Rapid detection method for cadmium residual quantity in aquatic product based on magnetic qualitative separation and polarized light distinguishing and quantifying

Abstract

The invention relates to a rapid detection method for cadmium residual quantity in aquatic products based on magnetic qualitative separation and polarized light distinguishing and quantifying. The method comprises the following steps: coupling a biotinylated cadmium monoclonal antibody through streptavidin magnetic microspheres, and specifically enriching trace cadmium in the aquatic product digestive juice to form a magnetic compound; a muller matrix microscope is used for collecting a polarization microscopic image of a compound, and a deep learning model (such as RESNET34) is combined to carry out binary classification and particle counting on the image, so that rapid and high-sensitivity quantitative detection of cadmium can be realized. The method is easy and convenient to operate, the target object magnetic separation time is within 2 min, the particle distinguishing accuracy rate is higher than 84%, the quantitative correlation Rreaches 0.9 or above, and the method is suitable for rapid monitoring of trace cadmium (several micrograms / L) in aquatic products.

Description

Technical Field

[0001] This invention belongs to the field of heavy metal detection technology in water, specifically relating to a rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation. Background Technology

[0002] Rapid global urbanization and industrialization may lead to increasingly severe environmental pollution, with large amounts of waste being released into the environment, including harmful heavy metals. Heavy metals can accumulate in the liver, heart, kidneys, and brain tissue in the human body, disrupting their normal biological functions. Continuous monitoring of heavy metal contamination in aquatic products is crucial for ensuring food safety systems.

[0003] Cadmium (ion) is generally estimated to have an environmental concentration of less than 1 μg·L⁻¹. -1 Cadmium is recognized as having "direct ecotoxicological importance." Its presence in water bodies leads to selective burden on aquatic microorganisms, directly impacting the survival of aquatic life and damaging the human nervous system, kidneys, and bones. Due to its toxicity, global drinking water and wastewater discharge standards strictly limit cadmium levels. Because of its non-degradable nature, heavy metals can remain in sediments for extended periods and accumulate in the human body through the food chain.

[0004] However, traditional detection methods such as atomic absorption spectrometry and ICP-MS are cumbersome to operate, rely on large instruments and equipment, and have complicated pretreatment processes, making it impossible to achieve rapid and accurate enrichment and separation of trace residual heavy metals in water. Summary of the Invention

[0005] Technical problem solved: In view of the problems of cumbersome operation procedures and inability to accurately and specifically separate and enrich trace amounts of heavy metal cadmium in aquatic products in existing technologies, this invention proposes a method for quantifying trace amounts of heavy metal cadmium in aquatic products by using magnetic microspheres coupled with antibodies and polarization differentiation. The method is easy to operate and can specifically adsorb trace amounts (a few μg / L) of heavy metal cadmium in aquatic products.

[0006] Technical Solution: A rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light differentiation for quantitative analysis, the steps of which are as follows:

[0007] Step 1: Add the conjugate MBs@Ab to the digestion solution of the aquatic product to be tested to enrich cadmium. After magnetic separation, the cadmium-bound complex is obtained and denoted as MBs@Ab@Cd.

[0008] Step 2: Prepare samples of the cadmium-bound complex MBs@Ab@Cd and the unbound cadmium conjugate MBs@Ab, place them under a Müller matrix microscope for polarization microscopy image acquisition, and obtain the Müller matrix diagrams of MBs@Ab and MBs@Ab@Cd based on the acquired images.

[0009] Step 3: Use the deep learning model RESNET34 to analyze the Müller matrix images of MBs@Ab and MBs@Ab@Cd, distinguish and count the particulate matter of the two materials, and obtain the number of MBs@Ab@Cd particulate matter / (number of MBs@Ab + number of MBs@Ab@Cd particulate matter), which is recorded as the cadmium detection ratio.

[0010] Step 4: Take cadmium standard solution and plot a standard curve: The gradients of the standard curve are 10 wt%, 30 wt%, 50 wt%, 70 wt%, and 100 wt% of cadmium added to the cadmium standard solution. Based on the methods in Steps 1-3, calculate the number of particles for each and the cadmium detection ratio for each. Plot the cadmium addition ratio on the x-axis and the cadmium detection ratio on the y-axis. Substitute the cadmium detection ratio of the sample to be tested obtained in Step 3 into the standard curve to obtain the cadmium addition ratio of the sample to be tested. Multiply this ratio by the amount of cadmium added in the standard solution to achieve quantification of Cd.

[0011] The preparation method of the conjugate MBs@Ab is as follows: activate magnetic microspheres to obtain activated magnetic microspheres, denoted as MBs; react cadmium monoclonal antibody with biotin, and after purification, obtain biotinylated cadmium monoclonal antibody, denoted as Ab; conjugate the biotinylated cadmium monoclonal antibody and the activated magnetic microspheres to form a conjugate, denoted as MBs@Ab.

[0012] Preferably, the magnetic microspheres are streptavidin magnetic microspheres with a particle size of 1-3 µm and a concentration of 10 mg / mL, and the streptavidin magnetic microspheres are activated using PBST Nuffer.

