Albumin nanoparticles loaded with hemin as peroxidase mimics for immunoassay

Contemporary immunoassays commonly used in clinical diagnostics mostly utilize enzymes, such as horseradish peroxidase, for signal generation. Numerous research is dedicated to the development of artificial peroxidase-mimicking catalysts with lower cost, high activity, better operational stability, and tunable properties. Herein we synthesized hemin-loaded bovine serum albumin (BSA) nanoparticles and applied them as catalytic labels (nanozymes) in colorimetric immunoassay of anti-tetanus antibodies. Hemin is a key part of the peroxidase catalytic center, possessing peroxidase like-activity. Albumin nanoparticles were loaded with multiple hemin molecules and decorated with Streptococcal protein G. Resulting nanozymes possessed good colloidal stability and allowed for antibody detection in blood serum. The sensitivity of antibody detection was sufficient for the assessment of post-vaccination immunity.

A novel colorimetric immunoassay for sensitive monitoring of ochratoxin A based on an enzyme-controlled citrate-iron(III) chelating system

Biotoxins present in foods represent a major source of contamination. Although different analytical methods had been developed, a new detection method for identification and quantification of these toxins is necessary...

Generation of Rat Monoclonal Antibody to Detect Hydrogen Sulfide and Polysulfides in Biological Samples

Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective H2S measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples. We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM). We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively. The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM. By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 μM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.

Further Advance of Gambierdiscus Species in the Canary Islands, with the First Report of Gambierdiscus belizeanus

Ciguatera Poisoning (CP) is a human food-borne poisoning that has been known since ancient times to be found mainly in tropical and subtropical areas, which occurs when fish or very rarely invertebrates contaminated with ciguatoxins (CTXs) are consumed. The genus of marine benthic dinoflagellates Gambierdiscus produces CTX precursors. The presence of Gambierdiscus species in a region is one indicator of CP risk. The Canary Islands (North Eastern Atlantic Ocean) is an area where CP cases have been reported since 2004. In the present study, samplings for Gambierdiscus cells were conducted in this area during 2016 and 2017. Gambierdiscus cells were isolated and identified as G. australes, G. excentricus, G. caribaeus, and G. belizeanus by molecular analysis. In this study, G. belizeanus is reported for the first time in the Canary Islands. Gambierdiscus isolates were cultured, and the CTX-like toxicity of forty-one strains was evaluated with the neuroblastoma cell-based assay (neuro-2a CBA). G. excentricus exhibited the highest CTX-like toxicity (9.5–2566.7 fg CTX1B equiv. cell−1) followed by G. australes (1.7–452.6.2 fg CTX1B equiv. cell−1). By contrast, the toxicity of G. belizeanus was low (5.6 fg CTX1B equiv. cell−1), and G. caribaeus did not exhibit CTX-like toxicity. In addition, for the G. belizeanus strain, the production of CTXs was evaluated with a colorimetric immunoassay and an electrochemical immunosensor resulting in G. belizeanus producing two types of CTX congeners (CTX1B and CTX3C series congeners) and can contribute to CP in the Canary Islands.

Single-Atom Nanozymes Linked Immunosorbent Assay for Sensitive Detection of Aβ 1-40: A Biomarker of Alzheimer’s Disease

Single-atom nanozymes (SANs) possess unique features of maximum atomic utilization and present highly assembled enzyme-like structure and remarkable enzyme-like activity. By introducing SANs into immunoassay, limitations of ELISA such as low stability of horseradish peroxidase (HRP) can be well addressed, thereby improving the performance of the immunoassays. In this work, we have developed novel Fe-N-C single-atom nanozymes (Fe-Nx SANs) derived from Fe-doped polypyrrole (PPy) nanotube and substituted the enzymes in ELISA kit for enhancing the detection sensitivity of amyloid beta 1-40. Results indicate that the Fe-Nx SANs contain high density of single-atom active sites and comparable enzyme-like properties as HRP, owing to the maximized utilization of Fe atoms and their abundant active sites, which could mimic natural metalloproteases structures. Further designed SAN-linked immunosorbent assay (SAN-LISA) demonstrates the ultralow limit of detection (LOD) of 0.88 pg/mL, much more sensitive than that of commercial ELISA (9.98 pg/mL). The results confirm that the Fe-Nx SANs can serve as a satisfactory replacement of enzyme labels, which show great potential as an ultrasensitive colorimetric immunoassay.