Interactive response layered superlattice nanoprobe as well as synthesis method and application thereof

Abstract

The invention discloses an interactive response layered superlattice nanoprobe as well as a synthesis method and application thereof, and belongs to the technical field of nanomaterials and biosensing. The nanoprobe is synthesized through a ligand regulation and control self-assembly strategy, specifically, a platinum source, n-caprylic acid and molybdenum hexacarbonyl are mixed and heated for a reaction, and after the reaction is finished, aftertreatment is conducted to obtain the interactive response layered superlattice nanoprobe. The nanoprobe has a highly-ordered layered superlattice structure, and can generate synergistic change of structure and catalytic activity under different H2O2 concentrations, so that specific interactive response to H2O2 is realized. The nanoprobe is controllable in synthesis process and stable in structure, the interactive response performance of the nanoprobe provides core material support for threshold type detection of biomarkers, the technical problems that a traditional nanomaterial is single in response to substrate concentration change and insufficient in sensitivity are solved, and the nanoprobe has important application value in the fields of biosensing, clinical detection and the like.

Description

An interactive responsive layered superlattice nanoprobe, its synthesis method, and its applications. Technical Field

[0001] This invention relates to the fields of nanomaterial synthesis and biosensing technology, specifically to an interactive responsive layered superlattice nanoprobe, its synthesis method, and its applications. Background Technology

[0002] In clinical diagnosis, the metabolic processes of many disease-related biomarkers are accompanied by the generation of H2O2, and the concentration of H2O2 often has a characteristic threshold (such as the H2O2 threshold of L-tryptophan metabolite, a metabolic marker associated with tuberculous meningitis, is 0.2 μM). Therefore, if materials that can produce specific interactive responses to the H2O2 concentration threshold are developed, precise monitoring of disease progression can be achieved.

[0003] Currently, the detection methods for H2O2 mainly rely on colorimetric methods (such as horseradish peroxidase, HRP), fluorescence methods, chemiluminescence methods, and electrochemical methods based on natural enzymes. However, natural enzymes have inherent drawbacks such as high extraction costs, poor stability, and sensitivity to environmental factors leading to easy inactivation, which limit their reliability and large-scale use in practical applications.

[0004] To overcome these limitations, a series of nanomaterials with peroxidase (POD) mimicry activity, known as "nanozymes," have been widely developed for H2O2 detection in recent years. Compared with traditional natural enzymes, nanozymes have significant advantages such as simple preparation, low cost, high stability, and ease of surface functionalization. However, the response of traditional nanozymes to substrates (such as H2O2) is mostly linearly dependent, lacking the ability to specifically recognize substrate concentration thresholds, making it difficult to meet the detection needs of nonlinear concentration changes of biomarkers in complex biological systems.

[0005] Therefore, designing an interactive nanoprobe with tunable structure and strong response specificity is key to overcoming the limitations of traditional nanozyme detection. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing probes by providing a method for synthesizing an interactive-response layered superlattice nanoprobe that can generate a specific interactive response to an H2O2 concentration threshold, so as to be applied to the monitoring of complex physiological diseases.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] In a first aspect, the present invention provides a method for synthesizing interactive responsive layered superlattice nanoprobes, comprising the following steps:

[0009] A platinum source was dissolved in n-caprylic acid, molybdenum hexacarbonyl was added, and a protective gas was introduced to remove oxygen from the system, resulting in a mixed solution.

[0010] The mixed solution was heated in an oil bath to form a layered superlattice structure through self-assembly. After the reaction was completed, the precipitate was collected by centrifugation, washed, and dried to obtain the interactive responsive layered superlattice nanoprobes (Pt MALs).

[0011] Furthermore, the platinum source is bis(acetylacetone)platinum Pt(acac)2.

[0012] Furthermore, the mass-to-volume ratio of platinum source to octanoic acid is 20-30 mg: 15-20 mL.

[0013] Furthermore, the mass ratio of platinum source to molybdenum hexacarbonyl is 0.67-6:1.

[0014] Furthermore, the oil bath heating temperature is 90-110℃, and the time is 1-3 hours.

[0015] Secondly, this invention provides an interactive responsive layered superlattice nanoprobe, prepared using the method described above. The nanoprobe has an interlayer period of 2.20 nm and a monolayer thickness of approximately 0.40 nm, with Pt element uniformly dispersed within the material. This nanoprobe exhibits peroxidase (POD) activity, catalyzing the generation of reactive oxygen species (ROS) from H₂O₂, wherein the ROS includes superoxide anion (O₂O₂). ·- Singlet oxygen () 1 O2) and hydroxyl radicals (·OH).

