Photosensitive microspheres for optical chemiluminescence detection

Abstract

The application relates to a photosensitive microsphere for photochemical chemiluminescence detection. The photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, the photosensitive amount Ps of the photosensitive microsphere is between 1.34 and 16.28; the photosensitive amount Ps=OD λ1 / C2*10 3 , wherein: OD λ1 is an absorbance value corresponding to a maximum absorption peak of a wavelength-absorbance curve obtained by performing full-wavelength scanning on the photosensitive microsphere with a concentration C2 in a visible light range of 300-800 nm, lambda 1 is a wavelength corresponding to the maximum absorption peak, and C2 is the concentration of the photosensitive microsphere in photochemical chemiluminescence detection. The scheme provided by the application can regulate the photosensitive microsphere according to the determined numerical range of the photosensitive amount, so that the test results of the photosensitive microsphere in clinical application have consistency and repeatability, and the test results have high accuracy and precision.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of photochemical luminescence, in particular to a photosensitive microsphere for photochemical luminescence detection. BACKGROUND

[0002] Photochemical luminescence is a typical homogeneous immunoassay technique, and its luminescence system is composed of "luminescent microspheres" and "photosensitive microspheres". The two kinds of microspheres have good suspension characteristics in the liquid phase, and the microspheres meet the liquid dynamics characteristics when meeting antigens or antibodies in the liquid phase. Based on the antigens or antibodies coated on the surface of the nanometer microspheres forming an immune complex in the liquid phase, the luminescent microspheres and the photosensitive microspheres are pulled close. Under the excitation of excitation light, singlet oxygen transfer occurs between the luminescent microspheres and the photosensitive microspheres, and then the luminescent microspheres produce high-energy red light, i.e. chemical luminescence signal. The number of photons in the red light can be converted into the concentration of target molecules in the sample to be tested by photon counter and mathematical fitting. When the sample to be tested does not contain target molecules, the immune complex cannot be formed between the two kinds of microspheres, and the distance between the two kinds of microspheres exceeds the propagation range of singlet oxygen. Therefore, the singlet oxygen is rapidly quenched in the liquid phase, and no high-energy red light signal is generated during photochemical luminescence detection. Therefore, the photosensitive reagent containing photosensitive microspheres is one of the indispensable important components of the detection reagent of the photochemical luminescence analysis system. Its role is that the photosensitive microspheres in the reagent can produce singlet oxygen after being excited by external excitation light. The singlet oxygen transfers energy to the luminescent microspheres within a distance of 200 nm from the photosensitive microspheres, so that the luminescent microspheres can produce a chemical luminescence signal. By collecting the chemical luminescence signal, the number of photons is converted into the concentration of target molecules by using a photon counter and mathematical fitting, so as to realize the detection of target molecules in the sample to be tested.

[0003] Therefore, how to obtain a photosensitive microsphere with good consistency and meeting the needs of clinical testing is a problem to be solved at present. SUMMARY

[0004] To solve or partially solve the problems in the related art, the present application provides a photosensitive microsphere, which can standardize the photosensitive microsphere according to the determined numerical range of the photosensitive amount, so that the test results of the photosensitive microsphere in clinical application have consistency and repeatability, and ensure that the test results have high accuracy and precision.

[0005] The present application provides a photosensitive microsphere, which comprises a carrier and a photosensitive substance carried by the carrier, and the photosensitive amount Ps of the photosensitive microsphere is between 1.34 and 16.28; the photosensitive amount Ps=OD λ1 / C 2*10 3 , wherein:

[0006] OD λ1is the absorbance value corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained after full wavelength scanning of the photosensitive microspheres in the visible light range of 300 nm to 800 nm at a concentration of C 2; C 2 is the concentration of the photosensitive microspheres when the photochemical luminescence detection is performed, C 2 is in ug / ml.

[0007] In an embodiment, the concentration of the photosensitive microspheres ;

[0008] wherein, k is the corresponding slope in the linear relationship of the carrier concentration-absorbance curve, b is the corresponding intercept in the linear relationship of the carrier concentration-absorbance curve; OD λ2 is the absorbance value corresponding to the photosensitive microspheres at a wavelength of λ 2, and the carrier concentration-absorbance curve is obtained by using a plurality of carriers with different concentrations at a wavelength of λ 2; the wavelength λ 2 is the wavelength corresponding to the same or similar absorbance value of the photosensitive microspheres and the carrier with the same concentration in the wavelength-absorbance curve.

[0009] In an embodiment, C 2 is selected from 10 ug / ml to 200 ug / ml.

[0010] In an embodiment, the linear relationship of the carrier concentration-absorbance curve is y = kx + b wherein,

[0011] x is the different concentration of the carrier with a preset particle size, y is the absorbance value of the carrier at the corresponding concentration, k is the slope, b is the intercept.

[0012] In an embodiment, the wavelength λ 2 is selected from any wavelength value within the ratio of OD 感光微球 / OD 载体 of 0.85 to 1.15, and the wavelength λ 2 is not equal to the wavelength λ 1.

[0013] wherein, OD 感光微球 and OD 载体respectively, are absorbance values corresponding to the same wavelength value in the range of 300 nm to 800 nm of each of the photosensitive microspheres and the carrier at the same concentration.

[0014] In an embodiment, the wavelength λ 2 is 400 nm to 600 nm.

[0015] In an embodiment, the photosensitive microspheres are carriers filled with photosensitive substances, wherein the wavelength λ 1 is a wavelength corresponding to a maximum absorption peak in a wavelength-absorbance curve obtained by full wavelength scanning of the photosensitive substances in a visible light range of 300 nm to 800 nm of the photosensitive substances.

[0016] In an embodiment, the wavelength λ 1 is 600 nm to 700 nm.

[0017] In an embodiment, the photosensitive microspheres are prepared according to a mass ratio of the carrier to the photosensitive substance of 10: (0.04-4).

[0018] In an embodiment, the carrier has a particle size of 190 nm to 280 nm.

[0019] The technical solution provided in the present application can have the following beneficial effects:

[0020] After determining the absorbance value OD λ1 and the value of the concentration C 2, the photosensitive amount Ps of the photosensitive microspheres can be determined according to the ratio of the absorbance value OD λ1 and the concentration C2. When the value of the photosensitive amount Ps of the photosensitive microspheres is between 1.34 and 16.28, the application of the photosensitive microspheres in the photosensitive reagent for mixing reaction with the luminescent microspheres can make the intensity of the chemiluminescence signal of the luminescent microspheres meet the requirements in the light-induced chemiluminescence detection, reduce the fluctuation of the detection results caused by the influence of other interference factors on the chemiluminescence signal, make the detection results have consistency and repeatability in clinical application, and make the detection results have more clear distinguishability and higher precision. The photosensitive microspheres of the present application, through the clear numerical limitation, give the performance standard that can be executed when the photosensitive microspheres are applied in light-induced chemical detection, have clear operability, and are suitable for the promotion of industry standards.

