Nitrofuran Metabolites Research Articles

Rapid Apta-Chromogenic Detection Method for Nitrofuran Metabolite Determination.

Nitrofuran (NF) contamination in food products is a global problem resulting in the banned utilization and importation of nitrofuran contaminated products. A novel chromogenic detection method using a specific DNA aptamer with high affinity and specificity to nitrofurans was developed. Single-stranded DNA aptamers specific to nitrofuran metabolites, including 3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), were isolated using magnetic bead-SELEX. The colorimetric detection of nitrofurans using gold nanoparticles (AuNPs) exhibited an AOZ detection range of 0.01-0.06 ppb with a limit of detection (LOD) of 0.03 ppb. At the same time, this system could detect AMOZ and AHD at a range of 0.06 ppb and 10 ppb, respectively. The fast nitrofuran extraction method was optimized for food, such as fish tissues and honey, adjusted to be completed within 3-6 h. This novel apta-chromogenic detection method could detect NF metabolites with a sensitivity below the minimum required performance limit (MPRL). This analysis will be valuable for screening, with a shortened time of detection for aquaculture products such as shrimp and fish muscle tissues.

Determination of 12 prohibited veterinary drug residues in pig urine by ultra high performance liquid chromatography-tandem mass spectrometry

A method was established for the simultaneous detection of 12 prohibited veterinary drugs, including β2-receptor agonists, nitrofuran metabolites, nitroimidazoles, chlorpromazine, and chloramphenicol, in pig urine. The sample was pretreated by enzymolysis, acid hydrolysis/derivatization, and liquid-liquid extraction combined with solid-phase extraction. Detection was performed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Ammonium acetate solution (0.2 mol/L, 4.5 mL) and β-glucuronidase/aryl sulfatase (40 μL) were added to the sample, which was subsequently enzymolized at 37 ℃ for 2 h. Then, 1.5 mL of 1.0 mol/L hydrochloric acid solution and 100 μL of 0.1 mol/L o-nitrobenzaldehyde solution were added to the sample. The mixture was incubated at 37 ℃ for 16 h, and the analytes were extracted with 8 mL of ethyl acetate by liquid-liquid extraction. The lower aqueous phase obtained after extraction was extracted and purified using a mixed cation-exchange solid-phase extraction column. The extracts were combined, the extraction solution was blow-dried with nitrogen, and the residue was redissolved for determination. The samples were analyzed under multiple-reaction monitoring mode with both positive and negative electrospray ionization, and quantified using an isotope internal standard method. The correlation coefficients (r) of the 12 compounds were >0.99. The limits of detection (LODs) and quantification (LOQs) of chloramphenicol were 0.05 and 0.1 μg/L, respectively, and the LODs and LOQs of the other compounds were 0.25 and 0.5 μg/L, respectively. The mean recoveries and RSDs at 1, 2, and 10 times the LOQ were 83.6%-115.3% and 2.20%-12.34%, respectively. The proposed method has the advantages of high sensitivity, good stability, and accurate quantification; thus, it is suitable for the simultaneous determination of the 12 prohibited veterinary drug residues in pig urine.

Determination of nitrofuran metabolites and nifurpirinol in animal tissues and eggs by ultra-high performance liquid chromatography-tandem mass spectrometry validated according to Regulation (EU) 2021/808

In this work, an analytical method for the determination of eight non-allowed nitrofurans, including nifurpirinol and the metabolites of furazolidone, furaltadone, nitrofurantoin, nitrofurazone, nifursol, nitrovin, and nifuroxazide in animal tissues, including muscle (poultry, bovine, ovine, porcine, rabbit, and fish), kidney (bovine, ovine, porcine), and eggs, has been developed and validated according to Regulation (EU) 2021/808. The method was based on derivatization with 2-nitrobenzaldehyde in acid medium, followed by vortex-assisted liquid-liquid extraction and solid phase extraction for sample purification prior to ultra-high performance liquid chromatography-tandem mass spectrometry. Under selected conditions, the method was validated showing satisfactory relative matrix effects (CV ≤ 20 %), linearity (R2 ≥ 0.98), trueness (≤20 %, expressed as bias), accuracy (83–120 %), repeatability (1.7–19.9 %), reproducibility (1.9–25.7 %), specificity (blank signal ≤30 % at the LCL), and ruggedness. The decision limit for confirmation (CCα) for the target analytes ranged from 0.27 to 0.35 μg kg−1, all below the current reference point for action (RPA) of 0.5 μg kg−1 for the studied compounds. This validated method is currently accredited according to UNE-EN ISO/IEC 17025 by the Spanish National Accreditation Body (ENAC) to be implemented for official control analyses in the Public Health Laboratory of Valencia (Spain).

