Detection method for detecting parathyroid hormone by utilizing aggregation-induced emission liquid crystal-polymer composite membrane

Abstract

The invention discloses a dual-channel qualitative and quantitative detection method for a small-molecule to-be-detected substance based on an aggregation-induced emission liquid crystal-polymer composite membrane, and application of the dual-channel qualitative and quantitative detection method to detection of parathyroid hormone. According to the method, TPE-PPE molecules are introduced on the basis of traditional liquid crystal detection, the luminous liquid crystal is prepared to be used for substance detection, and detection results of fluorescent optical dual channels are compared. An optical image can only detect parathyroid hormone with the concentration of 10 mu g / mL or above, and the change of the aggregation state of AIE is realized by utilizing the sensitivity of fluorescence to liquid crystal molecule orientation, so that the fluorescence detection of AIE-LCs (AIE-LCs) can realize the detection of 1ng / mL. Besides, 5wt% of prepolymer is introduced, ultraviolet irradiation is carried out for 8s, an amorphous structure is formed on the surface of the protein, a detection signal is amplified, and the optical detection limit and the fluorescence detection limit are synchronously reduced and respectively reach 0.01 mu g / mL and 50pg / mL.

Description

Technical Field

[0001] The invention relates to the field of parathyroid hormone detection, in particular to a detection method for parathyroid hormone based on utilizing an aggregation-induced luminescence liquid crystal-polymer composite film. Background Art

[0002] Parathyroid hormone is a basic single-chain polypeptide hormone secreted by parathyroid chief cells. It is composed of 84 amino acids, with a molecular weight of about 9500Da and a molecular formula of C 408 H 674 N 126 O 126 S2. The role of parathyroid hormone involves maintaining blood calcium levels. Its main effects include promoting calcium absorption by bones and inhibiting calcium excretion by renal tubules. Parathyroid adenomas, hyperplasia, adenocarcinomas, etc. can all cause abnormal increases in parathyroid hormone secretion, leading to metabolic disorders such as hypercalcemia and hypophosphatemia. Long-term hemodialysis patients have chronic renal failure, vitamin and mineral metabolism disorders, which lead to low blood calcium and high blood phosphorus in the body, stimulating the parathyroid glands, thereby increasing the synthesis and secretion of parathyroid hormone, which can lead to secondary hyperparathyroidism in severe cases. Therefore, controlling parathyroid hormone levels can provide important guidance for the continuous clinical management of patients with chronic kidney disease; in addition, monitoring parathyroid hormone levels 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 biosensors, spectral property detection, and immunoassay chromatography boxes. The results show that each generation of methods has certain limitations, such as the need for markers and complex structures. Although traditional liquid crystal detection has gotten rid of these defects, it can only achieve qualitative and simulated quantitative detection of analytes. This patent uses aggregation-induced luminescent liquid crystals for accurate quantification of parathyroid hormone, and uses the luminescent liquid crystal-polymer film formed under ultraviolet light of the prepolymer to reduce the detection limit. Summary of the invention

[0004] The object of the present invention is to provide a method for detecting parathyroid hormone using an aggregation-induced emission liquid crystal-polymer composite film, so as to solve the problems raised in the above-mentioned background technology.

[0005] In order to solve the above technical problems, the present invention discloses a dual-channel method for qualitative and quantitative detection of small molecule analytes based on aggregation-induced emission liquid crystal-polymer composite film.

[0006] A detection method for parathyroid hormone using an aggregation-induced luminescent liquid crystal-polymer composite film comprises using the luminescent liquid crystal-polymer composite film to perform dual-channel qualitative and quantitative detection on a small molecule analyte.

[0007] Furthermore, the method for preparing the luminescent liquid crystal-polymer composite film includes filling a liquid crystal empty box with prepolymer-luminescent liquid crystal, and obtaining the luminescent liquid crystal-polymer composite film by ultraviolet light irradiation.

[0008] Furthermore, the preparation method of the liquid crystal empty box includes cleaning, immersing the upper glass sheet in a DMOAP solution, immersing the lower glass sheet in a mixed aqueous solution of APTES and DMOAP, and then in a glutaraldehyde aqueous solution, preparing a biorecognition layer, and sealing to form a liquid crystal empty box.

[0009] Furthermore, the preparation method of the prepolymer-luminescent liquid crystal includes mixing nematic liquid crystal and aggregation-induced emission molecules, and then adding prepolymer to obtain the prepolymer-luminescent liquid crystal.

[0010] Furthermore, the mass ratio of the aggregation-induced emission molecules to the nematic liquid crystal is 0.1 wt %; and the mass ratio of the prepolymer to the luminescent liquid crystal is 5 wt %.

