CN111912977A - Photosensitive detection system and manufacturing method and application thereof - Google Patents
Photosensitive detection system and manufacturing method and application thereof Download PDFInfo
- Publication number
- CN111912977A CN111912977A CN202010578325.3A CN202010578325A CN111912977A CN 111912977 A CN111912977 A CN 111912977A CN 202010578325 A CN202010578325 A CN 202010578325A CN 111912977 A CN111912977 A CN 111912977A
- Authority
- CN
- China
- Prior art keywords
- solution
- ligand
- microsphere
- photosensitive
- microspheres
- 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
Links
Images
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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
- G01N2405/04—Phospholipids, i.e. phosphoglycerides
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a photosensitive detection system and a manufacturing method and application thereof. The preparation method of the detection system comprises the steps of preparing a photosensitizer, preparing photosensitive microspheres and preparing ligand microspheres. The invention also discloses an application method of the photosensitive detection system in preparing a measuring reagent for the anti-cardiolipin antibody IgA. The photosensitizer in the photosensitive detection system has the advantages of firm structure, difficult decomposition, good stability, good photosensitive effect of the system, high sensitivity and good market value.
Description
Technical Field
The invention relates to the technical field of biomolecules, in particular to a photosensitive detection system and a manufacturing method and application thereof.
Background
A homogeneous phase chemiluminescence method of Lumigen company, use chemiluminescence label to mark antigen or antibody, use peroxidase to mark another antigen or antibody at the same time, go on detecting the analyte in the sample together; the chemiluminescence is generated under the action of laser in a pure liquid phase environment, so that a quantitative detection means is achieved. Although the method achieves the detection purpose of homogenization chemiluminescence and has the advantage of better stability when detecting low-concentration reagents, the linear range is narrower, the method has limitations and is not favorable for wide popularization, and the hook effect is easy to generate for detecting proteins with larger molecular weight.
The earliest photosensitizers were traced to ancient egypt, who had taken photosensitizers containing drugs and then used to treat vitiligo by exogenous laser irradiation, and iron porphyrin photosensitizers were discovered in recent times. Photosensitizers are frequently used in clinical treatment and disease detection at present, but problems are found in application, such as: easy decomposition in liquid-image environment, relatively delayed energy conversion, poor uniformity, etc.
Disclosure of Invention
The present invention is to overcome the above problems and to provide a photosensitizer synthesized by using a polymer compound in which two different wavelengths are combined, and a detection system made of the photosensitizer, and the scheme of the present invention is as follows:
a photosensitive detection system, comprising:
(1) photosensitive microspheres coated with a specific photosensitizer;
(2) ligand energy conversion microspheres (hereinafter referred to as ligand microspheres);
the preparation method of the photosensitizer comprises the following steps: adding 50-100 parts of copper tetraaminophthalocyanine, 2.12-3.15 parts of tetrafluorophthalonitrile, 0.02-0.10 part of copper (II) chloride, 30-90 parts of ethyl glycolate, 50-120 parts of potassium carbonate and 800-1200 parts of pyridine into 15000 parts of toluene in parts by weight, and heating and refluxing for reaction for 4-8 hours under the protection of nitrogen; and (3) evaporating the solvent in vacuum, dissolving and filtering by using chloroform, and recrystallizing in n-hexane after reducing pressure and drying the filtrate.
The invention discloses a manufacturing method of a photosensitive detection system, which comprises the following steps:
s1, preparing a photosensitizer, wherein the preparation method of the photosensitizer is the same as that of the photosensitizer;
s2, preparing photosensitive microspheres
Washing carboxylated microspheres with water and ethanol, centrifuging at 30000rpm for 15-20 minutes, adding aminoethanol, heating at 105 ℃ by adopting an oil bath method under the condition of stirring, heating for 10 minutes, adding S1 photosensitizer, and adding sodium hydroxide 5 minutes after adding the photosensitizer; then slowly reducing the temperature to the room temperature within two hours; after cooling, the mixture was diluted with ethanol, then centrifuged at 30000rpm for 30min, the supernatant was discarded, and the precipitate was resuspended in ethanol by ultrasound; repeatedly centrifuging, resuspending the precipitate with water, and then resuspending with ethanol water solution to obtain photosensitive microspheres;
s3 preparation of ligand microspheres
The method comprises the following steps: preparing a ligand microsphere energy agent; labeling avidin of the substance to be detected with a ligand microsphere energy agent; ligand microspheres are prepared using a ligand microsphere energy agent that labels avidin, the substance to be detected.
The invention also discloses application of the photosensitive detection system, and the photosensitive detection system is used for preparing a reagent for measuring the anti-cardiolipin antibody IgA.
As an improvement, the method for preparing the ligand microsphere energy agent in S3 comprises the following steps:
firstly, preparing a precursor a of ligand microsphere energy agent
To 500ml of anhydrous methanol were added 10 to 23.0 g of ammonium acetate, 16.3 g of N- [ (2' -pyridyl) -2-oxoethyl ] pyridine iodide, and 10.76 g of a solution of 1- (3-pyridyl) -3- (dimethylamino) -2-propen-1-one under reflux with stirring for 24 hours. Cooling the reaction liquid to room temperature, standing at-15 ℃ for 1 hour, filtering, collecting precipitate, fully washing with cold methanol (-10-20 ℃), and recrystallizing the product with acetonitrile to obtain a ligand microsphere energy agent precursor a;
② preparation of ligand microsphere energy agent precursor b
Adding 4.5 g of ligand microsphere energy agent precursor a into a mixed solution of 45ml of sulfuric acid, 45ml of acetic acid and 12ml of water; stirring and reacting at 75-80 ℃ for 48 hours, adding the reaction solution into 300ml of ice water, filtering and collecting precipitate, fully washing with water, and then washing with absolute ethyl alcohol; obtaining 4.8 g of hydrolysate after vacuum drying; adding 8 g of thionyl chloride into 400ml of anhydrous methanol while cooling with ice water, stirring for 15 minutes, adding 4.80 g of the hydrolysate, stirring and refluxing the reaction solution for 8 hours, continuously stirring for 16 hours at room temperature, and drying with anhydrous sodium sulfate; after evaporation of the solvent, the product was chromatographed on a silica gel column, developed with methylene chloride-methanol (99:1), and the first fraction eluted first was collected. After the solvent is evaporated, recrystallizing the product by using toluene, and drying in vacuum to obtain a ligand microsphere energy agent precursor b;
preparing ligand microsphere energy agent precursor c
Adding 1.94 g of ligand microsphere energy agent precursor b (2.8mmol) into 120ml of ethanol, then adding 4 g of potassium hydroxide and 10ml of water, and stirring and refluxing the reaction solution for 3 hours; evaporating the solvent under reduced pressure, dissolving the resultant in 150ml of water, filtering to remove trace insoluble substances, and slowly dropping a trifluoroacetic acid aqueous solution into the stirred solution; fully washing and drying in vacuum to obtain a ligand microsphere energy agent precursor c;
preparing ligand microsphere energy agent
Putting the precursor c of ligand microsphere energy agent in a container P2O5After sufficiently drying in a vacuum drier, 181.7mg of the dried N, N-dimethylformamide was dissolved in 5ml of dry N, N-dimethylformamide, and 34.5mg of N-hydroxysuccinamide (NHS) and 61.9mg of N, N' -Dicyclohexylcarbodiimide (DCC) were added with stirring, and after stirring at room temperature for 24 hours, insoluble matter was removed by filtration; and (3) after the filtrate is decompressed and concentrated to remove the solvent, washing a product with a small amount of isopropanol, and drying in vacuum to obtain the ligand microsphere energy agent.
