Fluorescence polarization assay to detect the presence of traces of ciprofloxacin

Abstract

Detection of ciprofloxacin residues in milk by sensitive and rapid methods is of great interest due to its use in the treatment of dairy livestock health. Current analytical approaches to antibiotics detection, are laboratory-based methods and they are time-consuming and require trained personnel. To cope this problem, we propose an assay, based on fluorescence polarization principle, able to detect the presence of ciprofloxacin in diluted milk sample without any pre-treatment. The proposed method is based on the use of ciprofloxacin-protein conjugate labeled with near infrared fluorescence dye, which upon binding to specific antibody causes an increase of the fluorescence polarization emission signal. The developed assay allows for the detection of ciprofloxacin at a concentration of 1ppb, which represents an amount lower than the maximum residual limit (MRL) of ciprofloxacin in milk, as set by the European Union regulation (100 ppb).The antibiotics treatment plays a crucial role in dairy livestock health 1 . It is being used for therapeutic purposes as well as for improving breeding efficiency. However, this treatment can leave residues of antibiotics in foods of animal origin (milk and meat), and consequently an increase of the human exposure to antibiotics 2 . While assessing the usage of antibiotic residues, the exceptional use of such drugs was legally considered 3 . Hence, the risk of the presence of antibiotic residues in milk and dairy products could increase in case that a required elimination period of them in food is not properly defined.Antibiotic residues in milk and dairy products present detrimental consequences on the consumer health, causing disturbances in the intestinal flora as well as allergic reactions that could lead to anaphylaxis 4 . Moreover, antibiotic residues might increase microbial resistance, a major health risk 5 . Among anti-bacterial agents used for the management of infections in dairy livestock, ciprofloxacin, belonging to the fluoroquinolone class, is the common antibiotic used in case of pulmonary, urinary and digestive infections 6 . However, ciprofloxacin could cause hypersensitivity in humans 7 . Due to the importance of considering the presence of ciprofloxacin in milk, a residue surveillance of anti-microbial presence is crucial to support food-farming practices, upraising consequently the food safety level. It is to be noted that the maximum residual limit (MRL) for ciprofloxacin in milk has been fixed at 100 ppb (Council Regulation EEC/2377/90). In Lebanon, few studies have investigated the presence of antibiotic residues in milk [8][9][10] . A recent study showed that ciprofloxacin and oxytetracycline are mostly found antibiotics in milk in Lebanon (personal communication).Currently, laboratory-based methods, such as liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC) [11][12][13] and microbiological assays 14 are the common used approaches for ciprofloxacin detection. These methods have different restrictions that make the...

Emergent Biosensing Technologies Based on Fluorescence Spectroscopy and Surface Plasmon Resonance

“…The results obtained suggest that the method could be applied directly in milk without interference. For the PenG detection, the LoD of the method was 1.0 nmol/L, which is much less than the required maximum residual limit (MRL) in EU regulations (12.0 nmol/L) [134] while the obtained results for the ciprofloxacin reached a sensitivity of 1 ppb [135], 100 times lower than MRL of ciprofloxacin in milk, as fixed by the European Union regulation (100 ppb).…”

“…A microarray of different spots was printed on a modified glass chip. Recently, it has been developed a fluorescence polarization method to detect directly in milk the presence of PenG [134] and Ciprofloxacin [135]. This methodology is based on the increase of fluorescence polarization emission of a fluorescence-labeled compound derivative upon their binding to specific antibodies.…”

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“…To determine the antibody titer, an indirect ELISA test was performed as described by El Kojok et al [36], with slight modifications. The ELISA plate was coated with 50 µL/well of recombinant antigen (from 1 µg/mL to 0.001 µg/mL) and/or virus sample (from 1:10 to 1:1000) diluted in 0.05 M carbonate buffer at pH 9.6 and incubated overnight at 4 • C. As a negative control, some wells were coated by coating buffer.…”

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“…In recent years, QDs have been used in FPIA to detect tumor markers [ 77 , 78 ], cysteine and mercury ions [ 25 ], antithrombin [ 79 ], and adenosine triphosphate (ATP) [ 80 ], among others. In addition to QDs, copper nanoclusters have been used in FPIA to detect the Citrus Tristeza virus [ 81 ], AuNPs have been applied as tracers for the sensitive detection of silver ions [ 23 ], silica nanoparticles—for thrombin detection [ 82 ], and carbon nanoparticles combined with aptamer—for real-time FA detection of apyrase [ 83 ], as well as in near-infrared fluorescence dye to detect ciprofloxacin [ 84 ].…”