinium ester and make it emit light. Therefore, acridinium ester will Background fluorescence is present. If the fluorescence of acridinium ester is used as the measured fluorescence, the background fluorescence will interfere with the measured fluorescence. Therefore, in order to further improve the sensitivity of detection and reduce the measurement error, the embodiments of the present application do not use the fluorescence of acridinium ester as the measured fluorescence.
  • the chemiluminescent substrate of acridinium ester is an alkaline solution of H 2 O 2 , also known as an oxidizing agent.
  • H 2 O 2 an alkaline solution of H 2 O 2
  • acridinium ester and an alkaline solution of H 2 O 2 coexist, the molecules of acridinium ester are attacked by hydrogen peroxide ions, and acridinium ester can form unstable dioxyethane with hydrogen peroxide (H 2 O 2 ). , the subsequent decomposition of dioxyethane emits fluorescence.
  • the maximum emission wavelength of the first fluorescent group acridinium ester is 430 nm.
  • the second fluorophore can be a quantum dot.
  • the maximum absorption wavelength of the second fluorescent group quantum dot may be in the range of 420 nm to 520 nm, for example, it may be 470 nm.
  • the maximum emission wavelength of the second fluorescent group may be in the range of 595 nm to 615 nm, for example, it may be 605 nm. Therefore, the first fluorescent group and the second fluorescent group in the embodiments of the present application can produce fluorescence resonance energy transfer effect.
  • the quantum dots are core-shell structure quantum dots, the core layer material of which is selected from one or more of CdSe, CdS, CdTe, CdSeTe, CdZnS, ZnTe, CdSeS, PbS and PbTe, and the shell layer material of which One or more selected from ZnS, ZnSe, ZnSeS, PbS and PbSeS.
  • the particle size of the quantum dots may range from 3 to 5 nm, for example, may be 4.1 nm.
  • G in the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule can be replaced by iso G, and C can be replaced by iso C. Because when the test solution comes from blood samples, etc., natural nucleic acids also exist in these samples, so by introducing non-natural base pairs ( iso G and iso C), the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid can be avoided The molecules bind non-specifically to nucleic acids in the blood sample, thereby avoiding measurement errors due to DNA mismatches.
  • these unnatural base pairs do not affect the mutual pairing of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule, and can still form a stable neck-loop structure, thereby not affecting the generation of the second fluorescence.
  • G is replaced by iso G and C is replaced by iso C in the full-length sequence of the first nucleic acid molecule.
  • G is replaced by iso G and C is replaced by iso C in the full-length sequence of the second nucleic acid molecule.
  • G in the full-length sequence of the third nucleic acid molecule is replaced by iso G, and C is replaced by iso C.
  • G in the first hybridization region and the sixth hybridization region paired with each other are replaced by iso G, and C is replaced by iso C.
  • the first hybridization region is iso G iso CT iso GA iso GTT from the 5' end to the 3' end
  • the sixth hybridization region is AA iso CT iso CA iso G iso C from the 5' end to the 3' end.
  • G is replaced by iso G and C is replaced by iso C in the paired second hybridization region and the third hybridization region.
  • the second hybridization region is iso CAA iso C iso GA iso C from the 5' end to the 3' end
  • the third hybridization region is iso GT iso C iso GTT iso G from the 5' end to the 3' end.
  • G is replaced by iso G and C is replaced by iso C in the fourth hybridization region and the fifth hybridization region paired with each other.
  • the fourth hybridization region is iso G iso CT iso GA iso GAT from the 5' end to the 3' end
  • the fifth hybridization region is AT iso CT iso CA iso G iso C from the 5' end to the 3' end.
  • the structural formula of iso C can be:
  • the bonding method of iso G and iso C can be:
  • iso G and iso C can produce complementary pairing, which does not affect the mutual pairing of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule, but can prevent the first nucleic acid molecule, the second nucleic acid molecule from , the third nucleic acid molecule is mismatched with the natural nucleic acid in the solution to be tested. Therefore, the above embodiment can reduce the measurement error caused by the mismatch between the single-stranded DNA and the natural nucleic acid molecule.
  • the oxidation inhibiting reagent includes: an antioxidant used for the first fluorescent group to be oxidized to emit the first fluorescence.
  • an antioxidant used for the first fluorescent group to be oxidized to emit the first fluorescence.
  • the first conjugate, the second conjugate, the third conjugate and the oxidation inhibition reagent need to be added to the solution to be tested, and then mixed to form a sample to be tested.
  • the first fluorescent group acridinium ester will not emit fluorescence because Antioxidants inhibit the oxidative luminescence of acridinium esters. Therefore, antioxidants need to be removed from the sample to be tested before testing.
  • the oxidation inhibitor reagent in order to achieve smooth removal of antioxidants from the sample to be tested, further includes a carrier molecule and binds the antioxidant to the surface of the carrier molecule.
  • the carrier molecule has a larger molecular weight, it is easier to remove from the sample to be tested. Simply removing the carrier molecules simultaneously removes the antioxidants.
  • the carrier molecule may be graphene oxide (GO).
  • the carboxyl group on graphene oxide is combined with the hydroxyl group on the antioxidant through a sulfoxide condensation agent, and the carboxyl group on graphene oxide is bonded through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Binds to amino groups on antioxidants.
  • the first conjugate, the second conjugate, the third conjugate and the oxidation inhibitor reagent are added to the solution to be tested to form the sample to be tested.
  • the third nucleic acid molecule is adsorbed on the surface of graphene oxide through ⁇ - ⁇ stacking.
  • the terminally labeled acridinium ester cannot oxidize and emit light due to the presence of antioxidants. Even if a small part of the acridinium ester chemiluminesces, that is, produces background fluorescence with a wavelength of 430nm, it will not affect the measurement results. Make an impact. Because the fluorescence obtained during detection comes from the second fluorophore quantum dot and not from acridinium ester, you can set a filter to only obtain the fluorescence emitted by the quantum dot and filter out the background of acridinium ester. Fluorescence.
  • the embodiments of the present application can minimize the impact of the background fluorescence of acridinium ester on the measurement results, thereby reducing measurement errors.
  • the fluorescence emitted by the quantum dots can be obtained through a filter and a photomultiplier tube (PMT).
  • PMT photomultiplier tube
  • the antioxidant can be selected from any one or more of cannabidiol, vitamin C, vitamin E, tea polyphenols, and glutathione.
  • the oxidizing agent may include an alkaline solution of hydrogen peroxide. After removing the antioxidant, the added oxidizing agent can cause the first fluorescent group acridinium ester to emit fluorescence.
  • Embodiments of the present application use immune reactions to enable antibodies to recognize small molecule substances, thereby shortening the distance between single-stranded DNAs that can be complementary paired, thereby triggering DNA assembly, triggering cascade DNA assembly, and causing fluorescence resonance between two fluorescent molecules.
  • the detection signal is generated by the energy transfer effect.
  • Each embodiment of the present application uses acridinium ester (AE) as the fluorescence energy donor (donor), fluorescent quantum dots (QDs) as the fluorescence energy acceptor (acceptor), and is based on the chemiluminescence resonance energy transfer system. Fluorescence signal amplification is achieved through immune reaction and DNA self-assembly, and the detection of the content of small molecule substances is converted into the detection of fluorescence signal intensity.
  • the methods of the embodiments of the present application can be performed homogeneously. Compared with the traditional fluorescence resonance energy transfer analysis method, the detection method of the embodiment of the present application is simple and fast, and does not require expensive lasers.
  • the second fluorescence is used as the detection fluorescence, which reduces the background interference of the first fluorescence.
  • the embodiments of the present application use immune reaction to enrich small molecule substances, which has good selectivity for small molecule substances and high detection sensitivity.
  • some embodiments of the present application provide a detection method for small molecule substances, and the detection method can use the above-mentioned reagent combination. Characteristics regarding the reagent combinations described above may be incorporated into this section.
  • the detection method of the above embodiment includes the following steps:
  • the first conjugate is formed by coupling at least a first nucleic acid molecule and an antibody.
  • the second conjugate is composed of at least a substrate protein conjugate, a second nucleic acid molecule and a second fluorescent group coupled in sequence.
  • the third conjugate is formed by coupling at least a first fluorescent group and a third nucleic acid molecule.
  • the antibody and the substrate protein conjugate form an immune complex
  • the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule form a neck-ring structure, so that the third nucleic acid molecule forms a neck-ring structure.
  • the first fluorescent group and the second fluorescent group are located on the same side of the neck ring structure.
  • the oxidation inhibiting reagent contains an antioxidant for inhibiting the first fluorescent group from being oxidized by oxidizing substances in the solution to be measured. Since the first fluorescent group does not emit the first fluorescence in the presence of an antioxidant, the antioxidant needs to be removed before adding the oxidant. In addition, the first fluorescent group will be protected by antioxidants before being oxidized, thereby also reducing the background fluorescence emitted by itself due to the influence of other factors.
  • the first fluorescent group will produce the first fluorescence after being oxidized.
  • a filter is used to filter out the first fluorescence.
  • the filter only allows the second fluorescent group.
  • the relative intensity refers to the remaining fluorescence intensity value after excluding the background fluorescence intensity of the control group from the intensity of the second fluorescence signal.
  • the working concentration of the first conjugate in the sample to be tested is 1 nM to 20 nM.
  • Working concentration refers to the concentration of each reagent during actual detection, that is, the final concentration of each reagent in the sample to be tested. The working concentration is not equal to the storage concentration of each reagent in the small molecule detection kit.
  • the working concentration of the second conjugate in the sample to be tested is 1 nM to 20 nM.
  • the working concentration of the third conjugate in the sample to be tested is 0.05 nM to 0.2 nM.
  • the working concentration of the oxidation inhibition reagent in the sample to be tested is 15 ⁇ g/ml to 25 ⁇ g/ml.
  • the blood sample of the solution to be tested is from a whole blood sample, a serum sample or a plasma sample.
  • the mixing time is 5 minutes to 10 minutes. In some embodiments, the mixing temperature is 36 to 37 degrees.
  • the volume of oxidant is 200 ⁇ L.
  • the oxidizing agent is alkaline hydrogen peroxide solution with a pH value of 8.0.
  • the alkaline hydrogen peroxide solution is prepared by dissolving hydrogen peroxide in TBS buffer, where the final concentration of hydrogen peroxide is 0.1M and the final concentration of TBS is 10mM.
  • the small molecule substance includes triiodothyronine, tetraiodothyronine, or progesterone.
  • the small molecule substances are not limited to the above types.
  • the antibody of the present application can specifically bind to small molecule substances and form a stable immune complex.
  • An antibody can only bind to one small molecule substance or small molecule substrate.
  • relatively stable immune complexes are formed.
  • the immune complex will pull the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule closer together to form a neck ring structure.
  • the first fluorescent group and the second fluorescent group are located on the same side of the neck ring structure.
  • the first fluorescent group can excite the second fluorescent group to produce the second fluorescence. Since the small molecule substance or small molecule substrate has a one-to-one correspondence with the antibody and the second fluorophore, and the strongest fluorescence degree of the second fluorophore is known, then the number of small molecule substances is related to the second fluorophore.
  • the degree of fluorescence quenching is a linear relationship, based on which the relationship between the number of small molecule substances and the remaining fluorescence intensity can also be obtained.
  • the content of the small molecule substance can be calculated based on the functional relationship between the intensity of the second fluorescence and the content of the small molecule substance contained in the standard curve and the obtained fluorescence intensity of the second fluorescent group.
  • the specific detection steps are as follows: combining the detection reagents (first conjugate, second conjugate, third conjugate and oxidation inhibition reagent) with the blood sample to be tested containing small molecule substances Or mix whole blood samples or serum samples or plasma samples, incubate the reaction on a chemiluminescence detector at 37°C for 5-10 minutes, add chemiluminescence substrates hydrogen peroxide, sodium hydroxide and TBS buffer as oxidants, The chemiluminescence fluorescence signal generated is collected through the PMT detection module in the chemiluminescence detector. The chemiluminescence detector automatically calls the standard curve, and reports the concentration of small molecule substances in the sample to be tested based on the functional relationship between fluorescence intensity and small molecule substance content contained in the standard curve.
  • the small molecule substance detection kit includes: a first storage tube, a second storage tube, a third storage tube, a fourth storage tube, a fourth storage tube, and a fifth storage tube.
  • the first storage tube stores the conjugate of the first nucleic acid molecule and the antibody.
  • the first nucleic acid molecule contains 55 bases, and the 3' end is modified with an NH2C7 group.
  • the 3'C7 amino-modified primer was purchased from Genscript Biotechnology Co., Ltd.
  • the NH2C7 group is covalently connected to the amino group of the antibody through the first coupling agent disuccinimide suberate sodium salt (BS3), thereby forming a conjugate of the first nucleic acid molecule and the antibody.
  • BS3 first coupling agent disuccinimide suberate sodium salt
  • the second storage tube stores the substrate protein conjugate, the second nucleic acid molecule and the conjugate of the second fluorescent group.
  • Antibodies and substrate protein conjugates form immune complexes in the absence or presence of small amounts of small molecules.
  • the second nucleic acid molecule contains 53 bases, with a thiol group modified at the 3' end and an NH2C6 group modified at the 5' end.
  • Thiol modification reagent 3'SH C6 and 5'Aminolinker (C6) modified primers were purchased from Genscript Biotechnology Co., Ltd.
