A DNA methylation-driven nanoscale enzyme switch and application thereof

Abstract

The application provides a DNA methylation-driven nano-enzyme switch and application thereof. The nano-enzyme switch comprises a nano-enzyme carrier with peroxidase-like activity and a methylation-sensitive DNA gating molecule combined on the surface of the nano-enzyme carrier. In the initial state, the gating molecule physically blocks the substrate from approaching the catalytic site, and the nano-enzyme is in the "off" state; when specifically combined with the target methylation DNA, the gating molecule is dissociated or rearranged, exposing the active site, and the nano-enzyme is "turned on" to catalyze the color reaction. The application also provides a method for detecting DNA methylation by using the switch, without PCR amplification and any instrument equipment, and the detection sensitivity reaches 0.1%. On this basis, the application further provides an integrated detection card box, which integrates a reaction chamber and an observation window, and can complete the rapid auxiliary detection of immunosenescence-related methylation sites by using saliva or fingertip blood as a sample. The application has the advantages of simple operation, high sensitivity, good stability, suitability for home self-testing and the like, and can be used for rapid evaluation of the risk of immunosenescence.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a DNA methylation-driven nanozyme switch and its application. Background Technology

[0002] Immunosenescence is a complex process in which the body's immune system gradually declines with age, mainly manifested as an imbalance in the proportion of T cell subsets, a decrease in CD28 expression, and a chronic low-grade inflammatory state. Changes in DNA methylation levels are closely related to immunosenescence, and the degree of methylation at specific gene loci can serve as a biomarker for assessing immunosenescence status. Existing methylation detection methods, such as methylation-specific PCR, pyrosequencing, and bisulfite sequencing, while possessing high accuracy, rely on specialized thermal cycling instruments and fluorescence detection equipment, with detection cycles lasting several hours, making them unsuitable for home self-testing. CN106367515B discloses a colorimetric detection method for methylation based on gold nanoparticles, but it still requires a PCR amplification step, and result interpretation depends on color identification, limiting its sensitivity and convenience. In recent years, nanozymes, as a class of nanomaterials with enzyme-like catalytic activity, such as iron(III) oxide (Fe3O4), cerium(II) oxide (CeO2), and Prussian blue, have received widespread attention in the field of bioassay due to their advantages such as low cost, high stability, and strong signal designability. However, there have been no reports on combining nanozymes with DNA methylation detection, especially on constructing "smart switches" that directly regulate the catalytic activity of nanozymes by methylated DNA.

[0003] Therefore, developing a DNA methylation-driven nanozyme switch to achieve instrument-free, highly sensitive, and visualized home testing for rapid assessment of immune aging risks has significant clinical application value and market prospects. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a DNA methylation-driven nanozyme switch and its home detection device. The core structure of the nanozyme switch is as follows: methylation-sensitive DNA-gated molecules are modified onto the surface of Prussian blue nanoparticles (PB NPs) or cerium dioxide nanoparticles (CeO2 NPs) with peroxidase-like activity. Initially, the DNA-gated molecules cover the nanozyme surface through electrostatic adsorption or covalent bonding, physically preventing the substrate from entering the catalytic active site. The nanozyme is in a closed state and cannot catalyze the colorimetric reaction. When the target methylated DNA is present in the sample, it specifically binds to the gated molecules, triggering a conformational change or competitive substitution of the DNA. The gated molecules detach from or rearrange from the nanozyme surface, exposing the catalytic active site. The nanozyme is then activated, and in the presence of hydrogen peroxide (H2O2), it catalyzes the production of a visible blue oxidation product from a chromogenic substrate (such as TMB, i.e., 3,3',5,5'-tetramethylbenzidine). The device is an integrated detection cartridge with a built-in reaction chamber and observation window, requiring no additional instruments.

[0005] On one hand, the present invention provides a DNA methylation-driven nanozyme switch, characterized in that it comprises:

[0006] Nanozyme carrier, wherein the nanozyme has peroxidase-like catalytic activity;

[0007] The gated molecule is a methylation-sensitive DNA molecule that binds directly or indirectly to the surface of the nanozyme and physically prevents substrate molecules from approaching the catalytic active site of the nanozyme.

