CN117517664A

CN117517664A - Troponin T detection kit for photoexcitation chemiluminescence detection

Info

Publication number: CN117517664A
Application number: CN202310109720.0A
Authority: CN
Inventors: 杨阳; 李建武; 康蔡俊; 洪琳; 黄正铭; 李临
Original assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Current assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Priority date: 2023-02-13
Filing date: 2023-02-13
Publication date: 2024-02-06

Abstract

The application relates to a troponin T detection kit for photoexcitation chemiluminescence detection, which comprises a luminescent microsphere and a photosensitive microsphere, wherein the luminescent microsphere comprises a first carrier and a luminescent substance carried by the first carrier, the surface of the luminescent microsphere is connected with a troponin antibody, and the troponin antibody can be specifically combined with troponin T; the photosensitive microsphere comprises a second carrier and a photosensitive substance carried by the second carrier, and the photosensitive quantity Ps of the photosensitive microsphere is between 1.34 and 16.28; light sensing amount ps=od _λ1 /C ₂ *103, wherein: OD (optical density) _λ1 The visible light region of 300-800 nm has a specific concentration of C ₂ Wavelength-absorbance curve obtained after full wavelength scanning of photosensitive microspheresAbsorbance values corresponding to the maximum absorption peak of the line. In the scheme of the application, the light sensing quantity Ps is 1.34<PS<16.28 to detect troponin T antibodies, can make the test result in clinical application have uniformity and repeatability, and the test result has higher accuracy and precision.

Description

Troponin T detection kit for photoexcitation chemiluminescence detection

Technical Field

The invention relates to the technical field of light-activated chemiluminescence, in particular to a troponin T detection kit for light-activated chemiluminescence detection.

Background

Cardiac troponin T (cTnT) is a characteristic contractile protein on cardiac muscle fibers, which is one of three subunits of troponin (I, T, C), which, together with tropomyosin, binds actin via the myofilaments of myofibrils. Cardiac troponin T (cTnT) is a specific and highly sensitive marker of myocardial injury, rises rapidly After Myocardial Infarction (AMI), and can last for two weeks, and is clinically mainly used for auxiliary diagnosis of myocardial infarction.

The basic principle of photoexcitation is a homogeneous immune response. The method is based on antigens or antibodies coated on the surfaces of microspheres, immune complexes are formed in a liquid phase, and the photosensitive microspheres and the luminous microspheres are pulled up. Under the excitation of light, singlet oxygen is transferred between the two kinds of microspheres, so that the luminescent microspheres generate high-energy red light, and photon numbers are converted into target molecule concentration in a sample through a single photon counter and mathematical fitting. When the sample does not contain target molecules, immune complexes cannot be formed between the two microspheres, the distance between the two microspheres exceeds the propagation range of singlet oxygen, the singlet oxygen is rapidly quenched in a liquid phase, and no high-energy red light is generated during detection.

The photosensitive reagent is one of indispensable important components in a commodity kit, and has the function that the photosensitive microsphere in the reagent can generate singlet oxygen after being excited by external excitation light, and the singlet oxygen transfers energy to the luminescent microsphere within 200nm from the photosensitive microsphere, so that a chemiluminescent signal can be finally generated, and the detection of an unknown in a sample is realized. The final clinical application and chemiluminescence detection results of the photo-activated chemiluminescence detection kit product are affected by the concentration selection of the photosensitizer filled in the photosensitive microspheres, the efficiency and time of generating singlet oxygen in the liquid phase of the photosensitive microspheres, the production cost of the photosensitive reagents and the like. Currently, the prior art lacks high performance, photosensitive reagents that meet clinical testing requirements.

Therefore, it is of great practical significance to provide troponin T assay kits and methods of use thereof.

Disclosure of Invention

In view of the above, in order to solve or partially solve the problems in the related art, the present application provides a troponin T detection kit for photo-activated chemiluminescence detection, which can make the test results in clinical applications consistent and repeatable, and ensure that the test results have higher accuracy and precision.

The invention provides a troponin T detection kit for photoexcitation chemiluminescence detection, which comprises a luminescent microsphere and a photosensitive microsphere, wherein the luminescent microsphere comprises a first carrier and a luminescent substance carried by the first carrier, the surface of the luminescent microsphere is connected with a troponin antibody, and the troponin antibody can be specifically combined with troponin T;

the photosensitive microsphere comprises a second carrier and a photosensitive substance carried by the second 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 one embodiment, the concentration of the photosensitive microspheres

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.

