•We designed and synthesized Chromotrope FB (Chr FB) hapten with an amino group.•A highly sensitive and specific chemiluminescence immunoassay (CLIA) for Chr FB was developed.•The CLIA method is more sensitive than ELISA and HPLA method.•The method could be successfully used in rapid detection of Chr FB in yoghurt hard candy, vitamin drink and bread.Chromotrope FB (Chr FB) is a synthetic azo dye permitted for use in foods and medicines. An acceptable daily intake (ADI) of Chr FB was 0–0.5 mg/kg in China. In this study, we synthesized a Chr FB hapten with an amino group to prepare its artificial immunogen. Polyclonal antibodies obtained from New Zealand rabbits were applied to develop an indirect competitive chemiluminescent immunoassay (icCLIA) to detect Chr FB in foods. A horseradish peroxidase (HRP)-luminol-H2O2 system was used to yield CL signal with p-iodophenol as an enhancement reagent. The method showed good specificity towards Chr FB and could detect as low as 0.02 ng mL−1 Chr FB in buffer, 0.07 ng g−1 in yoghurt candy, 0.07 ng g−1 in vitamin drink and 0.13 ng g−1 in bread. Compared with HPLC method, the proposed method is more sensitive by two orders of magnitude. The accuracy and precision of this method are acceptable and comparable with HPLC method. Therefore, the proposed method could be used for rapid screening of Chr FB in the mentioned foodstuffs.

In this research, we developed a multianalyte fluorescence sensing system through a carbon dots (CDs)-based fluorescent probe that can specifically recognize Fe(III) by fluorescence quenching. The CDs prepared using black tea by a hydrothermal method show outstanding properties like low cytotoxicity, high photostability, excellent biocompatibility, and high sensitivity. It was found that the fluorescence of CDs can be quenched by micromolar concentrations of Fe(III) in both aqueous solutions as well as living cells. It is well known that glucose can be oxidized by glucose oxidase (GOx) to release H2O2, which, in turn, can oxidize Fe(II) to Fe(III). Based on this consideration, a multianalyte sensing system was established. Therefore the quantitative analysis of Fe(III), H2O2, and glucose with detection limits of 0.25 μM, 0.82 μM, and 1.71 μM, respectively, was achieved by the simple and cost-effective multianalyte CDs sensing system constructed. The sensing system showed high photostability and negligible cytotoxicity toward HeLa cells, which enables it to be applied in the visualization of Fe(III) or H2O2 in living cells. The system was further applied in the detection of Fe(III) or glucose in human serum, and satisfactory results were obtained.

•Enantioselective immunogen and S-amlodipine specific antibody were prepared.•A magnetism based immunosensor was assembled.•S, R-amlodipine could be purely separated and collected from racemic amlodipine.•Captured S-amlodipine could be accurately quantitated with a LOD of 0.04 ng mL−1.•The magnetism based immunosensor is simple, reliable and multi-functional.Amlodipine is one of the most important anti-hypertension agents and S-amlodipine is 1000 folds more efficient than R-amlodipine. However, chiral separation of amlodipine remains a challenge. Here we present an effective strategy for chiral resolution of amlodipine using anti-S-amlodipine antibody for ultra-specific capture and magnetic beads for convenient collecting. The collected enantiomers are ultra-pure based on the chiral HPLC identification. Simultaneously, the captured S-amlodipine could be quantitatively detected with a linear range from 0.1 to 1000 ng mL−1 (R2 = 0.9937), along with a limit of detection of 0.04 ng mL−1. The advantages of the developed magnetic electrochemical biosensor for amlodipine include specific chiral separation, sensitive quantitation, easy-to-operate procedures and time-saving protocols. This method is demonstrated as a novel tool for highly confident separation and quantification of amlodipine enantiomers, in this study. The magnetic biosensor may open up new avenues for intensive study of amlodipine enantiomers and thus be potential in clinical pharmacy and pharmacokinetics studies.Download high-res image (86KB)Download full-size image

Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL–1 SAH with little cross-reaction (3.8%) to SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine, and 5-Aza-2′-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 μM, and 0.48 μM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNA MTase activity and screening its inhibitors.

