Quenching type supramolecular fluorescent probe for pepsin detection as well as preparation method and application of quenching type supramolecular fluorescent probe

Abstract

The invention discloses a quenching type supramolecular fluorescent probe for pepsin detection as well as a preparation method and application of the quenching type supramolecular fluorescent probe. The quenching type supramolecular fluorescent probe is obtained by virtue of supramolecular interaction of a luminous monomer and a protein molecule; wherein the protein molecule is selected from any one or a combination of more of human serum albumin, bovine serum albumin, casein, hemoglobin and collagen; the luminous monomer comprises an alpha-cyano styrene derivative. The quenching type supramolecular fluorescent probe is easy to synthesize, has good water solubility, can be used as a quenching type fluorescent probe of pepsin, has good selectivity and anti-interference capability, and can well meet the requirements of high sensitivity and accurate detection of pepsin.

Description

Technical Field

[0001] The present invention relates to a "quenched type" supramolecular fluorescent probe for pepsin detection, its preparation method and application, belonging to the technical field of biological monitoring. Background Art

[0002] In critically ill patients (such as severe trauma, shock, multiple organ failure, etc.), under strong stress, the barrier function is often damaged due to insufficient gastric mucosal blood perfusion, which is likely to cause acute gastric mucosal lesions or even stress ulcer bleeding. At this time, pepsin detection can be used as an important indicator for evaluating gastric mucosal damage - an increase in pepsin activity indicates enhanced gastric acid attack, while abnormal pepsin secretion reflects gastric mucosal dysfunction. In the management of critically ill patients, monitoring the change of pepsin level helps to early identify patients at high risk of digestive tract bleeding, guide timely clinical intervention, and provide a reference basis for evaluating the severity of the disease and prognosis.

[0003] At present, a variety of analytical methods for pepsin detection have been developed, mainly including enzyme-linked immunosorbent assay (ELISA), molecular imprinting analysis, electrochemical immunosensor method, etc. These methods are relatively mature and have certain detection sensitivity, but there are still many obvious defects: First, these methods generally require complex sample pretreatment processes, which are cumbersome to operate and invasive to patients; Second, the accuracy and repeatability of quantitative detection need to be improved, especially with large errors in low-concentration samples; In addition, these methods have high requirements for the experimental environment and operators, and the detection cost is expensive, facing major challenges in clinical promotion and market acceptance. These technical bottlenecks seriously restrict the popularization and application of pepsin detection in clinical practice, especially in scenarios such as emergency and intensive care that require rapid decision-making. Summary of the Invention

[0004] The main purpose of the present invention is to provide a "quenched type" supramolecular fluorescent probe for pepsin detection, its preparation method and application, so as to overcome the deficiencies in the prior art.

[0005] To achieve the foregoing invention purpose, the technical solutions adopted by the present invention include:

[0006] The embodiment of the present invention provides a "quenched type" supramolecular fluorescent probe for pepsin detection, and the "quenched type" supramolecular fluorescent probe is obtained by the supramolecular interaction between a luminescent monomer and a protein molecule; wherein, the protein molecule is selected from any one or a combination of human serum albumin, bovine serum albumin, casein, hemoglobin, collagen; the luminescent monomer includes an α-cyanostyrene derivative, and the α-cyanostyrene derivative has a structure shown in formula (I):

[0007]

[0008] Among them, R is selected from diethylamino or octahydroquinolizine, X is selected from a sulfur atom, a nitrogen atom or an oxygen atom, and n is selected from any integer in the range of 10 to 12.

[0009] The embodiment of the present invention also provides a preparation method of the foregoing "quenching type" supramolecular fluorescent probe, which includes:

[0010] Dissolve the luminescent monomer (BCPY) in a buffer solution to obtain a luminescent monomer solution;

[0011] Dissolve the protein molecule (M) in a buffer solution to obtain a protein molecule solution;

[0012] And, perform ultrasonic treatment on the luminescent monomer solution, and then mix it with the protein molecule solution for reaction to obtain a "quenching type" supramolecular fluorescent probe (BCPY-M).

[0013] The embodiment of the present invention also provides the use of the foregoing "quenching type" supramolecular fluorescent probe in the detection of pepsin.

