NL2028742A - Fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites, preparation method and application thereof - Google Patents
Fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites, preparation method and application thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention provides a fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites, a preparation method and an application thereof, and belongs to the technical 5 field of antibiotic detection. The kit comprises a chromatographic test strip and quantum dot probes. A nitrocellulose membrane of the chromatographic test strip is provided with test lines T1, T2, T3, and T4 and a control line C; the test lines T1, T2, T3, and T4 are 10 spray—coated with envelope antigens 2—NPAOZ—BSA, 2—NPAHD— BSA, 2—NPSEM—BSA, and 2—NPAMOZ—BSA, respectively; and the quantum dot probes comprise red light quantum dot—coupled 2—NPAOZ antibody, yellow light quantum dot-coupled 2-NPAHD antibody, green light quantum dot—coupled 2—NPSEM 15 antibody, and orange light quantum dot—coupled 2—NPAMOZ antibody. The kit of the present invention can simultaneously detect four kinds of nitrofuran metabolites, with high sensitivity.
Description
[01] The present invention belongs to the technical field of antibiotic detection, in particular to a fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites, a preparation method and an application thereof.
[02] Nitrofurans are a kind of broad-spectrum antibiotics, mainly including furazolidone, nitrofurantoin, furaltadone, and nitrofurazone. Because of low price and good antibacterial effect, nitrofurans are widely used in the prevention and treatment of bacterial diseases of aquatic animals. Nitrofurans have been banned in animal food production in many countries including China because of their mutagenicity and carcinogenicity. However, incidents of excessive residues of nitrofurans still occur from time to time, so the detection of the residues of nitrofurans is of great significance.
[03] Nitrofurans are metabolized rapidly when they enter the animal’s body, so nitrofuran metabolites are mainly detected in food detection. At present, instrumental method is the main method of detecting this kind of drugs. However, instrumental method requires expensive instrument and complex operation, so it is not suitable for rapid on-site detection.
[04] Colloidal gold immunochromatography is a new type of immunolabeling technique which uses colloidal gold as a tracer marker to be applied to antigens and antibodies.
Because of its advantages such as convenience and quickness, visible results to naked eyes, and applicability to a variety of detection conditions, it has been used in the on- site detection and rapid screening of nitrofurans. However, the biggest problem with the technology 1s that the sensitivity needs to be improved.
[05] In view of this, the present invention aims to provide a fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites, a preparation method and an application thereof. The fluorescence immunochromatographic kit uses four kinds of quantum dots as markers to respectively label four kinds of nitrofuran metabolite antibodies. By combining the advantages of quantum dots such as long fluorescence lifetime, high quantum yield and good light stability with immunochromatography, the detection sensitivity is improved on the basis of the realization of simultaneous detection of four kinds of nitrofuran metabolites.
[06] In order to realize the objective of the present invention, the present invention provides the following technical solutions.
[07] The present invention provides a fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites, characterized in comprising a chromatographic test strip and quantum dot probes;
[08] The chromatographic test strip comprises a PVC backing pad, and further comprises a sample pad, a nitrocellulose membrane (NC membrane) and an absorbent pad which are sequentially overlapped and fixed on the PVC backing pad, the nitrocellulose membrane is provided with test lines Tl, T2, T3, and T4 and a control line C; the test lines Tl, TZ, T3, and T4 are spray-coated with envelope antigens 2-NPAQZ- BSA, 2-NPAHD-BSA, 2-NPSEM-BSA, and 2-NPAMOZ-BSA, respectively; the control line C is spray-coated with rabbit anti-mouse IgG; and
[09] the quantum dot probes comprise a red light quantum dot- coupled 2-NPA0Z antibody, a yellow light quantum dot-coupled
2-NPAHD antibody, a green light quantum dot-coupled 2-NPSEM antibody, and an orange light quantum dot-coupled 2-NPAMOZ antibody.
[10] Preferably, the concentrations of the envelope antigens 2-NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA and 2-NPAMOZ-BSA are
0.1-0.5 mg/mL, 0.05-0.3 mg/mL, 0.2-0.6 mg/mL and 0.5-2 mg/mL, respectively; the spraying amounts of the envelope antigens 2-NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA and 2- NPAMOZ-BSA are 0.8-1.2 uL/cm, respectively.
[11] Preferably, the concentration of the rabbit anti-mouse IgG is 0.08-0.12 mg/mL; the spraying amount of the rabbit anti-mouse IgG is 0.8-1.2 uL/cm.
[12] Preferably, the red light quantum dot-coupled 2-NPAOZ antibody, yellow light quantum dot-coupled 2-NPAHD antibody, green light quantum dot-coupled 2-NPSEM antibody, and orange light quantum dot-coupled 2-NPAMOZ antibody are applied in a volume ratio of (4-6): (4-6): (8-12): (4-6).
[13] Preferably, the kit further comprises Tween-20.
[14] Preferably, the sample pad overlaps with the nitrocellulose membrane by 1-2 mm, and the absorbent pad overlaps the nitrocellulose membrane by 1-2 mm.
