Preparation method and application of metal-containing nanomaterial, protein and bioenzyme composite nanomaterial

Abstract

The application relates to the technical field of nanomaterials, in particular to a preparation method and application of a metal-containing nanomaterial, a protein and a biological enzyme composite nanomaterial, which comprises the following steps: taking a tyrosine (Tyr) enantiomer as a research object, taking D-cysteine (D-Cys) as a chiral source, and synthesizing gold nanoparticle-D-cysteine (AuNP-D-Cys) with chiral recognition capability; AuNP-D-Cys is compounded with bovine serum albumin (BSA) with opposite chiral recognition capability through a gold-sulfur bond to form AuNP-D-Cys / BSA, so that the chiral recognition capability of the composite material AuNP-D-Cys / BSA is reversed; and the specific hydrolysis of trypsin (Trypsin) to BSA causes the recovery of the chiral recognition capability of the composite material AuNP-D-Cys / BSA / Trypsin. Through precise regulation of the concentration of Trypsin, the chiral recognition capability of the metal nanomaterial can be controlled and regulated, the purpose of regulating the chiral recognition capability of the metal nanomaterial according to needs is achieved, and the application has important application value in the fields of chiral recognition and electrochemistry.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of nanomaterials, in particular to a preparation method and application of a metal-containing nanomaterial, a protein and a biological enzyme composite nanomaterial. BACKGROUND

[0002] Chirality refers to the phenomenon that a real object cannot overlap with its mirror image, and it is a phenomenon widely existing in nature. A pair of enantiomers has similar physical and chemical properties, but has completely different pharmacological activities in a chiral environment. The most famous example is the "clomiphene" event. Therefore, chiral recognition is of great significance to human health. It is well known that the amino acids in structural proteins are L-amino acids, and naturally occurring sugars are D-forms. Because the same chiral molecules have different affinities when combined with different chiral materials, it is particularly important to regulate the chirality of materials as needed according to the chirality of target substances.

[0003] At present, the regulation methods of the chiral recognition ability of materials include temperature-induced regulation, pH-induced regulation and light-induced regulation. In order to more conveniently apply materials with chiral recognition ability to in-vivo detection and treatment and other fields, a more mild and effective chiral regulation method is expected to be proposed. SUMMARY

[0004] The present disclosure provides a preparation method and application of a metal-containing nanomaterial, a protein and a biological enzyme composite nanomaterial. The biological enzyme has the advantages of good biocompatibility, mild action conditions, strong reaction specificity and high catalytic efficiency, and the present application is a novel, mild and efficient regulation method.

[0005] In a first aspect, the present disclosure provides a preparation method of a metal-containing nanomaterial, a protein and a biological enzyme composite nanomaterial, comprising the following steps:

[0006] (1) Preparation of a gold nanoparticle-D-cysteine (AuNP-D-Cys) solution: uniformly mix a chloroauric acid solution and a trisodium citrate solution to obtain a mixed solution, dropwise add a sodium borohydride solution to the mixed solution, then add a D-cysteine solution, stir at room temperature in the dark for 0.5-5 h until the solution changes from red to deep purple, and obtain the AuNP-D-Cys solution;

[0007] (2) Preparation of a gold nanoparticle-D-cysteine / bovine serum albumin (AuNP-D-Cys / BSA) solution: use a pipette to take the AuNP-D-Cys solution obtained in step (1), uniformly mix the AuNP-D-Cys solution with a phosphate buffer solution of bovine serum albumin, stir at room temperature in the dark for 5-20 h, and obtain the AuNP-D-Cys / BSA solution;

[0008] (3) Preparation of AuNP-D-Cys / BSA / Trypsin solution: The AuNP-D-Cys / BSA solution obtained in step (2) was mixed with a trypsin solution, incubated at 30-50℃ for 1-10 h, centrifuged, and washed with ultrapure water to obtain the AuNP-D-Cys / BSA / Trypsin solution.