[0013] Preferably, the molar ratio of the cadmium monoclonal antibody to biotin is 1:30, and the reaction conditions are as follows: biotin is added to the cadmium monoclonal antibody, and the mixture is placed in a mixer at room temperature in the dark for reaction. After the reaction is completed, the mixture is blocked at room temperature and purified by chromatography to obtain biotinylated cadmium monoclonal antibody.

[0014] Preferably, the ratio of the biotinylated cadmium monoclonal antibody to the activated magnetic microspheres is 10~20 µg:1 mg, and the coupling conditions are as follows: add the biotinylated cadmium monoclonal antibody to the activated magnetic microspheres, add PBS buffer to make the total volume 100 µL, then vortex mix for 10 s, and then place it in a roller mixer for 60 min, controlling the temperature at 25~37℃. At this point, the coupling is completed.

[0015] Preferably, the preparation process of the digestive solution of the aquatic product to be tested in step one is as follows: Weigh 0.2~0.5 g of solid sample or 0.50~3.00 ml of liquid sample into a microwave digestion vessel, add 5 ml of nitric acid, let stand for 30 min, and cover with the safety valve; place the digestion vessel into the microwave digestion system to digest the residual heavy metal cadmium in the aquatic product into the solution, thereby obtaining the digestive solution of the aquatic product.

[0016] Preferably, the enrichment of cadmium in step one is as follows: the conjugate MBs@Ab is directly added to the digestion liquid of aquatic products for extraction, and the extract is incubated in a mixer with the temperature set at 25-37℃ for 30 min. After incubation, the supernatant is removed by magnetic separation to obtain the cadmium-bound complex MBs@Ab@Cd.

[0017] Preferably, in step two, the Müller matrix microscope uses a green light source, a 40× objective lens, and acquires four images for each sample.

[0018] Preferably, in step two, the Müller matrix is ​​obtained through a polarizer (PSG) and an analyzer (PSA), and the Müller matrix of the sample is calculated for classification.

[0019] Preferably, in step three, the deep learning model is RESNET34, the ratio of training set to test set is 80%:20%, binary classification is performed, and a sample confusion classification matrix is ​​output based on the test set results, and the particles of the two materials are marked and counted with different colors.

[0020] Preferably, in step four, the volume of the cadmium standard solution is 500 μL and the amount of cadmium added is 2 ng.

[0021] Preferably, the aquatic products include fish, shellfish, crustaceans and their processed products.

[0022] This invention utilizes immunomagnetic bead separation technology, which features high specificity, high sensitivity, and fast separation speed. It also boasts strong anti-interference capabilities and is non-destructive to samples, enabling rapid and highly sensitive detection of trace targets. By modifying the biotin on antibodies, streptavidin magnetic microspheres can be linked to antibodies via a biotin affinity system to synthesize immunomagnetic microspheres. This allows for precise capture of trace targets, while leveraging the excellent magnetic separation properties of the complex and the specificity of the antibody-antigen pair to avoid matrix interference, achieving qualitative separation of target pollutants. Optical detection can be applied to particulate matter classification. Polarization is a fundamental property of light. Polarized light irradiation of particulate matter can reveal its microscopic morphological characteristics, offering advantages such as non-destructive, non-contact, and high information content. A Müller matrix microscope can measure the Müller matrix of a sample, which characterizes the sample's polarization optical properties. Based on the optical properties of different particulate matter, different particles can be classified and counted, achieving quantitative analysis of different particles.

[0023] Beneficial Effects: This invention utilizes a magnetic microsphere-coupled antibody method to construct a detection method combining magnetic immunoadsorption and polarization quantification, achieving specific enrichment and quantitative detection of trace cadmium in aquatic products. This invention is simple to operate, capable of specifically adsorbing trace amounts (a few µg / L) of cadmium, with magnetic separation time of less than 2 minutes and particle differentiation efficiency exceeding 84%. Quantitative detection results: R 2 It reaches 0.9 or higher. Attached Figure Description

[0024] Figure 1 Zeta potential plots for MBs@Ab and MBs@Ab@Cd;

[0025] Figure 2 Immunoelectron micrographs of MBs@Ab (a) and MBs@Ab@Cd (b);

[0026] Figure 3 The UV-Vis absorption spectra are for MBs@Ab and MBs@Ab@Cd.

[0027] Figure 4 Müller matrix diagrams for MBs@Ab(a) and MBs@Ab@Cd(b) and their classification confusion matrix diagram (c);

[0028] Figure 5 Flowchart for classifying MBs, MBs@Ab, and MBs@Ab@Cd;

[0029] Figure 6 A quantitative standard curve for cadmium (Cd). Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, all raw materials and equipment used in the embodiments of this specification are from commercially available products.

[0032] In this instruction manual, cadmium specifically refers to divalent cadmium ions.

[0033] One embodiment of this specification provides a rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light-based quantitative differentiation. The steps are as follows:

[0034] Step 1: Cadmium-specific enrichment: Residual cadmium in aquatic products was digested with a reagent (nitric acid) to obtain a solution containing the target analyte. The conjugate MBs@Ab was directly added to the solution containing the target analyte for extraction. The extract was incubated in a mixer set at 25-37°C for 30 minutes. After incubation, the supernatant was removed by magnetic separation, and the microspheres were washed with PBS to obtain cadmium-bound MNP@Ab, i.e., MBs@Ab@Cd.

