CN113358861A - Method for detecting chloramphenicol based on quantum dot fluorescent microsphere probe - Google Patents
Method for detecting chloramphenicol based on quantum dot fluorescent microsphere probe Download PDFInfo
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- DRDVZXDWVBGGMH-UHFFFAOYSA-N zinc;sulfide Chemical class [S-2].[Zn+2] DRDVZXDWVBGGMH-UHFFFAOYSA-N 0.000 description 1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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Abstract
The invention relates to a method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe, which comprises the following steps: (1) preparing carboxylated Mn-ZnS quantum dot fluorescent nano microspheres; (2) preparing a Mn-ZnS quantum dot nano microsphere-chloramphenicol fluorescent probe; (3) a construction and detection method of a chloramphenicol fluorescent test strip; (4) mixing and dripping the Mn-ZnS quantum dot-chloramphenicol fluorescent probe and the sample to be detected in the step (2) on a sample pad; (5) and obtaining a detection result according to the fluorescence intensity of the test strip after 4-5 min. The invention improves the stability and the sensitivity of the fluorescent probe; the Mn-ZnS quantum dot which is synthesized in one step and modified by carboxyl and amino improves the dispersibility and stability of the quantum dot in an aqueous solvent, increases the sensitivity and stability of the fluorescent probe after the Mn-ZnS quantum dot is coupled with a chloramphenicol antibody, and has good stability, high sensitivity and good commercial application value.
Description
Technical Field
The invention belongs to the cross fields of material, biology, biological detection and the like, and particularly relates to a method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe.
Background
Chloramphenicol (CAP) is a broad-spectrum amide alcohol antibiotic drug, has good inhibition effect on pathogenic microorganisms such as bacillus anthracis, pneumococcus, streptococcus, listeria, staphylococcus, chlamydia and rickettsia, and can be used for treating and preventing typhoid fever, meningitis, urinary tract infection and other diseases. However, CAP has major toxic and side effects on human body, such as suppression of bone marrow hematopoiesis. In addition, CAP is extremely stable in physicochemical properties, is not easily degraded, and can accumulate in the human body through the food chain, thereby causing serious damage to the human health.
Quantum Dots (QDs) are widely used as fluorescent probes with excellent performance in various fields such as biomarkers and environmental monitoring, and are popular among scientists. The manganese-doped zinc sulfide quantum dots retain the original fluorescence characteristics of host ZnS, and the luminescence life of the doped manganese ions reaches ms level (about 1.1ms), so that the interference of background fluorescence and scattered light can be effectively avoided.
The Mn-ZnS QDs has high quantum yield, good dispersibility and high stability, but the quantum dots can not be directly applied to a biological system due to the hydrophobic property of the surface, ligand exchange is needed to be carried out on the quantum dots, and then the prepared water-soluble quantum dots are applied to the biological system, so that the steps are complicated.
Disclosure of Invention
Object of the Invention
Aiming at the lack of a method for quickly, simply, conveniently, highly sensitively and accurately detecting chloramphenicol at present, the invention provides a method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe. The method has the advantages of simple process, high sensitivity, rapidness and good stability, and can be widely popularized and used for chloramphenicol detection.
Technical scheme
A method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe is characterized by comprising the following steps:
(1) preparing carboxylated Mn-ZnS quantum dot fluorescent nano microspheres; the diameter of the carboxylated Mn-ZnS quantum dot fluorescent nano microsphere is 4 +/-0.5 nm;
the carboxylated Mn-ZnS quantum dot fluorescent nano microsphere is prepared by the following steps:
ZnSO is added4、MnCl2Dissolving glutathione in ultrapure water, adjusting pH to 11 with 0.01mol/L NaOH, magnetically stirring under nitrogen for 30-40min, adding Na2Continuously stirring the S solution for 20-30min, raising the temperature to 55-60 ℃, stopping introducing nitrogen, and continuously reacting for 2-4 hours; ZnSO4、MnCl2And glutathione at a molar ratio of 2.5: 0.1: 5; MnCl2With Na2The molar ratio of S is 1: 25; MnCl2The molar volume ratio to ultrapure water is 0.01: 50 (mmol: mL);
after the reaction is finished, adding equal volume of ethanol to precipitate the synthesized quantum dots, performing centrifugal separation at the rotation speed of 10000-12000rpm to remove liquid to obtain a product, washing the product with deionized water for more than or equal to 5 times to remove unreacted raw materials, and drying the product at low temperature for later use.
