Yongjun Wu

Determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay

A chemiluminescence enzyme immunoassay (CLEIA) based on the HRP-luminol-H₂O₂ chemiluminescence system for highly sensitive detection of enrofloxacin (ENR) was proposed in this study. Key factors that affect the precision and accuracy for the determination of ENR residues were optimised. Under the optimal conditions, the proposed method showed an excellent performance. The linearity range for method developed for determination of ENR was 0.35-1.0 ng/mL with a correlation coefficient greater than 0.994. The limit of detection was 0.03 ng/mL and the relative standard deviations (RSDs) were less than 9.4% and 13.0% for intra-day and inter-day assays. The proposed method was satisfactorily applied to determine ENR in milk, eggs, and honey samples at three spiked levels (0.4, 0.7, and 1.0 ng/mL) and the recoveries ranged from 92.4% to 104.2% for milk, 93.8% to 103.2% for eggs and 94.1% to 105.0% for honey, respectively. Compared the results of CLEIA with those of ELISA and HPLC, the advantages of the CLEIA were further confirmed. Moreover, one 96-well microtiter plate coated with anti-ENR can be used to detect multiple samples at the same time, which indicated that the CLEIA using HRP-luminol-H₂O₂ system was a sensitive, high throughput and real-time method for ENR residues analysis.

Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine

Sulfamethoxydiazine (SMD), which is often used for animal disease treatment, is harmful to human health. No SMD residue should be detected in food in some countries, such as USA and Japan. Therefore, it is significant to develop a high-throughput, high-sensitivity and accurate method for the determination of the content of SMD in food. In this paper, chemiluminescence enzyme immunoassay (CLEIA) was developed for quantification of SMD. For this method, the limit of detection was 3.2 pg/ml, the linear range was from 10 to 2000 pg/ml, the within-day and inter-day precision were below 13% and below 18%, respectively, and the recovery was from 85% to 105%. Milk and egg were selected as samples to be examined with this method, and the result indicated that this CLEIA method was suitable for screening and quality control of food.

Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay

In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4°C with no additive up to 30days. These data were valuable for establishing CLEIA to quantify enrofloxacin.

Study on the reaction mechanism and the static injection chemiluminescence method for detection of acetaminophen

Acetaminophen, also called paracetamol, is found in Tylenol, Excedrin and other products as over-the-counter medicines. In this study, acetaminophen as a luminol signal enhancer was used in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP) for the first time. The use of acetaminophen in the luminol-HRP-H2O2 system affected not only the intensity of the obtained signal, but also its kinetics. It was shown that acetaminophen was to be a potent enhancer of the luminol-HRP-H2O2 system. A putative enhancement mechanism for the luminol-H2O2-HRP-acetaminophen system is presented. The resonance of the nucleophilic amide group and the benzene ring of acetaminophen structure have a great effect on O-H bond dissociation energy of the phenol group and therefore on phenoxyl radical stabilization. These radicals act as mediators between HRP and luminol in an electron transfer reaction that generates luminol radicals and subsequently light emission, in which the intensity of CL is enhanced in the presence of acetaminophen. In addition, a simple method was developed to detect acetaminophen by static injection CL based on the enhanced CL system of luminol-H2O2-HRP by acetaminophen. Experimental conditions, such as pH and concentrations of substrates, have been examined and optimized. The proposed method exhibited good performance, the linear range was from 0.30 to 7.5 mM, the relative standard deviation was 1.86% (n = 10), limit of detection was 0.16 mM and recovery was 99 ± 4%.

An ultrasensitive chemiluminescence immunoassay for fumonisin B1 detection in cereals based on gold-coated magnetic nanoparticles: Nanoparticles-based chemiluminescence immunoassay for fumonisin B1 detection

