A Hybrid Osmium Nanocluster with Glucose Oxidase and Peroxidase Activities

Abstract

The present invention discloses a hybrid osmium nanocluster with both glucose oxidase and peroxidase activities, which is characterized in that a hybrid osmium nanocluster is formed by mediating the growth of osmium nanozyme based on the glucose oxidase skeleton. The hybrid osmium nanocluster has a small particle size (1.3 nm) and simultaneously has peroxidase activity (19.4 U / mg) and glucose oxidase activity (91.2 U / mg). The hybrid osmium nanocluster can achieve self-cascade catalysis of glucose, and its effect is significantly better than that of adding glucose oxidase and osmium nanocluster step by step. Given its excellent overall stability and the ability to quickly achieve self-cascade catalytic reaction of glucose, the hybrid osmium nanocluster can be used as a very promising material in the fields of biosensing and medical treatment, etc.

Description

Technical Field

[0001] The present invention discloses a hybrid osmium nanocluster with both glucose oxidase activity and peroxidase activity, belonging to the fields of synthetic biology, nanotechnology and bionics. Background Art

[0002] Nanozymes are a class of nanomaterials with enzyme-like activities, which have attracted extensive attention from researchers in various fields due to their high stability, batch production, economy and applicability. However, due to the problems of low catalytic activity and poor catalytic selectivity of nanozymes, the strategy of combining natural enzymes with nanozymes is still widely used. In the past ten years or more, researchers have developed a variety of applications based on the natural enzyme-nanozyme strategy. However, most of the work realizes cascade catalysis by adding nanozymes and natural enzymes step by step. For example, glucose oxidase is added to the system first to catalyze glucose to generate hydrogen peroxide, and then the peroxidase activity of the nanozyme is used to catalyze the reaction of hydrogen peroxide with the substrate. From the perspective of operation, this method is relatively cumbersome, and multiple reagent additions and reaction condition optimizations are required between each step of the reaction. From the perspective of application, this method has low catalytic efficiency and the defects of natural enzymes and nanozymes respectively. Therefore, how to integrate natural enzymes and nanozymes to prepare nanomaterials with dual enzyme activities and further improve the self-cascade effect and overall stability is an urgent problem to be solved.

[0003] In the field of nanozymes, noble metal nanozymes play an important role due to their unique optical properties, excellent enzyme-like activities, good stability and biocompatibility. Different from other noble metals, osmium has relatively few development applications in various fields. Recently, it has been reported that osmium nanomaterials can be used as nanozymes and have highly efficient and specific peroxidase activity. In addition, it has also been reported that proteins can mediate the growth of osmium nanozymes under mild conditions. These works have inspirations for the design and property exploration of osmium nanozymes. Based on the above, the present invention forms a hybrid osmium nanocluster (GOx-OsNCs) by mediating the growth of osmium nanozymes based on the glucose oxidase backbone. GOx-OsNCs can efficiently and stably exhibit glucose oxidase and peroxidase activities and realize glucose self-cascade catalytic reactions. The "hybrid osmium nanocluster with both glucose oxidase activity and peroxidase activity" provided by the present invention has great application potential and commercial value in the fields of biosensing and medical treatment. Summary of the Invention

[0004] The object of the present invention is to form a hybrid osmium nanocluster (GOx-OsNCs) based on the glucose oxidase framework-mediated growth of osmium nanozymes. The aqueous solution of GOx-OsNCs has good dispersibility and high stability, and the particle size of OsNCs is small and evenly distributed (1.3 nm). GOx-OsNCs have both glucose oxidase and peroxidase activities. Based on the dual enzyme activities, GOx-OsNCs can catalyze glucose to generate hydrogen peroxide, and at the same time, GOx-OsNCs can rapidly catalyze the oxidation of hydrogen peroxide to substrates, thus realizing an efficient glucose self-cascade catalytic reaction.

