CN116577499A - Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof - Google Patents

Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof Download PDF

Info

Publication number
CN116577499A
CN116577499A CN202310863500.7A CN202310863500A CN116577499A CN 116577499 A CN116577499 A CN 116577499A CN 202310863500 A CN202310863500 A CN 202310863500A CN 116577499 A CN116577499 A CN 116577499A
Authority
CN
China
Prior art keywords
group
donor
probe
acceptor
luminescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202310863500.7A
Other languages
Chinese (zh)
Other versions
CN116577499B (en
Inventor
张兵波
郝良文
杨维涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tongji University
Original Assignee
Tongji University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tongji University filed Critical Tongji University
Priority to CN202310863500.7A priority Critical patent/CN116577499B/en
Publication of CN116577499A publication Critical patent/CN116577499A/en
Application granted granted Critical
Publication of CN116577499B publication Critical patent/CN116577499B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • G01N33/531—Production of immunochemical test materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the technical field of immunoassay, and particularly relates to a homogeneous catalysis chemiluminescence kit, a preparation method and a use method thereof. The kit comprises a donor material linked to a first recognition molecule, an acceptor material linked to a second recognition molecule and a catalytic substrate hydrogen peroxide (H 2 O 2 ) The donor material may catalyze H 2 O 2 Producing singlet oxygen 1 O 2 ) The method comprises the steps of carrying out a first treatment on the surface of the The acceptor material has 1 O 2 Triggering the luminescence characteristic. The kit of the invention adopts homogeneous phase immunity technology, can be used with the existing luminescence detector to determine the content of target molecules in the sample to be detected, and has no need of light excitation, no need of special detection instrument, high sensitivity and enzyme-linked immunosorbent assay (ELISA) compared with the light excitation chemiluminescence (AlphaLISA) technologyCompared with heterogeneous immunoassay such as an accessory (ELISA) technology, the method has the advantages of no washing step, simple operation, wide detection linear range, short detection process time, high sensitivity and high accuracy.