As an improvement, the preparation method of the ligand microsphere in S3 comprises the following steps: 4ml of 20% concentration 80nm carboxylated microspheres (400mg) are washed 2 times with 4ml of water and 4ml of absolute ethyl alcohol, centrifuged at 30000rpm for 15 minutes, diluted with 3ml of aminoethanol in a 25ml round flask for reaction, and a magnetic stirrer is arranged in the flask; heating the oil to 105 ℃, stirring for 10min, then adding 40mg of ligand microsphere energy agent solution for marking the avidin to be detected, and stirring for 5 min; then 1ml of 0.1M NaOH was added slowly over 5 minutes, during which the temperature of the oil solution was kept at 105 ℃ and then slowly lowered to room temperature over 2 hours; after cooling, the mixture was diluted to 20m with ethanol, centrifuged at 30000rpm for 30min to remove the supernatant, and the pellet was resuspended in ethanol by sonication. Centrifugation was repeated and the pellet was resuspended in water and the pellet was resuspended to a volume of 40ml in 5ml of 10% aqueous ethanol. To this solution were added 20mg BSA and 25mg NaN3And then, freezing and storing at the temperature of minus 20 ℃ to prepare the ligand microsphere.
As an improvement, the application method of the photosensitive detection system comprises the steps of labeling streptavidin by using photosensitive microspheres, detecting anticardiolipin antigen by using ligand microsphere labels and preparing mouse anticardiolipin antigen
As an improvement, the method for labeling streptavidin by using photosensitive microspheres comprises the following steps:
step one, photosensitive microsphere suspension treatment: absorbing a certain amount of photosensitive microspheres in a high-speed refrigerated centrifuge for centrifugation, removing supernatant, adding a certain amount of MES buffer solution, resuspending on an ultrasonic cell disruption instrument, and adding the MES buffer solution to adjust the concentration of the photosensitive microspheres to 200 mg/ml;
step two, preparing a streptavidin solution: weighing a certain amount of streptavidin, and dissolving the streptavidin into 10mg/ml MES buffer solution;
step three, mixing: mixing the treated photosensitive microsphere suspension, 8mg/ml streptavidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution;
step four, reaction: MES buffer solution is prepared into 25mg/ml NHS solution, added according to the volume ratio of 1:25 to the reaction solution, and rapidly mixed evenly. Rotating and reacting at 37 ℃ for 24 hours;
step five, sealing: preparing 75mg/ml Gly solution by MES buffer solution, rapidly mixing 250mg/ml BSA solution and reaction solution at a volume ratio of 5:10, and reacting at 37 ℃ for 12 hours;
step six, cleaning: adding MES buffer solution into the reacted solution, centrifuging with a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending, centrifuging again, washing for three times, finally suspending with a small amount of photosensitive reagent buffer solution, measuring the solid content, and adjusting the concentration to 10mg/ml with photosensitive microsphere reagent buffer solution.
As an improvement, the method for labeling avidin of the analyte to be detected by using the ligand microsphere energy agent as described in S3 is
Dissolving 5mg of Streptavidin (SA) in 10ml of 0.1mol/L sodium bicarbonate buffer solution with pH value of 9.1, adding 10mg of ligand microsphere energy agent, stirring at room temperature for reaction for 3 hours, and reacting the solution at 4 ℃ with a solution containing 0.25 g of NaN3Dialyzing the solution with 0.1mol/L sodium bicarbonate solution for three times, each for 24 hours, removing unreacted marker substance, and adding EuCL to the solution3Obtaining the labeled streptomyces after the solutionSolution of avidin; wherein EuCL3The mass ratio of the ligand microsphere energy agent to the ligand microsphere energy agent is 1.5: 2.
As an improvement, the method for detecting the anticardiolipin antigen by using the ligand microsphere label comprises the following steps:
step one, suspension treatment of ligand microspheres: centrifuging a certain amount of ligand microspheres in a high-speed refrigerated centrifuge, discarding supernatant, adding a timed MES buffer solution, ultrasonically crushing until the particles are resuspended, and adding the MES buffer solution to adjust the concentration of the particles to 100 mg/ml;
step two, antibody treatment: dialyzing the antigen with 0.05M MES buffer solution with pH of 6.0, determining the concentration after dialysis, and adjusting the concentration to 8 mg/ml;
step three, reaction: MES buffer solution is prepared into EDAC solution of 40mg/ml, added according to the proportion of 100mg/ul of microspheres and EDAC, and rapidly reacted and uniformly mixed for 16 hours;
step four, sealing: adding 200mg/ml casein solution (MES buffer solution) with the volume ratio of 10:8 to the reaction solution, rapidly mixing, and performing rotary reaction at 37 deg.C for 16 hr;
step five, cleaning: and (4) centrifugally washing the mixture for four times by using MES buffer solution, finally suspending the mixture by using reagent buffer solution, measuring the particle size and the solid content, and adjusting the concentration to 10 mg/ml.
As an improvement, the preparation method of the mouse anti-human cardiolipin antigen comprises the following steps:
step one, absorbing a certain amount of mouse anti-human cardiolipin antigen, dialyzing at the temperature of 2-8 ℃ at 0.1M and pH of 8.5NaHCO3A solution; sampling the dialyzed mouse anti-human cardiolipin antigen to determine the antigen concentration; 10mg/ml Biotin solution was prepared in DMSO.
