CN116430033A - Photo-excitation chemiluminescence detection kit and application method thereof - Google Patents
Photo-excitation chemiluminescence detection kit and application method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The invention relates to the technical field of light excitation chemiluminescence, in particular to a light excitation chemiluminescence detection kit and a use method thereof. The photosensitive microsphere in the kit comprises a carrier and a photosensitive substance carried by the carrier, wherein the photosensitive quantity Ps of the photosensitive microsphere is between 1.34 and 16.28; light sensing amount ps=od λ1 /C 2 *10 3 Wherein: OD (optical density) λ1 The visible light region of 300-800 nm has a specific concentration of C 2 Absorbance value, lambda corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained after full wavelength scanning of the photosensitive microspheres 1 Is the wavelength corresponding to the maximum absorption peak; c (C) 2 The concentration of the photosensitive microsphere in the process of photo-excitation chemiluminescence detection can be improved, and the performance of the photo-excitation chemiluminescence detection kit and the clinical detection result can be improved.
Description
Technical Field
The invention relates to the technical field of light excitation chemiluminescence, in particular to a light excitation chemiluminescence detection kit and a use method thereof.
Background
Photo-activated chemiluminescence is a typical homogeneous immunoassay method, which is characterized by a "double sphere". The double-sphere is that the luminous system is composed of luminous reagent containing luminous microsphere and photosensitive reagent containing photosensitive microsphere, and the two microspheres have good suspension property in liquid phase. The liquid phase of the microsphere and the antigen or antibody meet completely meet the liquid dynamic characteristics. Immune complexes are formed in the liquid phase based on the two antigens or antibodies coated on the surfaces of the nano-microspheres, thereby drawing the two nano-microspheres closer. Under the excitation of light, singlet oxygen transfer occurs between the two nano-microspheres, so that high-energy red light is generated, and the photon number is converted into the target molecule concentration through a photon counter and mathematical fitting. When the sample to be detected does not contain target molecules, immune complexes cannot be formed between the two nano-microspheres, at the moment, the distance between the two nano-microspheres exceeds the singlet oxygen transmission range (200 nm), the singlet oxygen is rapidly quenched in a liquid phase, and no high-energy level red light signal is generated during detection. The method has the characteristics of rapidness, homogeneous phase (no flushing), high sensitivity and simple operation.
The photoexcitation chemiluminescence detection kit comprises a luminous reagent, a biotin reagent and a photosensitive reagent, wherein the photosensitive reagent is an important component of a photoexcitation chemiluminescence analysis system, singlet oxygen can be generated after the photoexcitation of external excitation light, the singlet oxygen transmits energy to luminous microspheres within 200nm from the photosensitive microspheres, and finally, a chemiluminescent signal can be generated, so that the detection of an unknown substance is realized. The final clinical application and chemiluminescence detection results of the photo-activated chemiluminescence detection kit product are affected by the selection of the concentration of the dye filled in the photosensitive microspheres in the photosensitive reagent, the efficiency and time of generating singlet oxygen in the liquid phase of the photosensitive microspheres, and the like. Currently, luminescent reagents and biotin reagents are mature, and the prior art lacks high-performance photosensitive reagents meeting clinical test requirements.
Therefore, the provision of the photo-excitation chemiluminescence detection kit has important practical significance.
Disclosure of Invention
In view of the above, the invention provides a light-activated chemiluminescence detection kit, which is based on the principle of light-activated chemiluminescence and is composed of a luminescent reagent, a biotin reagent and a photosensitive reagent by applying a light-activated chemiluminescence immunoassay method; the luminous reagent is antigen or antibody protein coated on luminous microspheres, the luminous reagent is biotin-marked antigen or antibody protein as a reagent 1, the biotin reagent is biotin-marked antigen or antibody protein as a reagent 2, the universal liquid is a photosensitive reagent, and the photosensitive reagent is SA coated on the photosensitive microspheres, so that the luminous reagent is combined with the biotin reagent, and most importantly, the photosensitive quantity is 1.34< PS <16.28; based on the photosensitive reagent with the photosensitive quantity of 1.34< PS <16.28, the performance and the clinical test result of the photo-excitation chemiluminescence detection kit can be improved.
In order to achieve the above object, the present invention provides the following technical solutions:
in a first aspect, the invention provides the use of a photosensitive microsphere in the preparation of a microsphere composition, a reagent composition, a kit, a detection system and/or a detection device for photo-activated chemiluminescence detection,
the photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, wherein the photosensitive quantity Ps of the photosensitive microsphere is between 1.34 and 16.28; the sensitization amount ps=od λ1 /C 2 *10 3 Wherein:
the OD is λ1 The visible light region of 300-800 nm has a specific concentration of C 2 The lambda is the absorbance value corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained after full wavelength scanning of the photosensitive microsphere 1 Is the wavelength corresponding to the maximum absorption peak; the C is 2 Is the concentration of photosensitive microsphere in light-activated chemiluminescence detection, C 2 In ug/ml.
In some embodiments of the present invention,
concentration of the photosensitive microsphere
Wherein k is the linear relationship of carrier concentration-absorbance curveA corresponding slope, b is the corresponding intercept in the linear relationship of the carrier concentration-absorbance curve; OD (optical density) λ2 Is photosensitive microsphere at wavelength lambda 2 The corresponding absorbance value is lower; the carrier concentration-absorbance curve is at wavelength lambda using multiple carriers of different concentrations 2 A curve obtained below; the wavelength lambda 2 The photosensitive microsphere and the carrier which have the same concentration have the same or similar absorbance values corresponding to the wavelength-absorbance curve.
In some embodiments of the invention, the C 2 Selected from 10ug/ml to 200ug/ml.
In some embodiments of the invention, the linear relationship of carrier concentration-absorbance curve is y=kx+b, wherein:
x is different concentrations of the carrier with preset particle size, y is absorbance value of the carrier at the corresponding concentration, k is slope, and b is intercept.
In some embodiments of the invention, the wavelength λ 2 Selected from OD Photosensitive microsphere /OD Carrier body A ratio of any one of the wavelength values within 0.85 to 1.15, and a wavelength lambda 2 Not equal to wavelength lambda 1 ；
Wherein OD Photosensitive microsphere And OD (optical density) Carrier body The absorbance values corresponding to the same wavelength values of the photosensitive microspheres and the carriers with the same concentration in the range of 300 nm-800 nm are respectively utilized.
In some embodiments of the invention, the wavelength λ 2 400nm to 600nm.
In some embodiments of the present invention, the photosensitive microsphere is a carrier filled with a photosensitive material, wherein the wavelength λ 1 The wavelength corresponding to the maximum absorption peak in a wavelength-absorbance curve obtained by scanning the photosensitive material in the full wavelength range of 300-800 nm in the visible light region of the photosensitive material.
In some embodiments of the invention, the wavelength λ 1 600nm to 700nm.
In some embodiments of the invention, the photosensitive microsphere is prepared according to a mass ratio of the carrier to the photosensitive substance of 10 (0.04-4).
In some embodiments of the invention, the carrier has a particle size of 190nm to 280nm.
In some embodiments of the invention, the surface of the photosensitive microsphere is not coated with a polysaccharide, and the surface of the photosensitive microsphere is connected with an avidin, wherein the avidin is selected from the group consisting of ovalbumin, egg yolk avidin, streptavidin, neutravidin and avidin-like molecules, preferably streptavidin.
In some embodiments of the invention, the photoexcitation chemiluminescence detects a chemiluminescent signal generated by a luminescent complex formed from a luminescent microsphere-immunocomplex-photosensitive microsphere;
the luminescent microsphere comprises a carrier and a luminescent substance carried by the carrier, wherein the luminescent substance can react with singlet oxygen to generate chemiluminescence;
The photosensitive microsphere can generate singlet oxygen under the excitation of light.
In a second aspect, the invention also provides a microsphere composition for photoexcitation chemiluminescence detection, including luminescent microspheres and the photosensitive microspheres.
In a third aspect, the present invention also provides a reagent combination for photoexcitation chemiluminescent detection, comprising a luminescent reagent and a photoactive reagent;
the photosensitive agent comprises a buffer solution and the photosensitive microspheres stored in the buffer solution;
the luminescent reagent comprises luminescent microspheres and substances capable of reacting with the to-be-detected substances.
