A dual-mode nanocapsule and its preparation method
By modifying mesoporous silica nanoparticles with gold nanoparticles and fluorescence quenchers, and combining fluorescent molecules and Raman reporter molecules, a dual-mode nanocapsule was prepared, which solves the problem that existing technologies cannot simultaneously monitor ATP and H2O2, and achieves highly sensitive intracellular species detection, which is suitable for early disease diagnosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XUZHOU NORMAL UNIVERSITY
- Filing Date
- 2023-10-16
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies lack chemical tools capable of simultaneously monitoring ATP and H2O2 species in vitro and in vivo. In particular, the difficulty in preparing nanomaterials that combine SERS probes with fluorescent probes makes it impossible to achieve simultaneous and independent imaging of H2O2 and ATP.
A dual-mode nanocapsule was prepared by modifying gold nanoparticles and fluorescence quenchers onto mesoporous silica nanoparticles, combining fluorescent molecules and Raman reporter molecules to form a nucleic acid aptamer-encapsulated nanocapsule, and using fluorescence and SERS technology to achieve simultaneous monitoring of ATP and H2O2.
It achieves simultaneous monitoring of intracellular ATP and H2O2 with high sensitivity and selectivity, making it suitable for early disease diagnosis and possessing broad application prospects.
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Figure CN117398936B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioanalytical materials, specifically to a dual-mode nanocapsule and its preparation method. Background Technology
[0002] Adenosine-5′-triphosphate (ATP) plays a crucial role in regulating fundamental cellular functions such as neurotransmission, cell movement, and ion channel function. ATP concentrations are typically between 1 and 10 mM, with a typical ATP-to-adenosine diphosphate (ADP) ratio of 1000:1. Disruption of ATP homeostasis is associated with various diseases, including Parkinson's disease, ischemia, and hypoglycemia, often caused by oxidative stress, which generates highly reactive oxygen species (ROS) and reactive nitrogen species (RNS). Hydrogen peroxide (H₂O₂), in particular, is a typical ROS and is known to inhibit ATP production by oxidatively inactivating mitochondrial ATP synthase. Pathological events show a correlation between ATP and H₂O₂ concentrations, indicating a need for a chemical tool capable of simultaneously monitoring these species in vitro and in vivo.
[0003] Surface-enhanced Raman scattering (SERS) technology, as a technique that can provide molecular fingerprint information, has attracted much attention for non-invasive and real-time detection of biomarkers in cellular and animal applications. However, due to the difficulty in preparing nanomaterials that combine SERS probes with fluorescent probes, mainly due to the difficulty in controlling substrate stability, no dual-mode probe materials capable of simultaneous and independent imaging of H2O2 and ATP have been reported to date. Summary of the Invention
[0004] Based on this, the present invention provides a dual-mode nanocapsule and its preparation method to solve the problem of the lack of chemical tools in the prior art for simultaneously monitoring ATP or H2O2 species in vitro and in vivo.
[0005] To achieve the above objectives, the present invention provides a method for preparing bimodal nanocapsules, comprising the following steps:
[0006] 1) Prepare a solution of mesoporous silica (MSNs) with deionized water, add chloroauric acid (HAuCl4) solution and stir to mix, then add sodium borohydride (NaBH4) solution under ice bath stirring conditions to react and obtain gold nanoparticle-modified mesoporous silica, denoted as nanomaterial MSN@Au NPs;
[0007] 2) Using ultrapure water as a solvent, prepare a solvent with the gold nanoparticle-modified mesoporous silica (MSN@AuNPs) prepared in step 1). Add a fluorescence quencher to react and obtain mesoporous silica modified with the fluorescence quencher, denoted as nanomaterial MSN-NaFL@Au NPs; then add fluorescent molecules and Raman reporter molecules 4-mercaptophenylboronic acid (4-MA) in sequence to react and obtain bimolecularly modified nanoprobes, denoted as nanomaterial MSN-NaFL / Au NPs-MA;
[0008] 3) Using ultrapure water as a solvent, a solution of adenosine-5′-triphosphate nucleic acid aptamer was mixed with a nanoprobe (MSN-NaFL / Au NPs-MA) to obtain a bimodal nanocapsule encapsulated with nucleic acid aptamer, denoted as the nanomaterial MSN-NaFL@AuNPs-4-MA@ATP. apt .
[0009] As a further preferred technical solution of the present invention, in step 1), mesoporous silica is mixed with deionized water and sonicated continuously for 15-20 min. Chloroauric acid solution is added and stirred for 0.5-1 h. After centrifugation and washing, sodium borohydride solution is added under ice bath stirring conditions to react. After centrifugation, ultrapure water is washed and dried to obtain gold nanoparticle-modified mesoporous silica.