  • the 5’ end modification is added to the 5’ sugar ring in the form of phosphorite amine through a ⁇ -cyanoethyl chemical reaction in the last step of the synthesis cycle, rather than to the last base.
  • the model of the amino-modified water-soluble quantum dots can be amino-water-soluble quantum dots (PEG)-605, which is composed of CdSe/ZnS and is purchased from Xi'an Qiyue Biotechnology Co., Ltd.
  • PEG-605 has a maximum absorption peak wavelength of 470 nm, a maximum emission wavelength of 605nm, and a particle size of 4.1nm.
  • the amino group of the amino-modified quantum dot is coupled with the thiol group at the 3' end of the second nucleic acid molecule to form a conjugate of the second nucleic acid molecule and the quantum dot.
  • the NH2C6 group at the 5' end of the second nucleic acid molecule is covalently bonded to the amino group on the substrate protein conjugate through the second coupling agent BS3, forming a coupling between the substrate protein conjugate, the second nucleic acid molecule and the second fluorescent group.
  • the third storage tube stores the conjugate of the first fluorescent group and the third nucleic acid molecule; the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule can form a neck-loop structure under the conditions of immune complex formation; the first The fluorescent group and the second fluorescent group can be located on the same side of the neck ring structure.
  • the third nucleic acid molecule contains 22 bases, and the 5' end is modified with an NH2C6 group.
  • the modified primer 5'Aminolinker (C6) is added to the sugar ring at the 5' end of the third nucleic acid molecule through a ⁇ -cyanoethyl chemical reaction in the form of phosphorus amine in the last step of the synthesis cycle, instead of being added to the last base.
  • the modified primer was purchased from Genscript Biotechnology Co., Ltd.
  • the conjugate of the first fluorescent group and the third nucleic acid molecule is formed by adding acridinium ester (NSP-DMAE-NHS).
  • the first fluorescent group acridinium ester was purchased from Suzhou Yake Technology Co., Ltd., CAS number is 194357 -64-7.
  • the 3 to 10 base positions starting from the 5' end of the third nucleic acid molecule are the same as the 3 to 10 base positions starting from the 3' end of the second nucleic acid molecule (i.e., the 44th to 51st bases starting from the 5' end). base site) are completely complementary.
  • the 5 to 12 base positions starting from the 3' end of the third nucleic acid molecule i.e., the 11th to 18th base positions starting from the 5' end
  • the bases at each base site are completely complementary, as shown in Figure 1.
  • the fourth storage tube stores antioxidants capable of inhibiting oxidation of the first fluorescent group.
  • Antioxidants can bind to the surface of carrier molecules.
  • the carrier molecule can be graphene oxide.
  • the fifth storage tube stores an oxidant capable of oxidizing the first fluorescent group to emit the first fluorescence.
  • the detection principle of the small molecule substance kit in the above embodiment is as follows:
  • the third nucleic acid molecule has 8 bases that are complementary to the first nucleic acid molecule and the second nucleic acid molecule, and the natural base pairs G and C are replaced with unnatural base pairs iso G and iso C.
  • the third nucleic acid molecule is adsorbed on the surface of graphene oxide (GO) through ⁇ - ⁇ stacking, and its end-labeled acridinium ester (AE) is resistant to It cannot oxidize and emit light due to the presence of oxidant. Even if a small part of acridinium ester produces background fluorescence, the chemiluminescence of acridinium ester (430nm wavelength) cannot be detected by PMT due to the instrument's filter (only allowing 605nm light to pass through). detected.
  • the antibody and substrate protein conjugate can form an immune complex. Since the antibody is coupled to the first nucleic acid molecule, and the substrate protein conjugate is coupled to the second nucleic acid molecule, the immune complex brings the first nucleic acid molecule and the second nucleic acid molecule close enough to form an ortho-complex, and can interact with The third nucleic acid molecule hybridizes, so that the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule form a neck ring structure, and the first fluorescent group and the second fluorescent group are located on the same side of the neck ring structure.
  • the complex formed by the antibody and DNA is basically not adsorbed by the carrier molecule graphene oxide, so the antioxidant coupled to the carrier molecule will basically not affect the luminescence of acridinium ester.
  • the added oxidizing agent can oxidize the first fluorescent group and cause it to emit the first fluorescence.
  • the first fluorescence excites the second fluorescent group to emit the second fluorescence (for example, 605nm) based on fluorescence resonance energy transfer. ), in order to obtain the content of small molecule substances based on the intensity of the second fluorescence.
  • This application provides a small molecule substance detection system, which includes: a reaction container, a micro-syringe pump, an optical filter and a calculation module.
  • the reaction vessel has a holding chamber capable of holding the solution to be tested.
  • the microinjection pump is connected to the accommodation chamber of the reaction vessel through the injection pipeline, and injects the mixture of the first conjugate, the second conjugate, the third conjugate and the oxidation inhibitor reagent into the accommodation chamber;
  • the first A conjugate is formed by coupling at least a first nucleic acid molecule and an antibody
  • a second conjugate is formed by at least conjugating a substrate protein conjugate, a second nucleic acid molecule and a second fluorescent group in sequence
  • a third coupling is The substance is at least formed by coupling a first fluorescent group and a third nucleic acid molecule
  • the oxidation inhibiting reagent contains an antioxidant that inhibits the first fluorescent group from being oxidized and emitting the first fluorescence.
  • the filter is disposed on the exit light path of the first fluorescence and allows the transmission of the second fluorescence that is the same as the maximum emission wavelength of the second fluorescence group.
  • the light signal detection module is located on the downstream side of the filter in the emission light path of the fluorescence signal, and acquires the second fluorescence transmitted through the filter.
  • the calculation module converts the second fluorescence into a digital signal and obtains the content of the small molecule substance in the solution to be tested based on the functional relationship between the fluorescence intensity and the content of the small molecule substance.
  • the standard curve contains a standard equation, which contains a one-to-one correspondence between the digital signal and the content of the small molecule substance.
  • the embodiments of this application provide a simple, fast, and sensitive homogeneous chemiluminescence immunoassay protein through the dual quenching mechanism of graphene oxide coupled antioxidants and filters, combined with chemiluminescence resonance energy transfer and immunoassay technology. Detection method. Compared with existing immunoassay methods, some embodiments of the present application have the following characteristics:
  • Some embodiments of the present application are homogeneous immunoassay methods, which are simple to operate and greatly shorten the turnover time (TAT) of clinical test specimens. There is no need for centrifugation of blood samples and whole blood can be loaded in about 5 minutes. Issue a test report.
  • the acridinium ester emits oxidation light
  • the quantum dots emits excitation light, which can realize self-excited emission between acridinium ester and quantum dots without the need to set up complex external excitation light.
  • the system can effectively reduce the complexity and cost of measuring instruments. As the requirements for supporting testing equipment are reduced, the modules are reduced, the cost is reduced, and the failure rate is also greatly reduced. Automatic testing or miniaturized portable point-of-care testing (POCT) can be achieved.
  • POCT portable point-of-care testing
  • the acridinium ester-quantum dot luminescence system of some embodiments of the present application introduces a dual quenching mechanism (graphene oxide-reducing agent and filter), with lower background fluorescence and higher detection sensitivity, and is suitable for Higher sensitivity detection.
  • Some embodiments of the present application induce the switching of the graphene oxide-antioxidant quenching mechanism through an immune reaction, and with the introduction of optical filters, the switching efficiency reaches 100%. Combined with the chemiluminescence resonance energy transfer effect, the quantum Spot luminescence eliminates the need for separation and cleaning steps.
  • the DNA molecules in some embodiments of the present application contain non-natural base pairs (isoG and isoC) to avoid non-specific binding to nucleic acids in the sample.
  • This preparation example provides a method for preparing a conjugate of a second nucleic acid molecule and a quantum dot, which includes the following steps:
  • SMCC solution Weigh 10 mg of 4-(N-Maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester , SMCC) was dissolved in 1mL DMF. SMCC was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., the item number is N159712, and the CAS number is 64987-85-5.
  • This preparation example provides a method for preparing a conjugate of acridinium ester (AE) and a third nucleic acid molecule, which includes the following steps:
  • NHS-AE solution Weigh 4 mg of acridinium ester (NSP-DMAE-NHS) and dissolve it in 1 mL of purified water.
  • Acridinium ester was purchased from Suzhou Yake Technology Co., Ltd., CAS number 194357-64-7.
  • This preparation example provides a method for preparing a conjugate of a first nucleic acid molecule and an antibody, which includes the following steps:
  • BS3 solution Weigh 10 mg of disuccinimide suberate sodium salt (BS3) and dissolve it in 1 mL of purified water.
  • BS3 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number S304724.
  • Dialysis Aspirate the conjugate of the first nucleic acid molecule and antibody from the EP tube, add it to the dialysis bag (100kd specification), tie the dialysis bag and put it into a beaker containing 2-3L PBS solution, and perform dialysis ( Soak the dialysis bag in advance), change the dialysate once every 2-3 hours, and dialyze three times in total. After dialysis, collect the liquid in the dialysis bag into a centrifuge tube and store it at 2-8°C for later use.
  • This preparation example provides a method for preparing a conjugate of a substrate protein conjugate, a second nucleic acid molecule, and a second fluorescent group, which includes the following steps:
  • BS3 solution Weigh 10 mg of disuccinimide suberate sodium salt (BS3) and dissolve it in 1 mL of purified water.
  • BS3 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
  • Activation Take out the packaged substrate protein conjugate (conjugate of small molecule substrate and scaffold protein), thaw, centrifuge and mix. Add 3 ⁇ L of BS3 solution for every 1 mg of substrate protein conjugate, add 6.5 ⁇ L of the conjugate of the second nucleic acid molecule and quantum dots, mix evenly in the EP tube, and incubate at 37°C for 30 minutes.
  • substrate protein conjugate conjugate of small molecule substrate and scaffold protein
  • Dialysis Aspirate the conjugate of the substrate protein conjugate, the second nucleic acid molecule and the second fluorescent group from the EP tube, add it to the dialysis bag (specification 100kd), tie the dialysis bag and put it into the dialysis bag containing 2- In a 3L PBS solution beaker, perform dialysis (soak the dialysis bag in advance), change the dialysate once every 2-3 hours, and dialyze three times in total. After dialysis, collect the liquid in the dialysis bag into a centrifuge tube and store it at 2-8°C. stand-by.
  • This example uses a homogeneous immunoassay method based on graphene oxide-antioxidant quenching and acridinium ester chemiluminescence to detect free triiodothyronine (FT3) in serum.
  • the antibody was purchased from Bioventix, with the clone number 17c6r;
  • the substrate protein conjugate also known as the scaffold small molecule
  • the antibodies, substrate protein conjugates and corresponding DNA molecules are coupled through the above preparation examples.
  • the specific detection method includes the following steps:
  • chemiluminescent substrate After incubation, add 200 ⁇ L of chemiluminescent substrate through the HSCL-10000 chemiluminescence instrument.
  • PMT photomultiplier tube
  • Table 1 is a comparison table of the detection values of this embodiment and the Roche detection method.
  • This example uses a homogeneous immunoassay method based on graphene oxide-antioxidant quenching and acridinium ester chemiluminescence to detect tetraiodothyronine (serum T4) in serum.
  • the antibody was purchased from Nanjing Okai Biotechnology Co., Ltd., with the clone number K88a6, and the substrate protein conjugate (also known as scaffold small molecule) was purchased from Shanghai Hanzun Biotechnology, with the clone number T4-BSA-HT-020561.
  • the antibody, substrate protein conjugate and corresponding DNA are coupled through the above preparation example.
  • the specific detection method includes the following steps:
  • PMT photomultiplier tube
  • the detection limit of serum FT4 in this example is 0.5-100 pmol/L. After testing 40 clinical samples, the error between the FT4 detection value of this embodiment and the Roche test value was -1.66%, indicating that the detection method of this embodiment has high accuracy.
  • the test results are shown in Table 2 below.
  • the Roche test refers to the Roche FT4 kit.
  • Table 2 is a comparison table of the detection values of this embodiment and the Roche detection method.
  • This embodiment uses a homogeneous immunoassay method based on graphene oxide-antioxidant quenching and acridinium ester chemiluminescence to detect progesterone (Prog) in serum.
  • the antibody was purchased from Biospacific, with the clone number PPOGESTERONE MAB, PURE/A25050;
  • the substrate protein conjugate also known as scaffold small molecule was purchased from Biospacific, with the clone number PPOGESTERONE-11-BSA/V56050.
  • the antibody, substrate protein conjugate and corresponding DNA are coupled through the above preparation example.
  • the specific detection method includes the following steps:
  • PMT photomultiplier tube
  • the detection limit of Prog in this example is 0.05-60ng/mL. After testing 40 clinical samples, the error between the Prog detection value of this embodiment and the Roche test value was 2.63%, indicating that the detection method of this embodiment has high accuracy.
  • the test results are shown in Table 3 below.
  • Roche test refers to the Roche Prog test kit.
  • Table 3 is a comparison table of the detection values of this embodiment and the Roche detection method.