In some embodiments of the invention, the sugar content in the buffer solution is 1 g.+ -. 0.2g per liter of volume.
In some embodiments of the invention, the reagent combination further comprises a labeling reagent comprising a label and the substance capable of reacting with the test substance;
the photosensitizing agent also includes a substance capable of binding to the label;
preferably, the substance capable of reacting with the analyte includes, but is not limited to: an antigen or antibody;
preferably, the markers include, but are not limited to: biotin;
preferably, the substance capable of binding to the label includes, but is not limited to: streptavidin.
In a fourth aspect, the invention also provides a kit for photo-activated chemiluminescent detection, comprising one or more of any of the following, and a substance capable of reacting with an analyte, acceptable adjuvant, adjuvant or carrier;
(I) The photosensitive microsphere; or (b)
(II) the microsphere composition; or (b)
(III) the reagent combination;
such carriers include, but are not limited to: reagent bottles, reagent cards, test strips or chips.
In some embodiments of the invention, the kit comprises a luminescent reagent, a labeling reagent, and a photosensitizing reagent;
the luminescent agents include, but are not limited to: antibody-coated luminescent particles;
such labeling agents include, but are not limited to: a biotin-labeled antibody;
the photosensitizing agent includes, but is not limited to: streptavidin coated photosensitive microsphere.
In a fifth aspect, the present invention also provides a system or apparatus for photoexcitation chemiluminescent detection, including one or more of any of the following, as well as acceptable modules or components;
(I) The photosensitive microsphere; or (b)
(II) the microsphere composition; or (b)
(III) the reagent combination; or (b)
(IV) the kit.
The beneficial effects of the invention include, but are not limited to:
In the technical scheme of the application, the absorbance value OD is determined λ1 And concentration C 2 After the value of (2), according to suctionPhotometric value OD λ1 And concentration C 2 The ratio of the ratio to the photosensitive microsphere is determined. When the value of the sensitization quantity Ps of the sensitization microsphere is between 1.34 and 16.28, the sensitization microsphere is applied to the sensitization reagent to carry out a mixed reaction with the luminous microsphere, so that the intensity of a chemiluminescent signal of the luminous microsphere can meet the requirements in photoexcitation chemiluminescence detection, the fluctuation of a detection result caused by the influence of other interference factors on the chemiluminescent signal is reduced, the detection result has consistency and repeatability in clinical application, and the detection result has more definite distinction and higher precision. The photosensitive microsphere provided by the application has the advantages that the performance standard for execution in the application of the photosensitive microsphere to the photo-excitation chemical detection is given through definite numerical limitation, the operability is definite, and the photosensitive microsphere is suitable for popularization of industry specifications.
Based on the research, the light excitation chemiluminescence detection kit provided by the invention is based on the light excitation chemiluminescence principle, and a kit formed by applying a light excitation chemiluminescence immunoassay method comprises a luminescence reagent, a biotin reagent and a photosensitive reagent; the luminous reagent is antigen or antibody protein coated on luminous microspheres, the luminous reagent is biotin-marked antigen or antibody protein as a reagent 1, the biotin reagent is biotin-marked antigen or antibody protein as a reagent 2, the universal liquid is a photosensitive reagent, and the photosensitive reagent is SA coated on the photosensitive microspheres, so that the luminous reagent is combined with the biotin reagent, and most importantly, the photosensitive amount is 1.34< PS <16.28; based on the photosensitive reagent with the photosensitive quantity of 1.34< PS <16.28, the performance and the clinical test result of the photo-excitation chemiluminescence detection kit can be improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a graph showing the particle size results of a 20ug/ml carrier measured by a particle sizer as shown in the specific examples of the present application;
FIG. 2 is a graph showing the particle size results of 20ug/ml photosensitive microspheres measured by a particle size meter according to an embodiment of the present application;
FIG. 3 shows a wavelength-absorbance curve of a photosensitive material shown in an embodiment of the present application;
FIG. 4 shows wavelength-absorbance curves for different concentrations of carrier as shown in the specific embodiments of the present application;
FIG. 5 shows wavelength-absorbance curves for various concentrations of photosensitive microspheres as shown in the specific embodiments of the present application;
FIG. 6 shows a wavelength-absorbance curve for 10 μg/ml of carrier and photosensitive microspheres as shown in the specific examples herein;
FIG. 7 shows a carrier concentration-absorbance curve for a carrier at a wavelength of 500nm as shown in the specific embodiments of the present application;
fig. 8 shows a concentration-amount of photosensitizing substance curve shown in the embodiment of the present application.
Detailed Description
The invention discloses a photo-excitation chemiluminescence detection kit and a use method thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.
It should be understood that although the terms "first," "second," "third," etc. may be used herein to describe various information, these information should not be limited by these terms. These terms are only used to distinguish one type of information from another. For example, a first message may also be referred to as a second message, and similarly, a second message may also be referred to as a first message, without departing from the scope of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
The luminescent microspheres may be polymer particles filled with a luminescent compound formed by coating a functional group on a first support, and may be referred to as luminescent microspheres or luminescent particles. The surface of the luminous microsphere is provided with hydrophilic carboxyl glucan, and the interior of the luminous microsphere is provided with a chemical hair composition capable of reacting with active oxygen (such as singlet oxygen). In some embodiments of the invention, the chemical composition undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate, which may decompose while or subsequently emit light. In some preferred embodiments of the present invention, the luminescent composition, such as europium complexes, is merely illustrative and not limiting.
In the present application, the photosensitive microsphere includes a second carrier and a photosensitive substance carried by the carrier. The second carrier (also referred to as blank microspheres) may be polymer particles, and the photosensitive material may be coated on the surface of the carrier and/or filled in the second carrier. The photosensitive material may be capable of generating active oxygen (e.g., singlet oxygen) under light excitation, and the polymer particles may be polystyrene microspheres, or may be microspheres of other materials for detection, which is not limited herein. The photosensitive substance may be, for example, a photosensitizer or a photosensitive dye, which may be a photosensitive substance known in the art, such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll, and is not limited thereto. The photosensitive microspheres may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other sensitizers include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.
The immune complex refers to a complex obtained by combining an antibody with an antigen, and the immune complex is an antigen-antibody, antigen-antibody-antigen, antibody-antigen-antibody, antigen-antibody-secondary antibody and the like. The luminous compound refers to a compound formed by directly or indirectly connecting luminous microspheres and photosensitive microspheres, and can receive laser irradiation with specific wavelength to generate chemical reaction to emit detectable light.
In one embodiment, the photosensitive microsphere for photoexcitation chemiluminescence detection has a photosensitive amount Ps between 1.34 and 16.28; the light sensing amount Ps of the photosensitive microsphere is determined according to the following formula (1).
Ps＝ OD λ1 /C 2 *10 3 (1)
Wherein OD λ1 The visible light region of 300-800 nm has a specific concentration of C 2 Absorbance value, lambda corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained after full wavelength scanning of the photosensitive microspheres 1 Is the wavelength corresponding to the maximum absorption peak; c (C) 2 Is the concentration of photosensitive microsphere in light-activated chemiluminescence detection, C 2 The unit is ug/ml.
In the present application, the photosensitive microsphere includes a carrier and a photosensitive substance carried by the carrier. The carrier can be polymer particles, and the photosensitive substance can be coated on the surface of the carrier and/or filled in the carrier. The photosensitive material may be capable of generating active oxygen (e.g., singlet oxygen) under light excitation, and the polymer particles may be polystyrene microspheres, or may be microspheres of other materials for detection, which is not limited herein. The photosensitive substance may be, for example, a photosensitizer or a photosensitive dye, which may be a photosensitive substance known in the art, such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll, and is not limited thereto. The photosensitive microspheres may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other sensitizers include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.
