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CN-120847407-B - Fluorescent resonance energy transfer probe system and method for high-sensitivity homogeneous phase immunity detection of milbemycetin in whole blood

CN120847407BCN 120847407 BCN120847407 BCN 120847407BCN-120847407-B

Abstract

The invention discloses a fluorescence resonance energy transfer probe system and a method for detecting milbemycetin in whole blood by high-sensitivity homogeneous phase immunity. According to the invention, naYbF 4 of a surface-labeled BA monoclonal antibody is used as a donor probe, tm nanocrystalline and NaDyF 4 nanocrystalline of a surface-labeled BA antigen are used as an acceptor probe to construct a fluorescence resonance energy transfer probe system, the probe system is used for qualitative and quantitative detection of the milbemycetin in whole blood, the donor probe and a whole blood sample to be detected are incubated and tested for the fluorescence intensity emitted at 800 nm, then the acceptor probe is added for incubation and tested for the fluorescence intensity emitted at 800 nm, and the ratio of the two fluorescence intensities is compared with a control group or a standard curve to qualitatively or quantitatively detect the milbemycetin in the whole blood. The probe system can effectively overcome the background interference of a whole blood sample and realize high-sensitivity homogeneous phase immunity detection of BA in whole blood.

Inventors

  • QIN YIRU
  • WEI WEI
  • CHEN YU
  • WU BANGHUA
  • RONG WEIFENG

Assignees

  • 广东省职业病防治院(广东省职业卫生检测中心)
  • 华南师范大学

Dates

Publication Date
20260505
Application Date
20250729

Claims (11)