Step two, marking: according to the requirement of n/10 × 2ul (n is the labeling ratio of 20) of Biotin (DMSO) for every 1mg of antigen, the mixture is rapidly mixed, and the mixture is rotated and mixed at the temperature of 2-8 ℃ for reaction overnight.
Step three, dialysis: and dialyzing the reacted biotin-labeled antigen in a biotin-labeled dialysis buffer solution at the temperature of 2-8 ℃.
Step four, volume fixing: and sucking the dialyzed protein out and transferring the dialyzed protein into a container, sampling and determining the protein concentration, and obtaining the test result to obtain the protein concentration.
The invention has the advantages that:
the photosensitizer disclosed by the invention has the advantages of firm structure, difficulty in decomposition, good stability, electron transfer in a low-oxygen environment and no influence of high temperature of pH value.
The detection system disclosed by the invention fully utilizes the characteristics of the photosensitizer to complex the photosensitizer on the surface of silicon dioxide or polystyrene to form a photosensitive microsphere. The photosensitizer coated by the photosensitive microsphere releases oxygen molecules under the action of exciting light, then generates a synergistic effect with lanthanide coated by the ligand microsphere to generate active oxygen, and the active oxygen is transited from a ground state to a singlet excited state and then transited to a triplet excited state through intersystem crossing, the triplet excited photosensitizer can generate different active oxygen after reacting in one or two forms, and when the photosensitizer is mainly divided into an I type generating oxygen free radicals and a II type generating singlet oxygen, the photosensitizer irradiates the photosensitive microsphere with light with a certain wavelength, and the photosensitive microsphere absorbs light energy and transits from the ground state to a first excited state; the photosensitive microsphere in the first excited state can oxidize biological macromolecules and organelles in cells and initiate apoptosis to achieve the effect of killing the cells by emitting oxygen of high-energy radicals to jump to a ground state, and oxygen absorbs active oxygen released by singlet oxygen when energy is transited from the ground state to the singlet state, and the photosensitive microsphere can also be used for detecting proteins, NDAs, receptors and tumor factors.
Drawings
FIG. 1 is a graph comparing a measured value of a reagent of the present invention with a measured value of Roche.
Detailed Description
The present invention will be described in detail and specifically with reference to the following examples so as to facilitate the understanding of the present invention, but the following examples do not limit the scope of the present invention.
Example 1
The embodiment discloses a preparation process of a photosensitizer, which comprises the following steps:
copper tetraaminophthalocyanine (0.05-0.1 g), tetrafluorophthalonitrile (2.12-3.15 mg), copper chloride (/ 0.02-0.10 mg), ethyl glycolate (30-90 mg), K2C03 (0.050-0.1 mg)2g) And pyridine (1mL) was added to toluene (15 mL) in a kettle under N2Heating and refluxing for reaction for 6h under protection. After the solvent was evaporated in vacuo, it was dissolved in about 60mL of chloroform and filtered. The filtrate was evaporated to dryness under reduced pressure and recrystallized from n-hexane to give 0.042g of indigo product in about 61% yield.
Example 2
The embodiment discloses a preparation method of a photosensitive detection system and application of the photosensitive detection system in preparation of an anti-cardiolipin antibody IgA detection reagent, which comprises the following steps:
s1, preparing a photosensitizer, wherein the preparation method of the photosensitizer is the same as that of the embodiment 1.
S2, preparing photosensitive microspheres
Washing 4ml of 20% carboxylated microsphere (400mg) washed at 150nm for 2 times with a mixed solution of 4ml of water and 4ml of absolute ethyl alcohol; centrifuging at 30000rpm for 15 min, diluting with 3ml aminoethanol, reacting in 25ml round flask, and placing a magnetic stirrer in the flask; the oil was heated to 105 ℃ and stirred for 10min, then 40mg of photosensitizer was added and stirred for 5min, then 1ml of 0.1M NaOH was added slowly over the next 5min, in the course of which the temperature of the oil solution was kept at 105 ℃ and then slowly lowered to room temperature over 2 hours. After cooling, the mixture is diluted to 20ml by ethanol, centrifuged at 30000rpm for 30min, the supernatant is discarded, and the precipitate is resuspended in ethanol by ultrasound; repeatedly centrifuging, and resuspending the precipitate with water; the re-precipitation was resuspended to a volume of 40ml in 5ml of 10% aqueous ethanol to obtain photosensitive microspheres.
S3 preparation of ligand microspheres
The method comprises the following steps: preparing a ligand microsphere energy agent; labeling avidin of a substance to be detected by using a ligand microsphere energy agent (specifically labeling streptavidin by using the ligand microsphere energy agent); ligand microspheres are prepared using a ligand microsphere energy agent that labels avidin, the substance to be detected.
The method for preparing the ligand microsphere energy agent comprises the following steps:
firstly, preparing a precursor a of ligand microsphere energy agent
To 500ml of anhydrous methanol were added 10 to 23.0 g of ammonium acetate, 16.3 g of N- [ (2' -pyridyl) -2-oxoethyl ] pyridine iodide, and 10.76 g of a solution of 1- (3-pyridyl) -3- (dimethylamino) -2-propen-1-one under reflux with stirring for 24 hours. Cooling the reaction liquid to room temperature, standing at-15 ℃ for 1 hour, filtering, collecting precipitate, fully washing with cold methanol (-10-20 ℃), and recrystallizing the product with acetonitrile to obtain a ligand microsphere energy agent precursor a;
② preparation of ligand microsphere energy agent precursor b
Adding 4.5 g of ligand microsphere energy agent precursor a into a mixed solution of 45ml of sulfuric acid, 45ml of acetic acid and 12ml of water; stirring and reacting at 75-80 ℃ for 48 hours, adding the reaction solution into 300ml of ice water, filtering and collecting precipitate, fully washing with water, and then washing with absolute ethyl alcohol; obtaining 4.8 g of hydrolysate after vacuum drying; adding 8 g of thionyl chloride into 400ml of anhydrous methanol while cooling with ice water, stirring for 15 minutes, adding 4.80 g of the hydrolysate, stirring and refluxing the reaction solution for 8 hours, continuously stirring for 16 hours at room temperature, and drying with anhydrous sodium sulfate; after evaporation of the solvent, the product was chromatographed on a silica gel column, developed with methylene chloride-methanol (99:1), and the first fraction eluted first was collected. After the solvent is evaporated, recrystallizing the product by using toluene, and drying in vacuum to obtain a ligand microsphere energy agent precursor b;
preparing ligand microsphere energy agent precursor c
Adding 1.94 g of ligand microsphere energy agent precursor b (2.8mmol) into 120ml of ethanol, then adding 4 g of potassium hydroxide and 10ml of water, and stirring and refluxing the reaction solution for 3 hours; evaporating the solvent under reduced pressure, dissolving the resultant in 150ml of water, filtering to remove trace insoluble substances, and slowly dropping a trifluoroacetic acid aqueous solution into the stirred solution; fully washing and drying in vacuum to obtain a ligand microsphere energy agent precursor c;
preparing ligand microsphere energy agent
Putting the precursor c of ligand microsphere energy agent in a container P2O5After sufficiently drying in a vacuum drier, 181.7mg of the dried N, N-dimethylformamide was dissolved in 5ml of dry N, N-dimethylformamide, and 34.5mg of N-hydroxysuccinamide (NHS) and 61.9mg of N, N' -Dicyclohexylcarbodiimide (DCC) were added with stirring, and after stirring at room temperature for 24 hours, insoluble matter was removed by filtration; after the filtrate is decompressed and concentrated to remove the solvent,and washing the product with a small amount of isopropanol, and drying in vacuum to obtain the ligand microsphere energy agent.