[0021] It should be understood that the foregoing general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which like reference characters refer to like parts throughout the figures, and in which:

[0023] Figure 1 is a particle size result graph of 20 ug / ml carrier measured by the particle size instrument in the specific embodiment of the present application;

[0024] Figure 2 is a particle size result graph of 20 ug / ml photosensitive microspheres measured by the particle size instrument in the specific embodiment of the present application;

[0025] Figure 3 is a wavelength-absorbance curve of photosensitive substance in the specific embodiment of the present application;

[0026] Figure 4 is a wavelength-absorbance curve of carrier of different concentrations in the specific embodiment of the present application;

[0027] Figure 5 is a wavelength-absorbance curve of photosensitive microspheres of different concentrations in the specific embodiment of the present application;

[0028] Figure 6 is a wavelength-absorbance curve of 10 ug / ml carrier and photosensitive microspheres in the specific embodiment of the present application;

[0029] Figure 7 is a carrier concentration-absorbance curve of carrier at wavelength 500 nm in the specific embodiment of the present application;

[0030] Figure 8 is a mass ratio-photosensitive amount curve of photosensitive substance in the specific embodiment of the present application. DETAILED DESCRIPTION

[0031] Embodiments of the present application will be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the application are shown. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.

[0032] 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 herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0033] It should be understood that, although the terms "first," "second," "third," etc. can be used herein to describe various information, these terms are not intended to denote a particular order or hierarchy among the information. These terms are merely used to distinguish one category of information from another. For example, a first information can also be termed a second information, and, similarly, a second information can also be termed a first information, without departing from the scope of the present application. Therefore, the use of the terms "first," "second," etc., can not necessarily indicate a particular order or hierarchy among the information. In the description of the present application, the meaning of "a plurality" is two or more, unless otherwise explicitly defined specifically.

[0034] The photosensitive microspheres for photo-induced chemiluminescence detection in an embodiment have a photosensitivity Ps between 1.34 and 16.28; the photosensitivity Ps of the photosensitive microspheres is determined according to the following formula (1).

[0035] Ps=OD λ1 / C 2*10 3 (1)

[0036] wherein, OD λ1 is an absorbance value corresponding to a maximum absorption peak of a wavelength-absorbance curve obtained by full-wavelength scanning of the photosensitive microspheres with a concentration of 2 in a visible light range of 300 nm to 800 nm, C 1 is a wavelength corresponding to the maximum absorption peak, λ 2 is a concentration of the photosensitive microspheres when photo-induced chemiluminescence detection is performed, and C 2 is a unit of ug / ml. C

[0037] In the present application, the photosensitive microspheres include a carrier and a photosensitive substance carried by the carrier. The carrier can be a high-molecular micro-particle, and the photosensitive substance can be coated on the surface of the carrier and / or filled in the interior of the carrier. The photosensitive substance can generate active oxygen (e.g., singlet oxygen) under photo-excitation, and the high-molecular micro-particle can be a polystyrene microsphere, but can also be a microsphere of other material that can be detected, without limitation. The photosensitive substance can be, for example, a photosensitizer or a photosensitive dye, which can be a photosensitive substance known in the art, such as methylene blue, rose bengal, porphyrin, phthalocyanine and chlorophyll, without limitation. The photosensitive microspheres can also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide into singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc., which release singlet oxygen when heated or directly absorb light.

[0038] ​Furthermore, the concentration of the substance dissolved in the buffer solution was further analyzed using visible light in the 300nm~800nm ​​range via a spectrophotometer and other equipment. C The photosensitive microspheres were scanned across the entire wavelength range, and the absorbance values ​​corresponding to different wavelength scans were read. After generating the corresponding wavelength-absorbance curves, the wavelength... λ 1. The wavelength corresponding to the maximum characteristic peak (i.e., the maximum absorption peak) in the wavelength-absorbance curve of the photosensitive microsphere is selected; correspondingly, by selecting the absorbance value corresponding to the maximum characteristic peak in the wavelength-absorbance curve, the concentration can be determined. C 2 photosensitive microspheres at wavelength λ The absorbance value OD corresponding to 1 λ1 Furthermore, in order to ensure the wavelength λ To ensure the accuracy of the value of 1, in one embodiment, the wavelength-absorbance curves of photosensitive microspheres with different concentrations but the same photosensitive substance can be measured in advance. The wavelength corresponding to the maximum characteristic peak in the wavelength-absorbance curves of the photosensitive microspheres at each concentration can then be selected as the wavelength. λ The value of 1. Through experiments described below, it is verified that photosensitive microspheres with the same photosensitive material have the same maximum characteristic peak at different concentrations, and the maximum characteristic peak of the photosensitive microsphere is the same as the maximum characteristic peak of the photosensitive material it carries. For example, taking copper phthalocyanine as the photosensitive material, the wavelengths of the photosensitive material and the photosensitive microsphere... λ 1. All values ​​are 680 nm. Therefore, by selecting the wavelength corresponding to the maximum characteristic peak of the wavelength-absorbance curve of the photosensitive microspheres... λ 1. Then determine the corresponding absorbance value OD. λ1 This ensures the accuracy of the calculated photosensitivity. When different photosensitive materials are used for the photosensitive microspheres, the visible light region in the 300nm~800nm ​​range can be adaptively scanned again to determine the wavelength. λ The specific value of 1.

[0039] It should be noted that before use, photosensitive microspheres are typically stored in a lyophilized solid or refrigerated liquid state. When the photosensitive microspheres are in a solid state, a buffer solution needs to be added for reconstitution. The concentration of the reconstituted photosensitive microsphere solution is the initial concentration. C 1. When the photosensitive microspheres are stored as a liquid, their concentration at this point is the initial concentration. C 1. In photochemiluminescence detection, the photosensitive microspheres may be used at an initial concentration directly for detection; that is, the concentration of the photosensitive microspheres at the time of detection. C 2 equals C 1; or, it can also be used for those with an initial concentration C The photosensitive microspheres were diluted before being used in the detection; that is, the concentration of the photosensitive microspheres used in the detection was [missing information]. C 2 does not equalC 1, C The value of 2 corresponds to the actual concentration after dilution from the initial concentration. This can be understood as the concentration of the photosensitive microspheres being determined once the specific value of wavelength λ1 is determined. C When 2 changes, the corresponding absorbance value OD λ1 The corresponding differences may exist; OD λ1 The value is determined based on actual measurements. The absorbance value OD is determined accordingly. λ1 and concentration C After obtaining the value of 2, based on the absorbance value OD λ1 and concentration C The ratio of 2 can determine the photosensitivity Ps of the photosensitive microsphere. When the photosensitivity Ps of the photosensitive microsphere is between 1.34 and 16.28, applying the photosensitive microsphere to the photosensitive reagent to react with the luminescent microsphere ensures that the intensity of the chemiluminescence signal of the luminescent microsphere meets the requirements for photo-induced chemiluminescence detection. This reduces fluctuations in detection results caused by interference factors, resulting in consistent and repeatable detection results in clinical applications, and providing more precise discrimination and accuracy. The photosensitive microsphere of this application, through a clearly defined numerical limit on photosensitivity, provides an executable performance standard for its application in photo-induced chemiluminescence detection, demonstrating clear operability and suitability for the promotion of industry standards.