(2) Adding a chloramphenicol monoclonal antibody into the carboxylated Mn-ZnS quantum dot fluorescent nanospheres prepared in the step (1) to prepare Mn-ZnS quantum dot nanosphere-chloramphenicol fluorescent probes;
the prepared carboxylated Mn-ZnS quantum dot fluorescent nano-microsphere and chloramphenicol monoclonal antibody are used for preparing the Mn-ZnS quantum dot nano-microsphere-chloramphenicol fluorescent probe, and the method comprises the following steps:
adding Mn-ZnS quantum dots into a brown small bottle containing a phosphate buffer solution, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution, fully mixing, quickly placing the small bottle into an ultrasonic cleaner for ultrasonic treatment for 30-40min, adding a chloramphenicol monoclonal antibody, reacting for 90-100min under the action of magnetic stirring at room temperature to obtain an Mn-ZnS quantum dot nano microsphere-chloramphenicol fluorescent probe of an Mn-ZnS QDs-CAP conjugate, and storing at 4 ℃ for later use; the mass ratio of Mn-ZnS quantum dots, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5: 18: 15; the mass-to-volume ratio of the Mn-ZnS quantum dots to the phosphate buffer solution is 0.5: 1 mg: mL; the mass ratio of the Mn-ZnS quantum dots to the chloramphenicol monoclonal antibody is 10: 1.
(3) preparing a detection line T line by using a chloramphenicol-bovine serum albumin conjugate, preparing a quality control line C line by using a goat anti-mouse secondary antibody, spraying the quality control line C line on a nitrocellulose membrane, and assembling a glass fiber sample pad, the nitrocellulose membrane provided with the T line and the C line and a water absorption pad which are sequentially overlapped and stuck on a hard backing plate to obtain a chromatography test paper layer; cutting into test strips with the width of 4-5 mm;
(4) respectively adding 5-7 microliters of the nanoparticle-chloramphenicol fluorescent probe obtained in the step (2) and a sample to be detected into a hole of an enzyme-labeled plate; incubating in oven at 37 deg.C for 20-25min to obtain solution D;
(5) inserting the chromatography test strip prepared in the step (3) into the hole of the ELISA plate containing the solution D in the step (4), immersing one end of the glass fiber sample pad into the liquid in the hole of the ELISA plate, starting timing when the liquid begins to perform chromatography on the nitrocellulose membrane, observing under an ultraviolet analyzer after 4-5min, and performing quantitative judgment through the color change of a T line.
Advantages and effects
The invention has the following advantages and beneficial effects:
glutathione is added as a stabilizer during the synthesis of the Mn-ZnS quantum dots, the Mn-ZnS quantum dots modified by carboxyl and amino are synthesized in one step, the dispersibility and the stability of the quantum dots in an aqueous phase solvent are improved, and the sensitivity and the stability of the fluorescent probe after the Mn-ZnS quantum dots are coupled with a chloramphenicol antibody are improved.
Drawings
FIG. 1 shows Mn-ZnS quantum dot fluorescent nanospheres.
Detailed Description
The invention is described in detail below with reference to the attached drawing figures:
a method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe is characterized by comprising the following steps:
(1) preparing carboxylated Mn-ZnS quantum dot fluorescent nano microspheres; the diameter of the carboxylated Mn-ZnS quantum dot fluorescent nano microsphere is 4 +/-0.5 nm;
the carboxylated Mn-ZnS quantum dot fluorescent nano microsphere is prepared by the following steps:
ZnSO is added4、MnCl2Dissolving glutathione in ultrapure water, adjusting pH to 11 with 0.01mol/L NaOH, magnetically stirring under nitrogen for 30-40min, adding Na2S, after continuously stirring for 20-30min, stopping introducing nitrogen when the temperature is raised to 55-60 ℃, and continuously reacting for 2-4 hours; ZnSO4、MnCl2And glutathione at a molar ratio of 2.5: 0.1: 5; MnCl2With Na2The molar ratio of S is 1: 25; MnCl2The molar volume ratio to ultrapure water is 0.01: 50 (mmol: mL).
