High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices

In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL⁻¹ to 1600 pg mL⁻¹, and the measured low limit of detection (LOD) was 3.2 pg mL⁻¹. No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future.     

Micellar electrokinetic chromatography and laser induced fluorescence detection of botulinum neurotoxin type A activity using a dual-labelled substrate

The activity of Botulinum neurotoxin type A (BoNT A) can be measured by monitoring the toxin's endopeptidase reaction with its peptide substrate. In this report, a sensitive and simple capillary electrophoresis (CE) method for analysing BoNT A activity was developed using a peptide substrate labelled with Fluorescein isothiocynate (FITC) at the N-terminal and biotin at the C-terminal. This dual labelling enables not only highly sensitive laser induced fluorescence (LIF) detection of the reaction product, but also good analytical separation of the product from the peptide substrate by Micellar Electrokinetic Chromatography (MEKC). The separation between the product peak and the substrate peak was approximately 5 min using the dual-labelled substrate, while just about 1 min using the FITC-labelled substrate without biotinylation. Using the current assay method, BoNT A with concentration as low as 0.1 ng ml^?1 (3.6 U mL^?1 in mouse LD₅₀) in water was detected with a S:N ratio of 3 (RSD <19%) and a linear range of four orders of magnitude. With CE's advantages of very small sample volume needed, this method may find particular applications as in assays of BoNT A activity in water samples and kinetic analyses of toxin activity.     

Sensitive and rapid chemiluminescence enzyme immunoassay for microcystin-LR in water samples

A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR). Several physicochemical parameters such as the chemiluminescent assay mediums, the dilution ratio of MC-LR-OVA conjugate, monoclonal antibody concentration, and peroxidase labeled antibody concentration were studied and optimized. Under optimum conditions, calibration curve obtained for MC-LR had detection limits of 0.032 ± 0.003 μg L⁻¹, the 50% inhibition concentration (IC₅₀) was 0.20 ± 0.02 μg L⁻¹ and the quantitative detection range was 0.062-0.65 μg L⁻¹. The proposed methods was successfully applied to the monitoring of MC-LR in spiked water samples without significant effect of the matrix, and the recovery of MC-LR added to water samples at different concentrations ranged from 80% to 115% with the coefficients of variation (CVs) less than 9%. The LOD attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of CLEIA as a screening tool for the analysis of MC-LR in environmental samples.     

Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14 fM (1.4 pg mL⁻¹). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min.     

Inter-laboratory validation of the fluorescent protein phosphatase inhibition assay to determine diarrhetic shellfish toxins: intercomparison with liquid chromatography and mouse bioassay

Toxic episodes of diarrhetic shellfish toxins (DSP) in shellfish harvesting areas have serious economic and public health implications, where fluorescent protein phosphatase inhibition assay (FPPIA) may be a highly useful tool for monitoring purposes. This paper presents results from the first inter-laboratory study to validate the assay. Three laboratories participated in the design and development of the inter-laboratory work. Standard solutions and spiked samples of the main toxin, okadaic acid, were used at the beginning of the validation exercise to avoid cross-inhibition of other toxins that would otherwise deteriorate the quantitative significance of the data. HPLC with fluorimetric detection of okadaic acid was also submitted to inter-laboratory validation to be subsequently used as a quantitative reference method. FPPIA results from spiked samples were free of systematic bias in any laboratory and determinations repeated over 3 days showed that the classic “repeatability” was the main within-laboratory source of variability (15-26% R.S.D. depending on the sample).After the inter-laboratory validation of both HPLC and FPPIA methods, 83 samples of mussel hepatopancreas collected during a toxic DSP episode were analyzed over 9 weeks. Toxic levels determined with FPPIA were in line with mouse bioassay results, highlighting the lack of false negative results of the FPPIA test: 98.7% of samples whose concentration of okadaic acid equivalents was over 0.8 μg/g hep., provided positive bioassay results within 24 h of observation time. The reliability and the quantitativeness of the FPPIA method in naturally contaminated samples was demonstrated by intercomparison with mouse bioassay and HPLC.     

