Florfenicol Binding to Molecularly Imprinted Polymer Nanoparticles in Model and Real Samples

Abstract

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent (ELISA) assay to determine and quantify florfenicol (FF) in real food samples such as liquid milk and salmon muscle is presented here. The nanoMIPs were synthesized by a solid-phase approach with an immobilized FF (template) and characterized using dynamic light scattering, a SPR-2 biosensor system and transmission electron microscopy. Immobilization of nanoMIPs was conducted by preparing a homogenous solution of FF-nanoMIPs in water mixed with polyvinyl alcohol (PVA) 0.2% (w/v) in each well of a microplate. The detection of florfenicol was achieved in competitive binding experiments with a horseradish peroxidase−florfenicol (FF-HRP) conjugate. The assay made it possible to measure FF in buffer and in real samples (liquid milk and salmon muscle) within the range of 60−80 and 90-100 ng/mL, respectively. The immobilized nanoMIPs were stored for six weeks at room temperature and at 5 • C. The results indicate good signal recovery for all FF concentrations in spiked milk samples, without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.with florfenicol (FF) being the main antibiotic used (52%), followed by oxytetracycline, flumequine and oxolinic acid (44%, 1% and 1%, respectively) [5,6]. FF, a fluorinated derivative of thiamphenicol, is a new generation of fenicol drugs [7]. Apart from in the aquaculture industry, FF is widely used as chloramphenicol alternative to prevent and treat bacterial diseases in pigs [8], bovine [9], poultry [10] and animal edible tissues [11]. Several countries adopted maximum permissible residue limits (MRLs) of antibiotics in foods of animal origin, such as meat, milk and eggs [12]. In Chile, the maximum permissible residue limit of FF in muscle and skin of salmon is 1000 µg/kg [13]. At present, no MRL has been set for FF in milk. It is however of vital importance to develop a reliable method for FF analysis in milk products to avoid potential harmful effects to consumers [14]. Therefore, there is a demand for relevant sensitive, selective, fast and inexpensive analytical protocols from legislation, health authorities and companies operating in the food market [15]. Various analytical techniques have been reported for the analysis of FF, including high performance liquid chromatography, gas chromatography, liquid chromatography-Tandem mass spectrometry [16], gas chromatography-Mass spectrometry [17], and enzyme-linked immunosorbent assay (ELISA) [18]. The manufacture of molecular imprinting polymers (MIP) can be very valuable for those selective and sensitive methods that are needed. Molecular impression is a technique for producing in a polymer matrix selective binding sites that recognize a particular molecule. These polymers are very sta...

A molecularly imprinted polymer nanoparticle-based surface plasmon resonance sensor platform for antibiotic detection in river water and milk

“…As seen in Figure 2, the generated materials were also assessed for their specificity towards their target molecules. As Table 1 shows, the nanoMIPs, when loaded with a non-target antibiotic, produce KD values into the µM range, thus showing a 100-fold decrease in affinity and demonstrating target specificity, consistent with similarly produced nanoMIPs (37,38). This is a significant demonstration of selectivity given the similarity in structure shown in Figure 1.…”

“…The KD values obtain within this work are similar to that of the KD values previously obtained for this type of small molecule imprinted nanoMIPs. Work by Caro et al produced a nanoMIP for the antibiotic Florfenicol, that obtained a KD value of 75 nM and in practical terms resembled monoclonal antibodies (38).…”

Highly Selective Aptamer‐Molecularly Imprinted Polymer Hybrids for Recognition of SARS‐CoV‐2 Spike Protein Variants

“…This is consistent with prior work, [ 32 , 33 ] and is comparable to that of monoclonal antibodies. [ 37 ] It is also in the same order of magnitude (10 −8 M) as the aptamer generated in this study.…”

Imprinted Polymers as Synthetic Receptors in Sensors for Food Safety

“…They are a bit more expensive than paper-based assays, but they offer a higher degree of robustness. Chip-based devices have been combined with various read-out principles including colorimetric and SPR for the detection of the whole range of food contaminants, from drugs to insecticides and even whole-cell bacteria [ 199 , 228 , 229 ]. Devices such as these could accelerate the application of imprinting technology in the food-safety value chain from a technological perspective.…”