CN111999277B - Structure for realizing fluorescence resonance transfer and application thereof - Google Patents
Structure for realizing fluorescence resonance transfer and application thereof Download PDFInfo
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Abstract
The invention is suitable for the technical fields of biology and chemistry. The invention provides a structure for realizing fluorescence resonance transfer and application thereof, and particularly designs a brand new structure of an energy donor and an energy acceptor, which can improve the efficiency of fluorescence resonance energy transfer. The fluorescence resonance transfer method can be applied to the detection of the content of various substances (organic molecules, inorganic groups, biological molecules, proteins and the like). The invention can also be applied to the immune reaction process, and the energy acceptors and the energy donors are respectively arranged on the solid carriers or the microspheres, so that the efficiency of fluorescence resonance energy transfer can be improved, separation-free and washing-free in the immune reaction process can be realized, and the immunological reaction can be simply and rapidly completed.
Description
Technical Field
The invention belongs to the technical field of biology and chemistry, and particularly relates to a structure for realizing fluorescence resonance transfer and application thereof.
Background
The fluorescence resonance transfer technology is important in immunological detection, but the fluorescence resonance transfer efficiency is key to the technology to realize high-sensitivity and high-accuracy detection. Typically, when energy transfer is achieved using singlet oxygen, the distance between the donor and acceptor of the singlet oxygen needs to be within about 200nm, otherwise the efficiency is greatly reduced, thereby reducing the sensitivity of detection.
The current method for solving the problem is realized by changing the sizes of a singlet oxygen donor and an acceptor, and the efficiency of capturing the singlet oxygen by the acceptor can be greatly improved under the condition that the size of the singlet oxygen donor is smaller than that of the acceptor, so that the detection sensitivity and the detection precision are improved. However, such a method requires high precision in controlling the sizes of donor and acceptor microspheres to be on the order of hundreds or hundreds of nanometers, and is very costly; and the mother solution of the liquid to be detected cannot be separated from the fluorescent resonator during detection, so that the background signal is further enhanced.
Therefore, the realization of a simple and low-cost fluorescence resonance system is of great significance to the immunological detection of the technology.
Disclosure of Invention
The embodiment of the invention aims to provide a structure for realizing fluorescence resonance transfer, and aims to solve the problems in the background technology.
The embodiment of the invention is realized in such a way that a structure for realizing fluorescence resonance transfer comprises an energy donor and an energy acceptor; one of the energy donor and the energy acceptor is in a planar coating structure, and the other is in a free granular structure.
As a preferred embodiment of the present invention, one of the energy donor and the energy acceptor is cured on the surface of the solid support; the structure for realizing fluorescence resonance transfer specifically comprises:
fluorescent microspheres or photosensitive microspheres with free granular structures, wherein fluorescent materials are connected to the surfaces of the fluorescent microspheres in a chemical bond mode, and photosensitive materials are connected to the surfaces of the photosensitive microspheres in a chemical bond mode; and
the photosensitization carrier with a planar coating structure is used as a carrier of the fluorescent microsphere, and the surface of the photosensitization carrier is connected with a photosensitive material in a chemical bond mode; or a fluorescent carrier with a planar coating structure, which is used as a carrier of the photosensitive microsphere, and the surface of the fluorescent carrier is connected with a fluorescent material in a chemical bond mode.
As another preferable mode of the embodiment of the invention, the preparation method of the photosensitized carrier or the fluoresced carrier includes the steps of:
mixing organic molecules with a solvent to obtain a solution A; the mass concentration of the solution A is 0.01% -1%;
mixing a photosensitive material or a fluorescent material with the solution A to obtain a solution B; the mass concentration of the photosensitive material or the fluorescent material in the solution B is 0.01-1%;
spraying a solution B containing a photosensitive material or a fluorescent material on the surface of the substrate material with the surface treated by functionalization, so as to obtain the photosensitized carrier or the fluorescent carrier.
