Nanobodies for Accurate Recognition of Iso-tenuazonic Acid and Development of Sensitive Immunoassay for Contaminant Detection in Foods.

The accurate analysis of chemical isomers plays an important role in the study of their different toxic effects and targeted detection of pollutant isomers in foods. The Alternaria mycotoxins tenuazonic acid (TeA) and iso-tenuazonic acid (ITeA) are two isomer mycotoxins with the lack of single analysis methods due to the similar structures. Antibody-based immunoassays exhibit high sensitivity and superior application in isomer-specific determination. Previously, various kinds of antibodies for TeA have been prepared in our group. Herein, highly specific nanobodies (Nbs) against ITeA mycotoxin were selected from immune nanobody phage display library, and one of Nbs, namely Nb(B3G3) exhibited excellent affinity, thermal stability as well as organic solvent tolerance. By molecular simulation and docking technology, it was found that stronger interaction between Nb(B3G3) and ITeA lead to higher affinity than that for its isomer TeA. Furthermore, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was established with a limit of detection (LOD) of 0.09 ng/mL for ITeA mycotoxin. The recovery rate of ITeA in spiked samples was analyzed with 84.8%-89.5% for rice, 78.3%-96.3% for flour, and 79.5%-90.7% for bread. A conventional LC-MS/MS method was used to evaluate the accuracy of this proposed icELISA, which showed a satisfactory consistent correlation. Since the convenient strategy for nanobody generation by phage display technology, this study provide new biorecognition elements and sensitive immunoassay for analysis of ITeA in foods.

The Most Active Oxidase-Mimicking Mn2 O3 Nanozyme for Biosensor Signal Generation.

Oxidase-mimicking nanozymes are more desirable than peroxidase-mimicking ones since H2 O2 can be omitted. However, only a few nanomaterials are known for oxidase-like activities. In this work, we compared the activity of Mn2 O3 , Mn3 O4 and MnO2 and found that Mn2 O3 had the highest oxidase activity. Interestingly, the activity of Mn2 O3 was even inhibited by H2 O2 . The oxidase-like activity of Mn2 O3 was not much affected by the presence of proteins such as bovine serum albumin (BSA), but the physisorption of antibodies to Mn2 O3 was not strong enough to withstand the displacement by BSA. We then treated Mn2 O3 with 3-aminopropyltriethoxysilane to graft an amine group, which was used to conjugate antibodies using glutaraldehyde as a crosslinker. A one-step indirect competitive ELISA (icELISA) was developed for the detection of isocarbophos, and an IC50 of 261.7 ng/mL was obtained, comparable with the results of the standard two-step assay using horseradish peroxidase (HRP)-labeled antibodies. This assay has the advantage of significant timesaving for rapid detection of large amounts of samples. This work has discovered a highly efficient oxidase-mimicking nanozyme useful for various nano- and analytical applications.

Visual upconversion nanoparticle-based immunochromatographic assay for the semi-quantitative detection of sibutramine.

Immunochromatographic assay (ICA) has been used widely for the onsite monitoring of illegal additives due to its simplicity, speed, and low cost. However, a scanner is commonly required for ICA to achieve quantitative results. In this work, we developed a visual semi-quantitative ICA for sibutramine, a banned additive in diet foods, without the need for a scanner for measurement. Monoclonal antibodies specific for sibutramine were raised and conjugated with upconversion nanoparticles (UCNPs) as the luminescent tracer. ICA was developed by employing multiple test lines to achieve the semi-quantitative detection of sibutramine. Based on the optimal conditions, the cutoff levels (limit of quantitation, LOQ) of T1 line, T2 line, T3 line, and T4 line were 0.02 µg/mL, 0.15 µg/mL, 1.0 µg/mL, and 7.5 µg/mL, respectively, in buffer system. The ICA demonstrated a LOQ at 0.2 mg/kg for sibutramine in diet food samples. The assay (including pretreatment) can be finished within 30 min without the aid of other instruments, except a laser pen. No false positive or false negative results were observed. The results indicated that the proposed method was reliable, simple, and rapid for the screening of sibutramine abuse in diet food samples.