[0035] Step 2: Polarized observation of cadmium: Prepare cadmium-bound MBs@Ab@Cd and MBs@Ab solutions using a glass slide. Set the Müller matrix microscope to green light and the magnification to 40×. After preparation, place the prepared MBs@Ab and MBs@Ab@Cd samples under the Müller matrix microscope for observation. Select areas with uniform particle distribution and clear field of view for image acquisition, and acquire images four times for each sample.

[0036] Step 3: Binary classification of MBs@Ab and MBs@Ab@Cd: The polarizer PSA of the Müller matrix detects the transmitted light passing through the sample, with waveplate R1 rotating four times for four data acquisitions; the polarizer PSG generates four sets of illumination light with different polarization states, whose Stokes vectors form a 4×4 matrix; the PSA correspondingly obtains four sets of transmitted light Stokes vectors, forming a 4×4 matrix. The Müller matrix of the sample is calculated using the following formula. Each Müller matrix contains 16 array element images (M... 11 M 12 M 13 M 14 M 21 M 22 M 23 M 24 M 31 M 32 M 33 M 34 M 41 M 42 M 43 M 44 The pixel coordinates of the image in each array element correspond to the spatial location of the same sample. The sample is classified by the Müller matrix of the sample. The Müller matrix of the sample is calculated by the following formula (4).

[0037] (4)

[0038] Among them, S PSA The PSA corresponds to a 4×4 matrix composed of four sets of transmitted light Stokes vectors; S PSG It is a 4×4 matrix composed of four groups of Stokes vectors of illumination light with different polarization states in the PSG.

[0039] Step 4: Calculate the cadmium detection ratio: After collecting and classifying samples under a Müller matrix microscope, polarized microscopic images of MBs@Ab@Cd and MBs@Ab containing cadmium and their respective Müller matrix diagrams were obtained. RESNET34 was used to analyze the image data, and the particles of the two materials were counted using different color markers. The result was the number of MBs@Ab@Cd particles / (number of MBs@Ab + number of MBs@Ab@Cd particles), recorded as the cadmium detection ratio.

[0040] Step 5, Cadmium Quantification: Take cadmium standard solution and plot a standard curve: The gradients of the standard curve are 10 wt%, 30 wt%, 50 wt%, 70 wt%, and 100 wt% of cadmium added in the cadmium standard solution. Based on the methods in Steps 1-3, calculate the amount of particulate matter for each and the cadmium detection ratio for each. Plot the standard curve with the cadmium addition ratio as the x-axis and the cadmium detection ratio as the y-axis. Substitute the cadmium detection ratio of the sample to be tested obtained in Step 3 into the standard curve to obtain the cadmium addition ratio of the sample to be tested. Multiply this ratio by the amount of cadmium added in the standard solution to achieve cadmium quantification.

[0041] The preparation method of the conjugate MBs@Ab is as follows: Activate magnetic microspheres to obtain activated magnetic microspheres, denoted as MBs; react cadmium monoclonal antibody with biotin, and after purification, obtain biotinylated cadmium monoclonal antibody, denoted as Ab; conjugate the biotinylated cadmium monoclonal antibody and the activated magnetic microspheres to form a conjugate, denoted as MBs@Ab, as detailed below:

[0042] Activation of magnetic microspheres: Take streptavidin magnetic microspheres into a centrifuge tube, vortex to mix, then mix on a roller mixer, remove the supernatant by magnetic separation, add PBST Buffer, mix on a roller mixer at room temperature, remove the supernatant after magnetic separation, repeat the washing with PBST Buffer several times, remove the supernatant for later use, and the activation is complete;

[0043] Biotinylated cadmium monoclonal antibody: Biotin was added to the cadmium monoclonal antibody, and the mixture was placed in the dark on a mixer at room temperature. After the reaction was completed, the mixture was blocked at room temperature and then purified by PD10 chromatography column to obtain biotinylated cadmium monoclonal antibody.

[0044] Preparation of magnetic microsphere-conjugated antibodies: Add biotinylated cadmium monoclonal antibody to activated magnetic microspheres, add PBS buffer, vortex mix, and then incubate in a roller mixer at a temperature of 25-37°C. The conjugation is now complete. The conjugate is represented as MBs@Ab, where MBs represents magnetic microspheres, Ab represents antibody, and @ represents conjugation.

[0045] As a further preferred embodiment of the present invention, the particle size of the streptavidin magnetic microspheres is 1-3 µm.

[0046] As a further preferred embodiment of the present invention, the streptavidin magnetic microspheres are placed in a centrifuge tube, vortexed for 10 s, then mixed on a roller mixer for 15 min, magnetically separated on a magnetic separator for 1 min, the supernatant is removed, PBST Buffer is added, and the mixture is mixed on a roller mixer at room temperature for 5 min. After magnetic separation, the supernatant is removed, and the washing is repeated twice with PBST Buffer. The supernatant is then removed and the mixture is ready for use, thus completing the activation process.