Simultaneous determination of sex hormones in egg products by ZnCl2 depositing lipid, solid-phase extraction and ultra performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method was developed for simultaneous determination of residues of 17 sex hormones in egg products. Target compounds were extracted from samples with methanol in an ultrasonic bath, effectively separated from lipids in the extracts by ZnCl₂ depositing filtration and purified using a C₁₈ solid-phase extraction (SPE) and followed by NH₂ SPE cartridge. The analytes were quantified by liquid chromatography using a BEH C₁₈ column coupled to an electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS) operating in negative mode for estrogens and in positive multiple reaction monitoring mode for androgens. The parameters of the mass spectrometer and the composition of mobile phase and additives were also optimized to enhance detection sensitivity. Average recoveries of the target compounds varied from 70.0% to 121.0% with relative standard deviations ranging from 2.3% to 11.2% at two fortification levels. The limits of detection (LOD) of the method were from 0.002 μg kg⁻¹ to 0.23 μg kg⁻¹ and the limits of quantification (LOQ) were in the range of 0.007-0.76 μg kg⁻¹.     

Magnetic Fe3O4@Au composite-enhanced surface plasmon resonance for ultrasensitive detection of magnetic nanoparticle-enriched α-fetoprotein

Small molecules or analytes present at low concentrations are difficult to detect directly using conventional surface plasmon resonance (SPR) techniques because only small changes in the refractive index of the medium are typically induced by the binding of these analytes. Here, we present an amplification technique using core–shell Fe₃O₄@Au magnetic nanoparticles (MNPs) for an SPR bioassay. To evaluate this amplification effect, a novel SPR sensor based on a sandwich immunoassay was developed to detect α-fetoprotein (AFP) by immobilizing a primary AFP antibody (Ab₁) on the surface of a 3-mercapto-1-propanesulfonate/chitosan-ferrocene/Au NP (MPS/CS-Fc/Au NP) film employing Fe₃O₄@Au–AFP secondary antibody conjugates (Fe₃O₄@Au–Ab₂) as the amplification reagent. The stepwise fabrication of the biosensor was characterized using UV-vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. A calibration curve of Fe₃O₄@Au–Ab₂ conjugates amplification for AFP detection was obtained to yield a correlation in the range of 1.0–200.0 ng mL⁻¹ with a detection limit of 0.65 ng mL⁻¹, and a significant increase in sensitivity was therefore afforded through the use of Fe₃O₄@Au–Ab₂ conjugates as an amplifier. This magnetic separation and amplification strategy has great potential for the detection of other biomolecules of interest with low interference and high sensitivity by changing the antibody label used in the Fe₃O₄@Au–antibody conjugates.     

Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements

A surface plasmon resonance (SPR) immunobiosensor assay was developed and validated to detect microcystin toxins in Spirulina and Aphanizomenon flos-aquae blue-green algae (BGA) food supplements. A competitive inhibition SPR-biosensor was developed using a monoclonal antibody to detect microcystin (MC) toxins. Powdered BGA samples were extracted with an aqueous methanolic solution, centrifuged and diluted in HBS-EP buffer prior to analysis. The assay was validated in accordance with the performance criteria outlined in EU legislation 2002/657/EC. The limit of detection (LOD) of the assay was calculated from the analysis of 20 known negative BGA samples to be 0.561 mg kg⁻¹. The detection capability (CCβ) of the assay was determined to be ≤0.85 mg kg⁻¹ for MC-LR. The biosensor assay was successfully applied to detect MC-LR toxins in BGA samples purchased on the Irish retail market. MC-LR was detected in samples at levels ranging from <0.5 to 2.21 mg kg⁻¹. The biosensor results were in good agreement with an established LC-MS/MS assay. The assay is advantageous because it employs a simple clean-up procedure compared to chemical assays and allows automated unattended analysis of samples unlike ELISA.     