Further, the concentration of the buffer solution dissolved in the solution is C by using visible light in the range of 300nm to 800nm through a spectrophotometer or the like 2 The photosensitive microsphere of the fluorescent lamp is scanned in full wavelength, the absorbance values corresponding to different wavelength scans are read, and after a corresponding wavelength-absorbance curve is generated, the wavelength lambda is obtained 1 The wavelength corresponding to the maximum characteristic peak (namely the maximum absorption peak) of the photosensitive microsphere in the wavelength-absorbance curve is selected; correspondingly, the concentration can be determined to be C by selecting the absorbance value corresponding to the maximum characteristic peak in the wavelength-absorbance curve 2 At wavelength lambda 1 Corresponding absorbance value OD λ1 . Further, to ensure the wavelength lambda 1 In one embodiment, the wavelength-absorbance curves of the photosensitive microspheres with different known concentrations and the same photosensitive substance can be measured in advance, and the wavelength corresponding to the maximum characteristic peak is selected from the wavelength-absorbance curves of the photosensitive microspheres with different concentrations as lambda 1 Is a value of (a). Experiments in the following prove that the maximum characteristic peaks corresponding to the photosensitive microspheres with the same photosensitive substance under different concentrations are the same, and the maximum characteristic peaks of the photosensitive microspheres are the same as the maximum characteristic peaks of the photosensitive substance carried by the photosensitive microspheres, for example, the photosensitive substance is taken as copper phthalocyanine, and the wavelength lambda of the photosensitive substance and the photosensitive microspheres is the same as the maximum characteristic peaks of the photosensitive substance carried by the photosensitive substance 1 All were 680nm. Therefore, by selecting the wavelength lambda corresponding to the maximum characteristic peak of the wavelength-absorbance curve of the photosensitive microsphere 1 Then, the corresponding absorbance value OD is determined λ1 Accuracy of the calculation result of the light sensing amount can be ensured. When the photosensitive microsphere adopts photosensitive substances with different materials, the visible light region in the range of 300 nm-800 nm can be adaptively reused for scanning so as to determine the wavelength lambda 1 Specific values of (2).
Before use, the initial state of the photosensitive microspheres during storage is generally a lyophilized solid substance or a refrigerated liquid. When photosensitive microsphereWhen the compound is solid, buffer solution is added for re-dissolution, and the concentration of the re-dissolved photosensitive microsphere solution is the initial concentration C 1 . When the photosensitive microsphere is stored as liquid, the concentration at the moment is the initial concentration C 1 . In the light-activated chemiluminescence detection, the photosensitive microsphere may directly participate in the detection by adopting the initial concentration, namely the concentration C when the photosensitive microsphere participates in the detection 2 Equal to C 1 The method comprises the steps of carrying out a first treatment on the surface of the Alternatively, the initial concentration C may be 1 The photosensitive microsphere is diluted and then participates in detection, namely the concentration C of the photosensitive microsphere during the detection 2 Not equal to C 1 ，C 2 The value of (2) is the true concentration after the corresponding initial concentration is diluted. It can be appreciated that when the wavelength lambda is 1 After determining the specific value of (C) the concentration of photosensitive microspheres 2 When changing, the corresponding absorbance value OD λ1 May be correspondingly different, OD λ1 Is determined from the actual measurement. At the determination of absorbance value OD λ1 And concentration C 2 After the value of (2), according to the absorbance value OD λ1 And concentration C 2 The amount of sensitization Ps of the sensitization microsphere can be determined. When the value of the sensitization quantity Ps of the sensitization microsphere is between 1.34 and 16.28, the sensitization microsphere is applied to the sensitization reagent to react with the luminous microsphere, so that the intensity of a chemiluminescent signal of the luminous microsphere can meet the requirement in photoexcitation chemiluminescence detection, the fluctuation of a detection result caused by the influence of other interference factors on the chemiluminescent signal is reduced, the detection result has consistency and repeatability in clinical application, and the detection result has more definite distinction and higher precision. The photosensitive microsphere has definite operability and is suitable for popularization of industry specifications, and the performance standard of the photosensitive microsphere for execution in the photo-excitation chemical detection is given through definite numerical limitation of the photosensitive quantity.
Further, to facilitate the definition of the concentration C of the photosensitive microspheres 2 Is the concentration C of photosensitive microsphere 2 Determined according to the following formula (2).
Where k is the corresponding slope in the linear relationship of carrier concentration-absorbance curve, b is the corresponding intercept, OD, in the linear relationship of the carrier concentration-absorbance curve λ2 Is photosensitive microsphere at wavelength lambda 2 The corresponding absorbance value is the carrier concentration-absorbance curve at wavelength lambda using carriers of different concentrations 2 A curve obtained below; wavelength lambda 2 The photosensitive microsphere with the same concentration and the carrier have the same or similar absorbance value corresponding to the wavelength-absorbance curve.
Specifically, in order to obtain the concentration C of the photosensitive microsphere satisfying the range of the photosensitive amount Ps 2 Experiments can be carried out by using carriers with the same material and particle size as the photosensitive microspheres to determine the concentration C of the photosensitive microspheres 2 Is not limited in terms of the range of (a). It should be noted that, due to the limitation of the manufacturing process of the photosensitive microspheres and the carrier, the definition of "the same particle size", etc. in the present application means that the difference in particle size between the microspheres is ±5nm, and such a small difference in particle size between the microspheres can be regarded as the same particle size. Wherein, a plurality of carriers with different concentrations and the same particle size can be prepared in advance, and the particle size of the carrier is selected from 190 nm-280 nm. And adopts the same wavelength lambda 2 The absorbance values corresponding to the carriers of each concentration are respectively scanned and measured, so that a relation curve of the carrier concentration and the absorbance can be established, and then a linear relation of the carrier concentration and the absorbance is obtained, and the linear relation can be expressed by the following formula (3).
y＝kx+b (3)
Wherein x is different concentrations of carriers with preset particle sizes, y is an absorbance value of the carriers at the corresponding concentration, k is a slope in the formula (2), and b is an intercept in the formula (2). That is, by the correlation calculation of the formula (3), the values of k and b in the formula (2) can be determined, and thus the concentration C of the photosensitive microspheres can be determined 2 . Further, to determine the values of k and b, in one embodiment, the wavelength λ 2 Selected from OD Photosensitive microsphere /OD Carrier body Any wave having a ratio of 0.85 to 1.15Long value, and wavelength lambda 2 Not equal to wavelength lambda 1 The method comprises the steps of carrying out a first treatment on the surface of the Wherein OD Photosensitive microsphere And OD (optical density) Carrier body The absorbance values corresponding to the same wavelength values of the photosensitive microspheres and the carriers with the same concentration in the range of 300nm to 800nm are respectively utilized. In the present embodiment, the wavelength lambda 2 Not equal to wavelength lambda 1 I.e. wavelength lambda 2 The influence of the absorbance value of the photosensitive substance on the absorbance value of the carrier is reduced for the wavelength corresponding to the non-characteristic peak in the wavelength-absorbance curve, namely, the wavelength avoiding the characteristic peak of the photosensitive substance. It will be appreciated that the absorbance OD of the photosensitive microsphere at the same concentration over the full wavelength range can be obtained by scanning the photosensitive microsphere and the carrier, respectively, at the same microsphere concentration using the same wavelength in the range of 300nm to 800nm Photosensitive microsphere And obtaining the absorbance value OD of the carrier at the concentration corresponding to the same wavelength in the full wavelength range Carrier body . The applicant researches show that specific experimental data can be checked to obtain the following related contents, and OD is selected Photosensitive microsphere /OD Carrier body The ratio satisfies a wavelength in the range of 0.85 to 1.15 as lambda 2 The concentration C of the photosensitive microsphere can be determined more accurately by the carrier concentration-absorbance curve of the carrier than by selecting wavelengths outside the above-mentioned ratio range 2 Is a range of values. It is found from experiments that the wavelength lambda 2 The absorbance value OD of the photosensitive microsphere is within the range of 440nm to 580nm Photosensitive microsphere And carrier absorbance value OD Carrier body The ratio of (2) is within 0.85 to 1.15, which means that the content of the photosensitive substance has less influence on the measurement of the concentration of the microspheres, otherwise. In one embodiment, the wavelength λ 2 May be 440nm to 580nm. For example, wavelength lambda 2 May be 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm. It should be understood that when the photosensitive materials selected by the photosensitive microspheres are different, the maximum characteristic peak of the photosensitive materials will be changed correspondingly due to the influence of the properties of the photosensitive materials, and the wavelength lambda can be known in the same manner 1 And wavelength lambda 2 Correspondingly adjust OD λ1 And OD (optical density) λ2 And also adjusted accordingly.