[0016] Thirdly, the present invention provides a method for detecting hydrogen peroxide threshold using the above-mentioned interactive-responsive layered superlattice nanoprobe, comprising the following steps:

[0017] The interactive-responsive layered superlattice nanoprobe dispersion was mixed thoroughly with the hydrogen peroxide solution and 3,3',5,5'-tetramethylbenzidine (TMB) solution, and then incubated by heating. After incubation, the absorbance of the reaction solution at 652 nm was measured using a UV spectrophotometer. The concentration range or threshold of hydrogen peroxide in the test solution was determined by comparing it with a preset concentration-absorbance standard relationship. The concentration of the interactive-responsive layered superlattice nanoprobe dispersion was 1-2 mg / mL.

[0018] Furthermore, during the comparison, when the hydrogen peroxide concentration was in the range of 0-0.2 μM, the absorbance increased linearly with increasing concentration. At this time, the nanoprobe maintained a layered superlattice structure, the POD activity was stable, and the ability to catalyze the decomposition of H2O2 continued to increase. When the hydrogen peroxide concentration exceeded 0.2 μM, the coordination bonds of the nanoprobe broke, the layered structure disintegrated, and nanoclusters (Pt GLs) were formed, which then agglomerated into nanoparticles (Pt NAs). The POD activity gradually decreased with the degree of structural damage, and the absorbance no longer followed the aforementioned linear increase relationship.

[0019] Furthermore, the volume ratio of the interactive response layered superlattice nanoprobe dispersion, the hydrogen peroxide solution to be tested, and the TMB solution is 1-2:1:1.

[0020] Furthermore, the incubation temperature was 37 ℃, and the incubation time was 20-30 min.

[0021] Fourthly, this invention provides the application of the above-mentioned interactive responsive layered superlattice nanoprobe in the preparation of biomarker hydrogen peroxide detection products.

[0022] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

[0023] Advantages of the synthesis process: The nanoprobe of this invention adopts a ligand-controlled self-assembly strategy. Using octanoic acid as a solvent and ligand, Pt atoms and octanoic acid self-assemble through coordination bonds and van der Waals forces to achieve one-step synthesis of Pt MALs nanoprobes. The synthesis method is simple, the reaction conditions are mild (dissolution at room temperature, reaction at 90°C), the raw materials are readily available, the cost is low, and the reaction parameters (raw material ratio, reaction temperature, time, centrifugation speed, etc.) are clear and controllable, with good reproducibility, making it suitable for large-scale industrial production.

[0024] Unique structure: The nanoprobes Pt MALs prepared in this invention have a highly ordered layered superlattice structure with uniformly dispersed Pt elements and Pt-O and Pt-Pt bonds, which endow the material with excellent colloidal stability and POD catalytic activity.

[0025] High response specificity: The nanoprobe of this invention has a concentration threshold specific response to H2O2 (threshold 0.2 μM), exhibiting synergistic changes in structure and catalytic activity before and after the threshold. This solves the technical problems of traditional nanozymes having a single response and lacking threshold recognition ability. It can be used directly for H2O2 detection, or indirectly for the detection of biomarkers generated based on H2O2 (such as L-tryptophan, glucose, etc.). It is especially suitable for clinical diagnostic scenarios that require differentiation of concentration thresholds (such as early diagnosis and disease assessment of tuberculous meningitis), and has important practical application value. Attached Figure Description

[0026] Figure 1 shows the effect of different concentrations of H2O2 on the absorbance of the TMB oxidation reaction catalyzed by Pt MALs nanoprobes.

[0027] Figure 2 is a TEM image of the PtMALs solution prepared in Example 1.

[0028] Figure 3 is a TEM image of the PtMALs solution prepared in Example 2.

[0029] Figure 4 shows the structural changes of Pt MALs after reaction with 0.05 μM H2O2 in Application Example 1 (TEM).

[0030] Figure 5 shows the structural changes of Pt MALs after reaction with 0.5 μM H2O2 in Application Example 1 (TEM). Detailed Implementation

[0031] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0032] As mentioned above, the interactive responsive layered superlattice nanoprobes (Pt MALs) provided by this invention are synthesized through a ligand-regulated self-assembly strategy. The synthesis and usage steps are as follows:

[0033] Step (1): Take 20-30 mg of Pt(acac)2 as the platinum source, and 15-20 mL of n-caprylic acid as the solvent and ligand. Dissolve Pt(acac)2 completely in n-caprylic acid at room temperature.

