Sensor based on supramolecular calixarene and preparation method and application thereof

Abstract

The invention relates to a sensor based on supramolecular calixarene and a preparation method and application thereof. Comprising calixarene and a fluorescent indicator, and the molar ratio of the calixarene to the fluorescent indicator is 1: (3.5-4.5). The calixarene is tert-butyl calixarene (BFT) or calixarene monoboric acid derivative (BFTPS), and the fluorescent indicator is 6, 7-dihydroxy coumarin. The invention also provides a preparation method of the sensor and application of the sensor in detection of carbohydrate antigens, glycoproteins, cancer cells and serum. The calixarene used in the invention has a unique three-dimensional cavity molecular structure, and can selectively adsorb guest molecules of different sizes. Then calixarene is modified, boric acid derivatization groups are introduced, calixarene mono-boric acid derivatives are used as host molecules, XDS is used as a fluorescent indicator, and the sensor based on the supramolecular calixarene is formed and can be used for detecting biological samples such as carbohydrate antigens, glycoproteins, cancer cells and serum.

Description

Technical Field

[0001] The present invention relates to a supramolecular calixarene-based sensor, a preparation method thereof and an application thereof, belonging to the technical fields of biomedicine and analytical chemistry. Background Art

[0002] In recent years, with the in-depth study of the pathogenesis of cancer, tumor-associated carbohydrate antigens have attracted much attention as important markers for early cancer diagnosis. In the field of supramolecular chemistry, calixarenes, due to their unique molecular structure and excellent host-guest recognition properties, provide new opportunities for the detection of biomolecules.

[0003] During the occurrence and development of cancer, abnormal glycosylation is extremely common. Glycosylation, as a process of attaching sugars to proteins or lipids under the action of glycosyltransferases, widely exists in living organisms and is highly sensitive to the physiological environment. During the evolution of diseases, glycosylation will change significantly, making it a valuable disease marker. Many cancers exhibit abnormal glycosylation characteristics, mainly manifested in two types of changes: one is the emergence of new glycosylation patterns, such as Lewis antigens, polysialic acids, abnormal core fucosylation, and increased N-glycan branching; the other is the incomplete synthesis of glycan groups, especially prominent in O-glycosylation, resulting in the exposure of Tn antigens, sTn antigens, and T antigens.

[0004] These tumor-associated carbohydrate antigens can be detected in most cancers but are usually absent in healthy tissues, becoming key targets for cancer diagnosis. For example, under normal physiological conditions, T and Tn antigens can attach more monosaccharides to form complex glycan chains; however, due to the imbalance in the expression of two key glycan synthesis enzymes, Cosmc and sTn synthase (ST6GalNAc-I), the above three antigens are exposed on the cell surface, promoting more aggressive cancer cell behavior and accelerating processes such as tumor growth, extracellular matrix adhesion, migration, invasion, and metastasis. With the advancement of precision medicine, it has been found that tumors of epithelial origin highly express truncated O-glycans. However, at the individual level, carbohydrate antigens are not expressed 100%, and the expression rates of the three antigens vary. Among different subtypes of the same cancer, there are significant differences in the expression of truncated O-glycans, and most individual tumors show intratumoral heterogeneity in the expression patterns of Tn, sTn, and T antigens. Given the close association between carbohydrate antigens and cancer, the accurate detection of them is of great significance for early cancer diagnosis, disease monitoring, and treatment strategy formulation.

[0005] Current methods for detecting tumor carbohydrate biomarkers mainly include immunoassay and lectin assay, etc., but they all have certain defects. Immunoassay utilizes the specific interaction between antibodies and antigens to detect glycoproteins based on protein structure. However, this method simply measures the concentration of glycoproteins without considering abnormal glycosylation, and false positive results are prone to occur. Moreover, antibodies have drawbacks such as high price, difficult acquisition, and short shelf life, which limit their wide application. Although lectins can selectively recognize and bind to glycan structures through hydrogen bonds, metal coordination, van der Waals forces, and hydrophobic interactions to achieve detection or separation from complex matrices, they have deficiencies in specificity and numerous interfering factors. In addition, some carbohydrates lack specific lectins, which have a greater impact on the accuracy and sensitivity of detection results and are difficult to meet the clinical requirements for highly sensitive and highly specific detection of tumor carbohydrate biomarkers.

[0006] Therefore, there is an urgent need to develop a highly sensitive and highly selective detection sensor and detection method to meet the urgent needs of clinical early cancer diagnosis and promote the further development of biodietection technology in the medical field. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a sensor based on supramolecular calixarene, its preparation method and application. It is used for the detection of tumor-related carbohydrate antigens to overcome the deficiencies of existing detection methods and achieve accurate, sensitive, and high-throughput detection of tumor-related carbohydrate antigens, providing strong support for the early diagnosis, condition monitoring, and treatment plan formulation of cancer.

[0008] The technical solution of the present invention is as follows:

[0009] A sensor based on supramolecular calixarene, comprising calixarene and a fluorescent indicator;

[0010] The molar ratio of the calixarene to the fluorescent indicator is 1:(3.5 - 4.5).

[0011] Preferably according to the present invention, the calixarene is tert-butylcalixarene (BFT) or calixarene monoborate derivative (BFTPS).

[0012] More preferably, the tert-butylcalixarene is tert-butylcalix[4]arene, tert-butylcalix[6]arene or tert-butylcalix[8]arene; the calixarene monoborate derivative is calix[4]arene monoborate derivative, calix[6]arene monoborate derivative or calix[8]arene monoborate derivative.

[0013] Preferably according to the present invention, the fluorescent indicator is 6,7-dihydroxycoumarin.

[0014] Preferably according to the present invention, the molar ratio of the calixarene to the fluorescent indicator is 1:4.

[0015] Preferably according to the present invention, the calixarene monoboric acid derivative is prepared by the following method:

[0016] (1) Add calixarene, 4-(bromomethyl)phenylboronic acid pinacol ester, and anhydrous potassium carbonate to ultradry acetonitrile in sequence, reflux and react at 80 - 90 °C under nitrogen protection for 4 - 7 h to obtain a borate compound;

[0017] (2) Add the borate compound to an aqueous solution of tetrahydrofuran, add sodium periodate and hydrochloric acid in sequence, react overnight at 20 - 30 °C for 12 - 18 h, and after purification, obtain the calixarene monoboric acid derivative.