During the determinationFixed wavelength lambda 2 Then, can adopt the same wavelength lambda 2 Scanning a plurality of carriers with known different concentrations x and the same particle size to obtain corresponding absorbance values y, thereby establishing an equation according to a formula (3) to calculate and obtain values of k and b, and calculating the concentration C of the photosensitive microsphere with the same particle size as the carrier according to a formula (2) 2 . Further, the loading amount of carriers of different particle diameters at the same concentration to the photosensitive substance may be different, thereby affecting the absorbance value, i.e., the carrier concentration-absorbance curve is also related to the particle diameter of the carrier. Therefore, in order to establish an accurate and reliable carrier concentration-absorbance curve, in one embodiment, carriers with particle diameters within 190nm to 290nm are selected to establish a corresponding carrier concentration-absorbance curve to control the concentration C of the photosensitive microspheres 2 The calculated result of (2) and the actual concentration are within 10%. For example, the predetermined particle size of the carrier may be 190nm, 200nm, 220nm, 240nm, 260nm, 280nm or 290nm. For example, a corresponding carrier concentration-absorbance curve can be established using 190nm of carrier, thereby establishing an equation according to equation (3) to calculate the values for k and b. Preferably C 2 Selected from 10ug/ml to 200ug/ml.
Specifically, the k, b and λ are known from the above calculation in the range of 1.34 to 16.28 for the known light sensing amount Ps 2 On the premise of (a), conversely, the concentration C of the photosensitive microspheres 2 I.e. can be adjusted according to formulas (1) and (2). That is, in the actual photo-activated chemical detection process, after arbitrarily disposing the photosensitive microspheres with unknown concentration, the photosensitive microspheres with unknown concentration are respectively subjected to the wavelength lambda under the condition that the specific value of the unknown concentration is unknown 1 And wavelength lambda 2 Scanning and obtaining the corresponding absorbance value, namely OD λ1 And OD (optical density) λ2 Calculating the specific value of the unknown concentration by the formula (2), and if the value range of the unknown concentration falls within 10ug/ml to 200ug/ml, calculating the concentration C 2 Substituting the value of Ps between 1.34 and 16.28 to calculate the amount of light sensing Ps in formula (1) indicates that the concentration of the disposed photosensitive microsphere can be applied to photo-induced chemiluminescence detection.
In summary, it can be seen that in the known photosensitive microsphereConcentration C 2 Under the condition that the value range is 10 ug/ml-200 ug/ml, the photosensitive value Ps corresponding to the photosensitive microsphere can be obtained directly according to the formula (1) without using the formulas (2) and (3); similarly, at unknown photosensitive microsphere concentration C 2 On the premise of the specific numerical value of the photosensitive microsphere, the corresponding photosensitive value Ps of the photosensitive microsphere can be determined according to the mode. If the calculated value of Ps is between 1.34 and 16.28, the photosensitive microsphere can achieve the above effect, namely the photosensitive microsphere can be applied to photo-excitation chemiluminescence detection, and the accuracy and precision of the detection result meet the clinical application requirements.
Further, in order to reduce the effect of substances other than the carrier and the photosensitive substance on the absorbance value, in one embodiment, the surface of the photosensitive microsphere is not coated with the polysaccharide; or the polysaccharide content of the photosensitive microsphere is not higher than 25mg per gram of mass. Wherein polysaccharide refers to carbohydrates containing three or more unmodified or modified monosaccharide units, such as dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran, aminodextran, and the like. The interference on the measurement result of the absorbance value is reduced by not adding the polysaccharide or controlling the content of the polysaccharide, so that the detection result in clinical application is more accurate.
The main equipment comprises:
TABLE 1
| Device name | Device model | Manufacturer' s |
| High-speed refrigerated centrifuge | CR-21G or CR-21N | HITACHI |
| Electronic balance | ALC-2100.2 or BSA2202S | Sartorius |
| Analytical balance | AL204-01 | METTLER |
| Medical refrigerator | HYC-310S | Sea Er |
| Magnetic stirrer | H03-A or 85-2A | Shanghai Mei Yingpu |
| Constant speed stirrer | MYP2011-100 | Shanghai Mei Yingpu |
The main raw materials are as follows:
TABLE 2
| Material name | Storage conditions |
| Luminescent particles FG | Light-shielding and refrigerating at 2-8 |
| Antibody | |
| 1 | Freezing at- |
| Antibody | |
| 2 | Freezing at-15deg.C |
| Biotin | Freezing at-20deg.C |
In the light-activated chemiluminescence detection kit and the application method thereof, the related raw materials or reagents can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 preparation of luminescent reagent
1. Dialyzing the raw materials, placing 1mg of antibody 1 in a 3.5KD dialysis bag, and dialyzing in 0.05M CB buffer (pH 9.6) for 3 times;
2. concentration measurement, the protein recovered by dialysis was aspirated and transferred to a clean centrifuge tube, and the concentration was measured by BCA method and found to be 2.42mg/ml.
3. Particle treatment, taking 1ml of FG particles with the concentration of 10mg/ml in a centrifuge tube, centrifuging for 1 hour by 30000G, discarding the supernatant, carrying out ultrasonic resuspension by using 0.05M CB buffer (pH 9.6), repeating the operation once, and fixing the volume of 50mg/ml for standby.
4. Coupling reaction, according to particles: mixing FG and antigen protein at a mass ratio of 10:1, performing coupling reaction by using a rotary mixing mode, and performing rotary reaction at 37 ℃ overnight; an 8mg/mL NaBH4 solution was prepared using 0.05M CB buffer, and immediately added to each reaction tube, and the reaction was spun for 2 hours.
5. Constant volume, cleaning and constant volume: the centrifugation and washing were repeated twice to remove the excess protein, and finally the volume was fixed to 10mg/ml in the luminescent reagent buffer.
6. Preparing a luminous reagent, namely diluting the prepared luminous concentrated solution to 80ug/ml by using 50ml of luminous reagent buffer solution, and balancing for 2 hours for later use.
EXAMPLE 2 preparation of Biotin reagent
1. Dialyzing the raw material, placing 1mg of antibody 2 in a 3.5KD dialysis bag, and dialyzing 3 times in 0.1M NaHCO3 buffer (pH8.5);
2. concentration measurement, the protein recovered by dialysis was aspirated and transferred to a clean centrifuge tube, and the concentration was measured by BCA method and found to be 1.81mg/ml.
3. Labeling reaction, preparing 20mg/ml biotin NHS active ester with DMSO according to protein: labeling reaction is carried out by using a rotary mixing mode according to the biotin mol ratio of 1:30, and the labeling reaction is carried out by using a mixing mode, and the mixing reaction is carried out for 16 hours.
4. Dialysis of the marked product, product dialysis, dialysis bag specification: 3500D, dialysis buffer: 0.02MHEPES buffer (pH 8.0), dialysis time and number of times: 3 times of dialysis for 3 hours each time; the protein recovered by dialysis was aspirated and transferred to a clean centrifuge tube, and the protein concentration was measured using BCA method at 1.64mg/ml for use.
5. The biotin reagent is prepared, 50ml of biotin reagent buffer is used, and the prepared marked product is diluted to 1ug/ml and balanced for 2 hours for standby.
EXAMPLE 3 preparation of photosensitizing agent
1. Preparation of microspheres
Preparation of the (one) Carrier
a) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added to the three-necked flask, followed by stirring for 10 minutes, and N was introduced into the three-necked flask 2 30min。
b) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water, respectively, to prepare aqueous solutions. Adding the aqueous solution into the reaction system of the three-neck flask in the step a), and continuously introducing N 2 30min。
c) The reaction system was heated to 70℃and reacted for 15 hours to obtain an emulsion.
d) The emulsion after the reaction is cooled to room temperature and filtered by a suitable filter cloth. And washing the emulsion obtained after filtration by using deionized water through centrifugal sedimentation until the conductivity of the centrifuged supernatant approaches to that of the deionized water, diluting the emulsion by using water, and preserving the emulsion.
e) The average particle diameter of the gaussian distribution of the particle size of the latex microspheres in the emulsion was 190nm as measured by a nanoparticle sizer.
