Application of supramolecular sensing array in detection of glue traditional Chinese medicine

Abstract

The invention belongs to the field of biochemical technical detection, and particularly relates to application of a supramolecular sensing array in detection of glue type traditional Chinese medicines. Sensing units in the supramolecular sensing array are at least one of QAC5AEY, QAAC4AEY, QAAC4A12CEY and WTP3Fl, and a glue traditional Chinese medicine solution can effectively compete and replace dye molecules in a macrocyclic host cavity in the host-guest system, so that dye is free in the solution, and the dye molecules in the macrocyclic host cavity in the host-guest system are removed. Further, high-sensitivity detection and effective distinguishing of the glue traditional Chinese medicines can be realized by changing the fluorescence intensity, the operation is simple and convenient, the response is quick, the problems of insufficient selectivity and the like when the glue traditional Chinese medicines with similar structures are identified by a traditional analysis method are effectively solved, and the method is suitable for popularization and application. And a new thought and a new method with good application prospects are provided for rapid authenticity identification and quality control of glue traditional Chinese medicines.

Description

Technical Field

[0001] This invention belongs to the field of biochemical detection technology, and in particular relates to the application of a supramolecular sensing array in the detection of gelatinous traditional Chinese medicine. Background Technology

[0002] Gelatinous traditional Chinese medicines are solid gelatinous blocks made from animal hides, bones, shells, or horns through a process of boiling, concentrating, and drying. These gelatinous medicines contain animal protein, peptides, amino acids, and abundant trace elements, and possess effects such as nourishing blood, yin, moisturizing dryness, and stopping bleeding. Based on the source of the animal raw materials used, gelatinous traditional Chinese medicines are divided into the following categories: hide gelatin, such as donkey-hide gelatin (Ejiao), cowhide gelatin (Huangmingjiao), and pigskin gelatin (Xin Ejiao); horn gelatin, such as deer antler gelatin; bone gelatin, such as deer bone gelatin; and shell gelatin, such as tortoise shell gelatin and turtle shell gelatin. Due to its significant clinical efficacy and tonic value, the market demand for donkey-hide gelatin (Ejiao) continues to grow. However, donkey hide, its main raw material, is not a major livestock product, and the surge in demand further exacerbates the raw material shortage. Furthermore, counterfeit products adulterated with donkey-hide gelatin are common in the market, seriously affecting the market reputation and clinical safety of Ejiao.

[0003] Currently, the identification of gelatinous traditional Chinese medicines mainly relies on spectroscopic methods, chromatographic methods, and their coupling with mass spectrometry. In spectroscopic analysis, visible-near-infrared spectroscopy (VIS-NIR) can be used to distinguish donkey-hide gelatin from bovine-hide gelatin; Fourier transform infrared spectroscopy (FTIR) combined with two-dimensional correlation analysis (2D-IR) can reveal the microscopic differences between donkey-hide gelatin and yellow gelatin at the molecular level; near-infrared spectroscopy (NIR) technology can also be used to identify donkey-hide gelatin, yellow gelatin, deer antler gelatin, and tortoise shell gelatin. In chromatographic analysis, high-performance liquid chromatography (HPLC) fingerprinting is applied to distinguish donkey-hide gelatin from tortoise shell gelatin. Based on characteristic peptide detection, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC / Q-TOF-MS) and high performance liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS) can be used to distinguish various types of gelatin. In addition, proteomics analysis, gel electrophoresis, and DNA molecular identification methods are also gradually being applied in the identification of gelatinous medicinal materials. Existing identification techniques can analyze the characteristics of gelatinous Chinese medicines from multiple dimensions (such as the characteristic peptide detection method included in the Chinese Pharmacopoeia), but these methods lack versatility, rely on large and expensive equipment, and are complex and time-consuming. There is an urgent need to develop more efficient and convenient identification techniques for gelatinous Chinese medicines. Summary of the Invention

[0004] To address the above technical problems, this invention provides an application of supramolecular sensing arrays in the detection of gelatinous traditional Chinese medicines. This invention utilizes a host-guest system constructed from macrocyclic host molecules QAC5A, QAAC4A, QAAC4A12C, and WTP3 with fluorescent dyes EY and Fl for the identification and analysis of gelatinous traditional Chinese medicines. The method is simple to operate, has a rapid response, and high sensitivity, providing a new method for the rapid identification of gelatinous traditional Chinese medicines.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides an application of a supramolecular sensing array in the detection of gelatinous traditional Chinese medicine, wherein the sensing unit in the supramolecular sensing array is at least one of QAC5A (5,11,17,23,29-penta(trimethylammonium)-31,32,33,34,35-penta(4-methylpentoxy)calix[5] aryl)•EY (eosin Y disodium salt), QAAC4A (quaternary ammonium azocalix[4] aryl)•EY, QAAC4A12C (quaternary ammonium azocalix[4] aryl dodecyloxy ether)•EY and WTP3 (water-soluble terphenyl[3] aryl)•Fl (fluorescein sodium).