[0018] More preferably, in step (1), the molar ratio of the calixarene, 4-(bromomethyl)phenylboronic acid pinacol ester, and anhydrous potassium carbonate is 1:(0.9 - 1.2):(0.9 - 1.2), and the volume of anhydrous acetonitrile is 80 - 140 mL.

[0019] Most preferably, when the calixarene is calix[4]arene, the molar ratio of calix[4]arene, 4-(bromomethyl)phenylboronic acid pinacol ester, and anhydrous potassium carbonate is 1:0.9:0.9, and the volume of anhydrous acetonitrile is 80 mL; when the calixarene is calix[6]arene, the molar ratio of calix[6]arene, 4-(bromomethyl)phenylboronic acid pinacol ester, and anhydrous potassium carbonate is 1:1.2:1.2, and the volume of anhydrous acetonitrile is 100 mL; when the calixarene is calix[8]arene, the molar ratio of calix[8]arene, 4-(bromomethyl)phenylboronic acid pinacol ester, and anhydrous potassium carbonate is 1:1.2:0.9, and the volume of anhydrous acetonitrile is 140 mL.

[0020] More preferably, in step (2), the aqueous solution of tetrahydrofuran is obtained by mixing tetrahydrofuran (THF) and water according to a volume ratio of 4:1.

[0021] More preferably, in step (2), the mass ratio of the borate compound, the aqueous solution of tetrahydrofuran, sodium periodate, and hydrochloric acid is 1:(2.5 - 3.5):(1 - 2).

[0022] Most preferably, the molar ratio of the borate compound, the aqueous solution of tetrahydrofuran, sodium periodate, and hydrochloric acid is 1:3:1.5.

[0023] The preparation method of the above-mentioned supramolecular calixarene-based sensor includes the following steps:

[0024] Prepare a calixarene monoboric acid derivative solution and a fluorescent indicator solution respectively, and then mix the calixarene monoboric acid derivative solution and the fluorescent indicator solution evenly so that the molar ratio of the calixarene monoboric acid derivative to the fluorescent indicator is 1:(3.5 - 4.5) to obtain a supramolecular calixarene-based sensor.

[0025] The application of the above-mentioned supramolecular calixarene-based sensor in detecting carbohydrate antigens, glycoproteins, cancer cells, and serum.

[0026] Preferably according to the present invention, the carbohydrate antigen is sTn carbohydrate antigen, Tn carbohydrate antigen or T carbohydrate antigen; the glycoprotein is BSM glycoprotein, aBSM glycoprotein or αFet glycoprotein; the cancer cells are leukemia cells Jurkat, KG-1, K562, gastric cancer cells MKN45 or breast cancer cells MDA-MB-231.

[0027] A method for detecting carbohydrate antigens, glycoproteins, cancer cells, and serum using the above-mentioned supramolecular calixarene-based sensor for non-diagnostic purposes, comprising the following steps:

[0028] Add different volumes of the sample solution to be detected to the supramolecular calixarene-based sensor solution to make the final concentration of the sample to be detected 0.5 nM to 20 μM, incubate at 37 °C for 15 to 25 min, measure the fluorescence emission spectrum of the mixed solution, and collect the fluorescence response signal F at F464 to obtain fluorescence change data; then perform dimensionality reduction processing on the fluorescence change data by linear discriminant analysis (LDA) to visualize the fluorescence change data; finally, qualitatively distinguish the sample to be detected according to the distribution of the sample to be detected in the visualized fluorescence change data.

[0029] Those not detailed in the present invention can be carried out according to the prior art.

[0030] Advantageous effects:

[0031] 1. The calixarene used in the present invention has a unique three-dimensional cavity molecular structure, which can selectively adsorb guest molecules of different sizes. Then, by modifying the calixarene and introducing a boronic acid derivatization group, a series of calix[4]arene monoboric acid derivatives (B4FTPS), calix[6]arene monoboric acid derivatives (B6FTPS) or calix[8]arene monoboric acid derivatives (B8FTPS) are synthesized. Finally, using the calixarene monoboric acid derivative as the host molecule and 6,7-dihydroxycoumarin (XDS) as the fluorescent indicator, a supramolecular calixarene-based sensor is formed, and this sensor can be used for the detection of biological samples such as carbohydrate antigens, glycoproteins, cancer cells, and serum.

[0032] 2. The present invention provides the use of a supramolecular calixarene-based sensor for detecting biological samples such as carbohydrate antigens, glycoproteins, cancer cells, and serum. Among them, calixarene monoborate derivatives can specifically interact with tumor-associated carbohydrate antigens. 6,7-dihydroxycoumarin (XDS) has an ortho-dihydroxy structure, can specifically bind to boric acid, and its molecular size and structure are similar to those of carbohydrate antigens. In the detection system, the calixarene derivative and XDS first form a host-guest complex. When a carbohydrate antigen is added, due to the competitive effect of the carbohydrate antigen and XDS on the binding site of the calixarene, XDS will partially dissociate from the binding site of the calixarene, thus causing a change in the fluorescence signal. Different calixarene derivatives have different binding abilities to different carbohydrate antigens, so that when each carbohydrate antigen interacts with the sensor array, each sensing unit generates different degrees of fluorescence changes, forming a unique "fluorescence fingerprint". By collecting and analyzing these "fluorescence fingerprints" and using pattern recognition algorithms such as linear discriminant analysis (LDA), different carbohydrate antigens can be accurately distinguished, realizing the highly sensitive and highly selective detection of tumor-associated carbohydrate antigens.

