CN116376535A - Luminous composition and preparation method and application thereof - Google Patents
Luminous composition and preparation method and application thereof Download PDFInfo
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- CN116376535A CN116376535A CN202211724052.4A CN202211724052A CN116376535A CN 116376535 A CN116376535 A CN 116376535A CN 202211724052 A CN202211724052 A CN 202211724052A CN 116376535 A CN116376535 A CN 116376535A
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 82
- 229960002685 biotin Drugs 0.000 claims description 64
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 4
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- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
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- SADPCIINLASURY-UHFFFAOYSA-N 2-morpholin-4-ylethanesulfonic acid;sodium Chemical compound [Na].OS(=O)(=O)CCN1CCOCC1 SADPCIINLASURY-UHFFFAOYSA-N 0.000 description 1
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- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical group C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 1
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
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- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
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- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
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- 238000004020 luminiscence type Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Urology & Nephrology (AREA)
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- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Cell Biology (AREA)
- Optics & Photonics (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a luminous composition, a preparation method and application thereof. The luminescent composition comprises luminescent particles and PEG coupled to the luminescent particles; the luminescent particles are capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal; the PEG is at least one selected from PEG 2000-PEG 4000. The luminous composition can prevent nonspecific adsorption on the surface of the luminous composition and reduce the background. The light-activated chemiluminescence detection reagent containing the luminous composition is beneficial to identifying an object to be detected and improves signal value and reagent sensitivity.
Description
The invention claims a light-emitting particle modified by PEG, a preparation method and application of the light-emitting particle modified by PEG, and priority of the patent, wherein the application number is CN202111671509.5, the application date is 2021, and the application date is 12, 31.
Technical Field
The invention belongs to the field of photo-excitation chemiluminescence detection, and particularly relates to a luminous composition, a preparation method and application thereof.
Background
The photoexcitation chemiluminescence method is one of the common methods of chemiluminescence analysis technology, and can be used for researching the interaction between biomolecules. The technology integrates the research of the related fields of polymer particle technology, organic synthesis, protein chemistry, clinical detection and the like. The method combines the photosensitive particles and the luminous composition in a certain range under the action of biotin and streptavidin to generate the transfer of ionic oxygen energy and emit optical signals, so that the sample to be detected is detected. Compared with the traditional ELISA method, the method has the characteristics of homogeneous phase, high sensitivity, simple operation, easy automation and the like. Therefore, the application prospect is very broad.
In the prior art, the detection reagent for detecting the sample to be detected by the photo-excitation chemiluminescence method has the defects of low sensitivity and the like. Therefore, it is required to provide a detection reagent for photoexcitation chemiluminescent detection which can improve the detection sensitivity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a luminous composition, which can reduce non-specific adsorption on the surface of the luminous composition and remarkably improve the sensitivity of a detection reagent when being applied to light-activated chemiluminescence detection.
To this end, a first aspect of the present invention provides a luminescent composition comprising luminescent particles and PEG coupled to the luminescent particles; the luminescent particles are capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal; the PEG is at least one selected from PEG 2000-PEG 4000.
In some embodiments of the invention, the luminescent particles are selected from at least one of carboxyl-modified luminescent particles, amine-modified luminescent particles, aldehyde-modified luminescent particles, and thiol-modified luminescent particles; preferably carboxyl-modified luminescent particles.
In some embodiments of the invention, the luminescent microparticles are coupled to PEG via an amide bond.
In some embodiments of the invention, the luminescent composition is coupled to biotin.
In some embodiments of the invention, the luminescent composition is coupled to biotin via an amide bond.
In a second aspect, the present invention provides a method of preparing a light-emitting composition according to the first aspect of the invention, comprising: the luminescent particles are mixed with PEG reagent and then reacted to obtain the luminescent composition.
In some embodiments of the invention, the method comprises the steps of:
s1, activating the luminescent particles to obtain activated luminescent particles;
s2, mixing the activated luminescent particles with a PEG reagent with an amino group, and reacting to obtain the luminescent composition.
In some embodiments of the invention, the molar ratio of functional groups on the surface of the activated luminescent particles to amine groups in the PEG reagent is 1 (10-15).
In some embodiments of the invention, the functional groups on the surface of the activated luminescent particles are selected from at least one of carboxyl groups, aldehyde groups, amine groups, and thiol groups; preferably a carboxyl group.