[0006] This invention reveals that gelatinous traditional Chinese medicine solutions can effectively competitively displace dye molecules in the macrocyclic host cavity of the QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl host-guest systems, leading to dye release in the solution. This, in turn, enables rapid detection of gelatinous traditional Chinese medicines through changes in fluorescence intensity. Furthermore, methodological validation shows that the precision and repeatability of the above host-guest systems for detecting donkey-hide gelatin test solutions meet the requirements.

[0007] Preferably, the gelatinous traditional Chinese medicine includes donkey-hide gelatin, yellow gelatin, deer antler gelatin, tortoise shell gelatin, turtle shell gelatin, deer bone gelatin, and new donkey-hide gelatin.

[0008] Preferably, the solvent of the sensing unit is a HEPES buffer solution with a HEPES concentration of 10 mM and a pH of 7.4.

[0009] More preferably, the sensor array in the supramolecular sensing array is QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl. The classification model composed of the quaternary supramolecular sensing matrix formed by the above four subject-guest pairs, combined with linear discriminant analysis, achieves an overall classification accuracy of 92.86% for donkey-hide gelatin, yellow gelatin, deer antler gelatin, tortoise shell gelatin, turtle shell gelatin, deer bone gelatin, and new donkey-hide gelatin, demonstrating good recognition ability.

[0010] More preferably, the gelatinous traditional Chinese medicine is donkey-hide gelatin, new donkey-hide gelatin, turtle shell gelatin, and deer bone gelatin, as well as tortoise shell gelatin or deer antler gelatin. The quaternary supramolecular sensing array model composed of the above four host-guest pairs has certain limitations in distinguishing between deer antler gelatin and tortoise shell gelatin, and the accuracy in distinguishing between the two is not good, but it has good accuracy in distinguishing between the two and other gelatinous traditional Chinese medicines.

[0011] More preferably, when using QAC5A•EY to identify the gelatinous traditional Chinese medicine, the final concentration of QAC5A in the system is 0.4 μM, and the final concentration of EY in the sensing unit solution is 0.5 μM.

[0012] More preferably, when using QAAC4A•EY to identify the gelatinous traditional Chinese medicine, the final concentration of QAAC4A in the system is 1 μM, and the final concentration of EY in the sensing unit solution is 1 μM.

[0013] More preferably, when using QAAC4A12C•EY to identify the gelatinous traditional Chinese medicine, the final concentration of QAAC4A12C in the system is 0.6 μM, and the final concentration of EY in the sensing unit solution is 1 μM.

[0014] More preferably, when using WTP3•Fl to identify the gelatinous traditional Chinese medicine, the final concentration of WTP3 in the system is 0.3 μM, and the final concentration of Fl in the sensing unit solution is 0.6 μM.

[0015] The second aspect of this invention provides a method for comparing the concentration of donkey-hide gelatin in different samples, specifically including the following operations: forming a supramolecular sensing array from host-guest pair solutions; adding the sample solution to be tested to each host-guest pair solution; obtaining a data matrix by observing changes in fluorescence signals; and comparing the concentration of donkey-hide gelatin in different sample solutions by linear discriminant analysis; wherein the host-guest pair solution is selected from at least one of QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl solutions.

[0016] Preferably, the solvent of the host-guest pair solution is a HEPES buffer solution with a HEPES concentration of 10 mM and a pH of 7.4.

[0017] Preferably, when the host-guest pair solution is QAC5A•EY, QAAC4A•EY or QAAC4A12C•EY, the excitation wavelength for detecting the fluorescence signal is 517 nm.

[0018] Preferably, when the host-guest pair solution is WTP3•Fl, the excitation wavelength for detecting the fluorescence signal is 500 nm.

[0019] Preferably, when the host-guest pair solution is QAC5A•EY, the final concentration of QAC5A in the host-guest pair solution is 0.4 μM, and the final concentration of EY in the system is 0.5 μM.

[0020] Preferably, when the host-guest pair solution is QAAC4A, the final concentration of QAAC4A in the host-guest pair solution is 1 μM, and the final concentration of EY in the system is 1 μM.

[0021] Preferably, when the host-guest pair solution is QAAC4A12C•EY, the final concentration of QAAC4A12C in the host-guest pair solution is 0.6 μM, and the final concentration of EY in the system is 1 μM.

[0022] Preferably, when the host-guest pair solution is WTP3•Fl, the final concentration of WTP3 in the host-guest pair solution is 0.3 μM, and the final concentration of Fl in the system is 0.6 μM.

[0023] More preferably, the host-guest pair solutions constituting the supramolecular sensing array are QAAC4A•EY and WTP3•Fl. The host molecules of QAAC4A•EY and WTP3•Fl can effectively encapsulate the chemical components in the donkey-hide gelatin solution, and the fluorescence response intensity of the sensing system has a clear correlation with the sample concentration. The classification model composed of the binary supramolecular sensing matrix of QAAC4A•EY and WTP3•Fl combined with linear discriminant analysis can achieve a 100% classification accuracy for donkey-hide gelatin test solutions of different concentrations, demonstrating high reliability.