[0009] The AuNP-D-Cys used in the present application has high recognition ability for D-tyrosine, and the bovine serum albumin has high affinity for L-tyrosine. Therefore, the chiral recognition ability of the composite material AuNP-D-Cys / BSA is reversed, and the chiral recognition of the amino acid enantiomers is dominated by bovine serum albumin. Since trypsin can specifically hydrolyze bovine serum albumin, the chiral recognition ability of AuNP-D-Cys / BSA / Trypsin can be restored by incubation with different concentrations of trypsin, and the high recognition of D-tyrosine is re-exhibited.

[0010] Biological enzymes have the advantages of good biocompatibility, mild reaction conditions, strong reaction specificity, and high catalytic efficiency. The method for precisely regulating the chiral recognition ability of metal nanomaterials based on biological enzymes proposed in the present application is a novel, mild, and efficient regulation method. The AuNP-D-Cys, AuNP-D-Cys / BSA, and AuNP-D-Cys / BSA / Trypsin involved in the present application all have excellent chiral recognition ability, which lays a foundation for the construction of subsequent regulation processes.

[0011] Preferably, in step (1), the molar ratio of chloroauric acid, trisodium citrate, sodium borohydride, and D-cysteine is (5-20):(5-20):(1-2):(3-8).

[0012] Preferably, in step (1), the concentration of the aqueous chloroauric acid solution is 10-50 mmol / L, the concentration of the aqueous trisodium citrate solution is 10-50 mmol / L, the concentration of the aqueous sodium borohydride solution is 50-200 mmol / L, and the concentration of the aqueous D-cysteine solution is 10-50 mmol / L.

[0013] Preferably, in step (2), the concentration of the phosphate buffer solution is 0.01-1 mol / L, and the pH is 6.0-8.0.

[0014] Preferably, in the step (2), the volume ratio of the AuNP-D-Cys solution to the phosphate buffer solution of bovine serum albumin is (1-2):(5-10), and the concentration of the phosphate buffer solution of bovine serum albumin is 1-100 mg / mL.

[0015] Preferably, in the step (3), the volume ratio of the AuNP-D-Cys / BSA / Trypsin solution to the trypsin aqueous solution is (1-2):(1-10), and the concentration of the trypsin aqueous solution is 0.001-100 mg / mL.

[0016] In a second aspect, the present disclosure provides an application of a composite nanomaterial containing a metal nanomaterial, a protein and a biological enzyme, which is applied in electrochemical chiral recognition of amino acid enantiomers, and the differential pulse voltammetry is used to recognize the amino acid enantiomers.

[0017] The present application can realize accurate regulation of the chiral recognition ability of the metal nanomaterial by accurately regulating the concentration of the biological enzyme, and then realize chiral regulation of the metal nanomaterial based on the biological enzyme, so as to achieve the purpose of regulating the chiral recognition ability of the metal nanomaterial on demand. The application of the biological enzyme in the field of electrochemical chiral regulation is expanded, which can provide a reference idea for subsequent on-demand regulation of chiral materials and open up a new way for constructing an intelligent and convenient chiral sensing platform with variable enantiomer selectivity.

[0018] Preferably, the following steps are included: taking the AuNP-D-Cys, the AuNP-D-Cys / BSA and the AuNP-D-Cys / BSA / Trypsin modified glassy carbon electrode as a working electrode, taking a platinum sheet electrode as a counter electrode, taking silver / silver chloride as a reference electrode, respectively placing the working electrode, the counter electrode and the reference electrode in a phosphate buffer solution of L-tyrosine and D-tyrosine for incubation, and placing them under an electrochemical window for testing and recognition by differential pulse voltammetry.

[0019] Preferably, the concentration of the L-tyrosine and the D-tyrosine solution is 0.1-1 mmol / L, and the volume is 20-30 mL.

[0020] Preferably, the concentration of the phosphate buffer solution is 0.1-1 mol / L, and the pH is 6.0-8.0; and the voltage of the electrochemical window is 0.4-1.4 V.