[0033] 3. The present invention has confirmed that the supramolecular calixarene-based sensor can effectively detect and distinguish BSM glycoprotein, aBSM glycoprotein, and αFet glycoprotein rich in Tn antigen, T antigen, and sTn antigen glycan structures, can effectively detect and distinguish leukemia cell KG-1, gastric cancer cell MKN45, and breast cancer cell MDA-MB-231, can detect and distinguish different leukemia subtype cells Jurkat, KG-1, and K562, and can detect and distinguish serum containing carbohydrate antigens, indicating that the sensor has good recognition ability and potential application prospects in complex biological matrices, and proving its potential application value in the analysis of complex biological samples. Brief Description of the Drawings

[0034] Figure 1 1H NMR spectrum of calixarene monoborate derivatives (B4FTPS / B6FTPS / B8FTPS);

[0035] In the figure, (a) is B4FTPS; (b) is B6FTPS; (c) is B8FTPS.

[0036] Figure 2 Fluorescence results and binding constant results of calixarene and fluorescent indicator;

[0037] In the figure, (a) is the fluorescence result; (b) is the binding constant curve.

[0038] Figure 3 Fluorescence and binding constant results of the supramolecular calixarene-based sensor for detecting Tn carbohydrate antigen.

[0039] In the figure, (a) shows the fluorescence results; (b) shows the binding constant curve.

[0040] Figure 4 They are the fluorescence and binding constant results of the sensor based on supramolecular calixarene for detecting T carbohydrate antigen.

[0041] In the figure, (a) shows the fluorescence results; (b) shows the binding constant curve.

[0042] Figure 5 They are the fluorescence and binding constant results of the sensor based on supramolecular calixarene for detecting sTn carbohydrate antigen.

[0043] In the figure, (a) shows the fluorescence results; (b) shows the binding constant curve.

[0044] Figure 6 They are the statistical results of the binding constants of the sensor based on supramolecular calixarene for detecting Tn carbohydrate antigen, T carbohydrate antigen and sTn carbohydrate antigen.

[0045] Figure 7 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting single-component glycoproteins at the same concentration.

[0046] Figure 8 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting single glycoproteins at different concentrations.

[0047] Figure 9 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting protein mixtures.

[0048] Figure 10 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting single-component cells at the same concentration.

[0049] Figure 11 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting single cells at different concentrations.

[0050] Figure 12 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting cell mixtures.

[0051] Figure 13 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting single-component leukemia subtype cells at the same concentration.

[0052] Figure 14 They are the visualization results of the fluorescence change data of the sensor based on supramolecular calixarene for detecting single leukemia subtype cells at different concentrations.

[0053] Figure 15Visualization results of fluorescence changes in the detection of a single leukemia subtype cell mixture by a supramolecular calixarene-based sensor.

[0054] Figure 16 Visualization results of fluorescence changes in the detection of sera containing different concentrations of a single-component sugar antigen by a supramolecular calixarene-based sensor.

[0055] Figure 17 Visualization results of fluorescence changes in the detection of sera containing the same concentration of a single-component sugar antigen by a supramolecular calixarene-based sensor.

[0056] Figure 18 Visualization results of fluorescence changes in the detection of sera containing a mixed sugar antigen by a supramolecular calixarene-based sensor. Detailed implementation mode

[0057] The following further illustrates the solution of the present invention through specific examples and drawings, but does not limit the scope of protection required by the present invention. All raw materials used in the examples are conventional raw materials, and all equipment used is conventional equipment, which can be purchased commercially.

[0058] Example 1

[0059] 1. A preparation method of a calix[4]arene monoborate derivative, the steps are as follows:

[0060] 1) Weigh 1 mmol (0.64891 g) of p-tert-butylcalix[4]arene, 0.9 mmol (0.2673 g) of 4-bromomethylphenylboronic acid pinacol ester, and 0.9 mmol (0.1244 g) of anhydrous potassium carbonate, dissolve them in 80 mL of anhydrous acetonitrile, stir at room temperature until the particles are evenly dispersed, and then heat to 85 °C under nitrogen protection for 4 h; monitor the reaction process by thin-layer chromatography (TLC) to confirm the formation of new substances, the appearance of the target compound, and the completion of the reaction. After the reaction is completed, concentrate the reaction solution under vacuum, filter it under reduced pressure, wash the filter cake with dichloromethane to obtain a relatively clear yellow liquid; then extract and separate it with 1N HCl (2 × 50 mL) and distilled water (1 × 40 mL), take the lower-layer product solution, remove water with anhydrous sodium sulfate and then filter, concentrate the filtrate to dryness under vacuum, dissolve the precipitate in dichloromethane, and preliminarily purify it by column chromatography (petroleum ether: ethyl acetate 5:1) to obtain 865.0 mg of a pale yellow powder, namely the calix[4]arene monoborate compound;

[0061] 2) Add 1.0 mmol (866.1 mg) of calix[4]arene monoborate ester derivative and 3.0 mmol (639.0 mg) of sodium periodate to the reaction system of tetrahydrofuran THF:water (4:1). After 30 min, add 1.5 mmol (1.5 mL) of 1N HCl, and stir at room temperature overnight. The solution turns orange-yellow; rotary evaporate to remove THF, extract with ethyl acetate, and rotary evaporate to recover the precipitate; then purify by column chromatography (dichloromethane:ethyl acetate 1:1) to obtain 238.7 mg of pale yellow crystals, which is calix[4]arene monoboric acid derivative (B4FTPS).

[0062] 2. Prepare calix[6]arene monoboric acid derivative (B6FTPS) using tert-butylcalix[6]arene (B6FT) as the raw material and calix[8]arene monoboric acid derivative (B8FTPS) using tert-butylcalix[8]arene (B8FT) as the raw material according to the same method.

[0063] In the preparation process of calix[6]arene monoboric acid derivative (B6FTPS), the molar ratio of calix[6]arene, 4-bromomethylphenylboronic acid pinacol ester, and anhydrous potassium carbonate is 1:1.2:1.2, the volume of anhydrous acetonitrile is 100 mL, and other parameters are the same as the above method.

[0064] In the preparation process of calix[8]arene monoboric acid derivative (B8FTPS), the molar ratio of calix[8]arene, 4-bromomethylphenylboronic acid pinacol ester, and anhydrous potassium carbonate is 1:1.2:0.9, the volume of anhydrous acetonitrile is 140 mL, and other parameters are the same as the above method.

[0065] 3. The 1H NMR results of B4FTPS, B6FTPS, and B8FTPS prepared in this example are as Figure 1 shown.

