Enzyme cascade application of micro-nano particle immobilized enzyme based on zinc coordination compound
Through the method of zinc-based coordination compound nanoparticle solid-carrying enzyme, the problems of enzyme instability and ease of inactivation in the prior art are solved, and the enzyme activity and stability are significantly improved, and are used for efficient detection of dopamine, glucose and lactose.
Patent Information
- Application Number
- CN202311176712.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-13
- Publication Date
- 2025-03-14
AI Technical Summary
The existing immobilized enzyme technology has problems such as enzyme instability, easy desorption and shedding, covalent coupling reactions lead to enzyme denaturation, and cross-linking methods to inactivate enzymes, which limits its wide application.
The zinc-based coordination compound nanoparticles ZnNBs and ZnNPs were used as carriers to synthesize nanospheres and nanoparticles through particle-sphere directional self-assembly method, and the single enzyme, double enzyme or triple enzyme were respectively solidified to construct an enzyme cascade reaction system to improve the activity and stability of the enzyme.
It significantly improves the activity and stability of the enzyme, and realizes sensitive detection of dopamine, glucose and lactose. It has a wide detection range, low detection limit, good specificity. The preparation method of solid-loaded enzymes is simple, with high catalytic activity, good stability, long service life, and can be reused.
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Abstract
Description
Technical Field
[0001] The invention relates to an enzyme cascade application based on zinc coordination compound micro-nano particles immobilizing enzymes, belonging to the technical field of nano-biosensor technology. Background Art
[0002] As an interdisciplinary technology, immobilized enzyme technology has been widely used in the fields of life science, biomedicine, food science, chemistry and chemical engineering, and environmental science. Immobilized enzyme technology is a technology developed based on the shortcomings of free enzymes, such as instability, easy loss, difficulty in separation from substrates, difficulty in reuse, and high cost of single use. It overcomes the above shortcomings of free enzymes by immobilizing the enzyme on a carrier, and constantly seeks more perfect immobilization methods and better immobilization carriers. At present, enzyme immobilization methods can be divided into four categories: adsorption method, covalent coupling method, cross-linking method, and embedding method. Although the adsorption method has the advantages of mild conditions, simple operation, and repeated use of adsorbents, its adsorption force is weak and it is easy to desorb and fall off. The covalent coupling method shows good stability, but the covalent coupling reaction easily denatures and inactivates the enzyme; the cross-linking method is more likely to inactivate the enzyme; the embedding method can obtain higher enzyme activity recovery, but its disadvantage is that it is not suitable for mass transfer of high molecular weight substrates, and there are often problems such as diffusion limitation. The shortcomings of the above-mentioned methods of enzyme immobilization limit their wide application. Therefore, the development of simple and practical immobilization methods and the design, synthesis and application of new enzyme immobilization materials with excellent performance are one of the focuses of current research on immobilized enzymes.
[0003] To overcome these shortcomings, we used zinc nitrate hexahydrate and Hmtz as raw materials to synthesize a nanosphere ZnNBs and a nanoparticle ZnNPs by particle-sphere directed self-assembly, proposed a new strategy for the efficient combination of ZnNBs and ZnNPs with enzymes (enzyme@ZnNFs; enzyme@ZnNPs as signal reporting unit), and constructed an immobilized enzyme bio-microreactor, providing new materials for the preparation of immobilized enzyme bio-microreactors. Summary of the invention
[0004] The technical problem solved by the present invention is: in order to overcome the shortcomings of the prior art, an enzyme cascade application based on zinc coordination compound micro-nano particles immobilizing enzymes is proposed, which greatly improves the activity and stability of natural enzymes and is used for sensitive detection of dopamine, glucose and lactose. Zinc-based coordination compound nanoparticles ZnNBs and ZnNPs are used as carriers to immobilize different enzymes (HRP, HRP / GOx, or HRP / GOx / β-Gal), and the experimental signal is then output as a fluorescent signal through a DA-DHA-H2O2 system to construct a fluorescent signal microreactor, which solves the shortcomings of free enzymes being unstable and difficult to reuse, improves enzyme activity, and is used to detect dopamine, glucose and lactose; the detection range is wide, the detection limit is low, and the specificity is good. In addition, the preparation method of zinc coordination compound micro-nano particles immobilizing enzymes is simple, the catalytic activity is high, the stability is good, the service life is long, and it can be reused.
[0005] In order to solve the technical problem of the present invention, the proposed technical scheme is: an enzyme cascade application based on zinc coordination compound micro-nanoparticles immobilized enzymes, the zinc coordination compound micro-nanoparticles ZnNBs or ZnNPs respectively immobilize an enzyme (HRP) to form HRP@ZnNBs; HRP@ZnNPs complex, respectively immobilize two enzymes (HRP / GOx) to form HRP / GOx@ZnNBs; HRP / GOx@ZnNPs complex, respectively immobilize three enzymes (HRP / GOx / β-Gal) to form HRP / GOx / β-Gal@ZnNBs; HRP / GOx / β-Gal@ZnNPs complex, and then the immobilized enzyme replaces the free enzyme to catalyze the three enzyme cascade reactions to produce fluorescence, the above three enzyme cascade reaction systems all generate yellow fluorescent compound AFC, the fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer, and the excitation wavelength and emission wavelength of AFC are 490nm and 550nm, respectively.
[0006] Zinc-based coordination compound nanoparticles ZnNBs and ZnNPs were respectively immobilized with an enzyme (HRP) to form HRP@ZnNBs; HRP@ZnNFs were added with Tris-HCl (pH=7.4) buffer solution containing dopamine DA, 1,5-naphthalenediol (DHA) and oxygenated water H2O2 at different concentrations. The combined action of HRP and H2O2 promoted the rapid in situ fluorescence reaction of DA and DHA to generate the fluorescent compound AFC; the fluorescence signal of the system was analyzed and detected by a fluorescence spectrometer, and the quantitative detection of dopamine was achieved according to the change in fluorescence intensity.
[0007] Zinc-based polymer nanoparticles ZnNBs and ZnNPs were respectively immobilized with two enzymes (HRP / GOx) to form HRP / GOx@ZnNBs; dopamine DA, 3 1,5-naphthalene diol DHA and Tris-HCl (pH=7.4) buffer solution of different concentrations of glucose were added to the HRP / GOx@ZnNPs complex, and glucose was oxidized to gluconic acid and H2O2 by GOx. The combined action of HRP and H2O2 promoted the rapid in situ fluorescence reaction of DA and DHA to generate the fluorescent compound AFC; the fluorescence signal of the system was analyzed and detected by a fluorescence spectrometer, and the quantitative detection of glucose was achieved according to the change of fluorescence intensity.
[0008] Zinc-based polymer nanoparticles ZnNBs and ZnNPs were immobilized with three enzymes (HRP / GOx / β-Gal) to form HRP / GOx / β-Gal@ZnNBs; dopamine, DHA and Tris-HCl (pH=7.4) buffer solution of different concentrations of lactose were added to the HRP / GOx / β-Gal@ZnNPs complex. β-Gal catalyzed the hydrolysis of lactose to produce glucose and galactose, and GOx catalyzed the oxidation of glucose to produce gluconic acid and H2O2. The produced H2O2, as a substrate of HRP, catalyzed the DA-DHA reaction to produce fluorescent AFC; β-Gal catalyzed the hydrolysis of lactose to produce glucose and galactose, and GOx catalyzed the oxidation of glucose to produce gluconic acid and H2O2. The produced H2O2, as a substrate of HRP, catalyzed the reaction of dopamine (DA)-1,5-dihydroxynaphthalene (DHA) to produce fluorescent AFC; the fluorescence signal of the system was analyzed and detected by a fluorescence spectrometer, and the quantitative detection of lactose was achieved according to the change of fluorescence intensity.