[0024] The third aspect of this invention provides a method for identifying gelatinous traditional Chinese medicines, specifically including the following operations: a supramolecular sensing array composed of host-guest pair solutions; adding a gelatinous traditional Chinese medicine test solution to each host-guest pair solution; obtaining a data matrix by observing changes in fluorescence signals; and identifying the type of gelatinous traditional Chinese medicine contained in the gelatinous traditional Chinese medicine test solution by linear discriminant analysis; wherein the gelatinous traditional Chinese medicines include donkey-hide gelatin, yellow gelatin, deer antler gelatin, tortoise shell gelatin, turtle shell gelatin, deer bone gelatin, and new donkey-hide gelatin; wherein the host-guest pair solutions are selected from at least one of QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl solutions.

[0025] Preferably, the solvent of the host-guest pair solution is a HEPES buffer solution with a HEPES concentration of 10 mM and a pH of 7.4.

[0026] Preferably, when using QAC5A•EY, QAAC4A•EY or QAAC4A12C•EY to identify the gelatinous traditional Chinese medicine, the excitation wavelength for detecting the fluorescence signal is 517 nm.

[0027] Preferably, when using WTP3•Fl to identify the gelatinous traditional Chinese medicine, the excitation wavelength for detecting the fluorescence signal is 500 nm.

[0028] Preferably, when using QAC5A•EY to identify the gelatinous traditional Chinese medicine, the final concentration of QAC5A in the system is 0.4 μM, and the final concentration of EY in the host-guest pair solution is 0.5 μM.

[0029] Preferably, when using QAAC4A•EY to identify the gelatinous traditional Chinese medicine, the final concentration of QAAC4A in the system is 1 μM, and the final concentration of EY in the host-guest pair solution is 1 μM.

[0030] Preferably, when using QAAC4A12C•EY to identify the gelatinous traditional Chinese medicine, the final concentration of QAAC4A12C in the system is 0.6 μM, and the final concentration of EY in the host-guest pair solution is 1 μM.

[0031] Preferably, when using WTP3•Fl to identify the gelatinous traditional Chinese medicine, the final concentration of WTP3 in the system is 0.3 μM, and the final concentration of Fl in the host-guest pair solution is 0.6 μM.

[0032] More preferably, the host-guest pair solutions constituting the supramolecular sensing array are QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl.

[0033] More preferably, the gelatinous traditional Chinese medicine is donkey-hide gelatin, new donkey-hide gelatin, turtle shell gelatin, and deer bone gelatin, as well as tortoise shell gelatin or deer antler gelatin.

[0034] Compared with existing technologies, this invention has the following advantages: This invention innovatively applies supramolecular sensing technology to the identification and analysis of gelatinous traditional Chinese medicines. By utilizing a dynamic host-guest recognition system constructed from macrocyclic main molecules QAC5A, QAAC4A, QAAC4A12C, and WTP3 with fluorescent dyes EY and Fl, it achieves highly sensitive detection and effective differentiation of aqueous extracts of various gelatinous traditional Chinese medicines. The operation is simple and the response is rapid, effectively overcoming the problems of insufficient selectivity faced by traditional analytical methods when identifying structurally similar gelatinous traditional Chinese medicines. The method for detecting the concentration of donkey-hide gelatin and the method for identifying gelatinous traditional Chinese medicines provided by this invention utilize the cross-response mechanism of the sensor array, resulting in highly reliable detection results. This provides a new approach and method with promising application prospects for the rapid identification and quality control of genuine and counterfeit gelatinous traditional Chinese medicines. Attached Figure Description

[0035] Figure 1The figures show the fluorescence response of different host-guest systems to the donkey-hide gelatin test solution in Example 1; the host-guest system in Figure A is QAC5A•EY (0.4 μM / 0.5 μM), the host-guest system in Figure B is QAAC4A•EY (1 μM / 1 μM), the host-guest system in Figure C is QAAC4A12C•EY (0.6 μM / 1 μM), and the host-guest system in Figure D is WTP3•Fl (0.3 μM / 0.6 μM). Figure 2 The figures show the fluorescence response of different host-guest systems to the test solution in Example 1; Figure A shows the host-guest system as QAC5A•EY (0.4 μM / 0.5 μM), Figure B shows the host-guest system as QAAC4A•EY (1 μM / 1 μM), Figure C shows the host-guest system as QAAC4A12C•EY (0.6 μM / 1 μM), and Figure D shows the host-guest system as WTP3•Fl (0.3 μM / 0.6 μM). Figure 3 To verify the intraday precision used in Example 1 ( n = 6) (Figure A), daytime precision ( n = 3, each point represents the average of six repetitions on the same day) (Chart B) and repeatability ( n = 6) (Figure C) fluorescence response ( I / I 0); Figure 4 The fluorescence response of different concentrations of donkey-hide gelatin test solutions in Example 2 ( n =6)(Figure A) and the results of linear discriminant analysis (95% confidence interval) (Figure B); Figure 5 The fluorescence response of the gelatinous traditional Chinese medicine in Example 3 ( n =6)(Figure A) and the results of linear discriminant analysis (95% confidence interval) (Figure B); Figure 6 To compare the effects of the donkey-hide gelatin test solution on other dye guests in Examples 1 and 2, and to assess the fluorescence response of the other host-guest systems to the donkey-hide gelatin test solution. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0037] Gelatinous traditional Chinese medicines share similar origins and have complex compositions, making accurate identification difficult. Existing identification methods mainly rely on spectroscopic methods, chromatographic methods, and their coupling with mass spectrometry. These methods are cumbersome to operate and analyze, and still face many challenges in terms of versatility and practicality. There is an urgent need to develop more efficient and convenient identification techniques for gelatinous traditional Chinese medicines.