[0040] Furthermore, in order to facilitate the determination of the concentration of photosensitive microspheres... C The adjustment range of 2, the concentration of photosensitive microspheres C 2 is determined according to the following formula (2).

[0041] (2)

[0042] in, k It is the slope corresponding to the linear relationship of the carrier concentration-absorbance curve. b It is the intercept corresponding to the linear relationship of the carrier concentration-absorbance curve, OD. λ2 It is the photosensitive microspheres at wavelength λ The absorbance values ​​corresponding to point 2, and the carrier concentration-absorbance curves show the absorbance values ​​at different wavelengths using different carrier concentrations. λ The curve obtained in step 2; wavelength λ 2 represents the wavelengths corresponding to the same or similar absorbance values ​​of photosensitive microspheres and carriers with the same concentration in the wavelength-absorbance curve.

[0043] Specifically, in order to obtain a concentration of photosensitive microspheres that meets the sensitivity range of Ps... C 2. Experiments can be conducted using a carrier with the same material and particle size as the photosensitive microspheres to determine the concentration of the photosensitive microspheres. C2. It is to be noted that, based on the limitation of the manufacturing process of the photosensitive microspheres and the carrier, the definition of "same particle size" in the present application refers to the particle size difference between the microspheres being ± 5 nm, which is so small that the microspheres can be regarded as having the same particle size. In this embodiment, a plurality of carriers having different concentrations and the same particle size can be prepared in advance, and the particle size of the carrier is selected from 190 nm to 280 nm. The absorbance value of each concentration of the carrier is measured by scanning with the same wavelength λ2, so as to establish the relationship curve between the concentration of the carrier and the absorbance, and then obtain the linear relationship between the concentration of the carrier and the absorbance, which can be represented by the following formula (3).

[0044] y kx b (3)

[0045] x y k b k b C 2. Further, in order to determine the values of k and b , in an embodiment, the wavelength λ 2 is selected from any wavelength value within the ratio of OD 感光微球 / OD 载体 being 0.85 to 1.15, and the wavelength λ 2 is not equal to the wavelength λ 1; wherein OD 感光微球 and OD 载体 are the absorbance values of the photosensitive microspheres and the carrier respectively at the same wavelength within the range of 300 nm to 800 nm. In this embodiment, the wavelength λ2 is not equal to the wavelength λ 1, i.e. the wavelength λ 2 is the wavelength corresponding to the non-characteristic peak in the wavelength-absorbance curve, i.e. avoiding the wavelength of the characteristic peak of the photosensitive substance, so as to reduce the influence of the absorbance value of the photosensitive substance itself on the absorbance value of the carrier. It can be understood that, by scanning the photosensitive microspheres and the carrier with the same wavelength within the range of 300 nm to 800 nm, the absorbance value OD 感光微球 of the photosensitive microspheres at the full wavelength range under the same concentration can be obtained, and the absorbance value OD 载体 of the carrier at the same wavelength within the full wavelength range under the same concentration can be obtained.​​​​​​​​. The applicant found that the specific experimental data can be seen in the relevant content below, select OD 感光微球 / OD 载体 The ratio of the wavelength in the range of 0.85 to 1.15 is selected as λ 2, compared to the wavelength outside the above ratio range, the concentration C2 of the photosensitive microspheres can be more accurately determined by the carrier concentration-absorbance curve of the carrier. Experiments show that when the wavelength λ 2 is in the range of 440nm~580nm, the absorbance value OD 感光微球 of the photosensitive microspheres and the absorbance value OD 载体 of the carrier are within the range of 0.85 to 1.15, indicating that the content of the photosensitive substance has little effect on the determination of the microsphere concentration, otherwise. In an embodiment, the wavelength λ 2 can be 440nm~580nm. For example, the wavelength λ 2 can be 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm. It should be understood that when the photosensitive substance of the photosensitive microspheres is selected, the maximum characteristic peak of the photosensitive substance will change accordingly due to the influence of the nature of the photosensitive substance. Similarly, the wavelength λ 1 and the wavelength λ 2 are adjusted accordingly, and the OD λ1 and the OD λ2 are also adjusted accordingly.

[0046] After determining the wavelength λ 2, the same wavelength λ 2 can be used to scan multiple carriers with different known concentrations x but the same particle size to obtain the corresponding absorbance values y , so as to establish an equation according to formula (3) to calculate the values of k and b , so that the concentration C 2 of the photosensitive microspheres with the same particle size as the carrier can be calculated according to formula (2). Further, the same concentration of carriers with different particle sizes can have different loadings of photosensitive substances, thereby affecting the absorbance value, i.e. the carrier concentration-absorbance curve is also related to the particle size of the carrier. Therefore, in order to establish an accurate and reliable carrier concentration-absorbance curve, in an embodiment, carriers with a particle size of 190nm~290nm are selected to establish the corresponding carrier concentration-absorbance curve, so as to control the concentration CThe deviation of the calculated result of 2 from the true concentration is within 10%. For example, the preset particle size of the carrier can be 190 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, or 290 nm. For example, a carrier of 190 nm can be used to establish a corresponding carrier concentration-absorbance curve, and an equation can be established according to formula (3) to calculate the value of k and b . Preferably, C 2 is selected from 10 ug / ml to 200 ug / ml.

[0047] Specifically, in the case where the known photosensitive amount Ps is in the range of 1.34 to 16.28, and the values of 2 are known according to the above calculation, k 、 b and λ 2, in the reverse direction, the concentration of the photosensitive microspheres C 2 can be adjusted according to formulas (1) and (2). That is, in the actual photochemical detection process, after configuring photosensitive microspheres of unknown concentration, in the case where the specific value of the unknown concentration is unknown, the unknown concentration of the photosensitive microspheres is scanned at the wavelength λ 1 and the wavelength λ 2, and the corresponding absorbance values, i.e., OD λ1 and OD λ2 , are obtained. After the specific value of the unknown concentration is calculated by formula (2), if the value of the unknown concentration falls within the range of 10 ug / ml to 200 ug / ml, the calculated concentration C 2 can be substituted into formula (1) to calculate the photosensitive amount Ps. If the value of Ps is in the range of 1.34 to 16.28, it indicates that the concentration of the configured photosensitive microspheres can be applied to photochemical luminescence detection.