After the reaction is finished, adding equal volume of ethanol to precipitate the synthesized quantum dots, performing centrifugal separation at the rotation speed of 10000-12000rpm to remove liquid to obtain a product, washing the product with deionized water for more than or equal to 5 times to remove unreacted raw materials, and drying the product at low temperature for later use.
(2) Adding a chloramphenicol monoclonal antibody into the carboxylated Mn-ZnS quantum dot fluorescent nanospheres prepared in the step (1) to prepare Mn-ZnS quantum dot nanosphere-chloramphenicol fluorescent probes;
the prepared carboxylated Mn-ZnS quantum dot fluorescent nano-microsphere and chloramphenicol monoclonal antibody are used for preparing a nano-microsphere-chloramphenicol fluorescent probe, and the preparation method comprises the following steps:
adding Mn-ZnS quantum dots into a brown small bottle containing a phosphate buffer solution, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution, fully mixing, quickly placing the small bottle into an ultrasonic cleaner for ultrasonic treatment for 30-40min, finally adding a chloramphenicol monoclonal antibody, reacting for 90-100min under the action of magnetic stirring at room temperature to obtain the Mn-ZnS quantum dot nano microsphere-chloramphenicol fluorescent probe of the Mn-ZnS QDs-CAP conjugate, and storing at 4 ℃ for later use. The mass ratio of Mn-ZnS quantum dots, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5: 18: 15; the mass-to-volume ratio of the Mn-ZnS quantum dots to the phosphate buffer solution is 0.5: 1 (mg: mL); the mass ratio of the Mn-ZnS quantum dots to the chloramphenicol monoclonal antibody is 10: 1.
(3) preparing a detection line T line by using a chloramphenicol-bovine serum albumin conjugate, preparing a quality control line C line by using a goat anti-mouse secondary antibody, spraying, and packaging to obtain a chromatography test strip; the chromatographic test paper strip is formed by sequentially overlapping and sticking a nitrocellulose membrane, a glass fiber sample pad, a nitrocellulose membrane with a T line and a C line, and a water absorption pad on a hard backing plate, and then cutting into a test paper strip with a width of 4-5 mm.
(4) Respectively adding 5-7 microliters of the nanoparticle-chloramphenicol fluorescent probe prepared in the step (2) and a sample to be detected into a hole of an enzyme-labeled plate; incubating in oven at 37 deg.C for 20-25min to obtain solution D;
(5) inserting the chromatographic test strip prepared in the step (3) into the hole of the ELISA plate containing the solution D in the step (4), immersing one end of the glass fiber sample pad into the liquid in the hole of the ELISA plate, timing when the liquid begins to carry out chromatography to the nitrocellulose membrane, observing under an ultraviolet analyzer after 4-5min, and carrying out quantitative judgment through the color change of a T line.
In the invention, glutathione is added as a stabilizer when the Mn-ZnS quantum dot is synthesized, and the added glutathione enables the surface of the Mn-ZnS quantum dot to have amino and carboxyl (the amino and the carboxyl are hydrophilic groups), so that the Mn-ZnS quantum dot with carboxyl and amino modification is synthesized in one step, compared with Mn-ZnS synthesized by other methods, the dispersity and the stability of the quantum dot in an aqueous phase solvent are improved, and the sensitivity and the stability of a fluorescent probe after the Mn-ZnS quantum dot is coupled with a chloramphenicol antibody are improved; this is also concluded from the use of Mn-ZnS quantum dots containing amino and carboxyl groups in this invention.
