A fluorescent detection method for protein markers based on cyclodextrin-adamantanized iridium complex host-guest assembly labeling

Abstract

The present invention relates to a protein marker fluorescence detection method based on cyclodextrin-adamantanized iridium complex host-guest assembly labeling, which belongs to the application technology of organic iridium complex hydrogenation catalysis technology in the field of biological fluorescence sensing. In order to solve the numerous biocompatibility problems such as easy thiol deactivation faced by organic iridium complex hydrogenation catalysts in biological applications, the present invention adopts the following technical scheme: (1) preparation of a nano material M co-labeled with an organic iridium complex with high catalytic hydrogenation activity and a labeled antibody, and successfully solves the irreversible problem of easy thiol deactivation of the metal iridium complex in protein labeling detection by regulating the modification sequence of the labeled antibody, β-cyclodextrin, a blocking agent and adamantane functionalized metal iridium complex during the material assembly process; (2) coating an antibody A on a carrier; (3) adding a detected antigen biological protein marker; (4) adding the organic iridium complex and the nano material M co-labeled with the labeled antibody; and (5) adding a fluorescent detection liquid containing a hydrogenation regulation probe, wherein the iridium complex can catalyze the probe molecule to produce a fluorescence signal change, and quantitative detection of the biological protein marker to be detected can be achieved by testing the fluorescence intensity change of the detection liquid. This technology realizes the rapid and sensitive analysis of biological protein markers and has the advantages of simple operation and good thermal stability.

Description

Technical Field

[0001] The present invention relates to the field of organometallic complex catalysis, functional material preparation and bioluminescent sensing technology, and in particular to organometallic iridium complex catalytic hydrogenation reaction, host-guest self-assembly of gold nanoparticles by organometallic iridium complex and supramolecular compound β-cyclodextrin, and further to the application of organometallic iridium complex functionalized gold nanoparticles in the detection of biological protein markers. Background Art

[0002] Faced with the increasingly complex health and safety issues of mankind, as well as people's pursuit of high quality for their own health, it is particularly important and urgent to develop more sensitive, stable, and adaptable to a variety of complex situations. Among the existing disease detection technologies, Enzyme-Linked ImmunoSorbent Assay (ELISA) has been used in clinical applications of almost all diseases due to its low detection cost and simple operation. It is especially suitable for the initial qualitative diagnosis of diseases and large-scale physical examinations. Although the ELISA detection method has many advantages, it is limited by the enzyme's own catalytic activity, thermal stability and catalytic substrates. This method still faces many shortcomings in detection sensitivity and stability, especially for the early diagnosis of cancer. There is a relative lack of products. Based on this, on the basis of the ELISA detection method, exploring highly active and highly stable enzyme replacement products will hopefully develop more sensitive and stable disease detection methods.

[0003] The present invention develops a series of highly stable and highly catalytically active cyclodextrin-adamantane iridium complex self-assembly labeling strategies to label nanoparticles, and uses them to replace the enzymes in traditional ELISA detection methods. By strictly controlling the modification order of the nanomaterials by the labeled antibody and the adamantane functionalized iridium complex, the irreversible problem of easy sulfhydryl deactivation of the metal iridium complex in the construction of the detection method is successfully solved. Further, combined with a hydrogenation-regulated fluorescent probe that specifically responds to the metal iridium complex, and combined with traditional ELISA detection technology, a highly stable protein marker fluorescence detection method is constructed. Summary of the invention

[0004] In order to solve the key technical problem that the thiol poisoning of organometallic iridium complex hydrogenation catalyst is easy in the application of disease marker detection, the present invention studies a self-assembly labeling strategy based on cyclodextrin-adamantane iridium complex, with gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), polystyrene microspheres (PS) or silica microspheres (SiO 2) as a carrier and a post-modification strategy of organic metal iridium complex were adopted to successfully solve the influence of conventional blocking agent fetal bovine serum albumin (BSA) on the catalyst. Furthermore, a highly sensitive response hydrogenation-regulated fluorescent probe and traditional ELISA analysis technology were combined to construct a new type of disease marker fluorescence detection method.

[0005] To achieve the above object, the present invention adopts the following technical solutions.

[0006] A fluorescence detection method for protein markers labeled by cyclodextrin-adamantanized iridium complex host-guest assembly.

[0007] The method comprises the following steps: (1) preparing a nano material co-labeled with a cyclodextrin-adamantanized iridium complex and a labeled antibody Ab1; (2) using an ELISA plate or magnetic beads as a carrier, coating the carrier with a coating antibody, blocking the carrier with a blocking agent, and then binding the carrier with a corresponding antigen to be detected; (3) adding a quantitative and excessive amount of the nano material obtained in step (1) to the antigen carrier material obtained in step (2), and removing the excess nano material after incubation; (4) adding a fluorescent detection liquid containing a hydrogenation-regulated fluorescent probe to the labeled material obtained in step (3), wherein the metal iridium complex catalyst can catalyze the fluorescent probe molecule to generate a fluorescent signal, and then the quantitative detection of the antigen biological protein marker to be detected can be achieved by detecting the fluorescence intensity of the detection liquid.

[0008] Step (1) Preparation of nanomaterials co-labeled with cyclodextrin-adamantane iridium complex and labeled antibody Ab1, comprising the following steps: 1) using gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), polystyrene microspheres (PS) or silica microspheres (SiO 2 ) as a carrier, first directly mixed with the labeled antibody Ab1 in a certain ratio for modification or activated with a condensing agent (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDCI, N-hydroxysuccinimide NHS) and then coupled to obtain Ab1-labeled nanomaterials AuNPs@Ab1 or AgNPs@Ab1 or PS@Ab1 or SiO 2 @Ab1; 2) AuNPs@Ab1 or AgNPs@Ab1 or PS@Ab1 or SiO 2 @Ab1 was further mixed with thiol- or disulfide- or amino-functionalized β-cyclodextrin for 2 to 24 hours, and then incubated with blocking agent fetal bovine serum albumin (BSA) or skim milk powder or casein or Tween to obtain cyclodextrin-functionalized Ab1-labeled gold nanoparticles AuNPs-Ab1@CD or AgNPs-Ab1@CD or PS-Ab1@CD or SiO 2-Ab1@CD; 3) AuNPs-Ab1@CD or AgNPs-Ab1@CD or PS-Ab1@CD or SiO 2 -Ab1@CD was incubated in adamantane iridium complex solution for 1-24 hours to obtain cyclodextrin-adamantane host-guest modified iridium complex labeled nanomaterials AuNPs-Ab1@CD@Ir or AgNPs-Ab1@CD@Ir or PS-Ab1@CD@Ir or SiO 2 -Ab1@CD@Ir;

[0009] The analysis of the corresponding protein markers includes the following steps:

[0010] 4) Loading the coated antibody Ab2 on the carrier ELISA plate or magnetic nanoparticles, and incubating and blocking with blocking protein BSA to obtain the Ab2-loaded carrier material S@Ab2;

[0011] 5) Add the test protein AG to a certain concentration of S@Ab2 buffer solution, so that the test protein AG specifically binds to the coated antibody Ab2 on the carrier to obtain S-Ab2@AG;

[0012] 6) Add the iridium complex labeled nanoparticles AuNPs-Ab1@CD@Ir or AgNPs-Ab1@CD@Ir or PS-Ab1@CD@Ir or SiO2 into S-Ab2@AG. 2 -Ab1@CD@Ir, realizes the labeling of the protein AG to be tested by iridium complex.

[0013] 7) Add a fluorescent detection solution containing a redox fluorescent probe and a hydrogen source (formic acid or sodium formate or potassium formate or ammonium formate or triethylamine formate or nicotinamide adenine dinucleotide disodium salt (reduced form) NADH) to the labeled material obtained in step 6, incubate the reaction at a temperature of 0-100 degrees Celsius for 0-120 minutes, and then test the change in fluorescence intensity of the solution to achieve quantitative detection of the protein AG to be tested.

