CN118483430A - Microsphere composition and kit for myocardial troponin T immunoassay - Google Patents
Microsphere composition and kit for myocardial troponin T immunoassay Download PDFInfo
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Classifications
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Urology & Nephrology (AREA)
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- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
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Abstract
The invention relates to a microsphere composition and a kit for myocardial troponin T immunoassay. The microsphere composition comprises a luminescent microsphere and a photosensitive microsphere, wherein the luminescent microsphere is coated with a cardiac troponin T antibody, the photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, the photosensitive substance can generate singlet oxygen under the excitation of light, and the photosensitive quantity Ps of the photosensitive microsphere is 1.34-16.28. The photosensitive microsphere in the microsphere composition for myocardial troponin T immunodetection provided by the invention has specific photosensitive amount Ps. When the microsphere composition or the kit comprising the microsphere composition is used for carrying out immunodetection on the cardiac troponin T, better analysis sensitivity can be obtained, and the analysis sensitivity can be less than 3pg/mL.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a microsphere composition and a kit for myocardial troponin T immunodetection.
Background
Cardiac troponin T (cTnT) is a characteristic contractile protein on cardiac muscle fibers, with a molecular weight of 39.7kDa, which is one of three subunits of troponin (I, T, C), which, together with tropomyosin, binds actin via the myofilaments of myofibrils.
CTnT is a specific and highly sensitive marker of myocardial injury, rises rapidly After Myocardial Infarction (AMI), and can last for two weeks. The method is mainly used for clinically auxiliary diagnosis of the necrosis of the acute coronary syndrome, such as acute myocardial infarction. And is also an index of risk stratification in patients with acute coronary syndromes and myocardial risk in patients with chronic renal failure.
The sensitivity of the detection of the cardiac troponin T is improved, the early diagnosis of acute myocardial infarction can be obviously improved in clinical application, and the detection time is shortened; improving early risk stratification and prognosis evaluation of acute coronary syndrome; more patients with unstable angina previously diagnosed are diagnosed as non-ST elevation myocardial infarction (nstemii), which is beneficial to take corresponding therapeutic measures earlier and more aggressively, and also makes it possible to identify chronic structural myocardial damage.
The photo-excitation chemiluminescence method has the advantages of high sensitivity, high specificity, simple and quick operation, easy standardized operation, short time consumption, good reagent stability, no pollution and the like, as the other chemiluminescence methods. In addition, by introducing the laser technology and the nano microsphere technology, the reaction is carried out in a homogeneous phase, so that the reaction speed is increased, repeated separation and cleaning steps are avoided, the detection background value can be effectively reduced, the reaction time is shortened, and the automatic operation can be realized.
Photosensitive microspheres are microspheres that can be exposed to laser light to activate ambient oxygen. The microsphere contains phthalocyanine substances, and various functional groups are provided on the surface of the microsphere for coupling biomolecules, such as avidin molecules. If the photosensitive microsphere is irradiated by excitation light (680 nm), the phthalocyanine in the coating can instantaneously generate high-energy singlet oxygen ions (oxygen molecules with one excited electron), and the luminescent microsphere can be induced to generate optical signals. In order to further improve the sensitivity of cTnT detection, the excitation amount of singlet oxygen ions can be changed by changing the filling amount of the photosensitive substances in the photosensitive microspheres, so that the light signal intensity generated by the luminescent microspheres is changed, and further higher analysis sensitivity is obtained.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a microsphere composition for the immune detection of cardiac troponin T, and better analysis sensitivity can be obtained when the microsphere composition or a kit comprising the microsphere composition is used for the immune detection of cardiac troponin T.
To this end, the first aspect of the present invention provides a microsphere composition for cardiac troponin T immunoassay, comprising a luminescent microsphere and a photosensitive microsphere, wherein the luminescent microsphere is coated with cardiac troponin T antibodies, the photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, the photosensitive substance is capable of generating singlet oxygen under light excitation, and the photosensitive amount Ps of the photosensitive microsphere is 1.34 to 16.28.
In some embodiments of the invention, the amount of sensitization ps= (OD λ1/C2)×103; wherein the OD λ1 is the absorbance value at wavelength λ1 corresponding to a sensitization microsphere having a mass concentration of C2, the C2 being the mass concentration of the sensitization microsphere used in performing a cardiac troponin T immunoassay in μg/ml.
In other embodiments of the present invention, λ1 is the wavelength to which the photosensitive microsphere has a maximum absorbance value in the visible light range of 300 to 800 nm; preferably, the lambda 1 is 600-700 nm; more preferably, the λ1 is 680nm.
In some embodiments of the invention, the photosensitive microsphere has a mass concentration c2= (OD λ2 -b)/k; wherein k and b are respectively the slope and intercept corresponding to a standard curve of carrier concentration-absorbance values at a wavelength of lambda 2, OD λ2 is the absorbance value corresponding to the photosensitive microsphere at the wavelength of lambda 2, and the carrier concentration-absorbance curve is a curve obtained by adopting a plurality of carriers with different concentrations at the wavelength of lambda 2; the wavelength lambda 2 is a wavelength corresponding to the absorbance value of the photosensitive microsphere with the same concentration and the carrier with the same or similar absorbance value in the wavelength-absorbance curve.
In other embodiments of the present invention, the λ2 is selected from any absorption wavelength value with the ratio of OD Photosensitive microsphere /OD Carrier body within 1±15%, the OD Photosensitive microsphere and OD Carrier body are absorbance values corresponding to the same wavelength value of the photosensitive microsphere and the carrier in the visible light region of 300-800 nm, respectively, and the wavelength λ2 is not equal to the wavelength λ1; preferably, the lambda 2 is 400-600 nm; more preferably, the λ2 is 500nm.