Description

Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof
Technical Field
The invention relates to the field of homogeneous immunoassay, in particular to a homogeneous catalysis chemiluminescence immunoassay kit, and also relates to a preparation method and a use method thereof.
Background
While homogeneous phase immunoassay is an analytical method in which an antigen and an antibody are immunoreacted in the same medium, heterogeneous phase immunoassay is an analytical method in which an antibody or an antigen is immobilized on a solid phase carrier surface, and a desired antigen or antibody is bound to the solid phase surface by a specific immunoreaction to form an antigen-antibody immune complex. The current immunoassay technology is mainly based on heterogeneous reaction modes of microwell plates. This type of technique requires not only the entrapment of antibodies or antigens, but also a slow immune reaction process, requiring multiple washes, which is not conducive to automation. The homogeneous immunoassay technology does not need embedding of antibodies or antigens, has rapid reaction, does not need washing, is easy to realize high throughput and automation, and is therefore more and more interesting and favored at present.
The photoexcitation chemiluminescence immunoassay (amplified luminescent proximity homogeneous assay linked immunosorbent assay, alphaLISA) technique is a new homogeneous immunoassay technique, which is an organic combination of two disciplines, chemical and biological: in chemistry, the technology employs donor nanomaterials containing photosensitive materials (e.g., phthalocyanines) and acceptor nanomaterials containing luminescent materials (e.g., rubrene, europium chelates, etc.). Under 680 and nm excitation light irradiation, oxygen molecules around the photosensitive material in the donor nano material are excited to convert into high-energy state 1 O 2 。 1 O 2 Has a half-life of about 4 [ mu ] s, which determines a diffusion diameter of about 200 nm, if outside this range 1 O 2 Will fall back to the ground state. Generated by 1 O 2 Out-diffusion, the receptor nano material in the immune sandwich compound can be received 1 O 2 And inside of 1 O 2 Triggering the photo reagent to react to generate energy, the energy is rapidly transferred to the luminescent reagent in the receptor nano material, and the luminescent reagent generates emitted light after receiving the energy for instrument detection, so the photo-activated chemiluminescence immunoassay technology is providedThe bottom is very low, and the signal to noise ratio is remarkably improved.
However, in the alphaLISA mode, the donor material still needs an external light source excitation and precise light source control system, which has high requirements on hardware such as instruments and equipment, and the detection cost is also improved; the other surface light source has limited penetration capability, and generates 1 O 2 Is not efficient. Thus, an in situ generation by chemical catalytic reaction without external light excitation is provided 1 O 2 To excite the acceptor material to emit light, and to realize efficient and sensitive homogeneous detection.
Disclosure of Invention
Aiming at the defects of heterogeneous detection and the AlphaLISA technology, the invention provides a homogeneous catalysis chemiluminescence immunoassay kit, a preparation method and a use method thereof, and the method utilizes the catalysis triggering luminescence principle, so that the method has the advantages of no need of external light excitation, no need of washing steps, low instrument requirement, high sensitivity and the like.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a homogeneous catalytic chemiluminescent kit comprising: donor probe, acceptor probe and hydrogen peroxide (H) 2 O 2 ) The method comprises the steps of carrying out a first treatment on the surface of the The donor probe comprises a linked donor material and a first recognition molecule, and the acceptor probe comprises a linked acceptor material and a second recognition molecule; wherein the donor material is used for catalyzing hydrogen peroxide to generate singlet oxygen 1 O 2 ) The method comprises the steps of carrying out a first treatment on the surface of the The first recognition molecule is one or more of an antibody, a nano antibody, an antigen, a recognition peptide, an aptamer, DNA or RNA; the acceptor material has singlet oxygen triggering cycle luminescence characteristics; the acceptor material comprises a singlet oxygen triggered luminescent reagent; the singlet oxygen-triggered luminescence reagent comprises N, N-dimethyl-4- (6-phenyl-2, 3-dihydro-1, 4-oxetan-5-yl) aniline as a chemiluminescent substance and 2- ((5- (4- (diphenylamino) phenyl) thiophen-2-yl) methylene) malononitrile as a luminescence enhancer; the second recognition molecule is an antibody, nanobody, antigen, recognition peptide, aptamer, DNA orOne or more of the RNAs; the first recognition molecule and the second recognition molecule are configured to specifically bind to a target molecule.
Further, it also includes: a standard for the target molecule; the target molecule is an antigen, peptide, antibody, amino acid, DNA, RNA, virus, hormone, drug or metabolite.
Further, the donor material is one of sodium molybdate, sodium hypochlorite, molybdenum stearate, molybdenum sulfide, molybdenum phosphate, manganese oxide-based material and derivatives thereof; the surface of the donor material is also connected with a chemical group, and the chemical group is at least one of epoxy group, chloromethyl group, sulfydryl group, amino group, hydroxyl group, maleic amine group, sulfonic group, carboxyl group and aldehyde group.
Further, the acceptor material further comprises a spherical carrier, and the singlet oxygen trigger luminescence reagent is embedded in the spherical carrier; wherein the spherical carrier is mesoporous silica, dendrimer, polystyrene microsphere, metal organic frame, covalent organic frame or liposome; the surface of the receptor material is also connected with a chemical group, and the chemical group is at least one of epoxy group, chloromethyl group, sulfydryl group, amino group, hydroxyl group, maleic amine group, sulfonic group, carboxyl group and aldehyde group.
Further, the average particle size of the donor material is 5 nm to 1000 nm, that is, the average particle size of the donor material may be 5 nm, 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm or 1000 nm. The average particle size of the acceptor material is 5 nm-1000 nm, that is, the average particle size of the acceptor material may be 5 nm, 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm or 1000 nm.
The second aspect of the present invention provides a method for preparing the homogeneous catalysis chemiluminescent kit, which comprises the following steps: a chemical coupling method is adopted to connect the first recognition molecule to the surface of the donor material, so as to prepare a donor probe; a second recognition molecule is connected to the surface of the receptor material by adopting a chemical coupling method to prepare a receptor probe; and dissolving hydrogen peroxide in ultrapure water at a concentration of 5 mu M-5M for catalytic reaction to obtain the homogeneous catalysis chemiluminescent kit.
Further, the chemical coupling method includes a diazo method, a glutaraldehyde method, a glutaric anhydride method or a carbodiimide method; the molar ratio of the first recognition molecule to the donor material is (10-1000): 1, a step of; the molar ratio of the second recognition molecule to the receptor material is (10-1000): 1.
the third aspect of the present invention provides a method for using the homogeneous catalysis chemiluminescent kit, which is characterized in that: the using method comprises the following steps: (1) Respectively adding the donor probe and the acceptor probe into a sample solution to be detected to form a mixture to be detected; when the sample to be detected contains target molecules, forming donor probe-target molecule-acceptor probe immune complexes; when the sample to be detected does not contain target molecules, the donor probe and the acceptor probe are in a separation state, so that immune complexes are not formed; (2) Adding hydrogen peroxide into the mixture to be tested, and decomposing the hydrogen peroxide into singlet oxygen and H by a donor material 2 O; when an immune complex is formed, singlet oxygen diffuses to the acceptor probe, producing chemiluminescence; when no immune complex exists in the mixture to be tested, no chemiluminescence is generated; (3) And (3) reading the chemiluminescent luminescence signal by using a luminescence detector, so as to realize quantitative detection of the concentration of the target molecule in the sample to be detected.
Further, the reaction temperature in the step (1) is 20-50 ℃ and the reaction time is 3-30 minutes; in the step (3), a luminescence detector is used for reading the luminescence signal of the chemiluminescence for 1-5 minutes; and (3) the emission wavelength of the chemiluminescence in the step (3) is 400-2000 nm.
The beneficial effects of the invention are as follows:
the invention provides a homogeneous catalysis chemiluminescence kit, and a preparation method and a use method thereof. The kit comprises a donor material linked to a first recognition molecule to form a donor probe, an acceptor material linked to a second recognition molecule to form an acceptor probe, and a catalytic substrate hydrogen peroxide, the donor material being capable of catalyzing H 2 O 2 Generating singlet oxygen; the acceptor material has 1 O 2 Triggering the luminescence characteristic. The kit provided by the invention adopts a homogeneous phase immunity technology, can be matched with the existing luminescence detector to be used for measuring the content of target molecules in a sample to be measured. Compared with the photo-excitation chemiluminescence technology, the method has the advantages that photo-excitation is not needed, a special detection instrument is not needed, and the sensitivity is high; compared with heterogeneous immunoassay such as enzyme-linked immunosorbent (enzyme linked immunosorbent assay, ELISA) technology, the method has the advantages of no washing step, simple operation, wide detection linear range, short detection process time, high sensitivity and high accuracy.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings, which are not to be construed as limiting the embodiments unless specifically indicated otherwise.
FIG. 1 is a schematic diagram of a homogeneous catalytic chemiluminescent kit and detection method therefor;
FIG. 2 is a schematic diagram of a cyclically enhanced chemiluminescence;
FIG. 3 is a standard curve of a homogeneous catalytic chemiluminescent kit for detecting Alpha Fetoprotein (AFP) in human serum and a detection method thereof;
FIG. 4 is a standard curve of a homogeneous catalytic chemiluminescent kit for the detection of the amino terminal brain natriuretic peptide precursor (NT-proBNP) in human serum and the detection method thereof;
FIG. 5 is a standard curve of a homogeneous catalytic chemiluminescent kit for detection of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP) in a human nasal/pharyngeal swab and method of detection thereof;
FIG. 6 is a standard curve of a homogeneous catalytic chemiluminescent kit for detecting Lactoferrin (LF) in milk samples and a detection method thereof;
FIG. 7 is a standard curve of a homogeneous catalysis chemiluminescent kit and detection method for detecting mutant DNA of a patient with lung adenocarcinoma in human serum;
Fig. 8 is a graph showing the light emission time of example 1 of the present invention.
Detailed Description
In a first aspect the invention provides a homogeneous catalytic chemiluminescent kit as shown in figure 1, said kit comprising: donor probe, acceptor probe and hydrogen peroxide (H) 2 O 2 ) The method comprises the steps of carrying out a first treatment on the surface of the The donor probe comprises a linked donor material and a first recognition molecule, and the acceptor probe comprises a linked acceptor material and a second recognition molecule; wherein the donor material is used for catalyzing hydrogen peroxide to generate singlet oxygen 1 O 2 ) The method comprises the steps of carrying out a first treatment on the surface of the The first recognition molecule is one or more of an antibody, a nano antibody, an antigen, a recognition peptide, an aptamer, DNA or RNA; the acceptor material has singlet oxygen trigger luminescence characteristics; the second recognition molecule is one or more of an antibody, a nanobody, an antigen, a recognition peptide, an aptamer, DNA or RNA. The first recognition molecule and the second recognition molecule are different substances in the same class. For example: the first recognition molecule and the second recognition molecule are both antibodies, but are different antibodies. In addition, hydrogen peroxide is herein a catalytic substrate.
The second aspect of the present invention provides a method for preparing the homogeneous catalysis chemiluminescent kit, which comprises the following steps:
A chemical coupling method is adopted to connect the first recognition molecule to the surface of the donor material, so as to prepare a donor probe;
a second recognition molecule is connected to the surface of the receptor material by adopting a chemical coupling method to prepare a receptor probe;
and dissolving hydrogen peroxide in ultrapure water at a concentration of 5 mu M-5M for catalytic reaction to obtain the homogeneous catalysis chemiluminescent kit. That is, in the course of practical use, the kit of the present invention comprises a donor probe solution, an acceptor probe solution, and a hydrogen peroxide solution, in total, three solutions stored separately.
The third aspect of the present invention provides a method for using the homogeneous catalysis chemiluminescent kit, which is characterized in that: the using method comprises the following steps:
(1) Respectively adding the donor probe and the acceptor probe into a sample solution to be detected to form a mixture to be detected;
if the sample to be tested contains target molecules, forming donor probe-target molecule-acceptor probe immune complexes; if the sample to be detected does not contain target molecules, the donor probe and the acceptor probe are in a separation state, so that immune complexes are not formed;
(2) Adding hydrogen peroxide into the mixture to be tested, and decomposing the hydrogen peroxide into singlet oxygen and H by a donor material 2 O；
If an immune complex is formed, singlet oxygen diffuses to the acceptor probe, producing chemiluminescence; if no immunocomplexes are present in the test mixture, no chemiluminescence will occur;
(3) And (3) reading the chemiluminescent luminescence signal by using a luminescence detector, so as to realize quantitative detection of the concentration of the target molecule in the sample to be detected.
The present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The term "sample to be measured" as used herein refers to a mixture to be measured containing or suspected of containing a target molecule. Samples to be tested that may be used in the present invention include body fluids such as blood (which may be anticoagulated blood as is commonly found in collected blood samples), plasma, serum, urine, semen, saliva, sweat, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be measured can be diluted with a diluent as required before use. For example, in order to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before on-machine testing and then tested on a testing instrument.
The term "target molecule" as used herein refers to a substance in a sample to be tested during detection. A recognition molecule having a specific binding affinity for the target molecule will be used to detect the target molecule. The target molecule may be an antigen, peptide, antibody, amino acid, DNA, RNA, virus, hormone, drug, metabolite, or the like.
Example 1
The donor material is molybdenum-sulfur compound-protein nano-composite, the protein is Ovalbumin (OVA), and the average particle size is 5 nm; the receptor material is polystyrene microsphere embedded 1 O 2 A reagent for triggering luminescence, wherein the surface of the polystyrene microsphere is connected with carboxyl, and the average particle size is 200 nm; can be used for 1 O 2 The reagent triggering luminescence consists of a chemiluminescent substance N, N-dimethyl-4- (6-phenyl-2, 3-dihydro-1, 4-oxetan-5-yl) aniline (SO) and a luminescence enhancer 2- ((5- (4- (diphenylamino) phenyl) thiophen-2-yl) methylene) malononitrile (TTMN); the detection principle is as follows: 1 O 2 can attack SO to form an unstable intermediate of SO, and the intermediate can release light with the wavelength of 470 nm from the excited state to the ground state, the light with the wavelength band can be used as a light source to re-excite the adjacent light-emitting enhancer TTMN to make the TTMN emit 650 nm light, and further generate 1 O 2 Attack the SO, thereby forming a cyclically enhanced chemiluminescence which can be detected and collected as an optical signal by a luminescence detector for quantitative detection of the target molecule, see FIG. 2; the target molecule is alpha fetoprotein (alpha fetoprotein, AFP); the first recognition molecule is an anti-AFP antibody 1; the second recognition molecule is an anti-AFP antibody 2; the sample to be tested is human serum.
The use of the classical singlet oxygen generation method, sodium molybdate + hydrogen peroxide, resulted in the generation of significant amounts of singlet oxygen in the solution, which in turn resulted in the cycling enhanced chemiluminescence of polystyrene microsphere embedded SO and TTMN (STPS), which was seen to last 1200 seconds (20 minutes) as shown in fig. 8.
The specific implementation steps are as follows:
1. preparation of molybdenum sulfur Compound-OVA nanocomposite (MoO)
Weighing OVA 60 mg, adding into ultrapure water at 30 mL, magnetically stirring to dissolve into clear transparent water solution, and adding Na 2 MoO 4 •2H 2 O (66.9 mg) and Na 2 S•9H 2 O (66.6, mg) was stirred and mixed to obtain a mixed solution. The molar ratio of Mo to S under this condition is 1:1. subsequently, an aqueous solution of NaOH (1M) was added to adjust the pH of the mixture to 11 (about 400. Mu.L of NaOH was consumed), and an aqueous solution of hydrochloric acid (1M) was slowly added dropwise to adjust the pH to 6.5 (about 800. Mu.L was consumed) with magnetic stirring, and a slow yellowing of the solution color was observed during the dropwise addition. After the pH was adjusted, the reaction was protected from light by reaction 1 h, and the reaction solution was transferred to a dialysis bag (molecular weight cut-off 3200) for dialysis 15 h to remove salt small molecule impurities generated by the reaction. The dialyzate is freeze-dried to obtain pale yellow solid powder, and the pale yellow solid powder is stored at the temperature of minus 20 ℃ for standby.
2. Preparation of donor Probe by anti-AFP antibody 1 connected with MoO
(1) Weighing 10 mg MoO, dissolving in 1mL ultrapure water, adding 0.2 mL new 0.1M NaIO 4 The solution was reacted at room temperature for 20 minutes in the dark.
(2) The solution was packed in a dialysis bag, dialyzed against 1 mM, pH 4.4 sodium acetate buffer, and left overnight at 4 ℃.
(3) The hydroformylation pH was raised to 9.0 to 9.5 by adding 20. Mu.L of 0.2. 0.2M pH 9.5 carbonate buffer, and then immediately adding 5 mg anti-AFP antibody 1 to 1mL of 0.01M carbonate buffer, and gently stirring at room temperature for 2 hours in the absence of light.