(II) preparation of photosensitive microspheres
a) A25 ml round bottom flask was prepared, 0.11g of copper phthalocyanine (i.e., photosensitive substance) was added, 10ml of N, N-dimethylformamide was stirred uniformly by magnetic force, and the round bottom flask was heated to 75℃in a water bath to obtain a photosensitive substance solution.
b) A 100ml three-necked flask was prepared, 10ml of 95% ethanol, 10ml of water and 10ml of the carrier prepared by 1.e) above having a concentration of 10% were added thereto, and the mixture was stirred uniformly by magnetic force, and the three-necked flask was warmed to 70℃in a water bath.
c) Slowly dripping the photosensitive substance solution in the step a) into the three-neck flask in the step b), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling to obtain emulsion. It will be appreciated that the mass ratio of the carrier of step b) and the photosensitive material solution of step a) may be adjusted according to the requirements of the subsequent experiments.
d) Centrifuging the emulsion obtained in the step c) for 1 hour according to a centrifugal force of 30000G, discarding supernatant after centrifugation, and re-suspending by using 50% ethanol. After three repeated centrifugal washes, the microspheres were resuspended to the desired concentration in the subsequent experiments with 50mmol/L CB buffer at ph=10.
2. Determination of photosensitive microsphere concentration C 2 Method for evaluating application range
The experiment adopts the preset particle diameter to establish the linear relation between the carrier concentration and the absorbance value, thereby obtaining the absorbance value OD in the formula (2) λ2 Obtaining the concentration C of the photosensitive microsphere 2 . This experiment is used to explain the establishment procedure of the above-described formula (2) and formula (3). 1. Full wavelength scanning and particle size detection of microspheres
Particle size detection was performed in advance to ensure uniformity of particle size of the microspheres used in the experiment. The main materials and equipment involved in the experiment are shown in table 3.
TABLE 3 Table 3
| Raw materials and instruments | Specification and model | Manufacturer' s |
| Carrier body | Particle diameter of 190nm | Boyang |
| Photosensitive microsphere | Particle diameter of 190nm | Boyang |
| Mixing instrument | - | - |
| Particle diameter instrument | M0DEL380 | PSS.NICOMP |
| Ultraviolet spectrophotometer | UV-1600PC | MADAPA |
| Deionized water | - | - |
The experimental process is specifically as follows:
1.1 selection of microsphere particle size
In this experiment, in order to ensure the consistency of data, subsequent experiments can be performed by uniformly using photosensitive microspheres and carriers having the same particle size, for example, 190 nm.
1.2 preparation of different concentrations of Carrier and photosensitive microspheres
The prepared photosensitive microspheres and blank microspheres are respectively diluted by deionized water to prepare carriers and photosensitive microspheres with different concentrations, wherein the prepared concentrations are respectively 10ug/ml,20ug/ml,30ug/ml,40ug/ml,50ug/ml,60ug/ml,70ug/ml,80ug/ml,90ug/ml,100ug/ml and the like. Namely, respectively configuring 10 concentrations of carriers and 10 concentrations of photosensitive microspheres; in addition, 5ug/ml of photosensitive substance solution was prepared.
2. Microsphere particle size detection
The particle size meter was turned on and the particle size of the support and photosensitive microspheres of 20ug/ml was taken as an example for detection.
Wherein, experimental data are shown in fig. 1 and 2. The average particle size of the carrier was 187.1nm, and the average particle size of the photosensitive microspheres was 190.9nm.
The detection result of the particle size meter shows that the particle sizes of the carrier and the photosensitive microspheres are about 190nm, the wave crest is narrower, the particle sizes of the microspheres are more uniform, and the microspheres can be used as microspheres required by subsequent experiments.
3. Selecting wavelengths
Opening an ultraviolet spectrophotometer, preheating for 30min, adjusting the ultraviolet spectrophotometer, setting the wavelength to 300-800 nm, setting the step length to 1nm, calibrating zero by using deionized water, and sequentially detecting the photosensitive microspheres, the carrier and the photosensitive substance solution with the concentrations configured in the step 1.2. It is understood that the wavelength setting needs to be greater than 300nm because the scanned wavelength is susceptible to interference from absorbance values of other substances, thereby affecting the accuracy and precision of the detection result.
4. Experimental data
4.1 photosensitive materials
As shown in FIG. 3, FIG. 3 shows the wavelength-absorbance curve of the photosensitive material after scanning at 300nm to 800 nm. As can be seen from fig. 3, the photosensitive dye has distinct peaks at 360nm, 610nm, 650nm and 680nm, respectively, wherein 680nm is the main peak, i.e. the maximum characteristic peak of the photosensitive substance.
4.2 vectors
FIG. 4 is a graph showing the wavelength-absorbance curves of 10 carriers at different concentrations after scanning at 300nm to 800 nm. As can be seen from FIG. 4, the carriers with different concentrations have no characteristic peak in the graph after being scanned by visible light of 300nm to 800 nm. Meanwhile, as can be seen from fig. 4, the carriers with different concentrations have different absorbance values after scanning, and the microsphere concentration of the carrier is positively correlated with the absorbance value.
4.3 photosensitive microspheres
Fig. 5 shows the absorbance curves of 10 photosensitive microspheres with different concentrations at the wavelength of 300 nm-800 nm, and as can be seen from fig. 5, the photosensitive microspheres with different concentrations show obvious peaks at 360nm, 610nm, 650nm and 680nm after being scanned by visible light with 300 nm-800 nm, wherein 680nm is the main peak, i.e. the maximum characteristic peak of the photosensitive microspheres is the same as the maximum characteristic peak of the photosensitive substance. I.e. the photosensitive substance filled with photosensitive microspheres will directly affect the wavelength value corresponding to the maximum characteristic peak. As can be seen from fig. 5, the absorbance values of the photosensitive microspheres at different concentrations after scanning are different, and the microsphere concentration of the photosensitive microspheres is positively correlated with the absorbance value. Thus, different concentrations of photosensitive microspheres affect the magnitude of the amount of light sensed Ps.
4.4 wavelength lambda 1 And wavelength lambda 2 Is determined by (a)
The wavelength-absorbance curves of the same concentration of photosensitive microspheres and carrier were compared. As shown in FIG. 6, taking photosensitive microspheres and carriers with the concentration of 10 mug/ml as an example, after the carrier and the photosensitive microspheres with the same concentration scan the wavelength of 300 nm-800 nm, 680nm characteristic peak of the photosensitive microspheres is the maximum characteristic peak of the photosensitive substance. Therefore, the wavelength corresponding to the maximum characteristic peak can be selected as lambda by reading the absorbance value of 680nm to most reflect the content of the photosensitive substance in the photosensitive microsphere 1 I.e. lambda 1 680nm. It can be understood that the photosensitive material sampled in the experiment is copper phthalocyanine, and when the photosensitive dye is other raw materials, the maximum characteristic peaks may be different, and the corresponding wavelength lambda 1 And determining according to actual conditions.
For wavelength lambda 2 Is determined with the aim ofFor according to the corresponding absorbance value OD λ2 To determine the concentration C of the photosensitive microspheres 2 . Therefore, for measuring the concentration of the photosensitive microspheres based on the absorbance value, it is necessary to avoid the maximum characteristic peak of the photosensitive substance, i.e., wavelength lambda 2 And lambda is 1 Different. Such a design is because, when a region where a peak appears in the photosensitive substance is selected, the absorbance value at the wavelength corresponding to the peak contains the absorbance value of the carrier itself plus the absorbance value of the photosensitive substance, thereby affecting the concentration test of the photosensitive microsphere. As can be seen from FIG. 6, λ is selected between 400nm and 600nm 2 Optimally, there is no characteristic peak in this wavelength region. Although the characteristic peak of the photosensitive substance does not exist in the wavelength range of 300-330 nm, the wavelength is easily influenced by protein substances in other samples to be tested, and the clinical application is influenced. The absorbance value corresponding to the wavelength of 700 nm-800 nm is lower, so that the detection sensitivity is low, the fluctuation of the test result is large, and the absorbance value corresponding to the wavelength of 400 nm-600 nm is more consistent, therefore, the wavelength lambda 2 Selected from 400nm to 600 nm.
Further, for accurate determination of the wavelength lambda 2 Is analyzed using the absorbance of one of the photosensitive microspheres and the carrier at the same concentration. Photosensitive microspheres and carriers at a concentration of 50ug/ml were used as examples and are shown in Table 4.