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

[0012] Furthermore, the preparation of the biorecognition layer includes sequentially assembling avidin, parathyroid hormone antibody and parathyroid hormone.

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

[0014] Furthermore, the luminescent liquid crystal-polymer composite film is obtained by irradiating with ultraviolet light for 8 seconds.

[0015] Furthermore, the prepolymer is epoxy resin glue.

[0016] In a second aspect, the present invention also provides a method for preparing an aggregation-induced emission liquid crystal-polymer composite film, wherein nematic liquid crystal, aggregation-induced emission molecules, and prepolymers are mixed to prepare a prepolymer-luminescent liquid crystal mixture, which is then cured by ultraviolet light to form a film.

[0017] The nematic liquid crystal is SLC1717 (melting point Tm is -20°C, clearing point (clearing point, the temperature at which the liquid crystal state changes to the isotropic state) T NI =92.0°C, the dielectric anisotropy parameter at room temperature (25°C) is Δε=12.2, and the birefringence is 0.22); the aggregation-induced emission molecule is TPE-PPE.

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

[0019] (1) Immerse the clean glass in a mixed aqueous solution of APTES and DMOAP and react at a constant temperature; then immerse it in a glutaraldehyde aqueous solution and react at room temperature;

[0020] (2) adding avidin dropwise to the aldehyde-modified lower glass surface;

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

[0022] (4) Add parathyroid hormone dropwise onto the glass slide with the antibody fixed thereon;

[0023] (5) The treated glass surface is made into a liquid crystal box and tested using a polarizing microscope and a fluorescence spectrometer.

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

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

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

[0027] Furthermore, the optimal ultraviolet light irradiation time is 8s;

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

[0029] (1) The present invention introduces aggregation-induced emission molecules, and utilizes their sensitivity to the arrangement of liquid crystal molecules to reduce the detection limit, while achieving accurate quantification of the analyte and realizing the change of the aggregation state of AIE, so that the fluorescence detection of AIE liquid crystals (AIE-LCs) can achieve 1 ng / mL detection;

[0030] (2) The present invention designs and synthesizes an aggregation-induced luminescent liquid crystal-polymer composite film, which uses prepolymer to amplify the detection signal of the analyte to further reduce the detection limit; in the future, this material is expected to break through single-point detection and achieve continuous detection of the analyte;

[0031] (3) The present invention also introduces 5 wt% of prepolymer and irradiates with ultraviolet light for 8 s. Due to the formation of an amorphous structure on the protein surface, the detection signal is amplified, and the simultaneous decrease of the optical and fluorescence detection limits is achieved, reaching 0.01 μg / mL and 50 pg / mL, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The optical images and fluorescence curve images of the prepared prepolymer-luminescent liquid crystal system under different concentrations and UV irradiation times;

[0033] Figure 2 Optical images and fluorescence curve images at different avidin concentrations;

[0034] Figure 3 Comparison of contact angle images and fluorescence images at different avidin concentrations;

[0035] Figure 4 Optical images and fluorescence curve images at different parathyroid hormone antibody concentrations;

[0036] Figure 5 Contact angle images at different antibody concentrations;

[0037] Figure 6 The optical images and fluorescence curve images of the glass surface immobilized with 1 μg / mL antibody at different UV irradiation times after filling the prepolymer-luminescent liquid crystal;

[0038] Figure 7 Optical images and fluorescence curve images of different parathyroid hormone concentrations before and after ultraviolet irradiation;

[0039] Figure 8 Optical images and fluorescence curve images at different bovine serum albumin concentrations. DETAILED DESCRIPTION

[0040] Further embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the further embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0041] In addition, in the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes, and do not specifically refer to the order or sequence, nor are they used to limit the present invention. They are only used to distinguish components or operations described with the same technical terms, and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions and technical features between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[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 (AR) unless otherwise specified. Parts are parts by weight unless otherwise specified, and the materials are purchased from commercial channels.

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

[0044]

[0045] The prepolymer is epoxy resin glue purchased from Shenzhen Tegu New Materials Co., Ltd., brand: Lantian LEAFTOP, epoxy value equivalent: 195±10g / mol, amine value: 280±10mgKOH / g;

[0046] DMOAP and APTES were purchased from SigmaAldrich.

[0047] Buffer was purchased from Thermo Fisher Scientific, and avidin was purchased from Abcam (Shanghai) Trading Co., Ltd.

