Method for detecting parathyroid hormone using aggregation-induced emission liquid crystal-polymer composite film

Abstract

The application discloses a method for detecting small molecules based on an aggregation-induced emission liquid crystal-polymer composite film, and applies the method to the detection of parathyroid hormone. The method introduces TPE-PPE molecules on the basis of traditional liquid crystal detection, prepares luminescent liquid crystals for the detection of substances, and compares the detection results of the fluorescence optical double channel. The optical image can only detect parathyroid hormone above 10 ug / mL, while the sensitivity of the fluorescence to the orientation of the liquid crystal molecules realizes the change of the aggregation state of the AIE, so that the fluorescence detection of the AIE liquid crystal (AIE-LCs) can realize the detection of 1 ng / mL. In addition, 5wt% of the prepolymer is introduced, and ultraviolet irradiation is performed for 8 s. Since the amorphous structure is formed on the surface of the protein, the detection signal is amplified, and the optical and fluorescence detection limits are simultaneously reduced to 0.01 ug / mL and 50 pg / mL respectively.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of parathyroid hormone detection, in particular a detection method based on the use of aggregation-induced emission liquid crystal-polymer composite film for detecting parathyroid hormone. BACKGROUND

[0002] Parathyroid hormone is a basic single-chain polypeptide hormone secreted by parathyroid chief cells, which is composed of 84 amino acids, has a molecular weight of about 9500 Da, and a molecular formula of C 408 H 674 N 126 O 126 S2. The function of parathyroid hormone is related to the maintenance of blood calcium levels, and its main effects include promoting bone absorption of calcium, inhibiting calcium excretion in the renal tubule, etc. Parathyroid gland tumors, hyperplasia, adenocarcinoma, etc. can cause abnormal increase of parathyroid hormone secretion, leading to metabolic disorders such as high blood calcium and low blood phosphorus in the body. Chronic hemodialysis patients due to chronic renal failure, vitamin and mineral metabolism disorders, leading to decreased blood calcium and increased blood phosphorus in the body, stimulating the parathyroid gland, thereby causing an increase in parathyroid hormone synthesis and secretion, and in severe cases, can lead to secondary hyperparathyroidism. Therefore, controlling the level of parathyroid hormone can provide important guidance for the continuous clinical management of chronic kidney disease patients; in addition, monitoring the level of parathyroid hormone is also the basis for the success or failure of parathyroid surgery.

[0003] With the development of science and technology, many researchers have explored many new methods, including electrochemical biosensing, spectral property detection, and immunoreagent chromatography boxes, etc. The results show that each generation of methods has certain limitations, such as the need for markers, complex structure, etc. While the traditional liquid crystal detection has indeed overcome these defects, it can only achieve qualitative and simulated quantitative detection of the analyte. In this patent, aggregation-induced emission liquid crystal is used for precise quantitative detection of parathyroid hormone, and the light-emitting liquid crystal-polymer film formed under ultraviolet light of the pre-polymer is used to reduce the detection limit. SUMMARY

[0004] The purpose of the present application is to provide a detection method for detecting parathyroid hormone by using aggregation-induced emission liquid crystal-polymer composite film, in order to solve the problems raised in the background art.

[0005] To solve the above technical problems, the present application discloses a method for double-channel qualitative and quantitative detection of small molecule test substances based on aggregation-induced emission liquid crystal-polymer composite film.

[0006] A detection method for detecting parathyroid hormone by using aggregation-induced emission liquid crystal-polymer composite film, comprising double-channel qualitative and quantitative detection of small molecule test substances by using light-emitting liquid crystal-polymer composite film.

[0007] Further, the preparation method of the light-emitting liquid crystal-polymer composite film comprises filling the prepolymer-light-emitting liquid crystal into a liquid crystal empty box, and irradiating the liquid crystal empty box with ultraviolet light to obtain the light-emitting liquid crystal-polymer composite film.