[0014] The embodiment of the present invention also provides a method for detecting pepsin, which includes:

[0015] Provide the foregoing "quenching type" supramolecular fluorescent probe;

[0016] And, mix the "quenching type" supramolecular fluorescent probe with a sample to be tested to form a liquid-phase mixing system, and based on the change in the fluorescence intensity of the "quenching type" supramolecular fluorescent probe and the liquid-phase mixing system, the detection of pepsin is realized.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) The "quenching type" supramolecular fluorescent probe provided by the present invention has excellent photostability, pH stability and good water solubility. At the same time, the preparation method of the probe is simple, mainly relying on intermolecular interactions, and the cost is low.

[0019] (2) The "quenching type" supramolecular fluorescent probe provided by the present invention has high affinity and specificity for pepsin, and can have a strong interaction with pepsin, resulting in a significant change in the fluorescence signal;

[0020] (3) The specific recognition between the "quenching type" supramolecular fluorescent probe provided by the present invention and pepsin enables it to effectively distinguish pepsin from other proteins and biomolecules, has good selectivity and anti-interference ability, and can well meet the requirements of highly sensitive and accurate detection of pepsin. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 is the fluorescence spectrum of BCPY-M with different concentrations of pepsin in Example 1 of the present invention;

[0023] Figure 2 is the linear relationship diagram between the difference between the fluorescence intensity (F) of BCPY-M after adding pepsin and the original fluorescence intensity (F0) of BCPY-M and the concentration of pepsin at a wavelength of 560 nm in Example 1 of the present invention;

[0024] Figure 3 is the test result diagram of the selective recognition ability of BCPY-M for pepsin in Example 1 of the present invention;

[0025] Figure 4 is the test result diagram of the anti-interference ability of BCPY-M for different substances in Example 1 of the present invention. Detailed implementation manners

[0026] In view of the defects of the prior art, the inventors of this case have proposed the technical solutions of the present invention through long-term research and a large number of practices. To facilitate the understanding of this application, the following will describe this application in more detail. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0027] Specifically, as an aspect of the technical solution of the present invention, it relates to a "quenching type" supramolecular fluorescent probe for pepsin detection, and the "quenching type" supramolecular fluorescent probe is obtained by the supramolecular interaction between a luminescent monomer and a protein molecule; wherein, the protein molecule is selected from any one or a combination of human serum albumin (HSA), bovine serum albumin (BSA), casein, hemoglobin, and collagen; the luminescent monomer includes an α-cyanostyrene derivative, and the α-cyanostyrene derivative has a structure shown in formula (I):

[0028]

[0029] Among them, R is selected from diethylamino or octahydroquinolizinyl, X is selected from a sulfur atom, a nitrogen atom or an oxygen atom, and n is selected from any integer in the range of 10 to 12.

[0030] As another aspect of the technical solution of the present invention, the preparation method of the aforementioned "quenching type" supramolecular fluorescent probe involved therein includes:

[0031] Dissolve the luminescent monomer in a buffer solution to obtain a luminescent monomer solution;

[0032] Dissolve the protein molecule in a buffer solution to obtain a protein molecule solution;

[0033] And, subject the luminescent monomer solution to ultrasonic treatment, and then mix it with the protein molecule solution for reaction to obtain a "quenching type" supramolecular fluorescent probe.

[0034] In some preferred embodiments, the concentration of the luminescent monomer solution is 5×10 -6 mol / L to 1×10 -3 mol / L.

[0035] In some preferred embodiments, the concentration of the protein molecule solution is 5×10 -6 mol / L to 1×10 - 3 mol / L.

[0036] In some preferred embodiments, the molar ratio of the luminescent monomer to the protein molecule is 0.1 to 10:1.

[0037] In some preferred embodiments, the buffer solution includes any one of sodium dihydrogen phosphate-phosphate buffer solution, citric acid-sodium citrate buffer solution, glycine-hydrochloric acid buffer solution, and is not limited thereto.