[15] The present invention provides a preparation method of the kit, comprising preparation of the chromatographic test strip and preparation of the quantum dot probes;
[16] the preparation of the chromatographic test strip comprises the following steps:
[17] soaking the sample pad with a pretreatment solution, and drying to obtain a treated sample pad;
[18] overlapping and fixing the treated sample pad, the nitrocellulose membrane, and the absorbent pad on the PVC backing pad sequentially; and
[19] spray-coating the envelope antigens 2-NPAOZ-BSA, 2- NPAHD-BSA, 2-NPSEM-BSA, and 2-NPAMOZ-BSA on the test lines Tl, T2, T3, and T4, respectively; spray-coating the rabbit anti-mouse IgG on the control line C; and
[20] the preparation of the quantum dot probes comprises the following steps:
[21] diluting the red light CdSe/ZnS QDs, the yellow light CdSe/ZnS QDs, the green light CdSe/ZnS QDs, and the orange light CdSe/ZnS QDs with MES buffer to 0.08-0.12 mg/mL, respectively, to obtain a diluted red light CdSe/ZnS QDs solution, a yellow light CdSe/ZnS QDs solution, a green light CdSe/ZnS QDs solution, and an orange light CdSe/ZnS QDs solution;
[22] mixing the diluted red light CdSe/ZnS QDs solution, the diluted yellow light CdSe/ZnS QDs solution, the diluted green light CdSe/ZnS QDs solution, and the diluted orange light CdSe/ZnS QDs solution with EDC and NHS respectively for activation to obtain an activated red light CdSe/ZnS QDs solution, an activated yellow light CdSe/ZnS QDs solution, an activated green light CdSe/ZnS QDs solution, and an activated orange light CdSe/ZnS QDs solution; and
[23] mixing the activated red light CdSe/ZnS QDs solution, the activated yellow light CdSe/ZnS (QDs solution, the activated green light CdSe/ZnS (QDs solution, and the activated orange light CdSe/ZnS QDs solution with 2-NPAOZ antibody, 2-NPAHD antibody, 2-NPSEM antibody, and 2-NPAMOZ antibody respectively to obtain the red light quantum dot- coupled 2-NPA0Z antibody, the yellow light quantum dot- coupled 2-NPAHD antibody, the green light quantum dot- coupled 2-NPSEM antibody, and the orange light quantum dot- coupled Z-NPAMOZ antibody.
[24] Preferably, the pretreatment solution uses PBS as a solvent, and further comprises: sucrose with a mass percentage of 1.5%-2.5%, BSA with a mass percentage of 0.4%-
0.6%, and Tween-20 with a volume percentage of 0.04%-0.06%.
[25] The present invention provides a method for using the kit, comprising the following steps:
[26] mixing the target analyte solution, the quantum dot probes and Tween-20, incubating to obtain a reaction solution, and then dipping the sample pad of the chromatographic test strip into the reaction solution , and reading a result.
[27] The present invention provides the use of the kit in the detection of residues of nitrofurans.
[28] Beneficial effects of the present invention: The fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites provided 5 in this invention comprises a chromatographic test strip and quantum dot probes. The quantum dot probes use four kinds of quantum dots as markers to label four kinds of nitrofuran metabolite antibodies, respectively. By combining the advantages of quantum dots such as long fluorescence lifetime, high quantum yield and good light stability with immunochromatography, the sensitivity of detection is improved on the basis of the realization of simultaneous detection of four kinds of nitrofuran metabolites. According to the records of the embodiments, the detection lines of the four kinds of nitrofuran metabolites are as follows: the detection limit of AOZ (Tl) is 0.4 pg/mL; the detection limit of AHD (T2) is 0.1 pg/mL; the detection limit of SEM (T3) is 0.4 pg/mL; the detection limit of AMOZ (T4) is 0.5 ng/mL
[29] FIG. 1 shows the detection results of nitrofuran metabolites at different concentrations by using the fluorescence immunochromatographic kit; wherein,
[30] in the target analyte solution contacted by the test strip 0, the concentrations of AQZ, AHD, SEM, and AMOZ are all 0 ng/mL;
[31] in the target analyte solution contacted by the test strip 1, the concentration of AHD is 20 ng/mL, and the concentrations of AQZ, SEM, and AMOZ are all 50 ng/mL;
[32] in the target analyte solution contacted by the test strip 2, the concentration of AHD is 40 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 100 ng/mL;
[33] in the target analyte solution contacted by the test strip 3, the concentration of AHD is 60 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 200 ng/mL;
[34] in the target analyte solution contacted by the test strip 4, the concentration of AHD is 80 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 300 ng/mL;
[35] in the target analyte solution contacted by the test strip 5, the concentration of AHD is 100 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 400 ng/mL;
[36] in the target analyte solution contacted by the test strip 6, the concentration of AHD is 150 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 500 ng/mL;
