WO2022193689A1 - ***的时间分辨荧光、显色双信号试纸条及其制备方法与应用 - Google Patents

***的时间分辨荧光、显色双信号试纸条及其制备方法与应用 Download PDF

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WO2022193689A1
WO2022193689A1 PCT/CN2021/130133 CN2021130133W WO2022193689A1 WO 2022193689 A1 WO2022193689 A1 WO 2022193689A1 CN 2021130133 W CN2021130133 W CN 2021130133W WO 2022193689 A1 WO2022193689 A1 WO 2022193689A1
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estrogen
test strip
fluorescence
time
bsa
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French (fr)
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张毅
何永熙
王文龙
王凌凌
严秀平
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江南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the invention belongs to the technical field of analysis and detection, and in particular relates to a detection method of environmental estrogen, in particular to a time-resolved fluorescence and color development double-signal immunochromatographic test strip of estrogen and a preparation method and application thereof.
  • Estrogen (E) is a substance that promotes the development of secondary sexual characteristics and the maturation of sexual organs in female animals, and is secreted by the ovary and placenta of female animals. Estrogen has extensive and important physiological roles, not only promoting and maintaining the physiological role of female reproductive organs and secondary sexual characteristics, but also significantly affecting endocrine, cardiovascular, metabolic systems, bone growth and maturation, and skin. . Natural estrogens are mainly estradiol E2, estrone E1, and estriol Estriol; most of the estrogen drugs commonly used in clinical use are synthetic derivatives of estradiol as the parent, such as diethylstilbestrol, ethinylestradiol, etc.
  • 17 ⁇ -estradiol is the most active natural estrogen of all endocrine disruptors, and is often used as an endocrine-regulating drug for menopausal women.
  • estrogen when estrogen accumulates in the human body through drinking water and food beyond safe thresholds, it can damage human health, disrupt body balance, and even endanger offspring.
  • the detection methods of estrogen mainly include chromatography, electrochemical method, surface-enhanced Raman spectroscopy (SERS) method, etc.
  • Chromatography has high sensitivity and good stability, but cannot be used for on-site detection; electrochemical method and SERS method have fast response and sensitivity, and can be used for on-site detection, but have low stability and reproducibility.
  • Test strip is a paper-based rapid detection technology, which is low-cost, easy to operate, and can realize on-site detection. It has been widely used in clinical diagnosis, food safety, environmental monitoring and other fields. Colloidal gold is commonly used as a color probe for test strips because its color results are visible to the naked eye, but its sensitivity is low and the readings are subjective. In order to improve the sensitivity of the test paper, gold nanoflowers/gold nanorods or secondary labeling of colloidal gold can be used, or fluorescent lateral flow analysis test paper can be developed, such as small molecule organic dyes, quantum dots, upconversion nanoparticles, etc. as signal probes
  • the above-mentioned fluorescent lateral flow analysis test paper has disadvantages such as interference of fluorescent background of the test paper or the need for a high-energy laser light source.
  • the purpose of the present invention is to provide a preparation method and application of a novel, specific and rapid estrogen TRF and color-developing double-signal immunochromatographic test strip according to the current technical deficiencies and detection needs.
  • the present invention is based on the principle of immune recognition and fluorescence resonance energy transfer, and uses long afterglow particles (PLPs) coupled with estrogen-bovine serum albumin (E-BSA) as the fluorescence donor, and is modified with estrogen monoclonal antibody.
  • PRPs long afterglow particles
  • E-BSA estrogen-bovine serum albumin
  • Colloidal gold CG-mAb
  • TRF competitive time-resolved fluorescence
  • a low-power ultraviolet lamp is used as the excitation light source
  • the continuous shooting function of a smartphone is used to realize the acquisition of the fluorescence image of the detection area of the test paper in TRF mode, which can effectively remove the fluorescence background interference of the test paper and improve the signal-to-noise ratio.
  • the fluorescent signal source and its non-migrating property on nitrocellulose membrane fix the estrogen competitor in the detection area, which cleverly solves the contradiction between the nano-level limitation of the size of the signal material by the conventional test paper and the short afterglow of the small-sized and long-lasting particles; TRF and chromogenic dual-signal detection combine naked-eye rapid screening with precise quantification while increasing the confidence of the results.
  • the method can be used for rapid and sensitive detection of estrogen.
  • the first object of the present invention is to provide a time-resolved fluorescence and color-developing double-signal test strip for estrogen, the test strip structure is that a sample pad, nitrocellulose are stacked and pasted on a PVC bottom plate in sequence along the horizontal direction
  • the plain film is the NC film and the absorbing pad.
  • the two ends of the NC film are located in the overlapping lower layer; the NC film is used to separate and detect the analyte and other substances in the sample, the sample pad is used for sample loading, and the absorbing pad is used to absorb excess Liquid, PVC bottom plate provides physical support for the test strip;
  • the NC film includes a detection zone, namely a T zone, and a quality control zone, namely a C zone;
  • Estrogen-bovine serum albumin-long afterglow particles E-BSA-PLPs are fixed on the detection area, and the estrogen is an estrogen compound with phenolic hydroxyl group and estrogen effect and an estrogen-like compound, including estradiol (E2), estriol (E3), estrone (E1), bisphenol A (BPA), diethylstilbestrol, ethinyl estradiol, etc; Physiological effects similar to estrogen, also known as estrogen-like effects.