[0066] It can be Figure 1 seen that the calixarene reacted with 4-bromomethylphenylboronic acid pinacol ester and deprotected to obtain the boric acid derivative of calixarene, which is reflected in the characteristic peak at chemical shift 5. In addition, according to the chemical integration, by calculating the ratio of the number of hydrogen atoms of the methylene group (chemical shift 2 - 5) on phenylboronic acid to the benzene ring (chemical shift 7 - 8) of calixarene, it is deduced that calix[4]arene, calix[6]arene, and calix[8]arene are all monosubstituted, that is, calix[4]arene monoboric acid derivative, calix[6]arene monoboric acid derivative, or calix[8]arene monoboric acid derivative are all successfully prepared.

[0067] Example 2

[0068] 1. Prepare the solution

[0069] Weigh 16.22 mg of calix[4]arene (B4FT), 24.33 mg of calix[6]arene (B6FT), 32.44 mg of calix[8]arene (B8FT), 19.57 mg of calix[4]arene monoborate derivative (B4FTPS), 7.68 mg of calix[6]arene monoborate derivative (B6FTPS), or 35.79 mg of calix[8]arene monoborate derivative (B8FTPS), and dissolve them separately in 25 mL of THF to obtain 6 stock solutions of calixarene compounds with a concentration of 1 mM each; then dilute the stock solutions of calixarene compounds into gradient concentration calixarene compound solutions of 0.25 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM. Among them, B8FTPS is diluted into gradient concentration solutions of 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 10 μM, and 20 μM.

[0070] Weigh 1.78 mg of XDS and dissolve it in 250 mL of water to obtain a stock solution of XDS with a concentration of 40 mM; then dilute the stock solution of XDS into a 1 μM XDS solution.

[0071] 2. Determine the optimal binding ratio

[0072] According to a volume ratio of 1:1, gradually add dropwise the gradient concentration calixarene compound solutions (B4FT / B6FT / B8FT / B4FTPS / B6FTPS are all 0, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM; B8FTPS is 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 10 μM, 20 μM) to the XDS solution (1 μM) respectively. During the dropping process, gently stir continuously to ensure uniform mixing to obtain a mixed solution; then suck the mixed solution into a fluorescence cuvette and record the fluorescence spectrum in the wavelength range of 400 - 650 nm to collect the fluorescence response signal data; then import the fluorescence response signal data into Origin 2024b software to analyze the influence of the gradient concentration calixarene compound solutions on the fluorescence intensity of XDS; finally, calculate the binding constants between different calixarene hosts and different sugar antigens through the Benesi-Hildebrand equation, and screen out the optimal binding ratio of the calixarene compound solution and XDS. The results are as Figure 2 shown.

[0073] As Figure 2 can be seen, when the molar ratio of calixarene to the fluorescent indicator is 1:4, a clearly distinguishable fluorescence response can be generated in the mixed solution. This molar ratio is used as the basic construction scheme of the sensor solution, providing reliable technical support for the detection and analysis of subsequent carbohydrate antigens.

[0074] 3. Detect free sugar antigens

[0075] The calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS) were mixed in PBS buffer according to a molar ratio of 1:4, and the mixture was vigorously shaken up and down using a vortex oscillator to promote self-assembly to form a supramolecular complex, obtaining a sensor solution based on supramolecular calixarene; then, Tn saccharide antigen, T saccharide antigen, and sTn saccharide antigen solutions were added to the sensor solution, respectively, so that the final concentrations of different saccharide antigens (Tn, T, and sTn) were all 0.5 nM to 20 μM (specific gradient concentrations are shown in Figures 3 - 5 ), obtaining a glycogen detection system; the glycogen detection system was incubated at 37 °C for 20 min to ensure sufficient reaction between the saccharide antigen and the sensing unit to form a stable binding state; then, the fluorescence emission spectrum of the glycogen detection system was measured using a fluorescence spectrophotometer, and the fluorescence response signal F at 464 nm was collected; based on the data of the change in the fluorescence response signal F of XDS caused by different concentrations of saccharide antigens, a double reciprocal plot of the corresponding concentration C and the fluorescence change value F - F0 was made, and combined with the Benesi-Hildebrand equation, the binding constants between the calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS) and XDS and between the supramolecular calixarene-based sensor (BFT(PS)-XDS) and the three saccharide antigens could be calculated. The results are shown in Figures 3 - 5 ; statistical analysis of the above binding constant data was performed, and the results are shown in Figure 6 .

[0076] As can be seen from Figures 3 - 5 , when the three saccharide antigens (Tn, T, and sTn) act on the supramolecular calixarene-based sensor (BFT(PS)-XDS) respectively, the fluorescence of XDS all shows a recovery phenomenon, and there are differences in the fluorescence effects of different saccharide antigens on the sensing system, indicating that competitive displacement has occurred between the saccharide antigen and XDS, resulting in the release of XDS and causing fluorescence recovery.

[0077] As can be seen from Figure 6 , the binding constants of the three saccharide antigens (Tn, T, and sTn) with the calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS) are much larger than the binding constants of XDS with the calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS). This indicates that the three saccharide antigens have a stronger binding ability to calixarene than XDS, which is the basis of the IDA method. At the same time, the differences in these binding constants further support the rationality and feasibility of the sensing array designed based on IDA and can be used to distinguish different saccharide antigens.

[0078] Example 3

[0079] Detect and distinguish BSM glycoprotein, aBSM glycoprotein and αFet glycoprotein rich in Tn antigen, T antigen and sTn antigen glycan structures.

[0080] In this glycoprotein detection and discrimination experiment, referring to the steps of detecting free sugar antigens in Example 2, multi-level detection was performed on three glycoproteins (BSM, αBSM and αFet), including detection of single-component glycoproteins at the same concentration, single glycoproteins at different concentrations, and glycoprotein mixtures, to evaluate the application ability of the supramolecular calixarene-based sensor of the present invention in complex biological samples.

[0081] 1. Detection of single-component glycoproteins at the same concentration

[0082] Select BSM glycoprotein, aBSM glycoprotein and αFet glycoprotein at three concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL) as target analytes.