[0021] In summary, the present application has the following beneficial effects:

[0022] 1. In this application, AuNP-D-Cys, which has a high recognition ability for D-tyrosine, was selected as the base material. Then, it was compounded with bovine serum albumin, which has a high affinity for L-tyrosine. This caused the chiral recognition ability of the composite material AuNP-D-Cys / BSA to be reversed, exhibiting enantiomeric chiral recognition dominated by bovine serum albumin. Since trypsin can specifically hydrolyze bovine serum albumin, the chiral recognition ability of AuNP-D-Cys / BSA / Trypsin can be restored after incubation with different concentrations of trypsin, and it once again exhibits high-efficiency recognition of D-tyrosine.

[0023] 2. The bioenzymes in this application have advantages such as good biocompatibility, mild reaction conditions, strong reaction specificity, and high catalytic efficiency. The method proposed in this application for precisely regulating the chiral recognition ability of metal nanomaterials based on bioenzymes is a novel, mild, and efficient regulation method. The AuNP-D-Cys, AuNP-D-Cys / BSA, and AuNP-D-Cys / BSA / Trypsin involved all have excellent chiral recognition ability, which lays the foundation for the construction of subsequent regulation processes.

[0024] 3. This application can achieve precise control of the chiral recognition ability of metal nanomaterials by precisely controlling the concentration of biological enzymes, thereby realizing the chiral control of metal nanomaterials based on biological enzymes. This achieves the goal of controlling the chiral recognition ability of metal nanomaterials on demand, expands the application of biological enzymes in the field of electrochemical chiral control, provides a reference for the on-demand control of chiral materials, and opens up new avenues for building intelligent and convenient chiral sensing platforms with variable enantioselectivity.

[0025] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the scope of protection of this disclosure. Attached Figure Description

[0026] Figure 1 This is the extinction spectrum of AuNP-D-Cys in this application;

[0027] Figure 2 The AuNP-D-Cys / bare glassy carbon electrode (GCE), AuNP-D-Cys / BSA / GCE, AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE, AuNP-D-Cys / BSA / Trypsin (1 mg / mL) / GCE, and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) / GCE of this application are in the presence of 5 mM [Fe(CN)6] 4- / 3- Cyclic voltammograms in 0.1 MkCl solution;

[0028] Figure 3 is the Nyquist plot of AuNP-D-Cys / GCE, AuNP-D-Cys / BSA / GCE, AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE, AuNP-D-Cys / BSA / Trypsin (1 mg / mL) / GCE, AuNP-D-Cys / BSA / Trypsin (10 mg / mL) / GCE of the present application in 0.1 M KCl solution containing 5 mM [Fe(CN)6] 4- / 3-

[0029] Figure 4 is the differential pulse voltammogram of unmodified GCE of the present application recognizing tyrosine enantiomers;

[0030] Figure 5 is the differential pulse voltammogram of AuNP-D-Cys / GCE of the present application recognizing tyrosine enantiomers;

[0031] Figure 6 is the differential pulse voltammogram of BSA / GCE of the present application recognizing tyrosine enantiomers;

[0032] Figure 7 is the differential pulse voltammogram of AuNP-D-Cys / BSA / GCE of the present application recognizing tyrosine enantiomers;

[0033] Figure 8 is the differential pulse voltammogram of AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE of the present application recognizing tyrosine enantiomers;

[0034] Figure 9 is the differential pulse voltammogram of AuNP-D-Cys / BSA / Trypsin (1 mg / mL) / GCE of the present application recognizing tyrosine enantiomers;

[0035] Figure 10 is the differential pulse voltammogram of AuNP-D-Cys / BSA / Trypsin (10 mg / mL) / GCE of the present application recognizing tyrosine enantiomers;

[0036] Figure 11 is the result stability plot of AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE, AuNP-D-Cys / BSA / Trypsin (1 mg / mL) / GCE AuNP-D-Cys / BSA / Trypsin (10 mg / mL) / GCE of the present application recognizing tyrosine enantiomers on different electrodes. DETAILED DESCRIPTION ​

[0037] The application will be further described in detail in connection with the following examples, it is particularly pointed out that: the following examples are not specified conditions, according to the conventional conditions or manufacturer's recommended conditions, the following examples of raw materials used in the special note, except that can be sourced from ordinary market.

