Hydrogen-bonded organic framework immobilized glucose oxidase composite and dual optical signal detection method for alpha-glucosidase

Abstract

The application discloses a hydrogen-bonded organic framework immobilized glucose oxidase composite material and a double-optical-signal detection method of alpha-glucosidase. Through one-step assembly of 4,4',4'',4'''-methane tetraphenylformamidine (TAPM), glucose oxidase (GOx) and 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBApy), a hydrogen-bonded organic framework immobilized GOx composite material GOx@HOFs with dual functions of enzyme immobilization carrier and intrinsic fluorescence signal unit is prepared. Alpha-glucosidase catalyzes the hydrolysis of the substrate pNPG to generate glucose and p-nitrophenol, the former is catalyzed by GOx@HOFs to realize colorimetric detection through a catalytic cascade reaction, and the latter can weaken the intrinsic fluorescence of GOx@HOFs to realize fluorescence detection. The application realizes double-mode detection of 'one enzyme-catalyzed reaction and double signal output', and has the advantages of simple operation, high sensitivity and good reliability.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and bioanalysis detection technology, specifically relating to a method for detecting α-glucosidase activity by dual optical signals of glucose oxidase immobilized in a hydrogen-bonded organic framework, an immobilized glucose oxidase composite material, its preparation method, and its application. Background Technology

[0002] α-glucosidase (α-Glu) is a class of small-molecule hydrolases that catalyze the cleavage of glycosidic bonds to release glucose, playing a crucial role in glucose metabolism regulation, functional enzyme research, drug screening, and related bioanalysis. Changes in α-glucosidase activity are closely related to glucose metabolism studies and enzyme inhibitor evaluation; therefore, establishing a sensitive, stable, and reliable method for detecting α-glucosidase activity is of great significance for both basic research and applied analysis.

[0003] Currently, α-glucosidase activity detection typically employs colorimetric, fluorescence, electrochemical, and chromatographic methods. Colorimetric methods are advantageous due to their simplicity and low cost, while fluorescence methods offer high sensitivity and intuitive response, thus both are widely used in enzyme activity detection. However, existing α-glucosidase detection methods still have certain limitations in practical applications. For example, the application of dual-signal output detection modes in α-glucosidase activity analysis requires further research. Furthermore, some detection methods rely on natural enzymes for catalysis or cascade reactions, but natural enzymes often suffer from poor stability and susceptibility to environmental influences, affecting the repeatability and applicability of the detection system. Immobilizing natural enzymes using suitable framework materials can not only improve their stability but also potentially promote signal amplification during enzyme cascade processes. Therefore, developing an α-glucosidase activity detection method based on immobilized natural enzymes and incorporating dual-signal output is of significant research importance.

[0004] In recent years, hydrogen-bonded organic frameworks (HOFs), as an emerging class of organic framework materials, have shown great application potential in bioanalysis and sensing due to their mild assembly conditions, strong structural designability, and ease of functional modulation. Existing research shows that HOFs can not only serve as carriers of functional components but also participate in signal response processes. Therefore, developing a method for detecting α-glucosidase activity of immobilized natural enzymes based on hydrogen-bonded organic frameworks is of great significance for expanding the application of HOFs in biosensing.

[0005] Among the published patents related to α-glucosidase detection, patent CN121090624A discloses a photoelectrochemical biosensor and its preparation method and application. This patent employs a three-electrode system, using an ITO electrode modified with a silane small molecule probe and a β-cyclodextrin-functionalized graphite carbon nitride composite material as the working electrode to construct a photoelectrochemical biosensing platform for the detection of α-glucosidase and L-histidine. The sensor detects α-glucosidase concentrations in the range of 5-100 U / L, exhibiting certain specificity, stability, and reproducibility, and can be applied in clinical diagnosis and drug screening. This patent provides a new analytical approach for α-glucosidase detection; however, the sensor construction process is relatively complex, and specialized instruments are required for detection.