(4) Adding 0.1 mL newly prepared 4.4 mg/mL NaBH 4 The solution was thoroughly mixed, left in the dark at 4℃for two hours, and dialyzed overnight at 4℃in 0.15. 0.15M (pH 7.4) PBS.
(5) After the dialysis was completed, an equal volume of saturated ammonium sulfate solution was added, and the solution was left to stand at 4℃for 2 hours, centrifuged at 3000/r/min for 30 minutes, and the precipitate was taken out, washed again with a half-saturated ammonium sulfate solution 2 times, and then dissolved in PBS (pH 7.4) of 0.15M.
(6) Dialyzing to remove salt, and storing at 4deg.C for use.
3. Preparation of polystyrene microsphere-embedded SO and TTMN (STPS)
STPS was synthesized by the swelling method. First 5 mg TTMN and 3 mg SO were dissolved in 3 mL propylene glycol methyl ether solution and preheated to 70 ℃. Subsequently, 1mL carboxylated polystyrene microspheres (200 nm,100 mg/mL) were added and reacted for 30 minutes. The heating was then immediately stopped and the solution was cooled to room temperature to produce STPS. The synthesized STPS was washed three times with deionized water, centrifuged at 13500 rpm for 15 minutes, and the residue was redispersed in 4 mL deionized water. The prepared STPS solution was stored in a dark environment at 4℃for future use.
4. Preparation of receptor probes by linking anti-AFP antibody 2 with STPS
AFP antibody 2 was attached to the STPS surface using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in a one-step procedure.
(1) Transferring 10 μl of STPS, dissolving in 1 mL phosphate buffer (PBS buffer) (0.01M, pH=7.4), adding 10 μg of anti-AFP antibody 2, electrostatic adsorbing for 30 min, adding 5 μg of EDC, stirring, reacting for 30 min, repeating for 3 times, and observing aggregation phenomenon of STPS;
(2) Subsequently, 100. Mu.L of bovine serum albumin (Bovine Serum Albumin, BSA) (10 mg/mL) was added, and after electrostatic adsorption for 30 min, 5. Mu.g of EDC was added to react for 30 min;
(3) 5. Mu.g of EDC was again added and reacted for 30 min, centrifuged and the precipitate was redissolved in 100. Mu.L of the redissolved solution.
The components of the complex solution are as follows: 0.01M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
5. Drawing of a standard curve for detection of AFP antigen
AFP standards were diluted with PBS (0.01M, pH 7.4) buffer to different concentration gradients, specifically: 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.3 ng/mL, 15.6 ng/mL, 7.8 ng/mL, 3.9 ng/mL, 1.95 ng/mL, 0.98 ng/mL, 0.49 ng/mL, 0.245 ng/mL, 0.12 ng/mL, 0.06 ng/mL, and 0 ng/mL. 200. Mu.L of AFP standard with different concentrations is taken respectively, 6. Mu.L of donor probe and 6. Mu.L of acceptor probe are added simultaneously, and after reaction for 10 min at 37 ℃, 10. Mu.L of H of 0.1M is added 2 O 2 After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. Obtaining the concentration of AFP in the sample to be detected by substituting the average value into a standard curve, taking the obtained luminous intensity as an ordinate, and taking the concentration of AFP as the ordinateAnd (ng/mL) drawing a standard curve for the abscissa, and calculating a linear equation. The specific experimental results are as follows: the linear standard curve is y= 4.693ln (x) +3.2038, r=0.991, see fig. 3. The minimum detection limit of the method is defined as the average luminous intensity +3 times standard deviation of the results obtained when 20 AFP concentrations are detected to be 0, and the minimum detection line is calculated to be 0.12 by the standard curveng/mL。
6. Detection of AFP content in serum samples
The invention relates to a homogeneous catalysis chemiluminescence immunoassay kit, a preparation method and a use method thereof, which are implemented by the following steps when being used for detecting the AFP content in a human serum sample:
firstly diluting a serum sample by 10 times, then adding 6 mu L of donor probe and 6 mu L of acceptor probe into the diluted sample simultaneously, reacting for 10 min at 37 ℃, adding 10 mu L of H of 0.1M 2 O 2 ，H 2 O 2 And MoO reaction, in situ generation 1 O 2 Generated by 1 O 2 The acceptor probe may be attacked to make its internal luminescence enhancer TTMN emit 650 nm light, and after 2 min, the luminescence intensity of the solution is measured with a luminescence detector. Substituting the resulting luminescence intensity into the standard curve according to 5 in example 1, and calculating the concentration of the corresponding sample, i.e. the actual concentration of AFP in the serum sample.
Example 2
The donor material is manganese oxide-based MnOx nanosheets with an average particle size of 1000 nm; the receptor material is polystyrene microsphere embedded 1 O 2 The surface of the polystyrene microsphere is connected with carboxyl, and the average particle size of the polystyrene microsphere is 100 nm; can be used for 1 O 2 The reagent for triggering luminescence is prepared from chemiluminescent substance SO and luminescence enhancer (1-10-phenanthroline) tris [4, 4-trifluoro-1- (2-thienyl) -1, 3-butanedione]Europium (III) (EuIII); emission wavelength of EuIII is 615 nm; the target molecule is an amino terminal brain natriuretic peptide precursor (NT-proBNP); the first recognition molecule is NT-proBNP aptamer 1; the second recognition molecule is NT-proBNP aptamer 2; the sample to be tested is human serum.
The specific implementation steps are as follows:
1. preparation of metal oxide nanoplatelets MnOx
In preparing the MnOx nanosheets, naBH is added 4 (6 mg/mL,150 mL) aqueous solution was added to KMnO 4 In the solution (0.5 mg/mL,15 mL), after 1 hour of intense ultrasonic treatment, it was centrifuged at 11000 rpm for 15 minutes, and the precipitate was redissolved in 10 mL ultra pure water.
2. Preparation of donor probe by connecting NT-proBNP aptamer 1 with MnOx nanosheets
(1) 10 mg of SH-PEG2000-COOH was weighed out and dissolved in 1 mL of MnOx (1 mg/mL) and reacted overnight at room temperature. 10000 Centrifugation at rpm for 15 min, the pellet was redissolved in 1 mL PBS buffer (0.01 m, ph=7.4).
And connecting the NT-proBNP aptamer 1 on the surface of the MnOx nanosheet by adopting an EDC one-step method.
(2) Transferring 10 mu L of MnOx nanosheets, dissolving in 1 mL of PBS buffer solution (0.01M, pH=7.4), adding 10 mu M of NT-proBNP aptamer 1, carrying out electrostatic adsorption for 30 min, then adding 5 mu g of EDC, stirring and reacting for 30 min, repeating for 3 times, and observing the aggregation phenomenon of the MnOx nanosheets or not;
(3) Subsequently, 100. Mu.L of BSA (10 mg/mL) was added, and after 30 min of electrostatic adsorption, 5. Mu.g of EDC was added for reaction for 30 min;
(4) Again, 5. Mu.g of EDC was added and reacted for 30 min, centrifuged at 13500 rpm for 15 min, and the precipitate was reconstituted into 100. Mu.L of reconstituted solution.
The components of the complex solution are as follows: the 0.01M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
3. Preparation of polystyrene microsphere-embedded SO and EuIII (SEuPS)
SEuPS was synthesized by the swelling method. First, 5 mg EuIII and 3 mg SO were dissolved in 3 mL propylene glycol methyl ether solution and preheated to 70 ℃. Subsequently, 1 mL carboxylated polystyrene microspheres (100 nm,100 mg/mL) were added and reacted for 30 minutes. The heating was then immediately stopped and the solution was cooled to room temperature. The synthesized SEuPS was washed three times with deionized water, centrifuged at 13500 rpm for 15 minutes, and the residue was redispersed in 4 mL deionized water. The prepared seu ps solution was stored in a dark environment at 4 ℃ for future use.
4. Preparation of receptor probes by linking NT-proBNP aptamer 2 with SEuPS
The NT-proBNP aptamer 2 was attached to the surface of SEuPS using the EDC one-step method.
(1) Transferring 10 mu L of SEuPS, dissolving in 1 mL of PBS buffer solution (0.01M, pH=7.4), adding 10 mu M of NT-proBNP aptamer 2, carrying out electrostatic adsorption for 30 min, then adding 5 mu g of EDC, stirring and reacting for 30 min, repeating for 3 times, and observing the aggregation phenomenon of SEuPS with/without;
(2) Subsequently, 100. Mu.L of BSA (10 mg/mL) was added, and after 30 min of electrostatic adsorption, 5. Mu.g of EDC was added for reaction for 30 min;
(3) 5. Mu.g of EDC was again added and reacted for 30 min, centrifuged and the precipitate was redissolved in 100. Mu.L of the redissolved solution.
The components of the complex solution are as follows: the 0.01M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
5. Drawing of a standard curve for the detection of NT-proBNP antigen
NT-proBNP standard was diluted with PBS (0.01M, pH 7.4) buffer to different concentration gradients, specifically: 10000. 5000, 2500, 1250, 625, 313, 156, 78, 39, 19.5, 9.8 and 0 fg/mL. Respectively taking 200 mu L of NT-proBNP standard substances with different concentrations, simultaneously adding 6 mu L of donor nano material coated with antibody 1 and 6 mu L of acceptor nano material coated with antibody 2, reacting at 37 ℃ for 10 min, and adding 10 mu L of 0.1M H 2 O 2 After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. And drawing a standard curve by taking the obtained light intensity as an ordinate and the concentration of NT-proBNP (pg/mL) as an abscissa, and calculating a linear equation. The specific experimental results are as follows: the linear standard curve is y= 5.6953ln (x) -17.131, r= 0.9849, see fig. 4. The minimum detection limit of the method is defined as the average luminous intensity +3 times standard deviation of the results obtained when the concentration of 20 NT-proBNP is 0, and the minimum detection line is calculated to be 10 by the standard curvefg/mL。
6. Detection of NT-proBNP content in serum samples