[0038] The AuNP-D-Cys, AuNP-D-Cys modified electrode, BSA modified electrode, AuNP-D-Cys / BSA modified electrode, AuNP-D-Cys / BSA / Trypsin(0.1mg / mL) modified electrode, AuNP-D-Cys / BSA / Trypsin(1mg / mL) modified electrode, AuNP-D-Cys / BSA / Trypsin(10mg / mL) modified electrode were used to identify the tyrosine enantiomers according to the following method: identification efficiency = I L / I D ;I L represents the oxidation peak current of the modified electrode in the L-tyrosine solution; I D represents the oxidation peak current of the modified electrode in the D-tyrosine solution.

[0039] Preparation example of raw materials and / or intermediates

[0040] Preparation example 1

[0041] A preparation method of AuNP-D-Cys, comprising the following steps:

[0042] 1mL of 20mM chloroauric acid solution and 1mL of 20mM trisodium citrate solution were uniformly mixed, then 20μL of 100mM sodium borohydride solution was added dropwise to the mixed solution. Subsequently, 334μL of 30mM D-cysteine solution was immediately added. Stirring at room temperature for 2h in the dark until the solution changed from red to deep purple, to obtain AuNP-D-Cys solution.

[0043] As shown in Figure 1 , the UV-visible extinction spectrum of AuNP-D-Cys was characterized, and the extinction waveband of AuNP-D-Cys below 300nm was attributed to D-Cys, and the extinction waveband of AuNP-D-Cys around 560nm was attributed to the surface plasmon resonance peak of AuNP. Through the UV-visible extinction spectrum, it was confirmed that the chiral source cysteine was successfully introduced into the synthesis of AuNP.

[0044] Example

[0045] Example 1

[0046] A preparation method of a metal-containing nanomaterial, protein and biological enzyme composite nanomaterial and its electrochemical characterization, comprising the following steps:

[0047] (1) Preparation of AuNP-D-Cys modified electrode: 1 mL of 20 mM chloroauric acid solution and 1 mL of 20 mM trisodium citrate solution were mixed uniformly, and then 20 μL of 100 mM sodium borohydride solution was added dropwise to the mixed solution. Subsequently, 334 μL of 30 mM D-cysteine solution was immediately added. The solution was stirred at room temperature for 2 h in the dark until the solution changed from red to deep purple, obtaining AuNP-D-Cys solution. Then, 150 μL of the prepared AuNP-D-Cys solution was removed with a pipette and diluted to 1500 μL with ultrapure water. 5 μL of the diluted AuNP-D-Cys solution was dropped on the electrode surface and naturally air-dried to obtain the AuNP-D-Cys modified electrode.

[0048] (2) Preparation of AuNP-D-Cys / BSA modified electrode: 150 μL of AuNP-D-Cys solution obtained in step (1) was removed with a pipette and mixed uniformly with 750 μL of 10 mg / mL bovine serum albumin in 0.1 mol / L phosphate buffer solution with pH = 7.0, 600 μL of ultrapure water, and stirred at room temperature for 10 h in the dark, obtaining AuNP-D-Cys / BSA solution. 5 μL of AuNP-D-Cys / BSA solution was dropped on the electrode surface and naturally air-dried to obtain the AuNP-D-Cys / BSA modified electrode.

[0049] (3) Preparation of AuNP-D-Cys / BSA / Trypsin modified electrode: 100 μL of AuNP-D-Cys / BSA solution obtained in step (2) was mixed with 100 μL of 0.1, 1, 10 mg / mL trypsin solution, and incubated at 37°C for 3 h. The mixed solution was centrifuged at 10000 rpm for 10 min, and resuspended with 100 μL of ultrapure water, finally obtaining AuNP-D-Cys / BSA / Trypsin solution with different concentrations. 5 μL of AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL), AuNP-D-Cys / BSA / Trypsin (1 mg / mL), and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) solution was dropped on the electrode surface respectively, and naturally air-dried to obtain AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL), AuNP-D-Cys / BSA / Trypsin (1 mg / mL), and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) modified electrode.