[0006] Patent CN122188086A discloses a bipyridine covalent organic framework with peroxidase-like biosensing activity and its application in α-glucosidase detection. This patent describes the preparation of two bipyridine COF materials through a Schiff base condensation reaction of 4,4',4'',4'''-(pyrene-1,3,6,8-tetraalkyl)-tetraphenylamine with either 2,2'-bipyridine-5,5'-dicarboxaldehyde or 3,3'-bipyridine-6,6'-dicarboxaldehyde. One of these materials was used to construct a colorimetric biosensor for α-Glu. The constructed colorimetric detection system achieved a detection limit of 0.24 U / L, enabling the detection of α-glucosidase. This patent demonstrates the application potential of organic framework materials in α-glucosidase detection; however, its technical approach mainly focuses on a single colorimetric detection mode. Further research is needed to expand the use of framework materials to immobilize natural enzymes and construct dual-optical signal output detection systems. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by proposing a hydrogen-bonded organic framework-immobilized glucose oxidase composite material and a dual optical signal detection method for α-glucosidase. First, a hydrogen-bonded organic framework-immobilized GOx composite material (GOx@HOFs) is prepared through a one-step assembly of an amidine-containing organic building block, glucose oxidase (GOx), and a carboxyl-containing organic building block, serving as both an enzyme immobilization carrier and an intrinsic fluorescent signal unit. Then, the products generated from the hydrolysis of the substrate catalyzed by α-glucosidase trigger colorimetric and fluorescence signal changes, respectively, to detect α-glucosidase activity.

[0008] The technical solution to achieve the objective of this invention is:

[0009] A method for preparing a hydrogen-bonded organic framework-immobilized glucose oxidase composite material includes the following steps:

[0010] Step 1: Dissolve the amidine-containing organic building blocks in ultrapure water to obtain the first solution;

[0011] Step 2: Dissolve glucose oxidase in ultrapure water, then add the resulting glucose oxidase solution dropwise to the first solution obtained in Step 1, and react under stirring conditions to obtain the second solution;

[0012] Step 3: Dissolve the carboxyl-containing organic building blocks in ultrapure water, then add ammonia to deprotonate them, and obtain the third solution;

[0013] Step 4: Add the third solution obtained in Step 3 to the second solution obtained in Step 2, and react under light-protected conditions to assemble the hydrogen-bonded organic framework and simultaneously immobilize glucose oxidase, thus obtaining a hydrogen-bonded organic framework immobilized glucose oxidase composite material.

[0014] Step 5: Centrifuge the product obtained in Step 4, wash it, redisperse it in ultrapure water, and store it at low temperature for later use.

[0015] Further, in step 1, the amidine-containing organic building block is 4,4',4'',4'''-methanetetraphenylamidine (TAPM), the amount of TAPM is 2.5 mg-100 mg, and the volume of ultrapure water is 5 mL-200 mL.

[0016] Furthermore, in step 2, the amount of glucose oxidase used is 1 mg-30 mg, the volume of ultrapure water used to dissolve the glucose oxidase is 1 mL-20 mL, and the stirring reaction time after the glucose oxidase solution is added dropwise to the first solution is 10 min-60 min.

[0017] Further, in step 3, the carboxyl-containing organic building block is 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (abbreviated as H4TBApy), the amount of H4TBApy is 2.5 mg-100 mg, the volume of ultrapure water is 2 mL-30 mL, the volume of added ammonia is 10 μL-1000 μL, and the concentration of ammonia is 0.1%-10%.

[0018] Furthermore, in step 4, the third solution is mixed with the second solution and reacted in the dark for 10 min-12 h.

[0019] Furthermore, in step 5, the centrifugation speed is 3000 rpm-12000 rpm, the centrifugation time is 5 min-20 min, the washing number is 3-8 times, and the obtained product is finally dispersed in 1 mL-20 mL of ultrapure water and stored at 2-8℃.

[0020] Using the above preparation method, the hydrogen-bonded organic framework immobilized glucose oxidase composite material has the dual functions of glucose oxidase immobilization carrier and intrinsic fluorescent signal response unit, and has better storage stability, temperature resistance and higher residual activity compared with free glucose oxidase.

[0021] The present invention further provides the application of the prepared hydrogen-bonded organic framework immobilized glucose oxidase composite material in the detection of α-glucosidase activity.

[0022] Specifically, the prepared hydrogen-bonded organic framework-immobilized glucose oxidase composite material was used to detect α-glucosidase activity, including colorimetric detection methods and fluorescence detection methods.

