Enzyme immobilization method mediated by metal-G4DNAzyme nucleic acid framework and application

Abstract

The invention discloses a metal-G4DNAzyme nucleic acid framework mediated enzyme immobilization method and an application of the metal-G4DNAzyme nucleic acid framework mediated enzyme immobilization method. The method comprises the following steps: co-incubating an enzyme and a G-quadruplex-containing nucleic acid sequence in a PBS buffer solution, adding divalent metal ions, placing in a centrifuge tube, oscillating, carrying out ultrasonic treatment, centrifuging a supernatant, washing with deionized water, redissolving and precipitating by adopting the PBS buffer solution, co-incubating with hyperchlorheme, centrifuging to remove the supernatant, and washing away redundant hyperchlorheme, thereby obtaining the G-quadruplex-containing nucleic acid sequence. And the metallo-G4DNAzyme nucleic acid framework mediated enzyme is obtained. The problems that an existing functional protein and nucleic acid coupling method is complex in process and harsh in condition, and activity and functions of protein are difficult to retain are solved. Coupling of enzyme and nucleic acid is achieved through biomimetic mineralization, and the formed functional metal nucleic acid framework immobilized enzyme has the dual functions of target recognition and signal amplification and is applied to detection of small molecular targets.

Description

Technical Field

[0001] The invention belongs to the field of enzyme immobilization, and in particular relates to a metal-G4DNAzyme nucleic acid framework-mediated enzyme immobilization method and application. Background Art

[0002] Protein-nucleic acid conjugates obtained by coupling functional proteins with nucleic acids that have both programmability and recognition capabilities have been widely used in the fields of biological detection, drug delivery, molecular biology research, etc. In order to meet the needs of many applications and achieve efficient coupling, not only are proteins and nucleic acids required to have available functional groups and mild coupling conditions, but the formed conjugates must also have the functions and activities of free proteins. However, the modification of additional functional groups in nucleic acids increases the cost of coupling; some non-specific modifications close to or located at the active site of the protein can easily inactivate the conjugate. Although, the stoichiometric ratio of the coupling and the location of the modification can be precisely controlled by regulating the expression of tag fusion proteins through genetic engineering. However, the need for special equipment and additional chemical reactions still limit its widespread application. Summary of the invention

[0003] The purpose of the present invention is to solve the problems of the existing functional protein and nucleic acid coupling methods, such as the complex process, harsh conditions, and difficulty in retaining the activity and function of the protein. The present invention aims to provide a metal-G4zyme nucleic acid framework-mediated enzyme immobilization method. In the presence of metal ions and nucleic acids containing G4DNAzyme sequences, the present invention realizes the coupling of enzymes and nucleic acids through biomimetic mineralization. The formed functional metal nucleic acid framework has the dual functions of target recognition and signal amplification, and realizes the application of small molecule target detection.

[0004] In order to achieve the above object, the present invention provides a metal-G4 DNAzyme nucleic acid framework-mediated enzyme immobilization method, comprising the following steps:

[0005] Step S1: co-incubate the enzyme and the nucleic acid sequence containing the G quadruplex in a PBS buffer solution, add 100-500 mM divalent metal ions, place in a centrifuge tube and shake, react at room temperature for a certain period of time and then sonicate, centrifuge at 10000 rpm to remove the supernatant, wash the precipitate with deionized water for 3 times, and re-dissolve the precipitate with a PBS buffer solution to obtain a functional metal nucleic acid framework-immobilized enzyme for use;

[0006] Step S2): After the prepared functional metal nucleic acid framework immobilized enzyme is incubated with hemin for 1 hour, the supernatant is removed by centrifugation, and the excess hemin is washed away to obtain the metal-G4 DNAzyme nucleic acid framework-mediated enzyme, which is placed at 4° C. for use.

[0007] The present invention also provides an application of a metal-G4 DNAzyme nucleic acid framework-mediated enzyme for electrochemical detection of enrofloxacin. Specifically, the method comprises the following steps:

[0008] Step 1): preparing a metal-G4 DNAzyme nucleic acid framework-mediated enzyme;

[0009] Step 2): pre-treating the glassy carbon electrode;

[0010] Step 3): anneal the thiol-modified enrofloxacin split aptamer 2 at 95° C. for 10 min, incubate the annealed 2 μM enrofloxacin split aptamer 2 with the pretreated glassy carbon electrode for 3 h, fix it on the electrode through the Au-S bond, and then incubate it with 2 mM MCH for 1 h to block nonspecific sites;

[0011] Step 4): the glassy carbon electrode modified with the enrofloxacin split aptamer 2 prepared in step 3) is incubated with the metal nucleic acid framework-immobilized enzyme and different concentrations of enrofloxacin for 1 hour to form a "sandwich" structure through the target and the two split aptamers;

[0012] Step 5): The modified electrode prepared in step 4) is subjected to DPV detection, and a mixed solution of TMB and H2O2 is used as an electrocatalytic substrate to establish a relationship between the electrochemical signal intensity of oxTMB formed by oxidation and the concentration of enrofloxacin.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The "one-pot method" is used to prepare functional metal nucleic acid framework-immobilized enzymes, which solves the problem that the existing functional protein and nucleic acid coupling methods are complex, have harsh conditions, and are difficult to retain the activity and function of the protein.