[0014] Preferably, the nanoparticles in step 1) are all monodisperse nanoparticles with a diameter of 1 nm to 5000 nm and a spherical, rod-shaped or diamond-shaped morphology. Nanomaterials include gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), polystyrene microspheres (PS) or silica microspheres (SiO 2 ), wherein the surfaces of polystyrene microspheres and silica microspheres must contain active functional groups such as amino, carboxyl, ethylene oxide, and N-hydroxysuccinimide.

[0015] Preferably, the adamantane functionalized iridium complex described in step 3) contains an adamantane functional group in its structure, which can form a strong host-guest binding body with β-cyclodextrin. The anion form of the metal catalyst is a fluoroborate ion (BF 4- ) or hexafluorophosphate ion (PF 6- ) or sulfate ion (SO 4 2- ) or nitrate ion (NO 3- ), fluoride ion (F - ) or chloride ion (Cl - ) or bromide ion (Br - ) or iodide ion (I - ), the metal catalyst ligands include but are not limited to nitrogen-nitrogen ligands (1,10-phenanthroline or 2,2'-bipyridine or 2,2'-bipyrimidine or 2-pyridinebenzamide or 2-pyridinebenzamide or 2-quinolinebenzamide or 2-quinolinebenzamide) and carbon-nitrogen ligands (1-phenylpyrazole or phenylimine or phenylpyridine or phenylimidazole or benzoquinoline).

[0016] The structure of the adamantane metal iridium complex comprises one or more of the following structures:

[0017]

[0018] Preferably, the generation and amplification of the fluorescent signal is that the iridium complex catalyzes the hydrogenation reduction reaction of the redox fluorescent probe, and the fluorescent probe molecules all contain oxidized functional groups (benzoquinone or naphthoquinone or anthraquinone or nitro or carbonyl or nicotinamide salt or aldehyde or pyridinium salt), and when the oxidized functional groups in the fluorescent probe molecules are hydrogenated and reduced to the corresponding reduced state (benzenediol or naphthalene diol or anthracene diol or amino or hydroxyl or nicotinamide salt or methylhydroxyl or dihydropyridine), the probe will produce obvious fluorescence enhancement or weakening. Fluorescent chromophores mainly include: coumarins, rhodamine, xanthenes, naphthalene diimides, benzoxazoles, stilbenes, thiophene dicarboxylic acid amides, condensed aromatic hydrocarbons, and perylene tetracarboximides.

[0019] The hydrogenation reduction regulated fluorescent probe mainly comprises one or a combination of the following structures:

[0020]

[0021] Preferably, the order of step 3 and step 4 cannot be reversed, and the adamantane functionalized metal iridium complex can be introduced only after the blocking with fetal bovine serum albumin (BSA) or skimmed milk powder or casein or Tween is completed.

[0022] Preferably, the composition of the redox fluorescent detection solution in step 7) is: 10-90% (V / V) of organic solvent (including dimethyl sulfoxide DMSO or N,N-dimethylformamide DMF or ethanol or methanol or acetonitrile or dioxane), 90-10% (V / V) of water, and the volume sum of the organic solvent and water is 100% (V / V); redox fluorescent probe molecules with a final concentration of 1-1000 μM; formic acid with a final concentration of 10-1000 mM, sodium formate with a final concentration of 10-1000 mM; pH is 1-12.

[0023] Beneficial Effects

[0024] The present invention utilizes the host-guest interaction between adamantane and cyclodextrin, and by adjusting the modification order of the metal iridium complex catalyst on the labeling material, effectively solves the problem of the inhibitory inactivation of the metal catalyst by the blocking agent. Further combined with the hydrogenation-regulated fluorescent probe and the traditional ELISA detection strategy, a sensitive and stable fluorescent detection method for biological protein markers is established.

[0025] The purpose of the present invention is to address the deficiencies in traditional immunoassay technology, break through the technical bottleneck of the application of organic metal complexes in the construction of disease detection methods, and develop a new type of protein marker fluorescence detection method. Based on the traditional protein immune response, nanomaterials labeled with high hydrogenation activity and high stability metal iridium complexes are used to replace the enzymes in the commercial method, and a sensitive hydrogenation reduction fluorescence regulation system is introduced to provide a disease marker fluorescence detection method based on cyclodextrin-adamantanized iridium complex labeling. This detection method can achieve sensitive and stable detection of test samples, and has the advantages of simple operation, rapid detection, good accuracy, and low cost. It can play an important role in in vitro detection of biomarker proteins and clinical research. Its main advantages are:

[0026] (1) The host-guest coupling strategy of cyclodextrin-adamantane iridium complex and the post-modification strategy of metal iridium complex can successfully solve the problem of complex catalyst poisoning caused by blocking agent. This breaks through the technical bottleneck of the application of metal iridium complex hydrogenation catalyst in the construction of disease detection methods.

[0027] (2) Using stable and highly active metal iridium complexes to catalyze hydrogenation reactions instead of traditional enzyme catalysis processes. Since metal catalysts have better thermal stability and smaller molecular structures, they can shorten the reaction time by adjusting the reaction temperature, playing an important role in the in vitro detection of biological protein markers.

[0028] The present invention is based on an efficient and stable metal iridium complex catalyzed hydrogenation reduction reaction, rationally introduces a cyclodextrin-adamantane iridium complex host-guest coupling strategy, and solves the problem of complex catalyst poisoning caused by a blocking agent; further combined with a hydrogenation-regulated fluorescent probe, a new method for fluorescent detection of disease markers based on cyclodextrin-adamantane iridium complex labeling is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The flowchart of the preparation of nanomaterials co-labeled with organic iridium complex and labeled antibody, the numbers in the figure are:

[0030] 1. Labeled nanomaterials; 2. Labeled antibodies; 3. Labeled antibody-modified nanomaterials; 4. 6-monothiol β-cyclodextrin; 5. Labeled antibodies and cyclodextrin co-modified nanomaterials; 6. Blocking agent; 7. Blocked labeled antibodies and cyclodextrin co-modified nanomaterials; 8. Diamond-functionalized organic iridium complexes; 9. Nanomaterials co-labeled with organic iridium complexes and labeled antibodies.

[0031] Figure 2 This is an application flow chart of the fluorescent detection method of disease markers based on cyclodextrin-adamantane iridium complex labeling using magnetic beads as coated antibodies and antigen carriers. The numbers in the figure are:

[0032] 1. Magnetic beads; 2. Coated antibodies; 3. Antigens;

[0033] 4. Nanomaterials co-labeled with organic iridium complexes and labeled antibodies; 5. Hydrogenation-regulated fluorescent probes.

[0034] Figure 3 This is an application flow chart of the fluorescence detection method of disease markers based on cyclodextrin-adamantane iridium complex labeling, using ELISA plate as a coated antibody and antigen carrier. The numbers in the figure are:

[0035] 1. ELISA plate; 2. Coated antibody; 3. Antigen;

[0036] 4. Nanomaterials co-labeled with organic iridium complexes and labeled antibodies; 5. Hydrogenation-regulated fluorescent probes. DETAILED DESCRIPTION

[0037] The following is a specific embodiment of a method for fluorescent detection of disease markers based on cyclodextrin-adamantanized iridium complex labeling according to the present invention, which is described in conjunction with the accompanying drawings. It should be noted that the implementation of the present invention is not limited to the embodiments described below.

[0038] 1) Figure 1The process shown is specifically as follows: the nanomaterial 1 is first directly mixed with the labeled antibody 2 in a certain ratio for modification or activated with a condensing agent (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDCI, N-hydroxysuccinimide NHS) and then coupled to obtain the labeled antibody modified nanomaterial 3; further mixed with thiol or disulfide bond or amino functionalized β-cyclodextrin 4 and oscillated for 2 to 24 hours to obtain the labeled antibody and cyclodextrin co-modified nanomaterial 5; then the blocking agent fetal bovine serum albumin (BSA) or skimmed milk powder or casein or Tween 6 is used for incubation and blocking to obtain the blocked labeled antibody and cyclodextrin co-modified nanomaterial 7; finally, the obtained nanomaterial 7 is incubated in a solution of adamantane iridium complex 8 for 1-24 hours to obtain the organic iridium complex and labeled antibody co-labeled nanomaterial 9.

