METHOD FOR CONSTRUCTING CHROMATOGRAPHY TEST STRIP FOR TRIAZOPHOS BASED ON MOLECULAR IMPRINTING AND ELECTROSPINNING

Abstract

The present invention relates to a method for constructing a chromatography test strip for triazophos based on molecular imprinting and electrospinning. The present invention combines electrospinning, molecular imprinting and the immunochromatography test strip technology. Molecularly imprinted T-line (detection limit) is prepared on an NC membrane by electrospinning, and goat anti-mouse IgG is used as C-line (quality control line). With fluorescence changes occurring when triazophos hapten-murine IgG/fluorescein isothiocyanate conjugate (THBu-IgG-FITC) fluorescent probe directly competes with the target triazophos to bind to the molecularly imprinted binding site, a chromatography-fluorescence detection method for triazophos based on molecular imprinting and electrospinning is established. The functional material adsorbing triazophos provided by the present invention adopts a virtual template to avoid template leakage, and can be used in immunochromatography to replace a biological antibody. The functional material has higher selectivity, higher stability, longer service life, and stronger resistance to adverse environment.

Claims

1. (canceled)

2. A method for constructing a chromatography test strip for triazophos comprising the steps of: constructing a chromatography test strip, comprising: treating a sample pad with a sample pad treatment solution; drying the sample pad; cutting the sample pad into strips; drawing a secondary antibody on a nitrocellulose (NC) membrane at a predetermined flow rate by a scriber; drying the NC membrane at a first temperature; assembling the test strip, comprising: cutting the NC membrane along a line a prescribed dimension below a quality control line (C-line); placing an aluminum foil strip between the cut NC membranes including an upper NC membrane and a lower NC membrane; pasting the upper NC membrane, the lower NC membrane, and the middle aluminum foil on to a fluorescent board, with a test line (T-line) being separated by a prescribed distance from each other; and pasting an absorbent pad and the sample pad on upper NC membrane and the lower NC membrane, respectively, with each of the absorbent pad and the sample pad overlapping the NC membrane by a prescribed overlap distance; preparing, by electrospinning, a molecularly imprinted T-line on the NC membranes, comprising: preparing an electrospinning solution, comprising: preparing a cellulose acetate (CA) electrospinning matrix solution, comprising: adding a weighted measure of a CA powder to acetone for CA-acetone solution; agitating the CA-acetone solution at a second prescribed temperature in a water bath for a set duration, the CA being dissolved in the CA-acetone solution; and preparing a molecularly imprinted polymer (MIP) dispersion solution for triazolone, comprising: adding a weighted measure of MIP to acetone; subjecting the MIP dispersion solution to ultrasonic dispersion at a third prescribed temperature, the MIP being dissolved and evenly dispersed in the acetone; and mixing a prescribed volume of the CA electrospinning matrix solution with the MIP dispersion solution and being agitated in a water bath a third prescribed temperature to yield the electrospinning solution; drawing the electrospinning solution into a syringe of an electrospinning device; placing the assembled test strip on to a receiving plate of the electrospinning device; and clamping a first end of the T-line to a negative electrode, molecularly imprinted nanofibers evenly covering the T-line without covering other non-conductive parts of the test strip.

3. The method of claim 2, wherein the sample pad treatment solution is a 0.5% polysorbate surfactant buffer.

4. The method of claim 2, wherein the predetermined flow rate for drawing the secondary antibody on the NC membrane is 1 μL/cm.

5. The method of claim 2, wherein the secondary antibody is a goat anti-mouse IgG.

6. The method of claim 2, wherein the first temperature is 37° C.

7. The method of claim 2, wherein the prescribed dimension below the quality control line is 5 mm.

8. The method of claim 2, wherein the aluminum foil strip is 1 mm in width.

9. The method of claim 2, wherein the prescribed distance between the C-line and the T-line is 5 mm.

10. The method of claim 2, wherein the prescribed overlap distance is 1 mm.

11. The method of claim 2, wherein the CA-acetone solution has a 120 mg/ml CA concentration.

12. The method of claim 2, wherein the second prescribed temperature is 50° C.

13. The method of claim 2, wherein the MIP dispersion solution has a 20 mg/ml MIP concentration.

14. The method of claim 2, wherein mixing the prescribed volume of the CA electrospinning matrix solution with the MIP dispersion solution includes: adding 111 μL of the MIP dispersion to 1 ml of the CA electrospinning matrix solution; and adding 7 μL of a 10% polysorbate surfactant buffer.

