Optimization of an anti-HER2 nanobody expression using the Taguchi method

Despite being widely used in immunotherapy of cancer, whole antibodies are limited by several disadvantages. This has led to the advent of novel biomolecules such as nanobodies. Taguchi method is a statistical experimental design to study the effect of multiple variables in biological processes. In an effort to overexpress a recombinant anti-human epidermal growth factor receptor type 2 (HER2) nanobody, we performed a detailed study to find optimal condition of temperature, induction, culture media, vector, and host strain, using Taguchi methodology. A total of 16 various experiments were designed. Total protein of the formulated cultures were assessed by Bradford test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by size exclusion high performance liquid chromatography to quantify the relative concentration of the nanobody in different expression settings. Western blotting was performed to confirm the expression of the anti-HER2 nanobody. When, individually, optimum parameters determined by Taguchi were applied, including SHuffle strain cultured in LB medium, induced with 0.4?mM isopropyl-β-D-thio-galactoside for 18?h at 24°C, production yield further increased by about 9% (25.4?mg/L), compared to the highest expression setting. Flow cytometry and enzyme-linked immunosorbent assay result indicated improved protein binding in optimized conditions. Overall, our findings provide a basis for further investigations on economical production of recombinant nanobodies to improve production yield and activity.     

Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO₂ capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with k_cat values of up to 10⁶?s^?1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO₂. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (α-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe₃O₄ nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe₃O₄.     

Study of cryopreservation of articular chondrocytes using the Taguchi method

This study evaluates the effect of control factors on cryopreservation of articular cartilage chondrocytes using the Taguchi method. Freeze-thaw experiments based on the L₈(2⁷) two-level orthogonal array of the Taguchi method are conducted, and ANOVA (analysis of variables) is adopted to determine the statistically significant control factors that affect the viability of the cell. Results show that the type of cryoprotectant, freezing rate, thawing rate, and concentration of cryoprotectant (listed in the order of influence) are the statistically significant control factors that affect the post-thaw viability. The end temperature and durations of the first and second stages of freezing do not affect the post-thaw viability. Within the ranges of the control factors studied in this work, the optimal test condition is found to be a freezing rate of 0.61 ± 0.03 °C/min, a thawing rate of 126.84 ± 5.57 °C/min, Me₂SO cryoprotectant, and a cryoprotectant concentration of 10% (v/v) for maximum cell viability. In addition, this study also explores the effect of cryopreservation on the expression of type II collagen using immunocytochemical staining and digital image processing. The results show that the ability of cryopreserved chondrocytes to express type II collagen is reduced within the first five days of monolayer culture.     

Quantification of non‐specific binding of magnetic micro‐ and nanoparticles using cell tracking velocimetry: Implication for magnetic cell separation and detection

The maturation of magnetic cell separation technology places increasing demands on magnetic cell separation performance. While a number of factors can cause sub‐optimal performance, one of the major challenges can be non‐specific binding of magnetic nano‐ or microparticles to non‐targeted cells. Depending on the type of separation, this non‐specific binding can have a negative effect on the final purity, the recovery of the targeted cells, or both. In this work, we quantitatively demonstrate that non‐specific binding of magnetic nanoparticles can impart a magnetization to cells such that these cells can be retained in a separation column and thus negatively impact the purity of the final product and the recovery of the desired cells. Through experimental data and theoretical arguments, we demonstrate that the number of MACS magnetic particles needed to impart a magnetization that is sufficient to cause non‐targeted cells to be retained in the column to be on the order of 500–1,000 nanoparticles. This number of non‐specifically bound particles was demonstrated experimentally with an instrument, cell tracking velocimeter, CTV, and it is demonstrated that the sensitivity of the CTV instrument for Fe atoms contained in magnetic nanoparticles on the order of 1 × 10^?15 g/mL of Fe. Biotechnol. Bioeng. 2010;105: 1078–1093. © 2009 Wiley Periodicals, Inc.     