[0039] 2) Figure 2 The process shown is as follows:

[0040] First, the coated antibody 2 is connected to the magnetic bead 1 through a chemical reaction; then, the magnetic bead fixed with the coated antibody 2 can specifically bind to the antigen 3 to be tested, and then combine with the nanomaterial 4 co-labeled with the organic iridium complex and the labeled antibody; through the corresponding relationship between the organic iridium complex and the antigen, more antigens can be combined with more transition metal catalysts, and at the same time, a single organic iridium complex can catalyze a large number of fluorescent probe molecules to produce fluorescent signals, thereby achieving antigen labeling and signal amplification; finally, a detection solution is added to the magnetic bead 1 labeled with the organic iridium complex (the detection solution contains hydrogenation reduction fluorescent "on-off" type probe molecules 5, and hydrogen source formic acid or sodium formate or ammonium formate), and the quantitative detection of the antigen 3 can be achieved by detecting the fluorescence intensity of the detection solution.

[0041] Figure 3 The process shown is as follows:

[0042] First, the coated antibody 2 is connected to the ELISA plate 1 through chemical adsorption; then, the ELISA plate fixed with the coated antibody 2 can specifically bind to the antigen 3 to be tested, and then combine with the nanomaterial 4 co-labeled with the organic iridium complex and the labeled antibody; through the corresponding relationship between the organic iridium complex and the antigen, more antigens can be combined with more transition metal catalysts, and at the same time, a single organic iridium complex can catalyze a large number of fluorescent probe molecules to produce fluorescent signals, thereby achieving antigen labeling and signal amplification; finally, a detection solution (the detection solution contains hydrogenation reduction fluorescent "on-off" type probe molecules 5, and a hydrogen source of formic acid or sodium formate or ammonium formate) is added to the ELISA plate 1 labeled with the organic iridium complex, and the quantitative detection of the antigen 3 can be achieved by detecting the fluorescence intensity of the detection solution.

[0043] In the following embodiments, with reference to the above figures, the processes are the same as those shown in the corresponding figures.

[0044] Example 1

[0045] Application of a fluorescence detection method for disease markers labeled with cyclodextrin-adamantane iridium complex in the detection of alpha-fetoprotein (AFP) (the implementation process can be referred to Figure 1 and Figure 2 ). refer to Figure 1 The nanomaterial 1 is a gold nanoparticle with a diameter between 5 nm and 200 nm, the labeled antibody is a polyclonal AFP antibody 2, the assembled cyclodextrin 4 is 6-monothiol-β-cyclodextrin, the blocking agent 6 is fetal bovine serum albumin BSA, and the adamantane functionalized metal iridium complex is [(η 5 -C 5 Me 5 )Ir(Adamantan-2yl-picolinamide)Cl]Ir-1. refer to Figure 2 The magnetic beads 1 used are 50-500nm carboxyl magnetic beads, amino magnetic beads, epoxy magnetic beads and streptavidin magnetic beads, etc. The coated antibody 2 used is a monoclonal AFP antibody, and the fluorescent probe used is Probe-1 (after hydrogenation reduction, the probe excitation wavelength is 550nm and the emission wavelength is 676nm). Antigen 3 uses AFP antigen, and the detection solution is a fluorescent detection solution containing hydrogenation-regulated fluorescent probe Probe-1.

[0046] The following is combined with Figure 1 Explain the specific steps:

[0047] (1) Preparation of nanomaterials co-labeled with organic iridium complexes and labeled antibodies (using gold nanoparticles as an example)

[0048] ① Take 2.0 mL of gold nanoparticles 1 and transfer them to a 5 mL centrifuge tube, separate and remove the supernatant, then add 50-200 μg of labeled antibody 2 to the centrifuge tube containing gold nanoparticles 1 and incubate at 25°C for 2 to 10 hours.

[0049] ② Add 2 mL of 0.5 mM 6-monomercapto-β-cyclodextrin aqueous solution to the gold nanoparticle solution, and continue incubating at 25° C. for 2-10 hours.

[0050] ③ Without separation, continue to add 2 mL of 2% blocking agent fetal bovine serum albumin BSA to the above solution, continue incubation at 25°C for 2 hours, and then transfer to a 4°C refrigerator and store overnight;

[0051] ④ The blocked gold nanoparticles were centrifuged and washed with phosphate buffer solution (PBST) containing Tween 20 (3-6 times), and then 2 mL of 0.1 mM adamantane iridium complex Ir-1 aqueous solution was added, and the mixture was incubated at 25°C for 24 hours. The excess iridium complex was removed by centrifugation, and the mixture was transferred to a 4°C refrigerator for storage.

[0052] The preparation process of the organic iridium complex prepared by using other adamantane iridium complexes Ir-2, Ir-3, Ir-4, Ir-5, Ir-6, Ir-7, Ir-8, Ir-9, Ir-10, Ir-11, Ir-12, Ir-13, Ir-14, Ir-15, and Ir-16, and the preparation process of the nanomaterial co-labeled with the labeled antibody is the same as the above steps, except that:

[0053] ④ The blocked gold nanoparticles were centrifuged and washed with phosphate buffer solution (PBST) containing Tween 20 (3-6 times), and then 2 mL of 0.1 mM adamantane iridium complex Ir-2 or Ir-3 or Ir-4 or Ir-5 or Ir-6 or Ir-7 or Ir-8 or Ir-9 or Ir-10 or Ir-11 or Ir-12 or Ir-13 or Ir-14 or Ir-15 or Ir-16 solution (containing 10-20 v / v% dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) in aqueous solution) was added, and the mixture was incubated at 25°C for 24 hours, and the excess iridium complex was removed by centrifugation, and the mixture was transferred to a 4°C refrigerator for storage.

[0054] The following is combined with Figure 2 Explain the specific steps:

[0055] (2) Preparation of coated antibody-coupled magnetic beads (using amino magnetic beads as an example)

[0056] ① Take 500 μL of amino magnetic beads 1 and transfer them to a 1.5 mL centrifuge tube. Remove the supernatant by magnetic separation.

[0057] Add 1.0 mL of 10%-20% glutaraldehyde aqueous solution, shake at 25°C for 0.5-10 hours in a dark environment, and keep the magnetic beads 1 suspended during this period;

[0058] ② Remove excess glutaraldehyde by magnetic separation and wash with water three times, then add 50-200 μg of coated antibody to the centrifuge tube containing magnetic beads 1 (0.01-0.05% Tween 20 can be added to improve the dispersibility of magnetic beads 1), shake gently to mix, and place at 25°C for 0.5-10 hours, during which time the magnetic beads 1 should be kept suspended;

[0059] ③ Without separation, add 100 μL-1000 μL of bovine serum albumin (BSA) and phosphate buffer solution (pH=7.0-8.0, containing 1-10% BSA) to the magnetic bead solution to resuspend the magnetic beads 1, and react at 25°C for 1.0-24 hours to block the non-specific reaction sites on the surface of the magnetic beads 1; after blocking, place the centrifuge tube containing the magnetic beads on a magnetic separation rack to remove the supernatant by magnetic separation, wash with 1000 μL of phosphate buffer solution PBST containing 0.1% Tween 20 for more than 3 times each time, resuspend in the preservation solution, and store at 4°C for use.