[0034] Step (2): Take 5-30 mg of Mo(CO)6 as a reducing agent, add it to the solution in step (1) and stir until dissolved. Then, introduce a protective gas to remove oxygen from the system and avoid oxygen interfering with the reduction reaction.

[0035] Step (3): Transfer the mixed solution from step (2) into an oil bath at 90-110℃ and heat for 1-3 h. Mo(CO)6 decomposes upon heating to release CO, which reduces Pt(acac)2 to Pt atoms. The Pt atoms and n-caprylic acid self-assemble through coordination bonds and van der Waals forces to form a layered superlattice structure.

[0036] Step (4): After the reaction is complete, the precipitate is collected by centrifugation, and washed 2-3 times alternately with n-hexane and ethanol to remove unreacted raw materials and impurities. After drying, pure Pt MALs nanoprobes are obtained and diluted in ethanol solution to obtain a dispersion.

[0037] Step (5): Take a 1-2 mg / mL Pt MALs nanoprobe dispersion and place it in a 0.5 mL centrifuge tube. Add H2O2 standard solutions of different concentrations (0.05, 0.2, 0.5, 2 μM) and TMB solution (10 mM). Vortex for 5 s to mix thoroughly. Incubate in a 37℃ water bath for 20-30 min. Measure the absorbance of each reaction solution at 652 nm using a UV spectrophotometer and record the absorbance values ​​and trends, as shown in Figure 1. The volume ratio of Pt MALs, H2O2, and TMB solutions is 1-2:1:1. During detection:

[0038] ① When the H2O2 concentration is between 0 and 0.2 μM, Pt MALs maintain a complete layered superlattice structure, and the absorbance of the blue product generated by the catalytic oxidation of TMB shows a good linear relationship with the H2O2 concentration.

[0039] ②When the H2O2 concentration exceeds 0.2 μM, the coordination bond between the Pt atom and the n-caprylic acid breaks, the layered structure disintegrates, the linear relationship of absorbance disappears, and it gradually decreases with increasing concentration.

[0040] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0041] Example 1

[0042] A method for preparing an interactive responsive layered superlattice nanoprobe includes the following steps:

[0043] (1) Dissolve 20 mg Pt (acac)2 in 16 mL n-caprylic acid at room temperature and stir vigorously until dissolved to obtain a uniformly mixed light yellow solution.

[0044] (2) Add 10 mg Mo (CO)6 to the solution obtained in step (1), stir and continuously introduce argon gas for 0.5 h.

[0045] (3) Place the solution from step (2) in an oil bath at 90°C, stir and keep warm for 2 hours, and then let it cool naturally.

[0046] (4) After the reaction was completed, the precipitate was collected by centrifugation, washed twice with hexane and ethanol alternately to remove unreacted raw materials and impurities, and dried to obtain pure Pt MALs nanoprobes. Its TEM image is shown in Figure 2, and the whole structure appears as vesicles.

[0047] Example 2

[0048] A method for preparing an interactive responsive layered superlattice nanoprobe includes the following steps:

[0049] (1) Dissolve 30 mg Pt (acac)2 in 16 mL n-caprylic acid at room temperature and stir vigorously until dissolved to obtain a uniformly mixed light yellow solution.

[0050] (2) Add 30 mg Mo (CO)6 to the solution obtained in step (1), stir and continuously introduce argon gas for 0.5 h.

[0051] (3) Place the solution from step (2) in an oil bath at 110°C, stir and keep warm for 2 hours, and then let it cool naturally.

[0052] (4) After the reaction was completed, the precipitate was collected by centrifugation, and washed three times alternately with n-hexane and ethanol to remove unreacted raw materials and impurities. After drying, pure Pt MALs nanoprobes were obtained. Its TEM image is shown in Figure 3, which shows an overall independent bubble structure.

[0053] Application Example 1

[0054] Take 20 μL of Pt MALs (1 mg / mL) nanoprobe dispersion and place it in a 0.5 mL centrifuge tube. Add H2O2 standard solution (0.05 μM) and then 20 μL of TMB solution (10 mM). Vortex for 5 s to mix thoroughly and incubate in a 37℃ water bath for 30 min. Collect the precipitate by centrifugation and wash twice with hexane and ethanol alternately. The TEM image is shown in Figure 4, and its structure remains intact.