[0005] In order to achieve the object of the above detection method, the present invention adopts the following technical solutions:

[0006] A hybrid osmium nanocluster with both glucose oxidase and peroxidase activities, characterized in that it is prepared by the following method: Take 0.5 mL of 25 mg / mL glucose oxidase, and add 0.5 mL of 20 mmol / L potassium hexachloroosmate and 50 µL of 0.8 mol / L NaOH respectively, stir and mix, and then place it in a water bath at 37 °C for reaction for 3 h; The average particle size of the obtained GOx-OsNCs is 1.3 nm, the lattice spacing corresponding to the 101 crystal plane of Os is 0.207 nm, and it consists of 71% Os 0 and 29% Os 4+ Composition.

[0007] The GOx-OsNCs obtained in the present invention can catalyze the oxidation of hydrogen peroxide by TMB and have a strong absorption value at 652 nm in the ultraviolet range. Therefore, GOx-OsNCs have peroxidase activity, and the enzyme activity is 19.4 U / g.

[0008] The GOx-OsNCs obtained in the present invention can catalyze the oxidation of glucose by TMB and have a strong absorption value at 652 nm in the ultraviolet range. Therefore, GOx-OsNCs have glucose oxidase activity, and the enzyme activity is 91.2 U / mg; The glucose oxidase activity of GOx-OsNCs can also be confirmed by the reaction of glucose with 3,5-dinitrosalicylic acid.

[0009] The GOx-OsNCs obtained in the present invention can catalyze glucose to produce hydrogen peroxide and then catalyze its oxidation of TMB to develop color, proving that it has both glucose oxidase activity and peroxidase activity; Based on the dual enzyme activities, GOx-OsNCs can catalyze glucose to generate hydrogen peroxide, and at the same time, GOx-OsNCs can rapidly catalyze the oxidation of hydrogen peroxide to substrates, thus realizing an efficient glucose self-cascade catalytic reaction; The self-cascade catalytic effect will be better than the effect of adding GOx and OsNCs step by step for reaction.

[0010] The GOx-OsNCs obtained in the present invention can detect glucose through the change of color signal, and the linear response range is 0.1 - 3.2 mmol / L for glucose concentration, which proves that GOx-OsNCs can achieve glucose self-cascade catalytic reaction.

[0011] The GOx-OsNCs obtained in the present invention have good stability, and the self-cascade catalytic effect can be maintained above 90%.

[0012] Specifically, the technical solution adopted in the present invention is as follows:

[0013] (I) Synthesis of GOx-OsNCs: Using the method of controlling variables, the glucose oxidase, potassium hexachloroiridate, sodium hydroxide, temperature and time are respectively fixed, and the self-cascade catalytic effect of the GOx-OsNCs-glucose-substrate system is tested, and the optimal synthesis conditions are obtained. The optimal synthesis steps of GOx-OsNCs are as follows: Prepare glucose oxidase (25 mg / mL, 0.5 mL), and add potassium hexachloroiridate (20 mmol / L, 0.5 mL) and NaOH (0.8 mol / L, 50 μL) respectively, stir and mix, and place in a 37 °C water bath for reaction for 3 h. During the reaction process, the color of the solution gradually changes from light yellow to black. The solution obtained above is further purified, centrifuged and ultrafiltered in a centrifuge at a speed of 7000 revolutions per minute for 20 minutes, and washed with deionized water, and the ultrafiltration is repeated until the impurity content is less than 1%. After the ultrafiltration is completed, take the upper black liquid, dilute and make up the volume to the original volume to obtain GOx-OsNCs (14.4 mg / mL), and store it in a 4 °C refrigerator for standby.

[0014] (II) Peroxidase activity and enzyme activity of GOx-OsNCs: Take the GOx-OsNCs (1.44 mg / mL, 20 μL) synthesized in the technical solution (I), add phosphate buffer (pH = 4, 20 mmol / L, 2.78 mL), hydrogen peroxide (6 mmol / L, 1 mL), 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB) (6 mmol / L, 0.2 mL), mix them, and scan the absorption spectrum after reacting in a 37 °C water bath for 30 minutes. The above content is used to verify the peroxidase activity of GOx-OsNCs.

