TIME-RESOLVED FLUORESCENCE AND CHROMOGENIC DUAL-SIGNAL TEST STRIP FOR ESTROGEN AND ITS PREPARATION METHOD AND APPLICATION

Abstract

A time-resolved fluorescence and chromogenic dual-signal test strip for estrogen is based on the principle of immune recognition and fluorescence resonance energy transfer. In time-resolved fluorescence mode, estrogen-BSA-persistent luminescence particle complex is the fluorescence donor, and colloidal gold modified with estrogen monoclonal antibody is the fluorescence receptor, which is also chromogenic signal unit in the chromogenic mode. The photos of strips in both modes are obtained with smart phones. The time-resolved fluorescence intensity of the test strip test zone is positively correlated with estrogen content, and the chromogenic intensity is negatively correlated with estrogen content. The competitive time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strips can accurately and quickly detect estrogen and estrogen-like compounds.

Claims

1. A time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strip for estrogen comprises a sample pad, a nitrocellulose membrane that is NC membrane with two ends, and an absorbent pad on the PVC bottom plate in order along a horizontal direction, and the two ends of the NC membrane are located in a lower layer where the NC film overlaps with the sample pad and absorption pad; the NC membrane comprises a test zone, namely T zone, and a quality control zone, namely C zone; the T zone is fixed with estrogen-bovine serum albumin-persistent luminescent particles (E-BSA-PLPs), and the estrogen is an estrogen compound or an estrogen-like compound with phenolic hydroxyl groups and an estrogen effect, including estradiol, estriol, estrone, bisphenol A, diethylstilbestrol, and ethinyl estradiol; secondary antibody is fixed on the quality control zone.

2. The time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strip for estrogen of claim 1 wherein the secondary antibody fixed in the C zone includes, but is not limited to, goat anti-mouse antibody, rabbit anti-mouse antibody, goat anti-rabbit antibody and donkey anti-rabbit antibody.

3. The method of preparing the time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strip for estrogen of claim 1 comprising: (i) synthesizing estrogen-bovine serum albumin conjugate E-BSA; (ii) carboxyl modification of PLPs; (iii) preparing the complex of E-BSA and persistent luminescent particles E-BSA-PLPs; (iv) constructing the test strips: dropping E-BSA-PLPs in T zone, dropping the secondary antibody against the source of the primary antibody in C zone, and storing the strips in vacuum bags after drying.

4. The method of preparing the time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strip for estrogen of claim 3 wherein the active ester method for biological coupling is used in the step (iii), specifically, ultrasonically disperse the carboxyl modified PLPs into PBS, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and NHS with stirring at room temperature, and then centrifuge to remove the supernatant and wash the precipitate with PBS, and then add E-BSA and continue stirring overnight, and then collect the precipitate coupling product by centrifugation, wash with PBS and re-dissolve in PBS for later use.

5. The method of preparing the time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strip for estrogen of claim 3 wherein in the step (iv), the T zone is dripped with a 5-20 mM PBS solution containing 0.5-5 mg/mL E-BSA-PLPs, and the C zone is dripped with a 5-20 mM PBS solution containing 0.05-0.5 mg/mL secondary antibody.

6. The method of preparing the time-resolved fluorescence and chromogenicdual-signal immunochromatographic test strip for estrogen of claim 3 wherein in the step (ii), E-BSA is synthesized by the estrogen carboxymethyl ether (E-CME) and BSA with the active ester method, and in the step (iii), the PLPs and carboxyl-ethyl-silanetriol sodium salt (CES) are subjected to a condensation reaction to obtain carboxyl-modified PLPs.

7. A detection device for detecting estrogen comprising a test strip and an estrogen monoclonal antibody modified colloidal gold, that is, CG-mAb, and the test strip is the time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strip for estrogen of claim 1 or 2, or test strips prepared by the method of any one of claims 3-6, and the detection device uses E-BSA-PLPs complex on the test strip as fluorescent donors and CG-mAb as fluorescent acceptors.

8. The detection device for estrogen of claim 7 wherein the C zone is used as a reference for verifying the validity of the test strip, and it always shows red and no fluorescence; the time-resolved fluorescence intensity of the T zone is positively correlated with the estrogen content in the sample, and the chromogenic intensity is negatively correlated with the estrogen content in the sample, specifically, when the sample does not contain estrogen, the T zone becomes red, and the fluorescence is quenched; when the sample contains estrogen, the T zone appears light red or even colorless, and the fluorescence shows up.