Determination of nitrofuran metabolites in muscle by LC-MS/MS and LC-MS/MS/MS: Alternative validation according to Commission Implementing Regulation (EU) 2021/808

Nitrofurans are banned in food of animal origin in the European Union. A rugged liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and LC-MS/MS cubed (LC-MS/MS/MS) analytical method allowing the confirmation of five metabolites of nitrofuran antibacterial agents, furaltadone, furazolidone, nifursol, nitrofurantoin and nitrofurazone in animal muscle tissue was developed and validated. The validation of 1-aminohydantoin (AHD), 3-amino-5-methyl-morpholino-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), 3,5-dinitrosalicylic acid hydrazide (DNSH) and semicarbazide (SEM) was carried out according to Commission Implementing Regulation (EU) 2021/808, by the adoption of a matrix-comprehensive in-house validation concept and the standard addition calibration. Validation was successfully performed, starting from 0.4 µg kg−1. Decision limit (CCα), recovery, repeatability, within-laboratory reproducibility and uncertainty of measurement were determined together with a factorial effect analysis. The procedure was verified through the analysis of two certified reference materials. The sample preparation consisted in acid hydrolysis and derivatization steps, followed by a liquid-liquid extraction of the analytes with ethylacetate.

Exploration of porous imine-based covalent organic framework for solid-phase extraction of five trace sulfonamides in food samples.

In this article, a highly crystalline porous imine-based covalent organic framework was synthesized at room temperature and used as solid-phase extraction (SPE) adsorbent for the purification and enrichment of trace sulfonamides (SAs) from food samples. The structure of the obtained material was characterized and studied in detail. The extraction process was optimized and the final elution was determined by the ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry method. Low limits of detection (0.02-0.19μg/kg) were obtained under optimal conditions, with the recoveries ranging from 70.5% to 105.3% when spiked at different levels. The adsorption process of the material for SAs was fitted by the Langmuir and Freundlich adsorption isotherm model, and the extraction capacity for Nitrofuran metabolites from food samples was also investigated for comparison. The results demonstrated that the framework was a good candidate SPE adsorbent that can be used for the enrichment of drug residues in complex matrix, and the work may provide a systematic study method for the development of porous adsorbents.

Original-structure-based hapten design strategy and development of a derivatization-free gold-based paper lateral-flow immunoassays for detecting furaltadone metabolite in shrimp

Veterinary drug residues in food-producing animals pose a threat to human health and public safety, and accurate measurement of these drug residues is necessary. However, detecting nitrofuran metabolite residues is challenging because of their low molecular weight and the long derivatisation time required for their indirect detection. Current antibodies produced against these metabolites have limited accuracy and sensitivity owing to the traditional and ineffective hapten design based on the reaction of 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) with nitrobenzaldehyde. In this study, a new antigen strategy was adopted using an original-structure-based hapten design combined with a new carrier protein with immunosynergistic effects to produce a monoclonal antibody that could recognise the metabolite of furaltadone (AMOZ) directly without derivatisation with excellent analytical performance. Finally, a gold-based paper lateral flow immunoassay for AMOZ without derivatisation was established with a visual limit of detection of 5 ng/mL, IC50 of 0.97 ng/mL and linear range (IC20∼IC80) of 0.45–2.07 ng/mL. The method was sensitive and accurate, and detection was completed within 10 min. This new antigen design strategy provides a theoretical basis and reference for antibody production of other ultrasmall molecules.