A rapid and simple fluorescence enzyme-linked immunosorbent assay for tetrabromobisphenol A in soil samples based on a bifunctional fusion protein.

Tetrabromobisphenol A (TBBPA) is the largest brominated flame retardant which can be released to environment and cause long-term hazard. In this work, we developed a rapid and highly sensitive fluorescence enzyme-linked immunosorbent assay (FELISA) for monitoring of TBBPA in soil samples. TBBPA specific nanobody derived from camelid was fused with alkaline phosphatase to obtain the bi-functional fusion protein, which enable the specific binding of TBBPA and the generation of detection signal simultaneously. The assay showed an IC50 of 0.23 ng g-1, limit detection of 0.05 ng g-1 and linear range from 0.1 to 0.55 ng g-1 for TBBPA in soil samples. Due to the high resistance to organic solvents of the fusion protein, a simple pre-treatment by using 40% dimethyl sulfoxide (DMSO) as extract solvent can eliminate matrix effect and obtain good recoveries (ranging from 93.4% to 112.4%) for spiked soil samples. Good relationship between the results of the proposed FELISA and that of liquid chromatography tandem mass spectrometry (LC-MS/MS) was obtained, which indicated it could be a powerful analytical tool for determination of TBBPA to monitor human and environmental exposure.

The Constipation-Relieving Property of d-Tagatose by Modulating the Composition of Gut Microbiota.

d-tagatose, a monosaccharide as well as a dietary supplement, has been reported as having a wide range of applicability in the food industry, however, the prebiotic activity, anticonstipation effects, and related mechanisms are still unclear. In this study, using the loperamide-induced constipation Kunming mice as the animal model, the effects of d-tagatose for the prevention of constipation were evaluated by gastrointestinal transit experiment and defecation experiment. Furthermore, the underlying mechanism was clarified by evaluating the change of the biochemical indicators and analyzing 16S rRNA amplicon of gut microbiota among the different mice groups. The results showed that the gastrointestinal transit rate, fecal number, and weight in six hours were significantly enhanced after the administration of d-tagatose. In addition, d-tagatose significantly increased the serum levels of acetylcholine (Ach) and substance P (SP), whereas the serum levels of nitric oxide (NO) were significantly decreased. Moreover, the 16S rRNA sequencing analysis revealed that the changes in the gut microbiota caused by constipation were restored by d-tagatose treatment. In conclusion, this study indicated that the administration of d-tagatose as a dietary supplement can effectively prevent and relieve constipation in Kunming mice, and it is a promising prebiotic candidate with constipation-relieving properties.

Production of Antigen-Binding Fragment against O,O-Diethyl Organophosphorus Pesticides and Molecular Dynamics Simulations of Antibody Recognition.

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.

Physicochemical Properties of Bovine Serum Albumin-Glucose and Bovine Serum Albumin-Mannose Conjugates Prepared by Pulsed Electric Fields Treatment.

The pulsed electric fields (PEF) treatment is a novel method for obtaining glycated proteins by way of a Maillard reaction between proteins and polysaccharides but its effect on the preparation of protein-monosaccharide conjugate has not been explored. This study aimed to prepare bovine serum albumin (BSA)-glucose and BSA-mannose conjugates using PEF in pH 10.0 at an intensity of 10 or 20 kV/cm, frequency of 1 kHz, pulse width of 20 µs and 73.5 pulses. The conjugates were evaluated for physicochemical properties. The results indicated that PEF not only promoted Maillard reaction between BSA and glucose or mannose but also alleviated the undesirable browning. PEF treatment favored the increased surface hydrophobicity and emulsifying activity in BSA but reduced surface hydrophobicity and foaming stability and improved foaming capacity in BSA-glucose and BSA-mannose conjugates. These findings provided useful considerations in the application of PEF treatment as a potential method to prepare BSA-monosaccharide conjugates by Maillard reaction.

Nutritional Composition and Antioxidant Properties of the Fruits of a Chinese Wild Passiflora foetida.