In one embodiment, the C ₂ Selected from 10ug/ml to 200ug/ml.

In one embodiment, the linear relationship of carrier concentration-absorbance curve is y=kx+b, wherein:

x is different concentrations of carriers with preset particle sizes, y is an absorbance value of the carrier at the corresponding concentration, k is a slope, and b is an intercept; wherein the concentration of the second carrier is selected from 10ug/ml to 100ug/ml.

In one embodiment, the wavelength λ ₂ Selected from OD _{Photosensitive microsphere} /OD _{Second carrier} 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) _{Second carrier} 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 one embodiment, the wavelength λ ₁ 600nm to 700nm, the wavelength lambda ₂ 440nm to 580nm.

In one embodiment, the photosensitive microsphere is prepared according to the mass ratio of the second carrier to the photosensitive substance of 10 (0.04-4).

In one embodiment, the particle size of the second support is selected from 190nm to 280nm.

In one embodiment, the photosensitive microspheres are stored in a buffer solution having a sugar content of 1 g.+ -. 0.2g per liter of volume of the buffer solution.

In one embodiment, the pH of the buffer solution is between 6 and 10.

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 the absorbance value OD _λ1 And concentration C ₂ The ratio of the ratio to define the amount of the photosensitive agent Ps. When the value of the sensitization quantity Ps of the sensitization microsphere is between 1.34 and 16.28, the luminous reagent is added into the sensitization reagent to react with the sensitization microsphere, so that the propagation efficiency and time of singlet oxygen can be ensured to be stable, the intensity of a chemiluminescence signal of the luminous microsphere can be ensured to meet the requirement in the photoexcitation chemiluminescence detection, the fluctuation of a detection result caused by the influence of other interference factors on the chemiluminescence signal is reduced, the detection result has consistency and repeatability in clinical application, and the detection result has more definite distinction and higher precision. In addition, the photosensitive quantity of the photosensitive reagent is limited in a definite numerical range, so that the material cost is saved to the maximum extent, and the reliability of the photo-excitation chemiluminescence detection result can be ensured.

Based on a troponin T detection kit with a light sensing amount Ps of 1.34< PS <16.28 to detect troponin T antibodies, the method has the advantages of high sensitivity and good precision.

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 second 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 is a wavelength-absorbance curve of a photosensitizer shown in an embodiment of the present application;

FIG. 4 is a graph of wavelength versus absorbance for different concentrations of a second carrier shown in the specific embodiments of the present application;

FIG. 5 is a graph of wavelength versus absorbance for various concentrations of photosensitive microspheres shown in the specific examples herein;

FIG. 6 is a wavelength-absorbance curve of 10 μg/ml of a second carrier and photosensitive microspheres as shown in the specific examples herein;

FIG. 7 is a second carrier concentration-absorbance curve for a second carrier at a wavelength of 500nm as shown in the specific embodiments of the present application;

FIG. 8 is a graph of photosensitizing material concentration versus photosensitizing amount as shown in an embodiment of the application;

FIG. 9 shows the method of using the troponin T assay kit of the present application in light-activated chemiluminescence detection.

Detailed Description

The invention discloses a troponin T 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 specification. 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.

Term interpretation:

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.

In some embodiments of the invention, the photosensitive microsphere for photoexcitation chemiluminescence detection has a photosensitivity amount Ps of 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 Is the absorbance value corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained by scanning the photosensitive microsphere in the full wavelength range of 300-800 nm, lambda ₁ Is the wavelength corresponding to the maximum absorption peak; c (C) ₂ Is the concentration (C) of the photosensitive microsphere during the photo-induced chemiluminescence detection ₂ Units are ug/ml).