[0015] 2. The obtained functional metal nucleic acid framework immobilized enzyme has the dual functions of target-specific recognition (aptamer) and signal amplification (enzyme and G4DNAzyme).

[0016] 3. This strategy can be extended to the immobilization of other enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure is a schematic diagram of the preparation process of the functional metal nucleic acid framework immobilized enzyme of the present invention.

[0018] Figure 2 The influence of the presence or absence of G4 on the properties of the prepared immobilized enzyme.

[0019] Figure 3 DPV spectra of immobilized enzymes with different compositions.

[0020] Figure 4Comparative results of preparing metal nucleic acid framework-immobilized enzymes using glucose. DETAILED DESCRIPTION

[0021] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0022] like Figure 1 As shown, a metal-G4 DNAzyme nucleic acid framework-mediated enzyme immobilization method comprises the following steps:

[0023] Step S1: co-incubate the enzyme and the nucleic acid sequence containing the G quadruplex in a PBS buffer solution, add 100-500 mM divalent metal ions, place in a centrifuge tube and shake, react at room temperature for a certain period of time and then sonicate, centrifuge at 10000 rpm to remove the supernatant, wash the precipitate with deionized water for 3 times, and re-dissolve the precipitate with a PBS buffer solution to obtain a functional metal nucleic acid framework-immobilized enzyme for use;

[0024] Step S2): After the prepared functional metal nucleic acid framework immobilized enzyme is incubated with hemin for 1 hour, the supernatant is removed by centrifugation, and the excess hemin is washed away to obtain the metal-G4 DNAzyme nucleic acid framework-mediated enzyme, which is placed at 4° C. for use.

[0025] Preferably, the enzyme in step S1 of the present invention comprises at least one of horseradish peroxidase, glucose oxidase and laccase.

[0026] Preferably, the pH concentration of PBS in step S1 of the present invention is 6.8.

[0027] Preferably, the divalent metal ion in step S1 of the present invention is Ca 2+ 、Co 2+ 、Ni 2+ 、Zn 2+ , Mn 2+ At least one of .

[0028] The enzyme mediated by the metal-G4 DNAzyme nucleic acid framework of the present invention is used for electrochemical detection of enrofloxacin.

[0029] The enzyme mediated by the metal-G4 DNAzyme nucleic acid framework of the present invention is used for electrochemical detection of enrofloxacin, comprising the following steps:

[0030] Step 1): preparing a metal-G4 DNAzyme nucleic acid framework-mediated enzyme;

[0031] Step 2): pre-treating the glassy carbon electrode;

[0032] Step 3): anneal the thiol-modified enrofloxacin split aptamer 2 at 95° C. for 10 min, incubate the annealed 2 μM enrofloxacin split aptamer 2 with the pretreated glassy carbon electrode for 3 h, fix it on the electrode through the Au-S bond, and then incubate it with 2 mM MCH for 1 h to block nonspecific sites;

[0033] Step 4): the glassy carbon electrode modified with the enrofloxacin split aptamer 2 prepared in step 3) is incubated with the metal nucleic acid framework-immobilized enzyme and different concentrations of enrofloxacin for 1 hour to form a "sandwich" structure through the target and the two split aptamers;

[0034] Step 5): The modified electrode prepared in step 4) is subjected to DPV detection, and a mixed solution of TMB and H2O2 is used as an electrocatalytic substrate to establish a relationship between the electrochemical signal intensity of oxTMB formed by oxidation and the concentration of enrofloxacin.

[0035] Example 1

[0036] A metal-G4 DNAzyme nucleic acid framework-mediated enzyme immobilization method:

[0037] 0.1 mg horseradish peroxidase (HRP) and 100 μM G4-ENR-split1 containing a G-quadruplex sequence were co-incubated with 3.3 mM (pH 6.8) PBS buffer solution, 200 mM calcium chloride (CaCl2) was added, and the mixture was shaken in a centrifuge tube and reacted at room temperature for 12 h, then sonicated for 10 min. The product was centrifuged at 10,000 rpm for 10 min to remove the supernatant, washed 3 times with deionized water, incubated with hemin for 1 h, centrifuged to remove excess hemin, and then stored at 4°C.