[0048] Example 1

[0049] Preparation of prepolymer-luminescent liquid crystal:

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

[0051] Preparation of liquid crystal empty box:

[0052] Ultrasonic clean the upper and lower glass sheets of the liquid crystal box with detergent for 30 minutes, ultrasonic clean with the prepared 7% NaOH solution for 15 minutes, and ultrasonic clean with deionized water and ethanol alternately for 5 minutes. Immerse the clean upper glass sheet of the liquid crystal box in 0.2wt% DMOAP solution, let it stand at room temperature for 30 minutes, clean and blow dry, and dry at 100℃ for 15 minutes. Immerse the clean lower glass sheet of the liquid crystal box in a mixed aqueous solution of 4wt% APTES and 1wt% DMOAP, keep it at 80℃ for 1 hour, clean and blow dry, and dry at 110℃ for 1 hour. Then immerse it in a 2% (v / v) glutaraldehyde aqueous solution, react at room temperature for 1 hour, clean and blow dry. Prepare avidin solutions of different concentrations with buffer. Take 1μL of avidin and add it dropwise to the aldehyde-treated lower glass sheet, react at room temperature for 2 hours, clean, soak it in 0.1M NaBH3CN for 5 minutes, and clean and blow dry. Dissolve the biotinylated parathyroid hormone antibody in a buffer solution to prepare parathyroid hormone antibody solutions of different concentrations. Take 1 μL of the antibody solution and drop it on the surface of the lower glass sheet assembled with avidin, react at room temperature for 2 hours, wash and blow dry. Dissolve the parathyroid hormone in a buffer solution to prepare parathyroid hormone solutions of different concentrations. Take 1 μL of the parathyroid hormone solution and drop it on the glass surface fixed with the parathyroid hormone antibody, react at room temperature for 2 hours, wash and blow dry. The upper glass sheet after self-assembly and the lower glass sheet after immune reaction are matched face to face, separated by a 12 μm polyester gasket in the middle to form a liquid crystal box. Subsequently, seal both sides with ultraviolet curing glue, fix both sides with long-tailed ticket clips, and irradiate with ultraviolet light for 5 minutes to obtain a liquid crystal empty box.

[0053] Preparation of luminescent liquid crystal-polymer composite films:

[0054] The prepolymer-luminescent liquid crystal was filled into the empty liquid crystal box, and the luminescent liquid crystal-polymer composite film was prepared by irradiating the box with ultraviolet light for 8 seconds.

[0055] Example 2

[0056] The difference from Example 1 is the preparation of the prepolymer-luminescent liquid crystal system: by weight, 99.9 parts of SLC1717 and 0.1 parts of TPE-PPE are mixed to obtain a luminescent liquid crystal system, 3 wt% of a prepolymer is added to the prepared luminescent liquid crystal system, the prepared prepolymer-luminescent liquid crystal is filled into a liquid crystal box, and ultraviolet light is irradiated for 30 seconds to obtain a luminescent liquid crystal-polymer composite film. The preparation steps of the empty liquid crystal box are consistent with those in Example 1.

[0057] Example 3

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

[0059] Performance Testing

[0060] like Figure 1 As shown, the prepared luminescent liquid crystal-polymer composite film was exposed to 5 mW / cm 2 After ultraviolet irradiation for 0s, 10s, 20s, and 30s, the optical image and fluorescence curve image of the prepolymer-luminescent liquid crystal were presented. The prepolymer concentrations were 3wt%, 4wt%, and 5wt%, respectively. The doping with a lower concentration (3wt%) maintained a dark texture until 30s, without showing any obvious changes. However, 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. The prepolymer concentration and UV time were then optimized through fluorescence data to control the degree of polymerization of the prepolymer. Figure 1 A, the optimal prepolymer concentration is 5wt%, and the optimal UV irradiation time is 10s.

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

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

[0063] To obtain the optimal biotinylated PTH antibody concentration, different concentrations of the antibody were further investigated, e.g. Figure 4 As shown in A. When the PTH antibody concentration reaches 1 μg / mL, its optical image shows a dark area, but when the antibody concentration is further increased, a large bright area can be clearly seen. Figure 4 B shows the fluorescence curve image, and the fluorescence intensity is the highest when the antibody concentration is 1 μg / mL. In order to fix as many antibodies as possible on the substrate to provide more bioactive groups and maintain a high fluorescence intensity to improve the sensitivity of subsequent detection of parathyroid hormone, the antibody concentration was finally fixed at 1 μg / mL.

