A fluorescence resonance energy transfer probe system and a method for high-sensitivity homogeneous immunoassay of mycophenolic acid in whole blood

Abstract

The application discloses a fluorescence resonance energy transfer probe system and a method for high-sensitivity homogeneous immuno-detection of mycophenolic acid in whole blood. The application uses NaYbF4:Tm nanocrystals marked with BA monoclonal antibody on the surface as a donor probe, NaDyF4 nanocrystals marked with BA antigen on the surface as an acceptor probe, and constructs a fluorescence resonance energy transfer probe system. The probe system is used for qualitative and quantitative detection of mycophenolic acid in whole blood. The donor probe is incubated with a whole blood sample to be detected, and the fluorescence intensity emitted at 800 nm is tested. Then, the acceptor probe is added for joint incubation, and the fluorescence intensity emitted at 800 nm is tested. The ratio of the fluorescence intensity of the two times is compared with a control group or a standard curve, so that the mycophenolic acid in the whole blood can be qualitatively or quantitatively detected. The probe system can effectively overcome the background interference of the whole blood sample, and realize high-sensitivity homogeneous immuno-detection of BA in the whole blood.

Description

Technical Field

[0001] This invention belongs to the field of immunological detection, specifically relating to a fluorescence resonance energy transfer probe system and a method for highly sensitive homogeneous immunoassay of bongkrekic acid in whole blood. Background Technology

[0002] Bongkrekic acid (BA) is a highly toxic metabolite produced by *Pseudomonas cocovenenans* subspecies *Pseudomonas aeruginosa*, first discovered by Dutch scholars Mertens and Vanveen in 1930. BA is an unsaturated tricarboxylic acid fatty acid commonly found in fermented grains, spoiled white fungus, black fungus, and other spoiled starchy foods. BA is a potent respiratory toxin that can cause acute poisoning in humans, leading to damage to the nervous, digestive, and urinary systems. Severe BA poisoning can rapidly cause liver and kidney damage, resulting in multiple organ failure and a high mortality rate. Due to its stable molecular structure, common food processing techniques such as high-temperature heating, washing, and freezing are unlikely to remove it. Therefore, the detection of BA is of great significance for human health in preventing poisoning and effectively responding to rapid treatment after poisoning.

[0003] Currently, the detection of BA poisoning mainly focuses on exogenous samples such as suspected spoiled food or vomit, but this often faces problems such as sample shortage or loss of representativeness. Whole blood samples can directly reflect the degree of toxin exposure in the human body, and real-time blood drug concentrations can provide a basis for dosage adjustment of treatment measures such as blood purification. Therefore, whole blood samples are ideal for BA poisoning assessment and treatment monitoring. Establishing a rapid and accurate technology for detecting BA in whole blood is crucial for improving the efficiency of poisoning treatment and reducing mortality.

[0004] Achieving high-sensitivity detection of BA in whole blood faces several challenges. First, the low concentration of BA in blood places high demands on the sensitivity of the detection method. Second, whole blood contains proteins, lipids, and other endogenous substances, which can easily affect the accuracy and specificity of the detection. While the commonly used high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for BA detection meets the requirements for quantification and high sensitivity, its complex sample pretreatment process, expensive equipment, and long detection time make it unsuitable for rapid on-site detection. Homogeneous detection methods, which do not require complex steps such as sample separation and washing, offer advantages such as ease of operation and fast detection speed, making them an ideal strategy for BA detection in whole blood. However, whole blood exhibits strong absorption and emission in the visible light region. To achieve homogeneous immunoassay for BA in whole blood, it is necessary to overcome problems such as strong background interference and low sensitivity. Traditional fluorescent probes cannot overcome background interference such as the autofluorescence and light scattering of whole blood, leading to reduced sensitivity and specificity. Therefore, a technology for rapid, sensitive, and homogeneous detection of BA in whole blood without background interference remains to be developed. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide a method for preparing a fluorescence resonance energy transfer (NIR-NIR FRET) probe system based on near-infrared excitation and emission. This invention involves modifying NaYbF4:Tm nanocrystals with carboxyl groups and binding them with BA monoclonal antibodies to obtain NaYbF4:Tm nanocrystals surface-labeled with BA monoclonal antibodies. Similarly, modifying NaDyF4 nanocrystals with carboxyl groups and binding them with BA antigens to obtain NaDyF4 nanocrystals surface-labeled with BA antigens. The NaYbF4:Tm nanocrystals surface-labeled with BA monoclonal antibodies serve as donor probes, and the NaDyF4 nanocrystals surface-labeled with BA antigens serve as acceptor probes, thus obtaining a fluorescence resonance energy transfer (NIR-NIR FRET) probe system based on near-infrared excitation and emission.

[0006] Another objective of this invention is to provide a near-infrared excitation and emission-based fluorescence resonance energy transfer (NIR-NIR FRET) probe system obtained by the above preparation method. This NIR-NIR FRET probe system can effectively overcome background interference in whole blood samples and achieve highly sensitive homogeneous immunodetection of BA in whole blood.

[0007] Another object of the present invention is to provide the application of the above-mentioned fluorescence resonance energy transfer (NIR-NIR FRET) probe system based on near-infrared excitation and emission in the detection of BA in whole blood.

[0008] Another objective of this invention is to provide a highly sensitive homogeneous immunoassay method for detecting bongkrekic acid (BA) in whole blood. This method employs a NIR-NIR FRET nanoprobe that is excited at 980 nm and emits at 800 nm, avoiding the strong absorption and emission of whole blood in the visible light region. This overcomes the background interference problem inherent in traditional fluorescent probes in complex samples like whole blood, achieving highly sensitive and specific detection of trace amounts of BA in whole blood without requiring complex sample pretreatment. Furthermore, detecting BA in whole blood samples allows for direct analysis of the toxin exposure level, more accurately reflecting the poisoning state in the human body, and providing a reliable basis for poisoning diagnosis, assessment of the degree of poisoning, and subsequent drug treatment.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing a fluorescence resonance energy transfer (NIR-NIR FRET) probe system, comprising the following steps:

[0011] (1) NaYbF4:Tm nanocrystals labeled with BA monoclonal antibody

[0012] S1 mixes and dissolves ytterbium acetate, thulium acetate, 1-octadecene and oleic acid to obtain a precursor solution, adds sodium hydroxide and ammonium fluoride, and heats to react, to obtain NaYbF4:Tm nanocrystals with oleic acid as the surface ligand;

[0013] S2 removes the oleic acid ligand from NaYbF4:Tm nanocrystals with oleic acid as the surface ligand, and then reacts them with polyacrylic acid to obtain polyacrylic acid modified NaYbF4:Tm nanocrystals.

[0014] S3 dispersed polyacrylic acid-modified NaYbF4:Tm nanocrystals in 4-morphine ethanesulfonic acid (MES) buffer, added ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (Sulfo-NHS) to activate the carboxyl groups on the polyacrylic acid, and then combined with BA monoclonal antibody to obtain NaYbF4:Tm nanocrystals with surface-labeled BA monoclonal antibody;

[0015] (2) NaDyF4 nanocrystals labeled with BA antigen

[0016] S4 reacts dysprosium oxide (Dy2O3) with trifluoroacetic acid by heating to obtain dysprosium trifluoroacetate. Sodium trifluoroacetate (CF3COONa), 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate are reacted by heating to obtain NaDyF4 nanocrystals with oleic acid and oleylamine as surface ligands.

[0017] S5 removes the oleic acid and oleylamine ligands from NaDyF4 nanocrystals with surface ligands of oleic acid and oleylamine, and then reacts them with polyacrylic acid to obtain polyacrylic acid modified NaDyF4 nanocrystals.

[0018] S6 dispersed polyacrylic acid-modified NaDyF4 nanocrystals in 4-morphine ethanesulfonic acid (MES) buffer, added ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (Sulfo-NHS) to activate the carboxyl groups on the polyacrylic acid, and then combined with BA antigen to obtain NaDyF4 nanocrystals with BA antigen labeled on the surface.

[0019] A fluorescence resonance energy transfer (NIR-NIR FRET) probe system was constructed using NaYbF4:Tm nanocrystals labeled with BA monoclonal antibody as donor probes and NaDyF4 nanocrystals labeled with BA antigen as acceptor probes.

[0020] The probe system described in this invention emits fluorescence at 800 nm under near-infrared light excitation at 980 nm for NaYbF4:Tm nanocrystals. When the antibody in the NaYbF4:Tm nanocrystals labeled with BA monoclonal antibody binds to the antigen in the NaDyF4 nanocrystals labeled with BA antigen, fluorescence resonance energy transfer (FRET) occurs between the NaYbF4:Tm and NaDyF4 nanocrystals, quenching the fluorescence of NaYbF4:Tm at 800 nm. Therefore, the probe system described in this invention can achieve BA detection based on FRET.

[0021] Preferably, the NaYbF4:Tm nanocrystals in S1 have a hexagonal phase crystal structure and a particle size of 20-30 nm (e.g., 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, etc.), more preferably 24.3±0.8 nm.

[0022] Preferably, in the NaYbF4:Tm nanocrystals described in S1, the molar ratio of Yb to Tm is 95:5 to 99.5:0.5 (e.g., 99.5:0.5, 99:1, 98:2, 97:3, 96:4, and 95:5, etc.).