[0038] This invention provides an application of a supramolecular sensing array in the identification of colloidal traditional Chinese medicines. The sensing units in the supramolecular sensing array are QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl. The colloidal traditional Chinese medicine solution can effectively competitively displace dye molecules in the macrocyclic host cavity of the aforementioned host-guest system, causing the dye to become free in the reaction system. The identification of colloidal traditional Chinese medicines can then be achieved by observing changes in fluorescence intensity.

[0039] This invention also provides a method for comparing the concentration of donkey-hide gelatin in different samples.

[0040] This invention also provides a method for identifying gelatinous traditional Chinese medicines.

[0041] The technical solution of the present invention will be further described below with reference to several embodiments.

[0042] The medicinal materials used in the following examples: donkey-hide gelatin ( Colla Corii Asini CCA), yellow gelatin ( Colla Corii Bovis CCB), deer antler glue ( Colla Cornus Cervi CCC), Tortoise shell glue ( Colla Carapacis et Plastri Testudinis The (CCPT) was purchased from Dong-E-E-Jiao Co., Ltd., and was identified as a donkey (Equus equina). Equus asinus L. Solid gelatinous blocks made by boiling and concentrating the hide of cattle (Bovidae family). Bos taurus domesticus Gmelin Solid gelatinous blocks made from the skin of the sika deer, a species of deer, through boiling and concentration. Cervus nippon Temminck Or red deer Cervus elaphus Linnaeus Solid gelatinous blocks made by boiling and concentrating the horns in water, and turtles (a type of turtle). Chinemys reevesii (Gray) A solid gelatinous block made by boiling and concentrating the carapace and plastron of turtle shell in water. Colla Carapacis Trionychis The substance (CCT) was purchased from Henan Laojuntang Pharmaceutical Co., Ltd., and was identified as a soft-shelled turtle (Trionyx sinensis). Trionyx sinensis Wiegmann Deer bone glue is a solid gelatinous substance made by boiling and concentrating the carapace of deer in water. Colla Ossis Cervi The item (COC) was purchased from the Bozhou Traditional Chinese Medicine Market in Anhui Province and identified as a sika deer (Cervidae). Cervus nippon Temminck Or red deer Cervus elaphus LinnaeusIt is a solid gelatinous block made by boiling and concentrating bones in water. New donkey-hide gelatin (New donkey-hide gelatin) Colla Corii Suilli The substance (CCS) was purchased from the Anguo Traditional Chinese Medicine Market in Hebei Province and identified as a pig (Pig). Sus scrofa domestica Brisson It is a solid gelatinous block made by boiling and concentrating the skin.

[0043] The main pharmaceuticals and reagents used in the following examples are: eosin Y disodium salt (EY, MKCG1738). β -Cyclodextrin ( β -clodextrins, β -CD, Z31J11Y119887), methyl- β -Cyclodextrin (Me- β -clodextrins, Me- β-CD, Q11O10H99426), cucurbituril[7] (CB[7], MKCF0481), and cucurbituril[8] (CB[8], MKCB7158) were all purchased from Sigma-Aldrich, USA. Methylene blue (MB, K50873270003) was purchased from Merck GmbH, Germany. Berberine hydrochloride (BC, Q11O10H99426) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Sodium fluorescein (Fl, SHBL7023) was purchased from Wuhan AmyJet Technology Co., Ltd. Neutral red (NR, MKCG8905) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 100+H05500K101) were purchased from Tianjin Xiens Biochemical Technology Co., Ltd. Sodium hydroxide (NaOH, analytical grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 5,11,17,23,29-penta(trimethylammonium)-31,32,33,34,35-penta(4-methylpentoxy)calix[5]arene (QAC5A), quaternary-ammonium modified azocalix[4]arene (QAAC4A), and quaternary-ammonium-modified azocalix[4]arene dodecyloxy ether (QAAC4A12C) were obtained from Nankai University. Water-soluble terphen[3]arene (WTP3) was obtained from Tianjin Normal University. Ultrapure water was prepared using a multifunctional ultrapure water system.

[0044] All other reagents and materials used in the following examples can be purchased commercially. Unless otherwise specified, all reagents and materials are used directly after purchase without further processing.