[0038] Example 2

[0039] A metal-G4 DNAzyme nucleic acid framework-mediated enzyme immobilization method synthesized metal nucleic acid framework for electrochemical detection of enrofloxacin:

[0040] 1) Pretreatment of glassy carbon electrode.

[0041] The glassy carbon electrode was polished for 3 min in a solution containing 0.3 μm and 0.05 μm alumina, respectively. Next, the electrode was ultrasonically treated for 5 min in ultrapure water, ethanol, and nitric acid solutions, respectively. The electrode was then activated by CV scanning in a potassium ferrocyanide solution. Finally, gold nanoparticles were deposited on the glassy carbon electrode using the It method, and finally dried with nitrogen for later use.

[0042] 2) Electrochemical sensing device based on metal nucleic acid framework-immobilized enzyme.

[0043] The annealed ENR split 2-SH was incubated with the pretreated electrode for 3 h, and then the modified glassy carbon electrode was immersed in 2 mM MCH (6-mercaptohexanol) for 1 h, and then the electrode was immersed in the composite solution formed by the metal nucleic acid framework immobilized enzyme and different concentrations of ENR for 1 h, and finally the modified electrode was subjected to DPV measurement.

[0044] Example 3

[0045] A metal-G4 DNAzyme nucleic acid framework-mediated glucose oxidase immobilization method and its glucose detection application:

[0046] 1) GOx and G4DNAzyme synthesize enzymes immobilized on metal nucleic acid frameworks:

[0047] A certain proportion of GOx was mixed with G4, and 200 mM CaCl2 and PBS (pH 6.8) were added for buffering. The mixture was placed at room temperature (25°C) for 12 h, ultrasonicated for 10 min, centrifuged at 1000 rpm for 10 min, washed three times with ultrapure water, incubated with hemin for 1 h, and then centrifuged to remove excess hemin and stored at 4°C.

[0048] 2) Metal nucleic acid framework immobilized enzyme detection of glucose:

[0049] The metal nucleic acid framework-immobilized enzyme was mixed with TMB, NaHAC buffer (pH 4.0) and glucose solutions of different concentrations, and UV measurements were performed after standing for 25 minutes. Different concentrations of glucose corresponded to different UV absorbance values.

[0050] 3) Selectivity of glucose detection:

[0051] The metal nucleic acid framework-immobilized enzyme was mixed with TMB, NaHAC buffer (pH 4.0) and D-fructose, sucrose, D-mannose, L-cysteine, and glutathione solution and allowed to stand for 25 min before UV measurement.

[0052] like Figure 4 As shown, the comparison results of the metal nucleic acid framework immobilized enzyme prepared using glucose are shown. In the figure (A), the feasibility of the metal nucleic acid framework immobilized enzyme formed by glucose oxidase and G4 is shown. The enzyme activity is improved after glucose oxidase and G4 are co-immobilized. In the figure (B), it is shown that the immobilized enzyme has a certain linear relationship in detecting glucose. In the figure (C), it is shown that the immobilized enzyme has a certain selectivity in detecting glucose.

[0053] Figure 2The effect of the presence or absence of G4 on the properties of the prepared immobilized enzyme is shown. Figure (A) shows the atomic force microscopy characterization of the prepared metal nucleic acid framework immobilized enzyme, with a thickness of 2.5nm. Figure (B) shows the characteristic fluorescence spectrum of riboflavin (ThT) indicating the G4 sequence on the surface of the immobilized enzyme, indicating that G4 is successfully immobilized on the metal nucleic acid framework. Figure (C) shows the catalytic activity of different immobilized enzymes. HRP, immobilized enzyme without G4 and immobilized enzyme containing G4 all show the absorption peak of TMB at 652nm, and the catalytic activity of the immobilized enzyme containing G4 is the highest.

[0054] Figure 3 The DPV spectra of immobilized enzymes with different components. (A) in the figure is the DPV spectrum of the metal nucleic acid framework immobilized enzyme with or without G4 formation to detect enrofloxacin, showing that the signal gain of the metal nucleic acid framework immobilized enzyme containing G4 is relatively large. (B) in the figure is the DPV spectrum of the metal nucleic acid framework immobilized enzyme detecting different concentrations of enrofloxacin, showing that within a certain enrofloxacin concentration range, as the enrofloxacin concentration increases, the DPV signal gradually increases. (C) in the figure is the linear relationship of the immobilized enzyme detecting enrofloxacin, indicating that within a certain enrofloxacin concentration range, the concentration of enrofloxacin is linearly related to the signal of oxTMB. (D) in the figure shows that the immobilized enzyme has a certain selectivity and anti-interference ability in detecting enrofloxacin.