  • a secondary antibody is immobilized on the quality control area.
  • the secondary antibodies immobilized in the quality control area include but are not limited to goat anti-mouse secondary antibodies, rabbit anti-mouse secondary antibodies, goat anti-rabbit secondary antibodies, and donkey anti-rabbit secondary antibodies.
  • Another object of the present invention is to provide a method for preparing a time-resolved fluorescence and colorimetric double-signal test strip for estrogen, the preparation steps are as follows:
  • test paper drop E-BSA-PLPs in the T area, drop the secondary antibody in the C area, and store in a vacuum bag after drying for future use.
  • the step (3) uses the active ester method to carry out biological coupling, and the specific method is as follows: ultrasonically dispersing the long afterglow particles modified by carboxyl groups into phosphate buffered solution PBS; then adding 1-3-dimethylformaldehyde Aminopropyl-3-ethylcarbodiimide hydrochloride EDC HCl and N-hydroxysuccinimide NHS, continue to stir at room temperature, then centrifuge, remove the supernatant, and wash with PBS; then add E-BSA , stirring was continued overnight, and the coupling product was collected by centrifugation, washed with PBS, and then reconstituted in PBS for use.
  • the specific method is as follows: ultrasonically dispersing the long afterglow particles modified by carboxyl groups into phosphate buffered solution PBS; then adding 1-3-dimethylformaldehyde Aminopropyl-3-ethylcarbodiimide hydrochloride EDC HCl and N-
  • the 5-20mM PBS solution containing 0.5-5mg/mL E-BSA-PLPs was added dropwise to the T area, and the 5-20mM PBS solution containing 0.05-0.5mg/mL secondary antibody was added dropwise to the C area. PBS solution.
  • step (2) adopts estrogen carboxymethyl ether E-CME and BSA to synthesize E-BSA by active ester method
  • described step (3) adopts long afterglow particles PLPs, sodium carboxyethylsilane trioxide CES, carboxyl-modified long afterglow particles were obtained by carboxylation.
  • Another object of the present invention is to provide a detection device for detecting estrogen, comprising a test strip and a colloidal gold CG-mAb modified by an estrogen monoclonal antibody, wherein the test strip is the above-mentioned estrogen
  • the detection device uses the estrogen-bovine serum albumin-long afterglow particle complex on the test strip as the fluorescence donor, and the colloidal gold modified by the estrogen monoclonal antibody as the fluorescence donor. fluorescent receptors.
  • the method for preparing estrogen monoclonal antibody-modified colloidal gold is as follows: preparing colloidal gold and modifying the primary antibody, namely estrogen monoclonal antibody, to obtain estrogen monoclonal antibody-modified colloidal gold CG-mAb, which is used for modification
  • Sources of monoclonal antibodies for colloidal gold include, but are not limited to, mice, rats, and rabbits.
  • the estrogen monoclonal antibody-modified colloidal gold is stored in a closed container including a centrifuge tube in the state of solution or lyophilized powder.
  • the secondary antibody immobilized in the quality control area is a secondary antibody derived from the primary antibody.
  • the C area is used as a reference to verify the validity of the test paper results, which is always red and has no fluorescence;
  • the time-resolved fluorescence intensity of the T area is positively correlated with the estrogen content in the sample, and the color intensity is negatively correlated with the estrogen content in the sample.
  • the T region is red and the fluorescence is quenched
  • the T region appears light red or even disappears, and the fluorescence is enhanced.
  • the application method of the detection device is as follows: the sample to be tested and the CG-mAb are placed in the running buffer and mixed for 3-10min, put into the test strip, and the color development result is read after 15-30min; the running buffer The solution is 8-15mmol/L buffer containing 5-15% sucrose, 6-10% BSA, 0.15-0.30% Tween-20 at pH 6.5-8.0, the buffer includes but not limited to phosphate buffer, borate buffer, carbonate buffer;
  • test strip After the color development is completed, the test strip is placed under an ultraviolet lamp to excite, and a fluorescence image acquisition device is used to obtain the fluorescence images before and after the ultraviolet light source is turned off, and read the fluorescence results.
  • the fluorescence image acquisition device includes a smartphone, and the smartphone acquires fluorescence images before and after the ultraviolet light source is turned off in a continuous shooting mode, and obtains time-resolved fluorescence photos.
  • the detection principle of the estrogen rapid time-resolved fluorescence and color-developing dual-signal immunochromatographic test strip of the present invention is explained as follows by taking estradiol as an example: Area C is used as a reference for verifying the validity of the test strip results, and it always shows red and does not fluoresce .
  • the sample solution and CG-mAb were premixed and moved towards the direction of the absorbent paper under the action of capillary action.
  • the CG-mAb was captured by the E2-BSA-PLPs in the T region, and the T region was red, and E2 -FRET between BSA-PLPs and CG-mAb quenches the fluorescence of long afterglow particles, and the excess CG-mAb will be captured by the secondary antibody in the C region, so that the C region also turns red; when the sample contains estradiol, estrogen The diol binds to part of the CG-mAb to reduce the total amount of CG-mAb captured in the T region.
  • the T region can be seen to be light red or even eliminated, while the FRET between E2-BSA-PLPs and CG-mAb is reduced or even disappeared.