[0048] As can be seen from the above, in the case where the concentration of the photosensitive microspheres C 2 is known and the value range is 10 ug / ml to 200 ug / ml, formulas (2) and (3) can not be used, and the corresponding photosensitive amount Ps value of the photosensitive microspheres can be calculated directly according to formula (1). Similarly, in the case where the specific value of the unknown concentration of the photosensitive microspheres C 2 is known, the corresponding photosensitive amount Ps value of the photosensitive microspheres can be determined according to the above method. If the calculated result Ps value is in the range of 1.34 to 16.28, it is determined that the photosensitive microspheres can achieve the above effect, i.e., can be applied to photochemical luminescence detection, and the accuracy and precision of the detection result meet the requirements of clinical application.

[0049] Further, in order to reduce the influence of substances other than the carrier and the photosensitive substance on the absorbance value, in an embodiment, the surface of the photosensitive microsphere is not coated with polysaccharide; or the polysaccharide content of the photosensitive microsphere is not more than 25 mg per gram of mass. The polysaccharide refers to a carbohydrate containing three or more unmodified or modified monosaccharide units, such as dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran, and aminodextran, etc. By not adding polysaccharide or controlling the content of polysaccharide, the interference with the determination result of the absorbance value is reduced, and the detection result in clinical application is more accurate.

[0050] The photosensitive amount of the photosensitive microsphere of the present application is described below in combination with specific experimental data.

[0051] I. Preparation of microspheres

[0052] 1. Preparation of carrier

[0053] a) Prepare a 100ml three-necked flask, and add 40mmol of styrene, 5mmol of propylene aldehyde, and 10ml of water into the three-necked flask, respectively. After stirring for 10min, introduce N2 into the three-necked flask for 30min.

[0054] b) Weigh 0.11g of ammonium persulfate and 0.2g of sodium chloride, and dissolve them in 40ml of water to prepare an aqueous solution. Add the aqueous solution into the reaction system of the three-necked flask of step a), and continue to introduce N2 for 30min.

[0055] c) Increase the temperature of the reaction system to 70℃, and react for 15h to obtain an emulsion.

[0056] d) After the reaction is completed, cool the emulsion to room temperature, and then filter the emulsion with a suitable filter cloth. Wash the filtered emulsion with deionized water by centrifugal sedimentation until the conductivity of the supernatant is close to that of deionized water, and then dilute with water. Store in the form of emulsion.

[0057] e) The Gaussian distribution average particle size of the latex microspheres in the emulsion is 190nm measured by a nanoparticle size analyzer.

[0058] 2. Preparation of photosensitive microspheres

[0059] a) Prepare a 25ml round-bottom flask, and add 0.11g of copper phthalocyanine (i.e. photosensitive substance) and 10ml of N,N-dimethylformamide into the round-bottom flask. Stir uniformly by magnetic stirring, and perform water bath heating to 75℃ to obtain a photosensitive substance solution.

[0060] b) Prepare a 100ml three-necked flask, add 10ml 95% ethanol, 10ml water and 10ml carrier prepared in 1.e) above with a concentration of 10% respectively, mix uniformly by magnetic stirring, and warm the three-necked flask to 70°C in a water bath.

[0061] c) Slowly add the photosensitive substance solution in step a) to the three-necked flask in step b), stop stirring after 2 hours of reaction at 70°C, and naturally cool to obtain an emulsion. It can be understood that the mass ratio of the carrier in step b) and the photosensitive substance solution in step a) can be adjusted according to the subsequent experimental requirements.

[0062] d) Centrifuge the emulsion obtained in step c) at a centrifugal force of 30000G for 1 hour, discard the supernatant after centrifugation, and resuspend with 50% ethanol. After repeating the centrifugation and washing three times, resuspend the photosensitive microspheres in 50mMol / L CB buffer with a pH value of 10 to the required concentration for subsequent experiments.

[0063] II. Method for determining the applicable range of the value of photosensitive microsphere concentration C2

[0064] In this experiment, the linear relationship between carrier concentration and absorbance value is established by presetting the particle size, so that the absorbance value OD λ2 obtains the concentration C2 of the photosensitive microspheres. This experiment is used to explain the establishment process of the above formulas (2) and (3).

[0065] 1. Full wavelength scanning and particle size detection of microspheres

[0066] In order to ensure the consistency of the particle size of the microspheres used in the experiment, particle size detection is performed in advance.

[0067] Among them, the main raw materials and equipment involved in the experiment are shown in Table 1.

[0068] Table 1

[0069]

[0070] The experimental process is as follows:

[0071] 1.1 Selection of microsphere particle size

[0072] In this experiment, in order to ensure the consistency of the data, photosensitive microspheres and carriers with the same particle size, such as a preset particle size of 190nm, can be uniformly used for subsequent experiments.

[0073] 1.2 Preparation of carriers and photosensitive microspheres with different concentrations

[0074] The prepared photosensitive microspheres and carriers were diluted with deionized water to prepare carriers and photosensitive microspheres with different concentrations, and 10 concentrations were prepared, i.e. 10 ug / ml, 20 ug / ml, 30 ug / ml, 40 ug / ml, 50 ug / ml, 60 ug / ml, 70 ug / ml, 80 ug / ml, 90 ug / ml, 100 ug / ml, etc. Namely, the above-mentioned 10 concentrations of carriers and the above-mentioned 10 concentrations of photosensitive microspheres were prepared respectively; and a photosensitive substance solution with a concentration of 5 ug / ml was prepared.

[0075] 2. Microsphere particle size detection

[0076] The particle size instrument was turned on, and the particle sizes of the carriers and photosensitive microspheres with a concentration of 20 ug / ml were detected as an example.

[0077] The experimental data are shown in Figure 1 and Figure 2 The average particle size of the carriers is 187.1 nm, and the average particle size of the photosensitive microspheres is 190.9 nm.

[0078] From the detection results of the particle size instrument, it can be seen that the particle sizes of the carriers and photosensitive microspheres are about 190 nm, and the wave peak is relatively narrow, and the microspheres have a uniform particle size, which can be used as the microspheres required in subsequent experiments.

[0079] 3. Selection of wavelength

[0080] The ultraviolet spectrophotometer was turned on and preheated for 30 min, the ultraviolet spectrophotometer was adjusted, the wavelength was set to 300 nm-800 nm, the step length was 1 nm, deionized water was used to calibrate zero, and the photosensitive microspheres, carriers and photosensitive substance solution with various concentrations prepared in step 1.2 were detected in turn. It should be understood that when the scanned wavelength is less than 300 nm, it is easy to be interfered by the absorbance values of other substances, thereby affecting the accuracy and precision of the detection results, therefore the wavelength setting needs to be greater than 300 nm.