[37] in the target analyte solution contacted by the test strip 7, the concentration of AHD is 200 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 600 ng/mL;
[38] FIG. 2 shows the non-specific detection results of the fluorescence immunochromatographic kit in the present invention; wherein,
[39] in the target analyte solution contacted by the test strip 1, AOQZ, AHD, SEM and AMOZ are all negative;
[40] in the target analyte solution contacted by the test strip 2, AOZ is positive, while AHD, SEM and AMOZ are all negative;
[41] in the target analyte solution contacted by the test strip 3, AHD is positive, while AOZ, SEM and AMOZ are all negative;
[42] in the target analyte solution contacted by the test strip 4, SEM is positive, while AOZ, AHD and AMOZ are all negative;
[43] in the target analyte solution contacted by the test strip 5, AMOZ is positive, while AOZ, AHD and SEM are all negative;
[44] in the target analyte solution contacted by the test strip 6, AQZ, AHD, SEM and AMOZ are all positive;
[45] FIG. 3 shows the detection results of furazolidone metabolites at different concentrations based on colloidal gold chromatography;
[46] FIG. 4 shows the detection results of furantoin metabolites at different concentrations based on colloidal gold chromatography;
[47] FIG. 5 shows the detection results of nitrofurazone
: metabolites at different concentrations based on colloidal gold chromatography;
[48] FIG. 6 shows the detection results of furaltadone metabolites at different concentrations based on colloidal gold chromatography;
[49] FIG. 7 is a schematic diagram of the detection principle of the chromatographic test strip in the fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites provided by the invention.
[50] A fluorescence immunochromatographic kit for simultaneous detection of four kinds of mnitrofuran metabolites is provided in this invention, comprising a chromatographic test strip and quantum dot probes. The chromatographic test strip comprises a PVC backing pad, and further comprises a sample pad, a nitrocellulose membrane and an absorbent pad which are sequentially overlapped and fixed on the PVC backing pad, the nitrocellulose membrane is provided with test lines Tl, T2, T3, and T4 and a control line C; the test lines T1, T2, T3, and T4 are spray-coated with envelope antigens Z-NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM- BSA, and 2-NPAMQZ-BSA, respectively; the control line C is spray-coated with rabbit anti-mouse IgG; and the quantum dot probes comprise red quantum dot-coupled 2-NPA0Z antibody, yellow quantum dot-coupled 2-NPAHD antibody, green quantum dot-coupled 2-NPSEM antibody, and orange quantum dot-coupled 2-NPAMOZ antibody.
[51] In the present invention, the chromatographic test strip comprises a sample pad, the sample pad is preferably made of glass fiber, and the sample pad is overlapped on the nitrocellulose membrane, with an overlap length preferably of 1-2 mm.
[52] In the present invention, the chromatographic test strip comprises a nitrocellulose membrane, the nitrocellulose membrane is provided with test lines Tl, T2, T3, T4, and a control line C; the test lines Tl, T2, T3, and T4 are spray-
coated with envelope antigens Z2-NPAOZ-BSA, 2-NPAHD-BSA, 2- NPSEM-BSA, and 2-NPAMOZ-BSA, respectively. In the present invention, the concentrations of the envelope antigens 2- NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA, and 2-NPAMOZ-BSA are preferably 0.1-0.5 mg/mL, 0.05-0.3 mg/mL, 0.2-0.6 mg/mL,
0.5-2 mg/mL, respectively; more preferably 0.2-0.4 mg/mL,
0.08-0.12 mg/mL, 0.3-0.5 mg/mL, 0.8-1.2 mg/mL; most preferably 0.3 mg/mL, 0.1 mg/mL, 0.4 mg/mL, 1 mg/mL. In the present invention, the spraying amounts of the envelope antigens (source: Shandong Landu Biotechnology Co., Ltd.) 2-NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA, and 2-NPAMOZ-BSA are preferably 0.8-1.2 uL/cm, respectively; more preferably 0.9-
1.1 uL/cm, most preferably 1.0 uL/cm. In the present invention, the control line C is spray-coated with rabbit anti-mouse IgG, the concentration of the rabbit anti-mouse IgG is preferably 0.08-0.12 mg/mL, more preferably 0.09-
0.11 mg/mL, most preferably 0.1 mg/mL; the spraying amount of the rabbit anti-mouse IgG is preferably 0.8-1.2 uL/cm, more preferably 0.9-1.1 uL/cm, most preferably 1.0 uL/cm.
[53] In the present invention, the chromatographic test strip comprises an absorbent pad, the absorbent pad is preferably a filter paper; and the absorbent pad is overlapped on the nitrocellulose membrane, with an overlap length preferably of 1-2 mm.
[54] In the present invention, the chromatographic test strip comprises a PVC backing pad, on which a sample pad, a nitrocellulose membrane and an absorbent pad are overlapped and fixed.
[55] In the present invention, the width of the chromatographic test strip is preferably 3.4-3.6cm, more preferably 3.5cm.