N-alkylpyridinium quaternization combined with liquid chromatography–electrospray ionization-tandem mass spectrometry: A highly sensitive method to quantify fatty alcohols in thyroid tissues

A highly sensitive method was developed for the identification and quantification of fatty alcohols in biological tissues. In the presence of pyridine-d₀ and triflic anhydride (Tf₂O), fatty alcohols were converted into permanently charged N-alkylpyridinium ions. Stable isotope-labeled derivatives were generated by pyridine-d₅ and added as internal standard (IS). The mixture was analyzed by liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). This method was optimized and validated in terms of reaction time, derivatization efficiency, stability, desalting, and ion suppression effect. Besides, fatty alcohols exhibited good linear relationship (r² > 0.993) over the concentration range of 10 ng mL⁻¹–1 μg mL⁻¹. The limits of detection (LODs) were lowered from previously reported 0.1 ng mL⁻¹ to 0.25 pg mL⁻¹. Precision (RSD% < 15.6%), accuracy (93.0–107.2%), matrix effect, and recovery (in thyroid tissues) were validated as well. Finally, this method was applied for the analysis of ten even carbon-numbered fatty alcohols (C₈–C₂₄) in human thyroid carcinoma and para-carcinoma tissues, revealing a significant decrease of fatty alcohols (free and esterified) in thyroid carcinoma tissues (p < 0.05).     

Open-sandwich immunoassay for sensitive and broad-range detection of a shellfish toxin gonyautoxin

At present, the analytical method for paralytic shellfish poisoning (PSP) toxins in shellfish is the mouse bioassay (MBA), which is an official method of the Association of Analytical Communities (AOAC [8]). However, the low sensitivity and concerns over the number of live animals required for testing have been cited as the major reason for seeking its replacement. In this report, we employed an open-sandwich immunoassay (OS-IA) to detect gonyautoxin (GTX2/3), a kind of PSP toxins. OS-IA, which utilizes the antigen-induced enhancement of antibody V_H/V_L interaction, can measure a small molecule antigen in a noncompetitive format. Hence it has a wider working range and shorter measurement time. We isolated anti-GTX2/3 antibody gene from a hybridoma GT-13A by screening a Fab-displaying phage library. Then the vectors for OS-IA were constructed, and examined for antigen concentration-dependency of the V_H/V_L interaction by OS-ELISA. As a result, in each case, signal intensity increases notably in a wide concentration range (0.1 to >1000 ng mL⁻¹) of free GTX2/3, which was enough to cover its regulation value (80 μg 100 g⁻¹) in many countries. So OS-IA will be widely applicable to detect PSP toxins in shellfish meats and in drinking water.     

Sensitive detection of type G botulinum neurotoxin through Endopep-MS peptide substrate optimization

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A–G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD₅₀ toxin present in buffer solution.

     

Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum

Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an enzymatic and mass spectrometric assay called Endopep-MS. However, no comprehensive validation of the method has been presented. The main purpose of this work was to perform a validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, and F in serum. The limit of detection (LOD), selectivity, precision, stability in matrix and solution, and correlation with the MBA were evaluated. The LOD was equal to or even better than that of the MBA for BoNT-A, B, D/C, E, and F. Furthermore, Endopep-MS was for the first time successfully used to differentiate between BoNT-C and D and their mosaics C/D and D/C by different combinations of antibodies and target peptides. In addition, sequential antibody capture was presented as a new way to multiplex the method when only a small sample volume is available. In the comparison with the MBA, all the samples analyzed were positive for BoNT-C/D with both methods. These results indicate that the Endopep-MS method is a valid alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, and speed and that it does not require experimental animals.     

Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in shellfish matrixes

The detection of toxins in shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent α-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of α-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these shellfish extracts with the α-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6 ± 7.8% and 89.6 ± 3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 μg kg⁻¹ and 85-700 μg kg⁻¹ of shellfish meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in shellfish as a screening assay.     