Example 1:
a method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe comprises the following steps:
(1) preparing carboxylated Mn-ZnS quantum dot fluorescent nano microspheres:
taking 0.25mmol of ZnSO4、0.01mmol MnCl2Dissolving 0.5mmol glutathione in 50mL ultrapure water, adding into 50mL three-neck flask, mixing, adjusting pH to 11 with 0.01mol/L NaOH, magnetically stirring under nitrogen for 40min, adding 0.25mmol Na2S, after continuously stirring for 30min, stopping introducing nitrogen when the temperature is raised to 60 ℃, and continuously reacting for 2 hours;
after the reaction is finished, adding ethanol with the same volume to precipitate the synthesized quantum dots, performing centrifugal separation at the rotating speed of 12000rpm, removing liquid to obtain a product, washing the product with deionized water for 5 times to remove unreacted raw materials, and drying the product at low temperature for later use;
(2) preparing a Mn-ZnS quantum dot nano microsphere-chloramphenicol fluorescent probe:
adding 0.5mg of Mn-ZnS quantum dots prepared in the step (1) into a brown small bottle containing 1mL of phosphate buffer solution, adding 1.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.5mg of N-hydroxysuccinimide, fully mixing, quickly placing the small bottle into an ultrasonic cleaner for ultrasonic treatment for 40min, finally adding 0.05mg of chloramphenicol monoclonal antibody, reacting for 100min under the action of magnetic stirring at room temperature to obtain the Mn-ZnS quantum dot nanosphere-chloramphenicol fluorescent probe of the Mn-ZnS QDs-CAP conjugate, and storing at 4 ℃ for later use;
(3) preparation of immunochromatography test strip
Selecting a nitrocellulose membrane with the model of Pall 170(CN 170) as a carrier, dissolving chloramphenicol-BSA coating source in 0.01mol/L phosphate buffer solution with the pH value of 7.4 and containing 0.05 percent Tween-20, determining the final concentration to be 1mg/mL to obtain a membrane spraying solution A, and spraying the membrane spraying solution A on a 10mm position above the nitrocellulose membrane by using a membrane scribing instrument (Shanghai gold mark) to form a T line (detection line);
dissolving the goat anti-rabbit secondary antibody in 0.01mol/L phosphate buffer solution containing 0.05% Tween-20 and having pH of 7.4, determining the final concentration to be 1mg/mL to obtain a spray solution B, and spraying the spray solution B on a position 5mm above a detection line T line by using a film scratching instrument (Shanghai gold mark) to form a C line (quality control line);
placing the sprayed nitrocellulose membrane in a constant-temperature vacuum drying oven at 37 ℃ for drying, and storing in a room-temperature drying environment for later use;
splicing and sticking on a hard board in sequence: a glass fiber sample pad, a nitrocellulose membrane with a cut C, T line, and absorbent paper. Cutting into 4mm wide test strip after assembly to obtain immunochromatography test strip, storing in sealed dry bag for use (the storage time for temporary determination is within 2 months, the detection result has no influence)
(4) Detection of Chloramphenicol
Taking 6 microliters of the nanoparticle-chloramphenicol fluorescent probe prepared in the step (2), adding the nanoparticle-chloramphenicol fluorescent probe into 100 microliters of a sample to be tested, incubating the sample in an oven at 37 ℃ for 20min, and taking out the sample to obtain a solution D;
accurately preparing a chloramphenicol standard substance, wherein the diluent is deionized water without chloramphenicol, the concentrations of the deionized water are respectively 0, 100, 150, 500 and 1000ng/mL, and the determination is carried out in the step (4);
(5) inserting the chromatographic test strip prepared in the step (3) into the hole of the ELISA plate containing the solution D in the step (4), immersing one end of the glass fiber sample pad into the liquid in the hole of the ELISA plate, timing when the liquid begins to carry out chromatography to the nitrocellulose membrane, observing under an ultraviolet analyzer after 5min, and carrying out quantitative judgment through the color change of a T line.
The example is repeated for 10 times, and the result shows that the visual inspection limit of the chloramphenicol fluorescent immunochromatographic test strip is 150ng/mL, the color of the T line gradually becomes lighter to disappear with the increase of the concentration gradient under the irradiation of a 365nm ultraviolet lamp, the color gradient change of the T line is more consistent, the linear range is better, the requirement of quantitative detection is met, and the sensitivity is improved compared with that of a colloidal gold immunochromatographic test strip.
As shown in figure 1, the Mn-ZnS quantum dot fluorescent nano microsphere has the diameter of 4 +/-0.5 nm and good dispersibility.