In some embodiments of the present invention, the method of determining the amount of light sensing Ps of the photosensitive microsphere comprises the steps of:
s1, establishing a standard curve of carrier concentration-absorbance value at a wavelength lambda 2 according to a carrier with a series of concentrations, and further obtaining a slope k and an intercept b of the standard curve;
S2, detecting an absorbance value OD λ2 corresponding to the photosensitive microsphere at a wavelength lambda 2, and calculating the mass concentration C2 of the photosensitive microsphere according to the slope k and the intercept b obtained in the step S1;
s3, detecting an absorbance value OD λ1 corresponding to the photosensitive microsphere at the wavelength lambda 1, and calculating the photosensitive quantity Ps of the photosensitive microsphere according to the C2 obtained in the step S2.
In other embodiments of the invention, the carrier is polystyrene microspheres that do not contain photosensitive material.
In some embodiments of the invention, the carrier particle size is 150 to 250nm; preferably 180-200 nm; more preferably 190nm.
In some embodiments of the invention, the photosensitive microsphere is coated with streptavidin.
In some embodiments of the invention, the photoactive material comprises at least one of methylene blue, rose bengal, porphyrin, and phthalocyanine.
In a second aspect the invention provides a kit for cardiac troponin T immunoassay comprising a microsphere composition according to the first aspect of the invention.
In some embodiments of the invention, the kit further comprises:
Agent 1 comprising luminescent microspheres coated with cardiac troponin T antibodies;
reagent 2 comprising a biotin-labeled cardiac troponin T antibody.
In some embodiments of the invention, the kit specifically comprises:
A universal photosensitive microsphere liquid, which comprises photosensitive microspheres coated by streptavidin;
Agent 1 comprising luminescent microspheres coated with cardiac troponin T antibodies;
reagent 2 comprising a biotin-labeled cardiac troponin T antibody.
In some embodiments of the invention, the photosensitive microsphere has a photosensitivity Ps of 1.34 to 16.28.
In some embodiments of the invention, the concentration of the streptavidin-coated photosensitive microsphere is 50 μg/mL; and/or the concentration of the luminescent microsphere coated by the cardiac troponin T antibody is 100 mug/mL; and/or, the biotin-labeled cardiac troponin T antibody concentration is 2 μg/mL.
In some embodiments of the invention, the luminescent microspheres are polymeric microspheres that are coated on a substrate with functional groups to form a matrix filled with luminescent compounds and lanthanoids.
The beneficial effects of the invention are as follows: the photosensitive microsphere in the microsphere composition for myocardial troponin T immunodetection provided by the invention has specific photosensitive amount Ps. When the microsphere composition or the kit comprising the microsphere composition is used for carrying out immunodetection on the cardiac troponin T, better analysis sensitivity can be obtained, and the analysis sensitivity can be less than 3pg/mL.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a graph showing the particle size results of a 20ug/ml carrier measured by a particle sizer according to an embodiment 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 according to an embodiment of the application;
FIG. 4 shows wavelength-absorbance curves for different concentrations of carrier according to embodiments of the application;
FIG. 5 shows wavelength-absorbance curves for various concentrations of photosensitive microspheres according to an embodiment of the application;
FIG. 6 shows a wavelength-absorbance curve of 10 μg/ml of carrier and photosensitive microspheres according to an embodiment of the application;
FIG. 7 shows a carrier concentration-absorbance curve of a carrier at a wavelength of 500nm according to an embodiment of the application;
FIG. 8 shows a concentration-amount of photosensitive material curve for a specific embodiment of the present application;
FIG. 9 shows a graph of the linear dependence of the concentration of samples measured using the Roche troponin T assay kit and the same samples measured using the kit of example 4 of the present invention.
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 microsphere composition for myocardial troponin T immunoassay according to the first aspect of the present invention comprises a luminescent microsphere and a photosensitive microsphere, wherein the luminescent microsphere is coated with myocardial troponin T antibody, the photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, the photosensitive substance can generate singlet oxygen under the excitation of light, and the photosensitive quantity Ps of the photosensitive microsphere is 1.34-16.28.
In some embodiments of the present application, the photosensitive microsphere may have a photosensitive amount Ps of 1.34、2.5、2.51、3、3.5、4.05、4.5、4.99、6、7、8、9、10.03、11.12、12、13、14.93、15、16、16.28、17、17.6、18、19、20.19、21、22、23、24 or 25, etc. In some more preferred embodiments of the present application, the photosensitive microsphere has a photosensitivity Ps of 1.34 to 16.28. 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 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 the definite numerical limitation of the photosensitive quantity, the operability is definite, and the photosensitive microsphere is suitable for popularization of industry specifications.
In the invention, the photosensitive microsphere comprises 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. Under the excitation of red laser (600-700 nm), the photosensitive microsphere releases active oxygen (such as singlet oxygen) with high energy state. When the distance between the photosensitive microsphere and the luminous microsphere is close enough, the singlet oxygen released by the photosensitive microsphere can reach the luminous microsphere and emit light with high energy level of 520-620 nm through a series of chemical reactions, so that the light is detected by an instrument.
In the present invention, the carrier may be polystyrene microspheres, but may be microspheres made of other materials for detection, which is not limited thereto.