[0008] Further, the preparation method of the liquid crystal empty box comprises cleaning, immersing the upper glass sheet into a DMOAP solution, immersing the lower glass sheet into a mixed APTES and DMOAP aqueous solution first, and then immersing the lower glass sheet into a glutaraldehyde aqueous solution, preparing a biological recognition layer, and sealing to obtain the liquid crystal empty box.

[0009] Further, the preparation method of the prepolymer-light-emitting liquid crystal comprises mixing the nematic liquid crystal and the aggregation-induced emission molecule, and then adding the prepolymer to obtain the prepolymer-light-emitting liquid crystal.

[0010] Further, the mass ratio of the aggregation-induced emission molecule to the nematic liquid crystal is 0.1wt%, and the mass ratio of the prepolymer to the light-emitting liquid crystal is 5wt%.

[0011] Further, the weight percentage ratio of the APTES and the DMOAP is 4:1, and the glutaraldehyde aqueous solution is 2% v / v.

[0012] Further, the preparation of the biological recognition layer comprises sequentially assembling avidin, parathyroid hormone antibody, and parathyroid hormone.

[0013] Further, the concentration of the avidin is 1 μg / mL, and the concentration of the parathyroid hormone antibody is 1 μg / mL.

[0014] Further, the light-emitting liquid crystal-polymer composite film is obtained by irradiating the liquid crystal empty box with ultraviolet light for 8 seconds.

[0015] Further, the prepolymer is an epoxy resin glue.

[0016] In a second aspect, the present application further provides a preparation method of an aggregation-induced emission liquid crystal-polymer composite film, which comprises mixing nematic liquid crystal, aggregation-induced emission molecule, and prepolymer to prepare a prepolymer-light-emitting liquid crystal mixture, and then performing ultraviolet light curing to form a film.

[0017] The nematic liquid crystal is SLC1717 (melting point Tm=-20℃, clearing point Tc=92.0℃, dielectric anisotropy parameter Δε=12.2 at room temperature (25℃), and birefringence 0.22), and the aggregation-induced emission molecule is TPE-PPE. NI

[0018] The method for detecting parathyroid hormone by fluorescence-optical dual-channel detection comprises the following steps:

[0019] ​(1) immerging clean glass into APTES and DMOAP mixed aqueous solution, constant temperature reaction; then immerging it into glutaraldehyde aqueous solution, room temperature reaction;

[0020] (2) adding avidin to the aldehyde group treated lower glass surface;

[0021] (3) adding biotinylated parathyroid hormone antibody to the avidin assembled lower glass surface;

[0022] (4) adding parathyroid hormone to the glass piece fixed with antibody;

[0023] (5) making the treated glass surface into a liquid crystal cell, and detecting by using a polarizing microscope and a fluorescence spectrometer.

[0024] Further, the weight percentage ratio of APTES and DMOAP is 4:1.

[0025] Further, the optimal avidin concentration is 1 μg / mL.

[0026] Further, the optimal antibody concentration is 1 μg / mL.

[0027] Further, the optimal ultraviolet irradiation time is 8 s.

[0028] Compared with the prior art, the present application provides a method for double-channel qualitative and quantitative detection of small molecule test substances based on an aggregation-induced emission liquid crystal-polymer composite film, which has the following beneficial effects:

[0029] (1) The present application introduces aggregation-induced emission molecules, which are sensitive to the arrangement of liquid crystal molecules, reduce the detection limit, and realize accurate quantification of test substances. The change of the aggregation state of AIE realizes the fluorescence detection of AIE liquid crystals (AIE-LCs) at 1 ng / mL.

[0030] (2) The present application designs and synthesizes an aggregation-induced emission liquid crystal-polymer composite film, which amplifies the detection signal of test substances by using a prepolymer, further reducing the detection limit. In the future, this material is expected to break through single-point detection and realize continuous detection of test substances.

