Preparation and application of novel Fe monatomic nano-enzyme

Abstract

According to the preparation and application of the novel Fe monatomic nano enzyme, mesoporous silica is used as a substrate material, the surface of the substrate material is coated with a layer of dopamine and hemin, then high-temperature calcination is performed to form the monatomic nano enzyme Fe-N-C, then the monatomic nano enzyme Fe-N-C is used as a signal reporter molecule of a lateral flow chromatography test strip, and the application of the monatomic nano enzyme Fe-N-C in the lateral flow chromatography test strip is realized. A labeled antibody is connected to the surface of the antibody, so that rapid detection of cardiac troponin I (cTnI) is realized. In the monatomic nano-enzyme, dopamine mainly provides a carbon source, heme mainly provides Fe and a nitrogen source, the larger specific surface area of mesopores provides abundant sites for the monatomic nano-enzyme, and the contact area of the monatomic nano-enzyme and a substrate is increased, so that the enzyme activity is improved. In conclusion, the material has a large specific surface area and relatively high enzyme activity. And finally, quickly detecting the biomarker cTnI after the test strip platform is integrated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the preparation and application of a novel Fe single-atom nanozyme. Background Art

[0002] Lateral flow immunoassay (LFIA) is an effective POCT detection method and has been widely reported for monitoring and diagnosing various specific targets

[10] . However, the disadvantage of low sensitivity limits its further practical application. In the past few decades, the continuous development of nanotechnology has brought vitality to the development of various nanomaterials with enzyme-like activity. Since 2015, these nanomaterials have been introduced as labels for signal amplification, enabling LFIA to achieve high-sensitivity detection, which greatly facilitates trace detection. Researchers have reported that LFIA connected to enzyme-like nanomaterials can enhance the signal amplification of biosensors. The intrinsic peroxidase activity of nanomaterials can catalyze substrates and produce obvious color changes on the relevant line, thereby greatly improving the detection performance. Although the stability of enzyme-like nanomaterials is much better than that of natural nanomaterials, it is still a huge challenge to significantly improve the catalytic activity of such nanomaterials. Adjusting the size of nanomaterials, especially shrinking the catalytic active sites on the materials to a few or even single atoms, is considered a potential way to address this challenge. Single-atom site catalysts (SASCs) have attracted widespread attention in recent years due to their isolated active metal sites and unique electronic / geometric structures. Unlike traditional nanomaterials whose catalytic activity mainly comes from surface atoms, the improvement of SASCs catalytic ability comes almost 100% from the utilization of active metal atoms. Most importantly, the coordination structure of Fe-Nx sites on SASCs vividly mimics the active sites of natural enzymes. Based on these, SASCs not only show countless advantages in electrocatalytic applications, but also exhibit high-performance enzyme-like properties and great potential in biosensing applications.

[0003] Nanozymes have the characteristics of low cost, high stability, customizable surface properties, and easy synthesis and storage. They have enzyme-like activities and are widely used in the field of disease diagnosis and treatment. However, compared with natural enzymes, nanozymes usually have lower activity and unsatisfactory specificity, which seriously limits their practical applications. Fortunately, natural metalloenzymes (cytochrome c oxidase (CcO), superoxide dismutase, etc.) have a well-defined local coordination environment and electronic structure, which provides us with an ingenious blueprint for the rational design of nanozymes. Among various nanozymes, single-atom metal nanozymes have a structure similar to the active metal center of natural metalloenzymes, with the chemical, geometric and electronic configuration of atomically dispersed metals bonded to nitrogen-doped carbon supports (MNCs), and are considered to be alternatives to natural metalloenzymes.

[0004] Single-atom nanozymes (SAzymes) are a new type of nanozymes whose active sites are composed of independent metal atoms, similar to the active sites of natural metalloenzymes. SAzymes have a single-core metal site and a clear coordination structure, and are good at mimicking the active center of enzymes, which makes the activity of SAzymes close to or even exceeds that of natural enzymes in industrial reactions.

