Nanometer enzyme catalysis enhanced quantum precision measurement method

Abstract

The invention discloses a nano-enzyme catalysis enhanced quantum precision measurement method, which is characterized in that hydrogen peroxide is catalyzed by a nano-enzyme composite system to generate free radicals, the paramagnetism and spin noise characteristics of the free radicals can generate physical disturbance on the NV color center quantum state, and the change of the concentration of the free radicals can obviously cause the deviation of the peak value of a diamond NV color center ODMR spectrogram. Therefore, a strategy for quantum precision measurement is established; the method comprises the following analysis steps: introducing nano-enzyme to catalyze and generate free radicals to change the ODMR spectrogram change of the NV color center; the fluorescence difference before and after nano enzyme modification and catalysis and the change of ODMR spectrogram peak offset are collected to realize accurate determination of the detection signal intensity; according to the method, the NV color center fluorescence can be enhanced, the detection sensitivity is improved, and the technology has wide application prospects in the precise detection fields such as biological single molecule detection and in-situ analysis of living bodies.

Description

Technical Field

[0001] This invention belongs to the field of quantum measurement and biodetection technology, specifically relating to a method for enhancing quantum precision measurement through nanozyme catalysis. Background Technology

[0002] Diamond nitrogen-vacancy (NV) centers, as promising quantum sensors, have demonstrated outstanding performance in the nanoscale precision measurement of physical quantities such as magnetic fields, electric fields, and temperature, as well as in biomolecular imaging. Based on photodetector magnetic resonance (ODMR) technology, NV centers can achieve non-invasive detection of micro-Tesla-level magnetic fields and achieve high-resolution readout of magnetic field signals through photofluorescence and microwave modulation. However, current technological bottlenecks significantly limit their application expansion: on the one hand, the fluorescence signal intensity of a single NV center is relatively low, resulting in insufficient signal-to-noise ratio in weak signal scenarios such as low-concentration sample detection; on the other hand, environmental noise interference limits further improvement in its quantum measurement accuracy. Establishing a nanozyme-NV center system combines the catalytic signal amplification capability of nanozymes with the physical limit sensitivity of quantum sensors. Designing nanozymes based on gold nanoparticles can enhance the fluorescence intensity of NV centers while simultaneously initiating catalytic reactions to generate free radicals and spin noise, achieving ultrasensitive detection of low-concentration biomolecules.

[0003] Chinese patent CN202311098137.0 describes a fluorescent nanodiamond / polyethyleneimine / gold nanoparticle composite material and its preparation method. This composite material can enhance the fluorescence performance of fluorescent nanodiamonds by utilizing the surface plasmon resonance domain of gold nanoparticles. Chinese patent 202410453030.1 describes a method for preparing one-dimensional photonic crystals and nano-noble metals to enhance the fluorescence of diamond NV-centers on the diamond surface using a template method and magnetron sputtering. However, the above analytical methods are complex to operate in practice and can only regulate the fluorescence performance of NV-centers. Summary of the Invention

[0004] To address the aforementioned issues, this invention discloses a method for enhancing quantum precision measurement through nanozyme catalysis. By catalyzing hydrogen peroxide to generate free radicals through a nanozyme composite system, changes in the concentration of free radicals significantly shift the peak values ​​of the ODMR spectrum of diamond NV color centers, thereby significantly improving detection accuracy.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: A method for enhancing quantum precision measurement through nanozyme catalysis includes the following steps: (a) Selecting and preparing nanozymes with high catalytic activity; (b) Constructing a nanozyme probe-miRNA-fluorescent nanodiamond probe system; (c) Hydrogen peroxide is added to the nanozyme probe-miRNA-fluorescent nanodiamond probe system to catalyze the reaction and generate free radicals. Quantum precision measurement is achieved by enhancing the fluorescence intensity of the NV center and changing the ODMR spectrum of the NV center.

[0006] Furthermore, in step (a), the nanozyme is Au-Co / NC. Preparation steps: S1. Cobalt chloride hexahydrate solution was slowly added to 2-methylimidazole solution, and after stirring continuously for 24 h, the metal-organic framework material ZIF67 was obtained by centrifugation, washing and drying. S2. ZIF67 was subjected to high-temperature annealing in a nitrogen atmosphere to obtain Co / NC; S3. Add chloroauric acid solution and sodium citrate reducing agent to Co / NC solution to grow Au nanoparticles in situ to obtain nanozyme Au-Co / NC.