15. The method of claim 2, wherein the electrospinning solution is subject to ultrasonic dispersion for a predetermined duration at room temperature.

16. The method of claim 2, wherein the electrospinning device includes an automatic microflow pump, the syringe, a height-adjusting frame, the jet needle (22 G), the receiving plate, and a high-voltage power supply. Before spinning, the grounding is checked, and the temperature and humidity are recorded;

17. The method of claim 16, further comprising: adjusting the distance between the jet needle and a receiving plate of the electrospinning device to 13 cm; adjusting the flow rate of the microflow pump to 12 μL/min; and adjusting the high-voltage power supply to 12.0 kV.

18. The method of claim 2, further comprising: drying the test strips upon the molecularly imprinted nanofibers covering the T-line; and cutting the test strip into a plurality of smaller strips.

19. The method of claim 18, wherein the smaller strips have a width dimension of 3.5 mm.

20. The method of claim 18, further comprising storing the smaller strips in a dessicator at room temperature.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is a structure diagram of molecularly imprinted electrospinning test strip (a) and a flow chart of directly-competitive fluorescence detection (b); and

[0026] FIG. 2 is a standard curve of a test strip for triazophos.

DETAILED DESCRIPTION

[0027] To enable a person skilled in the art to better understand the present invention, the technical solutions of the present invention is further described below with reference to the accompanying drawings and examples.

[0028] 1. Synthesis of Hapten

(1) Synthesis of O-ethyl dichlorothiophosphate (TZM-1): 68 g (about 0.4 mol) of thiophosphoryl chloride (PSCl3) was weighed and added to a three-necked flask with a low-temperature thermometer, and the liquid was cooled to −10° C. to −5° C. in an ice-brine bath. 55 g (about 1.2 mol) of absolute ethyl alcohol was added dropwise with vigorous stirring at a rate that was strictly controlled so that the temperature of the reaction solution was always not higher than 0° C. After the dropwise addition was completed, the reaction was continued at 10° C. for 2 h. After the reaction was completed, the reaction solution was washed with (0±5)° C. distilled water (100 ml×2). The oil phase was separated, dried over anhydrous Na2SO4, and then distilled under reduced pressure with a water aspirator. Fraction at 65° C. to 75° C. was collected to obtain a colorless, transparent and oily liquid (51.8 g; yield 72.3%, calculated based on thiophosphoryl chloride).
(2) Synthesis of O-ethyl-O-[3-(1-phenyl-1, 2, 4-triazolyl)chlorothiophosphate (TZM-2): 36 g (about 0.2 mol) of TZM-1 was weighed and added to a 250 ml three-necked flask. About 16 g (about 0.1 mol) of 1-phenyl-1, 2, 4-triadimenol was added with stirring, and then about 15 ml of TEA and 80 ml of DCM were added. After all solids were dissolved, the resulting solution was cooled to a temperature lower than 20° C. in an ice water bath. Then a trace amount of catalyst was added, and 55 ml of a 2 mol/L NaOH aqueous solution was added dropwise. The reaction continued for 1 h. After the reaction was completed, 50 ml of 5% NaOH iced aqueous solution was added. The resulting solution was shaken, and the water phase was removed. The oil phase was washed with ice water to neutrality, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a small amount of brown oily substance. Petroleum ether (50 ml×2) was added to the oily substance for extraction, and the extract was concentrated under reduced pressure to obtain a yellow liquid (10.6 g; yield 35%, calculated based on triadimenol).
(3) Synthesis of triazophos hapten: 1.03 g (about 10 mmol) of 4-aminobutyric acid was weighed and dissolved in 10 ml of a NaOH solution (1 mol/L), and the resulting solution was cooled to 0° C. to 10° C. in an ice water bath. 1.51 g (about 5 mmol) of TZM-2 dissolved in 10 ml of dioxane was slowly added with stirring, a trace amount of catalyst was added, and 10 ml of a NaOH aqueous solution (1 mol/L) was added dropwise. The solution was warmed to 15° C. to 25° C. for 4 h of reaction. After the reaction was completed, 50 ml of water was added, and the reaction solution was washed with petroleum ether (40 ml×2), and the petroleum ether phase was removed. pH of the water phase was adjusted to about 3 with 2 mol/L HCL, and ethyl acetate (40 ml×2) was added for extraction. The extract was washed with a small amount of water, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was sealed and stored overnight at 4° C., and a colorless product was precipitated. The precipitate was recrystallized with an ethyl acetate-petroleum ether system, filtered out, and dried to obtain 0.52 g of a white solid (THBu, yield 27%, calculated based on intermediate TZM-2).