  • the estrogen rapid time-resolved fluorescence and color development double-signal immunochromatographic test strip of the present invention is based on the principle of fluorescence resonance energy transfer, and uses long afterglow particles with excellent afterglow and micron size as the fixed fluorescent probe in the T zone, and low power Ultraviolet lamp excitation combined with smartphone photography realizes fluorescence acquisition in TRF mode, which effectively removes the background interference of test strip fluorescence and improves the signal-to-noise ratio.
  • this method has a detection limit (up to 0.1 mg/mL) lower by two orders of magnitude, better specificity, less light background pollution than similar fluorescent detection test paper, and fast detection speed, which is suitable for field applications. Screening.
  • FIG. 1 is a schematic diagram of a test strip time-resolved fluorescence and color-developing double-signal lateral flow chromatography of the present invention
  • Fig. 2 is the response of the test strip of the present invention to the spiked drinking water sample: color development mode (A), TRF mode (B)
  • Fig. 3 is the working curve of the test strip of the present invention: color rendering mode (A), TRF mode (B);
  • Figure 4 is a graph showing the response of the test strip of the present invention to different concentrations of estradiol: color development mode (A), TRF mode (B).
  • estradiol as an example of estrogen, and fully describe in detail the preparation of the whole test strip and the use of the detection device.
  • Example 1 Preparation of estradiol rapid time-resolved fluorescence, coloration dual-signal immunochromatographic test strip and its detection device, and water sample detection
  • Colloidal gold was prepared by sodium citrate reduction method, and CG-mAb was prepared by coupling with estradiol monoclonal antibody by adsorption. /L borate buffer containing 5% sucrose, 1% BSA, pH 8.2) dissolved and stored for later use.
  • estradiol and 0.5 g of KCl were weighed and dissolved in 6 mL of dimethyl sulfoxide (DMSO), and the ultrasonic reaction was carried out for 5 min. Under stirring conditions, 100 mg of bromoacetic acid was added, and 50 mL of ice water was added to terminate the reaction after 2 h of reaction. Extraction with ethyl acetate to collect unreacted estradiol, add 2mol/L hydrochloric acid dropwise to the aqueous phase to acidify, followed by white precipitate, remove the supernatant after centrifugation, and wash the precipitate with ultrapure water to pH value After about 7, vacuum freeze-drying to obtain E2-CME.
  • DMSO dimethyl sulfoxide
  • the active ester method was used to synthesize E2-BSA.
  • the specific experimental operation was as follows: 3.3 mg of E2-CME was weighed and dissolved in 0.5 mL of DMSO, 6 mg of NHS and 7 mg of EDC ⁇ HCl were added, and the reaction was stirred for 12 h. 20 mg of BSA was weighed and dissolved in 4 mL of carbonate buffer solution (CBS) (50 mM, pH 9.6), and the activated E2-CME solution was slowly added dropwise to the CBS solution to continue stirring for 12 h. After the reaction, the mixture was transferred to a dialysis bag and dialyzed into 1 L of PBS (10 mM, pH 7.4) solution. The dialysate was replaced every 12h for 3 consecutive days. After the dialysis, the E2-BSA conjugates were obtained by aliquoting them for cryopreservation.
  • E2-BSA-PLPs complexes were prepared by coupling E2-BSA with carboxyl modified long afterglow particles by active ester method.
  • the specific method was as follows: ultrasonically disperse the carboxyl modified long afterglow particles into phosphate buffered saline solution PBS ; Then add 6mg 1-3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride EDC HCl and 15mg N-hydroxysuccinimide NHS, continue to stir at room temperature and then centrifuge, remove the supernatant Then, 1 mL of E2-BSA with a concentration of 1 mg/mL was added, and the mixture was continuously stirred overnight. The coupled product was collected by centrifugation, washed with PBS, and then reconstituted in PBS for use. The product was stored in PBS solution at 2 mg/mL (calculated as long afterglow particle concentration) until use.
  • E2-BSA-PLPs were diluted to 2 mg/mL with 10 mM PBS solution.
  • the goat anti-mouse secondary antibody was diluted to 0.1 mg/mL with 10 mM PBS solution.
  • the concentration is the detection limit of the chromogenic method; in TRF mode, the negative T region has no obvious fluorescence, and the T region emits weak light when the E2 concentration is 0.1 ng/mL ( Figure 4B), and this concentration is the detection limit of the TRF method. Therefore, the detection limit of the constructed TRF test paper can reach 0.1 mg/mL.
  • the C area is used as a reference to verify the validity of the test strip results, and it always shows red and does not fluoresce.
  • Embodiment 2 The detection of milk by estradiol rapid time-resolved fluorescence and color development double-signal immunochromatographic test strips includes the following steps:
  • test solution with 20 ⁇ L of CG-mAb and 10 ⁇ L of running buffer (10 mmol/L phosphate buffer, containing 10% sucrose, 8% BSA, 0.25% Tween-20, pH 7.4) in a centrifuge tube for 5 min. Then insert the test strip into the centrifuge tube, and read and display the result after 25 minutes. After the color development is completed, place the test strip under an ultraviolet light to excite, and use a smartphone to obtain fluorescence images before and after the ultraviolet light source is turned off, and read the fluorescence results. Results are shown to be below the limit of quantification, ie, below the limit of detection.
