Cartridges with molecularly imprinted recognition elements for antibiotic residues monitoring in milk

Microfabricated optical cells reported for fluorescence based analysis generally offer an optical path length limited to the substrate thickness, i.e. a reduced sample volume. Larger volume are highly desirable to increase the overall analysis sensitivity. The proposed optical cell consists of an array of open channels. The cell is illuminated along its length, that is perpendicular instead of normal to the cell plane. The optical path is defined by the channel and the probed volume determined by the number of channels composing the array. The menisci formed at the open end of the channels provide a direct air-liquid interface with the probed volume. With this 90° excitation/detection arrangement, stray light coupling can be largely reduced. This cell format is particularly suitable for analytical cartridges based on capillary filling.

Libraries of molecularly imprinted polymers (MIPs) targeted for penicillin G have been synthesized and screened. Chemometric analysis was done to identify synthesis parameters of importance for the capacity and the selectivity of the synthesized MIPs. MIPs selective for penicillin G have been identified and studied in a radioligand competitive binding assay for cross-reactivity against other antibiotics. Some cross-reactivity was seen for 4-ppb antibiotics (i.e., ampicillin and amoxicillin), 6-aminopenicillanic acid (6-APA), and penicillin V. Low cross-reactivity was seen for 30-ppb antibiotics (i.e., oxacillin, nafcillin, and cloxacillin). No cross-reactivity was seen for other antibiotics (i.e., erythromycin, dapsone, tetracycline, chloramphenicol, and cephapirin). Molecular recognition studies in aqueous acetonitrile indicated that 1% buffer pH 7 gave the best selective recognition of both penicillin G and a fluorescent beta-lactam analog (pyreneacetic aminopenicillanic acid). These synthetic recognition elements are expected to be useful in assays, sensors, and instrumentation aiming at analysing penicillin G. The material can also be used for solid-phase extraction of penicillin G. Matrices of interest containing penicillin G include milk, meat, and fermentation broths. The recognition event (i.e., penicillin G binding to the MIP) has to take place in aqueous acetonitrile at low water content in order to maintain binding capacity and selectivity.

Geometrically defined gas-liquid and liquid-liquid micro-interfaces are of fundamental interest for a large variety of applications, from fine chemical synthesis and analysis to electronic chip heat exchangers. The evaporation device developed in this project offers such interface. Our device could in particular be conveniently used in multiphase mixing or interface a Solid Phase Micro Extraction (SPME) analysis system in which pressure, temperature and volume can be easily controlled. Another exploitation route lays in device cooling by means of the evaporation-condensation process used in heat pipes and capillary pumped loops (CPL). A CPL system is a two-phase thermal control system that uses surface tension forces to circulate a cooling fluid. In place of a mechanical pump, the pressure difference required to circulate the fluid comes from a capillary pressure rise across the meniscus separating the vapour and liquid phases in the fine pore wick located in the evaporator. Evaporation of a liquid requires high amounts of energy taken from the environment. Reducing the size of a liquid droplet increases the evaporation rate and hence the cooling rate.

Seven novel fluorescent pyrene- and dansyl-labelled beta-lactam antibiotics (BLAs) and two analogues of penicillin-G bearing a bromine atom and a dimethylamino group, respectively, have been synthesised and characterised to act as substrates in competitive-type assays based on organic polymers (MIPs) molecularly imprinted against the target (unlabelled) antibiotics. Pyrene- and dansyl-labelled acrylate monomers have also been synthesised together with several fluorescent imprinted polymers that would allow the analysis of antibiotic targets containing in their molecular structure a functional group intrinsically able to quench (or enhance in some cases) the fluorescence of the polymer. The fluorescent BLAs are neither limited nor restricted to that application but they may be used in other fields such as competitive immunoassays, the determination of the activity of beta-lactamase enzymes, the study of bacterial membrane proteins, the analysis of microbial fermentation broths or the development of sensitive analytical methods to detect and quantify antibiotics in biological fluids, pharmaceutical formulas and animal-source foods. Compared to the isolated pyrene and dansyl structures, most of the new molecules have demonstrated to keep intact their unique absorption and fluorescent features, including wavelength maximums, emission quantum yields and emission lifetimes. Moreover, they are well recognised by synthetic polymers molecularly imprinted against penicillin G so that competitive-type analyses of beta-lactam antibiotics have proved to be feasible.

Competitive binding assays using MIPs as recognition elements (MIA, molecularly imprinted sorbent assay) have been developed for penicillin G (PenG) monitoring in synthetic samples. The assay is based on a novel highly fluorogenic derivative of the beta-lactam antibiotic (BLA) labelled with pyrene, namely pyreneacetic aminopenicillanic acid (PAAP), and penicillin G selective polymers. The competitive binding studies indicate the existence of cross-reactivity with antibiotics derived from the 6-aminopenicillanic acid, particularly those with an allowed 4-ppb maximum residue level (MRL), so that the assay could be applied to the determination of this group of compounds. Other antibiotics, such as chloramphenicol, tetracycline or cephapirine do not compete with PAAP for binding to the imprinted polymer. The stability of PAAP has been demonstrated in concentrated acetonitrile solutions at -20°C for over 6 moths and in solid form for over two years. The assay is expected to be useful for the analysis of beta-lactams in milk, animal tissues, medicated foodstuffs or fermentation broth. The assay must be carried out in 1% water in acetonitrile to keep the appropriate binding capacity and selectivity.

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