[0050] (4) Using the AuNP-D-Cys modified electrode, AuNP-D-Cys / BSA modified electrode, AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) modified electrode, AuNP-D-Cys / BSA / Trypsin (1 mg / mL) modified electrode, and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) modified electrode prepared in Preparation Example 1 as the working electrode, the platinum sheet electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode, the electrodes were placed in a solution containing 5 mM [Fe(CN)6]. 4- / 3- Cyclic voltammetry was performed in 0.1 mM KCl solution within an electrochemical window of –0.2–0.6 V and a scan rate of 100 mV / s. Under the same conditions, the AuNP-D-Cys modified electrode, AuNP-D-Cys / BSA modified electrode, AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) modified electrode, AuNP-D-Cys / BSA / Trypsin (1 mg / mL) modified electrode, and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) modified electrode prepared in Preparation Example 1 were used as working electrodes, a platinum sheet electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode. The electrodes were then placed in a solution containing 5 mM [Fe(CN)6]. 4- / 3- In a 0.1 MkCl solution, the open-circuit potential is ~0.24 V, and the frequency range is 10. 5 Electrochemical impedance spectroscopy was performed at -0.01 Hz.

[0051] like Figure 2 As shown, when the electrode surface is modified with AuNP-D-Cys, a pair of distinct [Fe(CN)6] atoms are observed. 4- / 3- The redox peaks are attributed to the solution [Fe(CN)6]. 4- / 3- The redox process. Furthermore, due to the good conductivity of AuNP-D-Cys, AuNP-D-Cys / GCE [Fe(CN)6] 4- / 3- The redox peak current values ​​are relatively large. When AuNP-D-Cys is complexed with BSA, the AuNP-D-Cys / BSA / GCE [Fe(CN)6]... 4- / 3- The redox peak current decreased significantly, which is due to the poor conductivity of the BSA coated on the AuNP surface. When GCE was modified with AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL), Au-D-Cys / BSA / Trypsin (1 mg / mL), and Au-D-Cys / BSA / Trypsin (10 mg / mL), respectively, [Fe(CN)6] 4- / 3-The peak current of the redox peak current of AuNP-D-Cys / BSA is different from that of Au-D-Cys / BSA, and the degree of enhancement is directly related to the concentration of trypsin. With the increase of the concentration of trypsin, due to the specific hydrolysis of trypsin to BSA, BSA on the surface of AuNP-D-Cys is gradually hydrolyzed into amino acids, and the [Fe(CN)6] 4- / 3- The redox peak current gradually increases with the increase of the concentration of trypsin.

[0052] Figure 3 The AC impedance diagrams of different modified electrodes show the following change trend according to the size of charge transfer impedance: Au-D-Cys / BSA / GCE (364.4Ω)>Au-D-Cys / BSA / Trypsin (0.1mg / mL) / GCE (298.7Ω)>Au-D-Cys / BSA / Trypsin (1mg / mL) / GCE (212.4Ω)>Au-D-Cys / BSA / Trypsin (10mg / mL) / GCE (207.3Ω)>AuNP-D-Cys / GCE (82.43Ω).

[0053] The AC impedance test results of different electrodes are consistent with the cyclic voltammetry results of each electrode.

[0054] Example 2

[0055] The metal nanomaterial, protein and biological enzyme composite nanomaterial prepared according to the method of Example 1 is different in that the Au-D-Cys modified electrode, AuNP-D-Cys / BSA modified electrode prepared in Preparation Example 1, and the GCE electrode, 5μL of 5mg / mL bovine serum albumin in 0.1mol / L pH=7.0 phosphate buffer solution modified electrode are used as the working electrode, the platinum sheet electrode is used as the counter electrode, and the silver / silver chloride electrode is used as the reference electrode. They are respectively placed in 25mL of 0.1M pH=7.0 phosphate buffer solution containing 1mM L-tyrosine, D-tyrosine, and tested by differential pulse voltammetry in the electrochemical window of 0.4-1.3V.