  • Embodiment 3 The detection of pork by estradiol rapid time-resolved fluorescence and color development double-signal immunochromatographic test strips includes the following steps:
  • Example 1 illustrates that the test strip can detect spiked E2 at 0.1 ng/mL and above. Moreover, the analysis can be completed in about 30 minutes, and the detection time is short.
  • the test strips and related detection materials have mild storage conditions, can be used in a wide range of scenarios, and can be used in different occasions, such as Examples 2 and 3.
  • the test strip and the corresponding detection device of the present invention have good estradiol detection performance and applicability.
  • estradiol which has the same phenolic hydroxyl structure as estradiol and has estrogenic effects.
  • Hormones and estrogen-like hormones such as estriol, estrone, bisphenol A, diethylstilbestrol, ethinyl estradiol, etc., replace the corresponding estrogen monoclonal monomer with the estrogen monoclonal monomer corresponding to each estrogen. That's it.
  • the phenolic hydroxyl group in these estrogen structures can be derived from a carboxyl group through a series of chemical reactions, and then the estrogen can be coupled to the carrier through this carboxyl group.

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Abstract

一种***的时间分辨荧光、显色双信号试纸条及其制备方法与应用，***为具有酚羟基、***效应的***化合物以及类***化合物，包括***、雌三醇、雌酮、双酚A、己烯雌酚、炔雌醇等。***的时间分辨荧光、显色双信号试纸条基于免疫识别和荧光共振能量转移原理，以***-牛血清蛋白-长余辉粒子为荧光供体并将其固定在试纸检测区，以***单克隆抗体修饰的胶体金为荧光受体，以智能手机拍照方式获得时间分辨荧光照片，试纸检测区的时间分辨荧光强度与***含量呈正相关，显色强度与***含量呈负相关，建立了***的竞争型时间分辨荧光、显色双信号免疫层析试纸条，可对具有羟基、***效应的***以及类***进行准确快速的检测。

Description

***的时间分辨荧光、显色双信号试纸条及其制备方法与应用 技术领域
本发明属于分析检测技术领域，具体涉及环境***的检测方法，特别是***的时间分辨荧光、显色双信号免疫层析试纸条及其制备方法与应用。
背景技术
***(Estrogen,E)是促进雌性动物***发育及性器官成熟的物质，由雌性动物卵巢和胎盘分泌产生。***具有广泛而重要的生理作用，不仅有促进和维持女性生殖器官和***的生理作用，并对内分泌、心血管、代谢***、骨骼的生长和成熟，皮肤等均有明显的影响。天然***主要是***E2、雌酮E1、雌三醇Estriol；目前临床上常用的***类药物多是以***为母体人工合成的衍生物，如己烯雌酚、炔雌醇等。17β-***是所有内分泌干扰物中具有最强活性的天然***，常被用作更年期女性的内分泌调节药物。然而，当***通过饮用水和食物在人体内积累超过安全阈值时，会损害人类健康、破坏机体平衡，甚至危害后代。
目前，***的检测方法主要有色谱法、电化学法、表面增强拉曼光谱(SERS)法等。色谱法灵敏度高、稳定性好，但无法应用于现场检测；电化学法和SERS法响应快速、灵敏，均可用于现场检测，但稳定性和重现性较低。
