A light-driven micro-robot for real-time multi-site hydrogen peroxide detection and a preparation method thereof

Abstract

The present application belongs to the technical field of micro robot, and particularly relates to a light-driven micro robot for real-time multi-site hydrogen peroxide detection and a preparation method thereof. The present application is manufactured through a simple film blowing method, and a light-driven micro robot with a composite Leba structure is invented, which integrates UCNPs, OVG and PEG-NH2. The muBot can detect H2O2 by analyzing the luminescence intensity ratio (LIR) from UCNPs and OVG through luminescence resonance energy transfer (LRET). By using a scanning optical tweezer, the muBot can be captured by 1064 nm near-infrared light, and the light of this wavelength is basically non-damaging to biological cells. The spatial resolution of the muBot is 0.6 mu m, and the detection limit of the H2O2 concentration is 0.15*10 ‑6 M, and normal, deformed and apoptotic red blood cells can be detected in real time and at multiple sites.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of micro robots, and particularly relates to a light-driven micro robot for real-time multi-site hydrogen peroxide detection and a preparation method thereof. BACKGROUND

[0002] Hydrogen peroxide (H2O2) is an effective biomarker of cell physiological activities, regulates cell proliferation, differentiation and metabolism, and plays a role in signal transduction, intracellular defense and detoxification. Changes in H2O2 levels are closely related to the occurrence and development of various diseases. On the one hand, its accumulation can lead to oxidative stress, inflammation and cancer; on the other hand, its reduction can affect various signaling molecules related to cell apoptosis, autophagy, cytokines and mRNA expression, and can further affect the physiological functions and metabolic processes of cells. Therefore, the detection of H2O2 is crucial for health monitoring and disease diagnosis. H2O2 detection methods, such as probes and molecular fluorescent probes, have greatly promoted the development of this field. Although the needle-shaped probe is convenient to carry and has stable performance, it is invasive to biological tissues, and the larger size (several tens of microns) limits the improvement of spatial resolution. Molecular fluorescent probes have the ability to achieve non-invasive and sub-micron spatial resolution. However, their activity is usually insufficient because they rely on passive staining of biological tissues and passive transport characteristics of target groups anchored. In addition, they also pollute cells. Therefore, there is an urgent need to develop a real-time, non-invasive, high spatial resolution, strong activity and environmentally friendly H2O2 detection method for early diagnosis and prevention of diseases.

[0003] Micro robots (μBots) are a kind of small robotic systems that can move and perform specific tasks at the micron scale by collecting chemical fuels or external energy sources (such as magnetic fields, acoustic fields and light fields). However, for chemical fuel-driven μBots, the energy generated by chemical reactions (such as the decomposition of H2O2) is used for navigation, but the residual fuel causes biological toxicity, and the low driving precision (error > 5 μm) limits the precise movement of μBots. Magnetically driven μBots can perform fine motion control in complex biological environments, but they face technical challenges in maintaining the size of small robots, and may leave residual magnetic materials. Acoustically driven μBots are biologically safe within a certain frequency range, but long-term driving may face challenges such as cavitation effect and low fluorescence imaging. In contrast, light-driven μBots use a light source to achieve on-off control and directional movement. This driving method not only realizes fast, flexible and precise navigation, but also does not require additional chemicals or magnetism. In addition, light-driven microstructures exhibit environmentally friendly characteristics and biocompatibility when performing biosensing, providing a potential solution for real-time H2O2 detection. SUMMARY

[0004] The present application aims at the existing problems, and provides a light-driven micro robot for real-time multi-site hydrogen peroxide detection and a preparation method thereof.

[0005] The present application is realized by the following technical solutions:

[0006] A light-driven micro robot for real-time multi-site hydrogen peroxide detection and a preparation method thereof, comprising the following steps:

[0007] (1) PEG-NH2 and OVG are respectively fully dissolved in CH2Cl2 and acetone to prepare PEG-NH2 solution and OVG solution;

[0008] (2) PEG-NH2 solution, UCNPs solution and OVG solution are mixed together, ultrasonic treatment is carried out to obtain a uniform dispersion solution, then nitrogen gas flow is blown into the reaction bottle, and the mixed solution is blown onto the wall of the rotating reaction container until the solvent is blown dry to form a film;

[0009] (3) Deionized water is added to the film and ultrasonic treatment is carried out to form a uniform suspension, the suspension is purified by centrifugation and washed with anhydrous ethanol, and the centrifugation is repeated after each washing, finally, the obtained μBot is resuspended in 1 mL deionized water for subsequent test use.

[0010] Further, the concentration of the PEG-NH2 solution in step (1) is 5 mg / mL.

[0011] Further, the concentration of the OVG solution in step (1) is 1 mg / mL.

[0012] Further, the cyclohexane solution of the UCNPs solution in step (2) is a solution of upconversion nanoparticles, and the concentration is 1 mmol / 10 mL.

[0013] Further, the adding amount of the PEG-NH2 solution, the UCNPs solution and the OVG solution in step (2) is respectively: 1 mL of PEG-NH2 solution, 20 μL of UCNPs solution and 50 μL of OVG solution.

[0014] Further, the ultrasonic treatment time in step (2) is 5 min.

[0015] Further, the ultrasonic treatment time in step (3) is 10 min, the centrifugation speed is 8000 rpm each time, the centrifugation time is 5 min each time, and the number of anhydrous ethanol is 2.

[0016] Further, the μBot of the present application adopts a composite Leba structure design Figure 1), including the following core components: sensing layer: upconversion nanoparticles (UCNPs, β-NaYF4:1%Tm 3+ ) as donors, emitting blue light (475 nm) by 980 nm excitation; H2O2-responsive probe OxiVision Green (OVG) as acceptor, generating green fluorescence (512 nm) by luminescence resonance energy transfer (LRET) mechanism. Encapsulation layer: polyethylene glycol amine (PEG-NH2) wraps the sensing layer by electrostatic adsorption, thickness 50-100 nm, preventing fluorescence group leakage and improving biocompatibility.

[0017] Further, a scanning optical tweezers (SOTs) system (Tweez250si, Aresis, Europe) was used, as shown in Figure 2 The system was equipped with a solid-state continuous wave Nd: YAG (1064 nm) infrared laser (8) and an inverted microscope. In terms of optical imaging and manipulation, a water immersion objective (4) was used, characterized by a magnification factor of 60x and a numerical aperture of 1.0. The 1064 nm laser beam emitted from the laser source was introduced into an acousto-optic deflector (AOD) unit (7) with a maximum switching rate of 100 kHz and connected to a computer. This device facilitated spatial scanning of optical traps by precise control of the laser beam. A beam expander (6) was used to widen the laser beam, producing a wide collimated laser beam. Subsequently, the laser beam was reflected by a dichroic mirror (5) and effectively focused by the water immersion objective (4), thereby generating optical traps within the sample chamber (3) with individual trap powers ranging from 0 to 100 mW, enabling simultaneous capture, movement, and rotation control of the μBot. The integrated illumination system used a condenser (2) to concentrate light onto the sample. Images within the sample plane were collected by the objective, then passed through the dichroic mirror (5), an additional reflector (9), and finally focused onto a standard Charge-coupled Device (CCD) (11) camera through a filter (10).

[0018] The present invention has the following advantages compared to the prior art:

[0019] The present invention is manufactured by a simple blowing film method, and invents a light-driven microrobot (μBot) with a composite Leba structure (Leba is a common bread in northern China), which integrates upconversion nanoparticles (UCNPs, β-NaYF4:1%Tm 3+), OxiVision Green (OVG) and polyethylene glycol-amino (PEG-NH2). μBot can detect H2O2 by luminescence resonance energy transfer (LRET) analysis of luminescence intensity ratio (LIR) from UCNPs and OVG. With scanning optical tweezers, μBot can be captured by 1064 nm near-infrared light, and the light of this wavelength is basically non-damaging to biological cells. The spatial resolution of μBot is 0.6 μm, and the detection limit of H2O2 concentration is 0.15×10 -6 M, real-time and multi-site detection of normal, deformed and apoptotic red blood cells can be performed. The detection ability of H2O2 of the μBot of the present application can promote early disease screening, and is suitable for cell physiological activity monitoring, early disease diagnosis and biomarker detection. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Schematic diagram of light-driven micro robot;

[0021] Figure 2 Schematic diagram of experimental device;

[0022] Figure 2 In the middle: 1, illumination light source, 2, condenser, 3, sample chamber, 4, objective lens, 5, dichroic mirror, 6, beam expander, 7, AOD acousto-optic deflector, 8, 1064 nm laser, 9, reflector, 10, optical filter, 11, CCD charge-coupled device;

[0023] Figure 3 Schematic diagram of the process of preparing μBots by blown film method;

[0024] Figure 4 SEM image of prepared μBot, the scale is 300 nm;

[0025] Figure 5 In the middle, (a) photoluminescence spectrum of UCNPs and absorption and emission spectrum of OVG in H2O2 environment (λex = 980 nm; λex = 490 nm); (b) LRET energy transfer mechanism from UCNPs to OVG in H2O2 environment; (c) schematic diagram of H2O2 detection process;

[0026] Figure 6 Lifetime decay curve of μBot and UCNPs at 475 nm emission wavelength;

[0027] Figure 7Optical microscopy images (bright and dark field) of trapped μBots in (a) deionized water and (b) H2O2 environment;

[0028] Figure 8 Optical microscopy images (dark field) of linear (a) and circular motion (b) of microbots in H2O2 environment; (c) optical dark field microscopy images of the distance between two adjacent μBots in H2O2 environment without interfering with each other's light emission, laser power for each optical trap: 20 mW;

[0029] Figure 9 In the presence of (a) μBots in different concentrations (40, 60 and 80 μΜ) of H2O2 solution, optical microscopy images (dark field) of green and blue channels; (b) the relationship between LIR of μBots and concentration of H2O2 solution in the range of 20-100 μΜ; (c) optical stability of μBots in 20 μΜ H2O2 solution within 10 min; (d, e) specificity evaluation of μBots under different concentrations (5-25 μΜ) of Cys and GSSG solution, laser power for each optical trap: 20 mW;

[0030] Figure 10 In the presence of (a1-3) optical microscopy images (bright and dark field) of μBots rotating around RBC for real-time H2O2 detection; (b) LIR of μBots rotating around RBC at 0°, 90°, 180°, 270° and 360°; (c) optical microscopy images (bright and dark field) of four μBots trapped by four optical traps for real-time detection at sites 1, 2, 3 and 4 around the RBC membrane surface; (d) LIR of four μBots at sites 1, 2, 3 and 4 around RBC, laser power for each optical trap: 20 mW;

[0031] Figure 11 In the presence of (a1-4) optical microscopy images (bright and dark field) of μBots for dynamic H2O2 detection during the process of red blood cell deformation; (b) LIR of μBots during the process of red blood cell deformation;

[0032] Figure 12 In the presence of (a1-4) optical microscopy images (bright and dark field) of μBots for dynamic detection of H2O2 during the process of red blood cell apoptosis; (b) LIR of μBots when red blood cells are in the process of apoptosis. Laser power to induce red blood cell apoptosis: 60 mW. DETAILED DESCRIPTION

[0033] In order to further explain the present application, the following specific examples are described below.

[0034] Example 1: μBot morphology characterization and verification of luminescence resonance energy transfer

[0035] The morphology was further characterized by scanning electron microscopy (SEM) images. Figure 4 The prepared μBot has a Leba structure with an average length of 380 nm and a width of 255 nm. In order to determine the donor-acceptor relationship between UCNPs (energy donor) and OVG (energy acceptor), the PL spectrum of UCNPs (λex: 980 nm) and the absorption and emission spectra of OVG (λex: 490 nm) were characterized ( Figure 5 (a). Two strong blue emission bands in the wavelength range of 432-496 nm indicate that UCNPs can be excited by 980 nm laser light. Furthermore, the absorption band of OVG (black line) significantly overlaps with the blue emission band of UCNPs. Furthermore, when oxidized in the presence of H2O2, OVG exhibits a strong green emission band in the wavelength range of 470-700 nm, indicating that LRET can occur from UCNPs to OVG. The LRET process in the presence of H2O2 is shown in Figure 2. Figure 5 b. UCNPs are excited by 980 nm laser and undergo a three-photon or four-photon process from the ground state ( 3 H6) transitions to the excited state ( 1 G4 and 1 D2). Then, they 1 D2 and 1 G4 emits blue light with wavelengths of 450 nm and 475 nm. 1 A portion of the energy of G4 is transferred to OVG, causing a transition from its ground state (S0) to an excited state (S1), and resulting in green light emission at a wavelength of 512 nm. Through the LRET process, the change in the intensity ratio of the blue light emission from UCNPs to the green light emission from OVG can indicate the presence of H2O2. Such a process can be used to achieve H2O2 detection ( Figure 5 c) In the absence of H2O2, the designed μBot only emits blue fluorescence. However, when exposed to H2O2, it mainly emits green fluorescence, while the intensity of blue light is weakened due to the oxidation of OVG.

[0036] To further demonstrate this, the lifetime decay curve of μBot was tested at an emission wavelength of 475 nm ( Figure 6 The calculated lifetime of μBot is 193.91 μs. The lifetime decay curve of UCNPs at an emission wavelength of 475 nm was also measured for comparison. The lifetime of μBot at 475 nm is shorter than that of UCNPs (τ UCNPs= 361.28 μs). This is because the UCNPs transfer energy to the OVG in the μBot. Therefore, it can be determined that there is a LRET process from the UCNPs to the OVG in the μBot.

[0037] Example 2: Performance of μBot in moving and positioning experiments under scanning optical tweezers

[0038] The trapping laser of SOTs is a laser beam with a central wavelength of 1064 nm. The UCNPs in this study can be excited by the 1064 nm laser to achieve luminescence emission, and the trapping light can also serve as the excitation light for the μBot. Figure 7 Figure 1a shows the optical microscopic images (bright field and dark field) of the μBot trapped in deionized water (the laser power used is within the biological safety level of 20 mW). The trapped μBot emits blue light, indicating that the 1064 nm laser can effectively excite the μBot. At the same time, the trapped μBot emits green light in the H2O2 environment (Figure 1b), confirming that the 1064 nm laser can achieve μBot excitation without affecting the μBot's H2O2 detection. Figure 7

[0039] The micro-robot trapped by SOTs can be manipulated by dynamic optical traps. Figure 8 Figures 2a-b show the optical microscopic images of the linear and circular motion of the μBot. The linear and rotational motion speeds of the micro-robot are set to 3.6 μm / s and 0.25 rps, respectively. At t = 1, 2 and 3 s, the μBot moves linearly to 3.6, 7.2 and 10.8 μm (Figure 2a), respectively. At t = 0.9, 2 and 3 s, the μBot rotates counterclockwise by 81°, 180° and 270° (Figure 2b), respectively. In addition, two micro-robots are stably trapped by two optical traps, which can achieve a spatial resolution of about 0.6 μm by reducing the distance between the two μBots without interfering with each other's light emission (Figure 2c). Therefore, the μBot can perform a series of movements and positioning to achieve real-time multi-point detection of H2O2. Figure 8 Figure 8 Figure 8

[0040] Example 3: Performance experiment of μBot in detecting H2O2

[0041] The detection performance of the μBot was studied, and the optical microscopic images (dark field) of the μBot green and blue channels showed (Figure 3a-b) that the μBot could detect H2O2 in the range of 0-1000 μM. The detection limit of the μBot was 0.1 μM (S / N = 3) (Figure 3c). Figure 9 ​​​​a) with the increase of H2O2 solution concentration, the luminescence signal of green channel was enhanced, while the luminescence signal of blue channel was gradually weakened. Here, the optical images of green and blue channels were obtained by processing and separating the optical microscopic images of μBots using ImageJ software. The signal from the green channel can be used as a detection signal, and the signal from the blue channel can be used as a reference signal. The average gray value of the luminescence image can directly reflect the luminescence signal. By calculating the average gray value of the green and blue channel images, the luminescence intensity ratio (LIR) of green light to blue light depending on the H2O2 concentration can be obtained. Figure 9 b shows the linear relationship between LIR and H2O2 concentration, and the correlation coefficient R 2 = 0.988. The lowest concentration of H2O2 that can be detected from the background signal (denoted as LOD): LOD = 3σ / S; where σ represents the standard error of 10 consecutive scans of the blank sample, and S is the slope of the linear fitting. The calculated value of LOD of μBot is 0.15 × 10 -6 M, which is lower than the value 0.27 × 10 -6 M reported in the literature (Adv. Mater. 2022, 34, 2205760). In order to ensure the accuracy of the detection, the luminescence stability of μBot was monitored by measuring the LIR of μBot over time (c) of FIG. 1. The results show that LIR hardly changes with time. Figure 9 In addition, cysteine (Cys) and glutathione disulfide (GSSG) were selected as the main redox buffer in cells to evaluate the specificity of μBot, and a 20-μM H2O2 solution was selected as the signal background. The relationship between the LIR of μBot and the concentration (5, 10, 15, 20 and 25 μM) of Cys and GSSG is shown in d, e of FIG. 1. With the change of cysteine and GSSG concentration, LIR remains unchanged, which confirms the specificity of μBot to H2O2 solution. Therefore, the μBot prepared in the present application has a low detection limit, good optical stability and specificity, and can be used for accurate detection of H2O2. Figure 9

[0042] Example 4: Experiment of μBot for real-time detection of H2O2 concentration in red blood cells (RBC)

[0043] ​Mature red blood cells were chosen as the experimental model because of their simple structure, lack of organelles such as nucleus, good rigidity, and easy deformation. In addition, red blood cells carry a large amount of hemoglobin that participates in chemical reactions and rapidly generates H2O2. First, a red blood cell and a μBot were captured by two optical traps, respectively. Then, in order to ensure the detection accuracy and the smooth rotation of the μBot, the μBot was driven to be as close as possible to the RBC membrane surface without sticking, maintaining a distance of about 0.4 μm. Next, the μBot rotated around the RBC at a rotation speed of 0.1 rps and a rotation radius of 2.5 μm. At T = 0 s, the captured μBot was at the current position (θ = 0°) and was ready to start rotating. Figure 10 As time increased, the μBot rotated to different positions; at t = 5 s and 10 s, the μBot rotated to θ = 180° and 360°, respectively (a2, 3 of Figure 10 By this method, the LIR of the μBot at each position can be obtained, indicating the distributed concentration of H2O2 around the RBC. The LIR measured by the μBot at different positions (0°, 90°, 180°, 270°, and 360°) remained around 1.26 (b of Figure 10 , indicating that the distribution of H2O2 around the RBC was relatively uniform. The measured H2O2 concentration around the RBC was in line with the normal RBC expression range (10.65-11.98 x 10 -6 M), further reflecting the normal physiological state of the RBC. This indicates that the μBot can effectively achieve real-time detection of H2O2 in red blood cells.

[0044] In addition to the above method, a μBot array composed of multiple μBots with sub-micron spatial resolution can also be used for real-time multi-site H2O2 detection of RBCs. Four μBots captured by four optical traps for real-time detection at sites 1, 2, 3, and 4 around the RBC membrane surface. Optical microscopy images (bright field) show that the four μBots are located at positions 1, 2, 3, and 4 around the normal RBC (c of Figure 10 , and the four μBots emit green light in dark field. The LIR of positions 1, 2, 3, and 4 remained around 1.26 (d of Figure 10 , indicating that the H2O2 concentration was uniformly distributed at these positions.

[0045] Example 5: μBot for real-time H2O2 detection in the dynamic process of red blood cells

[0046] Under external stimuli (such as tension), RBCs deform, potentially increasing or decreasing their H2O2 expression. To induce RBC deformation, an optical force is applied to pull and deform the RBC, while the μBot simultaneously records H2O2 levels for real-time monitoring. The method first establishes an optical trap (Trap 1) to simultaneously manipulate the RBC and the μBot (t = 0). The μBot is placed above the center of the RBC to prevent displacement from the initial detection position due to changes in cell morphology during RBC manipulation. After 1 minute of detection, another optical trap (Trap 2) is added to secure the edge of the RBC and pull it outward, causing the RBC to deform within seconds and maintain deformation until Trap 2 is removed at t = 5 minutes. Thereafter, the RBC gradually recovers its original shape due to its inherent elasticity. Figure 11 Figure a shows the optical microscopic images (bright field and dark field) at t = 0, 2, 5, and 7 min. At t = 0 min, the round RBC and the luminescent μBot on it are in the initial state ( Figure 11 At t = 2 min, the RBC deforms from a circular shape to an elliptical shape, and the luminescence intensity increases ( Figure 11 At t = 5 min, the RBC maintains its elliptical shape, but the luminescence intensity becomes stronger ( Figure 11 At t = 7 min, after the optical force is withdrawn, the RBC almost recovers its original shape, and its luminescence intensity decreases to the intensity observed at t = 0 min ( Figure 11 The relationship between μBot's LIR and time indicates that RBCs are engaged in normal physiological activities and produce normal levels of H2O2 ( Figure 11 b). The red background area indicates the dynamic H2O2 detection of RBCs after the addition of Trap 2. After pulling the RBCs, the μBot's LIR increased, reaching a maximum of 3.01 at t = 4 min. The estimated concentration of H2O2 was 34.96 × 10 -6 M. This indicates that the internal physiological activities of red blood cells have undergone drastic changes, disrupting the balance of intracellular catalase decomposition of H2O2, ultimately leading to an increase in H2O2 concentration. After 5 minutes, when the pulling force was withdrawn, the LIR of the μBot gradually decreased to 1.26 at t = 7 minutes, which is consistent with the situation at t = 0 minutes. This is attributed to the re-establishment of the balance of intracellular catalase decomposition of H2O2 when the RBC gradually returned to its initial shape from the elliptical shape. The ability of μBot to dynamically detect H2O2 around deformed red blood cells can help in the early detection of tumors and early warning of metastasis. These results indicate that the light-driven μBot of the present invention will be able to identify changes at specific sites in diseased cells, thereby providing evidence for exploring the pathogenesis of diseases such as cancer, discovering early diagnostic markers, and identifying therapeutic targets.

[0047] Example 6: μBot real-time detection experiment during erythrocyte apoptosis

[0048] To induce erythrocyte apoptosis, a high laser power of 60 mW was applied to the RBCs. Two optical traps were set up to manipulate the RBCs (Trap 1) and the μBot (Trap 2), respectively, bringing the μBot into contact with the RBC membrane surface. After maintaining stability for 1 minute, the laser power for trapping the RBCs was increased to 60 mW, causing photothermal damage to the RBCs and further inducing rapid apoptosis. Figure 12 Figure a shows optical microscopic images (bright field and dark field) of dynamic H2O2 detection of RBCs during apoptosis. At t = 0 min, the RBCs are plump and smooth, and the μBot next to them emits green light normally ( Figure 12 At t = 2 min, the RBCs shrank slightly and the luminescence intensity of the μBot became stronger, indicating the onset of RBC apoptosis ( Figure 12 At t = 4 min, RBC contraction became more obvious and holes began to appear. The luminescence intensity of μBot was the strongest, indicating that RBC was in the middle stage of apoptosis ( Figure 12 At t = 5 min, the holes on the RBC become more obvious, but the luminescence intensity of the μBot decreases ( Figure 12 a4), indicating that RBC is in the late stage of apoptosis. The relationship between μBot LIR and time shows ( Figure 12 (b) The LIR remained at around 1.26 with little change in the first minute, indicating normal RBC activity. After 1 minute, the LIR began to increase (t = 2 and 3 min) and reached a maximum of 6.15 at t = 4 min. The estimated concentration of H2O2 was 76.84 × 10 -6 This is because during erythrocyte apoptosis, intracellular catalase disrupts the balance of H2O2 decomposition; as the erythrocyte membrane dissolves, its contents are rapidly released, leading to a rapid increase in H2O2 concentration. At t = 5 and 6 minutes, the LIR decreases as H2O2 diffuses into the surrounding environment, causing the concentration around the RBC to gradually decrease. Therefore, the μBot can indicate redox homeostasis during normal cellular apoptosis and provide early warning of potential disease risks caused by abnormal apoptosis for health monitoring and early disease screening.

[0049] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a light-driven microrobot for real-time multi-site hydrogen peroxide detection, characterized in that: The steps include: (1) Dissolve PEG-NH2 and OVG in CH2Cl2 and acetone to prepare PEG-NH2 solution and OVG solution respectively; (2) The PEG-NH2 solution, UCNPs solution, and OVG solution were mixed together and ultrasonically treated to obtain a uniform dispersion solution. Then, a nitrogen stream was blown into the reaction flask to blow the mixed solution onto the wall of the rotating reaction vessel until the solvent was blown dry to form a thin film; The UCNPs solution is a cyclohexane solution of upconversion nanoparticles with a concentration of 1 mmol / 10 mL; The ultrasonic treatment time is 5 min; (3) Deionized water was added to the membrane and ultrasonicated to form a uniform suspension. The suspension was purified by centrifugation and washed with anhydrous ethanol. Centrifugation was repeated after each wash. Finally, the obtained μBot was resuspended in 1 mL of deionized water for subsequent testing. The ultrasonic treatment time was 10 min, the centrifugal speed was 8000 rpm for each time, the centrifugal time was 5 min, and the number of times of anhydrous ethanol was 2 times.

2. The method for preparing a light-driven microrobot for real-time multi-site hydrogen peroxide detection according to claim 1, characterized in that: The concentration of the PEG-NH2 solution described in step (1) is 5 mg / mL.

3. The method for preparing a light-driven microrobot for real-time multi-site hydrogen peroxide detection according to claim 1, characterized in that: The concentration of the OVG solution described in step (1) is 1 mg / mL.

4. The method for preparing a light-driven microrobot for real-time multi-site hydrogen peroxide detection according to claim 1, characterized in that: The amounts of PEG-NH2 solution, UCNPs solution and OVG solution added in step (2) are: 1 mL of PEG-NH2 solution, 20 μL of UCNPs solution and 50 μL of OVG solution, respectively.

5. A light-driven microrobot for real-time multi-site hydrogen peroxide detection, characterized in that: The invention is prepared by the method according to any one of claims 1 to 4.

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