Contaminated Milk Samples Research Articles

A highly sensitive aptamer–antibody birecognized ECL sensing platform based on the cascaded reaction between CeO2@mrGO and Co-SAC@NC for E. coli O157:H7 in untreated milk

Rapid, sensitive and specific detection of foodborne pathogens is of great significance to the early warning and prevention of foodborne diseases and environmental pollution. Here, an electrochemiluminescent (ECL) biosensor with the cascaded reaction between two nanozymes was developed for the detection of E. coli O157:H7. Cobalt single-atom catalyst (Co-SAC@NC) with oxidase (OD) -like activity, as a co-reaction accelerator of O2, catalyzed oxygen to reactive oxygen species (ROSs) and boosted ECL of luminol-O2 system. Another nanozyme CeO2@mrGO with excellent phosphatase-like activity by the introduction of Fe was use to label Ab. CeO2@mrGO captured E. coli O157:H7, and then combined with Ap on the electrode surface through the affinity of aptamer and antibody to form a sandwich structure on the gold electrode (CeO2@mrGO-Ab/E. coli O157:H7/Ap-AuE). CeO2@mrGO converted AAP into AA, which consumed ROS and quenched ECL emission of luminol-O2 system. As a consequence, this ECL sensor had a linear response range of 17–1.7 × 105 CFU/mL with LOD of 2.78 CFU/mL. This aptamer-antibody birecognized system was able to be applied to assay E. coli O157:H7 in untreated contaminated milk samples with satisfactory results.

Fate of aflatoxin M1 in milk during various processing treatments

The focus of this research was to investigate the fate of aflatoxin M1 (AFM1) in naturally contaminated milk samples during various processing treatments such as skimming, boiling, sterilisation, pasteurisation and fermentation. The raw milk samples were collected, and AFM1‐contaminated samples were subjected to different processing treatments and analysed using AFM1 quantitative rapid test strip. Processing treatments like skimming and pasteurisation did not change the AFM1 content in milk, whereas high temperature heat treatments like boiling and sterilisation reduced AFM1 content up to 20%. Fermentation of milk with Streptococcus thermophilus reduced AFM1 up to 8%.

Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL-1, and the detection limit of the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU mL-1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.

Lithium acetate mediated paper-based assay for absorbance analysis of E. coli concentrations

Paper-based assays have revolutionized POC diagnostics, offering significant values across diverse sectors due to their simplicity, cost-effectiveness, and portability. Detecting E. coli is critical for public health but faces challenges in traditional methods, prompting ongoing exploration of alternative approaches. To address the need for heightened sensitivity, this study introduces lithium acetate as a novel element in a paper-based E. coli detection assay, accompanying absorbance analysis using microplate readers. Utilization of lithium acetate as a lysis reagent and carrying out absorbance measurements in paper microtiter plates provide a significant stride in improving sensitivity and assay efficiency. The assay substantially reduced the analysis time to 4 hours with a lower quantification limit of 103 cfu/ml for detecting E. coli in pure cultures. The results suggest that the assay could determine 10 cfu/ml of E. coli in a contaminated milk sample with 4 hours of enrichment time. Thus, the study contributes towards improving E. coli detection with lower quantification limits and shorter analysis times. It is anticipated that the diagnostic methodology would circumvent the challenges in conventional E. coli detection methods, placing emphasis on absorbance analysis to achieve a streamlined, accurate, and rapid paper-based detection technique.

Reduction and binding of aflatoxin M1 in milk by Lactobacillus plantarum and Lactobacillus brevis isolated from Siahmazgi cheese