试纸条是一种纸基快速检测技术，低成本、易操作、可实现现场检测，目前已广泛应用于临床诊断、食品安全、环境监测等领域。胶体金因其显色结果肉眼可见而常用作试纸条显色探针，但其灵敏度较低且读数结果具有主观性。为提高试纸灵敏度，可以采用金纳米花/金纳米棒或对胶体金二次标记，或开发荧光型侧流分析试纸，如以小分子有机染料、量子点、上转换纳米粒子等为信号探针，但上述荧光型侧流分析试纸存在试纸荧光背景干扰或需要高能量激光光源等弊端。
发明内容
本发明的目的在于根据目前技术不足及检测需要，提供一种新型、特异、快速的***TRF和显色双信号免疫层析试纸条的制备方法和应用。
本发明基于免疫识别和荧光共振能量转移原理，以偶联***-牛血清蛋白(E-BSA)的长余辉粒子(persistent luminescent particles，PLPs)为荧光供体，以***单克隆抗体修饰的胶体金(CG-mAb)为荧光受体，建立了***的竞争型时间分辨荧光 (Time-resolved fluorescence，TRF)、显色双信号试纸条。本方法以低功率紫外灯为激发光源，以智能手机连拍功能实现TRF模式采集试纸的检测区荧光图像，有效去除试纸荧光背景干扰进而提高信噪比；以余辉优良的大尺寸长余辉粒子为荧光信号源并利用其在硝酸纤维素膜上不迁移的特性将***竞争物固定于检测区，巧妙解决了常规试纸对信号材料尺寸的纳米级别限制和小尺寸长余辉粒子余辉短暂的矛盾；TRF和显色双信号检测将裸眼快筛与精密定量相结合，同时提高了结果的可信度。本方法可以用于***的快速、灵敏检测。
本发明的目的可通过以下技术方案实现：
本发明的第一个目的是，提供一种***的时间分辨荧光、显色双信号试纸条，所述试纸条结构为PVC底板上沿水平方向依次交叠粘贴有样品垫、硝酸纤维素膜即NC膜、吸收垫，所述NC膜两端位于交叠下层；NC膜用于实现样品中分析物与其他物质的分离和检测，样品垫用于上样，吸收垫用于吸收过量液体，PVC底板为试纸提供物理支撑；
所述NC膜包括检测区即T区、质控区即C区；
所述检测区上固定有***-牛血清蛋白-长余辉粒子E-BSA-PLPs，所述***为具有酚羟基且具有***效应的***化合物以及类***化合物，包括***(E2)、雌三醇(E3)、雌酮(E1)、双酚A(BPA)、己烯雌酚、炔雌醇等；所述***效应是指能够与***受体作用、对内分泌***产生类似***的生理效应，也称***样效应。
所述质控区上固定有二抗。
进一步的，所述质控区固定的二抗包括但不限于羊抗鼠二抗、兔抗鼠二抗、羊抗兔二抗、驴抗兔二抗。
本发明的另一个目的是，提供一种***的时间分辨荧光、显色双信号试纸条的制备方法，制备步骤如下：
(1)合成***-牛血清蛋白偶联物E-BSA；
(2)对长余辉粒子PLPs进行羧基修饰；
(3)制备偶联E-BSA的长余辉粒子复合物E-BSA-PLPs，在PBS溶液中储存备用；
(4)构建试纸：T区滴加E-BSA-PLPs，C区滴加二抗，干燥后于真空袋中保存备用。
进一步的，所述的步骤(3)使用活性酯法进行生物偶联，具体方法为：将被羧基修饰过的长余辉粒子超声分散至磷酸盐缓冲溶液PBS中；然后加入1-3-二甲氨基丙基-3-乙基碳二亚胺盐酸盐EDC·HCl和N-羟基琥珀酰亚胺NHS，室温条件下持续搅拌后离心，除去上清液，并用PBS洗涤；再加入E-BSA，持续搅拌过夜，通过离心收集偶联产物，随后用PBS洗涤后再复溶于PBS中备用。
进一步的，所述步骤(4)中，T区滴加含有0.5-5mg/mL E-BSA-PLPs的5-20mM PBS溶液，C区滴加含有0.05-0.5mg/mL二抗的5-20mM PBS溶液。
进一步的，所述的步骤(2)采用***羧甲基醚E-CME、BSA通过活性酯法合成E-BSA，所述步骤(3)采用长余辉粒子PLPs、羧乙基硅烷三醇钠CES，进行羧合反应得羧基修饰的长余辉粒子。
本发明的另一种目的在于，提供一种用于检测***的检测装置，包括试纸条和***单克隆抗体修饰的胶体金CG-mAb，所述试纸条为上述的***的时间分辨荧光、显色双信号试纸条，所述检测装置以试纸条上的***-牛血清蛋白-长余辉粒子复合物为荧光供体，以***单克隆抗体修饰的胶体金为荧光受体。
进一步的，制备***单克隆抗体修饰的胶体金的方法为：制备胶体金并修饰一抗即***单克隆抗体，得到***单克隆抗体修饰的胶体金CG-mAb，所述用于修饰胶体金的单克隆抗体来源包括但不限于小鼠、大鼠、兔。所述***单克隆抗体修饰的胶体金以溶液或冻干粉末的状态保存于包括离心管在内的密闭容器中。对应的，所述质控区固定的二抗是针对一抗来源的二抗。
进一步的，C区作为验证试纸结果有效性的参照，始终显红色、无荧光；T区的时间分辨荧光强度与样品中***含量呈正相关，显色强度与样品中***含量呈负相关，具体为：
当样品不含***时，T区显红色、荧光猝灭；
当样品中含有***时，T区显浅红色甚至消线、荧光增强。
进一步的，该检测装置的应用方法如下：将待测样品与CG-mAb置于运行缓冲液中混合3-10min，放入试纸条，15-30min后读取显色结果；所述运行缓冲液为8-15mmol/L包含5-15％蔗糖、6-10％BSA、0.15-0.30％吐温-20的pH 6.5-8.0的缓冲液，所述缓冲液包括但不限于磷酸盐缓冲液、硼酸盐缓冲液、碳酸盐缓冲液；