[0081] 4. Experimental data

[0082] 4.1 Photosensitive substance

[0083] As shown in Figure 3 , Figure 3 is the wavelength-absorbance curve of the photosensitive substance after scanning at 300 nm-800 nm. As can be seen from Figure 3 , the photosensitive substance has obvious wave peaks at 360 nm, 610 nm, 650 nm and 680 nm, among which the wave peak at 680 nm is the main peak, i.e. the maximum characteristic peak of the photosensitive substance.

[0084] 4.2 Carrier

[0085] Figure 4The wavelength-absorbance curves of the carrier at 10 different concentrations were obtained after scanning at 300 nm to 800 nm. Figure 4 It can be seen that, after scanning with visible light from 300nm to 800nm, the curves for different concentrations of the carrier do not show characteristic peaks. Meanwhile, from Figure 4 It can be seen that the absorbance values ​​after scanning are different for carriers of different concentrations, and the microsphere concentration of the carrier is positively correlated with the absorbance value.

[0086] 4.3 Photosensitive microspheres

[0087] Figure 5 The absorbance curves of photosensitive microspheres at 10 different concentration points were scanned at wavelengths ranging from 300 nm to 800 nm. Figure 5 It can be seen that photosensitive spheres of different concentrations, after being scanned with visible light from 300nm to 800nm, changed from... Figure 5 It can be seen that the photosensitive microspheres exhibit distinct peaks at 360nm, 610nm, 650nm, and 680nm, with the 680nm peak being the dominant peak. This means the maximum characteristic peak of the photosensitive microspheres is the same as that of the photosensitive material. Therefore, the photosensitive material filling the photosensitive microspheres directly affects the wavelength value corresponding to the maximum characteristic peak. Furthermore, from... Figure 5 It can be seen that different concentrations of photosensitive microspheres result in different absorbance values ​​after scanning, and the concentration of photosensitive microspheres is positively correlated with the absorbance value. Therefore, different concentrations of photosensitive microspheres do not affect the value of photosensitivity Ps.

[0088] 4.4 Determination of wavelengths λ1 and λ2

[0089] In step 4.4, the wavelength-absorbance curves of photosensitive microspheres and carriers at the same concentration point are compared. For example... Figure 6 As shown, taking photosensitive microspheres and a carrier with a concentration of 10 μg / ml as an example, after scanning wavelengths from 300 nm to 800 nm, the 680 nm characteristic peak of the photosensitive microspheres is the maximum characteristic peak of the photosensitive substance. Therefore, reading the absorbance value at 680 nm best reflects the content of the photosensitive substance in the photosensitive microspheres, and the wavelength corresponding to the maximum characteristic peak can be selected as λ1, i.e., λ1 is 680 nm. It can be understood that the photosensitive substance sampled in this experiment is copper phthalocyanine. When the photosensitive substance is other raw materials, the maximum characteristic peak may be different, and the corresponding wavelength λ1 will be determined according to the actual situation.

[0090] The purpose of determining the wavelength λ2 is to use it in conjunction with the corresponding absorbance value OD. λ2To determine the concentration C2 of the photosensitive microspheres, it is necessary to avoid the maximum characteristic peak of the photosensitive substance, i.e., the wavelength λ2 differs from λ1. This design is because when selecting the region where the photosensitive substance exhibits a peak, the absorbance value at the corresponding wavelength includes the absorbance value of the carrier itself plus the absorbance value of the photosensitive substance, thus affecting the concentration test of the photosensitive microspheres. Figure 6 It can be seen that λ2 is optimally selected within the 400nm~600nm range, as there are no characteristic peaks in this wavelength range. Although there are also no characteristic peaks of photosensitive substances in the 300nm~330nm range, this wavelength is easily affected by protein substances in other test samples, affecting its clinical application. The absorbance values ​​corresponding to wavelengths between 700nm~800nm ​​are relatively low, resulting in low detection sensitivity and large fluctuations in test results. The absorbance values ​​corresponding to wavelengths between 400nm~600nm are more consistent. Therefore, the wavelength λ2 is selected from the 400nm~600nm range.

[0091] Furthermore, to accurately determine the value of wavelength λ2, the absorbance of one of the photosensitive microspheres and the carrier with the same concentration was analyzed. Taking a photosensitive microsphere and carrier with a concentration of 50 μg / ml as an example, as shown in Table 2 below.

[0092] Table 2

[0093]

[0094] To minimize the influence of the photosensitive material on the measured microsphere concentration, the selected wavelength λ2 needs to be within the same OD range. 感光微球 / OD 载体 The ratio is within the range of 0.85 to 1.15, that is, (1 + 15%). As shown in Table 2 below, when the wavelength λ2 is between 440nm and 580nm, the absorbance value OD of 50ug / ml photosensitive microspheres is... 感光微球 The absorbance value OD of the carrier at 50ug / ml 载体 The ratio is within the range of 0.85 to 1.15, indicating that the absorbance value of the photosensitive material corresponding to the wavelength in this range has little impact on the absorbance values ​​of the photosensitive microspheres and the carrier. However, when the OD... 感光微球 / OD 载体 When the ratio is greater than 1.15, it indicates that the content of the photosensitive substance affects the determination of the concentration of the photosensitive microspheres. Preferably, when the wavelength is 500nm and 510nm, OD 感光微球 / OD 载体 The ratios are all 1.05, and a constant ratio indicates that the influence of the photosensitive material is constant. Therefore, in this embodiment, the preferred wavelength is... λ2 represents 500 nm. It is understandable that when using photosensitive materials of different types to prepare photosensitive microspheres, the wavelength can be re-determined using the method described above. λ 2.

[0095] 5. Establishment of carrier concentration-absorbance curves

[0096] In this embodiment, by selecting a carrier with the same preset particle size at a wavelength... λ 2. The absorbance values ​​of different concentrations of the carrier were tested to establish a carrier concentration-absorbance curve.

[0097] In step 4.4 above, the wavelength is determined. λ After step 2, the wavelength selected in this experiment was... λ 2. A carrier with a particle size of approximately 190 nm and a wavelength of 500 nm was used in the study. The carrier concentration-absorbance curve is shown below. Figure 7 As shown.