Rapid multi-residue LC-MS/MS determination of nitrofuran metabolites, nitroimidazoles, amphenicols, and quinolones in honey with ultrasonic-assisted derivatization – magnetic solid-phase extraction

A rapid multi-residue LC-MS/MS method for the identification and determination of banned veterinary drugs in honey was developed. A total of 31 investigated veterinary drugs belonging to 4 classes including nitrofurans metabolites, nitroimidazoles, amphenicols, and quinolones were quantified by LC-MS/MS with ESI using one single injection. The sample preparation included treatment with 5-nitro-2-furaldehyde (5-NFA) in a thermostated ultrasonic bath (80 °C, 0.5М НСl, 20 min) to liberate matrix-bound residues of nitrofurans. Magnetic hypercrosslinked polystyrene (HCP/Fe3O4) was proposed for the solid-phase extraction and clean-up of target analytes prior to LC-MS/MS analysis. To evaluate and validate the performance of method, the criteria of the Decision (EC) no 2002/657 were applied. The LOQs of the examined analytes range from 0.3 to 1 μg kg−1, which indicates good sensitivity to quantify the target compounds in honey. The recoveries of veterinary drugs from 1 g of honey with 50 mg of the sorbent are 97–109% for nitrofuran metabolites, 84–115% for nitroimidazoles, 86–103% for amphenicols, and 97–118% for quinolones. The relative standard deviations of intra-day and inter-day precision analyses (RSD) are less than 16%. This methodology was applied to real honey samples and trace levels of some veterinary drugs were detected.

Exposure Assessment of Nitrofuran Metabolites in Fish and Honey Produced in Armenia: A Pilot Investigation.

In Armenia, the presence of nitrofuran residues in food products is unacceptable for both domestic sales and export. However, food may contain nitrofuran metabolites (NMs) due to the illegal use of these drugs in the agrofarming practice. This study aimed to identify NMs as the marker residues for nitrofurans in fish and honey produced in Armenia and assess the potential health risks associated with consuming these foods. The commodities studied were natural honey and three species of farmed fish produced by various regions nationwide. Concentrations of the marker metabolites (3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), and semicarbazide (SEM)) were determined through an enzyme-linked immunosorbent assay (ELISA) and verified using liquid chromatography-mass spectrometry (LC-MS/MS). Consumer groups were identified based on their average daily intake of foods. Health risk was assessed by calculating the margin of exposure (MOE). Reference values for health risk assessment were obtained from the European Food Safety Authority (EFSA). Results showed that 33.3% of fish samples and 44.4% of honey samples contained NMs, the mean concentrations ranging from 0.05 μg/kg to 0.52 μg/kg. All MOE values obtained were over 10,000, indicating that the detected concentrations of NMs in fish and honey produced in Armenia pose no health risk to consumers. However, these results highlight the illicit use of highly toxic substances and the need for improved control of farming practices.

Residue analysis of nitrofuran metabolites in five food commodities from Croatia using ultra-high performance liquid chromatography–tandem mass spectrometry

The aim of the current study was to validate a screening and confirmatory method for the determination of nitrofuran residues: 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), 3,5-dinitrosalicylic acid hydrazine (DNSAH), nifurpirinol (NPIR), nifuroxazide (metabolite 4-HBH), and nitrovin (metabolite AMG) in muscle, milk, eggs, honey, and casings, in accordance with the legislation 2002/657/EC and 2021/808/EC. Sample preparation included hydrolysis and derivatization, followed by ethyl acetate extraction and measurement by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Validation parameters specified in this method are specificity/selectivity, trueness, precision, decision limit (CCα), and detection capability (CCβ). In the screening method, the CCβ value ranged from 0.275 to 0.582 µg kg-1, while in the confirmatory method, the CCα value ranged from 0.264 to 0.407 µg kg-1. The recovery values ranged from 93.5% to 127.5%. A total of 497 routine samples from the National Residue Monitoring Control Plan (NRCP) and border inspection were analysed from 2021 to 2022. SEM was detected in a total of 11 samples. Only four samples had a concentration above the Reference Point for Action (RPA = 0.5 µg kg-1). The validated method is suitable for monitoring nitrofuran residues at levels below the recently established RPA.