显色完成后，将试纸条置于紫外灯下激发，使用荧光图像采集设备获取紫外光源关 闭前后的荧光图像，读取荧光结果。
更进一步的，所述荧光图像采集设备包括智能手机，所述智能手机以连拍模式获取紫外光源关闭前后的荧光图像，获得时间分辨荧光照片。
本发明的***快速时间分辨荧光、显色双信号免疫层析试纸条检测原理，以***为例解释如下：C区作为验证试纸结果有效性的参照，始终显红色、不发荧光。样品溶液和CG-mAb预混合后在毛细作用下向吸水纸方向移动，当样品中没有***时，CG-mAb被T区E2-BSA-PLPs捕获，肉眼可见T区显红色，而E2-BSA-PLPs和CG-mAb之间发生FRET使长余辉粒子荧光猝灭，多余的CG-mAb会被C区的二抗捕获从而C区也显红色；当样品中含有***时，***与部分CG-mAb结合从而减少了T区捕获的CG-mAb总量，肉眼可见T区显浅红色甚至消线，而E2-BSA-PLPs和CG-mAb之间的FRET减少甚至消失，使长余辉粒子的荧光恢复。随着样品中***浓度的增大，被捕获于T区的CG-mAb越来越少，T区变淡，与分析物浓度呈反比，而长余辉粒子的亮度逐渐增大，与分析物浓度呈正比。
有益效果：本发明的***快速时间分辨荧光、显色双信号免疫层析试纸条基于荧光共振能量转移原理，以余辉优良、微米尺寸的长余辉粒子为T区固定荧光探针，低功率紫外灯激发结合智能手机拍摄实现TRF模式荧光采集，有效去除试纸荧光背景干扰进而提高信噪比。该方法具有比胶体金显色法低两个数量级的检测限(可达到0.1mg/mL)和较好的特异性，较同类荧光检测试纸光背景污染更少，且检测速度快，适用于现场筛查。
附图说明
图1为本发明试纸条时间分辨荧光、显色双信号侧流层析示意图；
图2为本发明试纸条对加标饮用水水样的响应：显色模式(A)，TRF模式(B)
图3为本发明试纸条的工作曲线：显色模式(A)，TRF模式(B)；
图4为本发明试纸条对不同浓度***的响应图：显色模式(A)，TRF模式(B)。
具体实施方式
下面结合附图和实施例对本发明作更进一步的说明。
实施例
根据下述实施例，可以更好的理解本发明。然而，本领域的技术人员容易理解，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会 限制权利要求书中所详细描述的本发明。
以下实施例以***作为***的示例，对整个试纸条的制备、检测装置的使用进行充分详细的说明。
实施例1：***快速时间分辨荧光、显色双信号免疫层析试纸条及其检测装置的制备及水样检测
1.试纸材料的制备
1.1制备胶体金
采用柠檬酸钠还原法制备胶体金，通过吸附作用与***单克隆抗体偶联制备CG-mAb，离心(30min，4℃，10000r/min)后的产物以0.2mL重悬缓冲液(20mmol/L硼酸盐缓冲液含5％蔗糖，1％BSA，pH 8.2)溶解储存备用。
1.2合成***羧甲基醚(E2-CME)和E2-BSA偶联物
称取100mg***和0.5g KCl溶解于6mL二甲基亚砜(DMSO)中，超声反应5min。在搅拌条件下，加入100mg溴乙酸，反应2h后加入50mL冰水终止反应。用乙酸乙酯萃取收集未反应的***，向水相中逐滴加入2mol/L盐酸酸化，随之出现白色沉淀，离心后去除上清液，将沉淀用超纯水离心洗涤至pH值为7左右后真空冷冻干燥，得E2-CME。
采用活性酯法合成E2-BSA，具体实验操作为：称取3.3mg E2-CME溶于0.5mL DMSO中，并加入6mg NHS和7mg EDC·HCl，搅拌反应12h。称取20mg BSA溶解于4mL碳酸盐缓冲溶液(CBS)(50mM，pH 9.6)，将活化好的E2-CME溶液缓慢滴加到CBS溶液中继续搅拌反应12h。反应结束后，将混合物转移到透析袋中并放入1L PBS(10mM，pH7.4)溶液中进行透析。每12h更换一次透析液，连续透析3天。透析结束后，分装冷冻保存，即得E2-BSA偶联物。
1.3长余辉粒子修饰
称取30mg长余辉粒子PLPs，并加5mL无水乙醇于10mL离心管中超声分散。在50mL圆底烧瓶中加入20mL无水乙醇，5mL水，0.13mL正硅酸四乙酯(TEOS)，在搅拌条件下迅速将长余辉无水乙醇溶液加入到圆底烧瓶中，超声10min使得溶液充分混匀。然后向溶液中加入0.5mL氨水(28-30％)，超声30min后转移到磁力搅拌器上搅拌反应7.5h。反应结束后，用无水乙醇离心(10000rpm，10min)洗涤3次，所得的产物真空干燥，备用。将25mg二氧化硅封装后的长余辉粒子超声分散至25mL水 中，在电磁搅拌条件下，向反应液中加入100μL羧乙基硅烷三醇钠(carboxyethylsilanetriol sodium salt，CES)，然后在室温下持续搅拌24h。搅拌结束后离心分离，沉淀用超纯水洗涤2次，无水乙醇洗涤2次，室温真空干燥，得羧基修饰的长余辉粒子，备用。
1.4E2-BSA-PLPs复合物制备