[0056] As shown in Figure 4 , the bare GCE can be observed to have an oxidation peak current of tyrosine at about 0.65V after being placed in the L-tyrosine, D-tyrosine solution, which proves that the oxidation of tyrosine occurs on the surface of the bare GCE electrode. However, since the GCE surface does not have chiral sites, it is not possible to effectively distinguish the enantiomers of tyrosine, Figure 4The oxidation peak currents of L-tyrosine and D-tyrosine are almost equal, and their differential pulse voltammetric curves almost completely overlap, indicating that the bare GCE cannot recognize the tyrosine enantiomer. This proves that the subsequent signal difference originates solely from the material, rather than from interference from the bare GCE.

[0057] like Figure 5 As shown, when AuNP-D-Cys is modified on the GCE surface, because D-Cys has a better adsorption effect with the isomorphic D-tyrosine, more D-tyrosine is oxidized on the electrode surface. It can be observed that the oxidation peak current signal of D-tyrosine is stronger than that of L-tyrosine (IL>ID), and the peak current ratio (IL / ID) is 0.74, indicating that Au-D-Cys can efficiently recognize D-tyrosine. When differential pulse voltammetry is performed using BSA / GCE, because BSA has natural chiral sites, it can recognize tyrosine enantiomers, such as... Figure 6 It can be observed that the oxidation peak current signal of L-tyrosine is significantly stronger than that of D-tyrosine (ID>IL), and the peak current ratio (IL / ID) reaches 1.24. Furthermore, due to the poor conductivity of BSA, the current signal of BSA / GCE is relatively weak. It is worth noting that when AuNP-D-Cys is complexed with BSA (e.g., ...), ... Figure 7 The peak current ratio (IL / ID) changed to 1.20 (IL>ID). This demonstrates that the chiral recognition effect was reversed after AuNP-D-Cys was combined with BSA, laying the foundation for subsequent regulation of the chiral recognition ability of metal nanomaterials based on biological enzymes. Furthermore, a comparison of the oxidation peak current maps shows that the conductivity of AuNP-D-Cys is significantly higher than that of BSA. Therefore, the conductivity of AuNP-D-Cys / BSA, formed by combining AuNP-D-Cys with BSA, is weaker than that of AuNP-D-Cys but slightly higher than that of BSA, thus confirming the successful combination of AuNP-D-Cys and BSA.

[0058] Example 3

[0059] The composite nanomaterials containing metal nanomaterials, proteins, and bioenzymes were prepared according to the method of Example 1. The difference was that the AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE, AuNP-D-Cys / BSA / Trypsin (1 mg / mL) / GCE, and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) / GCE prepared in Example 1 were used as working electrodes, a platinum sheet electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode. They were placed in 25 mL of 0.1 M phosphate buffer solution (pH 7.0) containing 1 mL of M-tyrosine and D-tyrosine, respectively. Differential pulse voltammetry was used for testing within an electrochemical window of 0.4–1.3 V, and the changes in the oxidation peak current of tyrosine were recorded using differential pulse voltammetry.

[0060] like Figure 8 When GCE was modified with Au-D-Cys / BSA / Trypsin (0.1 mg / mL), the hydrolysis of BSA led to the exposure of highly conductive Au-D-Cys, which then interacted with the attached... Figure 7 In comparison, the oxidation peak current intensity was slightly improved; however, since BSA was only partially hydrolyzed, its oxidation peak current signal was still much lower than that of AuNP-D-Cys. The amount of trypsin added at a concentration of 0.1 mg / mL was insufficient to reverse the chiral recognition effect of Au-D-Cys / BSA / Trypsin (0.1 mg / mL). Therefore, the chirality of Au-D-Cys / BSA / Trypsin (0.1 mg / mL) was dominated by BSA, which still had better selectivity for L-tyrosine (IL>ID), and the recognition efficiency (IL / ID) decreased to 1.13.