[0038] In some more specific embodiments, the preparation method of the "quenching type" supramolecular fluorescent probe includes the following steps:

[0039] 1) Dissolve the luminescent monomer (BCPY) in a buffer solution to obtain a luminescent monomer solution with a concentration range of 5×10 -6 mol / L to 1×10 -3 mol / L;

[0040] 2) Dissolve the protein molecule M in a buffer solution to obtain a protein molecule M solution with a concentration range of 5×10 -6 mol / L to 1×10 -3 mol / L;

[0041] The protein molecule M is any one or a mixture of any proportion of human serum albumin (HSA), bovine serum albumin (BSA), casein, hemoglobin or collagen;

[0042] 3) After ultrasonically treating the luminescent fluorescent molecule solution for 5 - 15 min, add the protein molecule M solution to obtain a "quenching type" supramolecular fluorescent probe solution, wherein the molar ratio of the luminescent fluorescent molecule solution to the protein molecule M solution is 0.1 - 10.

[0043] In some preferred embodiments, the preparation method of the luminescent monomer includes:

[0044] React a first mixed reaction system containing a first compound, a basic substance, and a second compound to obtain an intermediate product; wherein, the first compound includes 8 - hydroxyjulolidine - 9 - formaldehyde and / or 4 - (diethylamino)salicylaldehyde; the second compound includes any one or a combination of more than one of 1,10 - dibromodecane, 1,11 - dibromoundecane, and 1,12 - dibromododecane;

[0045] And, react a second mixed reaction system containing at least the intermediate product, a third compound, and piperidine to obtain a luminescent monomer; wherein, the third compound includes any one or a combination of more than one of benzothiazole - 2 - acetonitrile, benzoxazole - 2 - acetonitrile, and benzimidazole - 2 - acetonitrile.

[0046] In some preferred embodiments, the preparation method specifically includes:

[0047] Mix the first compound with the basic substance and perform a vacuum treatment, then add a first solvent and the second compound and reflux and stir at 70 - 120 °C for 12 - 24 h to obtain a first mixed solution;

[0048] And, perform reduced pressure distillation, extraction, water removal, reduced pressure distillation, and column chromatography on the first mixed solution to obtain an intermediate product.

[0049] Further, the molar ratio of the first compound to the second compound is 1∶2 - 1∶5.

[0050] Further, the molar ratio of the first compound to the basic substance is 1∶3 - 1∶6.

[0051] Further, the first solvent includes any one or a combination of more than one of acetonitrile, isopropanol, and tetrahydrofuran, and is not limited thereto.

[0052] In some preferred embodiments, the preparation method specifically includes: Dissolve the intermediate product and the third compound in ethanol, then add piperidine and react at room temperature for 12 h to obtain a second mixed solution, then filter, wash, and recrystallize, and then, under nitrogen protection, dissolve in a second solvent and react at 80 - 130 °C for 12 - 24 h to obtain a luminescent monomer.

[0053] Further, the molar ratio of the intermediate product, the third compound to the second solvent is 1:1 - 3:1 - 5.

[0054] Further, the second solvent includes any one or a combination of pyridine, dioxane, isopropanol, etc., and is not limited thereto.

[0055] In some preferred embodiments, the preparation method of the luminescent monomer includes the following steps:

[0056] 1) Mix compound L1 (the aforementioned "first compound") and potassium carbonate in a molar ratio of 1:5. After evacuating the air, add 20 - 30 mL of anhydrous acetonitrile. Under stirring at room temperature, add compound L2 (the aforementioned "intermediate product"), and heat to reflux and stir at a temperature above 80 °C for 12 - 24 h to obtain a mixed solution 1;

[0057] Among them, the compound L1 is 8 - hydroxydjulolidine - 9 - carbaldehyde or 4 - (diethylamino) salicylaldehyde, and the compound L2 is 1,10 - dibromodecane or 1,11 - dibromoundecane or 1,12 - dibromododecane;

[0058] The molar ratio of the compound L2 to the compound L1 is 2:1;

[0059] 2) Distill the mixed solution 1 obtained in step 1) under reduced pressure, extract it with a dichloromethane / water system to obtain an organic phase, add an appropriate amount of anhydrous sodium sulfate to remove water, and distill under reduced pressure to obtain a solid product 1;