以活性酯法将E2-BSA与修饰了羧基的长余辉粒子进行偶联制备E2-BSA-PLPs复合物，具体方法为：将被羧基修饰过的长余辉粒子超声分散至磷酸缓冲盐溶液PBS中；然后加入6mg 1-3-二甲氨基丙基-3-乙基碳二亚胺盐酸盐EDC·HCl和15mg N-羟基琥珀酰亚胺NHS，室温条件下持续搅拌后离心，除去上清液，并用PBS洗涤；再加入1mL浓度为1mg/mL的E2-BSA，持续搅拌过夜，通过离心收集偶联产物，随后用PBS洗涤后再复溶于PBS中备用。产物以2mg/mL(以长余辉粒子浓度计算)在PBS溶液中储存备用。
1.5检测区(T区)溶液的配制
用10mM PBS溶液将E2-BSA-PLPs稀释成2mg/mL。
1.6质控区(C区)溶液的配制
用10mM PBS溶液将羊抗鼠二抗稀释成0.1mg/mL。
2.试纸条的制备
按照图1的模组合方式，将NC膜粘贴在PVC底板中间，样品垫与吸水垫分别搭接于NC膜左右两端，将搭建好的大卡切割成3mm宽度的纸条，即得到空白试纸条。分别在图1中T区和C区滴加1.0μL T区溶液和0.5μL C区溶液。将点样之后的试纸条放在烘箱中，在37℃条件下干燥30min，于真空袋中保存备用。
3.工作曲线绘制
用甲醇配置10mL 1mg/mL E2标准溶液，用10mM PBS稀释到浓度为0mg/mL、0.1mg/mL、0.5mg/mL、1mg/mL、5mg/mL、10mg/mL、20mg/mL，作为待测液备用。将80μL待测液与20μL CG-mAb和10μL运行缓冲液(10mmol/L磷酸盐缓冲液，含10％蔗糖，8％BSA，0.25％吐温-20，pH 7.4)在离心管中混合5min，然后将试纸条***离心管，25min后读取结果，显色模式的结果如图4A，相应的工作曲线为图3A，TRF模式的结果如图4B，相应的工作曲线为图3B。结果显示，显色模式下，阴性和低浓度阳性试纸T区呈现明显***，E2浓度增加至5ng/mL时T区颜色变淡，10ng/mL时 T区颜色消失(图4A)，以此浓度为显色法检测限；TRF模式下，阴性T区无明显荧光，E2浓度为0.1ng/mL时T区发出微弱的光(图4B)，以此浓度为TRF法检测限。因此，所构建的TRF型试纸的检测限可以达到0.1mg/mL。C区作为验证试纸结果有效性的参照，始终显红色、不发荧光。
4.样品预处理
量取1mL液相色谱-质谱联用(HPLC-MS)检测结果显示未检出***的饮用水水样，分别加入800μL浓度为0mg/mL、0.10mg/mL、1mg/mL、10mg/mL的E2标准溶液，作为待测液备用。
5.样品检测
将80μL待测液与20μL CG-mAb和10μL运行缓冲液(10mmol/L磷酸盐缓冲液，含10％蔗糖，8％BSA，0.25％吐温-20，pH 7.4)在离心管中混合5min，然后将试纸条***离心管，25min后读取显色结果。显色完成后，将试纸条置于紫外灯下激发，使用智能手机获取紫外光源关闭前后的荧光图像，读取荧光结果。结果如图2所示，可以检出0.1ng/mL及以上加标E2。而且，分析可在约30分钟内完成。
实施例2：***快速时间分辨荧光、显色双信号免疫层析试纸条检测牛奶，包括以下步骤：
1.试纸材料的制备
同实施例1
2.试纸条的制备
同实施例1
3.工作曲线绘制
同实施例1
4.样品预处理
牛奶样品(HPLC-MS检测结果显示未检出***)在10℃经过8000r/min离心10min后弃掉上层乳脂，获得脱脂牛奶。用去离子水按照体积比1:20稀释，过滤，量取4份(每份1mL)于10mL的离心管中，作为待测液备用。
5.样品检测
将80μL待测液与20μL CG-mAb和10μL运行缓冲液(10mmol/L磷酸盐缓冲液，含10％蔗糖，8％BSA，0.25％吐温-20，pH 7.4)在离心管中混合5min，然后将试纸 条***离心管，25min后读取显示结果。显色完成后，将试纸条置于紫外灯下激发，使用智能手机获取紫外光源关闭前后的荧光图像，读取荧光结果。结果显示为低于定量限，即低于检出限。
实施例3：***快速时间分辨荧光、显色双信号免疫层析试纸条检测猪肉，包括以下步骤：
1.试纸材料的制备
同实施例1
2.试纸条的制备
同实施例1
3.工作曲线绘制
同实施例1
4.样品预处理
称取剔除了骨、猪皮的猪肉(HPLC-MS检测结果显示未检出***)肌肉组织样本100g，用搅碎机将猪肉样品搅碎到一定程度后取适量肉样于组织捣碎匀浆机中搅成肉糜状，分别称取肉糜状的猪肉4份(每份1g)于离心管中，加入乙酸乙酯3ml，无水氧化钙0.1g，用漩涡振荡器于最大振荡速度下振荡2min，离心机在4000rpm离心min，移取上清液于另一试管，氮气吹干，加入1mL正丁烷溶解残渣，加入PH＝7.0的PBS-甲醇溶液(3:2)0.5mL，漩涡振荡器振荡2min，再用离心机在4000rpm离心10min，吸取除去上层正丁烷层，取下层溶液作为待测液。
5.样品检测