[0010] As a further preferred technical solution of the present invention, in step 2), after adding fluorescent molecules to react, centrifuge, wash with ultrapure water, and then prepare a solution again with ultrapure water as solvent; after adding 4-mercaptophenylboronic acid to react, centrifuge, wash with ultrapure water, and obtain bimolecular modified nanoprobes.
[0011] As a further preferred technical solution of the present invention, in step 3), the adenosine-5′-triphosphate nucleic acid aptamer solution is mixed with the nanoprobe using ultrapure water as a solvent, and the mixture is continuously shaken for 0.5-1 h, centrifuged, and washed with ultrapure water to obtain a dual-mode nanocapsule encapsulated with nucleic acid aptamer.
[0012] As a further preferred embodiment of the present invention, the aptamer base sequence of the adenosine-5′-triphosphate is ACC TGG GGG AGT ATT GCG GAG GAA GGT GTC ACA(A). 10 .
[0013] As a further preferred embodiment of the present invention, the fluorescence quencher is a black hole quencher (BHQ series), 5-carboxytetramethylrhodamine, or succinimide ester.
[0014] As a further preferred embodiment of the present invention, the fluorescent molecule is sodium fluorescein, fluorescein, or potassium fluorescein.
[0015] According to another aspect of the present invention, the present invention also provides a dual-mode nanocapsule that can be used to simultaneously monitor the levels of hydrogen peroxide and adenosine-5′-triphosphate (ATP) in cells.
[0016] The preparation method of this invention is simple, the reaction is mild, environmentally friendly, the materials are readily available, the production cost is low, and it is easy to carry out large-scale industrial production.
[0017] The dual-mode nanocapsules prepared by this invention are composite nanomaterials with high sensitivity, good selectivity, and excellent stability. Most importantly, they can simultaneously monitor the levels of hydrogen peroxide and adenosine-5′-triphosphate (ATP) in cells using fluorescence and surface-enhanced Raman scattering (SERS) techniques, so as to be applied to the early monitoring of diseases.
[0018] In the presence of ATP, the ATP nucleic acid aptamer detaches from the surface of the silica spheres in the bimodal nanocapsules of this invention, causing the release of fluorescent molecules (such as sodium fluorescein, NaFL) from the mesopores, generating a fluorescent signal and enabling ATP detection. In the presence of H₂O₂, the boric acid functional group on the Raman reporter molecule 4-mercaptophenylboronic acid (4-MA) on the surface of AuNPs is converted to a hydroxyl group, thereby achieving selective SERS sensing for H₂O₂. This bimodal nanocapsule utilizes SERS and fluorescence to simultaneously monitor intracellular hydrogen peroxide and ATP levels, effectively meeting the needs of early disease diagnosis, thus possessing broad application prospects and potentially becoming a new disease detection tool. Attached Figure Description
[0019] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0020] Figure 1 This is a scanning electron microscope image of the mesoporous silica modified with Au NPs in Example 1.
[0021] Figure 2 This shows the SERS changes of the nanoprobe in Example 1 before and after the addition of hydrogen peroxide.
[0022] Figure 3 This describes the stability of the nanocapsules in Example 1 under different pH conditions.
[0023] Figure 4 This is the change in SERS spectral intensity after adding different concentrations of hydrogen peroxide in Example 1.
[0024] Figure 5 This is the change in fluorescence intensity before and after the addition of ATP in Example 1.
[0025] Figure 6 This is the change in SERS spectral intensity before and after the addition of ATP in Example 1.
[0026] Figure 7 This is the fluorescence intensity change after adding different concentrations of ATP in Example 1.
[0027] Figure 8 This is the change in SERS spectral intensity after adding different concentrations of ATP in Example 1.
[0028] Figure 9 This is Raman confocal imaging of the dual-mode nanocapsules after they enter the cells, as described in Example 1.
[0029] Figure 10 The fluorescence signal intensity of the dual-mode nanocapsule (Group A) and the unencapsulated nanoprobe (Group B) in Example 1 after entering the cell changes over time.
[0030] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0031] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.
[0032] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.
[0033] Example 1
[0034] MSN-NaFL / Au NPs-MA-ATP apt Preparation method:
[0035] Step (1): 2.5 mg of mesoporous silica (MSNs) powder was dispersed in 2.5 mL of deionized water and sonicated for 20 min to obtain an MSNs solution. Then, 37.5 μL of HAuCl4 solution (20 mM) was added to the uniformly dispersed MSNs solution and stirred for 30 min before centrifugation and washing. Then, under ice bath conditions, the washing product was added to 187.5 μL of NaBH4 solution (0.1 M) and stirred vigorously for 1 h. The solution gradually turned purple. After centrifugation, the product was washed three times with ultrapure water and dried overnight in a vacuum drying oven to finally obtain the nanomaterial MSN@Au NPs.