[0083] Mix calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS and B8FTPS) in PBS buffer at a molar ratio of 1:4 respectively, and use a vortex oscillator to oscillate up and down sufficiently to promote their self-assembly to form a supramolecular complex, obtaining a supramolecular calixarene-based sensor solution; then add BSM glycoprotein, aBSM glycoprotein and αFet glycoprotein solutions at different concentrations to the sensor solution respectively to obtain a glycoprotein detection system; incubate the glycoprotein detection system at 37 °C for 20 min to ensure that the glycoprotein reacts fully with the sensing unit to form a stable binding state; then measure the fluorescence emission spectrum of the glycogen detection system with a fluorescence spectrophotometer and collect the fluorescence response signal F at 464 nm.

[0084] Five repeated experiments were carried out for each of the 3 glycoproteins (BSM, aBSM and αFet), constructing a training matrix containing 6 sensing units (B4FT, B6FT, B8FT, B4FTPS, B6FTPS and B8FTPS), 3 target substances (BSM, aBSM and αFet) and 5 repeated measurements. The data was processed by linear discriminant analysis (LDA) for dimensionality reduction, and two of the most important discriminant factors were selected to visualize the fluorescence change data. The results are as Figure 7 shown.

[0085] As Figure 7 can be seen, the fluorescence change data of the same type of glycoprotein are clustered together, while the fluorescence change data of different types of glycoproteins are completely distinguished, indicating that the supramolecular calixarene-based sensor provided by the present invention can distinguish three glycoproteins at various concentrations.

[0086] 2. Detection of single glycoproteins at different concentrations

[0087] Prepare BSM glycoprotein, aBSM glycoprotein and αFet glycoprotein solutions with gradient concentrations of 0.05, 0.1, 0.5, 1, 5, 10 μg / mL as the target analytes.

[0088] According to the method in point 1 of this example, use the supramolecular calixarene-based sensor to detect the target analytes respectively, and the results are as Figure 8 shown.

[0089] It can be seen from Figure 8 that the supramolecular calixarene-based sensor provided by the present invention can accurately distinguish single-component glycoproteins with different concentrations. This indicates that the sensor has good performance in the quantitative analysis of complex glycoproteins and can provide reliable technical support for the detection of cancer-related carbohydrate antigens.

[0090] 3. Detection of protein mixtures

[0091] Prepare binary protein mixtures, namely: 75% αBSM + 25% BSM, 25% αBSM + 75% BSM, 50% αBSM + 50% BSM; 75% αBSM + 25% αFet, 25% αBSM + 75% αFet, 50% αBSM + 50% αFet; 75% αFet + 25% BSM, 25% αFet + 75% BSM, 50% αFet + 50% BSM;

[0092] Prepare ternary protein mixtures, namely: 10% αBSM + 10% αFet + 80% BSM, 30% αBSM + 30% αFet + 40% BSM, 20% αBSM + 20% αFet + 60% BSM;

[0093] The total concentration of all the above protein mixtures is accurately set to 1 μg / mL as the target analyte, and "%" is the mass percentage.

[0094] According to the method in point 1 of this example, use the supramolecular calixarene-based sensor to detect the target analytes respectively, and at the same time use the solutions of individual BSM glycoprotein, aBSM glycoprotein and αFet glycoprotein as the controls for each protein mixture. The results are as Figure 9 shown.

[0095] It can be seen from Figure 9 that the supramolecular calixarene-based sensor provided by the present invention produces different response patterns for each mixture, and both binary protein mixtures and ternary protein mixtures can be clearly distinguished. This shows that the supramolecular calixarene-based sensor provided by the present invention can accurately identify glycoprotein mixtures with different ratios, demonstrating its potential application value in the analysis of complex biological samples.

[0096] Example 4

[0097] Detect and distinguish leukemia cells KG-1, gastric cancer cells MKN45, and breast cancer cells MDA-MB-231.

[0098] In this cell detection and discrimination experiment, referring to the steps of detecting free sugar antigens in Example 2, three types of cells (leukemia cells KG-1, gastric cancer cells MKN45, and breast cancer cells MDA-MB-231) were detected at multiple levels, including detecting single-component cells at the same concentration, single cells at different concentrations, and cell mixtures, to evaluate the application ability of the sensor based on supramolecular calixarene of the present invention in complex biological samples.

[0099] 1. Detection of single-component cells at the same concentration

[0100] Select gastric cancer cells MKN45, breast cancer cells MDA-MB-231, and leukemia cells KG-1 at four concentrations (4×10, 2×10, 1×10, 4×10 3 cells / mL) as the target analytes.

[0101] According to a molar ratio of 1:4, the calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS) were mixed in PBS buffer respectively, and the vortex oscillator was used to fully oscillate up and down to promote their self-assembly to form a supramolecular complex, obtaining a sensor solution based on supramolecular calixarene; then different concentrations of gastric cancer cells MKN45, breast cancer cells MDA-MB-231, and leukemia cells KG-1 were added to the sensor solution respectively to obtain a cell detection system; the cell detection system was incubated at 37°C for 20 min to ensure that the cells reacted fully with the sensing unit to form a stable binding state; then the fluorescence emission spectrum of the cell detection system was measured with a fluorescence spectrophotometer, and the fluorescence response signal F at 464 nm was collected.

[0102] Five repeated experiments were carried out for each of the three types of cells (gastric cancer cells MKN45, breast cancer cells MDA-MB-231, and leukemia cells KG-1), constructing a training matrix containing 6 sensing units (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS), 3 target analytes (gastric cancer cells MKN45, breast cancer cells MDA-MB-231, and leukemia cells KG-1t), and 5 repeated measurements. The data was processed by linear discriminant analysis (LDA) for dimensionality reduction, and two of the most important discriminant factors were selected to visualize the fluorescence change data. The results are as Figure 10 shown.

[0103] Figure 10Among them, 4w corresponds to 4×10 cells / mL, 2w corresponds to 2×10 cells / mL, 1w corresponds to 1×10 cells / mL, and 4k corresponds to 4×10 3 cells / mL.

