Bovine Serum Albumin Conjugate Research Articles

Preparation of a thioxoimidazolidin-linked sialoside BSA conjugate for the inhibition of influenza virus

A novel thioxoimidazolidin-linked sialoside bovine serum albumin (WM-BSA) conjugate was synthesized and evaluated as an inhibitor of influenza virus hemagglutinin (HA) and neuraminidase (NA). The multivalent conjugate was prepared by the attachment of thioxoimidazolidin-sialoside monomer (WM) to BSA via adipate linker. Surface plasmon resonance analysis revealed that WM-BSA exhibited potent binding to recombinant influenza HA and NA proteins, with dissociation constants in the submicromolar range. WM-BSA also demonstrated inhibitory activities in the low micromolar range against HA and NA proteins from multiple influenza strains. Investigation of cytopathic effects in infected MDCK cells indicated that WM-BSA possessed antiviral activity with EC50 values of 35–55 μM. The multivalent presentation of sialosides on the BSA scaffold significantly enhanced both the binding affinity and degree of inhibition compared to the monomeric compound WM. These results demonstrate the potential of multivalent sialoside-protein conjugate as a platform for developing novel anti-influenza agent.

The resistance of BSA-Dex conjugate nanoemulsions to depletion flocculation induced by excessive dextran in Maillard reaction

Protein nanoemulsions are excellent carriers of nutrients due to their unique surface activity and structural properties, however, poor stability has limited their application. The Maillard reaction can enhance protein emulsification, but an excess of dextran formed during the reaction can trigger detrimental effect. In this study, the influence of unbound dextran following the Maillard reaction on nanoemulsions composed of bovine serum albumin (BSA) and dextran conjugates was studied. The results showed that saturation grafting occurred at a 1:7 M ratio of BSA to dextran during the Maillard reaction. Rheological analysis revealed that the relative amount of difference between the loss modulus (G″) and storage modulus (G′) in the nanoemulsions was most pronounced within the ratio of 1:7–1:11. This difference decreased when the dextran content exceeded 1:11, indicating a reduction in the system’s ability to prevent aggregation. Nanoemulsions within the range of ratios 1:7–1:11 showed no droplet aggregation, particle size, or zeta-potential alterations after 3-months of storage. They remained stable at 90 °C, with NaCl concentrations within 75 mM, and at pH 3–8. Small amounts of free dextran remaining from the Maillard reaction are deemed acceptable in subsequent nanoemulsions preparation. This study offers a scientific foundation for devising cost-effective strategies in industrial production.

Diseleno-albumin, a native bio-inspired drug free therapeutic protein induces apoptosis in lung cancer cells through mitochondrial oxidation

Macromolecular therapeutic is the emerging concept in the fields of drug delivery and drug discovery. The present study reports the design and development of a serum albumin based macromolecular chemotherapeutic by conjugating bovine serum albumin (BSA) with 3,3′-diselenodipropionic acid (DSePA), a pharmacologically active organo-diselenide (R-Se-Se-R). The reaction conditions were optimised to achieve the controlled conjugation of BSA with DSePA without causing any significant alteration in its physico-chemical properties or secondary structure and crosslinking. The chemical characterisation of the reaction product through various spectroscopic techniques viz., FT-IR, Raman, XPS, AAS and MALDI-TOF-MS, established the conjugation of about ∼5 DSePA molecules per BSA molecule. The DSePA conjugated BSA (Se-Se-BSA) showed considerable stability in aqueous and lyophilized forms. The cytotoxicity studies by involving cell lines of cancerous and non-cancerous origins indicated that Se-Se-BSA selectively inhibited the proliferation of cancerous cells. The cellular uptake studies by physically labelling Se-Se-BSA with curcumin and following its intracellular fluorescence confirmed that uptake efficiency of Se-Se-BSA was almost similar to that of native BSA. Finally, studies on the mechanism of action of Se-Se-BSA in the A549 (lung adenocarcinoma) cells revealed that it induced mitochondrial ROS generation followed by mitochondrial dysfunction, activation of caspases and apoptosis. Together, these results demonstrate a bio-inspired approach of exploring diselenide (-Se-Se-) grafted serum albumin as the potential drug free therapeutic for anticancer application.