[0060] The steps of coupling the coated antibody to the carboxyl magnetic beads are basically the same as the steps of coupling the ligand to the amino magnetic beads (2), except that:

[0061] ① After the purchased carboxyl modified magnetic beads are mixed evenly, 500 μL of carboxyl magnetic beads 1 are transferred to a 1.5 mL centrifuge tube, the supernatant is removed by magnetic separation, and magnetic separation and washing are performed twice with 200 μL PBS solution (50 mM PBS, pH = 7.4). Then, 500 μL of freshly prepared N-hydroxysuccinimide aqueous solution (0.1-0.5 M) NHS and 500 μL of N-hydroxy 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution (0.1-1 M) EDCI are added to the centrifuge tube, vortexed to mix well to fully suspend the magnetic beads 1, and then placed at 25°C for reaction for 1-5 hours, during which the magnetic beads 1 should be kept suspended.

[0062] The contents of steps ② and ③ are the same as step (2) of coupling the amino magnetic beads with the coated antibody.

[0063] The steps for coupling epoxy magnetic beads and streptavidin magnetic beads to ligands are the same, and the difference from step (2) for coupling amino magnetic beads to ligands is that the contents of steps ② and ③ are the same as step (2) for coupling amino magnetic beads to coated antibodies.

[0064] (3) Prepare the test solution.

[0065] ①Using Probe-1 as a hydrogenation-regulated fluorescence probe, the fluorescence regulation mechanism of the probe molecule Probe-1 is as follows:

[0066]

[0067] The fluorescence of the probe Probe-1 fluorophore is quenched due to the intramolecular electron transfer of the oxidized functional group trimethyl-p-benzoquinone in its structure; however, when the quinone in its molecular structure is reduced to p-phenol, the amide bond in the probe molecule will break, and the intramolecular electron transfer fluorescence quenching effect of the fluorescent substrate dicyanoisophorone in the probe molecule will be destroyed, and the molecule will produce a strong fluorescence signal.

[0068] Note: The fluorescence response mechanism of probes Probe-2, Probe-4, Probe-5, Probe-7, and Probe-8 is similar to that of Probe-1, except that the fluorescent substrates of the probes are different. The fluorescent substrate of Probe-2 is phenolic hydroxydicyanoisophorone, the fluorescent substrate of Probe-4 is 4-methyl-7-aminocoumarin, the fluorescent substrate of Probe-5 is 7-hydroxycoumarin, the fluorescent substrate of Probe-7 is rhodamine 110, and the fluorescent substrate of Probe-8 is naphthalene diimide.

[0069] ②Configuration of detection liquid.

[0070] The probe molecule Probe-1 is dissolved in a mixed solvent of dioxane and water (containing 10%-90% dioxane) to keep the final concentration of the probe molecule Probe-1 at 1-1000 μM. Then, formic acid, sodium formate or ammonium formate is added to the mixed solvent to keep the final concentration of formate ions in the solution between 10-1000 mM. The pH of the mixed solution is adjusted to 3-10, and the probe solution is stored at 4°C for future use.

[0071] (4) Immunoassay using alpha-fetoprotein antigen (AFP) as an example

[0072] ① Take 10 μL (10 mg / mL) of magnetic beads 1 modified with AFP monoclonal coated antibody 2 and transfer to a 100 μL centrifuge tube. After washing 1-2 times with PBST solution, add 100 μL of antigen AFP 3 of different concentrations and incubate at 37°C for 1-3 hours.

[0073] ② Wash the magnetic beads prepared in step ① 1 2-5 times with PBST solution, add 50-200 μL of the organic iridium complex prepared in step (1) and the nanomaterial co-labeled with the labeled antibody, and incubate at 37° C. for 1-3 hours.

[0074] ③ Wash the magnetic beads in step ② 1 2-5 times with PBST solution, add 100 μL of the detection solution prepared in step (3), react at 30-60°C for 5-60 minutes, and then perform fluorescence test using a fluorescence spectrometer or fluorescence microplate reader.

[0075] The significance of fluorescence intensity in Example 1

[0076] (1) The higher the concentration of alpha-fetoprotein antigen AFP, the more organic iridium complexes and nanomaterials co-labeled with labeled antibodies can be bound by the carrier magnetic beads. After adding the detection solution, under the same conditions, more probe molecules Probe-1 can be catalyzed to undergo hydrogenation reduction, the fluorescence is turned on, and a stronger fluorescence intensity is generated.

[0077] (2) When the concentration of alpha-fetoprotein antigen AFP is low or zero, the concentration of the organic iridium complex bound to the carrier magnetic beads and the nanomaterial co-labeled with the labeled antibody will also be relatively low or zero, so that only a small amount of the probe molecule Probe-1 can undergo hydrogenation reduction or no probe molecule Probe-1 can be reduced, and the fluorescence intensity generated by the detection solution will also be relatively weak or no fluorescence.

[0078] (3) Therefore, the present invention can utilize nanomaterials co-labeled with organic iridium complexes and labeled antibodies to realize fluorescence detection in the detection of liver cancer marker alpha-fetoprotein antigen (AFP).

[0079] Example 2

[0080] Application of a fluorescence detection method for disease markers based on cyclodextrin-adamantane iridium complex labeling in actual serum sample detection (the process can be referred to the attached Figure 2 ), amino magnetic beads 1 are used as carrier materials, the coated antibody 2 is a monoclonal AFP coated antibody, the labeled antibody in the nanomaterial co-labeled with an organic iridium complex and a labeled antibody is a polyclonal AFP labeled antibody, the organic iridium complex is Ir-2, the probe used is Probe-3, the antigen 3 is a human serum sample, and the detection liquid is a fluorescent detection liquid containing Probe-3. Figure 2 Explain the specific steps:

[0081] The steps are basically the same as in Example 1.

[0082] The difference between Example 2 and Example 1 is that the basic contents of steps (3) and (4) of Example 2 are:

[0083] (3) Prepare the test solution.

[0084] ①Using Probe-3 as a hydrogenation-regulated fluorescence probe, the fluorescence regulation mechanism of the probe molecule Probe-3 is as follows:

[0085]

[0086] The fluorescence of the probe Probe-3 fluorophore is quenched due to the intramolecular electron transfer of the oxidized functional group N-methylpyridinium salt in its structure; however, when the N-methylpyridinium salt in its molecular structure is reduced to N-methyl-4-dihydropyridine, the fluorescence quenching effect of the probe fluorescent substrate intramolecular electron transfer is destroyed, and the molecule produces a strong fluorescence signal.

[0087] ②Configuration of detection liquid.

[0088] The probe molecule Probe-3 is dissolved in an aqueous solution to maintain the final concentration of the probe molecule Probe-3 at 1-1000 μM, and then formic acid or sodium formate or ammonium formate is added to the mixed solvent to maintain the final concentration of formate ions in the solution between 10-1000 mM, and the pH of the mixed solution is adjusted to 6-10. The probe solution is stored at 4°C for future use.

[0089] (4) Immunoassay using human serum samples as an example

[0090] ① Take 10 μL (10 mg / mL) of magnetic beads 1 modified with AFP monoclonal antibody 2 and transfer it to a 100 μL centrifuge tube. After washing 1-2 times with PBST solution, add 100 μL of human serum of different concentrations and incubate at 37°C for 1-3 hours.

[0091] The contents of steps (4) ② and ③ are the same as those in Example 1.

[0092] Characterization significance of fluorescence intensity in detection example 2

[0093] (1) Positive samples: When the AFP content in human serum samples is high (>25μg / L), it shows a strong fluorescence intensity, which indicates that the serum sample may come from a liver cancer patient. Negative samples: When the AFP content in human serum samples is low (<25μg / L), the fluorescence intensity is very weak due to the low AFP content, which indicates that the serum sample should come from a normal person.

[0094] (2) By comparing with the concentration standard curve of alpha-fetoprotein, the present invention can more conveniently and accurately detect the content of alpha-fetoprotein in each serum sample to be tested.

[0095] (3) Example 2 proves that in the immunosorbent assay, the change in the fluorescence intensity of the detection solution caused by the nanomaterials co-labeled with organic iridium complexes and labeled antibodies at different concentrations can realize the detection of actual serum samples.