To reduce the effect of the photosensitive material on the concentration of the microspheres measured, the wavelength lambda is selected 2 The range of (2) requires an OD at the same wavelength Photosensitive microsphere /OD Carrier body The ratio of (2) is within 0.85 to 1.15, i.e., (1.+ -. 15%). As can be seen from Table 4, when the wavelength lambda is 2 At 440nm to 580nm, the absorbance OD of the photosensitive microsphere of 50ug/ml Photosensitive microsphere Absorbance value OD with 50ug/ml carrier Carrier body The ratio of (2) is within the range of 0.85 to 1.15, thereby indicating that the absorbance value of the photosensitive material corresponding to the wavelength in the interval has less influence on the absorbance value of the photosensitive microsphere and the carrier, and when the OD is Photosensitive microsphere /OD Carrier body When the ratio of (2) is greater than 1.15, it is indicated that the content of the photosensitive substance affects the determination of the concentration of the photosensitive microspheres. Preferably, the OD is at wavelengths of 500nm and 510nm Photosensitive microsphere /OD Carrier body The ratio of (2) was 1.05, and a constant ratio indicates a constant influence of the photosensitive substance. Therefore, in the present embodiment, the wavelength λ is preferable 2 500nm. It will be appreciated that when photosensitive materials of different materials are selected to prepare photosensitive microspheres, the wavelength lambda can be determined by referring to the above method 2 。
TABLE 4 Table 4
5. Establishment of Carrier concentration-absorbance curve
In this embodiment, the carrier with the same particle size is selected to have the wavelength lambda 2 The absorbance values of the carriers at different concentrations were then tested to establish a carrier concentration-absorbance curve.
Determining the wavelength lambda at step 4.4 above 2 The experiment then selects wavelength lambda 2 The concentration-absorbance curve of the carrier was examined at 500nm and a particle diameter of about 190nm, and is shown in FIG. 7.
Specifically, in order to obtain carriers with different concentrations, the mass of the carrier is obtained by a traditional drying method, deionized water is added into the carrier with known mass to prepare 10mg/ml of carrier, and the carrier is further diluted by the deionized water to prepare 10 kinds of carriers with the concentration of 10ug/ml,20ug/ml,30ug/ml,40ug/ml,50ug/ml,60ug/ml,70ug/ml,80ug/ml,90ug/ml,100ug/ml and the like. Then scanning the carrier of each concentration by a wavelength of 500nm to obtain an absorbance value OD corresponding to each concentration 500 A linear relationship of carrier concentration to absorbance value y=kx+b can be established. At a known concentration x, when lambda 2 At 500nm, the absorbance value y can be directly measured by a spectrophotometer, and thus k of 0.0021 and b of 0.0359 can be calculated. After determining the values of k and b, the method is carried out according to formula (2), i.e. the carrier concentration x= (OD λ2 -b)/k＝(OD λ2 -0.0359)/0.0021。
Since the above experiment obtained the values of k and b by measuring the absorbance values of different concentrations using only carriers having a particle diameter of 190nm, the applicant used carriers having different particle diameters for verification in order to verify the accuracy of the calculation result of the above carrier concentration x. Carriers with different particle sizes are prepared respectively, and the particle sizes of the carriers comprise 7 particle sizes such as 190nm,200nm,220nm,240nm,260nm,280nm and 300 nm. And preparing the carrier with each particle size into theoretical concentration values of 40ug/ml,50ug/ml and 60ug/ml according to the mass of the carrier calculated by a drying method. The absorbance OD of the carrier at each concentration per particle diameter according to the wavelength of 500nm on the premise of knowing the theoretical concentration value λ2 And (5) detecting. To obtain accurate results, each concentration of each particle size was divided into 3 parts for detection of absorbance values. At the time of obtaining the absorbance value OD corresponding to each concentration of each particle diameter of each carrier λ2 After that, according to (OD λ2 -0.0359)/0.0021, and comparing the calculation result with the theoretical concentration value, the deviation of the calculation result is determined. The results are shown in Table 5:
TABLE 5
As can be seen from the data in Table 5, when the particle diameter of the carrier is 280nm or less, the recovery deviation of the concentration is within 10%, i.e., according to (OD λ2 The deviation between the carrier concentration value x calculated by 0.0359)/0.0021 and the theoretical concentration value is within 10 percent, which shows that the method for determining the concentration of the photosensitive microsphere according to the absorbance value adopted by the application has better accuracy. Thus, the values of k and b determined by the above method can be applied to the concentration C of the photosensitive microspheres in the formula (2) 2 Is calculated by the computer.
As can be seen from FIG. 7, in this experiment, when C 2 When the value of (2) is 10 ug/ml-100 ug/ml, the linear relation is better. The number of experiments is limited, C 2 The value of (2) is not limited to 10ug/ml to 100ug/ml, C can be further determined in combination with the following experiment 2 Is a range of values.
3. Comparing the influence of photosensitive microspheres with different mass ratios on the quantity of photosensitive light
1. Preparation of photosensitive microspheres with different mass ratios
According to the preparation method of the photosensitive microsphere in the first step and the second step, the corresponding photosensitive microsphere is prepared by adopting different mass ratios of the carrier to the photosensitive substance, namely, 6 photosensitive microspheres with the mass ratios of the carrier to the photosensitive substance of 10:4, 10:2, 10:1, 10:0.2, 10:0.04 and 10:0 are prepared firstly, namely, the microspheres 1 to 6 in the table 4 and the table 5. Wherein 10:0 represents that the photosensitive microsphere contains no photosensitive substance and is only an empty carrier. And then respectively diluting the prepared photosensitive microspheres with different mass ratios by deionized water, namely respectively diluting the prepared photosensitive microspheres corresponding to the six mass ratios by 500 times, 1000 times and 2000 times, wherein the photosensitive microspheres with each mass ratio obtain 3 diluted photosensitive microspheres with different concentrations. Scanning the diluted photosensitive microspheres by an ultraviolet spectrophotometer to obtain a wavelength lambda 1 680nm and wavelength lambda 2 An absorbance value OD corresponding to 500nm, and a corresponding concentration value C is obtained by calculation according to the formula (2) 2 And calculating according to the formula (2) to obtain the corresponding sensitization quantity Ps. Specific data are shown in table 6 below. It is to be noted that, as shown in fig. 5, at the wavelength λ 1 The photosensitive microsphere has a strong absorption peak, namely a maximum characteristic peak, and the corresponding absorbance value can most reflect the concentration of the photosensitive substance. The absorbance value of the photosensitive microsphere contains the absorbance values of the carrier and the photosensitive substance, and thus the true absorbance value OD of the photosensitive substance λ1 photosensitive material For OD λ1 photosensitive microsphere -OD λ1 vector 。
TABLE 6
Further, corresponding average value of the sensitization amount and CV value of the variation coefficient are calculated according to each mass ratio of the sensitization amount of the sensitization materials with different dilution factors in the table 6, wherein CV value is the ratio of standard deviation to average value, and specific calculation results can refer to the related data in the table 7.
As is clear from Table 6, the concentration value C of the photosensitive microsphere 6 at a dilution factor of 500X 2 The calculated value according to equation (2) is 199, which is very close to the corresponding theoretical concentration value 198. Thus, C was supplemented with experiment II above 2 The value range of (C) 2 The range of values may be 10ug/ml to 200ug/ml.
TABLE 7
As can be seen from table 6, the photosensitive materials with the same mass ratio are more consistent in calculated photosensitive amount after being diluted by different multiples; as can be seen from table 7, the CV values of the light-sensitive amounts of the light-sensitive substances of different mass ratios were all within 10%, which indicates that the calculation results of the light-sensitive amounts determined according to the formulas (1) and (2) were less fluctuated and the calculation was more accurate. And the photosensitive substances with the same mass ratio are described, and the photosensitive quantity is related to the corresponding dilution factor, namely the concentration of the photosensitive microspheres.