[0005] At present, single-atom nanozymes are mainly used in antibacterial, antiviral and tumor treatment. The pioneering work of a team elucidated the high capacity of FeN5 sites restricted by carbon nanoframes (FeN5 SA / CNF) to produce toxic OH·, which was used for high-efficiency antibacterial and mouse wound disinfection performance in vitro. Other researchers have developed a biomembrane microenvironment (BME)-activated FePN SAzyme nanotherapy platform to treat bacterially infected wounds by consuming the high GSH levels of BME. In 2022, a research team synthesized a single-atom nanozyme with anti-tumor effects both in vivo and in vitro. In the same year, another team synthesized a single-atom nanozyme with Co as the metal active center, which can decompose endogenous H 2 O 2 Generate O 2 , then O 2 It is converted into cytotoxic superoxide free radicals, thereby killing tumor cells. There are also studies that apply single-atom nanozymes to biosensors, and some scholars use the peroxidase activity of single-atom nanozymes to achieve H 2 O 2 , glucose and ascorbic acid detection. Summary of the invention

[0006] The specific technical solutions of the present invention are as follows:

[0007] A novel preparation and application of Fe single-atom nanozyme, characterized in that it comprises the following steps:

[0008] S1. Dissolve cetyltrimethylammonium bromide (CTAB) in deionized water, add triethanolamine (TEA), and stir;

[0009] S2. Add a mixture of ethyl silicate (TEOS) and cyclohexane to the above solution and stir;

[0010] S3. Collect by centrifugation and wash with deionized water and anhydrous ethanol three times;

[0011] S4. The washed material is dispersed in acetone and refluxed (to remove CTAB);

[0012] S5. Wash with anhydrous ethanol 3 times;

[0013] S6. vacuum drying to obtain MSNs;

[0014] S7. Add MSNs to the flask, and then add anhydrous ethanol and Tris-Hcl (10 mM, pH = 8.5);

[0015] S8. Dopamine hydrochloride (PDA-Hcl) is dissolved in deionized water and added dropwise to the above solution with stirring;

[0016] S9. Dissolve hemin in dimethyl sulfoxide (DMSO), weigh glucose and dissolve it in water, and stir;

[0017] S10. Wash with ethanol 3 times;

[0018] S11. Vacuum drying to obtain MSNs-PDA-Hemin;

[0019] S12. Place the dried MSNs-PDA-Hemin into a tube furnace and calcine at high temperature to form single-atom nanozyme Fe-NC.

[0020] Furthermore, in the S1, the mass ratio of CTAB to TEA is 11.0-12.0, the mass ratio of CTAB to deionized water is 0.05, the mass ratio of TEA to deionized water is 0.0045, the stirring temperature is 60° C., and the stirring time is 1 h.

[0021] Furthermore, the volume ratio of TEOS to cyclohexane in S2 is 0.25, the stirring temperature is 60° C., and the stirring time is 24 h.

[0022] Furthermore, the temperature of the S6 vacuum drying is 45°C.

[0023] Furthermore, the mass ratio of MSNs, dopamine hydrochloride and hemin described in S7, S8 and S9 is 4:15:5, the mass ratio of hemin chloride to glucose is 0.2, dopamine hydrochloride provides the carbon source, and chlorohemoglobin provides the iron source and nitrogen source.

[0024] Furthermore, the stirring time described in S8 is 5 hours.

[0025] Furthermore, the stirring time described in S9 is 24 hours.

[0026] Furthermore, the pyrolysis rate described in S12 is 5°C min-1, the pyrolysis temperature is 800°C, and the pyrolysis time is 2h.

[0027] Preparation of Fe-NC-Ab:

[0028] ① 0.8 mg of Fe-NC was redissolved in 1 ml PBS (0.1 M pH = 7.4);

[0029] ② Add 63 μg EDC·HCl and 30 μg cTnI antibody and shake for 2 hours;

[0030] ③ Add 50 μl (10%) casein solution and shake for 2 h, then add 50 μl (10%) BSA solution and shake for 2 h;

[0031] ④ The obtained Fe-NC-Ab was centrifuged at 12000 rpm for 10 min, washed with BBS three times, and then dispersed in 1 mg / ml BSA-BBS solution.