In the present invention, the photosensitive substance may be, for example, a photosensitizer or a photosensitizing 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.
In the present invention, 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.
In some embodiments of the invention, the photosensitive microsphere has a photosensitivity ps= (OD λ1/C2)×103; wherein OD λ1 is the absorbance value at wavelength λ1 corresponding to a photosensitive microsphere having a mass concentration of C2, and C2 is the mass concentration of the photosensitive microsphere used in performing the cardiac troponin T immunoassay in μg/ml.
In other embodiments of the present invention, λ1 is the wavelength to which the photosensitive microsphere has a maximum absorbance value in the visible light range of 300 to 800 nm.
In the present invention, the method for determining λ1 may be: full-wavelength scanning is carried out on the photosensitive microsphere with the concentration of C2 dissolved in the buffer solution by using visible light in a range of 300-800 nm through a spectrophotometer and other equipment, absorbance values corresponding to different wavelength scanning are read, and after a corresponding wavelength-absorbance curve is generated, the wavelength lambda 1 is selected from the wavelength corresponding to the maximum characteristic peak (namely the maximum absorption peak) of the photosensitive microsphere in the wavelength-absorbance curve; correspondingly, the absorbance value corresponding to the maximum characteristic peak in the wavelength-absorbance curve is selected, so that the absorbance value OD λ1 of the photosensitive microsphere with the concentration of C2 corresponding to the wavelength lambda 1 can be determined.
In some preferred embodiments of the invention, the λ1 is 600 to 700nm.
In some most preferred embodiments of the invention, the λ1 is 680nm.
In order to ensure accuracy of the value of the wavelength λ1, in some embodiments, the wavelength-absorbance curves of the photosensitive microspheres having different known concentrations and the same photosensitive substance may be measured in advance, and the wavelength corresponding to the maximum characteristic peak may be selected from the wavelength-absorbance curves of the photosensitive microspheres at each concentration as the value of λ1. 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 1 of the photosensitive substance and the photosensitive microspheres is 680nm. Therefore, by selecting the wavelength λ1 corresponding to the maximum characteristic peak of the wavelength-absorbance curve of the photosensitive microsphere and then determining the corresponding absorbance value OD λ1, the accuracy of the calculation result of the amount of photosensitive light can be ensured. When the photosensitive microspheres adopt 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 specific value of the wavelength lambda 1.
In some embodiments of the invention, to facilitate defining the adjustment range of the concentration C2 of the photosensitive microsphere, the mass concentration c2= (OD λ2 -b)/k of the photosensitive microsphere; wherein k and b are respectively the slope and intercept corresponding to a standard curve of carrier concentration-absorbance values at a wavelength of lambda 2, OD λ2 is the absorbance value corresponding to the photosensitive microsphere at the wavelength of lambda 2, and the carrier concentration-absorbance curve is a curve obtained by adopting a plurality of carriers with different concentrations at the wavelength of lambda 2; the wavelength lambda 2 is a wavelength corresponding to the absorbance value of the photosensitive microsphere with the same concentration and the carrier with the same or similar absorbance value in the wavelength-absorbance curve.
Before use, the initial state of the photosensitive microspheres during storage is generally a lyophilized solid substance or a refrigerated liquid. When the photosensitive microsphere is a solid substance, a buffer solution is required to be added for re-dissolution, and the concentration of the re-dissolved photosensitive microsphere solution is the initial concentration C1. When the photosensitive microsphere is stored as a liquid, the concentration at this time is the initial concentration C1. When the photo-excitation chemiluminescence detection is carried out, the photosensitive microsphere can directly participate in the detection by adopting the initial concentration, namely the concentration C2 of the photosensitive microsphere during the detection is equal to C1; or the photosensitive microsphere with the initial concentration C1 can be diluted and then participates in detection, namely, the concentration C2 of the photosensitive microsphere during the detection at the moment is not equal to C1, and the value of C2 is the true concentration after the corresponding initial concentration is diluted. It will be appreciated that when the concentration C2 of the photosensitive microsphere is changed after the specific value of the wavelength λ1 is determined, the corresponding absorbance value OD λ1 may be correspondingly different, and the value of OD λ1 is determined according to the actual measurement. After the values of the absorbance value OD λ1 and the concentration C2 are determined, the light sensing amount Ps of the light sensing microsphere can be determined according to the ratio of the absorbance value OD λ1 and the concentration C2.
In the present application, in order to obtain the concentration C2 of the photosensitive microsphere satisfying the range of the light sensing amount Ps, experiments may be performed using the same material and particle size carrier as the photosensitive microsphere to determine the range of the concentration C2 of the photosensitive microsphere. The loading amount of carriers with different particle diameters at the same concentration on the photosensitive substance can be different, so that the absorbance value is influenced, namely the carrier concentration-absorbance curve is also related to the particle diameter of the carrier. 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", and the like 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 carriers is selected from 190 nm-280 nm; and the absorbance values corresponding to the carriers with the concentrations are respectively scanned and measured by adopting visible light with the same wavelength lambda 2, so that an accurate and reliable relation curve of the carrier concentration and the absorbance is established; 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 carrier to control the calculated concentration C2 of the photosensitive microsphere to be within 10% of the true concentration. A linear relationship of carrier concentration versus absorbance is then obtained, which can be expressed as y=kx+b. Wherein x is different concentrations of the carrier with preset particle size, y is an absorbance value of the carrier at the corresponding concentration, k is a slope in formula c2= (OD λ2 -b)/k, and b is an intercept in formula c2= (OD λ2 -b)/k. That is, by the correlation calculation of the formula y=kx+b, the values of k and b in the formula c2= (OD λ2 -b)/k can be determined, and thus the concentration C2 of the photosensitive microsphere can be determined. Preferably, C2 is selected from 10ug/ml to 200ug/ml.