[0007] Furthermore, in step (b), the diamond is a nanodiamond containing nitrogen vacancy centers, denoted as FNDs; the average particle size of the FNDs is approximately 100 nm; and the surface of the FNDs is modified with carboxyl groups.

[0008] In step S1, the concentration of the cobalt chloride hexahydrate solution is 50–100 mM, and the concentration of the 2-methylimidazole solution is 200–400 mM, such that the molar ratio of the cobalt chloride hexahydrate solution to the 2-methylimidazole solution is 1:4–1:8.

[0009] In step S2, the nitrogen flow rate is 70–80 mL / min. –1 The heating rate is 2-5℃ / min, the annealing temperature is 600-800℃, and the holding time is 1.5-2h.

[0010] In step S3, the mass ratio of Au nanoparticles to Co / NC is 1:4, and the molar ratio of sodium citrate to chloroauric acid is 1:3 to 1:5.

[0011] In step (b), the diamond is a nanodiamond containing nitrogen vacancy centers, denoted as FNDs; the average particle size of the FNDs is about 100-200 nm; and the surface of the FNDs is modified with carboxyl groups.

[0012] In step (b), the specific process for constructing the nanozyme probe-miRNA-fluorescent nanodiamond probe system is as follows: S4. Preparation of nanozyme probes; S5. Preparation of fluorescent nanodiamond probes; S6, Prepare a mixed solution containing a coupling structure of nanozyme probe-miRNA and fluorescent nanodiamond probe.

[0013] The specific steps in S4 are as follows: Add 5 μL of 1M tris(2-carboxyethyl)phosphine solution to the thio-modified DNA probe, let it stand for half an hour to fully reduce the thio groups, then add diluted Au-Co / NC and incubate for 3 hours to couple Au-Co / NC and DNA probe through gold-sulfur bonds. After centrifugation and washing, add 6-mercapto-1-hexanol and bovine serum albumin to block the excess sites on the nanozyme, and then resuspend in 100-400 μL of deionized water to obtain the nanozyme probe solution.

[0014] The specific steps in S5 are as follows: Take the surface-carboxylated fluorescent nanodiamonds, add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide, mix well and let stand for 3 hours, then add the amino-modified DNA probe, incubate for 3-6 hours, centrifuge and wash, and dissolve in 300 μL of deionized water to obtain the fluorescent nanodiamond probe solution.

[0015] S6 specifically involves the following steps: Take the nanozyme probe solution and the fluorescent nanodiamond probe in deionized water, add the miRNA to be tested, react on a shaker for 6-12 hours, centrifuge and wash three times to obtain a mixed solution containing the coupled structure of nanozyme probe, miRNA and fluorescent nanodiamond probe.

[0016] In step (c), the method for precise quantum measurement of NV color centers catalyzed by nanozymes according to claim 1 is characterized in that its working principle is to introduce nanozymes to regulate the fluorescence intensity of NV color centers of fluorescent nanodiamonds and to introduce nanozymes to catalyze quantitative hydrogen peroxide to regulate the quantum spin state of fluorescent nanodiamonds, and to collect and analyze the fluorescence differences before and after nanozyme modification and the ODMR spectrum changes of hydrogen peroxide catalyzed by nanozymes to achieve accurate determination of the intensity of the detection signal.

[0017] The beneficial effects of this invention are as follows: Nanozymes combined with fluorescent nanodiamonds catalyze the generation of free radicals from hydrogen peroxide, causing a shift in the ODMR peak of the NV center. Different concentrations of miRNA are coupled with different amounts of nanozyme probes and FND probes to catalyze the generation of different amounts of free radicals from quantitative hydrogen peroxide, causing different degrees of shift in the ODMR peak of the NV center. This method utilizes the high catalytic activity of nanozymes and the excellent fluorescence and tunable properties of the NV center to reduce interference from environmental factors during the detection process, significantly improving detection accuracy and achieving ultrasensitive detection of miRNA. Attached Figure Description

[0018] Figure 1 This is an experimental schematic diagram of a method for enhancing quantum precision measurement through nanozyme catalysis as described in this invention.

[0019] List of identifiers in attached diagrams: 1 is an Au-Co / NC nanozyme probe, 2 is a fluorescent nanodiamond probe, 3 is a hydrogen peroxide molecule, and 4 is a free radical catalyzing the generation of hydrogen peroxide.

[0020] Figure 2 The graph shows the change in fluorescence intensity of fluorescent nanodiamonds before and after modification with nanozymes.