The invention relates to a catalytic homogeneous phase chemiluminescence immunoassay kit and a detection method thereof, which are implemented by the following steps when being used for detecting the content of NT-proBNP in a serum sample:
firstly diluting a serum sample by 10 times, then adding 6 mu L of donor probe and 6 mu L of acceptor probe into the diluted sample simultaneously, reacting for 10 min at 37 ℃, adding 10 mu L of 0.1M H 2 O 2 ，H 2 O 2 And MnOx reaction in situ generation 1 O 2 Generated by 1 O 2 The acceptor material may be attacked to cause it to emit 615 nm chemiluminescence. After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. Substituting the resulting luminescence intensity into the standard curve of example 2, 5, and calculating the concentration of the corresponding sample, i.e. the actual concentration of NT-proBNP in the serum sample.
Example 3
The donor material is porous molybdenum phosphate nano particles (MoPO) with an average particle size of 500 nm; the acceptor material is mesoporous silica Microsphere (MSN) embedded cocoa 1 O 2 A reagent that triggers luminescence; the surface of the MSN is connected with carboxyl, and the average particle size is 100 nm; can be used for 1 O 2 The reagent for triggering luminescence is prepared from chemiluminescent substance azide-methyl methacrylate-styrene-adamantane (AMPA) and luminescence enhancer semiconductor quantum dot Ag 2 S is formed;
Ag 2 the emission wavelength of S is 2000 nm; the target molecule is a novel coronavirus nucleocapsid protein (SARS-CoV-2 NP) antigen; the first recognition molecule is anti-SARS-CoV-2 NP antibody 1; the second recognition molecule is anti-SARS-CoV-2 NP antibody 2; the sample to be tested is a human nasal/pharyngeal swab sample.
The specific implementation steps are as follows:
1. preparation of MoPO nanoparticles
Molybdenum trioxide, tetramethylammonium hydroxide, phosphoric acid and water are used as raw materials, and the raw materials are synthesized under the hydrothermal condition. Firstly, adding 0.5 mL of water and 0.5 mL (2 mg/mL) of molybdenum trioxide into a polytetrafluoroethylene lining, uniformly mixing, then adding 0.1 mL (mg/mL) of phosphoric acid, fully and uniformly stirring, then adding 1 mL of tetramethylammonium hydroxide, further uniformly stirring, sealing, putting the reaction kettle into an oven, and crystallizing for 3 days at 220 ℃. And taking out the reaction kettle after the reaction is finished, cooling, centrifuging, filtering, washing with distilled water to be neutral, and drying at room temperature to obtain the MoPO nano particles.
2. Preparation of donor Probe by connecting anti-SARS-CoV-2 NP antibody 1 with MoPO
(1) 10mg of SH-PEG2000-COOH was weighed into 1mL of MoPO (1 mg/mL) and reacted overnight at room temperature. 10000 Centrifugation at rpm for 15 min, the pellet was redissolved in 1mL PBS buffer (0.01 m, ph=7.4).
EDC one-step method is adopted to connect anti SARS-CoV-2 NP antibody 1 on the surface of MoPO nano-sheet.
(2) Transferring 10 mu L of MoPO nano-sheets, dissolving in 1mL of PBS buffer solution (0.01M, pH=7.4), adding 10 mu M of anti-SARS-CoV-2 NP antibody 1, carrying out electrostatic adsorption for 30 min, then adding 5 mu g of EDC, stirring and reacting for 30 min, repeating for 3 times, and observing the aggregation phenomenon of MoPO nano-sheets;
(3) Subsequently, 100. Mu.L of BSA (10 mg/mL) was added, and after 30 min of electrostatic adsorption, 5. Mu.g of EDC was added for reaction for 30 min;
(4) Again, 5. Mu.g of EDC was added and reacted for 30 min, centrifuged at 13500 rpm for 15 min, and the precipitate was reconstituted into 100. Mu.L of reconstituted solution.
The components of the complex solution are as follows: the 0.01M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
3. MSN microsphere embedding AMPA and Ag 2 Preparation of S (MAA)
First, 5 mg of Ag is added 2 S and 3 mg SO were dissolved in 3 mL propylene glycol methyl ether solution and preheated to 70 ℃. Subsequently, 1mL carboxylated MSN (100 nm,100 mg/mL) was added and reacted for 30 min. The heating was then immediately stopped and the solution was cooled to room temperature. The synthesized MSA was washed three times with deionized water, centrifuged at 13500 rpm for 15 min, and the residue was redispersed in 4 mL deionized water. The prepared MSA solution was stored in a dark environment at 4℃for future use.
4. Preparation of acceptor probe by anti-SARS-CoV-2 NP antibody 2 linking MAA
SARS-CoV-2 NP antibody 2 was attached to the MAA surface using the EDC one-step method.
(1) Transferring 10 μl of MAA, dissolving in 1 mL PBS buffer (0.01M, pH=7.4), adding 10 μg of anti-SARS-CoV-2 NP antibody 2, electrostatic adsorbing for 30 min, adding 5 μg of EDC, stirring, reacting for 30 min, repeating for 3 times, and observing aggregation phenomenon of MAA;
(2) Subsequently, 100. Mu.L of BSA (10 mg/mL) was added, and after 30 min of electrostatic adsorption, 5. Mu.g of EDC was added for reaction for 30 min;
(3) 5. Mu.g of EDC was again added and reacted for 30 min, centrifuged and the precipitate was redissolved in 100. Mu.L of the redissolved solution.
The components of the complex solution are as follows: 0.01 M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
5. Drawing standard curve for detecting SARS-CoV-2 NP antigen
SARS-CoV-2 NP standard was diluted with PBS (0.01M, pH 7.4) buffer to different concentration gradients, specifically: 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.13 ng/mL, 1.56 ng/mL, 0.78 ng/mL, 0.39 ng/mL, 0.195 ng/mL, 0.098 ng/mL, 0.049 ng/mL, and 0 ng/mL. 200. Mu.L of SARS-CoV-2 NP standard substance with different concentrations are respectively taken, 6. Mu.L of donor probe and 6. Mu.L of acceptor probe are simultaneously added, and after reaction for 10 min at 37 ℃, 10. Mu.L of H of 0.1M is added 2 O 2 After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. And drawing a standard curve by taking the obtained luminous intensity as an ordinate and the concentration (ng/mL) of SARS-CoV-2 NP as an abscissa, and calculating a linear equation. The specific experimental results are as follows: the linear standard curve is y= 1.4917 x+1.3295, r= 0.9908, see fig. 5. The minimum detection limit of the method is defined as the average luminous intensity +3 times standard deviation of the results obtained when 20 SARS-CoV-2 NP concentrations are detected to be 0, and the minimum detection line is calculated to be 0.195 by the standard curveng/mL。
6. Detection of SARS-CoV-2 NP content in nasal/pharyngeal swab sample
The invention relates to a homogeneous catalysis chemiluminescence immune detection kit, a preparation method and a use method thereof, which are implemented by the following steps when the kit is used for detecting the SARS-CoV-2 NP content in a human nose/throat swab sample:
first, a sample of a nasal/pharyngeal swab is takenDiluting 10 times, adding 6. Mu.L of donor probe and 6. Mu.L of acceptor probe to the diluted sample at the same time, reacting at 37 ℃ for 10 min, adding 10. Mu.L of 0.1M H 2 O 2 ，H 2 O 2 And MoPO reaction, in situ generation 1 O 2 Generated by 1 O 2 Can attack the receptor probe to make its internal Ag 2 S emits 2000 nm light, and after 2 min, the luminescence intensity of the solution is measured by a luminescence detector. Substituting the resulting luminescence intensity into the standard curve of example 3, 5, and calculating the concentration of the corresponding sample, namely the actual concentration of SARS-CoV-2 NP in the nasal/pharyngeal swab sample.
Example 4
The donor material is polystyrene microsphere surface coated molybdenum sulfate-BSA nano-composite (PS-MoB), and the average grain diameter is 800 nm; the receptor material is Liposome (LPO) embedded cocoa 1 O 2 A reagent that triggers luminescence; the surface of the LPO is connected with carboxyl, and the average particle size is 1000 nm; can be used for 1 O 2 The reagent for triggering luminescence consists of a chemiluminescent substance SO and luminescence enhancer carbon quantum dots (CDs); the emission wavelength of CDs is 980 nm; target molecule Lactoferrin (LF); the first recognition molecule is an anti-LF antibody 1; the second recognition molecule is an anti-LF antibody 2; the sample to be tested is a milk sample.
The specific implementation steps are as follows:
1. preparation of donor probe by connecting anti-LF antibody 1 with PS-MoB nano-composite
(1) Adding BSA 60 mg into ultrapure water at 30: 30 mL, magnetically stirring to obtain clear and transparent aqueous solution, and adding Na 2 MoO 4 •2H 2 O (66.9 mg) and Na 2 S•9H 2 O (66.6, mg) was stirred and mixed to obtain a mixed solution. The molar ratio of Mo to S under this condition is 1:1. subsequently, an aqueous solution of NaOH (1M) was added to adjust the pH of the mixture to 11 (about 400. Mu.L of NaOH was consumed), and an aqueous solution of hydrochloric acid (1M) was slowly added dropwise to adjust the pH to 6.5 (about 800. Mu.L was consumed) with magnetic stirring, and a slow yellowing of the solution color was observed during the dropwise addition. After adjusting the pH, the reaction is carried out in a dark place for 1 h, the reaction solution is transferred into a dialysis bag (the molecular weight cut-off is 3200) for dialysis for 15 h, To remove salt small molecule impurities generated by the reaction. The dialyzate is freeze-dried to obtain pale yellow solid powder MoB, and the pale yellow solid powder MoB is stored at the temperature of minus 20 ℃ for standby.
(2) Transferring 10 mu L of polystyrene microsphere (800 nm,100 mg/mL) to dissolve in 1 mL PBS buffer (0.01M, pH=7.4), adding 10 mu g of anti-LF antibody 2, performing electrostatic adsorption for 30 min, then adding 5 mu g of EDC, stirring and reacting for 30 min, repeating for 3 times, and observing the aggregation phenomenon of PS or not;