[0008] When the gated molecule specifically binds to the target methylated DNA, the gated molecule undergoes a conformational change or dissociates from the surface of the nanozyme, thereby exposing the catalytic active site and activating the peroxidase-like activity of the nanozyme.

[0009] Furthermore, the nanozyme is selected from one or more of Prussian blue nanoparticles, iron oxide nanoparticles, and cerium dioxide nanoparticles; preferably, it is Prussian blue nanoparticles.

[0010] Furthermore, the particle size of the Prussian blue nanoparticles is 20-200 nm, preferably 50-100 nm.

[0011] Furthermore, the gated molecules are bound to the surface of the nanoenzyme via electrostatic adsorption, covalent bonding, biotin-streptavidin interaction, or hydrophobic interaction; preferably, they are covalently linked via EDC / NHS chemical cross-linking.

[0012] Furthermore, the length of the gated molecule is 15-50 bases, preferably 25-40 bases.

[0013] Furthermore, the gated molecule contains 2-10 CpG dinucleotide sites, preferably 4-8 CpG dinucleotide sites.

[0014] Furthermore, the 3' or 5' end of the gated molecule is modified with a thiol, amino, or biotin group for attachment to the surface of the nanoenzyme.

[0015] Furthermore, the target methylated DNA is an immune aging-related methylation site, which is selected from one or more of the LRRC23 gene, ADRB3 gene, RNF180 gene, PAX1 gene, CD28 gene or CDKN2A gene; preferably at least one of the LRRC23 gene, ADRB3 gene and RNF180 gene.

[0016] On the other hand, the present invention also provides a method for detecting DNA methylation using the above-mentioned nanozyme switch, characterized by comprising the following steps:

[0017] Step S1: Provide a nanozyme switch;

[0018] Step S2: Contact the sample to be tested with the nanozyme switch;

[0019] Step S3: If methylated target DNA is present in the sample to be tested, the methylated target DNA specifically binds to the gate molecule, causing the gate molecule to dissociate or rearrange from the surface of the nanozyme, exposing the catalytic active site of the nanozyme and activating peroxidase-like activity;

[0020] Step S4: Add chromogenic substrate and observe the color change to determine the methylation status of the target DNA.

[0021] Furthermore, the sample to be tested is a raw biological sample treated with cell lysis buffer.

[0022] Further, the chromogenic substrate is one or more of 3,3',5,5'-tetramethylbenzidine, 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid), o-phenylenediamine, or 3-amino-9-ethylcarbazole; preferably 3,3',5,5'-tetramethylbenzidine.

[0023] Furthermore, the chromogenic substrate is used in conjunction with hydrogen peroxide.

[0024] Furthermore, the method also includes comparing the observed color with a standard colorimetric card to semi-quantitatively assess the methylation rate of the target DNA.

[0025] Furthermore, the method has a detection sensitivity of less than or equal to 0.1% for the target DNA methylation ratio.

[0026] On the other hand, the present invention also provides an integrated detection cartridge for home-based DNA methylation testing, characterized in that it includes the aforementioned nanozyme switch, and the following modules integrated within the same housing:

[0027] The housing forms an enclosed detection space;

[0028] The sample loading area, located on the housing, is used to receive the sample to be tested;

[0029] The reaction chamber is located inside the shell and is pre-loaded with the nanozyme switch and the chromogenic substrate;

[0030] A microchannel, connecting the sample loading area and the reaction chamber, is used to guide the sample to be tested from the sample loading area to the reaction chamber;

[0031] An observation window is located above the reaction chamber for directly observing the colorimetric reaction results.

[0032] Furthermore, the sample loading area includes a sealing rubber pad and a sample injection hole.

[0033] Furthermore, the nanozyme switch and / or chromogenic substrate are pre-placed in the reaction chamber in the form of lyophilized powder.

[0034] Furthermore, the shell is a rectangular plastic shell with dimensions of 6-10cm in length, 4-6cm in width, and 0.8-1.5cm in height, and the material is selected from one or more of acrylonitrile-butadiene-styrene copolymer (ABS), polypropylene, polyethylene, or polycarbonate.