[56] In the present invention, the kit further comprises quantum dot probes; the quantum dot probes comprise the red light quantum dot-coupled 2-NPAQOZ antibody, the yellow light quantum dot-coupled 2-NPAHD antibody, the green light quantum dot-coupled 2-NPSEM antibody, and the orange light quantum dot-coupled 2-NPAMOZ antibody. In the present invention, the red light quantum dot-coupled 2-NPAOZ antibody, the yellow light quantum dot-coupled 2-NPAHD antibody, the green light quantum dot-coupled 2-NPSEM antibody, and the orange light quantum dot-coupled 2-NPAMOZ antibody are used in a volume ratio of {(4-6):{(4-6):{8- 12):{4-6)}), more preferably 5:5:10:5.
[57] In the present invention, the kit further comprises Tween-20, the function of Tween-20 is to increase the dispersion of labeled antibody in the chromatography system, and the chromatography speed, and to reduce the non- specificity. The present invention further provides a preparation method of the kit, comprising preparation of the chromatographic test strip and preparation of the quantum dot probes.
[58] In the present invention, the preparation of the chromatographic test strip comprises the following steps: soaking the sample pad with a pretreatment solution, and drying to obtain a treated sample pad; overlapping and fixing the treated sample pad, the nitrocellulose membrane, and the absorbent pad on the PVC backing pad sequentially; spray-coating the envelope antigens 2-NPAOZ-BSA, 2-NPAHD- BSA, 2-NPSEM-BSA, and 2-NPAMOZ-BSA on the test lines Tl, T2, T3, and T4, respectively; and spray-coating the rabbit anti- mouse IgG on the control line C.
[59] In the present invention, soaking the sample pad with a pretreatment solution and drying to obtain the treated sample pad. In the present invention, the pretreatment solution uses PBS as a solvent, and further comprises: sucrose with a mass percentage of 1.5%-2.5%, BSA with a mass percentage of 0.43-0,63, and Tween-20 with a volume percentage of 0.04%-0.06%; more preferably, the pretreatment solution uses PBS as a solvent, and further comprises: sucrose with a mass percentage of 2%, BSA with a mass percentage of 0.5%, and Tween-20 with a volume percentage of 0.05%. In the present invention, the soaking time is preferably 4-6 min, more preferably 5 min. The invention has no special restrictions on the drying method and time, and the conventional drying method and time in the art can be adopted.
[60] In the present invention, the treated sample pad, the nitrocellulose membrane and the absorbent pad are sequentially overlapped and fixed on the PVC backing pad. In the present invention, the treated sample pad, the nitrocellulose membrane and the absorbent pad may preferably be pasted on the PVC backing pad; the sample pad and the absorbent pad are overlapped on the nitrocellulose membrane, respectively, with an overlap length preferably of 1-2 mm.
[61] In the present invention, the test lines Tl, T2, T3, and T4 are spray-coated with envelope antigens 2-NPAQZ-BSA, 2- NPAHD-BSA, 2-NPSEM-BSA, and 2-NPAMOZ-BSA, respectively. In the present invention, the envelope antigens Z2-NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA, and Z-NPAMOZ-BSA and the rabbit anti-mouse IgG may preferably be spray-coated by a Biostrip Dispenser. In the present invention, the spray-coated chromatographic test strip may preferably be dried and then cut. In the present invention, the drying temperature is preferably 35-40°C, more preferably 37°C; the drying time is preferably 1.5-2.5 h, more preferably 2 h. The invention has no special limitations on the specific operation of the cutting, and the conventicnal cutting method in the art can be adopted.
[62] In the present invention, the preparation of the quantum dot probes comprises the following steps:
[63] diluting the red light CdSe/ZnS QDs, the yellow light CdSe/ZnS QDs, the green light CdSe/ZnS QDs, and the orange light CdSe/ZnS QDs with MES buffer to 0.08-0.12 mg/mL, respectively, to obtain diluted red light CdSe/ZnS QDs, yellow light CdSe/ZnS QDs, green light CdSe/ZnS QDs, and orange light CdSe/ZnS QDs solutions; In the present invention, the concentrations of the red light CdSe/ZnS QDs solution, the yellow light CdSe/ZnS QDs solution, the green light CdSe/ZnS QDs solution, and the orange light CdSe/ZnS QDs solution are preferably 8-12 mg/mL, respectively, more preferably 10 mg/mL. The red light CdSe/ZnS QDs, the yellow light CdSe/ZnS QDs, green light CdSe/ZnS QDs, and orange light CdSe/ZnS QDs are preferably purchased from Shanghai Kundao Biotechnology Co., Ltd. In the present invention, the red light CdSe/ZnS (QDs, yellow light CdSe/ZnS QDs, green light CdSe/ZnS QDs, and orange light CdSe/ZnS QDs are respectively mixed with MES buffer for dilution. In the present invention, the dilution may preferably further comprise ultrasonic treatment, the power of the ultrasonic treatment is preferably 25-35 kHz, more preferably 30 kHz; the time of the ultrasonic treatment is preferably 0.5-1.5 min, and more preferably 1 min.