In some embodiments of the invention, the amine-bearing PEG reagents include, but are not limited to, linear bifunctional PEG reagents and multi-arm bifunctional PEG reagents.
In some embodiments of the invention, the linear bifunctional PEG reagent is selected from NH 2 -PEG-COOH、NH 2 -PEG-OH、NH 2 -PEG-Male、NH 2 -PEG-N 3 Any one of the following.
In other embodiments of the present invention, the multi-arm bifunctional PEG reagent is selected from the group consisting of
And
In some embodiments of the invention, the PEG reagent bearing an amine group is NH 2 -PEG-COOH。
In some embodiments of the invention, the method further comprises the steps of: s3, mixing the luminous composition with a biotin reagent and then reacting.
In some embodiments of the invention, the biotin reagent is NH 2 -PEG-biotin reagent, preferably NH 2 PEG2000-biotin reagent, NH 2 -PEG4000-biotin reagent.
In some embodiments of the invention, the reaction conditions in step S2 and step S3 are: the temperature is 35-40 ℃, the time is 2-4 hours, and the rotating speed is 10-50 rpm.
In a third aspect the present invention provides a photoexcited chemiluminescent detection reagent comprising a luminescent composition according to the first aspect of the present invention or prepared by a method according to the second aspect.
In some embodiments of the invention, the detection reagent further comprises a bioactive molecule coupled to the luminescent composition, the bioactive molecule being capable of specifically binding to the analyte.
In some embodiments of the invention, the biologically active molecule is an antibody or antigen.
In a fourth aspect, the present invention provides a photo-activated chemiluminescent detection kit comprising a photo-activated chemiluminescent detection reagent according to the third aspect of the present invention.
In some embodiments of the invention, the kit further comprises the following components:
a biotin-labeled further biologically active molecule capable of specifically binding to the analyte to be detected; and/or the number of the groups of groups,
avidin coated photosensitive particles capable of generating active oxygen in an excited state.
In some embodiments of the invention, the kit specifically comprises:
component a1 comprising a biotin-labeled luminescent composition coupled to a bioactive molecule, said bioactive molecule being capable of specifically binding to an analyte;
component b1 comprising a biotin-labeled further biologically active molecule, which further biologically active molecule is capable of specifically binding to the object to be detected;
component c1 comprising avidin-coated photosensitive particles capable of generating active oxygen in an excited state.
In some embodiments of the invention, the photosensitive particles are polymer particles coated on a substrate by a functional group to form a filled photosensitive compound.
In some embodiments of the invention, the active oxygen is singlet oxygen.
In some embodiments of the invention, the biologically active molecule is an antibody or antigen.
In a fifth aspect, the present invention provides a luminescent composition according to the first aspect or a luminescent composition according to the second aspect or a detection reagent according to the third aspect or a detection kit according to the fourth aspect for use in photo-activated chemiluminescence detection.
The beneficial effects of the invention are as follows: the luminous composition is coupled with PEG with stronger hydrophilicity on the surface of luminous particles, so that a hydration layer is formed on the surface of the luminous composition, the nonspecific adsorption of the surface of the luminous composition is prevented, and the background is reduced. The detection reagent containing the luminous composition reduces the steric hindrance effect combined with the object to be detected (such as antigen to be detected or antibody to be detected) because the linear flexible chain of PEG leads bioactive molecules (such as antibody or antigen) to be far away from the surface of the particles, thereby being beneficial to the identification of the object to be detected and improving the signal value and the sensitivity of the reagent.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
The luminescent composition according to the first aspect of the present invention comprises luminescent particles and PEG coupled to the luminescent particles; the luminescent particles are capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal; the PEG is at least one selected from PEG 2000-PEG 4000.
In the present invention, the term "PEG" is polyethylene glycol (Polyethylene glycol), the chemical formula is HO (CH) 2 CH 2 O) n H, has no irritation, slightly bitter taste, good water solubility and good compatibility with a plurality of organic components.
In the present invention, the term "luminescent particles" is polymer particles filled with a luminescent compound and a lanthanoid element, which can be formed on a substrate by coating functional groups. It undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose with or subsequent to luminescence.