This study aimed to diagnose the ability of two probiotic strains isolated from Siahmazgi cheese to reduce the concentration of aflatoxin M1 (AFM1), which was able to reduce it up to 50%. Two strains of Lactobacillus isolated from local Siahmazgi cheese, were inoculated into raw milk containing 10 ng/mL AFM1. AFM1 levels were assessed by HPLC at 0, 24, 48 and 72 h. Also, for the assessment of the possible reduction of AFM1 by non-viable bacterial cells, heat treatment was applied in milk. In general, significant differences were observed between the control group and AFM1 contaminated milk samples containing probiotics during 72 h of refrigeration (p < 0.05). In the presence of Lactobacillus brevis, Lactobacillus plantarum and mixture probiotics, the concentrations of AFM1 ranged from 0.102 ± 0.004 ng/mL to 0.052 ± 0.002 ng/mL, 0.114 ± 0.002 ng/mL to 0.074 ± 0.003 ng/mL and 0.108 ± 0.003 ng/mL to 0.065 ± 0.002 ng/mL, respectively. Also, in the heated group, just a decrease was observed in the first 24 h. The results showed that native Iranian strains of Lactobacillus have good potential in reducing AFM1 in milk. The time factor is effective in reducing AFM1 by living bacterial cells.

Multiplex Aptamer-Based Fluorescence Assay Using Magnetism-Encoded Nanoparticles for Simultaneous Detection of Multiple Pathogenic Bacteria.

Multiplex assay has emerged as a robust and versatile method for the simultaneous detection of multiple analytes in a single test. However, challenges in terms of poor accuracy and complexity remained. In this work, we developed a multiplex aptamer-based fluorescence assay using magnetism-encoded nanoparticles for the simultaneous detection of multiple pathogenic bacteria. The encapsulation of different amounts of Fe3O4 nanoparticles in zeolitic imidazolate framework-90 (ZIF-90) leads to the formation of Fe3O4@ZIF-90 (FZ) composites with distinct magnetism strengths. By functionalizing a specific aptamer on the surface of the FZ composites, target bacteria can be specifically and precisely separated from a mixed sample in a sequential manner. This property allows for the simultaneous quantitative analysis of multiple target bacteria by using a single-color fluorescence label, thereby resulting in minimal spectral crosstalk interference and improved accuracy. The successful determination of multiple bacteria in contaminated milk samples demonstrates the applicability of this multiplex assay in complex biological matrices. Compared to conventional multiplex fluorescence assays, this approach offers distinct advantages of simplicity, efficiency, and implementation. We believe that this study can provide valuable insights into the development of the multiplex assay while introducing a new method for the simultaneous detection of multiple bacteria.

Experimental Protocol to Toxoplasma gondii Detection in Fresh Goat Milk.

Toxoplasma gondii is a zoonotic parasite with global distribution capable of infecting homeothermic animals. Transmission of protozoan to humans includes ingestion of water and raw food contaminated with sporulated oocysts, ingestion of raw or undercooked meat with tissue cysts, and tachyzoites' transplacental transmission. Fresh goat milk intake has already been linked to human toxoplasmosis outbreaks, but little is known about the infectious potential of this biological sample. Accordingly, the aim of the present study is to assess the survival and infectivity of T. gondii tachyzoites in fresh goat milk samples through an experimental protocol to detect this parasite via bioassay carried out with a murine model, DNA amplification, and serology. Swiss Webster mice were inoculated with fresh goat milk samples contaminated with different T. gondii RH strain tachyzoite concentrations per milliliter and stored for different refrigeration times. Animals showing clinical signs compatible to toxoplasmosis were euthanized. Milk samples contaminated with high parasitic loads and kept for a shorter refrigeration time were the most lethal ones. No significant differences were observed between mean death rates recorded for different goat milk contamination concentrations (p = 0.1888), and for the refrigeration time, contaminated milk samples were kept under (p = 0.9440). T. gondii DNA was amplified in all contaminated milk samples, but only one of the surviving mice was serologically positive. Results of the present study have shown T. gondii survival and infectivity in fresh goat milk samples, and it highlights its significant risk for public health. Therefore, molecular methods must be the tests of choice when milk samples are used to assess infection caused by protozoan in goats' dairy products.