[64] In the present invention, the diluted red light CdSe/ZnS QDs, yellow light CdSe/ZnS QDs, green light CdSe/ZnS QDs, and orange light CdSe/ZnS QDs solutions are respectively mixed with EDC and NHS for activation to obtain an activated red light CdSe/ZnS QDs solution, an activated yellow light CdSe/ZnS QDs solution, an activated green light CdSe/ZnS QDs solution, and an activated orange light CdSe/ZnS QDs solution. In the present invention, the volume ratios of the diluted red CdSe/ZnS QDS solution, the diluted yellow CdSe/ZnS ODS solution, the diluted green CdSe/ZnS QDS solution and the diluted orange CdSe/ZnS QDS solution to EDC and NHS are preferably (48-52):1:1, respectively; more preferably 50:1:1. In the present invention, the concentration of EDC is preferably 0.8-1.2 mg/mL, more preferably 1 mg/mL; the concentration of NHS is preferably
1.5-2.5 mg/mL, more preferably 2 mg/mL. In the present invention, the activation is preferably carried out on a shaker, the rotational speed of the activation is preferably 180-220rpm, more preferably 200rpm; the activation time is preferably 25-35 min, more preferably 30 min; the activation is preferably carried out in the dark.
[65] In the present invention, the activated red light CdSe/ZnS QDs is coupled with 2-NPAOZ antibody to obtain red light quantum dot-coupled 2-NPA0Z antibody; the activated yellow light CdSe/ZnS QDs is coupled with 2-NPAHD antibody to obtain yellow light quantum dot-coupled 2-NPAHD; the activated green light CdSe/ZnS QDs is coupled with 2-NPSEM antibody to obtain green light quantum dot-coupled 2-NPSEM, and the activated orange light CdSe/ZnS QDs is coupled with 2-NPAMQZ antibody to obtain orange light quantum dot-coupled 2-NPAMOZ antibody. In the present invention, the volume ratio of the 2-NPA0Z antibody to the original red light CdSe/ZnS QDs is preferably (1-4):1; the volume ratio of the 2-NPAHD antibody to the original yellow light CdSe/ZnS QDs is preferably (1-4):1; the volume ratio of the 2-NPSEM antibody to the original green light CdSe/ZnS QDs is preferably (1-4):1; the volume ratio of the 2-NPAMOZ antibody to the original orange light CdSe/ZnS QDs is preferably (1-4):1. In the present invention, the coupling is preferably carried out under shaking, the temperature of coupling is preferably 20-25°C, the time of coupling is preferably 1.5-2.5 h, more preferably 2 h; the coupling is preferably carried out on a shaker. In the present invention, after the coupling, the product is preferably blocked with a BSA solution and then stored; the mass percentage of the BSA solution is preferably 0.8-1.2%, more preferably 14; the time of blocking is preferably 1 h, the temperature of blocking is preferably 3-5°C, more preferably 4°C. In the present invention, the 2-NPAOZ antibody, 2-NPAHD antibody, 2-NPSEM antibody, and 2-NPAMOZ antibody are preferably purchased from Shandong Landu Biotechnology Co., Ltd.
[66] In the present invention, the red light quantum dot- coupled 2-NPA0Z antibody, the yellow light quantum dot- coupled 2-NPAHD antibody, the green light quantum dot- coupled 2-NPSEM antibody, and the orange light quantum dot- coupled Z-NPAMOZ antibody are used in a volume ratio of (4- 6): (4-6) :(8-12):(4-¢), more preferably 5:5:10:5.
[67] In the present invention, the prepared chromatographic test strip and quantum dot probes are assembled to obtain the kit. In the present invention, there is no special limitation to the packaging materials and standards of the chromatographic test strip and the quantum dot probes. The conventional packaging materials for chromatographic test strip and the quantum dot probes in the art can be used. In the present invention, Tween-20 may preferably be individually packaged and assembled into the kit.
[68] The present invention further provides a method for using the kit, comprising the following steps: mixing the target analyte solution, the quantum dot probes and Tween- 20, incubating the mixture to obtain a reaction solution, and then contacting the reaction solution with the sample pad on the chromatographic test strip, and reading a result under an ultraviolet lamp.
[69] In the present invention, the target analyte solution, the quantum dot probes and Tween-20 are mixed and incubated to obtain a reaction solution. The volume ratio of the target analyte solution, the quantum dot probes and Tween-20 is preferably 100:25:10. The temperature of incubating is preferably 36-38°C, more preferably 37°C; the time of incubating is preferably 10-20 min, more preferably 15 min.
In the present invention, the mixing and incubating may be preferably carried out in a 96-well reaction vessel. In the present invention, the preparation method of the target analyte solution refers to the sample treatment method in the national standard/T 21311-2007.