[0060] 3) Subject the solid product 1 obtained in step 2) to silica gel column chromatography, and the eluent is dichloromethane and petroleum ether, where dichloromethane∶petroleum ether = 1∶10, to separate and obtain compound A (the aforementioned "second compound");

[0061] 4) Dissolve compound A and compound L3 (the aforementioned "third compound") in 40 mL of ethanol, add an appropriate amount of piperidine, and react at room temperature for 12 h to obtain a mixed solution 2;

[0062] Among them, the compound L3 is benzothiazole - 2 - acetonitrile or benzoxazole - 2 - acetonitrile or benzimidazole - 2 - acetonitrile;

[0063] The ratio of compound A∶compound L3∶piperidine = 1∶1.5∶1;

[0064] 5) Filter the mixed solution 2 to obtain a red powder, wash it with ice ethanol multiple times, and perform 2 recrystallizations with ethanol to obtain a solid product 2;

[0065] 6) Under nitrogen protection, dissolve the solid product 2 in 50 mL of anhydrous pyridine, react at 110 °C for 12 - 24 h, after cooling to room temperature, distill off pyridine under reduced pressure, and wash 3 times in a dichloromethane / ether system to obtain a fluorescent molecule, that is, a luminescent monomer.

[0066] As another aspect of the technical solution of the present invention, it also relates to the use of the aforementioned "quenching type" supramolecular fluorescent probe in the detection of pepsin.

[0067] As another aspect of the technical solution of the present invention, it also relates to a method for detecting pepsin, which includes:

[0068] Providing the aforementioned "quenching type" supramolecular fluorescent probe;

[0069] And, mixing the "quenching type" supramolecular fluorescent probe with a sample to be tested to form a liquid-phase mixing system, and detecting pepsin according to the change in the fluorescence intensity of the "quenching type" supramolecular fluorescent probe in the liquid-phase mixing system.

[0070] Specifically, the method for detecting pepsin includes: uniformly mixing the "quenching type" supramolecular fluorescent probe with a sample to be tested containing pepsin to form a liquid-phase mixing system, reacting under certain conditions for an appropriate time, and observing the change in the fluorescence intensity of the fluorescent probe before and after mixing with the sample to be tested, so as to detect pepsin in the sample to be tested.

[0071] The "quenching type" supramolecular fluorescent probe in the present invention is easy to synthesize, has good water solubility, can be used as a "quenching type" fluorescent probe for pepsin, and at the same time, the probe has good selectivity and anti-interference ability, which can well meet the requirements of high-sensitivity and accurate detection of pepsin.

[0072] The present invention is further illustrated by the following examples: According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0073] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following examples are well-known in the art.

[0074] Example 1

[0075] Dissolve the fluorescent molecule BCPY (the structure shown in formula (I)), where R is diethylamino, X is an O atom, and n is 10, in a buffer solution to obtain BCPY solution 1, and its concentration is: 5×10 -6mol / L. Dissolve HSA in the buffer to obtain Solution 2, whose concentration is: 1×10 -5 mol / L. After ultrasonicating Solution 1 for 5 - 15 min, add Solution 2 to obtain the BCPY-M solution, where the molar ratio of the fluorescent molecule BCPY to HSA is 0.5∶1. Additionally, prepare a series of pepsin standard solutions with different concentrations using the buffer.

[0076] Before detecting pepsin, first determine the optimal conditions for the system reaction. First, control conditions such as time and temperature to be the same, change the pH of the BCPY-M solution obtained in Example 1, add an equal amount of pepsin, and obtain the optimal reaction pH of 3 by recording the change in fluorescence emission. Next, under the condition of pH = 3, add an equal amount of pepsin to the BCPY-M solution and react for the same time at different temperatures. Obtain the optimal reaction temperature of 40 °C by recording the change in fluorescence emission. Subsequently, under the conditions of pH = 3 and temperature of 37 °C, add an equal amount of pepsin to the BCPY-M solution and react for different times. Obtain the optimal reaction time of 30 min by recording the change in fluorescence emission. Therefore, the optimal reaction conditions for the system are pH = 3, reaction temperature 37 °C, and reaction time 30 min. Under the optimal reaction conditions, add the pepsin solution to the BCPY-M solution and control the final concentration of pepsin to be 0, 0.01, 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.00, 1.50, 2.00 μmol / L. After the reaction is completed, perform fluorescence emission spectroscopy testing and record its fluorescence spectrum, as Figure 1 It can be seen that the BCPY-M of this example is used as a "quenching type" supramolecular fluorescence probe.