[0029] 2. Preparation of THBu-IgG-FITC fluorescent probe

(1) 9.43 mg of triazophos hapten (0.025 mmol) was weighed and dissolved in 0.5 ml of DMF.
(2) 8.63 mg of NHS (0.075 mmol) was weighed and added to the solution prepared in step 1, and the resulting mixture was stirred at room temperature for 15 min.
(3) 7.73 mg of DCC (0.0375 mmol) was weighed and dissolved in 0.5 ml of DMF, and the obtained solution was added to the solution prepared in step 2 dropwise. The resulting mixture was stirred overnight at room temperature and then centrifuged at 4,000 rpm/min for 10 min.
(4) 200 μL of the supernatant in step 3 was pipetted and slowly added to 1 ml of CBS solution (0.01 mol/L) in which 10 mg of mouse IgG was dissolved, and the resulting solution was stirred at 20° C. for 4 h.
(5) 2.95 mg of FITC was weighed and dissolved in 2.95 ml of CBS (0.05 mol/L, pH=9.6), the obtained solution was added to the reaction solution in step 4 dropwise in the dark. Then the reaction solution was slowly stirred at 4° C. for 8 h in the dark.
(6) The synthesized THBu-IgG-FITC fluorescent probe was dialyzed in a 0.01 mol/L PBS (pH=7.4) solution at 4° C. until the dialysate was clear, and stored at 4° C. The fluorescent probe is not suitable for long-term storage, and should be used as soon as possible.

[0030] 3. Preparation of molecularly imprinted microspheres: 29.4 mg (0.1 mmol) of triazolone (template) was weighed and added to a 100 ml round-bottom flask, and 20 ml of acetonitrile (pore-forming agent) was added to dissolve the template. Then 51 μL (0.6 mmol) of MAA (functional monomer) was added, and the mixture was shaken at room temperature for 30 min of prepolymerization. 319.3 μL (1.0 mmol) of TRIM (crosslinking agent) and 30 mg of AIBN (initiator) were then added, and the tube was sealed immediately after 2 min of nitrogen charge. The polymerization reaction was conducted in a 60° C. water bath for 24 h. After the polymerization was completed, the reaction solution was taken out and centrifuged, and the supernatant was removed. Then the resulting precipitate was dispersed in methanol and then centrifuged to remove the unreacted reactant. The obtained polymer was wrapped with a filter paper, and placed in a Soxhlet extractor for extracting the template with a methanol:acetic acid (9:1, v/v) solution.

[0031] 4. Construction of chromatography test strip: A sample pad was treated with a sample pad treatment solution (0.5% Tween-0.02 M pH 7.2 PB buffer), then dried, and cut into strips. A secondary antibody (goat anti-mouse IgG) was drawn on an NC membrane at a flow rate of 1 μL/cm by a scriber and dried at 37° C. Then a test strip was assembled as follows: as T-line needed to be spun on an aluminum foil (NC membrane is non-conductive), the NC membrane was cut along a line 5 mm below C-line, and an aluminum foil of 1 mm width was placed between the obtained two NC membranes; the upper and lower NC membranes and the middle aluminum foil were pasted on a black fluorescent board, with T-line and C-line being 5 mm apart from each other; and then an absorbent pad and the sample pad were pasted on the upper and lower sides of the NC membrane respectively, with each pad overlapping with the NC membrane by 1 mm. The assembled test strip is shown in FIG. 1 (a).