[70] In the present invention, after mixing and incubating, a reaction solution is obtained, and then the reaction solution is contacted with the sample pad on the chromatographic test strip, and a result is read with an ultraviolet lamp. In the present invention, the chromatographic test strip is preferably inserted into the sample hole for chromatography and then the result is read with an ultraviolet lamp, the time of the chromatography is preferably 25-35 min, more preferably 30 min.
[71] In the present invention, when the test line Tl, T2, T3 or T4 shows a red, yellow, green or orange fluorescence band, it indicates that the detection sample does not contain a corresponding nitrofuran metabolite or the content of the corresponding nitrofuran metabolite is below the detection limit. When the fluorescence of the test line TI, T2, T3 or T4 disappears completely, it indicates that the content of a corresponding nitrofuran metabolite in the sample to be detected is higher than the detection limit; the weaker the fluorescence on the T line, the higher the corresponding nitrofuran metabolite residue in the detection sample.
[72] The invention further provides the use of the kit in detection of residues of nitrofurans. In the present disclosure, there is no special limitation to the type of sample to be tested, any sample that may contain residual nitrofurans may be adopted, for example, food.
[73] The technical solutions provided by the present invention will be described in detail below in combination with examples. However, these examples should not be construed as limiting the claimed scope of the present disclosure.
[74] Example 1
[75] Preparation of a chromatographic test strip:
[76] A sample pad was soaked in a pretreatment solution (using PBS as the solvent, containing sucrose with a mass percentage of 2%, BSA with a mass percentage of 0.5% and Tween-20 with a volume percentage of 0.05%) for 5 min and then dried in an oven at 37°C for 2 h. The dried sample pad, a nitrocellulose membrane, and a water absorbent pad were successively pasted on a PVC backing pad, where the sample pad and the conjugate pad overlapped with the nitrocellulose membrane by a length of 1.5 mm, respectively. Four kinds of envelope antigens 2-NPAQZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA, and 2-NPAMOZ-BSA and rabbit anti-mouse IgG (all purchased from Shandong Landu Biotechnology Co., Ltd.) were fixed on the nitrocellulose membrane in a unit spray amount of 1 uL/cm using a Biostrip Dispenser and marked as test lines Tl, T2, T3, and T4 and a control line C. The concentration of the antibody spraying on the test line was 0.3 mg/mL, 0.1 mg/mL, 0.4 mg/mL, and 1 mg/mL, respectively, and the concentration of the control line C was 0.1 mg/mL. The prepared test strip was dried in an oven at 37°C for 2 h. Then the test strip was cut into 3.5 cm-wide test strips for later use.
[77] Preparation of quantum dot probes:
[78] 5 pL of 10 mg/mL red light CdSe/ZnS QDs was diluted with 495 uL of MES buffer, and then was thoroughly mixed and subjected to ultrasonic treatment at 30 kHz for 1 min. Then uL of freshly prepared EDC (1 mg/mL) and 10 pL of freshly 10 prepared NHS (2 mg/mL) were added separately for activation, and reacted in the dark for 30 min on a shaker at 200 rpm. An activated quantum dot solution was subjected to ultrasonic treatment for 1 min, then 10 pL of 1 mg/mL 2- NPAOZ antibody (purchased from Shandong Landu Biotechnology Co., Ltd.) was added for coupling, and the resulting mixture was shaken on a shaker at 25°C for 2 h, and 20 pL of 1% BSA was added for blocking for 1 h, and the resulting mixture was stored in a refrigerator at 4°C for further use.
[79] The four kinds of quantum dot fluorescent probes were synthesized in the same way, and the difference was that the 2-NPAOZ antibody was labeled by red light CdSe/ZnS QDs, the 2-NPAHD antibody was labeled by yellow light CdSe/ZnS QDs, the 2-NPSEM antibody was labeled by green light CdSe/ZnS QDs, and the 2-NPAMOZ antibody was labeled by orange light CdSe/ZnS QDs.
[80] 100 pL of target analyte solution, 5 ulL of red light quantum dot-coupled 2-NPAQZ antibody, 5 uL of yellow quantum dot-coupled 2-NPAHD antibody, 10 uL of green quantum dot- coupled 2-NPSEM antibody, 5 ul of orange quantum dot-coupled 2-NPAMOZ antibody, and 10 |L of Tween-20 were mixed, and the resulting mixture was thoroughly shaken and incubated at 37°C for 15 min; and then the test strip was inserted into a sample hole to perform chromatography for 30 min, and the result was read using a UV lamp. |81] Determination of results
[82] Negative (-): when the test line Tl, T2, T3 or T4 shows a red, yellow, green or orange fluorescence band, it indicates that the amount of the corresponding residual nitrofuran metabolite in the sample is below the detection limit or the sample does not contain the corresponding nitrofuran metabolite residue.
[83] Positive (+): if the fluorescence on the test line Tl, T2, T3 or T4 disappears completely, it indicates that the residual amount of the corresponding nitrofuran metabolite in the sample is higher than the detection limit. The weaker the fluorescence at the T-line is, the higher the content of the corresponding residual nitrofuran metabolite in the sample is.