Further, a graph of different mass fractions of the photosensitive material shown in fig. 8 obtained with the calculated photosensitive material can be plotted according to table 7. As can be seen from fig. 8, the correlation between the amount of light sensed by the photosensitive microsphere per unit concentration and the mass ratio of the photosensitive substance is detected based on the absorbance value, that is, the larger the mass ratio of the photosensitive substance is, the higher the concentration of the photosensitive substance is, the larger the amount of light sensed is. Meanwhile, when the mass ratio of the carrier to the photosensitive substance is smaller than 10:1, the linear relation between the photosensitive quantity and the photosensitive concentration is better; when the mass ratio of the carrier to the photosensitive material is 10 (2-4), the increase of the photosensitive quantity is obviously reduced, which indicates that the ratio of the photosensitive material, namely the quantity of the photosensitive material filled in the carrier is gradually increased to be saturated. Such trend changes correspond to the change in the amount of light actually sensed by the photosensitive microspheres. In addition, when the mass ratio of the carrier to the photosensitive substance is 10:4, the photosensitive quantity of the obtained photosensitive microsphere reaches the peak value of 20.12, and even if the mass ratio of the photosensitive substance is continuously improved, the photosensitive quantity of the photosensitive microsphere is not further increased, so that the material cost is saved by controlling the mass ratio of the carrier to the photosensitive substance. Effect example comparing the performances of photosensitive microspheres with different light-sensitive amounts in clinical application
1. Preparing photosensitive reagents with different light-sensitive amounts according to the photosensitive microspheres with different light-sensitive amounts
a) And (3) photosensitive microsphere suspension treatment: sucking a certain amount of the photosensitive microspheres prepared in the step one and the step 2 in the embodiment 3, centrifuging in a high-speed refrigerated centrifuge, removing the supernatant, adding a certain amount of MES buffer, oscillating the microspheres on an ultrasonic cell disruption instrument by ultrasonic waves to re-suspend the microspheres, and adding the MES buffer to adjust the concentration of the photosensitive microspheres to 100mg/ml.
b) Avidin solution preparation: a quantity of streptavidin was weighed and dissolved to 8mg/ml in MES buffer.
c) Mixing: mixing the processed 100mg/ml photosensitive microsphere suspension, 8mg/ml avidin and MES buffer solution in the volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.
d) The reaction: 25mg/ml NaBH is prepared by adopting MES buffer solution 3 CN solution, naBH 3 The CN solution and the reaction solution were rapidly and uniformly mixed in a volume ratio of 1:25. The reaction was carried out at 37℃for 48 hours with rotation.
e) Closing: 75mg/ml Gly glycine solution and 25mg/ml NaBH are prepared by adopting MES buffer solution 3 CN solution, gly glycine solution and NaBH 3 Preparing a mixed solution of the CN solution and the reaction solution according to the volume ratio of 2:1:10, adding the mixed solution into the solution obtained after the reaction in the step d), uniformly mixing, and rotating at the constant temperature of 37 ℃ for 2 hours for reaction. Then adding 200mg/ml BSA solution (MES buffer solution) and the mixed solution with the volume ratio of the reaction solution being 5:8, quickly mixing evenly, and carrying out rotary reaction at the constant temperature of 37 ℃ for 16 hours.
f) Cleaning: and e, adding MES buffer solution into the solution reacted in the step e, centrifuging by a high-speed refrigerated centrifuge, removing supernatant, adding fresh MES buffer solution, re-suspending by an ultrasonic method, centrifuging again, repeatedly washing for 3 times, suspending by a small amount of MES buffer solution, and determining the solid content to be 10mg/ml.
g) Preparing a photosensitive reagent: the universal buffer solution of the photosensitive reagent is used for preparing the photosensitive microspheres coated with streptavidin and adopting the 6 different photosensitive amounts in mass proportion, thereby preparing the photosensitive reagent with 6 different photosensitive amounts. The sensitization amount of the 6 sensitization agents is shown in table 8.
TABLE 8
| Name of the name | Light sensing amount |
| |
0.77 |
| |
1.34 |
| |
4.07 |
| |
11.49 |
| Photosensitive agent 5 | 16.28 |
| Photosensitive agent 6 | 20.12 |
2. Evaluation of the Properties of the photosensitizing Agents of 6 different photosensitizers
The above 6 kinds of photosensitive reagents having different amounts of light are applied to the detection of clinical samples, thereby evaluating the basic performance of the photosensitive reagents having different amounts of light in the clinical application to the detection of samples.
Experimental raw materials and equipment:
TABLE 9
| Instrument for measuring and controlling the intensity of light | Specification and model | Manufacturer' s |
| LiCA detector | HT | BEYOND DIAGNOSTICS (SHANGHAI) Co.,Ltd. |
| Hepatitis B surface antigen detection kit | HBsAg | BEYOND DIAGNOSTICS (SHANGHAI) Co.,Ltd. |
2.1 testing the sensitivity of 6 different light-sensitive reagents
Samples cal1 to cal6 of the kit using 6 kinds of HBsAg having known different target molecule concentrations, and reagents 1 to 6 using 6 kinds of different amounts of the above prepared reagents were used. Firstly, each sample is respectively added into a corresponding reaction container, then a luminescent reagent and a biotin reagent are respectively added into each reaction container in sequence, and each reaction container is combined by incubation at 37 ℃ to form a first compound luminescent microsphere-antibody-antigen-antibody-biotin. And then respectively adding corresponding photosensitive reagents into each reaction container, and carrying out photo-excitation chemiluminescence detection by using a LiCA detector to obtain corresponding chemiluminescence signal values. The data of each photosensitizing agent corresponding to the detected chemiluminescent signal values are shown in the third through eighth columns of Table 10. Wherein the target molecule concentration of the sample cal1 is 0, i.e. the sample contains no hepatitis B surface antigen, the sample cal1 is a negative sample, and the corresponding measured value can be used as the measuring standard of the signal value of each photosensitive reagent.
Table 10
Wherein the theoretical values in the first column in table 10 are the corresponding known target molecule concentrations in the 6 samples. The third column through the eighth column in table 10 are chemiluminescent signal values measured in the LiCA detector for samples of various target molecule concentrations for each kit comprising a photosensitizing reagent and a luminescent reagent. According to the data in the table, for the samples with the same target molecule concentration, the light sensitivity of the light sensitive reagent is between 1.34 and 16.28, and the larger the light sensitivity is, the larger the data of the measured signal value is; when the light sensing amount of the photosensitive agent reaches 20.12, a signal drop phenomenon occurs instead. Therefore, 1.34 to 16.28 are selected as the range of the value of the light sensing amount Ps.
Further, as shown in table 11, for the same photosensing agent, as the ratio of signal values corresponding to samples with different target molecule concentrations, when the photosensing amount of the photosensing agent is lower than 1.34, the numerical distinction degree between the signal values corresponding to the photosensing agent 1 is very low, i.e. the detection sensitivity is low, and samples with different target molecules cannot be distinguished according to the signal values. While the corresponding signal value of the photosensitizing agent 2 is lower than that of the photosensitizing agents 3 to 6, a certain distinction can be made for target molecules of different concentrations. The corresponding signal values of the photosensitizing agents 3 to 6 show definite signal values for both low-concentration target molecules and higher-concentration target molecules; meanwhile, the same photosensitive reagent has obvious difference of signal value values corresponding to the target molecule concentrations with different sizes, and shows good differentiation, so that the concentration interval of the target molecules can be judged according to the signal values.
TABLE 11
| Kit for detecting a substance in a sample | |
|
|
|
Photosensitive agent 5 | Photosensitive agent 6 |
| cal2/call | 2.43 | 1.25 | 2.21 | 2.25 | 2.21 | 1.92 |
| cal6/call | 10.57 | 1086.25 | 2220.23 | 1759.43 | 1705.22 | 1816.37 |
2.2 testing the accuracy of the detection results of 5 photosensitive Agents with different amounts of light
Preparation of experimental samples:
selecting target molecules with known same mass and diluting into three mass control samples sp1, sp2 and sp3 with known different concentrations; 10 samples S1 to S10 with target molecule concentrations decreasing linearly are selected, and the target molecules of all samples are HBsAg; 4 negative samples N1, N2, N3, N4 were selected without target molecule.
The total of 17 different samples were tested for target molecule concentration with 5 different amounts of photosensitizing reagents 2 to 6, respectively. The corresponding third to seventh columns of concentration data are converted from the chemiluminescent signals measured by the LiCA detector, and as shown in Table 12, the first column is referred to as the theoretical value, which is the true concentration value in each sample.