[0023] Preferably, the ratio of the total molar amount of ytterbium acetate and thulium acetate, the molar amount of sodium hydroxide, the molar amount of ammonium fluoride, the volume of 1-octadecene, and the volume of oleic acid in S1 is 1-3 mmol: 2.5-5 mmol: 3-5 mmol: 15-20 mL: 7.5-10 mL. Within this ratio range, the total molar amount ratio of ytterbium acetate and thulium acetate can be 1 mmol, 1.5 mmol, 2 mmol, 2.5 mmol, 3 mmol, etc.; the molar amount ratio of sodium hydroxide can be 2.5 mmol, 3 mmol, 3.5 mmol, 4 mmol, 4.5 mmol, 5 mmol, etc.; the molar amount ratio of ammonium fluoride can be 3 mmol, 3.5 mmol, 4 mmol, 4.5 mmol, 5 mmol, etc.; and the volume ratio of 1-octadecene can be 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, etc. The volume ratio of oleic acid can be 7.5 mL, 8 mL, 8.5 mL, 9 mL, 9.5 mL, 10 mL, etc., for example, 1 mmol: 2.5 mmol: 4 mmol: 15 mL: 10 mL, etc.

[0024] Preferably, the ytterbium acetate, thulium acetate, 1-octadecene and oleic acid in S1 are mixed and dissolved at 150-170 °C (e.g. 150 °C, 155 °C, 160 °C, 165 °C, 170 °C, etc.) and heated until the solid is completely dissolved.

[0025] Preferably, the operations in S1 are all performed in an inert gas atmosphere, which is at least one of nitrogen, argon and helium.

[0026] Preferably, in step S1, sodium hydroxide and ammonium fluoride are added to the precursor solution as sodium hydroxide methanol solution and ammonium fluoride methanol solution, respectively. Before heating the reaction, the system is heated to 120-140 °C (e.g., 120 °C, 125 °C, 130 °C, 135 °C, 140 °C, etc.) to remove water and methanol from the reaction system.

[0027] The sodium hydroxide methanol solution has a sodium hydroxide molar concentration of 0.25-0.5 mmol / mL (e.g., 0.25 mmol / mL, 0.3 mmol / mL, 0.35 mmol / mL, 0.4 mmol / mL, 0.45 mmol / mL, 0.5 mmol / mL, etc.), and the ammonium fluoride methanol solution has an ammonium fluoride molar concentration of 0.3-0.5 mmol / mL (e.g., 0.3 mmol / mL, 0.35 mmol / mL, 0.4 mmol / mL, 0.45 mmol / mL, 0.5 mmol / mL, etc.).

[0028] Preferably, the heating reaction in S1 is carried out at a temperature of 290-310 °C (e.g., 290 °C, 295 °C, 300 °C, 305 °C, 310 °C, etc.) and for a time of 50-70 min (e.g., 50 min, 55 min, 60 min, 65 min, 70 min, etc.).

[0029] Preferably, after the heating reaction described in S1 is completed, the mixture is cooled to below 90 °C, and the precipitate is collected by centrifugation to obtain NaYbF4:Tm nanocrystals. The nanocrystals are then dispersed in cyclohexane for later use. The concentration of NaYbF4:Tm nanocrystals dispersed in cyclohexane is 0.1-0.2 mmol / mL (e.g., 0.1 mmol / mL, 0.12 mmol / mL, 0.15 mmol / mL, 0.18 mmol / mL, 0.2 mmol / mL, etc.).

[0030] Preferably, the method for removing oleic acid ligands from NaYbF4:Tm nanocrystals with oleic acid as the surface ligand in S2 is as follows:

[0031] Add hydrochloric acid aqueous solution and ethanol to the cyclohexane dispersion of NaYbF4:Tm nanocrystals, shake to mix, and let stand for 6-12 h (e.g., 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, etc.) to remove oleic acid ligands on the surface of the nanocrystals. Then centrifuge to collect the ligand-free NaYbF4:Tm nanocrystals and disperse them in water.

[0032] The concentration of the NaYbF4:Tm nanocrystalline cyclohexane dispersion is 0.1-0.2 mmol / mL (e.g., 0.1 mmol / mL, 0.12 mmol / mL, 0.15 mmol / mL, 0.18 mmol / mL, 0.2 mmol / mL, etc.); the concentration of the hydrochloric acid aqueous solution is 0.05-0.2 mol / L (e.g., 0.05 mol / L, 0.1 mol / L, 0.12 mol / L, 0.15 mol / L, 0.18 mol / L, 0.2 mol / L, etc.); the volume ratio of the NaYbF4:Tm nanocrystalline cyclohexane dispersion, hydrochloric acid aqueous solution, and ethanol is 1:1:1.

[0033] Preferably, in step S2, the NaYbF4:Tm nanocrystals with oleic acid as the surface ligand are removed, resuspended in water, and then added dropwise to an aqueous polyacrylic acid solution for mixing and stirring. The concentration of the ligand-free NaYbF4:Tm nanocrystals resuspended in water is 0.1-0.2 mmol / mL (e.g., 0.1 mmol / mL, 0.12 mmol / mL, 0.15 mmol / mL, 0.18 mmol / mL, 0.2 mmol / mL, etc.), and the concentration of the aqueous polyacrylic acid solution is 10-20 mg / mL (e.g., 10 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, etc.).

[0034] Preferably, the polyacrylic acid in S2 and S5 has a molecular weight of 1500-2500 (e.g., 1500, 1800, 2000, 2200, 2500, etc.).

[0035] Preferably, the mass ratio of the polyacrylic acid to the molar amount of NaYbF4:Tm nanocrystals in S2 is 100-300 mg: 1-3 mmol. Within this range, the mass ratio of the polyacrylic acid can be 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 220 mg, 250 mg, 280 mg, 300 mg, etc., and the molar amount ratio of the NaYbF4:Tm nanocrystals can be 1 mmol, 1.5 mmol, 2 mmol, 2.5 mmol, 3 mmol, etc., for example, 100 mg: 1 mmol, 150 mg: 3 mmol, etc.

[0036] Preferably, the reaction with polyacrylic acid in S2 is carried out at room temperature for 30-60 min (e.g., 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0037] Preferably, after the reaction with polyacrylic acid in step S2 is completed, the solid is collected by centrifugation and dispersed in water to obtain a polyacrylic acid-modified NaYbF4:Tm nanocrystal aqueous dispersion with a concentration of 0.1-0.4 mmol / L (e.g., 0.1 mmol / mL, 0.15 mmol / mL, 0.2 mmol / mL, 0.25 mmol / mL, 0.3 mmol / mL, 0.35 mmol / mL, 0.4 mmol / mL, etc.).

[0038] Preferably, the molar amount of the polyacrylic acid-modified NaYbF4:Tm nanocrystals in S3, the mass of ethyl-(3-dimethylaminopropyl)carbodiimide, the mass of N-hydroxysuccinimide, and the mass ratio of BA monoclonal antibody are 0.1 mmol: 2-6 mg: 5-9 mg: 0.03-50 μg. Within this range, the mass ratio of ethyl-(3-dimethylaminopropyl)carbodiimide can be 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, etc., the mass ratio of N-hydroxysuccinimide can be 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, etc., and the mass ratio of BA monoclonal antibody can be 0.03 μg, 0.1 μg, 0.3 μg, 0.9 μg, 2.7 μg, 8.1 μg, 50 μg, etc., for example, 0.1 mmol: 4 mg: 7 mg: 2.7 μg, etc.

[0039] Preferably, the polyacrylic acid-modified NaYbF4:Tm nanocrystals described in S3 are dispersed in 4-morphine ethanesulfonic acid (MES) buffer at a concentration of 0.02-0.2 mmol / mL (e.g., 0.02 mmol / mL, 0.05 mmol / mL, 0.08 mmol / mL, 0.11 mmol / mL, 0.14 mmol / mL, 0.17 mmol / mL, 0.2 mmol / mL, etc.).

[0040] Preferably, the activation time of the carboxyl groups on the polyacrylic acid in S3 is 30-60 min (e.g., 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0041] Preferably, after activating the carboxyl groups on the polyacrylic acid in step S3, the solid is collected by centrifugation and redispersed in 4-morphine ethanesulfonic acid (MES) buffer at a concentration of 0.02-0.2 mmol / mL (e.g., 0.02 mmol / mL, 0.05 mmol / mL, 0.08 mmol / mL, 0.11 mmol / mL, 0.14 mmol / mL, 0.17 mmol / mL, 0.2 mmol / mL, etc.).

[0042] Preferably, the method for binding with BA monoclonal antibody as described in S3 is as follows: NaYbF4:Tm nanocrystals with activated carboxyl groups on polyacrylic acid are dispersed in 4-morphine ethanesulfonic acid (MES) buffer, BA monoclonal antibody is added and shaken for 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.), and then excess bovine serum albumin is added to block the activated carboxyl sites that have not bound to the antibody. After centrifugation and washing, NaYbF4:Tm nanocrystals with surface-labeled BA monoclonal antibody are obtained.

[0043] Preferably, the NaYbF4:Tm nanocrystals labeled with BA monoclonal antibody described in S3 are dispersed in a glycine buffer solution containing 1-5% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), etc.) bovine serum albumin, and stored at 4°C for later use.

[0044] Preferably, the NaDyF4 nanocrystals in S4 have a cubic phase crystal structure and a particle size of 5-8 nm (e.g., 5 nm, 6 nm, 7 nm, 8 nm, etc.), more preferably 5.8 ± 0.6 nm.

[0045] Preferably, the molar ratio of dysprosium oxide (Dy2O3) to the volume of trifluoroacetic acid in S4 is 0.5-1.5 mmol: 6-12 mL. Within this ratio range, the molar ratio of dysprosium oxide (Dy2O3) can be 0.5 mmol, 0.75 mmol, 1 mmol, 1.25 mmol, 1.5 mmol, etc., and the volume ratio of trifluoroacetic acid can be 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, etc., for example, 0.5 mmol: 12 mL, etc.