A Zanamivir-protein conjugate mimicking mucin for trapping influenza virion particles and inhibiting neuraminidase activity

Influenza viruses contribute significantly to the global health burden, necessitating the development of strategies against transmission as well as effective antiviral treatments. The present study reports a biomimetic strategy inspired by the natural antiviral properties of mucins. A bovine serum albumin (BSA) conjugate decorated with the multivalent neuraminidase inhibitor Zanamivir (ZA-BSA) was synthesized using copper-free click chemistry. This synthetic pseudo-mucin exhibited potent neuraminidase inhibitory activity against several influenza strains. Virus capture and growth inhibition assays demonstrated its effective absorption of virion particles and ability to prevent viral infection in nanomolar concentrations. Investigation of the underlying antiviral mechanism of ZA-BSA revealed a dual mode of action, involving disruption of the initial stages of host-cell binding and fusion by inducing viral aggregation, followed by blocking the release of newly assembled virions by targeting neuraminidase activity. Notably, the conjugate also exhibited potent inhibitory activity against Oseltamivir-resistant neuraminidase variant comparable to the monomeric Zanamivir. These findings highlight the application of multivalent drug presentation on protein scaffold to mimic mucin adsorption of viruses, together with counteracting drug resistance. This innovative approach has potential for the creation of antiviral agents against influenza and other viral infections.

Chemoenzymatic synthesis of sialyl-α2,3-lactoside–functionalized BSA conjugate inhibits influenza infection

Influenza remains a global public health threat, and the development of new antivirals is crucial to combat emerging drug-resistant influenza strains. In this study, we report the synthesis and evaluation of a sialyl lactosyl (TS)–bovine serum albumin (BSA) conjugate as a potential multivalent inhibitor of the influenza virus. The key trisaccharide component, TS, was efficiently prepared via a chemoenzymatic approach, followed by conjugation to dibenzocyclooctyne-modified BSA via a strain-promoted azide-alkyne cycloaddition reaction. Biophysical and biochemical assays, including surface plasmon resonance, isothermal titration calorimetry, hemagglutination inhibition, and neuraminidase inhibition, demonstrated the strong binding affinity of TS-BSA to the hemagglutinin (HA) and neuraminidase (NA) proteins of the influenza virus as well as intact virion particles. Notably, TS-BSA exhibited potent inhibitory activity against viral entry and release, preventing cytopathic effects in cell culture. This multivalent presentation strategy highlights the potential of glycocluster-based antivirals for combating influenza and other drug-resistant viral strains.

Altering the competitive environment of B cell epitopes significantly extends the duration of antibody production.

Persistent immunoglobulin G (IgG) production (PIP) provides long-term vaccine protection. While variations in the duration of protection have been observed with vaccines prepared from different pathogens, little is known about the factors that determine PIP. Here, we investigated the impact of three parameters on the duration of anti-peptide IgG production, namely amino acid sequences, protein carriers, and immunization programs. We show that anti-peptide IgG production can be transformed from transient IgG production (TIP) to PIP, by placing short peptides (Pi) containing linear B cell epitopes in different competitive environments using bovine serum albumin (BSA) conjugates instead of the original viral particles. When goats were immunized with the peste des petits ruminants (PPR) live-attenuated vaccine (containing Pi as the constitutive component) and BSA-Pi conjugate, anti-Pi IgG production exhibited TIP (duration < 60 days) and PIP (duration > 368 days), respectively. Further, this PIP was unaffected by subsequent immunization with the PPR live-attenuated vaccine in the same goat. When goats were coimmunized with PPR live-attenuated vaccine and BSA-Pi, the induced anti-Pi IgG production showed a slightly extended TIP (from ~60 days to ~100 days). This discovery provides new perspectives for studying the fate of plasma cells in humoral immune responses and developing peptide vaccines related to linear neutralizing epitopes from various viruses.

Efficient Sequential Detection of Two Antibiotics Using a Fiber-Optic Surface Plasmon Resonance Sensor.

Antibiotic residues have become a worldwide public safety issue. It is vital to detect multiple antibiotics simultaneously using sensors. A new and efficient method is proposed for the combined detection of two antibiotics (enrofloxacin (Enro) and ciprofloxacin (Cip)) in milk using surface plasmon resonance (SPR) sensors. Based on the principle of immunosuppression, two antibiotic antigens (for Enro and Cip) were immobilized on an optical fiber surface with conjugates of bovine serum albumin using dopamine (DA) polymerization. Each single antigen was bound to its corresponding antibody to derive standard curves for Enro and Cip. The fiber-optic sensor's sensitivity was 2900 nm/RIU. Detection limits were calculated to be 1.20 ng/mL for Enro and 0.81 ng/mL for Cip. The actual system's recovery rate was obtained by testing Enro and Cip in milk samples; enrofloxacin's and ciprofloxacin's mean recoveries from the milk samples were 96.46-120.46% and 96.74-126.9%, respectively. In addition, several different regeneration solutions were tested to analyze the two target analytes' regeneration ability; NaOH and Gly-HCl solutions were found to have the best regeneration ability.