[0015] The peroxidase activity of GOx-OsNCs was calculated based on the above reaction system (the hydrogen peroxide concentration was changed to 4 mol / L). The absorbance of the reaction system at 652 nm in the ultraviolet range was detected every 10 seconds (within 1 minute) and calculated through the Lambert-Beer law. One enzyme unit (U) was defined as follows: the amount required for GOx-OsNCs to catalyze the production of 1 μmol of product per minute under the conditions of pH = 4 and 37 °C. The enzyme activity can be defined as U per gram of GOx-OsNCs.

[0016] (III) Glucose oxidase activity and enzyme activity of GOx-OsNCs: Take the GOx-OsNCs synthesized in Technical Solution (I) (1.44 mg / mL, 20 μL), add phosphate buffer (pH = 5, 20 mmol / L, 2.78 mL), glucose (6 mmol / L, 1 mL), and 3,3’,5,5’-tetramethylbenzidine hydrochloride (TMB, 6 mmol / L, 0.2 mL). After mixing, scan the absorption spectrum after a 30-minute water bath at 37 °C. The above content was used to verify the glucose oxidase activity of GOx-OsNCs.

[0017] The calculation of the glucose oxidase activity of GOx-OsNCs is as follows: Sequentially add phosphate buffer (pH = 5.6, 20 mmol / L, 1 mL), glucose (2 - 7 mmol / L, 0.5 mL), and GOx / GOx-OsNCs (6.25 mg / mL, 0.5 mL) into an EP tube. After mixing, place it in a reaction at 37 °C for 10 min. Subsequently, add 1 mL of 3,5-dinitrosalicylic acid to the above mixture, react in a 100 °C water bath for 10 min, immediately cool it on ice for 5 min, and measure the absorbance at 540 nm after diluting it by a factor of two. A standard curve was established with different concentrations of glucose as the abscissa and their absorbance values at 540 nm as the ordinate to determine the amount of glucose consumed. One enzyme unit (U) was defined as follows: the amount required for GOx-OsNCs to catalyze 1 μmol of glucose per minute under the conditions of pH = 5.6 and 37 °C. According to U (mol / min) = (Abs 葡萄糖 -Abs 葡萄糖+GOx ) / k × t, UA (U / mg) = U / m was used to calculate the enzyme activity in GOx-OsNCs. Among them, Abs 葡萄糖 is the absorbance of the control group, k is the slope of the glucose standard curve, t is the reaction time (minutes), and m is the mass of the enzyme.

[0018] (4) Glucose sensing achieved through the self-cascade catalytic reaction based on GOx-OsNCs: Take the GOx-OsNCs synthesized in Technical Solution (1) (1.44 mg / mL, 20 μL), add phosphate buffer (pH = 5, 20 mmol / L, 2.78 mL), glucose (different concentrations, 1 mL), and TMB (2 mmol / L, 0.2 mL). After mixing, detect the absorbance at 652 nm under ultraviolet light after a 7-minute water bath at 37 °C, and determine the response range of glucose based on its linear range.

[0019] (5) Stability experiment of GOx-OsNCs: Take the day when GOx-OsNCs are synthesized as the 0th day, and measure the absorbance of the catalytic system at 652 nm under ultraviolet light after the material has been placed for one week, two weeks, three weeks, and four weeks respectively according to the operation steps in Technology (3). Take the absorbance on the 0th day as the original absorbance (100%) and calculate the relative activity.

[0020] Advantages of the present invention:

[0021] (1) The synthesis conditions of the hybrid osmium nanoclusters are green, environmentally friendly, and reasonably optimized;

[0022] (2) The hybrid osmium nanoclusters have good aqueous solution dispersibility and high stability, and the osmium nanoclusters have small particle size and uniform distribution;

[0023] (3) The hybrid osmium nanoclusters have both glucose oxidase and peroxidase activities, and can achieve self-cascade catalysis of glucose, which has great advantages compared with the step-by-step reaction. Description of the Drawings

[0024] Figure 1 It is a synthesis flow chart.