[0046] Preferably, the heating reaction of dysprosium oxide (Dy2O3) with trifluoroacetic acid in S4 is performed as follows: dysprosium oxide (Dy2O3) is added to an aqueous solution of trifluoroacetic acid and heated to 90-110 °C (e.g., 90 °C, 95 °C, 98 °C, 100 °C, 105 °C, 108 °C, 110 °C, etc.) until the solid is completely dissolved and the solution becomes clear and transparent. Then, the solution is heated until it is completely evaporated to dryness, yielding dysprosium trifluoroacetate solid powder.

[0047] Preferably, the ratio of sodium trifluoroacetate (CF3COONa), 1-octadecene, oleic acid, oleylamine, and dysprosium trifluoroacetate in step S4 is 1-3 mmol: 10-20 mL: 5-10 mL: 5-10 mL: 1-3 mmol. Within this ratio range, the dosage of sodium trifluoroacetate can be 1 mmol, 1.5 mmol, 2 mmol, 2.5 mmol, 3 mmol, etc.; the dosage of 1-octadecene can be 10 mL, 12 mL, 15 mL, 18 mL, 20 mL, etc.; the dosage of oleic acid can be 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, etc.; the dosage of oleylamine can be 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, etc.; and the dosage of dysprosium trifluoroacetate can be 1 mmol, 1.5 mmol, 2 mmol, 2.5 mmol, 3 mmol, etc., for example, 1 mmol: 10 mL. mL: 5 mL: 5 mL: 1 mmol, etc.

[0048] Preferably, the heating reaction of sodium trifluoroacetate (CF3COONa), 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate in S4 is carried out at a temperature of 250-300 °C (e.g., 250 °C, 260 °C, 270 °C, 280 °C, 290 °C, 300 °C, etc.) for a time of 25-45 min (e.g., 25 min, 28 min, 30 min, 35 min, 38 min, 40 min, 45 min, etc.).

[0049] Preferably, the operations in S4 are all performed in an inert gas atmosphere, which is at least one of nitrogen, argon and helium.

[0050] Preferably, before the sodium trifluoroacetate (CF3COONa), 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate in S4 are heated to react, they are kept at 120-150 °C (e.g. 120 °C, 130 °C, 140 °C, 150 °C, etc.) for 30-50 min (e.g. 30 min, 35 min, 40 min, 45 min, 50 min, etc.) to remove moisture.

[0051] Preferably, the surface ligands of S4, NaDyF4 nanocrystals, are oleic acid and oleylamine dispersed in cyclohexane at a concentration of 0.1-0.2 mmol / mL (e.g., 0.1 mmol / mL, 0.12 mmol / mL, 0.15 mmol / mL, 0.18 mmol / mL, 0.2 mmol / mL, etc.).

[0052] Preferably, the method for removing oleic acid and oleylamine ligands from NaDyF4 nanocrystals with oleic acid and oleylamine as surface ligands as described in S5 is as follows:

[0053] Add hydrochloric acid aqueous solution and ethanol to the cyclohexane dispersion of NaDyF4 nanocrystals, shake to mix, and let stand for 6-12 h (e.g., 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, etc.) to remove oleic acid and oleylamine ligands on the surface of the nanocrystals. Then centrifuge to collect the ligand-free NaDyF4 nanocrystals and disperse them in water.

[0054] The concentration of the NaDyF4 nanocrystalline cyclohexane dispersion is 0.1-0.2 mmol / mL (e.g., 0.1 mmol / mL, 0.12 mmol / mL, 0.15 mmol / mL, 0.18 mmol / mL, 0.2 mmol / mL, etc.); the concentration of the hydrochloric acid aqueous solution is 0.05-0.2 mol / L (e.g., 0.05 mol / L, 0.1 mol / L, 0.12 mol / L, 0.15 mol / L, 0.18 mol / L, 0.2 mol / L, etc.); the volume ratio of the NaDyF4 nanocrystalline cyclohexane dispersion, hydrochloric acid aqueous solution, and ethanol is 1:1:1.

[0055] Preferably, in step S5, the NaDyF4 nanocrystals with oleic acid and oleylamine as surface ligands are removed, resuspended in water, and then added dropwise to an aqueous polyacrylic acid solution for mixing and stirring. The concentration of the ligand-free NaDyF4 nanocrystals resuspended in water is 0.1-0.2 mmol / mL (e.g., 0.1 mmol / mL, 0.12 mmol / mL, 0.15 mmol / mL, 0.18 mmol / mL, 0.2 mmol / mL, etc.), and the concentration of the aqueous polyacrylic acid solution is 10-20 mg / mL (e.g., 10 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, etc.).

[0056] Preferably, the mass ratio of polyacrylic acid to NaDyF4 nanocrystals in S5 is 100-300 mg: 1-3 mmol. Within this range, the mass ratio of polyacrylic acid can be 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 220 mg, 250 mg, 280 mg, 300 mg, etc., and the molar ratio of NaDyF4 nanocrystals can be 1 mmol, 1.5 mmol, 2 mmol, 2.5 mmol, 3 mmol, etc., for example, 100 mg: 1 mmol, 150 mg: 3 mmol, etc.

[0057] Preferably, the reaction with polyacrylic acid in step S5 is carried out at room temperature for 30-60 minutes (e.g., 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, etc.).

[0058] Preferably, after the reaction with polyacrylic acid in step S5 is completed, the solid is collected by centrifugation and dispersed in water to obtain a polyacrylic acid-modified NaDyF4 nanocrystal aqueous dispersion with a concentration of 0.1-0.4 mmol / L (e.g., 0.1 mmol / mL, 0.15 mmol / mL, 0.2 mmol / mL, 0.25 mmol / mL, 0.3 mmol / mL, 0.35 mmol / mL, 0.4 mmol / mL, etc.).

[0059] Preferably, the molar amount of the polyacrylic acid-modified NaDyF4 nanocrystals in S6, the mass ratio of ethyl-(3-dimethylaminopropyl)carbodiimide, the mass ratio of N-hydroxysuccinimide to the mass ratio of BA antigen is 0.1 mmol: 2-6 mg: 5-9 mg: 0.02-50 μg. Within this range, the mass ratio of ethyl-(3-dimethylaminopropyl)carbodiimide can be 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, etc., the mass ratio of N-hydroxysuccinimide can be 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, etc., and the mass ratio of BA antigen can be 0.02 μg, 0.08 μg, 0.32 μg, 1.28 μg, 5.12 μg, 20.48 μg, 50 μg, etc., for example, 0.1 mmol: 4 mg: 7 mg: 5.12 μg, etc.

[0060] Preferably, the polyacrylic acid-modified NaDyF4 nanocrystals described in S6 are dispersed in 4-morphine ethanesulfonic acid (MES) buffer at a concentration of 0.02-0.2 mmol / mL (e.g., 0.02 mmol / mL, 0.05 mmol / mL, 0.08 mmol / mL, 0.11 mmol / mL, 0.14 mmol / mL, 0.17 mmol / mL, 0.2 mmol / mL, etc.).

[0061] Preferably, the activation time of the carboxyl groups on the polyacrylic acid in S6 is 30-60 min (e.g., 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.).

[0062] Preferably, after activating the carboxyl groups on the polyacrylic acid in step S6, the solid is collected by centrifugation and redispersed in 4-morphine ethanesulfonic acid (MES) buffer at a concentration of 0.02-0.2 mmol / mL (e.g., 0.02 mmol / mL, 0.05 mmol / mL, 0.08 mmol / mL, 0.11 mmol / mL, 0.14 mmol / mL, 0.17 mmol / mL, 0.2 mmol / mL, etc.).

[0063] Preferably, the method for binding with BA antigen described in S6 is as follows: NaDyF4 nanocrystals with activated carboxyl groups on polyacrylic acid are dispersed in 4-morphine ethanesulfonic acid (MES) buffer, BA antigen is added and shaken for 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.), then excess bovine serum albumin is added to block the activated carboxyl sites that have not bound to the antibody, and after centrifugation and washing, NaDyF4 nanocrystals with BA antigen labeled on the surface are obtained.

[0064] Preferably, the NaDyF4 nanocrystals labeled with BA antigen described in S6 are dispersed in a glycine buffer solution containing 1-5% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), etc.) bovine serum albumin, and stored at 4 °C for later use.

[0065] Secondly, the present invention provides a fluorescence resonance energy transfer (NIR-NIR FRET) probe system, which is obtained by the above preparation method.

[0066] Thirdly, the present invention provides the application of the above-mentioned fluorescence resonance energy transfer (NIR-NIR FRET) probe system in the detection of BA in whole blood.

[0067] Fourthly, this invention provides a highly sensitive homogeneous immunoassay method for detecting bongkrekic acid in whole blood, a qualitative method comprising the following steps:

[0068] (1) Establishing a negative control group: Add whole blood samples without BA to the donor probe buffer of the above fluorescence resonance energy transfer probe system. After the first incubation, the fluorescence intensity emitted at 800 nm is measured under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I ;

[0069] (2) Detection group: Add the whole blood sample to be tested to the donor probe buffer of the above fluorescence resonance energy transfer probe system, and after the first incubation, test the fluorescence intensity emitted at 800 nm under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I ;

[0070] If the fluorescence intensity ratio in the test group I / I 0 Equal to the ratio of fluorescence intensity in the negative control group I / I 0 If the whole blood sample to be tested does not contain BA; if the fluorescence intensity ratio in the test group is... I / I 0 The fluorescence intensity ratio greater than that in the negative control group I / I 0 If the whole blood sample to be tested contains BA;

[0071] The quantitative method includes the following steps:

[0072] (1) Establishing the standard curve and fitting equation: A series of BA standard whole blood samples with different concentration gradients were added to the donor probe buffer of the above fluorescence resonance energy transfer probe system. After the first incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I The concentration of BA standard whole blood samples was used as the x-axis, and the fluorescence intensity ratio was... I / I 0 Using the ordinate as the vertical axis, establish a standard curve and fit the equation;

[0073] (2) Detection: Add the whole blood sample to be tested to the donor probe buffer of the above fluorescence resonance energy transfer probe system. After the first incubation, measure the fluorescence intensity emitted at 800 nm under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I The ratio of fluorescence intensity I / I 0Substitute the standard curve and / or equation from step (1) to calculate the BA content in the standard curve.