[0077] The limit of detection (LOD) of BCPY-M for pepsin can be calculated by the formula defined by IUPAC. The specific formula (1) is as follows:

[0078] LOD = 3σ / k (1)

[0079] In the formula, σ represents the standard deviation of the fluorescence intensity of 10 blank samples (BCPY-M solution without added pepsin) at 560 nm, and k represents the slope of the linear fitting curve. The standard deviation σ can be calculated by formula (2):

[0080]

[0081] n represents the number of samples, x i represents the fluorescence intensity value of each blank sample, Represents the average fluorescence intensity of 10 tests. The calculated LOD is 0.75 nmol / L, which indicates that BCPY-M is sensitive enough for the detection of pepsin. Figure 2 Shows the linear relationship between the difference between the fluorescence intensity (F) of BCPY-M after adding pepsin and the original fluorescence intensity (F0) of BCPY-M and the pepsin concentration at a wavelength of 560 nm.

[0082] As Figure 3 and Figure 4 shown, when K + , Na + , Ca 2+ , Fe 3+ , Fe 2+ , SO4 2- , CO3 2- , HCO3 - , NO3 - , NO2 - , PO4 2- , trypsin, lysozyme, cholic acid, α-amylase, L-lysine, L-glutamic acid, L-cysteine, DL-serine, glycine and other different potential interfering substances coexist, the fluorescence intensity at 560 nm does not change significantly, only the presence of pepsin will cause a significant decrease in the fluorescence intensity of BCPY-M. When pepsin and 50-fold equivalent of other interfering substances are added to BCPY-M together, the ratio of fluorescence emission fluctuates within a small range, indicating that BCPY-M has excellent selectivity and anti-interference ability as a probe for detecting pepsin. Therefore, BCPY-M can be used as a "quenching type" supramolecular fluorescence probe to detect pepsin.

[0083] For the practical application of this probe, using artificial acidic saliva as the matrix, and adding BCPY-M and pepsin of this example, the final concentrations of pepsin are 0.2, 0.4 and 0.6 μmol / L respectively. The detected values calculated according to the standard curve show good consistency with the added values, indicating that BCPY-M of this example can be used as a quenching type supramolecular fluorescence probe to realize the detection of pepsin in artificial acidic saliva.

[0084] Example 2

[0085] Dissolve the fluorescent molecule BCPY, where R is octahydroquinazoline, X is an O atom, and n is 12, in a buffer solution to obtain BCPY solution 1 with a concentration of: 5×10 -5 mol / L. Dissolve BSA in a buffer solution to obtain solution 2 with a concentration of: 5×10 -5mol / L. After sonication of Solution 1 for 5 - 15 min, Solution 2 was added to obtain the BCPY-M solution, where the molar ratio of the fluorescent molecule BCPY to BSA was 1:1. Additionally, a series of pepsin standard solutions with different concentrations were prepared using a buffer solution.

[0086] First, the optimal reaction conditions of the system were determined. First, with the time, temperature and other conditions controlled to be the same, the pH of the BCPY-M solution was changed, and an equal amount of pepsin was added. By recording the changes in fluorescence emission, the optimal reaction pH was found to be 2.2. Next, under the condition of pH = 2.2, an equal amount of pepsin was added to the BCPY-M solution, and the reaction was carried out at different temperatures for the same time. By the changes in fluorescence emission, the optimal reaction temperature was found to be 37 °C. Subsequently, under the conditions of pH = 2.2 and temperature of 37 °C, an equal amount of pepsin was added to the BCPY-M solution, and the reaction was carried out for different times. By the changes in fluorescence emission, the optimal reaction time was found to be 25 min. Therefore, the optimal reaction conditions of the system were pH = 2.2, reaction temperature 37 °C, and reaction time 25 min. Under the optimal reaction conditions, a pepsin solution was added to the BCPY-M solution, and the final concentrations of pepsin were controlled to be 0, 0.01, 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.00, 1.50, 2.00 μmol / L. After the reaction was completed, fluorescence emission spectroscopy was performed.