[0009] The synthesis conditions of ZnNBs are as follows: 2 mL of 0.2 mM zinc nitrate solution is poured into 2 mL of 0.5 mM Hmtz solution, and the solvent is ethanol. The mixture is vortexed at 1000 rpm for 1 min, and the mixed solution is kept at 4°C for 3 hours to obtain a mixed solution containing white precipitated ZnNBs, which is then washed for use.
[0010] The synthesis conditions of ZnNPs are as follows: 2 mL of zinc nitrate solution 0.4 mM is added in batches of 200 μL to 2 mL of Hmtz solution 2.0 mM (all solvents are ultrapure water, vortexed while adding), and placed in a constant temperature environment of 4°C for 24 hours to obtain a mixed solution of white precipitated ZnNPs.
[0011] Preferably, the steps of the enzyme immobilization method are:
[0012] 13 mg of ZnNBs and ZnNPs were weighed and dissolved in 6 mL of HRP (0.25 mg / mL) aqueous solution, and gently shaken at 200 rpm and 37 °C for 6 h to form a single enzyme-immobilized HRP@ZnNBs; HRP@ZnNPs complex;
[0013] 13 mg of ZnNBs and ZnNPs were weighed and dissolved in 6 mL of aqueous solution containing HRP / GOx (HRP 0.25 mg / mL, GOx 0.125 mg / mL), respectively, and gently shaken at 200 rpm and 37 °C for 6 h to form a dual-enzyme immobilized HRP / GOx@ZnNBs; HRP / GOx@ZnNPs complex;
[0014] 13 mg of ZnNBs and ZnNPs were weighed and dissolved in 6 mL of aqueous solution containing HRP / GOx / β-Gal (HRP 0.25 mg / mL, GOx 0.125 mg / mL, β-Gal 0.125 mg / mL), respectively, and gently shaken at 200 rpm and 37 °C for 6 h to form a HRP / GOx / β-Gal@ZnNBs; HRP / GOx / β-Gal@ZnNPs complex with three enzymes immobilized;
[0015] After preparation, the above six complexes were placed at 4°C for use.
[0016] Preferably, a series of dopamine, DHA and H2O2 at different concentrations are added to a Tris-HCl buffer solution, and the reaction is catalyzed by HRP@ZnNBs and HRP@ZnNPs complexes, respectively. After reacting at room temperature for 5 minutes, fluorescence spectral analysis is performed; the HRP concentration is 0.25 mg / mL, and the system is subjected to fluorescence signal analysis and detection by a fluorescence spectrometer, and quantitative detection of dopamine is achieved based on changes in fluorescence intensity.
[0017] Preferably, a series of different concentrations of glucose are incubated with HRP / GOx@ZnNBs; HRP / GOx@ZnNPs complexes at 37°C for 30 minutes, and then DA and DHA are added. After reacting at room temperature for 5 minutes, fluorescence spectral analysis is performed; the HRP concentration is 0.25 mg / mL, and the GOx concentration is 0.125 mg / mL. The system is subjected to fluorescence signal analysis and detection by a fluorescence spectrometer, and quantitative detection of glucose is achieved based on changes in fluorescence intensity.
[0018] Preferably, a series of different concentrations of lactose are incubated with HRP / GOx / β-Gal@ZnNBs; HRP / GOx / β-Gal@ZnNPs complexes at 37°C for 30 minutes, and then DA and DHA are added. After reacting at room temperature for 5 minutes, fluorescence spectral analysis is performed; the HRP concentration is 0.25 mg / mL, and the concentrations of GOx and β-Gal are both 0.125 mg / mL. The system is subjected to fluorescence signal analysis and detection by a fluorescence spectrometer, and quantitative detection of lactose is achieved based on changes in fluorescence intensity.
[0019] Zinc-based coordination compound nanoparticles ZnNBs and ZnNPs were respectively immobilized with an enzyme (HRP) to form HRP@ZnNBs; HRP@ZnNFs, and 300μL of Tris-HCl (10mM, pH=7.4) buffer solution containing 400μM dopamine DA, 30μM 1,5-naphthalenediol (DHA) and 60μM hydrogen peroxide H2O2 was added. The combined action of HRP and H2O2 promoted the rapid in situ fluorescence reaction of DA and DHA to generate the fluorescent compound AFC. Zinc-based polymer nanoparticles ZnNBs and ZnNPs were respectively immobilized with two enzymes (HRP / GOx) to form HRP / GOx@ZnNBs; HRP / GOx@ZnNPs complex, 300μL of Tris-HCl (10mM, pH=7.4) buffer solution containing 400μM dopamine DA, 30μM 1,5-naphthalenediol DHA and 5mM glucose was added, and glucose was oxidized to gluconic acid and H2O2 by GOx. The combined action of HRP and H2O2 promoted the rapid in situ fluorescence reaction of DA and DHA to generate the fluorescent compound AFC. The zinc-based coordination compound nanoparticles ZnNBs and ZnNPs were immobilized with three enzymes (HRP / GOx / β-Gal) to form HRP / GOx / β-Gal@ZnNBs; HRP / GOx / β-Gal@ZnNPs complexes, and 300μL of Tris-HCl (10mM, pH=7.4) buffer solution containing 400μM DA, 300μM DHA and 5mM lactose was added. β-Gal catalyzed the hydrolysis of lactose to produce glucose and galactose, and GOx catalyzed the oxidation of glucose to produce gluconic acid and H2O2. The produced H2O2, as a substrate for HRP, catalyzed the DA-DHA reaction to produce fluorescent AFC. The three enzyme cascade reaction systems all produced yellow fluorescent compounds AFC. The fluorescence intensity of the synthesized AFC was detected by fluorescence spectrometer. The excitation wavelength and emission wavelength of AFC were 490nm and 550nm, respectively.
[0020] The obtained HRP@ZnNBs and HRP@ZnNBs complexes were added to Tris-HCl buffer solution, and a series of different concentrations of dopamine were added, followed by DHA and H2O 2，,The reaction was catalyzed by HRP@ZnNBs and HRP@ZnNPs respectively. After the reaction was carried out at room temperature for 5 minutes, fluorescence spectrum analysis was performed. The fluorescence signal of the system was analyzed and detected by fluorescence spectrometer.
[0021] A method for detecting glucose using a fluorescence channel sensor based on ZnNBs and ZnNPs.
[0022] The obtained ZnNBs and ZnNPs were mixed with HRP / GOx (0.25 mg / mL, 0.125 mg / mL) for 6 h, centrifuged and washed to obtain HRP / GOx@ZnNBs and HRP / GOx@ZnNPs complexes;
[0023] The obtained HRP / GOx@ZnNBs and HRP / GOx@ZnNPs complexes were added to Tris-HCl buffer solution, and a series of different concentrations of glucose were added, and incubated at 37°C for 30 minutes. Then DA and DHA were added, and the reaction was carried out at room temperature for 5 minutes before fluorescence spectrum analysis. The fluorescence signal analysis and detection of the system were carried out by fluorescence spectrometer.
[0024] A method for detecting lactose based on a fluorescence channel sensor constructed by ZnNBs and ZnNPs.