[0032] Preparation of Fe-NC based chromatography test strips:

[0033] The test strip consists of four main components: sample pad, absorbent pad, NC membrane, on which T and C lines are generated by dispensing cTnI capture antibody (1 mg mL -1 ) and goat anti-mouse IgG (mg mL -1 ) solution, density is 0.74 mL cm -1 The sample pad and the absorbent pad were sequentially attached to the NC membrane with an overlap of about 2 mm to form a complete test strip. The test strip was then cut into 3.9 mm wide pieces and stored at room temperature for subsequent use.

[0034] Using Fe-NC test paper to detect cTnI:

[0035] 20 μL serum cTnI antigen solution was mixed with 65 μL probe solution containing 5 μL Fe-NC single-atom nanozymes and 60 μL BBS buffer (2% BSA, 2% sodium chloride, 0.1% B66 and 0.1% casein). The resulting Fe-NC-Ab complex was added to the sample pad of the test strip. The reaction was continued at room temperature for 15 minutes. Then a mobile phone camera was used to capture photos of the test strip at the T line and C line.

[0036] The advantages of this application are as follows:

[0037] A novel Fe single-atom nanozyme obtained by the present invention, the nanozyme uses mesoporous silica as a base material, a layer of dopamine and hemin chloride is coated on its surface, and then calcined at high temperature to form a single-atom nanozyme Fe-NC, which has excellent peroxidase activity, and then it is used as a signal reporter molecule of a lateral flow chromatography test strip, and a labeled antibody is connected on its surface to achieve rapid detection of cardiac troponin I (cTnI). In the present invention, dopamine mainly provides a carbon source, hemin mainly provides Fe and nitrogen sources, and the larger specific surface area of ​​the mesopores provides it with abundant attachment sites, which increases the contact area between the single-atom nanozyme and the substrate, thereby improving the enzyme activity. In summary, the material has a large specific surface area and a high enzyme activity. Therefore, the rapid detection of the biomarker cTnI is achieved after the test strip platform is integrated. This method combines single-atom nanozymes with lateral flow chromatography, broadens the application scenarios of single-atom nanozymes, and opens up a new path for the use of lateral flow chromatography.

[0038] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiments of the present application in conjunction with the accompanying drawings as follows.

[0039] Based on the detailed description of the specific embodiments of the present application in combination with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art description are briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In all drawings, similar elements or parts are generally identified by similar reference numerals.

[0041] Figure 1 Schematic diagram of the synthesis scheme of the new Fe single-atom nanozyme

[0042] Figure 2 This is a working principle diagram of a novel Fe single-atom nanozyme combined with lateral flow chromatography technology to achieve rapid detection of cTnI in the present invention

[0043] Figure 3 Image of a new type of Fe single-atom nanozyme

[0044] Figure 4 Infrared spectrum

[0045] Figure 5 Schematic diagram of enzyme activity test of each part of the synthesis process of a novel Fe single-atom nanozyme of the present invention

[0046] Figure 6 The results of enzyme activity test of a novel Fe single-atom nanozyme at different pH values ​​in this invention are shown in FIG.

[0047] Figure 7 Schematic diagram of the results of rapid detection of cTnI by combining a novel Fe single-atom nanozyme with lateral flow chromatography technology of the present invention DETAILED DESCRIPTION

[0048] We first used mesoporous silica with a high specific surface area as the substrate material, and then coated a layer of dopamine and hemin on its surface in the presence of a buffer solution, followed by high-temperature calcination to form Fe-NC single-atom nanozymes, which were then dispersed in deionized water to form a uniformly dispersed Fe single-atom nanozyme solution, and then anti-cTnI-specific antibodies were connected to its surface, and then Fe-NC-based test strips were prepared, and finally Fe-NC single-atom nanozymes were used as probes to achieve rapid detection of cTnI.