[0096] Example 3

[0097] Application of a fluorescence detection method for disease markers labeled with cyclodextrin-adamantane iridium complex in the detection of carcinoembryonic antigen (CEA) (the process can be referred to Figure 3 ), the ELISA plate 1 is a polystyrene ELISA plate, the antibody 2 is a monoclonal CEA coated antibody, the blocking agent 6 is fetal bovine serum albumin BSA, and the adamantane functionalized metal iridium complex is [(η5-C 5 Me 5 )Ir(Ada-quinoline-2-amide)Cl]Ir-3. refer to Figure 2The ELISA plate is used as a carrier of carcinoembryonic antigen and antibody, the coated antibody 2 is a monoclonal CEA antibody, and the fluorescent probe is Probe-6 (after hydrogenation reduction, the probe excitation wavelength is 470nm and the emission wavelength is 530nm). Antigen 3 uses CEA antigen, and the detection solution is a detection solution containing the hydrogenation-regulated fluorescent probe Probe-6.

[0098] The following is combined with Figure 3 Explain the specific steps:

[0099] (1) The preparation process of nanomaterials co-labeled with organic iridium complex and labeled antibody is as follows Figure 1 (Taking amino silica microspheres as an example for illustration)

[0100] ① Take 2.0 mL of the amino-modified silica nanoparticles 1 and transfer them to a 5 mL centrifuge tube. Centrifuge to remove the supernatant, add 3 mL of a 10%-20% glutaraldehyde aqueous solution, incubate at 25° C. for 2 to 10 hours, centrifuge to remove excess glutaraldehyde, and wash with water for more than 3 times;

[0101] ② Add 50-200 μg of CEA labeled antibody 2 to the centrifuge tube containing silica microspheres 1 obtained in ①, and continue incubating at 25°C for 2 to 10 hours;

[0102] ③ Add 2.0 mL of 0.5 mM 6-monoamino-β-cyclodextrin aqueous solution to the centrifuge tube containing silica microspheres 1 obtained in ②, and continue incubating at 25° C. for 2-10 hours;

[0103] ④ After centrifugation to remove excess cyclodextrin and labeled antibodies, add 2.0 mL of 2% blocking agent fetal bovine serum albumin BSA to solution ③, continue incubation at 25°C for 2 hours, and then transfer to a 4°C refrigerator and store overnight;

[0104] ⑤ The blocked silica nanoparticles were centrifuged and washed with phosphate buffer solution (PBST) containing Tween 20 (3-6 times), and then 2 mL of 0.1 mM adamantane iridium complex Ir-3 solution (containing 10%-90% organic solvent DMF) was added, and the mixture was incubated at 25°C for 24 hours. The excess iridium complex was removed by centrifugation and the mixture was transferred to a 4°C refrigerator for storage.

[0105] The preparation process of the organic iridium complex prepared by using other adamantane iridium complexes Ir-1, Ir-2, Ir-4, Ir-5, Ir-6, Ir-7, Ir-8, Ir-9, Ir-10, Ir-11, Ir-12, Ir-13, Ir-14, Ir-15, and Ir-16, and the preparation process of the nanomaterial co-labeled with the labeled antibody is the same as the above steps, except that:

[0106] ④ The blocked gold nanoparticles were centrifuged and washed with phosphate buffer solution (PBST) containing Tween 20 (3-6 times), and then 2 mL of 0.1 mM adamantane iridium complex Ir-1 or Ir-2 or Ir-4 or Ir-5 or Ir-6 or Ir-7 or Ir-8 or Ir-9 or Ir-10 or Ir-11 or Ir-12 or Ir-13 or Ir-14 or Ir-15 or Ir-16 solution (containing 10-20 v / v% dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) in aqueous solution) was added, and the mixture was incubated at 25°C for 24 hours, and the excess iridium complex was removed by centrifugation, and the mixture was transferred to a 4°C refrigerator for storage.

[0107] (2) An ELISA plate 1 is used as a carrier of carcinoembryonic antigen (CEA) antigen and antibody. The ELISA plate 1 can be a 96-well ELISA plate, a 48-well ELISA plate, a 384-well ELISA plate, or other ELISA plates with different numbers of wells. The ELISA plates include two types: detachable and non-detachable. The colors of the ELISA plates include black, white, and colorless and transparent colors.

[0108] (3) Prepare the test solution.

[0109] ①Using Probe-6 as a hydrogenation-regulated fluorescence probe, the fluorescence regulation mechanism of the probe molecule Probe-6 is as follows:

[0110]

[0111] The fluorescence of the probe Probe-6 fluorophore is strong due to the intramolecular electron resonance effect of the oxidized functional group aldehyde in its structure; however, when the aldehyde group in its molecular structure is reduced to methyl hydroxyl group, the intramolecular electron resonance effect of the probe fluorescent substrate is weakened, and the molecular fluorescence signal is weakened.

[0112] ②Configuration of detection liquid.

[0113] The probe molecule Probe-6 is dissolved in an aqueous solution (containing 1.0%-20% organic solvent DMF) to keep the final concentration of the probe molecule Probe-6 at 1-1000 μM, and then formic acid or sodium formate or ammonium formate is added to the mixed solvent to keep the final concentration of formate ions in the solution between 10-1000 mM. The pH of the mixed solution is adjusted to 6-10, and the probe solution is stored at 4°C for future use.

[0114] (4) Immunoassay using carcinoembryonic antigen (CEA) as an example

[0115] ① Monoclonal antibody 2CEA (1.0-4.0 μg mL -1 ) was dissolved in carbonate buffer at pH = 9.6, and 50-150 μL of the above solution was added to each well of ELISA plate 1 and incubated at 4°C overnight;

[0116] ② Wash the ELISA plate in step ① 3-5 times with phosphate-buffered saline (PBST), and then add 100-400 μL 1% bovine serum albumin (BSA) for blocking;

[0117] ③ Wash the ELISA plate in step ② 3-5 times with phosphate-buffered saline Tween buffer (PBST), add different concentrations of carcinoembryonic antigen (AFP), and incubate at 37° C. for 1-3 hours.

[0118] ④ Wash the ELISA plate prepared in step ③ 3-5 times with phosphate-buffered saline (PBST), add 100-200 μL of the organic iridium complex prepared in step (1) and the nanomaterial co-labeled with the labeled antibody, and incubate at 37° C. for 1-3 hours;

[0119] ⑤ Wash the ELISA plate prepared in step ④ 3-5 times with phosphate-buffered saline (PBST), add 100 μL of the detection solution prepared in step (2), react at 20-100° C. for 10-100 min, and then perform quantitative detection using a fluorescence spectrometer or a fluorescence ELISA reader.

[0120] Characterization significance of fluorescence intensity in detection example 3 (detection principle)

[0121] (1) When the concentration of carcinoembryonic antigen (CEA) is high, it means that the carrier magnetic beads can bind more organic iridium complexes and nanomaterials co-labeled with labeled antibodies. After adding the detection solution, under the same conditions, it can catalyze more probe molecules Probe-6 to undergo hydrogenation reduction, turn on the fluorescence, and produce a stronger fluorescence intensity.

[0122] (2) When the concentration of carcinoembryonic antigen CEA is low or zero, the concentration of the organic iridium complex bound to the carrier magnetic beads and the nanomaterial co-labeled with the labeled antibody will also be relatively low or zero, so that only a small amount of the probe molecule Probe-6 can undergo hydrogenation reduction or no probe molecule Probe-6 can be reduced, and the fluorescence intensity generated by the detection solution will also be relatively weak or no fluorescence.

[0123] (3) Therefore, the present invention can utilize nanomaterials co-labeled with organic iridium complexes and labeled antibodies to realize fluorescence detection in the detection of CEA, a broad-spectrum cancer marker.

[0124] Example 4

[0125] Application of a fluorescence detection method for disease markers based on cyclodextrin-adamantane iridium complex labeling in actual serum sample detection (the process can be referred to the attached Figure 3), using ELISA plate 1 as carrier material, the coated antibody 2 used is monoclonal CEA coated antibody, the labeled antibody in the nanomaterial co-labeled with organic iridium complex and labeled antibody is polyclonal CEA labeled antibody, the organic iridium complex is Ir-3, the probe used is Probe-6, the antigen 3 uses human serum sample, and the detection liquid is a fluorescent detection liquid containing Probe-6. Figure 2 Explain the specific steps:

[0126] The steps are basically the same as those in Example 3.