[0025] Figure 2 It is the optimization of the potassium osmate hexachloride concentration in the synthesis conditions.

[0026] Figure 3 It is the optimization of the sodium hydroxide concentration in the synthesis conditions.

[0027] Figure 4 It is the optimization of the reaction temperature in the synthesis conditions.

[0028] Figure 5 It is the optimization of the reaction time in the synthesis conditions.

[0029] Figure 6 It is the ultraviolet scanning spectrum and pictures of glucose oxidase, potassium osmate hexachloride, and GOx-OsNCs.

[0030] Figure 7 It is the Gaussian distribution diagram of particle size.

[0031] Figure 8It is a transmission electron microscope image (including high-resolution transmission electron microscope images).

[0032] Figure 9 It is the X-ray photoelectron spectroscopy of Os element (4f orbital).

[0033] Figure 10 It is the peroxidase activity of GOx-OsNCs.

[0034] Figure 11 It is the peroxidase activity of GOx-OsNCs.

[0035] Figure 12 It is the glucose oxidase activity of GOx-OsNCs.

[0036] Figure 13 It is the glucose oxidase activity of GOx-OsNCs.

[0037] Figure 14 It is the comparison of the glucose self-cascade catalytic reaction based on GOx-OsNCs and the glucose stepwise catalytic reaction based on GOx + OsNCs.

[0038] Figure 15 It is the realization of glucose sensing by the self-cascade catalytic reaction based on GOx-OsNCs.

[0039] Figure 16 It is the stability experiment of GOx-OsNCs. Embodiment

[0040] Example 1: Using the control variable method, fix glucose oxidase, potassium hexachloroiridate, sodium hydroxide, temperature and time respectively, test the self-cascade catalytic effect of the GOx-OsNCs-glucose-substrate system, and obtain the optimal synthesis conditions (as Figure 2-5 shown). The optimal synthesis conditions are as follows: Prepare glucose oxidase (25 mg / mL, 0.5 mL), add potassium hexachloroiridate (20 mmol / L, 0.5 mL) and NaOH (0.8 mol / L, 50 µL) respectively, stir and mix, then place it in a 37 °C water bath for 3 h. During the reaction, the color of the solution gradually changes from light yellow to black. Purify the obtained solution, centrifuge and ultrafilter it in a centrifuge at 7000 revolutions per minute for 20 minutes, wash it with deionized water, and repeat ultrafiltration until the impurities are less than 1%. After ultrafiltration, take the upper black liquid, dilute and make up the volume to the original volume to obtain hybrid osmium nanoclusters (GOx-OsNCs, 14.4 mg / mL), and store it in a 4 °C refrigerator for later use. The synthesis flowchart is as Figure 1 shown.

[0041] Example 2: Glucose oxidase (12.5 mg / mL), potassium hexachloroosmate (10 mmol / L), and the GOx-OsNCs prepared in Example 1 (14.4 mg / mL) were each diluted 40 times and then scanned in the ultraviolet range of 200 - 800 nm. The synthesis of GOx-OsNCs was preliminarily judged based on the changes in the ultraviolet absorption peaks. As Figure 6 shown, the absorption peaks of GOx-OsNCs compared with potassium hexachloroosmate (Os 4+ ) disappeared at 220 nm, 255 nm, 340 nm, 370 nm, and 420 nm, indicating that the valence state of osmium had changed. At the same time, GOx-OsNCs retained the absorption peak of glucose oxidase at 280 nm, and a resonance ion absorption peak of osmium also appeared after 450 nm. These phenomena indicated the successful synthesis of hybrid osmium nanoclusters. As Figure 7 shown by transmission electron microscopy, the average particle size of 50 GOx-OsNCs was obtained as 1.3 nm using a Gaussian distribution. As Figure 8 shown, the interplanar spacing of the obtained OsNCs was 0.207 nm, corresponding to the (101) plane of the osmium crystal. After freeze-drying GOx-OsNCs to obtain a powder, X-ray photoelectron spectroscopy characterization was performed. As Figure 9 shown, the Os element in GOx-OsNCs consisted of 71% Os 0 and 29% Os 4+ .