[0049] Experimental Materials:

[0050] Hexadecyltrimethylammonium bromide (CTAB), dopamine hydrochloride, and tetraethyl orthosilicate (TEOS) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., triethanolamine (TEA), cyclohexane, anhydrous ethanol, acetone, and dimethyl sulfoxide (DMSO) were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd., hemin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and TMB was purchased from Shanghai Beyotime Biotechnology Co., Ltd. All aqueous solutions were made of deionized water.

[0051] The following experimental instruments are combined with the accompanying drawings and specific embodiments to further describe the technical solution of the present invention. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope that the present invention is intended to protect.

[0052] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0053] Experimental instruments:

[0054] 1200℃ open vacuum tube furnace, electric stirrer, SUC ultrasonic cleaning machine, heat collecting constant temperature magnetic stirring bath, flask, Tecan microplate reader, CNC high-speed chopper, gold spraying line scoring machine, ultracentrifuge.

[0055] Embodiment 1:

[0056] This example introduces a method for preparing a novel Fe single-atom nanozyme. Figure 1 and Figure 2 As shown, Figure 1 Flow chart for the preparation of new Fe single-atom nanozymes. Figure 2 The novel Fe single-atom nanozyme is applied to a lateral flow chromatography test strip, comprising the following steps:

[0057] S1 Hexadecyltrimethylammonium bromide (CTAB) was dissolved in deionized water, triethanolamine (TEA) was added, and stirred;

[0058] S2: adding a mixture of tetraethyl orthosilicate (TEOS) and cyclohexane to the above solution and stirring;

[0059] S3 was collected by centrifugation and washed three times with deionized water and anhydrous ethanol;

[0060] S4: Dispersing the washed material in acetone and refluxing (removing CTAB);

[0061] S5 washed with anhydrous ethanol 3 times;

[0062] S6 vacuum drying; obtaining MSNs;

[0063] S7: Add MSNs to the flask, then add anhydrous ethanol and Tris-Hcl (10 mM, pH = 8.5);

[0064] S8 dopamine hydrochloride (PDA-Hcl) was dissolved in deionized water and added dropwise to the above solution with stirring;

[0065] S9 Hemin was dissolved in dimethyl sulfoxide (DMSO), glucose was weighed and dissolved in water, and stirred;

[0066] S10 was washed three times with ethanol;

[0067] S11 vacuum drying; obtaining MSNs-PDA-Hemin;

[0068] S12 puts the dried MSNs-PDA-Hemin into a tube furnace and calcines it at high temperature to form single-atom nanozyme Fe-NC.

[0069] Furthermore, in the S1, the mass ratio of CTAB to TEA is 11.0-12.0, the mass ratio of CTAB to deionized water is 0.05, the mass ratio of TEA to deionized water is 0.0045, the stirring temperature is 60° C., and the stirring time is 1 h.

[0070] Furthermore, the volume ratio of TEOS to cyclohexane in S2 is 0.25, the stirring temperature is 60° C., and the stirring time is 24 h.

[0071] Furthermore, the temperature of the S6 vacuum drying is 45°C.

[0072] Furthermore, the mass ratio of MSNs, dopamine hydrochloride and hemin described in S7, S8 and S9 is 4:15:5, the mass ratio of hemin chloride to glucose is 0.2, dopamine hydrochloride provides the carbon source, and chlorohemoglobin provides the iron source and nitrogen source.

[0073] Furthermore, the stirring time described in S8 is 5 hours.

[0074] Furthermore, the stirring time described in S9 is 24 hours.

[0075] Furthermore, the pyrolysis rate described in S12 is 5°C min-1, the pyrolysis temperature is 800°C, and the pyrolysis time is 2h.

[0076] Preparation of Fe-NC-Ab

[0077] ① 0.8 mg of Fe-NC was redissolved in 1 ml PBS (0.1 M pH = 7.4);

[0078] ② Add 63 μg EDC·HCl and 30 μg cTnI antibody and shake for 2 hours;

[0079] ③ Add 50 μL (10%) casein solution and shake for 2 h, then add 50 μL (10%) BSA solution and shake for 2 h;

[0080] ④ The obtained Fe-NC-Ab was centrifuged at 12000 rpm for 10 min, washed with BBS three times, and then dispersed in 1 mg / ml BSA-BBS solution.