[0036] Step (2): While stirring continuously, disperse 10 mg of MSN@Au NPs obtained in step (1) into 5 mL of ultrapure water. Add 1.3 mg of fluorescence quencher (BHQ) powder, or uniformly disperse 1.3 mg of fluorescence quencher (BHQ) powder in 5 mL of ultrapure water, and then mix and stir the fluorescence quencher with the purple solution from step (1). Finally, due to electrostatic interaction, BHQ is modified in the mesopores of MSN to obtain an MSN / Au NPs solution modified with fluorescence quencher.
[0037] Step (3): First, mix 500 μL of MSN / Au NPs solution with 1 mL of sodium fluorescein (NaFL, 10... -5 After mixing with M), centrifuging and washing to remove excess NaFL, the mixture was uniformly dispersed in 500 μL of ultrapure water. Then, 1 mL of Raman reporter molecule 4-mercaptophenylboronic acid (4-MA) solution (10 mg) was added. After centrifugation and washing to remove excess 4-MA solution, MSN-NaFL@Au NPs-4-MA bimolecularly modified nanoprobes were obtained. These nanoprobes were then dispersed in 1 mL of ultrapure water to obtain the MSN-FL / Au NPs-MA nanoprobe solution. In this step, the fluorescence signal of sodium fluorescein (NaFL) entering the mesopores is quenched by the fluorescence quencher modified in MSN. Because the Raman reporter molecule 4-MA forms Au-S bonds with the Au NPs surface, it is anchored to the Au NPs surface.
[0038] Step (4): Add 100 μL of adenosine-5′-triphosphate (ATP) nucleic acid aptamer solution to the 50 μL MSN-FL / Au NPs-MA nanoprobe solution obtained in step (3), stir continuously, centrifuge and wash to obtain MSN-NaFL@Au NPs-4-MA-ATP apt Dual-mode nanocapsules. The aptamer base sequence of adenosine-5′-triphosphate (ATP) is ACC TGGGGG AGT ATT GCG GAG GAA GGT GTC ACA(A). 10 .
[0039] The final product, the bimodal nanocapsule, and the intermediate product prepared in Example 1 were subjected to the following tests, respectively: Figure 1-10 As shown.
[0040] Figure 1 This is a scanning electron microscope image of MSN modified with Au NPs, demonstrating the successful growth of gold nanoparticles (MSN / AuNPs) on the surface of mesoporous silica via in-situ reduction.
[0041] Figure 2The changes in SERS of the nanoprobe before and after the addition of hydrogen peroxide can be observed at 1590-1600 cm⁻¹. -1 The presence of a peak indicates that the boric acid group on the Raman reporter molecule 4-MA in its nanoprobe reacts with H2O2.
[0042] Figure 3 The SERS spectra of the nanocapsules under different pH conditions show that the effect of pH on the bimodal nanocapsules is negligible.
[0043] Figure 4 The images show SERS spectra after adding different concentrations of H2O2. The H2O2 concentration decreases sequentially from top to bottom, and a value of 1594 cm⁻¹ can be observed. -1 / 1581cm -1 The peak ratio gradually decreases.
[0044] Figure 5 The images show the fluorescence emission spectra before and after the addition of ATP. The dual-mode nanocapsules initially exhibit low fluorescence signal intensity due to encapsulation. Upon ATP addition, the nanocapsules are induced to open, releasing a fluorescence signal with increased intensity.
[0045] Figure 6 The images show the SERS spectra before and after ATP addition. The dual-mode nanocapsules exhibit low SERS signal intensity due to encapsulation. Upon ATP addition, the nanocapsules are induced to open and release 4-MA, resulting in enhanced SERS signal intensity.
[0046] Figure 7 The fluorescence emission spectra are those obtained after adding different concentrations of ATP. As the ATP concentration increases, the molecular weight of the sodium fluorescein released by the dual-mode nanocapsules increases, and the fluorescence signal gradually strengthens.
[0047] Figure 8 The SERS light was obtained after adding different concentrations of ATP. As the ATP concentration increased, the molecular weight of 4-MA released by the dual-mode nanocapsules increased, and the SERS signal gradually strengthened.
[0048] Figure 9 This is Raman confocal imaging after the nanocapsules enter the cell. Four points are selected from the image to scan the SERS spectrum. The SERS spectrum corresponding to the blue square area can be seen... Figure 2 SERS spectrum of the reaction between nanoprobe and H2O2 Figure 1 This study demonstrates the SERS sensing capability of the bimodal nanocapsule for H2O2 content. Compared to current common methods for detecting H2O2, such as fluorescence, photoelectrochemical, and spectrophotometry, existing methods are limited by expensive fluorescent agents, complex operating procedures, and low sensitivity. In contrast, the bimodal nanocapsule of this invention has a stable substrate and high sensitivity, meeting the requirements for early disease diagnosis.