The method for labeling streptavidin by using ligand microsphere energy agent comprises the following steps:
dissolving 5mg of Streptavidin (SA) in 10ml of 0.1mol/L sodium bicarbonate buffer solution with pH value of 9.1, adding 10mg of ligand microsphere energy agent, stirring at room temperature for reaction for 3 hours, and reacting the solution at 4 ℃ with a solution containing 0.25 g of NaN3Dialyzing the solution with 0.1mol/L sodium bicarbonate solution for three times, each for 24 hours, removing unreacted marker substance, and adding EuCL to the solution3Obtaining labeled streptavidin solution; wherein EuCL3The mass ratio of the ligand microsphere energy agent to the ligand microsphere energy agent is 1.5: 2.
The method for preparing the ligand microsphere by using the ligand microsphere energy agent marked with streptavidin comprises the following steps:
4ml of 20% concentration 80nm carboxylated microspheres (400mg) are washed 2 times with 4ml of water and 4ml of absolute ethyl alcohol, centrifuged at 30000rpm for 15 minutes, diluted with 3ml of aminoethanol in a 25ml round flask for reaction, and a magnetic stirrer is arranged in the flask; heating the oil to 105 ℃, stirring for 10min, then adding 40mg of ligand microsphere energy agent solution for labeling streptavidin, and stirring for 5 min; then 1ml of 0.1M NaOH was added slowly over 5 minutes, during which the temperature of the oil solution was kept at 105 ℃ and then slowly lowered to room temperature over 2 hours; after cooling, the mixture was diluted to 20m with ethanol, centrifuged at 30000rpm for 30min to remove the supernatant, and the pellet was resuspended in ethanol by sonication. Centrifugation was repeated and the pellet was resuspended in water and the pellet was resuspended to a volume of 40ml in 5ml of 10% aqueous ethanol. To this solution were added 20mg BSA and 25mg NaN3And then, freezing and storing at the temperature of minus 20 ℃ to prepare the ligand microsphere.
The method for preparing the anti-cardiolipin antibody IgA assay reagent by using the photosensitive detection system comprises the following steps:
(1) labeling streptavidin (denoted as component X) with photosensitive microspheres;
(2) labeling and detecting anticardiolipin antigen (marked as component Y) by using ligand microsphere;
(3) a murine anti-human cardiolipin antigen (denoted as component Z) was prepared.
The method for labeling streptavidin by using photosensitive microspheres comprises the following steps:
step one, photosensitive microsphere suspension treatment: absorbing a certain amount of photosensitive microspheres in a high-speed refrigerated centrifuge for centrifugation, removing supernatant, adding a certain amount of MES buffer solution, resuspending on an ultrasonic cell disruption instrument, and adding the MES buffer solution to adjust the concentration of the photosensitive microspheres to 200 mg/ml;
step two, preparing a streptavidin solution: weighing a certain amount of streptavidin, and dissolving the streptavidin into 10mg/ml MES buffer solution;
step three, mixing: mixing the treated photosensitive microsphere suspension, 8mg/ml streptavidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution;
step four, reaction: MES buffer solution is prepared into 25mg/ml NHS solution, added according to the volume ratio of 1:25 to the reaction solution, and rapidly mixed evenly. Rotating and reacting at 37 ℃ for 24 hours;
step five, sealing: preparing 75mg/ml ethanolamine solution and 250mg/ml casein solution by MES buffer solution, quickly and uniformly mixing the solution and the reaction solution according to the volume ratio of 5:10, and reacting for 12 hours at 37 ℃;
step six, cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, washing for three times, finally suspending by a small amount of photosensitive reagent buffer solution, measuring the solid content, and adjusting the concentration to 10mg/ml by using photosensitive microsphere reagent buffer solution.
The method for detecting the anticardiolipin antigen by using ligand microsphere labeling comprises the following steps:
step one, suspension treatment of ligand microspheres: centrifuging a certain amount of ligand microspheres in a high-speed refrigerated centrifuge, discarding supernatant, adding a timed MES buffer solution, ultrasonically crushing until the particles are resuspended, and adding the MES buffer solution to adjust the concentration of the particles to 100 mg/ml;
step two, antibody treatment: dialyzing the antigen with 0.05M MES buffer solution with pH of 6.0, determining the concentration after dialysis, and adjusting the concentration to 8 mg/ml;
step three, reaction: MES buffer solution is prepared into EDAC solution of 40mg/ml, added according to the proportion of 100mg/ul of microspheres and EDAC, and rapidly reacted and uniformly mixed for 16 hours;
step four, sealing: adding 200mg/ml casein solution (MES buffer solution) with the volume ratio of 10:8 to the reaction solution, rapidly mixing, and performing rotary reaction at 37 deg.C for 16 hr;
step five, cleaning: and (4) centrifugally washing the mixture for four times by using MES buffer solution, finally suspending the mixture by using reagent buffer solution, measuring the particle size and the solid content, and adjusting the concentration to 10 mg/ml.