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Abstract

一种检测小分子物质的试剂组合、试剂盒、检测***及检测方法。其中，在待测溶液中没有含有或含有少量小分子物质的条件下，抗体和底物蛋白结合物相结合并形成免疫复合物，多个核酸分子之间的互补配对产生颈环结构，第一荧光基团基于荧光共振能量转移激发第二荧光基团发射第二荧光，并计算出小分子物质的含量。在待测溶液中含有大量小分子物质的条件下，无法形成免疫复合物和颈环结构，第二荧光基团无法发射第二荧光，故判断出待测溶液中含有大量小分子物质。本申请的检测方法简单、背景干扰小、灵敏度高、测量误差小。

Description

检测小分子物质的试剂组合、试剂盒、检测***及检测方法
相关申请的交叉引用
本申请要求于2022年06月23日提交中国专利局、申请号为202210718352.5、发明名称为“检测小分子物质的试剂组合、试剂盒、检测***及检测方法”的中国专利申请的优先权，其全部内容通过引用的方式并入本文中。
技术领域
本申请属于化学发光检测技术领域，具体涉及一种检测小分子物质的试剂组合、试剂盒、检测***及检测方法。
背景技术
人体的血液是个复杂的多组分体系，含有多种调控因子、激素等小分子物质。其中，部分小分子物质在健康组织中不分泌，而在病变组织中才分泌。这些异常分泌的小分子物质会进入血液中，因此，可以根据血液中是否存在该类小分子物质来判断人体中是否存在健康组织或病变组织。检测血液中是否存在小分子物质可以采用化学发光免疫分析法(Chemiluminescence immunoassay，CLIA)。该方法将一个发光基团与抗小分子物质的抗体相连，以制成检测试剂。当血液样品中不存在小分子物质时，抗体无法与血液中的小分子物质特异性结合，此时，发光基团一般不具有发光活性，除了本底荧光之外，不会产生较强的荧光强度。当待测溶液中存在小分子物质时，抗体能够与小分子物质特异性结合，发光基团在发光底物的作用下发射荧光。故可以通过采用化学发光检测仪器检测待测溶液中是否存在荧光来判断小分子物质血液中是否存在小分子物质。
然而，上述方法采用单一荧光基团来发射荧光，并根据该单一荧光基团是否发射荧光来判断是否存在小分子物质。由于单一荧光基团在没有小分子物质的存在下也会发射本底荧光，该本底荧光会影响对荧光的获取，从而在测试小分子物质的含量时造成较大的误差。
发明内容
本申请的一些实施例的目的在于提供一种检测小分子物质的试剂组合、试剂盒、检测 ***及检测方法，能够避免现有的化学发光检测法的单一荧光基团的本底荧光对检测结果造成影响。本文中提到的一些实施例可以来自相同的实施例，也可以来自不同的实施例。
为了解决上述技术问题，本申请的一些实施例提供了一种用于检测小分子物质的试剂组合。该试剂组合至少包括：第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂。
其中，第一偶联物至少由第一核酸分子和抗体偶联而成。第一核酸分子含有第一杂交区和第二杂交区。第一杂交区和第二杂交区并非直接相邻，而是相隔2个或5个核苷酸。抗体的互补决定区(Complementarity determining region，CDR)能与小分子物质特异性结合。在本申请的各个实施例中，核酸分子均为单链DNA。在本申请中，在描述各个单链DNA时，其连接方式从左到右是从5’端到3’端。
第二偶联物至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成。底物蛋白结合物至少由小分子底物与支架蛋白偶联而成。小分子底物与待测的小分子物质是相同的物质。为了区分起见，与支架蛋白相结合的物质称为小分子底物，游离于待测溶液中的物质称之为小分子物质。第二核酸分子具有第三杂交区和第四杂交区。第三杂交区与第四杂交区直接相连，中间并没有间隔核苷酸。第三杂交区与第二杂交区互补。对于抗体而言，小分子底物仅有一个抗原表位，该抗原表位能与抗体的CDR特异性结合，并且该结合是可逆的，能被游离于待测溶液中的小分子物质逆转结合。也即，处于游离状态的小分子物质与处于结合状态的小分子底物能够与抗体的抗原表位竞争性结合。每个抗体的CDR只能同时结合1个小分子底物或者1个小分子物质。
第三偶联物至少由第一荧光基团和第三核酸分子偶联而成。第三核酸分子含有第五杂交区和第六杂交区。第五杂交区与第六杂交区直接相连，中间并没有间隔核苷酸。第五杂交区与第四杂交区互补，第六杂交区与第一杂交区互补。第一核酸分子、第二核酸分子和第三核酸分子均为单链DNA。通过上述单链DNA分子之间的互补配对，第一核酸分子、第二核酸分子和第三核酸分子能够组装成颈环结构(Stem-loop structure)。颈部的结构为⊥形，互相配对的区域呈现双螺旋结构。
在一些实施例中，在抗体与底物蛋白结合物中的小分子底物相结合而并非与待测溶液中游离的小分子物质相结合的情况下，第一荧光基团在能被氧化剂氧化并且在不存在抗氧化剂的条件下发出第一荧光，第一荧光作为激发光在第一核酸分子、第二核酸分子和第三核酸分子相互配对的条件下基于荧光共振能量转移效应激发第二荧光基团发出第二荧光， 以便根据第二荧光的强度获得小分子物质的含量。如果完全没有获得第二荧光的强度，那么说明小分子物质的含量太高，全部的抗体均与游离的小分子物质相结合，而并非与底物蛋白结合物中的小分子底物相结合，超过了检测限，此时，无法根据荧光强度得到小分子物质的含量。
在一些实施例中，当待测溶液中小分子物质的浓度较低时，小分子物质与抗体的结合程度较弱，而小分子底物与抗体的结合程度较强，那么第二荧光的强度越强。因此，待测溶液中小分子物质的浓度与第二荧光的强度呈反比关系。可以事先获得第二荧光的强度与小分子物质的含量之间的函数关系式，从而在二者之间建立起一一对应的数值关系，因此，就可以将对小分子物质的含量的测量转换为对荧光强度的数值的测量。
氧化抑制试剂包括抗氧化剂。该抗氧化剂能够抑制第一荧光基团被氧化而发出第一荧光。本申请各个实施例中的第一荧光基团为氧化式发光，而并非受到激发光的照射而发荧光，因此，需要尽量降低第一荧光基团被待测溶液中的氧化性物质氧化而产生背景荧光。背景荧光属于噪音的一种，会对荧光测量的真实值产生影响，进而影响小分子物质含量的测量结果的准确性。抗氧化剂与氧化剂不能同时存在于待测样品中，以免氧化剂对第一荧光基团的氧化发光作用被抗氧化剂中和。因此，需要先移除抗氧化剂再加入氧化剂。
在本申请的一些实施例中，第一荧光基团与第二荧光基团之间发生荧光共振能量转移效应需要具备以下基本条件：第一核酸分子、第二核酸分子、第三核酸分子在待测溶液中不存在小分子物质的条件下完全互补配对，此时产生的第二荧光的强度最大。第一核酸分子、第二核酸分子、第三核酸分子在待测溶液中存在较低浓度的小分子物质在的条件下部分互补配对，此时也会产生第二荧光。互补配对会形成颈环结构，使第一荧光基团与第二荧光基团的间距小于能够发生荧光共振能量转移的极限间距，例如，在70埃至99埃的范围内，或者在7nm至10nm的范围内。
在本申请的一些实施例中，第一荧光基团可以为吖啶酯，第二荧光基团可以为量子点。
在本申请的一些实施例中，第一荧光基团的最大发射波长为430nm。
第二荧光基团的最大吸收波长可以在420nm至520nm的范围内，如可以为470nm。第二荧光基团的最大发射波长可以在595nm至615nm的范围内，如可以605nm。在本申请的一些实施例中，量子点为核壳结构量子点，其核层材料选自CdSe、CdS、CdTe、CdSeTe、CdZnS、ZnTe、CdSeS、PbS和PbTe中的一种或多种，其壳层材料选自ZnS、ZnSe、ZnSeS、 PbS和PbSeS中的一种或多种。
在本申请的一些实施例中，量子点的粒径范围可以为3nm至5nm，也可以为4.1nm至4.2nm。
在本申请的一些实施例中，第一核酸分子的3’末端的糖环通过第一偶联剂与抗体的氨基共价连接。第一核酸分子的5’末端的糖环没有修饰。可选地，第一核酸分子的3’末端的糖环经过NH2C7修饰基团修饰，NH2C7修饰基团通过第一偶联剂与抗体的氨基共价连接。可选地，第一偶联剂为双琥珀酰亚胺辛二酸酯钠盐。
在本申请的一些实施例中，第二核酸分子的3’末端与第二荧光基团连接。可选地，第二核酸分子的3’末端的糖环经过巯基修饰，第二荧光基团的表面经过氨基修饰，巯基通过第三偶联剂与第二荧光基团的表面的氨基共价连接。可选地，第三偶联剂为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯。
在本申请的一些实施例中，第二核酸分子的5’末端的糖环通过第二偶联剂与底物蛋白结合物的支架蛋白的氨基共价连接。可选地，第二核酸分子的5’末端的糖环经过NH2C6修饰基团修饰，NH2C6修饰基团通过第二偶联剂与支架蛋白的氨基共价连接。可选地，第二偶联剂为双琥珀酰亚胺辛二酸酯钠盐。
在本申请的一些实施例中，第三核酸分子的5’末端与第一荧光基团共价连接。可选地，第三核酸分子的5’末端的糖环经过NH2C6修饰基团修饰，NH2C6修饰基团与第一荧光基团共价连接。
在本申请的一些实施例中，第一核酸分子中，按照从5’端到3’端的顺序，第一杂交区位于第二杂交区的上游。第二核酸分子中，按照从5’端到3’端的顺序，第三杂交区位于第四杂交区的上游。第三核酸分子中，按照从5’端到3’端的顺序，第五杂交区位于第六杂交区的上游。
在本申请的一些实施例中，第一核酸分子具有55个核苷酸，第一杂交区覆盖第一核酸分子从5’端开始的第3至10个碱基位点，第二杂交区覆盖第一核酸分子从5’端开始的第13至19个碱基位点。第二核酸分子具有53个核苷酸，第三杂交区覆盖第二核酸分子从5’端开始的37至43个碱基位点，第四杂交区覆盖第二核酸分子从5’端开始的第44至51个碱基位点。第三核酸分子具有22个核苷酸，第五杂交区覆盖第三核酸分子从5’端开始的第3至10个碱基位点，第六杂交区覆盖第三核酸分子从5’端开始的第11至18个碱基位点。 六个DNA序列之间的互补配对使得第一核酸分子、第二核酸分子、第三核酸分子两两杂交而形成颈环结构，颈部的形状为⊥形。
在本申请的一些实施例中，第一杂交区从5’端到3’端为GCTGAGTT，第六杂交区从5’端到3’端为AACTCAGC。第二杂交区从5’端到3’端为CAACGAC，第三杂交区从5’端到3’端为GTCGTTG。第四杂交区从5’端到3’端为GCTGAGAT，第五杂交区从5’端到3’端为ATCTCAGC。每个杂交区都是单链DNA，而并非双链DNA，同一条单链DNA内的两个DNA序列并不相互配对，而是与另外的单链DNA的DNA序列相互配对。
在本申请的一些实施例中，第一核酸分子的全长序列如SEQ ID No：1所示，第二核酸分子的全长序列如SEQ ID No：2所示，第三核酸分子的全长序列如SEQ ID No：3所示。
在本申请的一些实施例中，第一核酸分子、第二核酸分子和/或第三核酸分子中的G可以被 isoG替代，C可以被 isoC替代。 isoG和 isoC为非天然碱基对。采用非天然碱基对进行配对可以有效避免第一核酸分子、第二核酸分子和/或第三核酸分子与待测溶液中的天然核酸进行错配，从而避免错配影响颈环结构的形成，进而避免错配所导致的测量误差。
其中， isoG的结构式为：
Figure PCTCN2022114296-appb-000001
折线表示连接位点。
isoC的结构式为：
Figure PCTCN2022114296-appb-000002
isoG与 isoC的键合方式为：
Figure PCTCN2022114296-appb-000003
Figure PCTCN2022114296-appb-000004
表示与DNA分子上的脱氧核糖相连接。
在本申请的一些实施例中，第一杂交区和第六杂交区中的G被 isoG替代，C被 isoC替代。第一杂交区从5’端到3’端为 isoG isoCT isoGA isoGTT，第六杂交区从5’端到3’端为AA isoCT isoCA isoG isoC。
在本申请的一些实施例中，第二杂交区和第三杂交区中的G被 isoG替代，C被 isoC替代。第二杂交区从5’端到3’端为 isoCAA isoC isoGA isoC，第三杂交区从5’端到3’端为 isoGT isoC isoGTT isoG。
在本申请的一些实施例中，第四杂交区和第五杂交区中的G被 isoG替代，C被 isoC替代。第四杂交区从5’端到3’端为 isoG isoCT isoGA isoGAT，第五杂交区从5’端到3’端为AT isoCT isoCA isoG isoC。
在本申请的一些实施例中，第一核酸分子的全长序列为：