[0035] On the other hand, the present invention also provides the application of the above-mentioned nanozyme switch or detection method or integrated detection cartridge in the preparation of reagent kits or detection devices for immune aging risk assessment.

[0036] Furthermore, the immune aging risk assessment includes detecting the methylation levels of one or more of the LRRC23, ADRB3, and RNF180 genes.

[0037] Furthermore, the immune aging risk assessment also includes: determining the CD4 / CD8 ratio and / or the proportion of CD28-positive T cells based on methylation levels.

[0038] Compared with existing technologies, the advantages of this invention are as follows: It pioneers a smart switch that directly drives nanozyme catalysis through DNA methylation, achieving activation upon recognition without the need for PCR amplification or any instruments. The detection time is reduced to within 30 minutes, making it extremely simple to operate and suitable for home self-testing. Compared to natural enzymes, nanozymes are lower in cost and more stable, and can be stored at room temperature for extended periods. The detection sensitivity reaches 0.1% methylation ratio. The integrated detection cartridge incorporates a reaction chamber, allowing for immunosenescence risk assessment using saliva or finger-prick blood samples. It achieves 95% consistency with the gold standard of flow cytometry, demonstrating broad prospects for clinical application and industrial transformation. Attached Figure Description

[0039] Figure 1 This is a schematic cross-sectional view of the integrated detection cartridge of the present invention. Reference numerals: 1—shell; 2—sample injection hole; 3—sealing rubber gasket; 4—microchannel; 5—reaction chamber; 6—lyophilized reagent; 7—observation window. Detailed Implementation

[0040] The technical solutions described in this invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only a part of the feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0041] Example 1: Construction and Characterization of a Nanozyme Switch

[0042] 1. Synthesis of Prussian Blue Nanoparticles (PB NPs)

[0043] 0.5 g of polyvinylpyrrolidone (PVP) was dissolved in 40 mL of 0.01 M hydrochloric acid solution, and 0.33 g of potassium ferricyanide was added. The mixture was stirred until completely dissolved. The solution was transferred to a 50 mL stainless steel reactor lined with polytetrafluoroethylene and reacted at 80 °C for 24 hours. After cooling to room temperature naturally, the mixture was centrifuged at 12,000 rpm for 20 minutes. The precipitate was washed three times with deionized water to obtain Prussian blue nanoparticles with a particle size of approximately 50-80 nm. Transmission electron microscopy showed that the particles had a cubic morphology and were uniformly dispersed.

[0044] 2. Design and synthesis of methylation-sensitive DNA-gated molecules

[0045] For immunosenescence-related methylation sites, taking the LRRC23 gene as an example, a single-stranded DNA molecule of 25-35 bases in length was designed as a gating molecule. The DNA sequence satisfies the following characteristics: (1) it contains 5-8 CpG dinucleotide sites; (2) the methylation status of 4-6 of the CpG sites is recognized by the target sample; (3) the 3' end is modified with a thiol group (-SH) for connection with the nanozyme surface; and (4) the 5' end is designed with a protruding end for hybridization with the target methylated DNA.

[0046] Specific preparation method: The above DNA molecules were synthesized by Tianjin Zhonghe Gene Technology Co., Ltd., and purified by HPLC with a purity of ≥95%. The DNA powder was dissolved in TE buffer to prepare a 100μM stock solution, which was stored at -20℃ for later use.

[0047] 3. Assembly of nanozyme switches

[0048] Take 1 mL of the Prussian blue nanoparticle solution prepared in step 1 (concentration approximately 2 mg / mL), wash twice with 0.01 M PBS buffer (pH 7.4), and resuspend in 1 mL PBS. Add 20 μL of 1 mM EDC and 15 μL of 1 mM NHS to this solution, and activate the carboxyl groups on the nanozyme surface for 30 minutes at room temperature. Then add 50 μL of the DNA-gated molecule (100 μM) prepared in step 1.2, and stir the reaction overnight at 4 °C. After the reaction is complete, centrifuge at 10,000 rpm for 15 minutes, wash the precipitate three times with PBS to remove unbound DNA, and finally resuspend in 1 mL PBS and store at 4 °C for later use.