[0074] This invention employs a competitive method for detecting BA. During detection, when BA is present in the whole blood sample, it binds to the BA monoclonal antibody labeled on the surface of NaYbF4:Tm nanocrystals, forming an antigen-antibody complex. Subsequently, when NaDyF4 nanocrystals labeled with BA antigen are added, the antigen-antibody complex can no longer bind to the BA antigen on the NaDyF4 nanocrystals. Therefore, no fluorescence resonance energy transfer (FRET) occurs between NaYbF4:Tm and NaDyF4, and the fluorescence intensity of NaYbF4:Tm at 800 nm remains unchanged. If there is no BA in the whole blood, when NaDyF4 nanocrystals labeled with BA antigen are added again, the BA monoclonal antibody on NaYbF4:Tm binds to the BA antigen on NaDyF4, forming an antigen-antibody complex. FRET occurs between NaYbF4:Tm and NaDyF4, causing the fluorescence of NaYbF4:Tm at 800 nm to be quenched.

[0075] Preferably, in the qualitative method, the concentration of the donor probe is the same in the first incubation system of steps (1) and (2), and the concentration of the whole blood sample is also the same; the concentration of the donor probe is the same in the second incubation system of steps (1) and (2), and the concentration of the recipient probe is also the same.

[0076] Preferably, in the quantitative method, the concentration of the donor probe is the same in the first incubation system of steps (1) and (2), and the concentration of the whole blood sample is also the same; the concentration of the donor probe is the same in the second incubation system of steps (1) and (2), and the concentration of the recipient probe is also the same.

[0077] Preferably, in the qualitative and quantitative methods, the molar ratio of the donor probe and the recipient probe in steps (1) and (2) is 1:0.5-1:2 (e.g., 1:0.5, 1:1, 1:1.5, 1:2, etc.); the ratio of the amount of donor probe to whole blood sample in steps (1) and (2) is 0.1-0.3 mmol: 1-3 mL. Within this ratio range, the amount of donor probe can be 0.1 mmol, 0.15 mmol, 0.2 mmol, 0.25 mmol, 0.3 mmol, etc., and the amount of whole blood sample can be 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, etc., for example, 0.1 mmol: 1 mL, etc.

[0078] Preferably, in the qualitative and quantitative methods, the concentration of the donor probe buffer in steps (1) and (2) is 0.02-0.2 mmol / mL (e.g., 0.02 mmol / mL, 0.05 mmol / mL, 0.08 mmol / mL, 0.11 mmol / mL, 0.14 mmol / mL, 0.17 mmol / mL, 0.2 mmol / mL, etc.), and the concentration of the recipient probe buffer is 0.02-0.2 mmol / mL (e.g., 0.02 mmol / mL, 0.05 mmol / mL, 0.08 mmol / mL, 0.11 mmol / mL, 0.14 mmol / mL, 0.17 mmol / mL, 0.2 mmol / mL, etc.). The buffer is a glycine buffer solution containing 1-5% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), etc.) bovine serum albumin.

[0079] Preferably, in both qualitative and quantitative methods, the incubation refers to standing at 37°C for 5-25 minutes (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, etc.).

[0080] Preferably, in the quantitative method, the concentration of the BA standard whole blood sample in step (1) is 0-100 ng / mL (e.g., 0.0 ng / mL, 1.0 ng / mL, 2.5 ng / mL, 5.0 ng / mL, 7.5 ng / mL, 10.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, 75.0 ng / mL, 100.0 ng / mL, etc.).

[0081] Preferably, in the qualitative and quantitative methods, the molar ratio of NaYbF4:Tm nanocrystals in the donor probe to the mass of BA monoclonal antibody is 0.1 mmol:0.03-50 μg (e.g., 0.1 mmol:0.03 μg, 0.1 mmol:0.1 μg, 0.1 mmol:0.3 μg, 0.1 mmol:0.9 μg, 0.1 mmol:2.7 μg, 0.1 mmol:8.1 μg, 0.1 mmol:50 μg, etc.); the molar ratio of NaDyF4 nanocrystals in the recipient probe to the mass of BA antibody is 0.1 mmol:0.02-50 μg (e.g., 0.1 mmol:0.02 μg, 0.1 mmol:0.08 μg, 0.1 mmol:0.32 μg, 0.1 mmol:1.28 μg, 0.1 mmol:5.12 μg, 0.1 mmol:0.1 mmol:0.02 μg, ... mmol: 20.48 μg, 0.1 mmol: 50 μg, etc.).

[0082] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0083] This invention discloses a method for detecting BA in whole blood based on an NIR-NIR FRET probe system, which enables highly sensitive homogeneous detection of BA in untreated whole blood samples without background interference. Compared with existing BA detection methods, the specific advantages are as follows:

[0084] (1) Compared with the detection of suspected food or vomit samples, which has problems such as missing samples and inability to accurately represent the degree of poisoning of patients, the present invention detects BA in whole blood samples, which can directly analyze the exposure level of toxins in whole blood, more accurately reflect the poisoning state of the human body, and provide a reliable basis for poisoning diagnosis, judgment of the degree of poisoning and subsequent drug treatment.

[0085] (2) This invention uses a NIR-NIR FRET nanoprobe that is excited by 980 nm light and emits at 800 nm, thus avoiding the strong absorption and emission of whole blood in the visible light region (e.g., Figure 1 This method overcomes the background interference problem of traditional fluorescent probes in complex samples such as whole blood, thus achieving highly sensitive and specific detection of trace amounts of BA in whole blood without the need for complex sample pretreatment.

[0086] (3) The present invention adopts a homogeneous immunoassay method. The detection process does not require complex steps such as sample separation and washing. Whole blood samples can be directly added to the detection system, which greatly simplifies the detection process and realizes rapid detection of BA in whole blood samples. This shortens the diagnosis time and saves valuable time for effective treatment, thereby improving the efficiency of treating patients with BA poisoning.

[0087] In summary, this invention enables direct detection of BA in whole blood, overcoming the challenges of strong background interference and complex sample processing in whole blood BA detection. It not only improves the sensitivity, speed, and accuracy of the detection method but also enables direct and rapid assessment of BA exposure levels in patients, providing technical support for the immediate diagnosis and precise treatment of BA poisoning. Attached Figure Description

[0088] Figure 1 The absorption and emission spectra of whole blood and the excitation and emission spectra of NaYbF4:Tm;

[0089] Figure 2 X-ray diffraction pattern of NaYbF4:Tm2% nanocrystals;

[0090] Figure 3 This is a transmission electron microscope (TEM) image of NaYbF4:Tm2% nanocrystals (the inset in the upper right corner is the corresponding particle size distribution histogram).

[0091] Figure 4 For different concentrations of Tm 3+ Emission spectrum of doped NaYbF4:Tmx% nanocrystals under 980 nm excitation (the inset in the lower left corner is the corresponding line graph);

[0092] Figure 5 X-ray diffraction pattern of NaDyF4 nanocrystals;

[0093] Figure 6 Transmission electron microscopy (TEM) image of NaDyF4 nanocrystals (the inset in the upper right corner is the corresponding particle size distribution histogram).

[0094] Figure 7 The absorption spectrum of NaDyF4 nanocrystals;

[0095] Figure 8 The image shows a comparison of the emission spectra of NaYbF4:Tm2% nanocrystals before and after coupling with NaDyF4 nanocrystals under 980 nm excitation. The lower left image is a schematic diagram of energy transfer between NaYbF4:Tm2% and NaDyF4.

[0096] Figure 9 The image shows a comparison of the fluorescence lifetime of NaYbF4:Tm2% nanocrystals before and after coupling with NaDyF4 nanocrystals under 980 nm excitation.

[0097] Figure 10 The figure shows the optimization of the amount of BA monoclonal antibody modified on the surface of NaYbF4:Tm2% nanocrystals, the amount of BA antigen modified on the surface of NaDyF4 nanocrystals, and the incubation time.

[0098] Figure 11 A schematic diagram of the NaYbF4:Tm2%-NaDyF4FRET probe system for detecting BA;

[0099] Figure 12 Standard curve for detecting BA in the NaYbF4:Tm2%-NaDyF4FRET probe system;

[0100] Figure 13 For the detection of BA using the NaYbF4:Tm2%-NaDyF4FRET probe system I / I 0 result.