[0025] The obtained ZnNBs and ZnNPs were mixed with HRP / GOx / β-Gal (0.25 mg / mL, 0.125 mg / mL, 0.125 mg / mL) for 6 h, centrifuged and washed to obtain HRP / GOx / β-Gal@ZnNB and HRP / GOx / β-Gal@ZnNP complexes.
[0026] The obtained HRP / GOx / β-Gal@ZnNB and HRP / GOx / β-Gal@ZnNP complexes were added to Tris-HCl buffer solution, and a series of different concentrations of lactose were added, and incubated at 37°C for 30 minutes. Then DA and DHA were added, and the reaction was carried out at room temperature for 5 minutes before fluorescence spectrum analysis. The fluorescence signal analysis and detection of the system was carried out by fluorescence spectrometer.
[0027] Zn(NO3)2 and Hmtz were reacted at 4℃ for 3 hours to synthesize ZnNBs. The directional self-assembly process of ZnNBs was characterized and analyzed in detail by scanning electron microscopy (SEM), energy dispersive element analyzer (EDS), etc. It was inferred that ZnNBs are zinc-based coordination compounds formed by directional self-assembly of particles-spheres.
[0028] In order to explore the formation mechanism of ZnNBs nanosphere materials, the products at different stages of the ZnNBs formation process were characterized by SEM. As the substrate reaction time increases, SEM results show that ZnNBs exist in two states: particles and spheres.
[0029] In order to verify the ability of ZnNBs to bind to enzymes, ZnNBs were incubated with HRP and centrifuged to obtain HRP@ZnNBs complex. We performed Fourier transform infrared tests on HRP@ZnNBs and HRP, and the results showed that the HRP@ZnNBs complex contained a carboxyl stretching band of the amide function in HRP, which was not present in ZnNBs lacking enzyme units, preliminarily proving that ZnNBs successfully immobilized the HRP enzyme.
[0030] In order to verify the ability of ZnNBs to bind to enzymes, we conducted X-ray diffraction tests on HRP@ZnNBs and ZnNBs. The results showed that HRP@ZnNBs and ZnNBs components have similar crystal structures, but the XRD spectrum intensity of the synthesized HRP@ZnNBs is weakened relative to that of ZnNBs. This is because the amorphous enzyme is encapsulated in the ZnNBs structure, which weakens the ZnNBs spectrum intensity, proving that ZnNBs successfully immobilizes the HRP enzyme.
[0031] To verify the ability of ZnNBs to bind to enzymes, we performed thermogravimetric analysis (TGA) on HRP@ZnNBs and ZnNBs. The results showed that there was a weight loss of enzyme in HRP@ZnNBs, which once again confirmed the presence of enzymes in HRP@ZnNBs.
[0032] To verify the ability of ZnNBs to bind to the enzyme, we performed Zeta potential tests on HRP@ZnNBs, ZnNBs, and HRP. The results showed that the potential of HRP@ZnNBs moved from the positive value of ZnNBs (+6.8 mV) to +1.52 mV, further proving that ZnNBs successfully immobilized the HRP enzyme.
[0033] The method for detecting dopamine based on the microreactor constructed by ZnNBs and ZnNPs is as follows: 5 μL of HRP@ZnNBs or HRP@ZnNPs complex solution is added to a Tris-HCl (10 mM, pH = 7.4) buffer solution containing 300 μM DHA and 600 μM H2O2, followed by adding a series of dopamine at different concentrations. Finally, the solution is left to react at room temperature for 5 minutes and then the fluorescence spectrum (Ex = 490 nm) is measured.
[0034] The method for detecting glucose using a microreactor constructed based on ZnNBs and ZnNPs is as follows: 5 μL of HRP / GOx@ZnNBs or HRP / GOx@ZnNPs complex solution is added to a Tris-HCl (10 mM, pH = 7.4) buffer solution containing a series of different glucose concentrations, and the mixture is gently shaken at 200 rpm and 37 °C for 30 min. Then, 300 μM DHA and 400 μM DA are added. Finally, the solution is left to react at room temperature for 5 minutes and then the fluorescence spectrum (Ex = 490 nm) is measured.
[0035] The method for detecting lactose using a microreactor constructed based on ZnNBs and ZnNPs is as follows: 5 μL of HRP / GOx / β-Gal@ZnNBs or HRP / GOx / β-Gal@ZnNPs complex solution is added to a Tris-HCl (10 mM, pH = 7.4) buffer solution containing a series of different lactose concentrations, and the mixture is gently shaken at 200 rpm and 37 °C for 30 min. Then, 300 μM DHA and 400 μM DA are added. Finally, the solution is left to react at room temperature for 5 minutes and then the fluorescence spectrum (Ex = 490 nm) is measured.
[0036] Beneficial effects:
[0037] The method of the present invention uses zinc nitrate hexahydrate and Hmtz as raw materials to synthesize a particle-sphere directed self-assembled ZnNBs and a nanoparticle ZnNPs, respectively, and proposes a method of using ZnNBs and ZnNPs as carriers to immobilize enzymes, thereby improving the activity and stability of the enzymes, and constructing an enzyme cascade reaction to achieve the detection of dopamine, glucose, and lactose. ZnNB and ZnNPs can be prepared in advance in large quantities, the material preparation operation is simple, the reaction conditions are mild, the raw materials are cheap, the immobilized enzyme preparation method is simple, the catalytic activity is high, the stability is good, the service life is long, and it can be reused.
[0038] The invention also carries out detailed characterization and analysis on the directional self-assembly process of ZnNBs by means of SEM, EDS and the like.
[0039] In order to further explore the influence of other factors on the morphology of ZnNBs, the products with different substrate concentrations were characterized by SEM. 2+ :Hmtz=1:2.5,Zn 2+ :0.01, 0.02, 0.0.025, 0.1, 0.2 mM) on ZnNBs. SEM results show that the overall concentration of the substrate plays a key role in the formation of ZnNBs. 2+ Only when Hmtz is 0.2mM and Hmtz is 0.5mM can nanospheres with uniform size and independent structure be formed.
[0040] In order to further explore the influence of other factors on the morphology of ZnNBs, the products of changing the synthesis temperature were characterized by SEM. The other conditions were fixed to explore the influence of different synthesis temperatures (4, 37, 50, 70℃) on ZnNBs. The SEM results showed that the reaction temperature also played a key role in the formation of ZnNBs. With the increase of reaction temperature, the spikes on the surface of ZnNBs became sharper and the shell inside ZnNBs became thicker. It can be seen from the X-ray diffraction pattern that in the reaction at 70℃, the intensity of the diffraction peak was the strongest, indicating the formation and crystallization process of highly crystalline ZnNBs. The detection comparison of dopamine, glucose and lactose based on the three enzyme cascade reactions constructed by ZnNBs synthesized at different synthesis temperatures (4, 37, 50, 70℃) was also studied. It was found that the three enzyme cascades constructed based on ZnNBs synthesized at 4℃ had the widest detection range, the lowest detection limit and the highest activity, so the subsequent synthesis temperature was selected as 4℃.
[0041] HRP can induce a rapid in situ fluorescence reaction between DA and DHA in the presence of H2O2. The prepared HRP@ZnNBs replace free HRP to construct a fluorescence channel sensor for detecting dopamine. HRP@ZnNBs has higher catalytic activity than free HRP and the relative fluorescence intensity shows a good linear relationship with dopamine concentration.