[0047] As a further preferred embodiment of the present invention, the PBST Buffer is PBS with 0.05wt% Tween20 added and the pH adjusted to 7.4.

[0048] As a further preferred embodiment of the present invention, the molar ratio of the cadmium monoclonal antibody to biotin is 1:30.

[0049] As a further preferred embodiment of the present invention, the biotin is pretreated before addition, and dissolved in DMSO after equilibration at room temperature. The cadmium monoclonal antibody is diluted with labeling buffer, and then the dissolved biotin is added. The mixture is wrapped in aluminum foil to protect it from light and placed on a mixer to react at room temperature for 3 hours. After the reaction is completed, blocking solution is added, and the mixture is placed on a mixer to block at room temperature for 30 minutes. Then, it is purified by PD10 chromatography column to obtain biotinylated cadmium monoclonal antibody.

[0050] As a further preferred embodiment of the present invention, the concentration of the streptavidin magnetic microspheres is 10 mg / mL, and the ratio of biotinylated cadmium monoclonal antibody to streptavidin magnetic microspheres is 10~20 µg:100 µL.

[0051] As a further preferred embodiment of the present invention, the biotinylated cadmium monoclonal antibody is added to the activated magnetic microspheres, and PBS buffer is added to make the total volume 100µL. Then, the mixture is vortexed for 10 s and then incubated in a roller mixer for 60 min at a temperature of 37°C. Thus, the coupling is completed.

[0052] As a further preferred embodiment of the present invention, the coupling agent MBs@Ab is directly added to the water sample after digestion of aquatic products containing cadmium for extraction. The extract is placed in a mixer with the temperature set at 37°C and incubated for 30 minutes. After incubation, the supernatant is removed by magnetic separation. Specifically, the supernatant is placed on a magnetic rack and left to stand for 3 minutes.

[0053] As a further preferred embodiment of the present invention, in step two, samples combining cadmium (MBs@Ab@Cd) and MBs@Ab are prepared and placed under a 40x objective lens of a Müller matrix microscope for image acquisition. Each sample is acquired four times (to ensure the accuracy of subsequent analysis data).

[0054] As a further preferred embodiment of the present invention, the polarizer PSA in step three comprises two linearly polarized cameras (DoFP CCD1, DoFP CCD2), one non-polarizing beam splitter prism, and one quarter-wave plate R2 with a fixed angle. The PSA detects the transmitted light passing through the sample, and R1 rotates four times to collect data four times.

[0055] In a further preferred embodiment of the present invention, the polarizer PSG in step three contains a fixed-angle polarizer P1 and a rotatable quarter-wave plate R1. By rotating R1, light with different polarization states can be obtained. The resulting four sets of illumination lights with different polarization states form a 4×4 matrix with their Stokes vectors.

[0056] In a further preferred embodiment of the present invention, step four involves analyzing the data using deep learning RESNET34 to identify MBs@Ab and MBs@Ab@Cd particles. The sample dataset is divided into a training set (80%) and a test set (20%). A sample confusion classification matrix is ​​output based on the test set results, and different particulate matter is counted to achieve specific quantification of cadmium.

[0057] The specific implementation method is as follows:

[0058] Example 1

[0059] This embodiment provides a rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light-based quantitative differentiation. The steps are as follows:

[0060] (1) Selection and activation of magnetic microspheres: Streptavidin magnetic microspheres (purchased from Suzhou Weidu Biotechnology Co., Ltd.) with a particle size of 1-3 µm were selected. 100 µL (concentration of 10 mg / mL) of streptavidin magnetic microspheres were placed in a 1.5 mL centrifuge tube, vortexed for 10 s, mixed on a roller mixer for 15 min, and then magnetically separated on a magnetic separator for 1 min. The supernatant was removed. 400 µL of PBST Buffer (PBS with 0.05 wt% Tween 20 added, pH adjusted to 7.4) was added; the mixture was mixed on a roller mixer for 5 min at room temperature (20-25℃), and after magnetic separation, the supernatant was removed. The microspheres were washed twice with PBST Buffer and then the supernatant was removed for later use. Activation was completed, and the activated magnetic microspheres were designated as MBs.

[0061] (2) Biotinylation of cadmium monoclonal antibody: After equilibration at room temperature (22-25℃), weigh an appropriate amount of biotin and dissolve it in DMSO at a ratio of 1 mg: 25 µL. Take 1-2 mg / mL of cadmium monoclonal antibody (purchased from the antibody company (Beijing Biotyscience Technology Co., Ltd., not manufactured by us), and then add the dissolved biotin (cadmium monoclonal antibody: biotin = 1:30, molar ratio). Wrap it in aluminum foil to protect it from light and place it on a mixer to react at room temperature for 3 h. After the reaction, block it on a mixer at room temperature for 30 min. Purify it using a PD10 chromatography column. Collect the purified antibody-biotin-labeled product, store it at 4℃ for later use, and label it Ab.