The method for preparing the mouse anti-human cardiolipin antigen comprises the following steps:
step one, absorbing a certain amount of mouse anti-human cardiolipin antigen, dialyzing at the temperature of 2-8 ℃ at 0.1M and pH of 8.5NaHCO3A solution; sampling the dialyzed mouse anti-human cardiolipin antigen to determine the antigen concentration; 10mg/ml Biotin solution was prepared in DMSO.
Step two, marking: according to the requirement of n/10 × 2ul (n is the labeling ratio of 20) of Biotin (DMSO) for every 1mg of antigen, the mixture is rapidly mixed, and the mixture is rotated and mixed at the temperature of 2-8 ℃ for reaction overnight.
Step three, dialysis: and dialyzing the reacted biotin-labeled antigen in a biotin-labeled dialysis buffer solution at the temperature of 2-8 ℃.
Step four, volume fixing: and sucking the dialyzed protein out and transferring the dialyzed protein into a container, sampling and determining the protein concentration, and obtaining the test result to obtain the protein concentration.
The following describes a method of measuring the anti-cardiolipin antibody IgA measurement reagent prepared in this example, and the results are analyzed for accuracy.
5ul of sample is added into the reaction well, and then 5ul of component Y and 5ul of component Z are added in sequence. Then put into an instrument (Perkin Elmer Victor model 1420 time-resolved fluorometer) and automatically operated according to the following steps by the instrument; after the incubation is carried out for 20 minutes under vibration at 37 ℃, 37ul of the component A is automatically added, and the instrument automatically generates laser to irradiate the micropores for calculating the light quantum after the incubation is carried out for 15 minutes at 37 ℃.
The lowest detection limit is:
and detecting the 20-well calibrator 1(0APLU/ml), calculating the RLU mean value (AVE) and the Standard Deviation (SD) of the calibrator, and inversely substituting AVE +2SD into a standard curve to obtain a concentration value, namely the analytical sensitivity of the kit.
| Reagent batch number | 4 degree reagent | 37 degree reagent |
| Minimum detection limit (0APLU/ml) | 0.0352 | 0.0526 |
And (4) evaluating conclusion: the minimum detection limits of the 4-degree and 37-degree reagents reach the requirements, wherein the minimum detection limit of the 4-degree reagent is slightly higher than that of the 37-degree reagent.
Internal precision:
the quality control products QC L and QCH are detected by using anti-cardiolipin antibody detection kits at 4 degrees and 37 degrees, and 10 holes are respectively detected. The following results were obtained:
batch precision:
2 batches of experiments were performed each day, 8-well detection was performed on QCL and QCH with 4-degree stored anti-cardiolipin antibody IgA kit for each batch of experiments for a total of 5 days, and the inter-batch precision of the reagents was calculated. The following results were obtained:
and (4) conclusion: the precision of the product in batches is less than 10 percent, the precision between batches is less than 15 percent, the precision is good, and the product meets the requirements.
Linearity
High-value samples (required measurement value is between 200APLU/ml and 300 APLU/ml) are selected as high-concentration serum samples. The low sample (required value <10APLU/ml) was selected as the low sample. 8 samples with the same concentration level are prepared by using the high-low concentration samples, and the specific preparation process is as follows:
the detection results of the 8 samples with the concentration measured by the 4-degree reagent and the 37-degree reagent are as follows:
4 degree reagent:
reagent at 37 degrees:
and (3) performing linear fitting by taking the detection result as a Y axis and the dilution ratio as an X axis, and calculating to obtain a correlation coefficient r of the linear fitting: 0.9989
And (4) conclusion: the correlation coefficients r of the detection results of the 4-degree kit and the 37-degree kit are respectively 0.9997 and 0.9989, and are more than 0.99, which indicates that the reagent linearity meets the expected requirements.
Mixed recovery rate
The mixed recovery experiment was performed with 4-degree stored reagents, 4 samples were selected for uniform distribution, the higher value sample and the lower value sample were mixed in equal volume, the mixed sample and 4 samples were measured with 4-degree stored reagents, three wells were measured for each sample, and the average was taken, the results are as follows:
the average recovery rate is 91.89%
And (4) conclusion: the average recovery rate of the detection result is between 90% and 110%, one of the average recovery rate is slightly lower than 90%, and the measured value is basically accurate and meets the requirement.
Hook experiment
The test was repeated 5 times for the Hook spot and high concentration calibrator 6 required in the DIR and the mean and SD calculated. The results of the tests performed on the above sample groups using 4 and 37 degrees anti-cardiolipin antibody IgA reagents are shown in the following table:
and (4) conclusion: no HOOK phenomenon was observed at 2000APLU/ml in the 4 degree and 37 degree kits, and HOOK-3SD > Cal6+3 SD.
Interference test
1500mg/dL triglyceride (ROCHE), 20mg/dL bilirubin, 1000mg/dL hemoglobin, 2000IU/mL RF, 600ng/mL HAMA, 500U/mL ANA and a blank without additive were added to # 1 and # 2 samples, respectively, and the above sample groups were tested using a 4-degree storage anti-cardiolipin antibody IgA kit, with the results shown in the following table:
and (4) conclusion: the results of 1500mg/dL triglyceride, 20mg/dL bilirubin, 1000mg/dL hemoglobin, 2000IU/mL RF, 600ng/mL HAMA and 500U/mL ANA do not exceed 10% of the product, indicating that the effect of the above-mentioned interfering substances at this concentration is not obvious.
Clinical relevance and compliance rates
(A) Clinical relevance
In the embodiment, the reagent measurement value and the Roche measurement value are compared and plotted to draw a correlation curve, a linear regression method is adopted to obtain a linear regression equation, and a correlation coefficient r is calculated. 210 parts of clinical samples detected by an anti-cardiolipin antibody IgA kit of ROCHE company are collected, and the samples are detected by the anti-cardiolipin antibody IgA detection kit developed by self. The results were analyzed and are shown in FIG. 1.
The regression equation is: y is 1.010x-0.148
The correlation coefficient r is 0.9975, slope is 1.010, YInt is-0.148.
And (4) conclusion: the correlation r between the detection result of the anti-cardiolipin antibody IgA detection kit developed by the method and the ROCHE result is as follows: 0.9975, greater than 0.99, the regression equation slope is: 1.010 is in the range of 0.95-1.05, intercept is: -0.148 is in the range of-3.0-3.0. The reagent and foreign similar products have good coincidence rate to clinical samples, and the method is equivalent to the prior art.
The embodiments of the present invention have been described in detail above, but they are merely exemplary, and the present invention is not equivalent to the above described embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, it is intended that all equivalent alterations and modifications be included within the scope of the invention, without departing from the spirit and scope of the invention.