[0055] Application Example 2

[0056] Take 20 μL of Pt MALs (1 mg / mL) nanoprobe dispersion and place it in a 0.5 mL centrifuge tube. Add H2O2 standard solution (0.5 μM) and then 20 μL of TMB solution (10 mM). Vortex for 5 s to mix thoroughly and incubate in a 37℃ water bath for 30 min. Collect the precipitate by centrifugation and wash twice with hexane and ethanol alternately. The TEM image is shown in Figure 5. The structure shows partial collapse, forming clustered aggregates.

[0057] In summary, the interactive responsive layered superlattice nanoprobes (Pt MALs) provided by this invention were successfully synthesized through an innovative ligand-regulated self-assembly strategy. These probes not only possess a highly ordered layered superlattice structure and excellent peroxidase (POD) activity, but also exhibit a specific interactive response to hydrogen peroxide (H2O2) concentration thresholds. When the H2O2 concentration is below 0.2 μM, the Pt MALs maintain a stable layered structure, exhibiting good catalytic activity and a linear response relationship. However, when the H2O2 concentration exceeds 0.2 μM, the probe structure disintegrates, the catalytic activity decreases, and the absorbance no longer increases linearly, showing a significant synergistic change in structure and activity. This unique H2O2 concentration threshold response characteristic makes the nanoprobes of this invention valuable and promising for applications in complex physiological disease monitoring, particularly in clinical diagnostic scenarios requiring concentration threshold differentiation, such as the early diagnosis and disease assessment of tuberculous meningitis. Furthermore, the synthesis method of the present invention is simple, low-cost, and uses readily available raw materials, and has good repeatability and stability, making it suitable for large-scale industrial production and further enhancing its application potential in fields such as biosensing, disease diagnosis, and environmental monitoring.

[0058] The embodiments and application examples described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for synthesizing an interactive responsive layered superlattice nanoprobe, characterized in that, The synthesis method includes the following steps: dissolving a platinum source in octanoic acid, adding molybdenum hexacarbonyl, and introducing a protective gas to remove oxygen from the system to obtain a mixed solution; heating the mixed solution in an oil bath to form a layered superlattice structure; after the reaction is complete, collecting the precipitate by centrifugation, washing and drying to obtain the interactive responsive layered superlattice nanoprobe.

2. The synthesis method according to claim 1, characterized in that, The platinum source used is di(acetylacetone)platinum Pt(acac)2.

3. The synthesis method according to claim 2, characterized in that, The mass-to-volume ratio of platinum source to octanoic acid is 20-30 mg: 15-20 mL, and the mass ratio of platinum source to molybdenum hexacarbonyl is 0.67-6:

1.

4. The synthesis method according to claim 1, characterized in that, The oil bath heating temperature is 90-110℃, and the time is 1-3 hours.

5. An interactive responsive layered superlattice nanoprobe, characterized in that, It is prepared by the method described in any one of claims 1-4.

6. A method for detecting hydrogen peroxide threshold using the interactive-response layered superlattice nanoprobe as described in claim 5, characterized in that, Includes the following steps: The interactive-responsive layered superlattice nanoprobe dispersion was mixed thoroughly with the hydrogen peroxide solution and the 3,3',5,5'-tetramethylbenzidine solution, and then incubated by heating. After incubation, the absorbance of the reaction solution at 652 nm was measured using a UV spectrophotometer. The concentration range or threshold of hydrogen peroxide in the test solution was determined by comparing it with a preset concentration-absorbance standard relationship. The concentration of the interactive-responsive layered superlattice nanoprobe dispersion was 1-2 mg / mL.

7. The method according to claim 6, characterized in that, During the comparison, when the hydrogen peroxide concentration was in the range of 0-0.2 μM, the absorbance increased linearly with increasing concentration; when the hydrogen peroxide concentration exceeded 0.2 μM, the absorbance did not follow a linear increasing relationship.

8. The method according to claim 6, characterized in that, The volume ratio of the interactive response layered superlattice nanoprobe dispersion, the hydrogen peroxide solution to be tested, and the TMB solution is 1-2:1:

1.

9. The method according to claim 6, characterized in that, The incubation temperature is 37 ℃ and the incubation time is 20-30 min.

10. The application of the interactive responsive layered superlattice nanoprobe as described in claim 5 in the preparation of a biomarker hydrogen peroxide concentration threshold detection product.

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