9. The detection device for estrogen of claim 7 wherein the application method of the detection device is as follows: put the sample to be tested and the CG-mAb in the running buffer and mix for 3-10 min, put the test strip into the mixture and read the chromogenic result in 15-30 min; the running buffer is 8-15 mmol/L of which pH is 6.5-8.0, containing 5-15% sucrose, 6-10% BSA, 0.15-0.30% Tween-20, and the buffer comprises phosphate buffer, borate buffer, and carbonate buffer; after the chromogenic detection is completed, place the test strip under the ultraviolet light to excite, use the fluorescent image acquisition device to obtain the fluorescent images before and after the ultraviolet light source is turned off, and read the fluorescent results.

10. The detection device for estrogen of claim 9 wherein the fluorescence image acquisition device comprises a smart phone, and the smart phone obtains fluorescence images before and after the ultraviolet light source is turned off in a continuous shooting mode and obtains time-resolved fluorescence photos.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a schematic diagram of the time-resolved fluorescence and chromogenic dual-signal immunochromatographic test strip for estrogen.

[0033] FIG. 2 is the response of the test strip to the spiked drinking water samples: chromogenic mode (A), TRF mode (B).

[0034] FIG. 3 is the standard curve of the test strip: chromogenic mode (A), TRF mode (B).

[0035] FIG. 4 is the response of the test strip to different concentrations of estradiol: chromogenic mode (A), TRF mode (B).

DESCRIPTION OF PREFERRED EMBODIMENTS

[0036] The detailed implementation of the invention is further described as follows. The following embodiments are used to illustrate the invention, but not to limit the scope of the invention. Technicians in this field can clearly understand the characteristics and efficacy of the invention from the contents explained in this specification, and the invention can also be implemented or applied in other specific ways. Except for special instructions, the materials and reagents etc. used in the invention can be obtained from commercial sources.

[0037] The following examples take estradiol as an example of estrogen to fully describe the preparation of the entire test strip and the use of the detection device.

EXAMPLE 1

[0038] The construction of TRF and chromogenic dual-signal immunochromatographic test strip for estrogen and its application in a drinking water sample are as follows:

[0039] 1. Preparation of Test Strip Materials

[0040] 1.1. Preparation of Colloidal Gold (CG) and CG Modified by Monoclonal Antibody Against Estradiol (CG-mAb)

[0041] CG is prepared by sodium citrate reduction method, and CG-mAb is prepared by coupling CG with estradiol monoclonal antibody by adsorption, and the product after centrifugation (30 min, 4° C., 10000 r/min) is dissolved in 0.2 mL buffer (20 mmol/L borate buffer containing 5% sucrose, 1% BSA, pH 8.2) and stored for later use.

[0042] 1.2. Synthesis of Estradiol Carboxymethyl Ether (E2-CME) and E2-BSA Conjugate

[0043] Dissolving 100 mg of estradiol and 0.5 g of KCl in 6 mL of dimethyl sulfoxide (DMSO), and ultrasonicating for 5 min. Under stirring, 100 mg of bromoacetic acid is added. After 2 h of reaction, 50 mL of ice water is added to terminate the reaction. Extracted the unreacted estradiol with ethyl acetate, add 2 mol/L hydrochloric acid dropwise to the aqueous phase to acidify the solution until the white precipitate appeared. After centrifugation, the supernatant is removed, and the precipitate is washed with ultrapure water to pH≈7 and then vacuum freeze-dried to obtain E2-CME.

[0044] E2-BSA is synthesized by the active ester method. Specifically, dissolve 3.3 mg of E2-CME in 0.5 mL DMSO, adding 6 mg NHS and 7 mg EDCHCl, and stir for 12 h to react. Dissolve 20 mg of BSA in 4 mL of carbonate buffer solution (CBS) whose concentration is 50 mM and pH is 9.6, and add the activated E2-CME solution slowly to the CBS solution and continue to stir for 12 h. After the reaction, transfer the mixture to a dialysis bag and put in 1 L of PBS for dialysis whose concentration is 10 mM and pH is 7.4. Change the pressure fluid every 12 h for 3 consecutive days. After the dialysis, the E2-BSA conjugate is obtained and frozen for further use.

[0045] 1.3. Modification of PLPs

[0046] Dissolve 30 mg of PLPs in 5 mL of absolute ethanol under ultrasonication in a 10 mL centrifuge tube. Add 20 mL of absolute ethanol, 5 mL of water, and 0.13 mL of tetraethylorthosilicate (TEOS) to a 50 mL round-bottom flask. Under stirring, put the PLPs-ethanol solution into the round-bottom flask quickly and sonicate for 10 minutes to thoroughly mix the solution. Add 0.5 mL ammonia (28-30%) to the mixture, sonicating for 30 min and then stir the mixture for 7.5 h. After the reaction, Centrifuge and wash 3 times with absolute ethanol under the centrifugal conditions of 10000 r/min, 10 min. Dry the product under vacuum to get silica-encapsulated PLPs.