  1. 1. A method for detecting the rice fermentation acid in whole blood by non-diagnostic homogeneous phase immunity is characterized in that, A qualitative method comprising the steps of: (1) Setting up a negative control group, namely adding a whole blood sample without BA into a donor probe buffer solution of a fluorescence resonance energy transfer probe system, performing first incubation, testing the fluorescence intensity I 0 emitted at the position of 800 nm under the excitation of 980 nm light, adding an acceptor probe buffer solution, performing second incubation, and testing the fluorescence intensity I emitted at the position of 800 nm under the excitation of 980 nm light; (2) The detection group comprises the steps of adding a whole blood sample to be detected into a donor probe buffer solution of a fluorescence resonance energy transfer probe system, performing first incubation, testing the fluorescence intensity I 0 emitted at the position 800 nm under 980 nm light excitation, adding an acceptor probe buffer solution, performing second incubation, and testing the fluorescence intensity I emitted at the position 800 nm under 980 nm light excitation; If the fluorescence intensity ratio I/I 0 in the detection group is equal to the fluorescence intensity ratio I/I 0 in the negative control group, the whole blood sample to be detected does not contain BA, and if the fluorescence intensity ratio I/I 0 in the detection group is greater than the fluorescence intensity ratio I/I 0 in the negative control group, the whole blood sample to be detected contains BA; a method of quantification comprising the steps of: (1) Setting up a standard curve and a fitting equation, namely respectively adding a series of BA standard whole blood samples with concentration gradients into donor probe buffer solution of a fluorescence resonance energy transfer probe system, after first incubation, testing the fluorescence intensity I 0 emitted at 800 nm under 980 nm light excitation, respectively adding acceptor probe buffer solution, after second incubation, testing the fluorescence intensity I emitted at 800 nm under 980 nm light excitation, taking the concentration of the BA standard whole blood samples as an abscissa and the fluorescence intensity ratio I/I 0 as an ordinate, setting up a standard curve and fitting the equation; (2) Adding a whole blood sample to be detected into a donor probe buffer solution of a fluorescence resonance energy transfer probe system, performing first incubation, testing the fluorescence intensity I 0 emitted at the position of 800 nm under 980 nm light excitation, adding an acceptor probe buffer solution, performing second incubation, testing the fluorescence intensity I emitted at the position of 800 nm under 980 nm light excitation, and substituting the fluorescence intensity ratio I/I 0 into the standard curve and/or equation of the step (1) to calculate the BA content 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 NaYbF 4 of a BA monoclonal antibody with a surface marked with Tm nanocrystalline, and the acceptor probe is NaDyF 4 nanocrystalline of a BA antigen with a surface marked with the acceptor probe; The NaYbF 4 of the surface-labeled BA monoclonal antibody is characterized in that the crystal form of Tm nanocrystalline is hexagonal phase, and the grain size is 20-30 nm; The NaDyF 4 nanocrystalline with the BA antigen marked on the surface has a cubic phase and a grain diameter of 5-8 nm.
  2. 2. The method for homogeneous immunodetection of milbemycetin in whole blood for non-diagnostic purposes according to claim 1, wherein in the qualitative method, the concentration of the donor probe in the first incubation system is the same in step (1) and step (2), and the concentration of the whole blood sample is the same in the second incubation system, wherein the concentration of the donor probe is the same in step (1) and step (2), and the concentration of the acceptor probe is the same in the second incubation system; and/or, in the quantitative method, the concentration of the donor probe in the first incubation system of the step (1) is the same as that of the whole blood sample, and the concentration of the donor probe in the second incubation system of the step (1) is the same as that of the acceptor probe; And/or in the qualitative method and the quantitative method, the molar ratio of the donor probe to the acceptor probe in the step (1) and the step (2) is 1:0.5-1:2, and the dosage ratio of the donor probe to the whole blood sample in the step (1) and the step (2) is 1-3 mmol:1-3 mL; and/or, in the NaYbF 4 :Tm nanocrystalline of the surface-labeled BA monoclonal antibody, the molar ratio of Yb to Tm is 95:5-99.5:0.5; And/or, the ratio of the molar quantity of NaYbF 4 to the mass of the BA monoclonal antibody is 0.1 mmol:0.03-50 mug; And/or, the molar amount of NaDyF 4 nanocrystals to mass of BA antibody is 0.1 mmol:0.02-50 μg.
  3. 3. The method for homogeneous immunodetection of milbemycetin in whole blood for non-diagnostic purposes according to claim 1 or 2, characterized in that in both the qualitative and quantitative methods, the concentration of the donor probe buffer in step (1) and in step (2) is 0.02-0.2 mmol/mL, the concentration of the acceptor probe buffer is 0.02-0.2 mmol/mL, and the buffer is glycine buffer containing 1-5% (w/v) bovine serum albumin; and/or, in both qualitative and quantitative methods, the incubation refers to resting at 37 ℃ for 5-25 min; and/or, in the quantitative method, the concentration of the BA standard whole blood sample in the step (1) is 0-100 ng/mL.