In the present invention, the "luminescent composition" includes luminescent particles and PEG coupled to the luminescent particles. The luminescent particles are capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal.
In some embodiments of the invention, the PEG may be PEG2000, PEG4000, or the like.
In some embodiments of the invention, the luminescent particles are selected from at least one of carboxyl-modified luminescent particles, amine-modified luminescent particles, aldehyde-modified luminescent particles, and thiol-modified luminescent particles; preferably carboxyl-modified luminescent particles.
In some embodiments of the invention, the luminescent microparticles are coupled to PEG via an amide bond.
In some embodiments of the invention, the luminescent composition is coupled to biotin.
In some embodiments of the invention, the luminescent composition is coupled to biotin via an amide bond.
A second aspect of the invention relates to a method for preparing a light-emitting composition according to the first aspect of the invention, comprising: the luminescent particles are mixed with PEG reagent and then reacted to obtain the luminescent composition.
In some embodiments of the invention, the method comprises the steps of:
s1, activating the luminescent particles to obtain activated luminescent particles;
s2, mixing the activated luminescent particles with a PEG reagent with an amino group, and reacting to obtain the luminescent composition.
In some embodiments of the invention, the luminescent particles are activated by EDC/NHS activation.
In some embodiments of the invention, the molar ratio of functional groups on the surface of the activated luminescent particles to amine groups in the PEG reagent is 1 (10-15).
In some embodiments of the present invention, the molar ratio of carboxyl groups on the surface of the activated luminescent particles to amine groups in the PEG reagent may be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc. In some preferred embodiments of the invention, the molar ratio of carboxyl groups on the surface of the activated luminescent particles to amine groups in the PEG reagent is 1:15.
In some embodiments of the invention, the functional groups on the surface of the activated luminescent particles are selected from at least one of carboxyl groups, aldehyde groups, amine groups, and thiol groups; preferably a carboxyl group.
In some embodiments of the invention, the amine-bearing PEG reagents include, but are not limited to, linear bifunctional PEG reagents and multi-arm bifunctional PEG reagents.
In some embodiments of the invention, the linear bifunctional PEG reagent is selected from NH 2 -PEG-COOH、NH 2 -PEG-OH、NH 2 -PEG-Male and NH 2 -PEG-N 3 Any one of the following.
In some embodiments of the invention, the multi-arm bifunctional PEG reagent is selected from the group consisting of
And
In some embodiments of the invention, the PEG reagent bearing an amine group is NH 2 -PEG-COOH(amide-PEG-acid)。
In the present invention, the term "amine group" is an organic compound in which the hydrogen atom of ammonia is replaced with an alkyl group.
In some embodiments of the invention, the method further comprises the steps of: s3, mixing the luminous composition with a biotin reagent and then reacting.
In some embodiments of the invention, the biotin reagent is NH 2 -PEG-biotin reagent, preferably NH 2 PEG2000-biotin reagent, NH 2 -PEG4000-biotin reagent.
In some embodiments of the invention, the reaction conditions in step S2 and step S3 are: the temperature is 35-40 ℃, the time is 2-4 hours, and the rotating speed is 10-50 rpm.
In some preferred embodiments of the present invention, the reaction conditions in step S2 and step S3 are: the temperature was 37℃for 4 hours and the rotational speed was 30rpm.
A third aspect of the invention relates to a photo-activated chemiluminescent detection reagent comprising a luminescent composition according to the first aspect of the invention or prepared by a method according to the second aspect.
In some embodiments of the invention, the detection reagent further comprises a bioactive molecule coupled to the luminescent composition, the bioactive molecule being capable of specifically binding to the analyte.
In some embodiments of the invention, the biologically active molecule is an antibody or antigen. When the bioactive molecule is an antibody, the corresponding object to be detected is an antigen; when the bioactive molecule is an antigen, the corresponding analyte is an antibody. In the present invention, the term "antibody" is used in its broadest sense to include antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
In the present invention, the term "antigen" (Ag) refers to a substance that stimulates the body to produce an immune response and binds to antibodies and sensitized lymphocytes, which are immune response products, in vivo and in vitro, and generates an immune effect. The antigenic substance has two important properties: immunogenicity (immunogenicity) and immunoreactivity (immunoreactivity). Immunogenicity refers to the ability of an antigen to induce a specific immune response in the body to produce antibodies and/or sensitized lymphocytes; immunoreactivity refers to the ability to specifically bind to a corresponding immune effector substance (antibody or sensitized lymphocyte) in vivo or in vitro.