[0062] (3) Evaluation of the conjugation effect of the magnetic microspheres with the antibody: Add 10 µg of the purified and collected biotinylated cadmium monoclonal antibody from step (2) to the centrifuge tube from step (1), control the single variable, add PBS buffer to make the total volume 100 µL, vortex mix for 10 s, and then incubate it in a roller mixer for 60 min at a temperature of 37 °C. The conjugation is now complete. The conjugate is represented as MBs@Ab, where MBs represents the activated magnetic microspheres, Ab represents the biotinylated cadmium monoclonal antibody, and @ represents conjugation.

[0063] After the centrifuge tubes in step (3) have been coupled, place them on a magnetic rack and separate for 3 minutes, then collect the supernatant.

[0064] This study selected the following methods to evaluate the coupling effect:

[0065] ① The concentration of monoclonal antibody in the supernatant was determined using the BCA method. Each supernatant was prepared in triplicate, and the average value was taken to obtain the amount of antibody remaining in the supernatant. Then, the specific amount of antibody conjugation was calculated by the differential method. The calculation formulas are as follows: (1) and (2):

[0066] (1);

[0067] (2);

[0068] The units for antibody conjugation amount, antibody addition amount, and antibody residue amount are all μg.

[0069] The specific coupling quantity calculation results are shown in the table below:

[0070]

[0071] ② The potential and particle size changes of the magnetic microspheres before and after coupling were measured using a Zeta potential analyzer. See the results below. Figure 1As can be seen from the figure, the Zeta potential of MBs@Ab after adding antibody decreased from -9.63 mV to -20.5 mV compared with magnetic microspheres MBs. The cadmium antibody has a negative charge on its surface, and the binding increases the negative charge on the surface of the microspheres. The increase in negative charge proves the successful coupling of the antibody.

[0072] ③ Immunoelectron microscopy was used to examine the labeling of colloidal gold secondary antibody on the surface of the magnetic microspheres before and after conjugation, which can determine the antibody conjugation status. Results are as follows: Figure 2 As shown in the figure, compared with the magnetic microspheres MBs, the MBs@Ab with added antibody has black colloidal gold particles labeled with antibody on its surface, while the original microspheres have no antibody on their surface and are therefore unlabeled, proving the successful conjugation of the antibody.

[0073] ④ The UV-Vis absorption spectra of the magnetic microspheres before and after coupling were measured using UV-Vis absorption spectroscopy. The coupling effect of the antibody was determined based on the changes in the absorption peaks. See the results below. Figure 3 As can be seen from the figure, MBs@Ab and MBs have similar absorption peaks in their UV-Vis spectra, but the absorption peak of MBs@Ab is higher than that of MBs.

[0074] (4) Before measuring aquatic products, the samples need to be microwave digested. Weigh 0.2~0.5g of solid sample or 0.50~3.00ml of liquid sample into a microwave digestion vessel, add 5ml of nitric acid, let stand for 30min, and close the safety valve. Place the digestion vessel into the microwave digestion system to digest the residual heavy metal cadmium in the aquatic products into the solution, and obtain the aquatic product digest solution.

[0075] In this embodiment, a simulated aquatic product digestion solution (cadmium-concentrated water sample with a concentration of 4 ppb) was used. 1 mg of the conjugate MBs@Ab, evaluated for its coupling effect, was directly added to 100 µL of cadmium-concentrated water sample for extraction. To ensure thorough mixing and reaction, 4 times the volume of the cadmium-concentrated water sample was added to maintain a suitable reaction environment. The mixture was then placed in a roller mixer set to 37°C and incubated for 30 minutes. After incubation, the centrifuge tubes were placed on a magnetic rack and allowed to stand for 3 minutes. During this time, MBs@Ab, due to its magnetism, aggregated on one side of the centrifuge tube and tightly bound to the target analyte. Thus, using magnetic separation technology, the cadmium-bound MBs@Ab could be easily separated from the supernatant.

[0076] The cadmium in the supernatant was filtered through a 0.22 µm filter membrane, and its concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS). The binding rate (%) was calculated based on the change in cadmium mass concentration before and after the experiment, and can be calculated using the following formula (3):

[0077] (3).

[0078] In this embodiment, the initial amount of cadmium was 2 ng, and the remaining amount of cadmium in the supernatant was 0.5 ng, with a binding rate calculated to be 75%. The binding rate reflects the efficiency of MBs@Ab in adsorbing the target analyte cadmium, thus determining that the maximum amount of Cd that 1 mg of MBs@Ab can adsorb is 1.5 ng.

[0079] (5) Assembling the Müller matrix microscope: A Müller matrix microscope is obtained by adding a polarizer PSA and a polarizer PSG to a commercial transmission microscope. The PSG has a fixed-angle polarizer P1 and a rotatable quarter-wave plate R1. By rotating R1, different polarization states of light can be obtained. The PSA includes two linearly polarized cameras (DoFP CCD1, DoFP CCD2), one non-polarizing beam splitter prism, and one fixed-angle quarter-wave plate R2. The PSA detects the transmitted light passing through the sample. R1 is rotated four times (±45° and ±19.6°) to perform four data acquisitions. The PSG generates four sets of illumination light with different polarization states, and their Stokes vectors form a 4×4 matrix S. PSG The PSA correspondingly yields four sets of transmitted light Stokes vectors, forming a 4×4 matrix S. PSA The Müller matrix of the sample is calculated using the following formula (4).