Occurence of antimicrobial residues in milk and labneh consumed in Lebanon

ABSTRACT Antimicrobials are administered in livestock for different uses leading to milk contamination and several undesirable effects. Because there is a lack of surveillance of antimicrobial residues (AMRs) in milk and dairy products in Lebanon, this study aims to determine the occurrence of AMRs in 90 Lebanese samples of milk and labneh (concentrated yoghurt). Multi-residue screening methods with suitable sample preparations were applied to detect 71 AMRs in milk and labneh, respectively, using LC-MS/MS. Of the total number of samples, 71% was contaminated with AMRs and (fluoro)quinolones and macrolides were the most detected families. Additional confirmation tests proved that 6.7% of the milk samples were non-compliant for the macrolides tilmicosin, tulathromycin and spiramycin. Moreover, some labneh prepared from contaminated milk samples was analysed to determine the fate of AMRs during the manufacturing process. The results showed that some AMRs could be concentrated, eliminated or degraded, based on their physicochemical characteristics.

Box-Behnken Design for Assessing the Efficiency of Aflatoxin M1 Detoxification in Milk Using Lactobacillus rhamnosus and Saccharomyces cerevisiae.

Milk contaminated with aflatoxin can lead to liver cancer. Aflatoxin B1 (AFB1), a serious animal feed contaminant, is transformed into Aflatoxin M1 (AFM1) and secreted in milk. In this study, a biological method using probiotic bacteria, Lactobacillus rhamnosus (L. rhamnosus) in combination with Saccharomyces cerevisiae (S. cerevisiae), was used to assess their antiaflatoxigenic effect in animal milk. A Box-Behnken design was used to establish the optimal ratio of L. rhamnosus and S. cerevisiae, incubation time, and temperature for efficient AFM1 detoxification from milk. To achieve this, the primary, interaction, and quadratic effects of the chosen factors were investigated. To investigate the quadratic response surfaces, a second-order polynomial model was built using a three-factor, three-level Box-Behnken design. The quantity of AFM1 was detected by the ELISA technique. The results of these experiments obtained an optimum condition in AFM1 detoxification of the three tested factors in order to maximize their effect on AFM1 detoxification in milk. The model was tested in three highly contaminated milk samples to assure the efficacy of the model. AFM1 detoxification was up to 98.4% in contaminated milk samples. These promising results provide a safe, low-cost, and low-time-consuming solution to get rid of the problem of milk contamination with AFM1.

Development of a CRISPR/Cas9-integrated lateral flow strip for rapid and accurate detection of Salmonella

Rapid and accurate detection of foodborne pathogens is crucial for safeguarding human health. Herein, we constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-integrated lateral flow strip (Cas9-LFS) for detecting Salmonella . A primer set targeting the fimA gene of Salmonella spp. was used to amplify all Salmonella serovars. After extraction, the genomic DNA of Salmonella was amplified with unlabelled forward primer and FITC-labelled reverse primer, thus producing numerous FITC-labelled amplicons that can be precisely recognized by Cas9 complexed with a specific single-guide RNA (sgRNA). When loaded on the LFS, the complexes of amplicons and Cas9/sgRNA can act as bonds to anchor gold nanoparticles to the test line, resulting in a visible color change. This design makes Cas9-LFS more powerful than conventional LFS in eliminating false-positives caused by primer dimers. By using Salmonella Typhimurium as a reference strain, Cas9-LFS achieved a limit of detection as low as 10 2 CFU/mL and showed good accuracy in distinguishing Salmonella -contaminated milk. We expect this unique strategy to open a new window for building versatile pathogen detection tools. • A CRISPR/Cas9-integrated LFS (Cas9-LFS) was constructed for accurate and visual detection of Salmonella. • Primer-dimer interference that is inevitable in conventional amplifications can be totally overcome with the method. • As low as 10 2 CFU/mL of Salmonella can be well measured with the constructed method. • Salmonella -contaminated milk samples have been measured with considerable accuracy.

An Electrochemical Sensor for the Detection of Albendazole Using Glassy Carbon Electrode Modified with Platinum-Palladium Nanocomposites.