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 prove that the maximum characteristic peak corresponding to the photosensitive microsphere with the same photosensitive substance under different concentrations is the same, and the maximum characteristic peak of the photosensitive microsphere is the same as the maximum characteristic peak of the filled photosensitive substance,for example, taking a photosensitive substance as copper phthalocyanine as an example, the wavelength lambda of the photosensitive substance and the photosensitive microsphere 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 the photosensitive microsphere 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 the chemiluminescent signal of the luminous microsphere can meet the requirement in the photoexcitation chemiluminescent detection, and the chemiluminescent signal is reducedThe number is influenced by other interference factors to cause fluctuation of the detection result, so that the detection result has consistency and repeatability in clinical application, and the detection result has more definite distinction degree and higher precision. That is, in order to control the material cost, after the concentration of the photosensitive microspheres is regulated and controlled, the regulated photosensitive amount of the photosensitive microspheres is only required to be ensured to be 1.34-16.28, and Ps is not less than 1.34 and not more than 16.28, so that the material cost is saved to the maximum extent, and the reliability of the photo-excitation chemiluminescence detection result is ensured. 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 the second carrier concentration-absorbance curve, b is the corresponding intercept, OD, in the linear relationship of the second carrier concentration-absorbance curve _λ2 Is photosensitive microsphere at wavelength lambda ₂ The corresponding absorbance value is that the concentration-absorbance curve of the second carrier adopts the second carrier with different concentrations at the wavelength lambda ₂ A curve obtained below; wavelength lambda ₂ The photosensitive microsphere with the same concentration and the second carrier have the same or similar absorbance value corresponding wavelength in 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 performed to determine the concentration C of the photosensitive microspheres by using a second carrier with the same material and particle size as the photosensitive microspheres ₂ Is not limited in terms of the range of (a). It should be noted that, based on the limitation of the manufacturing process of the photosensitive microsphere and the second carrier, the definition of "the same particle size", etc. in the present application refers to the microsphereThe difference in particle size was.+ -. 5nm, and the difference in particle size was small, and the same was found. The particle size of the second carrier is selected from 190 nm-280 nm. Wherein, a plurality of second carriers with different concentrations and same particle size can be prepared in advance and the same wavelength lambda is adopted ₂ The absorbance value corresponding to the second carrier of each concentration is scanned and measured respectively, so that a relation curve of the second carrier concentration and the absorbance can be established, and then a linear relation of the second 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 the different concentration of the second carrier with the same particle diameter, y is the absorbance value of the second carrier at the corresponding concentration, k is the slope in the formula (2), and b is the 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 _{Second carrier} 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 The method comprises the steps of carrying out a first treatment on the surface of the Wherein OD _{Photosensitive microsphere} And OD (optical density) _{Second carrier} The absorbance values corresponding to the same wavelength values of the photosensitive microspheres with the same concentration and the second carrier within the range of 300 nm-800 nm 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 itself on the absorbance value of the second carrier is reduced for the wavelength corresponding to the non-characteristic peak in the wavelength-absorbance curve, i.e., the wavelength avoiding the characteristic peak of the photosensitive substance. It is understood that the absorbance OD of the photosensitive microsphere at the same concentration in the full wavelength range can be obtained by scanning the photosensitive microsphere and the second carrier at the same microsphere concentration with the same wavelength in the range of 300nm to 800nm _{Photosensitive microsphere} And obtaining the absorbance value OD of the second carrier at the concentration corresponding to the same wavelength in the full wavelength range _{Second carrier} . Through research by the applicantNow, specific experimental data can be checked for the following related content, and OD is selected _{Photosensitive microsphere} /OD _{Second carrier} 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 more accurately determined by the second carrier concentration-absorbance curve of the second carrier than by selecting wavelengths outside the above 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 a second carrier absorbance value OD _{Second carrier} 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.

At a determined wavelength lambda ₂ Then, can adopt the same wavelength lambda ₂ Scanning a plurality of second 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 microspheres with the same particle size as the second carriers according to a formula (2) ₂ . Further, the loading amount of the photosensitive material by the second carrier having different particle diameters at the same concentration may be different, thereby affecting the absorbance value, i.e., the second carrier concentration-absorbance curve is also related to the particle diameter of the second carrier. Therefore, in order to establish an accurate and reliable second carrier concentration-absorbance curve, in one embodiment, a second carrier having a particle size within 190nm to 290nm is selected to establish a corresponding second carrier concentration-absorbance curve. For example, the particle size of the second carrier may be 190nm, 200nm, 220nm, 240nm, 260nm, 280nm or 290nm to control photosensitivityConcentration of microsphere C ₂ The calculated result of (2) and the actual concentration are within 10%. For example, a corresponding second carrier concentration-absorbance curve can be established using a 190nm second carrier to establish 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 at a known photosensitive microsphere concentration 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 secure stability of the photosensitizing agent, the pH of the buffer solution in the photosensitizing agent is 6 to 10, preferably pH 8. The buffer solution may be a Mes buffer solution, hepes buffer solution, tris buffer solution. Preferably, the buffer solution may be Hepes buffer solution having a pH of 8. By setting the pH value of the buffer system in the photosensitive reagent, the stability of the photosensitive reagent can be ensured, and the stability of the detection result in clinical application can be ensured.