[0101] Figure 14 A specificity evaluation diagram for the detection of BA using the NaYbF4:Tm2%-NaDyF4FRET probe system. Detailed Implementation

[0102] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0103] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0104] Example 1: Preparation and characterization of NaYbF4:Tm2% nanocrystals

[0105] 0.98 mmol of ytterbium acetate (Yb(CH3COO)3), 0.02 mmol of thulium acetate (Tm(CH3COO)3), 15 mL of 1-octadecene (ODE), and 10 mL of oleic acid (OA) were added to a 100 mL three-necked flask. The mixture was stirred vigorously for 5 min under a nitrogen atmosphere, and then heated to 100 °C for 15 min to remove water from the reaction system. The mixture was then heated to 160 °C for 30 min until the solid was completely dissolved. After the reaction system cooled to room temperature, 10 mL of a methanol solution containing 2.5 mmol of sodium hydroxide (NaOH) was slowly added, and the mixture was stirred vigorously for 30 min under a nitrogen atmosphere. Then, 10 mL of a methanol solution containing 4 mmol of ammonium fluoride (NH4F) was slowly added, and the mixture was stirred vigorously for 50 min under a nitrogen atmosphere. The reaction system was then heated to 130 °C for 30 min to remove water and methanol from the system. The temperature was then raised to 300 °C and the reaction was carried out for 60 min. After the reaction was completed, the solution was cooled to room temperature and transferred to a 50 mL centrifuge tube. 10 mL of anhydrous ethanol was added, and the mixture was centrifuged at 7500 rpm for 5 min. The solid was then washed 1-2 times with anhydrous ethanol. NaYbF4:Tm2% nanocrystals were then obtained and dispersed in 10 mL of cyclohexane for later use.

[0106] Tm was prepared using the same method described above. 3+ NaYbF4:Tmx% with different doping concentrations, where x values ​​are 0.5, 1, 3, 4 and 5 respectively.

[0107] First, X-ray single-crystal diffraction analysis was performed on the NaYbF4:Tm2% nanocrystals prepared in this embodiment. The results are as follows: Figure 2 As shown, the crystal structure of NaYbF4:Tm2% nanocrystals is consistent with the standard card JCPDS 16-0334, proving that the crystal form of the nanocrystals is hexagonal.

[0108] Secondly, the NaYbF4:Tm2% nanocrystals prepared in this embodiment were subjected to transmission electron microscopy (TEM) testing, from... Figure 3The TEM images show that the NaYbF4:Tm2% nanocrystals have regular morphology, uniform size, and relatively uniform particle size. The inset in the upper right corner (particle size statistics) shows that the particle size of the NaYbF4:Tm2% nanocrystals is 24.3±0.8 nm.

[0109] Emission spectra of NaYbF4:Tmx% (x=0.5,1,2,3,4,5) nanocrystals were analyzed under 980 nm light excitation. Figure 4 As shown, with Tm 3+ With increasing doping concentration, the emission intensity of the nanocrystals at 800 nm first increases and then decreases, at Tm 3+ The emission is strongest at a doping concentration of 2%. This is because increasing Tm... 3+ The doping concentration will increase Tm 3+ sensitizer Yb 3+ The energy-grabbing ability is enhanced, resulting in increased luminescence; simultaneously, high concentrations of Tm 3+ Doping leads to Tm 3+ Enhanced cross-relaxation between molecules consumes energy that should be used for radiative relaxation, resulting in reduced luminescence.

[0110] Example 2: Preparation and Characterization of NaDyF4 Nanocrystals

[0111] Add 0.5 mmol of dysprosium oxide (Dy₂O₃) to a 100 mL three-necked flask, then add 10 mL of ultrapure water and 6 mL of trifluoroacetic acid (CF₃COOH). Heat the reaction system to 98 °C to completely dissolve the solid. After the solution becomes completely transparent, continue heating to evaporate to dryness, obtaining solid dysprosium trifluoroacetate. Cool to room temperature. Add 1 mmol of sodium trifluoroacetate (CF₃COONa), 10 mL of ODE, 5 mL of OA, and 5 mL of oleylamine (OM) to the same three-necked flask. Under a nitrogen atmosphere, heat the reaction system to 130 °C and react for 40 min to remove water. Then, heat to 300 °C and react for 30 min. After the reaction is complete, allow the solution to cool to room temperature and transfer to a 50 mL centrifuge tube. Add 10 mL of anhydrous ethanol and centrifuge at 7500 rpm for 5 min. Wash the solid 1-2 times with anhydrous ethanol. NaDyF4 nanocrystals were then obtained and dispersed in 10 mL of cyclohexane for later use.

[0112] First, XRD diffraction analysis was performed on the NaDyF4 nanocrystals prepared in this embodiment. For example... Figure 5 As shown, the crystal structure of NaDyF4 nanocrystals is basically consistent with JCPDS 27-1426, proving that the crystal form of the nanocrystals is cubic.

[0113] Secondly, the NaDyF4 nanocrystals prepared in this embodiment were subjected to transmission electron microscopy (TEM) testing, from... Figure 6 The TEM image shows that the NaDyF4 nanocrystals are uniform in size and uniform in diameter. The inset in the upper right corner (particle size statistics) shows that the particle size of the NaDyF4 nanocrystals is 5.8 ± 0.6 nm.

[0114] The NaDyF4 nanocrystals prepared in this embodiment were subjected to ultraviolet absorption testing, such as... Figure 7 As shown, the characteristic absorption peak of NaDyF4 nanocrystals is located at 800±10 nm.

[0115] Example 3: Coupling and characterization of NaYbF4:Tm2% nanocrystals and NaDyF4 nanocrystals

[0116] 1. Modification of NaYbF4:Tm2% nanocrystalline surface based on polyacrylic acid (PAA)

[0117] Add 10 mL of hydrochloric acid solution (0.1 mol / L) and 10 mL of ethanol to the NaYbF4:Tm2% nanocrystalline cyclohexane dispersion (10 mL, 0.1 mmol / mL) prepared in Example 1. Shake the mixture for 10 min and let it stand for 8 h to remove the oleic acid ligands on the surface of the nanocrystals. Then centrifuge (11000 rpm, 30 min) to collect the ligand-free NaYbF4:Tm2% nanocrystals, wash them 1-2 times with ultrapure water, and redisperse them in 10 mL of ultrapure water. Next, the NaYbF4:Tm2% nanocrystals dispersed in ultrapure water were added dropwise to 10 mL of ultrapure water containing 100 mg of PAA (molecular weight 2000). The mixture was stirred vigorously at room temperature for 60 min. After the reaction was completed, the mixture was centrifuged (11000 rpm, 30 min) to collect the PAA-modified NaYbF4:Tm2% nanocrystals (PAA-NaYbF4:Tm2%). The mixture was washed 1-2 times with ultrapure water and then redispersed in 10 mL of ultrapure water to obtain the PAA-NaYbF4:Tm2% nanocrystal dispersion (0.1 mmol / mL).

[0118] The same method was used to modify the surface of NaDyF4 nanocrystals to obtain PAA-NaDyF4 nanocrystal dispersion.

[0119] 2. Modification of NaDyF4 nanocrystal surface based on O-phosphorylethanolamine (PEA)

[0120] After removing oleic acid and oleylamine ligands from the NaDyF4 nanocrystal cyclohexane dispersion (10 mL, 0.1 mmol / mL) prepared in Example 2 using the same method described above, the ligand-free NaDyF4 nanocrystal dispersion was added dropwise to 10 mL of ultrapure water containing 100 mg PEA. The mixture was stirred vigorously for 60 min, centrifuged (11000 rpm, 30 min) to collect the PEA-modified NaDyF4 nanocrystals (PEA-NaDyF4). The nanocrystals were washed 1-2 times with ultrapure water and then redispersed in 5 mL of ultrapure water to obtain the PEA-NaDyF4 nanocrystal dispersion (0.2 mmol / mL), which was stored at 4 °C for later use.

[0121] 3. Take 1 mL of PAA-NaYbF4:Tm2% nanocrystal dispersion (0.1 mmol / mL) into a 2 mL EP tube, add 1 mL of MES buffer (pH=6) to wash the nanocrystals, centrifuge (16500 rpm, 15 min), discard the supernatant, repeat the washing 2-3 times, and then redisperse the nanocrystal solid in 1 mL of MES buffer to obtain a 0.1 mmol / mL nanocrystal MES dispersion. Add 400 μL of MES buffer containing 4 mg EDC and 7 mg Sulfo-NHS, and react on a mixer for 30 min. Then wash 2-3 times with MES buffer, centrifuge (16500 rpm, 15 min), discard the supernatant, and redisperse the nanocrystal solid in 500 μL of MES buffer. Then, 500 μL of PEA-NaDyF4 nanocrystal dispersion (0.2 mmol / mL) was added, and the mixture was slowly shaken for 3 h to obtain NaYbF4:Tm2% nanocrystals coupled with NaDyF4 nanocrystals.

[0122] The emission spectra of NaYbF4:Tm2% nanocrystals without coupling and with coupling NaDyF4 nanocrystals were measured under 980 nm light excitation. Figure 8 As shown, the emission intensity of NaYbF4:Tm2% nanocrystals at 800 nm decreases after coupling with NaDyF4 nanocrystals. A schematic diagram of energy transfer between NaYbF4:Tm2% and NaDyF4 is shown below. Figure 8 As shown in the lower left corner.

[0123] Simultaneously, lifetime tests were performed on NaYbF4:Tm2% nanocrystals (980 nm photoexcitation, 800 nm emission), such as... Figure 9 As shown, the fluorescence lifetime of NaYbF4:Tm2% nanocrystals decreased significantly after coupling with NaDyF4 nanocrystals.