[0061] When GCE is modified with Au-D-Cys / BSA / Trypsin (1 mg / mL), as Figure 9As shown, the oxidation peak current value was slightly higher than that of Au-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE. This is because further hydrolysis of BSA gradually exposed the surface of Au-D-Cys, leading to a gradual recovery of its conductivity. Furthermore, when Au-D-Cys / BSA / Trypsin (1 mg / mL) modified GCE, the oxidation peak current curves of the tyrosine enantiomers almost completely overlapped, making it impossible to effectively distinguish between them. This phenomenon may be because when the trypsin concentration is 1 mg / mL, the amount of trypsin is just enough to completely hydrolyze the excess BSA, causing the chiral recognition effect of the remaining BSA on the tyrosine enantiomers to cancel out the chiral recognition effect of Au-D-Cys itself. This results in the adsorption of equal amounts of L-tyrosine and D-tyrosine on the surface of Au-D-Cys / BSA / Trypsin (1 mg / mL) / GCE. Therefore, for Au-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE, the oxidation peak currents of the tyrosine enantiomers are almost equal (IL≈ID), making it impossible to perform chiral recognition of the tyrosine enantiomers. The peak current ratio (IL / ID) is 1.03, and the recognition efficiency is close to 1.

[0062] like Figure 10 As shown, even with a BSA concentration as high as 10 mg / mL, the BSA coating on the Au-D-Cys surface was not completely hydrolyzed, and its oxidation peak current signal was still lower than that of AuNP-D-Cys itself. When the electrode was modified with Au-D-Cys / BSA / Trypsin (10 mg / mL), due to the hydrolysis of a large amount of BSA on the Au-D-Cys surface, the recognition effect became dominated by AuNP-D-Cys. Therefore, compared with L-tyrosine, Au-D-Cys / BSA / Trypsin (10 mg / mL) / GCE showed a higher oxidation peak current intensity in D-tyrosine solution, exhibiting better selectivity for D-tyrosine, with a recognition efficiency (IL / ID) of 0.893.

[0063] Application examples

[0064] Application Example 1

[0065] In order to investigate the stability of the metal-containing nanomaterial, protein and biological enzyme composite nanomaterial, AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL), AuNP-D-Cys / BSA / Trypsin (1 mg / mL) and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) prepared in Example 1 were respectively drop-coated on the surface of three electrodes, and the change of the oxidation peak current of tyrosine enantiomers was recorded by differential pulse voltammetry, and the average value was taken.

[0066] As shown in Figure 11 , the recognition effect (I L / I D average value) of AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL), AuNP-D-Cys / BSA / Trypsin (1 mg / mL) and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) on the three electrodes was 1.13, 1.03 and 0.89, respectively, and the relative standard deviation was 4.16%, 2.44% and 5.23%, respectively. Compared with AuNP-D-Cys / BSA / Trypsin (0.1 mg / mL) / GCE, AuNP-D-Cys / BSA / Trypsin (1 mg / mL) / GCE and AuNP-D-Cys / BSA / Trypsin (10 mg / mL) / GCE, the stability of the recognition effect was reversed and recovered, which proved that the chiral recognition ability of the metal nanomaterial could be controlled by precise regulation of the concentration of trypsin, indicating the reliability of the method for precise regulation of the chiral recognition ability of the metal nanomaterial based on biological enzymes.

[0067] The above is only an exemplary specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present disclosure, which should be covered within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.