An electroanalytical electrode for the detection of albendazole (ABZ) active ingredient in pharmaceutical dosage form and in contaminated animal-derived products was developed using a glassy carbon electrode modified with platinum-palladium nanoparticles. The electro-catalytic performance of the bimetallic-modified glassy carbon electrode was compared with its bare counterpart. Under optimized conditions, the modified electrode revealed two well-resolved anodic peak currents at 1.10 and 1.23 V using differential pulse voltammetry. Pure ABZ, as well as ABZ in spiked foods (milk and chicken), were detected with little interference from the food matrix. This electrode demonstrated high sensitivity and applicability, with a lower limit of detection of 0.08 µmol L-1 in aqueous solution and 10 µmol L-1 in the contaminated ground chicken and 100 µmol L-1 in the contaminated milk sample. The fabricated sensor is low in cost and appropriate for the estimation of albendazole in tablet dosage forms and biological samples, and so can act as a quality control tool in the pharmaceutical and food industry.

Raw goat's milk fermented Anbaris from Lebanon: insights into the microbial dynamics and chemical changes occurring during artisanal production, with a focus on yeasts.

Anbaris is a raw goat milk product naturally fermented in terracotta jars. The aim of this research paper was to follow the dynamics underlying an artisanal production to understand the concomitant evolution of the microbial populations in relation to the chemical changes occurring within the product, make sure of the sanitary conditions prevailing during the production and uncover for the first time its culturable yeast populations. Throughout the fermentation process, Anbaris was endowed with high acidity and included high microbial populations counts of LAB and yeasts that were rapidly installed within the product and maintained as regular new milk additions were made, contributing to lipolytic and proteolytic activities. Salt content varied according to the arbitrary salt additions made during the process but was high in the end product while protein and fat contents varied inversely to moisture. Frequent additions of Enterobacteriaceae and Coliforms contaminated milk samples seemingly fueled a contamination of the product during its manufacturing and in the final fresh Anbaris. Seven species of culturable yeasts, Pichia kudriavzevii, Kluyveromyces marxianus, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Debaryomyces hansenii, Candida parapsilosis and Kazachstania exigua were found during the production. The first two dominated the process in terms of frequency of occurrence and abundance at the different stages and might be signature species of the product. The same lineage of K. marxianus isolates was maintained throughout the fermentation and sample specific patterns were observed. Strains of this species exhibited low diversity within our product, and more globally in the Lebanese dairy products we studied.

Decrease of activity of antioxidant enzymes, lysozyme content, and protein degradation in milk contaminated with heavy metals (cadmium and lead)

The aim of this study was to evaluate the effect of added Cd and Pb to milk on its stability by determining antioxidant enzymatic activities, lysozyme content, and protein degradation. Antioxidant enzymatic activities were spectrophotometrically determined by superoxide dismutase, catalase, xanthine oxidase, and glutathione peroxidase assays; lysozyme was identified and quantified by HPLC-UV analysis, and protein degradation was investigated by spectrophotometric analysis of advanced oxidation protein products (AOPP) and dityrosine content. In this study, contaminated milk samples showed a significant reduction in activity of all studied enzymes compared with control milk. The contamination of milk also led to a significant reduction in the lysozyme content; lysozyme content was decreased about 22% and 18% in Pb milk and Cd milk, respectively, compared with control milk. The presence of the contaminants in the milk resulted in a significant increase of both dityrosine concentration and AOPP compared with the control milk. Moreover, between types of contaminated milk, dityrosine and AOPP values were significantly higher in the Pb milk than in the Cd milk. Therefore, it is important to monitor the presence of these toxic elements in milk for the damage they cause to consumer health both directly due to their ingestion and indirectly due to loss of milk stability.

