Light-driven micro-robot for real-time multi-site hydrogen peroxide detection and preparation method of light-driven micro-robot

Abstract

The invention 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 of the light-driven micro-robot. The light-driven micro-robot is manufactured through a simple film blowing method, the light-driven micro-robot of the composite column bar Leba structure is invented, and UCNPs, OVG and PEG-NH2 are integrated. The [mu] Bot can detect H2O2 by analyzing the luminous intensity ratio (LIR) from UCNPs and OVG by luminescent resonance energy transfer (LRET). By means of scanning optical tweezers, the muBot can be captured by 1064nm near-infrared light, and the light with the wavelength basically has no damage to biological cells. The spatial resolution of muBot is 0.6 mu m, the detection limit of H2O2 concentration is 0.15 * 10 <-6 > M, and real-time and multi-site detection can be carried out on normal, deformed and apoptotic red blood cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of microrobots, and in particular relates to a light-driven microrobot for real-time multi-site hydrogen peroxide detection and a preparation method thereof. Background Art

[0002] Hydrogen peroxide (H2O2) is a potent biomarker of cellular physiological activity, regulating cell proliferation, differentiation, and metabolism, and playing a role in signal transduction, intracellular defense, and detoxification. Changes in H2O2 levels are closely associated with the development and progression 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 apoptosis, autophagy, cytokine expression, and mRNA expression, potentially further impacting cellular physiological functions and metabolic processes. Therefore, H2O2 detection is crucial for health monitoring and disease diagnosis. H2O2 detection methods, such as probes and molecular fluorescent probes, have greatly advanced this field. While needle-shaped probes are portable and stable, they are invasive to biological tissues, and their large size (tens of microns) limits improvements in spatial resolution. Molecular fluorescent probes offer the potential for non-invasiveness and submicron spatial resolution. However, their activity is often insufficient because they rely on passive staining of biological tissues and the passive transport properties of the anchored target moiety. Furthermore, they can contaminate 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] Microrobots (μBots) are tiny robotic systems capable of moving at the micrometer scale and performing specific tasks by harvesting chemical fuels or external energy sources (such as magnetic fields, acoustic fields, and optical fields). However, chemical-fuel-driven μBots use energy generated by chemical reactions (such as the decomposition of H₂O₂) to navigate, but residual fuel can be biotoxic, and low actuation accuracy (error >5 μm) limits precise movement. Magnetic-field-driven μBots can achieve fine motion control in complex biological environments, but they face technical challenges in maintaining a small robot size and may leave residual magnetic materials. Acoustic-field-driven μBots are biosafe within a certain frequency range, but long-term actuation may face challenges such as cavitation and low fluorescence imaging. In contrast, light-driven μBots use a single light source for on-off control and directional movement. This actuation method not only enables fast, flexible, and precise navigation, but also does not require additional chemicals or magnets. Furthermore, light-driven microstructures exhibit environmentally friendly properties and biocompatibility for biosensing, providing a potential solution for real-time H₂O₂ detection. Summary of the Invention

[0004] The purpose of the present invention is to address the existing problems and provide a light-driven microrobot for real-time multi-site hydrogen peroxide detection and a preparation method thereof.

[0005] The present invention is achieved through the following technical solutions: A light-driven microrobot for real-time multi-site hydrogen peroxide detection and a preparation method thereof, comprising the following steps: (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 flow 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; (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.

[0006] Furthermore, the concentration of the PEG-NH2 solution in step (1) is 5 mg / mL.

[0007] Furthermore, the concentration of the OVG solution in step (1) is 1 mg / mL.

[0008] Furthermore, the UCNPs solution described in step (2) is converted into a cyclohexane solution of nanoparticles at a concentration of 1 mmol / 10 mL.

[0009] Furthermore, the amounts of the 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.

[0010] Furthermore, the ultrasonic treatment time in step (2) is 5 min.

[0011] Furthermore, the ultrasonic treatment time in step (3) is 10 min, the centrifugal speed is 8000 rpm, the centrifugal time is 5 min, and the number of times of anhydrous ethanol is 2 times.

[0012] Furthermore, the μBot of the present invention adopts a composite Leba structure design ( Figure 1 ), including the following core components: Sensing layer: upconversion nanoparticles (UCNPs, β-NaYF4:1%Tm 3+) serves as a donor, emitting blue light (475 nm) upon excitation at 980 nm. The H2O2-responsive probe OxiVision Green (OVG) serves as an acceptor, generating green fluorescence (512 nm) via luminescence resonance energy transfer (LRET). Encapsulation layer: Polyethylene glycol amine (PEG-NH2) is electrostatically adsorbed around the sensing layer, with a thickness of 50-100 nm, to prevent leakage of the fluorescent group and enhance biocompatibility.

[0013] Furthermore, a scanning optical tweezers (SOTs) system (Tweez250si, Aresis, Europe) was used, as Figure 2 As shown. The system is equipped with a solid-state continuous-wave Nd: YAG (1064 nm) infrared laser (8) and an inverted microscope. For optical imaging and manipulation, a water immersion objective (4) is used, which features a magnification factor of 60× and a numerical aperture of 1.0. The 1064 nm laser beam emitted from the laser source is directed into an acousto-optic deflector (AOD) unit (7), which has a maximum switching rate of 100 kHz and is connected to a computer. This device facilitates spatial scanning of the optical trap by precisely controlling the laser beam. A beam expander (6) is used to widen the laser beam to produce a wide collimated laser beam. Subsequently, the laser beam is reflected by a dichroic mirror (5) and effectively focused by a water immersion objective (4), thereby generating an optical trap in the sample chamber (3). The power of a single optical trap is 0-100 mW, which can simultaneously achieve the capture, movement and rotation control of the μBot. The integrated illumination system uses a condenser (2) to focus the light onto the sample. The image in the sample plane is collected by the objective lens, then focused through a dichroic mirror (5), an additional reflector (9), and finally through a filter (10) onto a standard charge-coupled device (CCD) (11) camera.

[0014] Compared with the prior art, the present invention has the following advantages: The present invention uses a simple film blowing method to create 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 analyzing the luminescence intensity ratio (LIR) from UCNPs and OVG through luminescence resonance energy transfer (LRET). Using scanning optical tweezers, μBot can be trapped by 1064nm near-infrared light, which is basically harmless to biological cells. μBot has a spatial resolution of 0.6 μm and a detection limit of 0.15×10 -6 M, can perform real-time and multi-site detection of normal, deformed, and apoptotic red blood cells. The μBot's ability to detect H2O2 can facilitate early disease screening and is suitable for monitoring cell physiological activities, early disease diagnosis, and biomarker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of a light-driven microrobot; Figure 2 Schematic diagram of the experimental setup; Figure 2 Middle: 1. Illumination 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. Filter, 11. CCD charge-coupled device; Figure 3 Schematic diagram of the process of preparing μBots by film blowing; Figure 4 SEM image of the prepared μBot, scale bar is 300 nm; Figure 5 (a) Photoluminescence spectrum of UCNPs and absorption and emission spectra 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; Figure 6 The lifetime decay curves of μBot and UCNPs at 475 nm emission wavelength; Figure 7 Optical microscope images (bright and dark field) of the captured μBot in (a) deionized water and (b) H2O2 environments. Figure 8Optical microscopy images (dark field) of the linear (a) and circular (b) motion of the microrobot in a H2O2 environment; (c) Optical dark field microscopy image of the distance between two adjacent μBots in a H2O2 environment without interfering with each other's light emission. Laser power per light trap: 20 mW. Figure 9 (a) Optical microscopy images (dark field) of the green and blue channels of μBot in H2O2 solutions with different concentrations (40, 60, and 80 μM); (b) Relationship between the LIR of μBot and the H2O2 solution concentration in the range of 20-100 μM; (c) Optical stability of μBot in 20 μM H2O2 solution within 10 min; (d, e) Specificity evaluation of μBot in Cys and GSSG solutions with different concentrations (5-25 μM), laser power per optical trap: 20 mW; Figure 10 (a1-3) Optical microscopic images (bright field and dark field) of the μBot rotating around the RBC for real-time H2O2 detection; (b) LIR of the μBot when it rotates around the RBC at 0°, 90°, 180°, 270°, and 360°; (c) Optical microscopic images (bright field and dark field) of four μBots captured 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 the RBC, laser power of each optical trap: 20 mW; Figure 11 Middle, (a1-4) Optical microscopic images (bright field and dark field) of μBot dynamic H2O2 detection during RBC deformation; (b) LIR of μBot during RBC deformation; Figure 12 Middle, (a1-4) Optical microscopic images (bright field and dark field) of dynamic detection of H2O2 by the μBot during erythrocyte apoptosis; (b) LIR of the μBot during erythrocyte apoptosis. Laser power for inducing erythrocyte apoptosis: 60 mW. DETAILED DESCRIPTION

[0016] In order to further explain the present invention, it is described below with reference to the following specific embodiments.

[0017] Example 1: μBot morphology characterization and verification of luminescence resonance energy transfer The morphology was further characterized by scanning electron microscopy (SEM) images. Figure 4The 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.

[0018] 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 UCNPs transfer energy to OVG in μBot. Therefore, it can be determined that there is a LRET process from UCNPs to OVG in μBot.

[0019] Example 2: μBot's performance under scanning optical tweezers: movement and positioning experiments The SOTs' trapping laser is a laser beam with a central wavelength of 1064 nm. The UCNPs in this study can be excited by the 1064 nm laser, achieving luminescence, and the trapped light can also serve as the excitation light for the μBot. Figure 7 Figure a shows optical microscopic images (bright field and dark field) of a μBot trapped in deionized water (the laser power used was within the biosafety 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 7 b), confirming that 1064 nm laser can achieve μBot excitation without affecting μBot's H2O2 detection.

[0020] Microrobots trapped by SOTs can be manipulated by dynamic optical trapping. Figure 8 Figures a and b show optical microscopy images of the μBot's linear and circular motions. The microrobot's linear and rotational motion speeds were set at 3.6 μm / s and 0.25 rps, respectively. At t = 1, 2, and 3 s, the μBot moved linearly to 3.6, 7.2, and 10.8 μm, respectively. Figure 8 At t = 0.9, 2, and 3 s, μBot rotates counterclockwise by 81°, 180°, and 270°, respectively ( Figure 8 b). In addition, the two microrobots were stably captured by two optical traps, which could achieve a spatial resolution of approximately 0.6 μm by reducing the distance between the two μBots without interfering with each other's light emission ( Figure 8 c). Therefore, μBot can perform a series of movements and positioning to achieve real-time multi-point detection of H2O2.

[0021] Example 3: μBot H2O2 Detection Performance Experiment The detection performance of μBot was studied, and the optical microscopic images (dark field) of the green and blue channels of μBot showed ( Figure 9 (a) As the H2O2 solution concentration increases, the luminescence signal in the green channel increases, while the luminescence signal in the blue channel gradually decreases. Here, optical images of the green and blue channels were obtained by processing and separating optical microscopy images of the μBot using ImageJ software. The signal from the green channel serves as the detection signal, and the signal from the blue channel serves as the reference signal. The average grayscale value of the luminescence image directly reflects the luminescence signal. By calculating the average grayscale value of the green and blue channel images, the luminescence intensity ratio (LIR) of green to blue light, which depends on the H2O2 concentration, can be obtained. Figure 9b shows the linear relationship between LIR and H2O2 concentration, with the correlation coefficient R 2 = 0.988. The lowest concentration of H2O2 that can be detected from the background signal (expressed 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 fit. The calculated LOD value of μBot is 0.15×10 -6 M, which is lower than the value of 0.27×10 reported in the literature (Adv. Mater. 2022, 34, 2205760). -6 To ensure the accuracy of the detection, the LIR of μBot was monitored over a period of time to measure the luminescence stability of μBot ( Figure 9 c), showing that the LIR barely changes over time. Furthermore, cysteine ​​(Cys) and glutathione disulfide (GSSG) were selected as the main redox buffers in cells to evaluate the specificity of μBot, and a 20-μM H2O2 solution was used as the signal background. The relationship between the LIR of μBot and the concentrations of Cys and GSSG (5, 10, 15, 20, and 25 μM) is shown in Figure 2. Figure 9 d and e. The LIR remained constant with changes in cysteine ​​and GSSG concentrations, confirming the μBot's specificity for H2O2 solutions. Therefore, the μBot prepared in this invention exhibits a low detection limit, excellent optical stability, and specificity, enabling accurate detection of H2O2.

[0022] Example 4: μBot real-time detection of H2O2 concentration in red blood cells (RBC) Mature red blood cells were chosen as the experimental model because they have a simple structure, lack organelles such as the nucleus, have good rigidity, and are easily deformed. In addition, red blood cells carry a large amount of hemoglobin that participates in chemical reactions and quickly produces H2O2. First, a red blood cell and a μBot were captured by two optical traps respectively. Then, to ensure the detection accuracy and smooth rotation of the μBot, the μBot was driven as close to the red blood cell membrane surface as possible without adhesion, maintaining a distance of approximately 0.4 μm. Next, the μBot rotated around the RBC with a rotation speed of 0.1 rps and a rotation radius of 2.5 μm. At T = 0 s, the captured μBot was at its current position (θ = 0°) and was ready to start rotating ( Figure 10 As time goes by, μBot rotates to different positions; at t = 5 s and 10 s, μBot rotates to θ = 180° and 360°, respectively ( Figure 10a2, 3). In this way, the LIR of μBot at each position can be obtained, indicating the distribution concentration of H2O2 around RBC. The LIR measured by μBot at different positions (0°, 90°, 180°, 270° and 360°) remains around 1.26 ( Figure 10 b), indicating that the distribution of H2O2 around RBCs is relatively uniform. The H2O2 concentration measured around RBCs is consistent with the normal RBC expression range (10.65–11.98×10 -6 M), further reflecting the normal physiological state of RBC. This shows that μBot can effectively realize the real-time detection of H2O2 in red blood cells.

[0023] In addition to the above methods, a μBot array consisting of multiple μBots with submicron spatial resolution can also be used for real-time multi-site H2O2 detection of RBCs. Four μBots captured by four optical traps were used for real-time detection at sites 1, 2, 3, and 4 around the RBC membrane surface. Optical microscopy images (bright field) showed that the four μBots were located at sites 1, 2, 3, and 4 around the normal RBC ( Figure 10 c), and the four μBots emit green light in the dark field. The LIR of positions 1, 2, 3, and 4 remains around 1.26 ( Figure 10 d), indicating that the H2O2 concentration is uniformly distributed at these locations.

[0024] Example 5: μBot performs real-time H2O2 detection during the dynamic process of red blood cells 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 11At 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.

[0025] Example 6: μBot real-time detection experiment during erythrocyte apoptosis 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 12At 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.

[0026] 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; (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.

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 UCNPs solution described in step (2) is a cyclohexane solution of upconverted nanoparticles with a concentration of 1 mmol / 10 mL.

5. 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.

6. The method for preparing a light-driven microrobot for real-time multi-site hydrogen peroxide detection according to claim 1, characterized in that: The duration of the ultrasonic treatment in step (2) is 5 min.

7. The method for preparing a light-driven microrobot for real-time multi-site hydrogen peroxide detection according to claim 1, characterized in that: The ultrasonic treatment time in step (3) was 10 min, the centrifugal speed was 8000 rpm, the centrifugal time was 5 min, and the number of times of anhydrous ethanol was 2 times.

8. 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 7.

Images