[0098] Specifically, to obtain carriers of different concentrations, the mass of the carrier was first determined using a traditional drying method. Deionized water was then added to the known mass of carrier to prepare a 10 mg / ml carrier solution. This 10 mg / ml carrier solution was further diluted with deionized water to prepare carriers of 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, and 100 μg / ml, for a total of 10 concentrations. Next, the absorbance (OD) value for each concentration was obtained by scanning the carrier at a wavelength of 500 nm. 500 This allows the establishment of a linear relationship between carrier concentration and absorbance value. y = kx + b At a known concentration x The value when λ 2. Absorbance value at 500nm y It can be directly measured by a spectrophotometer, and thus can be calculated. k It is 0.0021. b It is 0.0359. (In determining...) k and b After obtaining the numerical value, according to formula (2), that is, the carrier concentration x =(OD λ2 -b) / k =(OD λ2 -0.0359) / 0.0021.

[0099] The above experiments only used a carrier with a particle size of 190 nm to measure the absorbance values ​​at different concentrations. k and bThe numerical value was used to verify the concentration of the aforementioned carrier. x To verify the accuracy of the calculation results, the applicant used carriers of different particle sizes. First, carriers of different particle sizes were prepared, including seven sizes: 190nm, 200nm, 220nm, 240nm, 260nm, 280nm, and 300nm. Then, based on the carrier mass calculated by the drying method, theoretical concentrations of 40ug / ml, 50ug / ml, and 60ug / ml were prepared for each particle size. Given the known theoretical concentrations, the absorbance (OD) values ​​of each concentration of carrier with each particle size were measured at a wavelength of 500nm. λ2 The analysis was conducted. To obtain accurate results, each concentration of each particle size was divided into three portions for absorbance measurement. The absorbance values ​​(OD values) for each concentration of each particle size of the carrier were then obtained. λ2 Then, according to (OD) λ2 The concentration value was calculated by dividing the result by -0.0359 by 0.0021, and the calculated result was compared with the theoretical concentration value to determine the deviation of the calculation result. The results are shown in Table 3 below:

[0100] Table 3

[0101]

[0102] As can be seen from the data in Table 3, when the particle size of the carrier is less than or equal to 280 nm, the concentration recovery deviation is within 10%, i.e., according to (OD) λ2 The carrier concentration value calculated as (-0.0359) / 0.0021 x The deviation from the theoretical concentration value is within 10%, indicating that the method used in this application to determine the concentration of photosensitive microspheres based on absorbance values ​​has good accuracy. Therefore, the concentration determined by the above method can be used... k and b The numerical value is applied to the concentration of photosensitive microspheres in formula (2). C Calculation of 2. From Figure 7 It can be seen that in this experiment, when C A good linear relationship was observed when the value of 2 ranged from 10 ug / ml to 100 ug / ml. It should be noted that, due to the limited number of experiments conducted, C The value of 2 is not limited to 10 ug / ml to 100 ug / ml, and can be further determined by combining the following experiments. C The range of values ​​for 2.

[0103] III. Comparison of the effects of different mass ratios of photosensitive microspheres on photosensitivity

[0104] 1. Preparation of photosensitive microspheres with different mass ratios

[0105] First, following the preparation methods for photosensitive microspheres in steps one and two above, photosensitive microspheres were prepared using different mass ratios of carrier to photosensitive substance. Specifically, six types of photosensitive microspheres were prepared with mass ratios of 10:4, 10:2, 10:1, 10:0.2, 10:0.04, and 10:0, as shown in Tables 4 and 5 (microspheres 1 to 6). Here, 10:0 indicates that the photosensitive microsphere contains no photosensitive substance and is merely an empty carrier. Next, the prepared photosensitive microspheres with different mass ratios were diluted with deionized water, specifically diluted 500 times, 1000 times, and 2000 times for each of the six mass ratios. This resulted in three different concentrations of photosensitive microspheres after each dilution. The diluted photosensitive microspheres were scanned using a UV spectrophotometer to obtain the absorbance values ​​OD corresponding to wavelengths λ1680nm and λ2500nm. The corresponding concentration value C2 was calculated using formula (2) above, and the corresponding photosensitivity Ps was also calculated using formula (2) above. Specific data are shown in Table 4 below. It should be noted that... Figure 5 It is known that the photosensitive microspheres have a strong absorption peak at wavelength λ1, which is the maximum characteristic peak, and the corresponding absorbance value best reflects the concentration of the photosensitive substance. The absorbance value of the photosensitive microspheres includes the absorbance values ​​of both the carrier and the photosensitive substance; therefore, the true absorbance value OD of the photosensitive substance is... λ1感光物质 For OD λ1感光微球 -OD λ1载体 .

[0106] Table 4

[0107]

[0108] Furthermore, based on the photosensitivity of each mass ratio of photosensitive material at different dilution ratios in Table 4 above, the mean photosensitivity and coefficient of variation (CV) value are calculated. The CV value is the ratio of the standard deviation to the mean. The specific calculation results can be found in the relevant data in Table 5 below.

[0109] As shown in Table 4 above, when the dilution factor is 500X, the concentration value C2 of the photosensitive microspheres 6 is calculated according to formula (2) as 199, which is very close to the corresponding theoretical concentration value of 198. Therefore, the range of C2 in Experiment 2 above is supplemented, that is, the range of C2 can be 10 ug / ml to 200 ug / ml.

[0110] Table 5

[0111]

[0112] As shown in Table 4 above, the photosensitivity obtained by diluting the same mass proportion of photosensitive material by different factors is relatively consistent. As shown in Table 5, the CV values ​​of photosensitivity for different mass proportions of photosensitive material are all within 10%, indicating that the calculation results of photosensitivity determined by formulas (1) and (2) have small fluctuations and are relatively accurate. It also shows that for the same mass proportion of photosensitive material, its photosensitivity is related to the corresponding dilution factor, i.e., the concentration of photosensitive microspheres.

[0113] Furthermore, based on Table 5, the following can be plotted: Figure 8 The diagram shows the curves illustrating the relationship between different mass percentages of the photosensitive material and the calculated photosensitivity. From... Figure 8 It can be seen that the correlation between the photosensitivity of a unit concentration of photosensitive microspheres and the mass ratio of the photosensitive substance is consistent with the absorbance value; that is, the higher the mass ratio of the photosensitive substance and the higher the concentration of the photosensitive substance, the greater the photosensitivity. Meanwhile, when the mass ratio of carrier to photosensitive substance is less than 10:1, the linear relationship between photosensitivity and photosensitivity concentration is better; when the mass ratio of carrier to photosensitive substance is between 10:(2~4), the increase in photosensitivity becomes significantly smaller, indicating that the proportion of photosensitive substance, i.e., the amount of photosensitive substance filled in the carrier, gradually increases until it approaches saturation. This trend is consistent with the actual changes in the photosensitivity of the photosensitive microspheres. Furthermore, when the mass ratio of carrier to photosensitive substance is 10:4, the photosensitivity of the obtained photosensitive microspheres reaches a peak of 20.12. Even if the mass ratio of the photosensitive substance is further increased, the photosensitivity of the photosensitive microspheres will not increase further. Therefore, by controlling the mass ratio of carrier to photosensitive substance, material costs can be saved.