[0042] Example 3: Take the GOx-OsNCs prepared in Example 1 (1.44 mg / mL, 20 µL), add phosphate buffer (pH = 4, 20 mmol / L, 2.78 mL), hydrogen peroxide (6 mmol / L, 1 mL), and 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB, 6 mmol / L, 0.2 mL). After mixing, it was incubated in a water bath at 37 °C for 30 minutes and then scanned to obtain its absorption spectrum. The results are as Figure 10 shown. It can be seen from the figure that: there was no visual color change after the reaction of TMB + hydrogen peroxide and TMB + GOx-OsNCs. After adding hydrogen peroxide to the TMB + GOx-OsNCs system, the color turned blue and an absorption peak appeared at 652 nm, proving that GOx-OsNCs had peroxidase activity.

[0043] Take the GOx-OsNCs prepared in Example 1 (1.44 mg / mL, 20 µL), add phosphate buffer (pH = 4, 20 mmol / L, 2.78 mL), hydrogen peroxide (4 mol / L, 1 mL), 3,3’,5,5’-tetramethylbenzidine hydrochloride (TMB, 6 mmol / L, 0.2 mL), detect the absorbance at 652 nm every 10 seconds (within a total of 1 minute), and calculate its enzyme activity to be 19.4 U / mg through the Lambert-Beer law. The results are as Figure 11 shown.

[0044] Example 4: Take the GOx-OsNCs prepared in Example 1 (1.44 mg / mL, 20 µL), add phosphate buffer (pH = 4, 20 mmol / L, 2.78 mL), glucose (5 mmol / L, 1 mL), 3,3’,5,5’-tetramethylbenzidine hydrochloride (TMB, 6 mmol / L, 0.2 mL), mix them, and scan the absorption spectrum after a 30-minute water bath at 37 °C. The results are as Figure 12 shown. When the system is TMB + glucose, the color of the solution does not change. However, after the reaction of TMB + glucose + GOx-OsNCs, the solution turns blue and a characteristic absorption peak appears at 652 nm, indicating that GOx-OsNCs can catalyze glucose to first generate hydrogen peroxide, and further catalyze hydrogen peroxide to generate hydroxyl radicals, thereby oxidizing TMB to form a blue product, proving that GOx-OsNCs have glucose oxidase activity.

[0045] Example 5: Sequentially add phosphate buffer (pH = 5.6, 20 mmol / L, 1 mL), glucose (2 - 7 mmol / L, 0.5 mL), and the GOx / GOx-OsNCs prepared in Example 1 (6.25 mg / mL, 0.5 mL) into an EP tube, mix well, and place it in a 37 °C reaction for 10 min. Then add 1 mL of 3,5-dinitrosalicylic acid to the above mixture, react in a 100 °C water bath for 10 min, immediately cool it on ice for 5 min, dilute it by one-fold, and measure the absorbance at 540 nm. Through the redox reaction of glucose with different concentrations of 3,5-dinitrosalicylic acid to generate 3-amino-5-nitrosalicylic acid, this product will turn brownish-red when heated. Use the linear relationship between its color depth and glucose concentration, as Figure 13 shown. After a certain amount of glucose reacts with GOx (glucose oxidase) and GOx-OsNCs for a fixed time, the consumption of glucose is carried out. Substitute the measured absorbance value into the formula U (mol / min) = (Abs 葡萄糖 -Abs 葡萄糖+GOx) / k×t, the activity of GOx-OsNCs was calculated to be 91.2 U / mg by UA (U / mg) = U / m.

[0046] Example 6: Take the GOx-OsNCs prepared in Example 1 and inactivate the activity of its GOx (glucose oxidase) (70 o C for 1 hour), and OsNCs with unaffected peroxidase activity can be obtained. Referring to the operation method of Example 4, take inactivated GOx OsNCs (1.44 mg / mL, 20 µL), add GOx (glucose oxidase) (114 U / mg, 20 µL) to react with the glucose-TMB system, and scan the ultraviolet-visible spectrum. From Figure 14 The results show that the self-cascade catalytic effect of GOx-OsNCs is better than that of adding GOx and OsNCs separately.

