New immunosensor for β-lactam antibiotics determination in river waste waters

Highlights

• A selective and sensitive single use amperometric immunosensor for penicillin G was developed.

• This immunosensor device used two different competitive formats.

• LOD was of the order of 10⁻¹⁰ M and the K_aff value about 10⁸ M⁻¹.

• The immunosensor displayed a good selectivity toward different classes of antibiotics.

• The immunosensor was used to test penicillin G recovery in river waste water.

Abstract

The aim of the present research was to develop a single use, simple but highly sensitive amperometric immunosensor for penicillin G and other β-lactam antibiotics based on a “competitive assay”. The immunosensor developed uses an amperometric electrode for hydrogen peroxide as transducer and the peroxidase enzyme as marker. The results demonstrate the full validity of this immunosensor method which was optimized by comparing two different competitive operating formats. LOD was of the order of 10⁻¹⁰ M. The immunosensor developed displayed low selectivity toward all β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics. In addition the K_aff value (about 10⁸ M⁻¹) was evaluated. Lastly, the immunosensor was used to test a β-lactam antibiotic “pool” and to recover penicillin G in common real matrices such as river waste water, obtaining good recoveries.

Introduction

Several kinds of analytical methods for clinical, environmental and food control purposes involving antibiotic analysis were developed in recent decades [1], [2], [3], [4], [5], and some of these methods are now commercially used and available [6], [7]. The methods described in the literature now include a good number of immunological methods [8], [9], [10] in view of the possibility of generating a large number of antibodies for the analysis of numerous chemical species. Originally, if a low molecular weight chemical substance produced by microorganisms showed growth-inhibitory effects on other microorganisms in high dilution it was defined as an antibiotic. However, in the present context any chemical substance, either of microbial, synthetic, or semi-synthetic origin, used in the chemical treatment of infections, is called an antibiotic. The discovery of penicillin by Alexander Fleming in 1928 and the subsequent isolation of the compound by other researchers opened up vast horizons in the pharmaceutical field. Penicillin was used on a large scale in the years following its discovery up until recent times. However, within four years after its introduction, its widespread use on humans and animals induced bacterial resistance infections. It thus became increasingly necessary to monitor on a large scale the presence of this and other β-lactam antibiotics in waste water, river water and even in surface water tables [11], [12]. A number of analytical methods for penicillin control were developed, especially involving chromatographic, capillary electrophoresis and MS techniques [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], but also based on microbial screening [24]. Finally, penicillin G, amoxicillin and other β-lactam antibiotics were determined in serum, urine and calf's milk using a microbial reception assay, or a microbial growth inhibition assay [25]. Lastly innovative immunological analytical methods, based for instance on the interaction of novel fluorescent labeled β-lactams within polymer materials, molecularly imprinted (MI) with penicillin G, has also been studied, using both radioactive and fluorescent competitive assay [26]; alternatively, a molecularly imprinted solid-phase extraction (MISPE) coupled HPLC method has also been proposed [27]. On the other hand, automated molecularly imprinted sorbent based assay (MIA) for the sensitive analysis of β-lactam antibiotics has been described [28]. Nevertheless, the need for rapid tests with low LOD values (at least of the order of 10⁻⁸ to 10⁻¹⁰ M) has also encouraged the use of sensors and biosensor methods. In previous years, for instance, also some of the authors of the present paper developed ISEs for use in analyzing β-lactam antibiotics [29] and other authors proposed several potentiometric or impedimetric sensors based on a polyaniline coated electrode [30], although their sensitivity and selectivity was usually not particularly high. In recent years therefore more specific enzymatic biosensors have been developed for the analysis of penicillin G and other β-lactam antibiotics using penicillinase as enzyme and different types of sensor for pH measurement as transducer: potentiometric (glass [31], [32], ISEs, or polymeric membrane electrodes [33], operating also in a flow or flow injection format [34], [35], [36]). Lastly numerous others enzymatic sensors have been described: enzyme-FET [37], or BioFET [38], hybrid microbial growth carbon dioxide biosensor [39] and optical (i.e. optode based fluorescence [40], [41], [42], or absorbance [43], or evanescent wave [44]). The difficulty encountered in developing this type of enzymatic sensor is that found in all those biosensors in which a pH variation produced by an enzymatic reaction is to be measured even when having to operate practically in a pH buffered solution. The availability of antibodies for β-lactam antibiotics on the market is thus now encouraging the development of innovative immunological analytical methods that do not have the drawback of the above-cited enzymatic methods and which usually have better selectivity and offer the advantage of being able to analyze real samples without any need for pre-treatment. For instance, an electrochemical paper immunosensor against neomycin based on carbon nanotubes coated on the filtration paper by dip-dry cycles and the chronoamperometric measurement method has been described [45]; other fluorescent immunoassay devices, developed using a competitive format for the analysis of β-lactam antibiotics, have also been reported in the literature [46], [47]; lastly a label free impedimetric flow injection immunosensor for the direct detection of penicillin G has been proposed [48] and biosensors based on surface plasmon resonance (SPR) assays for the detection of β-lactam antibiotics in milk and other matrixes have been reported [49], [50]. Also a parallel affinity sensor array based on chemiluminescence [51], [52], and an indirect competitive chemiluminescence microarray immunoassay (CL-MIA) [53] have been described. In the case of the latter interesting immunological methods, however, special equipment is needed which is not always readily available in ordinary analytical laboratories. Our intention was instead to develop not a complex device suitable for an array system but on the contrary an inexpensive immunosensor able for a single use that was easy to build as it was made of materials readily obtainable on the market, using an amperometric transducer for hydrogen peroxide, a device that was almost always available in the laboratory. The present paper thus describes the development of a new classical, but highly sensitive electrochemical competitive immunosensor assay for penicillin G (or β-lactam antibiotics “pool”) analysis. In actual fact, two different competitive formats were used for penicillin determination in which the antigen (penicillin) or the antibody (anti-penicillin), respectively, were conjugated with horseradish peroxidase enzyme using a biotinylation process. After optimizing the “competitive” measurement format, the penicillin immunosensor was used to determine a β-lactam antibiotics “pool” present in two central Italy river waste waters [54], [55]. Lastly, selectivity vis-à-vis other types of antibiotics and the affinity constant values were determined and discussed.