[84] Table 1 Determination of results N= = TT = 1 = — Lt LL be pe pee pe ms | [sr etortess [Green | ALL [eee
[85] Eight test strips were used to detect 8 kinds of target analyte solutions, respectively, and the results are shown in FIG. 1, wherein in the target analyte solution contacted by the test strip 0, the concentrations of AOZ (3-amino-2- oxazolidinone), AHD ({1- aminohydantoin), SEM (semicarbazide), and AMOZ (3-amino-5- morpholinomethyl-2-oxazolidinone) are all 0 ng/mL.
[86] In the target analyte solution contacted by the test strip 1, the concentration of AHD is 20 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 50 ng/mL.
[87] In the target analyte solution contacted by the test strip 2, the concentration of AHD is 40 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 100 ng/mL.
[88] In the target analyte solution contacted by the test strip 3, the concentration of AHD is 60 ng/mL, and the concentrations of AQZ, SEM, and AMOZ are all 200 ng/mL.
[89] In the target analyte solution contacted by the test strip 4, the concentration of AHD is 80 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 300 ng/mL.
[90] In the target analyte solution contacted by the test strip 5, the concentration of AHD is 100 ng/mL, and the concentrations of AOZ, SEM, and AMOZ were all 400 ng/mL;
[91] In the target analyte solution contacted by the test strip 6, the concentration of AHD is 150 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 500 ng/mL.
[92] In the target analyte solution contacted by the test strip 7, the concentration of AHD is 200 ng/mL, and the concentrations of AOZ, SEM, and AMOZ are all 600 ng/mL.
[93] Test results are as follows: the detection limit of AOZ (Tl) is 0.4 pg/mL; the detection limit of AHD (T2) is 0.1 ng/mL; the detection limit of SEM (T3) is 0.4 pg/mL; and the detection limit of AMOZ (T4) is 0.5 pg/mL.
[94] Specificity test:
[95] A specificity test method was conducted as follows: 5 HL of red light quantum dot-coupled 2-NPAQZ antibody, 5 nL of yellow light quantum dot-coupled 2-NPAHD antibody, 10 uL of green light quantum dot-coupled 2-NPSEM antibody, 5 uL of orange quantum dot-coupled 2-NPAMOZ antibody, and 10 uL of Tween-20 were added to the target analyte solution without A0Z, AHD, SEM, and AMOZ, to the solutions containing any one of AOZ, AHD, SEM, and AMOZ, and to the target analyte solution containing AQZ, AHD, SEM, and AMOZ, respectively. The resulting mixtures were thoroughly shaken and incubated at 37°C for 15 min, then the test strip was inserted into a sample hole to conduct chromatography for 30 min, and results were read using a UV lamp.
[96] Test results are shown in Table 2 and FIG. 2.
[97] In the target analyte solution contacted by the test strip 1, AOZ, AHD, SEM and AMOZ are all negative, thus the test lines on the test strip show a red, yellow, green and orange fluorescent band, respectively.
[988] In the target analyte solution contacted by the test strip 2, AOZ is positive, while AHD, SEM and AMOZ are all negative, thus the fluorescent band on the test line corresponding to AOZ disappears, other test lines show a vellow, green and orange fluorescent band, respectively.
[99] In the target analyte solution contacted by the test strip 3, AHD is positive, while AOZ, SEM and AMOZ are all negative, thus the fluorescent band on the test line corresponding to AHD disappears, other test lines show a red, green and orange fluorescent band, respectively.
[100] In the target analyte solution contacted by the test strip 4, SEM is positive, while AOZ, AHD and AMOZ are all negative, thus the fluorescent band on the test line corresponding to SEM disappears, other test lines show a red, yellow and orange fluorescent band, respectively;
[101] In the target analyte solution contacted by the test strip 5, AMOZ is positive, while A0OZ, AHD and SEM are all negative, thus the fluorescent band on the test line corresponding to AMOZ disappears, other test lines show a red, yellow and green flucrescent band, respectively.
[102] In the target analyte solution in contacted by the test strip 6, AOZ, AHD, SEM and AMOZ are all positive, thus all fluorescent bands on four test lines on the test strip disappear.
[103] Test results show that there is no cross-reaction among four kinds of target ananlyte, indicating that the test strip has good specificity.
