Preparation method of controllable NV color center micro brake, product of controllable NV color center micro brake and application of controllable NV color center micro brake in biological detection

Abstract

The invention discloses a preparation method of a controllable NV color center micro brake, a product of the controllable NV color center micro brake and application of the controllable NV color center micro brake in biological detection. According to the micro brake, active control, enrichment, release and the like of the fluorescent nano-diamond NV color center can be achieved, an efficient active control sensing mechanism is constructed, the transmission efficiency of a target object is greatly improved, and more sensitive judgment can be provided for diagnosis of diseases such as tumors and cancers. The Janus particle has a hollow porous structure and a unilaterally distributed gold film, can directionally and rapidly move under the action of H2O2 and near-infrared laser, and can accurately capture and enrich fluorescent nano-diamond particles modified with a detection probe; then adding to-be-detected substances MUC1 with different concentrations to compete with the complementary DNA chain so as to realize release of the fluorescent nano-diamond from the surface of the micro brake, and finally realizing ultra-sensitive detection through a fluorescence spectrum.

Description

Technical Field

[0001] The present invention relates to a preparation method of a manipulable NV color center micro-brake, its product and application in biological detection, belonging to the field of micro-nano brakes. Background Art

[0002] Micro / nano motors / brakes, also known as micro-nano robots, refer to a class of micro-nano scale intelligent bionic materials with dimensions in the micro-nano level that can convert various forms of energy (such as light energy, electrical energy, magnetic energy, ultrasonic energy, and chemical energy, etc.) into kinetic energy to complete special tasks. Micro / nano motors have the advantages of high stability, simple preparation process, wide application range, etc., and show great application potential in the fields of biosensing, drug delivery, disease diagnosis and treatment, bionic materials, and environmental remediation.

[0003] Exosomes (EXO) are extracellular vesicles with a diameter of 30 - 150 nm, containing a variety of proteins, peptides, nucleic acids, lipids, and metabolites. A large number of studies have shown that exosomes, as non-invasive biomarkers, are of great significance for the diagnosis, treatment monitoring, and prognosis evaluation of breast cancer. The most commonly used methods for exosome membrane protein analysis are enzyme-linked immunosorbent assay (ELISA), Western Blot (WB), and nano flow cytometry (nFC). However, the practical application of these methods is hindered due to the need for complex instruments or laborious procedures or limited sensitivity. Currently, new technologies are needed for cancer diagnosis based on the detection of exosome proteins. Traditional fluorescence detection means such as using dye molecules have disadvantages such as weak signals, photobleaching, and low stability. At the same time, since the detection probe belongs to passive diffusion during the detection process, the efficiency of capturing target substances such as protein polypeptides in complex biological samples is low, which greatly limits the accurate quantification of target substances. The NV color center of fluorescent nanodiamonds (FNDs) has characteristics such as high-quality fluorescence, low biotoxicity, and stable physical and chemical properties; at the same time, micro-nano brakes, due to their inherent autonomous movement characteristics and the ability to convert the signal of recognition events into a visual analysis signal according to different sensing strategies, and enhance the mass transfer rate, turning passive diffusion into active movement, realizing stable and efficient ultrasensitive detection.

[0004] In the prior art, the Chinese patent "Application No.: 202311155602.X" reported a method for realizing high-efficiency enzyme-linked immunosorbent assay by a magnetic field-driven micro-nano robot, which involves the preparation of micro-nano robot MNRs-Ab1 and the rapid and instant detection of antibodies. At the same time, separation is achieved spatially through magnetic control drive and guidance, improving the detection efficiency. The Chinese patent "Application No.: 202311532914.8" reported a leukocyte membrane biomimetic coating micro-motor fluorescence probe, its preparation method and application. The catalytic active molecule glucose oxidase in the leukocyte membrane biomimetic coating micro-motor fluorescence probe decomposes glucose to provide driving force, and captures target tumor cells through the specific targeting ability of its surface aptamer to epithelial cell adhesion molecule on the surface of tumor cells, thereby realizing the rapid enrichment and fluorescence quantitative detection of tumor cells. The photothermal micro-brake has advantages such as wireless and remote manipulation and good biocompatibility, and can realize start-stop function by controlling light irradiation, and has strong drug loading and photothermal therapy capabilities. However, a single driving mode such as chemical driving has a single motion behavior and cannot achieve more complex motion modes. Currently, there is no report on a composite braking sensor based on a chemical + photothermal dual-mode driven micro-brake and fluorescent nanodiamond. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a preparation method and product of a manipulable NV color center micro-brake and its application in biological detection, realizing the simple manipulation of FNDs and ultrasensitive biological detection.

[0006] Technical Solution: To solve the above technical problems, the present invention provides a preparation method of a manipulable NV color center micro-brake, including the following steps:

[0007] (1) Dissolve the purified polystyrene (PS) microspheres in a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution containing dopamine hydrochloride (DA), and reduce at room temperature to obtain PS@PDA;

[0008] (2) Add the PS@PDA in step (1) to a potassium permanganate (KMnO4) solution, continuously stir, centrifuge, dry and then calcine and anneal to obtain a hollow MnO2 shell;

[0009] (3) Ultrasonically disperse the hollow MnO2 shell prepared in step (2) in a mixed solution of absolute ethanol and distilled water, spin-coat it on a silicon wafer, dry and fix it, and then carry out a thermal evaporation reaction, and centrifuge and enrich to obtain the manipulable NV color center micro-brake.

[0010] Among them, the particle size of the PS microspheres in step (1) is 1 - 3 μm.

[0011] Among them, in the 10 mM Tris-HCl solution containing DA in step (1), the DA concentration is 0.5 - 4 mg / mL, and the reaction rate of DA is controlled by adjusting the pH within the range of 8.0.

[0012] Among them, in step (2), the concentration of the KMnO4 solution is 2.5 - 15 mg / mL, the stirring time is 10 - 60 min, the annealing temperature is 450 - 650 °C, and the time is 0.5 - 4 h.

[0013] Among them, in step (3), the silicon wafer is treated with a plasma cleaner at medium intensity for 60 - 240 s.

[0014] Among them, in the thermal evaporation reaction in step (3), 0.02 - 0.05 g of Au seeds are added to the tungsten boat in advance, the vacuum is pumped in advance, and the thermal evaporation reaction rate is set to The reaction time is set to 20 min - 60 min, and the reaction current is set to 65 - 73 A.

[0015] The present invention also provides a manipulable NV color center micro-brake prepared by the above preparation method. In this micro-brake sensor, the thiol aptamer probe is assembled on the H-MnO2 particles to form H-MnO2@Au-apt, and the DNA aptamer complementary probe with amino groups is assembled on the fluorescent nanodiamonds (FNDs) to form FNDs-DNA. The H-MnO2@Au-apt / MCH and FNDs-DNA are assembled to form the H-MnO2-apt / DNA-FNDs complex. Among them, the thiol aptamer sequence is used to specifically capture the MUC1 protein, and the FNDs in the FNDs-DNA serve as signal tags; when the MUC1 protein specifically recognizes the H-MnO2@Au-apt / MCH, the FNDs-DNA falls off, resulting in a change in the detectable fluorescence signal.

[0016] The present invention also provides the application of the manipulable NV color center micro-brake in detecting the MUC1 protein.

[0017] The present invention also provides a method for detecting the MUC1 protein, including the following steps:

[0018] Step S1: Prepare a Janus micro-brake modified with a capture probe;

[0019] Step S2: Prepare FNDs modified with a detection probe;

[0020] Step S3: Assemble the Janus micro-brake and FNDs to form a complex (1).

[0021] Step S4: Add different concentrations of MUC1 to the complex and then wash and enrich the competitive binding product (2).

[0022] Step S5: Detect the fluorescence intensities of the complex (1) and the competitive product (2).

[0023] Furthermore, the above steps specifically include:

[0024] (1) Add the thiol-containing aptamer probe pre-activated with tris(2-carboxyethyl)phosphine (TCEP) to the solution containing the manipulable NV color center micro-brake, incubate, centrifuge, wash, and then add MCH to block the part of the Au shell that is not connected to the aptamer to avoid specific adsorption, obtaining the Janus micro-brake modified with capture probes (H-MnO2@Au-apt);

[0025] (2) Mix the surface-carboxylated fluorescent nanodiamonds with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and add the aptamer complementary DNA probe modified at the 5'-end, incubate at room temperature, obtaining FNDs containing detection probes (FNDs-DNA);

[0026] (3) Mix the H-MnO2@Au-apt described in step (1) and the FNDs-DNA described in step (2), add H2O2 and irradiate with a NIR laser to obtain the H-MnO2@Au-apt-DNA-FNDs complex;

[0027] (4) Mix the H-MnO2@Au-apt-DNA-FNDs complex described in step (3) with the test sample to obtain a mixture containing different H-MnO2@Au-apt-DNA-FNDs and the test sample;

[0028] (5) Focus the H-MnO2@Au-apt-DNA-FNDs described in step (3) and the mixture described in step (4) clearly, excite with a 532 nm excitation light, and collect the fluorescence signal at 632 nm; when the fluorescence signal in the mixture decreases relative to the H-MnO2@Au-apt-DNA-FND, it indicates that the competition of MUC1 causes the detachment of FNDs-DNA, that is, there is MUC1 protein in the test sample.

[0029] Among them, according to the linear relationship between the fluorescence signal at 632 nm and the concentration of MUC1 protein, the concentration of MUC1 protein is calculated; among them, the linear relationship is: y = 0.685x + 9.186, R 2 = 0.9423, y represents the fluorescence signal at 632 nm, and x represents Log(concentration of MUC1 protein).

[0030] Among them, the nucleotide sequence of the aptamer probe containing a mercapto group described in step (1) is shown in SEQ ID NO.1, and it can be used to specifically recognize and bind to the MUC1 protein.

[0031] Among them, the nucleotide sequence of the aptamer complementary DNA probe containing an amino group described in step (2) is shown in SEQ ID NO.2, and it competes with the MUC1 protein for shedding during the sensing detection process, realizing the detection strategy of fluorescence weakening.

[0032] Among them, the mercapto aptamer probe is 0.5 - 1 OD, and the concentration is 50 - 200 μM.

[0033] Among them, the volume of the TECP is 5 - 10 μL, and the concentration is 0.5 - 2 M.

[0034] Among them, the time of ultrasonic treatment at room temperature is 30 minutes.

[0035] Among them, the concentration of MCH is 5 mM - 20 mM.

[0036] Among them, under the dual driving action of H2O2 and NIR laser, the competitive binding of MUC1 protein and FNDs - DNA is utilized to achieve the accelerated enrichment and release of FNDs on the surface of the micro - brake, thereby realizing the manipulation and ultrasensitive fluorescence detection of FNDs.

[0037] The micro - brake has dual - mode manipulation motion of chemical concentration field and light field. The micro - brake has a Janus structure including a hollow MnO2 porous shell layer and a single - side - distributed Au film outside the shell layer, with a particle size of 1 - 5 μm. Among them, the MnO2 shell layer catalyzes the decomposition of H2O2 to produce O2 and H2, and the diffusion of microparticles and bubbles near the hemisphere of the micro - brake jointly promote the movement of the brake. Au undergoes photothermal conversion under the action of NIR to generate a thermophoretic force to drive the movement of the brake.

[0038] Using the method of the present invention, the preparation of a manipulable NV - color - center micro - brake can be realized, and the obtained micro - brake has broad application prospects for the detection of cancer markers by utilizing the highly stable fluorescence characteristics of NV - color centers in FNDs.

[0039] The principle of the present invention: As As shown in Figure 1As shown, the Janus micro-brakes modified with capture probes in vascular cancer cells first actively search for and pair with the detection probe FNDs under the dual drive of NIR and H2O2; then MUC1 mucin is added. Since the binding force between MUC1 mucin and apt in the micro-brake is stronger than that of FNDs-DNA, the competitive pairing of MUC1 mucin and FNDs-DNA causes the shedding of FNDs-DNA, which also realizes the simple manipulation of the active capture and release of FNDs by the micro-brake; 532 nm in the figure is the green excitation wavelength of the NV color center fluorescence in FNDs, and 680 nm is the red emission wavelength. The competition between FNDs-DNA and MUC1 mucin causes FNDs-DNA to fall off from the micro-brake, so the signal transduction of the concentration of MUC1 mucin is experimentally determined according to the decrease in fluorescence intensity relative to H-MnO2@Au-apt-DNA-FND.

[0040] The micro-brake of the present invention has a Janus structure including a hollow MnO2 porous shell layer and a single-sided distributed Au film outside the shell layer. Among them, the MnO2 shell layer catalyzes the decomposition of H2O2 to produce O2 and H2, and the diffusion of microparticles and bubbles near the hemisphere of the micro-brake jointly promote the movement of the brake. Au undergoes photothermal conversion under the action of NIR to generate a thermophoretic force to drive the movement of the brake. The FNDs containing NV color centers are 100 nm.

[0041] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: (1) The present invention provides a manipulable NV color center micro-brake, which can be used for the ultrasensitive detection of cancer-related marker protein polypeptides; the Janus micro-brake has a hollow structure and an Au layer with a photothermal effect, and can move directionally under the action of H2O2 and NIR laser, and can quickly and efficiently capture FNDs and the target through the dual drive effect; (2) The biological detection method of the present invention constructs an active control sensing mechanism, overcomes the bottleneck of low transmission efficiency of the traditional incubation method, and greatly shortens the incubation time; (3) The present invention uses the competitive binding of FNDs and protein polypeptide markers to the micro-brake, which can efficiently enrich and release FNDs, and realizes the ultrasensitive detection of protein polypeptide markers while simply manipulating FNDs; (4) The micro-brake can realize the active manipulation, enrichment and release of the fluorescent nanodiamond NV color center, construct an efficient active control sensing mechanism, greatly improve the transmission efficiency of the target, and can provide a more sensitive judgment for the diagnosis of diseases such as tumor cancer. Description of the Drawings

[0042] Figure 1 is The experimental schematic diagram of the preparation of a manipulable NV color center micro-brake and its biological detection method of the present invention;

[0043] Figure 2Schematic diagram for the preparation of a manipulable NV - color - center micro - actuator and its probe modification, where: 1 is a PS micro - sphere, 2 is PS@PDA@MnO2, 3 is hollow MnO2, 4 is hollow MnO2 after Au plating (micro - actuator), 5 is a micro - actuator modified with a probe, 6 is FNDs modified with a probe, 7 is a conjugate of Janus micro - actuator, FNDs and a coupling agent, 8 is a conjugate of Janus micro - actuator and a target object.

[0044] Figure 3 Linear relationship of the detection results in Example 1. Detailed implementation manners

[0045] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0046] Example 1

[0047] Preparation of a manipulable NV - color - center micro - actuator and its biological detection method ( Figure 2 ) is as follows:

[0048] (1) Preparation of Janus micro - actuator

[0049] a. Preparation of MnO2 by dopamine reduction method at room temperature:

[0050] First, take 200 μL of 3 - μm polystyrene (PS) micro - spheres and dissolve them in 20 mL of triple - distilled water. After centrifugation and washing to remove impurities, they are enriched to 2 mL. Prepare a 10 mM Tris - HCl (tris - hydroxymethylaminomethane hydrochloride) solution containing 2 mg / mL DA (dopamine hydrochloride). Add the washed 2 - mL PS (polystyrene) micro - sphere solution to 20 mL of the above - mentioned Tris - HCl solution containing DA, adjust the pH to about 8, and reduce at room temperature for 6 h. After the reaction, a PS@PDA solution is obtained, which is centrifuged and washed three times with distilled water and then re - enriched to 2 mL.

[0051] Add the obtained 2 - mL PS@PDA solution to a KMnO4 solution with a concentration of 15 mg / mL, continuously stir for 20 min, then centrifuge and wash the obtained PS@PDA@MnO2 three times, and place it in an oven at 70 °C for drying after enrichment. Finally, anneal the completely dried PS@PDA@MnO2 in air at 550 °C for 2 h to obtain a hollow MnO2 shell.

[0052] b. Preparation of Janus dual - drive micro - actuator

[0053] Weigh the prepared hollow MnO₂ shell in step a, ultrasonically disperse it in a mixed solution of absolute ethanol and distilled water (volume ratio 1:1) for standby; treat the cut smooth silicon wafer (10 * 10 mm) with a plasma cleaner at medium intensity for 120 s, then spin-coat the dispersed hollow MnO₂ shell on the treated silicon wafer, and then transfer it to a heating table at 40 °C for drying and fixing.

[0054] Paste the silicon wafer spin-coated with the hollow MnO₂ shell on the thermal evaporation stage. Add 0.05 g of Au seeds in advance in the tungsten boat, evacuate the vacuum in advance, and set the thermal evaporation reaction rate to Set the reaction time to 30 min and the reaction current to 73 A. After the reaction is completed and cooled, take out the silicon wafer, ultrasonically make the particles fall off, and then centrifuge and enrich to obtain the Janus dual-drive micro-brake (H-MnO₂@Au).

[0055] (2) Biological detection method:

[0056] a. Dissolve 0.5 OD of thiol MUC1 aptamer probe (sequence: SEQ ID NO.1: 5’-SH-(CH2)6-GCAGTTGATCCTTTGGATACCCTGG-3’) to 100 μM, add 5 μL of 1 M tris(2-carboxyethyl)phosphine (TECP), mix well and ultrasonically treat at room temperature for 30 minutes to obtain the activated DNA probe containing thiol; dilute the above-prepared Janus dual-drive micro-brake in 1 mL of distilled water, add the above-activated DNA probe containing thiol and incubate for 6 h, centrifuge and wash, then add MCH for capping, and finally resuspend in 0.5 mL of distilled water to obtain the Janus micro-brake modified with capture probe (H-MnO₂@Au-apt).

[0057] b: Take 100 μg of 100 nm surface-carboxylated fluorescent nanodiamonds (manufacturer: Adamas Nanotechnologies), add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide, mix well and let stand, then add the aptamer complementary DNA probe modified at the 5’ end with amino group (sequence: SEQ ID NO.2: 5’-NH2-(CH2)6-TTTTTTATCCAAAGA-3’), incubate at room temperature for 3 hours, then centrifuge and wash three times, and resuspend in 1 mL of deionized water to obtain the FNDs modified with detection probe (FNDs-DNA).

[0058] c: Take 30 μL of the Janus microactuators modified with capture probes and 10 μL of the FNDs modified with detection probes, dilute them in distilled water, then add 1 mM H2O2 and adjust the power of the NIR laser to 1.5 W. Use the dual-driving effect to accelerate the incubation rate for 20 min. Finally, centrifuge and wash three times to obtain the complex assembled by Janus microactuators and FNDs (H-MnO2@Au-apt-DNA-FNDs).

[0059] d: Add the H-MnO2@Au-apt-DNA-FNDs complex to 2 mL of 1 μM MUC1 protein (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.). Use the dual-driving effect to accelerate the incubation rate. Finally, centrifuge and wash to obtain a mixture containing H-MnO2@Au-apt-DNA-FNDs and H-MnO2@Au-apt-MUC1.

[0060] e: Take the H-MnO2@Au-apt-DNA-FNDs and the mixture, respectively, drop-coat them on the washed silicon wafer, dry them at room temperature, then place them under the 40X objective lens of the fluorescence spectrometer in the confocal system for clear focusing. Use 532 nm excitation light for excitation, collect the fluorescence signal at 632 nm. By comparing the different fluorescence signals, sensitive detection of the MUC1 mucin concentration at the 0.16 pM level can be achieved. The results are as shown Figure 3 as follows.

[0061] Example 2

[0062] The preparation of a manipulable NV color center microactuator and its biological detection method are as follows:

[0063] (1) Preparation of Janus microactuators

[0064] a. Prepare MnO2 by the room-temperature dopamine reduction method:

[0065] First, take 300 μL of 1-μm polystyrene (PS) microspheres and dissolve them in 20 mL of triple-distilled water. After centrifuging and washing to remove impurities, enrich them to 2 mL. Prepare a 10 mM Tris-HCl solution containing 4 mg / mL DA. Dissolve the washed PS microspheres in 20 mL of the Tris-HCl solution containing DA, adjust the pH to about 8, and reduce them at room temperature for 6 h. After the reaction, centrifuge and wash the PS@PDA solution three times with distilled water and re-enrich it to 2 mL.

[0066] The obtained 2 mL of PS@PDA solution was added to a KMnO4 solution with a concentration of 2.5 mg / mL. After continuous stirring for 60 min, the obtained PS@PDA@MnO2 was centrifuged and washed three times, and then placed in an oven at 70 °C for drying after enrichment. Finally, the completely dried PS@PDA@MnO2 was subjected to air annealing at 650 °C for 0.5 h to obtain a hollow MnO2 shell.

[0067] b. Preparation of Janus dual-driven micro-brake

[0068] Weigh the hollow MnO2 shell prepared in step a and disperse it ultrasonically in a mixed solution of absolute ethanol and distilled water (volume ratio 1:1) for standby; the cut smooth silicon wafer (10 * 10 mm) was treated with a plasma cleaner at medium intensity for 120 s, and then the dispersed hollow MnO2 shell was spin-coated on the treated silicon wafer, and then transferred to a heating table at 40 °C for drying and fixing.

[0069] Paste the silicon wafer spin-coated with the hollow MnO2 shell on the thermal evaporation stage. Add 0.05 g of Au seeds in advance in the tungsten boat, evacuate the vacuum in advance, and set the thermal evaporation reaction rate to The reaction time was set to 60 min and the reaction current was set to 70 A. After the reaction ended and cooled, the silicon wafer was taken out, and the particles were detached by ultrasonic treatment, and then centrifuged and enriched to obtain the Janus dual-driven micro-brake.

[0070] (2) Biological detection method:

[0071] a. Dissolve 1 OD of thiol-modified aptamer probe (SEQ ID NO.1) to 200 μM, add 5 μL of 2 M TECP, mix well and ultrasonicate at room temperature for 30 minutes to obtain an activated DNA probe containing thiol; dilute the above-prepared Janus micro-brake in 1 mL of distilled water, add the above-activated DNA probe containing thiol and incubate for 6 h, centrifuge and wash, then add MCH for capping, and finally resuspend in 0.5 mL of distilled water to obtain a Janus micro-brake modified with a capture probe.

[0072] b: Take 100 μg of 100 nm fluorescent nanodiamonds carboxylated on the surface, add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide, mix well and let stand, then add an aptamer complementary DNA probe (SEQ ID NO.2) modified at the 5'-end with an amino group, incubate at room temperature for 5 hours, then centrifuge and wash three times, and resuspend in 1 mL of deionized water to obtain FNDs modified with a detection probe.

[0073] c: Take 30 μL of the Janus microbrakes modified with capture probes and 10 μL of the FNDs modified with detection probes, dilute them in distilled water, then add 2 mM H2O2 and adjust the power of the NIR laser to 2.0 W. Use the dual-driving effect to accelerate the incubation rate for 15 min, and finally centrifuge and wash three times to obtain the assembled complex (H-MnO2@Au-apt-DNA-FNDs).

[0074] d: Add the H-MnO2@Au-apt-DNA-FNDs complex to 2 mL of 0.1 μM MUC1 protein, use the dual-driving effect to accelerate the incubation rate, and finally centrifuge and wash to obtain a mixture containing H-MnO2@Au-apt-DNA-FNDs and H-MnO2@Au-apt-MUC1.

[0075] e: Take H-MnO2@Au-apt-DNA-FNDs and the mixture and drop-coat them on the cleaned silicon wafer respectively. After drying at room temperature, place them under the 40X objective lens of the fluorescence spectrometer in the confocal system for focusing clearly. Use a 532 nm excitation light for excitation and collect the fluorescence signal at 632 nm. By comparing the differences in fluorescence signals, sensitive detection of the MUC1 mucin concentration at the 0.45 pM level can be achieved.

[0076] Example 3

[0077] The preparation of a manipulable NV color center microbrake and its biological detection method are as follows:

[0078] (1) Preparation of Janus microbrakes

[0079] a. Prepare MnO2 by the room-temperature dopamine reduction method:

[0080] First, take 300 μL of 1 μm polystyrene (PS) microspheres and dissolve them in 20 mL of triple-distilled water. After centrifuging and washing to remove impurities, enrich them to 2 mL; prepare a 10 mM Tris-HCl solution containing 0.5 mg / mL DA. Dissolve the washed PS microspheres in 20 mL of the Tris-HCl solution containing DA, adjust the pH to about 8, and reduce them at room temperature for 6 h. After the reaction, centrifuge and wash the PS@PDA solution three times with distilled water and re-enrich it to 2 mL.

[0081] Add the obtained 2 mL of PS@PDA solution to a 5 mg / mL KMnO4 solution, continuously stir for 20 min, then centrifuge and wash the obtained PS@PDA@MnO2 three times, and place it in an oven at 70 °C for drying after enrichment. Finally, anneal the completely dried PS@PDA@MnO2 in air at 450 °C for 4 h to obtain a hollow MnO2 shell.

[0082] b. Preparation of Janus dual-driven micro-brake

[0083] Weigh the prepared hollow MnO₂ shell in step a and ultrasonically disperse it in a mixed solution of absolute ethanol and distilled water (volume ratio 1:1) for standby; treat the cut smooth silicon wafer (10*10 mm) with a plasma cleaner at medium intensity for 60 s, then spin-coat the dispersed hollow MnO₂ shell on the treated silicon wafer, and then transfer it to a heating table at 40 °C for drying and fixing.

[0084] Paste the silicon wafer spin-coated with the hollow MnO₂ shell on the thermal evaporation stage. Add 0.02 g of Au seeds in advance in the tungsten boat, evacuate the vacuum in advance, and set the thermal evaporation reaction rate to Set the reaction time to 20 min and the reaction current to 65 A. After the reaction is completed and cooled, take out the silicon wafer, ultrasonically remove the particles, and then centrifuge and enrich to obtain the Janus dual-driven micro-brake.

[0085] (2) Biological detection method:

[0086] a. Dissolve 0.5 OD of thiol-modified aptamer probe (SEQ ID NO.1) to 50 μM, add 10 μL of 0.5 M TECP, mix well and ultrasonically treat at room temperature for 30 minutes to obtain an activated DNA probe containing thiol; dilute the above-prepared Janus micro-brake in 1 mL of distilled water, add the above-activated DNA probe containing thiol and incubate for 6 h, centrifuge and wash, then add MCH for capping, and finally resuspend in 0.5 mL of distilled water to obtain the Janus micro-brake modified with capture probe.

[0087] b: Take 100 μg of 100-nm fluorescent nanodiamonds carboxylated on the surface, add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide, mix well and let stand, then add the aptamer complementary DNA probe (SEQ ID NO.2) modified at the 5'-end, incubate at room temperature for 5 hours, then centrifuge and wash three times, and resuspend in 1 mL of deionized water to obtain FNDs containing detection probe.

[0088] c: Take 30 μL of the Janus micro-brake modified with capture probe and 10 μL of the FNDs modified with detection probe and dilute them in distilled water, then add 2 mM H₂O₂ and adjust the power of the NIR laser to 2.0 W, use the dual-driven effect to accelerate the incubation rate for 15 min, and finally centrifuge and wash three times to obtain the complex assembled by Janus micro-brake and FNDs (H-MnO₂@Au-apt-DNA-FNDs).

[0089] d: Add the H-MnO2@Au-apt-DNA-FNDs complex to 2 mL of 1 nM MUC1 protein, and use the dual driving effect to accelerate the incubation rate. Finally, centrifuge and wash to obtain a mixture of H-MnO2@Au-apt-DNA-FNDs and H-MnO2@Au-apt-MUC1.

[0090] e: Take the H-MnO2@Au-apt-DNA-FNDs and the mixture, respectively, drop-coat them on a cleaned silicon wafer, dry them at room temperature, then place them under the 40X objective lens of the fluorescence spectrometer in the confocal system for clear focusing. Use a 532 nm excitation light for excitation, collect the fluorescence signal at 632 nm, and by comparing the different fluorescence signals, sensitive detection of the MUC1 mucin concentration at the 1.3 pM level can be achieved.

[0091] Example 4

[0092] The preparation of a controllable NV color center microactuator and its biological detection method are as follows:

[0093] (1) Preparation of Janus microactuator

[0094] a. Prepare MnO2 by the room temperature dopamine reduction method:

[0095] First, dissolve 300 μL of 2 μm polystyrene (PS) microspheres in 20 mL of triple-distilled water, centrifuge and wash to remove impurities, and enrich to 2 mL; prepare a 10 mM Tris-HCl solution containing 2 mg / mL DA. Dissolve the washed PS microspheres in 20 mL of the Tris-HCl solution containing DA, adjust the pH to about 8, and reduce at room temperature for 6 h. After the reaction, the PS@PDA solution is centrifuged and washed three times with distilled water and re-enriched to 2 mL.

[0096] Add the obtained 2 mL of PS@PDA solution to a 5 mg / mL KMnO4 solution, continuously stir for 30 min, then centrifuge and wash the obtained PS@PDA@MnO2 three times, enrich it, and place it in an oven at 70 °C for drying. Finally, anneal the completely dried PS@PDA@MnO2 in air at 550 °C for 0.5 h to obtain a hollow MnO2 shell.

[0097] b. Preparation of Janus dual-driving microactuator

[0098] Weigh the prepared hollow MnO₂ shell in step a and ultrasonically disperse it in a mixed solution of absolute ethanol and distilled water (volume ratio 1:1) for standby; treat the cut smooth silicon wafer (10*10 mm) with a plasma cleaner at medium strength for 240 s, then spin-coat the dispersed hollow MnO₂ shell on the treated silicon wafer, and then transfer it to a heating table at 40 °C for drying and fixing.

[0099] Paste the silicon wafer spin-coated with the hollow MnO₂ shell on the thermal evaporation stage. Add 0.05 g of Au seeds in the tungsten boat in advance, evacuate to a good vacuum in advance, and set the thermal evaporation reaction rate to Set the reaction time to 50 min and the reaction current to 70 A. After the reaction is completed and cooled, take out the silicon wafer, ultrasonically remove the particles, and then centrifuge and enrich to obtain the Janus dual-driven micro-brake.

[0100] (2) Biological detection method:

[0101] a. Dissolve 1 OD of thiol-modified aptamer probe (SEQ ID NO.1) to 100 μM, add 5 μL of 1 M TECP, mix well and ultrasonically treat at room temperature for 30 minutes; dilute the prepared Janus micro-brake in 1 mL of distilled water, add the above-activated DNA probe containing thiol and incubate for 6 h, centrifuge and wash, then add MCH for capping, and finally resuspend in 0.5 mL of distilled water to obtain the Janus micro-brake modified with capture probe.

[0102] b: Take 100 μg of 100 nm surface-carboxylated fluorescent nanodiamonds, add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide, mix well and let stand, then add the aptamer-complementary DNA probe (SEQ ID NO.2) modified at the 5'-end, incubate at room temperature for 5 hours, then centrifuge and wash three times, and resuspend in 1 mL of deionized water to obtain FNDs containing detection probe.

[0103] c: Take 30 μL of the Janus micro-brake modified with capture probe and 10 μL of the FNDs modified with detection probe and dilute them in distilled water, then add 2 mM H₂O₂ and adjust the power of the NIR laser to 2.0 W, and use the dual-drive effect to accelerate the incubation rate for 15 min. Finally, centrifuge and wash three times to obtain the H-MnO₂@Au-apt-DNA-FNDs complex.

[0104] d: Add the H-MnO2@Au-apt-DNA-FNDs complex to 2 mL of 0.1 nM MUC1 protein, and use the dual driving effect to accelerate the incubation rate. Finally, centrifuge and wash to obtain a mixture containing H-MnO2@Au-apt-DNA-FNDs and H-MnO2@Au-apt-MUC1.

[0105] e: After dropping and drying the H-MnO2@Au-apt-DNA-FNDs and the mixture on the cleaned silicon wafer at room temperature respectively, place them under the 40X objective lens of the fluorescence spectrometer in the confocal system for clear focusing. Use a 532 nm excitation light for excitation, collect the fluorescence signal at 632 nm, and by comparing the differences in fluorescence signals, sensitive detection of MUC1 mucin concentration at the 0.32 pM level can be achieved.

Claims

1. A preparation method of a controllable NV color center micro-brake, characterized in that, It includes the following steps: (1) Dissolve the purified polystyrene microspheres in a Tris-HCl solution containing DA, and reduce them at room temperature to obtain PS@PDA; (2) Add the PS@PDA described in step (1) to a KMnO4 solution, continuously stir, centrifuge, dry, and then perform calcination annealing to obtain a hollow MnO2 shell; (3) Ultrasonically disperse the hollow MnO2 shell prepared in step (2) in a mixed solution of absolute ethanol and distilled water, spin-coat it on a silicon wafer, dry and fix it, and then perform a thermal evaporation reaction. After centrifugal enrichment, the controllable NV color center microactuator is obtained.

2. The preparation method according to claim 1, wherein In step (1), the particle size of the polystyrene is 1-3 μm.

3. The preparation method according to claim 1, characterized in that, In step (1), the concentration of DA is 0.5-4 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the KMnO4 solution is 2.5-15 mg / mL, and the annealing temperature is 450-650 °C.

5. The preparation method according to claim 1, characterized in that, In the thermal evaporation reaction in step (3), 0.02 - 0.05 g of Au seeds are added to the tungsten boat in advance, the vacuum is pumped in advance, and the thermal evaporation reaction rate is set to The reaction time is set to 20 min - 60 min, and the reaction current is set to 65 - 73 A.

6. A controllable NV color center microactuator prepared by the preparation method according to any one of claims 1-5.

7. Use of the controllable NV color center microactuator according to claim 6 in detecting MUC1 protein.

8. A method for detecting MUC1 protein, characterized in that, It includes the following steps: (1) Add an activated aptamer probe containing a thiol group to the solution containing the controllable NV color center microactuator described in claim 4, incubate, centrifuge and wash, and then add 6-mercapto-1-hexanol to obtain H-MnO2@Au-apt; the nucleotide sequence of the aptamer probe containing a thiol group is as shown in SEQ ID NO.1; (2) Mix surface-carboxylated fluorescent nanodiamonds with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and add an aptamer complementary DNA probe modified with an amino group at the 5' end, and incubate at room temperature to obtain FNDs-DNA; (3) Mix the H-MnO2@Au-apt described in step (1) and the FNDs-DNA described in step (2), add H2O2 as a fuel and perform wireless driving with NIR to obtain H-MnO2@Au-apt-DNA-FNDs; (4) After mixing and incubating the H-MnO2@Au-apt-DNA-FNDs described in step (3) with the test sample, centrifuge and wash, add the H-MnO2@Au-apt-DNA-FNDs described in step (3), perform focusing clarity, and excite with 532 nm excitation light, and collect the fluorescence signal at 632 nm; when the fluorescence signal in the mixture decreases relative to H-MnO2@Au-apt-DNA-FND, it indicates that MUC1 protein exists in the test sample.

9. The method according to claim 8, wherein According to the linear relationship between the fluorescence signal at 632 nm and the MUC1 protein concentration, the MUC1 protein concentration was calculated; wherein, the linear relationship is: y = 0.685x + 9.186, R 2 = 0.9423, where y represents the fluorescence signal at 632 nm and x represents Log(MUC1 protein concentration).

10. The method according to claim 8, wherein In step (2), the nucleotide sequence of the aptamer complementary DNA probe containing an amino group is as shown in SEQ ID NO.2.

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