The aim of this work was to evaluate the main nutrients and their antioxidant properties of a Chinese wild edible fruit, Passiflora foetida, collected from the ecoregion of Hainan province, China. The analytical results revealed that P. foetida fruits were rich in amino acids (1097 mg/100 g in total), minerals (595.75 mg/100 g in total), and unsaturated fatty acids (74.18 g/100 g in total fat). The lyophilized powder of edible portion contained the higher polyphenols content than the inedible portion powder. The UPLC-Q-TOF-MSE analysis of the extractable and non-extractable phenolics indicated the presence of 65 compounds including 39 free phenolics, 14 insoluble-glycoside-phenolics, and 22 insoluble-ester-phenolics. In addition, the non-extractable phenolics obtained by alkali hydrolysis showed significant antioxidant activities by/through DPPH and ABTS radical scavenging. These findings of P. foetida fruits, for the first time, suggest that these polyphenol-rich fruits may have potential nutraceutical efficacies.

Electronic halocyclization and radical haloazidation of benzene-linked 1,7-dienes for the synthesis of functionalized 3,1-benzoxazines.

A novel electronic halocyclization and radical haloazidation of benzene-linked 1,7-dienes for the formation of functionalized 3,1-benzoxazines has been achieved by using TMSN3 as an azido source and NBS as a halogen source. This methodology is highlighted by its mild conditions and wide substrate scope, which concomitantly introduces one C-N and two C-halogen bonds into one molecule.

Catalytic topological insulator Bi2Se3 nanoparticles for invivo protection against ionizing radiation.

Bi2Se3 nanoparticles (NPs) have attracted wide interests in biological and medical applications. Layer-like Bi2Se3 with high active surface area is promising for free radical scavenging. Here, we extended the medical applications of Bi2Se3 NPs further to in vivo protection against ionizing radiation based on their superior antioxidant activities and electrocatalytic properties. It was found that Bi2Se3 NPs can significantly increase the surviving fraction of mice after exposure of high-energy radiation of gamma ray. Additionally, the Bi2Se3 NPs can help to recover radiation-lowered red blood cell counts, white blood cell counts and platelet levels. Further investigations revealed that Bi2Se3 NPs behaved as functional free radical scavengers and significantly decreased the level of methylenedioxyamphetamine. In vivo toxicity studies showed that Bi2Se3 NPs did not cause significant side effects in panels of blood chemistry, clinical biochemistry and pathology.

Storage of gold nanoclusters in muscle leads to their biphasic in vivo clearance.

Ultrasmall gold nanoclusters (Au NCs) show great potential in biomedical applications. Long-term biodistribution, retention, toxicity, and pharmacokinetics profiles are pre-requisites in their potential clinical applications. Here, the biodistribution, clearance, and toxicity of one widely used Au NC species-glutathione-protected Au NCs or GSH-Au NCs-are systematically investigated over a relatively long period of 90 days in mice. Most of the Au NCs are cleared at 30 days post injection (p.i.) with a major accumulation in liver and kidney. However, it is surprising that an abnormal increase of the Au amount in the heart, liver, spleen, lung, and testis is observed at 60 and 90 days p.i., indicating that the injected Au NCs form a V-shaped time-dependent distribution profile in various organs. Further investigations reveal that Au NCs are steadily accumulating in the muscle in the first 30 days p.i., and the as-stored Au NCs gradually release into the blood in 30-90 days p.i., which induces a re-distribution and re-accumulation of Au NCs in all blood-rich organs. Further hematology and biochemistry studies show that the re-accumulation of Au NCs still causes some liver toxicity at 30 days p.i. The muscle storage and subsequent release may give rise to the potential accumulation and toxicity risk of functional nanomaterials over long periods of time.

Preparative resolution of gatifloxacin enantiomers with pre-column esterification strategy and comparing their enantioselectivity to bacteria and antibody.