[0023] The activity of α-glucosidase was detected by combining the prepared hydrogen-bonded organic framework-immobilized glucose oxidase composite material with a colorimetric method. The detection method included the following steps:

[0024] Step A: Mix the α-glucosidase substrate solution with NaAc-HAc buffer and pre-incubate;

[0025] Step B: Add the α-glucosidase solution to be tested and the prepared hydrogen-bonded organic framework immobilized glucose oxidase composite material dispersion, and incubate the reaction.

[0026] Step C: Add NaAc-HAc buffer, copper ion solution and chromogenic substrate solution, and continue incubation for color development;

[0027] Step D: Measure the absorption spectrum of the reaction system and use the absorbance at a specific wavelength to perform quantitative analysis of α-glucosidase activity.

[0028] Further, in step A, the α-glucosidase substrate is p-nitrophenyl-α-D-glucopyranoside (pNPG), the substrate solution volume is 5 μL-100 μL, the concentration is 0.1 mmol / L-50 mmol / L, the NaAc-HAc buffer volume is 10 μL-200 μL, the concentration is 1 mmol / L-100 mmol / L, the pre-incubation temperature is 30-60℃, and the pre-incubation time is 5-30 min.

[0029] Further, in step B, the volume of the α-glucosidase solution to be tested is 5 μL-100 μL, the volume of the dispersion of the hydrogen-bonded organic framework immobilized glucose oxidase composite material is 5 μL-100 μL, the reaction temperature is 30-60℃, and the reaction time is 10-60 min.

[0030] Further, in step C, the NaAc-HAc buffer solution has a volume of 50 μL-500 μL and a concentration of 1 mmol / L-100 mmol / L; the copper ion solution has a volume of 5 μL-100 μL and a concentration of 0.1 mmol / L-20 mmol / L; the chromogenic substrate solution has a volume of 5 μL-100 μL and a concentration of 0.1 mmol / L-20 mmol / L; the copper ions are derived from copper nitrate trihydrate; the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB); the chromogenic temperature is 30-50℃; and the chromogenic time is 10-50 min.

[0031] Furthermore, in step D, the absorption spectrum detection range is 400-800 nm, the absorbance at 652 nm is used as the quantitative analysis signal, and the blank sample without α-glucosidase is measured under the same conditions.

[0032] In the colorimetric detection method, α-glucosidase catalyzes the hydrolysis of pNPG to produce glucose, and the hydrogen-bonded organic framework-immobilized glucose oxidase composite material catalyzes the oxidation of glucose to produce H2O2. H2O2 then reacts with Cu... 2+ The presence of this substance promotes the oxidation of TMB to oxTMB, thereby generating an absorption signal at 652 nm.

[0033] The activity of α-glucosidase was detected by combining the prepared hydrogen-bonded organic framework-immobilized glucose oxidase composite material with fluorescence detection. The detection method included the following steps:

[0034] Step a: Mix the α-glucosidase substrate solution, the α-glucosidase solution to be tested, and PBS buffer, and then proceed with the reaction;

[0035] Step b: Add the prepared hydrogen-bonded organic framework immobilized glucose oxidase composite material dispersion and mix;

[0036] Step c: Measure the fluorescence signal of the system and use the change in fluorescence intensity to achieve quantitative analysis of α-glucosidase activity.

[0037] Further, in step a, the α-glucosidase substrate is p-nitrophenyl-α-D-glucopyranoside, the substrate solution volume is 5 μL-100 μL, the concentration is 0.1 mmol / L-50 mmol / L, the α-glucosidase solution to be tested has a volume of 5 μL-100 μL, the PBS buffer volume is 50 μL-500 μL, the concentration is 1 mmol / L-100 mmol / L, the reaction temperature is 30-60℃, and the reaction time is 5-60 min.

[0038] Further, in step b, the volume of the hydrogen-bonded organic framework immobilized glucose oxidase composite material dispersion added is 1 μL-100 μL.

[0039] Further, in step c, the fluorescence excitation wavelength is 300-420 nm, the fluorescence signal in the range of 420-520 nm is analyzed, and the fluorescence intensity change value at 460 nm is used as the quantitative analysis signal. The fluorescence intensity change value is F0-F, where F0 is the fluorescence intensity of the system without α-glucosidase, and F is the fluorescence intensity of the system with α-glucosidase.