[0104] From Figure 10 it can be seen that there is good parallelism within the same cell type, while there are significant differences between different cell types. The sensor based on supramolecular calixarene provided by the present invention can completely distinguish gastric cancer cells MKN45, breast cancer cells MDA-MB-231, and leukemia cells KG-1 at four concentrations (4×10, 2×10, 1×10, 4×10 3 cells / mL).

[0105] 2. Detection of different concentrations of the same cancer cell

[0106] Select 12 concentrations (4×10, 3.5×10, 3×10, 2.5×10, 2×10, 1.5×10, 1×10, 8×10 3 , 6×10 3 , 4×10 3 , 2×10 3 , 1×10 3 cells / mL) of gastric cancer cells MKN45, breast cancer cells MDA-MB-231, and leukemia cells KG-1 as target analytes.

[0107] According to the method in point 1 of this example, use the sensor based on supramolecular calixarene to detect the target analytes respectively, and the results are as Figure 11 shown.

[0108] From Figure 11 it can be seen that the data clusters corresponding to different concentrations of cancer cells are completely separated, indicating that the sensor based on supramolecular calixarene provided by the present invention can accurately distinguish cancer cells at different concentrations. It proves the high sensitivity and accuracy of the sensor based on supramolecular calixarene provided by the present invention in the detection of cancer cell concentration, and provides reliable technical support for the detection of cancer cells in practical applications.

[0109] 3. Detection of cell mixtures

[0110] Prepare binary cancer cell mixtures, namely: 75% MKN45 + 25% KG-1, 25% MKN45 + 75% KG-1, 50% MKN45 + 50% KG-1; 75% MKN45 + 25% MDA-MB-231, 25% MKN45 + 75% MDA-MB-231, 50% MKN45 + 50% MDA-MB-231; 75% KG-1 + 25% MDA-MB-231, 25% KG-1 + 75% MDA-MB-231, 50% KG-1 + 50% MDA-MB-231;

[0111] Prepare ternary cancer cell mixtures, 10% KG-1 + 10% MKN45 + 80% MDA-MB-231, 30% KG-1 + 30% MKN45 + 40% MDA-MB-231, 20% KG-1 + 20% MKN45 + 60% MDA-MB-231;

[0112] The above cell mixtures are used as target analytes, and "%" is the percentage of the number of cells.

[0113] According to the method in point 1 of this example, use the supramolecular calixarene-based sensor to detect the target analytes respectively, and use single leukemia cells KG-1, gastric cancer cells MKN45, and breast cancer cells MDA-MB-231 as controls for each cell mixture. The results are as Figure 12 shown.

[0114] As can be Figure 12 seen, the supramolecular calixarene-based sensor provided by the present invention produces different response patterns for each cell mixture, and binary and ternary cancer cell mixtures can be clearly distinguished. This shows that the supramolecular calixarene-based sensor provided by the present invention can accurately identify cell mixtures with different proportions, demonstrating its potential application value in the analysis of complex biological samples.

[0115] Example 5

[0116] Detect and distinguish different leukemia subtype cells Jurkat, KG-1, and K562.

[0117] In this cell detection and discrimination experiment, referring to the steps of detecting free sugar antigens in Example 2, perform multi-level detection on 3 leukemia subtype cells (leukemia subtype cells Jurkat, KG-1, and K562), including detecting single-component cells at the same concentration, single cells at different concentrations, and cell mixtures, to evaluate the application ability of the supramolecular calixarene-based sensor of the present invention in complex biological samples.

[0118] 1. Detection of single-component cells at the same concentration

[0119] Four concentrations (4×10, 2×10, 1×10, 2×10 3 cells / mL) of leukemia subtype cells Jurkat, KG-1, and K562 were selected as target analytes.

[0120] According to a molar ratio of 1:4, calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS) were mixed in PBS buffer, and vortexed thoroughly up and down to promote self-assembly to form supramolecular complexes, obtaining sensor solutions based on supramolecular calixarenes; then, different concentrations of leukemia subtype cells Jurkat, KG-1, and K562 were added to the sensor solutions respectively to obtain cell detection systems; the cell detection systems were incubated at 37 °C for 20 min to ensure sufficient reaction between the cells and the sensing units to form a stable binding state; then, the fluorescence emission spectra of the cell detection systems were measured with a fluorescence spectrophotometer, and the fluorescence response signal F at 464 nm was collected.

[0121] Five repeated experiments were performed on each of the 3 types of cells (leukemia subtype cells Jurkat, KG-1, and K562), constructing a training matrix containing 6 sensing units (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS), 3 target analytes (leukemia subtype cells Jurkat, KG-1, and K562), and 5 repeated measurements. The data was dimensionally reduced by linear discriminant analysis (LDA), and two of the most important discriminant factors were selected to visualize the fluorescence change data, and the results are as Figure 13 shown.

[0122] Figure 13 In it, 4w corresponds to 4×10 cells / mL, 2w corresponds to 2×10 cells / mL, 1w corresponds to 1×10 cells / mL, and 2k corresponds to 2×10 3 cells / mL.

[0123] It can be Figure 13 seen that good parallelism is presented within the same cell type, while significant differences exist between different cell types, indicating that the sensor based on supramolecular calixarenes provided by the present invention can completely distinguish leukemia subtype cells Jurkat, KG-1, and K562 at four concentrations (4×10, 2×10, 1×10, 2×10 3 cells / mL).

[0124] 2. Detection of different concentrations of the same cancer cell

[0125] Select 12 concentrations (4×10, 3.5×10, 3×10, 2.5×10, 2×10, 1.5×10, 1×10, 8×10 3 , 6×10 3 , 4×10 3 , 2×10 3 , 1×10 3 cells / mL) of leukemia subtype cells Jurkat, KG-1, and K562 as target analytes.

[0126] According to the method in point 1 of this example, use the supramolecular calixarene-based sensor to detect the target analytes respectively, and the results are as Figure 14 shown.

[0127] Figure 14 Among them, 4w, 3.5w, 3w, 2.5w, 2w, 1.5w, 1w, 8k, 6k, 4k, 2k, 1k correspond to 12 concentrations.