In this study, a specific antibody against sirolimus was produced by its well synthesized immunogen and a competitive chemiluminescence immunoassay (CLIA) was established. The sirolimus molecule was modified with succinic anhydride (hapten 1) and carboxymethoxylamine (hapten 2). Hapten 1 was conjugated with bovine serum albumin to serve as the immunogen whilst hapten 2 was conjugated with ovalbumin for the coating antigen. A polyclonal antibody was prepared by immunizing rabbits with the purified immunogen. A CLIA method was established using the antibody for the analysis of sirolimus in phosphate-buffered saline and plasma. The limits of detection of the method in the two samples could reach 0.20 and 0.25 ng mL−1, respectively. Furthermore, this immunoassay was successfully applied to the pharmacokinetic study of sirolimus and the calculation of its pharmacokinetic parameters. The results suggest that this immunoassay has significant potential to be developed as a kit which can provide a sensitive, specific and inexpensive approach to therapeutic drug monitoring of sirolimus.

For routine monitoring of the pharmacokinetic behavior of anticancer drug methotrexate (MTX), polyclonal antibodies for MTX were originally produced, and a sensitive chemiluminescence immunoassay (CLIA) was developed for the determination of plasma MTX. Three kinds of coupling reagents (EDC, CDI and isobutyl chloroformate) were utilized to synthesize MTX immunogens. The coupling ratio, titer and sensitivity of polyclonal antibodies for each immunogen were evaluated. Consequently, MTX–EDC–cBSA was found to be the optimal immunogen since it showed the highest coupling ratio and yielded antibodies with the highest sensitivity. Under optimal conditions, the developed CLIA showed a limit of detection (LOD) of 4.3 ng mL−1 in buffer and 9.1 ng mL−1 in plasma with acceptable coefficients of variations (<14.9%). The method exhibited no cross-reaction with the MTX metabolite (7-OH MTX) and structural analogs. When applied in a pharmacokinetic study, the CLIA results were statistically consistent with the HPLC method in measuring key pharmacokinetic parameters (t1/2, Cmax, AUC0–12 and MRT0–12). In conclusion, the CLIA method showed advantages of simple sample preparation, low cost, high sensitivity and good reproducibility. These properties make it a potential tool in the rapid detection of MTX for therapeutic drug monitoring (TDM).

•A 2p-HF-LPME based chiral SFC for 1,4-DHP was developed.•Several 1,4-DHP enantiomers could be well separated.•The method is rapid, practical, effective and environmentally friendly.Chiral separation of seven commonly used 1,4-dihydropyridines (1,4-DHP) was achieved by supercritical fluid chromatography (SFC) on a immobilised polysaccharide chiral selectors coated with cellulose-tris(3,5-dichlorophenylcarbamate) (Chiralpak IC). In this method, isopropanol was used as a modifier and a maximum resolution of 13.38 was resulted. Nimodipine content of actual samples was determined through two-phase hollow fibre-based liquid-phase microextraction (2p-HF-LPME). Under optimal conditions, the limits of detection of the two nimodipine enantiomers were 0.3 and 0.5 μg cm−3. Recoveries of 80.0–99.8% were achieved. The developed SFC technique coupled with 2p-HF-LPME is a rapid, effective and environment-friendly method for separating and quantifying 1,4-DHP enantiomers.

As one of the rarely allowable azo dyes, ponceau 4R can be added in some foods in some countries. However, it is necessary to develop a credible and rapid analytical method for its monitoring, because of its potentially harmful risk. The hapten of ponceau 4R was first designed and synthesized by introducing a primary amine group into the structure of ponceau 4R. Based on the well-prepared hapten, the immunogen of ponceau 4R was prepared using glutaraldehyde to link ponceau 4R to the carrier protein. The triggered polyclonal antibody was obtained and tested by ELISA to optimize the proper dilution. An icELISA was developed for ponceau 4R, and the IC50 of the method is 36.82 ng/mL. The limit of detection is 0.80 ng/mL, and the linear range is 1–10000 ng/mL. Five selected structural analogues have no cross-reactivity with the anti-ponceau 4R polyclonal antibody (<0.3). In three food samples (grape juice, carbonated beverage, and RIO cocktail), the assay exhibits good stability and reproducibility with a recovery range of 93.87–103.77%, and the intra- and interassay coefficients of variation were <11.73%. The results indicate that the proposed icELISA is sensitive, accurate, specific, and simple, which provides an alternative for the detection of ponceau 4R in foods.