(3) Subsequently, 100. Mu.L of MoB (10 mg/mL) was added, and after 30 min of electrostatic adsorption, 5. Mu.g of EDC was added for reaction for 30 min;
(4) 5. Mu.g of EDC was again added and reacted for 30 min, centrifuged and the precipitate was redissolved in 100. Mu.L of the redissolved solution.
The components of the complex solution are as follows: the 0.01M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
2. Preparation of Liposomes (LPO) embedding SO and CDs (LSC)
First 5 mg CDs and 3 mg SO were dissolved in 3 mL propylene glycol methyl ether solution. Subsequently, 1 mL carboxyl LPO (1000 nm,100 mg/mL) was added and reacted for 30 minutes. The heating was then immediately stopped and the solution was cooled to room temperature. The synthesized LSC was washed three times with deionized water, centrifuged at 13500 rpm for 15 minutes, and the residue was redispersed in 4 mL deionized water. The prepared LSC solution was stored in a dark environment at 4 ℃ for future use.
3. Preparation of receptor probe by anti-LF antibody 2 connected with LSC
(1) Removing 10 μl of LSC, dissolving in 1 mL PBS buffer (0.01M, pH=7.4), adding 10 μg of anti-LF antibody 2, electrostatic adsorbing for 30 min, adding 5 μg of EDC, stirring, reacting for 30 min, repeating for 3 times, and observing aggregation phenomenon of LSC;
(2) Subsequently, 100. Mu.L of BSA (10 mg/mL) was added, and after 30 min of electrostatic adsorption, 5. Mu.g of EDC was added for reaction for 30 min;
(3) 5. Mu.g of EDC was again added and reacted for 30 min, centrifuged and the precipitate was redissolved in 100. Mu.L of the redissolved solution.
The components of the complex solution are as follows: the 0.01M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
4. Drawing of standard curve for detecting LF
LF standards were diluted with PBS (0.01M, pH 7.4) buffer to different concentration gradients, specifically: 1000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.3 ng/mL, 15.6 ng/mL, 7.8 ng/mL, 3.9 ng/mL, 1.95 ng/mL, 0.98 ng/mL, and 0 ng/mL. 200 mu L of LF standard substances with different concentrations are respectively taken, 6 mu L of donor probe and 6 mu L of acceptor probe are simultaneously added, after reaction is carried out for 10 min at 37 ℃, 10 mu L of H of 0.1M is added 2 O 2 After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. And drawing a standard curve by taking the obtained luminous intensity as an ordinate and the LF concentration (ng/mL) as an abscissa, and calculating a linear equation. The specific experimental results are as follows: the linear standard curve is y=0.0793 x+4.2983, r= 0.9956, see fig. 6. The minimum detection limit of the method is defined as the average luminous intensity +3 times standard deviation of the results obtained when 20 LF concentrations are detected to be 0, and the minimum detection line is calculated to be 3.9 through the standard curve ng/mL。
5. Detection of LF content in milk sample
The invention relates to a homogeneous catalysis chemiluminescence immunoassay kit, a preparation method and a use method thereof, which are implemented by the following steps when being used for detecting the LF content in a milk sample:
firstly diluting milk sample by 10 times, then adding 6 mu L of donor probe and 6 mu L of acceptor probe into the diluted sample simultaneously, reacting at 37 ℃ for 10 min, adding 10 mu L of 0.1M H 2 O 2 ，H 2 O 2 And MoO reaction, in situ generation 1 O 2 Generated by 1 O 2 The acceptor probe may be attacked such that its internal CDs molecules are oxidized to emit 980 nm light. After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. Substituting the resulting luminous intensity into the standard curve of 4 in example 4, and calculating the concentration of the corresponding sample, namely the actual concentration of LF in the milk sample.
Example 5
The donor material is molybdenum oxide nano particles (MoO) 3 ) An average particle diameter of 50 nm; acceptor materialEmbedding for Metal Organic Frameworks (MOFs) 1 O 2 A reagent that triggers luminescence; the surface of the MOF is connected with carboxyl, and the average particle size is 50 nm; can be used for 1 O 2 The reagent for triggering luminescence consists of a chemiluminescent substance AMPA and a luminescence enhancer Fluorescein Isothiocyanate (FITC); the emission wavelength of FITC is 520 nm; mutant DNA of a target molecule of a lung adenocarcinoma patient; the first recognition molecule is DNA1 paired with the mutant DNA; the second recognition molecule is DNA2 paired with the mutant DNA; the sample to be tested is human serum.
The specific implementation steps are as follows:
1. molybdenum oxide nanoparticle (MoO) 3 ) Is prepared from
Adding 2.5-g sodium molybdate dihydrate into a three-neck flask, adding 50-mL distilled water, stirring to dissolve, performing ultrasonic treatment for 30 min, adding potassium borohydride solution dropwise in ultrasonic treatment, reacting for 30 min, filtering, and freeze-drying to obtain molybdenum oxide nanoparticles.
2. Mutant DNA sequences
GTTGGAGCTAGTGGCGTAG
3. DNA1 sequence
AGCTCCAAC-COOH
3. DNA2 sequence
AAAAAAAAAAAAAAAAAAAATTTT CTACGCCACT-NH 2
4. DNA1 ligation MoO 3 Preparation of donor probes
(1) 10 mg SH-PEG2000-NH was weighed 2 1mL MoO was dissolved 3 (1 mg/mL) was allowed to react overnight at room temperature. 10000 Centrifugation at rpm for 15 min, the pellet was reconstituted in 1mL PBS buffer (0.01M, pH=7.4),
(2) DNA1 was dissolved in 0.5. 0.5 mL ultrapure water, and then 35. Mu.M of DNA1 was added to a solution containing 1mL of PBS (pH 7.4).
(2) EDC 0.1. 0.1 mM and N-hydroxysuccinimide (N-Hydroxy succinimide, NHS) 0.4. 0.4 mM were added to react 4 h.
(3) 20 mg of MoO was added and reaction 4 h was dialyzed overnight against 0.15M (pH 7.4) PBS at 4deg.C.
(5) After the dialysis was completed, the solution was stored at 4℃for further use.
5. Preparation of Metal Organic Frameworks (MOFs) embedding AMPA and FITC (MAFs)
First 5 mg FITC and 3 mg AMPA were dissolved in 3 mL propylene glycol methyl ether solution. Subsequently, 1mL carboxylated MOF (50 nm,100 mg/mL) was added and reacted for 30 minutes. The heating was then immediately stopped and the solution was cooled to room temperature. The synthesized MAF was washed three times with deionized water, centrifuged at 13500 rpm for 15 minutes, and the residue was redispersed in 4 mL deionized water. The prepared MAF solution was stored in a dark environment at 4℃for future use.
6. Preparation of acceptor probes by DNA2 ligation of MAF
(1) Transferring 10 μl of MAF in 1 mL PBS buffer (0.01M, pH=7.4), adding 35 μM DNA2, electrostatic adsorbing for 30 min, adding 5 μg EDC, stirring, reacting for 30 min, repeating for 3 times, and observing MAF aggregation;
(2) Then 100 mu L of mg of glucosamine (10 mg/mL) is added, electrostatic adsorption is carried out for 30 min, and 5 mu g of EDC is added for reaction for 30 min;
(3) 5. Mu.g of EDC was again added and reacted for 30 min, centrifuged and the precipitate was redissolved in 100. Mu.L of the redissolved solution.
The components of the complex solution are as follows: the 0.01M PBS buffer (ph=7.4) contained 25% sucrose with 0.1% sodium azide, 1% BSA,1% tween 20.
7. Drawing of standard curve for detecting mutant DNA of lung adenocarcinoma patient
The mutant DNA standard was diluted with PBS (0.01M, pH 7.4) buffer to different concentration gradients, specifically: 25000 pg/mL, 5000 pg/mL, 1000 pg/mL, 200 pg/mL, 40 pg/mL, 8 pg/mL, 1.6 pg/mL, 0.32 pg/mL, 0.064 pg/mL, 0 pg/mL. Respectively taking 200 mu L of mutant DNA standard substances with different concentrations, simultaneously adding 6 mu L of donor probe and 6 mu L of acceptor probe, reacting at 37 ℃ for 10 min, and adding 10 mu L of 0.1M H 2 O 2 After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. And drawing a standard curve by taking the obtained luminous intensity as an ordinate and the mutation DNA concentration (pg/mL) as an abscissa, and calculating a linear equation. The specific experimental results are as follows: the linear standard curve is y= 4.3221ln (x) +4.1995, r= 0.9929, see fig. 7. The minimum detection limit of the method is defined as the average luminous intensity +3 times standard deviation of the results obtained when the concentration of 20 mutant DNA is 0, and the minimum detection line is calculated to be 0.16 by the standard curvepg/mL。
8. Detecting content of mutant DNA of lung adenocarcinoma patient in human serum sample
The application relates to a homogeneous catalysis chemiluminescence immunoassay kit, a preparation method and a use method thereof, which are implemented by the following steps when the kit is used for detecting the content of mutant DNA of a lung adenocarcinoma patient in a human serum sample:
firstly diluting human serum sample by 10 times, then adding 6 mu L of donor probe and 6 mu L of acceptor probe into the diluted sample simultaneously, reacting for 10 min at 37 ℃, adding 10 mu L of H of 0.1M 2 O 2 ，H 2 O 2 And MoO reaction, in situ generation 1 O 2 Generated by 1 O 2 The acceptor probe may be attacked to cause its internal FITC molecule to emit 520 nm light. After 2 min, the luminescence intensity of the solution was measured with a luminescence detector. Substituting the resulting luminescence intensity into the standard curve according to 7 in example 5, and calculating the concentration of the corresponding sample, namely the actual concentration of the mutant DNA of the lung adenocarcinoma patient in the human serum sample.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the application and that various changes in form and details may be made therein without departing from the spirit and scope of the application. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the application, and the scope of the application is therefore intended to be limited only by the appended claims.