A isoC isoG isoCT isoGA isoGTTAT isoCAA isoC isoGA isoCTTTTTTTAT isoCA isoCAT isoCA isoG isoG isoCT isoCTA isoG isoC isoGTAT isoG isoCTATT isoG。在本申请的另一些实施例中，第一核酸分子的全长序列可以为如SEQ ID No：1所示的序列中至少部分或全部G被 isoG替代并且至少部分或全部C被 isoC替代所形成的序列。
在本申请的一些实施例中，第二核酸分子的全长序列为：
TA isoC isoGT isoC isoCA isoGAA isoCTTTA isoC isoCAAA isoC isoCA isoCA isoC isoC isoCTTTTTTT isoGT is oC isoGTT isoG isoG isoCT isoGA isoGATT isoC。在本申请的另一些实施例中，第二核酸分子的全长序列为如SEQ ID No：2所示的序列中至少部分或全部G被 isoG替代并且至少部分或全部C被 isoC替代所形成的序列。
在本申请的一些实施例中，第三核酸分子的全长序列为：
isoC isoGAT isoCT isoCA isoG isoCAA isoCT isoCA isoG isoCA isoG isoC isoG。在本申请的另一些实施例中，第三核酸分子的全长序列为如SEQ ID No：3所示的序列中至少部分或全部G被 isoG替代并且至少部分或全部C被 isoC替代所形成的序列。
在本申请的一些实施例中，氧化抑制试剂还包括载体分子，载体分子的表面结合抗氧化剂。因为第一荧光基团为氧化式发光，而非受激式发光，一旦有氧化性物质的存在，第一荧光基团便可能发出荧光。抗氧化剂的作用主要是防止第一荧光基团被这些具有氧化能力的物质氧化，从而产生对测量结果可能造成影响的背景荧光。这些具有氧化能力的物质可以来自于待测溶液或血液样本等。抗氧化剂选自***二酚、维生素C、维生素E、茶多酚、谷胱甘肽中的任意一种或几种。在本申请的一些实施例中，氧化剂包括过氧化氢的碱性溶液。氧化剂又可称为第一荧光基团的化学发光底物，因为这些底物本身就具有氧化性。
在本申请的一些实施例中，载体分子可以为氧化石墨烯。氧化石墨烯上的一部分羧基通过氧化亚砜缩合剂与抗氧化剂上的羟基结合，氧化石墨烯上的一部分羧基通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与抗氧化剂上的氨基结合，从而抗氧化剂附着于氧化石墨烯上。因为抗氧化剂和氧化剂不能同时加入待测溶液中，否则氧化剂就不能使第一荧光基团氧化发光，所以在加入氧化剂之前需要从待测溶液中去除抗氧化剂。将抗氧化剂结合于氧化石墨烯载体上更有利于去除抗氧化剂。
在本申请的各个实施例中，采用两个荧光基团来产生用于测量的第二荧光，第一荧光基团为氧化型发光，第二荧光基团为光激发型发光，并且第一荧光基团发射的第一荧光能够基于荧光共振能量转移激发第二荧光基团发射第二荧光。在采集到第二荧光的情况下，即可判断出待测溶液中是否存在小分子物质或者得到小分子物质的浓度，若第二荧光的强度达到最大值，说明游离状态的小分子物质不会与结合状态的小分子底物竞争性结合抗体，那么待测溶液中完全不含有小分子物质。另外，因为小分子物质的含量与第二荧光的强度呈反比关系，根据第二荧光的强度也能得到小分子物质的含量。该种检测方法排除了第一荧光基团的本底荧光对于测量结果的影响，提高了检测的灵敏度和准确性。另外，本申请各个实施例的两个荧光基团无需设置额外的激发光光源就能发射荧光信号，也降低了检测***的复杂性，能够实现可移动式检测。再者，本申请的各个实施例中的第一荧光基团为闪光型发光，检测的时间很短，能够快速得到检测结果。
本申请的一些实施例还提供了一种小分子物质的检测方法，其包括如下步骤：
(1)、在待测溶液中加入第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂，混合后形成待测样品。
在一些实施例中，在步骤(1)中，第一偶联物至少由第一核酸分子和抗体偶联而成， 第一核酸分子含有第一杂交区和第二杂交区，抗体能与小分子物质特异性结合。第二偶联物至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成，第二核酸分子具有第三杂交区和第四杂交区，第三杂交区与第二杂交区互补。底物蛋白结合物的小分子底物能与抗体的互补决定区特异性结合。第三偶联物至少由第一荧光基团和第三核酸分子偶联而成。第三核酸分子含有第五杂交区和第六杂交区，第五杂交区与第四杂交区互补，第六杂交区与第一杂交区互补。在待测溶液中没有含有游离的小分子物质或者含有的小分子物质的浓度较低的条件下，抗体通过与小分子物质相结合而与底物蛋白结合物形成免疫复合物，第一核酸分子、第二核酸分子和第三核酸分子形成颈环结构，第一荧光基团与第二荧光基团位于颈环结构的同侧，故两个荧光分子之间能够发生荧光共振能量转移，由此，第二荧光基团被激发而发射第二荧光。
(2)、从待测样品中去除用于抑制第一荧光基团被氧化而发射荧光的氧化抑制试剂，加入用于氧化第一荧光基团而发射第一荧光的氧化剂并按照第二荧光基团的最大发射波长收集第二荧光，即排除了最大发射波长之外的其它波长的荧光。
(3)、在收集到第二荧光的情况下，根据第二荧光的强度和荧光强度与小分子物质含量的一一对应关系获得小分子物质的含量。
在收集第二荧光的强度时，为了避免第一荧光对第二荧光产生干扰，可以采用滤光片过滤掉第一荧光基团被氧化后产生的荧光，只允许第二荧光透过该滤光片，由此收集第二荧光基团发出的第二荧光，并根据第二荧光的强度获得小分子物质的含量。
待测溶液可以来自于血液样本。血液的成分比较复杂，会含有多种氧化性物质，如果第一荧光基团被这些氧化性物质氧化，便会产生本底荧光，从而影响测量结果的准确性，因此，本申请为了降低本底荧光对测量结果的影响，并不采用第一荧光基团发射的第一荧光作为检测荧光信号，而采用第二荧光基团发射的第二荧光作为检测荧光信号。
在本申请的一些实施例中，待测样品中，第一偶联物的工作浓度可以为1nM至20nM。第二偶联物的工作浓度可以为1nM至20nM。第三偶联物的工作浓度可以为0.05nM至0.2nM。氧化抑制试剂的工作浓度可以为15μg/ml至25μg/ml。
在本申请的一些实施例中，待测溶液来自全血样本、血清样本或者血浆样本等血液样本。
在本申请的一些实施例中，混合的时间可以为5分钟至10分钟。
在本申请的一些实施例中，混合的温度可以为36度至37度。
在本申请的一些实施例中，氧化剂的体积可以为200μL，氧化剂为碱性过氧化氢溶液，pH值为8.0。该碱性过氧化氢溶液是将双氧水溶解于TBS缓冲液中进行的，其中，过氧化氢的终浓度为0.1M，TBS的终浓度为10mM。
在本申请的一些实施例中，小分子物质包括三碘甲状腺原氨酸、四碘甲状腺原氨酸、或孕酮。
在本申请的一些实施例中，经过混合后的待测样品中，在待测溶液中不含有或含有少量小分子物质的条件下，第一偶联物中的抗体与小分子底物形成免疫复合物，该免疫复合物使三条单链DNA的互补序列的距离拉进，两两互补配对并实现DNA之间的组装，从而形成颈环结构。
本申请的一些实施例还提供了一种小分子物质检测试剂盒，其包括：第一储存管、第二储存管、第三储存管、第四储存管和第五储存管。
其中，第一储存管至少储存第一核酸分子和抗体的偶联物。
第二储存管至少储存底物蛋白结合物、第二核酸分子和第二荧光基团的偶联物。抗体和底物蛋白结合物在小分子物质不存在或存在少量小分子物质的条件下能与小分子底物形成免疫复合物。相反，如果存在过量的小分子物质，那么小分子物质就会竞争性结合原本与小分子底物结合的抗体，不会形成免疫复合物，从而就不会引发单链DNA之间的组装。
第三储存管至少储存第一荧光基团和第三核酸分子的偶联物。第一核酸分子、第二核酸分子和第三核酸分子在免疫复合物形成的条件下基于邻近效应而组装成颈环结构，颈环结构的空间构象使得第一荧光基团与第二荧光基团自发排列于颈环结构的同侧，并且二者之间的空间距离小于能够发生荧光共振能量转移的极限距离，例如，在70埃至99埃的范围内，这为两个荧光基团之间发生荧光共振能量转移提供了条件。
第四储存管至少储存能够抑制第一荧光基团被氧化的抗氧化剂。可选地，抗氧化剂可以修饰于载体分子上，从而成为氧化抑制试剂。可选地，载体分子可以选自氧化石墨烯。
第五储存管至少储存氧化剂。该氧化剂能够氧化第一荧光基团以发射第一荧光。氧化剂与抗氧化剂并不同时存在于待测样品中。在加入氧化剂之前，抗氧化剂和载体分子需要从待测样品中移除。因为抗氧化剂修饰于载体分子上，而载体分子氧化石墨烯呈现片层网络结构，极易从待测样品中移除，所以可以通过移除载体分子来实现对抗氧化剂的移除。 另外，氧化石墨烯也能吸附游离的第三核酸分子，从而吸附第一荧光基团和第三核酸分子的偶联物，使得抗氧化剂抑制处于游离状态的第一荧光基团被不当氧化，从而抑制第一荧光基团不当发射背景荧光。
在本申请的实施例中，第一核酸分子含有第一杂交区和第二杂交区；第二核酸分子具有第三杂交区和第四杂交区；第三核酸分子含有第五杂交区和第六杂交区；第三杂交区与第二杂交区互补，第五杂交区与第四杂交区互补，第六杂交区与第一杂交区互补。第一杂交区、第二杂交区、第三杂交区、第四杂交区、第五杂交区和第六杂交区至少部分或者全部含有碱基 isoG和 isoC。由于采用 isoG替代了天然碱基G， isoC替代了天然碱基C，从而降低了错配的可能性。
在本申请的实施例中，小分子物质检测试剂盒的检测原理如下所示。
在待测溶液中不含有小分子物质或者含有少量小分子物质的条件下，抗体和底物蛋白结合物全部或部分形成免疫复合物。第一核酸分子、第二核酸分子和第三核酸分子的距离接近，发生杂交，并自组装成颈环结构。三条核酸分子(即单链DNA)中的六个DNA序列(即杂交区)两两互补配对形成颈环结构的颈部，其余的非DNA序列形成颈环结构的环部。每条单链DNA内部的两个DNA序列不能互补配对，只能与另外的单链DNA的互补序列配对。颈环结构的空间构象使得第一荧光基团与第二荧光基团位于颈环结构的同侧并且二者的距离能够保证二者之间发生荧光共振能量转移。在移除氧化抑制试剂的条件下，氧化剂能氧化第一荧光基团并使其发射第一荧光，第一荧光基于荧光共振能量转移效应激发第二荧光基团发出第二荧光，以便根据第二荧光的强度获得小分子物质的含量。
在待测溶液中含有过量的小分子物质的条件下，免疫复合物和颈环结构无法形成，即便移除了抗氧化剂并且随后加入的氧化剂使第一荧光基团发射第一荧光，但是第一荧光基团与第二荧光基团的距离大于能发生荧光共振能量转移的极限距离，那么第一荧光基团就无法激发第二荧光基团发射第二荧光。无法获得第二荧光的强度说明待测溶液中含有过量的小分子物质。
本申请的实施例中第二荧光基团在激发光的激发作用下才能发射第二荧光，如果没有第一荧光的激发作用，那么第二荧光基团就不会发射用于测量小分子物质含量的第二荧光，因此，第二荧光基团作为检测基团基本不会发射背景荧光，这保证了采用第二荧光作为检测荧光的可靠性。
本申请的一些实施例提供了一种小分子物质检测***，其包括：反应容器、微量注射泵、滤光片、光信号检测模块和计算模块。
其中，反应容器具有能容纳待测溶液的容置腔室。
微量注射泵通过注射管道与容置腔室相连通，将第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂的混合物通过注射管道注射入容置腔室中，以便与待测溶液进行混合；第一偶联物至少由第一核酸分子和抗体偶联而成，第二偶联物至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成，第三偶联物至少由第一荧光基团和第三核酸分子偶联而成，氧化抑制试剂含有抑制第一荧光基团被氧化而发射第一荧光的抗氧化剂。
滤光片设于第一荧光的出射光路上，允许与第二荧光基团的最大发射波长具有相同波长的第二荧光透过。
光信号检测模块设于第一荧光的出射光路上并位于滤光片的下游侧，获取从滤光片透射的第二荧光。第二荧光的强度值可以为0，此时表示待测溶液中存在过量的小分子物质，因为第二荧光基团为受激发光，而并非氧化发光，在没有激发光激发的条件下，第二荧光基团本身是不发射第二荧光的，那么其也就不会产生背景荧光噪音，故在存在过量的小分子物质的条件下，第二荧光的强度能够达到零，而在不存在小分子物质或存在少量小分子物质的条件下，会存在第二荧光。第二荧光的强度值与小分子物质的含量呈反比。而如果采用第一荧光作为检测荧光，由于第一荧光为氧化发光，而待测溶液中会存在氧化性物质，那么第一荧光会被这些氧化性物质氧化而发射背景荧光噪音。因此，采用第二荧光作为检测荧光比采用第一荧光作为检测荧光更准确，误差更小。
计算模块，将所述第二荧光转换为数字信号并且根据荧光强度和小分子物质含量的函数关系式获得待测溶液中小分子物质的含量。
由于采用以上技术方案，本申请的一些实施例能够取得以下技术效果：
本申请的一些实施例采用免疫反应和荧光共振能量转移效应相结合来检测待测溶液中是否含有小分子物质。具体为：