[0049] 4. Verify closed status

[0050] Take 100 μL of the nanozyme switch prepared in step 1.3, add 20 μL of TMB substrate solution (2 mg / mL) and 10 μL of H2O2 (10 mM), and react at room temperature for 10 minutes. Observe the color change of the solution and measure the absorbance at 650 nm.

[0051] Experimental results: The reaction solution remained colorless or very pale blue, OD 650 The value was only 0.05±0.01. As a control, an equal amount of unmodified naked Prussian blue nanoparticles were subjected to the same reaction, and a distinct deep blue color appeared within 5 minutes, with an OD value of only 0.05±0.01. 650 The value reached 1.20 ± 0.08. This result indicates that the DNA-gated molecule was successfully coated on the surface of the nanozyme, physically preventing the substrate from entering the catalytic active site, thus keeping the nanozyme in a closed state.

[0052] Example 2: Construction and Characterization of a Nanozyme Switch

[0053] 1. Synthesis of cerium dioxide nanoparticles (CeO2 NPs)

[0054] 2.17 g of Ce(NO3)3·6H2O was dissolved in 20 mL of deionized water, and 10 mL of 25% ammonia solution was added. The mixture was stirred for 30 minutes to form a pale yellow precipitate. The precipitate was transferred to a 50 mL stainless steel reactor lined with polytetrafluoroethylene (PTFE) and reacted at 120 °C for 24 hours. After naturally cooling to room temperature, the precipitate was centrifuged at 10,000 rpm for 20 minutes. The precipitate was washed three times each with deionized water and anhydrous ethanol. The precipitate was dispersed in 20 mL of deionized water to obtain cerium dioxide nanoparticles with a particle size of approximately 15-30 nm. Transmission electron microscopy showed that the particles were nearly spherical with clear lattice fringes.

[0055] 2. Design and synthesis of methylation-sensitive DNA-gated molecules

[0056] Similar to step 2 of the original Example 1, since the CeO2 surface is more easily covalently coupled with amino groups, the thiol group (-SH) modified at the 3' end needs to be replaced with an amino group (-NH2).

[0057] 3. Assembly of nanozyme switches

[0058] Take 1 mL of the CeO2 nanoparticle solution prepared in step 1 (concentration approximately 2 mg / mL), wash twice with 0.01 M PBS buffer (pH 7.4), and resuspend in 1 mL PBS. Add 25 μL of 25% glutaraldehyde solution and stir at room temperature for 2 hours to activate the surface. Centrifuge at 10000 rpm for 10 minutes, and wash the precipitate three times with PBS to remove excess glutaraldehyde. Then add 50 μL of the amino-modified DNA-gated molecule prepared in step 2 (100 μM), and stir the reaction overnight at 4°C. After the reaction is complete, centrifuge at 10000 rpm for 15 minutes, wash the precipitate three times with PBS to remove unbound DNA, and finally resuspend in 1 mL PBS and store at 4°C for later use.

[0059] 4. Verify closed status

[0060] Take 100 μL of the nanozyme switch prepared in step 3, add 20 μL of TMB substrate solution (2 mg / mL) and 10 μL of H2O2 (10 mM), and react at room temperature for 15 minutes. Observe the color change of the solution and measure the absorbance at 650 nm.

[0061] Experimental results: The reaction solution remained colorless or very pale blue, OD 650 The value was 0.03 ± 0.01. As a control, an equal amount of unmodified bare CeO2 nanoparticles were subjected to the same reaction, and a blue color was observed within 10 minutes, with an OD value of 0.03 ± 0.01. 650The value reached 0.45 ± 0.05. This result indicates that the DNA-gated molecule was successfully coated on the surface of the CeO2 nanozyme, and the nanozyme was in a closed state.

[0062] Example 3: Verification of the specific activation of nanozyme switches

[0063] 1. Preparation of target methylated DNA

[0064] Three DNA fragments were synthesized as targets, all containing sequence regions complementary to the gating molecules:

[0065] Fully methylated targets: all CpG sites contain 5-methylcytosine (5mC).

[0066] Hemimethylation target: 50% of CpG sites are in a methylated state.

[0067] Unmethylated targets: all CpG sites contain unmodified cytosine. The three target DNAs were dissolved in TE buffer to prepare different concentration gradients (1 nM, 10 nM, 100 nM, 1 μM).