Self-assembly of maltose-albumin nanoparticles for efficient targeting delivery and therapy in liver cancer

The effective delivery and targeted release of drugs within tumor cells are critical factors in determining the therapeutic efficacy of nanomedicine. To achieve this objective, a conjugate of maltose (Mal) and bovine serum albumin (BSA) was synthesized by the Maillard reaction and self-assembled into nanoparticles with active-targeting capabilities upon pH/heating induction. This nanoparticle could be effectively loaded with doxorubicin (DOX) to form stable nanodrugs (Mal-BSA/DOX) that were sensitive to low pH or high glutathione (GSH), thereby achieving a rapid drug release (96.82 % within 24 h). In vitro cell experiments indicated that maltose-modified BSA particles efficiently enhance cellular internalization via glucose transporters (GLUT)-mediated endocytosis, resulting in increased intracellular DOX levels and heightened expression of γ-H2AX. Consequently, these results ultimately lead to selective tumor cells death, as evidenced by an IC50 value of 3.83 μg/mL in HepG2 cells compared to 5.87 μg/mL in 293t cells. The efficacy of Mal-BSA/DOX in tumor targeting therapy has been further confirmed by in vivo studies, as it effectively delivered a higher concentration of DOX to tumor tissue. This targeted delivery approach not only reduces the systemic toxicity of DOX but also effectively inhibits tumor growth (TGI, 75.95 %). These findings contribute valuable insights into the advancement of targeting-albumin nanomedicine and further support its potential in tumor treatment.

Targeted treatment of atherosclerosis with protein–polysaccharide nanoemulsion co-loaded with photosensitiser and upconversion nanoparticles

Macrophages are the most abundant cell group in atherosclerosis (AS) lesions and play a vital role in all stages of AS progression. Recent research has shown that reactive oxygen species (ROS) generation from photodynamic therapy (PDT) induces macrophage autophagy to improve abnormal lipid metabolism and inflammatory environment. Especially in macrophage-derived foam cells, which has become a potential strategy for the treatment of AS. In this study, we prepared the conjugate (DB) of dextran (DEX) and bovine serum albumin (BSA). The DB was used as the emulsifier to prepare nanoemulsion loaded with upconversion nanoparticles (UCNPs) and chlorin e6 (Ce6) (UCNPs-Ce6@DB). The DEX modified on the surface of the nanoemulsion can recognise and bind to the scavenger receptor class A (SR-A) highly expressed on macrophages and promote the uptake of macrophage-derived foam cells in AS plates through SR-A-mediated endocytosis. In addition, UCNPs-Ce6@DB-mediated PDT enhanced ROS generation and induced autophagy in macrophage-derived foam cells, enhanced the expression of ABCA1, a protein closely related to cholesterol efflux, and inhibited the secretion of pro-inflammatory cytokines. Ultimately, UCNPs-Ce6@DB was shown to inhibit plaque formation in mouse models of AS. In conclusion, UCNPs-Ce6@DB offers a promising treatment for AS.

Para-Cresyl Sulfate and BSA Conjugation for Developing Aptasensor: Spectroscopic Methods and Molecular Simulation

Para-cresol sulfate (PCS), a protein-bound uremic toxin, is gut microbiota derived metabolite and known for its role in human physiology and pathology. After PCS formation, it goes to blood stream and interact with serum albumin with high affinity. Normally, PCS is cleared by the kidney but if it gets accumulated then leads to chronic kidney disease (CKD). Present study is an attempt to understand the nuances of interaction wherein bovine serum albumin (BSA) has been taken as model protein to understand this interaction. Absorption spectroscopy result indicated that in presence of PCS (ranging from 2 μM to 200 μM), the absorbance of BSA gradually decreased due to complex formation between them and obtained detection limit of 0.42 μM with a linearity of 0.95. Fourier transform infra-red and circular spectroscopy results demonstrated change in secondary structure of BSA upon interaction of PCS. These interaction studies reveal that PCS binds on two sites of BSA. Docking studies represent binding score of −5.6 Kcal mol−1, demarking that PCS is involved in interaction with BSA via amino acid residues, forming the stable complex. This study helps in PCS aptamers synthesis by using this conjugate, to fabricate apta-sensors for early detection of CKD.