[0128] It can be seen from Figure 14 that the supramolecular calixarene-based sensor provided by the present invention can accurately distinguish and has good discrimination ability for three leukemia subtype cells in a certain concentration range (6×10 3 ~4×10 cells / mL). It is proved that the supramolecular calixarene-based sensor provided by the present invention has certain sensitivity and accuracy in the detection of leukemia subtype cells. Only when the concentration is lower than 6×10 3 cells / mL, there will be partial overlap between different leukemia subtype cells, providing reliable technical support for the detection of leukemia subtype cells in practical applications.

[0129] 3. Detection of cell mixtures

[0130] Prepare binary leukemia subtype cell mixtures, namely: 75% K562 + 25% KG-1, 25% K562 + 75% KG-1, 50% K562 + 50% KG-1; 75% K562 + 25% Jurkat, 25% K562 + 75% Jurkat, 50% K562 + 50% Jurkat; 75% KG-1 + 25% Jurkat, 25% KG-1 + 75% Jurkat, 50% KG-1 + 50% Jurkat;

[0131] Prepare ternary leukemia subtype cell mixtures, 10% KG-1 + 10% K562 + 10% Jurkat, 30% KG-1 + 30% K562 + 40% Jurkat, 20% KG-1 + 20% K562 + 60% Jurkat;

[0132] The above cell mixture is used as the target analyte, and "%" represents the percentage of the number of cells.

[0133] According to the method in point 1 of this example, the target analyte was detected separately using the supramolecular calixarene-based sensor. At the same time, the individual leukemia subtype cells Jurkat, KG-1, and K562 were used as controls for each cell mixture. The results are as Figure 15 shown.

[0134] As Figure 15 can be seen, the supramolecular calixarene-based sensor provided by the present invention produced different response patterns for each mixture, and the binary leukemia subtype cell mixture and the ternary leukemia subtype cell mixture could be clearly distinguished. This shows that the supramolecular calixarene-based sensor provided by the present invention can accurately identify leukemia subtype cell mixtures with different ratios, demonstrating its potential application value in the analysis of complex biological samples.

[0135] Example 6

[0136] Detect and distinguish serum samples containing carbohydrate antigens.

[0137] In this serum detection experiment, referring to the steps for detecting free carbohydrate antigens in Example 2, different serum samples were detected at multiple levels, including sera containing different concentrations of single-component carbohydrate antigens, sera containing the same concentration of single-component carbohydrate antigens, and sera containing mixed carbohydrate antigens, to evaluate the application ability of the supramolecular calixarene-based sensor of the present invention in complex biological samples.

[0138] 1. Detection of sera containing different concentrations of single-component carbohydrate antigens

[0139] The Tn carbohydrate antigen, sTn carbohydrate antigen, and T carbohydrate antigen were respectively added to the serum to make their concentrations in a gradient of 0.6, 1, 1.25, 2, 3.3, and 10 nM, obtaining sera containing different concentrations of single-component carbohydrate antigens as the target analyte.

[0140] The calixarene compound solutions (B4FT, B6FT, B8FT, B4FTPS, B6FTPS, and B8FTPS) were mixed in PBS buffer according to a molar ratio of 1:4, and the vortex oscillator was used to fully oscillate up and down to promote their self-assembly to form a supramolecular complex, obtaining the supramolecular calixarene-based sensor solution; then sera containing different concentrations of single-component carbohydrate antigens were respectively added to the sensor solution to obtain a serum detection system; the serum detection system was incubated at 37 °C for 20 min to ensure that the carbohydrate antigen fully reacted with the sensing unit to form a stable binding state; then the fluorescence emission spectrum of the serum detection system was measured with a fluorescence spectrophotometer, and the fluorescence response signal F at 464 nm was collected.

[0141] Five repeated experiments were carried out for each of the 6 concentrations (0.6, 1, 1.25, 2, 3.3, 10 nM), constructing a training matrix containing 6 sensing units (B4FT, B6FT, B8FT, B4FTPS, B6FTPS and B8FTPS), 18 analytes (3 carbohydrate antigens × 6 concentrations) and 5 repeated measurements. The data was dimensionally reduced by linear discriminant analysis (LDA), two of the most important discriminant factors were selected, and the fluorescence change data was visualized. The results are as Figure 16 shown.

[0142] As Figure 16 can be seen, the sensor based on supramolecular calixarene provided by the present invention produced different fluorescence response patterns for three carbohydrate antigens at six different concentrations. Although the confounding effect of serum had a certain impact on the samples, resulting in the data clusters of samples at each concentration being closer in spatial distance, the sensor based on supramolecular calixarene was still able to distinguish these carbohydrate antigens at different concentrations due to its sensitive response to signals. This indicates that the sensor has good recognition ability and potential application prospects in complex biological matrices.

[0143] 2. Detection of serum containing single-component carbohydrate antigen at the same concentration

[0144] Tn carbohydrate antigen, sTn carbohydrate antigen and T carbohydrate antigen were respectively added to the serum to make their concentrations at the gradient of 0.6, 1, 2, 10 nM, obtaining serum containing single-component carbohydrate antigen at the same concentration as the target analyte.

[0145] According to the method in point 1 of this example, the sensor based on supramolecular calixarene was used to detect the target analytes respectively. The results are as Figure 17 shown.

[0146] As Figure 17 can be seen, in serum, carbohydrate antigens at different concentrations triggered differential responses of the sensor based on supramolecular calixarene provided by the present invention. At four concentrations, the three carbohydrate antigens could be accurately classified with an accuracy rate of 100%, and three non-overlapping groups were formed. This result indicates that the sensor has the ability to accurately distinguish three carbohydrate antigens in serum samples.

[0147] 3. Detection of serum containing mixed carbohydrate antigens

[0148] Serum containing binary carbohydrate antigens was prepared, namely Tn:T = 3:1, Tn:T = 1:3, Tn:T = 1:1, Tn:sTn = 1:1, Tn:sTn = 1:3, Tn:sTn = 3:1, T:sTn = 1:3, T:sTn = 1:1 and T:sTn = 3:1;

[0149] Prepare sera containing ternary sugar antigens, namely Tn∶T∶sTn = 1∶1∶8, Tn∶T∶sTn = 2∶2∶3, and Tn∶T∶sTn = 3∶3∶4;

[0150] The above sera containing mixed sugar antigens are used as target analytes.