[0040] In the fluorescence detection method, α-glucosidase catalyzes the hydrolysis of pNPG to generate p-nitrophenol (p-NP). p-NP weakens the intrinsic fluorescence of the hydrogen-bonded organic framework-immobilized glucose oxidase composite material, resulting in a decrease in fluorescence intensity. The change in fluorescence intensity is used for quantitative analysis.

[0041] The beneficial effects of this invention are as follows:

[0042] This invention integrates enzyme immobilization and optical signal response by constructing a hydrogen-bonded organic framework-immobilized glucose oxidase composite material. This composite material serves both as an immobilization carrier for glucose oxidase and as an intrinsic fluorescent signal response unit, eliminating the need for additional fluorescent markers and simplifying the detection system construction process.

[0043] Based on the different products generated by the hydrolysis of substrates catalyzed by α-glucosidase, this invention establishes two detection modes: colorimetric and fluorescence. This allows a single enzymatic reaction to be converted into two different optical signal outputs, achieving dual-mode detection. Compared with single-signal detection, dual-mode signals help improve the reliability of detection results. Simultaneously, the immobilized glucose oxidase composite material exhibits better storage stability, temperature resistance, and residual activity than free glucose oxidase.

[0044] The detection method established in this invention has the advantages of simple operation, high sensitivity, and wide linear range, and has good application prospects in α-glucosidase activity analysis, biosensor detection, and screening of related inhibitors. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the preparation process of the hydrogen-bonded organic framework immobilized glucose oxidase composite material in Example 1.

[0046] Figure 2 This is a schematic diagram illustrating the principle of dual optical signal detection of α-glucosidase activity in Example 1.

[0047] Figure 3 This is a feasibility analysis diagram of the colorimetric detection mode in the embodiment;

[0048] in, Figure 3 A is a schematic diagram of the colorimetric detection principle;

[0049] Figure 3 B is the absorption spectrum used to verify the feasibility of the colorimetric reaction with glucose as an intermediate substrate;

[0050] Figure 3 C is a feasibility analysis diagram for colorimetric detection under the condition of missing components;

[0051] Figure 3 D is the colorimetric response diagram under different concentrations of α-glucosidase.

[0052] Figure 4 This is a graph showing the quantitative analysis results of α-glucosidase in the colorimetric detection mode in the examples;

[0053] in, Figure 4 A shows the absorption spectra corresponding to different concentrations of α-glucosidase;

[0054] Figure 4 B is the response curve of absorbance at 652 nm as a function of α-glucosidase concentration;

[0055] Figure 4 C represents the linear relationship between absorbance at 652 nm and α-glucosidase concentration.

[0056] Figure 5 This is a feasibility analysis diagram of the fluorescence detection mode in the examples;

[0057] in, Figure 5 A is a schematic diagram of the fluorescence detection principle;

[0058] Figure 5 B is a verification diagram showing the changes in the fluorescence response of GOx@HOFs induced by p-nitrophenol;

[0059] Figure 5 C is a diagram showing the feasibility analysis of fluorescence detection under different component conditions;

[0060] Figure 5 D is the fluorescence response diagram under different concentrations of α-glucosidase.

[0061] Figure 6 This is a graph showing the quantitative analysis results of α-glucosidase under the fluorescence detection mode in the examples;

[0062] in, Figure 6 A shows the fluorescence emission spectra corresponding to different concentrations of α-glucosidase;

[0063] Figure 6B is the response curve of fluorescence intensity change as a function of α-glucosidase concentration;

[0064] Figure 6 C represents the linear relationship between the change in fluorescence intensity at 460 nm and the concentration of α-glucosidase.

[0065] Figure 7 for Figure 1 Figure showing the stability analysis results of the hydrogen-bonded organic framework-immobilized glucose oxidase composite material.

[0066] in, Figure 7 A is a comparison of the relative activities of GOx@HOFs and free GOx under room temperature storage;

[0067] Figure 7 B is a comparison graph of the relative activities of GOx@HOFs and free GOx at different temperatures.