  4. 4. The method for homogeneous immunodetection of milbemycetin in whole blood for non-diagnostic purposes according to claim 1 or 2, characterized in that the NaYbF 4 :tm nanocrystals of the surface-labeled BA monoclonal antibody are obtained by the following method: (1) Removing ligand oleic acid from NaYbF 4 :Tm nanocrystalline with oleic acid as a surface ligand, and reacting with polyacrylic acid to obtain polyacrylic acid modified NaYbF 4 :Tm nanocrystalline; (2) Dispersing polyacrylic acid modified NaYbF 4 :Tm nanocrystalline in buffer solution, adding ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to activate carboxyl on polyacrylic acid, adding BA monoclonal antibody to make oscillation reaction, finally adding excessive bovine serum albumin to block activated carboxyl site which is not combined with antibody, so as to obtain NaYbF 4 :Tm nanocrystalline of surface-marked BA monoclonal antibody; And/or, the ratio of the mass of the polyacrylic acid in the step (1) to the molar quantity of NaYbF 4 :Tm nanocrystalline is 100-300 mg:1-3 mmol; And/or, the molar amount of polyacrylic acid modified NaYbF 4 :Tm nanocrystalline, the mass of ethyl- (3-dimethylaminopropyl) carbodiimide, the mass of N-hydroxysuccinimide and the mass of BA monoclonal antibody in the step (2) are 0.1 mmol:2-6 mg:5-9 mg:0.03-50 mug; and/or, the NaDyF 4 nanocrystalline with the BA antigen marked on the surface is obtained by the following method: (1) Removing the NaDyF 4 nano-crystal with oleic acid and oleylamine serving as the surface ligands, and reacting with polyacrylic acid to obtain NaDyF 4 nano-crystal modified by polyacrylic acid; (2) Dispersing NaDyF 4 nano-crystals modified by polyacrylic acid in a buffer solution, adding ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to activate carboxyl groups on polyacrylic acid, adding BA antigen to carry out oscillation reaction, and finally adding excessive bovine serum albumin to block activated carboxyl sites which are not combined with the antigen, thus obtaining NaDyF 4 nano-crystals with the surface marked with the BA antigen; And/or, the ratio of the mass of the polyacrylic acid in the step (1) to the molar quantity of NaDyF 4 nano-crystals is 100-300 mg:1-3 mmol; and/or the molar amount of the polyacrylic acid modified NaDyF 4 nano-crystal, the mass of the ethyl- (3-dimethylaminopropyl) carbodiimide and the ratio of the mass of the N-hydroxysuccinimide to the mass of the BA antigen in the step (2) are 0.1 mmol:2-6 mg:5-9 mg:0.02-50 mug.
  5. 5. A method of preparing a fluorescence resonance energy transfer probe system according to any one of claims 1-4, comprising the steps of: (1) NaYbF 4 Tm nanocrystalline surface marked BA monoclonal antibody S1, ytterbium acetate, thulium acetate, 1-octadecene and oleic acid are mixed and dissolved to obtain a precursor solution, sodium hydroxide and ammonium fluoride are added, and heating reaction is carried out to obtain NaYbF 4 with oleic acid as a surface ligand, namely Tm nanocrystalline; S2, removing ligand oleic acid from NaYbF 4 :Tm nanocrystalline with oleic acid as a surface ligand, and reacting with polyacrylic acid to obtain polyacrylic acid modified NaYbF 4 :Tm nanocrystalline; S3, dispersing polyacrylic acid modified NaYbF 4 :Tm nanocrystalline in a buffer solution, adding ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to activate carboxyl on polyacrylic acid, and then combining with a BA monoclonal antibody to obtain NaYbF 4 :Tm nanocrystalline of the surface-marked BA monoclonal antibody; (2) NaDyF 4 nanocrystalline surface-labeled BA antigen S4, dysprosium oxide and trifluoroacetic acid are heated and reacted to obtain dysprosium trifluoroacetate, sodium trifluoroacetate, 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate are heated and reacted to obtain NaDyF 4 nano-crystals with oleic acid and oleylamine as surface ligands; S5, removing the NaDyF 4 nano-crystal with the surface ligand of oleic acid and oleylamine by using the ligand of oleic acid and oleylamine, and reacting with polyacrylic acid to obtain a polyacrylic acid modified NaDyF 4 nano-crystal; S6, dispersing the polyacrylic acid modified NaDyF 4 nano-crystals in a buffer solution, adding ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to activate carboxyl groups on polyacrylic acid, and then combining with BA antigen to obtain NaDyF 4 nano-crystals with the surface marked with BA antigen; The NaYbF 4 of the surface-marked BA monoclonal antibody takes Tm nanocrystalline as a donor probe, and the NaDyF 4 nanocrystalline of the surface-marked BA antigen as an acceptor probe, thus forming a fluorescence resonance energy transfer probe system.
  6. 6. The method for preparing a fluorescence resonance energy transfer probe system according to claim 5, wherein in the NaYbF 4 :Tm nanocrystalline, the molar ratio of Yb to Tm is 95:5-99.5:0.5; And/or the total molar amount of ytterbium acetate and thulium acetate, the molar amount of sodium hydroxide, the molar amount of ammonium fluoride, the volume ratio of 1-octadecene to the volume ratio of oleic acid is 1-3 mmol:2.5-5 mmol:3-5 mmol:15-20 mL:7.5-10 mL; And/or the operations in S1 are all performed under an inert gas atmosphere, wherein the inert gas atmosphere is at least one of nitrogen atmosphere, argon atmosphere and helium atmosphere; And/or the temperature of the heating reaction in S1 is 290-310 ℃, and the time is 50-70 min; and/or S1, wherein the crystal form of the NaYbF 4 :Tm nanocrystalline is hexagonal phase, and the grain diameter is 20-30 nm; And/or S4, wherein the crystal form of the NaDyF 4 nano-crystal is cubic phase, and the grain size is 5-8 nm; and/or the ratio of the molar amount of dysprosium oxide to the volume of trifluoroacetic acid of S4 is 0.5-1.5 mmol:6-12 mL; and/or S4, wherein the dosage ratio of the sodium trifluoroacetate, the 1-octadecene, the oleic acid, the oleylamine and the dysprosium trifluoroacetate is 1-3 mmol:10-20 mL:5-10 mL:5-10 mL:1-3 mmol; and/or S4, wherein the temperature of the heating reaction of sodium trifluoroacetate, 1-octadecene, oleic acid, oleylamine and dysprosium trifluoroacetate is 250-300 ℃ and the time is 25-45 min; And/or the operations in S4 are all performed under an inert gas atmosphere, which is at least one of a nitrogen atmosphere, an argon atmosphere, and a helium atmosphere.