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 may not be 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 following description is made with reference to specific embodiments.

Experimental raw material and equipment

TABLE 1

Material name	Storage conditions and expiration dates
		Luminous microsphere (FG)	2-8deg.C, sealed and light-proof
Troponin T antibody I (marker)	≤-15℃
		Troponin T antibody II (marker)	≤-15℃
Biotin	≤-15℃

TABLE 2

In the troponin T detection kit and the application method thereof provided by the invention, the raw materials and the reagents used can be purchased from the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of a luminescent concentrated solution

1. Dialyzing the raw materials, placing 1mg of troponin T antibody I in a 3.5KD dialysis bag, and dialyzing in 0.05MCB buffer solution (pH9.6) 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 2.71mg/ml.

3. Microsphere treatment, taking 1ml of FG microspheres with the concentration of 10mg/ml in a centrifuge tube, centrifuging for 30 minutes, discarding the supernatant, carrying out ultrasonic resuspension by using 0.05M CB buffer, repeating the operation once, and fixing the volume of 50mg/ml for standby.

4. Coupling reaction, according to microsphere: mixing FG microsphere and antibody protein with the mass ratio of protein being 10:0.5, performing coupling reaction by using a rotary mixing mode, and performing rotary reaction for 20 hours; 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 set to 10mg/ml of the luminescent concentrated solution.

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 Biotin reagent (i.e., labeling reagent)

1. Dialysis of the raw materials, taking 1mg of antibody respectivelyII, placing in 3.5KD dialysis bags, respectively, at 0.1MNaHCO ₃ Dialyzing the buffer solution 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 1.84mg/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 by BCA method, 1.64mg/ml of concentrated biotin solution, for further use.

5. The biotin reagent is prepared, 50ml of biotin reagent buffer solution is used, and the prepared biotin concentrated solution is diluted to 1ug/ml and balanced for 2 hours for standby.

EXAMPLE 3 preparation of photosensitizing agent

A. Preparation of microspheres

Preparation of (one) the second Carrier

a) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced ₂ 30min。

b) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step a), and continuing to introduce N ₂ 30min。

c) The reaction system was warmed to 70℃and reacted for 15 hours.

d) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. The obtained emulsion is washed by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, and then diluted with water, and is stored in the form of emulsion.

e) The average particle size of the Gaussian distribution of the particle size of the latex microsphere is 190nm as measured by a nanometer particle sizer.

(II) preparation of photosensitive microspheres

a) A25 ml round bottom flask was prepared, 0.11g of copper phthalocyanine (i.e., photosensitive material) and 10ml of N, N-dimethylformamide were added thereto, and the mixture was stirred uniformly by magnetic force, and the round bottom flask was heated to 75℃in a water bath to obtain a photosensitive material solution.

b) A 100ml three-necked flask was prepared, 10ml of 95% ethanol, 10ml of water and 10ml of the second carrier prepared by 1.e) above with a concentration of 10% were added, respectively, 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 second carrier of step b) to the photosensitive material solution of step a) may be adjusted according to the requirements of 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 photosensitive microspheres were resuspended to the desired concentration in the subsequent experiments with 50mMol/L of CB buffer at ph=10.

B. Measurement of the amount of sensitization

The light-sensitive amount of the light-sensitive microspheres is measured by an OD method:

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 Is the absorbance value corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained by scanning the photosensitive microsphere at full wavelength in the visible light region of 300-800 nm, lambda ₁ Is the wavelength corresponding to the maximum absorption peak; c (C) ₂ Is the concentration (C) of the photosensitive microsphere dissolved in the buffer solution before the photo-activated chemiluminescence detection ₂ Units are ug/ml).

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).