Claims (10)
1. A photosensitive detection system, comprising:
(1) photosensitive microspheres coated with a specific photosensitizer;
(2) ligand energy conversion microspheres (hereinafter referred to as ligand microspheres);
the preparation method of the photosensitizer comprises the following steps: adding 50-100 parts of copper tetraaminophthalocyanine, 2.12-3.15 parts of tetrafluorophthalonitrile, 0.02-0.10 part of copper (II) chloride, 30-90 parts of ethyl glycolate, 50-120 parts of potassium carbonate and 800-1200 parts of pyridine into 15000 parts of toluene in parts by weight, and heating and refluxing for reaction for 4-8 hours under the protection of nitrogen; and (3) evaporating the solvent in vacuum, dissolving and filtering by using chloroform, and recrystallizing in n-hexane after reducing pressure and drying the filtrate.
2. A method of making a photosensitive detection system according to claim 1, comprising the steps of:
s1, preparing a photosensitizer, wherein the preparation method of the photosensitizer is the same as the method in the claim 1;
s2, preparing photosensitive microspheres
Washing carboxylated microspheres with water and ethanol, centrifuging at 30000rpm for 15-20 minutes, adding aminoethanol, heating at 105 ℃ by adopting an oil bath method under the condition of stirring, heating for 10 minutes, adding S1 photosensitizer, and adding sodium hydroxide 5 minutes after adding the photosensitizer; then slowly reducing the temperature to the room temperature within two hours; after cooling, the mixture was diluted with ethanol, then centrifuged at 30000rpm for 30min, the supernatant was discarded, and the precipitate was resuspended in ethanol by ultrasound; repeatedly centrifuging, resuspending the precipitate with water, and then resuspending with ethanol water solution to obtain photosensitive microspheres;
s3 preparation of ligand microspheres
The method comprises the following steps: preparing a ligand microsphere energy agent; labeling avidin of the substance to be detected with a ligand microsphere energy agent; ligand microspheres are prepared using a ligand microsphere energy agent that labels avidin, the substance to be detected.
3. The method of claim 2, wherein the step of preparing the ligand microsphere energy agent in S3 comprises:
firstly, preparing a precursor a of ligand microsphere energy agent
To 500ml of anhydrous methanol were added 10 to 23.0 g of ammonium acetate, 16.3 g of N- [ (2' -pyridyl) -2-oxoethyl ] pyridine iodide, and 10.76 g of a solution of 1- (3-pyridyl) -3- (dimethylamino) -2-propen-1-one under reflux with stirring for 24 hours. Cooling the reaction liquid to room temperature, standing at-15 ℃ for 1 hour, filtering, collecting precipitate, fully washing with cold methanol (-10-20 ℃), and recrystallizing the product with acetonitrile to obtain a ligand microsphere energy agent precursor a;
② preparation of ligand microsphere energy agent precursor b
Adding 4.5 g of ligand microsphere energy agent precursor a into a mixed solution of 45ml of sulfuric acid, 45ml of acetic acid and 12ml of water; stirring and reacting at 75-80 ℃ for 48 hours, adding the reaction solution into 300ml of ice water, filtering and collecting precipitate, fully washing with water, and then washing with absolute ethyl alcohol; obtaining 4.8 g of hydrolysate after vacuum drying; adding 8 g of thionyl chloride into 400ml of anhydrous methanol while cooling with ice water, stirring for 15 minutes, adding 4.80 g of the hydrolysate, stirring and refluxing the reaction solution for 8 hours, continuously stirring for 16 hours at room temperature, and drying with anhydrous sodium sulfate; after evaporation of the solvent, the product was chromatographed on a silica gel column, developed with methylene chloride-methanol (99:1), and the first fraction eluted first was collected. After the solvent is evaporated, recrystallizing the product by using toluene, and drying in vacuum to obtain a ligand microsphere energy agent precursor b;
preparing ligand microsphere energy agent precursor c
Adding 1.94 g of ligand microsphere energy agent precursor b (2.8mmol) into 120ml of ethanol, then adding 4 g of potassium hydroxide and 10ml of water, and stirring and refluxing the reaction solution for 3 hours; evaporating the solvent under reduced pressure, dissolving the resultant in 150ml of water, filtering to remove trace insoluble substances, and slowly dropping a trifluoroacetic acid aqueous solution into the stirred solution; fully washing and drying in vacuum to obtain a ligand microsphere energy agent precursor c;
preparing ligand microsphere energy agent
Putting the precursor c of ligand microsphere energy agent in a container P2O5After sufficiently drying in a vacuum drier, 181.7mg of the dried N, N-dimethylformamide was dissolved in 5ml of dry N, N-dimethylformamide, and 34.5mg of N-hydroxysuccinamide (NHS) and 61.9mg of N, N' -Dicyclohexylcarbodiimide (DCC) were added with stirring, and after stirring at room temperature for 24 hours, insoluble matter was removed by filtration; and (3) after the filtrate is decompressed and concentrated to remove the solvent, washing a product with a small amount of isopropanol, and drying in vacuum to obtain the ligand microsphere energy agent.
4. The method of claim 2, wherein the ligand microsphere of S3 is prepared by: 4ml of 20% strength carboxylated microspheres (400mg) in 80nm, washed 2 times with 4ml of water +4ml of absolute ethanol, centrifuged at 30000rpm for 15 minutes and diluted with 3ml of aminoethanol in a 25ml round flaskReacting, and placing a magnetic stirrer in the bottle; heating the oil to 105 ℃, stirring for 10min, then adding 40mg of ligand microsphere energy agent solution for marking the avidin to be detected, and stirring for 5 min; then 1ml of 0.1M NaOH was added slowly over 5 minutes, during which the temperature of the oil solution was kept at 105 ℃ and then slowly lowered to room temperature over 2 hours; after cooling, the mixture was diluted to 20m with ethanol, centrifuged at 30000rpm for 30min to remove the supernatant, and the pellet was resuspended in ethanol by sonication. Centrifugation was repeated and the pellet was resuspended in water and the pellet was resuspended to a volume of 40ml in 5ml of 10% aqueous ethanol. To this solution were added 20mg BSA and 25mg NaN3And then, freezing and storing at the temperature of minus 20 ℃ to prepare the ligand microsphere.