[0045] Example 1 This embodiment investigates the interference of donkey-hide gelatin and other gelatinous traditional Chinese medicines on fluorescence signals in different host-guest systems.

[0046] 1. Solution preparation 1.1 Preparation of HEPES (10 mmol / L, pH 7.4) buffer solution Weigh 2.38 g of HEPES and dissolve it in 0.9 L of ultrapure water. At 25 °C, adjust the pH of the solution to 7.4 with sodium hydroxide solution (0.1 mol / L) and add ultrapure water to 1 L to obtain a 10 mmol / L buffer solution.

[0047] 1.2 Preparation of Sample Solution 1.2.1 Preparation of solutions by host and guest components Accurately weigh the macrocyclic host (QAC5A, QAAC4A, QAAC4A12C, WTP3) and dye guest (EY, Fl) sample powders, and prepare macrocyclic host and dye guest stock solutions of appropriate concentrations using HEPES buffer (10 mM, pH = 7.4), respectively. Store at 4 ℃ for later use. Take appropriate amounts of macrocyclic host and dye guest stock solutions, and dilute them with HEPES buffer (10 mM, pH = 7.4) to appropriate concentrations and mix well to obtain host-guest pair solutions: QAC5A•EY (final concentration, 0.4 μM / 0.5 μM), QAAC4A•EY (final concentration, 1 μM / 1 μM), QAAC4A12C•EY (final concentration, 0.6 μM / 1 μM), and WTP3•Fl (final concentration, 0.3 μM / 0.6 μM).

[0048] 1.2.2 Preparation of the test solution Take 0.25 g of donkey-hide gelatin sample powder (passed through a No. 2 sieve), place it in a 10 mL centrifuge tube, add 5 mL of pure water, and sonicate at 40 ℃ for 30 min. Cool to room temperature, centrifuge at 14000 rpm for 15 min, and filter the supernatant through a 0.45 μm filter membrane to obtain the donkey-hide gelatin test solution. Prepare the test solutions of yellow gelatin, deer antler gelatin, tortoise shell gelatin, turtle shell gelatin, deer bone gelatin, and new donkey-hide gelatin using the same method.

[0049] 2. Fluorescence spectrophotometer detection method Two mL of the host-guest pair solution was placed in a quartz cuvette (Agilent Technologies, Inc., USA) with a path length of 10 mm. Five μL of the test solution was added. Fluorescence was detected using a Varian Cary Eclipse fluorescence spectrometer (Agilent Technologies, Inc., USA) equipped with a Cary Single-cuvette Peltier cuvette temperature control device at excitation wavelengths of EY (λex = 517 nm) and Fl (λex = 500 nm), respectively. The fluorescence titration was performed at 25 °C.

[0050] Record the fluorescence emission spectra of the host and guest components when the solution reaches equilibrium, and denote the fluorescence intensity at the maximum emission wavelength as . I0. After adding the test solution, record the fluorescence emission spectrum when equilibrium is reached. The fluorescence intensity at the maximum emission wavelength is recorded as . I .

[0051] 3. Fluorescence response of different host-guest systems to the test solution like Figure 1 As shown, the addition of the donkey-hide gelatin test solution caused a significant increase in fluorescence intensity in the host-guest systems QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl, with fluorescence intensity changes of 131, 264, 211, and 481, respectively. This indicates that the donkey-hide gelatin test solution can effectively competitively displace the dye molecules EY and Fl from the macrocyclic host cavity, leading to the free dye in the solution and an increase in the fluorescence intensity of the system.

[0052] The fluorescence emission spectra of the test solutions of donkey-hide gelatin (CCA), yellow gelatin (CCB), deer antler gelatin (CCC), tortoise shell gelatin (CCPT), turtle shell gelatin (CCT), deer bone gelatin (COC), and new donkey-hide gelatin (CCS) added to the above system are as follows: Figure 2 As shown, there are significant differences in the degree of fluorescence signal enhancement caused by different types of gelatinous Chinese medicine samples, indicating that each host-guest system has the potential to distinguish between various types of gelatinous Chinese medicine.

[0053] Test Example 1 This test example provides a methodological investigation of the method used in Example 1 to detect the donkey-hide gelatin test solution using the QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl host-guest system.

[0054] 1. Intra-day precision test The donkey-hide gelatin test solution and the host-guest pair solution were prepared according to the method in "1.2" of Example 1. 5 μL of the donkey-hide gelatin test solution was added to each host-guest pair solution. The fluorescence spectrophotometer detection method described in Example 1 was repeated 6 times, and the fluorescence response value of the sensing unit to the test solution was calculated. P = I / I 0), calculate the mean and RSD of the six sets of data. The results are as follows: Figure 3 As shown in Figure A, the relative standard deviation (RSD) of the response signal does not exceed 2.35%, indicating that the intraday precision and instrument precision of the method are good.