Gatifloxacin (GFX) is a kind of chiral fluoroquinolones compound due to the methyl group at the C-3 position of the piperazine ring[1]. Although the enantiomers of GFX show similar levels of antimicrobial activity and pharmacokinetics[2], the other biological activities (i.e., toxicity or enantioselective recognition to various receptors in vivo) of GFX enantiomers have not yet been studied. With this in mind, we developed a rapid and cost-effective high performance liquid chromatographic (HPLC) separation procedure for GFX enantiomers with a pre-column esterification strategy. With significant enhancement of drug solubility and optimization for chromatographic conditions, the proposed method was scaled up to preparative HPLC to obtain optical active S-(-)- and R-(+)-GFX. The antibacterial activities of GFX enantiomers after preparative separation were further verified by measuring the Minimum Inhibitory Concentration (MIC) values against Escherichia coli ATCC 25922. In addition, the binding selectivity of GFX enantiomers to protein receptor were evaluated by antibody using enzyme-linked immunosorbent assay (ELISA) for the first time.

Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris.

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons encoding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L⁻¹ in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL⁻¹, respectively.

Optimization of the extraction of anthocyanins from the fruit skin of Rhodomyrtus tomentosa (Ait.) Hassk and identification of anthocyanins in the extract using High-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS).

Anthocyanins are naturally occurring polyphenols that impart bright color to fruits, vegetables and plants. In this study, the extraction of anthocyanins from freeze-dried fruit skin of downy rose-myrtle (Rhodomyrtus tomentosa (Ait.) Hassk var. Gangren) was optimized using response surface methodology (RSM). Using 60% ethanol containing 0.1% (v/v) hydrochloric acid as extraction solvent, the optimal conditions for maximum yields of anthocyanin (4.358 ± 0.045 mg/g) were 15.7:1 (v/w) liquid to solid ratio, 64.38 °C with a 116.88 min extraction time. The results showed good fits with the proposed model for the anthocyanin extraction (R(2) = 0.9944). Furthermore, the results of high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis of the anthocyanins extracted from the fruit skin of downy rose-myrtle revealed the presence of five anthocyanin components, which were tentatively identified as delphinidin-3-glucoside, cyanidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside and malvidin-3-glucoside.

Ultrasensitive and rapid colorimetric detection of paraquat via a high specific VHH nanobody.

Rapid and quantitative detection of paraquat is crucial because of its high toxicity. Here, we developed an ultrasensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip based on our synthesized variable domain of heavy chain antibody (VHH, also called Nanobody) for paraquat detection. Briefly, the specific immunogen selected from six designed antigens was employed to immunize alpaca, and a high-efficiency capacity of 1.6 × 1013 pfu mL-1 phage display nanobody library was established for biopanning against paraquat. The selected nanobody exhibited high sensitivity (limit of detection (LOD) was 0.0090 ng mL-1 and IC50 was 0.0588 ng mL-1 in buffer) and stability to high temperatures and denaturants. The molecular docking results indicated that the π-π, cation-π, and hydrogen bond interactions between paraquat and the pocket-like structures of complementarity-determining regions (CDRs) in VHH played a critical role in the antibody-paraquat recognition, competition, and affinity processes. The constructed TRFICA recognized paraquat through a quantitative analysis using the strip reader, and showed no cross-reactivity with other herbicides, and a semi-quantitative analysis using the naked eye. Notably, the potential practical applications of the TRFICA evaluated by performing a quantitative analysis of paraquat in food samples (vegetables, fruits, and grain products) and biological samples (blood and urine) showed a recovery rate range between 76.7% and 133.3% with inter-assay coefficient variation lower than 18.5%. The nanobody from phage display libraries was effective for small molecule recognition and detection, and it is a vital tool for immunoassay.

The Simultaneous Determination of Chlorpyrifos-Ethyl and -Methyl with a New Format of Fluorescence-Based Immunochromatographic Assay.