Wherein k is the corresponding slope in the linear relationship of the second carrier concentration-absorbance curve, b is the corresponding intercept in the linear relationship of the second carrier concentration-absorbance curve, OD _λ2 Is photosensitive microsphere at wavelength lambda ₂ The corresponding absorbance value is that the second carrier concentration-absorbance curve adopts different concentrations of the second carrier at the wavelength lambda ₂ A curve obtained below; the wavelength lambda ₂ The photosensitive microsphere and the second carrier having the same concentration have the same or similar absorbance value corresponding wavelength in 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 performed to determine the concentration C of the photosensitive microspheres by using a second carrier with the same material and particle size as the photosensitive microspheres ₂ Is not limited in terms of the range of (a). Wherein, a plurality of second carriers with different concentrations and same particle size can be prepared in advance and the same wavelength lambda is adopted ₂ The absorbance value corresponding to the second carrier of each concentration is scanned and measured respectively, so that a relation curve of the second carrier concentration and the absorbance can be established, and then a linear relation of the second carrier concentration and the absorbance is obtained, and the linear relation can be expressed by the following formula (3).

y＝kx+b (3)

Specifically, in the method for measuring the sensitization of the sensitization microsphere, each parameter is optimally determined as follows:

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
			Second carrier	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

For photosensitive microspheres and a second carrier, because the linear relation between the absorbance of the microspheres with different particle diameters and the concentration of the microspheres is inconsistent, the particle diameters of the microspheres need to be concerned to be estimated when the concentration of the microspheres is measured by using absorbance values. In this experiment, in order to ensure consistency of data, subsequent experiments may be performed uniformly using photosensitive microspheres having the same particle size, for example, 190nm, and a second carrier.

1.2 preparation of microspheres at different concentrations

And respectively diluting the prepared photosensitive microsphere and the second carrier by deionized water to prepare second 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 second 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 second carrier and the photosensitive microspheres was measured at 20 ug/ml. Wherein, experimental data are shown in fig. 1 and 2. The average particle size of the second 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 second carrier and the photosensitive balls are about 190nm, the wave crest is narrower, the particle sizes of the microspheres are 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 second 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 materials respectively show distinct peaks at 360nm, 610nm, 650nm and 680nm, wherein 680nm is the main peak, namely the maximum characteristic peak of the photosensitive materials.

4.2 second Carrier

FIG. 4 is a graph showing the wavelength-absorbance curves of 10 second carriers at different concentrations after scanning at 300nm to 800 nm. As can be seen from fig. 4, the second carrier at different concentrations has 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 absorbance values of the second carriers with different concentrations are different after scanning, and the microsphere concentration of the second 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 fig. 5 shows that the photosensitive microspheres with different concentrations show obvious peaks at 360nm, 610nm, 650nm and 680nm after being scanned by visible light with the wavelength of 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 a 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)

In this step 4.4, the wavelength-absorbance curves of the photosensitive microspheres and the second carrier at the same concentration are compared. As shown in FIG. 6, the photosensitive microsphere and the second carrier each having a concentration of 10. Mu.g/ml are exemplified, the same concentrationAfter scanning the wavelength of 300 nm-800 nm, the 680nm characteristic peak of the photosensitive microsphere is the largest 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 this experiment is copper phthalocyanine, and when the photosensitive material 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 For determining, based on 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 second carrier itself plus the absorbance value of the photosensitive substance, thereby having an influence on 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 second carrier at the same concentration. Photosensitive microspheres and a second carrier at a concentration of 50ug/ml are exemplified as shown in Table 4.

It can be understood that when the selected photosensitive materials are different, different characteristic peaks are generated, and the absorbance values corresponding to the characteristic peaks may be different, so that the measurement is performedA large deviation occurs in the concentration of the fixed photosensitive microspheres. 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 _{Second carrier} 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 of second carrier _{Second carrier} 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 a smaller influence on the absorbance values of the photosensitive microsphere and the second carrier, and when the OD is _{Photosensitive microsphere} /OD _{Second carrier} 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 _{Second carrier} 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 redetermined by reference to the above method ₂ 。

TABLE 4 Table 4

In this embodiment, the second carrier with the same preset particle size is selected at the wavelength lambda ₂ The absorbance values of the second carrier at different concentrations were then tested to establish a second carrier concentration-absorbance curve.

5. Establishment of a second Carrier concentration-absorbance Curve

In this embodiment, the second carrier with the same particle size is selected to have a wavelength lambda ₂ The absorbance values of the second carrier at different concentrations were then tested to establish a second carrier concentration-absorbance curve.