Wherein the surface of the substrate material is subjected to functionalization treatment by using H to the surface of the substrate material 2 O 2 Cleaning with hydrochloric acid, naOH solution, ethanol, etc., to expose-OH, -NH on its surface 2 Or COOH and other functional groups.
As another preferable scheme of the embodiment of the invention, the preparation method of the fluorescent microsphere or the photosensitive microsphere comprises the following steps:
taking a polymer microsphere with a surface subjected to functionalization treatment, and mixing the polymer microsphere with a solvent to obtain a solution C; the mass concentration of the solution C is 0.01 to 5 percent
Mixing the photosensitive material or fluorescent material and organic molecules with the solution C, and centrifuging and cleaning to obtain the fluorescent microsphere or the photosensitive microsphere.
Wherein, the polymer microsphere with the surface treated by functionalization can be introduced into-OH, -NH by carrying out surface modification, oxidation, acid-base treatment and the like on the polymer microsphere (with the diameter of 60-500 nm) 2 -COOH and the like.
As another preferable scheme of the embodiment of the invention, the photosensitive material is any one of quantum dots, rhodamine, fluorescent yellow, methylene blue, rose bengal, porphyrin and derivatives thereof.
As another preferable mode of the embodiment of the invention, the fluorescent material is any one of 4, 5-dimethyl sulfide-4' - [2- (9-anthracenyl) ethylsulfide ] tetrathiafulvalene, phthalocyanine, ATTA-Eu complex and derivatives thereof.
As another preferable aspect of the embodiment of the present invention, the base material is a block or a sheet structure.
As another preferable mode of the embodiment of the invention, the base material is any one of glass, an alumina substrate, a mica sheet, quartz, a nitrocellulose film, and an aluminum sheet.
As another preferable scheme of the embodiment of the invention, the organic molecule is at least one of polyethylene oxide, starch, acacia, polystyrene, agarose, amino sugar, maltose, isocyanate and organic dibasic acid.
As another preferable scheme of the embodiment of the invention, the solvent is at least one of water, ethanol, acetone, chloroform and dimethylformamide.
Another object of the embodiments of the present invention is to provide a method for implementing fluorescence resonance transfer by adopting the above structure, which includes the following steps:
attaching a label to a photosensitizing carrier or a fluorescer carrier;
attaching a label to the fluorescent microsphere or the photosensitive microsphere;
mixing a photosensitized carrier or a fluoresced carrier connected with a marker, a fluorescent microsphere or a photosensitive microsphere connected with the marker and a sample to be detected, and then detecting a fluorescent signal; or mixing the fluorescent microsphere or photosensitive microsphere connected with the marker with the sample to be detected, and then placing the mixture on a photosensitized carrier or a fluoresced carrier connected with the marker for fluorescence signal detection.
According to the structure for realizing fluorescence resonance transfer, provided by the embodiment of the invention, the energy acceptors and the energy donors are respectively arranged on the solid carriers or the microspheres, so that the efficiency of fluorescence resonance energy transfer can be improved, and the structure can be applied to fluorescence resonance transfer, so that separation-free and washing-free in the immune reaction process can be realized, and the immunological reaction can be simply and rapidly completed. Specifically, singlet oxygen or other excited states can be generated through the photosensitive material by utilizing excitation light irradiation, and meanwhile, the singlet oxygen or fluorescence energy can be efficiently transferred to the fluorescent microsphere or the fluorescent carrier to realize low-background fluorescence emission, and a reaction signal is obtained after the fluorescent signal is received by the fluorescent signal collector. During testing, the coupled photosensitive material and fluorescent material can be simply separated from a mother solution system to reduce the interference of a mother solution background signal, such as natural sedimentation, clean water cleaning and the like; when the interference is not strong, direct detection can be performed without separation.
Drawings
FIG. 1 is a spectrum obtained in example 1 of the present invention.