In the invention, the obtaining modes of the OD Photosensitive microsphere and the OD Carrier body are as follows: the spectrophotometry is utilized to scan the full wavelength of the photosensitive microsphere and the carrier with the same concentration in the visible light region with the wavelength of 300-800 nm, and then the absorbance curves of the wavelength-absorbance values of the photosensitive microsphere and the carrier are respectively obtained; the absorbance values corresponding to the same wavelength values on the two obtained absorbance curves are OD Photosensitive microsphere and OD Carrier body , and when the ratio of the OD Photosensitive microsphere /OD Carrier body is within + -15%, any absorbance wavelength value is lambda 2.
In some preferred embodiments of the invention, the λ2 is 400 to 600nm.
In some more preferred embodiments of the invention, the λ2 is 500nm.
In one embodiment of the present invention, the wavelength λ2 may be 440nm to 580nm. For example, the wavelength λ2 may be 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm.
In the present invention, to determine the values of k and b, in some embodiments, wavelength λ2 is selected from any wavelength value for which the ratio of OD Photosensitive microsphere /OD Carrier body is within 0.85 to 1.15, and wavelength λ2 is not equal to wavelength λ1; wherein, OD Photosensitive microsphere and OD Carrier body are absorbance values corresponding to the same wavelength value of the photosensitive microsphere and the carrier with the same concentration in the range of 300nm to 800nm respectively. It is understood that the photosensitive microsphere and the carrier having the same microsphere concentration are scanned at the same wavelength in the range of 300nm to 800nm, respectively, and the absorbance value OD Photosensitive microsphere of the photosensitive microsphere at the concentration in the full wavelength range and the absorbance value OD Carrier body of the carrier at the concentration in the full wavelength range can be obtained.
The applicant researches show that specific experimental data can be checked in the following related content, and the wavelength with the OD Photosensitive microsphere /OD Carrier body ratio meeting the range of 0.85 to 1.15 (namely 1+/-15%) is selected as lambda 2, so that compared with the wavelength outside the range of the ratio, the value range of the concentration C2 of the photosensitive microsphere can be more accurately determined through the carrier concentration-absorbance curve of the carrier. Experiments show that when the wavelength lambda 2 is in the range of 440-580 nm, the ratio of the absorbance value OD Photosensitive microsphere of the photosensitive microsphere to the absorbance value OD Carrier body of the carrier is within 0.85-1.15, which indicates that the content of the photosensitive substance has less influence on the measurement of the microsphere concentration, otherwise. 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 λ1 and the wavelength λ2 are correspondingly adjusted, and the OD λ1 and the OD λ2 are correspondingly adjusted. After determining the wavelength λ2, a plurality of carriers with known different concentrations x and the same particle size can be scanned by adopting the same wavelength λ2 to obtain a corresponding absorbance value y, so that an equation is established according to a formula y=kx+b to calculate and obtain values of k and b, and thus the concentration C2 of the photosensitive microsphere with the same particle size as the carrier can be calculated according to a formula c2= (OD λ2 -b)/k.
In some embodiments of the present invention, the wavelength λ2 is not equal to the wavelength λ1, i.e., the wavelength λ2 is a wavelength corresponding to a non-characteristic peak in the wavelength-absorbance curve, i.e., a wavelength that avoids a characteristic peak of the photosensitive material, reducing the effect of the absorbance value of the photosensitive material itself on the absorbance value of the carrier.
In the present invention, under the premise that the known light sensing amount Ps ranges from 1.34 to 16.28, and k, b and λ2 are known according to the above calculation, conversely, the concentration C2 of the photosensitive microsphere can be adjusted according to the formula ps= (OD λ1/C2)×103 and c2= (OD λ2 -b)/k. That is, after the photosensitive microsphere with unknown concentration is arbitrarily configured in the actual photo-excitation chemical detection process, the photosensitive microsphere with unknown concentration is scanned by the wavelength λ1 and the wavelength λ2 respectively to obtain the corresponding absorbance values, namely OD λ1 and OD λ2, and then the specific value of the unknown concentration is calculated by the formula c2= (OD λ2 -b)/k. If the value range of the unknown concentration falls within the range of 10ug/ml to 200ug/ml, the calculated concentration C2 is substituted into the formula ps= (OD λ1/C2)×103 to calculate the light sensing amount Ps, if the Ps value is between 1.34 and 16.28, the configured concentration can be detected by the photo-excitation light sensing microsphere.
In summary, under the condition that the concentration C2 of the photosensitive microsphere is known and the value range is 10ug/ml to 200ug/ml, the value of the photosensitive quantity Ps corresponding to the photosensitive microsphere can be directly obtained by calculation according to the formula Ps= (OD λ1/C2) without using the formula C2= (OD λ2 -b)/k and y=kx+b; similarly, on the premise of unknown specific numerical value of the photosensitive microsphere concentration C2, the photosensitive quantity Ps value corresponding to 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.