Example 2:
a method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe comprises the following steps:
(1) preparing carboxylated Mn-ZnS quantum dot fluorescent nano microspheres:
taking 0.25mmol of ZnSO4、0.01mmol MnCl2Dissolving 0.4mmol glutathione in 50mL ultrapure water, adding into 50mL three-neck flask, mixing, adjusting pH to 11 with 0.01mol/L NaOH, magnetically stirring under nitrogen for 30min, adding 0.25mmol Na2S, after continuously stirring for 20min, stopping introducing nitrogen when the temperature is raised to 55 ℃, and continuously reacting for 4 hours;
after the reaction is finished, adding ethanol with the same volume to precipitate the synthesized quantum dots, performing centrifugal separation at the rotating speed of 12000rpm, removing liquid to obtain a product, washing the product with deionized water for 8 times to remove unreacted raw materials, and drying the product at low temperature for later use;
(2) preparing a Mn-ZnS quantum dot nano microsphere-chloramphenicol fluorescent probe:
adding 0.4mg of Mn-ZnS quantum dots prepared in the step (1) into a brown small bottle containing 1mL of phosphate buffer solution, adding 1.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.5mg of N-hydroxysuccinimide, fully mixing, quickly placing the small bottle into an ultrasonic cleaner for ultrasonic treatment for 30min, finally adding 0.05mg of chloramphenicol monoclonal antibody, reacting for 90min under the action of magnetic stirring at room temperature to obtain the Mn-ZnS quantum dot nanosphere-chloramphenicol fluorescent probe of the Mn-ZnS QDs-CAP conjugate, and storing at 4 ℃ for later use;
(3) preparation of immunochromatography test strip
Selecting a nitrocellulose membrane with the model of Pall 170(CN 170) as a carrier, dissolving chloramphenicol-BSA coating source in 0.01mol/L phosphate buffer solution with the pH value of 7.4 and containing 0.05 percent Tween-20, determining the final concentration to be 1mg/mL to obtain a membrane spraying solution A, and spraying the membrane spraying solution A on a 10mm position above the nitrocellulose membrane by using a membrane scribing instrument (Shanghai gold mark) to form a T line (detection line);
dissolving the goat anti-rabbit secondary antibody in 0.01mol/L phosphate buffer solution containing 0.05% Tween-20 and having pH of 7.4, determining the final concentration to be 1mg/mL to obtain a spray solution B, and spraying the spray solution B on a position 5mm above a detection line T line by using a film scratching instrument (Shanghai gold mark) to form a C line (quality control line);
placing the sprayed nitrocellulose membrane in a constant-temperature vacuum drying oven at 37 ℃ for drying, and storing in a room-temperature drying environment for later use;
splicing and sticking on a hard board in sequence: a glass fiber sample pad, a nitrocellulose membrane with a cut C, T line, and absorbent paper. Cutting into test strips with width of 5mm after assembly to obtain immunochromatography test strip, storing in sealed dry bag for use (the storage time for temporary determination is within 2 months, the detection result has no influence)
(4) Detection of Chloramphenicol
Taking 5 microliters of the nanoparticle-chloramphenicol fluorescent probe prepared in the step (2), adding the nanoparticle-chloramphenicol fluorescent probe into 100 microliters of a sample to be tested, incubating the sample in an oven at 37 ℃ for 25min, and taking out the sample to obtain a solution D;
accurately preparing a chloramphenicol standard substance, wherein the diluent is deionized water without chloramphenicol, the concentrations of the deionized water are respectively 0, 100, 150, 500 and 1000ng/mL, and the determination is carried out in the step (4);
(5) inserting the chromatographic test strip prepared in the step (3) into the hole of the ELISA plate containing the solution D in the step (4), immersing one end of the glass fiber sample pad into the liquid in the hole of the ELISA plate, timing when the liquid begins to carry out chromatography to the nitrocellulose membrane, observing under an ultraviolet analyzer after 5min, and carrying out quantitative judgment through the color change of a T line.
The example is repeated for 10 times, and the result shows that the visual inspection limit of the chloramphenicol fluorescent immunochromatographic test strip is 150ng/mL, the color of the T line gradually becomes lighter to disappear with the increase of the concentration gradient under the irradiation of a 365nm ultraviolet lamp, the color gradient change of the T line is more consistent, the linear range is better, the requirement of quantitative detection is met, and the sensitivity is improved compared with that of a colloidal gold immunochromatographic test strip.
As shown in figure 1, the Mn-ZnS quantum dot fluorescent nano microsphere has the diameter of 4 +/-0.5 nm and good dispersibility.