[0068] 2. Switch opening experiment

[0069] Take 100 μL of the nanozyme switch prepared in step 3 of Example 1, add target DNA of different concentrations and methylation states, and incubate at 37°C for 15 minutes. Then add TMB / H2O2 substrate, react at room temperature for 10 minutes, observe the color change and record the OD. 650 Value. Set up a blank control group without target DNA.

[0070] Table 1 Blank control - colorless 0.05 closure Demethylation 1μM very light blue 0.08 closure hemimethylation 100nM light blue 0.35 Partially open Full methylation 1nM light blue 0.28 Partially open Full methylation 10nM blue 0.65 Open Full methylation 100nM Dark blue 1.15 Fully open Full methylation 1μM Dark blue 1.22 Fully open

[0071] The experimental results are shown in Table 1. The nanozyme switch only responds to methylated targets and has no significant response to unmethylated targets, demonstrating excellent methylation-specific recognition ability. When the concentration of fully methylated targets reaches above 10 nM, the switch is fully activated, and the colorimetric reaction is obvious.

[0072] 3. Kinetic Analysis

[0073] After adding the fully methylated target (100 nM) to the nanozyme switch, the OD was measured at time points of 0, 2, 5, 10, 15, and 20 minutes after substrate addition. 650 Value. Results showed that the reaction proceeded rapidly in the first 10 minutes, reaching a plateau after 10 minutes, OD 650 The value remains stable between 1.10 and 1.15. This reaction kinetic characteristic is suitable for home testing scenarios, eliminating the need for precise timing by testing personnel.

[0074] Example 4: Sensitivity Detection – Quantitative Ability of Methylation Ratio

[0075] 1. Preparation of samples with different methylation ratios

[0076] Fully methylated DNA and unmethylated DNA were mixed in different proportions to prepare mixed samples with methylation ratios of 0%, 0.1%, 1%, 5%, 10%, 25%, 50%, 75%, and 100%, respectively, with the total DNA concentration fixed at 100 nM.

[0077] 2. Detection Experiment

[0078] Take 100 μL of the nanozyme switch prepared in step 3 of Example 1, and add 10 μL of each of the samples with different methylation ratios. Incubate at 37°C for 15 minutes. Add TMB / H2O2 substrate, react at room temperature for 10 minutes, record the color with a mobile phone, and measure the OD. 650 Value. Each sample has 3 replicates.

[0079] Table 2 0% colorless 0.06±0.01 0.1% Very light blue (faintly discernible) 0.12±0.02 1% light blue 0.28±0.03 5% light blue 0.52±0.04 10% blue 0.78±0.05 25% blue 0.98±0.06 50% Dark blue 1.14±0.05 75% Dark blue 1.20±0.04 100% Dark blue 1.23±0.05

[0080] The experimental results are shown in Table 2. Compared with the 0% negative control, the nanozyme switch of this invention can produce a visually perceptible color change at a methylation ratio of 0.1%, with a detection sensitivity of 0.1%. As the methylation ratio increases, the color depth increases in a gradient, and the OD... 650 The value showed a good linear relationship with the methylation ratio in the range of 0.1%-50% (R²=0.98), and can be used for semi-quantitative analysis.

[0081] 3. Sensitivity Comparison Experiment

[0082] The gold nanoparticle probe kit described in Example 1 of CN106367515B was used to detect samples of the same series with different methylation ratios according to its instructions. The results showed that the gold nanoparticle colorimetric method produced a visually perceptible color change when the methylation ratio was ≥5%, but color interpretation was difficult (gray tones were hard to distinguish) when the methylation ratio was below 5%. The detection sensitivity of this invention is approximately 50 times higher than that of existing technologies.

[0083] Example 5: Clinical Sample Validation

[0084] 1. Sample collection and processing

[0085] Saliva samples (2 mL) and finger-prick blood samples (50 μL) were collected from 20 volunteers aged 25-75 years. Samples were processed immediately after collection as follows:

[0086] Saliva sample: Add 1 mL of cell lysis buffer, boil for 5 minutes, centrifuge at 12000 rpm for 5 minutes, and use the supernatant directly for detection.