In some embodiments of the present invention, the method of determining the amount of light sensing Ps of the photosensitive microsphere comprises the steps of:
s1, establishing a standard curve of carrier concentration-absorbance value at a wavelength lambda 2 according to a carrier with a series of concentrations, and further obtaining a slope k and an intercept b of the standard curve;
S2, detecting an absorbance value OD λ2 corresponding to the photosensitive microsphere at a wavelength lambda 2, and calculating the mass concentration C2 of the photosensitive microsphere according to the slope k and the intercept b obtained in the step S1;
s3, detecting an absorbance value OD λ1 corresponding to the photosensitive microsphere at the wavelength lambda 1, and calculating the photosensitive quantity Ps of the photosensitive microsphere according to the C2 obtained in the step S2.
In some embodiments of the present invention, the step S1 specifically includes: preparing carriers with different concentration gradients and series concentrations, detecting the absorbance value of the carriers with the series concentrations at the wavelength lambda 2 by using a spectrophotometer, and then establishing a standard curve of the carrier concentration-absorbance value according to the obtained absorbance value.
In some embodiments of the invention, the carrier is polystyrene microspheres that do not contain photosensitive material.
In the invention, the polystyrene microsphere is a common polymer microsphere material. The polystyrene microsphere has the universality of high molecular microsphere materials, such as small particle size, large specific surface area, strong adsorptivity, good dispersibility, easy modification and the like.
In the invention, the particle size of the carrier is 150-250 nm; preferably 180-200 nm; more preferably 190nm.
In some embodiments of the invention, the photosensitive microsphere is coated with streptavidin.
In the present invention, "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding to a biotin.
The second aspect of the invention relates to a kit for cardiac troponin T immunoassay comprising a microsphere composition according to the first aspect of the invention.
In some embodiments of the invention, the kit further comprises:
Agent 1 comprising luminescent microspheres coated with cardiac troponin T antibodies;
reagent 2 comprising a biotin-labeled cardiac troponin T antibody.
In the present invention, the "luminescent microsphere (FG)" is a polymeric microsphere that may be coated on a substrate with functional groups to form a matrix filled with luminescent compounds and lanthanoids. 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 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 invention, the biotin is widely existed in animal and plant tissues, and has two cyclic structures on the molecule, namely an imidazolone ring and a thiophene ring, wherein the imidazolone ring is a main part combined with avidin. 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.
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.
Example 1 preparation of luminescent reagent
1. Dialyzing the raw material, taking 1mg of antigen 1, placing in a 3.5KD dialysis bag, and dialyzing 3 times in 0.02M HEPES buffer solution (pH 8.0);
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 2.78mg/ml.
3. Particle treatment, 10mg/mlFG particles 1ml in a centrifuge tube, after centrifugation for 30 minutes, the supernatant was discarded, and the suspension was sonicated with 0.05M MES buffer, repeated, and 20mg/ml of each of NHS and EDAC 25ul was added. Rapidly and uniformly mixing to obtain an activating solution, putting the activating solution into a multi-tube adjustable rotary mixer, centrifuging for 30 minutes, and discarding the supernatant for later use;
4. Coupling reaction, according to particles: mixing FG and antigen protein according to the mass ratio of the protein of 10:0.2, performing coupling reaction by using a rotary mixing mode, and performing rotary reaction for 2 hours; then, 0.1ml 75mg/ml Gly solution, 0.1ml 100mg/ml BSA solution was added thereto, and the mixture was stirred and reacted for 20 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 50ug/ml by using 50ml of luminous reagent buffer solution, and balancing for 2 hours for later use.
EXAMPLE 2 preparation of Biotin reagent
1. Raw material dialysis, namely respectively taking 1mg of antigen 2, respectively placing the antigen 2 into 3.5KD dialysis bags, and dialyzing for 3 times in 0.1M NaHCO3 buffer solution;
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. Labeling reaction, preparing 20mg/ml biotin NHS active ester according to protein: the labeling reaction is carried out by adopting a rotary mixing mode according to the biotin mol ratio of 1:30, and the reaction is carried out for 20 hours.
4. Dialysis of the marked product, product dialysis, dialysis bag specification: 3500D, dialysis buffer: 0.02MHEPES buffer, dialysis temperature: 2-8 ℃, dialysis time and 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 2.12mg/ml for further use.
5. The biotin reagent is prepared, 50ml of biotin reagent buffer is used, and the prepared biotin concentrated solution is diluted to 0.5ug/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 each of the three-necked flask, and after stirring for 10 minutes, N 2 min was introduced into the three-necked flask.
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. The aqueous solution was added to the reaction system of the three-necked flask of step a), and N 2 min was continued.
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 above 1.e) of the prepared carrier were added, and the mixture was stirred uniformly by magnetic force, and the three-necked flask was heated 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. Method for determining value application range of photosensitive microsphere concentration C 2
The experiment adopts the preset particle size to establish the linear relation between the carrier concentration and the absorbance value, so that the concentration C 2 of the photosensitive microsphere is obtained according to the absorbance value OD λ2 in the formula (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 1.
TABLE 1
| 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 the carriers 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 diameter of the carrier is 187.1nm, and the average particle diameter of the photosensitive microsphere is 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 Determination of wavelength lambda 1 and wavelength lambda 2
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, by reading the absorbance value of 680nm to most reflect the content of the photosensitive substance in the photosensitive microsphere, the wavelength corresponding to the maximum characteristic peak can be selected as lambda 1, namely lambda 1 is 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 is determined according to the actual situation.
For the determination of the wavelength lambda 2, the purpose is to determine the concentration C 2 of the photosensitive microspheres from the corresponding absorbance value OD λ2. Therefore, for determining the concentration of the photosensitive microspheres based on the absorbance value, it is necessary to avoid the maximum characteristic peak of the photosensitive substance, that is, the wavelength λ 2 is different from λ 1. 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, lambda 2 is selected to be optimal between 400nm and 600nm, and no characteristic peak exists in the wavelength interval. 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 low, 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 consistent, therefore, the wavelength lambda 2 is selected from the wavelength of 400 nm-600 nm.