在待测溶液中不含有或含有少量小分子物质的条件下，抗体和底物蛋白结合物基于免疫反应而形成免疫复合物，使得第一核酸分子、第二核酸分子和第三核酸分子的间距拉进，引发DNA组装并形成颈环结构，颈环结构使得第一荧光基团与第二荧光基团位于该颈环结构的同侧，从而在二者之间产生荧光共振能量转移现象并使第二荧光基团发射第二荧光作 为待测荧光信号。根据预设的荧光强度和小分子物质含量的函数关系式和待测荧光信号的强度来获得待测溶液中小分子物质的含量。
在待测溶液中存在过量小分子物质的条件下，免疫复合物和颈环结构无法形成，第二荧光基团不会受到激发光(即第一荧光)的激发而发射第二荧光，那么就无法捕捉到待测荧光信号，说明待测溶液中存在过量的小分子物质。另外，即便第一荧光基团在某些氧化性物质的作用下发射背景荧光，由于颈环结构无法形成，那么此时第一荧光基团与第二荧光基团之间的空间距离大于荧光共振能量转移效应所要求的最小间距，两种荧光基团之间不会发生荧光共振能量转移效应，第二荧光基团也就不会基于该背景荧光发射第二荧光。至少由此实现对待测溶液中是否存在小分子物质的定性检测和对待测溶液中小分子物质含量的定量检测。
本申请的各个实施例采用不易产生背景荧光噪音的第二荧光基团发射的第二荧光作为待测荧光信号，而不采用通常存在背景荧光噪音的第一荧光基团发射的第一荧光作为待测荧光信号，能够大幅度降低背景荧光噪音对检测结果的影响，提高检测的灵敏度和降低检测的误差，使测得的蛋白质含量更接近于真实值。
附图说明
图1是本申请的一些实施例的免疫复合物与颈环结构的示意图。
图2是本申请的一些实施例的检测方法的示意图。图2中，当待测溶液中不存在小分子物质或存在少量的小分子物质(图2所示的Target)时，抗体和小分子底物会形成免疫复合物，单链DNA分子之间会形成颈环结构。在洗脱掉修饰了抗氧化剂的氧化石墨烯之后加入氧化剂(过氧化氢的碱液)，吖啶酯被氧化剂氧化而发射430nm的第一荧光，第一荧光激发量子点发射605nm的第二荧光，此时称为Signal on状态。当存在过量小分子物质时，没有免疫复合物和颈环结构的产生，第三核酸分子由于通过π-π堆积作用吸附在氧化石墨烯表面，在洗脱掉修饰了抗氧化剂的氧化石墨烯时，第三核酸分子的偶联物会随着氧化石墨烯一起被洗脱而留存在洗脱掉的液体中，吖啶酯会被液体中的氧化性物质氧化而产生背景荧光，而洗脱后留存下的液体中存在底物蛋白结合物、以及与小分子物质结合后的抗体。量子点至少由于没有激发光的激发而并不产生荧光，此时称为Signal off状态，说明液体中存在过量的小分子物质。
附图标记：
小分子物质1、抗体2、底物蛋白结合物3、第一核酸分子4、第二核酸分子5、第三核酸分子6、第一荧光基团7、第二荧光基团8、第一杂交区9、第二杂交区10、第三杂交区11、第四杂交区12、第五杂交区13、第六杂交区14、氧化石墨烯和抗氧化剂的复合物15(即氧化抑制试剂)、支架蛋白16、免疫复合物17。
具体实施方式
以下结合具体实施方式对本申请的各个实施例的技术进行详细描述。应当知道的是，以下具体实施方式仅用于帮助本领域技术人员理解本申请，而非对本申请的限制。另外，以下具体实施方式可以在不付出创造性劳动的前提下任意组合，以形成新的实施例。
[用于检测小分子物质的试剂组合]
本申请的一些实施例提供了一种试剂组合。该试剂组合用于检测待测溶液中是否存在过量的小分子物质，由此实现对小分子物质的定性检测。并且，该试剂组合还能够在待测溶液中存在少量小分子物质的前提下获得该小分子物质在待测溶液中的浓度，由此实现对小分子物质含量的定量检测。
在本申请的一些实施例中，待测溶液可以来自血液样本或其它非血液样本。其中，血液样本可以来自于全血样本、血清样本或者血浆样本。
在本申请的一些实施例中，待检测的小分子物质的种类没有特别的限制。示例性地，小分子物质可以为三碘甲状腺原氨酸、四碘甲状腺原氨酸、或孕酮等。在本申请的一些实施例中，待检测的小分子物质的分子量可以在100至5000KD的范围内，但是有些情况下也不限于上述范围。
在本申请的一些实施例中，试剂组合包括：第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂。
其中，第一偶联物至少由第一核酸分子和抗体偶联而成。第一核酸分子的3’末端的糖环经过NH2C7修饰基团修饰，由NH2C7修饰基团通过第一偶联剂与抗体的氨基共价连接。NH2C7修饰基团修饰的是第一核酸分子的3’末端的糖环，而并非碱基。第一偶联剂可以为双琥珀酰亚胺辛二酸酯钠盐。抗体中与第一偶联剂相连的氨基并非位于抗体的CDR上，否则抗体就不能与小分子物质特异性结合。第一核酸分子的3’末端没有修饰。
在一些实施例中，第一核酸分子的全长序列如SEQ ID No：1所示。具体序列可以为ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG，但是也 不限于上述序列。
在一些实施例中，第一核酸分子含有第一杂交区和第二杂交区。第一核酸分子中，按照从5’端到3’端的顺序，第一杂交区可以位于第二杂交区的上游。示例性地，第一杂交区从5’端到3’端可以为GCTGAGTT，第二杂交区从5’端到3’端为CAACGAC。但第一杂交区与第二杂交区并不发生互补配对。在本申请的各个实施例中，每一个单链DNA内含的两个DNA序列之间并不发生互补配对。不同试剂之间的单链DNA才发生互补配对，从而组装成颈环结构。
在一些实施例中，第一核酸分子具有55个核苷酸，第一杂交区覆盖第一核酸分子从5’端开始的第3至10个碱基位点，第二杂交区覆盖第一核酸分子从5’端开始的第13至19个碱基位点。
在一些实施例中，抗体能与小分子物质特异性结合。每个抗体分子只能与一个小分子物质相结合，而不能与多个小分子物质同时结合。并且抗体分子的CDR仅能识别小分子物质的一个抗原表位，因此，能够实现二者的特异性结合。
第二偶联物可以至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成。
在一些实施例中，底物蛋白结合物至少由小分子底物与支架蛋白偶联而成。小分子底物与小分子物质相同，故小分子底物与小分子物质能够竞争性结合同一个抗体。当小分子物质的浓度很高时，能够使得小分子底物与抗体的CDR基本不结合。由此，当待测溶液中不存在或存在少量的小分子物质时，抗体与底物蛋白结合物能够发生免疫反应而形成免疫复合物。
在一些实施例中，第二核酸分子的全长序列如SEQ ID No：2所示。具体序列可以为TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC，但是也不限于上述序列。
在一些实施例中，第二核酸分子具有第三杂交区和第四杂交区。第二核酸分子中，按照从5’端到3’端的顺序，第三杂交区位于第四杂交区的上游。示例性地，第三杂交区从5’端到3’端为GTCGTTG，第四杂交区从5’端到3’端为GCTGAGAT。第三杂交区与第二杂交区互补。第三杂交区和第四杂交区之间不存在互补关系，因此，降低了DNA链内错配的可能性。
在一些实施例中，第二核酸分子具有53个核苷酸。第三杂交区覆盖第二核酸分子从5’ 端开始的37至43个碱基位点，第四杂交区覆盖第二核酸分子从5’端开始的第44至51个碱基位点。
在一些实施例中，第二核酸分子的3’末端与第二荧光基团连接。示例性地，第二核酸分子的3’末端的糖环经过巯基修饰，第二荧光基团的表面经过氨基修饰，巯基通过第三偶联剂与第二荧光基团的表面的氨基共价连接。第三偶联剂可以为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯。
在一些实施例中，第二核酸分子的5’末端的糖环经过NH2C6修饰基团修饰，由NH2C6修饰基团通过第二偶联剂与底物蛋白结合物的氨基共价连接。第二偶联剂为双琥珀酰亚胺辛二酸酯钠盐。底物蛋白结合物中的支架蛋白与第二偶联剂相连的氨基并非位于支架蛋白与小分子底物的结合位点上，否则支架蛋白就不能与小分子底物特异性结合。
第三偶联物可以至少由第一荧光基团和第三核酸分子偶联而成。
在一些实施例中，第三核酸分子的5’末端的糖环经过NH2C6修饰基团修饰，NH2C6修饰基团与第一荧光基团共价连接。
在一些实施例中，第三核酸分子的全长序列如SEQ ID No：3所示。具体序列可以为CGATCTCAGCAACTCAGCAGCG，但是也不限于上述序列。
在一些实施例中，第三核酸分子含有第五杂交区和第六杂交区，第五杂交区和第六杂交区之间并不互补。
在一些实施例中，第三核酸分子中，按照从5’端到3’端的顺序，第五杂交区位于第六杂交区的上游。
在一些实施例中，第三核酸分子具有22个核苷酸，第五杂交区覆盖第三核酸分子从5’端开始的第3至10个碱基位点，第六杂交区覆盖第三核酸分子从5’端开始的第11至18个碱基位点。第五杂交区从5’端到3’端的序列可以为ATCTCAGC。第六杂交区从5’端到3’端的序列可以为AACTCAGC。
在一些实施例中，第五杂交区与第四杂交区互补，第六杂交区与第一杂交区互补，第三杂交区与第二杂交区互补。在待测溶液中不含有或者含有少量小分子物质时，抗体与底物蛋白结合物形成免疫复合物，该免疫复合物的形成会带动三条单链DNA之间发生构象变化和DNA组装，并借助上述DNA序列之间的互补配对关系使得三条单链DNA之间能够形成颈环结构，第一荧光基团与第二荧光基团位于颈环结构的同侧，并在两种荧光分子之 间发生荧光共振能量转移(Fluorescence resonance energy transfer，FRET)。
在一些实施例中，第一荧光基团在能被氧化剂氧化的条件下发出第一荧光，第一荧光在第一核酸分子、第二核酸分子和第三核酸分子相互配对的条件下根据荧光共振能量转移效应激发第二荧光基团发出第二荧光，以便根据第二荧光的强度获得小分子物质的含量。当待测溶液中不存在或存在少量小分子物质时，由于第一荧光基团和第二荧光基团之间的距离满足荧光共振能量转移的产生条件，因此，第二荧光基团会发生第二荧光，第二荧光的波长与第一荧光不同，因此，将第二荧光作为检测荧光不会受到第一荧光基团所发射的本底荧光的影响。如果从待测溶液中没有获取到第二荧光强度，那么说明待测溶液中存在过量的小分子物质。当待测溶液中不存在或存在少量小分子物质时，抗体与小分子物质相结合，会形成稳定的免疫复合物，三条单链DNA形成颈环结构。免疫复合物和颈环结构能够使得第一荧光基团和第二荧光基团的距离足以产生荧光共振能量转移效应，从而根据第二荧光基团发出的第二荧光的强度和强度含量之间的标准曲线确定小分子物质的含量。
在一些实施例中，第一荧光基团发射荧光无需激发光的照射，而是在氧化剂的氧化作用下发光。但是第二荧光基团发射荧光需要激发光的照射，若没有激发光的照射，那么第二荧光基团本身是不会主动发射荧光的，故也就不容易产生本底荧光，因此，本申请实施例的测量结果不受到第二荧光基团的本底荧光的干扰。相比只用一种荧光基团发射荧光的方法而言，本申请的各个实施例的方法能够有效避免荧光分子的本底荧光的干扰，故测量结果更加准确。
在一些实施例中，在检测时，第一荧光基团与第二荧光基团需要能够发生荧光共振能量转移，需要满足以下条件：第一偶联物、第二偶联物、第三偶联物在不存在小分子物质的条件下完全互补配对，在存在少量的小分子物质的条件下部分互补配对。互补配对能够形成颈环结构，使第一荧光基团与第二荧光基团的间距小于或等于100埃。此时，第一荧光基团发射的第一荧光能够成为第二荧光基团的激发光，从而激发第二荧光基团产生第二荧光(如图1和图2所示)。
在一些实施例中，第一荧光基团可以为吖啶酯。吖啶酯发射荧光并不需要激发光，而是在氧化剂的存在下实现氧化发光，由于待测溶液中可能存在氧化性物质，也能够氧化吖啶酯并使其发光，因此，吖啶酯会存在本底荧光。如果采用吖啶酯的荧光作为测量荧光，那么本底荧光会对测量荧光产生干扰，因此，为了进一步提高检测的灵敏度和降低测量误 差，本申请的实施例并非采用吖啶酯的荧光作为测量荧光。吖啶酯的化学发光底物为H 2O 2的碱性溶液，又称氧化剂。当吖啶酯与H 2O 2的碱性溶液共存时，吖啶酯的分子受到过氧化氢离子进攻，吖啶酯能与过氧化氢(H 2O 2)形成不稳定的二氧乙烷，二氧乙烷的随后分解便会发射荧光。
在一些实施例中，第一荧光基团吖啶酯的最大发射波长为430nm。在一些实施例中，第二荧光基团可以为量子点。第二荧光基团量子点的最大吸收波长可以在420nm至520nm的范围内，如可以为470nm。第二荧光基团的最大发射波长可以在595nm至615nm的范围内，如可以为605nm。因此，本申请实施例中的第一荧光基团和第二荧光基团能够发生荧光共振能量转移效应。在一些实施例中，量子点为核壳结构量子点，其核层材料选自CdSe、CdS、CdTe、CdSeTe、CdZnS、ZnTe、CdSeS、PbS和PbTe中的一种或多种，其壳层材料选自ZnS、ZnSe、ZnSeS、PbS和PbSeS中的一种或多种。在一些实施例中，量子点的粒径范围可以为3至5nm，示例性地，可以为4.1nm。