[0042] ZnNBs can be used to construct a fluorescent channel sensor for detecting glucose. ZnNBs immobilize two enzymes HRP / GOx to synthesize the HRP / GOx@ZnNBs complex, which can replace the free HRP / GOx to catalyze the enzyme cascade reaction. The reaction inside the ZnNBs microreactor will increase the local concentration of the intermediate product and shorten the distance between the enzyme and the reactant, thereby improving the catalytic activity. In the fluorescent channel sensor for detecting glucose based on ZnNBs, the relative fluorescence intensity has a good linear relationship with the glucose concentration.
[0043] ZnNBs can be used to construct a fluorescent channel sensor for detecting lactose. ZnNBs immobilize three enzymes HRP / GOx / β-Gal to synthesize the HRP / GOx / β-Gal@ZnNBs complex, which can replace the free HRP / GOx / β-Gal to catalyze the enzyme cascade reaction, which is consistent with the mechanism of the dual enzyme cascade reaction. The catalytic activity of HRP / GOx / β-Gal@ZnNBs is higher than that of HRP / GOx / β-Gal. In the fluorescent channel sensor for detecting lactose based on ZnNBs, the relative fluorescence intensity shows a good linear relationship with the lactose concentration.
[0044] The stability of the microreactor constructed based on ZnNBs was also studied. The effects of room temperature storage days, pH, temperature, enzyme cleavage, and organic solvents on the catalytic activity of immobilized enzymes and free enzymes were explored. It was found that after being treated under the same conditions, the activity of the immobilized enzyme was much higher than that of the free enzyme, and the immobilized enzyme maintained a high activity after multiple catalysis, proving that ZnNBs can protect enzymes and improve their activity and stability.
[0045] HRP can induce a rapid in situ fluorescence reaction between DA and DHA in the presence of H2O2. The prepared HRP@ZnNPs replace free HRP to construct a fluorescence channel sensor for detecting dopamine. The relative fluorescence intensity shows a good linear relationship with the dopamine concentration.
[0046] ZnNPs can be used to construct a fluorescent channel sensor for detecting glucose. ZnNPs immobilize two enzymes HRP / GOx to synthesize HRP / GOx@ZnNPs complex, which can replace free HRP / GOx to catalyze enzyme cascade reactions. The reaction inside the ZnNPs microreactor will increase the local concentration of intermediates and shorten the distance between the enzyme and the reactant, thereby improving the catalytic activity. In the fluorescent channel sensor for detecting glucose based on ZnNPs, the relative fluorescence intensity has a good linear relationship with the glucose concentration.
[0047] ZnNPs can be used to construct a fluorescent channel sensor for detecting lactose. ZnNBs immobilize three enzymes HRP / GOx / β-Gal to synthesize the HRP / GOx / β-Gal@ZnNPs complex, which can replace the free HRP / GOx / β-Gal to catalyze the enzyme cascade reaction, which is consistent with the mechanism of the dual enzyme cascade reaction. The catalytic activity of HRP / GOx / β-Gal@ZnNPs is higher than that of HRP / GOx / β-Gal. In the fluorescent channel sensor for detecting lactose based on ZnNPs, the relative fluorescence intensity shows a good linear relationship with the lactose concentration.
[0048] The stability of the microreactor constructed based on ZnNPs was also studied. After the same trypsin cleavage treatment, the catalytic activity of the immobilized enzyme was much higher than that of the free enzyme, and the immobilized enzyme maintained a high activity after multiple catalysis, proving that ZnNPs can also protect the enzyme and improve its activity and stability. BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The embodiments of the present invention will be further described below in conjunction with the accompanying drawings.
[0050] Figure 1 This is a scanning electron microscope image of the zinc-based coordination compound nanospheres ZnNBs prepared in Example 1;
[0051] Figure 2Elemental analysis diagram of the zinc-based coordination compound nanospheres ZnNBs prepared in Example 1;
[0052] Figure 3 Schematic diagram of the particle-sphere directed self-assembly of the zinc-based coordination compound nanospheres ZnNBs prepared in Example 2 and the corresponding scanning electron micrograph;
[0053] Figure 4 The SEM characterization of the zinc-based coordination compound nanospheres ZnNBs substrates with different molar concentrations prepared in Example 2;
[0054] Figure 5 The SEM characterization of the zinc-based coordination compound nanospheres ZnNBs prepared in Example 2 at different reaction temperatures;
[0055] Figure 6 The X-ray diffraction diagrams of the products obtained from the zinc-based coordination compound nanospheres ZnNBs prepared in Example 2 at different reaction temperatures;
[0056] Figure 7 is the Fourier transform infrared spectra of HRP and HRP@ZnNBs in Example 3;
[0057] Figure 8 is the X-ray diffraction pattern of ZnNBs and HRP@ZnNBs in Example 3;
[0058] Fig. 9 Thermogravimetric images of ZnNBs and HRP@ZnNBs in Example 3;
[0059] Fig.10 The Zeta point map of HRP, ZnNBs and HRP@ZnNBs in Example 3;
[0060] Fig.11 Schematic diagram of the microreactor constructed based on ZnNBs for detecting dopamine in Example 3
[0061] Fig.12 This is a fluorescence time scan diagram of the microreactor constructed based on ZnNBs to detect dopamine in Example 3;
[0062] Fig.13 The fluorescence emission spectrum of dopamine detected by the microreactor constructed based on ZnNBs in Example 3;
[0063] Fig.14 This is a quantitative relationship diagram of dopamine detection using a microreactor constructed based on ZnNBs in Example 3;
[0064] Fig.15 This is a schematic diagram of detecting glucose using a microreactor constructed based on ZnNBs in Example 4;
[0065] Fig.16 This is a fluorescence time scan diagram of glucose detection by the microreactor constructed based on ZnNBs in Example 4;
[0066] Fig.17 This is the fluorescence emission spectrum of glucose detected by the microreactor constructed based on ZnNBs in Example 4;
[0067] Fig.18 This is a quantitative relationship diagram of glucose detection by the microreactor constructed based on ZnNBs in Example 4;
[0068] Fig.19 This is a schematic diagram of the detection of lactose by the microreactor constructed based on ZnNBs in Example 5;
[0069] Fig. 20 This is a fluorescence time scan diagram of lactose detection by the microreactor constructed based on ZnNBs in Example 5;
[0070] Fig.21 This is the fluorescence emission spectrum of lactose detected by the microreactor constructed based on ZnNBs in Example 5;
[0071] Fig. 22 This is a quantitative relationship diagram of lactose detection by the microreactor constructed based on ZnNBs in Example 5;
[0072] Fig.23 This is a time stability diagram of the microreactor constructed based on ZnNBs in Example 7;
[0073] Fig.24 This is a pH stability diagram of the microreactor constructed based on ZnNBs in Example 7;
[0074] Fig.25 This is a temperature stability diagram of the microreactor constructed based on ZnNBs in Example 7;
[0075] Fig.26 This is a cyclic stability diagram of the microreactor constructed based on ZnNBs in Example 7;
[0076] Fig. 27 The enzymatic cleavage and organic solvent stability diagram of the microreactor constructed based on ZnNBs in Example 7;
[0077] Fig.28 Schematic diagram of the detection of dopamine by a microreactor constructed based on ZnNPs in Example 8;
[0078] Fig.29 The fluorescence emission spectrum of dopamine detected by the microreactor constructed based on ZnNPs in Example 8;
[0079] Fig.30 This is a quantitative relationship diagram of the detection of dopamine by the microreactor constructed based on ZnNPs in Example 8;
[0080] Fig.31 Schematic diagram of the detection of glucose by the microreactor constructed based on ZnNPs in Example 9;
[0081] Fig.32 The fluorescence emission spectra of the microreactors GOx / HRP@ZnNPs and HRP@ZnNPs+GOx@ZnNPs constructed based on ZnNPs in Example 9 for catalytic detection of glucose;
[0082] Fig.33 This is the fluorescence emission spectrum of glucose detected by the microreactor constructed based on ZnNPs in Example 9;
[0083] Fig.34 This is a quantitative relationship diagram of glucose detection by the microreactor constructed based on ZnNPs in Example 9;
[0084] Fig.35 Schematic diagram of the detection of lactose by the microreactor constructed based on ZnNPs in Example 10;
[0085] Fig.36 The fluorescence emission spectra of the microreactor GOx / HRP / β-Gal@ZnNPs and HRP@ZnNPs+GOx@ZnNPs+β-Gal@ZnNPs constructed based on ZnNPs in Example 10 for catalytic detection of lactose;
[0086] Fig.37 This is the fluorescence emission spectrum of lactose detected by the microreactor constructed based on ZnNPs in Example 10;
[0087] Fig.38 This is a quantitative relationship diagram of lactose detection by the microreactor constructed based on ZnNPs in Example 10;
[0088] Fig.39 This is a graph showing the cyclic stability of dopamine detected by the microreactor constructed based on ZnNPs in Example 11;
[0089] Fig.40 This is a graph showing the cyclic stability of glucose detected by the microreactor constructed based on ZnNPs in Example 11;
[0090] Fig.41 This is a graph showing the cyclic stability of lactose detected by the microreactor constructed based on ZnNPs in Example 11;
[0091] Fig.42 This is the enzyme cleavage stability diagram of the microreactor constructed based on ZnNPs in Example 11;
[0092] Fig.43 It is a schematic diagram of the present invention. DETAILED DESCRIPTION
[0093] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and process are given, but the protection scope of the present invention is not limited to the following embodiment.