Surveillance of Nitrofuran Metabolite Residue in Pacific White Shrimp (Litopenaeus vannamei) at Local Market in Bangkok

The study aimed to analyze nitrofuran metabolite residues in Pacific white shrimp (Litopenaeus vannamei) from the 5 local markets in Bangkok. The samples weighing from 9.20+0.79 to 31.00+0.67 g were analyzed for nitrofuran metabolites: 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) by using LC-MS/MS technique. The results showed that only SEM in the range 0.104-0.160 ug/kg was found. The correlation coefficient between weight and SEM showed the negative linearity (r = -0.6867). It was found that there were less SEM residues in large shrimps than small shrimps. Therefore, the size of white shrimp can be used as an index for purchasing shrimp that are safe for consumers.

Facile synthesis of triazine-based porous organic polymer for the extraction and determination of nitrofuran metabolites residues from meat samples with ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry

In this work, a novel triazine-based porous organic polymers, TAPT-BPDD, was firstly synthesized by a facile method at room temperature. After characterized by FT-IR, FE-SEM, XRPD, TGA, and nitrogen-sorption experiments, TAPT-BPDD was applied as solid-phase extraction (SPE) adsorbent for the extraction of four trace nitrofuran metabolites (NFMs) from meat samples. The key parameters including the adsorbent dosage, sample pH, type and volume of eluents, type of washing solvents were evaluated in the extraction process. Combined with ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS/MS) analysis, good linear relationship (1–50 µg·kg−1, R2>0.9925) and low limits of detection (LODs, 0.05–0.56 µg·kg−1) were obtained under the optimal conditions. When spiked at different level, the recoveries were in the range of 72.7–111.6%. The adsorption isothermal model and extraction selectivity of TAPT-BPDD were also studied in detail. The results showed that TAPT-BPDD was a kind of promising SPE adsorbent for the enrichment of organics in food samples.

Hydrophobic carboxymethyl cellulose as a clean-up sorbent in the determination of nitrofuran metabolites in animal-fat samples

Hydrophobic-CMC biopolymers were fabricated for the removal of fat from food sample matrices. The CMCs were synthesized via the esterification of CMC with three alcohols with carbon chains of different lengths, in the presence of sulfuric acid.

Simultaneous determination of five metabolites of nitrofurans including the metabolite nifursol in shrimp and fish by UPLC- MS/MS: in-house method validation according to commission implementing regulation (EU) 2021/808

For the simultaneous identification and quantification of five nitrofurans metabolites in farmed shrimp and fish, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoine (AHD), semicarbazide (SEM), and 3,5-dinitrosalicylic acid hydrazide (DNSH), an accurate, precise, and specific method was developed. The mixture of water and methanol (60/40; v/v) was found to be the final optimised solvent for injection. The analytical run duration was 7 min, and the mobile phase included 2 mM methanol and ammonium formate. The new reference point for action (RPA) of 0.50 µg kg−1 as per EC/1871/2019 was taken into consideration and evaluated for the performance characteristics as per the CIR (EC)/2021/808 criteria. Specificity, relative retention time (≤0.25%) relative ion ratio (≤40%), linearity (0.25 to 2.0 µg kg−1), trueness (between 82.8 and 118.1%), repeatability (RSDr ≤14%), within lab reproducibility (RSDwr ≤16.9%), CCα (0.32–0.36 μg kg−1), ruggedness and relative matrix effect (≤14.26%) achieved acceptable values.

Quantum Dot Nanobeads as Multicolor Labels for Simultaneous Multiplex Immunochromatographic Detection of Four Nitrofuran Metabolites in Aquatic Products

A multicolor immunochromatographic assay platform based on quantum dot nanobeads (QBs) for the rapid and simultaneous detection of nitrofuran metabolites in different aquatic products is documented. These metabolites include 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), and 3-amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ). QBs with emission colors of red, yellow, green, and orange were employed and functionalized with the corresponding antibodies to each analyte to develop a multicolor channel. The visual detection limits (cutoff values) of our method for AOZ, AHD, SEM, and AMOZ reached up to 50 ng/mL, which were 2, 20, 20, and 20 times lower than those of traditional colloidal gold test strips, respectively. The test strip is capable of detection within 10 min in real samples while still achieving good stability and specificity. These results demonstrate that the developed multicolor immunochromatographic assay platform is a promising technique for multiplex, highly sensitive, and on-site detection of nitrofuran metabolites.