Further, in order to accurately determine the value of the wavelength lambda 2, the absorbance of one of the photosensitive microspheres and the carrier with the same concentration is used for analysis. Photosensitive microspheres and carriers at a concentration of 50ug/ml were used as examples and are shown in Table 2.
To reduce the effect of the photoactive material on the concentration of the microsphere being measured, the range of wavelengths lambda 2 selected requires a ratio of OD Photosensitive microsphere /OD Carrier body within 0.85 to 1.15 at the same wavelength, i.e., (1+15%). As can be seen from table 2, the ratio of the absorbance value OD Photosensitive microsphere of the photosensitive microsphere of 50ug/ml to the absorbance value OD Carrier body of the carrier of 50ug/ml is within 0.85 to 1.15 when the wavelength lambda 2 is between 440nm and 580nm, thereby indicating that the absorbance value of the photosensitive substance corresponding to the wavelength in the interval has less influence on the absorbance values of the photosensitive microsphere and the carrier, and that the content of the photosensitive substance affects the determination of the concentration of the photosensitive microsphere when the ratio of OD Photosensitive microsphere /OD Carrier body is greater than 1.15. Preferably, the ratio of OD Photosensitive microsphere /OD Carrier body is 1.05 at wavelengths of 500nm and 510nm, the constant ratio indicating a constant effect of the photosensitive material. Therefore, in this embodiment, the wavelength lambda 2 is preferably 500nm. It will be appreciated that when photosensitive materials of different materials are selected to prepare photosensitive microspheres, the wavelength lambda 2 can be determined with reference to the above method.
TABLE 2
5. Establishment of Carrier concentration-absorbance curve
In this embodiment, the carrier concentration-absorbance curve is established by selecting carriers with the same particle size and testing the absorbance values of different concentrations of the carriers at the wavelength lambda 2.
After the wavelength lambda 2 was determined in the above step 4.4, the experiment was conducted by selecting a carrier having a wavelength lambda 2 of 500nm and a particle diameter of about 190nm, and the carrier concentration-absorbance curve 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. And scanning the carrier with each concentration by using a wavelength of 500nm to obtain an absorbance value OD 500 corresponding to each concentration, and establishing a linear relation y=kx+b between the carrier concentration and the absorbance value. At a known concentration x, when lambda 2 is 500nm, the absorbance y can be measured directly from the spectrophotometer, so that k of 0.0021 and b of 0.0359 can be calculated. After the values of k and b are determined, the carrier concentration x= (OD λ2-b)/k＝(ODλ2 -0.0359)/0.0021 is according to equation (2).
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. Firstly, respectively preparing carriers with different particle diameters, the particle size of each carrier comprises 190nm,200nm,220nm,240nm,260nm,280nm,300nm and the like. 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 value OD λ2 of the carrier at each concentration of each particle size was measured at a wavelength of 500nm, given the theoretical concentration value. To obtain accurate results, each concentration of each particle size was divided into 3 parts for detection of absorbance values. After obtaining the absorbance value OD λ2 corresponding to each concentration of each particle diameter of each carrier, the corresponding concentration value was calculated as (OD λ2 -0.0359)/0.0021, and the calculated result was compared with the theoretical concentration value, and the deviation of the calculated result was determined. The results are shown in Table 3:
TABLE 3 Table 3
As can be seen from the data in Table 3, when the particle diameter of the carrier is less than or equal to 280nm, the recovery deviation of the concentration is within 10%, that is, the deviation of the concentration value x of the carrier calculated according to (OD λ2 -0.0359)/0.0021 from the theoretical concentration value is within 10%, 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 using the above method can be applied to the calculation of the concentration C 2 of the photosensitive microspheres in the formula (2).
As can be seen from FIG. 7, in this experiment, the C 2 has a good linear relationship when the value is 10ug/ml to 100 ug/ml. It should be noted that, the value of C 2 is limited to 10ug/ml to 100ug/ml, and the value range of C 2 can be further determined by combining the following experiments.
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, namely, the microspheres 1 to 6 in the table 4. 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 absorbance values OD corresponding to the wavelength lambda 1 nm and the wavelength lambda 2 nm, calculating according to the formula (2) to obtain a corresponding concentration value C 2, and calculating according to the formula (2) to obtain a corresponding photosensitive quantity Ps. Specific data are shown in table 4 below. It is noted that, as shown in fig. 5, at the wavelength λ 1, the photosensitive microsphere has a strong absorption peak, i.e., a maximum characteristic peak, and the absorbance value corresponding to the absorption peak is the most capable of reflecting the concentration of the photosensitive substance. The absorbance values of the photosensitive microspheres include the absorbance values of the carrier and the photosensitive material, and thus the true absorbance value OD λ1 Photosensitive material of the photosensitive material is OD λ1 Photosensitive microsphere -ODλ1 Carrier body .
TABLE 4 Table 4
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 4, 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 5.
As can be seen from table 4, when the dilution factor is 500X, the calculated value of the concentration value C 2 of the photosensitive microsphere 6 according to the formula (2) is 199, which is very close to the corresponding theoretical concentration value 198. Therefore, the range of C 2 supplemented with the above experiment two, namely the range of C 2 can be 10ug/ml to 200ug/ml.