In the present invention, the term "specific binding" refers to the mutual recognition and selective binding reaction between two substances, namely the conformational correspondence between the corresponding reactants from a steric point of view.
The fourth aspect of the invention relates to a photo-activated chemiluminescent detection kit comprising a photo-activated chemiluminescent detection reagent according to the third aspect of the invention.
In some embodiments of the invention, the kit further comprises the following components:
a biotin-labeled further biologically active molecule capable of specifically binding to the analyte to be detected; and/or the number of the groups of groups,
avidin coated photosensitive particles capable of generating active oxygen in an excited state.
In some embodiments of the invention, the kit specifically comprises:
component a1 comprising a biotin-labeled luminescent composition coupled to a bioactive molecule, said bioactive molecule being capable of specifically binding to an analyte;
component b1 comprising a biotin-labeled further biologically active molecule, which further biologically active molecule is capable of specifically binding to the object to be detected;
component c1 comprising avidin-coated photosensitive particles capable of generating active oxygen in an excited state.
In the present invention, the term "photosensitive particles" is polymer particles filled with a photosensitive compound formed by coating a functional group on a substrate, and capable of generating active oxygen (e.g., singlet oxygen) under light excitation.
In the present invention, the term "active oxygen" refers to a general term for substances which are contained in the body or in the natural environment and are active in nature and which are composed of oxygen, mainly oxygen molecules in an excited state, including superoxide anions (O2— -) which are the product of one electron reduction of oxygen, hydrogen peroxide (H) 2 O 2 ) Hydroxyl radical (OH) of three-electron reduction product, nitric oxide and singlet oxygen (1O) 2 ) Etc.
In the invention, "Biotin" (Biotin), which is widely present in animal and plant tissues, has two cyclic structures on its molecule, namely an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main site for binding with streptavidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by protein cross-linking agents. The "avidin" molecule consists of 4 identical peptide chains, each of which is capable of binding one biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity.
The fifth aspect of the present invention relates to a luminescent composition according to the first aspect of the present invention or a luminescent composition according to the second aspect of the present invention or a detection reagent according to the third aspect or a kit according to the fourth aspect of the present invention for use in photo-activated chemiluminescence detection.
Examples
In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.
The experimental materials and experimental equipment used in examples 1-4 below are shown in tables 1 and 2, respectively.
Table 1: experimental raw materials
| Carboxyl group-modified luminescent particles | NH 2 -PEG16-COOH |
| NH 2 -PEG2000-COOH | NH 2 -PEG4000-COOH |
| EDC | NHS |
| NH 2 -PEG-Biotin | Gly |
| BSA | Luminous reagent cleaning liquid |
| Luminescent reagent buffer solution | Universal liquid |
| MES | Sodium chloride |
| Universal liquid reagent |
Table 2: experimental equipment
| Refrigerated centrifuge | LCD numerical control rotary mixing instrument |
| Ultrasonic cleaner | Electronic balance |
| Vortex oscillator | LiCA HT |
| Electric heating constant temperature incubator |
Example 1: preparation of PEG-modified carboxyluminescent compositions of different chain lengths
S1: taking a 2ml centrifuge tube, adding 20mg of CML-FG, supplementing 0.05M MES (0.05M NaCl) buffer to 2.0g, centrifuging at 15000rpm for 20min once, discarding the supernatant, adding 200 μl of MES (0.05M NaCl) buffer, and performing ultrasonic dispersion uniformly to obtain 25 μg/ml of particles;
s2: adding 100 μl of NHS (sodium chloride) with the concentration of 10mg/ml into the particle suspension after ultrasonic treatment, rapidly adding 100 μl of EDC with the concentration of 10mg/ml after vortex mixing, adding 0.05M MES (0.05M NaCl) after vortex mixing, and reacting for 30min at room temperature and vertical rotation at 30 rpm;
s3: adding purified water into a centrifuge tube to 2.0g, centrifuging at 15000rpm for 20 minutes, discarding the supernatant, and adding 200 μl of 0.02M HEPES buffer solution for ultrasonic dispersion uniformly;
s4: adding NH with different chain lengths into the activated particles respectively according to the mol ratio of carboxyl in the particles to amine in the PEG reagent of 1:15 2 -PEG-COOH reagent (NH 2 -PEG16-COOH、NH 2 -PEG2000-COOH and NH 2 PEG 4000-COOH), then 0.02M HEPES buffer was added to the mixture to a concentration of 20mg/ml of the microparticles in the system, and the mixture was subjected to a reaction at 37℃for 4 hours with a vertical rotation at 30 rpm;
s5: and (3) centrifugally washing the luminous reagent cleaning solution for 2 times, and fixing the volume of the luminous reagent buffer solution to the concentration of 10mg/ml to obtain PEG modified carboxyl luminous compositions with different chain lengths, namely PEG16-FG, PEG2000-FG and PEG4000-FG.