[0021] Figure 3 A comparison of ODMR peaks between FNDs and nanozyme probe-miRNA-FNDs probe after the introduction of nanozymes to initiate the catalytic reaction. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Example

[0023] Methods for enhancing quantum precision measurement through nanozyme catalysis, including I. Preparation of Au-Co / NC nanozymes, the specific steps are as follows: a: Using methanol as solvent, prepare a 200 mM 2-methylimidazole solution and a 50 mM cobalt chloride hexahydrate solution. Slowly add the cobalt chloride hexahydrate solution to the 2-methylimidazole solution at a volume ratio of 1:1. Then, stir the reaction at room temperature for 24 hours, collect the reaction mixture, centrifuge and wash, repeat three times, and collect the product after vacuum drying to obtain ZIF67.

[0024] b: In a nitrogen atmosphere, heat to 800℃ at a heating rate of 5℃ / min, hold for 2 hours, and use a gas flow rate of 80 mL / min. – 1 ZIF67 was subjected to high-temperature annealing to obtain Co / NC.

[0025] c: Add 10.15 mL of 1 mM chloroauric acid solution and 609 μL of 38.8 mM sodium citrate solution to 10 mg Co / NC, heat to boiling and react for 15 min, collect the product, centrifuge and wash 3 times to obtain the nanozyme Au-Co / NC.

[0026] II. Preparation of nanozyme probes, the specific steps are as follows: Dissolve a 0.5 OD DNA probe modified with thio groups to 100 μM, add 5 μL of 1 M tris(2-carboxyethyl)phosphine solution, let stand for half an hour to fully reduce the thio groups, then add 0.25 mg of Au-Co / NC and incubate for 3 hours to couple Au-Co / NC and DNA probe through gold-sulfur bonds. After centrifugation and washing, add 6-mercapto-1-hexanol and bovine serum albumin to block excess sites on the nanozyme, and then resuspend in 300 μL of deionized water to obtain the nanozyme probe solution.

[0027] III. Preparation of fluorescent nanodiamond probes, the specific steps are as follows: Take 100 μg of surface-carboxylated fluorescent nanodiamonds, add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide, mix well and let stand for 3 hours, then add amino-modified DNA probes, incubate at room temperature for 3 hours, centrifuge and wash three times, and dissolve in 1 mL of deionized water to obtain probe-modified fluorescent nanodiamond solution.

[0028] IV. Preparation of the conjugated structure of nanozyme probe, miRNA21, and fluorescent nanodiamond probe, with the following specific steps: Take 10 μL of nanozyme probe and 10 μL of fluorescent nanodiamond probe solution in 1 mL of deionized water, add the target miRNA21, and incubate on a shaker for 4 hours. After centrifugation and washing three times, dissolve in 100 μL of deionized water to obtain a mixed detection solution containing the coupled structure of nanozyme probe, miRNA21 and fluorescent nanodiamond probe.

[0029] V. Fluorescent detection of miRNA21, the specific steps are as follows: 20 μL of the above mixed detection solution was dropped onto a silicon wafer substrate and dried at room temperature. The substrate was then placed under the objective lens of a fluorescence spectrometer with a confocal system. A fluorescence signal near 632 nm was collected using 532 nm excitation light to achieve sensitive detection of miRNA21.

[0030] VI. Nanozymes regulate the fluorescence intensity of nanodiamonds and quantify the concentration of miRNA21. The specific steps are as follows: The luminescence efficiency of fluorescent nanodiamonds was altered by utilizing the surface plasmon resonance effect of nanozymes. Different concentrations of miRNA21 were coupled to these nanozymes, and the changes in fluorescence intensity at 632 nm were recorded using a fluorescence spectrometer. The results are shown in the attached figure. Figure 3 As shown, changes in fluorescence intensity can be used to analyze data and thus quantify the concentration of miRNA21. Example

[0031] The method for enhancing quantum precision measurement through nanozyme catalysis described in this embodiment differs from that in Embodiment 1 in that the detection method in the fifth step is different. Its function is to directly detect the electron spin quantum state to obtain the core quantum parameter ODMR spectrum, which has the sensing capability of higher sensitivity at the single-molecule level and stronger resistance to interference from complex environments.