[0068] Figure 8 for Figure 2 Schematic diagrams of the key substrates, intermediate products, and chromogenic substrates involved in the dual optical signal detection process;

[0069] in, Figure 8 A is a schematic diagram of the structural formula of p-nitrophenyl-α-D-glucopyranoside (pNPG);

[0070] Figure 8 B is a schematic diagram of the structural formula of p-nitrophenol (p-NP);

[0071] Figure 8 C is a schematic diagram of the structural formula of glucose;

[0072] Figure 8 D is a schematic diagram of the structural formula of 3,3',5,5'-tetramethylbenzidine (TMB). Detailed Implementation

[0073] The present invention will be further described below with reference to the embodiments and accompanying drawings, but this is not intended to limit the scope of the invention.

[0074] Example 1: Preparation of a hydrogen-bonded organic framework immobilized glucose oxidase composite material.

[0075] Reference Figure 1 and Figure 7 The preparation method of hydrogen-bonded organic framework immobilized glucose oxidase composite material includes the following steps:

[0076] Step 1: Weigh 11.25 mg TAPM and completely dissolve it in 15 mL of ultrapure water to obtain the first solution.

[0077] Step 2: Weigh 5 mg of glucose oxidase and dissolve it in 5 mL of ultrapure water. Then add the resulting glucose oxidase solution dropwise to the first solution obtained in Step 1. React under stirring conditions for 10 min to obtain the second solution.

[0078] Step 3: Weigh 10 mg H4TBApy, dissolve it in 8 mL of ultrapure water, and then add 100 μL of 1% ammonia water for deprotonation to obtain the third solution.

[0079] Step 4: Add the third solution obtained in Step 3 to the second solution obtained in Step 2, stir and mix continuously, and react in the dark for 1 h to allow the hydrogen-bonded organic framework to assemble and simultaneously achieve glucose oxidase immobilization.

[0080] Step 5: The product obtained in Step 4 was collected by centrifugation at 7000 rpm for 8 min, washed 6 times with ultrapure water, and finally redispersed in 6 mL of ultrapure water and stored at 4℃ for later use, to obtain the hydrogen-bonded organic framework immobilized glucose oxidase composite material, denoted as GOx@HOFs.

[0081] Reference Figure 2 and Figure 8 This invention constructs a dual-optical-signal detection system for α-glucosidase based on GOx@HOFs. α-glucosidase catalyzes the hydrolysis of p-nitrophenyl-α-D-glucopyranoside (pNPG) to produce glucose and p-nitrophenol (p-NP). The generated glucose enters the colorimetric detection pathway: glucose is oxidized to H2O2 by glucose oxidase immobilized in GOx@HOFs, and subsequently detected in Cu... 2+ In its presence, it promotes the oxidation of colorless TMB to generate blue oxTMB, thus producing an absorption signal at 652 nm. The generated p-nitrophenol enters the fluorescence detection pathway: p-NP attenuates the intrinsic fluorescence of GOx@HOFs, and the change in fluorescence intensity can be used to quantitatively analyze α-glucosidase activity.

[0082] Example 2: Construction and feasibility verification of colorimetric detection mode, referring to... Figure 3 .

[0083] Reference Figure 3 A. The colorimetric detection pathway is designed based on a glucose-mediated cascade reaction, initiated by the hydrolysis of pNPG catalyzed by α-glucosidase. α-glucosidase first hydrolyzes pNPG to produce glucose and p-nitrophenol; subsequently, the glucose is oxidized by GOx in GOx@HOFs, producing H2O2; in Cu... 2+In the presence of H2O2, colorless TMB is oxidized to blue oxTMB, which generates an absorption signal at 652 nm, thereby converting α-glucosidase activity into a measurable colorimetric output.

[0084] To verify the feasibility of the subsequent colorimetric reaction, glucose was first used as an intermediate substrate for verification. (Refer to...) Figure 3 B, after adding glucose, GOx@HOFs / Cu 2+ The TMB system produced a significant absorption signal at 652 nm, indicating that GOx retains its catalytic activity after being incorporated into the HOF matrix and can generate H2O2 for subsequent TMB oxidation, proving that the glucose-to-color signal conversion pathway is effective.