将80μL待测液与20μL CG-mAb和10μL运行缓冲液(10mmol/L磷酸盐缓冲液，含10％蔗糖，8％BSA，0.25％吐温-20，pH 7.4)在离心管中混合5min，然后将试纸条***离心管，25min后读取显色结果。显色完成后，将试纸条置于紫外灯下激发，使用智能手机获取紫外光源关闭前后的荧光图像，读取荧光结果。结果显示为低于定量限，即低于检出限。
实施例1说明了该试纸条可以检出0.1ng/mL及以上加标E2。而且，分析可在约30分钟内完成，检测时间较短。试纸条及相关检测用材料存放条件温和，可应用场景广泛，能适应不同的场合使用，如实施例2、3。
综上，本发明的试纸条及相应的检测装置具有良好的***检测性能及适用性，同 理，也可推广至与***具有相同的酚羟基结构且具有***效应的***以及类***，如雌三醇、雌酮、双酚A、己烯雌酚、炔雌醇等，使用时将对应***单克隆单体相应替换为与各***对应的***单克隆单体即可。这些***结构中的酚羟基可以通过一系列化学反应衍生出羧基，然后通过此羧基将***偶联在载体上。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种***的时间分辨荧光、显色双信号试纸条，其特征在于：所述试纸条结构为PVC底板上沿水平方向依次交叠粘贴有样品垫、硝酸纤维素膜即NC膜、吸收垫，所述NC膜两端位于交叠下层；
    所述NC膜包括检测区即T区、质控区即C区；
    所述检测区上固定有***-牛血清蛋白-长余辉粒子E-BSA-PLPs，所述***为具有酚羟基且具有***效应的***化合物以及类***化合物，包括***、雌三醇、雌酮、双酚A、己烯雌酚、炔雌醇；
    所述质控区上固定有二抗。
  2. 根据权利要求1所述的***的时间分辨荧光、显色双信号试纸条，其特征在于：所述质控区固定的二抗包括但不限于羊抗鼠二抗、兔抗鼠二抗、羊抗兔二抗、驴抗兔二抗。
  3. 根据权利要求1所述的***的时间分辨荧光、显色双信号试纸条的制备方法，其特征在于：制备步骤如下：
    (1)合成***-牛血清蛋白偶联物E-BSA；
    (2)对长余辉粒子PLPs进行羧基修饰；
    (3)制备偶联E-BSA的长余辉粒子复合物E-BSA-PLPs；
    (4)构建试纸：T区滴加E-BSA-PLPs，C区滴加针对一抗来源的二抗，干燥后于真空袋中保存备用。
  4. 如权利要求3所述的***的时间分辨荧光、显色双信号试纸条的制备方法，其特征在于，所述的步骤(3)使用活性酯法进行生物偶联，具体方法为：将被羧基修饰过的长余辉粒子超声分散至磷酸盐缓冲溶液PBS中；然后加入1-3-二甲氨基丙基-3-乙基碳二亚胺盐酸盐EDC·HCl和N-羟基琥珀酰亚胺NHS，室温条件下持续搅拌后离心，除去上清液，并用PBS洗涤；再加入E-BSA，持续搅拌过夜，通过离心收集偶联产物，随后用PBS洗涤后再复溶于PBS中备用。
  5. 如权利要求3所述的***的时间分辨荧光、显色双信号试纸条的制备方法，其特征在于：所述步骤(4)中，T区滴加含有0.5-5mg/mL E-BSA-PLPs的5-20mM PBS溶液，C区滴加含有0.05-0.5mg/mL二抗的5-20mM PBS溶液。
  6. 如权利要求3所述的***的时间分辨荧光、显色双信号试纸条的制备方法，其特征在于：所述的步骤(2)采用***羧甲基醚E-CME、BSA通过活性酯法合成E-BSA， 所述步骤(3)采用长余辉粒子PLPs、羧乙基硅烷三醇钠CES，进行羧合反应得羧基修饰的长余辉粒子。
  7. 一种用于检测***的检测装置，其特征在于：包括试纸条和***单克隆抗体修饰的胶体金CG-mAb，所述试纸条为根据权利要求1或2所述的，或权利要求3-6任一所述方法制备的***的时间分辨荧光、显色双信号试纸条，所述检测装置以试纸条上的***-牛血清蛋白-长余辉粒子复合物为荧光供体，以***单克隆抗体修饰的胶体金为荧光受体。
  8. 根据权利要求7所述的用于检测***的检测装置，其特征在于：C区作为验证试纸结果有效性的参照，始终显红色、无荧光；T区的时间分辨荧光强度与样品中***含量呈正相关，显色强度与样品中***含量呈负相关，具体为：
    当样品不含***时，T区显红色、荧光猝灭；
    当样品中含有***时，T区显浅红色甚至消线、荧光增强。
  9. 根据权利要求7所述的用于检测***的检测装置，其特征在于：该检测装置的应用方法如下：将待测样品与CG-mAb置于运行缓冲液中混合3-10min，放入试纸条，15-30min后读取显色结果；所述运行缓冲液为8-15mmol/L包含5-15％蔗糖、6-10％BSA、0.15-0.30％吐温-20的pH 6.5-8.0的缓冲液，所述缓冲液包括但不限于磷酸盐缓冲液、硼酸盐缓冲液、碳酸盐缓冲液；
    显色完成后，将试纸条置于紫外灯下激发，使用荧光图像采集设备获取紫外光源关闭前后的荧光图像，读取荧光结果。
  10. 根据权利要求9所述的用于检测***的检测装置，其特征在于：所述荧光图像采集设备包括智能手机，所述智能手机以连拍模式获取紫外光源关闭前后的荧光图像，获得时间分辨荧光照片。
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