[0064] Subsequently, the contact angles of glass surfaces immobilized with different concentrations of parathyroid hormone antibodies were tested, and the experimental results were as follows: Figure 5 As shown in Figure 2, the contact angle decreased with the increase of antibody concentration, which was consistent with the theoretical results, indicating that the antibody was successfully immobilized on the surface of the glass substrate.

[0065] To avoid false positive signals, it is necessary to examine the UV irradiation time of the immobilized antibody, control the degree of polymerization of the prepolymer, so that the antibody does not destroy the vertical orientation of the liquid crystal, and keep the optical texture dark and the fluorescence intensity good to the greatest extent in the absence of parathyroid hormone. Figure 6 As shown in A, the original optical texture without UV irradiation showed a complete dark area, and when the irradiation time lasted to 8s, it still showed a clear dark optical texture. However, when the irradiation time lasted to 9s, its optical texture became brighter, showing a large bright area, that is, the critical value was 8s. Similarly, the fluorescence intensity under different irradiation times was examined, and the experimental results are shown in Figure 6 B. When the optical texture is roughly the same, the fluorescence intensity is significantly different, which proves the sensitivity of fluorescence detection. In summary, when the antibody concentration is 1μg / mL and the irradiation time is 8s, it is most appropriate to keep the optical texture dark and the fluorescence intensity good at the same time.

[0066] Before UV irradiation, due to the small size of PTH molecules, when the PTH concentration is lower than 10 μg / mL, the optical texture presents a dark texture. The minimum PTH concentration for discerning the texture is 10 μg / mL. Figure 7 As shown in A. Due to the higher sensitivity of fluorescence, the detection limit of parathyroid hormone is reduced to 100pg / mL. Figure 7 As shown in B. When the UV irradiation intensity is 5mW / cm 2 After 8s of irradiation, both the optical and fluorescence detection limits decreased, e.g. Figure 7 C and 7D, which were decreased to 0.01 μg / mL and 50 pg / mL, respectively.

[0067] Several BSA concentrations were also tested, and the results were as follows: Figure 8 , there is no linear relationship between the fluorescence intensity and the concentration of bovine serum albumin, that is, the system is specific.

[0068] From the above, it can be seen that the detection method provided by the present invention using the aggregation-induced emission liquid crystal-polymer composite film not only couples the aggregation-induced emission characteristics of fluorescent molecules to quantitatively detect the substance to be tested, but also can amplify the detection signal of the substance to be tested through the polymer, further reducing the detection limit, and providing a possibility for the detection of small molecule substances.

[0069] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for detecting parathyroid hormone using an aggregation-induced luminescent liquid crystal-polymer composite film, comprising using the luminescent liquid crystal-polymer composite film to perform dual-channel qualitative and quantitative detection of small molecule analytes.

2. The detection method according to claim 1, characterized in that: The preparation method of the luminescent liquid crystal-polymer composite film comprises filling prepolymer-luminescent liquid crystal into a liquid crystal empty box, and obtaining the luminescent liquid crystal-polymer composite film by ultraviolet light irradiation.

3. The detection method according to claim 2, characterized in that: The preparation method of the liquid crystal empty box comprises cleaning, immersing the upper glass sheet in a DMOAP solution, immersing the lower glass sheet in a mixed aqueous solution of APTES and DMOAP, and then immersing the lower glass sheet in a glutaraldehyde aqueous solution, preparing a biorecognition layer, and sealing to form a liquid crystal empty box.

4. The detection method according to claim 2, characterized in that: The preparation method of the prepolymer-luminescent liquid crystal comprises mixing nematic phase liquid crystal and aggregation-induced luminescent molecules, and then adding prepolymer to obtain the prepolymer-luminescent liquid crystal.

5. The detection method according to claim 4, characterized in that: The mass ratio of the aggregation-induced emission molecules to the nematic liquid crystal is 0.1 wt %; the mass ratio of the prepolymer to the luminescent liquid crystal is 5 wt %.

6. The detection method according to claim 3, characterized in that: The weight percentage ratio of the APTES to DMOAP is 4:1, and the glutaraldehyde aqueous solution is 2% v / v.

7. The detection method according to claim 3, characterized in that: The preparation of the biological recognition layer includes sequentially assembling avidin, parathyroid hormone antibody and parathyroid hormone.

8. The detection method according to claim 7, characterized in that: The concentration of the avidin was 1 μg / mL, and the concentration of the parathyroid hormone antibody was 1 μg / mL.

9. The detection method according to claim 2, characterized in that: The luminescent liquid crystal-polymer composite film is obtained by irradiating with ultraviolet light for 8 seconds.

10. The detection method according to claim 2, characterized in that: The prepolymer is epoxy resin glue.

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