Determining the wavelength lambda at step 4.4 above ₂ The experiment then selects wavelength lambda ₂ 500nm, particle diameter of 190The second carrier around nm was studied, and the second carrier concentration-absorbance curve is shown in FIG. 7.

Specifically, in order to obtain the second carriers with different concentrations, the mass of the second carriers is obtained by a traditional drying method, deionized water is added into the second carriers with known mass to prepare 10mg/ml of the second carriers, and the second carriers with 10mg/ml are further diluted by the deionized water to prepare 10 concentrations of the second 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 second carrier with each concentration by wavelength of 500nm to obtain absorbance value OD corresponding to each concentration ₅₀₀ A linear relationship of the second carrier concentration to the 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 equation (2), i.e. the second 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 the second carrier having a particle diameter of 190nm, the applicant used the second carrier having a different particle diameter for verification in order to verify the accuracy of the calculation result of the above second carrier concentration x. The second carriers with different particle sizes are prepared respectively, and the particle sizes of the second carriers comprise 7 particle sizes such as 190nm,200nm,220nm,240nm,260nm,280nm and 300 nm. And preparing the second carrier with each particle size into theoretical concentration values of 40ug/ml,50ug/ml and 60ug/ml according to the mass of the second carrier calculated by a drying method. The absorbance value OD of the second carrier for each concentration of each 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 each concentration of each particle size of each second carrier, a corresponding absorbance value OD is obtained _λ2 After that, according to (OD _λ2 -0.0359)/0.0021 and correlating the result with the corresponding concentration valueThe theoretical concentration values are compared to determine the deviation of the calculated result. The results are shown in Table 5:

TABLE 5

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As can be seen from the data in Table 5, when the particle diameter of the second carrier is 280nm or less, the recovery deviation of the concentration is within 10%, i.e., according to (OD _λ2 The deviation of the second carrier concentration value x calculated by 0.0359)/0.0021 from 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 (C) is not limited to 10 ug/ml-100 ug/ml, and C can be further determined by combining the following experiments ₂ Is a range of values.

C. Verifying the sensitization of the sensitization microspheres

In order to verify the difference between the amount of light sensed Ps of the photosensitive microsphere determined according to formulas (1) and (2) and the amount of light sensed by the photosensitive microsphere, it can be determined according to the following experimental procedure.

1. Preparation of photosensitive microspheres with different mass ratios

Since the photosensitive microspheres are second carriers filled with photosensitive substances, for the photosensitive microspheres with the same concentration, if the contents of the photosensitive substances filled in the second carriers are different, namely the mass ratio of the second carriers to the photosensitive substances is different, the corresponding absorbance values are different. Therefore, the experiment needs to verify the effect of the photosensitive substance with different mass ratios on the calculation result of the amount of photosensitive light.

Firstly, according to the preparation method of the photosensitive microsphere in the step A and the step (II), the corresponding photosensitive microsphere is prepared by adopting different mass ratios of the second carrier to the photosensitive substance, namely, firstly, 6 photosensitive microspheres with the mass ratios of the second 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 6 and the table 7. Wherein 10:0 represents the second carrier which does not contain photosensitive substances and is only empty in the photosensitive microsphere. 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 comprises the absorbance value of the second 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 second vector} 。

TABLE 6

Further, corresponding average value of the photosensitive amount and CV value of the coefficient of variation are calculated according to the photosensitive amount of the photosensitive material at different dilution factors according to each mass ratio in table 6 above, wherein the CV value is the ratio of standard deviation to average value, and the specific calculation results can be referred to the related data in table 7 below.

As can be seen from Table 6 above, 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 above, 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. That is, in the case of specifying the specific value of the light-sensitive amount Ps, the mass ratio of the second carrier to the photosensitive substance need not be excessively focused, and the concentration of the photosensitive microsphere needs to be focused when manufacturing the photosensitive microsphere for clinical application, so that the material costs of the second carrier and the photosensitive substance can be controlled.

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 second 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 second carrier to the photosensitizer is 10 (2-4), the increase of the quantity of light sensitivity is obviously reduced, which indicates the proportion of the photosensitizer, namely the quantity of the photosensitizer filled in the second 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 second carrier to the photosensitizer 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 photosensitizer 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 second carrier to the photosensitizer.