[0047] Disperse 25 mg of silica-encapsulated PLPs into 25 mL of water ultrasonically, adding 100 μL of CES under electromagnetic stirring, and stir it at room temperature for 24 h. After centrifugation, wash the precipitate using ultrapure water and absolute ethanol twice each, and dry it in vacuum at room temperature to obtain carboxyl-modified PLPs.

[0048] 1.4. Preparation of E2-BSA-PLPs Complex

[0049] The E2-BSA-PLPs complex is prepared by coupling E2-BSA and carboxyl-modified PLPs by the active ester method. The specific method is: dispersing the carboxyl-modified PLPs into PBS ultrasonically, adding 6 mg of EDCHCl and 15 mg of NHS, stirring at room temperature and centrifuging to remove the supernatant, adding 1 mL of E2-BSA at a concentration of 1 mg/mL, stirring overnight, centrifuging and collecting the precipitate, washing the precipitate with PBS and dissolving the product E2-BSA-PLPs complex in PBS for later use. The product is stored in PBS solution at 2 mg/mL (calculated as the concentration of PLPs) for later use.

[0050] 1.5. Preparation of Test Zone (T Zone) Solution: E2-BSA-PLPs is Diluted to 2 mg/mL with 10 mM PBS solution.

[0051] 1.6. Preparation of Quality Control Zone (C Zone) Solution: the Goat Anti-Mouse Secondary Antibody is Diluted to 0.1 mg/mL with 10 mM PBS Solution.

[0052] 2. Preparation of Test Strips

[0053] According to FIG. 1, the NC film was pasted in the middle of the PVC baseplate, the sample pad and the absorbent pad are overlapped on the left and right ends of the NC film respectively, and then they are cut into strips of 3 mm width, obtaining blank test strips. 1.0 μL T zone solution and 0.5 μL C zone solution are dropwise added in the T zone and C zone of the test strips respectively. The test strips are dried at 37° C. for 30 min, and stored in a vacuum bag for later use.

[0054] 3. Drawing Working Curve

[0055] 10 mL of 1 mg/mL E2 standard stock solution is prepared with methanol and it was diluted with 10 mM PBS to concentrations of 0 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL of E2, and this series is the standard working solutions. 80 μL of the test solution, 20 μL CG-mAb and 10 μL running buffer (10 mmol/L phosphate buffer, 10% sucrose, 8% BSA, 0.25% Tween-20, pH 7.4) are mixed in a centrifuge tube for 5 min. A test strip is inserted into the centrifuge tube, and the result is analyzed after 25 minute. The result of chromogenic mode is shown in FIG. 4A, and the corresponding working curve is shown in FIG. 3A. The result of TRF mode is shown in FIG. 4B, and the corresponding working curve is shown in FIG. 3B. According to FIG. 4A, in the chromogenic mode, the T zone of the negative and low-concentration positive shows a clear purple-red color, whereas the color of the T zone becomes lighter when the E2 concentration increases to 5 ng/mL, and the color of the T zone disappears at 10 ng/mL. This concentration at which T zone disappears is the detection limit of the chromogenic method; in TRF mode, there is no obvious fluorescence in the negative T zone. When the E2 concentration is 0.1 ng/mL, the T zone emits weak fluorescence (FIG. 4B), and this concentration is the detection limit of the TRF method. Therefore, the detection limit of the constructed test strip can reach 0.1 mg/mL in TRF mode.

[0056] 4. Sample Pretreatment

[0057] First, the drinking water sample is filtered and detected with high performance liquid chromatography-mass spectrometry (HPLC-MS). It is estradiol-negative. Add 800 μL of E2 standard solutions with concentrations of 0 mg/mL, 0.10 mg/mL, 1 mg/mL, and 10 mg/mL, respectively, into 1 mL drinking water samples to get a series of spiked samples.

[0058] 5. Sample Detection

[0059] Mix 80 μL of the sample solution with 20 μL of CG-mAb and 10 μL of running buffer (10 mmol/L phosphate buffer, 10% sucrose, 8% BSA, 0.25% Tween-20, pH 7.4) in a centrifuge tube for 5 minute, and then inserted the test strip into the centrifuge tube, and read the chromogenic result after 25 minutes. After the chromogenic detection is completed, placed the test strip under an ultraviolet lamp to excite the fluorescence of PLPs, use a smartphone to obtain the fluorescence images before and after the ultraviolet light source is turned off, and analyze the image data. The results are shown in FIG. 2, indicating the test strip can detect at least 0.1 ng/mL of E2. Moreover, the analysis can be completed in about 30 minutes.