[0094] Example 1: The directed self-assembly synthesis process of ZnNBs was characterized and analyzed in detail by SEM, EDS and other methods. Pour 2mL of zinc nitrate (0.2mM) solution into 2mL of Hmtz (0.5mM) solution (the solvents are all ethanol) and vortex for 1min at 1000rpm. The mixed solution was then allowed to stand at a constant temperature of 4°C for 3h to obtain a mixed solution containing a white precipitate. After the reaction solution was centrifuged at 1000rpm for 1min, the precipitate was collected and washed three times with ultrapure water. The white precipitate was placed in an oven at 50°C and dried to obtain a solid powder of nanosphere ZnNBs. Figure 1 As shown in Figure 2, the macroscopic morphology of ZnNBs was characterized by scanning electron microscopy, and it was observed that ZnNBs were highly monodispersed, uniformly spherical, and the size was between 10-15 μm. The composition elements and element distribution state of ZnNBs were characterized by energy spectrum element analysis, and the element resolution diagram was drawn, as shown in Figure 2. Figure 2 The shown elements (Zn, C, N, O) are present and uniformly distributed in ZnNBs.
[0095] Example 2: 2 mL of zinc nitrate (0.2 mM) solution was poured into 2 mL of Hmtz (0.5 mM) solution (the solvent was ethanol), and vortexed at 1000 rpm for 1 min. The mixed solution was then kept at a constant temperature of 4°C, and the products of different reaction times (0 h, 0.5 h, 1 h, 2 h, and 3 h) during the synthesis of ZnNBs were selected and characterized by scanning electron microscopy to study the particle-sphere self-assembly synthesis mechanism of ZnNBs, such as Figure 3 As shown in the figure, in the early stage, Zn-Hmtz primary crystals are rapidly generated and appear in the form of nano-sized particles. At this stage, we speculate that Hmtz and Zn 2+ Mainly through the N and Zn in the Hmtzs main chain 2+The coordination effect forms coordination compounds, which provide locations for the nucleation of primary crystals and then agglomerate into small particles. In the second growth step, a large number of nanospheres formed by the agglomeration of small particles gradually appear in the reaction solution. The separation and gaps of these small particles lead to the rough surface and porous structure of the nanospheres. In the final stage, the "construction" of the nanosphere scaffold is basically completed, and the small particles assemble and agglomerate in a directional manner to form an independent nanosphere structure. As time goes by, the size of the nanospheres gradually increases, and finally three-dimensional ZnNBs with uniform size and stable morphology are assembled. When other conditions are fixed, different molar concentrations of the substrate (Zn 2+ :Hmtz=1:2.5,Zn 2+ :0.02, 0.025, 0.1, 0.2mM) on the effects of ZnNBs Figure 4 As shown. 2+ When the concentration is 0.02 mM, the product morphology is nanoparticles of 20.14 nm. 2+ When the concentration increased to 0.025 mM, micron particles with a size of about 1 μm began to appear in the product, which were assembled from many nanoparticles. 2+ concentration (0.1 mM), the newly generated self-assembled microparticles merged into more complete and independent microparticles. 2+ When the concentration reaches 0.2mM, the product morphology is a three-dimensional nanosphere with independent structure and uniform size of about 13μm. These results show that the overall concentration of the substrate plays a key role in the formation of ZnNBs, and the substrate can only form nanospheres with uniform size and independent structure within a specific concentration range.
[0096] By adjusting the synthesis temperature, the effect of temperature on the formation of ZnNBs was studied. Figure 5 As shown in the figure, the normally synthesized ZnNBs are spherical and porous on the surface. As the reaction temperature increases, the spikes on the surface of ZnNBs become sharper and sharper. The XRD of ZnNBs (4℃), ZnNBs (37℃), ZnNBs (50℃) and ZnNBs (70℃) were tested to study the crystallization process of nanospheres. Figure 6 As shown in Figure 2, the crystallinity of ZnNBs microparticles increases with increasing temperature. In the reaction at 70 °C, the intensity of the diffraction peak is the strongest, indicating the crystallization process and formation of highly crystalline ZnNBs.
[0097] Example 3: HRP@ZnNBs complex was obtained by incubation of HRP and ZnNBs nanospheres and then centrifuged and washed. Fourier transform infrared measurement ( Figure 7) showed that the HRP@ZnNBs complex contained the carboxyl stretching band of the amide function in HRP, which was not present in ZnNBs lacking enzyme units, preliminarily proving that ZnNBs successfully immobilized the HRP enzyme. X-ray diffraction pattern ( Figure 8 ) showed that HRP@ZnNBs and ZnNBs components have similar crystal structures, but the XRD spectrum intensity of the synthesized HRP@ZnNBs is weakened relative to that of ZnNBs. This is due to the encapsulation of amorphous enzymes in the ZnNBs structure, which weakens the ZnNBs intensity. Therefore, the XRD results indicate the successful synthesis of HRP@ZnNBs. Thermogravimetric analysis (TGA) showed the weight loss of the enzyme, which once again confirmed the presence of the enzyme in HRP@ZnNBs ( Fig. 9 The zeta potential of ZnNBs after HRP immobilization shifted from the positive value of ZnNBs (+6.8mV) to +1.52mV ( Fig.10 ). The prepared HRP@ZnNBs replaced the free HRP and constructed a microreactor based on ZnNBs to immobilize an enzyme ( Fig.11 ), DA-DHA was selected as the substrate of HRP-H2O2 for the fluorescence signal output of the microreactor. Fig.12 (a: free HRP, b: HRP@ZnNBs, c: ZnNBs) describes the time dependence of the catalytic reaction of equal amounts of free HRP and HRP@ZnNBs to produce AFC. Compared with free HRP, the biocatalytic activity in the ZnNBs reactor is almost the same, proving that ZnNBs do not affect the activity of individual enzymes. Control experiments show that ZnNBs cannot trigger fluorescence response. Dopamine is further quantified through the fluorescence channel, such as Fig.13 , Fig.14 As shown in the figure, as the dopamine concentration increases, the fluorescence intensity gradually increases. The relative fluorescence intensity and dopamine concentration show a good linear relationship in the range of 0-10 mM. The corresponding fitting linear equation is Y=102.42C DA +10.062(R 2 =0.9903), and the detection limit (LOD) was 1.67 μM. The experimental results showed that the developed fluorescence channel microreactor was feasible and had excellent performance for dopamine analysis.