[0085] Subsequently, a component deficiency experiment was conducted. (Refer to...) Figure 3 C, only when α-glucosidase, pNPG, GOx@HOFs, Cu 2+ When present with TMB, a significant absorption signal was observed only at 652 nm; removal of α-glucosidase or pNPG resulted in almost no signal, indicating that the color reaction originated from substrate hydrolysis triggered by α-glucosidase rather than non-specific oxidation; the reaction was significantly inhibited in the absence of GOx@HOFs, indicating that immobilized GOx plays a key role in glucose oxidation; the absence of Cu... 2+ The signal of the system weakened significantly with the addition of Cu, indicating that... 2+ It plays a promoting role in the H2O2-mediated oxidation of TMB.

[0086] Reference Figure 3 D. As the concentration of α-glucosidase increases, the absorption signal at 652 nm gradually increases, indicating that the constructed cascade system can transform the α-glucosidase-catalyzed pNPG hydrolysis process into a concentration-dependent colorimetric response.

[0087] Example 3: Quantitative detection of α-glucosidase in colorimetric mode, referring to... Figure 4 .

[0088] The following are the specific steps for detecting α-glucosidase activity using a GOx@HOFs-based colorimetric method:

[0089] Step A: Add 30 μL of pNPG solution (10 mmol / L) and 45 μL of NaAc-HAc buffer (10 mmol / L, pH 5.0) to a 1.5 mL centrifuge tube, and pre-incubate at 45°C in the dark for 5 min.

[0090] Step B: Add 30 μL of α-glucosidase solutions with different activities and 30 μL of GOx@HOFs dispersion, and incubate at 45℃ for 40 min.

[0091] Step C: Subsequently, add 205 μL NaAc-HAc buffer (10 mmol / L, pH 4.0) and 30 μL Cu. 2+ The solution (6 mmol / L, derived from copper nitrate trihydrate) and 30 μL TMB solution (8 mmol / L) were incubated at 45 °C for 40 min to develop color.

[0092] Step D: Record the UV-Vis absorption spectrum in the range of 400-800 nm, and use the absorbance at 652 nm as the quantitative analysis signal; measure the blank sample without α-glucosidase under the same conditions.

[0093] Reference Figure 4 A. As α-glucosidase activity increases, the absorption peak at 652 nm gradually strengthens. (Refer to...) Figure 4 B, the absorbance at 652 nm increases with increasing α-glucosidase activity. (Reference) Figure 4 C, within the range of 0.5-20 U / L, the absorbance at 652 nm showed a good linear relationship with α-glucosidase activity, and the linear regression equation was y = 0.01998x + 0.09195, R 2 =0.9926; the detection limit of this system was calculated to be 0.14 U / L based on the 3σ / k method.

[0094] The above results demonstrate that the colorimetric detection system based on GOx@HOFs can achieve sensitive quantitative analysis of α-glucosidase activity by utilizing the glucose-mediated cascade signal amplification effect.

[0095] Example 4: Construction and feasibility verification of fluorescence detection mode, referring to... Figure 5 .

[0096] Reference Figure 5 A fluorescence detection mode was constructed using the intrinsic fluorescence of GOx@HOFs as the signal response source and p-nitrophenol as a fluorescence attenuation factor. Unlike colorimetric detection, the fluorescence response does not depend on GOx-catalyzed glucose oxidation, but directly monitors the effect of p-NPs generated by α-glucosidase on the fluorescence of GOx@HOFs.

[0097] GOx@HOFs exhibit a broad blue emission band in the visible light region. The fluorescence intensity decreased significantly after the addition of p-NP, indicating that p-NP can effectively reduce the intrinsic fluorescence of GOx@HOFs.

[0098] To verify the feasibility of the fluorescence pathway, p-NP was first directly introduced into the GOx@HOFs solution. (Refer to...) Figure 5B, GOx@HOFs exhibited strong blue fluorescence, but the fluorescence intensity decreased significantly after the addition of p-NP, indicating that p-NP can effectively weaken the intrinsic fluorescence of the HOF matrix.