EXAMPLE 2

[0060] The construction of TRF and chromogenic dual-signal immunochromatographic test strip for estrogen and its application in a milk sample are as follows:

[0061] 1. Preparation of Test Strip Materials

[0062] Same as Example 1

[0063] 2. Preparation of Test Strips

[0064] Same as Example 1

[0065] 3. Drawing Working Curve

[0066] Same as Example 1

[0067] 4. Sample Pretreatment

[0068] First, the milk sample is detected with HPLC-MS and is found to be estradiol-negative. The milk sample is centrifuged by 8000 r/min at 10° C. for 10 min, and then the upper milk fat is discarded to obtain skim milk. Diluted the skim milk with deionized water at a volume ratio of 1:20, filtered, measured 4 portions, 1 mL each, and put them into four 10 mL centrifuge tubes, respectively, as the test solution for use.

[0069] 5. Sample Detection

[0070] Mix 80 μL of the sample solution with 20 μL of CG-mAb and 10 μL of running buffer (10 mmol/L phosphate buffer, 10% sucrose, 8% BSA, 0.25% Tween-20, pH 7.4) in a centrifuge tube for 5 minute, and then insert the test strip into the centrifuge tube, and read the chromogenic result after 25 minutes. After the chromogenic detection was completed, place the test strip under an ultraviolet lamp to excite the fluorescence of PLPs, use a smartphone to obtain the fluorescence images before and after the ultraviolet light source is turned off, and analyze the image data.

[0071] The concentration of E2 in the milk sample is found to be lower than the limit of quantification and the limit of detection.

EXAMPLE 3

[0072] The construction of TRF and chromogenic dual-signal immunochromatographic test strip for estrogen and its application in a pork sample of are as follows:

[0073] 1. Preparation of Test Strip Materials

[0074] Same as Example 1

[0075] 2. Preparation of Test Strips

[0076] Same as Example 1

[0077] 3. Drawing Working Curve

[0078] Same as Example 1

[0079] 4. Sample Pretreatment

[0080] First, the pork sample is detected with HPLC-MS and was found to be estradiol-negative. Weigh 100 g of the pork without bones and skin, crush and then homogenize the pork sample. Weigh 4 portions of homogenized pork sample, 1 g each, into centrifuge tubes, add 3 ml of ethyl acetate, 0.1 g of anhydrous calcium oxide, and shake for 2 min, centrifuge at 4000 r/min for 10 min, transfer the supernatant to another test tube, dry with nitrogen flow, add 1 mL of n-butane to dissolve the residue, added 0.5 mL of PBS-methanol solution (v:v=3:2, pH=7.0), shake for 2 min, then centrifuge at 4000 r/min for 10 min, remove n-butane in the upper layer, and take the solution in the lower layer as the test solution.

[0081] 5. Sample Detection

[0082] Mix 80 μL of the sample solution with 20 μL of CG-mAb and 10 μL of running buffer (10 mmol/L phosphate buffer, 10% sucrose, 8% BSA, 0.25% Tween-20, pH 7.4) in a centrifuge tube for 5 minute, and then insert the test strip into the centrifuge tube, and read the chromogenic result after 25 minute. After the chromogenic detection is completed, place the test strip under an ultraviolet lamp to excite the fluorescence of PLPs, use a smartphone to obtain the fluorescence images before and after the ultraviolet light source is turned off, and analyze the image data. The concentration of E2 in the milk sample is0 found to be lower than the limit of quantification and the limit of detection.

[0083] Example 1 shows that the test strip can detect spiked E2 at 0.1 ng/mL and above. Moreover, the analysis can be completed only in 30 minutes and the test strips and related testing materials are easy to store. The test strips can be used in a wide range of samples, such as Examples 2 and 3.

[0084] In summary, the test strip and the corresponding detection device of the present invention have good estradiol detection performance and applicability. Similarly, it can also be extended to estrogens and estrogen-like compounds, which has the same phenolic hydroxyl structure as estradiol and has an estrogenic effect, such as estriol, estrone, bisphenol A, diethylstilbestrol, ethinyl estradiol, etc. For each estrogen analyte, replace the E2's antibody with the corresponding primary antibody. The phenolic hydroxyl group in the estrogen structure can be derived to a carboxyl group through a series of chemical reactions, and then the estrogen can be coupled to BSA through this carboxyl group.

[0085] The foregoing is only the preferred embodiments of the present invention. It should be pointed out that for the common technical personnel in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also should be regarded as the protection scope of the present invention.