BACKGROUNDnIn the present study, a novel highly sensitive magnetic enzyme chemiluminescence immunoassay (MECLIA) was developed to detect fumonisin B1 (FB1 ) in cereal samples. The gold-coated magnetic nanoparticles (Fe3 O4 @Au, GoldMag) were used as solid phase carrier to develop a competitive CLIA for detecting FB1 , in which FB1 in samples would compete with FB1 -ovalbumin coated on the surface of Fe3 O4 @Au nanoparticles for binding with FB1 antibodies. Successively, horseradish peroxidase labeled goat anti-rabbit IgG (HRP-IgG) was conjugated with FB1 antibodies on the microplate. In substrate solution containing luminol and H2 O2 , HRP-IgG catalyzed luminol oxidation by H2 O2 , generating a high chemiluminescence signal. The FB1 immune GoldMag particles were characterized by Fourier transform infrared spectroscopy, scanning electron microscope and zeta potential analysis, etc. RESULTS: The concentrations and the reaction times of these immunoreagents were optimized to improve the performances of this method. The established method could detect as low as 0.027u2009ng mL-1 FB1 from 0.05u2009ng mL-1 to 25u2009ng mL-1 , demonstrating little cross-reaction (less than 2.4%) with other structurally related compounds. The average intrassay relative SD (RSD) (nu2009=u20096) was 3.4% and the average interassay RSD (nu2009=u20096) was 5.4%. This method was successfully applied for the determination of FB1 in corn and wheat and gave recoveries of between 98-110% and 91-105%, respectively.nnnCONCLUSIONnThe results of the present study suggest that the MECLIA approach has potential application for high-throughput fumonisin screening in cereals.

Detecting the polymorphism of TERF1 gene by an improved PCR-RFLP method

Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) is a common and mature method of detecting the single nucleotide polymorphism (SNP). But, for the polymorphism site rs3863242 of telomeric repeat binding factor 1(TERF1) gene, there is no appropriate restriction enzyme to recognize it, which limits the research between the variants of rs3863242 and human diseases.

Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

Alkaline phosphatase and horseradish peroxidase are the most commonly used reporter systems in chemiluminescent enzyme immunoassay (CLEIA). Which one, therefore, would be better when establishing a CLEIA method for a new target substance? There was no standard answer. In this study, both reporters were compared systematically including luminescence kinetics, conjugation methods, optimal condition and detection performance, using two common drugs, SD-methoxy-pyrimidine and enrofloxacin, as determination objects. The results revealed that there was much difference between the luminescence kinetics of the two systems. However, there was little difference between these systems when detecting the same substance, including in optimal conditions and determination of performance. Both reporters were suitable for establishing chemiluminescent enzyme immunoassays. Therefore, the choice of alkaline phosphatase or horseradish peroxidase as the reporter system in chemiluminescent enzyme immunoassays depends on availability. Conversely, these two report systems could be applied in simultaneous analysis of multicomponents due to their different optical behaviors and similar performances. But attention should be paid to conjugation method and coating buffer, which affected the luminescent intensity of different determination targets.

NF-E2-related factor 2 serves a key function in resistance to malignant transformation of BEAS-2B cells induced by coal tar pitch

Coal tar pitch (CTP) is a key factor in the development of occupational lung cancer. In order to investigate the function of the anti-oxidative signaling pathway regulated by NF-E2-related factor 2 (Nrf2) during cancer development, BEAS-2B cells were cultured with CTP extract for 30 passages. It was revealed that malignant transformation occurred in cells between the 20 and 30th passage. The expression levels of Nrf2 and NAD(P)H:quinone oxidoreductase 1 (NQO1) were promoted throughout the CTP exposure culture, and there was a positive linear correlation between the expression levels of Nrf2 and NQO1. Following knockdown of Nrf2 expression, the level of NQO1 decreased markedly and malignant transformation was more likely to occur. It was hypothesized that CTP may be toxic to BEAS-2B cells, which may lead to malignant transformation. Nrf2 was a quick response factor: Counteracting cytotoxicity by promoting the expression of anti-oxidative genes. Thus, Nrf2 was associated with the malignant transformation of BEAS-2B cells exposed to CTP and may be a potential therapeutic target.

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

Background DNA methyltransferase 1 (DNMT1), a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase) detection have focused on the prokaryote MTase and its activity. Methods A magnetic fluorescence-linked immunosorbent assay (FLISA) using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized. Results Under the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1–1,500 ng/mL, the recovery was 91.67%–106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%–11.29% and 7.03%–11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA) kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient r=0.866, p=0.001). The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter than ELISA kits. Conclusion These indicated that the proposed FLISA method was sensitive and high throughput and can quickly screen the level of DNMT1 in serum samples.