FIG. 2 is a spectrum obtained in example 2 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a structure for realizing fluorescence resonance transfer and application thereof, and a specific application method of the structure comprises the following steps:
s1, dissolving 0.01g of starch, 0.005g of isocyanate and 0.002g of succinic acid in 10mL of water to obtain a solution A; then adding 0.01g of rhodamine B, dissolving and shaking uniformly to obtain solution B; then, 0.2mL of solution B is spin-coated on the surface of a glass sheet with 2 square centimeters of surface treated and cleaned by 0.1M NaOH for 5 times, and then the glass sheet is irradiated by ultraviolet rays for 1 minute and cleaned by deionized water, so that the carboxylated photosensitized carrier is obtained.
S2, adding 1mg of isocyanate, 2mg of polyethylene oxide and 2mg of 4, 5-dimethyl-4' - [2- (9-anthraceneoxy) ethylthio ] tetrathiafulvalene to 20mL of aqueous solution C containing 200nm carboxylated polystyrene microspheres with concentration of 0.05w%, and fixing the volume to 100mL; next, the fluorescent microspheres were obtained by irradiating with ultraviolet rays and vigorously stirring for 1 minute, and then removing the unbound small molecules by centrifugal ultrasonic cleaning.
S3, respectively connecting the antibody or antigen to the photosensitized carrier and the fluorescent microsphere by using a surface functional group through a traditional immunological method, wherein the specific steps are as follows:
(1) Adding the photosensitization carrier into a beaker, adding 15mL of a marking buffer solution, and shaking and uniformly mixing;
(2) Adding 1mL of an activation buffer solution, and carrying out oscillation reaction at room temperature for 20 minutes;
(3) Adding 2mg of antibody 1 of C Reactive Protein (CRP), and performing shaking reaction at room temperature for 1 hour, wherein ultrasonic dispersion is performed for 20 seconds every 10 minutes;
(4) Adding 2mL of a sealing buffer solution, and uniformly mixing for 5 minutes at room temperature;
(5) Adding 5mL of a sealing buffer solution, and carrying out oscillation reaction for 3 hours at 37 ℃;
(6) Discarding the solution to obtain the labeled photosensitized carrier, airing at room temperature, and preserving at 5 ℃ for later use.
(7) Taking 1mL of the fluorescent microsphere, adding 4mL of a marking buffer solution, and uniformly mixing by ultrasonic;
(8) Adding 0.6mL of an activation buffer solution, and carrying out oscillation reaction at room temperature for 15 minutes, wherein ultrasonic dispersion is carried out for 40 seconds every 5 minutes;
(9) Adding 2mg of antibody 2 of C Reactive Protein (CRP), uniformly mixing by ultrasonic, and carrying out shaking reaction at 37 ℃ for 2 hours, wherein the ultrasonic dispersion is carried out for 20 seconds every 10 minutes;
(10) Adding 1mL of a blocking buffer solution, and uniformly mixing for 10 minutes at 37 ℃;
(11) Adding 5mL of a sealing buffer solution, and carrying out oscillation reaction for 1 hour at 37 ℃;
(12) Centrifuging for 30min by a low-temperature centrifuge, discarding supernatant, re-suspending by using a re-suspending buffer solution, performing ultrasonic dispersion for 3 times, and performing ultrasonic treatment for 40 seconds each time until the mixture is uniformly mixed to obtain the marked fluorescent microsphere.
The various buffers used are commercially available products and can be the same as the reagents used for the conventional immunological reaction markers in the prior art.
S4, taking the labeled photosensitized carrier as a capture site, mixing a serum sample (CRP concentration is 1 mg/L) with the labeled fluorescent microsphere, then dripping the mixture onto the labeled photosensitized carrier, and incubating the mixture at room temperature for 5 minutes to fully perform the reaction; then, the sample is placed into fluorescence detection equipment, after 532nm excitation, the concentration of the antigen to be detected is obtained through the fluorescence emission intensity of 420nm of 4, 5-dimethyl thio-4' - [2- (9-anthracenyl) ethylthio ] tetrathiafulvalene, and the obtained spectrogram is shown in figure 1. In addition, E dinburgh Instruments FLS-980 was used to detect that the fluorescence intensity in the energy transfer structure from the photosensitized carrier to the fluorescent microsphere was 7462.