Simultaneous Screening of Nitrofuran Metabolites in Honey Using Biochip Array Technology: Validation Study According to the Decision 2002/657/EC of the European Union.

Although no authorization is available for antibiotics to treat bee diseases, some veterinary compounds are used by beekeepers, and each country sets its own thresholds. Inappropriate and excessive use of these drugs can cause allergic reactions and antibiotic resistance in humans who consume the remaining antibiotic residues in honey and its products. It is, therefore, relevant to monitor the presence of antibiotic residues in this matrix. A rapid method for the simultaneous screening of nitrofuran metabolite residues in honey was validated according to Commission Decision 2002/657/EC (C.D 657) and the European guideline for the validation of screening methods for veterinary medicines. This multi-analytical screening method enables the simultaneous determination of four nitrofuran metabolites [3-amino-2-oxazolidone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-Aminohydantoin HCl (ADH), and semicarbazide (SEC)] from a single honey sample. Thirty-five honey samples were collected randomly as real samples for screening from Tehran, IR Iran, Germany, and the Netherlands in 2018. For all four antibiotic residues, the positivity threshold T was higher than the cut-off value Fm, and no false-positive results were obtained for three antibiotics (AOZ, AMOZ, and SEC). Detection capabilities (CCβ) of all compounds were under the minimum required performance limit (MRPL) authorized by the European Commission (currently 1 μg/kg). The screening results of 15 domestic and 20 imported honey samples showed that the levels of AOZ in 6.66% and 10% of the samples, the level of AMOZ in 13.33% and 0% of the samples, and the level of SEC in 33.33% and 40% of the samples were less than the cut-off ([in relative light units (RLUs)], respectively. This study found that this technique is valid for detecting and quantifying three antibiotic residues in honey samples at the measured validation levels. This method was simple, rapid, and capable of simultaneously screening three nitrofuran metabolites from a single honey sample.

Fast screening method to determine metabolites of nitrofurans in chicken meat using partitioned dispersive liquid–liquid microextraction combined with HPLC/DAD

Methods have been developed for the hydrolysis and derivatisation of protein-bound metabolites of nitrofurans and for the extraction of 2-nitrobenzaldehyde derivatives of the metabolites from chicken meat. In this work, the time needed for hydrolysis and derivatisation was reduced from the conventional 16 h to 90 min. Based on partitioned dispersed liquid–liquid microextraction, a method for extracting 2-nitrobenzaldehyde derivatives of metabolites from crude chicken meat has been developed. Under the optimised experimental conditions, enrichment factors (EFs) ranging from 92.8 to 208.9 were obtained. The method was linear over the range of 10–600 µg kg−1 with determination coefficients (r 2) between 0.9979 and 0.9996. Intraday and interday repeatability expressed as a percentage RSD, ranged from 2.2% to 11.2%, and 2.7% to 12.4%, respectively. LOD of 1.07–2.25 µg kg−1 and LOQ of 3.09–6.2 µg kg−1 were obtained. The proposed method was applied in the analysis of metabolites of nitrofurans in chicken meat obtained from farmers using them for their domestic consumption and proved free of the analytes. A recovery of 85.2–109.4% with a %RSD ranging between 3.4% and 13.7% was obtained at three spiking levels. The proposed method was successfully further applied for the analysis of target analytes in chicken meat samples purchased from different supermarkets around Roodeport, Gauteng (South Africa). There was no target analyte detected in the analysed samples. Therefore, the developed methods can be used for monitoring the corresponding metabolites of nitrofurans in chicken meat.