[0081] Preparation of Fe-NC-based chromatography test strips

[0082] The test strip consists of four main components: sample pad, absorbent pad, NC membrane, on which T and C lines are generated by dispensing cTnI capture antibody (1 mg mL -1 ) and goat anti-mouse IgG (mg mL -1 ) solution, density is 0.74 mL cm-1 The sample pad and the absorbent pad were sequentially attached to the NC membrane with an overlap of about 2 mm to form a complete test strip. The test strip was then cut into 3.9 mm wide pieces and stored at room temperature for subsequent use.

[0083] Detection of cTnI using Fe-NC test paper

[0084] 20 μL cTnI antigen solution was mixed with 65 μL probe solution containing 5 μL Fe-NC single-atom nanozymes and 60 μL BBS buffer (2% BSA, 2% sodium chloride, 0.1% B66 and 0.1% casein). The resulting Fe-NC-Ab complex was added to the sample pad of the test strip. The reaction was continued at room temperature for 15 minutes. The mobile phone camera was then used to capture photos of the test strip at the T line and C line.

[0085] Example 2

[0086] This example further introduces the transmission electron microscopy of nanozymes based on the above examples. Figure 3 , Figure 3 This is the image of nanozyme.

[0087] Figure 3 This is a transmission electron microscopy image of the Fe single-atom nanozyme formed after MSNs-PDA-Hemin was calcined at 800°C. It can be observed that the Fe single-atom nanozyme still maintains the mesoporous form of silica.

[0088] Example 3

[0089] This embodiment further introduces the above-mentioned embodiment. Figure 4 , Figure 4 is the infrared spectrum of MSNs-PDA-Hemin;

[0090] Figure 4 MSNs, MSNs-PDA and MSNs-PDA-Hemin were dried and ground into powder for infrared spectrum test. The results showed that MSNs-PDA had a peak at 2925 cm -1 The absorption peak of NH bond appeared, indicating that dopamine was successfully coated on mesoporous silica. The absorption peak of MSNs-PDA-Hemin was 1720 cm -1 The absorption peak of C=O bond appears, indicating that hemin is successfully coated on mesoporous silica.

[0091] Example 4

[0092] This embodiment further introduces the above embodiment. Figure 5 , Figure 5Schematic diagram of enzyme activity test of each part of the synthesis process of the new Fe single-atom nanozyme

[0093] Figure 5 During the synthesis of Fe-NC single-atom nanozymes, equal amounts of MSNs, MSNs-PDA, and MSNs-PDA-Hemin powders were dispersed in water, 50 μL of TMB solution was added and shaken for 1 minute, and the absorbance was measured using a microplate reader. The obtained data were then statistically analyzed and plotted into a curve graph. The results showed that before high-temperature calcination to form Fe-NC single-atom nanozymes, MSNs, MSNs-PDA, and MSNs-PDA-Hemin had almost no enzyme activity, indicating that the Fe active site is of great significance.

[0094] Example 5

[0095] This embodiment further introduces the above embodiment. Figure 6 , Figure 6 Schematic diagram of enzyme activity test of the new Fe single-atom nanozyme at different pH values

[0096] Figure 6 The synthesized Fe single-atom nanozyme was configured into a uniform solution of 2 mg / ml, and 2 μL of 2 mg / ml Fe-NC solution was added into centrifuge tubes containing 50 μL of sodium acetate buffer at pH=2, pH=4, pH=6, pH=8, and pH=10 and 50 μL of TMB solution, respectively. After shaking for 1 minute, the solution was added into a 96-well plate, and the absorbance was detected with a microplate reader. The obtained data were then statistically analyzed and plotted into a graph. The results showed that the newly synthesized Fe single-atom nanozyme had enzymatic activity at different pH values, and the enzyme activity was higher under acidic conditions, indicating that the Fe single-atom nanozyme had excellent low pH adaptability.