[0055] Table 1 shows the primer sequences of some raw materials used in the present invention.

[0056] Table 1 Primer sequences used

[0057]

[0058] Table 2 shows the enzyme kinetics of different immobilized enzymes. For the two substrates, the Km of the immobilized enzyme containing G4 is smaller than that without G4, indicating that the immobilized enzyme containing G4 has a good affinity for the substrate, and the Vmax of the immobilized enzyme containing G4 is larger than that without G4.

[0059] Table 2 Enzyme kinetics of different immobilized enzymes

[0060]

[0061] The present invention discloses a metal-G4DNAzyme nucleic acid framework-mediated enzyme immobilization method and application. The present invention mainly overcomes the problems of the existing functional protein and nucleic acid coupling methods being complicated in process, harsh in conditions, and difficult to retain the activity and function of the protein. The present invention realizes the coupling of enzyme and nucleic acid through bionic mineralization in the presence of metal ions and nucleic acids containing G4DNAzyme sequences. The functional metal nucleic acid framework-immobilized enzyme formed has the dual functions of target recognition and signal release, and is applied to the detection of small molecule targets. At the same time, the fixation strategy has a certain universality, and different types of enzymes can be replaced to expand to other small molecule detection applications.

[0062] The above embodiments of the present invention are merely examples for explaining the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes and modifications can be made based on the above description. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A metal-G4 DNAzyme nucleic acid framework-mediated enzyme immobilization method, characterized in that The following steps are involved: Step S1: co-incubate the enzyme and the nucleic acid sequence containing the G quadruplex in a PBS buffer solution, add 100-500 mM divalent metal ions, place in a centrifuge tube and shake, react at room temperature for a certain period of time and then sonicate, centrifuge at 10000 rpm to remove the supernatant, wash the precipitate with deionized water for 3 times, and re-dissolve the precipitate with a PBS buffer solution to obtain a functional metal nucleic acid framework-immobilized enzyme for use; Step S2): After the prepared functional metal nucleic acid framework immobilized enzyme is incubated with hemin for 1 hour, the supernatant is removed by centrifugation, and the excess hemin is washed away to obtain the metal-G4DNAzyme nucleic acid framework-mediated enzyme, which is placed at 4°C for use.

2. The method for enzyme immobilization mediated by metal-G4 DNAzyme nucleic acid framework according to claim 1, characterized in that The enzyme in the above step S1 includes at least one of horseradish peroxidase, glucose oxidase and laccase.

3. The method for enzyme immobilization mediated by metal-G4 DNAzyme nucleic acid framework according to claim 1, characterized in that The pH concentration of PBS in the above step S1 is 6.

8.

4. The method for enzyme immobilization mediated by metal-G4 DNAzyme nucleic acid framework according to claim 1, characterized in that The divalent metal ion in step S1 is Ca 2+ 、Co 2+ 、Ni 2+ 、Zn 2+ , Mn 2+ At least one of .

5. An application of an enzyme mediated by the metal-G4 DNAzyme nucleic acid framework according to any one of claims 1 to 4, characterized in that Used for electrochemical detection of enrofloxacin.

6. The use of the metal-G4 DNAzyme nucleic acid framework-mediated enzyme according to claim 6, characterized in that The electrochemical detection of enrofloxacin includes the following steps: Step 1): preparing a metal-G4 DNAzyme nucleic acid framework-mediated enzyme; Step 2): pre-treating the glassy carbon electrode; Step 3): anneal the thiol-modified enrofloxacin split aptamer 2 at 95° C. for 10 min, incubate the annealed 2 μM enrofloxacin split aptamer 2 with the pretreated glassy carbon electrode for 3 h, fix it on the electrode through the Au-S bond, and then incubate it with 2 mM MCH for 1 h to block nonspecific sites; Step 4): The glassy carbon electrode modified with the enrofloxacin split aptamer 2 prepared in step 3) was incubated with the metal nucleic acid framework-immobilized enzyme and different concentrations of enrofloxacin for 1 hour to form a "sandwich" structure through the target and the two split aptamers; Step 5): The modified electrode prepared in step 4) is subjected to DPV detection, and a mixed solution of TMB and H2O2 is used as an electrocatalytic substrate to establish a relationship between the electrochemical signal intensity of oxTMB formed by oxidation and the concentration of enrofloxacin.

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