Example 2: coupling of PEG-modified carboxyluminescent compositions with Biotin Agents
S1: taking 3 centrifuge tubes of 2ml, respectively adding 5mg of PEG16-FG, PEG2000-FG and PEG4000-FG luminous compositions, supplementing 0.05M MES (0.05M NaCl) buffer solution to 2.0g, centrifuging at 15000rpm for 20min once, discarding the supernatant, and adding 200ul of MES (0.05M NaCl) buffer solution for ultrasonic dispersion uniformly;
s2: adding 25 μl of NHS (polyethylene glycol) with the concentration of 10mg/ml into the microsphere suspension after ultrasonic treatment, rapidly adding 25 μl of EDC with the concentration of 10mg/ml into the microsphere suspension after vortex mixing, and vertically rotating at room temperature for 30rpm for reaction for 30min;
s3: adding purified water into a centrifuge tube to 2.0g, centrifuging at 15000rpm for 20 minutes, discarding the supernatant, and adding 200 μl of 0.02M HEPES buffer solution for ultrasonic dispersion uniformly;
s4: adding NH into the activated microsphere according to the mass ratio of the luminous composition to biotin of 10:0.5 2 -PEG16-Biotin、NH 2 -PEG2000-Biotin、NH 2 PEG4000-Biotin reagent, adding 0.02M HEPES buffer solution to the microsphere concentration of 20mg/ml in the system, and vertically rotating at 37 ℃ for 30rpm for reaction for 4 hours;
s5: gly (25. Mu.l) and (150 mg/ml) were added to the reaction system, and after vortexing, BSA (25. Mu.l) and (200 mg/ml) were added thereto, and after vortexing, the mixture was blocked at room temperature overnight.
S6: centrifuging and washing the luminous reagent cleaning solution for 4 times, and fixing the volume of the luminous reagent buffer solution to the concentration of 10mg/ml of microspheres to obtain a PEG modified carboxyl luminous composition coupled with a biotin reagent; FG-PEG16-Biotin, FG-PEG2000-Biotin and FG-PEG4000-Biotin, respectively.
Example 3: carboxyl luminescent particles coupled with biotin reagent
The procedure was essentially as in example 2, except that the carboxyl luminescent particles of unmodified PEG were added to the centrifuge tube in step S1. Finally obtaining the carboxyl luminescent particles coupled with Biotin reagent, namely FG-Biotin.
Example 4
The performance of the PEG-modified carboxyluminescent composition coupled to a biotin reagent prepared in example 2 and the carboxyluminescent microparticles coupled to a biotin reagent prepared in example 3 were tested on LiCA HT, respectively.
The kit provided in this example, comprising the PEG-modified carboxyluminescent composition coupled to a biotin reagent prepared in example 2, comprises:
reagent 1: a PEG-modified carboxyluminescent composition coupled to a biotin reagent;
reagent 2: avidin coated photosensitive microparticles.
The kit provided in this example, which includes the carboxyl luminescent microparticles coupled with a biotin reagent prepared in comparative example 3, comprises:
reagent 1: carboxyl luminescent particles coupled to a biotin reagent;
reagent 2: avidin coated photosensitive microparticles.
The specific kit components and test modes are as follows:
reagent 1: FG-PEG16-Biotin, FG-PEG2000-Biotin, FG-PEG4000-Biotin or FG-Biotin; (30. Mu.g/ml) 75. Mu.l;
reagent 2: 175. Mu.l of the universal liquid reagent (avidin-coated photosensitive microparticles).