Table 12
| Theoretical value | Sample of | |
|
|
Photosensitive agent 5 | Photosensitive agent 6 |
| 0.02 | sp1 | 0.0250 | 0.0235 | 0.0182 | 0.0186 | 0.0211 |
| 0.21 | sp2 | 0.2512 | 0.2028 | 0.2056 | 0.2015 | 0.2062 |
| 49.02 | sp3 | 49.8386 | 51.1355 | 48.3229 | 50.1592 | 50.8613 |
| 106.85 | S1 | 110.2827 | 112.8762 | 112.8134 | 115.0697 | 107.6863 |
| 54.32 | S2 | 57.5621 | 57.0891 | 57.5909 | 59.3762 | 60.6261 |
| 23.41 | S3 | 24.9368 | 25.3758 | 23.9947 | 24.9785 | 24.5732 |
| 7.37 | S4 | 8.3332 | 8.3079 | 7.9536 | 8.1843 | 8.5471 |
| 2.50 | S5 | 2.7045 | 2.7441 | 2.9045 | 2.9713 | 2.9642 |
| 0.23 | S6 | 0.2302 | 0.2549 | 0.2264 | 0.2332 | 0.2340 |
| 0.19 | S7 | 0.2390 | 0.2256 | 0.2184 | 0.2289 | 0.2355 |
| 0.10 | S8 | 0.1351 | 0.0968 | 0.0811 | 0.0827 | 0.1033 |
| 0.0315 | S9 | 0.0573 | 0.0357 | 0.0340 | 0.0352 | 0.0408 |
| 0.0256 | S10 | 0.0372 | 0.0268 | 0.0260 | 0.0268 | 0.0295 |
| / | N1 | 0.0207 | 0.0170 | 0.0050 | 0.0051 | 0.0123 |
| / | N2 | -0.0520 | 0.0144 | -0.0020 | -0.0030 | -0.0103 |
| / | N3 | -0.0162 | 0.0093 | 0.0000 | 0.0000 | -0.0017 |
| / | N4 | 0.0287 | -0.0036 | -0.0007 | -0.0006 | 0.0059 |
Because the light-sensitive amount of the light-sensitive reagent 1 is too low, the experiment does not need to continue to adopt the light-sensitive reagent 1 to participate in the performance test of accuracy. From the test data in table 12, it can be seen that the higher the exposure amount of the exposure agent, the closer the test data to the theoretical value, i.e., the higher the accuracy. The light sensing amount of the light sensing reagent 2 is the lowest, the concentration value measured for the sample of the low concentration target molecule fluctuates greatly from the theoretical value, and the concentration value measured for the sample of the high concentration target molecule is closer to the theoretical value, so the light sensing amount of the light sensing reagent 2 can be regarded as the lower limit of the light sensing amount. That is, when the light-sensitive amount of the light-sensitive microspheres is less than 1.34, the target molecules with various concentrations cannot be accurately detected, which is unfavorable for meeting the clinical detection requirements.
2.3 testing the accuracy of the detection results of 5 photosensitive Agents with different amounts of light
And continuously selecting samples sp1, sp2 and sp3 which are diluted to three known different concentrations and have target molecules with the same known mass in the 2.2, dividing the samples with each concentration into 10 parts, respectively testing with 5 photosensitive reagents to obtain corresponding concentration values, and obtaining a concentration Mean value Mean, standard deviation STDEV and variation coefficient CV of the 10 parts of samples. Specific values are shown in table 13.
TABLE 13
From the data in table 13, it is clear that the CV value of the same sample sp1 at the lowest concentration of the photosensitizing agent 2 is more than 10%, which means that the fluctuation of the test result is large and the accuracy is general, and therefore the photosensitizing amount 1.34 of the photosensitizing agent 2 can be used as the lower limit of the photosensitizing amount required for the photosensitizing microsphere in clinical detection.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (17)
1. The application of photosensitive microsphere in preparing microsphere composition, reagent combination, kit, detection system and/or detection device for light-activated chemiluminescence detection is characterized in that,
The photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, wherein the photosensitive quantity Ps of the photosensitive microsphere is between 1.34 and 16.28; the sensitization amount ps=od λ1 /C 2 *10 3 Wherein:
the OD is λ1 The visible light region of 300-800 nm has a specific concentration of C 2 The lambda is the absorbance value corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained after full wavelength scanning of the photosensitive microsphere 1 Is the wavelength corresponding to the maximum absorption peak; the C is 2 Is the concentration of the photosensitive microsphere during the photo-excitation chemiluminescence detection,C 2 in ug/ml.
2. The use according to claim 1, wherein,
concentration of the photosensitive microsphere
Where k is the corresponding slope in the linear relationship of carrier concentration-absorbance curve and b is the corresponding intercept in the linear relationship of the carrier concentration-absorbance curve; OD (optical density) λ2 Is photosensitive microsphere at wavelength lambda 2 The corresponding absorbance value is lower; the carrier concentration-absorbance curve is at wavelength lambda using multiple carriers of different concentrations 2 A curve obtained below; the wavelength lambda 2 The photosensitive microsphere and the carrier which have the same concentration have the same or similar absorbance values corresponding to the wavelength-absorbance curve.
3. The use according to claim 2, wherein said C 2 Selected from 10ug/ml to 200ug/ml.
4. The use according to claim 2, wherein the linear relationship of carrier concentration-absorbance curve is y = kx+b, wherein:
x is different concentrations of the carrier with preset particle size, y is absorbance value of the carrier at the corresponding concentration, k is slope, and b is intercept.
5. The use according to claim 2, wherein the wavelength λ is 2 Selected from OD Photosensitive microsphere /OD Carrier body A ratio of any one of the wavelength values within 0.85 to 1.15, and a wavelength lambda 2 Not equal to wavelength lambda 1 ；
Wherein OD Photosensitive microsphere And OD (optical density) Carrier body The absorbance values corresponding to the same wavelength values of the photosensitive microspheres and the carriers with the same concentration in the range of 300 nm-800 nm are respectively utilized.
6. The use according to claim 5, wherein the wavelength λ is 2 400nm to 600nm.
7. The use according to claim 1, wherein the photosensitive microsphere is a carrier filled with a photosensitive substance, wherein the wavelength λ 1 The wavelength corresponding to the maximum absorption peak in a wavelength-absorbance curve obtained by scanning the photosensitive material in the full wavelength range of 300-800 nm in the visible light region of the photosensitive material.
8. The use according to claim 7, wherein the wavelength λ is 1 600nm to 700nm.
9. The use according to claim 7, wherein said photosensitive microspheres are produced according to a mass ratio of said carrier to said photosensitive substance of 10 (0.04-4).
10. The use according to any one of claims 2 to 9, wherein the carrier has a particle size of 190nm to 280nm.
11. Use according to any one of claims 1 to 10, wherein the surface of the photosensitive microsphere is not coated with a polysaccharide and the surface of the photosensitive microsphere is attached with an avidin selected from the group consisting of ovalbumin, vitellin, streptavidin, neutravidin and avidin-like molecules, preferably streptavidin.
12. The use according to any one of claims 1 to 10, wherein the photoexcitation chemiluminescence detects a chemiluminescent signal generated by a luminescent complex formed by a luminescent microsphere-immunocomplex-photosensitive microsphere;
the luminescent microsphere comprises a carrier and a luminescent substance carried by the carrier, wherein the luminescent substance can react with singlet oxygen to generate chemiluminescence;
the photosensitive microsphere can generate singlet oxygen under the excitation of light.
13. Microsphere composition for photoexcited chemiluminescent detection, characterized by comprising luminescent microspheres and said photoactive microspheres in the use according to any one of claims 1 to 12.
14. The reagent combination for photo-activated chemiluminescence detection is characterized by comprising a luminescent reagent and a photosensitive reagent;
the photosensitizing agent comprising a buffer solution and the photosensitizing microspheres for use according to any one of claims 1 to 12 stored in the buffer solution;
the luminescent reagent comprises luminescent microspheres and substances capable of reacting with the to-be-detected substances.
15. The reagent combination of claim 13, wherein the sugar content per liter of volume of the buffer solution is 1g ± 0.2g.
16. The kit for detecting the photo-excitation chemiluminescence is characterized by comprising one or more of the following any items, and substances capable of reacting with an object to be detected, acceptable auxiliary materials, auxiliary agents or carriers;
(I) The photosensitive microsphere in the use according to any one of claims 1 to 12; or (b)
(II) the microsphere composition of claim 13; or (b)
(III), the combination of reagents of claim 14 or 15;
such carriers include, but are not limited to: reagent bottles, reagent cards, test strips or chips.
17. A system or apparatus for photoexcitation chemiluminescent detection, comprising one or more of any of the following, and an acceptable module or component;
(I) The photosensitive microsphere in the use according to any one of claims 1 to 12; or (b)
(II) the microsphere composition of claim 13; or (b)
(III), the combination of reagents of claim 14 or 15; or (b)
(IV) the kit of claim 16.
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