[0098] Example 4: HRP / GOx and ZnNBs nanospheres were incubated and then centrifuged and washed to obtain HRP / GOx@ZnNBs complex. The prepared HRP / GOx@ZnNBs replaced the free HRP / GOx to construct a ZnNBs-based microreactor for immobilizing two enzymes ( Fig.15). When the two enzymes (HRP / GOx) were immobilized on ZnNBs, GOx oxidized glucose to produce H2O2 and gluconic acid. The produced H2O2, as a substrate for HRP, catalyzed the DA-DHA reaction to generate fluorescent AFC, which was used for the fluorescent signal output of the microreactor. Fig.16 (a: HRP / GOx@ZnNBs, b: free HRP / GOx, c: HRP@ZnNBs, d: GOx@ZnNBs) describes the time dependence of the catalytic reaction of equal amounts of free HRP / GOx and HRP / GOx@ZnNBs to produce AFC. Compared with free HRP / GOx, the biocatalytic activity in the ZnNBs reactor is enhanced by about 9.5 times. Control experiments show that ZnNBs, HRP@ZnNBs and GOx@ZnNBs cannot trigger the fluorescence response of the dual enzyme cascade. Glucose is further quantified through the fluorescence channel, such as Fig.17 As shown in 18, with the increase of glucose concentration, the fluorescence intensity gradually increases, and the relative fluorescence intensity shows a good linear relationship with the glucose concentration in the range of 0-10 mM. The corresponding fitting linear equation is Y1=17.334+64.599C glucose (R1 2 =0.9888)(0.025-0.5mM), Y2=4.6676+33.855C glucose (R2 2 =0.9852)(0.5mM-10ng / mL), LOD is 8.33μM. The experimental results show that the developed fluorescence channel microreactor is feasible and has excellent performance for glucose analysis.
[0099] Example 5: HRP / GOx / β-Gal was incubated with ZnNBs nanospheres and then centrifuged and washed to obtain HRP / GOx / β-Gal@ZnNBs complex ( Fig.19 ). The prepared HRP / GOx / β-Gal@ZnNBs replaced the free HRP / GOx / β-Gal to construct a microreactor based on ZnNBs with three enzymes immobilized. When the three enzymes (HRP / GOx / β-Gal) were immobilized on ZnNBs, lactose was used as a matrix, β-Gal catalyzed the hydrolysis of lactose to produce glucose and galactose, and GOx catalyzed the oxidation of the obtained glucose to produce gluconic acid and H2O2. The produced H2O2, as a substrate for HRP, catalyzed the DA-DHA reaction to produce fluorescent AFC. Fig. 20(a: HRP / GOx / β-Gal@ZnNBs, b: free HRP / GOx / β-Gal, c: HRP / GOx@ZnNBs, d: HRPβ-Gal@ZnNBs) describes the time dependence of the catalytic reaction of equal amounts of free HRP / GOx / β-Gal and HRP / GOx / β-Gal@ZnNBs to produce AFC. Compared with free HRP / GOx / β-Gal, the biocatalytic activity in the ZnNBs reactor was enhanced by about 6.5 times. Control experiments showed that ZnNBs, HRP / GOx@ZnNBs and GOx / β-Gal@ZnNBs could not trigger the fluorescence response of the three-enzyme cascade. Glucose was further quantified by the fluorescence channel, such as Fig.21 As shown in Figure 22, as the glucose concentration increases, the fluorescence intensity gradually increases. The relative fluorescence intensity and lactose concentration show a good linear relationship in the range of 0-10 mM. The corresponding fitting linear equation is Y1=1.1308+39.134C lactuse (R1 2 =0.9919)(0.005-1mM) and Y2=39.025+3.6885C lactuse (R2 2 =0.9825)(1-10 mM), LOD was 1.67 μM. The experimental results showed that the developed fluorescence channel microreactor was feasible and had excellent performance for lactose analysis.
[0100] Example 6: Equal amounts of ZnNBs synthesized at different temperatures (4, 37, 50, 70°C) were immobilized with equal amounts of one enzyme, two enzymes, and three enzymes to obtain an immobilized enzyme mixed solution. Dopamine, glucose, and lactose were detected according to the above three enzyme cascade reactions, and their catalytic activities, detection ranges, and detection limits were compared.
[0101] As shown in Table 1, the ZnNBs immobilized enzyme obtained at 4°C had the highest catalytic activity, the widest detection range, and the lowest detection limit. The detection range of dopamine was 5-10 4 nM, the detection limit is 1.67nM, and the glucose detection range is 10-10 4 nM, detection limit is 3.33nM, lactose glucose detection range is 5-10 4 nM, and the detection limit was 1.67nM. The experimental results showed that the ZnNBs immobilized enzyme had the best performance when the synthesis temperature was 4℃.
[0102] Table 1 is a comparison table of the activities of microreactors constructed based on ZnNBs at different synthesis temperatures in Example 6
[0103]
[0104] Example 7: Take 2 mg of HRP@ZnNBs, HRP / GOx@ZnNBs and HRP / GOx / β-Gal@ZnNBs and dissolve them in 1 mL of ultrapure water to obtain a mixed solution of immobilized enzymes. Prepare HRP, HRP / GOx, and HRP / GOx / β-Gal free enzyme solutions with a concentration of 10 U / mL respectively, and place the three fixed enzymes and three free enzymes at room temperature for different periods of time (0, 2, 4, 6, 8, 10, 12, 14, 16, and 20 days). Take 5 μL from the three fixed enzymes and three free enzyme solutions to catalyze the fluorescence reaction of AFC, and compare the catalytic activity of the enzymes by comparing the changes in relative fluorescence intensity. Fig.23 As shown in the figure, due to the protective effect of ZnNBs on the active conformation of the enzyme, the residual activity of the immobilized enzyme was higher than 85% after being stored at room temperature for 20 days. The results show that the immobilized enzyme has high storage stability. The residual activity of the free enzyme was less than 10%. The increase in the residual activity of the immobilized enzyme compared with the free enzyme fully demonstrates the advantage of ZnNBs in stabilizing the protein structure.
[0105] According to the above operation, the three fixed enzymes and three free enzymes were treated in different pH aqueous solutions (3, 4, 5, 6, 7, 8, 9, 10, 11) for 30 minutes and then catalyzed to produce AFC fluorescence reaction. The catalytic activity of the enzymes was compared by the change of relative fluorescence intensity. Fig.24 As shown in the figure, when the pH reaches 3, the activity of the free enzyme decreases to about 20%, while the activity of the immobilized enzyme remains above 85%. When the pH reaches 11, the activity of the free enzyme decreases to about 25%, while the activity of the immobilized enzyme remains above 85%. Compared with the free enzyme, the immobilized enzyme has a higher tolerance to extreme pH values and can function normally in a wider pH range.