Example 2
The embodiment provides a structure for realizing fluorescence resonance transfer and application thereof, and a specific application method of the structure comprises the following steps:
s1, dissolving 1mg of isocyanate and 1mg of adipic acid in 10mL of water to obtain a solution A; then, 10mg of phthalocyanine derivative (octa (diethoxyphosphinylmethyl) phthalocyanine) was added to the mixture to dissolve and shake the mixture uniformly, thereby obtaining a solution B; then, 1mL of the solution B was taken and dropped 20 times onto the surface of an aluminum sheet which was spin-coated on the surface of 2 square cm and washed with 0.1M NaOH, and then irradiated with ultraviolet rays for 3 minutes, and washed with deionized water, to obtain a fluorescent support.
S2, adding 1mg of isocyanate, 5mg of hematoporphyrin magnesium and 5mL of dimethylformamide into 10mL of aqueous solution C containing 150nm carboxylated polystyrene microspheres with the concentration of 0.1w%, and fixing the volume to 100mL by using water; then, the mixture was irradiated with ultraviolet rays and vigorously stirred for 3 minutes, and then the unbound small molecules and the organic solvent were removed by centrifugal ultrasonic cleaning to obtain photosensitive microspheres.
S3, respectively connecting the antibody or antigen to the fluorescent carrier and the photosensitive microsphere by using a surface functional group through a traditional immunological method, wherein the specific steps are as follows:
(1) Adding the above-mentioned fluorescent carrier into beaker, adding 25mL of marking buffer solution, and uniformly shaking;
(2) Adding 2mL of an activation buffer solution, and carrying out oscillation reaction at room temperature for 15 minutes;
(3) Adding 3mg of Procalcitonin (PCT) antibody 1, and performing shaking reaction at room temperature for 2 hours, wherein ultrasonic dispersion is performed for 30 seconds every 15 minutes;
(4) Adding 4mL of a sealing buffer solution, and uniformly mixing for 10 minutes at room temperature;
(5) Adding 10mL of a blocking buffer solution, and carrying out oscillation reaction for 2 hours at 37 ℃;
(6) And (5) discarding the solution to obtain the labeled fluorescent carrier, airing at room temperature, and preserving at 5 ℃ for later use.
(7) Taking 3mL of the photosensitive microsphere, adding 10mL of a marking buffer solution, and uniformly mixing by ultrasonic;
(8) Adding 1mL of an activation buffer solution, and carrying out oscillation reaction at room temperature for 30 minutes, wherein ultrasonic dispersion is carried out for 30 seconds every 5 minutes;
(9) Adding 3mg of Procalcitonin (PCT) antibody 2, uniformly mixing by ultrasonic, and performing shaking reaction at 37 ℃ for 2 hours, wherein ultrasonic dispersion is performed for 40 seconds every 10 minutes;
(10) Adding 3mL of a blocking buffer solution, and uniformly mixing for 8 minutes at 37 ℃;
(11) Adding 10mL of a blocking buffer solution, and carrying out oscillation reaction for 2 hours at 37 ℃;
(12) Centrifuging for 40min by a low-temperature centrifuge, discarding supernatant, re-suspending by using a re-suspending buffer solution, performing ultrasonic dispersion for 5 times, and performing ultrasonic treatment for 30 seconds each time until the mixture is uniformly mixed, thus obtaining the marked photosensitive microsphere.
The various buffers used are commercially available products and can be the same as the reagents used for the conventional immunological reaction markers in the prior art.
S4, mixing a serum sample (PCT concentration is 1 mg/L) with the labeled photosensitive microspheres, then dripping the mixture onto the labeled fluorescent carrier, and incubating the mixture at 37 ℃ for 3 minutes to fully perform the reaction; then, the sample is placed into fluorescence detection equipment, and after 420nm irradiation, the concentration of the antigen to be detected is obtained through the 700nm fluorescence emission intensity of the phthalocyanine derivative, and the obtained spectrogram is shown in figure 2. In addition, edinburgh Instruments FLS-980 was used to detect that the fluorescence intensity in the energy transfer structure from the photosensitive microsphere to the fluorogenic carrier was 4862.