[0055] 2. Daytime precision test The donkey-hide gelatin test solution and the host-guest pair solution were prepared according to the method in "1.2" of Example 1. 5 μL of the donkey-hide gelatin test solution was added to each host-guest pair solution. The fluorescence spectrophotometer detection method described in Example 1 was repeated 6 times daily for three consecutive days. The fluorescence response value of the sensing unit to the test solution was calculated. P = I / I 0), calculate the average and RSD of the three-day test data. The results are as follows: Figure 3 As shown in Figure B, the RSD values ​​of the signals obtained from three consecutive days of measurement were all controlled within 2.43%, indicating that the method has good daytime precision.

[0056] 3. Repeatability test Six portions of donkey-hide gelatin test solution were prepared in parallel according to the method in "1.2.2" of Example 1. Macrocyclic host and dye guest stock solutions were prepared according to the method in "1.2.1" of Example 1. Then, each host-guest pair solution was diluted in parallel with HEPES buffer solution to obtain six portions according to the final concentration in "1.2.1". 5 μL of the donkey-hide gelatin test solution was added to each host-guest pair solution, and the fluorescence response value of the sensing unit to the test solution was calculated according to the fluorescence spectrophotometer detection method in Example 1. P = I / I 0), calculate the average and RSD values. The results are as follows: Figure 3 As shown in Figure C, the RSD values ​​of all sensing units are below 3.90%, indicating that the method has good repeatability.

[0057] Example 2 In this embodiment, QAAC4A•EY (1 μM / 1 μM) and WTP3•Fl (0.3 μM / 0.6 μM) were used to detect different concentrations of donkey-hide gelatin.

[0058] A 50 mg / mL solution of donkey-hide gelatin was prepared according to "1.2.2" in Example 1, and then diluted sequentially with pure water to obtain a series of solution concentrations of 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL. QAAC4A•EY (1 μM / 1 μM) and WTP3•Fl (0.3 μM / 0.6 μM), which showed significant responses to the donkey-hide gelatin solution, were selected to detect the five concentrations of donkey-hide gelatin solution. Three parallel samples were prepared for each concentration, and each sample was measured twice. The fluorescence intensity ratio was used as the analytical measure. P = I / IUsing 0 as the response signal, a 2×5×6 original data matrix was obtained. This matrix data was imported into Past 3 software and processed using the "Linear Discriminant Analysis (LDA)" module. The results are as follows... Figure 4 As shown in the figure, the linear discriminant analysis score plot shows that the variance contribution rate of the first discriminant factor is 99.55%, and within the 95% confidence interval, the five different concentrations of donkey-hide gelatin samples are well distinguished. This result indicates that the main molecules QAAC4A and WTP3 can effectively encapsulate the chemical components in the donkey-hide gelatin solution, and there is a clear correlation between the fluorescence response intensity of the sensing system and the sample concentration. To further verify the classification effect, the accuracy of the classification model composed of the binary supramolecular sensing matrix of QAAC4A•EY and WTP3•Fl combined with linear discriminant analysis was evaluated using a confusion matrix (Table 1). The results show that the classification accuracy of all concentration samples can reach 100%, indicating that the model has high reliability.

[0059] Table 1. Confusion matrix of LDA model of fluorescence response of donkey-hide gelatin at different concentrations

[0060] Example 3 This embodiment uses sensor arrays and pattern recognition to differentiate between different types of gelatinous traditional Chinese medicine.

[0061] The seven types of colloid-based traditional Chinese medicine test solutions were tested using QAC5A•EY (0.4 μM / 0.5 μM), QAAC4A•EY (1 μM / 1 μM), QAAC4A12C•EY (0.6 μM / 1 μM), and WTP3•Fl (0.3 μM / 0.6 μM). Three replicates of each colloid sample were prepared (preparation method as described in Section 1.2.2 of Example 1), and each replicate was measured twice. The fluorescence intensity ratio was used for analysis. P = I / I Using 0 as the response signal, a 4×7×6 original data matrix was obtained. This matrix data was imported into Past 3 software and analyzed using the "Linear Discriminant Analysis (LDA)" module. The results are as follows... Figure 5As shown in the linear discriminant analysis graph, the variance contribution rate of the first discriminant factor is 95.06%. Within the 95% confidence interval, the five types of samples—donkey-hide gelatin, new donkey-hide gelatin, turtle shell gelatin, deer bone gelatin, and tortoise shell gelatin (or deer antler gelatin)—can be effectively distinguished. The confidence intervals of yellow gelatin partially overlap with those of donkey-hide gelatin and new donkey-hide gelatin, and the confidence ellipses of deer antler gelatin and tortoise shell gelatin also basically coincide. This indicates that the classification model composed of the quaternary supramolecular sensing matrix consisting of QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl combined with linear discriminant analysis cannot yet accurately distinguish the above sample combinations. The reason for this may be that yellow gelatin, donkey-hide gelatin, and new donkey-hide gelatin all originate from mammalian hides, and the high similarity in their protein composition leads to similar response patterns generated by the sensor array. While deer antler gelatin and tortoise shell gelatin have different raw material sources, both are rich in collagen and various mineral elements, and their aqueous extracts may contain similar binding sites, exceeding the distinguishing capabilities of the current sensor array. This result objectively reflects the complexity of the chemical composition of gelatinous traditional Chinese medicines.