[0032] I. Preparation of Au-Co / NC nanozymes, the specific steps are as follows: a: Using methanol as solvent, prepare a 200 mM 2-methylimidazole solution and a 50 mM cobalt chloride hexahydrate solution. Slowly add the cobalt chloride hexahydrate solution to the 2-methylimidazole solution at a volume ratio of 1:1. Then, stir the reaction at room temperature for 24 hours, collect the reaction mixture, centrifuge and wash, repeat three times, and collect the product after vacuum drying to obtain ZIF67.

[0033] b: In a nitrogen atmosphere, heat to 800℃ at a heating rate of 5℃ / min, hold for 2 hours, and use a gas flow rate of 80 mL / min. – 1 ZIF67 was subjected to high-temperature annealing to obtain Co / NC.

[0034] c: Add 10.15 mL of 1 mM chloroauric acid solution and 609 μL of 38.8 mM sodium citrate solution to 10 mg Co / NC, heat to boiling and react for 15 min, collect the product, centrifuge and wash 3 times to obtain the nanozyme Au-Co / NC.

[0035] II. Preparation of nanozyme probes, the specific steps are as follows: Dissolve a 0.5 OD DNA probe modified with thio groups to 100 μM, add 5 μL of 1 M tris(2-carboxyethyl)phosphine solution, let stand for half an hour to fully reduce the thio groups, then add 0.25 mg of Au-Co / NC and incubate for 3 hours to couple Au-Co / NC and DNA probe through gold-sulfur bonds. After centrifugation and washing, add 6-mercapto-1-hexanol and bovine serum albumin to block excess sites on the nanozyme, and then resuspend in 300 μL of deionized water to obtain the nanozyme probe solution.

[0036] III. Preparation of fluorescent nanodiamond probes, the specific steps are as follows: Take 100 μg of surface-carboxylated fluorescent nanodiamonds, add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide, mix well and let stand for 3 hours, then add amino-modified DNA probes, incubate at room temperature for 3 hours, centrifuge and wash three times, and dissolve in 1 mL of deionized water to obtain probe-modified fluorescent nanodiamond solution.

[0037] IV. Preparation of the coupling structure of nanozyme, miRNA, and fluorescent nanodiamond, the specific steps are as follows: Take 10 μL of nanozyme probe and 10 μL of fluorescent nanodiamond probe solution in 1 mL of deionized water, add the target miRNA21, and incubate on a shaker for 4 hours. After centrifugation and washing three times, dissolve in 100 μL of deionized water to obtain a mixed detection solution containing the coupled structure of nanozyme probe, miRNA21 and fluorescent nanodiamond probe.

[0038] V. Quantum spin signal detection of miRNA21, the specific steps are as follows: 20 μL of the above mixed detection solution, 20 μL of 10 mM hydrogen peroxide solution, and 20 μL of pH 5.5 acetate-sodium acetate solution were drop-coated onto a silicon substrate and dried at room temperature. An antenna was then used to radiate 2.87 GHz pulsed microwaves under the action of an external radio frequency microwave source to alter the quantum spin population of nitrogen-vacancy color centers in the fluorescent nanodiamond nanoparticles in the mixed solution. The changes in the peak values ​​of the ODMR spectra of the mixed detection solution and pure diamond nanoparticles under microwave irradiation were collected to achieve sensitive detection of miRNA21. The results are attached. Figure 3 As shown.

[0039] VI. Nanozymes regulate the quantum spin state of nanodiamonds and quantify the concentration of miRNA21. The specific steps are as follows: Nanozymes were used to catalyze hydrogen peroxide to generate free radicals, thereby altering the magnetic field environment and the ODMR spectrum of fluorescent nanodiamonds. Nanozyme probes and fluorescent nanodiamonds were coupled with different concentrations of miRNA21, and the changes in ODMR spectrum peaks were recorded. The data were then analyzed to quantify the miRNA21 concentration.

[0040] Combining Examples 1 and 2, the low-abundance miRNA sensitive detection method based on quantum precision measurement of the present invention introduces nanozymes to regulate the fluorescence intensity of the fluorescent nanodiamond NV color center and introduces nanozymes to catalyze hydrogen peroxide to regulate the quantum spin state of FND. By collecting and analyzing the fluorescence difference before and after nanozyme modification and the shift of the ODMR spectrum peak of nanozyme-catalyzed hydrogen peroxide, the intensity of the detection signal is accurately determined. This method enhances quantum precision detection through nanozyme catalysis, enabling sensitive detection and precise concentration quantification of ultra-low concentration miRNAs. Compared with existing miRNA detection techniques, the present invention can achieve ultrasensitive single-molecule detection without complex molecular amplification techniques. In Example 2, quantum detection has higher sensitivity at the single-molecule level and stronger resistance to interference from complex environments, showing great application potential in the field of early biomarker detection for major diseases.

[0041] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A method for enhancing quantum precision measurement through nanozyme catalysis, characterized in that: Includes the following steps: (a) Selecting and preparing nanozymes with high catalytic activity; (b) Constructing a nanozyme probe-miRNA-fluorescent nanodiamond probe system; (c) Hydrogen peroxide is added to the nanozyme probe-miRNA-fluorescent nanodiamond probe system to catalyze the reaction and generate free radicals. Quantum precision measurement is achieved by enhancing the fluorescence intensity of the NV center and changing the ODMR spectrum of the NV center.

2. The method for enhancing quantum precision measurement through nanozyme catalysis according to claim 1, characterized in that, In step (a), the nanozyme is Au-Co / NC, and the preparation steps are as follows: S1. Cobalt chloride hexahydrate solution was added to 2-methylimidazole solution, and after stirring continuously for 24 h, the metal-organic framework material ZIF67 was obtained by centrifugation, washing and drying. S2. ZIF67 was subjected to high-temperature annealing in a nitrogen atmosphere to obtain Co / NC; S3. Add chloroauric acid solution and sodium citrate reducing agent to Co / NC solution to grow Au nanoparticles in situ to obtain nanozyme Au-Co / NC.

3. The method according to claim 2, characterized in that, In step S1, the concentration of cobalt chloride hexahydrate solution is 50-100 mM, the concentration of 2-methylimidazole solution is 200-400 mM, and the molar ratio of cobalt chloride hexahydrate to 2-methylimidazole solution is 1:4-1:

8.

4. The method according to claim 2, characterized in that, In step S2, the nitrogen flow rate is 70–80 mL / min. – 1 The heating rate is 2-5℃ / min, the annealing temperature is 600-800℃, and the holding time is 1.5-2h.

5. The method according to claim 2, characterized in that, In step S3, the mass ratio of Au nanoparticles to Co / NC is 1:4, and the molar ratio of sodium citrate to chloroauric acid is 1:3 to 1:

5.

6. The method according to claim 1, characterized in that, In step (b), the diamond is a nanodiamond containing nitrogen vacancy centers, denoted as FNDs; the average particle size of the FNDs is about 100-200 nm; and the surface of the FNDs is modified with carboxyl groups.

7. The method according to claim 1, characterized in that, In step (b), the method for constructing the nanozyme probe-miRNA-fluorescent nanodiamond probe system is characterized by the following specific process: S4. Preparation of nanozyme probes and fluorescent nanodiamond probes; S5. Prepare a mixed solution containing a conjugated structure of nanozyme probe-miRNA and fluorescent nanodiamond probe.

8. The method according to claim 7, characterized in that, S4 specifically involves: adding tris(2-carboxyethyl)phosphine to the thio-modified DNA probe to fully reduce the thio groups, then adding diluted Au-Co / NC to couple the Au-Co / NC and DNA probe via a gold-sulfur bond. After centrifugation and washing, 6-mercapto-1-hexanol and bovine serum albumin are added to block excess sites on the nanozyme, and then it is resuspended in 100-400 μL of deionized water to obtain the nanozyme probe solution. Take the surface-carboxylated fluorescent nanodiamonds, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, mix well and let stand for 3 hours, then add the amino-modified DNA probe, incubate for 3-6 hours, centrifuge and wash, and dissolve in 300 μL of deionized water to obtain the fluorescent nanodiamond probe solution.

9. The method according to claim 7, characterized in that, S5 specifically involves: taking the nanozyme probe solution and the fluorescent nanodiamond probe in deionized water, adding the miRNA to be tested, reacting on a shaker for 6–12 h, centrifuging and washing three times to obtain a mixed solution containing the coupled structure of nanozyme probe, miRNA, and fluorescent nanodiamond probe.

10. The method according to claim 1, characterized in that, In step (c), the working principle is to introduce nanozymes to regulate the fluorescence intensity of the NV color center of fluorescent nanodiamonds and to introduce nanozymes to catalyze quantitative hydrogen peroxide to regulate the quantum spin state of fluorescent nanodiamonds. The fluorescence difference before and after nanozyme modification and the ODMR spectrum change of hydrogen peroxide catalyzed by nanozymes are collected and analyzed to achieve accurate determination of the detection signal intensity.

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