Example 3
The embodiment provides a structure for realizing fluorescence resonance transfer, which comprises a photosensitizing carrier and fluorescent microspheres, wherein the preparation method of the photosensitizing carrier and the fluorescent microspheres is as follows:
s1, dissolving 0.01g of Arabic gum in a mixed solvent of 10mL of water and ethanol in an equal volume ratio to obtain a solution A; then adding 0.01g of quantum dots, dissolving and shaking uniformly to obtain a solution B; then, 0.2mL of solution B is spin-coated on the surface of a mica sheet with 2 square centimeters of surface treated and cleaned by 0.1M NaOH for 5 times, and then the mica sheet is irradiated by ultraviolet rays for 1 minute and cleaned by deionized water, so that the carboxylated photosensitized carrier is obtained.
S2, adding 0.5mg of isocyanate, 0.5mg of polystyrene and 2mg of ATTA-Eu complex into 20mL of aqueous solution C containing 200nm carboxylated polystyrene microspheres with the concentration of 0.01w percent, and fixing the volume to 100mL; next, the fluorescent microspheres were obtained by irradiating with ultraviolet rays and vigorously stirring for 1 minute, and then removing the unbound small molecules by centrifugal ultrasonic cleaning.
Example 4
The embodiment provides a structure for realizing fluorescence resonance transfer, which comprises a photosensitizing carrier and fluorescent microspheres, wherein the preparation method of the photosensitizing carrier and the fluorescent microspheres is as follows:
s1, dissolving 0.05g of agarose, 0.03g of aminosugar and 0.02g of maltose in 10mL of water to obtain a solution A; then adding 0.1g of porphyrin, dissolving and shaking uniformly to obtain a solution B; then, 0.2mL of solution B is spin-coated on the surface of an alumina substrate with 2 square centimeters of surface treated and cleaned by 0.1M NaOH for 5 times, and then the surface is irradiated by ultraviolet rays for 1 minute and cleaned by deionized water, so as to obtain the carboxylated photosensitized carrier.
S2, adding 1mg of isocyanate, 2mg of polyethylene oxide and 2mg of phthalocyanine into 20mL of aqueous solution C containing 200nm carboxylated polystyrene microspheres with the concentration of 5w percent, and fixing the volume to 100mL; next, the fluorescent microspheres were obtained by irradiating with ultraviolet rays and vigorously stirring for 1 minute, and then removing the unbound small molecules by centrifugal ultrasonic cleaning.
Example 5
The embodiment provides a structure for realizing fluorescence resonance transfer, which comprises a fluorescent carrier and photosensitive microspheres, wherein the preparation method of the fluorescent carrier and the photosensitive microspheres is as follows:
s1, dissolving 2mg of polyethylene oxide, 2mg of isocyanate and 1mg of adipic acid in 10mL of mixed solvent with equal volume ratio of water, ethanol and chloroform to obtain a solution A; then adding 10mg of phthalocyanine, dissolving and shaking uniformly to obtain a solution B; then, 1mL of the solution B was taken and applied dropwise 20 times to the surface of a quartz block which was spin-coated on the surface of 2 square cm and washed with 0.1M NaOH, and then irradiated with ultraviolet rays for 3 minutes, and washed with deionized water, to obtain a fluorescent support.
S2, adding 1mg of isocyanate, 0.5mg of polystyrene, 5mg of hematoporphyrin magnesium and 5mL of acetone into 10mL of aqueous solution C containing 150nm carboxylated polystyrene microspheres with the concentration of 2w%, and fixing the volume to 100mL by using water; then, the mixture was irradiated with ultraviolet rays and vigorously stirred for 3 minutes, and then the unbound small molecules and the organic solvent were removed by centrifugal ultrasonic cleaning to obtain photosensitive microspheres.