[0087] Example 3

[0088] The fluorescent molecule BCPY, where R is octahydroquinolizinium, X is a nitrogen atom, and n is 10, was dissolved in a buffer solution to obtain Solution 1 of BCPY, with a concentration of: 2×10 -4 mol / L. Casein was dissolved in a buffer solution to obtain Solution 2, with a concentration of: 1×10 - 4 mol / L. After sonication of Solution 1 for 5 - 15 min, Solution 2 was added to obtain the BCPY-M solution, where the molar ratio of the fluorescent molecule BCPY to casein was 2:1. Additionally, a series of pepsin standard solutions with different concentrations were prepared using a buffer solution.

[0089] First, determine the optimal conditions for the reaction of the system. First, control conditions such as time and temperature to be the same, change the pH of the BCPY-M solution, add an equal amount of pepsin, and obtain the optimal reaction pH of 2.6 by recording the change in fluorescence emission. Next, under the condition of pH = 2.6, add an equal amount of pepsin to the BCPY-M solution, react for the same time at different temperatures, and obtain the optimal reaction temperature of 40 °C by the change in fluorescence emission. Subsequently, under the conditions of pH = 2.6 and temperature of 40 °C, add an equal amount of pepsin to the BCPY-M solution, react for different times, and obtain the optimal reaction time of 35 min by recording the change in fluorescence emission. Therefore, the optimal reaction conditions for the system are pH = 2.6, reaction temperature 40 °C, and reaction time 35 min. Under the optimal reaction conditions, add a pepsin solution to the BCPY-M solution, control the final concentration of pepsin to be 0, 0.01, 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.00, 1.50, 2.00 μmol / L, and perform fluorescence emission spectroscopy testing after the reaction is completed.

[0090] Example 4

[0091] Dissolve the fluorescent molecule BCPY, where R is octahydroquinolizinium, X is S atom, and n is 12, in a buffer solution to obtain BCPY solution 1, and its concentration is: 4×10 -4 mol / L. Dissolve hemoglobin in a buffer solution to obtain solution 2, and its concentration is: 1×10 - 4 mol / L. After ultrasonicating solution 1 for 5 - 15 min, add solution 2 to obtain the BCPY-M solution, where the molar ratio of the fluorescent molecule BCPY to hemoglobin is 4:1. Additionally, prepare a series of standard pepsin solutions with different concentrations using a buffer solution.

[0092] First, determine the optimal conditions for the reaction of the system. First, control conditions such as time and temperature to be the same, change the pH of the BCPY-M solution obtained in Example 1, add an equal amount of pepsin, and obtain the optimal reaction pH of 2.5 by recording the change in fluorescence emission; next, under the condition of pH = 2.5, add an equal amount of pepsin to the BCPY-M solution, react for the same time at different temperatures, and obtain the optimal reaction temperature of 35 °C by recording the change in fluorescence emission; subsequently, under the conditions of pH = 2.5 and temperature of 35 °C, add an equal amount of pepsin to the BCPY-M solution, react for different times, and obtain the optimal reaction time of 35 min by recording the change in fluorescence emission. Therefore, the optimal reaction conditions for the system are pH = 2.5, reaction temperature 35 °C, and reaction time 35 min. Under the optimal reaction conditions, add a pepsin solution to the BCPY-M solution, control the final concentration of pepsin to be 0, 0.01, 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.00, 1.50, 2.00 μmol / L, and perform fluorescence emission spectroscopy testing after the reaction is completed.

[0093] Furthermore, the applicant also detected pepsin under the optimal reaction conditions using the BCPY-M probes prepared in Example 2, Example 3, and Example 4 with reference to the method of Example 1. The results showed that they can all be used as "quenching type" supramolecular fluorescence probes for detecting pepsin, and also exhibit excellent selectivity and anti-interference ability for pepsin, and can achieve specific and accurate detection of pepsin.

[0094] In addition, the inventor of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification with reference to the foregoing examples, and all obtained relatively ideal results.