[0097] Example 6

[0098] This embodiment further introduces the above-mentioned embodiment. Figure 7 , Figure 7 Schematic diagram of the results of applying the new Fe single-atom nanozyme to detect cTnI on lateral flow chromatography test strips

[0099] Figure 720 μL cTnI antigen solution was mixed with 65 μL probe solution, the probe solution contained 5 μL Fe-NC single-atom nanozyme and 60 μL BBS buffer (2% BSA, 2% sodium chloride, 0.1% B66 and 0.1% casein). The obtained Fe-NC-Ab complex was added to the sample pad of the test strip. The reaction was continued at room temperature for 15 minutes. Then the mobile phone camera was used to capture photos of the test strip at the T line and C line. The results showed that in the negative sample (no cTnI antigen, left picture), only the quality control line (C line) showed a color reaction, and in the positive sample (containing cTnI antigen), the C line and T line showed a color reaction at the same time, indicating that the Fe single-atom nanozyme synthesized in this application can achieve rapid detection of cTnI.

Claims

1. Preparation and application of a novel Fe single-atom nanozyme, characterized in that: The following steps are involved: S1. Dissolve cetyltrimethylammonium bromide (CTAB) in deionized water, add triethanolamine (TEA), and stir; S2. Add a mixture of ethyl silicate (TEOS) and cyclohexane to the above solution and stir; S3. Collect by centrifugation and wash with deionized water and anhydrous ethanol three times; S4. The washed material is dispersed in acetone and refluxed (to remove CTAB); S5. Wash with anhydrous ethanol 3 times; S6. vacuum drying to obtain MSNs; S7. Add MSNs to the flask, and then add anhydrous ethanol and Tris-Hcl (10 mM, pH = 8.5); S8. Dopamine hydrochloride (PDA-Hcl) is dissolved in deionized water and added dropwise to the above solution with stirring; S9. Dissolve hemin in dimethyl sulfoxide (DMSO), weigh glucose and dissolve it in water, and stir; S10. Wash with ethanol 3 times; S11. Vacuum drying to obtain MSNs-PDA-Hemin; S12. Place the dried MSNs-PDA-Hemin into a tube furnace and calcine at high temperature to form single-atom nanozyme Fe-NC.

2. The preparation and application of a novel Fe single-atom nanozyme according to claim 1, characterized in that: In the S1, the mass ratio of CTAB to TEA is 11.0-12.0, the mass ratio of CTAB to deionized water is 0.05, the mass ratio of TEA to deionized water is 0.0045, the stirring temperature is 60° C., and the stirring time is 1 h.

3. The preparation and application of a novel Fe single-atom nanozyme according to claim 1, characterized in that: The volume ratio of TEOS to cyclohexane in the S2 is 0.25, the stirring temperature is 60° C., and the stirring time is 24 h.

4. The preparation and application of a novel Fe single-atom nanozyme according to claim 1, characterized in that: The temperature of the S6 vacuum drying is 45°C.

5. The preparation and application of a novel Fe single-atom nanozyme according to claim 1, characterized in that: The mass ratio of MSNs, dopamine hydrochloride and hemin described in S7, S8 and S9 is 4:15:5, the mass ratio of hemin chloride to glucose is 0.2, dopamine hydrochloride provides the carbon source, and chlorohemoglobin provides the iron source and nitrogen source.

6. The preparation and application of a novel Fe single-atom nanozyme according to claim 1, characterized in that: The stirring time described in S8 is 5h.

7. The preparation and application of a novel Fe single-atom nanozyme according to claim 1, characterized in that: The stirring time described in S9 is 24h.

8. The preparation and application of a novel Fe single-atom nanozyme according to claim 1, characterized in that: The pyrolysis rate described in S12 is 5℃min -1 , the pyrolysis temperature is 800℃ and the pyrolysis time is 2h.

9. A novel Fe single-atom nanozyme prepared according to the method described in any one of claims 1-8.

10. Use of a novel Fe single-atom nanozyme according to claim 9 in cTnI detection.

Images