According to the above-mentioned method for measuring the amount of light sensitivity, the amounts of light sensitivity of the light-sensitive microspheres 2 to 5 prepared in this example were obtained, and corresponding light-sensitive reagents 1 to 4 were further prepared in order.

D. Preparation of photosensitive reagent

a) Microsphere suspension treatment: and (3) respectively sucking a certain amount of the photosensitive microspheres 2-5 prepared in the steps, centrifuging in a high-speed refrigerated centrifuge, removing the supernatant, adding a certain amount of MES buffer solution containing 1g/L sugar, performing ultrasonic treatment on the ultrasonic cell disruption instrument until particles are resuspended, and adding the MES buffer solution 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 is prepared by adopting MES buffer solution ₃ CN solution, naBH ₃ The CN solution is added according to the volume ratio of 1:25 with the reaction liquid, and is quickly and evenly mixed, and the mixture is subjected to rotary reaction at the constant temperature of 37 ℃ for 48 hours.

e) Closing: preparing 75mg/ml Gly solution and 25mg/ml NaBH in MES buffer ₃ CN solution is added into the solution according to the volume ratio of 2:1:10 with the reaction solution, and the mixture is uniformly mixed and rotated at 37 ℃ for 2 hours. 200mg/ml BSA solution (MES buffer) was added thereto, and the mixture was rapidly and uniformly mixed with the reaction mixture at a volume ratio of 5:8, and the mixture was subjected to rotary reaction at 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, discarding the supernatant, adding fresh MES buffer solution, suspending again by an ultrasonic method, centrifuging again, washing for 3 times, suspending by a small amount of MES buffer solution, measuring the solid content to be 10mg/ml, and preparing corresponding 4 photosensitive liquid concentrated solutions with different photosensitive amounts according to 4 photosensitive microspheres.

g) Preparing a photosensitive reagent, namely diluting the prepared 4 photosensitive solution concentrated solutions by using a photosensitive reagent buffer solution to prepare 50ug/mL photosensitive reagents, and preparing 200mL photosensitive reagents respectively to prepare the corresponding photosensitive reagents 1-4.

TABLE 8

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

Example 4 preparation of the kit

TABLE 9

Component name	Main composition of
		Troponin T reagent 1 (luminescent reagent)	Luminous microsphere coated troponin T antibody
Troponin T reagent 2 (labelling reagent)	Biotin-labeled troponin T antibodies
		Photosensitive agent	Photosensitive microsphere coated streptavidin

Kits were prepared according to the compositions shown in table 9.

Example 5 methods of Using the kits

Scaling: scaling was performed using matched troponin T calibrator samples by performing rogowski-valued sample detection and quality control detection.

Instrument: light machine chemiluminescence detector, model lica 500

The detection process is as shown in fig. 9:

the first step: taking a sample of 40ul troponin T calibrator in the reaction well;

and a second step of: adding 15ul of each of troponin T reagent 1 and troponin T reagent 2 to the reaction well in order to mix the luminescent reagent and the biotin reagent with the sample;

and a third step of: incubation, the immune reaction needs to be incubated for 15min at 37 ℃, so that the antigen-antibody is most fully combined, and a luminous microsphere-antigen-antibody-antigen-biotin complex is more easily formed;

fourth step: adding 175ul of photosensitive reagent, wherein the photosensitive amount is 1.34< PS <16.28, so that the photosensitive microsphere-SA avidin in the universal liquid is combined with biotin, if the photosensitive amount Ps of the photosensitive microsphere is less than 1.34, the photosensitive microsphere cannot release enough singlet oxygen when excited by 680nm excitation light, and the singlet oxygen can be absorbed by other proteins in the liquid phase in the transfer process, so that the insufficient singlet oxygen cannot be transferred to the luminescent microsphere, and cannot be absorbed by the luminescent microsphere, and the 610nm emitted light cannot be emitted; when the sensitization amount of the sensitization microsphere is 1.34< PS <16.28, the singlet oxygen energy can be ensured to be transferred to the luminous sphere and is absorbed by the luminous sphere to emit light of 610 nm;