[0151] According to the method in point 1 of this example, use the supramolecular calixarene-based sensor to detect the target analytes respectively. At the same time, use the sera containing individual sugar antigens as the control for the sera containing mixed sugar antigens. The results are as Figure 18 shown.

[0152] It can be seen from Figure 18 that the supramolecular calixarene-based sensor provided by the present invention can accurately distinguish sera containing mixed sugar antigens with different ratios, indicating the applicability of the sensor in detecting sugar antigen mixtures in complex samples, showing its high reliability in practical applications, and proving its potential application value in the analysis of complex biological samples.

[0153] In summary, the supramolecular calixarene-based sensor provided by the present invention can effectively detect and distinguish BSM glycoprotein, aBSM glycoprotein, and αFet glycoprotein rich in Tn antigen, T antigen, and sTn antigen glycan structures, can effectively detect and distinguish leukemia cell KG-1, gastric cancer cell MKN45, and breast cancer cell MDA-MB-231, can detect and distinguish different leukemia subtype cells Jurkat, KG-1, and K562, and can detect and distinguish sera containing sugar antigens, indicating that the sensor has good recognition ability and potential application prospects in complex biological matrices, and proving its potential application value in the analysis of complex biological samples.

Claims

1. A supramolecular calixarene-based sensor, characterized in that, It includes calixarene and a fluorescent indicator; The molar ratio of the calixarene to the fluorescent indicator is 1:(3.5 - 4.5).

2. The supramolecular calixarene-based sensor according to claim 1, characterized in that, The calixarene is tert-butyl calixarene (BFT) or calixarene monoboric acid derivative (BFTPS); Further preferably, the tert-butyl calixarene is tert-butyl calix[4]arene, tert-butyl calix[6]arene or tert-butyl calix[8]arene; the calixarene monoboric acid derivative is calix[4]arene monoboric acid derivative, calix[6]arene monoboric acid derivative or calix[8]arene monoboric acid derivative.

3. The supramolecular calixarene-based sensor according to claim 1, characterized in that, The fluorescent indicator is 6,7-dihydroxycoumarin; the molar ratio of the calixarene to the fluorescent indicator is 1:

4.

4. The sensor based on supramolecular calixarene according to claim 2, characterized in that, The calixarene monoboric acid derivative is prepared by the following method: (1) Add calixarene, 4-bromomethylphenylboronic acid pinacol ester and anhydrous potassium carbonate to ultradry acetonitrile in sequence, reflux and react at 80 - 90 °C under nitrogen protection for 4 - 7 h to obtain a borate compound; (2) Add the borate compound to an aqueous solution of tetrahydrofuran, add sodium periodate and hydrochloric acid in sequence, react overnight at 20 - 30 °C for 12 - 18 h, and after purification, obtain the calixarene monoboric acid derivative.

5. The supramolecular calixarene-based sensor according to claim 4, wherein In step (1), the molar ratio of the calixarene, 4-bromomethylphenylboronic acid pinacol ester and anhydrous potassium carbonate is 1:(0.9 - 1.2):(0.9 - 1.2), and the volume of anhydrous acetonitrile is 80 - 140 mL; Further preferably, when the calixarene is calix[4]arene, the molar ratio of calix[4]arene, 4-bromomethylphenylboronic acid pinacol ester and anhydrous potassium carbonate is 1:0.9:0.9, and the volume of anhydrous acetonitrile is 80 mL; when the calixarene is calix[6]arene, the molar ratio of calix[6]arene, 4-bromomethylphenylboronic acid pinacol ester and anhydrous potassium carbonate is 1:1.2:1.2, and the volume of anhydrous acetonitrile is 100 mL; when the calixarene is calix[8]arene, the molar ratio of calix[8]arene, 4-bromomethylphenylboronic acid pinacol ester and anhydrous potassium carbonate is 1:1.2:0.9, and the volume of anhydrous acetonitrile is 140 mL.

6. The supramolecular calixarene-based sensor according to claim 4, characterized in that, In step (2), the mass ratio of the borate compound, aqueous solution of tetrahydrofuran, sodium periodate and hydrochloric acid is 1:(2.5 - 3.5):(1 - 2); Further preferably, the molar ratio of the borate compound, aqueous solution of tetrahydrofuran, sodium periodate and hydrochloric acid is 1:3:1.

5.

7. The preparation method of the supramolecular calixarene-based sensor according to claim 1, characterized in that, The steps include as follows: Prepare a calixarene monoboric acid derivative solution and a fluorescent indicator solution respectively, and then mix the calixarene monoboric acid derivative solution and the fluorescent indicator solution evenly so that the molar ratio of the calixarene monoboric acid derivative to the fluorescent indicator is 1:(3.5 - 4.5) to obtain a sensor based on supramolecular calixarene.

8. Application of the sensor based on supramolecular calixarene according to claim 1 in detecting carbohydrate antigen, glycoprotein, cancer cell, serum.

9. The application according to claim 8, wherein The carbohydrate antigen is sTn carbohydrate antigen, Tn carbohydrate antigen or T carbohydrate antigen; the glycoprotein is BSM glycoprotein, aBSM glycoprotein or αFet glycoprotein; the cancer cells are leukemia cells Jurkat, KG-1, K562, gastric cancer cells MKN45 or breast cancer cells MDA-MB-231.

10. A method for detecting carbohydrate antigens, glycoproteins, cancer cells, and serum using the above-mentioned supramolecular calixarene-based sensor for non-diagnostic purposes, characterized in that, It includes the following steps: Add different volumes of the sample solution to be detected into the sensor solution based on supramolecular calixarene, so that the final concentration of the sample to be detected is 0.5 nM to 20 μM, incubate at 37 °C for 15 to 25 min, measure the fluorescence emission spectrum of the mixed solution, and collect the fluorescence response signal F at F464 to obtain fluorescence change data; then perform dimensionality reduction processing on the fluorescence change data by linear discriminant analysis (LDA) to visualize the fluorescence change data; finally, qualitatively distinguish the sample to be detected according to the distribution of the sample to be detected in the visualized fluorescence change data.

Images