Ex vivo fluorescence imaging for the identification of rhodamine-labeled bovine serum albumin and chitosan-coated gold and silver nanoparticles.

Therapeutic potential and toxic effects of in vivo administered gold nanoparticles (GNPs) and silver nanoparticles (SNP) depend on distribution in tissues. Rhodamine (Rho) labeled bovine serum albumin (BSA) and chitosan (Chi) were prepared by covalent conjugation and were characterized by fluorescence spectral analysis. GNP and SNP were coated with the labeled conjugates of BSA and chitosan by adsorption. The soluble Rho-BSA or Rho-Chi conjugates, uncoated, and conjugate-coated GNP, and SNP were orally administered into 8-week-old rats. After 24 h, rats were euthanized and the liver, kidney, spleen, and thymus were dissected. The tissues were examined ex vivo using a small animal in vivo imaging system. The liver, kidney, and thymus displayed higher fluorescence due to increased accumulation of Rho-BSA or Rho-Chi conjugate-coated nanoparticles (NPs) in the tissues as compared to the spleen where lower fluorescence was noticed. Tissues obtained from rats that were administered Rho-BSA or Rho-Chi conjugate-coated GNP and SNP showed tenfold higher fluorescence intensity as compared to tissues from rats that were given soluble conjugates or NP alone. The results strongly suggest significant tissue distribution of NP following oral administration.

Green Synthesis, Characterization, and Potential Biomedical Applications of AgNPs Using Coriander sativum and Olea europaea

Background: Over the last few decades, metallic nanoparticles, especially silver nanoparticles (AgNPs), have gained the focus of researchers globally due to their unique properties and a broad range of applications. Aim: This research study focused on the green synthesis of AgNPs by using Coriandrum sativum, Olea europaea leaf extract, and their bovine serum albumin conjugates. Materials and Methods: Biogenic AgNPs were characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, and scanning electron microscopy analysis. The potential biomedical applications of AgNPs and their conjugates were also evaluated through in vitro assays. AgNPs synthesis was confirmed by observing UV-visible absorption peaks at 380nm, 460nm (AgNPs derived from C.sativum and O. europaea respectively), 580nm, and 577nm (conjugates of particles from C.sativum and O. europaea respectively). Results: FTIR analysis revealed the presence of various functional groups on the surface of AgNPs. The average diameters of C. sativum and O. europaea derived AgNPs were 1025 d.nm and 134 d.nm, whereas the average size of AgNPs was 500nm, 200nm, 100nm, and 300nm with uniform morphology. Results of biomedical activity showed that AgNPs and their albumin conjugates were potential antidiabetic, anti-oxidant, and anti-hyperlipidemic with significant IC50 values compared to standard. The antimicrobial potential of AgNPs and their conjugates were tested against gram-positive and gram-negative bacterial strains and the best zone of inhibition of C. sativum derived conjugated AgNPs was observed against Salmonella enterica i.e. 29 mm. Conclusion: The research project provides an ecofriendly green synthesis method of AgNPs and their conjugates as well as their potential for the treatment of different diseases.

Adjuvant action of needle-shaped BC microfibrils.

Bacterial cellulose (BC) is an unbranched biopolymer produced by microorganisms and composed of glucopyranose units linked by β-1,4 bonds. This study investigates the adjuvant action of needle-shaped BC microfibrils (BCmFs) in vitro using bovine serum albumin (BSA) as a model antigen. BC produced by the static culture of Komagataibacter xylinus was then microparticled (1-5μm) by acid hydrolysis and characterized using Dynamic Light Scattering and Scanning Electron Microscopy. Subsequently, Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy, cytotoxicity, TNF-α (tumour necrosis factor-alpha) and IL-6 (interleukin-6) cytokine secretion, and cellular uptake of the BCmFs-BSA conjugate on the human monocyte cell line (U937) differentiated into macrophages were performed. The microfibrils were determined to be 1-5μm in size, needle-shaped, with a zeta potential of - 32mV. Their conjugation with the model antigen, BSA, was demonstrated by FTIR analysis. In the cytotoxicity assay, BCmFs-BSA in macrophage cells showed high viability (over 70%). Although the highest TNF-α cytokine level (113pg/ml) was obtained with BCmFs-BSA (Bovine serum albumin) conjugate (500µg/ml) and was statistically significant (p = 0.0001) compared to the positive control group (BSA-aluminium hydroxide), IL-6 cytokine levels were not statistically different from those in the control group as desired. It has been shown in macrophage-differentiated U937 cells that microbially synthesized BC in the form of needle-shaped microfibrils (BCmFs) has a high cellular uptake capacity and increases the immunogenicity of the antigen. These results demonstrate for the first time that BCmFs have the potential to serve as a vaccine adjuvant.