[0031] (3) The present application also introduces 5wt% of prepolymer, and ultraviolet irradiation for 8 s. Due to the formation of amorphous structure on the surface of the protein, the detection signal is amplified, and the optical and fluorescence detection limits are simultaneously reduced to 0.01 μg / mL and 50 pg / mL, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1Optical image and fluorescence curve image of the prepared prepolymer-luminescent liquid crystal system under different concentrations and UV irradiation time;

[0033] Figure 2 Optical image and fluorescence curve image under different concentrations of avidin;

[0034] Figure 3 Contact angle image and fluorescence image contrast under different concentrations of avidin;

[0035] Figure 4 Optical image and fluorescence curve image under different concentrations of parathyroid hormone antibody;

[0036] Figure 5 Contact angle image under different concentrations of antibody;

[0037] Figure 6 Optical image and fluorescence curve image of the glass surface with 1 μg / mL antibody after filling the prepolymer-luminescent liquid crystal under different UV irradiation time;

[0038] Figure 7 Optical image and fluorescence curve image under different concentrations of parathyroid hormone before and after UV irradiation;

[0039] Figure 8 Optical image and fluorescence curve image under different concentrations of bovine serum albumin. DETAILED DESCRIPTION

[0040] Further embodiments of the present application are described below with reference to the accompanying drawings, in which it is understood that the further embodiments described herein are merely illustrative and are not intended to limit the present application. It is to be understood that the drawings are not to scale and that, unless otherwise indicated, the drawings are intended to conceptually illustrate the structures, materials, and procedures described herein.

[0041] In addition, the descriptions such as "first", "second", etc. in the present application are only for the purpose of description, and do not mean to particularly indicate the order or sequence, nor to limit the present application. They are merely used to distinguish components or operations described by the same technical terms, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In addition, the technical solutions and technical features of various embodiments can be combined with each other, but it must be based on the realization of ordinary skilled in the art. When the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor within the scope of protection required by the present application.

[0042] Unless otherwise specified, the examples and comparative examples are parallel tests with the same components, component contents, preparation steps and preparation parameters. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are analytical reagents (A.R.) unless otherwise specified. The parts are weight parts unless otherwise specified. The materials are commercially available.

[0043] The chemical structure of TPE-PPE is as follows:

[0044]

[0045] The prepolymer is an epoxy resin adhesive, which is purchased from Shenzhen Tegon New Material Co., Ltd. with the brand: Lantian LEAFTOP, and the epoxy value equivalent is 195±10 g / mol, and the amine value is 280±10 mgKOH / g.

[0046] DMOAP and APTES are purchased from sigmaaldrich,

[0047] The buffer is purchased from Thermo Fisher Scientific Company, and the avidin is purchased from Abcam (Shanghai) Trading Co., Ltd.

[0048] Example 1

[0049] Preparation of the prepolymer-luminescent liquid crystal:

[0050] 99.9 parts of SLC1717 and 0.1 part of TPE-PPE are mixed to obtain a luminescent liquid crystal system by weight parts. A prepolymer is added to the prepared luminescent liquid crystal system in a proportion of 5wt%, to obtain a prepolymer-luminescent liquid crystal.

[0051] Preparation of the liquid crystal empty box:

[0052] The upper and lower glass sheets of the liquid crystal cell were cleaned by ultrasonic cleaning with detergent for 30 min, with 7% NaOH solution for 15 min, and with deionized water and ethanol for 5 min. The cleaned upper glass sheet was immersed in a 0.2 wt% DMOAP solution, and left to stand at room temperature for 30 min. The glass sheet was then cleaned, dried and dried at 100°C for 15 min. The cleaned lower glass sheet was immersed in a mixture of 4 wt% APTES and 1 wt% DMOAP, and left to stand at 80°C for 1 h. The glass sheet was then cleaned, dried and dried at 110°C for 1 h. The glass sheet was then immersed in a 2% (v / v) glutaraldehyde solution, and left to stand at room temperature for 1 h. The glass sheet was then cleaned, dried and dried at 100°C for 15 min. The biotin was dissolved in buffer to prepare a biotin solution of different concentrations. One μL of the biotin solution was dropped onto the aldehyde group-modified lower glass sheet, and left to stand at room temperature for 2 h. The glass sheet was then cleaned, soaked in 0.1 M NaBH3CN for 5 min, and dried. The parathyroid hormone antibody was dissolved in buffer to prepare a parathyroid hormone antibody solution of different concentrations. One μL of the parathyroid hormone antibody solution was dropped onto the surface of the biotin-modified lower glass sheet, and left to stand at room temperature for 2 h. The glass sheet was then cleaned and dried. The parathyroid hormone was dissolved in buffer to prepare a parathyroid hormone solution of different concentrations. One μL of the parathyroid hormone solution was dropped onto the surface of the parathyroid hormone antibody-modified glass sheet, and left to stand at room temperature for 2 h. The glass sheet was then cleaned and dried. The upper glass sheet was then matched with the immunoreacted lower glass sheet, with a 12 μm polyester spacer in between, to form a liquid crystal cell. The cell was then sealed with UV curing glue on both sides, and fixed with a long-tail clip. The cell was then irradiated with UV light for 5 min to obtain a liquid crystal cell.

[0053] Preparation of the light-emitting liquid crystal-polymer composite film

[0054] The pre-polymer-light-emitting liquid crystal was filled into the liquid crystal cell, and irradiated with UV light for 8 s to obtain the light-emitting liquid crystal-polymer composite film.

[0055] Example 2

[0056] The difference between this example and Example 1 is that the pre-polymer-light-emitting liquid crystal system was prepared by mixing 99.9 parts of SLC1717 with 0.1 part of TPE-PPE by weight. The prepared pre-polymer-light-emitting liquid crystal was filled into the liquid crystal cell, and irradiated with UV light for 30 s to obtain the light-emitting liquid crystal-polymer composite film. The preparation steps of the liquid crystal cell were the same as those of Example 1.

[0057] Example 3

[0058] Different from Example 1, the preparation of the prepolymer-luminescent liquid crystal system: 99.9 parts of SLC1717 and 0.1 part of TPE-PPE were mixed to obtain a luminescent liquid crystal system by weight parts, 4wt% of prepolymer was added to the prepared luminescent liquid crystal system, the prepared prepolymer-luminescent liquid crystal was filled into a liquid crystal cell, and a luminescent liquid crystal-polymer composite film was prepared by ultraviolet irradiation for 20s. The preparation steps of the liquid crystal empty box are consistent with those of Example 1.

[0059] Performance test

[0060] As Figure 1 shown, the prepared luminescent liquid crystal-polymer composite film was irradiated by ultraviolet light of 5mW / cm 2 for 0s, 10s, 20s and 30s, and the optical image and fluorescence curve image of the prepolymer-luminescent liquid crystal were presented, and the prepolymer concentration was 3wt%, 4wt% and 5wt% respectively. The lower concentration (3wt%) of doping still maintained the dark texture without obvious change until 30s. The optical texture of the luminescent liquid crystal-prepolymer system with a concentration of 4wt% and 5wt% became brighter at 30s and 20s respectively, showing a bright background signal. Then the prepolymer concentration and ultraviolet time were optimized to control the polymerization degree of the prepolymer through fluorescence data. Figure 1 A, the best prepolymer concentration is 5wt%, and the best ultraviolet irradiation time is 10s.