[0124] Example 4: NaYbF4:Tm2% nanocrystals labeled with BA monoclonal antibody

[0125] Take 100 μL of the PAA-NaYbF4:Tm2% nanocrystal dispersion (0.1 mmol / mL) from Example 3 into a 1.5 mL EP tube, add 500 μL of MES buffer (pH=6), wash 2-3 times, centrifuge (16500 rpm, 15 min), discard the supernatant, and redisperse the PAA-NaYbF4:Tm2% nanocrystals in 500 μL of MES buffer to obtain a 0.02 mmol / mL nanocrystal MES dispersion. Then add 40 μL of MES buffer containing 0.4 mg EDC and 70 μL of MES buffer containing 0.7 mg Sulfo-NHS, and gently vortex for 30 min. Subsequently, wash 2-3 times with MES buffer, centrifuge (16500 rpm, 15 min) to remove the supernatant, and redisperse the nanocrystal solid in 500 μL of MES buffer to obtain a 0.02 mmol / mL nanocrystal MES dispersion. Add 5 μg of BA monoclonal antibody (Antibody, Ab) (Ab) BA Add the solution and gently vortex for 2 h. Then add 500 μL of blocking buffer (containing 1% (w / v) bovine serum albumin (BSA) glycine buffer (pH=8, 0.1 mol / L)) and continue to gently vortex for 45 min to block other unreacted activation sites. Centrifuge (16500 rpm, 15 min) and remove the supernatant to obtain Ab. BA Coupled NaYbF4:Tm2% nanocrystals (NaYbF4:Tm2%-Ab) BA After washing the centrifuged solids 2-3 times with a final wash buffer (containing 5% (w / v) BSA glycine buffer (pH=8, 0.1 mol / L)), the solids were redispersed in 100 μL of the final wash buffer.

[0126] The BA antigen (Ag) was labeled on the surface of NaDyF4 nanocrystals using the same method. BA Replace the PAA-NaYbF4:Tm2% nanocrystalline dispersion with PAA-NaDyF4 nanocrystalline dispersion. BA Replace with Ag BA That is, NaDyF4-Ag is obtained. BA .

[0127] Example 5 NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA Optimization of BA detection

[0128] The amount of protein modified on the surface of NaYbF4:Tm2% nanocrystals and NaDyF4 nanocrystals affects the calibration of detection limits and economic aspects; simultaneously, reaction time affects time costs. Therefore, NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA BA testing needs to optimize the following three aspects:

[0129] (1) NaYbF4:Tm2% nanocrystalline surface Ab BA amount of modification

[0130] Following the method for labeling proteins on the nanocrystal surface in Example 4, 0.03 μg, 0.1 μg, 0.3 μg, 0.9 μg, 2.7 μg, and 8.1 μg of Ab were respectively labeled. BA Modification was applied to the surface of 0.1 mmol PAA-NaYbF4:Tm2% nanocrystals. Under otherwise unchanged conditions, the effects of six Ab on light excitation at 980 nm were tested. BA The luminescence intensity emitted by modified NaYbF4:Tm2% nanocrystals at 800 nm (denoted as ) I 0 ), and six Ab BA Modified NaYbF4:Tm2% nanocrystals were compared with NaDyF4-Ag obtained in Example 4. BA The luminescence intensity emitted at 800 nm by (0.1 mmol) after incubation at 37 °C for 30 min and excitation by 980 nm light (denoted as ) I In all tests, Ab BA The concentration of the modified NaYbF4:Tm2% nanocrystals was the same.

[0131] (2) Ag on the surface of NaDyF4 nanocrystals BA amount of modification

[0132] Following the method for labeling proteins on the nanocrystal surface in Example 4, 0.02 μg, 0.08 μg, 0.32 μg, 1.28 μg, 5.12 μg, and 20.48 μg of Ag were respectively labeled. BA The modified surface was coated with 0.1 mmol of PAA-NaDyF4 nanocrystals. Under otherwise identical conditions, the NaYbF4:Tm2%-Ab obtained in Example 4 was tested. BA The luminescence intensity emitted at 800 nm after excitation by 980 nm light (denoted as ) I 0 ), and six Ag BA Modified NaDyF4 nanocrystals were compared with NaYbF4:Tm2%-Ab obtained in Example 4.BA The luminescence intensity emitted at 800 nm by (0.1 mmol) after incubation at 37 °C for 30 min and excitation by 980 nm light (denoted as ) I In all tests, NaYbF4:Tm2%-Ab BA The concentrations are the same.

[0133] (3) Incubation time

[0134] When optimizing the incubation time, follow the optimized NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA Experiments were conducted to test NaYbF4:Tm2%-Ab BA The luminescence intensity emitted at 800 nm after excitation by 980 nm light (denoted as ) I 0 ), and test NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA The luminescence intensity emitted at 800 nm after incubation at 37 °C for 0 min, 5 min, 10 min, 15 min, 20 min, and 25 min at a molar ratio of 1:1 and followed by excitation with 980 nm light (denoted as ) is... I In all tests, NaYbF4:Tm2%-Ab BA The concentrations are the same.

[0135] like Figure 10 As shown, with the surface modification of NaYbF4:Tm2% nanocrystals with Ab... BA Increase in quality I / I 0 getting smaller and smaller, 2.7 μg Ab BA The results were almost identical to those of 8.1 μg modification, so Ab was ultimately chosen. BA The coupling amount was 2.7 μg / mL. Similarly, with the Ag modified on the surface of NaDyF4 nanocrystals... BA Increase in quality I / I 0 The size kept getting smaller, and finally the Ag on the surface of NaDyF4 nanocrystals was selected. BA The optimal modification amount was 5.12 μg / mL. After weighing time and reaction efficiency, 10 min was selected as the optimal incubation time.

[0136] Example 6 NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA Detection limit of BA

[0137] The optimized NaYbF4:Tm2%-Ab prepared in Example 5 BA (1 mL, 0.1 mmol / mL PAA-NaYbF4:Tm2% dispersion corresponds to Ab) BA Modification amount 2.7 μg) and NaDyF4-Ag BA (1 mL, 0.1 mmol / mL PAA-NaDyF4 dispersion corresponds to Ag) BA The modification amount was 5.12 μg), and 100 μL of 0.1 mmol / mL NaYbF4:Tm2%-Ab was added. BA The dispersion was added to Tris-HCl buffer (pH=7.4, 0.05 mol / L), followed by 100 μL of BA standard whole blood solution at a series of concentration gradients (0.0 ng / mL, 1.0 ng / mL, 2.5 ng / mL, 5.0 ng / mL, 7.5 ng / mL, 10.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, 75.0 ng / mL, 100.0 ng / mL). After incubation at 37 ℃ for 10 min, the emission intensity at 800 nm under 980 nm light excitation was measured (denoted as ). I 0 Then, 100 μL and 0.1 mmol / mL NaDyF4-Ag were added to the above system, respectively. BA The dispersion was incubated at 37 °C for 10 min, and the emission intensity at 800 nm after excitation by 980 nm light was measured again (denoted as ). I Three tests were conducted and the results were calculated for each point. I / I 0 The mean and standard deviation (SD) of the obtained 10 groups. I / I 0 By performing a fitting, we obtain the following: Figure 12 The results are shown.

[0138] NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA The BA detection uses a competitive method, the principle of which is as follows: Figure 11 As shown. NaYbF4:Tm2%-Ab BA Incubate the whole blood sample with Tris-HCl buffer at 37 °C for 10 min. If the sample contains BA, it will react with NaYbF4:Tm2%-Ab. BA Combine to form NaYbF4:Tm2%-Ab BAThe -BA complex was then further incubated with NaDyF4-AgBA at 37 °C for 10 min, resulting in NaYbF4:Tm2%-Ab BA -BA complex can no longer react with NaDyF4-Ag BA No fluorescence resonance energy transfer (FRET) occurs between NaYbF4:Tm2% and NaDyF4, and the fluorescence intensity emitted by NaYbF4:Tm2% at 800 nm remains unchanged. If BA is absent from the sample, adding NaDyF4-Ag again... BA At that time, NaYbF4:Tm2%-Ab BA NaDyF4-Ag BA Upon binding, FRET occurs between NaYbF4:Tm2% and NaDyF4, quenching the fluorescence emitted by NaYbF4:Tm2% at 800 nm.

[0139] like Figure 12 As shown, the concentration of BA and I / I 0 It exhibits good linearity in the range of 0-100 ng / mL, with a fitted relationship of y=0.82−0.33÷(1+(x÷13.31)). 0.93 ) (R 2 =0.99), according to the formula: LoD=Mean Blank +3SD Blank The detection limit was found to be 0.70 ng / mL.

[0140] Example 7 NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA Qualitative and quantitative detection of BA

[0141] The optimized NaYbF4:Tm2%-Ab prepared in Example 5 BA (1 mL, 0.1 mmol / mL PAA-NaYbF4:Tm2% dispersion corresponds to Ab) BA Modification amount 2.7 μg) and NaDyF4-Ag BA (1 mL, 0.1 mmol / mL PAA-NaDyF4 dispersion corresponds to Ag) BA The modification amount was 5.12 μg), and 100 μL of 0.1 mmol / mL NaYbF4:Tm2%-Ab was added. BAThe dispersion was added to Tris-HCl buffer (pH=7.4, 0.05 mol / L), followed by 100 μL of whole blood sample without BA (negative control group) and 100 μL of whole blood sample containing 5 ng / mL, 10 ng / mL, and 50 ng / mL BA (detection group). After incubation at 37 ℃ for 10 min, the emission intensity at 800 nm under 980 nm light excitation was measured (denoted as ). I 0 Then, 100 μL and 0.1 mmol / mL NaDyF4-Ag were added to the above system, respectively. BA The dispersion was incubated at 37 °C for 10 min, and the emission intensity at 800 nm after excitation by 980 nm light was measured again (denoted as ). I Three tests were conducted and the results for each group were calculated. I / I 0 The mean and standard deviation (SD) of the value.