The improper and excessive use in agriculture of chlorpyrifos-methyl (CPSM) and chlorpyrifos-ethyl (CPSE) may affect the health of human beings. Herein, a fluorescence-based immunochromatographic assay (FICA) was developed for the simultaneous determination of CPSM and CPSE. A monoclonal antibody (mAb) with equal recognition of CPSM and CPSE was generated by the careful designing of haptens and screening of hybridoma cells. Instead of labeling fluorescence with mAb, the probe was labeled with goat-anti-mouse IgG (GAM-IgG) and pre-incubated with mAb in the sample. The complex could compete with CPS by coating antigen in the test line. The new format of FICA used goat-anti-rabbit IgG (GAR-IgG) conjugated with rabbit IgG labeled with fluorescence microspheres as an independent quality control line (C line). The novel strategy significantly reduced nonspecific reactions and increased assay sensitivity. Under the optimal conditions, the proposed FICA showed a linear range of 0.015-64 mg/L and limit of detection (LOD) of 0.015 mg/L for both CPSE and CPSM. The average recoveries of CPS from spiked food samples by FICA were 82.0-110.0%. The accuracy was similar to the gas chromatography-tandem mass spectrometry (GC-MS/MS) results. The developed FICA was an ideal on-site tool for rapid screening of CPS residues in foods.

Conformational changes of hapten-protein conjugates resulting in improved broad-specificity and sensitivity of an ELISA for organophosphorus pesticides.

The type of hapten linkage to a carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon and a carboxamido spacer). These haptens were conjugated to bovine serum albumin (BSA) and used as immunogens to produce broad-specificity monoclonal antibodies (MAbs) to organophosphorus pesticides (OPs). Three-dimensional (3D) structures of hapten-lysine conjugates were optimized using molecular modeling (MM) to mimic conformations of hapten-BSA conjugates. The results from MM studies revealed a change of the 3D conformation and electrostatic potential of hapten 1 when the monocarboxylic acid linker was coupled to lysine. This result was consistent with the observed high-cross-reactivity of the corresponding MAb-H1 for the OPs. The competitive indirect enzyme-linked immunosorbent assay based on MAb-H1 is ideally suited to be used as a screening method for OP contaminants.

Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder.

A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3',6'-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL(-1) and the IC(50) (50% inhibition) value was 164.7 ng mL(-1). The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography-mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure.

Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples.

The development of easy-to-use and rapid-monitoring immunoassay methods for organic environmental pollutants in a class-selective manner is a topic of considerable environmental interest. In this work, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) based on a monoclonal antibody (MAb) with broad-specificity for organophosphorus pesticides (OPs) was applied to the detection of O,O-diethyl and O,O-dimethyl OPs in water samples. The ciELISA conditions were carefully optimized to obtain a three to five-fold improvement of sensitivity for most OPs, and thirteen OPs were determined at concentrations ranging from 0.017 to 30 ng mL(-1). The determination of spiked environmental water samples showed average recoveries from 81.5% to 115.1%, with the coefficient of variation (CV) ranging from 6.1% to 20.9%, which showed satisfactory reproducibility of the developed ciELISA. To overcome the negative aspect of broad-specificity immunoassays not providing qualitative and quantitative analysis of individual OPs in blind samples, we used "percent inhibition rate" to make the developed ciELISA a semi-quantitative method, which allows the monitoring of positive samples from hundreds of negative samples. The determination of OPs in blind water samples by the developed ELISA with confirmation by HPLC-MS/MS analysis demonstrated that the assay is ideally suited as a screening method for OP residues prior to chromatographic analysis.

First-principles investigation of Ag-doped gold nanoclusters.

Gold nanoclusters have the tunable optical absorption property, and are promising for cancer cell imaging, photothermal therapy and radiotherapy. First-principle is a very powerful tool for design of novel materials. In the present work, structural properties, band gap engineering and tunable optical properties of Ag-doped gold clusters have been calculated using density functional theory. The electronic structure of a stable Au(20) cluster can be modulated by incorporating Ag, and the HOMO-LUMO gap of Au(20-) (n)Ag(n) clusters is modulated due to the incorporation of Ag electronic states in the HOMO and LUMO. Furthermore, the results of the imaginary part of the dielectric function indicate that the optical transition of gold clusters is concentration-dependent and the optical transition between HOMO and LUMO shifts to the low energy range as the Ag atom increases. These calculated results are helpful for the design of gold cluster-based biomaterials, and will be of interest in the fields of radiation medicine, biophysics and nanoscience.