Impact of Adulteration on Physico-chemical Characteristics of Marketed Raw Milk

This study was conducted to determine the impact of adulteration on physico-chemical and coliforms content of marketed raw milk. For this purpose, 300 milk samples were collected from urban and peri-urban localities of Jhang, Pakistan. The results showed that 13.3%, 9.7%, 9%, 8.3% and 5.7% of the tested milk samples were positive for cane sugar, formalin, boric acid, H2O2 and urea, respectively besides the highest adulteration of water (95%). Other adulterants included soap (4.7%), sodium chloride (3.7%) and quaternary ammonium compounds (3.3%). The incidence of cane sugar and starch adulteration was highest in winter while formalin and H2O2 in summer season. The compositional quality of milk was also deteriorated by relatively lowered concentrations of lactose, fats, total solids, solids-not-fat (SNF) and proteins. Likewise, the pH and freezing point of contaminated milk samples were comparatively higher than the corresponding standard values. Furthermore, the adulterated milk samples were highly contaminated with E. coli (42.5%) and Enterobacter aerogenes (27.5%) as compared to non-adulterated milk. It was concluded that milk adulterationin the study area predominates round the year badly deteriorating the physico-chemical characteristic of milk besides contaminating it with injurious adulterants and water-borne pathogens posing potential health risks to the consumers.

Presence of Escherichia coli O157:H7 in Dairy Farms located in Najaf, Baghdad, Kirkuk, and Erbil, Iraq.

It has been approved that one of the most dangerous foodborne pathogenic bacteria is E. coli O157:H7, which is responsible for several infection and death cases worldwide. It is well documented that in the developing countries E. coli O157:H7 is considered the main causative pathogen of human gastrointestinal infections. Therefore, the current research was aimed to evaluate the prevalence of E. coli O157:H7 in dairy cattle's milk using a rapid method, in Iraq (Najaf, Baghdad, Kirkuk, and Erbil). Over a period of 6 months (During hot months) samples were obtained and investigated by culturing on selective media (CT-SMAC). The multiplex PCR (m-PCR) also used for milk sample direct investigation. Using biochemical tests the recorded data showed that, 2 recognized isolates were E. coli, while the recorded data obtained from m-PCR assay revealed that none of the isolated E. coli was toxigenic E.coli O157:H7. The results of m-PCR on the milk samples revealed that 45 milk samples contained at least one of the following genes: O157, H7, stx1, stx2 genes. Also the results of the m-PCR revealed that 2 samples (raw milk) were toxigenic O157:H7 positive. In conclusion, to the best of authors' knowledge, this investigation was the first report on the prevalence of E. coli O157:H7 in the raw milk samples in Iraq. The results showed that the proportion of contaminated milk samples contaminated with E. coli O157:H7 identified in the current survey were similar to that the results of the previously published research from different dairy products across different countries in the Middle East region.

Evaluation of Adulterants in Raw Cow Milk from Selale and around Addis Ababa City, Ethiopia

Addition of milk adulterants is a serious health problem and can negatively affect the nutritional quality of milk and milk products. In this study milk samples were collected from Selale, Debre Zeit and Sebeta areas and analyzed for formalin, starch, urea, salt and water contents. Out of 180 analyzed milk samples, 42.22% were adulterated with starch, salt and water. However, formalin and urea were not detected in any of the milk samples. Percentage of adulterated milk samples collected from Selale, Debre Zeit and Sebeta were 51.66%, 48.33% and 26.66% respectively. Debre Zeit had maximum percentage of water (41.66%) and starch (6.66%) followed by Selale water (38.33%) and starch (5%). While Sebeta had the lowest percentage of contaminated milk samples with starch (3.3%) and water (21.66%). Therefore, there is a need to set control mechanisms and aware producer, milk collectors, dairy processer and consumer on impacts of adulteration. Keywords: Adulterant, Formalin, Milk, Salt, Starch, Urea, Water DOI: 10.7176/JNSR/13-9-04 Publication date: May 31 st 2022

A genosensor platform based on DNA biofunctionalized SNCNCs coupled with “IS-primer” amplification reaction for sensitive and rapid Listeria monocytogenes detection