[0062] Further verification of classification accuracy was performed using a confusion matrix model (Table 2). The results showed that the overall classification accuracy was 92.86%, indicating that the sensor array has good recognition ability in distinguishing most types of gelatinous Chinese medicines. However, the discrimination effect on deer antler gelatin and tortoise shell gelatin still has certain limitations, and its classification accuracy needs to be further improved.

[0063] Table 2. Confusion matrix of LDA model for fluorescence response of different gelatinous traditional Chinese medicines

[0064] Comparative Example 1 This test case assessed the potential impact of the donkey-hide gelatin test solution on other dye guests. Take MB stock solution and dilute it to 10 µM with HEPES buffer (10 mM, pH = 7.4). Take 2 mL and place it in a quartz cuvette with an optical path of 10 mm. Add 5 μL of donkey-hide gelatin test solution. Use a Varian Cary Eclipse fluorescence spectrometer equipped with a Cary Single-cuvette Peltier cuvette temperature control device to detect fluorescence at the excitation wavelength of MB (λex = 664 nm). After the detection system stabilizes, no significant change in the fluorescence signal of the MB dye is observed. Figure 6 As shown in Figure A, the test solution of donkey-hide gelatin has no significant interference with MB.

[0065] Comparative Example 2 This comparative example provides the fluorescence response of other host-guest systems to the donkey-hide gelatin test solution.

[0066] 1. Solution preparation 1.1 Preparation of HEPES (10 mmol / L, pH 7.4) buffer solution: Same as in Example 1.

[0067] 1.2 Preparation of Sample Solution 1.2.1 Preparation of solutions by host and guest components Precisely weigh the large ring body ( β -CD、Me- β -CD, CB[7], CB[8]), and dye guest (MB, NR, BC) sample powders were prepared into macrocyclic host stock solutions and dye guest stock solutions of appropriate concentrations using HEPES buffer (10 mM, pH = 7.4), and stored at 4 ℃ for later use. Appropriate amounts of macrocyclic host stock solutions and dye guest stock solutions were taken and diluted with HEPES buffer solution (10 mM, pH = 7.4) to appropriate concentrations and mixed well to obtain host-guest pair solutions. β -CD•MB (final concentration, 1 mM / 10 μM), Me- β -CD•NR (final concentration, 0.4 mM / 10 μM), CB[7]•BC (final concentration, 12 μM / 10 μM), CB[8]•BC (final concentration, 3 μM / 10 μM).

[0068] 1.2.2 Preparation of the donkey-hide gelatin test solution: Same as in Example 1.

[0069] 2. Fluorescence spectrophotometer detection method Same as Example 1.

[0070] 3. Results like Figure 6 As shown, in 2 mL β 5 μL of donkey-hide gelatin test solution was added to the CD•MB (final concentration, 1 mM / 10 μM) system. After the system stabilized, the fluorescence intensity changed only slightly (|Δ I |= I - I 0=1), indicating that the donkey-hide gelatin test solution has difficulty removing MB from... β- Competition and displacement within the CD cavity reflects its relationship with... β -CD has a weak binding affinity, making this system unsuitable for fluorescence sensing analysis of donkey-hide gelatin; in 2 mL Me- β After adding 5 μL of donkey-hide gelatin test solution to CD•NR, CB[7]•BC and CB[8]•MB respectively, the change in fluorescence intensity (|Δ) I The values ​​for |) were 1, 7, and 5, respectively, none of which showed a significant signal response.

[0071] The above results indicate that none of the four host-guest systems showed good recognition compatibility with donkey-hide gelatin samples and are not suitable as recognition elements for their fluorescence sensing analysis.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of a supramolecular sensing array in the detection of colloidal traditional Chinese medicines, characterized in that, The sensing unit in the supramolecular sensing array is at least one of QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl.

2. The application according to claim 1, characterized in that, The gelatinous traditional Chinese medicines mentioned include donkey-hide gelatin, yellow gelatin, deer antler gelatin, tortoise shell gelatin, turtle shell gelatin, deer bone gelatin, and new donkey-hide gelatin; and / or The solvent of the sensing unit is a HEPES buffer solution with a HEPES concentration of 10 mM and a pH of 7.

4.

3. The application according to claim 2, characterized in that, The host-guest pair solution is QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl; and / or The gelatinous Chinese medicines mentioned include donkey-hide gelatin, new donkey-hide gelatin, turtle shell gelatin, and deer bone gelatin, as well as tortoise shell gelatin or deer antler gelatin.