[0114] IV. Comparison of the performance of photosensitive microspheres with different photosensitivity levels in clinical applications

[0115] 1. Prepare photosensitive reagents with different photosensitivity based on photosensitive microspheres with different photosensitivity.

[0116] a) Treatment of photosensitive microsphere suspension: A certain amount of the photosensitive microspheres prepared in steps one and two above were centrifuged in a high-speed refrigerated centrifuge. After discarding the supernatant, a certain amount of MES buffer was added. Then, the microspheres were resuspended by ultrasonic vibration on an ultrasonic cell disruptor. Finally, MES buffer was added to adjust the concentration of photosensitive microspheres to 100 mg / ml.

[0117] b) Preparation of streptavidin solution: Weigh a certain amount of streptavidin and dissolve it in MES buffer to 8 mg / ml.

[0118] c) Mixing: Mix the prepared 100 mg / ml photosensitive microsphere suspension, 8 mg / ml avidin and MES buffer at a volume ratio of 2:5:1 and mix quickly to obtain the reaction solution.

[0119] d) Reaction: A 25 mg / ml NaBH3CN solution was prepared using MES buffer. The NaBH3CN solution and the reaction solution were rapidly mixed at a volume ratio of 1:25 and the reaction was carried out at a constant temperature of 37°C for 48 hours by rotation.

[0120] e) Blocking: Prepare a 75 mg / ml Glycine solution and a 25 mg / ml NaBH3CN solution using MES buffer. Mix the Glycine solution, NaBH3CN solution, and reaction solution at a volume ratio of 2:1:10. Add the mixture to the solution after reaction in step d) above and mix thoroughly. After rotating and reacting at 37°C for 2 hours, add a 200 mg / ml BSA solution (MES buffer) to the mixture at a volume ratio of 5:8. Mix quickly and then rotate and react at 37°C for 16 hours.

[0121] f) Washing: Add MES buffer to the solution that has reacted in step e, centrifuge with a high-speed refrigerated centrifuge, discard the supernatant, add fresh MES buffer, and resuspend by sonication. Centrifuge again, repeat this washing process 3 times, and finally resuspend with a small amount of MES buffer. The solid content is measured to be 10 mg / ml.

[0122] g) Preparation of photosensitive reagents: Photosensitive microspheres coated with streptavidin in the above six mass ratios with different photosensitivity were prepared using a universal buffer solution for photosensitive reagents, thereby obtaining six photosensitive reagents with different photosensitivity. The photosensitivity of the six photosensitive reagents is shown in Table 6 below.

[0123] Table 6

[0124]

[0125] 2. Evaluate the performance of six photosensitive reagents with different photosensitivity levels.

[0126] The above six photosensitive reagents with different photosensitivity levels were applied to the detection of clinical samples to evaluate the basic performance of photosensitive reagents with different photosensitivity levels in clinical application for sample detection.

[0127] Experimental materials and equipment

[0128]

[0129] 2.1 Testing the sensitivity of six photosensitizing reagents with different light sensitivity levels

[0130] Six kit samples (cal1 to cal6) with known target molecule concentrations of hepatitis B surface antigen (HBsAg) and six photosensitive reagents (1 to 6) prepared above with different photosensitivity levels were used. First, each sample was added to its corresponding reaction vessel. Then, a luminescent reagent and a biotin reagent were added sequentially to each reaction vessel. Each reaction vessel was incubated at 37°C to form a first complex of luminescent microspheres-antibody-antigen-antibody-biotin. Next, the corresponding photosensitive reagent was added to each reaction vessel, and photo-induced chemiluminescence detection was performed using a LiCA detector to obtain the corresponding chemiluminescence signal values. The data of the detected chemiluminescence signal values ​​for each photosensitive reagent are shown in columns 3 to 8 of Table 7 below. Sample cal1 has a target molecule concentration of 0, i.e., it does not contain hepatitis B surface antigen; sample cal1 is a negative sample, and the corresponding measured value can be used as the benchmark for the signal values ​​of each photosensitive reagent.

[0131] Table 7

[0132]

[0133] In Table 7, the theoretical values ​​in the first column are based on the known target molecule concentrations in the six samples. Columns three through eight of Table 7 show the chemiluminescence signal values ​​measured by each kit containing a photosensitive reagent and a luminescent reagent in a LiCA detector for samples with various target molecule concentrations. According to the data in the table, for samples with the same target molecule concentration, the photosensitivity of the photosensitive reagent ranges from 1.34 to 16.28; the higher the photosensitivity, the larger the measured signal value. However, when the photosensitivity reaches 20.12, a signal decrease occurs. Therefore, a range of 1.34–16.28 is chosen as the range for the photosensitivity Ps.

[0134] Furthermore, as shown in Table 8 below, for the same photosensitive reagent, the ratio of signal values ​​corresponding to samples with different target molecule concentrations reveals that when the photosensitive reagent's light sensitivity is below 1.34, the numerical differentiation between the signal values ​​corresponding to photosensitive reagent 1 is extremely low, indicating low detection sensitivity. It is impossible to distinguish samples with different concentrations of target molecules based on the signal values. While the signal values ​​corresponding to photosensitive reagent 2 are lower than those of photosensitive reagents 3 to 6, they still exhibit some differentiation for different concentrations of target molecules. The signal values ​​corresponding to photosensitive reagents 3 to 6 show clear signal values ​​for both low and high concentrations of target molecules. Simultaneously, the signal values ​​corresponding to different sizes of target molecules with the same photosensitive reagent show significant differences, demonstrating good differentiation. Therefore, the concentration range of the target molecules can also be determined based on the signal values.

[0135] Table 8

[0136]

[0137] 2.2 Testing the accuracy of detection results using five different photosensitizing reagents.

[0138] Preparation of experimental samples:

[0139] Select target molecules with known equal mass and dilute them into three known different concentrations: sp1, sp2, and sp3. Select 10 samples S1 to S10 with target molecule concentrations decreasing linearly, and the target molecule in all samples is HBsAg. Select 4 negative samples N1, N2, N3, and N4 that do not contain target molecules.

[0140] The target molecule concentrations of the 17 different samples were tested using five different photosensitizing reagents, 2 to 6, with varying photosensitive levels. The concentration data in columns three through seven were obtained from the chemiluminescence signals measured by the LiCA detector, as shown in Table 9 below. The first column, representing the actual concentration values ​​(theoretical values) for each sample, was used as a reference.