[0061] Figure 2 A shows the optical signal images of different avidin concentrations. With the gradual decrease of avidin concentration, the liquid crystal texture gradually changes from bright to dark. To ensure the maximum antibody fixation rate, 1μg / mL of avidin is selected as the best. In addition, as Figure 2 B shows that when the avidin concentration increases, the disturbance to the liquid crystal molecules increases, resulting in a decrease in fluorescence intensity. It is worth noting that although the optical texture remains dark at an avidin concentration of 1μg / mL, the fluorescence intensity has already decreased to a certain extent, indicating that a slight change in the orientation of the liquid crystal molecules can cause a change in the fluorescence intensity, i.e. the detection of fluorescence is more sensitive than the detection of light.

[0062] In order to prove that avidin is indeed fixed on the glass surface, the contact angle data Figure 3 A) was measured. Due to the presence of hydrophilic amino groups on the surface of the protein, the higher the concentration of avidin, the smaller the contact angle of the glass surface. In addition, avidin (FITC) with a fluorescent molecule was purchased and fixed on the glass surface. Figure 3 B can be clearly seen that the glass surface with avidin shows a fluorescence image, while the blank glass surface has no fluorescence area.

[0063] To obtain the best concentration of biotinylated PTH antibody, further investigation was carried out on different concentrations of antibody, such as Figure 4 A. When the concentration of PTH antibody reached 1 μg / mL, the optical image showed dark regions, but when the concentration of antibody was further increased, large bright regions could be clearly seen. Figure 4 B shows the fluorescence curve image, and the fluorescence intensity is the highest when the concentration of antibody is 1 μg / mL. In order to fix as much antibody as possible on the substrate to provide more bioactive groups and maintain a high fluorescence intensity, the concentration of antibody is finally fixed at 1 μg / mL, so as to realize the improvement of the sensitivity of subsequent detection of parathyroid hormone.

[0064] Subsequently, the contact angle of the glass surface fixed with different concentrations of parathyroid hormone antibody was tested, and the experimental results are shown in Figure 5 A. With the increase of antibody concentration, the contact angle showed a downward trend, which was consistent with the theoretical results, indicating that the antibody was successfully fixed on the surface of the glass substrate.

[0065] In order to avoid false positive signals, it is necessary to investigate the ultraviolet irradiation time of the immobilized antibody, control the polymerization degree of the prepolymer, so that the antibody will not destroy the vertical alignment of the liquid crystal, and the optical texture dark and the fluorescence intensity are good to the greatest extent without parathyroid hormone. As shown in Figure 6 A, the original optical texture without irradiation of ultraviolet light shows a complete dark region, and when the irradiation time continues to 8 s, it still shows obvious dark optical texture. However, when the irradiation time continues to 9 s, the optical texture becomes bright, showing a large bright region, i.e. the critical value is 8 s. Similarly, the fluorescence intensity under different irradiation times was investigated, and the experimental results are shown in Figure 6 B. Under the condition that the optical texture is roughly the same, the fluorescence intensity is significantly different, which proves the sensitivity of fluorescence detection. In summary, the antibody concentration of 1 μg / mL and the irradiation time of 8 s are the most appropriate, which can maintain the optical texture dark and the fluorescence intensity good at the same time.

[0066] Before ultraviolet irradiation, because the parathyroid hormone molecule is small, when the concentration of parathyroid hormone is lower than 10 μg / mL, the optical texture shows a dark texture, i.e. the minimum concentration of parathyroid hormone that can be distinguished is 10 μg / mL, as shown in Figure 7 A. Due to the higher sensitivity of fluorescence, the detection limit of parathyroid hormone is reduced to 100 pg / mL, as shown in Figure 7 B. After 8 s of ultraviolet irradiation with an intensity of 5 mW / cm 2 The detection limits of optical and fluorescence both decreased, as shown in Figure 7 C and 7D, respectively, to 0.01 μg / mL and 50 pg / mL.

[0067] Several concentrations of bovine serum albumin were also tested, and the results are shown in Table 1. Figure 8 The fluorescence intensity is not linearly related to the concentration of bovine serum albumin, i.e., the system has specificity.

