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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 0-0.5mg/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-H 2 O 2 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 0.02ngmL -1 Chr FB in buffer, 0.07ngg -1 in yoghurt candy, 0.07ngg -1 in vitamin drink and 0.13ngg -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., (Copyright © 2016. Published by Elsevier B.V.)

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, we prepared polyclonal antibodies against anti-diabetic drug nateglinide (NTG), and established a sensitive chemiluminescent immunoassay (CLIA) to detect NTG in tablets and serum. Two kinds of immunogens were synthesized using ethylcarbodiimide (EDC)/hydroxysuccinimide (NHS) and carbonyldiimidazole (CDI)/4-dimethylaminopyridine (DMAP) as coupling reagents respectively. When activated by EDC/NHS, more molecules of NTG coupled with carrier protein in immunogens. A horseradish peroxidase (HRP)-luminol-H2O2 system with p-iodophenol enhancement was applied in the CLIA analysis. The antibodies in EDC/NHS group showed higher titer, sensitivity and wider detection linear range than those in CDI/DMAP group, and were chosen for next studies. The developed CLIA assay exhibited good selectivity towards NTG among structually similar analogs. The method could detect as low as 0.35 ng mL(-1) NTG in buffer, 2.1 ng mL(-1) NTG in serum and 0.84 ng mL(-1) NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400 µg mL(-1). The CLIA method could detect 78 samples in one assay, and the samples need only dilution in pretreatment. As a summary, this research offers a sensitive assay for high-throughout screening of NTG in formulation control and pharmacokinetic studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

For point-of-care testing of the illegal fortification of repaglinide (Rep) in natural dietary supplements, a competitive chemiluminescent immunoassay (CLIA) was established, using a horseradish peroxidase (HRP)-luminol-H2O2 system for signal amplification. Polyclonal antibodies for Rep were produced via immunization technique. Following optimization of the enzyme reaction time and concentrations of antibody and coating antigen, the method showed a limit of quantification (LOQ) of 1.0 ng/mL in PBS and limit of detection (LOD) of 8.3 ng/mL in serum and 6.0 ng/mL in blank tablets. When applied in natural dietary supplements, the method provided results consistent with those from HPLC, suggesting that the proposed method could be used for rapid screening of Rep in natural dietary supplements and detecting Rep in serum after administration.

We report a surprising discovery that Prussian blue (PB) can be employed as a highly sensitive and background-free resonant Raman reporter. Conventional Raman reporters show multiple spectral bands in the fingerprint region, which are generally overlapped with those from dominant endogenous biomolecules, and are thus difficult to be separated. Herein, we found that PB only possesses a strong and sharp single-band in the cellular Raman-silent region, where no Raman signals from biological species were observed. Therefore, the Raman spectra from PB and endogenous biomolecules are completely resolved without resorting to complicated spectral unmixing. Moreover, PB holds a strong UV-vis absorption band between 500 and 900 nm, which is resonant with the incident detection lasers, providing extremely high sensitivity. Through assembly of PB onto plasmonic cores, a new surface-enhanced resonance Raman scattering (SERRS) probe was achieved with a high signal-to-background ratio (SBR). We demonstrated the performance of the PB-based SERRS tags for high-sensitivity immunoassay and cancer cell imaging. [ABSTRACT FROM AUTHOR]

Fluoroquinolones are effective antimicrobial agents, but their residues in food may cause serious health issues. Hence, it is necessary to develop a rapid and simple assay for fluoroquinolones. In this study, monoclonal antibodies against ciprofloxacin with broad specificity to fluoroquinolones were prepared. The newly developed magnetic particle-based competitive enzyme immunoassay was performed in a homogeneous sample, and ciprofloxacin was quantitatively detected in the range of 0.3–24.3 ng mL−1. The IC50and LOD values of the method were 2.27 ng mL−1and 0.25 ng mL−1. In chicken muscle, the recoveries of spiked ciprofloxacin at 8, 20, and 40 ng mL−1were all higher than 79%. Additionally, the performance of the developed magnetic particle-based immunoassay exhibited an excellent correlation with commercial ELISA kits (r = 0.997). The anti-ciprofloxacin monoclonal antibodies provided high cross-reactivity with eight fluoroquinolones analogues: enrofloxacin (110.1%), sarafloxacin (99.7%), difloxacin (90.2%), danofloxacin (89.8%), norfloxacin (110.3%), lomefloxacin (65.2%), ofloxacin (75.1%), and flumequine (45.0%). This protocol provides an alternative immunoassay for ciprofloxacin with the advantages of simple separation, a homogeneous reaction, rapid detection (within 30 min), and stable results. Furthermore, it may be employed for the rapid general determination of fluoroquinolones in animal products. [ABSTRACT FROM PUBLISHER]

Here we present a fluorogenic multianalyte biosensor based on self-assembled tetraphenylethene (TPE) molecules by modification with diethylenetriamine (TPDA). The TPDA probe could specifically react with pyrophosphate (PPi) by hydrogen linkage. Simulation analysis results indicated that the coupling will lead to intermolecular cross-linking of TPDA molecules and turn on aggregated-induced emission at 470 nm. When alkaline phosphatase (ALP) was introduced, PPi was hydrolyzed and fluorescence emission was turned off. The proposed system allows sensitive detection for ALP activity (LOD = 0.09 mU mL −1 ) and PPi (LOD = 66.7 nM). For ALP assay, this is the first example using PPi to switch AIE phenomenon, which offers better pH selection (pH 7.4) than conventional methods (pH 8.0–9.6). For PPi analysis, this method does not require metal ions, indicating better stability with little interference. We further applied the TPDA-PPi complex as a new substrate for ALP in an immunoassay and proved that the substrate is more sensitive (LOD = 0.8 ng mL −1 ) than traditional one (LOD = 4.0 ng mL −1 ), which means that the complex is greatly potential in ultrasensitive immunoassay for biomedical diagnosis. [ABSTRACT FROM AUTHOR]

Polydiacetylene (PDA) vesicles-based chromatic biosensor could present a unique color change in response to a particular analyte or circumstance. However, its application in recognition of small molecules still remains a big challenge. In this paper, we describe a new strategy to build a competitive colorimetric immunoassay for detection of small molecules by employing hapten-modified PDA vesicles. A commonly used chemical food additive, sodium benzoate, was used as the model analyte. We coupled benzoate analog, 4-aminobenzioic acid, with PDA monomer to facilitate blue-to-red color transition after adding antibodies of sodium benzoate. In the presence of sodium benzoate, the interaction of its antibodies with 4-aminobenzioic acid would be inhibited and the extent of color change was reduced. The inhibition effect correlated well with sodium benzoate concentration, and the lowest distinguishable concentration of sodium benzoate was 100 ng mL −1 by naked-eye readout, and 0.01 ng mL −1 by UV–vis spectrometry. This assay was highly specific and as accurate as HPLC method. It could be also applied in foodstuffs of vinegar and soy sauce diluted by 100 or 200 times. The proposed method showed a great potential in benzoate analysis in these samples, and hapten-modified PDA vesicles was a promising approach in colorimetric detection of other chemical food additives. [ABSTRACT FROM AUTHOR]

Abstract: 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 0.3–20.0μgmL−1 for sodium benzoate with a detection limit of 0.26μgmL−1 in the borate buffer. In addition, the IC50 value was 2.48μgmL−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. [Copyright &y& Elsevier]