[0106] According to the above operation, the three fixed enzymes and three free enzymes were treated at different temperatures (20, 30, 40, 50, 60, 70°C) for 30 min to catalyze the fluorescence reaction of AFC. Fig.25 As shown in the figure, the catalytic activity of the immobilized enzyme gradually decreases with the increase of temperature, but the reduction rate of the immobilized enzyme is lower than that of the free enzyme. When the temperature rises to 70°C, the remaining activity of the free enzyme is only about 10%. However, the remaining activity of the immobilized enzyme is higher than 80%. This is because ZnNBs can form a "armor" on the surface of the enzyme, preventing the enzyme from direct contact with the high temperature environment, thereby avoiding the reduction of enzyme activity caused by high temperature.
[0107] The immobilized enzyme still retained more than 86% of its activity after 8 cycles of catalysis. Fig.26), which may be due to the limited influence of the ZnNBs framework. The above results show that the immobilized enzyme has high reusability, and the zinc-based coordination compound micro-nanoparticles ZnNBs can provide an appropriate microenvironment to stabilize the immobilized enzyme and significantly improve the activity and stability of the enzyme.
[0108] According to the above operation, the three fixed enzymes and three free enzymes were treated with trypsin (1 mg / mL) for 24 h to catalyze the fluorescence reaction of AFC. Fig. 27 As shown in Figure 3, after trypsin treatment, the immobilized enzyme still retained more than 84% of its activity. However, the free enzyme only retained about 15% of its activity under the same conditions. This suggests that ZnNBs can provide an effective shield for the enzyme against biodegradation.
[0109] At the same time, the three fixed enzymes and three free enzymes were treated with DMF for 30 min to catalyze the fluorescence reaction of AFC. Fig. 27 As shown in Figure 2, after DMF treatment, the immobilized enzyme still retained more than 80% of its activity. However, under the same conditions, the free enzyme only retained about 10% of its activity. This suggests that ZnNBs can provide an effective shield for enzymes against damage by organic solvents.
[0110] Embodiment 8:
[0111] The synthesis conditions of ZnNPs are as follows: 2 mL of zinc nitrate solution 0.4 mM is added in batches of 200 μL to 2 mL of Hmtz solution 2.0 mM (all solvents are ultrapure water, vortexed while adding), and placed in a constant temperature environment of 4°C for 24 hours to obtain a mixed solution of white precipitated ZnNPs.
[0112] HRP and ZnNPs nanoparticles were incubated and centrifuged to obtain HRP@ZnNPs complex. The prepared HRP@ZnNPs replaced the free HRP to construct a sensor based on ZnNPs immobilized with an enzyme. DA-DHA was selected as the substrate of HRP-H2O2 for the fluorescence signal output of the nanoreactor ( Fig.28 ). First, the enzyme (HRP) was immobilized on ZnNPs. On ZnNPs, the fluorescence intensity increased with the immobilization of different concentrations of HRP ( Fig.29 , 30). Two good linear relationships from 0-3mU / mL (F1 / F0=2.0136+7.4109C HRP , R 2 =0.9728) and 3-30 mU / mL (F1 / F0=22.4460+0.2008C HRP , R 2 =0.9646) was observed between the fluorescence intensity ratio and HRP concentration, indicating that the system can adsorb HRP on ZnNPs and quantify HRP within a certain range.
[0113] Example 9: HRP / GOx and ZnNPs nanoparticles were incubated and then centrifuged and washed to obtain HRP / GOx@ZnNBs complex. The prepared HRP / GOx@ZnNPss replaced the free HRP / GOx to construct a sensor based on ZnNPs immobilized with two enzymes ( Fig.31 ). When the two enzymes (HRP / GOx) were immobilized on ZnNPs, the oxidation of glucose by GOx produced H2O2 and gluconic acid. The produced H2O2, as a substrate of HRP, catalyzed the DA-DHA reaction to generate fluorescent AFC, which was used for the fluorescence signal output of the nanoreactor. Fig.32 As shown, the catalytic fluorescence of HRP / GOx@ZnNPs mixed with HRP@ZnNPs and GOx@ZnNPs containing the same concentration was enhanced by 1.8 times. The results show that the catalytic activity of the two enzymes (HRP and GOx) adsorbed on the same particle is better than that of the two enzymes adsorbed on different particles. This is because the cascade carried out in the limited volume of ZnNPs produces a high local concentration of H2O2 near HRP, resulting in an effective bio-enzyme cascade. Glucose was further quantified through the fluorescence channel, such as Fig.33 As shown in Figure 34, with the increase of glucose concentration, the fluorescence intensity gradually increases. The relative fluorescence intensity and glucose concentration show a good linear relationship in the range of 0-10mM, and the corresponding fitting linear equation is Y=2.5355+12.415C glucose (R 2 =0.9899)(0-0.5mM), LOD was 1.67μM. The experimental results showed that the developed fluorescence channel microreactor was feasible and had excellent performance for lactose analysis.
[0114] Example 10: HRP / GOx / β-Gal was incubated with ZnNPs nanoparticles and then centrifuged and washed to obtain HRP / GOx / β-Gal@ZnNBs complex ( Fig.35 ). The prepared HRP / GOx / β-Gal@ZnNPs replaced the free HRP / GOx / β-Gal to construct a sensor based on ZnNPs with three enzymes immobilized on them. When the three enzymes (HRP / GOx / β-Gal) were immobilized on ZnNPs, lactose was used as the matrix. β-Gal catalyzed the hydrolysis of lactose to produce glucose and galactose, and GOx catalyzed the oxidation of the obtained glucose to produce gluconic acid and H2O2. The produced H2O2 served as a substrate for HRP and catalyzed the DA-DHA reaction to produce fluorescent AFC. Finally, the efficiency of the biocatalyst was evaluated by quantifying lactose. When HRP / GOx / β-Gal@ZnNPs and HRP@ZnNPs were mixed with GOx@ZnNPs and β-Gal@ZnNPs at the same concentration, the catalytic fluorescence was enhanced by 1.7 times ( Fig.36As mentioned above, the increased catalytic activity of an enzyme cascade can be attributed to the high local concentration of a reactant produced by one enzyme. Fig.37 As shown in Figure 38, the relative fluorescence intensity showed a good linear relationship with the glucose concentration in the range of 0-0.5 mM, and the fitting linear equation was F1 / F0=1.6652+10.9350C lactose , R 2 =0.9808 (0-0.5 mM), LOD is 0.33 μM. The experimental results show that the developed fluorescence channel microreactor is feasible and has excellent performance for lactose analysis.
[0115] Example 11: HRP@ZnNPs, HRP / GOx@ZnNPs, and HRP / GOx / β-Gal@ZnNPs retained more than 86% of their activity after 6 cycles of continuous use ( Fig.39 , 40, 41), indicating that HRP@ZnNPs, HRP / GOx@ZnNPs, and HRP / GOx / β-Gal@ZnNPs can be reused. In addition, HRP@ZnNPs, HRP / GOx@ZnNPs, and HRP / GOx / β-Gal@ZnNPs were treated with trypsin (1 mg / mL) for 24 h. Fig.42 As shown in Figure 3, after trypsin treatment, HRP@ZnNPs, HRP / GOx@ZnZnNPs and HRP / GOx / β-Gal@ZnNPs still retained more than 90% of their activities, indicating that ZnNPs can provide an effective shielding for enzymes against biodegradation.
[0116] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.