Optimization of media composition for improving conversion of isoeugenol into vanillin with Pseudomonas sp. strain KOB10 using the Taguchi method

Abstract

The popular demand for natural food additives has resulted in a number of processes for producing natural vanillin. Although there are chemical procedures and plant sources for vanillin production, microbial bioconversions are being sought as a suitable ‘natural’ alternative. The present paper describes the conversion of isoeugenol to vanillin by a novel bacterial strain isolated from soil. The strain was identified as Pseudomonas sp. strain KOB10 based on morphological and physiochemical characteristics and its 16S rDNA gene sequence. We optimized medium composition for vanillin production using a Taguchi experimental design. Eight factors, i.e. isoeugenol, glycerol, tryptone, K₂HPO₄, KH₂PO₄, Cu²⁺, Mg²⁺ and Ca²⁺ concentrations, were selected and experiments based on an orthogonal array layout of L₁₈ (2² × 3⁶) were performed. Analysis of the experimental data using the Taguchi method indicated that Cu²⁺ and glycerol concentrations had the highest impact on isoeugenol conversion into vanillin at a substrate concentration of 0.9 g L^?1. Under the optimized conditions, growing cells of Pseudomonas sp. strain KOB10 produced 0.153 g vanillin L^?1 from 0.9 g isoeugenol L^?1, with a molar yield of 18.3% after incubation for 48 h. To improve the vanillin yield, the effect of other bioconversion parameters including time of isoeugenol addition, initial isoeugenol concentration and conversion time was studied; the results showed a maximum concentration of 3.14 g vanillin L^?1 after a total incubation time of 88 h with 15 g isoeugenol L^?1, which corresponded to a molar yield of 22.5%. Further standardization and optimization for vanillin production was challenging.     

Effect of heat dissipation of superparamagnetic nanoparticles in alternating magnetic field on three human cancer cell lines in magnetic fluid hyperthermia

ABSTRACT

Purpose: The purpose of this study was to propose a method for constructing the software setup required for investigating thermal effect of superparamagnetic nanoparticles on three human cell lines. This article aimed to examine the required nanoparticle dose, frequency, field intensity and the exposure time. Materials and methods: In the present study, first some general details were given about design and construction of the setup required for generating a safe magnetic field in order to examine the thermal effect of superparamagnetic nanoparticles on three human cancer cell lines, cultured under laboratory conditions. Next, a series of experimental tests were conducted to study the effect of magnetic field, on the cells. Finally, by applying three types of iron-based nanoparticles with mean diameters of 8, 15 and 20 nm, for 30 min, the temperature rise and specific absorption rate (SAR) were calculated. Results: By conducting experimental tests, the maximum temperature rise at the resonance frequency of the coil was reported to be 80 kHz, and it was observed that all the cells died when temperature of the cells reached 42°C/30 min. Based on the experiments, it was observed that magnetic field with intensity of 8 kA/m within the frequency range of 80–180 kHz did not have any effect on the cells. Conclusions: Based on the results, it can be concluded that the nanoparticle dose of 80 µg/ml with diameter of 8 nm at the resonance frequency of coil for 30 min was sufficient to destroy all the cancerous cells in the flask.     

High-efficiency bioaffinity separation of cells and proteins using novel thermoresponsive biotinylated magnetic nanoparticles

Thermoresponsive magnetic nanoparticles with an upper critical solution temperature (UCST) in aqueous solution were synthesized for the first time. Named Therma-Max, the material was synthesized by redox copolymerization of N-acryloyl glycinamide with a monomer form of biotin using methacrylated dextran-magnetite. While the resulting Therma-Max was completely dispersed at temperatures above the UCST (18°C) and could not be separated by a permanent magnet, it was rapidly flocculated when the temperature fell below the UCST and was easily separated by a permanent magnet. The flocculated particles dispersed completely when the temperature was raised to above the UCST. Because biotin was immobilized on the Therma-Max, avidin and antibodies were subsequently immobilized with good efficiency. Furthermore, transiently transfected Arabidopsis protoplasts, which have surface display of CD4 antigen, were efficiently captured and enriched by using a biotinylated anti-CD4 antibody in combination with avidin-conjugated Therma-Max. Also, the silkworm storage protein (SP2) was efficiently separated from the silkworm hemolymph by using biotinylated anti-IgG antibody and anti-SP2 antibody in combination with avidin-conjugated Therma-Max. In both cases, it was confirmed that specificity and adsorption capacity were markedly improved by converting the conventional micro-size fine magnetic particles to nano-size particles. These results show the potential of Therma-Max with a UCST in bioaffinity separation of cells and biomolecules.     