[0099] Further controlled experiments were conducted. (Refer to...) Figure 5 C. In the absence of pNPG, the GOx@HOFs / α-glucosidase system still maintains strong fluorescence, indicating that α-glucosidase itself does not cause significant fluorescence attenuation. After the addition of pNPG, the fluorescence intensity decreases to some extent. The complete system containing GOx@HOFs, pNPG and α-glucosidase shows a more obvious decrease in fluorescence, indicating that the fluorescence response is mainly related to the p-NP generated during the hydrolysis of pNPG catalyzed by α-glucosidase.

[0100] Reference Figure 5 D. As the concentration of α-glucosidase gradually increases, the fluorescence intensity of GOx@HOFs continuously decreases, indicating that the fluorescence response of the system is related to the activity of α-glucosidase.

[0101] Example 5: Quantitative detection of α-glucosidase in fluorescence mode, referring to... Figure 6 .

[0102] The specific steps for detecting α-glucosidase activity using GOx@HOFs combined with fluorescence method are as follows:

[0103] Step a: Add 30 μL of pNPG solution (10 mmol / L), 30 μL of α-glucosidase solution of different concentrations and 230 μL of PBS buffer (10 mmol / L, pH 7.0) to a 1.5 mL centrifuge tube, and incubate at 50 °C in the dark for 50 min.

[0104] Step b: Add 24 μL of GOx@HOFs dispersion and mix thoroughly.

[0105] Step c: Record the fluorescence emission spectrum with 375 nm as the excitation wavelength, and perform quantitative analysis using the fluorescence intensity change value F0-F at 460 nm, where F0 is the fluorescence intensity of the system without α-glucosidase, and F is the fluorescence intensity of the system with α-glucosidase.

[0106] Reference Figure 6 A. As the concentration of α-glucosidase increases, the fluorescence emission intensity of the system gradually decreases. (Reference) Figure 6 B, the change in fluorescence intensity increases with increasing α-glucosidase activity. (Refer to...) Figure 6C, within the range of 0.5-40 U / L, the fluorescence intensity variation F0-F at 460 nm showed a good linear relationship with α-glucosidase activity, with the linear regression equation being y=104.6x+569.9, R 2 =0.9938; the detection limit of this system was calculated to be 0.049 U / L based on the 3σ / k method.

[0107] This invention realizes dual-mode detection with "one enzyme-catalyzed reaction and dual signal output", which has the advantages of simple operation, high sensitivity and good reliability.

Claims

1. A method for preparing a hydrogen-bonded organic framework immobilized glucose oxidase composite material, characterized in that, Includes the following steps: Step 1: Dissolve the amidine-containing organic building blocks in ultrapure water to obtain the first solution; Step 2: Dissolve glucose oxidase in ultrapure water, then add the resulting glucose oxidase solution dropwise to the first solution obtained in Step 1, and react under stirring conditions to obtain the second solution; Step 3: Dissolve the carboxyl-containing organic building blocks in ultrapure water, then add alkali to deprotonate them, and obtain the third solution; Step 4: Add the third solution obtained in Step 3 to the second solution obtained in Step 2, and react under light-protected conditions to assemble the hydrogen-bonded organic framework and simultaneously immobilize glucose oxidase, thus obtaining a hydrogen-bonded organic framework immobilized glucose oxidase composite material. Step 5: Centrifuge the product obtained in Step 4, wash it, redisperse it in ultrapure water, and store it at low temperature.

2. The preparation method according to claim 1, characterized in that: In step 1, the amidine-containing organic building block is 4,4',4'',4'''-methanetetraphenylamidine; In step 3, the carboxyl-containing organic building unit is 1,3,6,8-tetra(4-carboxyphenyl)pyrene; In step 3, the alkali is ammonia.

3. The preparation method according to claim 1, characterized in that: In step 1, the amount of 4,4',4'',4'''-methanetetrabenzamide used is 2.5mg-100mg, and the volume of ultrapure water is 5mL-200mL; In step 2, the amount of glucose oxidase used is 1mg-30mg, the volume of ultrapure water used to dissolve the glucose oxidase is 1mL-20mL, and the stirring reaction time is 10min-60min. In step 3, the amount of 1,3,6,8-tetra(4-carboxyphenyl)pyrene used is 2.5mg-100mg, the volume of ultrapure water is 2mL-30mL, the amount of ammonia added is 10μL-1000μL, and the concentration of ammonia is 0.1%-10%.