Template-Based Porous Hydrogel Microparticles as Carriers for Therapeutic Proteins.

Hydrogels have been extensively researched for over 60 years for their limitless applications in biomedical research. In this study, porous hydrogel microparticles (PHMPs) made of poly(ethylene glycol) diacrylamide were investigated for their potential as a delivery platform for therapeutic proteins. These particles are made using hard calcium carbonate (CaCO3) templates, which can easily be dissolved under acidic conditions. After optimization of the synthesis processes, both CaCO3 templates and PHMPs were characterized using a wide range of techniques. Then, using an array of proteins with different physicochemical properties, the encapsulation efficiency of proteins in PHMPs was evaluated under different conditions. Strategies to enhance protein encapsulation via modulation of particle surface charge to increase electrostatic interactions and conjugation using EDC/NHS chemistry were also investigated. Conjugation of bovine serum albumin to PHMPs showed increased encapsulation and diminished release over time, highlighting the potential of PHMPs as a versatile delivery platform for therapeutic proteins such as enzymes or antibodies.

Amyloid-β/Tau burden and neuroinflammation dual-targeted nanomedicines synergistically restore memory and recognition of Alzheimer’s disease mice

Efficient multi-targeted intervention of complex pathogenic factors may hold promises for Alzheimer's disease (AD) treatment due to the continuous failure of single-targeted therapy in clinics. To this end, we developed two dual-targeted layered double hydroxide (LDH) nanoparticle (NP)-based nanomedicines in this research for simultaneously reducing amyloid β (Aβ)/hyperphosphorylated Tau burden and neuroinflammation. LDH NPs were first loaded with two therapeutics, i.e. Rutin and β-site amyloid precursor protein cleaving enzyme-1 (BACE1) or glycogen synthase kinase 3β (GSK3β) siRNA. The nanomedicines were further functionalized with Ang2 and RVG29 via adsorption of their bovine serum albumin (BSA) conjugates. In APP/PS1 and Tau.P301S AD mouse models, nanomedicines efficiently silenced the target genes and decreased the expression of abnormal Aβ and hyperphosphorylated Tau, respectively. Moreover, the co-delivered Rutin inhibited the Aβ aggregation and normalized the functions of microglia and astrocytes to reduce neuroinflammation, synergistically recovering the memory and cognitive deficits of AD mice, comparable to that of wide type mice. The successful AD treatment using the dual-targeted LDH nanomedicines provides a paradigm for the development of multi-targeted nanomedicines and warrants further study to combat AD with the complex pathogenic mechanisms.

Synthesis and characterization of surface-modified ultrafine titanium dioxide nanoparticles with an antioxidant functionalized biopolymer as a therapeutic agent: Anticancer and antimicrobial evaluation

Surface modified biodegradable protein-based nanoparticles have great potential in pharmaceutical applications. In the present study, we discuss the surface engineering of titanium dioxide nanoparticles (TiO2 NPs) with alpha-lipoic acid (ALA) functionalized bovine serum albumin (BSA) conjugate as a biocompatible therapeutic system. Sol-gel method is used to synthesize TiO2 NPs, which is characterized using ultraviolet (UV)/visible, Fourier transform infrared (FT-IR), X-ray diffraction analysis (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). BSA was functionalized with antioxidant ALA by Steglich esterification, and TiO2 NPs were loaded into this synthesized BSA-ALA conjugate. The finally developed product was studied using various characterization methods like UV-Visible, FT-IR, XRD, SEM-EDX, TEM, TG-DTG, DSC and fluorescence emission analysis. In vitro cytotoxicity of this system was examined by applying the trypan blue exclusion method, and antimicrobial analysis was implemented by well diffusion method against antibiotic-resistant bacterial and fungal strains.

Development of immunohistochemistry for detecting fluvoxamine in rat tissues using newly prepared monoclonal antibody: its precise localization in small intestine, kidney, and liver of rats.