[0127] The following is combined with Figure 3 Explain the specific steps:

[0128] (1) Preparation of nanomaterials co-labeled with organic iridium complex and labeled antibody (using aminosilica microspheres as carrier, as in Example 3);

[0129] (2) ELISA plate used (same as in Example 3)

[0130] (3) Prepare the test solution (same as in Example 3).

[0131] (4) Immunoassay using carcinoembryonic antigen (CEA) as an example

[0132] ① Monoclonal antibody 2CEA (1.0-4.0 μg mL -1 ) was dissolved in carbonate buffer at pH = 9.6, and 50-150 μL of the above solution was added to each well of ELISA plate 1 and incubated at 4°C overnight;

[0133] ② Wash the ELISA plate in step ① 3-5 times with phosphate-buffered saline (PBST), and then add 100-400 μL 1% bovine serum albumin (BSA) for blocking;

[0134] ③ Wash the ELISA plate in step ② 3-5 times with phosphate-buffered saline (PBST), add different concentrations of human serum, and incubate at 37° C. for 1-3 hours.

[0135] ④ Wash the ELISA plate prepared in step ③ 3-5 times with phosphate-buffered saline (PBST), add 100-200 μL of the organic iridium complex prepared in step (1) and the nanomaterial co-labeled with the labeled antibody, and incubate at 37° C. for 1-3 hours;

[0136] ⑤ Wash the ELISA plate prepared in step ④ 3-5 times with phosphate-buffered saline (PBST), add 100 μL of the detection solution prepared in step (2), react at 20-100° C. for 10-100 min, and then perform quantitative detection using a fluorescence spectrometer or a fluorescence ELISA reader.

[0137] Characterization significance of fluorescence intensity in detection example 2

[0138] (1) Positive samples: When the CEA content of human serum samples is high, it shows a strong fluorescence intensity, which can be judged that the serum sample may come from a cancer patient. Negative samples: When the CEA content of human serum samples is low, the fluorescence intensity is very weak due to the low CEA content, which can be judged that the serum sample should come from a normal person.

[0139] (2) By comparing with the concentration standard curve of carcinoembryonic antigen (CEA), the present invention can more conveniently and accurately detect the content of carcinoembryonic antigen (CEA) in each serum sample to be tested.

[0140] (3) Example 4 demonstrates that in an immunosorbent assay, the change in fluorescence intensity of the test solution caused by nanomaterials co-labeled with organic iridium complexes and labeled antibodies at different concentrations can achieve detection of actual serum samples.

[0141] Example 5

[0142] Application of a fluorescence detection method for disease markers labeled with cyclodextrin-adamantanized iridium complex in the detection of C-reactive protein (CRP) (the implementation process can be referred to Figure 1 and Figure 2 ). refer to Figure 1 The nanomaterial 1 is an amino polystyrene microsphere with a diameter between 5 nm and 200 nm, the labeled antibody is a polyclonal CRP antibody 2, the assembled cyclodextrin 4 is 6-amino-β-cyclodextrin, the blocking agent 6 is fetal bovine serum albumin BSA, and the adamantane functionalized metal iridium complex is [(η 5 -C 5 Me 5 )Ir(Adamantan-bipyridine)Cl]Ir-4. refer to Figure 2 The magnetic beads 1 used are 50-500nm carboxyl magnetic beads, amino magnetic beads, epoxy magnetic beads and streptavidin magnetic beads, etc. The coated antibody 2 used is a monoclonal CRP antibody, and the fluorescent probe used is Probe-9 (after hydrogenation reduction, the probe excitation wavelength is 465nm and the emission wavelength is 530nm). Antigen 3 uses CRP antigen, and the detection liquid is a fluorescent detection liquid containing hydrogenation-regulated fluorescent probe Probe-9.

[0143] The following is combined with Figure 1 Explain the specific steps:

[0144] (1) Preparation of nanomaterials co-labeled with organic iridium complex and labeled antibody (using amino polystyrene microspheres as an example)

[0145] ① Take 2.0 mL of amino polystyrene microspheres 1 and transfer them to a 5 mL centrifuge tube. Remove the supernatant by centrifugation. Add 3 mL of 10%-20% glutaraldehyde aqueous solution. Incubate at 25°C for 2 to 10 hours. Remove excess glutaraldehyde by centrifugation and wash with water for more than 3 times.

[0146] ② Add 50-200 μg of CRP labeled antibody 2 to the centrifuge tube containing polystyrene microspheres 1 obtained in ①, and continue incubating at 25°C for 2 to 10 hours;

[0147] ③ Add 2.0 mL of 0.5 mM 6-monoamino-β-cyclodextrin aqueous solution to the centrifuge tube containing the polystyrene microspheres 1 obtained in ②, and continue incubating at 25° C. for 2-10 hours;

[0148] ④ After centrifugation to remove excess cyclodextrin and labeled antibodies, add 2.0 mL of 2% blocking agent fetal bovine serum albumin BSA to solution ③, continue incubation at 25°C for 2 hours, and then transfer to a 4°C refrigerator and store overnight;

[0149] ⑤ The blocked polystyrene microspheres were centrifuged and washed with phosphate buffer solution (PBST) containing Tween 20 (3-6 times), and then 2 mL of 0.1 mM adamantane iridium complex Ir-4 aqueous solution was added, and the microspheres were incubated at 25°C for 24 hours. The excess iridium complex was removed by centrifugation and the microspheres were transferred to a 4°C refrigerator for storage.

[0150] The preparation process of the organic iridium complex prepared by using other adamantane iridium complexes Ir-1, Ir-2, Ir-3, Ir-5, Ir-6, Ir-7, Ir-8, Ir-9, Ir-10, Ir-11, Ir-12, Ir-13, Ir-14, Ir-15, and Ir-16, and the preparation process of the nanomaterials co-labeled with the labeled antibody is the same as the above steps, except that:

[0151] ⑤ The blocked gold nanoparticles were centrifuged and washed with phosphate buffer solution (PBST) containing Tween 20 (3-6 times), and then 2 mL of 0.1 mM adamantane iridium complex Ir-1 or Ir-2 or Ir-3 or Ir-5 or Ir-6 or Ir-7 or Ir-8 or Ir-9 or Ir-10 or Ir-11 or Ir-12 or Ir-13 or Ir-14 or Ir-15 or Ir-16 solution (containing 10-20 v / v% dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) in aqueous solution) was added, and the mixture was incubated at 25°C for 24 hours, and the excess iridium complex was removed by centrifugation, and the mixture was transferred to a 4°C refrigerator for storage.

[0152] (2) Preparation process of antibody-coupled magnetic beads (same as Example 1)

[0153] (3) Prepare the test solution.

[0154] ①Using Probe-9 as a hydrogenation-regulated fluorescence probe, the fluorescence regulation mechanism of the probe molecule Probe-9 is as follows:

[0155]

[0156] The fluorescence of the probe Probe-9 fluorophore is weak due to the intramolecular electron transfer effect of the oxidized functional group nitro in its structure; however, when the nitro in its molecular structure is reduced to amino, the intramolecular electron transfer effect of the probe fluorescent substrate is weakened and the molecular fluorescence signal is enhanced.

[0157] ②Configuration of detection liquid.

[0158] The probe molecule Probe-9 is dissolved in an aqueous solution (containing 1.0%-20% organic solvent DMSO) to keep the final concentration of the probe molecule Probe-9 at 1-1000 μM, and then formic acid or sodium formate or ammonium formate is added to the mixed solvent to keep the final concentration of formate ions in the solution between 10-1000 mM. The pH of the mixed solution is adjusted to 6-10, and the probe solution is stored at 4°C for future use.