Incubation at 37℃for 10min; liCA HT readings.
The test results are shown in Table 3.
TABLE 3 Table 3
| Reagent 1 | Signal value |
| FG-Biotin | 562174 |
| FG-PEG16-Biotin | 7806995 |
| FG-PEG2000-Biotin | 11373596 |
| FG-PEG4000-Biotin | 17800048 |
From the results of Table 3, it is clear that the signal value of the directly biotin-coupled carboxyluminescent particles is lowest, the signal value of the PEG-modified carboxyluminescent composition coupled to biotin can be increased up to 32-fold, and the increase in signal value is positively correlated with the increase in PEG chain length.
Example 5: preparation of luminescent reagent and performance test in HBsAg project
1. Experimental protocol:
the experimental group kit comprises: reagent 1:263sAb antibody coated PEG2000-FG luminescent composition (FG-PEG 16-263 sAb); reagent 2: biotin-labeled 04sAb antibody.
The control group kit comprises: reagent 1:263sAb antibody coated CML-FG luminescent microparticles (FG-263 sAb); reagent 2: biotin-labeled 04sAb antibody.
2. Experimental raw materials: as shown in table 4.
TABLE 4 Table 4
3. Experimental facilities: as shown in table 5.
TABLE 5
| Refrigerated centrifuge | LCD numerical control rotary mixing instrument |
| Ultrasonic cleaner | Electronic balance |
| Vortex oscillator | LiCA HT |
4. The experimental steps are as follows:
s1: taking 140 mu l of 263sAb, adding 140 mu l of PBS, uniformly mixing, washing a desalting column twice with PBS, 1500g, centrifuging for 1 time in 2min, collecting 230 mu l of liquid, and testing the concentration of the antibody by a BCA method;
s2: taking two 2ml centrifuge tubes, respectively adding 5mg of CML-FG luminescent particles and 5mg of PEG2000-FG luminescent composition, supplementing 0.05M MES (0.05M NaCl) buffer to 2.0g, centrifuging at 15000rpm for 20min once, discarding the supernatant, and adding 200 μl of MES (0.05M NaCl) buffer for ultrasonic dispersion uniformly;
s3: adding 25 μl of NHS (polyethylene glycol) with the concentration of 10mg/ml into the particle suspension after ultrasonic treatment, rapidly adding 25 μl of EDC with the concentration of 10mg/ml into the mixture after vortex mixing, and carrying out vortex mixing, wherein the final concentration of particles in the system is 20mg/ml, and carrying out vertical rotation at room temperature for 30rpm for reaction for 30min;
s4: adding purified water into a centrifuge tube to 2.0g, centrifuging at 15000rpm for 20min, discarding the supernatant, adding 200ul of purified water for ultrasonic re-dissolution, centrifuging once again, discarding the supernatant, and adding 0.02M PBS buffer solution for ultrasonic dispersion uniformly;
s5: adding 263sAb antibody into the activated particles according to the mass ratio of the particles to the antibody of 10:0.7, adding 0.02M PBS buffer solution to the concentration of 10mg/ml of the particles in the system, and vertically rotating at room temperature for 30rpm for reaction for 4 hours;
s6: gly (25. Mu.l) and (150 mg/ml) were added to the reaction system, and after vortexing, BSA (25. Mu.l) and (200 mg/ml) were added thereto, and after vortexing, the mixture was blocked at room temperature overnight.
S7: and (3) centrifugally washing the luminescent reagent cleaning solution for 4 times, and fixing the volume of the luminescent reagent buffer solution to the concentration of 10mg/ml to obtain the luminescent reagents in the HBsAg project, namely FG-PEG2000-263sAb and FG-263sAb.
5. Performance testing
The test pattern is as follows:
(1) 25ul of calibrator for HBsAg+25 ul of reagent 1 (70. Mu.g/ml) +25 ul of reagent 2;
(2) Incubation at 37℃for 15min;
(3) Add 175 μl of universal solution (avidin coated photosensitive microparticles);
(4) Incubation at 37℃for 10min;
(5) LiCA HT readings.
The test results are shown in Table 6.