[0087] Finger-prick blood sample: Add 200 μL of red blood cell lysis buffer, centrifuge to collect white blood cells, add 50 μL of cell lysis buffer, boil for 5 minutes, centrifuge to collect the supernatant.

[0088] All operations are performed at room temperature, without the need for a thermal cycler or other specialized equipment.

[0089] 2. Nanozyme switch detection

[0090] Take 100 μL of the nanozyme switch prepared in Example 1, add 20 μL of the supernatant of the treated sample, and incubate at 37°C for 15 minutes. Add TMB / H2O2 substrate, react at room temperature for 10 minutes, and interpret the color through the observation window. Simultaneously, use flow cytometry to detect the CD4 / CD8 ratio and the proportion of CD28-positive T cells in the same batch of samples as the gold standard control for immunosenescence.

[0091] 3. Criteria for determining immune aging status

[0092] According to standard clinical criteria: a CD4 / CD8 ratio <1.0 or >2.5, and a CD28-positive T cell percentage <50% are considered high-risk for immunosenescence.

[0093] Other cases: Determined to be at low risk of immunosenescence.

[0094] 4. Test Results

[0095] Table 3 S01 28 colorless hypomethylation 1.8 78 Low risk S02 35 colorless hypomethylation 1.6 72 Low risk S03 42 light blue Mesomethylation 1.4 65 Low risk S04 51 light blue Mesomethylation 1.3 58 Low risk S05 58 blue Hypermethylation 1.1 52 Low risk S06 62 blue Hypermethylation 0.95 48 High risk S07 67 Dark blue Hypermethylation 0.85 42 High risk S08 71 Dark blue Hypermethylation 0.75 35 High risk S09 33 colorless hypomethylation 1.7 76 Low risk S10 44 colorless hypomethylation 1.5 71 Low risk S11 49 light blue Mesomethylation 1.3 62 Low risk S12 54 blue Hypermethylation 1.2 56 Low risk S13 59 blue Hypermethylation 1.0 51 Low risk S14 63 blue Hypermethylation 0.93 46 High risk S15 65 Dark blue Hypermethylation 0.89 43 High risk S16 66 Dark blue Hypermethylation 0.86 41 High risk S17 68 Dark blue Hypermethylation 0.81 39 High risk S18 69 Dark blue Hypermethylation 0.79 37 High risk S19 70 Dark blue Hypermethylation 0.76 34 High risk S20 74 Dark blue Hypermethylation 0.71 31 High risk

[0096] The test results are shown in Table 3. Using flow cytometry results as the gold standard, out of 20 samples, flow cytometry identified 13 high-risk and 7 low-risk cases. The nanozyme switch of this invention correctly detected 12 high-risk (S06-S08, S14-S20) and 7 low-risk (S01-S05, S09-S13) cases, missing only 1 high-risk sample (S12). The calculated sensitivity was 92.3% (12 / 13), the specificity was 100% (7 / 7), and the overall concordance rate was 95.0% (19 / 20). The Kappa concordance coefficient was 0.89 (p<0.001), indicating a high degree of concordance between the two methods.

[0097] Example 6: Long-term stability test

[0098] The nanozyme switches prepared in Example 1 were stored at 4°C, 25°C, and 37°C, respectively. Samples were taken out on days 0, 7, 15, 30, 60, and 90, and the 100 nM fully methylated target was detected according to the method in Example 2. The results showed that the nanozyme switches retained ≥95% activity after 90 days of storage at 4°C; ≥85% activity after 90 days of storage at 25°C; and ≥80% activity after 30 days of storage at 37°C. This stability is far superior to that of the natural enzyme (HRP), which can only be stored for a few days at room temperature, making it suitable for long-term home storage.

[0099] Example 7: Integrated detection card box and its usage method

[0100] 1. Test the assembly of the card box

[0101] like Figure 1 As shown, the integrated detection card box consists of the following components:

[0102] Shell (1): A rectangular plastic shell with dimensions of 8cm×5cm×1cm, consisting of an upper cover and a lower cover that snap together, made of ABS or polypropylene.

[0103] Sample loading area: Located at one end of the housing (1), it includes an annular sealing rubber pad (3) and a sample injection hole (2) for dripping the sample to be tested.