Fifth step: the incubation is that the biotin-avidin combination is most sufficient for antigen-antibody combination at 37 ℃, so that the luminous microsphere-antibody-antigen-antibody-biotin-avidin-photosensitive microsphere complex is more easily formed; thereby the distance between the light sensing ball and the luminous ball is shortened;

sixth step: reading, namely irradiating the reaction hole by 680nm light, so that the photosensitive microsphere in the reaction hole emits singlet oxygen, when the distance between the photosensitive microsphere and the luminous microsphere is less than 200nm, the luminous microsphere can absorb the singlet oxygen, thereby emitting 610nm light, amplifying signals by a PMT (program guide) and collecting 610nm optical signal values; and calculating the concentration through a calibration curve to obtain a troponin T concentration value in the troponin T calibrator, and completing calibration when the calculated value is the same as the concentration of the calibrator.

Effect example

In this calibration experiment, troponin T calibrator cal1-cal6 of 6 different target molecule concentrations was used for the experiment according to the six steps described above. In the fourth step, each concentration of calibrator is combined with the prepared 4 photosensitive reagents with different light-sensing amounts respectively, the Roche assignment sample is detected, and the chemiluminescent signal value of the luminescent microspheres under each concentration of calibrator sample and the photosensitive reagents with different light-sensing amounts is measured. Specific experimental data are shown in table 10 below.

Table 10

From the data of table 10, the discrimination of test data between different light-sensing amounts was calculated. The calculation results are shown in table 11 below.

TABLE 11

As can be seen from the calculation data in table 11, the discrimination degree of the signal values corresponding to the participation of the photosensitive reagent 1 in the test is low, and the signal values corresponding to the participation of the photosensitive reagents 2 to 4 in the test are high and substantially consistent.

The chemiluminescent signal values were tested using 10 clinical rogowski samples of varying target molecule concentrations with a kit of 4 photosensitizing reagents. The test data are shown in table 12 below.

Table 12

As can be seen from the data in table 12, the photosensitizers with different photosensitizers have less influence on the measured values for the same clinical sample. That is, when the light sensing amount Ps is within a prescribed range, the clinical test requirements can be met, and the light-emitting microsphere can emit light with sufficient intensity.

Further, according to the above 10 samples, each sample was adjusted to a quality control of two concentration levels of low concentration and high concentration, and the influence of different light-sensing amounts on the accuracy of the measured concentration values was further tested. The test data are shown in table 13 below.

TABLE 13

The precision results show that the precision is poor when the photosensitive reagent 1 is used, the reproducibility CV of the photosensitive reagents 2 to 4 is within 10 percent, and the precision is good.

Conclusion: the photosensitizing reagent with the photosensitizing quantity of 1.34-16.28 can be suitable for the detection of the light-activated chemiluminescence troponin T detection kit, and the light-activated chemiluminescence troponin T detection kit with the photosensitizing quantity of 4.07-16.28 has better performance.

The embodiments of the present application have been described above, the foregoing description is exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A troponin T assay kit for photoexcitation chemiluminescent detection, comprising luminescent microspheres and photosensitive microspheres, wherein the luminescent microspheres comprise a first carrier and a luminescent substance carried by the first carrier, and troponin antibodies are attached to the surface of the luminescent microspheres, and are capable of specifically binding to troponin T;

2. The kit of claim 1, wherein the concentration of the photosensitive microspheres is

3. The kit of claim 1 or 2, wherein C ₂ Selected from 10ug/ml to 200ug/ml.

4. The kit of claim 2, wherein:

the linear relationship of carrier concentration-absorbance curve is y=kx+b, wherein:

5. The kit of claim 2, wherein:

the wavelength lambda ₂ Selected from OD _{Photosensitive microsphere} /OD _{Second carrier} 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 ；

6. The kit of claim 2, wherein:

the wavelength lambda ₁ 600nm to 700nm, the wavelength lambda ₂ 440nm to 580nm.

7. The kit according to claim 1, wherein the photosensitive microsphere is prepared according to a mass ratio of the second carrier to the photosensitive substance of 10 (0.04-4).

8. The kit of claim 1, wherein:

the particle size of the second carrier is selected from 190 nm-280 nm.

9. The kit of any one of claims 1 to 8, wherein:

the photosensitive microsphere is stored in a buffer solution, and the sugar content in the buffer solution is 1 g+/-0.2 g per liter of volume.

10. The kit of claim 9, wherein:

the pH value of the buffer solution is 6-10.