Ratiometric fluorescent immunochromatography for simultaneously detection of two nitrofuran metabolites in seafoods

Competitive immunochromatographic assay (ICA) has been widely used in food safety on-site monitoring. However, traditional competitive ICA relies on the change of single-signal intensity to discriminate the results, leading to limited sensitivity and accuracy of naked-eye visual inspection. In this study, we developed a sensitive ratiometric fluorescent immunochromatographic assay (RFICA) judging results based on the color changes of fluorescence. Two different color quantum dot nanobeads (QBs) were introduced as signal labels for rapid and accurate detection of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM) in seafoods. Under the optimal conditions, the visual detection limit for 2-NPAOZ and 2-NPSEM reached up to 1 and 0.5 ng/mL, which were 5- and 14-fold lower than that of traditional single-signal response fluorescent ICA strips. In addition, the RFICA strips were applied to real samples with acceptable recoveries of 81.33 ∼ 99.23 %. These results demonstrate that the developed RFICA platform possesses considerable potential in the on-site detection.

Rapid Hydrolysis and Derivatization of Nitrofuran Metabolites with a New Derivatizing Agent 5-Nitro-2-Furaldehyde in Their Determination in Chicken Meat by HPLC–MS/MS

Rapid Hydrolysis and Derivatization of Nitrofuran Metabolites with a New Derivatizing Agent 5-Nitro-2-Furaldehyde in Their Determination in Chicken Meat by HPLC–MS/MS

Dual-color aggregation-induced emission nanoparticles for simultaneous lateral flow immunoassay of nitrofuran metabolites in aquatic products

Nitrofurans such as furaltadone and nitrofurantoin are a type of synthetic broad-spectrum antibiotics. Various fluorescent nanomaterials have been used as labeling materials in immunochromatographic assays (ICAs) for nitrofurans detection. However, previous fluorescent nanomaterials can undergo aggregation-caused quenching, leading to a decrease in the detection sensitivity. In this study, we developed a multiplex immunochromatographic assay (mICA) based on dual-color aggregation-induced emission nanoparticles (AIENPs) as signal labels for the simultaneous detection of 3-amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ) and 1-aminohydantoin (AHD), which were the metabolites of furaltadone and nitrofurantoin, respectively. Under optimal conditions, the cut-off values of the mICA for derivatized AMOZ (2-NP-AMOZ) and AHD (2-NP-AHD) reached up to 3 and 5 ng/mL, respectively. These values are at least 166-and 200-fold higher than those of the commercial gold nanoparticles (GNPs)-based test strip, respectively. Furthermore, the test strip was successfully applied to the samples, with acceptable recoveries in the range of 83.0–98.2%.

A novel detection strategy for nitrofuran metabolite residues: Dual-mode competitive-type electrochemical immunosensor based on polyethyleneimine reduced graphene oxide/gold nanorods nanocomposite and silica-based multifunctional immunoprobe

Excessive residues of semicarbazide (SEM) can accumulate in animals after the original drug has been abused, posing a risk to human health. Herein, based on multifunctional silica-initiated dual mode signal response, a novel competitive-type immunosensor was constructed for ultrasensitive detection of SEM. As a preliminary signal amplification platform for immunosensors, polyethyleneimine reduced graphene oxide composite gold nanorods (PEI-rGO/AuNRs) modified gold electrodes (AuE) provide a high specific surface area and high electrical conductivity. The thionine-aminated silica nanospheres-AuPt (thi-SiO2@AuPt) were synthesized by a racile coprecipitation method for enzyme immobilization and redox species loading. The multifunctional silica nanosphere conjugated with labeling antibodies (Ab2) was employed as an immunoprobe. The per unit concentration target of SEM can be determined by differential pulse voltammetry (DPV) to detect the thi loaded on the immunoprobe, which can also be determined by square wave voltammetry (SWV) to detect the current generated by the reaction system of H2O2 and hydroquinone (HQ) catalyzed by the immunoprobe with peroxidase. Under optimal conditions, the proposed immunosensor displayed a wide linear range from 1 μg-0.01 ng/mL and low detection limits (S/N = 3) of 0.488 pg/mL and 0.0157 ng/mL, respectively. Ultimately, the developed method exhibits excellent performance in practical applications, providing promising probabilities for SEM detection.