Film analysis of activated sludge microbial discs by the Taguchi method and grey relational analysis

A biofilm model with substrate inhibition is proposed for the activated sludge growing discs of rotating biological contactor (RBC); this model is different from the steady-state biofilm model based on the Monod assumption. Both deep and shallow types of biofilms are examined and discussed. The biofilm models based on both Monod and substrate inhibition (Haldane) assumptions are compared. In addition, the relationships between substrate utilization rate, biofilm thickness, and liquid phase substrate concentration are discussed. The influence order of the factors that affect the biofilm thickness is studied and discussed by combining the Taguchi method and grey relational analysis. In this work, a Taguchi orthogonal table is used to construct the series that is needed for grey relational analysis to determine the influence priority of the four parameters S _B , kX _f , K _s, and K _i .     

Liquid-chromatographic and mass-spectrometric identification of lens proteins using microwave-assisted digestion with trypsin-immobilized magnetic nanoparticles

We used a newly developed method combining trypsin-immobilized magnetic nanoparticles (TIMNs) and microwave-assisted protein digestion to study the proteins of human lens tissue. The digested proteins were identified by liquid chromatography and mass spectrometry. The lens proteins were digested under optimized conditions (digestion time 1 min, microwave power 400 W, trypsin-to-protein ratio 1:5) determined using bovine serum albumin as the standard protein, before liquid-chromatographic and mass-spectrometric analysis. Twenty-six proteins were identified with the new digestion method compared with 11 proteins identified with traditional in-solution digestion (12 h). γ-Crystallin, β-crystallin, and superoxide dismutase 1 proteins, identified with the microwave-assisted method but not the traditional method, are related to cataract development according to some studies. The TIMNs were easily separated from the digestion products. This new digestion method could prove extremely useful for large-scale proteomic analyses.     

An intelligent approach to the discovery of luminescent materials using a combinatorial approach combined with Taguchi methodology

A significant advance made in combinatorial approach research was that the emphasis shifted from simple mixing to intelligent screening, so as to improve the efficiency and accuracy of discovering new materials from a larger number of diverse compositions. In this study, the long‐lasting luminescence of SrAl₂O₄, which is co‐doped with Eu²⁺, Ce³⁺, Dy³⁺, Li⁺ and H₃BO₃, was investigated based on a combinatorial approach in conjunction with the Taguchi method. The minimal number of 16 samples to be tested (five dopants and four levels of concentration) were designed using the Taguchi method. The samples to be screened were synthesized using a parallel combinatorial strategy based on ink‐jetting of precursors into an array of micro‐reactor wells. The relative brightness of luminescence of the different phosphors over a particular period was assessed. Ce³⁺ was identified as the constituent that detrimentally affected long‐lasting luminescence. Its concentration was optimized to zero. Li⁺ had a minor effect on long‐lasting luminescence but the main factors that contributed to the objective property (long‐lasting luminescence) were Eu²⁺, Dy³⁺ and H₃BO₃, and the concentrations of these dopants were optimized to 0.020, 0.030 and 0.300, respectively, for co‐doping into SrAl₂O₄. This study demonstrates that the utility of the combinatorial approach for evaluating the effect of components on an objective property (e.g. phosphorescence) and estimating the expected performance under the optimal conditions can be improved by the Taguchi method. Copyright © 2011 John Wiley & Sons, Ltd.     

Optimization of non-detergent treatment for enveloped virus inactivation using the Taguchi design of experimental methodology (DOE)

Abstract

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness—in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120?min), and temperature (25?°C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.     

Fast and efficient adsorption/desorption of protein by a novel magnetic nano-adsorbent

A novel magnetic nano-adsorbent was prepared by covalently binding polyacrylic acid (PAA) on Fe₃O₄ superparamagnetic nanoparticles (13.2 nm) via carbodiimide activation. The maximum weight ratio of PAA to Fe₃O₄ was 0.12 (i.e., average of two PAA molecules on a magnetic nanoparticle). The magnetic nano-adsorbent possessed a high ionic exchange capacity of 1.64 meq g^–1 and was efficient for the recovery of lysozyme. The lysozyme could be completely adsorbed in 0.1 M phosphate buffer at pH 3–5 and completely desorbed in NaSCN solution (>1 M) within 1 min, and retained 95% activity after adsorption/desorption. In addition, the adsorption behavior followed the Langmuir adsorption isotherm with a maximum adsorption amount of 0.224 mg mg^–1 and a Langmuir adsorption equilibrium constant of 10 ml mg^–1 at 25 °C. The change of enthalpy at 15–35 °C was –4.2 kJ ml mol^–1 mg^–1.     