TABLE 5
As can be seen from table 4, 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 5, CV values of the light-sensitive amounts of the light-sensitive substances of different mass ratios were all within 10%, indicating 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 5. 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.
Example 4 comparison of the Properties of photosensitive microspheres with different light-sensitive amounts in clinical applications
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 according to the volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.
D) The reaction: 25mg/ml NaBH 3 CN solution is prepared by adopting MES buffer solution, and the volume ratio of NaBH 3 CN solution to reaction solution is rapidly and evenly mixed with 1:25. The reaction was carried out at 37℃for 48 hours with rotation.
E) Closing: preparing 75mg/ml Gly glycine solution and 25mg/ml NaBH 3 CN solution by adopting MES buffer solution, preparing mixed solution by adopting Gly glycine solution, naBH 3 CN solution and reaction solution according to the volume ratio of 2:1:10, adding the mixed solution into the solution after the reaction in the step d), uniformly mixing, and rotating at the constant temperature of 37 ℃ for 2 hours. 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 uniformly, 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 photosensitive microspheres coated with streptavidin and adopting 5 different photosensitive amounts in the mass ratio of 10:4, 10:2, 10:1, 10:0.2 and 10:0.04, so as to prepare the photosensitive reagent for obtaining 5 different photosensitive amounts. The sensitization amount of the 5 sensitization agents is shown in table 6.
TABLE 6
| Name of the name | Light sensing amount |
| Photosensitive agent 1 | 1.34 |
| Photosensitive agent 2 | 4.07 |
| Photosensitive agent 3 | 11.49 |
| Photosensitive agent 4 | 16.28 |
| Photosensitive agent 5 | 20.12 |
2. Evaluation of the Properties of the photosensitizing Agents of 5 different photosensitizers
The above 5 kinds of photosensitizing agents having different photosensitizers were applied to the detection of clinical samples, thereby evaluating the basic properties of the photosensitizing agents having different photosensitizers in clinical applications to the detection of samples.
2.1 Test of Signal value of kit containing microsphere compositions with different amounts of light sensitivity
CTnT detection kits were prepared using the prepared 5 different amounts of photosensitizing reagents, as well as reagent 1 (including solution of luminescent microparticles coated with cardiac troponin T antibody (FG-cTnT antibody) at FG-cTnT antibody concentration of 100 μg/mL) and reagent 2 (including solution of Biotin-labeled cardiac troponin T antibody (Biotin-cTnT antibody) at Biotin-cTnT antibody concentration of 2 μg/mL), and 6 groups of cTnT samples with gradient elevation were tested.
The specific steps of detection are as follows:
(1) Adding 10uL of sample solution to be detected, 25uL of reagent 1 and 25uL of reagent 2 into a reaction container, uniformly mixing, and then incubating for 15min at 37 ℃;
(2) Adding 175 μl of the universal solution into the reaction vessel, mixing, incubating at 37deg.C for 10min, and applying The analyzer takes readings.
The test results are shown in Table 7.
TABLE 7
As can be seen from Table 7, as the amount of light sensed by the light sensing microspheres in the universal liquid increased from 1.34 to 16.28, the detection signal and sensitivity of the reagent increased. But there was no significant change in reagent performance when the amount of sensitization was greater than 16.28. The optimal light sensing amount of the photosensitive microsphere in the universal liquid is about 16.28.
2.2 Effect of sensitivity test
The kit of the invention comprises the following components:
General liquid: the solution comprises streptavidin coated photosensitive microsphere, the concentration of the streptavidin coated photosensitive microsphere in the general solution is 50 mug/mL, and the light sensing amount of the photosensitive microsphere is 16.28;
Reagent 1: a solution comprising luminescent microparticles coated with cardiac troponin T antibodies (FG-cTnT antibodies) at a FG-cTnT antibody concentration of 100 μg/mL;
reagent 2: a solution comprising Biotin-labeled cardiac troponin T antibody (Biotin-cTnT antibody) was used at a concentration of 2. Mu.g/mL.
(1) Blank (LoB) detection
5 Blank samples were tested, 4 wells each, for 3 days, and 60 total results were obtained, as shown in Table 8. Setting α=95%, i.e. LoB has a 95% possibility of containing the object to be detected, and sorting the results of the blank samples from small to large by adopting non-parametric test, wherein the 95 th percentile is LoB.
TABLE 8
As can be seen from table 8, loB =1.74 pg/mL was measured.
(2) Detection limit (LoD) detection
5 Low value samples were taken at concentrations ranging from 1LoB to 5LoB, each sample was tested for 4 wells for a total of 3 days, and 60 results were obtained as shown in Table 9. Results were analyzed for homogeneity of variance, with P > 0.05. The mean and standard deviation (SD i) of 60 wells were calculated and the limit of detection (LoD) was calculated according to the following formula:
Formula (1):
Formula (2):
Equation (3):
LoD＝LoB+cpSDL
Wherein:
SD L: SD of a dataset of J low value samples;
SD i: SD of all results for the i-th low value sample;
n i: all result numbers of the ith low value sample;
J: the number of low value samples;
c p: normally distributing multiplier factors of 95 percentiles;
L: total number of all low value sample results.
TABLE 9
As can be seen from table 9, lod=2.29 pg/mL was measured.
(3) Limit of quantification (LOQ) detection:
9 low-value samples are taken, the low value is near the detection limit, the high value is 6-7 times of the detection limit, and 7 samples are arranged according to the equal concentration point approximately during the period. Each sample concentration was measured 10 times for a total of 3 days, and the mean, SD and CV (%) of each well was calculated and then analyzed by precision curve analysis. The results are shown in Table 10.