[0142] The results are as follows Figure 13 Whole blood samples with BA concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, and 50 ng / mL I / I 0 The values ​​were 0.49, 0.59, 0.64, and 0.74, respectively, for the detection group. I / I 0 (0.59, 0.64, 0.74) were all greater than those in the negative group. I / I 0 (0.49) indicates that BA can be qualitatively detected in all whole blood samples.

[0143] Will Figure 13 The test results ( I / I 0 Substituting the values ​​(0.49, 0.59, 0.64, 0.74 respectively) into the equation of the standard curve in Example 6 (y = 0.82 − 0.33 ÷ (1 + (x ÷ 13.31)) 0.93 The calculated BA concentrations were 0 ng / mL, 5.35 ng / mL, 10.80 ng / mL, and 45.12 ng / mL. The BA concentrations calculated by this method had small deviations from the BA concentrations in the whole blood samples used in the experiment, indicating that the concentration of BA in whole blood samples can be quantitatively detected.

[0144] Example 8 NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA Specificity assay for detecting BA molecules

[0145] Specificity tests were performed on whole blood samples containing 100 ng / mL of BA and whole blood samples containing higher concentrations (200 ng / mL) of other substances, including lauric acid (LA), docosanoic acid (DA), linolenic acid (LNA), oleic acid (OA), glycine (Gly), lysine (Lys), proline (Pro), adenosine triphosphate (ATP), glucose, sodium chloride (NaCl), and potassium chloride (KCl).

[0146] The optimized NaYbF4:Tm2%-Ab prepared in Example 5 BA (1 mL, 0.1 mmol / mL PAA-NaYbF4:Tm2% dispersion corresponds to Ab) BA Modification amount 2.7 μg) and NaDyF4-Ag BA (1 mL, 0.1 mmol / mL PAA-NaDyF4 dispersion corresponds to Ag) BA The modification amount was 5.12 μg), and 100 μL of 0.1 mmol / mL NaYbF4:Tm2%-Ab was added. BA The dispersion was added to Tris-HCl buffer (pH=7.4, 0.05 mol / L), and 100 μL of whole blood sample containing 100 ng / mL BA or 200 ng / mL of a series of other substances (LA, DA, LNA, OA, Gly, Lys, Pro, ATP, Glucose, NaCl, and KCl) was added. After incubation at 37 °C for 10 min, the emission intensity at 800 nm under 980 nm light excitation was measured (denoted as ). I 0 Then, 100 μL and 0.1 mmol / mL NaDyF4-Ag were added to the above system, respectively. BA The dispersion was incubated at 37 °C for 10 min, and the emission intensity at 800 nm under 980 nm light excitation was measured again (denoted as ). I ).

[0147] like Figure 14 As shown, NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BAIt can selectively recognize BA and has no significant cross-reactivity with other substances (LA, DA, LNA, OA, Gly, Lys, Pro, ATP, Glucose, NaCl, and KCl), proving that NaYbF4:Tm2%-Ab BA and NaDyF4-Ag BA It has good specificity for detecting BA molecules.

[0148] Substituting BA=100 ng / mL from this example into the equation y=0.82−0.33÷(1+(x÷13.31)) in Example 6 0.93 The calculated value is y=0.78, which is consistent with the value measured in this embodiment. I / I 0 =0.79, which proves that the method has high accuracy in detecting BA in whole blood.

[0149] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for homogeneous immunoassay of bongkrekic acid in whole blood for non-diagnostic purposes, characterized in that, Qualitative methods include the following steps: (1) Establishing a negative control group: Add whole blood samples without BA to the donor probe buffer of the fluorescence resonance energy transfer probe system. After the first incubation, the fluorescence intensity emitted at 800 nm is measured under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I ; (2) Detection group: Add the whole blood sample to be tested to the donor probe buffer of the fluorescence resonance energy transfer probe system, perform the first incubation, and test the fluorescence intensity emitted at 800 nm under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I ; If the fluorescence intensity ratio in the test group I / I 0 Equal to the ratio of fluorescence intensity in the negative control group I / I 0 If the whole blood sample to be tested does not contain BA; if the fluorescence intensity ratio in the test group is... I / I 0 The fluorescence intensity ratio greater than that in the negative control group I / I 0 If the whole blood sample to be tested contains BA; The quantitative method includes the following steps: (1) Establishing the standard curve and fitting equation: A series of BA standard whole blood samples with different concentration gradients were added to the donor probe buffer of the fluorescence resonance energy transfer probe system. After the first incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I The concentration of BA standard whole blood samples was used as the x-axis, and the fluorescence intensity ratio was... I / I 0 Using the ordinate as the vertical axis, establish a standard curve and fit the equation; (2) Detection: Add the whole blood sample to be tested to the donor probe buffer of the fluorescence resonance energy transfer probe system. After the first incubation, measure the fluorescence intensity emitted at 800 nm under 980 nm light excitation. I 0 After adding receptor probe buffer and performing a second incubation, the fluorescence intensity emitted at 800 nm was measured under 980 nm light excitation. I The ratio of fluorescence intensity I / I 0 Substitute the standard curve and / or equation from step (1) to calculate the content of BA in the standard curve; The fluorescence resonance energy transfer probe system consists of a donor probe and an acceptor probe, wherein the donor probe is NaYbF4:Tm nanocrystals with surface-labeled BA monoclonal antibody, and the acceptor probe is NaDyF4 nanocrystals with surface-labeled BA antigen. The surface-labeled BA monoclonal antibody-containing NaYbF4:Tm nanocrystals have a hexagonal phase and a particle size of 20-30 nm. The NaDyF4 nanocrystals labeled with BA antigen have a cubic phase and a particle size of 5-8 nm.

2. The method for homogeneous immunoassay of bongkrekic acid in whole blood for non-diagnostic purposes according to claim 1, characterized in that, In the qualitative method, the concentrations of the donor probe and the whole blood sample are the same in the first incubation system in steps (1) and (2); the concentrations of the donor probe and the recipient probe are the same in the second incubation system in steps (1) and (2). And / or, in the quantitative method, the concentration of the donor probe is the same in the first incubation system of steps (1) and (2), and the concentration of the whole blood sample is also the same; the concentration of the donor probe is the same in the second incubation system of steps (1) and (2), and the concentration of the recipient probe is also the same. And / or, in qualitative and quantitative methods, the molar ratio of donor probe and recipient probe in steps (1) and (2) is 1:0.5-1:2; the ratio of donor probe to whole blood sample in steps (1) and (2) is 1-3 mmol:1-3 mL. And / or, in the NaYbF4:Tm nanocrystals labeled with BA monoclonal antibody, the molar ratio of Yb to Tm is 95:5 to 99.5:0.5; And / or, the molar ratio of NaYbF4:Tm nanocrystals to the mass ratio of BA monoclonal antibody is 0.1 mmol: 0.03-50 μg; And / or, the molar ratio of NaDyF4 nanocrystals to the mass of BA antibody is 0.1 mmol: 0.02-50 μg.

3. The method for homogeneous immunoassay of bongkrekic acid in whole blood for non-diagnostic purposes according to claim 1 or 2, characterized in that, In both qualitative and quantitative methods, the concentration of the donor probe buffer in steps (1) and (2) is 0.02-0.2 mmol / mL, and the concentration of the recipient probe buffer is 0.02-0.2 mmol / mL. The buffer is a glycine buffer solution containing 1-5% (w / v) bovine serum albumin. And / or, in qualitative and quantitative methods, the incubation refers to standing at 37 ℃ for 5-25 min; And / or, in the quantitative method, the concentration of the BA standard whole blood sample in step (1) is 0-100 ng / mL.

4. The method for homogeneous immunoassay of bongkrekic acid in whole blood for non-diagnostic purposes according to claim 1 or 2, characterized in that, The surface-labeled NaYbF4:Tm nanocrystals of BA monoclonal antibody were obtained by the following method: (1) After removing the oleic acid ligand from NaYbF4:Tm nanocrystals with oleic acid as the surface ligand, they were reacted with polyacrylic acid to obtain polyacrylic acid modified NaYbF4:Tm nanocrystals. (2) Disperse polyacrylic acid modified NaYbF4:Tm nanocrystals in buffer, add ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on polyacrylic acid, then add BA monoclonal antibody and shake the reaction, and finally add excess bovine serum albumin to block the activated carboxyl sites that are not bound to the antibody, to obtain NaYbF4:Tm nanocrystals with surface labeled BA monoclonal antibody; And / or, the mass ratio of the polyacrylic acid in step (1) to the molar amount of NaYbF4:Tm nanocrystals is 100-300 mg: 1-3 mmol; And / or, the molar amount of the polyacrylic acid-modified NaYbF4:Tm nanocrystals, the mass of ethyl-(3-dimethylaminopropyl)carbodiimide, the mass of N-hydroxysuccinimide and the mass of BA monoclonal antibody in step (2) are in the ratio of 0.1 mmol: 2-6 mg: 5-9 mg: 0.03-50 μg; And / or, the NaDyF4 nanocrystals with surface-labeled BA antigen are obtained by the following method: (1) After removing the ligands oleic acid and oleylamine from NaDyF4 nanocrystals with surface ligands oleic acid and oleylamine, they were reacted with polyacrylic acid to obtain polyacrylic acid modified NaDyF4 nanocrystals. (2) Disperse polyacrylic acid modified NaDyF4 nanocrystals in buffer, add ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on polyacrylic acid, then add BA antigen and shake the reaction, and finally add excess bovine serum albumin to block the activated carboxyl sites that are not bound to the antigen, to obtain NaDyF4 nanocrystals with BA antigen labeled on the surface. And / or, the mass ratio of the polyacrylic acid to the molar amount of NaDyF4 nanocrystals in step (1) is 100-300 mg: 1-3 mmol; And / or, the molar amount of the polyacrylic acid-modified NaDyF4 nanocrystals in step (2), the mass of ethyl-(3-dimethylaminopropyl)carbodiimide, the mass of N-hydroxysuccinimide and the mass of BA antigen are in the ratio of 0.1 mmol: 2-6 mg: 5-9 mg: 0.02-50 μg.