TABLE 6
| Sequence number | Target concentration ng/ml | FG-263sAb | FG-PEG2000-263sAb |
| Cal1 | 0 | 821 | 766 |
| Cal2 | 0.2 | 1160 | 1763 |
| Cal3 | 4 | 2357 | 3601 |
| Cal4 | 30 | 18900 | 29754 |
| Cal5 | 150 | 169392 | 255386 |
| Cal6 | 530 | 387769 | 722783 |
As can be seen from Table 6, after PEG modification, the negative sample signal slightly decreased, the positive sample signal increased by about 50% compared with the control, the reagent discrimination increased, and the detection sensitivity increased.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Claims (12)
1. A luminescent composition comprising luminescent microparticles and PEG coupled to the luminescent microparticles; the luminescent particles are capable of reacting with reactive oxygen species to produce a detectable chemiluminescent signal; the PEG is at least one selected from PEG 2000-PEG 4000.
2. The light-emitting composition according to claim 1, wherein the light-emitting microparticles are at least one selected from the group consisting of carboxyl-modified light-emitting microparticles, amine-modified light-emitting microparticles, aldehyde-modified light-emitting microparticles and thiol-modified light-emitting microparticles; preferably carboxyl-modified luminescent particles; preferably, the luminescent microparticles are coupled to PEG via an amide bond.
3. The luminescent composition according to claim 1 or 2, wherein the luminescent composition is coupled to biotin; preferably, the luminescent composition is coupled to biotin via an amide bond.
4. A method of preparing the luminescent composition of any one of claims 1-3, comprising the steps of:
s1, activating the luminescent particles to obtain activated luminescent particles;
s2, mixing the activated luminescent particles with a PEG reagent with an amino group, and reacting to obtain the luminescent composition.
5. The method according to claim 4, wherein the molar ratio of the functional groups on the surface of the activated luminescent particles to amine groups in the PEG reagent is 1 (10-15); preferably, the functional group on the surface of the activated luminescent particles is at least one selected from carboxyl, aldehyde, amine and mercapto groups; preferably a carboxyl group.
6. The method of claim 4 or 5, wherein the PEG reagent with amine groups includes, but is not limited to, a linear bifunctional PEG reagent and a multi-arm bifunctional PEG reagent; preferably, the linear bifunctional PEG reagent is selected from NH 2 -PEG-COOH、NH 2 -PEG-OH、NH 2 -PEG-Male、NH 2 -PEG-N 3 Any one of them; and/or the multi-arm bifunctional PEG reagent is selected from the group consisting of
Any one of the following.
7. The method according to any one of claims 4-6, characterized in that the method further comprises the steps of: s3, mixing the luminous composition with a biotin reagent and then reacting; preferably, the biotin reagent is NH 2 -PEG-biotin reagent.
8. A photoexcited chemiluminescent detection reagent comprising a luminescent composition according to any one of claims 1-3 or prepared by a method according to any one of claims 4-7; preferably, the detection reagent further comprises a bioactive molecule coupled to the luminescent composition, the bioactive molecule being capable of specifically binding to the analyte to be detected.
9. A photo-activated chemiluminescent detection kit comprising the photo-activated chemiluminescent detection reagent of claim 8.
10. The kit of claim 9, further comprising the following components:
a biotin-labeled further biologically active molecule capable of specifically binding to the analyte to be detected; and/or the number of the groups of groups,
avidin coated photosensitive particles capable of generating active oxygen in an excited state.
11. Kit according to claim 8 or 9, characterized in that it comprises in particular:
component a1 comprising a biotin-labeled luminescent composition coupled to a bioactive molecule, said bioactive molecule being capable of specifically binding to an analyte;
component b1 comprising a biotin-labeled further biologically active molecule, which further biologically active molecule is capable of specifically binding to the object to be detected;
component c1 comprising avidin-coated photosensitive particles capable of generating active oxygen in an excited state.
12. Use of a luminescent composition according to any one of claims 1-3 or a luminescent composition prepared according to any one of claims 4-7 or a detection reagent according to claim 8 or a kit according to any one of claims 9-11 in a photo-activated chemiluminescent assay.
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| CN117647644B (en) * | 2024-01-29 | 2024-05-28 | 北京万泰德瑞诊断技术有限公司 | Blocking agent and application thereof in immunodetection |
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