Example 6
The embodiment provides a structure for realizing fluorescence resonance transfer, which comprises a fluorescent carrier and photosensitive microspheres, wherein the preparation method of the fluorescent carrier and the photosensitive microspheres is as follows:
s1, dissolving 1mg of isocyanate and 1mg of adipic acid in 10mL of mixed solvent with equal volume ratio of water to acetone to obtain solution A; then, adding 10mg of 4, 5-dimethyl-thio-4' - [2- (9-anthracenyl) ethylthio ] tetrathiafulvalene, dissolving and shaking uniformly to obtain a solution B; then, 1mL of the solution B was taken and applied dropwise 20 times to the surface of a nitrocellulose membrane which had been spin-coated on a 2 square centimeter surface and washed with 0.1M NaOH, and then irradiated with ultraviolet light for 3 minutes, and washed with deionized water, to obtain a fluorescent support.
S2, adding 1mg of isocyanate, 5mg of hematoporphyrin magnesium and 10mL of chloroform into 10mL of aqueous solution C containing 150nm carboxylated polystyrene microspheres with the concentration of 1w%, and fixing the volume to 100mL by using water; then, the mixture was irradiated with ultraviolet rays and vigorously stirred for 3 minutes, and then the unbound small molecules and the organic solvent were removed by centrifugal ultrasonic cleaning to obtain photosensitive microspheres.
Example 7
This example provides a structure for achieving fluorescence resonance transfer and its application, and the specific method of using the structure differs from that of example 1 only in that the incubation method in step S4 is as follows:
mixing a serum sample (CRP concentration is 1 mg/L), the marked fluorescent microsphere and the marked photosensitized carrier, and then incubating at room temperature for 5 minutes to fully perform the reaction; then, the sample is placed in a fluorescence detection device for detection.
Example 8
This example provides a structure for achieving fluorescence resonance transfer and its application, and the specific method of using the structure differs from that of example 2 only in that the incubation method in step S4 is as follows:
mixing a serum sample (PCT concentration is 1 mg/L) with the labeled photosensitive microsphere and the labeled fluorescent carrier, and then incubating at 37 ℃ for 3 minutes to fully perform the reaction; then, the sample is placed in a fluorescence detection device for detection.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (2)
1. A structure for realizing fluorescence resonance transfer, which is characterized by comprising a photosensitized carrier and fluorescent microspheres; or the structure specifically comprises a fluorescent carrier and photosensitive microspheres;
the preparation method of the photosensitized carrier comprises the following steps:
starch, isocyanate and succinic acid are dissolved in water to obtain solution A; then adding rhodamine B, dissolving and shaking uniformly to obtain a solution B; then, the solution B is taken to be spin-coated on the surface of a glass sheet, the surface of which is cleaned by NaOH treatment, and then is irradiated by ultraviolet rays and is cleaned by deionized water, so as to obtain a carboxylated photosensitized carrier;
the preparation method of the fluorescent microsphere comprises the following steps:
adding isocyanate, polyethylene oxide and 4, 5-dimethyl sulfide-4' - [2- (9-anthraceneoxy) ethylsulfide ] tetrathiafulvalene into an aqueous solution C containing carboxylated polystyrene microspheres, and fixing the volume; then, irradiating with ultraviolet rays and stirring vigorously, and then removing unbound small molecules by centrifugal ultrasonic cleaning to obtain fluorescent microspheres;
the preparation method of the fluorescent carrier comprises the following steps:
dissolving isocyanate and adipic acid in water to obtain a solution A; then adding phthalocyanine derivative, dissolving and shaking uniformly to obtain solution B; then, the solution B is taken to be spin-coated on the surface of the aluminum sheet with the surface being cleaned by NaOH treatment, then ultraviolet irradiation is carried out, and deionized water is used for cleaning, thus obtaining a fluorescent carrier;
the preparation method of the photosensitive microsphere comprises the following steps:
adding isocyanate, hematoporphyrin magnesium and dimethylformamide into an aqueous solution C containing carboxylated polystyrene microspheres, and fixing the volume by water; then, the photosensitive microsphere is obtained by irradiating ultraviolet rays and vigorously stirring, and then removing unbound small molecules and organic solvent through centrifugal ultrasonic cleaning.