[0032] 5. Preparation of molecularly imprinted T-line on an NC membrane by electrospinning

(1) Preparation of an electrospinning solution Preparation of a CA electrospinning matrix solution: A certain amount of CA powder was weighed and added to acetone for preparing a 120 mg/ml CA-acetone solution, and the obtained solution was shaken at 50° C. in a water bath for 5 h until CA was completely dissolved. Preparation of an MIP dispersion solution for triazolone: A certain amount of MIPs was weighed and added to acetone for preparing a 20 mg/ml MIP dispersion solution, and the obtained solution was subjected to ultrasonic dispersion at room temperature for 50 min until MIPs were completely and evenly dispersed in acetone. Mixing of the CA electrospinning matrix solution with the MIP dispersion solution: 111 μL of 20 mg/ml MIP dispersion solution was added to 1 ml of 120 mg/ml CA electrospinning matrix solution, then 7 μL of 10% Tween solution was added, and the resulting solution was shaken in a 50° C. water bath for 120 min and subjected to ultrasonic dispersion for 30 min at room temperature to obtain an uniform MIP electrospinning solution for triazolone.
(2) Preparation of molecularly imprinted T-line on an NC membrane by electrospinning: An electrospinning device made in laboratory, with an automatic microflow pump, a 5 ml syringe, a height-adjusting frame, a jet needle (22 G), a receiving plate, and a high-voltage power supply, was adopted. Before spinning, the grounding was checked, and the temperature and humidity were recorded. The prepared MIP electrospinning solution was drawn into the syringe, the distance between the jet needle and the receiving plate was adjusted to 13 cm, and the flow rate of the microflow pump was set as 12 μL/min, and the high voltage as 12.0 kV. After the fiber extrusion was stable, the assembled test strip was placed on the receiving plate (ensuring that it was placed at the same position each time), and one end of the T-line aluminum foil was clamped with a negative electrode. 20 min later, molecularly imprinted nanofibers evenly covered T-line without covering other parts of the test strip that are not conductive. The obtained test strip was dried in an oven at 37° C., then cut into smaller strips having a width of 3.5 mm by a slitter, and stored in a desiccator at room temperature. The molecularly imprinted test strips were obtained.

[0033] 6. Experimental principle: A molecularly imprinted polymer, instead of an artificial antibody, is fixed on an NC membrane as T-line by electrospinning, and a secondary antibody is fixed as C-line by a scriber. As shown in FIG. 1 (b), when the target and the THBu-IgG-FITC fluorescent probe are added dropwise to the sample pad, the solution moves on the NC membrane by capillary action. Both the target triazophos and the triazophos hapten on the THBu-IgG-FITC probe can bind to the molecularly imprinted polymer on T-line, and IgG on the probe can bind to the secondary antibody on C-line. When moving to T-line, the target and the fluorescent probe compete to bind to the specific binding site on the molecularly imprinted polymer, causing the fluorescence intensity on T-line to be inversely proportional to the concentration of the target, and as the remaining target and probe continue to move to C-line, the IgG on the probe binds to the secondary antibody to achieve the quality control. A fluorescence immunoassay analyzer (wavelength for excitation: 450 nm to 470 nm, wavelength for receiving: 525 nm) is used to read the fluorescence values of C-line and T-line, and a qualitative and quantitative assay is performed according to the fluorescence intensity at T-line and the T/C value.

[0034] 7. Experimental process

(1) Preparation of test strips: Molecularly imprinted test strips were assembled according to step 4 and 5, and blocked with a blocking buffer (0.25% PVP+0.25% BSA+5% sucrose), dried at 37° C., and stored in a desiccator at room temperature.
(2) Competitive reaction: 100 μL of 10-fold-diluted THBu-IgG-FITC fluorescent probe (diluted with 0.01 M PBS) was added dropwise to the sample well of the test strip for chromatography, and 3 min later, the test strip was dried in a 37° C. oven for 15 min. Then 100 μL of triazophos standard solution or sample was added for chromatography, and 3 min later, the fluorescence detection was performed.
(3) Detection: The T/C value was read with a single-channel fluorescence immunoassay analyzer, and the content of triazophos was calculated according to a standard curve. It can be seen from FIG. 2 that the minimum detection limit of this assay method for triazophos is 20 μg/L, meeting the detection requirement.

[0035] The foregoing examples are merely illustrative of preferred implementations of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that several variations, improvements and replacements may be made by those of ordinary skill in the art without departing from the conception of the present invention, but such variations, improvements and replacements should fall within the protection scope of the present invention. Therefore, the patent protection scope of the present invention should be subject to the appended claims.