[0080] (4)

[0081] Among them, S PSA The PSA corresponds to a 4×4 matrix composed of four sets of transmitted light Stokes vectors; S PSG It is a 4×4 matrix composed of four sets of Stokes vectors of illumination light with different polarization states in the PSG; S PSG -1 For S PSG The inverse operation. Each pixel has one Müller matrix. For the sample, using the Müller matrix microscope and equation (4), a Müller matrix image can be obtained, which consists of 16 Müller matrix element images (denoted as M). 11 M 12 M 13 M 14 M 21 M 22 M 23 M 24 M 31 M 32 M 33 M 34 M 41 M 42 M 43 M 44Each element image is the same size, and the pixel coordinates of each element image correspond to the same sample space location.

[0082] (6) Wash the cadmium-bound conjugate MBs@Ab from step (4) with PBS solution, mix for 15 s, separate on a magnetic rack, remove the supernatant to obtain 1 mg of cadmium-bound MBs@Ab@Cd, and maintain the liquid environment with 1 ml of PBS solution. At the same time, take equal amounts of the conjugate MBs@Ab and MBs prepared in step (3) as controls, and maintain the liquid environment with the same volume of PBS solution. Adjust the light source of the Müller matrix microscope to green light and the magnification to 40×. Mix the cadmium-bound MBs@Ab@Cd, MBs@Ab and MBs solutions, respectively, and transfer one drop of each solution to a glass slide with a pipette. After stabilization, cover with a coverslip to complete the slide preparation. Place the prepared MBs@Ab, MBs@Ab@Cd and MBs slide samples under the Müller matrix microscope for observation and adjust the observation range. Select areas with uniform particle distribution and clear field of view for image acquisition, and acquire images four times for each sample.

[0083] (7) After sample collection, high-resolution polarization microscopic images of cadmium-bearing MBs@Ab@Cd, MBs@Ab, and MBs were obtained. The Müller matrix of the samples was calculated and output using a Müller matrix calculator. Pathol was used to analyze and classify the Müller matrix. The images of the 16 Müller matrix elements were used as analysis parameters (i.e., the 16 Müller matrix elements M). 11 M 12 M 13 M 14 M 21 M 22 M 23 M 24 M 31 M 32 M 33 M 34 M 41 M 42 M 43 M 44 This study uses the deep learning RESNET model as a classification model to extract the differential features of different particulate matter from the Müller matrix to achieve particulate matter classification. Specifically:

[0084] ① The classification analysis uses a 96×96×16 polarization microscopic image as the input layer. The size of the polarization microscopic image is 96×96 pixels, and each pixel has 16 channel parameters. Each parameter records information about different polarization directions, and the 16 array elements can characterize various polarization effects; among which M 11 For the light intensity information of the sample, M22 M 33 M represents the depolarization characteristics of the sample. 12 M 13 M represents the dichroic characteristics of the sample. 24 M 34 M 42 and M 43 The birefringence characteristic of the sample;

[0085] ② The convolutional layer is used to extract image features, magnify image details, and retain important information; the convolutional kernel is 7×7, the output has 64 channels, the stride is 2, and the activation function is ReLU. The 7×7 convolutional kernel can capture local features of the image, and the stride of 2 can reduce the size of the feature map while retaining important information.

[0086] ③ The pooling layer simplifies the feature map. By using max pooling, the maximum value in each region is selected, and the size of the feature map is halved (from 96×96 to 48×48), retaining the most important information.

[0087] ④ The next bottleneck block is a core component of ResNet, used for efficient feature extraction. Stage 1 has 164 channels and 3 bottleneck blocks, each consisting of three convolutional layers (1×1, 3×3, 1×1). Through these bottleneck blocks, higher-level features are extracted progressively. Stage 2 increases the number of channels in the feature map by 128, further extracting more complex features. Stage 3 increases the number of channels to 256, extracting even higher-level features. Stage 4 increases the number of channels in the feature map to 512, extracting the most complex features.

[0088] ⑤ The global average pooling layer averages the pixel values ​​of each feature map to generate a feature vector with a fixed length of 16.

[0089] ⑥ The fully connected layer classifies the particles based on the feature vectors, outputs the probability of each category, and finally determines which category the test particles belong to.

[0090] Following the steps described above, from the input layer to the convolutional layer, pooling layer, bottleneck block, global average pooling layer, and finally the fully connected layer, the entire model progressively extracts features, obtains the Müller matrix diagrams of different particles, and classifies the different particles. See [link to detailed calculation process] for more information. Figure 5 .

[0091] During the experiment, it was found that distinguishing only MBs@Ab and MBs@Ab@Cd microspheres without using MBs was as effective as the three-class classification method. Therefore, to optimize time and efficiency, a two-class classification method was selected for the quantitative detection of the heavy metal cadmium.