Example 3:
a method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe comprises the following steps:
(1) preparing carboxylated Mn-ZnS quantum dot fluorescent nano microspheres:
taking 0.25mmol of ZnSO4、0.01mmol MnCl20.6mmol of glutathione is dissolved in 50mL of ultrapure water, added into a 50mL three-neck flask and mixed,adjusting pH of the solution to 11 with 0.01mol/L NaOH, magnetically stirring for 35min under nitrogen gas, adding 0.25mmol Na2S, after continuously stirring for 25min, stopping introducing nitrogen when the temperature is raised to 58 ℃, and continuously reacting for 3 hours;
after the reaction is finished, adding ethanol with the same volume to precipitate the synthesized quantum dots, performing centrifugal separation at the rotating speed of 12000rpm, removing liquid to obtain a product, washing the product with deionized water for 10 times to remove unreacted raw materials, and drying the product at low temperature for later use;
(2) preparing a Mn-ZnS quantum dot nano microsphere-chloramphenicol fluorescent probe:
adding 0.6mg of Mn-ZnS quantum dots prepared in the step (1) into a brown small bottle containing 1mL of phosphate buffer solution, adding 1.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.5mg of N-hydroxysuccinimide, fully mixing, quickly placing the small bottle into an ultrasonic cleaner for ultrasonic treatment for 35min, finally adding 0.05mg of chloramphenicol monoclonal antibody, reacting for 95min under the action of magnetic stirring at room temperature to obtain the Mn-ZnS quantum dot nanosphere-chloramphenicol fluorescent probe of the Mn-ZnS QDs-CAP conjugate, and storing at 4 ℃ for later use;
(3) preparation of immunochromatography test strip
Selecting a nitrocellulose membrane with the model of Pall 170(CN 170) as a carrier, dissolving chloramphenicol-BSA coating source in 0.01mol/L phosphate buffer solution with the pH value of 7.4 and containing 0.05 percent Tween-20, determining the final concentration to be 1mg/mL to obtain a membrane spraying solution A, and spraying the membrane spraying solution A on a 10mm position above the nitrocellulose membrane by using a membrane scribing instrument (Shanghai gold mark) to form a T line (detection line);
dissolving the goat anti-rabbit secondary antibody in 0.01mol/L phosphate buffer solution containing 0.05% Tween-20 and having pH of 7.4, determining the final concentration to be 1mg/mL to obtain a spray solution B, and spraying the spray solution B on a position 5mm above a detection line T line by using a film scratching instrument (Shanghai gold mark) to form a C line (quality control line);
placing the sprayed nitrocellulose membrane in a constant-temperature vacuum drying oven at 37 ℃ for drying, and storing in a room-temperature drying environment for later use;
splicing and sticking on a hard board in sequence: a glass fiber sample pad, a nitrocellulose membrane with a cut C, T line, and absorbent paper. Cutting into 4.5mm wide test strip after assembly to obtain immunochromatography test strip, storing in sealed dry bag for use (the storage time for temporary determination is within 2 months, the detection result has no influence)
(4) Detection of Chloramphenicol
Taking 6 microliters of the nanoparticle-chloramphenicol fluorescent probe prepared in the step (2), adding the nanoparticle-chloramphenicol fluorescent probe into 100 microliters of a sample to be tested, incubating the sample in an oven at 37 ℃ for 23min, and taking out the sample to obtain a solution D;
accurately preparing a chloramphenicol standard substance, wherein the diluent is deionized water without chloramphenicol, the concentrations of the deionized water are respectively 0, 100, 150, 500 and 1000ng/mL, and the determination is carried out in the step (4);
(5) and (3) inserting the chromatographic test strip prepared in the step (3) into the hole of the ELISA plate containing the solution D in the step (4), immersing one end of the glass fiber sample pad into the liquid in the hole of the ELISA plate, timing when the liquid begins to carry out chromatography to the nitrocellulose membrane, observing under an ultraviolet analyzer after 5min, and carrying out quantitative judgment through the color change of the T line.
The example is repeated for 10 times, and the result shows that the visual inspection limit of the chloramphenicol fluorescent immunochromatographic test strip is 150ng/mL, the color of the T line gradually becomes lighter to disappear with the increase of the concentration gradient under the irradiation of a 365nm ultraviolet lamp, the color gradient change of the T line is more consistent, the linear range is better, the requirement of quantitative detection is met, and the sensitivity is improved compared with that of a colloidal gold immunochromatographic test strip.