Application of manganese (II) phthalocyanine synthesized in situ in the SiO2/SnO2 mixed oxide matrix for determination of dissolved oxygen by electrochemical techniques

This work describes the in situ immobilization of Mn(II) phthalocyanine (MnPc) in a porous SiO₂/SnO₂ mixed oxide matrix obtained by the sol gel processing method. The chemically modified matrix SiO₂/SnO₂/MnPc, possessing an estimated amount of 8 × 10⁻¹⁰ mol cm⁻² of MnPc on the surface, was used to prepare an electrode to analyze dissolved oxygen in water by an electrochemical technique. The electrode was prepared by mixing the material with ultrapure graphite and evaluated using differential pulse voltammetry. Dissolved O₂ was reduced at −0.31 V with a limit of detection (LOD) equal to 7.0 × 10⁻⁴ mmol L⁻¹. A mechanism involving four electrons in O₂ reduction was determined by the rotating disk electrode technique.     

Detection of Newcastle disease virus with quantum dots-resonance light scattering system

A sensitive QDs-based RLS assay method for the detection of Newcastle disease virus (NDV) antibody has been developed. CdTe quantum dots (QDs) were conjugated with Newcastle disease virus and used as RLS-based probes to detect NDV antibody. The electrostatic interaction between CdTe QDs and NDV resulted in enhanced resonance light scattering (RLS) signal characterized at 555 nm. Upon the addition of NDV antibody, QDs-NDV formed dispersive immunocomplex that can decrease the RLS signal. The decreased RLS intensity at 555 nm (ΔI_RLS) was linearly proportional to the concentration of NDV antibody (C_anti-NDV) in the range of 0.5-50 ng/mL, with correlation coefficient of 0.974 and detection limit of 0.1 ng/mL under the optimization conditions. The proposed method was applied to the determination of NDV antibody in spiked samples with satisfactory results.     

An electrochemical immunosensor for carcinoembryonic antigen enhanced by self-assembled nanogold coatings on magnetic particles

A quick and reproducible electrochemical-based immunosensor technique, using magnetic core/shell particles that are coated with self-assembled multilayer of nanogold, has been developed. Magnetic particles that are structured from Au/Fe₃O₄ core-shells were prepared and aminated after a reaction between gold and thiourea, and additional multilayered coatings of gold nanoparticles were assembled on the surface of the core/shell particles. The carcinoembryonic antibody (anti-CEA) was immobilized on the modified magnetic particles, which were then attached on the surface of solid paraffin carbon paste electrode (SPCE) by an external magnetic field. This is an assembly of a novel immuno biosensor for carcinoembryonic antigen (CEA). The sensitivity and response features of this immunoassay are significantly affected by the surface area and the biological compatibility of the multilayered nanogold. The linear range for the detection of CEA was from 0.005 to 50 ng mL⁻¹ and the limit of detection (LOD) was 0.001 ng mL⁻¹. The LOD is approximately 500 times more sensitive than that of the traditional enzyme-linked immunosorbent assay for CEA detection.     

Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V_H and V_L) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V_H and V_L genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 ± 0.03 and 0.02 ± 0.004 ng mL⁻¹, respectively, and the linear response range extended from 0.05 to 1.45 ng mL⁻¹. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS). The results showed a good correlation between the data of dc-CLEIA and HPLC–MS (R² > 0.99), indicating that the assay was an efficient analytical method for monitoring food safety.     