5. A method for preparing a fluorescence resonance energy transfer probe system according to any one of claims 1-4, characterized in that, Includes the following steps: (1) NaYbF4:Tm nanocrystals labeled with BA monoclonal antibody S1 mixes and dissolves ytterbium acetate, thulium acetate, 1-octadecene and oleic acid to obtain a precursor solution, adds sodium hydroxide and ammonium fluoride, and heats to react, to obtain NaYbF4:Tm nanocrystals with oleic acid as the surface ligand; S2 removes the oleic acid ligand from NaYbF4:Tm nanocrystals with oleic acid as the surface ligand, and then reacts them with polyacrylic acid to obtain polyacrylic acid modified NaYbF4:Tm nanocrystals. S3 disperses polyacrylic acid-modified NaYbF4:Tm nanocrystals in buffer solution, adds ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on polyacrylic acid, and then binds to BA monoclonal antibody to obtain NaYbF4:Tm nanocrystals with surface-labeled BA monoclonal antibody. (2) NaDyF4 nanocrystals labeled with BA antigen S4 reacts dysprosium oxide with trifluoroacetic acid by heating to obtain dysprosium trifluoroacetate. Sodium trifluoroacetate, 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate are reacted by heating to obtain NaDyF4 nanocrystals with oleic acid and oleylamine as surface ligands. S5 removes the oleic acid and oleylamine ligands from NaDyF4 nanocrystals with surface ligands of oleic acid and oleylamine, and then reacts them with polyacrylic acid to obtain polyacrylic acid modified NaDyF4 nanocrystals. S6 disperses polyacrylic acid-modified NaDyF4 nanocrystals in a buffer solution, adds ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on the polyacrylic acid, and then binds it with BA antigen to obtain NaDyF4 nanocrystals with surface-labeled BA antigen. NaYbF4:Tm nanocrystals labeled with BA monoclonal antibody were used as donor probes, and NaDyF4 nanocrystals labeled with BA antigen were used as acceptor probes to form a fluorescence resonance energy transfer probe system.

6. The method for preparing a fluorescence resonance energy transfer probe system according to claim 5, characterized in that, In the NaYbF4:Tm nanocrystals described in S1, the molar ratio of Yb to Tm is 95:5 to 99.5:0.5; And / or, the ratio of the total molar amount of ytterbium acetate and thulium acetate, the molar amount of sodium hydroxide, the molar amount of ammonium fluoride, the volume of 1-octadecene, and the volume of oleic acid in S1 is 1-3 mmol: 2.5-5 mmol: 3-5 mmol: 15-20 mL: 7.5-10 mL; And / or, all operations in S1 are performed in an inert gas atmosphere, which is at least one of nitrogen, argon and helium. And / or, the heating reaction described in S1 is carried out at a temperature of 290-310 °C for a time of 50-70 min; And / or, the NaYbF4:Tm nanocrystals described in S1 have a hexagonal phase and a particle size of 20-30 nm; And / or, the NaDyF4 nanocrystals described in S4 have a cubic phase crystal structure and a particle size of 5-8 nm; And / or, the molar ratio of dysprosium oxide to the volume of trifluoroacetic acid in S4 is 0.5-1.5 mmol: 6-12 mL; And / or, the ratio of sodium trifluoroacetate, 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate in S4 is 1-3 mmol: 10-20 mL: 5-10 mL: 5-10 mL: 1-3 mmol; And / or, the reaction of sodium trifluoroacetate, 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate in S4 is carried out at a temperature of 250-300 °C for a time of 25-45 min; And / or, the operations in S4 are all performed in an inert gas atmosphere, which is at least one of nitrogen, argon and helium.

7. The method for preparing a fluorescence resonance energy transfer probe system according to claim 5 or 6, characterized in that, The molecular weight of the polyacrylic acid described in S2 and S5 is 1500-2500; And / or, the mass ratio of the polyacrylic acid to the molar amount of NaYbF4:Tm nanocrystals in S2 is 100-300 mg: 1-3 mmol; And / or, the reaction with polyacrylic acid described in S2 is carried out at room temperature for 30-60 min; And / or, the molar amount of the polyacrylic acid-modified NaYbF4:Tm nanocrystals described in S3, the mass of ethyl-(3-dimethylaminopropyl)carbodiimide, the mass of N-hydroxysuccinimide, and the mass ratio of the BA monoclonal antibody are 0.1 mmol: 2-6 mg: 5-9 mg: 0.03-50 μg; And / or, the activation time of the carboxyl groups on the polyacrylic acid described in S3 is 30-60 min; And / or, the method of binding with BA monoclonal antibody described in S3 is as follows: NaYbF4:Tm nanocrystals with activated carboxyl groups on polyacrylic acid are dispersed in buffer, BA monoclonal antibody is added and shaken for 1-3 h, then excess bovine serum albumin is added to block the activated carboxyl sites that are not bound to the antibody, and after centrifugation and washing, NaYbF4:Tm nanocrystals with surface labeled with BA monoclonal antibody are obtained. And / or, the mass ratio of the polyacrylic acid to the molar amount of NaDyF4 nanocrystals in S5 is 100-300 mg: 1-3 mmol; And / or, the reaction with polyacrylic acid described in S5 is carried out at room temperature for 30-60 min; And / or, the molar amount of the polyacrylic acid-modified NaDyF4 nanocrystals described in S6, the mass of ethyl-(3-dimethylaminopropyl)carbodiimide, the mass of N-hydroxysuccinimide, and the mass ratio of the BA antigen are 0.1 mmol: 2-6 mg: 5-9 mg: 0.02-50 μg; And / or, the activation time of the carboxyl groups on the polyacrylic acid described in S6 is 30-60 min; And / or, the method of binding to BA antigen described in S6 is as follows: BA antigen is added to the NaDyF4 nanocrystal dispersion buffer after activating the carboxyl groups on polyacrylic acid and shaken for 1-3 h. Then, an excess of bovine serum albumin is added to block the activated carboxyl sites that have not bound to the antibody. After centrifugation and washing, NaDyF4 nanocrystals with BA antigen labeled on the surface are obtained.

8. The method for preparing a fluorescence resonance energy transfer probe system according to claim 5 or 6, characterized in that, S2 uses the following method to remove oleic acid ligands from NaYbF4:Tm nanocrystals with oleic acid as the surface ligand: Add hydrochloric acid aqueous solution and ethanol solution to the cyclohexane dispersion of NaYbF4:Tm nanocrystals, shake and mix, and let stand for 6-12 hours to remove oleic acid ligands on the surface of nanocrystals. Then centrifuge and collect the ligand-free NaYbF4:Tm nanocrystals and disperse them in water. And / or, the concentration of the NaYbF4:Tm nanocrystalline cyclohexane dispersion is 0.1-0.2 mmol / mL; the concentration of the hydrochloric acid aqueous solution is 0.05-0.2 mol / L; and the volume ratio of the NaYbF4:Tm nanocrystalline cyclohexane dispersion, hydrochloric acid aqueous solution, and ethanol is 1:1:

1. And / or, after removing the oleic acid ligand from the NaYbF4:Tm nanocrystals with oleic acid as the surface ligand in S2, they are resuspended in water and then added dropwise to an aqueous solution of polyacrylic acid for mixing and stirring. The concentration of the ligandless NaYbF4:Tm nanocrystals resuspended in water is 0.1-0.2 mmol / mL, and the concentration of the aqueous solution of polyacrylic acid is 10-20 mg / mL. And / or, the method described in S5 for removing oleic acid and oleylamine ligands from NaDyF4 nanocrystals with oleic acid and oleylamine surface ligands is as follows: Add hydrochloric acid aqueous solution and ethanol solution to the cyclohexane dispersion of NaDyF4 nanocrystals, shake and mix, and let stand for 6-12 h to remove oleic acid and oleylamine ligands on the surface of nanocrystals. Then centrifuge and collect the ligand-free NaDyF4 nanocrystals and disperse them in water. And / or, the concentration of the NaDyF4 nanocrystalline cyclohexane dispersion is 0.1-0.2 mmol / mL; the concentration of the hydrochloric acid aqueous solution is 0.05-0.2 mol / L; and the volume ratio of the NaDyF4 nanocrystalline cyclohexane dispersion, hydrochloric acid aqueous solution, and ethanol is 1:1:

1. And / or, in S5, after removing the ligands oleic acid and oleylamine from the NaDyF4 nanocrystals with surface ligands of oleic acid and oleylamine, they are resuspended in water and then added dropwise to an aqueous polyacrylic acid solution for mixing and stirring. The concentration of the ligand-free NaDyF4 nanocrystals resuspended in water is 0.1-0.2 mmol / mL, and the concentration of the aqueous polyacrylic acid solution is 10-20 mg / mL.

9. A fluorescence resonance energy transfer probe system according to any one of claims 1-4.

10. A fluorescence resonance energy transfer probe system prepared by the preparation method according to any one of claims 5-8.

11. The application of the fluorescence resonance energy transfer probe system according to any one of claims 9-10 in the detection of BA in whole blood for non-diagnostic purposes.

Images