[0068] As can be seen from the above, the detection method provided by the present application using the aggregation-induced emission liquid crystal-polymer composite film not only couples the aggregation-induced emission characteristics of the fluorescent molecules to quantitatively detect the to-be-detected substance, but also amplifies the detection signal of the to-be-detected substance through the polymer, further reduces the detection limit, and provides a possibility for the detection of small molecule substances.

[0069] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. If these modifications and variations of the present application belong to the scope of the claims of the present application and the equivalent technologies thereof, the present application also intends to include these modifications and variations.

Claims

1. A detection method for detecting parathyroid hormone by using an aggregation-induced emission liquid crystal-polymer composite film, comprising using the luminescent liquid crystal-polymer composite film to perform double-channel qualitative and quantitative detection on a small molecule to-be-detected substance. The preparation method of the aggregation-induced emission liquid crystal-polymer composite film comprises the following steps: (1) preparation of a prepolymer-emitting liquid crystal, comprising: 99.9 parts of SLC1717 and 0.1 part of TPE-PPE are mixed to obtain an emitting liquid crystal system, and 5 wt% of the prepolymer is added to the prepared emitting liquid crystal system to obtain a prepolymer-emitting liquid crystal; (2) preparation of a liquid crystal empty cell, comprising: The upper and lower glass sheets of the liquid crystal cell are ultrasonically cleaned with detergent for 30 min, ultrasonically cleaned with a prepared 7% NaOH solution for 15 min, and alternately ultrasonically cleaned with deionized water and ethanol for 5 min; the cleaned upper glass sheet of the liquid crystal cell is immersed in a 0.2 wt% DMOAP solution, and is left to stand at room temperature for 30 min, cleaned, dried, and dried at 100 ℃ for 15 min; the cleaned lower glass sheet of the liquid crystal cell is immersed in a mixed aqueous solution of 4 wt% APTES and 1 wt% DMOAP, and is kept at 80 ℃ for 1 h, cleaned, dried, and dried at 110 ℃ for 1 h; then the lower glass sheet is immersed in a 2% (v / v) glutaraldehyde aqueous solution, and is reacted at room temperature for 1 h, cleaned, and dried; avidin is prepared into different concentrations of avidin solutions by mixing with a buffer; 1 μL of the avidin is added dropwise to the aldehyde group-modified lower glass sheet, and is reacted at room temperature for 2 h, cleaned, soaked in 0.1 M NaBH3CN for 5 min, cleaned, and dried; parathyroid hormone antibodies are dissolved in a buffer to prepare different concentrations of parathyroid hormone antibody solutions; 1 μL of the antibody solution is added dropwise to the surface of the lower glass sheet assembled with avidin, and is reacted at room temperature for 2 h, cleaned, and dried; parathyroid hormone is dissolved in a buffer to prepare different concentrations of parathyroid hormone solutions; 1 μL of the parathyroid hormone solution is added dropwise to the surface of the glass sheet on which the parathyroid hormone antibodies are fixed, and is reacted at room temperature for 2 h, cleaned, and dried; the upper glass sheet after self-assembly is matched with the lower glass sheet after immunoreaction in a face-to-face mode, and a 12 μm polyester gasket is used to separate them to form a liquid crystal cell; then, the two sides are sealed with ultraviolet curing glue, and are fixed by long-tail clips, ultraviolet light is irradiated for 5 min, and the liquid crystal empty cell is obtained; (3) preparation of an emitting liquid crystal-polymer composite film: The prepolymer-emitting liquid crystal is filled into the liquid crystal empty cell, and an emitting liquid crystal-polymer composite film is prepared by irradiating ultraviolet light for 8 s.

2. The detection method according to claim 1, characterized in that, The concentration of the avidin is 1 μg / mL, and the concentration of the parathyroid hormone antibody is 1 μg / mL.

3. The method of claim 1, wherein, The prepolymer is an epoxy resin glue.

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