Modeling of the three-phase equilibrium in systems of the type water + nonionic surfactant + alkane

Liquid–liquid equilibria of systems water (A) + C_iE_j surfactant (B) + n-alkane (C) have been modeled by a mass-action law model previously developed and so far successfully applied to a series of binary water + C_iE_j systems and to the ternary system water + C₄E₁ + n-dodecane. These calculations provide the basis for the presented modeling. The aqueous systems give information about the association constants and the χ_AB-parameter of the Flory–Huggins theory and the ternary C₄E₁-system provides universal temperature functions for the χ_AC- and the χ_BC-parameter. The three-phase equilibrium for seven ternary C_iE_j systems (i = 6–12, j = 3–6) has been calculated by fitting one additional parameter for each of both temperature functions to the characteristic “fish-tail” point. The agreement with the experimental data is reasonably well. For systems with very small three-phase areas the results can considerably be improved by individual temperature functions that incorporate the experimental temperature maximum of the “fish” into the parameter fit. Based on the parameters of the system water + C₈E₄ + n-C₈H₁₈ the “fish-shaped” phase diagram of the system water + C₈E₄ + n-C₁₄H₃₀ was predicted reasonably well.     

A fast derivatization procedure for gas chromatographic analysis of perfluorinated organic acids

A rapid and simple derivatization procedure has been developed for gas chromatographic determination of perfluorinated organic acids (PFCAs, C₆–C₁₂), using isobutyl chloroformate (IBCF) to convert the acids into the more volatile isobutyl esters, under catalysis by pyridine. The procedure was optimized in an acetonitrile medium and applied to GC techniques with electron-capture detection (GC-ECD) and mass spectrometry with electron-impact ionization (GC-EI-MS); for the sake of comparison, HPLC with electrospray-ionization MS (HPLC-ESI(−)-MS) was also tested. The LOD and LOQ values obtained for these three techniques were compared, and the lowest LODs were obtained with GC-ECD (0.06–1.80 μg mL⁻¹). The procedure was further optimized in an aqueous medium, obtaining the best results in a phosphate buffer (pH 2.5, 50 mmol L⁻¹), in which the LOD and LOQ values were measured for GC-ECD a GC-EI-MS. The lowest LODs were found for GC-EI-MS (0.030–0.314 μg mL⁻¹). The practical applicability was tested on Vltava river water samples.     

A highly sensitive fluorescence resonance energy transfer aptasensor for staphylococcal enterotoxin B detection based on exonuclease-catalyzed target recycling strategy

An ultrasensitive fluorescence resonance energy transfer (FRET) bioassay was developed to detect staphylococcal enterotoxin B (SEB), a low molecular exotoxin, using an aptamer-affinity method coupled with upconversion nanoparticles (UCNPs)-sensing, and the fluorescence intensity was prominently enhanced using an exonuclease-catalyzed target recycling strategy. To construct this aptasensor, both fluorescence donor probes (complementary DNA₁–UCNPs) and fluorescence quencher probes (complementary DNA₂–Black Hole Quencher₃ (BHQ₃)) were hybridized to an SEB aptamer, and double-strand oligonucleotides were fabricated, which quenched the fluorescence of the UCNPs via FRET. The formation of an aptamer–SEB complex in the presence of the SEB analyte resulted in not only the dissociation of aptamer from the double-strand DNA but also both the disruption of the FRET system and the restoration of the UCNPs fluorescence. In addition, the SEB was liberated from the aptamer–SEB complex using exonuclease I, an exonuclease specific to single-stranded DNA, for analyte recycling by selectively digesting a particular DNA (SEB aptamer). Based on this exonuclease-catalyzed target recycling strategy, an amplified fluorescence intensity could be produced using different SEB concentrations. Using optimized experimental conditions produced an ultrasensitive aptasensor for the detection of SEB, with a wide linear range of 0.001–1 ng mL⁻¹ and a lower detection limit (LOD) of 0.3 pg mL⁻¹ SEB (at 3σ). The fabricated aptasensor was used to measure SEB in a real milk samples and validated using the ELISA method. Furthermore, a novel aptasensor FRET assay was established for the first time using 30 mol% Mn²⁺ ions doped NaYF₄:Yb/Er (20/2 mol%) UCNPs as the donor probes, which suggests that UCNPs are superior fluorescence labeling materials for food safety analysis.