Claims
1. An enzyme cascade application based on zinc coordination compound micro-nanoparticles immobilized enzyme, characterized in that: Zinc coordination compound micro-nanoparticles ZnNBs or ZnNPs are respectively immobilized with an enzyme (HRP) to form HRP@ZnNBs; HRP@ZnNPs complex, respectively immobilized two enzymes (HRP / GOx) to form HRP / GOx@ZnNBs; HRP / GOx@ZnNPs complex, three enzymes (HRP / GOx / β-Gal) were immobilized to form HRP / GOx / β-Gal@ZnNBs; HRP / GOx / β-Gal@ZnNPs complex, and then the immobilized enzyme replaced the free enzyme to catalyze the three enzyme cascade reactions to produce fluorescence. The above three enzyme cascade reaction systems all generated yellow fluorescent compounds AFC. The fluorescence intensity of the synthesized AFC was detected by fluorescence spectrometer. The excitation wavelength and emission wavelength of AFC were 490nm and 550nm, respectively. Zinc-based coordination compound nanoparticles ZnNBs and ZnNPs were respectively immobilized with an enzyme (HRP) to form HRP@ZnNBs; HRP@ZnNFs, added with different concentrations of dopamine DA, 1,5-naphthalene diol (DHA) and hydrogen peroxide H2O2 in Tris-HCl (pH=7.4) buffer solution, the combined action of HRP and H2O2 promoted the rapid in situ fluorescence reaction of DA and DHA to generate the fluorescent compound AFC; the fluorescence signal of the system was analyzed and detected by fluorescence spectrometer, and the quantitative detection of dopamine was achieved according to the change of fluorescence intensity; Zinc-based polymer nanoparticles ZnNBs and ZnNPs were respectively immobilized with two enzymes (HRP / GOx) to form HRP / GOx@ZnNBs; HRP / GOx@ZnNPs complex, dopamine DA, 3 1,5-naphthalene diol DHA and different concentrations of glucose in Tris-HCl (pH=7.4) buffer solution were added. Glucose was oxidized to gluconic acid and H2O2 by GOx. The combined action of HRP and H2O2 promoted the rapid in situ fluorescence reaction of DA and DHA to generate the fluorescent compound AFC. The fluorescence signal of the system was analyzed and detected by fluorescence spectrometer, and the quantitative detection of glucose was achieved according to the change of fluorescence intensity. Zinc-based polymer nanoparticles ZnNBs and ZnNPs were immobilized with three enzymes (HRP / GOx / β-Gal) to form HRP / GOx / β-Gal@ZnNBs; HRP / GOx / β-Gal@ZnNPs complex, dopamine, DHA and Tris-HCl (pH=7.4) buffer solution with different concentrations of lactose were added. β-Gal catalyzed the hydrolysis of lactose to produce glucose and galactose, and GOx catalyzed the oxidation of the obtained glucose to produce gluconic acid and H2O2. The produced H2O2, as a substrate of HRP, catalyzed the DA-DHA reaction to produce fluorescent AFC. β-Gal catalyzes the hydrolysis of lactose to produce glucose and galactose, and GOx catalyzes the oxidation of the obtained glucose to produce gluconic acid and H2O2. The produced H2O2, as a substrate of HRP, catalyzes the reaction of dopamine (DA)-1,5-dihydroxynaphthalene (DHA) to produce fluorescent AFC. The fluorescence signal of the system is analyzed and detected by a fluorescence spectrometer, and the quantitative detection of lactose is achieved according to the change of fluorescence intensity. The synthesis conditions of ZnNBs are as follows: 2 mL of 0.2 mM zinc nitrate solution is poured into 2 mL of 0.5 mM Hmtz solution, and the solvent is ethanol. The mixture is vortexed at 1000 rpm for 1 min, and the mixed solution is kept at 4°C for 3 hours to obtain a mixed solution containing white precipitated ZnNBs, which is then washed for use. The synthesis conditions of ZnNPs are as follows: 2 mL of zinc nitrate solution 0.4 mM is added in batches of 200 μL to 2 mL of Hmtz solution 2.0 mM (all solvents are ultrapure water, vortexed while adding), and placed in a constant temperature environment of 4°C for 24 hours to obtain a mixed solution containing white precipitated ZnNPs, which is then washed and set aside.
2. The enzyme cascade application based on zinc coordination compound micro-nanoparticles immobilized enzyme according to claim 1, characterized in that: The steps of the enzyme immobilization method are: 13 mg of ZnNBs and ZnNPs were weighed and dissolved in 6 mL of HRP (0.25 mg / mL) aqueous solution, respectively, and gently shaken at 200 rpm and 37 °C for 6 h to form single enzyme-immobilized HRP@ZnNBs. HRP@ZnNPs complex; 13 mg of ZnNBs and ZnNPs were weighed and dissolved in 6 mL of aqueous solution containing HRP / GOx (HRP 0.25 mg / mL, GOx 0.125 mg / mL), and gently shaken at 200 rpm and 37 °C for 6 h to form dual-enzyme immobilized HRP / GOx@ZnNBs. HRP / GOx@ZnNPs composite; 13 mg of ZnNBs and ZnNPs were weighed and dissolved in 6 mL of aqueous solution containing HRP / GOx / β-Gal (HRP 0.25 mg / mL, GOx 0.125 mg / mL, β-Gal 0.125 mg / mL), respectively, and gently shaken at 200 rpm and 37 °C for 6 h to form a HRP / GOx / β-Gal@ZnNBs; HRP / GOx / β-Gal@ZnNPs complex with three enzymes immobilized; After preparation, the above six complexes were placed at 4°C for use.
3. The enzyme cascade application based on zinc coordination compound micro-nanoparticle immobilized enzyme according to claim 1, characterized in that: A series of dopamine, DHA and H2O2 at different concentrations were added to Tris-HCl buffer solution, and the reactions were catalyzed by HRP@ZnNBs and HRP@ZnNPs complexes, respectively. Fluorescence spectroscopy analysis was performed after the reaction at room temperature for 5 minutes. The HRP concentration was 0.25 mg / mL, and the fluorescence signal analysis of the system was detected by fluorescence spectrometer, and the quantitative detection of dopamine was achieved based on the changes in fluorescence intensity.
4. The enzyme cascade application based on zinc coordination compound micro-nanoparticle immobilized enzyme according to claim 1, characterized in that: A series of different concentrations of glucose were respectively reacted with HRP / GOx@ZnNBs; The HRP / GOx@ZnNPs complex was incubated at 37°C for 30 min, and then DA and DHA were added. After reacting at room temperature for 5 min, fluorescence spectral analysis was performed; the HRP concentration was 0.25 mg / mL, and the GOx concentration was 0.125 mg / mL. The fluorescence signal analysis and detection of the system was performed by fluorescence spectrometer, and quantitative detection of glucose was achieved based on the change in fluorescence intensity.
5. The enzyme cascade application based on zinc coordination compound micro-nanoparticle immobilized enzyme according to claim 1, characterized in that: A series of different concentrations of lactose were respectively reacted with HRP / GOx / β-Gal@ZnNBs; The HRP / GOx / β-Gal@ZnNPs complex was incubated at 37°C for 30 min, and then DA and DHA were added. After reacting at room temperature for 5 min, fluorescence spectral analysis was performed; the HRP concentration was 0.25 mg / mL, and the concentrations of GOx and β-Gal were both 0.125 mg / mL. The fluorescence signal of the system was analyzed and detected by a fluorescence spectrometer, and the quantitative detection of lactose was achieved based on the changes in fluorescence intensity.