  7. 7. The method of claim 5 or 6, wherein the molecular weight of the polyacrylic acid in S2 and S5 is 1500-2500; And/or the ratio of the mass of the polyacrylic acid in S2 to the molar quantity of NaYbF 4 :Tm nanocrystalline is 100-300 mg:1-3 mmol; And/or S2, wherein the temperature of the reaction with polyacrylic acid is room temperature, and the time is 30-60 min; and/or, the molar quantity of the polyacrylic acid modified NaYbF 4 :Tm nanocrystalline, the mass of the ethyl- (3-dimethylaminopropyl) carbodiimide and the mass of the N-hydroxysuccinimide to the mass of the BA monoclonal antibody in the S3 are 0.1 mmol:2-6 mg:5-9 mg:0.03-50 mug; and/or, the time of activating carboxyl groups on the polyacrylic acid in S3 is 30-60 min; And/or S3, dispersing NaYbF 4 :Tm nanocrystalline after activating carboxyl on polyacrylic acid in buffer solution, adding BA monoclonal antibody and oscillating 1-3 h, then adding excessive bovine serum albumin to block activated carboxyl locus which is not combined with antibody, and centrifugally washing to obtain NaYbF 4 :Tm nanocrystalline of the surface marked BA monoclonal antibody; And/or the ratio of the mass of the polyacrylic acid in S5 to the molar quantity of NaDyF 4 nano-crystals is 100-300 mg:1-3 mmol; and/or S5, wherein the temperature of the reaction with polyacrylic acid is room temperature, and the time is 30-60 min; And/or the molar quantity of the polyacrylic acid modified NaDyF 4 nano-crystal, the mass of the ethyl- (3-dimethylaminopropyl) carbodiimide and the ratio of the mass of the N-hydroxysuccinimide to the mass of the BA antigen in the S6 is 0.1 mmol:2-6 mg:5-9 mg:0.02-50 mug; And/or, the time of activating carboxyl groups on the polyacrylic acid in S6 is 30-60 min; and/or S6, adding BA antigen into NaDyF 4 nano-crystal dispersion buffer solution after activating carboxyl on polyacrylic acid, oscillating 1-3 h, adding excessive bovine serum albumin to block activated carboxyl sites which are not combined with antibody, and centrifugally washing to obtain NaDyF 4 nano-crystal with surface marked BA antigen.
  8. 8. The method for preparing a fluorescence resonance energy transfer probe system according to claim 5 or 6, wherein the method for removing the ligand oleic acid from the Tm nanocrystals by S2 using the NaYbF 4 whose surface ligand is oleic acid is: Adding an aqueous hydrochloric acid solution and an ethanol solution into a NaYbF 4 :Tm nanocrystalline cyclohexane dispersion liquid, standing for 6-12: 12h after shaking and mixing, removing oleic acid ligand on the surface of the nanocrystalline, centrifuging, collecting NaYbF 4 :Tm nanocrystalline without ligand, and dispersing in water; And/or the concentration of the NaYbF 4 to Tm nanocrystalline cyclohexane dispersion liquid 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 NaYbF 4 to Tm nanocrystalline cyclohexane dispersion liquid to hydrochloric acid aqueous solution to ethanol is 1:1:1; and/or, removing ligand oleic acid from the NaYbF 4 with oleic acid serving as a surface ligand in S2, then re-suspending in water, and then dropwise adding the water into a polyacrylic acid aqueous solution for mixing and stirring, wherein the concentration of the ligand-free NaYbF 4 in water is 0.1-0.2 mmol/mL, and the concentration of the polyacrylic acid aqueous solution is 10-20 mg/mL; And/or, the method for removing the ligand oleic acid and oleylamine from NaDyF 4 nano-crystals with the surface ligand oleic acid and oleylamine in S5 comprises the following steps: Adding hydrochloric acid aqueous solution and ethanol solution into NaDyF 4 nanocrystalline cyclohexane dispersion, vibrating and mixing, standing for 6-12: 12 h, removing oleic acid and oleylamine ligand on the nanocrystalline surface, centrifuging, collecting NaDyF 4 nanocrystalline without ligand, and dispersing in water; And/or the concentration of NaDyF 4 nano-crystalline cyclohexane dispersion liquid is 0.1-0.2 mmol/mL, the concentration of hydrochloric acid aqueous solution is 0.05-0.2 mol/L, and the volume ratio of NaDyF 4 nano-crystalline cyclohexane dispersion liquid, hydrochloric acid aqueous solution and ethanol is 1:1:1; And/or, removing the ligand oleic acid and the oleylamine from NaDyF 4 nano crystals with the surface ligand oleic acid and the oleylamine in S5, then re-suspending in water, and then dropwise adding the water into a polyacrylic acid aqueous solution for mixing and stirring, wherein the concentration of the ligand-free NaDyF 4 nano crystals re-suspended in water is 0.1-0.2 mmol/mL, and the concentration of the polyacrylic acid aqueous solution is 10-20 mg/mL.
  9. 9. A fluorescence resonance energy transfer probe system according to any one of claims 1-4.
  10. 10. A fluorescence resonance energy transfer probe system made by the method of any one of claims 5-8.
  11. 11. Use of a fluorescence resonance energy transfer probe system according to any one of claims 9-10 for the detection of BA in whole blood for non-diagnostic purposes.