[0095] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A "quenching type" supramolecular fluorescence probe for pepsin detection, characterized in that: The "quenching type" supramolecular fluorescent probe is obtained by the supramolecular interaction between a luminescent monomer and a protein molecule; wherein, the protein molecule is selected from any one or a combination of human serum albumin, bovine serum albumin, casein, hemoglobin, and collagen; the luminescent monomer includes an α-cyanostyrene derivative, and the α-cyanostyrene derivative has the structure shown in formula (I): Wherein, R is selected from diethylamino or octahydroquinolizinyl, X is selected from a sulfur atom, a nitrogen atom or an oxygen atom, and n is selected from any integer from 10 to 12.

2. The preparation method of the "quenching type" supramolecular fluorescent probe according to claim 1, characterized in that, It includes: Dissolving the luminescent monomer in a buffer solution to obtain a luminescent monomer solution; Dissolving the protein molecule in a buffer solution to obtain a protein molecule solution; And, subjecting the luminescent monomer solution to ultrasonic treatment, and then mixing it with the protein molecule solution for reaction to obtain a "quenching type" supramolecular fluorescent probe.

3. The preparation method according to claim 2, characterized in that: The concentration of the luminescent monomer solution is 5×10 - 6 mol / L to 1×10 -3 mol / L; and / or, the concentration of the protein molecular solution is 5×10 -6 mol / L to 1×10 -3 mol / L; And / or, the molar ratio of the luminescent monomer to the protein molecule is 0.1 to 10:1; And / or, the buffer solution includes any one of sodium dihydrogen phosphate-phosphate buffer solution, citric acid-sodium citrate buffer solution, and glycine-hydrochloric acid buffer solution.

4. The preparation method according to claim 2, wherein The preparation method of the luminescent monomer includes: Reacting a first mixed reaction system containing a first compound, a basic substance, and a second compound to obtain an intermediate product; wherein, the first compound includes 8-hydroxyjulolidine-9-carbaldehyde and / or 4-(diethylamino)salicylaldehyde; the second compound includes any one or a combination of 1,10-dibromodecane, 1,11-dibromoundecane, and 1,12-dibromododecane; And, reacting a second mixed reaction system containing at least the intermediate product, a third compound, and piperidine to obtain a luminescent monomer; wherein, the third compound includes any one or a combination of benzothiazole-2-acetonitrile, benzoxazole-2-acetonitrile, and benzimidazole-2-acetonitrile.

5. The preparation method according to claim 2, characterized in that, Specifically includes: Mixing the first compound with a basic substance and performing a vacuum treatment, then adding a first solvent and the second compound and refluxing and stirring at 70-120 °C for 12-24 h to obtain a first mixed solution; And, performing reduced pressure distillation, extraction, water removal, reduced pressure distillation, and column chromatography on the first mixed solution to obtain an intermediate product.

6. The preparation method according to claim 5, wherein: The molar ratio of the first compound to the second compound is 1:2 - 1:5; And / or, the molar ratio of the first compound to the basic substance is 1:3 - 1:6; And / or, the first solvent includes any one or a combination of acetonitrile, isopropanol, and tetrahydrofuran.

7. The preparation method according to claim 2, characterized in that: Specifically includes: Dissolving the intermediate product and the third compound in ethanol, then adding piperidine and reacting at room temperature for 12 h to obtain a second mixed solution, then filtering, washing, and recrystallizing, and then dissolving in a second solvent under nitrogen protection and reacting at 80-130 °C for 12-24 h to obtain a luminescent monomer.

8. The preparation method according to claim 7, characterized in that: The molar ratio of the intermediate product, the third compound to the second solvent is 1:1 - 3:1 - 5; And / or, the second solvent includes any one or a combination of pyridine, dioxane, and isopropanol.

9. Use of the "quenching type" supramolecular fluorescent probe according to claim 1 in the detection of pepsin.

10. A method for detecting pepsin, characterized in that, Comprising: Providing the "quenching type" supramolecular fluorescent probe according to claim 1; And, mixing the "quenching type" supramolecular fluorescent probe with a sample to be measured to form a liquid-phase mixing system, and detecting pepsin according to the change in the fluorescence intensity of the "quenching type" supramolecular fluorescent probe and the liquid-phase mixing system.

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