在一些实施例中，第一核酸分子、第二核酸分子、第三核酸分子中的G可以被 isoG替代，C可以被 isoC替代。因为当待测溶液来自于血液样本等时，这些样本中也存在天然核酸，所以通过引入非天然碱基对( isoG和 isoC)能够避免第一核酸分子、第二核酸分子、第三核酸分子与血液样本中的核酸发生非特异性结合，从而避免由于DNA错配导致的测量误差。另外，这些非天然碱基对并不影响第一核酸分子、第二核酸分子、第三核酸分子之间的相互配对，依然能够形成稳定的颈环结构，从而不影响第二荧光的产生。示例性地，第一核酸分子的全长序列中的G被 isoG替代，C被 isoC替代。第二核酸分子的全长序列中的G被 isoG替代，C被 isoC替代。第三核酸分子的全长序列中的G被 isoG替代，C被 isoC替代。
在一些实施例中，相互配对的第一杂交区和第六杂交区中的G被 isoG替代，C被 isoC替代。示例性地，第一杂交区从5’端到3’端为 isoG isoCT isoGA isoGTT，第六杂交区从5’端到3’端为AA isoCT isoCA isoG isoC。
在一些实施例中，相互配对的第二杂交区和第三杂交区中的G被 isoG替代，C被 isoC替代。示例性地，第二杂交区从5’端到3’端为 isoCAA isoC isoGA isoC，第三杂交区从5’端到3’端为 isoGT isoC isoGTT isoG。
在一些实施例中，相互配对的第四杂交区和第五杂交区中的G被 isoG替代，C被 isoC替代。示例性地，第四杂交区从5’端到3’端为 isoG isoCT isoGA isoGAT，第五杂交区从5’端到3’ 端为AT isoCT isoCA isoG isoC。
在上述实施例中， isoG的结构式可以为：
Figure PCTCN2022114296-appb-000005
isoC的结构式可以为：
Figure PCTCN2022114296-appb-000006
isoG与 isoC的键合方式可以为：
Figure PCTCN2022114296-appb-000007
Figure PCTCN2022114296-appb-000008
表示与DNA分子上的脱氧核糖连接。
因此， isoG与 isoC之间能够产生互补配对，并不影响第一核酸分子、第二核酸分子、第三核酸分子之间的相互配对，但又能防止第一核酸分子、第二核酸分子、第三核酸分子与待测溶液中的天然核酸发生错配，因此，上述实施例能够降低因单链DNA与天然核酸分子发生错配而导致的测量误差。
氧化抑制试剂包括：用于第一荧光基团被氧化而发出第一荧光的抗氧化剂。在检测时，需在待测溶液中加入第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂，混合后形成待测样品。当待测样品中存在抗氧化剂时，即便第一偶联物、第二偶联物、第三偶联物形成了颈环结构，那么第一荧光基团吖啶酯也不会发射荧光，因为抗氧化剂会抑制吖啶酯的氧化发光。因此，在检测前需要从待测样品中移除抗氧化剂。
在一些实施例中，为了实现从待测样品中顺利移除抗氧化剂，氧化抑制试剂还包括载 体分子，并将抗氧化剂结合于载体分子的表面。此时，由于载体分子具有较大的分子量，比较容易从待测样品中移除。只要移除载体分子，便能实现同时移除抗氧化剂。
在一些实施例中，载体分子可以为氧化石墨烯(Graphene oxide，GO)。氧化石墨烯上的羧基通过氧化亚砜缩合剂与抗氧化剂上的羟基结合，氧化石墨烯上的羧基通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与抗氧化剂上的氨基结合。待测溶液中加入第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂后形成待测样品，第三核酸分子通过π-π堆积作用吸附在氧化石墨烯表面，其末端标记的吖啶酯(Acridinium ester，AE)由于抗氧化剂的存在而无法氧化发光，即使是少部分的吖啶酯发生化学发光，即产生波长为430nm的本底荧光，也不会对测量结果产生影响。因为在检测时所获取到的荧光来自于第二荧光基团量子点，而并非来自于吖啶酯，可以通过设置滤光片仅获取量子点发射的荧光，而过滤掉吖啶酯的本底荧光。另外，即便少部分的吖啶酯存在本底荧光，因为没有颈环结构的存在，该吖啶酯也不会基于荧光共振能量转移而激发量子点发射荧光。由此，本申请的实施例可以最大幅度降低吖啶酯的本底荧光对测量结果的影响，进而降低测量误差。
在一些实施例中，量子点发射的荧光可以通过滤光片和光电倍增管(Photomultiplier tube，PMT)来获得。示例性地，如果量子点发射的荧光为605nm，那么滤光片仅允许605nm波长的光通过。
在一些实施例中，抗氧化剂可以选自***二酚、维生素C、维生素E、茶多酚、谷胱甘肽中的任意一种或几种。氧化剂可以包括过氧化氢的碱性溶液。在移除抗氧化剂后，加入的氧化剂能够使第一荧光基团吖啶酯发射荧光。
本申请的实施例通过免疫反应使抗体识别小分子物质，从而拉近能够互补配对的单链DNA的间距，进而引发DNA组装、触发级联DNA组装，并使得两个荧光分子之间发生荧光共振能量转移效应而产生检测信号。
本申请的各个实施例以吖啶酯(AE)为荧光能量供体(donor)，荧光量子点(QDs)为荧光能量受体(acceptor)，基于化学发光共振能量转移***。通过免疫反应与DNA自组装实现荧光信号放大，将对小分子物质含量的检测转化为对荧光信号强度的检测。本申请的实施例的方法可均相进行。与传统的荧光共振能量转移分析方法相比，本申请的实施例的检测方法简单、快速，不需要昂贵的激光。本申请的实施例采用第二荧光作为检测荧光，降低了第一荧光的背景干扰。本申请的实施例采用免疫反应富集小分子物质，对小分子物质 的选择性好，检测灵敏度高。
[小分子物质的检测方法]
如图2所示，本申请的一些实施例提供了一种小分子物质的检测方法，该检测方法可以使用上述的试剂组合。关于上述的试剂组合的特征可以并入本部分。上述实施例的检测方法包括如下步骤：
(1)、在待测溶液中加入第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂，混合后形成待测样品。第一偶联物至少由第一核酸分子和抗体偶联而成。第二偶联物至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成。第三偶联物至少由第一荧光基团和第三核酸分子偶联而成。在待测溶液中不含有或含有少量小分子物质的条件下，抗体和底物蛋白结合物形成免疫复合物，第一核酸分子、第二核酸分子和第三核酸分子形成颈环结构，从而第一荧光基团与第二荧光基团位于颈环结构的同侧。在待测溶液中含有大量小分子物质的条件下，抗体与小分子物质特异性结合而与底物蛋白结合物中的小分子底物丧失结合，免疫复合物和颈环结构无法形成。
(2)、从待测样品中去除氧化抑制试剂，加入用于氧化第一荧光基团而发射第一荧光的氧化剂并按照第二荧光基团的最大发射波长收集第二荧光。氧化抑制试剂中含有用于抑制第一荧光基团被待测溶液中的氧化物质氧化的抗氧化剂。由于在抗氧化剂存在的条件下，第一荧光基团并不会发射第一荧光，那么在加入氧化剂之前，就需要事先去除抗氧化剂。另外，第一荧光基团在被氧化之前会受到抗氧化剂的抗氧化保护作用，从而也能够降低自身由于受到其它因素影响而发射的本底荧光。
(3)、在收集到第二荧光的情况下，根据第二荧光的强度和荧光强度与小分子物质含量的一一对应关系获得小分子物质的含量；在没有收集到第二荧光的情况下，说明待测溶液中含有过量的小分子物质，导致小分子底物与抗体完全不能结合。当然，后者属于极端情况，因此，本申请的各个实施例仍然能够根据第二荧光的强度获得小分子物质在待测溶液中的浓度。
本步骤中，第一荧光基团被氧化后会产生第一荧光，为了避免第一荧光对检测结果造成影响，采用滤光片过滤掉第一荧光，该滤光片仅允许第二荧光基团发出的第二荧光的透射，此时，根据第二荧光的强度获得小分子物质的含量。如果第二荧光的相对强度为0，说明待测溶液中含有过量的小分子物质。如果第二荧光的相对强度不为0，那么根据第二荧光 的相对强度和小分子物质含量的函数关系式获得待测溶液中小分子物质的含量。相对强度指的是从第二荧光信号的强度中排除了对照组的背景荧光强度之后剩余的荧光强度值。
在一些实施例中，在待测样品中，第一偶联物的工作浓度为1nM至20nM。工作浓度指的是在实际检测时各个试剂的浓度，也即各个试剂在待测样品中的终浓度。工作浓度不等于各个试剂在小分子物质检测试剂盒中的储存浓度。在一些实施例中，在待测样品中，第二偶联物的工作浓度为1nM至20nM。在一些实施例中，在待测样品中，第三偶联物的工作浓度为0.05nM至0.2nM。在一些实施例中，在待测样品中，氧化抑制试剂的工作浓度为15μg/ml至25μg/ml。
在一些实施例中，待测溶液血液样本，来自全血样本、血清样本或者血浆样本。
在一些实施例中，混合的时间为5分钟至10分钟。在一些实施例中，混合的温度为36度至37度。
在一些实施例中，氧化剂的体积为200μL。氧化剂为碱性过氧化氢溶液，pH值为8.0。该碱性过氧化氢溶液是将双氧水溶解于TBS缓冲液中进行的，其中，过氧化氢的终浓度为0.1M，TBS的终浓度为10mM。
在一些实施例中，小分子物质包括三碘甲状腺原氨酸、四碘甲状腺原氨酸、或孕酮。但是在其它的一些实施例中，小分子物质也不限于上述几种。只要本申请的抗体能与小分子物质特异性结合，并形成稳定的免疫复合物即可。一个抗体只能结合一个小分子物质或小分子底物。当抗体与小分子底物相结合时，会形成较稳定的免疫复合物。免疫复合物又会使第一核酸分子、第二核酸分子和第三核酸分子距离拉进而形成颈环结构，第一荧光基团与第二荧光基团位于颈环结构的同侧，第一荧光基团才能激发第二荧光基团产生第二荧光。由于小分子物质或小分子底物与抗体和第二荧光基团均为一一对应关系，并且第二荧光基团的最强荧光程度已知，那么小分子物质的数目与第二荧光基团的荧光猝灭程度是线性关系，据此也可以得到小分子物质的数目与剩余荧光强度的关系。可以根据标准曲线所含的第二荧光的强度和小分子物质含量的函数关系式和所获得的第二荧光基团的荧光强度计算出小分子物质的含量。
在一些实施例中，具体的检测步骤如下所示：将检测试剂(第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂)与含有小分子物质的待测血液样本或全血样本或血清样本或血浆样本相混合，在化学发光检测仪上于37℃温育反应5-10分钟，加入作为氧化剂的化学 发光底物过氧化氢、氢氧化钠和TBS缓冲液，通过化学发光检测仪中PMT检测模块采集产生的化学发光荧光信号。化学发光检测仪自动调用标准曲线，根据该标准曲线含有荧光强度和小分子物质含量的函数关系式报出待测样本中小分子物质的浓度。
[小分子物质检测试剂盒]
本申请的一些实施例提供了一种小分子物质检测试剂盒，该试剂盒使用上述的检测方法进行检测。小分子物质检测试剂盒包括：第一储存管、第二储存管、第三储存管、第四储存管、第四储存管、第五储存管。
其中，第一储存管储存第一核酸分子和抗体的偶联物。第一核酸分子含有55个碱基，3’端修饰有NH2C7基团。3'C7氨基修饰引物购买自金斯瑞生物科技股份有限公司。NH2C7基团通过第一偶联剂双琥珀酰亚胺辛二酸酯钠盐(BS3)与抗体的氨基共价连接，从而形成第一核酸分子和抗体的偶联物。
第二储存管储存底物蛋白结合物、第二核酸分子和第二荧光基团的偶联物。抗体和底物蛋白结合物在不存在或存在少量小分子物质的条件下形成免疫复合物。第二核酸分子含有53个碱基，3’端修饰巯基，5’端修饰NH2C6基团。巯基修饰试剂3'SH C6、5'Aminolinker(C6)修饰引物购买自金斯瑞生物科技股份有限公司。5’端修饰是在合成循环的最后一步以亚磷酸胺的形式通过β-氰乙基化学反应添加到5’糖环上的，而不是添加到最后一个碱基上。3’末端的巯基通过第三偶联剂4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)与氨基修饰的量子点QDs表面的氨基共价相连。在一些实施例中，氨基修饰的水溶性量子点的型号可以为氨基水溶性量子点(PEG)-605，成分为CdSe/ZnS，购买自西安齐岳生物科技有限公司。(PEG)-605的最大吸收峰波长470 nm，最大发射波长605nm，粒径为4.1nm。氨基修饰的量子点的氨基与第二核酸分子的3’端的巯基偶联，形成第二核酸分子和量子点的偶联物。第二核酸分子的5’端的NH2C6基团通过第二偶联剂BS3与底物蛋白结合物上的氨基共价结合，形成底物蛋白结合物、第二核酸分子和第二荧光基团的偶联物。