2. Use of the structure of claim 1 in fluorescence resonance transfer, comprising the steps of:
(1) Adding the photosensitization carrier into a beaker, adding a marking buffer solution, and vibrating and uniformly mixing;
(2) Adding an activation buffer solution, and carrying out oscillation reaction at room temperature;
(3) Adding antibody 1 of the C reaction protein, and carrying out oscillation reaction at room temperature;
(4) Adding a sealing buffer solution, and uniformly mixing at room temperature;
(5) Adding a sealing buffer solution, and carrying out oscillation reaction at 37 ℃;
(6) Discarding the solution to obtain a labeled photosensitized carrier, airing at room temperature and preserving for later use;
(7) Adding a marking buffer solution into the fluorescent microspheres, and uniformly mixing by ultrasonic waves;
(8) Adding an activation buffer solution, and carrying out oscillation reaction at room temperature;
(9) Adding antibody 2 of the C reaction protein, uniformly mixing by ultrasonic wave, and carrying out oscillation reaction at 37 ℃;
(10) Adding a sealing buffer solution, and uniformly mixing at 37 ℃;
(11) Adding a sealing buffer solution, and carrying out oscillation reaction at 37 ℃;
(12) Centrifuging by a low-temperature centrifuge, discarding supernatant, re-suspending by using a re-suspending buffer solution, and performing ultrasonic dispersion until the mixture is uniformly mixed to obtain labeled fluorescent microspheres;
(13) Mixing the serum sample with the labeled fluorescent microspheres by taking the labeled photosensitized carrier as a capture site, then dripping the mixture onto the labeled photosensitized carrier, and incubating the mixture at room temperature to fully perform the reaction; then placing the sample into fluorescence detection equipment, and obtaining the concentration of an antigen to be detected through excitation at 532nm and fluorescence emission intensity of 420nm of 4, 5-dimethyl sulfide-4' - [2- (9-anthracenyl) ethylsulfide ] tetrathiafulvalene;
alternatively, the application comprises the steps of:
(1) Adding the above-mentioned fluorescent carrier into beaker, adding the marking buffer solution, and uniformly mixing by shaking;
(2) Adding an activation buffer solution, and carrying out oscillation reaction at room temperature;
(3) Adding procalcitonin antibody 1, and performing oscillation reaction at room temperature;
(4) Adding a sealing buffer solution, and uniformly mixing at room temperature;
(5) Adding a sealing buffer solution, and carrying out oscillation reaction at 37 ℃;
(6) Discarding the solution to obtain a marked fluorescent carrier, airing at room temperature and preserving for later use;
(7) Adding a marking buffer solution into the photosensitive microspheres, and uniformly mixing by ultrasonic;
(8) Adding an activation buffer solution, and carrying out oscillation reaction at room temperature;
(9) Adding procalcitonin antibody 2, mixing with ultrasound, and performing shaking reaction at 37deg.C;
(10) Adding a sealing buffer solution, and uniformly mixing at 37 ℃;
(11) Adding a sealing buffer solution, and carrying out oscillation reaction at 37 ℃;
(12) Centrifuging by a low-temperature centrifuge, discarding supernatant, re-suspending by using a re-suspending buffer solution, and performing ultrasonic dispersion until the mixture is uniformly mixed to obtain labeled photosensitive microspheres;
(13) Mixing a serum sample with the labeled photosensitive microspheres, then dripping the mixture onto the labeled fluorescent carrier, and incubating the mixture at 37 ℃ for 3 minutes to fully perform the reaction; then, the sample is placed into fluorescence detection equipment, and the concentration of the antigen to be detected is obtained through the 700nm fluorescence emission intensity of the phthalocyanine derivative after 420nm irradiation.
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