5. Use of the photosensitive detection system of claim 1 to make an anti-cardiolipin antibody IgA assay reagent.
6. Use of a photosensitive detection system according to claim 5, comprising labeling streptavidin with photosensitive microspheres, detection of anti-cardiolipin antigens with ligand microsphere labels and preparation of murine anti-human cardiolipin antigens.
7. The use of the photosensitive detection system according to claim 6, wherein the labeling of streptavidin with photosensitive microspheres comprises the steps of:
step one, photosensitive microsphere suspension treatment: absorbing a certain amount of photosensitive microspheres in a high-speed refrigerated centrifuge for centrifugation, removing supernatant, adding a certain amount of MES buffer solution, resuspending on an ultrasonic cell disruption instrument, and adding the MES buffer solution to adjust the concentration of the photosensitive microspheres to 200 mg/ml;
step two, preparing a streptavidin solution: weighing a certain amount of streptavidin, and dissolving the streptavidin into 10mg/ml MES buffer solution;
step three, mixing: mixing the treated photosensitive microsphere suspension, 8mg/ml streptavidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution;
step four, reaction: MES buffer solution is prepared into 25mg/ml NHS solution, added according to the volume ratio of 1:25 to the reaction solution, and rapidly mixed evenly. Rotating and reacting at 37 ℃ for 24 hours;
step five, sealing: preparing 75mg/ml ethanolamine solution and 250mg/ml casein solution by MES buffer solution, quickly and uniformly mixing the solution and the reaction solution according to the volume ratio of 5:10, and reacting for 12 hours at 37 ℃;
step six, cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, washing for three times, finally suspending by a small amount of photosensitive reagent buffer solution, measuring the solid content, and adjusting the concentration to 10mg/ml by using photosensitive microsphere reagent buffer solution.
8. The use of a photosensitive detection system according to claim 2, 3 or 6, wherein the method of labeling avidin of the analyte with the ligand microsphere energy agent in S3 is
Dissolving 5mg of Streptavidin (SA) in 10ml of 0.1mol/L sodium bicarbonate buffer solution with pH value of 9.1, adding 10mg of ligand microsphere energy agent, stirring at room temperature for reaction for 3 hours, and reacting the solution at 4 ℃ with a solution containing 0.25 g of NaN3Dialyzing the solution with 0.1mol/L sodium bicarbonate solution for three times, each for 24 hours, removing unreacted marker substance, and adding EuCL to the solution3Obtaining labeled streptavidin solution; wherein EuCL3The mass ratio of the ligand microsphere energy agent to the ligand microsphere energy agent is 1.5: 2.
9. The use of a photosensitive detection system according to claim 6, wherein the method for detecting anticardiolipin antigen using ligand microsphere labeling comprises:
step one, suspension treatment of ligand microspheres: centrifuging a certain amount of ligand microspheres in a high-speed refrigerated centrifuge, discarding supernatant, adding a timed MES buffer solution, ultrasonically crushing until the particles are resuspended, and adding the MES buffer solution to adjust the concentration of the particles to 100 mg/ml;
step two, antibody treatment: dialyzing the antigen with 0.05M MES buffer solution with pH of 6.0, determining the concentration after dialysis, and adjusting the concentration to 8 mg/ml;
step three, reaction: MES buffer solution is prepared into EDAC solution of 40mg/ml, added according to the proportion of 100mg/ul of microspheres and EDAC, and rapidly reacted and uniformly mixed for 16 hours;
step four, sealing: adding 200mg/ml casein solution (MES buffer solution) with the volume ratio of 10:8 to the reaction solution, rapidly mixing, and performing rotary reaction at 37 deg.C for 16 hr;
step five, cleaning: and (4) centrifugally washing the mixture for four times by using MES buffer solution, finally suspending the mixture by using reagent buffer solution, measuring the particle size and the solid content, and adjusting the concentration to 10 mg/ml.
10. The use of a photosensitive detection system according to claim 6, wherein the murine anti-human cardiolipin antigen is prepared by:
step one, absorbing a certain amount of mouse anti-human cardiolipin antigen, dialyzing at the temperature of 2-8 ℃ at 0.1M and pH of 8.5NaHCO3A solution; sampling the dialyzed mouse anti-human cardiolipin antigen to determine the antigen concentration; 10mg/ml Biotin solution was prepared in DMSO.
Step two, marking: according to the requirement of n/10 × 2ul (n is the labeling ratio of 20) of Biotin (DMSO) for every 1mg of antigen, the mixture is rapidly mixed, and the mixture is rotated and mixed at the temperature of 2-8 ℃ for reaction overnight.
Step three, dialysis: and dialyzing the reacted biotin-labeled antigen in a biotin-labeled dialysis buffer solution at the temperature of 2-8 ℃.