[0047] Example 7: Take the GOx-OsNCs prepared in Example 1 (1.44 mg / mL, 20 µL), add phosphate buffer (pH = 5, 20 mmol / L, 2.78 mL), glucose (different concentrations, 1 mL), and TMB (2 mmol / L, 0.2 mL), mix them, and then detect the absorbance at 652 nm in the ultraviolet after a 7-minute water bath at 37 °C. Determine the response range of glucose through its linear range ( Figure 15 ), which proves that GOx-OsNCs can achieve a glucose self-cascade catalytic reaction.

[0048] Example 8: Count the day when GOx-OsNCs are prepared as the 0th day. On the 0th day, 1 week, 2 weeks, 3 weeks, and 4 weeks after placing the materials, take the GOx-OsNCs prepared in Example 1 (1.44 mg / mL, 20 µL), add phosphate buffer (pH = 5, 20 mmol / L, 2.78 mL), TMB (2 mmol / L, 0.2 mL), and glucose (5 mmol / L, 1 mL), mix them, and then detect the absorbance at 652 nm in the ultraviolet after a 7-minute water bath at 37 °C. Take the absorbance on the 0th day as the original absorbance and calculate the relative activity. From Figure 16 It can be seen that GOx-OsNCs has good stability, and its activity can be maintained above 90% after being placed for one month.

Claims

1. A hybrid osmium nanocluster with both glucose oxidase and peroxidase activities, characterized in that, Prepared by the following method: Take 0.5 mL of 25 mg / mL glucose oxidase, add 0.5 mL of 20 mmol / L potassium hexachloroosmate and 50 µL of 0.8 mol / L NaOH respectively, stir and mix them, and then place them in a 37 °C water bath for reaction for 3 h; The average particle size of the obtained GOx-OsNCs is 1.3 nm, the lattice spacing corresponding to the 101 crystal plane of Os is 0.207 nm, and it consists of 71% Os 0 and 29% Os 4+ ; The obtained GOx-OsNCs can catalyze the oxidation of TMB by hydrogen peroxide, showing a strong absorption value at 652 nm in the ultraviolet region. Therefore, GOx-OsNCs have peroxidase activity, and the enzyme activity is 19.4 U / g. The obtained GOx-OsNCs can catalyze the oxidation of glucose to TMB, showing a strong absorption value at 652 nm in the ultraviolet region. Therefore, GOx-OsNCs have glucose oxidase activity, and the enzyme activity is 91.2 U / mg. The glucose oxidase activity of GOx-OsNCs can also be confirmed by the reaction of glucose with 3,5-dinitrosalicylic acid.

2. A hybrid osmium nanocluster with both glucose oxidase and peroxidase activities according to claim 1, characterized in that, The obtained GOx-OsNCs can catalyze glucose to produce hydrogen peroxide and then catalyze its oxidation to TMB for color development, proving that it has both glucose oxidase activity and peroxidase activity. Based on the dual enzyme activities, GOx-OsNCs can catalyze glucose to generate hydrogen peroxide, and at the same time, GOx-OsNCs can rapidly catalyze the oxidation of hydrogen peroxide to the substrate, thus realizing an efficient glucose self-cascade catalytic reaction. The self-cascade catalytic effect will be better than the effect of adding GOx and OsNCs step by step.

3. A hybrid osmium nanocluster with both glucose oxidase and peroxidase activities according to claim 1 or 2, characterized in that, The obtained GOx-OsNCs can detect glucose through the change of color signal, and the linear response range is 0.1 - 3.2 mmol / L for glucose concentration, proving that GOx-OsNCs can realize the glucose self-cascade catalytic reaction.

4. A hybrid osmium nanocluster with both glucose oxidase and peroxidase activities according to claim 1 or 2, characterized in that, The obtained GOx-OsNCs have good stability, and the self-cascade catalytic effect can be maintained above 90%.

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