Efficient labeling of mesenchymal stem cells using cell permeable magnetic nanoparticles

For the purpose of successfully monitoring labeled cells, optimum labeling efficiency without any side effect is a prerequisite. Magnetic cellular imaging is a new and growing field that allows the visualization of implanted cells in vivo. Herein, superparamagnetic iron oxide (SPIO) nanoparticles were conjugated with a non-toxic protein transduction domain (PTD), identified by the authors and termed low molecular weight protamine (LMWP), to generate efficient and non-toxic cell labeling tools. The cells labeled with LMWP-SPIO presented the highest iron content compared to those labeled with naked SPIO and the complex of SPIO with poly-l-lysine, which is currently used as a transfection agent. In addition to the iron content assay, Prussian staining and confocal observation demonstrated the highest intracellular LMWP-SPIO presence, and the labeling procedure did not alter the cell differentiation capacity of mesenchymal stem cells. Taken together, cell permeable magnetic nanoparticles conjugated with LMWP can be suggested as labeling tools for efficient magnetic imaging of transplanted cells.     

Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability

Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe₃O₄ magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g^–1, only slightly lower than that of the naked ones (63 emu g^–1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol^–1, 0.23 mol min^–1 mg^–1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.     

Synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate by immobilized cells using amino acid-modified magnetic nanoparticles

Fe₃O₄-Arg was selected as the optimal carrier due to its high activity recovery of immobilized cells in the preparation of Fe₃O₄-Arg-Cells. The optimal immobilization conditions for the preparation of Fe₃O₄-Arg-Cells were 30 °C, 4 h, pH 7, and 3 g dry yeast. The activity recovery of immobilized cells reached 76.8 %. For a batch reduction in a shaker in an alternating magnetic field, Fe₃O₄-Arg-Cells were used as a catalyst to gain ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE). For further improvement in reduction productivity, a continuous reduction in the magnetic fluidized bed reactor system (MFBRS) was completed. Under their optimal transformation conditions, it took 24 h for Fe₃O₄-Arg-Cells to complete the conversion of ethyl 4-chloro-3-oxobutanoate (COBE) (0.8553 mol/L) in the shaker and only 8 h for the batch reduction in an alternating magnetic field. Continuous reduction in MFBRS provided new ideas for the efficient production of (R)-CHBE; 1.5882 mol/L (10 mL) of COBE can be completely converted in 6 h. The conversion and enantiomeric excess (e.e.) of (R)-CHBE were 100 % and above 99.9 % respectively, in the three reaction systems mentioned above.     

Trichoderma sp. spores and Kluyveromyces marxianus cells magnetic separation: Immobilization on chitosan-coated magnetic nanoparticles

In the present study, the interactions between chitosan-coated magnetic nanoparticles (C-MNP) and Trichoderma sp. spores as well as Kluyveromyces marxianus cells were studied. By Plackett–Burman design, it was demonstrated that factors which directly influenced on yeast cell immobilization and magnetic separation were inoculum and C-MNP quantity, stirring speed, interaction time, and volume of medium, while in the case of fungal spores, the temperature also was disclosed as an influencing factor. Langmuir and Freundlich models were applied for the mathematical analysis of adsorption isotherms at 30°C. For Trichoderma sp. spore adsorption isotherm, the highest correlation coefficient was observed for lineal function of Langmuir model with a maximum adsorption capacity at 5.00E?+?09 spores (C-MNP g^?1). Adsorption isotherm of K. marxianus cells was better adjusted to Freundlich model with a constant (K_f) estimated as 2.05E?+?08 cells (C-MNP g^?1). Both systems may have a novel application in fermentation processes assisted with magnetic separation of biomass.     