第三储存管储存第一荧光基团和第三核酸分子的偶联物；第一核酸分子、第二核酸分子和第三核酸分子在免疫复合物形成的条件下能形成颈环结构；第一荧光基团与第二荧光基团能位于颈环结构的同侧。第三核酸分子含有22个碱基，5’端修饰NH2C6基团。修饰引物5'Aminolinker(C6)是在合成循环的最后一步以亚磷酸胺的形式通过β-氰乙基化学反应 添加到第三核酸分子5’末端的糖环上，而不是添加到最后一个碱基上。该修饰引物购买自金斯瑞生物科技股份有限公司。通过加入吖啶酯(NSP-DMAE-NHS)形成第一荧光基团和第三核酸分子的偶联物，第一荧光基团吖啶酯购买自苏州亚科科技股份有限公司，CAS号为194357-64-7。第三核酸分子从5’端开始的3至10个碱基位点与第二核酸分子从3’端开始的3至10碱基位点(即从5’端开始的第44至51个碱基位点)的碱基完全互补。第三核酸分子从3’端开始的5至12个碱基位点(即从5’端开始的第11至18个碱基位点)与第一核酸分子从5’端开始的3至10个碱基位点的碱基完全互补，如图1所示。
第四储存管储存能够抑制第一荧光基团被氧化的抗氧化剂。抗氧化剂可以结合于载体分子的表面。在一些实施例中，载体分子可以为氧化石墨烯。
第五储存管储存能够氧化第一荧光基团以发射第一荧光的氧化剂。
其中，上述实施例中的小分子物质试剂盒的检测原理如下：
第三核酸分子分别有8个碱基与第一核酸分子、第二核酸分子互补配对，其中将天然碱基对G和C更换为非天然碱基对 isoG和 isoC。
在过量的小分子物质存在的条件下，不会形成颈环结构，第三核酸分子通过π-π堆积作用吸附在氧化石墨烯(GO)表面，其末端标记的吖啶酯(AE)由于抗氧化剂的存在而无法氧化发光，即使是少部分的吖啶酯产生背景荧光，吖啶酯的化学发光(430nm波长)也因仪器的滤光片(仅允许605nm的光透过)而无法被PMT检测到。
在不存在小分子物质或者存在少量小分子物质的条件下，抗体和底物蛋白结合物能形成免疫复合物。由于抗体和第一核酸分子相偶联，底物蛋白结合物和第二核酸分子相偶联，免疫复合物使得第一核酸分子和第二核酸分子足够接近而形成邻位复合物，并能与第三核酸分子杂交，从而第一核酸分子、第二核酸分子和第三核酸分子形成颈环结构，第一荧光基团与第二荧光基团位于颈环结构的同侧。抗体和DNA形成的复合物基本上不会被载体分子氧化石墨烯吸附，故偶联在载体分子上的抗氧化剂也基本上不会影响吖啶酯的发光。在移除抗氧化剂的条件下，所加入的氧化剂能氧化第一荧光基团并使其发射第一荧光，第一荧光基于荧光共振能量转移而激发第二荧光基团发出第二荧光(例如605nm)，以便根据第二荧光的强度获得小分子物质的含量。
[小分子物质检测***]
本申请提供了一种小分子物质检测***，其包括：反应容器、微量注射泵、滤光片和 计算模块。
其中，反应容器具有能容纳待测溶液的容置腔室。
微量注射泵通过注射管道与反应容器的容置腔室相连通，将第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂的混合物注射入容置腔室中；第一偶联物至少由第一核酸分子和抗体偶联而成，第二偶联物至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成，第三偶联物至少由第一荧光基团和第三核酸分子偶联而成，氧化抑制试剂含有抑制第一荧光基团被氧化而发射第一荧光的抗氧化剂。
滤光片设于第一荧光的出射光路上，允许与第二荧光基团的最大发射波长相同的第二荧光透过。
光信号检测模块设于位于荧光信号的出射光路的滤光片的下游侧，获取从滤光片透射的第二荧光。
计算模块将第二荧光转换为数字信号并且根据荧光强度和小分子物质含量的函数关系式获得待测溶液中小分子物质的含量。标准曲线中含有标准方程，其含有数字信号与小分子物质含量的一一对应关系。
本申请实施例通过氧化石墨烯偶联抗氧化剂和滤光片的双重淬灭机制，结合化学发光共振能量转移和免疫分析技术，提供了一种简单、快速、灵敏的均相化学发光免疫分析蛋白检测方法。相比于现有的免疫分析方法，本申请的一些实施例具有以下特点：
(1)、本申请的一些实施例为均相免疫分析方法，操作简单，同时大大缩短了临床检验标本周转时间(TAT)，无需血液标本的离心处理，可以全血上样，在5分钟左右出检测报告。
(2)、本申请的一些实施例的吖啶酯为氧化型发光，量子点为激发型发光，能够实现吖啶酯和量子点之间的自激发式发光，而无需设置复杂的外部激发光***，可以有效降低测量仪器的复杂度和成本。由于配套检测设备要求降低，模块减少，成本降低，故障率也大幅降低，能够实现自动化检测或小型化便携式床旁检测(POCT)。
(3)、本申请的一些实施例的吖啶酯-量子点发光体系引入双重淬灭机制(氧化石墨烯-还原剂和滤光片)，本底荧光更低，检测灵敏度更高，适用于较高灵敏度的检测。
(4)、本申请的一些实施例通过免疫反应诱发氧化石墨烯-抗氧化剂淬灭机制的开关，外加滤光片的引入，开关效率达到了100％，结合化学发光共振能量转移效应从而使得量子 点发光，无需分离和清洗步骤。
(5)本申请的一些实施例中的DNA分子含有非天然碱基对(isoG和isoC)，避免与样本中的核酸发生非特异性结合。
以下结合各个制备例和各个实施例对本申请的技术作进一步的说明。
制备例一第二核酸分子和第二荧光基团(量子点)的偶联物的制备
本制备例提供了第二核酸分子和量子点的偶联物的制备方法，其包括如下步骤：
1.配制SMCC溶液：称取10mg 4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(4-(N-Maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester，SMCC)溶于1mL DMF中。SMCC购买自上海阿拉丁生化科技股份有限公司，货号为N159712，CAS号为64987-85-5。
2.配制第二核酸分子溶液：取10μM第二核酸分子，加入1mL纯化水溶解。
3.制备第二核酸分子和量子点的偶联物：取100μL第二核酸分子溶液于EP管中，加入200μL QDs(购买自西安齐岳生物科技有限公司，型号为氨基水溶性量子点(PEG)-605，浓度为8μM)，加入3μL SMCC溶液，混合均匀，37℃孵育30min。QDs购买自西安齐岳生物科技有限公司，型号为氨基水溶性量子点(PEG)-605，浓度为8μM。
4.透析：将偶联好的第二核酸分子和量子点的偶联物从EP管中吸出，加入至透析袋(规格5kd)，扎好透析袋放入装有2-3L TE溶液(10mM Tris，1mM EDTA,PH＝8.0)的烧杯中，进行透析。透析之前将透析袋提前浸泡。2-3小时更换透析液一次，共透析三次，透析完成收取透析袋中的液体于离心管中，放入2-8℃保存待用。
制备例二第一荧光基团(吖啶酯)和第三核酸分子的偶联物的制备
本制备例提供了吖啶酯(AE)和第三核酸分子的偶联物的制备方法，其包括如下步骤：
1.配制第三核酸分子溶液：取20μM第三核酸分子，加入1mL纯化水溶解。
2.配制NHS-AE溶液：称取4mg吖啶酯(NSP-DMAE-NHS)，溶于1mL纯化水中。吖啶酯购买自苏州亚科科技股份有限公司，CAS号194357-64-7。
3.偶联：每1mg NHS-AE溶液中，加入10μL第三核酸分子溶液，于EP管中混合均匀，37℃孵育30min。
4.透析：将偶联好的产物从EP管中吸出，加入至透析袋(规格5kd)，扎好透析袋放入装有2-3L TE溶液(10mM Tris，1mM EDTA,PH＝8.0)的烧杯中，进行透析(将透析袋提 前浸泡)，2-3小时更换透析液一次，共透析三次，透析完成收取透析袋中的液体于离心管中，放入2-8℃保存待用。
制备例三第一核酸分子和抗体的偶联物的制备
本制备例提供了第一核酸分子和抗体的偶联物的制备方法，其包括如下步骤：
1.配制BS3溶液：称取10mg双琥珀酰亚胺辛二酸酯钠盐(BS3)溶于1mL纯化水中。BS3购买自上海阿拉丁生化科技股份有限公司，货号S304724。
2.活化：取出分装好的抗体，解冻，离心混匀。每1mg抗体中加入3μL BS3溶液，加入6.5μL第一核酸分子，于EP管中混合均匀，37℃孵育30min。
3.透析：将第一核酸分子和抗体的偶联物从EP管中吸出，加入至透析袋(规格100kd)，扎好透析袋放入装有2-3L PBS溶液的烧杯中，进行透析(将透析袋提前浸泡)，2-3小时更换透析液一次，共透析三次，透析完成收取透析袋中的液体于离心管中，放入2-8℃保存待用。
制备例四底物蛋白结合物、第二核酸分子和第二荧光基团的偶联物的制备
本制备例提供了底物蛋白结合物、第二核酸分子和第二荧光基团的偶联物的制备方法，其包括如下步骤：
1.配置BS3溶液：称取10mg双琥珀酰亚胺辛二酸酯钠盐(BS3)溶于1mL纯化水中。BS3购买自上海阿拉丁生化科技股份有限公司。
2.活化：取出分装好的底物蛋白结合物(小分子底物与支架蛋白的偶联物)，解冻，离心混匀。每1mg底物蛋白结合物中加入3μL BS3溶液，加入6.5μL第二核酸分子和量子点的偶联物，于EP管中混合均匀，37℃孵育30min。
3.透析：将底物蛋白结合物、第二核酸分子和第二荧光基团的偶联物从EP管中吸出，加入至透析袋(规格100kd)，扎好透析袋放入装有2-3L PBS溶液的烧杯中，进行透析(将透析袋提前浸泡)，2-3小时更换透析液一次，共透析三次，透析完成收取透析袋中的液体于离心管中，放入2-8℃保存待用。
实施例一
本实施例基于氧化石墨烯-抗氧化剂淬灭和吖啶酯化学发光的均相免疫分析方法对血清中游离的三碘甲状腺原氨酸(FT3)进行检测。其中，抗体购买自Bioventix，克隆号为17c6r；底物蛋白结合物(又称支架小分子)购买自南京拂晓生物科技，克隆号为T3-KLH.。通过 上述制备例对将抗体、底物蛋白结合物与相应的DNA分子进行偶联。具体检测方法包括如下步骤：
1、配制检测溶液：将第一核酸分子和抗体的偶联物(DNA1-抗体偶联物)、底物蛋白结合物、第二核酸分子和量子点的偶联物(支架蛋白-DNA2-QDs偶联物)、吖啶酯和第三核酸分子的偶联物、抗氧化剂修饰的氧化石墨烯(GO-AOD)混合，使它们的最终浓度分别为10nM、10nM、0.15μM和20μg/ml。
2、将50μL不同浓度的校准溶液或者含有三碘甲状腺原氨酸(血清T3)的血清样本与200μL检测溶液混合，37℃条件下温育5-10分钟。
3、在温育后，通过HSCL-10000化学发光仪加入200μL化学发光底物。化学发光底物包括：10mM TBS缓冲液、0.1M双氧水的碱液，pH＝8.0，并立即通过光电倍增管(PMT)检测该溶液的化学发光信号，检测时间3s。根据记录的化学发光值(RLU)，获得三碘甲状腺原氨酸的校准曲线和待测的血清样本中血清FT3的浓度。
经过检测40例临床样本，本实施例的FT3的检测值和罗氏测试值的误差为1.82％，说明本实施例的检测方法具有较高的准确度。检测结果如下表1所示。罗氏测试指的是罗氏FT3试剂盒。
表1为本实施例和罗氏检测方法的检测值的比较表
Figure PCTCN2022114296-appb-000009
实施例二
本实施例基于氧化石墨烯-抗氧化剂淬灭和吖啶酯化学发光的均相免疫分析方法对血清中四碘甲状腺原氨酸(血清T4)进行检测。其中，抗体购买自南京欧凯生物科技有限公司，克隆号为K88a6，底物蛋白结合物(又称支架小分子)购买自上海汉尊生物科技，克隆号为T4-BSA-HT-020561。通过上述制备例对抗体、底物蛋白结合物与相应的DNA进行偶联。具体检测方法包括如下步骤：
1、配制检测溶液：将第一核酸分子和抗体的偶联物、底物蛋白结合物、第二核酸分子和量子点的偶联物、吖啶酯和第三核酸分子的偶联物、抗氧化剂修饰的氧化石墨烯混合，使它们的最终浓度分别为10nM、10nM、0.15μM和20μg/ml。
2、将50μL不同浓度的校准溶液或者含有四碘甲状腺原氨酸的血清样本与200μL检测溶液混合，37℃条件下温育5-10分钟。
3、在温育后，通过HSCL-10000化学发光仪加入200μL化学发光底物。化学发光底物包括：10mM TBS缓冲液、0.1M双氧水的碱液，pH＝8.0。并立即通过光电倍增管(PMT)检测该溶液的化学发光信号，检测时间3s。根据记录的化学发光值(RLU)，获得血清FT4的校准曲线和待测的血清样本中血清T4的浓度。
经过多次检测，本实施例的血清FT4的检测限为0.5-100pmol/L。经过检测40例临床样本，本实施例的FT4的检测值和罗氏测试值的误差为-1.66％，说明本实施例的检测方法具有较高的准确度。检测结果如下表2所示。罗氏测试指的是罗氏FT4试剂盒。
表2为本实施例和罗氏检测方法的检测值的比较表
Figure PCTCN2022114296-appb-000010
实施例三
本实施例基于氧化石墨烯-抗氧化剂淬灭和吖啶酯化学发光的均相免疫分析方法对血清中孕酮(Prog)进行检测。其中，抗体购买自Biospacific，克隆号为PPOGESTERONE MAB,PURE/A25050；底物蛋白结合物(又称支架小分子)购买自Biospacific，克隆号为PPOGESTERONE-11-BSA/V56050。通过上述制备例对抗体、底物蛋白结合物与相应的DNA进行偶联。具体的检测方法包括如下步骤：