4. The preparation method according to claim 1, characterized in that: In step 4, the third solution is mixed with the second solution and reacted in the dark for 10 min-12 h. In step 5, the centrifugation speed is 3000rpm-12000rpm, the centrifugation time is 5min-20min, the washing number is 3-8 times, and the obtained product is finally dispersed in 1mL-20mL of ultrapure water and stored at 2-8℃.

5. The application of a hydrogen-bonded organic framework-immobilized glucose oxidase composite material prepared by the method of claim 1 in the detection of α-glucosidase activity, characterized in that, The applications include colorimetric detection methods and fluorescence detection methods.

6. The application according to claim 5, characterized in that, The colorimetric detection method includes the following steps: Step A: Mix the α-glucosidase substrate solution with NaAc-HAc buffer and pre-incubate; Step B: Add the α-glucosidase solution to be tested and the hydrogen-bonded organic framework immobilized glucose oxidase composite material dispersion prepared by the method described in claim 1, and carry out the incubation reaction; Step C: Add NaAc-HAc buffer, copper ion solution and chromogenic substrate solution, and continue incubation for color development; Step D: Measure the absorption spectrum of the reaction system and use the absorbance at 652 nm as the quantitative analysis signal to achieve quantitative analysis of α-glucosidase activity.

7. The application according to claim 6, characterized in that: In step A, the α-glucosidase substrate is p-nitrophenyl-α-D-glucopyranoside, the substrate solution volume is 5 μL-100 μL, the concentration is 0.1 mmol / L-50 mmol / L, the NaAc-HAc buffer volume is 10 μL-200 μL, the concentration is 1 mmol / L-100 mmol / L, the pre-incubation temperature is 30-60℃, and the pre-incubation time is 5-30 min. In step B, the volume of the α-glucosidase solution to be tested is 5 μL-100 μL, the volume of the composite material dispersion is 5 μL-100 μL, the reaction temperature is 30-60℃, and the reaction time is 10-60 min. In step C, the volume of the NaAc-HAc buffer solution is 50 μL-500 μL, the concentration is 1 mmol / L-100 mmol / L, the volume of the copper ion solution is 5 μL-100 μL, the concentration is 0.1 mmol / L-20 mmol / L, the volume of the chromogenic substrate solution is 5 μL-100 μL, and the concentration is 0.1 mmol / L-20 mmol / L. The copper ions are derived from copper nitrate trihydrate, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine, the chromogenic temperature is 30-50℃, and the chromogenic time is 10-50 min. In step D, the absorption spectrum detection range is 400-800 nm, and the absorbance at 652 nm is used as the quantitative analysis signal.

8. The application according to claim 5, characterized in that, The fluorescence detection method includes the following steps: Step a: Mix the α-glucosidase substrate solution, the α-glucosidase solution to be tested, and PBS buffer, and then proceed with the reaction; Step b: Add the hydrogen-bonded organic framework immobilized glucose oxidase composite material dispersion prepared by the method described in claim 1 and mix; Step c: Measure the fluorescence signal of the system and use the change in fluorescence intensity to achieve quantitative analysis of α-glucosidase activity.

9. The application according to claim 8, characterized in that: In step a, the α-glucosidase substrate is p-nitrophenyl-α-D-glucopyranoside, the substrate solution volume is 5 μL-100 μL, the concentration is 0.1 mmol / L-50 mmol / L, the α-glucosidase solution volume is 5 μL-100 μL, the PBS buffer volume is 50 μL-500 μL, the concentration is 1 mmol / L-100 mmol / L, the reaction temperature is 30-60℃, and the reaction time is 5-60 min. In step b, the volume of the composite material dispersion added is 1 μL-100 μL.

10. The application according to claim 8, characterized in that: In step c, the fluorescence excitation wavelength is 300-420nm, the fluorescence signal in the range of 420-520nm is analyzed, and the fluorescence intensity change value at 460nm is used as the quantitative analysis signal. The fluorescence intensity change value is F0-F, where F0 is the fluorescence intensity of the system without α-glucosidase, and F is the fluorescence intensity of the system with α-glucosidase.

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