[0092] (8) Quantitative detection of heavy metal cadmium was performed using a polarized Müller matrix microscope. After classification under the Müller matrix microscope, the Müller matrix diagrams of MBs@Ab@Cd and MBs@Ab bound to cadmium were obtained respectively. See [reference needed] Figure 4 (a) and Figure 4 (b). Each Müller matrix contains 16 array element images, where the pixel coordinates of the image in each array element correspond to the spatial location of the same sample. RESNET34 was used to analyze the image data, with 80% of the data selected as the training set for model training (i.e., database architecture) and 20% as the test set. MBs@Ab and MBs@Ab@Cd were classified into two classes. The classification parameter extraction process is shown in (7). Based on the test set results, a confusion classification matrix for the two samples was output. The sample classification results for the two classes are shown in [reference needed]. Figure 4 (c) As can be seen from the figure, the particle differentiation effect exceeds 84%.

[0093] Among the 16 Mueller matrix elements, MBS@Ab and MBS@Ab@Cd are most abundant in M... 11 M 21 M 22 M 44 No significant differences were observed in M. 12 M 24 A very significant difference was found in M. 13 M 14 M 23 M 31 M 32 M 33 M 34 M 41 M 42 M 43 Significant differences were found. Based on these differences, the two types of particulate matter were classified to identify their respective species. The effectiveness of polarization characterization in distinguishing these two types of particles was demonstrated. A cadmium standard solution (500 μL, Cd addition of 2 ng) was used, based on M... 12 M 24 M 13 M 14 M 23 M 31 M 32 M 33 M 34 M 41 M 42 M 43The particulate matter of the two materials was counted using different color markers. The standard curve gradients were 10%, 30%, 50%, 70%, and 100% of the Cd addition amount. Based on the above method, the number of particulate matter for each material was calculated. A standard curve was plotted with the Cd addition ratio as the x-axis and the Cd detection ratio (number of MBs@Ab@Cd particles / (number of MBs@Ab+MBs@Ab@Cd particles)) as the y-axis (see Figure 6 for the standard curve plotted in this embodiment). Samples with unknown concentrations can be substituted into the standard curve based on the detection results to calculate Cd quantification.

[0094] The quantitative R of the detection result in this embodiment 2 With a value of 0.9 or higher, it has certain advantages in detection limit and detection time compared with other detection methods.

[0095] Comparison with other existing technologies:

[0096] Existing methods for testing aquatic products include automated electrochemical detection (Federico Danilo Vallese, 2025), optical chemical sensors (Ali A. Ensafi, 2011), and ICP-MS (Fang Longxiang, 2024). The results comparing the testing time, detection limit, and upper detection limit with the present invention (MBs@Ab magneto-optical integrated synergistic detection technology) are shown below:

[0097]

[0098] As shown in the table, the magneto-optical integrated synergistic method for Cd detection, compared to the traditional ICP-MS method, reduces the detection time from 100-120 min to 1 min without significantly lowering the detection limit. Compared to automated electrochemical detection and optical chemical sensor detection methods, it offers advantages in both detection time and detection limit.

[0099] Comparative Example 1

[0100] Similar to Example 1, except that the magnetic microspheres selected in step (1) of this comparative example have a particle size of 600 nm. 100 μL (concentration 10 mg / mL) of the magnetic microspheres was transferred to a 1.5 mL centrifuge tube, vortexed for 10 s, mixed on a roller mixer for 15 min, and then magnetically separated on a magnetic separator for 1 min. The supernatant was removed. 900 μL of PBST Buffer (PBS with 0.05 wt% Tween 20 added, pH adjusted to 7.4) was added, and the mixture was mixed on a roller mixer for 5 min at room temperature (20-25℃). After magnetic separation, the supernatant was removed, and the mixture was washed twice with PBST Buffer, then the supernatant was removed and the mixture was ready for use. Activation was complete.

[0101] After activation, it is coupled with the biotinylated cadmium monoclonal antibody described in step (2). Due to the small particle size of the microspheres, the coupling rate is 69%, resulting in poor coupling effect.

[0102] Comparative Example 2

[0103] Similar to Example 1, except that in step (3) of this comparative example, 5 μg, 11 μg, and 15 μg of the biotinylated cadmium monoclonal antibody purified and collected in step (2) were respectively taken into the centrifuge tube of step (1). The conjugation rate was calculated, and the data are shown in the table below:

[0104]

[0105] As can be seen from the table, antibody conjugation is basically saturated at 10 μg. Considering the cost, 10 μg of antibody was selected for conjugation with 100 μL of magnetic microspheres.

[0106] Comparative Example 3

[0107] Similar to Example 1, the difference is that the reaction time in step (3) of this comparative example was selected as 30 min, 60 min, 90 min, and 120 min, respectively. The coupling rate was calculated, and the data are shown in the table below:

[0108]

[0109] As can be seen from the table, antibody conjugation is optimal when the reaction time is 60 min. Considering cost, the reaction time of the reaction system should be controlled at 60 min.

[0110] Comparative Example 4

[0111] Similar to Example 1, except that in step (3) of this comparative example, the reaction temperature was selected as 25°C for incubation of the magnetic microsphere antibody. After incubation, the supernatant was removed after magnetic separation, and the mixture was washed 1-2 times with PBST Buffer. The amount of antibody residue in the supernatant was then calculated, and the antibody conjugation effect was calculated.