[0159] (4) Immunoassay using C-reactive protein (CRP) as an example

[0160] ① Take 10 μL (10 mg / mL) of magnetic beads 1 modified with CRP monoclonal antibody 2 and transfer it to a 100 μL centrifuge tube. After washing 1-2 times with PBST solution, add 100 μL of antigen CRP 3 of different concentrations and incubate at 37°C for 1-3 hours.

[0161] ② Wash the magnetic beads prepared in step ① 1 2-5 times with PBST solution, add 50-200 μL of the organic iridium complex prepared in step (1) and the nanomaterial co-labeled with the labeled antibody, and incubate at 37° C. for 1-3 hours.

[0162] ③ Wash the magnetic beads in step ② 1 2-5 times with PBST solution, add 100 μL of the detection solution prepared in step (3), react at 30-60°C for 5-60 minutes, and then perform fluorescence test using a fluorescence spectrometer or fluorescence microplate reader.

[0163] The significance of fluorescence intensity in Example 5

[0164] (1) The higher the concentration of C-reactive protein antigen (CRP), the more organic iridium complexes and nanomaterials co-labeled with labeled antibodies can be bound by the carrier magnetic beads. After adding the detection solution, under the same conditions, more probe molecules Probe-9 can be catalyzed to undergo hydrogenation reduction, turn on fluorescence, and produce stronger fluorescence intensity.

[0165] (2) When the concentration of C-reactive protein antigen (CRP) is low or zero, the concentration of the organic iridium complex bound to the carrier magnetic beads and the nanomaterial co-labeled with the labeled antibody will also be relatively low or zero, so that only a small amount of the probe molecule Probe-9 can undergo hydrogenation reduction or no probe molecule Probe-9 can be reduced, and the fluorescence intensity generated by the detection solution will also be relatively weak or no fluorescence.

[0166] (3) Therefore, the present invention can utilize nanomaterials co-labeled with organic iridium complexes and labeled antibodies to realize fluorescence detection in the detection of the inflammatory marker C-reactive protein antigen (CRP).

[0167] Example 6

[0168] Application of a fluorescence detection method for disease markers based on cyclodextrin-adamantane iridium complex labeling in actual serum sample detection (the process can be referred to the attached Figure 2 ), magnetic beads 1 are used as carrier materials, the coated antibody 2 is a monoclonal CRP coated antibody, the labeled antibody in the nanomaterial co-labeled with an organic iridium complex and a labeled antibody is a polyclonal CRP labeled antibody, the organic iridium complex is Ir-4, the probe used is Probe-9, the antigen 3 is a human serum sample, and the detection liquid is a fluorescent detection liquid containing Probe-9. Figure 2 Explain the specific steps:

[0169] The steps are basically the same as in Example 5.

[0170] The following is combined with Figure 2 Explain the specific steps:

[0171] (1) Preparation of nanomaterials co-labeled with organic iridium complex and labeled antibody (using amino polystyrene microspheres as carrier, as in Example 5);

[0172] (2) ELISA plate used (same as in Example 5)

[0173] (3) Prepare the detection solution (same as in Example 5).

[0174] (4) Immunoassay using inflammatory markers (CRP) as an example

[0175] ① Take 10 μL (10 mg / mL) of magnetic beads 1 modified with CRP monoclonal antibody 2 and transfer it to a 100 μL centrifuge tube. After washing 1-2 times with PBST solution, add 100 μL of human serum samples of different concentrations and incubate at 37°C for 1-3 hours.

[0176] ② Wash the magnetic beads prepared in step ① 1 2-5 times with PBST solution, add 50-200 μL of the organic iridium complex prepared in step (1) and the nanomaterial co-labeled with the labeled antibody, and incubate at 37° C. for 1-3 hours.

[0177] ③ Wash the magnetic beads in step ② 1 2-5 times with PBST solution, add 100 μL of the detection solution prepared in step (3), react at 30-60°C for 5-60 minutes, and then perform fluorescence test using a fluorescence spectrometer or fluorescence microplate reader.

[0178] Characterization significance of fluorescence intensity in detection example 2

[0179] (1) Positive samples: When the C-reactive protein antigen (CRP) content of human serum samples is high, it shows a strong fluorescence intensity, which can be judged that the serum sample may come from an inflammatory patient. Negative samples: When the C-reactive protein antigen (CRP) content of human serum samples is low, the fluorescence intensity is very weak due to the low C-reactive protein antigen (CRP) content, which can be judged that the serum sample should come from a normal person.

[0180] (2) By comparing with the concentration standard curve of C-reactive protein antigen (CRP), the present invention can more conveniently and accurately detect the content of C-reactive protein antigen (CRP) in each serum sample to be tested.

[0181] (3) Example 6 proves that in the immunosorbent assay, the change in the fluorescence intensity of the detection solution caused by the nanomaterials co-labeled with organic iridium complexes and labeled antibodies at different concentrations can realize the detection of actual serum samples.

[0182] Example 7

[0183] Application of a fluorescence detection method for disease markers labeled with cyclodextrin-adamantane iridium complex in prostate specific antigen (PSA) detection (implementation process can be referred to Figure 1 and Figure 2 ). refer to Figure 1 The nanomaterial 1 is a gold nanoparticle with a diameter between 5 nm and 200 nm, the labeled antibody is a polyclonal PSA antibody 2, the assembled cyclodextrin 4 is 6-monothiol-β-cyclodextrin, the blocking agent 6 is fetal bovine serum albumin BSA, and the adamantane functionalized metal iridium complex is [(η 5 -C 5 Me 5 )Ir(Adamantan-2yl-picolinamide)Cl]Ir-1. refer to Figure 2The magnetic beads 1 used are 50-500nm carboxyl magnetic beads, amino magnetic beads, epoxy magnetic beads and streptavidin magnetic beads, etc. The coated antibody 2 used is a monoclonal PSA antibody, and the fluorescent probe used is Probe-1 (after hydrogenation reduction, the probe excitation wavelength is 550nm and the emission wavelength is 676nm). Antigen 3 uses PSA antigen, and the detection solution is a fluorescent detection solution containing hydrogenation-regulated fluorescent probe Probe-1.

[0184] The following is combined with Figure 1 Explain the specific steps:

[0185] (1) Preparation of nanomaterials co-labeled with organic iridium complex and labeled antibody (using gold nanoparticles as labeling material, as in Example 1);

[0186] (2) Preparation of coated antibody-coupled magnetic beads (using carboxyl magnetic beads as carrier, as in Example 1);

[0187] (3) Prepare the test solution (simultaneously with Example 1).

[0188] (4) Immunoassay using prostate-specific antigen (PSA) as an example

[0189] ① Take 10 μL (10 mg / mL) of magnetic beads 1 modified with PSA monoclonal coated antibody 2 and transfer to a 100 μL centrifuge tube. After washing 1-2 times with PBST solution, add 100 μL of antigen PSA 3 of different concentrations and incubate at 37°C for 1-3 hours.

[0190] ② Wash the magnetic beads prepared in step ① 1 2-5 times with PBST solution, add 50-200 μL of the organic iridium complex prepared in step (1) and the nanomaterial co-labeled with the labeled antibody, and incubate at 37° C. for 1-3 hours.

[0191] ③ Wash the magnetic beads in step ② 1 2-5 times with PBST solution, add 100 μL of the detection solution prepared in step (3), react at 30-60°C for 5-60 minutes, and then perform fluorescence test using a fluorescence spectrometer or fluorescence microplate reader.

[0192] The significance of fluorescence intensity in Example 7

[0193] (1) The higher the concentration of prostate-specific antigen (PSA), the more organic iridium complexes and nanomaterials co-labeled with labeled antibodies can be bound by the carrier magnetic beads. After adding the detection solution, under the same conditions, more probe molecules Probe-1 can be catalyzed to undergo hydrogenation reduction, fluorescence is turned on, and a stronger fluorescence intensity is produced.