4. The application according to claim 3, characterized in that, When using QAC5A•EY for the identification of the gelatinous traditional Chinese medicine, the final concentration of QAC5A in the sensing unit solution is 0.4 μM, and the final concentration of EY in the system is 0.5 μM; and / or When using QAAC4A•EY to identify the gelatinous traditional Chinese medicine, the final concentration of QAAC4A in the system is 1 μM, and the final concentration of EY in the sensing unit solution is 1 μM; and / or When using QAAC4A12C•EY for the identification of the gelatinous traditional Chinese medicine, the final concentration of QAAC4A12C in the system is 0.6 μM, and the final concentration of EY in the sensing unit solution is 1 μM; and / or When using WTP3•Fl to identify the gelatinous traditional Chinese medicine, the final concentration of WTP3 in the system is 0.3 μM, and the final concentration of Fl in the sensing unit solution is 0.6 μM.

5. A method for comparing the concentration of donkey-hide gelatin in different samples, characterized in that, Specifically, the operation includes the following steps: forming a supramolecular sensing array from host-guest pair solutions; adding the sample solution to be tested to each host-guest pair solution; obtaining a data matrix by observing changes in fluorescence signals; and comparing the concentration of donkey-hide gelatin in different sample solutions using linear discriminant analysis. The host-guest pair solutions are selected from at least one of QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl solutions.

6. The method according to claim 5, characterized in that, The solvent for the host-guest pair solution is a HEPES buffer solution with a HEPES concentration of 10 mM and a pH of 7.4; and / or When the host-guest pair solution is QAC5A•EY, QAAC4A•EY, or QAAC4A12C•EY, the excitation wavelength for detecting the fluorescence signal is 517 nm; and / or When the host-guest pair solution is WTP3•Fl, the excitation wavelength for detecting the fluorescence signal is 500 nm; and / or When the host-guest pair solution is QAC5A•EY, the final concentration of QAC5A in the host-guest pair solution is 0.4 μM, and the final concentration of EY in the system is 0.5 μM; and / or When the host-guest pair solution is QAAC4A, the final concentration of QAAC4A in the host-guest pair solution is 1 μM, and the final concentration of EY in the system is 1 μM; and / or When the host-guest pair solution is QAAC4A12C•EY, the final concentration of QAAC4A12C in the system is 0.6 μM, and the final concentration of EY in the host-guest pair solution is 1 μM; and / or When the host-guest pair solution is WTP3•Fl, the final concentration of WTP3 in the system is 0.3 μM, and the final concentration of Fl in the host-guest pair solution is 0.6 μM.

7. The method according to claim 5 or 6, characterized in that, The host-guest pair solutions constituting the supramolecular sensing array are QAAC4A•EY and WTP3•Fl.

8. A method for identifying gelatinous traditional Chinese medicines, characterized in that, Specifically, the operation includes the following steps: a supramolecular sensing array is formed by host-guest pair solutions; a gelatinous traditional Chinese medicine test solution is added to each host-guest pair solution; a data matrix is ​​obtained by observing changes in fluorescence signals; and linear discriminant analysis is used to identify the type of gelatinous traditional Chinese medicine contained in the test solution. The gelatinous traditional Chinese medicine includes donkey-hide gelatin, yellow gelatin, deer antler gelatin, tortoise shell gelatin, turtle shell gelatin, deer bone gelatin, and new donkey-hide gelatin. The host-guest pair solutions are selected from at least one of QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl solutions.

9. The identification method according to claim 8, characterized in that, The solvent for the host-guest pair solution is a HEPES buffer solution with a HEPES concentration of 10 mM and a pH of 7.4; and / or When using QAC5A•EY, QAAC4A•EY, or QAAC4A12C•EY to identify the gelatinous traditional Chinese medicine, the excitation wavelength for detecting the fluorescence signal is 517 nm; and / or When using WTP3•Fl for the identification of the gelatinous traditional Chinese medicine, the excitation wavelength for detecting the fluorescence signal is 500 nm; and / or When using QAC5A•EY for the identification of the gelatinous traditional Chinese medicine, the final concentration of QAC5A in the host-guest pair solution is 0.4 μM, and the final concentration of EY in the system is 0.5 μM; and / or When using QAAC4A•EY for the identification of the gelatinous traditional Chinese medicine, the final concentration of QAAC4A in the host-guest pair solution is 1 μM, and the final concentration of EY in the system is 1 μM; and / or When the QAAC4A12C•EY is used to identify the gelatinous traditional Chinese medicine, the final concentration of QAAC4A12C in the host-guest pair solution is 0.6 μM, and the final concentration of EY in the system is 1 μM. and / or When using WTP3•Fl to identify the gelatinous traditional Chinese medicine, the final concentration of WTP3 in the host-guest pair solution is 0.3 μM, and the final concentration of Fl in the system is 0.6 μM.

10. The identification method according to claim 8 or 9, characterized in that, The host-guest pair solutions constituting the supramolecular sensing array are QAC5A•EY, QAAC4A•EY, QAAC4A12C•EY, and WTP3•Fl; and / or The gelatinous Chinese medicines mentioned include donkey-hide gelatin, new donkey-hide gelatin, turtle shell gelatin, and deer bone gelatin, as well as tortoise shell gelatin or deer antler gelatin.

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