Step four, volume fixing: and sucking the dialyzed protein out and transferring the dialyzed protein into a container, sampling and determining the protein concentration, and obtaining the test result to obtain the protein concentration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010578325.3A CN111912977B (en) | 2020-06-23 | 2020-06-23 | Photosensitive detection system and manufacturing method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010578325.3A CN111912977B (en) | 2020-06-23 | 2020-06-23 | Photosensitive detection system and manufacturing method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111912977A true CN111912977A (en) | 2020-11-10 |
| CN111912977B CN111912977B (en) | 2023-09-12 |
Family
ID=73226484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010578325.3A Active CN111912977B (en) | 2020-06-23 | 2020-06-23 | Photosensitive detection system and manufacturing method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111912977B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114152742A (en) * | 2021-11-30 | 2022-03-08 | 深圳市易瑞生物技术股份有限公司 | Kit for light-activated chemiluminescence immunoassay containing magnetic luminescent microspheres and application thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5359056A (en) * | 1991-07-15 | 1994-10-25 | Nippon Shokubai Co., Ltd. | Phthalocyanine compounds, production method thereof, and near infrared ray absorption materials containing the same |
| WO1996029367A1 (en) * | 1995-03-23 | 1996-09-26 | Biosite Diagnostics Incorporated | Hybrid phthalocyanine derivatives and their uses |
| US5763189A (en) * | 1993-09-24 | 1998-06-09 | Biosite Diagnostics Incorporated | Fluorescence energy transfer and intramolecular energy transfer in particles using novel compounds |
| US5889181A (en) * | 1994-08-25 | 1999-03-30 | University Of Sherbrooke | Process for the syntheses of monosulphonated phthalocyanines, naphthobenzoporphyrazines and porphyrins via a modified meerwein reaction |
| JP2000095746A (en) * | 1998-09-24 | 2000-04-04 | Nippon Shokubai Co Ltd | Production of substituent-containing phthalonitrile or phthalocyanine compound |
| JP2009067848A (en) * | 2007-09-11 | 2009-04-02 | Nippon Shokubai Co Ltd | Aryl group-containing phthalocyanine compound and method for producing the same |
| US20120283430A1 (en) * | 2010-11-01 | 2012-11-08 | New Jersey Institute Of Technology | System and Method for Fluoroalkylated Fluorophthalocyanines With Aggregating Properties and Catalytic Driven Pathway for Oxidizing Thiols |
| US20190337958A1 (en) * | 2016-11-17 | 2019-11-07 | Tampereen Korkeakoulusäätiö SR | Photosensitizer |
| CN110736737A (en) * | 2018-07-18 | 2020-01-31 | 博阳生物科技（上海）有限公司 | microsphere composition for chemiluminescence detection and application thereof |
| WO2020034938A1 (en) * | 2018-08-13 | 2020-02-20 | 博阳生物科技（上海）有限公司 | Microsphere composition for chemiluminescence analysis and use thereof |
-
2020
- 2020-06-23 CN CN202010578325.3A patent/CN111912977B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5359056A (en) * | 1991-07-15 | 1994-10-25 | Nippon Shokubai Co., Ltd. | Phthalocyanine compounds, production method thereof, and near infrared ray absorption materials containing the same |
| US5763189A (en) * | 1993-09-24 | 1998-06-09 | Biosite Diagnostics Incorporated | Fluorescence energy transfer and intramolecular energy transfer in particles using novel compounds |
| US5889181A (en) * | 1994-08-25 | 1999-03-30 | University Of Sherbrooke | Process for the syntheses of monosulphonated phthalocyanines, naphthobenzoporphyrazines and porphyrins via a modified meerwein reaction |
| WO1996029367A1 (en) * | 1995-03-23 | 1996-09-26 | Biosite Diagnostics Incorporated | Hybrid phthalocyanine derivatives and their uses |
| JP2000095746A (en) * | 1998-09-24 | 2000-04-04 | Nippon Shokubai Co Ltd | Production of substituent-containing phthalonitrile or phthalocyanine compound |
| JP2009067848A (en) * | 2007-09-11 | 2009-04-02 | Nippon Shokubai Co Ltd | Aryl group-containing phthalocyanine compound and method for producing the same |
| US20120283430A1 (en) * | 2010-11-01 | 2012-11-08 | New Jersey Institute Of Technology | System and Method for Fluoroalkylated Fluorophthalocyanines With Aggregating Properties and Catalytic Driven Pathway for Oxidizing Thiols |
| US20190337958A1 (en) * | 2016-11-17 | 2019-11-07 | Tampereen Korkeakoulusäätiö SR | Photosensitizer |
| CN110736737A (en) * | 2018-07-18 | 2020-01-31 | 博阳生物科技（上海）有限公司 | microsphere composition for chemiluminescence detection and application thereof |
| WO2020034938A1 (en) * | 2018-08-13 | 2020-02-20 | 博阳生物科技（上海）有限公司 | Microsphere composition for chemiluminescence analysis and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| MEI-JIN LIN ET AL: "A convenient synthesis of a substituted phthalocyanine compound", vol. 59, no. 59, pages 607 * |
| 赵建社;郭荣荣;: "亚酞菁及其衍生物合成的研究进展" * |
| 赵建社;郭荣荣;: "亚酞菁及其衍生物合成的研究进展", 徐州工程学院学报(自然科学版), no. 01, pages 1 - 9 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114152742A (en) * | 2021-11-30 | 2022-03-08 | 深圳市易瑞生物技术股份有限公司 | Kit for light-activated chemiluminescence immunoassay containing magnetic luminescent microspheres and application thereof |
| CN114152742B (en) * | 2021-11-30 | 2024-05-28 | 深圳市易瑞生物技术股份有限公司 | Kit for photoexcitation chemiluminescence immunoassay containing magnetic luminescence microspheres and application of kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111912977B (en) | 2023-09-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1019722B1 (en) | Polymeric fluorophores enhanced by moieties providing a hydrophobic and conformationally restrictive microenvironment | |
| CN116539597A (en) | cTnI homogeneous phase chemiluminescence detection kit, detection method and device | |
| CN116413255A (en) | Homogeneous chemiluminescence detection kit and application thereof | |
| CN116337851A (en) | Homogeneous chemiluminescence detection kit and application thereof | |
| CN116577319A (en) | Microsphere composition for chemiluminescence detection and application thereof | |
| CN110736736B (en) | Homogeneous phase chemiluminescence POCT detection method and device using same | |
| CN114113582B (en) | Metal organic framework nanoenzyme biological probe and ELISA kit | |
| CN111912977B (en) | Photosensitive detection system and manufacturing method and application thereof | |
| CN111638363B (en) | 3-deoxyfructose rapid quantitative fluorescence detection device and preparation method thereof | |
| CN112014383B (en) | Photosensitizer for homogeneous chemiluminescence and preparation method and application thereof | |
| CN116559151A (en) | Microsphere composition for chemiluminescence detection and application thereof | |
| CN110274898B (en) | His@ZIF-8/Tb3+Method for preparing composite material | |
| CN116482083A (en) | Homogeneous phase chemiluminescence POCT detection method and device using same | |
| CN108760695B (en) | Method for quantitatively detecting thrombin by using phosphorescence probe based on PRET | |
| CN113125711B (en) | Acceptor reagent and application thereof | |
| CN114778828A (en) | Based on Co3O4SPR immunosensor of magnetic nanoparticle signal amplification probe | |
| CN101377499A (en) | Nerve specificity olefinic alcohol enzyme chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| CN113125705B (en) | Myoglobin homogeneous detection kit and application thereof | |
| CN113652391B (en) | Exosome capturing and analyzing integrated method based on boric acid directional coupling immunoaffinity | |
| CN113125703B (en) | Myoglobin homogeneous detection kit and application thereof | |
| CN113125700B (en) | Homogeneous detection kit for creatine kinase isozymes and application thereof | |
| CN113125721B (en) | Homogeneous detection kit for creatine kinase isozymes and application thereof | |
| CN107202893A (en) | A kind of small molecule chip, its construction method, its application and its detection method | |
| CN116559471A (en) | Procalcitonin homogeneous chemiluminescence detection kit, detection method and device | |
| CN113125730B (en) | Homogeneous detection kit for interleukin 6 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 |