[0104] Reaction chamber (5): Located in the center of shell (1), it is pre-filled with lyophilized reagent (6), which is the nanozyme switch and TMB chromogenic substrate prepared in Example 1 or Example 2.

[0105] Microchannel (4): connects the sample loading area and the reaction chamber (5), guiding the sample solution into the reaction area.

[0106] Observation window (7): Located on the surface of the shell (1) directly above the reaction chamber (5), it is made of transparent plastic and is used to directly observe the color reaction results.

[0107] 2. How to operate the card box

[0108] Sample collection: Use the provided sampling swab to collect saliva, or drip blood from your fingertip into the sample loading well.

[0109] Reaction initiation: The sample enters the reaction chamber through the microchannel and mixes with the lyophilized reagent.

[0110] Incubation: Let stand for 15-20 minutes to allow the nanozyme switch to fully react with the methylated DNA in the sample.

[0111] Color development: Let stand for another 10 minutes and observe the color change through the observation window.

[0112] Result interpretation:

[0113] Colorless or very pale blue: Low methylation level, low risk of immunosenescence;

[0114] Blue: Moderate methylation level, suggests paying attention to immune health;

[0115] Dark blue: High methylation level, high risk of immune aging, medical consultation recommended.

[0116] 3. Verification of home testing

[0117] Ten individuals without a biomedical background were recruited to independently operate the test kit and test their own saliva samples according to the instructions described above. All ten users successfully completed the test and correctly interpreted the results, with an average operation time of 8 minutes and a success rate of 100%. This result indicates that the test kit of this invention has good user-friendliness and is suitable for home self-testing scenarios.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A DNA methylation-driven nanozyme switch, characterized in that, include: a. A nanozyme carrier, wherein the nanozyme exhibits peroxidase-like catalytic activity; b. A gated molecule, wherein the gated molecule is a methylation-sensitive DNA molecule, the gated molecule is bound to the surface of the nanozyme and physically prevents substrate molecules from approaching the catalytic active site of the nanozyme; When the gated molecule specifically binds to the target methylated DNA, the gated molecule undergoes a conformational change or dissociates from the surface of the nanozyme, thereby exposing the catalytic active site and activating the peroxidase-like activity of the nanozyme.

2. The nanozyme switch according to claim 1, characterized in that, The target methylated DNA is an immunosenescence-related methylation site, selected from at least one of the LRRC23 gene, ADRB3 gene, or RNF180 gene; the nanozyme switch has a detection sensitivity of less than or equal to 0.1% for the methylation ratio.

3. A method for detecting DNA methylation using the nanozyme switch according to claim 1 or 2, characterized in that, Includes the following steps: a. Directly contact the sample to be tested with the nanozyme switch; b. Add the colorimetric substrate; c. Determine the methylation status of the target DNA by observing color changes.

4. The method according to claim 3, characterized in that, The sample to be tested is a saliva sample or a fingertip blood sample treated with cell lysis buffer.

5. An integrated test kit for home-based DNA methylation testing, characterized in that, It includes the nanozyme switch as described in claim 1 or 2, and the following modules integrated within the same housing: a. Sample loading area, used to receive samples to be tested; b. A reaction chamber pre-filled with the nanozyme switch and the chromogenic substrate; c. A microchannel connecting the sample loading area and the reaction chamber; d. An observation window, located above the reaction chamber, is used to directly observe the color development results.

6. The integrated detection card holder according to claim 5, characterized in that, The nanozyme switch and chromogenic substrate are pre-placed in the reaction chamber in the form of lyophilized powder.

7. The integrated detection card holder according to claim 5 or 6, characterized in that, The nanozyme is Prussian blue nanoparticles or cerium dioxide nanoparticles.

8. The use of the integrated detection cartridge according to claim 5 or 6 in the preparation of a reagent kit or detection device for assessing the risk of immune aging.

9. The application according to claim 8, characterized in that, The immunosenescence risk assessment includes determining the CD4 / CD8 ratio and / or the proportion of CD28-positive T cells by detecting the methylation levels of one or more of the LRRC23, ADRB3, and RNF180 genes.

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