As shown in figure 1, the Mn-ZnS quantum dot fluorescent nano microsphere has the diameter of 4 +/-0.5 nm and good dispersibility.
Claims (3)
1. A method for detecting chloramphenicol based on a quantum dot fluorescent microsphere probe is characterized by comprising the following steps:
(1) preparing carboxylated Mn-ZnS quantum dot fluorescent nano microspheres; the diameter of the carboxylated Mn-ZnS quantum dot fluorescent nano microsphere is 4 +/-0.5 nm;
(2) adding a chloramphenicol monoclonal antibody into the carboxylated Mn-ZnS quantum dot fluorescent nanospheres prepared in the step (1) to prepare Mn-ZnS quantum dot nanosphere-chloramphenicol fluorescent probes;
(3) preparing a detection line T line by using a chloramphenicol-bovine serum albumin conjugate, preparing a quality control line C line by using a goat anti-mouse secondary antibody, spraying the quality control line C line on a nitrocellulose membrane, and assembling a glass fiber sample pad, the nitrocellulose membrane provided with the T line and the C line and a water absorption pad which are sequentially overlapped and stuck on a hard backing plate to obtain a chromatography test paper layer; cutting into test strips with the width of 4-5 mm;
(4) respectively adding 5-7 microliters of the nanoparticle-chloramphenicol fluorescent probe obtained in the step (2) and a sample to be detected into a hole of an enzyme-labeled plate; incubating in oven at 37 deg.C for 20-25min to obtain solution D;
(5) inserting the chromatographic test strip prepared in the step (3) into the hole of the ELISA plate containing the solution D in the step (4), immersing one end of the glass fiber sample pad into the liquid in the hole of the ELISA plate, timing when the liquid begins to carry out chromatography to the nitrocellulose membrane, observing under an ultraviolet analyzer after 4-5min, and carrying out quantitative judgment through the color change of a T line.
2. The method for detecting chloramphenicol based on the quantum dot fluorescent microsphere probe as claimed in claim 1, characterized in that: the carboxylated Mn-ZnS quantum dot fluorescent nano microsphere in the step (1) is prepared by the following steps:
ZnSO is added4、MnCl2Dissolving glutathione in ultrapure water, adjusting pH to 11 with 0.01mol/L NaOH, magnetically stirring under nitrogen for 30-40min, adding Na2Continuously stirring the S solution for 20-30min, raising the temperature to 55-60 ℃, stopping introducing nitrogen, and continuously reacting for 2-4 hours; ZnSO4、MnCl2And glutathione at a molar ratio of 2.5: 0.1: 4-6; MnCl2With Na2The molar ratio of S is 1: 24-26; MnCl2The molar volume ratio to ultrapure water is 0.01: 45-55 (mmol: mL);
after the reaction is finished, adding equal volume of ethanol to precipitate the synthesized quantum dots, performing centrifugal separation at the rotation speed of 10000-12000rpm to remove liquid to obtain a product, washing the product with deionized water for more than or equal to 5 times to remove unreacted raw materials, and drying the product at low temperature for later use.
3. The method for detecting chloramphenicol based on the quantum dot fluorescent microsphere probe as claimed in claim 1, characterized in that: the carboxylated Mn-ZnS quantum dot fluorescent nanospheres and the chloramphenicol monoclonal antibody prepared in the step (2) are used for preparing Mn-ZnS quantum dot nanospheres-chloramphenicol fluorescent probes, and the method comprises the following steps:
adding Mn-ZnS quantum dots into a brown small bottle containing a phosphate buffer solution, adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution, fully mixing, quickly placing the small bottle into an ultrasonic cleaner for ultrasonic treatment for 30-40min, adding a chloramphenicol monoclonal antibody, reacting for 90-100min under the action of magnetic stirring at room temperature to obtain an Mn-ZnS quantum dot nano microsphere-chloramphenicol fluorescent probe of an Mn-ZnS QDs-CAP conjugate, and storing at 4 ℃ for later use; the mass ratio of Mn-ZnS quantum dots, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 4-6: 18: 15; the mass-to-volume ratio of the Mn-ZnS quantum dots to the phosphate buffer solution is 0.5: 1-1.5 mg: mL; the mass ratio of the Mn-ZnS quantum dots to the chloramphenicol monoclonal antibody is 9-11: 1.
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