[0141] Table 9

[0142]

[0143] Because the photosensitivity of photosensitive reagent 1 is too low, it is not necessary to continue using it in the accuracy performance test in this experiment. As shown in Table 9 above, the higher the photosensitivity of the photosensitive reagent, the closer the test data is to the theoretical value, i.e., the higher the accuracy. Photosensitive reagent 2 has the lowest photosensitivity; the concentration values ​​measured for samples with low concentrations of target molecules fluctuate significantly from the theoretical values, while the concentration values ​​measured for samples with high concentrations of target molecules are closer to the theoretical values. Therefore, the photosensitivity of photosensitive reagent 2 can be considered the lower limit of photosensitivity. In other words, when the photosensitivity of the photosensitive microspheres is below 1.34, it will be impossible to accurately measure target molecules of various concentrations, which is not conducive to meeting clinical testing needs.

[0144] 2.3 Testing the precision of detection results using five different photosensitizing reagents

[0145] Continuing from section 2.2, samples (sp1, sp2, and sp3) with the same known mass of target molecules, diluted to three known different concentrations, were selected. Each concentration sample was divided into 10 portions, and each portion was tested with one of the five photosensitive reagents. The corresponding concentration values, as well as the mean concentration (Mean), standard deviation (STDEV), and coefficient of variation (CV) of the 10 samples, were obtained. The specific values ​​are shown in Table 10 below.

[0146] Table 10

[0147]

[0148] As can be seen from the data in Table 10 above, the CV value of photosensitive reagent 2 for the same sample sp1 at the lowest concentration is greater than 10%, indicating that the test results fluctuate greatly and the precision is generally poor. Therefore, the photosensitivity of photosensitive reagent 2, 1.34, can be used as the lower limit of the photosensitivity required for photosensitive microspheres in clinical testing.

[0149] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A photosensitive microsphere for photo-induced chemiluminescence detection, the photosensitive microsphere comprising a carrier and a photosensitive substance carried by the carrier, characterized in that, The photosensitivity Ps of the photosensitive microspheres is between 1.34 and 16.28; the photosensitivity Ps = OD λ1 / C 2*10 3 ,in: The OD λ1 It is in the visible light region within the range of 300nm to 800nm ​​that the concentration is... C The absorbance value corresponding to the maximum absorption peak of the wavelength-absorbance curve obtained after full-wavelength scanning of the photosensitive microspheres described in 2, wherein... λ 1 is the wavelength corresponding to the maximum absorption peak; C 2 represents the concentration of the photosensitive microspheres during photo-induced chemiluminescence detection. C The unit for 2 is ug / ml; The concentration of the photosensitive microspheres ; in, k It is the slope corresponding to the linear relationship of the carrier concentration-absorbance curve. b It is the intercept corresponding to the linear relationship of the carrier concentration-absorbance curve; OD λ2 It is the photosensitive microspheres at wavelength λ The absorbance values ​​corresponding to 2; the carrier concentration-absorbance curve is obtained by using multiple carriers of different concentrations at wavelengths... λ The curve obtained under condition 2; the wavelength λ 2 refers to the wavelengths corresponding to the same or similar absorbance values ​​of the photosensitive microspheres and the carrier with the same concentration in the wavelength-absorbance curve; The surface of the photosensitive microspheres is not coated with polysaccharides; or the polysaccharide content of the photosensitive microspheres per gram of mass is not higher than 25 mg.

2. The photosensitive microspheres according to claim 1, characterized in that: The wavelength λ 2 is selected from OD 感光微球 / OD 载体 The ratio is any wavelength value between 0.85 and 1.15, and the wavelength λ 2 is not equal to wavelength λ 1; Among them, OD 感光微球 and OD 载体 These are the absorbance values ​​corresponding to the same wavelength value in the range of 300nm to 800nm ​​for the photosensitive microspheres and the carrier, respectively, using the same concentration.

3. The photosensitive microspheres according to claim 1 or 2, characterized in that: The wavelength λ 2 represents the wavelength corresponding to the non-characteristic peak in the wavelength-absorbance curve.

4. The photosensitive microsphere according to claim 3, characterized in that: The wavelength λ2 is 400nm~600nm.

5. The photosensitive microsphere according to claim 3, characterized in that: The wavelength λ2 is 440nm~580nm.

6. The photosensitive microspheres according to claim 1 or 2, characterized in that: The linear relationship of the carrier concentration-absorbance curve is as follows: y = kx + b ,in: x Different concentrations of carriers with preset particle sizes, y For the carrier at the corresponding concentration x The absorbance value at that time k The slope b This is the intercept.

7. The photosensitive microsphere according to claim 6, characterized in that: Using the same wavelength λ 2 pairs of multiple known different concentrations x However, when carriers of the same particle size are scanned, the corresponding absorbance values ​​are obtained. y According to the concentration x and absorbance value y Calculate to obtain the corresponding k and stated b The value.

8. The photosensitive microsphere according to claim 6, characterized in that: The preset particle size is 190 nm. k =0.0021, b =0.0359.

9. The photosensitive microsphere according to claim 1, characterized in that: The corresponding wavelength is determined based on the photosensitive material. λ 1. The photosensitive microspheres are carriers filled with photosensitive material, wherein the wavelength... λ 1 is the wavelength corresponding to the maximum absorption peak in the wavelength-absorbance curve obtained after scanning the photosensitive material in the visible light region of 300nm~800nm.

10. The photosensitive microsphere according to claim 9, characterized in that: The photosensitive substance is a photosensitizer or a photosensitive dye, and the wavelength is... λ 1 is selected from 600nm~700nm; or Wavelength-absorbance curves of photosensitive microspheres with different known concentrations and the same photosensitive substance were obtained in advance. The wavelength corresponding to the maximum characteristic peak in the wavelength-absorbance curves of photosensitive microspheres at each concentration was selected as the wavelength of the microspheres. λ The possible values ​​of 1.

11. The photosensitive microspheres according to any one of claims 1-2 and 10, characterized in that: The photosensitive microspheres are prepared according to a mass ratio of the carrier to the photosensitive substance of 10:(0.04~4).

12. The photosensitive microspheres according to any one of claims 1-2 and 10, characterized in that: The particle size of the carrier is 190nm~280nm.

13. The photosensitive microspheres according to any one of claims 1-2 and 10, characterized in that: The C 2 is selected from 10ug / ml to 200ug / ml; The polysaccharide is at least one of glucan, fructan, mannan, and galactan; When the photosensitive microspheres are stored as a solid, a buffer solution is added for reconstitution. The concentration of the reconstituted photosensitive microsphere solution is the same as the initial concentration. C 1; When the photosensitive microspheres are stored as a liquid, the concentration at this point is the initial concentration. C 1; When performing photo-induced chemiluminescence detection, if the photosensitive microspheres use an initial concentration... C 1. If it participates in the detection, then the concentration C 2 equals C 1; or, for those with an initial concentration C The photosensitive microspheres were diluted before being used in the detection. The concentration of the diluted photosensitive microspheres was then determined. C 2 does not equal C 1.

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