[0194] (2) When the concentration of prostate-specific antigen (PSA) is low or zero, the concentration of the organic iridium complex bound to the carrier magnetic beads and the nanomaterial co-labeled with the labeled antibody will also be relatively low or zero, so that only a small amount of the probe molecule Probe-1 can undergo hydrogenation reduction or no probe molecule Probe-1 can be reduced, and the fluorescence intensity generated by the detection solution will also be relatively weak or no fluorescence.

[0195] (3) Therefore, the present invention can utilize nanomaterials co-labeled with organic iridium complexes and labeled antibodies to realize fluorescence detection in the detection of prostate specific antigen (PSA), a prostate cancer marker.

[0196] Example 8

[0197] Application of a fluorescence detection method for disease markers based on cyclodextrin-adamantane iridium complex labeling in actual serum sample detection (the process can be referred to the attached Figure 2 ), amino magnetic beads 1 are used as carrier materials, the coated antibody 2 is a monoclonal PSA coated antibody, the labeled antibody in the nanomaterial co-labeled with an organic iridium complex and a labeled antibody is a polyclonal AFP labeled antibody, the organic iridium complex is Ir-1, the probe used is Probe-1, the antigen 3 is a human serum sample, and the detection liquid is a fluorescent detection liquid containing Probe-1. Figure 2 Explain the specific steps:

[0198] The steps are basically the same as those in Example 7.

[0199] The difference between Example 8 and Example 7 is that the basic contents of steps (3) and (4) of Example 7 are:

[0200] (3) preparing a test solution (same as in Example 7);

[0201] (4) Immunoassay using human serum samples as an example

[0202] ① Take 10 μL (10 mg / mL) of magnetic beads 1 modified with PSA monoclonal coated antibody 2 and transfer to a 100 μL centrifuge tube. After washing 1-2 times with PBST solution, add 100 μL of human serum of different concentrations and incubate at 37°C for 1-3 hours.

[0203] The contents of steps (4) ② and ③ are the same as those of Example 7.

[0204] Characterization significance of fluorescence intensity in detection example 8

[0205] (1) Positive samples: When the prostate-specific antigen (PSA) level in human serum is high, the fluorescence intensity is strong, which indicates that the serum sample may be from a prostate cancer patient. Negative samples: When the prostate-specific antigen (PSA) level in human serum is low, the fluorescence intensity is very weak due to the low content of iridium complex, which indicates that the serum sample should be from a normal person.

[0206] (2) By comparing with the standard curve of prostate-specific antigen (PSA) concentration, the present invention can more conveniently and accurately detect the content of prostate-specific antigen (PSA) in each serum sample to be tested.

[0207] (3) Example 8 proves that in the immunosorbent assay, the change in the fluorescence intensity of the detection solution caused by the nanomaterials co-labeled with organic iridium complexes and labeled antibodies at different concentrations can realize the detection of actual serum samples.

Claims

1. A method for fluorescent detection of protein markers based on host-guest assembly labeling of cyclodextrin-adamantanized iridium complex, characterized in that The preparation steps of the cyclodextrin-adamantane iridium complex labeling material are as follows: 1) Using gold nanoparticles AuNPs, silver nanoparticles AgNPs, polystyrene microspheres PS or silica microspheres SiO2 as carriers, first directly mixed with labeled antibody Ab1 for modification or activated with a condensing agent and then coupled to obtain Ab1-labeled nanomaterials AuNPs@Ab1 or AgNPs@Ab1 or PS@Ab1 or SiO2@Ab1; The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI, N-hydroxysuccinimide NHS; 2) AuNPs@Ab1 or AgNPs@Ab1 or PS@Ab1 or SiO2@Ab1 is further mixed and shaken with thiol- or disulfide- or amino-functionalized β-cyclodextrin for 2 to 24 hours, and then incubated and blocked with a blocking agent, fetal bovine serum albumin BSA or skimmed milk powder or casein or Tween, to obtain cyclodextrin-functionalized Ab1-labeled gold nanoparticles AuNPs-Ab1@CD or AgNPs-Ab1@CD or PS-Ab1@CD or SiO2-Ab1@CD; 3) Incubating AuNPs-Ab1@CD or AgNPs-Ab1@CD or PS-Ab1@CD or SiO2-Ab1@CD in an adamantane iridium complex solution for 1-24 hours to obtain cyclodextrin-adamantane host-guest modified iridium complex labeled nanomaterials AuNPs-Ab1@CD@Ir or AgNPs-Ab1@CD@Ir or PS-Ab1@CD@Ir or SiO2-Ab1@CD@Ir; The analysis of the corresponding protein markers includes the following steps: 4) Loading the coated antibody Ab2 on the carrier ELISA plate or magnetic nanoparticles, and incubating and blocking with blocking protein BSA to obtain the Ab2-loaded carrier material S@Ab2; 5) Add the test protein AG to the S@Ab2 buffer solution, so that the test protein AG specifically binds to the coated antibody Ab2 on the carrier to obtain S-Ab2@AG; 6) adding the iridium complex-labeled nanoparticles AuNPs-Ab1@CD@Ir or AgNPs-Ab1@CD@Ir or PS-Ab1@CD@Ir or SiO2-Ab1@CD@Ir obtained in step 3) to S-Ab2@AG to achieve labeling of the protein AG to be tested with the iridium complex; 7) Adding a fluorescent detection solution containing a redox fluorescent probe and a hydrogen source to the labeled material obtained in step 6, incubating the reaction at a temperature of 0-100 degrees Celsius for 0-120 minutes, and then testing the change in the fluorescence intensity of the solution, thereby achieving quantitative detection of the protein AG to be tested; The hydrogen source is formic acid, sodium formate, potassium formate, ammonium formate, triethylammonium formate, or reduced nicotinamide adenine dinucleotide disodium salt NADH.

2. According to the method for fluorescent detection of protein markers based on cyclodextrin-adamantane iridium complex host-guest assembly labeling according to claim 1, the nanoparticles in step 1) are all monodisperse nanoparticles with a diameter of 1nm-5000nm and a morphology including spherical, rod-shaped or diamond-shaped.

3. The method for fluorescent detection of protein markers based on cyclodextrin-adamantane iridium complex host-guest assembly labeling according to claim 1, characterized in that: The adamantane iridium complex described in step 3) contains an adamantane functional group in its structure and can form a strong host-guest binding body with β-cyclodextrin.

4. The method for fluorescent detection of protein markers based on cyclodextrin-adamantane iridium complex host-guest assembly labeling according to claim 1, characterized in that: The generation and amplification of the fluorescence signal is produced by the hydrogenation reduction reaction of the oxidized-reduced fluorescent probe catalyzed by the iridium complex. The fluorescent probe molecules all contain oxidized functional groups, and when the oxidized functional groups in the fluorescent probe molecules are hydrogenated and reduced to the corresponding reduced state, the probe will produce obvious fluorescence enhancement or reduction; The oxidizing functional group is benzoquinone, naphthoquinone, anthraquinone, nitro, carbonyl, nicotinamide, aldehyde or pyridinium; The oxidized functional group is reduced to hydroquinone, naphthalene diol, anthracene diol, amino, hydroxyl, nicotinamide, methylhydroxyl or dihydropyridine after hydrogenation; Fluorescent chromophores mainly include: coumarins, rhodamine, xanthenes, naphthalene diimides, benzoxazoles, stilbenes, thiophene dicarboxylic acid amides, condensed aromatic hydrocarbons, and perylene tetracarboximide.

5. The method for fluorescent detection of protein markers based on cyclodextrin-adamantane iridium complex host-guest assembly labeling according to claim 1, characterized in that: The structures of metal iridium complexes all contain adamantane functional groups.

6. The method for fluorescent detection of protein markers based on cyclodextrin-adamantane iridium complex host-guest assembly labeling according to claim 1, characterized in that: The order of step 3) and step 4) cannot be reversed. The adamantane iridium complex can only be introduced after the blocking with fetal bovine serum albumin BSA or skimmed milk powder or casein or Tween is completed.

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