Green synthesis of silver nanoparticles using Citrus limon peels and evaluation of their antibacterial and cytotoxic properties

The present work aimed to synthesis silver nanoparticles (AgNPs) using biological waste products Citrus limon peels, its characterization, antimicrobial activities and the cytotoxic effect of the synthesized green AgNPs. Characterization of the prepared AgNPs showed the formation of spherical, and few agglomerated AgNPs forms as measured by UV–visible spectrophotometer. The average size of the prepared AgNPs was 59.74 nm as measured by DLS technique. The spectrum of the synthesized AgNPs was observed at 3 KeV using the EDX. On the other hand, FTIR analysis of the green synthesized AgNPs showed the presence of alcohols, phenolics, mono-substituted alkynes, aliphatic primary amines, sodium salt, amino acid, or SiOH alcohol groups. The antimicrobial studies of the formed AgNPs showed positive activity against most of the studied human pathogenic bacteria with varying degrees. Finally, the evaluation of the cytotoxic effect of the green synthesized AgNPs were done using two types of cell lines, human breast cancer cell line (MCF-7) and human colon carcinoma cell line (HCT-116). The results revealed the concentration has a direct correlation with cell viability. The 50% inhibitory concentration (IC₅₀) of MCF-7 cell line was in of 23.5 ± 0.97 µL/100 µL, whereas the HCT-116 cell line was in 37.48 ± 5.93 µL/100 µL.     

Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays

Biosensors based on antibody recognition have a wide range of monitoring applications that apply to clinical, environmental, homeland security, and food problems. In an effort to improve the limit of detection of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently labeled protein, AlexaFluor647–chicken IgG (Alexa647–chick IgG). Antibody-labeled MNPs (Alexa647–chick–MNPs) were used to preconcentrate the target via magnetic separation and as the tracer to demonstrate binding to slides modified with anti-chicken IgG as a capture agent. A full optimization study of the antibody-modified MNPs and their use in the biosensor was performed. This investigation looked at the Alexa647–chick–MNP composition, MNP surface modifications, target preconcentration conditions, and the effect that magnetic extraction has on the Alexa647–chick–MNP binding with the array surface. The results demonstrate the impact of magnetic extraction using the MNPs labeled with fluorescent proteins both for target preconcentration and for subsequent integration into immunoassays performed under flow conditions for enhanced signal generation.     

Covalent immobilization of alkaline proteinase on amino-functionalized magnetic nanoparticles and application in soy protein hydrolysis

Magnetic nanoparticles (MNPs) were synthesized and surface modified with (3-Aminopropyl)triethoxysilane (APTES). The alkaline proteinase (AP) was covalently immobilized on the APTES-modified MNPs through glutaraldehyde linkage. The resulting AP-loaded MNPs have an average size of 84 nm in aqueous solution, and a magnetization of 40 emu/g, endowing the immobilized enzyme with excellent magnetic responsively and dispersity. The maximum amount of AP and catalytic activity immobilized 1.0 mg MNPs was 120 μg and 25.3 units, respectively. Immobilized AP showed maximum activity at pH 10.0 and 50°C. Compared with free enzyme, the immobilized AP exhibited better storage stability. Moreover, immobilized AP can be reused 10 times and still maintained about 50% of its initial activity. The degree of hydrolysis of soy protein hydrolysates for immobilized AP could reach 19.0%, which was closer to the value of free enzyme. The molecular weight (M.W.) analysis showed that the soy protein was hydrolyzed successfully into small peptides of two main fractions with an average M.W. of 742 and 2126 Da. This study indicated that the immobilized AP could be used to hydrolyze continuously soy protein for potential industry application. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2756, 2019.     

A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3O4@SiO2 magnetic nanoparticles

A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe₃O₄@SiO₂ magnetic nanoparticles (Fe–MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe–MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA‐CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe–MNPs (amine‐functionalized Fe₃O₄@SiO₂ and carboxylic acid‐functionalized Fe₃O₄@SiO₂). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC₅₀) values of the two methods were about 0.024 ng ml^?1 and 0.046 ng ml^?1 respectively and LODs were approximately 0.0002 ng ml^?1 and 0.001 ng ml^?1 respectively. These methods were much more sensitive than that of any traditional enzyme‐linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe–MNPs.