A monoclonal antibody (mAb) was produced against a fluvoxamine (FLV)-bovine serum albumin conjugate that was specific to both the conjugate and free form of FLV. The mAb enabled us to develop an immunohistochemistry (IHC) method for pharmacokinetic analysis of FLV at the cell and tissue levels. We demonstrated that IHC can be used to detect the localization of FLV in the small intestine, kidney, and liver 1 h after drug administration at the cell and tissue levels. Protease digestion is an important factor for obtaining appropriate IHC staining results for localization of drugs. In this study, precise FLV localization could be determined with only 1 h of protease digestion in the kidneys, but in the small intestine and liver, the staining results with two digestive conditions had to be merged. IHC provided new findings, such as (1) nerve cells are likely to uptake more FLV than other cells and tissues; (2) the ability of reabsorption and secretion in the kidney varies depending on the site, and the amount of FLV in the primary urine is regulated downstream of the proximal tubule S3 segment; and (3) some of the FLV is excreted in the bile.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00795-022-00337-6.

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size, shape, and salt concentration.

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose-based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

Simultaneous detection of foodborne pathogenic bacteria in milk by fluorescence immunoassay

Rapid, sensitive and simultaneous detection of multiple bacteria in foodborne is still a major challenge in public health field. Here, a fluorescence immunoassay that can achieve high-throughput detection of three Gram-positive foodborne pathogenic bacteria simultaneously was proposed. Vancomycin and bovine serum albumin conjugate (Van-BSA) was immobilized on a polycarbonate chip to capture three Gram-positive foodborne pathogenic bacteria, Staphylococcus aureus (S. aureus), Bacillus cereus (B. cereus) and Listeria monocytogenes (L. monocytogenes). CdSe/ZnS quantum dot modified antibodies (Ab-QD) were prepared by carbodiimide coupling chemistry. Due to the affinity reaction between antibodies and proteins on the bacterial surface, the simultaneous detection of multiple Gram-positive bacteria was achieved by monitoring the fluorescence signal of quantum dot by a portable microfluidic chip analyzer. Under optimal conditions, low detection limits was 18 CFU/well, 3 CFU/well and 36 CFU/well for S. aureus, B. cereus and L. monocytogenes, respectively. With satisfactory accuracy and precision, the proposed fluorescence immunoassay holds good prospects to detect pathogens in real food samples.

A Novel Lateral Flow Immunochromatographic Assay for Rapid and Simultaneous Detection of Aflatoxin B1 and Zearalenone in Food and Feed Samples Based on Highly Sensitive and Specific Monoclonal Antibodies.

Simultaneous aflatoxin (AFB1) and zearalenone (ZEN) contamination in agro-products have become widespread globally and have a toxic superposition effect. In the present study, we describe a highly sensitive and specific dual lateral flow immunochromatographic assay (dual test strip) for rapid and simultaneous detection of AFB1 and ZEN in food and feed samples based on respective monoclonal antibodies (mAbs). Two immunogens AFB1-BSA (an AFB1 and bovine serum albumin (BSA) conjugate) and ZEN-BSA (a ZEN and BSA conjugate) were synthesized in oximation active ester (OAE) and amino glutaraldehyde (AGA). The molecular binding ratio of AFB1:BSA was 8.64:1, and that of ZEN:BSA was 17.2:1, identified by high-resolution mass spectrometry (HRMS) and an ultraviolet spectrometer (UV). The hybridoma cell lines 2A11, 2F6, and 3G2 for AFB1 and 2B6, 4D9 for ZEN were filtered by an indirect non-competitive enzyme-linked immunosorbent assay (inELISA) and an indirect competitive enzyme-linked immunosorbent assay (icELISA), respectively. As AFB1 mAb 2A11 and ZEN mAb 2B6 had the lowest 50% inhibitive concentration (IC50) and cross-reactivity (CR), they were selected for subsequent experiments. By systematically optimizing the preparation condition of gold nanoparticles (AuNPs), AuNPs-labeled mAbs, and detection condition, the visual limit of detection (LOD) of the dual test strip was 1.0 μg/L for AFB1 and 5.0 μg/L for ZEN, whereas that of the test strip reader was 0.23 μg/L for AFB1 and 1.53 μg/L for ZEN. The high reproducibility and stability of the dual test were verified using mycotoxin-spiked samples. The dual test strips were highly specific and sensitive for AFB1 and ZEN, which were validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Thus, the proposed AFB1 and ZEN dual test strip is suitable for rapid and simultaneous detection of AFB1 and ZEN contamination in food and feed samples.