Accurate and timely diagnosis of Listeria monocytogenes (L. monocytogenes), a well-known foodborne pathogen with a serious threat to human life and health, is critical for managing bacterial infections and food quality. Here, we report a genosensor platform based on DNA biofunctionalized superparamagnetic nickel colloidal nanocrystal clusters (SNCNCs) coupling with “IS-primer” amplification reaction (ISAR) for rapid, ultrasensitive and specific L. monocytogenes detection. Specifically, the catalytic activity of SNCNCs was identified. After three-step modification, the signal unit was formed by immobilizing AuNPs and hairpin probes on the surface of SNCNCs, achieving enhanced catalytic activity, leading to the reduction of a colorimetric substrate 4-nitrophenol (4-NP). The in-situ signal amplification of target RNA on the surface of SNCNCs coupling with ISAR showed high sensitivity and a detection limit of 1.50 × 10-3 ng mL-1 under optimized conditions. As SNCNCs can bind and lyse bacteria, this genosensor can detect live L. monocytogenes without RNA extraction, with a detection limit of 3.80 × 102 CFU mL-1. Furthermore, the system could also detect L. monocytogenes in artificially contaminated milk samples within 6 h. These results indicate the great potential of our method for sensitive, specific, convenient and low-cost bacterial detection in food, environment and clinics.

Outer membrane protein A (OmpA) may be used as a novel target to enrich and detect Escherichia coli in milk samples

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 100 cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.

Efficiency of calcined Aluminum-Magnesium layered double hydroxide for adsorption of aflatoxin M1 from solution and matrix of milk.

Detoxification of aflatoxin M1 from solution and milk using layered double hydroxides was investigated. The Aluminum-Magnesium layered double hydroxide (Al-Mg LDH) and Iron-Magnesium layered double hydroxide (Fe-Mg LDH) were selected in their calcined and non-calcined forms to evaluate the effect of the calcination on detoxification. These materials were produced using the co-precipitation method. Preliminary adsorption tests confirmed use of Al-Mg LDH as the selected adsorbent. Characteristics of the adopted adsorbent were studied and confirmed by XRD, FTIR, SEM, and BET methods. Effects of the initial content of aflatoxin, amount of the adsorbents and detoxification time were investigated. Influence of the adsorbents on the nutritional aspects of milk were also studied. The study showed that while the non-calcined forms of LDH were not able to adsorb aflatoxin M1 more than 23%, the calcined form of Al-Mg LDH exhibited 100% adsorption in the solutions and about 70-100% in the contaminated milk samples. The reason is pointed to the fact that calcination of Al-Mg LDH considerably increased the surface area, the total pore volume, and the pore size of the material. Multivariate regression analysis and calculation of the Pearson correlation factor showed that the remained aflatoxin at each time was more strongly correlated with the initial amount of aflatoxin and the elapsed time and less strongly with the amount of the adsorbent. It was found that the adsorption isotherms fitted to the Freundlich equation with a high adsorption capacity of 555.5 mg g-1. PRACTICAL APPLICATION: This study is focused on examining ability of layered double hydroxides (LDH) for adsorbing AFM1 . LDHs are promising layered materials due to some of their interesting characteristics, such as ease of synthesis and uniqueness of structure. In practice, results of this study can be used for detoxification of aflatoxin, especially in milk, at high efficiency in shorter time durations.

Detection of 4 quinolone antibiotics by chemiluminescence based on a novel Nor-Biotin bifunctional ligand and SA-ALP technology.

A simple and effective direct competitive chemiluminescence immunoassay for the detection of 4 kinds of quinolone antibiotics in milk was established using Nor-Biotin (biotin-modified norfloxacin [NOR]) bifunctional ligand and alkaline phosphatase-conjugated streptavidin signal amplification technology. The polyclonal antibody was obtained after the immunization of New Zealand White rabbits using norfloxacin-derived antigen. "Click chemistry" was used for the rapid and facile synthesis of the Nor-Biotin bifunctional ligand. After the optimization of the incubation time and reaction buffer, the direct competitive chemiluminescence assay method was developed and used for sensitive detection of 4 kinds of quinolone drugs (NOR, pefloxacin, ciprofloxacin, and danofloxacin). The IC50 of the 4 kinds of quinolone drugs ranged from 7.35 to 24.27 ng/mL, and the lowest detection limits ranged from 0.05 to 0.16 ng/mL, which were below their maximum residue levels, approved by the EU for treatment of food-producing animals. To demonstrate the applicability of the assay, artificially contaminated milk samples with the 4 quinolone drugs were analyzed. The mean recovery rates of the drugs ranged from 86.31% to 112.11%.