[0049] Figure 10 The fluorescence signal intensity of the dual-mode nanocapsules (Group A) and the unencapsulated nanoprobes (Group B) after entering cells changed over time. It was observed that the fluorescence signal intensity of Group A increased with time, indicating a gradual increase in the molecular weight of the released sodium fluorescein. Conversely, the fluorescence signal intensity of Group B did not change significantly over time because the molecular weight of the sodium fluorescein exposed within the cells remained almost unchanged. This comparison further demonstrates the effectiveness of the nanocapsules in monitoring intracellular ATP levels. Most current techniques can only monitor either ATP or H₂O₂ individually, while the applicability of this invention lies in its ability to monitor both key species simultaneously in real time.
[0050] In summary, the fluorescent-SERS dual-mode nanocapsules prepared by this invention can sensitively monitor the levels of hydrogen peroxide and ATP in cells, and have broad application prospects in the biological field.
[0051] In this invention, the fluorescence quenchers and fluorescent molecules used are all conventional reagents. For example, the fluorescence quenchers 5-carboxytetramethylrhodamine or succinimide ester have the same effect as black hole quenchers (BHQ series) in this invention, and can quench the fluorescence signals of sodium fluorescein, fluorescein, or potassium fluorescein. This satisfies the following: by adding the fluorescence quencher to the MSN / Au NPs solution, the corresponding fluorescence quencher can be modified into the mesopores of the MSN due to electrostatic interactions, and the fluorescence signal of the fluorescent molecules entering the mesopores will be quenched by the fluorescence quencher modified in the MSN.
[0052] Example 2
[0053] Using the same preparation method as in Example 1, except that the fluorescence quencher was replaced with succinimide ester and the fluorescent molecule was replaced with potassium fluorescein, a bimodal nanocapsule was finally prepared. This bimodal nanocapsule was applied to monitor intracellular hydrogen peroxide and ATP levels, and the same testing method as in Example 1 yielded the same results.
[0054] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.
Claims
1. A method for preparing a dual-mode nanocapsule, characterized in that, Includes the following steps: 1) Prepare a solution of mesoporous silica and deionized water, add chloroauric acid solution and stir to mix, then add sodium borohydride solution under ice bath stirring conditions to react and obtain gold nanoparticle-modified mesoporous silica. 2) Using ultrapure water as a solvent, a solution was prepared with the gold nanoparticle-modified mesoporous silica prepared in step 1). A fluorescence quencher, a fluorescent molecule, and 4-mercaptophenylboronic acid were added sequentially to react and obtain bimolecularly modified nanoprobes. 3) Using ultrapure water as a solvent, a solution of adenosine-5′-triphosphate nucleic acid aptamer was mixed with a nanoprobe to obtain a dual-mode nanocapsule encapsulated with nucleic acid aptamer.
2. The method for preparing dual-mode nanocapsules according to claim 1, characterized in that, Step 1) specifically includes: mixing mesoporous silica with deionized water, sonicating continuously for 15-20 min, adding chloroauric acid solution and stirring for 0.5-1 h, centrifuging and washing; then adding sodium borohydride solution under ice bath stirring conditions to react, centrifuging separation, washing with ultrapure water, and drying to obtain gold nanoparticle-modified mesoporous silica.
3. The method for preparing dual-mode nanocapsules according to claim 1, characterized in that, In step 2), after adding fluorescent molecules and reacting, the mixture is centrifuged, washed with ultrapure water, and then prepared into a solution again with ultrapure water as the solvent. After adding 4-mercaptophenylboronic acid and reacting, the mixture is centrifuged, washed with ultrapure water, and bimolecularly modified nanoprobes are obtained.
4. The method for preparing dual-mode nanocapsules according to claim 1, characterized in that, In step 3), the adenosine-5′-triphosphate nucleic acid aptamer solution is mixed with the nanoprobe using ultrapure water as a solvent, and the mixture is continuously shaken for 0.5-1 h. After centrifugation and washing with ultrapure water, a dual-mode nanocapsule encapsulated with nucleic acid aptamer is obtained.
5. The method for preparing dual-mode nanocapsules according to claim 1, characterized in that, The fluorescent molecule is sodium fluorescein or potassium fluorescein.
6. The method for preparing dual-mode nanocapsules according to claim 1, characterized in that, The fluorescence quencher is a BHQ series black hole quencher, 5-carboxytetramethylrhodamine, or succinimide ester.
7. The method for preparing bimodal nanocapsules according to any one of claims 1 to 6, characterized in that, The aptamer base sequence of the adenosine-5′-triphosphate is ACC TGG GGG AGT ATT GCG GAG GAA GGT GTC ACA (A). 10 .
8. A dual-mode nanocapsule, characterized in that, Prepared by the method described in any one of claims 1-7.