[104] Table 2 Test results for non-specificity | : 6 omoz | - + |= | - 0 - camp |= = Low foe sm | = oo] + | mon | - | - | - | - : (+ means the presence of a target, and - means the absence of a target)
[105] 8 colloidal gold test strips (purchased from Shenzhen Baoankang Biotechnology Co., Ltd.) were used to test the furazolidone metabolite solutions at different concentrations, and results are shown in FIG. 3. The test strips were numbered as 0 to 7 sequentially from left to right;
[106] wherein, in the target analyte solution contacted by the test strip 0, the concentration of AOZ was 0 ng/mL;
[107] in the target analyte solution contacted by the test strip 1, the concentration of AOZ was 0.005 ng/mL;
[108] in the target analyte solution contacted by the test strip 2, the concentration of AOZ was 0.01 ng/mL;
[109] in the target analyte solution contacted by the test strip 3, the concentration of AOZ was 0.05 ng/mL;
[110] in the target analyte solution contacted by the test strip 4, the concentration of AOZ was 0.1 ug/mL;
[111] in the target analyte solution contacted by the test strip 5, the concentration of AOZ was 0.5 pg/mL;
[112] in the target analyte solution contacted by the test strip 6, the concentration of A0Z was 1 pg/mL;
[113] in the target analyte solution contacted by the test strip 7, the concentration of A0Z was 10 pg/mL.
[114] 8 colloidal gold test strips (purchased from Shenzhen
Baoankang Biotechnology Co., Ltd.) were used to test the furantoin metabolite solutions at different concentrations, and results are shown in FIG. 4. The test strips were numbered as 0 to 7 sequentially from left to right;
[115] wherein, in the target analyte solution contacted by the test strip 0, the concentration of AHD was 0 pg/mL;
[116] in the target analyte solution contacted by the test strip 1, the concentration of AHD was 0.005 pg/mL;
[117] in the target analyte solution contacted by the test strip 2, the concentration of AHD was 0.01 ug/mL;
[118] in the target analyte solution contacted by the test strip 3, the concentration of AHD was 0.05 ug/mL;
[119] in the target analyte solution contacted by the test strip 4, the concentration of AHD was 0.1 pg/mL;
[120] in the target analyte solution contacted by the test strip 5, the concentration of AHD was 0.5 ug/mL;
[121] in the target analyte solution contacted by the test strip 6, the concentration of AHD was 1 pg/mL;
[122] in the target analyte solution contacted by the test strip 7, the concentration of AHD was 10 ug/mL;
[123] 8 colloidal gold test strips (purchased from Shenzhen Baoankang Biotechnology Co., Ltd.) were used to test the nitrofurazone metabolite solutions at different concentrations, and results are shown in FIG. 5. The test strips were numbered as 0 to 7 sequentially from left to right;
[124] wherein, in the target analyte solution contacted by the test strip 0, the concentration of SEM was 0 ug/mL;
[125] in the target analyte solution contacted by the test strip 1, the concentration of SEM was 0.005 pg/mL;
[126] in the target analyte solution contacted by the test strip 2, the concentration of SEM was 0.01 ng/mL;
[127] in the target analyte solution contacted by the test strip 3, the concentration of SEM was 0.05 pg/mL;
[128] in the target analyte solution contacted by the test strip 4, the concentration of SEM was 0.1 pg/mL;
[129] in the target analyte solution contacted by the test strip 5, the concentration of SEM was 0.5 ug/mL;
[130] in the target analyte solution contacted by the test strip 6, the concentration of SEM was 1 pg/mL;
[131] in the target analyte solution contacted by the test strip 7, the concentration of SEM was 10 ug/mL;
[132] 8 colloidal gold test strips (purchased from Shenzhen Baoankang Biotechnology Co., Ltd.) were used to test the furaltadone metabolite solutions with different concentrations, and results are shown in FIG. 6. The test strips were numbered as 0 to 7 sequentially from left to right;
[133] wherein, in the target analyte solution contacted by the test strip 0, the concentration of AMOZ was 0 ng/mL;
[134] in the target analyte solution contacted by the test strip 1, the concentration of AMOZ was 0.005 ug/mL;
[135] in the target analyte solution contacted by the test strip 2, the concentration of AMOZ was 0.01 pg/mL;
[136] in the target analyte solution contacted by the test strip 3, the concentration of AMOZ was 0.05 pg/mL;
[137] in the target analyte solution contacted by the test strip 4, the concentration of AMOZ was 0.1 ng/mL;
[138] in the target analyte solution contacted by the test strip 5, the concentration of AMOZ was 0.5 ug/mL;
[139] in the target analyte solution contacted by the test strip 6, the concentration of AMOZ was 1 ug/mL;
[140] in the target analyte solution contacted by the test strip 7, the concentration of AMOZ was 10 ug/mL.
[141] Test results are as follows: the detection limit of AOZ is 10 ng/mL; the detection limit of AHD is 10 ug9/mL; the detection limit of SEM is 10 pg/mL; and the detection limit of AMOZ is 1 ug/mL. The sensitivity of the colloidal gold test strip is far lower than that of the test strip in the present invention.
[142] It can be known from the above examples that the fluorescence immunochromatographic kit for simultaneous detection of four kinds of nitrofuran metabolites provided by the present invention can realize the simultaneous detection of four kinds of nitrofuran metabolites, with improved sensitivity.
[143] The above descriptions are merely preferred implementations of the present invention. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present invention.
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| CN109239328A (en) * | 2018-09-26 | 2019-01-18 | 南京财经大学 | A kind of immunochromatographic method of four kinds of metabolites of nitrofuran of quick detection |
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