[0112] The effect after the reaction was poor, with the coupling rate only 49.24% at 25℃ due to the influence of reaction temperature.

[0113] Comparative Example 5

[0114] Similar to Example 1, the difference is that in step (6) of this comparative example, the observation magnification of the Müller matrix microscope is adjusted to 10×. Within the field of view, the microspheres are not clearly captured, and it is impossible to select areas with uniform particle distribution and clear field of view for image acquisition.

[0115] Comparative Example 6

[0116] Similar to Example 1, the difference is that in step seven of this comparative example, RESNET50 is used to analyze the image data, and different colors are used to mark and count particles of different materials. The classification prediction effect is 53%, the capacity of RESNET cannot be effectively utilized, and a long training period is required. This classification model has not fully converged. To improve the classification accuracy, RESNET34 is selected to classify and count particles.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation, characterized in that, The steps are as follows: Step 1: Add the conjugate MBs@Ab to the digestion solution of the aquatic product to be tested to enrich cadmium. After magnetic separation, the cadmium-bound complex is obtained and denoted as MBs@Ab@Cd. Step 2: Prepare slides of two pure samples, the cadmium-bound complex MBs@Ab@Cd and the unbound cadmium conjugate MBs@Ab, and acquire polarized microscopic images under a Müller matrix microscope. Based on the acquired images, obtain the Müller matrix diagrams of MBs@Ab and MBs@Ab@Cd. Step 3: Use the deep learning model RESNET34 to analyze the Müller matrix images of MBs@Ab and MBs@Ab@Cd, distinguish and count the particulate matter of the two materials, and obtain the number of MBs@Ab@Cd particulate matter / (number of MBs@Ab + number of MBs@Ab@Cd particulate matter), which is recorded as the cadmium detection ratio. Step 4: Take cadmium standard solution and plot the standard curve: The gradients of the standard curve are 10 wt%, 30 wt%, 50 wt%, 70 wt%, and 100 wt% of cadmium added in the cadmium standard solution. Based on the methods in Steps 1-3, calculate the number of particles for each and the cadmium detection ratio for each. Plot the standard curve with the cadmium addition ratio as the x-axis and the cadmium detection ratio as the y-axis. Substitute the cadmium detection ratio of the sample to be tested obtained in Step 3 into the standard curve to obtain the cadmium addition ratio of the sample to be tested. Multiply this ratio by the amount of cadmium added in the standard solution to achieve quantification of Cd. The preparation method of the conjugate MBs@Ab is as follows: activate magnetic microspheres to obtain activated magnetic microspheres, denoted as MBs; react cadmium monoclonal antibody with biotin, and after purification, obtain biotinylated cadmium monoclonal antibody, denoted as Ab; conjugate the biotinylated cadmium monoclonal antibody and the activated magnetic microspheres to form a conjugate, denoted as MBs@Ab.

2. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, The magnetic microspheres are streptavidin magnetic microspheres with a particle size of 1-3 µm and a concentration of 10 mg / mL. The streptavidin magnetic microspheres are activated using PBST Nuffer.

3. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, The molar ratio of cadmium monoclonal antibody to biotin is 1:

30. The specific reaction conditions are as follows: biotin is added to the cadmium monoclonal antibody, and the mixture is placed in a mixer at room temperature in the dark for reaction. After the reaction is completed, the mixture is blocked at room temperature and purified by chromatography to obtain biotinylated cadmium monoclonal antibody.

4. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, The ratio of the biotinylated cadmium monoclonal antibody to the activated magnetic microspheres is 10-20 µg: 1 mg. The coupling conditions are as follows: add the biotinylated cadmium monoclonal antibody to the activated magnetic microspheres, add PBS buffer to make the total volume 100 µL, then vortex mix for 10 s, and then incubate in a roller mixer for 60 min at a temperature of 25-37°C. The coupling is then complete.

5. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, The enrichment of cadmium in step one is as follows: the conjugate MBs@Ab is directly added to the aquatic product digestion liquid for extraction. The extract is placed in a mixer with the temperature set at 25-37℃ and incubated for 30 min. After incubation, the supernatant is removed by magnetic separation to obtain the cadmium-bound complex MBs@Ab@Cd.

6. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, In step two, the Müller matrix microscope uses a green light source and a 40× objective lens, and acquires four images for each sample.

7. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, In step two, the Müller matrix is ​​obtained through a polarizer (PSG) and an analyzer (PSA), and the Müller matrix of the sample is calculated for classification.

8. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, In step three, the ratio of the training set to the test set of the deep learning model RESNET34 is 80%:20%. It performs binary classification, outputs a sample confusion classification matrix based on the test set results, and marks and counts the particles of the two materials with different colors.

9. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, In step four, the volume of the cadmium standard solution is 500 μL, and the amount of cadmium added is 2 ng.

10. The rapid detection method for cadmium residues in aquatic products based on magnetic qualitative separation and polarized light quantitative differentiation according to claim 1, characterized in that, The aquatic products include fish, shellfish, crustaceans and their processed products.

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