Claims

1. A method for preparing a metal-containing nanomaterial, protein, and bioenzyme complex nanomaterial, characterized in that, The method comprises the following steps: (1) Preparation of gold nanoparticle-d-cysteine (AuNP-d-Cys) solution: uniformly mix a chloroauric acid solution and a trisodium citrate solution to obtain a mixed solution, dropwise add a sodium borohydride solution to the mixed solution, then add a d-cysteine solution, stir at room temperature and in the dark for 0.5-5 h until the solution changes from red to deep purple, and obtain the AuNP-d-Cys solution; (2) Preparation of gold nanoparticle-d-cysteine / bovine serum albumin (AuNP-d-Cys / BSA) solution: uniformly mix the AuNP-d-Cys solution obtained in step (1) with a phosphate buffer solution of bovine serum albumin, stir at room temperature and in the dark for 5-20 h, and obtain the AuNP-d-Cys / BSA solution; (3) Preparation of gold nanoparticle-d-cysteine / bovine serum albumin / trypsin (AuNP-d-Cys / BSA / Trypsin) solution: uniformly mix the AuNP-d-Cys / BSA solution obtained in step (2) with a trypsin solution, incubate at 30-50 °C for 1-10 h, centrifuge the mixed solution, and wash with ultrapure water, and finally obtain the AuNP-d-Cys / BSA / Trypsin solution. 2.The method of claim 1, wherein the metal-containing nanomaterial, protein, and bioenzyme composite nanomaterial is prepared by the method, and In step (1), the molar ratio of the chloroauric acid, the trisodium citrate, the sodium borohydride and the d-cysteine is (5-20):(5-20):(1-2):(3-8). ​ 3. The method for preparing a composite nanomaterial containing metal nanomaterials, proteins, and bioenzymes according to claim 1, characterized in that, In step (1), the concentration of the chloroauric acid solution is 10-50 mmol / L, the concentration of the trisodium citrate solution is 10-50 mmol / L, the concentration of the sodium borohydride solution is 50-200 mmol / L, and the concentration of the d-cysteine solution is 10-50 mmol / L. 4.The method of claim 1, wherein the metal-containing nanomaterial, protein, and bioenzyme composite nanomaterial is prepared by the method, and In step (2), the concentration of the phosphate buffer solution is 0.01-1 mol / L, and the pH is 6.0-8.

0. ​ 5.The method of claim 1, wherein the metal-containing nanomaterial, protein, and bioenzyme composite nanomaterial is prepared by the method, and In step (2), the volume ratio of the AuNP-d-Cys solution to the phosphate buffer solution of bovine serum albumin is (1-2):(5-10), and the concentration of the phosphate buffer solution of bovine serum albumin is 1-100 mg / mL. ​ 6.The method of claim 1, wherein the metal-containing nanomaterial, protein, and bioenzyme composite nanomaterial is prepared by the method, and In step (3), the volume ratio of the AuNP-d-Cys / BSA / Trypsin solution to the trypsin solution is (1-2):(1-10), and the concentration of the trypsin solution is 0.001-100 mg / mL. ​ 7. The use of the metal-containing nanomaterial, protein and bioenzyme composite nanomaterial prepared according to any one of claims 1-6, characterized in that, The composite nanomaterial is applied in electrochemical chiral recognition of amino acid enantiomers, and differential pulse voltammetry is used to recognize the amino acid enantiomers. 8.The application of claim 7, wherein the metal-containing nanomaterial, protein and bioenzyme composite nanomaterial is used for. The application comprises the following steps: taking the AuNP-d-Cys, the AuNP-d-Cys / BSA, the AuNP-d-Cys / BSA / Trypsin modified glassy carbon electrode as a working electrode, taking a platinum sheet electrode as a counter electrode, taking silver / silver chloride as a reference electrode, respectively placing the working electrode, the counter electrode and the reference electrode in l-tyrosine and d-tyrosine phosphate buffer solutions for incubation, and placing under an electrochemical window for test recognition by using a differential pulse voltammetry. 9.The application of claim 8, wherein the metal-containing nanomaterial, protein and bioenzyme composite nanomaterial is used for, The concentration of the l-tyrosine and the d-tyrosine solution is 0.1-1 mmol / L, and the volume is 20-30 mL.

10. The use of the metal-containing nanomaterial, protein and bioenzyme complex nanomaterial according to claim 8, characterized in that, The concentration of the phosphate buffer solution is 0.1-1 mol / L, and the pH is 6.0-8.0; the voltage of the electrochemical window is 0.4-1.4 V.

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