Claims (9)

1. A homogeneous catalytic chemiluminescent kit comprising: donor probes, acceptor probes, and hydrogen peroxide; the donor probe comprises a linked donor material and a first recognition molecule, and the acceptor probe comprises a linked acceptor material and a second recognition molecule;
wherein the donor material is used to catalyze hydrogen peroxide to produce singlet oxygen;
the first recognition molecule is one or more of an antibody, a nano antibody, an antigen, a recognition peptide, an aptamer, DNA or RNA;
the acceptor material has singlet oxygen triggering cycle luminescence characteristics; the acceptor material comprises a singlet oxygen triggered luminescent reagent;
the singlet oxygen-triggered luminescence reagent comprises N, N-dimethyl-4- (6-phenyl-2, 3-dihydro-1, 4-oxetan-5-yl) aniline as a chemiluminescent substance and 2- ((5- (4- (diphenylamino) phenyl) thiophen-2-yl) methylene) malononitrile as a luminescence enhancer;
the second recognition molecule is one or more of an antibody, a nano antibody, an antigen, a recognition peptide, an aptamer, DNA or RNA;
the first recognition molecule and the second recognition molecule are configured to specifically bind to a target molecule.
2. The homogeneous catalytic chemiluminescent kit of claim 1 further comprising: a standard for the target molecule;
The target molecule is an antigen, peptide, antibody, amino acid, DNA, RNA, virus, hormone, drug or metabolite.
3. The homogeneous catalysis chemiluminescent kit of claim 1 wherein the donor material is one of sodium molybdate, sodium hypochlorite, molybdenum stearate, molybdenum sulfide, molybdenum phosphate, manganese oxide-based materials and derivatives thereof;
the surface of the donor material is also connected with a chemical group, and the chemical group is at least one of epoxy group, chloromethyl group, sulfydryl group, amino group, hydroxyl group, maleic amine group, sulfonic group, carboxyl group and aldehyde group.
4. The homogeneous catalytic chemiluminescent kit of claim 1 wherein the acceptor material further comprises a spherical support, the singlet oxygen trigger luminescent reagent being embedded within the spherical support;
wherein the spherical carrier is mesoporous silica, dendrimer, polystyrene microsphere, metal organic frame, covalent organic frame or liposome;
the surface of the receptor material is also connected with a chemical group, and the chemical group is at least one of epoxy group, chloromethyl group, sulfydryl group, amino group, hydroxyl group, maleic amine group, sulfonic group, carboxyl group and aldehyde group.
5. The homogeneous catalysis chemiluminescent kit of claim 1 wherein the donor material has an average particle size of 5 nm to 1000 nm; the average particle size of the receptor material is 5 nm-1000 nm.
6. The method of preparing a homogeneous catalytic chemiluminescent kit according to any one of claims 1 to 5 comprising the steps of:
a chemical coupling method is adopted to connect the first recognition molecule to the surface of the donor material, so as to prepare a donor probe;
a second recognition molecule is connected to the surface of the receptor material by adopting a chemical coupling method to prepare a receptor probe;
and dissolving hydrogen peroxide in ultrapure water at a concentration of 5 mu M-5M for catalytic reaction to obtain the homogeneous catalysis chemiluminescent kit.
7. The method of preparing a homogeneous catalytic chemiluminescent kit of claim 6 wherein the chemical coupling process comprises a diazo process, glutaraldehyde process, glutaric anhydride process or carbodiimide process;
the molar ratio of the first recognition molecule to the donor material is (10-1000): 1, a step of;
the molar ratio of the second recognition molecule to the receptor material is (10-1000): 1.
8. use of a homogeneous catalytic chemiluminescent kit according to any one of claims 1 to 5 comprising the steps of:
(1) Respectively adding the donor probe and the acceptor probe into a sample solution to be detected to form a mixture to be detected;
when the sample to be detected contains target molecules, forming donor probe-target molecule-acceptor probe immune complexes; when the sample to be detected does not contain target molecules, the donor probe and the acceptor probe are in a separation state, so that immune complexes are not formed;
(2) Adding hydrogen peroxide into the mixture to be tested, and decomposing the hydrogen peroxide into singlet oxygen and H by a donor material 2 O；
When an immune complex is formed, singlet oxygen diffuses to the acceptor probe, producing chemiluminescent light; when no immune complex exists in the mixture to be tested, no chemiluminescence is generated;
(3) And (3) reading the chemiluminescent luminescence signal by using a luminescence detector, so as to realize quantitative detection of the concentration of the target molecule in the sample to be detected.
9. The method of claim 8, wherein the reaction temperature in step (1) is 20 ℃ to 50 ℃ for 3 to 30 minutes;
in the step (3), a luminescence detector is used for reading the luminescence signal of the chemiluminescence for 1-5 minutes;
And (3) the emission wavelength of the chemiluminescence in the step (3) is 400-2000 nm.
CN202310863500.7A 2023-07-14 2023-07-14 Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof Active CN116577499B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310863500.7A CN116577499B (en) 2023-07-14 2023-07-14 Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310863500.7A CN116577499B (en) 2023-07-14 2023-07-14 Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof

Publications (2)

Publication Number Publication Date
CN116577499A true CN116577499A (en) 2023-08-11
CN116577499B CN116577499B (en) 2023-09-19

Family

ID=87540037

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310863500.7A Active CN116577499B (en) 2023-07-14 2023-07-14 Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof

Country Status (1)

Country Link
CN (1) CN116577499B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340716A (en) * 1991-06-20 1994-08-23 Snytex (U.S.A.) Inc. Assay method utilizing photoactivated chemiluminescent label
US20050272108A1 (en) * 2004-05-21 2005-12-08 Bhanu Kalra Stabilized two component system for chemiluminescent assay in immunodiagnostics
US20160047816A1 (en) * 2014-07-25 2016-02-18 SeLux Diagnostics, Inc. Assay methods involving dissociable nanoparticles
CN108169497A (en) * 2018-02-11 2018-06-15 北京科美生物技术有限公司 Human prolactin detection kit and preparation method and application
CN112114130A (en) * 2019-06-21 2020-12-22 博阳生物科技（上海）有限公司 Receptor reagent for homogeneous phase chemiluminescence detection and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340716A (en) * 1991-06-20 1994-08-23 Snytex (U.S.A.) Inc. Assay method utilizing photoactivated chemiluminescent label
US20050272108A1 (en) * 2004-05-21 2005-12-08 Bhanu Kalra Stabilized two component system for chemiluminescent assay in immunodiagnostics
US20160047816A1 (en) * 2014-07-25 2016-02-18 SeLux Diagnostics, Inc. Assay methods involving dissociable nanoparticles
CN108169497A (en) * 2018-02-11 2018-06-15 北京科美生物技术有限公司 Human prolactin detection kit and preparation method and application
CN112114130A (en) * 2019-06-21 2020-12-22 博阳生物科技（上海）有限公司 Receptor reagent for homogeneous phase chemiluminescence detection and application thereof

Also Published As

Publication number Publication date
CN116577499B (en) 2023-09-19

Similar Documents

Publication Publication Date Title
CN110220888B (en) Preparation method of electrochemical luminescence sensor of ruthenium terpyridyl functionalized MOF
Tiwari et al. Recent advances and developments on integrating nanotechnology with chemiluminescence assays
Cheng et al. Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection
Ehsani et al. Comparison of CuO nanoparticle and CuO/MWCNT nanocomposite for amplification of chemiluminescence immunoassay for detection of the hepatitis B surface antigen in biological samples
WO2019223692A1 (en) Chemical luminescence analysis and measurement method, system using same, and kit
Wang et al. Highly sensitive rapid chemiluminescent immunoassay using the DNAzyme label for signal amplification
Liu et al. A novel aptamer-mediated CuInS 2 quantum dots@ graphene oxide nanocomposites-based fluorescence “turn off–on” nanosensor for highly sensitive and selective detection of kanamycin
Huang et al. A metal–organic framework nanomaterial as an ideal loading platform for ultrasensitive electrochemiluminescence immunoassays
CN116183901A (en) Protein marker fluorescence detection method based on cyclodextrin-adamantane iridium complex main object assembly labeling
CN116577499B (en) Homogeneous catalysis chemiluminescence kit, and preparation method and application method thereof
CN111693721A (en) Preparation method and application of enzyme-linked immunosorbent assay based on prussian blue nano enzyme label
CN113504371A (en) Separation-free chemiluminescence immunoassay method based on cyclodextrin inclusion
CN105181956B (en) Application of the fluorescence detection specifically responded based on metal ion in immune detection
Syamila et al. Bio-nanogate manipulation on electrode surface as an electrochemical immunosensing strategy for detecting anti-hepatitis B surface antigen
CN113777297A (en) Fluorescence differential rapid detection method based on magnetic nanoparticles
CN113109558A (en) Magnetic particle chemiluminescence kit for quantitatively detecting CYFRA21-1 and manufacturing method thereof
Lou et al. Biocompatible protein nanogels as robust signal labels for persistent chemiluminescence immunoassays
WO2020252871A1 (en) Receptor reagent for homogeneous chemiluminescent assay, and application thereof
CN110514646B (en) Chemiluminescence analysis determination method, system and kit using same
CN113125700B (en) Homogeneous detection kit for creatine kinase isozymes and application thereof
CN112114131A (en) Homogeneous phase chemiluminescence detection method and application thereof
CN113125703B (en) Myoglobin homogeneous detection kit and application thereof
CN110514647B (en) Chemiluminescence analysis determination method, system and kit using same
CN113125721B (en) Homogeneous detection kit for creatine kinase isozymes and application thereof
CN112240930A (en) Homogeneous phase chemiluminescence analysis method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant