Application of silver metallization microbubble in Hg < 2 + > detection and alloy microbubble nano-enzyme prepared from silver metallization microbubble in reductive drug detection

Abstract

The invention discloses application of silver metallized microbubbles in Hg < 2 + > detection and alloy microbubble nano-enzyme prepared from the silver metallized microbubbles in reductive drug detection, and belongs to the technical field of functional material preparation and nano-enzyme sensing. The invention aims to solve the problems of high background signal and poor reproducibility and stability of the existing carrier nano-enzyme. The method comprises the following steps: mixing silver metallization microbubbles with a TMB (Tetramethylbenzidine) solution, adding a sample to be detected, reacting, and standing until alloy microbubble nano-enzyme floats to the top end of the solution; determining the absorbance by using a microplate reader, or shooting a photo of the reaction solution by using handheld electronic equipment and extracting an RGB value; and calculating the concentration of Hg < 2 + > in the sample to be detected according to a pre-established concentration standard curve. According to the method, spontaneous separation is realized by utilizing micro-bubble buoyancy, quantitative analysis can be completed by combining a microplate reader or handheld electronic equipment, and the method is mainly used for on-site rapid detection of Hg < 2 + >.

Description

Technical Field

[0001] This invention belongs to the fields of functional material preparation technology, nanoenzyme sensing technology, and portable rapid detection technology, specifically relating to a silver-metallized microbubble in Hg 2+ Application of alloy microbubble nanozymes prepared therefrom in the detection of reducing drugs. Background Technology

[0002] Mercury ions, as a highly toxic heavy metal pollutant, are widely present in environmental water bodies and food matrices. Their residual levels directly affect ecological safety and human health, necessitating the development of convenient and accurate detection methods. Currently, conventional detection methods for mercury ions mostly rely on large-scale precision instruments such as inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS). While these methods offer high detection accuracy, they suffer from high equipment costs, complex operating procedures, long detection cycles, and reliance on specialized technical personnel. These limitations make them unsuitable for the practical needs of rapid on-site screening and high-throughput detection of large batches of samples, and are particularly unsuitable for widespread application in resource-constrained grassroots settings.

[0003] Colorimetric sensing technology based on nanomaterials has been increasingly applied to mercury ion detection research in recent years, attracting attention due to its advantages of visual response and relatively simple operation. Among them, nanozyme colorimetric sensing systems have become a research hotspot in this field due to their good material stability and relatively low preparation cost. However, existing nanozyme sensing systems still have significant shortcomings in practical applications: most nanozyme materials are uniformly dispersed in the detection reaction system, requiring additional steps such as centrifugation and filtration to separate the nanozyme from the reaction system after the reaction is completed in order to obtain stable signal readings. This not only increases the complexity of the detection operation but may also cause material loss or changes in the state of the reaction system during separation, thereby affecting the accuracy and batch-to-batch reproducibility of the detection results.

[0004] Hollow glass microbubbles, as low-density microcarriers, possess natural buoyancy and can theoretically achieve spontaneous separation after reaction through buoyancy, potentially simplifying separation steps in detection processes. However, the smooth surface and strong chemical inertness of hollow glass microbubbles make it difficult to directly achieve uniform and stable loading of functional recognition components and signal response components. Existing surface functionalization modification techniques for hollow glass microbubbles, especially surface metallization modification methods, often face problems such as uneven loading of metal components, weak bonding with the carrier, and easy detachment and aggregation. This results in significant batch-to-batch performance differences in the modified microbubble materials and insufficient signal response stability, making it difficult to meet the consistency requirements of material performance for routine mercury ion detection and hindering the practical application of such materials in the field of sensing detection. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0006] Another objective of this invention is to provide an Hg 2+ The colorimetric detection method can be based on silver and Hg. 2+ The specific amalgamation reaction enables selective recognition of the target analyte. Combined with absorbance measurement or RGB analysis using handheld electronic devices (such as smartphones or tablets), Hg analysis can be performed without the need for large instruments. 2+ Rapid on-site quantitative detection simplifies the testing process, lowers the operational threshold, and adapts to the practical application needs of scenarios such as water quality monitoring and environmental screening.

[0007] To achieve these objectives and other advantages of the present invention, an Hg is provided. 2+ The colorimetric detection method includes the following steps: Silver-metallized microbubbles were mixed with a TMB solution containing H2O2, and then Hg was added. 2+ The sample to be tested was reacted at room temperature for 10-15 min; After the reaction, the mixture is allowed to stand until the alloy microbubble nanoenzyme floats to the top of the solution. The absorbance at 652 nm was measured using an ELISA reader, or a handheld electronic device was used to photograph the reaction solution and extract the RGB values. Based on the pre-established absorbance-Hg 2+ Concentration standard curve or R / B value - Hg 2+ The concentration standard curve was used to calculate the Hg content in the sample. 2+ concentration.

[0008] In existing mercury ion detection technologies, large-scale precision instruments such as electrochemical workstations, atomic absorption spectrometers, or inductively coupled plasma mass spectrometry are widely used. While these methods offer high accuracy, they are expensive, involve complex procedures, have long detection cycles, and are difficult to implement rapidly in resource-constrained environments or under on-site conditions. To address this issue, this invention is based on the interaction of silver and Hg... 2+ The specific amalgamation reaction of TMB (tungsten mercury) generates a layer of silver-mercury alloy nanoparticles on the surface of microbubbles through in-situ amalgamation, thereby preparing alloy microbubble nanozymes. In this process, TMB not only participates in alloy formation as a reducing agent but also acts as a chromogenic substrate. The color change of the reaction system provides real-time feedback on the peroxidase-like activity of the formed alloy nanozymes, achieving integration of material preparation and catalytic performance evaluation. When Hg... 2+ When present, the generated alloy microbubble nanoenzymes exhibit peroxidase-like activity, catalyzing the oxidation of TMB to produce a blue product, and the change in absorbance or RGB value is related to Hg. 2+The concentrations showed a positive correlation. The entire detection process can be completed at room temperature, requiring no complex sample pretreatment or equipment operation, significantly simplifying the detection procedure. Simultaneously, the buoyancy-induced self-enrichment effect of microbubbles locally enriches catalytically active sites, enhancing detection sensitivity. Combined with RGB analysis using a handheld electronic device, rapid on-site quantitative detection can be achieved without specialized instruments, providing a simple and efficient technical means for mercury ion screening in water quality monitoring, environmental assessment, and food safety.

[0009] Preferably, the preparation of silver-metallized microbubbles includes the following steps: S1: Disperse hollow glass microbubbles in a piranha solution and stir the reaction at room temperature for 0.5-2 h. Then wash with ultrapure water until the washing solution is neutral and vacuum dry to obtain surface-activated hollow glass microbubbles. S2: Add the surface-activated hollow glass microbubbles to a polyethyleneimine solution with a concentration of 1-8 mg / mL, and rotate and mix at room temperature for 0.5-12 h. After the reaction, let it stand until the hollow glass microbubbles float to the top of the solution, remove the lower layer of solution, wash with ultrapure water, and vacuum dry to obtain hollow glass microbubbles coated with polyethyleneimine. S3: Add hollow glass microbubbles coated with polyethyleneimine to a buffer solution containing 1-1000 pmol of single-stranded DNA rich in cytosine bases, rotate and mix at room temperature for 0.5-4 h, and after the reaction, let stand until the complex floats to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain the intermediate product loaded with single-stranded DNA. S4: Mix the intermediate product loaded with single-stranded DNA with a silver nitrate solution of 0.1-50 mM, rotate and mix at room temperature for 10-120 min, and let stand until the complex floats to the top of the solution. Remove the lower layer of solution and wash with ultrapure water to obtain hollow glass microspheres loaded with silver ions. S5: Mix hollow glass microspheres loaded with silver ions with a sodium borohydride solution with a concentration of 0.1-50 mM, vortex for 10-200 min, and let stand until the product floats to the top of the solution. Remove the lower layer of solution and wash with ultrapure water to obtain silver metallized microbubbles.

[0010] When constructing a metal functional layer on the surface of hollow glass microbubbles, conventional metal deposition methods struggle to achieve uniform and stable metal coatings due to the smooth surface and chemical inertness of the microbubbles. This often results in particle agglomeration or detachment, leading to poor catalytic performance and significant batch-to-batch variations. To address this deficiency, this method first activates the surface of hollow glass microbubbles with a piranha solution to increase their surface reactivity. Then, utilizing the positive charge of polyethyleneimine, a uniform adsorption layer is constructed on the microbubble surface. Cytosine-rich single-stranded DNA is stably loaded onto the microbubble surface via electrostatic interactions. The specific coordination of cytosine bases with silver ions achieves ordered anchoring of the silver ions. Finally, reduction with sodium borohydride forms a uniformly distributed and firmly bound layer of silver nanoparticles on the microbubble surface.

[0011] Preferably, the step of taking a photograph of the reaction solution and extracting its RGB values ​​using a handheld electronic device includes: The reacted porous plate is placed on the imaging bracket. The non-detection area of ​​the porous plate is pre-set with a standard colorimetric bar, which contains at least one color block with a known RGB value. Use a handheld electronic device to simultaneously capture images of all reaction wells and standard colorimetric bars within the same field of view; Extract the R, G, and B values ​​of the central region of each reaction well in the image, and simultaneously extract the R, G, and B values ​​of the standard colorimetric bar; By utilizing the correspondence between the known RGB values ​​of the standard colorimetric bar and the extracted values, the image is color-corrected to obtain the corrected RGB values ​​of the reaction apertures; The R / B ratio was calculated based on the corrected RGB values ​​of the reaction wells, and then calculated according to the pre-established R / B value - Hg. 2+ Calculation of Hg in the sample by concentration standard curve 2+ concentration.

[0012] In practical applications, colorimetric detection based on handheld electronic devices often faces challenges such as changes in ambient lighting, differences in shooting angles, and inconsistent spectral responses of cameras from different mobile phone models. These factors can lead to systematic deviations in the extracted RGB values, thus affecting the accuracy and reproducibility of the detection results. To address this limitation, this invention further optimizes the RGB value extraction and correction process. During detection, the reacted multi-well plate is placed on a shooting bracket, and a standard colorimetric strip containing known RGB values ​​is preset in the non-detection area of ​​the multi-well plate. A handheld electronic device is used to simultaneously capture images containing all reaction wells and the standard colorimetric strip within the same field of view, ensuring consistent shooting conditions. The R, G, and B values ​​of the central region of each reaction well in the image are extracted, along with the R, G, and B values ​​of the standard colorimetric strip. A color correction model is established using the correspondence between the known RGB values ​​of the standard colorimetric strip and the actual extracted values ​​to perform overall color correction on the image, obtaining the corrected RGB values ​​of the reaction wells. Based on this, the R / B ratio is calculated, and the result is determined according to the pre-established R / B value - Hg. 2+ Calculation of Hg in the sample by concentration standard curve 2+ Concentration. This method, by introducing a standard colorimetric bar as a color reference, effectively eliminates color distortion caused by differences in shooting equipment, lighting environment, and shooting operation, making RGB data obtained from different batches and different devices comparable. It significantly improves the accuracy and data reproducibility of colorimetric detection using handheld electronic devices, providing a more reliable quantitative analysis method for rapid on-site detection.

[0013] Preferably, the non-detection area of ​​the multi-well plate is provided with multiple micro standard color blocks, which correspond one-to-one with the reaction wells of the multi-well plate and are arranged adjacent to the edge of the reaction well. When images are captured using a handheld electronic device, the image area of ​​each reaction well and the image area of ​​its corresponding micro standard color patch are captured simultaneously. Extract the R, G, and B values ​​of the central region of each reaction well, and simultaneously extract the R, G, and B values ​​of the micro standard color patch corresponding to that reaction well; By utilizing the correspondence between the known RGB values ​​and extracted values ​​of each micro-standard color patch, the RGB values ​​of the corresponding reaction wells are independently corrected to obtain the corrected R, G, and B values ​​for each reaction well. Based on the corrected RGB values ​​of each reaction well, the R / B ratio is calculated, and then adjusted according to the pre-established R / B value - Hg. 2+ Calculation of Hg in the sample by concentration standard curve 2+ concentration.

[0014] While using a single standard colorimetric bar to correct the overall image can eliminate some of the effects of differences in shooting conditions, for a detection system like a multi-well plate containing multiple independent reaction units, the reaction wells at different locations are often under different lighting environments. For example, the light intensity, angle, and potential shadow distribution of wells located at the edges differ from those at the center when illuminated. This spatial unevenness in lighting cannot be effectively corrected by a single standard colorimetric bar, resulting in systematic deviations in the RGB values ​​of different wells after correction, thus affecting the consistency and accuracy of the detection results for each well. To address this issue, this solution sets a corresponding miniature standard color block for each reaction well in the non-detection area of ​​the multi-well plate. These miniature color blocks are arranged near the edge of their respective reaction wells, ensuring that each color block and its corresponding reaction well are under almost identical lighting environments. When capturing images using a handheld electronic device, each reaction well and its adjacent micro-standard color patch are simultaneously captured. While extracting the RGB values ​​of the central region of each reaction well, the corresponding RGB values ​​of the micro-standard color patch are also extracted. Utilizing the correspondence between the known RGB values ​​of each micro-standard color patch and the actual extracted values, the RGB values ​​of the corresponding reaction wells are independently corrected to obtain the corrected R, G, and B values ​​for each reaction well. This local correction method effectively eliminates color distortion caused by differences in illumination between wells, ensuring that the color data of each reaction well is accurately reproduced based on the illumination conditions of its own environment. This significantly improves the consistency and precision of multi-well plate colorimetric detection, providing a more reliable data foundation for high-throughput sample analysis.

[0015] Preferably, the non-detection area of ​​the multi-hole plate is also provided with an information encoding area, which stores the precise color value data of each micro standard color block; Before taking pictures using a handheld electronic device, the information encoding area is read through the handheld electronic device to obtain the precise color value of each micro standard color block; After extracting the RGB values ​​of the micro standard color patches corresponding to each reaction well, the RGB values ​​of the corresponding reaction wells are independently corrected by using the correspondence between the precise color values ​​of the micro standard color patches and the extracted values, so as to obtain the corrected R, G, B values ​​of each reaction well. The R / B ratio is calculated based on the RGB values ​​of each reaction well after correction, and then based on the pre-established R / B value - Hg. 2+ Calculation of Hg in the sample by concentration standard curve 2+ concentration.

[0016] When using micro-standard color blocks to independently calibrate each reaction well, the accuracy of the calibration depends on the precision of the color value of the micro-standard color block itself. However, in actual production, whether micro-standard color blocks are produced through printing or material inlay, slight color differences may exist between different batches or even between color blocks in different positions within the same batch. Factors such as uneven ink thickness, fluctuations in dye concentration, or material aging can cause the actual color of the color block to not be completely consistent with the uniform nominal value specified at the factory. If all micro-standard color blocks are calibrated using the same fixed nominal value, this individual difference will be introduced as a systematic error into the calibration process of each reaction well, thus affecting the accuracy of the final test results. To address this issue, this solution further incorporates an information encoding area in the non-detection area of ​​the multi-well plate. This encoding area stores the precise color value data of each micro-standard color block measured by a precision instrument in the form of a QR code or similar format. Before capturing images using a handheld electronic device, the information encoding area is first scanned and read using a mobile phone to obtain the precise color value corresponding to each micro-standard color patch. After extracting the RGB values ​​of each reaction well and its corresponding micro-standard color patch, the RGB values ​​of the corresponding reaction well are independently corrected using the correspondence between the precise color value of each micro-standard color patch and the actual extracted value, obtaining the corrected R, G, and B values ​​for each reaction well. This method effectively eliminates the systematic bias caused by individual differences in micro-standard color patches, ensuring that the color correction of each reaction well is based on the precise reference value of its specific color patch, thereby achieving higher-precision color reproduction and further improving the accuracy and reliability of colorimetric detection using handheld electronic devices.

[0017] A colorimetric detection method for reducing drugs, comprising the following steps: Silver-metallized microbubbles were mixed with a TMB solution containing H2O2 and Hg. 2+ The solutions are mixed and reacted at room temperature for 10-15 minutes to allow Hg to react. 2+ When combined with silver-metallized microbubbles, an alloy microbubble nanoenzyme with peroxidase-like activity is produced. Add the test sample containing the reducing agent and react at room temperature for 10-15 min; After the reaction, the mixture is allowed to stand until the alloy microbubble nanoenzyme floats to the top of the solution. The absorbance at 652 nm was measured using an ELISA reader, or a handheld electronic device was used to photograph the reaction solution and extract the RGB values. The concentration of reducing drug in the sample to be tested is calculated based on the pre-established absorbance-drug concentration standard curve or R / B value-drug concentration standard curve.

[0018] In the routine detection of reducing drugs, large-scale precision instruments such as high-performance liquid chromatography (HPLC), mass spectrometry (MS / MS), or electrochemical workstations are the main analytical tools. While these methods can achieve accurate quantification, they are costly, involve complex sample pretreatment, have long detection cycles, and require professional operators, making them unsuitable for the practical needs of rapid on-site screening of food, pharmaceutical, and clinical samples. To address this technical bottleneck, this solution provides a colorimetric detection method for reducing drugs based on alloy microbubble nanoenzymes. The operational steps are as follows: first, silver metallized microbubbles are reacted with TMB solution and Hg... 2+ The solution is mixed and reacted at room temperature for 10 to 15 minutes to generate an alloy microbubble nanozyme with peroxidase-like activity. The test sample containing a reducing agent is then added, and the reaction continues at room temperature for another 10 to 15 minutes. After the reaction, the mixture is allowed to stand, where the microbubbles float spontaneously to the surface without centrifugation or filtration. Finally, the absorbance at 652 nm is measured using an ELISA reader, or an image is taken using a handheld electronic device and the RGB values ​​are extracted. The concentration of the reducing agent in the sample is calculated based on a pre-established absorbance-drug concentration standard curve or an R / B value-drug concentration standard curve. This method is based on the following principle: silver-metallized microbubbles and Hg... 2+ The resulting alloy microbubble nanozymes exhibit enhanced peroxidase-like activity, catalyzing the oxidation of TMB to blue oxTMB. When reducing agents are present in the system, these agents, acting as reducing agents, can competitively react with oxTMB or reactive oxygen species generated during the reaction, leading to a reduction in the blue product and a decrease in absorbance. The magnitude of this decrease is positively correlated with the concentration of the reducing agent. The entire detection process can be completed at room temperature, requiring no complex pretreatment or large-scale instrumentation. The buoyancy-induced self-enrichment effect of the microbubbles locally enriches the catalytically active sites, improving detection sensitivity. Combined with precise quantification using an ELISA reader or rapid RGB analysis using a handheld electronic device, this solution meets both the precise testing needs of laboratories and enables rapid screening under on-site conditions. It provides a simple and efficient technical solution for the quality control and on-site monitoring of reducing agents such as ascorbic acid and N-acetylcysteine.

[0019] Preferably, the reducing agent is one or more of ascorbic acid, N-acetylcysteine, D-penicillamine, dimercaptosuccinic acid, vitamin K3, edaravone, resveratrol, and curcumin.

[0020] A method for detecting Hg 2+ A portable colorimetric sensor, comprising a signal reporting unit, a colorimetric substrate, a reaction vessel, and a signal reading unit; The signal reporting unit is a silver-metallized microbubble; The chromogenic substrate is a mixed solution containing TMB and H2O2; The reaction vessel is a multi-well plate used to contain silver metallized microbubbles, chromogenic substrate, and the sample to be tested. The signal reading unit is a handheld electronic device used to capture images of the reaction solution and extract RGB values ​​for quantitative analysis.

[0021] In the context of rapid on-site detection of heavy metal ions and reducing agents, traditional methods often rely on large, sophisticated instruments such as ELISA readers, high-performance liquid chromatography (HPLC), or inductively coupled plasma mass spectrometry (ICP-MS). These methods suffer from high equipment costs, cumbersome operation, long detection cycles, and difficulty in meeting the demands of high-throughput on-site screening. To address this limitation, this technical solution constructs a portable colorimetric sensor based on silver-metallized microbubbles. This sensor uses silver-metallized microbubbles as the signal reporting unit, a mixed solution of TMB and H2O2 as the chromogenic substrate, a multi-well plate as the reaction vessel, and a handheld electronic device as the signal reading terminal. During detection, the silver-metallized microbubbles react with mercury ions in the sample to generate alloy microbubble nanozymes with peroxidase-like activity, catalyzing the oxidation of TMB for color development. After the reaction, the microbubbles, due to their low density, spontaneously float and aggregate to the liquid surface under buoyancy, forming a localized high-concentration micro-region. Signal reading and system separation can be achieved without centrifugation or filtration. Compared to a control system without buoyancy, this self-enrichment effect significantly enhances peroxidase-like activity, thereby significantly increasing detection sensitivity. After the colorimetric reaction, images of the reaction wells are captured using a handheld electronic device, and RGB values ​​are extracted for quantitative analysis. This allows for semi-quantitative screening through visual observation of color changes, or precise quantification based on the linear relationship between the R / B value and the target analyte concentration. This sensor integrates signal response, separation and enrichment, and detection readout, greatly simplifying the operation process and eliminating reliance on large equipment. It provides a simple, low-cost, and field-deployable rapid detection method for water quality monitoring, food safety, and pharmaceutical quality control.

[0022] An alloy microbubble nanoenzyme is prepared by the following steps: mixing silver metallized microbubbles with a TMB solution containing H2O2 and Hg at a concentration of 10-100 μM. 2+ The solutions were mixed and reacted at room temperature for 10-15 minutes. After the reaction, the mixture was washed with ultrapure water to obtain alloy microbubble nanoenzymes.

[0023] Silver-metallized microbubbles in Hg 2+ Application of alloy microbubble nanozymes prepared therefrom in the detection of reducing drugs.

[0024] The present invention has at least the following beneficial effects: This invention first provides a method for preparing alloy microbubble nanoenzymes. Through polyethyleneimine-mediated layer-by-layer assembly and in-situ amalgamation reaction, a uniform and stable loading of silver-mercury alloy nanoparticle layers is achieved on the surface of chemically inert hollow glass microbubbles. This effectively solves the problems of easy detachment and uneven distribution of metal coatings. Furthermore, the catalytic activity can be evaluated simultaneously during the preparation process, simplifying the process and improving the controllability and batch stability of the materials.

[0025] Secondly, based on the unique buoyancy-driven self-enrichment effect of silver metallized microbubbles, a portable colorimetric sensor was constructed, which allows the reacted material to spontaneously float and aggregate without centrifugation. This not only simplifies operation but also significantly enhances peroxidase-like activity and thus significantly improves detection sensitivity.

[0026] Furthermore, this sensor integrates the precise quantification of an ELISA reader with the RGB dual-mode reading function of a handheld electronic device, enabling Hg measurement without the need for large instruments. 2+ It enables rapid on-site detection of reducing drugs such as ascorbic acid, and demonstrates excellent accuracy and anti-interference capabilities in actual sample testing, providing a low-cost, high-throughput, and field-deployable general-purpose testing platform for environmental monitoring, food safety, and drug quality control.

[0027] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the preparation process of the alloy microbubble nanoenzyme of the present invention. Figure 2 SEM, HRSEM, and TEM characterization images of hollow glass microbubbles (GB), silver metallized microbubbles (B@Ag), and alloy microbubble nanoenzymes B@AgHg; Figure 3 For B@Ag to Hg 2+ The optical response detection results are shown; where a is the quantitative analysis and visual characterization of absorbance at 652 nm; b is the ultraviolet-visible absorption spectrum of the system. Figure 4 The graph shows the response of the B@AgHg system at 652 nm over time. Figure 5 Under the same experimental conditions, buoyant silver metallized microbubbles (B@Ag) and non-buoyant silica-supported silver microspheres (SiO2@Ag) were compared after the addition of Hg. 2+ Comparison of peroxidase-like activities before and after; Figure 6 The absorbance of the B@AgHg system at 652 nm and Hg2+ Linear calibration curve of concentration; Figure 7 The alloy microbubble nanoenzyme of the present invention is used for Hg 2+ A schematic diagram illustrating the construction of a portable colorimetric sensor for ascorbic acid; Figure 8 For the B@AgHg system against Hg 2+ Performance analysis of colorimetric sensors in smartphones; where a is the R / B value and Hg 2+ Linear relationship of concentration; b is the correlation analysis of R / B value and absorbance at 652 nm; Figure 9 The graph shows the selective response of the B@AgHg system to different substrate metal ions. Figure 10 R / B value and Hg based on standard colorimetric bar correction 2+ Concentration linear relationship graph; Figure 11 R / B value and Hg based on hole-color patch one-to-one correction 2+ Concentration linear relationship graph; Figure 12 R / B value and Hg corrected based on accurate color values ​​of the information encoding area 2+ Concentration linear relationship graph; Figure 13 The linear fitting curve of the absorbance of B@AgHg at 652 nm versus the concentration of ascorbic acid is shown. Figure 14 Performance analysis of the B@AgHg system for colorimetric sensing of ascorbic acid in smartphones; where a is the linear relationship between R / B value and ascorbic acid concentration; b is the correlation analysis between R / B value and absorbance at 652 nm. Figure 15 Statistical chart showing the anti-interference performance of the B@AgHg system against ascorbic acid. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0032] Experiment 1: Preparation and Characterization of Alloy Microbubbles B@AgHg according to Figure 1 The process shown illustrates the following steps for preparing alloy microbubbles B@AgHg: 1) Weigh 100 mg of hollow glass microbubbles (GB) pretreated with piranha solution (H2O2 / H2SO4=1:3, v / v), add 10 mL of polyethyleneimine solution (PEI solution: dissolved in ultrapure water to a concentration of 4 mg / mL), and mix at room temperature for 12 h (rotation speed of 5 rpm). After standing, the bubbles float to the top of the solution, the lower layer of solution is removed with a syringe, and then washed three times with ultrapure water. Finally, vacuum dry at room temperature to obtain PEI-encapsulated hollow glass microbubbles (B@PEI). 2) Accurately weigh 5 mg of B@PEI and add it to a 1.5 mL centrifuge tube, and add 1 mL of DNA buffer (200 pmol, single-stranded DNA rich in cytosine bases) to disperse it; after mixing and reacting at room temperature by rotating (5 rpm) for 0.5 h, the resulting B@PEI@DNA complex is washed three times with ultrapure water to remove free DNA. 3) Add 1 mL of AgNO3 solution (10 mM) to the B@PEI@DNA complex and shake to mix for 3 h. After the reaction is complete, wash three times with ultrapure water to remove free Ag. + Hollow glass microspheres loaded with silver ions (B@PEI@DNA@Ag) were obtained. + ); 4) Add B@PEI@DNA@Ag to 1 mL of freshly prepared ice-bathed NaBH4 solution (10 mM). + The complex was vortexed for 0.5 h, and B@Ag was collected. It was washed three times with ultrapure water to remove residual NaBH4 and reaction byproducts. After washing, B@Ag was redispersed in ultrapure water and stored in a refrigerator at 4 °C for later use.

[0033] 5) Take 4000 B@Ag nanoparticles into a 96-well plate, add 60 μL of TMB solution to disperse them, then add 10 μL of Hg(NO3)2 (dissolved in ultrapure water, 100 μM), and make up the total volume with ultrapure water to 100 μL; shake at room temperature for 10 min, let stand to separate, wash three times with ultrapure water, and collect the alloy microbubble nanoenzyme B@AgHg.

[0034] like Figure 2As shown, the morphology of GB, B@Ag, and B@AgHg was characterized using scanning electron microscopy (SEM), high-resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM). It can be observed that the smooth GB surface exhibits a uniform distribution of particles after metallization. After amalgamation, the silver amalgam aggregates into larger spherical particles or strip-like structures, which are firmly attached to the GB surface, confirming the successful construction of B@AgHg.

[0035] Experiment 2: The effect of B@AgHg system on Hg 2+ Colorimetric response test: Accurately pipette 4000 B@Ag molecules into a 96-well plate, add 60 μL of TMB solution containing H2O2 to each well for dispersion, then add 10 μL of Hg(NO3)2 (dissolved in ultrapure water, 100 μM), and bring the total volume to 100 μL with ultrapure water. After shaking at room temperature for 10 min, measure the absorbance of the B@AgHg system at 652 nm using a microplate reader, and observe the color change of the solution.

[0036] like Figure 3 a, Figure 3 As shown in b, add Hg 2+ Subsequently, TMB is oxidized to oxTMB, and the solution turns light blue; among which, Figure 3 The results showed that the absorbance at 652 nm was significantly different from that of the control group, which can achieve the detection of Hg. 2+ Quantitative analysis and visual representation, Figure 3 b is the UV-Vis absorption spectrum of the system, showing the effect of adding Hg. 2+ The system then exhibited a characteristic absorption peak at 652 nm, confirming that B@AgHg, generated by in-situ amalgamation of B@Ag, has enhanced peroxidase-like activity, while B@Ag has almost no nanozyme activity.

[0037] Experiment 3: Catalytic behavior of the B@AgHg system at different times: Accurately pipette 4000 B@Ag molecules into a 96-well plate, add 60 μL of TMB solution containing H2O2 to each well for dispersion, then add 10 μL of Hg(NO3)2 (dissolved in ultrapure water, 100 μM), and make up to 100 μL of ultrapure water; continuously monitor the absorbance of the B@AgHg system at 652 nm at different time points using a microplate reader.

[0038] The catalytic behavior of the B@AgHg system was evaluated using time-dependent absorbance curves. Figure 4As shown, in the initial stage of the reaction (0-10 min), the absorbance value at 652 nm increased rapidly, indicating that TMB was rapidly oxidized into a blue oxidation product. As time increased (10-30 min), the rate of increase in absorbance gradually slowed down and tended to plateau, which is attributed to the consumption of substrate or the accumulation of product reaching reaction equilibrium. This curve provides a basis for subsequently determining the optimal detection time (10 min).

[0039] Experiment 4: Contribution of buoyancy to the activity of B@AgHg nanoenzymes as peroxidases: Using solid SiO2 microspheres without buoyancy as a control, SiO2@Ag was prepared using the same method as B@Ag, and then SiO2@AgHg was obtained through amalgamation. 4000 B@Ag and 4000 SiO2@Ag particles were accurately pipetted into 96-well plates, and 60 μL of TMB solution was added to each well for dispersion. Then, 10 μL of Hg(NO3)2 (dissolved in ultrapure water, 100 μM) was added, and the volume was brought up to 100 μL with ultrapure water. After shaking at room temperature for 10 min, the absorbance at 652 nm was measured.

[0040] like Figure 5 As shown, under the same conditions, adding Hg 2+ Subsequently, the absorbance of the B@Ag-generated B@AgHg system was significantly higher than that of the SiO2@Ag-generated SiO2@AgHg system, and the background absorption of the B@Ag substrate was lower. This result indicates that the buoyancy-driven self-enrichment effect of hollow microbubbles can significantly enhance the peroxidase-like activity of B@AgHg.

[0041] Experiment 5: The effect of the B@AgHg system on Hg 2+ Quantitative detection performance test: Accurately pipette 4000 B@Ag molecules into a 96-well plate, add 60 μL of TMB solution to each well for dispersion, then add 10 μL of Hg(NO3)2 at different concentrations (0, 10, 20, 40, 60, 80, 100 μM), and make up to 100 μL with ultrapure water; after shaking at room temperature for 10 min, measure the absorbance of the B@AgHg system at 652 nm using a microplate reader.

[0042] like Figure 6 As shown, with Hg 2+ As the concentration increased from 10 μM to 100 μM, the absorbance of the system gradually increased, exhibiting a good linear relationship within this range. The linear regression equation was Y = 0.0075X - 0.022 (R²). 2 =0.998), and the detection limit calculated according to the 3σ / S rule is 0.36 μM. This good linearity indicates that the B@AgH system is effective against Hg. 2+The response is highly concentration-dependent and can be used for Hg. 2+ Precise quantitative analysis.

[0043] Example 1: Hg based on smartphone RGB analysis 2+ Portable detection 1. Construction of the detection system ( Figure 7 ) 4000 B@Ag molecules were placed in a 96-well plate, and 60 μL of TMB solution (commercially available TMB solution, acetate buffer containing TMB and H2O2, pH 6.5) and 10 μL of Hg(NO3)2 solution (100 μM) were added sequentially. Ultrapure water was then added to bring the total volume to 100 μL. The plate was incubated at room temperature with shaking for 10 min. The Hg... 2+ An in-situ amalgamation reaction with surface silver generates a silver-mercury alloy microbubble nanoenzyme (B@AgHg). Hg is then added into the pores. 2+ To prepare Hg containing different concentrations 2+ The reaction system consisted of 10, 20, 40, 60, 80, and 100 μM solutions, with four replicates for each concentration. After reacting at room temperature for 10 min, the solution was allowed to stand to allow the complex to float to the surface, and the solution color stabilized.

[0044] 2. Image Acquisition and RGB Analysis Place the 96-well plate under uniform lighting (standard light box or natural light with a white background), and take a vertical shot using the rear camera of your phone, maintaining a consistent shooting distance and angle. Import the photo into Adobe Photoshop 2020, use the rectangular marquee tool to select the center area of ​​each well (avoiding edge shadows), record the R, G, and B values, and calculate the R / B ratio.

[0045] 3. Establishing the standard curve Plotting R / B values ​​on the ordinate, Hg 2+ A standard curve is plotted with concentration on the x-axis. For example... Figure 8 As shown in figure a, within the range of 10-100 μM, the R / B value and Hg 2+ The concentration showed a good linear relationship, with the linear regression equation being Y = -0.0081X + 1.07 (R² = 0.988) and the detection limit being 1.84 μM.

[0046] 4. Correlation Validation Pearson correlation analysis was performed on the R / B values ​​of the same samples and the absorbance values ​​at 652 nm measured by the microplate reader in Example 1. The results showed a significant negative correlation between the two (r = -0.974). Figure 8 (b) This confirms that smartphone colorimetry can serve as a reliable alternative to ELISA readers and is suitable for rapid on-site testing.

[0047] 5. Selective evaluation Under the same experimental conditions, Hg was investigated. 2+ (100 μM) and other common metal ions (Ag) + Na + K + Zn 2+ Cd 2 + Fe 3+ Mn 2+ Mg 2+ Co 2+ Pb 2+ Ca 2+ Al 3+ Fe 2+ Zr 4+ Cu 2+ Ni 2+ The effect of concentrations (all at 100 μM) on the detection system. Figure 9 As shown, only Hg 2+ The method elicits a significant response, while other ions show no obvious interference, indicating that it is effective for Hg. 2+ It has excellent selectivity.

[0048] Example 2: Smartphone colorimetry based on standard colorimetric strip calibration for Hg 2+ accurate detection 1. Construction of the detection system Following the method in Example 1, preparations containing different concentrations of Hg were prepared. 2+ The reaction system consisted of 10, 20, 40, 60, 80, and 100 μM solutions, with four replicates for each concentration. After shaking the reaction at room temperature for 10 min, the solution was allowed to stand to allow the alloy microbubble nanoenzyme (B@AgHg) ​​to float to the surface, and the solution color stabilized.

[0049] 2. Perforated Plate Structure Design Using a specially designed 96-well plate, a standard colorimetric strip is pre-set in its non-test area (such as the edge of the plate or the blank area of ​​the plate surface). This colorimetric strip contains at least three color blocks with known RGB values ​​(such as light blue, medium blue, and dark blue) for subsequent color correction.

[0050] 3. Image Acquisition Place the reacted 96-well plate under standard lighting conditions (such as a standard light source box or natural light with a white background), and use the rear camera of a smartphone to take a vertical shot, ensuring that all reaction wells and standard colorimetric bars are captured in the same image simultaneously, while maintaining a consistent shooting distance and angle.

[0051] 4. RGB value extraction and color correction Import the image into Adobe Photoshop 2020 or a custom color analysis application: Extract the R, G, and B values ​​of the central region of each reaction well; Simultaneously extract the R, G, and B values ​​of each color block in the standard colorimetric bar; A linear correction model is established by utilizing the correspondence between the known RGB values ​​of the standard colorimetric bar and the actual extracted values; The RGB values ​​of each reaction well are calibrated to eliminate color deviations caused by factors such as shooting equipment, lighting environment, and shooting angle, and the calibrated R, G, and B values ​​are obtained.

[0052] 5. R / B value calculation and standard curve establishment Based on the corrected RGB values, the R / B ratio for each aperture is calculated. The R / B value is plotted on the ordinate, and Hg... 2+ A standard curve was plotted with concentration on the x-axis. The results are as follows: Figure 10 Within the concentration range of 10-100 μM, the corrected R / B value is similar to that of Hg. 2+ The concentration showed a good linear relationship, with the linear regression equation being (Y = -0.0083X + 1.08), (R²) 2 =0.993), with a detection limit of 1.72 μM, which significantly improves both the detection accuracy and reproducibility compared to the uncalibrated system.

[0053] Example 3: Smartphone colorimetry based on hole-color patch one-to-one correction for Hg 2+ High-precision detection Based on Example 2, this example further optimizes the color correction strategy by using micro standard color blocks that correspond one-to-one with the reaction wells for independent correction, so as to eliminate the systematic error caused by uneven illumination at different positions of the multi-well plate and improve the accuracy of detection and the consistency between wells.

[0054] 1. Construction of the detection system Following the method in Example 1, preparations containing different concentrations of Hg were made. 2+ The reaction system consisted of 10, 20, 40, 60, 80, and 100 μM solutions, with four replicates for each concentration. After shaking the reaction at room temperature for 10 min, the solution was allowed to stand to allow the alloy microbubble nanoenzyme (B@AgHg) ​​to float to the surface, and the solution color stabilized.

[0055] 2. Perforated Plate Structure Design A specially designed 96-well plate is used, with miniature standard color patches corresponding to each reaction well placed in its non-detection areas (such as the blank areas at the edges of each reaction well). Each miniature color patch is arranged close to the edge of its corresponding reaction well, ensuring that the color patch and its corresponding well are under almost identical lighting conditions (including light intensity, angle, and shadow distribution) during imaging. The color values ​​of all miniature color patches are pre-calibrated by the manufacturer using a high-precision spectrophotometer, and a uniform nominal value is provided (in this embodiment, no information encoding area is introduced, so all color patches use the same set of nominal values).

[0056] 3. Image Acquisition Place the reacted 96-well plate under standard lighting conditions (such as a standard light box or natural light against a white background) and take vertical photos using the rear camera of a smartphone, ensuring that each reaction well and its adjacent micro-standard color patch are captured simultaneously in the same image. Maintain a consistent distance and angle during shooting to ensure that all wells and color patches are clearly visible.

[0057] 4. RGB value extraction and independent calibration Import the image into image analysis software (such as Adobe Photoshop 2020): Extract the R, G, and B values ​​of the central region of each reaction well, and record them as follows: R 孔 , G 孔 , B 孔 ; Simultaneously, the R, G, and B values ​​of the micro-standard color patch corresponding to the hole are extracted and denoted as follows: R 色块 , G 色块 , B 色块 ; Using the known nominal RGB values ​​of each micro standard color patch ( R 标称 , G 标称 , B 标称 The proportional relationship between the actual extracted value and the actual extracted value was used to independently correct the RGB values ​​of the corresponding reaction wells: R 校正 = R 孔 × R 标称 / R 色块 ; G 校正 = G 孔 ×G 标称 / G 色块 ; B 校正 = B 孔 × B 标称 / B 色块 ; Obtain the corrected R, G, and B values ​​for each reaction well.

[0058] 5. R / B value calculation and standard curve establishment Based on the corrected RGB values, the R / B ratio for each aperture is calculated. The R / B value is plotted on the ordinate, and Hg... 2+ A standard curve was plotted with concentration on the x-axis. The results are as follows: Figure 11 As shown, within the concentration range of 10-100 μM, the corrected R / B value and Hg 2+ The concentration showed a good linear relationship, with the linear regression equation being Y = -0.0082X + 1.07, R0. 2 =0.996, detection limit is 1.68 μM.

[0059] Compared with the results of the overall standard colorimetric bar correction in Example 2, the linear correlation coefficient of this example is higher R. 2 The value increased from 0.993 to 0.996, and the relative standard deviation (RSD) between replicates of each concentration point decreased from 4.2% to 2.1%, indicating that one-to-one independent calibration of wells and color patches can effectively eliminate the difference in illumination between wells and significantly improve the precision and consistency of detection.

[0060] This embodiment overcomes the systematic errors caused by uneven illumination at different positions of the multi-well plate by introducing micro-standard color blocks that correspond one-to-one with the reaction wells for independent color correction, thus providing a more reliable data foundation for high-throughput colorimetric detection.

[0061] Example 4: Smartphone colorimetry based on precise color value correction of information encoding area for Hg 2+ High-precision detection Building upon Example 3, this example further introduces information encoding area technology. It stores the precise color value of each miniature standard color patch using a QR code, eliminating systematic errors caused by individual differences in the color patches and achieving Hg... 2+ Higher precision color reproduction and quantitative analysis in detection.

[0062] 1. Construction of the detection system Preparation of Hg with different concentrations 2+The reaction system consisted of 10, 20, 40, 60, 80, and 100 μM solutions, with four replicates for each concentration. After reacting at room temperature for 10 min, the solution was allowed to stand to allow the complex to float to the surface, and the solution color stabilized.

[0063] 2. Perforated Plate Structure Design Using a specially designed 96-well plate, its design includes the following features: Miniature standard color blocks: Each reaction hole has a dedicated miniature standard color block (approximately 2mm x 2mm) in the vicinity of its edge, ensuring that each color block is in the exact same lighting environment as its corresponding hole.

[0064] Information Encoding Area: A QR code is printed in a non-detection area of ​​the perforated plate (such as a blank area on the plate surface or the side of the plate). This QR code encodes and stores the precise RGB color value of each micro standard color patch measured by a high-precision spectrophotometer (i.e., the nominal reference value of each color patch, in the format: perforation number: R value, G value, B value). This QR code can be quickly scanned and read by a smartphone.

[0065] 3. Image acquisition and precise color patch value acquisition Before taking the photos, first use a smartphone to scan the QR code on the perforated plate to obtain and record the precise color value of each miniature standard color block. R 标称,i , G 标称,i , B 标称,i ),in i Indicates the hole position number.

[0066] After the reaction, the 96-well plate was placed under standard lighting conditions (such as a standard light source box or natural light with a white background), and the same smartphone rear camera was used to take vertical shots to ensure that all reaction wells and their corresponding micro standard color blocks were captured in the same image at the same time, while maintaining a consistent shooting distance and angle.

[0067] 4. RGB value extraction and independent calibration Import the image into image analysis software (such as Adobe Photoshop 2020): Extract the R, G, and B values ​​of the central region of each reaction well, and record them as follows: R 孔,i , G 孔,i , B 孔,i ; Simultaneously, the R, G, and B values ​​of the micro-standard color patch corresponding to the hole are extracted and denoted as follows: R 色块,i , G 色块,i ,B 色块,i ; Using the precise color value of the color block obtained from the QR code in step 3 R 标称,i , G 标称,i , B 标称,i The proportional relationship between the actual extracted value and the actual value was used to independently correct the RGB values ​​of the corresponding reaction wells: R 校正,i = R 孔,i × R 标,i称 / R 色块,i ; G 校正,i = G 孔,i × G 标称,i / G 色块,i ; B 校正,i = B 孔,i × B 标称,i / B 色块,i ; The corrected R, G, and B values ​​for each reaction well were obtained. This method not only eliminates the influence of uneven illumination between wells, but also corrects for individual color differences in the micro standard color blocks caused by printing batches, material aging, etc.

[0068] 5. R / B value calculation and standard curve establishment Based on the corrected RGB values, the R / B ratio for each aperture is calculated. The R / B value is plotted on the ordinate, and Hg... 2+ A standard curve was plotted with concentration on the x-axis. The results are as follows: Figure 12 Within the concentration range of 10-100 μM, the corrected R / B value is similar to that of Hg. 2+ The concentration showed a good linear relationship, with the linear regression equation being Y = -0.0081X + 1.07, R0. 2 =0.998, detection limit is 1.65 μM.

[0069] Compared with the results of the uniform nominal value correction in Example 3, the linear correlation coefficient R in this example is further improved. 2The relative standard deviation (RSD) between replicates of each concentration point decreased from 2.1% to 1.2%, which is an improvement from 0.996 to 0.998. This indicates that the introduction of an information coding area to store accurate color values ​​can effectively eliminate individual differences in color patches and achieve higher accuracy in color reproduction.

[0070] Example 5: Alloy microbubbles B@AgHg for portable detection of ascorbic acid (AA) 1. Construction of the detection system 4000 B@Ag molecules were placed in a 96-well plate, and 60 μL of TMB solution containing H2O2 and 10 μL of Hg(NO3)2 solution (100 μM) were added sequentially. Then, 20 μL of ascorbic acid (AA) standard solutions of different concentrations (0, 10, 20, 40, 60, 80, 100 μM) were added, and ultrapure water was added to bring the volume to 100 μL. After shaking at room temperature for 10 min, the mixture was allowed to stand to allow the complex to float.

[0071] 2. Microplate reader detection The absorbance at 652 nm was measured using an ELISA reader. For example... Figure 13 As shown, with increasing AA concentration, the blue color of the solution gradually lightens, and the absorbance value gradually decreases. Within the range of 10-100 μM, the absorbance value shows a good linear relationship with the AA concentration, and the linear regression equation is Y = -0.0062X + 0.71(R²). 2 =0.999), with a detection limit of 4.06 μM. AA, as a reducing agent, can competitively consume reactive oxygen species generated in the system or directly reduce oxTMB, thereby achieving quantitative detection.

[0072] 3. Smartphone colorimetric detection Place the 96-well plate under uniform lighting (standard light box or natural light with a white background), and take vertical photos using the rear camera of a mobile phone, maintaining a consistent shooting distance and angle. Import the photos into Adobe Photoshop 2020, use the rectangular marquee tool to select the center area of ​​each well (avoiding edge shadows), record the R, G, and B values, and calculate the R / B ratio; plot a standard curve with the R / B value on the ordinate and AA concentration on the abscissa. Figure 14 As shown in Figure a, within the range of 10-100 μM, the R / B value exhibits a good linear relationship with the AA concentration, and the linear equation is Y = 0.0065X + 0.21(R 2 =0.994), and the detection limit was 11.71 μM. Correlation analysis showed that the R / B value was significantly negatively correlated with the absorbance at 652 nm (r = -0.994, r = -0.994, r = -0.994). Figure 14 b).

[0073] 4. Selective assessment Under the same experimental conditions, the effects of AA (100 μM) and common interfering substances (D-arginine, D-lysine, L-phenylalanine, L-isoleucine, DL-proline, glucose, vitamin B2, vitamin B3, L-cysteine, and glutathione, all at a concentration of 100 μM) on the detection system were investigated. Figure 15 As shown, the absorbance decrease caused by the AA group was significantly greater than that caused by other interfering substances (P<0.0001), indicating that the B@AgHg system has good selectivity for AA and can be applied to the specific detection of AA in complex matrices.

[0074] 5. Actual sample testing Accurately weigh 20 tablets of each of the following vitamin C dosage forms: commercially available vitamin C tablets (labeled amount 100 mg / tablet), vitamin C chewable tablets (labeled amount 100 mg / tablet), and vitamin C effervescent tablets (labeled amount 1000 mg / tablet). Grind each tablet into a uniform fine powder. Accurately weigh the powder equivalent to the AA content of one tablet, transfer it to a test tube, and add an appropriate amount of ultrapure water to completely dissolve it. Filter the solution through a 0.22 μm microporous membrane to remove insoluble impurities. Dilute the filtrate with ultrapure water to ensure the AA concentration is within the linear detection range. The results showed that the measured values ​​for the three solid dosage forms were 100.92±2.09 mg / tablet, 98.26±1.90 mg / tablet, and 995.22±12.90 mg / tablet, respectively, which were consistent with the labeled values. Furthermore, the presence of color in the chewable and effervescent tablet solutions did not affect the detection of vitamin content. Commercially available vitamin C injection (labeled amount 250 mg / mL) was diluted with ultrapure water to ensure the AA concentration was within the linear detection range. The results showed that the measured value of vitamin C injection was 253.35 ± 7.61 mg / mL, which met the specified range (93%-107%) of the labeled amount in the Chinese Pharmacopoeia (2025 edition). This indicates that the method is accurate and reliable and can be used for the rapid detection of ascorbic acid in actual samples.

[0075] It should be noted that the above embodiments only use ascorbic acid as an example to describe the detection of reducing drugs in detail, but the colorimetric detection method based on alloy microbubble nanozymes provided by this invention is also applicable to the detection of other reducing drugs. According to the detection principle of this invention, Hg... 2+The activated alloy microbubble nanozyme exhibits enhanced peroxidase-like activity, catalyzing the oxidation and color development of TMB. When a reducing agent is present in the system, the agent can competitively consume reactive oxygen species or directly reduce oxTMB, leading to a decrease in absorbance, with the decrease being positively correlated with the drug concentration. Based on this universal detection mechanism, the method of this invention is also applicable to the colorimetric detection of reducing agents such as N-acetylcysteine, D-penicillamine, dimercaptosuccinic acid, vitamin K3, edaravone, resveratrol, and curcumin. Those skilled in the art can perform quantitative analysis of these drugs by referring to the detection method for ascorbic acid, which will not be elaborated here.

[0076] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A type of Hg 2+ The colorimetric detection method is characterized by, Includes the following steps: Silver-metallized microbubbles were mixed with a TMB solution containing H2O2, and then Hg was added. 2+ The sample to be tested was reacted at room temperature for 10-15 min; After the reaction, the mixture is allowed to stand until the alloy microbubble nanoenzyme floats to the top of the solution. The absorbance at 652 nm was measured using an ELISA reader, or a handheld electronic device was used to photograph the reaction solution and extract the RGB values. Based on the pre-established absorbance-Hg 2+ Concentration standard curve or R / B value - Hg 2+ The concentration standard curve was used to calculate the Hg content in the sample. 2+ concentration.

2. The Hg according to claim 1 2+ The colorimetric detection method is characterized by, The preparation of silver-metallized microbubbles includes the following steps: S1: Disperse hollow glass microbubbles in a piranha solution and stir the reaction at room temperature for 0.5-2 h. Then wash with ultrapure water until the washing solution is neutral and vacuum dry to obtain surface-activated hollow glass microbubbles. S2: Add the surface-activated hollow glass microbubbles to a polyethyleneimine solution with a concentration of 1-8 mg / mL, and rotate and mix at room temperature for 0.5-12 h. After the reaction, let it stand until the hollow glass microbubbles float to the top of the solution, remove the lower layer of solution, wash with ultrapure water, and vacuum dry to obtain hollow glass microbubbles coated with polyethyleneimine. S3: Add hollow glass microbubbles coated with polyethyleneimine to a buffer solution containing 1-1000 pmol of single-stranded DNA rich in cytosine bases, rotate and mix at room temperature for 0.5-4 h, and after the reaction, let stand until the complex floats to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain the intermediate product loaded with single-stranded DNA. S4: Mix the intermediate product loaded with single-stranded DNA with a silver nitrate solution of 0.1-50 mM, rotate and mix at room temperature for 10-200 min, and let stand until the complex floats to the top of the solution. Remove the lower layer of solution and wash with ultrapure water to obtain hollow glass microspheres loaded with silver ions. S5: Mix hollow glass microspheres loaded with silver ions with a sodium borohydride solution with a concentration of 0.1-50 mM, vortex for 10-120 min, and let stand until the product floats to the top of the solution. Remove the lower layer of solution and wash with ultrapure water to obtain silver metallized microbubbles.

3. The Hg according to claim 1 2+ The colorimetric detection method is characterized by, The steps for taking photos of the reaction solution and extracting RGB values ​​using a handheld electronic device include: The reacted porous plate is placed on the imaging bracket. The non-detection area of ​​the porous plate is pre-set with a standard colorimetric bar, which contains at least one color block with a known RGB value. Use a handheld electronic device to simultaneously capture images of all reaction wells and standard colorimetric bars within the same field of view; Extract the R, G, and B values ​​of the central region of each reaction well in the image, and simultaneously extract the R, G, and B values ​​of the standard colorimetric bar; By utilizing the correspondence between the known RGB values ​​of the standard colorimetric bar and the extracted values, the image is color-corrected to obtain the corrected RGB values ​​of the reaction apertures; The R / B ratio was calculated based on the corrected RGB values ​​of the reaction wells, and then calculated according to the pre-established R / B value - Hg. 2+ Calculation of Hg in the sample by concentration standard curve 2+ concentration.

4. The Hg according to claim 3 2+ The colorimetric detection method is characterized by, The non-detection area of ​​the multi-well plate is provided with multiple micro standard color blocks, which correspond one-to-one with the reaction wells of the multi-well plate and are arranged near the edge of the reaction well. When images are captured using a handheld electronic device, the image area of ​​each reaction well and the image area of ​​its corresponding micro standard color patch are captured simultaneously. Extract the R, G, and B values ​​of the central region of each reaction well, and simultaneously extract the R, G, and B values ​​of the micro standard color patch corresponding to that reaction well; By utilizing the correspondence between the known RGB values ​​and extracted values ​​of each micro-standard color patch, the RGB values ​​of the corresponding reaction wells are independently corrected to obtain the corrected R, G, and B values ​​for each reaction well. Based on the corrected RGB values ​​of each reaction well, the R / B ratio is calculated, and then adjusted according to the pre-established R / B value - Hg. 2+ Calculation of Hg in the sample by concentration standard curve 2+ concentration.

5. The Hg according to claim 4 2+ The colorimetric detection method is characterized by, The non-detection area of ​​the multi-hole plate is also equipped with an information encoding area, which stores the precise color value data of each micro standard color block; Before taking pictures using a handheld electronic device, the information encoding area is read through the handheld electronic device to obtain the precise color value of each micro standard color block; After extracting the RGB values ​​of the micro standard color patches corresponding to each reaction well, the RGB values ​​of the corresponding reaction wells are independently corrected by using the correspondence between the precise color values ​​of the micro standard color patches and the extracted values, so as to obtain the corrected R, G, B values ​​of each reaction well. The R / B ratio is calculated based on the RGB values ​​of each reaction well after correction, and then based on the pre-established R / B value - Hg. 2+ Calculation of Hg in the sample by concentration standard curve 2+ concentration.

6. A colorimetric detection method for reducing drugs, characterized in that, Includes the following steps: Silver-metallized microbubbles were mixed with a TMB solution containing H2O2 and Hg. 2+ The solutions are mixed and reacted at room temperature for 10-15 minutes to allow Hg to react. 2+ When combined with silver-metallized microbubbles, an alloy microbubble nanoenzyme with peroxidase-like activity is produced. Add the test sample containing the reducing agent and react at room temperature for 10-15 min; After the reaction, the mixture is allowed to stand until the alloy microbubble nanoenzyme floats to the top of the solution. The absorbance at 652 nm was measured using an ELISA reader, or a handheld electronic device was used to photograph the reaction solution and extract the RGB values. The concentration of reducing drug in the sample to be tested is calculated based on the pre-established absorbance-drug concentration standard curve or R / B value-drug concentration standard curve.

7. The Hg according to claim 1 2+ The colorimetric detection method is characterized by, Hg is achieved using a portable colorimetric sensor. 2+ The portable colorimetric sensor includes a signal reporting unit, a colorimetric substrate, a reaction vessel, and a signal reading unit for detection. The signal reporting unit is a silver-metallized microbubble; The chromogenic substrate is a mixed solution containing TMB and H2O2; The reaction vessel is a multi-well plate used to contain silver metallized microbubbles, chromogenic substrate, and the sample to be tested. The signal reading unit is a handheld electronic device used to capture images of the reaction solution and extract RGB values ​​for quantitative analysis.

8. An alloy microbubble nanoenzyme, characterized in that, Using the silver metallized microbubbles prepared according to claim 2 as raw materials, the following steps are performed: silver metallized microbubbles are reacted with a TMB solution containing H2O2 and Hg at a concentration of 10-100 μM. 2+ The solutions were mixed and reacted at room temperature for 10-15 minutes. After the reaction, the mixture was washed with ultrapure water to obtain alloy microbubble nanozymes.

9. The silver metallized microbubbles as described in claim 2 in Hg 2+ Applications in detection.

10. The application of the alloy microbubble nanoenzyme as described in claim 8 in the detection of reducing drugs, wherein the reducing drug is one or more of ascorbic acid, N-acetylcysteine, D-penicillamine, dimercaptosuccinic acid, vitamin K, edaravone, resveratrol, and curcumin.

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