Table 10
| Mean (pg/mL) | SDWL(pg/mL) | CV(％) | |
| Sample 1 | 1.92 | 0.20 | 10.4％ |
| Sample 2 | 3.98 | 0.33 | 8.2％ |
| Sample 3 | 5.95 | 0.30 | 5.0％ |
| Sample 4 | 8.03 | 0.30 | 3.8％ |
| Sample 5 | 10.07 | 0.31 | 3.1％ |
| Sample 6 | 12.02 | 0.26 | 2.2％ |
| Sample 7 | 13.98 | 0.25 | 1.8％ |
| Sample 8 | 15.95 | 0.24 | 1.5％ |
| Sample 9 | 17.99 | 0.25 | 1.4％ |
And (3) performing precision curve analysis by taking CV as an x axis and taking a sample mean value as a y axis, wherein coefficients of the obtained curves are as follows: y= 0.2917x -0.975
An acceptable standard of 10% of the imprecision will be allowed to enter the equation, yielding y=2.75 pg/mL. I.e., the limit of quantitation was 2.75pg/mL.
The blank limit of the troponin T detection kit (electrochemiluminescence method) Troponin T hs STAT is 3pg/mL, the detection limit is 5pg/mL, and the quantitative limit is 13pg/mL. The performance of the kit of the invention is better than that of the kit.
(4) Correlation comparison
162 Samples were selected, and linear regression analysis was performed with X as the concentration detected by the Roche troponin T assay kit and Y as the concentration detected by the kit of example 4 of the present invention, and the results are shown in FIG. 9.
As can be seen from fig. 9, the correlation r=0.99 between the two is good.
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 (10)
1. A microsphere composition for myocardial troponin T immunoassay, comprising luminescent microspheres and photosensitive microspheres, wherein the luminescent microspheres are coated with myocardial troponin T antibodies, the photosensitive microspheres comprise a carrier and a photosensitive substance carried by the carrier, the photosensitive substance can generate singlet oxygen under the excitation of light, and the photosensitive quantity Ps of the photosensitive microspheres is 1.34-16.28.
2. The microsphere composition of claim 1, wherein the amount of sensitization ps= (OD λ1/C2)×103; wherein OD λ1 is the absorbance value at wavelength λ1 for a sensitization microsphere having a mass concentration of C2, wherein C2 is the mass concentration of the sensitization microsphere used in performing a cardiac troponin T immunoassay in μg/ml.
3. Microsphere composition according to claim 2, characterized in that λ1 is the wavelength to which the photosensitive microsphere has a maximum absorbance value in the visible light range of 300-800 nm; preferably, the lambda 1 is 600-700 nm.
4. A microsphere composition according to claim 2 or 3, characterized in that the mass concentration of the photosensitive microspheres c2= (OD λ2 -b)/k; wherein k and b are respectively the slope and intercept corresponding to a standard curve of carrier concentration-absorbance values at a wavelength of lambda 2, OD λ2 is the absorbance value corresponding to the photosensitive microsphere at the wavelength of lambda 2, and the carrier concentration-absorbance curve is a curve obtained by adopting a plurality of carriers with different concentrations at the wavelength of lambda 2; the wavelength lambda 2 is a wavelength corresponding to the absorbance value of the photosensitive microsphere with the same concentration and the carrier with the same or similar absorbance value in the wavelength-absorbance curve.
5. The microsphere composition according to claim 4, wherein λ2 is selected from any absorption wavelength value with the ratio of OD Photosensitive microsphere /OD Carrier body being within 1±15%, the OD Photosensitive microsphere and OD Carrier body are absorbance values corresponding to the same wavelength value of the photosensitive microsphere and the carrier in the visible light region of 300-800 nm, respectively, and the wavelength λ2 is not equal to the wavelength λ1; preferably, the lambda 2 is 400-600 nm.
6. Microsphere composition according to any one of claims 1 to 5, characterized in that the method of determining the amount of light sensed Ps of the light sensitive microspheres comprises the steps of:
s1, establishing a standard curve of carrier concentration-absorbance value at a wavelength lambda 2 according to a carrier with a series of concentrations, and further obtaining a slope k and an intercept b of the standard curve;
S2, detecting an absorbance value OD λ2 corresponding to the photosensitive microsphere at a wavelength lambda 2, and calculating the mass concentration C2 of the photosensitive microsphere according to the slope k and the intercept b obtained in the step S1;
s3, detecting an absorbance value OD λ1 corresponding to the photosensitive microsphere at the wavelength lambda 1, and calculating the photosensitive quantity Ps of the photosensitive microsphere according to the C2 obtained in the step S2.
7. Microsphere composition according to any one of claims 1 to 6, characterized in that the carrier is polystyrene microspheres without photosensitive substances; preferably, the carrier particle size is 150-250 nm; preferably 180 to 200nm.
8. Microsphere composition according to any one of claims 1 to 7, characterized in that the photosensitive microspheres are coated with streptavidin; preferably, the photosensitive material includes at least one of methylene blue, rose bengal, porphyrin, and phthalocyanine.
9. A kit for cardiac troponin T immunoassay comprising a microsphere composition according to any one of claims 1 to 8.
10. The kit according to claim 9, wherein the concentration of streptavidin-coated photosensitive microspheres is 50 μg/mL; and/or the concentration of the luminescent microsphere coated by the cardiac troponin T antibody is 100 mug/mL.
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