1、配制检测溶液：将第一核酸分子和抗体的偶联物、底物蛋白结合物、第二核酸分子和量子点的偶联物、吖啶酯和第三核酸分子的偶联物、抗氧化剂修饰的氧化石墨烯混合，使它们的最终浓度分别为10nM、10nM、0.15μM和20μg/ml。
2、将50μL不同浓度的校准溶液或者含有孕酮的血清样本与200μL试剂溶液混合，37℃条件下温育5-10分钟。
3、在温育后，通过HSCL-10000化学发光仪加入200μL化学发光底物包括：10mM TBS缓冲液、0.1M双氧水的碱液，pH＝8.0。并立即通过光电倍增管(PMT)检测该溶液的化学发光信号，检测时间3s。根据记录的化学发光值(RLU)，获得Prog的校准曲线和待测血清样本中Prog的浓度。
经过多次检测，本实施例的Prog的检测限为0.05-60ng/mL。经过检测40例临床样本，本实施例的Prog的检测值和罗氏测试值的误差为2.63％，说明本实施例的检测方法具有较高的准确度。检测结果如下表3所示。罗氏测试指的是罗氏Prog检测试剂盒。
表3为本实施例和罗氏检测方法的检测值的比较表
Figure PCTCN2022114296-appb-000011
本申请已由上述相关实施例加以描述，然而上述实施例仅为实施本申请的范例。必需指出的是，已公开的实施例并未限制本申请的范围。相反地，包含于权利要求书的精神及范围的修改及均等设置均包括于本申请的范围内。

Claims (19)

  1. 一种用于检测小分子物质的试剂组合，其包括：
    第一偶联物，至少由第一核酸分子和抗体偶联而成；所述第一核酸分子含有第一杂交区和第二杂交区；所述抗体能与小分子物质特异性结合；
    第二偶联物，至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成；所述底物蛋白结合物至少由小分子底物与支架蛋白偶联而成，所述小分子底物与所述小分子物质相同；所述第二核酸分子具有第三杂交区和第四杂交区，所述第三杂交区与所述第二杂交区互补；
    第三偶联物，至少由第一荧光基团和第三核酸分子偶联而成；所述第三核酸分子含有第五杂交区和第六杂交区，所述第五杂交区与所述第四杂交区互补，所述第六杂交区与所述第一杂交区互补；所述第一荧光基团在所述抗体与所述底物蛋白结合物中的所述小分子底物相结合并且能被氧化剂氧化的条件下发出第一荧光，所述第一荧光在所述第一核酸分子、所述第二核酸分子和所述第三核酸分子相互配对的条件下激发所述第二荧光基团发出第二荧光，以根据所述第二荧光的强度和荧光强度与小分子物质含量的一一对应关系获得所述小分子物质的含量；
    氧化抑制试剂，包括：用于抑制所述第一荧光基团被氧化而发出所述第一荧光的抗氧化剂。
  2. 如权利要求1所述的用于检测小分子物质的试剂组合，其中，在待测溶液中不含有或含有少量所述小分子物质的条件下，所述抗体与所述底物蛋白结合物中的所述小分子底物特异性结合，从而所述抗体与所述底物蛋白结合物形成免疫复合物，所述第一核酸分子、所述第二核酸分子、所述第三核酸分子互补配对而形成颈环结构，使所述第一荧光基团与所述第二荧光基团的间距小于能够发生荧光共振能量转移的极限间距；
    在所述待测溶液中含有大量所述小分子物质的条件下，所述抗体与所述小分子物质特异性结合而与所述底物蛋白结合物中的所述小分子底物丧失结合，所述免疫复合物和所述颈环结构无法形成。
  3. 如权利要求2所述的用于检测小分子物质的试剂组合，其中，所述第一荧光基团为吖啶酯；所述第二荧光基团为量子点；所述第一核酸分子、所述第二核酸分子和所述第三 核酸分子为单链DNA分子。
  4. 如权利要求3所述的用于检测小分子物质的试剂组合，其中，所述量子点为核壳结构量子点，其核层材料选自CdSe、CdS、CdTe、CdSeTe、CdZnS、ZnTe、CdSeS、PbS和PbTe中的一种或多种，其壳层材料选自ZnS、ZnSe、ZnSeS、PbS和PbSeS中的一种或多种；所述量子点的最大吸收波长为470nm，最大发射波长为605nm；所述吖啶酯的最大发射波长为430nm。
  5. 如权利要求1至4任意一项所述的用于检测小分子物质的试剂组合，其中，所述第一杂交区从5’端到3’端为 isoG isoCT isoGA isoGTT，所述第六杂交区从5’端到3’端为AA isoCT isoCA isoG isoC；
    isoG的结构式为：
    Figure PCTCN2022114296-appb-100001
    isoC的结构式为：
    Figure PCTCN2022114296-appb-100002
  6. 如权利要求1至4任意一项所述的用于检测小分子物质的试剂组合，其中，所述第二杂交区从5’端到3’端为 isoCAA isoC isoGA isoC，所述第三杂交区从5’端到3’端为 isoGT isoC isoGTT isoG；
    isoG的结构式为：
    Figure PCTCN2022114296-appb-100003
    isoC的结构式为：
    Figure PCTCN2022114296-appb-100004
  7. 如权利要求1至4任意一项所述的用于检测小分子物质的试剂组合，其中，所述第四杂交区从5’端到3’端为 isoG isoCT isoGA isoGAT，所述第五杂交区从5’端到3’端为AT isoCT isoCA isoG isoC；
    isoG的结构式为：
    Figure PCTCN2022114296-appb-100005
    isoC的结构式为：
    Figure PCTCN2022114296-appb-100006
  8. 如权利要求1至4任意一项所述的用于检测小分子物质的试剂组合，其中，所述第一核酸分子的全长序列为如SEQ ID No：1所示的序列中至少部分或全部G被 isoG替代并且至少部分或全部C被 isoC替代所形成的序列；
    所述第二核酸分子的全长序列为如SEQ ID No：2所示的序列中至少部分或全部G被 isoG替代并且至少部分或全部C被 isoC替代所形成的序列；
    所述第三核酸分子的全长序列为如SEQ ID No：3所示的序列中至少部分或全部G被 isoG替代并且至少部分或全部C被 isoC替代所形成的序列；
    isoG的结构式为：
    Figure PCTCN2022114296-appb-100007
    isoC的结构式为：
    Figure PCTCN2022114296-appb-100008
  9. 如权利要求5至8中任意一项所述的用于检测小分子物质的试剂组合，其中，
    isoG与 isoC的键合方式为：
    Figure PCTCN2022114296-appb-100009
    表示与DNA分子中的脱氧核糖连接。
  10. 如权利要求1至9任意一项所述的用于检测小分子物质的试剂组合，其中，所述氧化抑制试剂还包括载体分子，所述载体分子的表面结合所述抗氧化剂；
    所述抗氧化剂选自***二酚、维生素C、维生素E、茶多酚、谷胱甘肽中的任意一种或几种；
    所述氧化剂包括过氧化氢的碱性溶液。
  11. 如权利要求10所述的用于检测小分子物质的试剂组合，其中，所述载体分子为氧化石墨烯；所述氧化石墨烯上的羧基通过氧化亚砜缩合剂与所述抗氧化剂上的羟基结合，所述氧化石墨烯上的羧基通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与所述抗氧化剂上的氨基结合。
  12. 一种小分子物质的检测方法，包括如下步骤：
    在待测溶液中加入第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂，混合后形成待测样品；所述第一偶联物至少由第一核酸分子和抗体偶联而成，所述抗体能与小分子 物质特异性结合；所述第二偶联物至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成，所述底物蛋白结合物至少由小分子底物与支架蛋白偶联而成，所述小分子底物与所述小分子物质相同；所述第三偶联物至少由第一荧光基团和第三核酸分子偶联而成；在所述待测溶液中不含有或含有少量所述小分子物质的条件下，所述抗体与所述底物蛋白结合物形成免疫复合物，所述第一核酸分子、所述第二核酸分子和所述第三核酸分子形成颈环结构，所述第一荧光基团与所述第二荧光基团位于所述颈环结构的同侧；
    从所述待测样品中去除用于抑制所述第一荧光基团被氧化的所述氧化抑制试剂，加入用于氧化所述第一荧光基团而发射第一荧光的氧化剂并按照所述第二荧光基团的最大发射波长收集第二荧光；
    在收集到所述第二荧光的情况下，根据所述第二荧光的强度和荧光强度与小分子物质含量的一一对应关系获得所述小分子物质的含量。
  13. 如权利要求12所述的小分子物质的检测方法，其中，所述待测样品中，所述第一偶联物的工作浓度为1nM至20nM；
    所述待测样品中，所述第二偶联物的工作浓度为1nM至20nM；
    所述待测样品中，所述第三偶联物的工作浓度为0.05nM至0.2nM；
    所述待测样品中，所述氧化抑制试剂的工作浓度为15μg/ml至25μg/ml。
  14. 如权利要求12所述的小分子物质的检测方法，其中，所述待测溶液来自全血样本、血清样本或者血浆样本。
  15. 如权利要求12所述的小分子物质的检测方法，其中，所述小分子物质包括三碘甲状腺原氨酸、四碘甲状腺原氨酸、或孕酮。
  16. 一种小分子物质检测试剂盒，其中，其包括：
    第一储存管，至少储存第一核酸分子和抗体的偶联物，所述抗体能与小分子物质特异性结合；
    第二储存管，至少储存底物蛋白结合物、第二核酸分子和第二荧光基团的偶联物；底物蛋白结合物至少由小分子底物与支架蛋白偶联而成，所述小分子底物与所述小分子物质相同；所述抗体与所述底物蛋白结合物在待测溶液中不存在或存在少量所述小分子物质的条件下形成免疫复合物；
    第三储存管，至少储存第一荧光基团和第三核酸分子的偶联物；所述第一核酸分子、所述第二核酸分子和所述第三核酸分子在所述免疫复合物形成的条件下能形成颈环结构， 所述第一荧光基团与所述第二荧光基团位于所述颈环结构的同侧，所述第一荧光基团发射的第一荧光能激发所述第二荧光基团发射第二荧光，在获取到所述第二荧光的强度的条件下根据所述第二荧光的强度获得所述小分子物质的含量；
    第四储存管，至少储存能够抑制所述第一荧光基团被氧化的氧化抑制试剂；以及
    第五储存管，至少储存能够氧化所述第一荧光基团以发射所述第一荧光的氧化剂。
  17. 如权利要求16所述的小分子物质检测试剂盒，其中，所述第一荧光基团为吖啶酯；所述第二荧光基团为量子点。
  18. 如权利要求16所述的小分子物质检测试剂盒，其中，第一核酸分子含有第一杂交区和第二杂交区；第二核酸分子具有第三杂交区和第四杂交区；第三核酸分子含有第五杂交区和第六杂交区；第三杂交区与第二杂交区互补，第五杂交区与第四杂交区互补，第六杂交区与第一杂交区互补；
    所述第一杂交区、所述第二杂交区、所述第三杂交区、所述第四杂交区、所述第五杂交区和所述第六杂交区至少部分或者全部含有碱基 isoG和 isoC；
    isoG的结构式为：
    Figure PCTCN2022114296-appb-100010
    isoC的结构式为：
    Figure PCTCN2022114296-appb-100011
  19. 一种小分子物质检测***，其中，包括：
    反应容器，具有能容纳待测溶液的容置腔室；
    微量注射泵，通过注射管道与所述容置腔室相连通，将第一偶联物、第二偶联物、第三偶联物和氧化抑制试剂的混合物通过所述注射管道注射入所述容置腔室中；所述第一偶 联物至少由第一核酸分子和抗体偶联而成，所述第二偶联物至少由底物蛋白结合物、第二核酸分子和第二荧光基团依次偶联而成，所述第三偶联物至少由第一荧光基团和第三核酸分子偶联而成，所述氧化抑制试剂含有抑制所述第一荧光基团被氧化而发射第一荧光的抗氧化剂；
    滤光片，设于所述第一荧光的出射光路上，允许所述第二荧光基团发射的第二荧光透过；
    光信号检测模块，设于所述第一荧光的出射光路上并位于所述滤光片的下游侧，获取从所述滤光片透射的所述第二荧光；
    计算模块，将所述第二荧光转换为数字信号，并且根据荧光强度和小分子物质含量的一一对应关系式获得所述待测溶液中所述小分子物质的含量。
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