•(E)-4,4'-(diazene-1,2-diyl)dibenzoic acid was selected as the hapten of benzoic acid.•The immunogen of benzoic acid was prepared by conjugating the hapten with protein.•A useful antibody against a small antigen – benzoic acid was obtained.•A homogeneous, simple and rapid FPIA was developed for sodium benzoate.A rapid and sensitive fluorescence polarization immunoassay (FPIA), based on a polyclonal antibody, has been developed for the detection of sodium benzoate in spiked samples. The immunogen and fluorescein-labeled analyte conjugate were successfully synthesized, and the tracer was purified by TLC. Under the optimal assay conditions, the FPIA shows a detection range of 0.3–20.0 μg mL−1 for sodium benzoate with a detection limit of 0.26 μg mL−1 in the borate buffer. In addition, the IC50 value was 2.48 μg mL−1, and the cross-reactivity of the antibodies with ten structurally and functionally related analogs were detected respectively. Four kinds of food samples (energy drink, candy, ice sucker, RIOTM cocktail) were selected to evaluate the application of FPIA in real systems. The recoveries were 96.68–106.55% in energy drink; 95.78–100.80% in candy, 86.97–102.70% in ice sucker, and 103.58–109.87% in benzoate contained sample RIOTM cocktail, and coefficients of variation of this method were all lower than 11.25%. Comparing with the detection results of HPLC, the developed FPIA has comparative performance in the real sample determination. The results suggest that the FPIA developed in this study is a rapid, convenient and simple method, which is suitable to be used as a screening tool for homogeneous detection of sodium benzoate in food products.

Amaranth (E 123) is a member of azo dyes, and it is allowed to use in various foods. The acceptable maximum addition of amaranth is strictly fixed because of its potential risk to physical health. The objective of this study was to prepare a specific anti-amaranth monoclonal antibody and develop an indirect competitive ELISA for amaranth quantification analysis. The immunogen and the coating antigen were designed by introducing a carboxyl group into amaranth for the conjugation with carrier proteins. Based on the immunogen, the monoclonal antibody exhibits satisfactory performances and the proposed ELISA shows an IC50 of 20.33 ng mL−1. The limit of detection is as low as 3.35 ng mL−1, and the linear standard curve of the method ranges from 3.0 to 243.0 ng mL−1. Additionally, the antibody reflects minimal cross-reactivity (<1 %) with six related food dyes (erythrosine, ponceau 4R, allura red, tartrazine, sunset yellow FCF, and brilliant blue). The recoveries of amaranth spiked beverage samples are in the range of 85.8–100.7 % with low coefficient of variation values (<11.5 %). The data shows that the developed ELISA provides a simple, sensitive, specific, and accurate alternative for amaranth determination and monitoring. Furthermore, it is the first time that icELISA of amaranth is developed based on monoclonal antibodies.

As a synthetic food dye, erythrosine is associated with serious toxicity in inducing thyroid tumors, and the use of erythrosine is strictly regulated in most counties including China. In this study, a direct enzyme-linked immunosorbent assay (ELISA) has been developed for analysis of erythrosine in food products. A highly specific monoclonal antibody (MAb) for erythrosine was produced using erythrosine–bovine serum albumin (BSA) conjugate as an artificial antigen, and horseradish peroxidase (HRP)-labeled MAb for erythrosine was utilized as the detection antibody. Under the optimized condition, the UV absorbance in microplate related well with erythrosine concentration in the range of 0.1–10.0 μg/g. The proposed method could be applied to determine erythrosine in beverage and cookie, with good recoveries (80 –103 %) for the three spiked levels (30, 50, and 100 μg/g), and the relative standard deviations of detected amount were <12.3 %.