Description

Fluorescent resonance energy transfer probe system and method for high-sensitivity homogeneous phase immunity detection of milbemycetin in whole blood Technical Field The invention belongs to the field of immunological detection, and particularly relates to a fluorescence resonance energy transfer probe system and a method for detecting milbemycetin in whole blood by high-sensitivity homogeneous phase immunity. Background Midamascoic acid (Bongkrekic acid, BA) is a highly toxic metabolite produced by the Pseudomonas species Cocois, the first time discovered by the Netherlands Mertens and Vanveen in 1930. BA is an unsaturated tricarboxylic fatty acid commonly found in fermented cereals, spoiled tremella, agaric and other spoiled starch-based foods. BA is a strong respiratory toxin, can cause acute poisoning of people, causes damage to nervous system, digestive system and urinary system, and serious BA poisoning can rapidly cause damage to liver and kidney, causes systemic multi-organ failure, and has high mortality rate. Because of its stable molecular structure, common food processing techniques such as high temperature heating, washing and freezing are difficult to remove. In order to prevent poisoning and effectively treat the poisoning quickly, the detection of BA has important significance on human health. At present, detection of BA poisoning is mainly focused on exogenous samples such as suspected spoiled foods or vomit, but the problems of sample missing or sample loss of representativeness are often faced. The whole blood sample can directly reflect the exposure degree of toxin in human body, and simultaneously, the real-time blood concentration can provide a dosage adjustment basis for therapeutic measures such as blood purification, so the whole blood sample is an ideal sample for BA poisoning evaluation and therapeutic monitoring. The establishment of a technology for rapidly and accurately detecting BA in whole blood is important for improving the poisoning treatment efficiency and reducing the mortality rate. Achieving high sensitivity BA detection in whole blood presents a number of challenges. First, the sensitivity of the detection method is highly desirable because of the low concentration of BA in the blood. Secondly, the whole blood contains proteins, lipids and other endogenous substances, which easily affect the accuracy and specificity of detection. The conventional high performance liquid chromatography-mass spectrometry (HPLC-MS) is used for detecting BA, but meets the requirements of quantification and high sensitivity, but has the disadvantages of complex sample pretreatment process, expensive equipment and long detection time, and is not suitable for on-site rapid detection. The homogeneous phase detection method does not need complex steps such as sample separation, washing and the like, has the advantages of simple operation, high detection speed and the like, and is an ideal strategy for detecting BA in whole blood. However, whole blood has strong absorption and strong emission in the visible light region, so that homogeneous immunodetection of BA in whole blood is required to be realized, the problems of strong signal background interference, low sensitivity and the like are required to be overcome, and the conventional fluorescent probe cannot overcome the problems of background interference such as autofluorescence and light scattering of whole blood, so that the sensitivity and the specificity are reduced. Therefore, a technology for realizing rapid and sensitive homogeneous detection of BA in whole blood without background interference has yet to be developed. Disclosure of Invention To solve the drawbacks and disadvantages of the prior art, a primary object of the present 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. According to the invention, naYbF 4:Tm nanocrystalline of the surface-marked BA monoclonal antibody is obtained by carrying out carboxyl modification on NaYbF 4:Tm nanocrystalline and binding of BA monoclonal antibody, naDyF 4 nanocrystalline of the surface-marked BA antigen is obtained by carrying out carboxyl modification on NaDyF 4 nanocrystalline and binding of BA antigen, naYbF 4:Tm nanocrystalline of the surface-marked BA monoclonal antibody is used as a donor probe, naDyF 4 nanocrystalline of the surface-marked BA antigen is used as an acceptor probe, and a fluorescence resonance energy transfer (NIR-NIR FRET) probe system based on near infrared excitation and emission is obtained. Another object of the present invention is to provide a fluorescence resonance energy transfer (NIR-NIR FRET) probe system based on near infrared excitation and emission, which is obtained by the above preparation method, and the NIR-NIR FRET probe system can effectively overcome the background interference of whole blood samples, and realize high-sensitivity