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We report the first results for the immunochemical determination of the β-lactam antibiotic drug amoxicillin (AMX) in water samples by ELISA (enzyme-linked immunosorbent assay). The applied indirect competitive ELISA using a commercially available mouse anti-AMX antibody enables testing for AMX with a limit of detection of around 1 µg/L. Calibration curves of calibrators prepared in different water samples showed equal shape. This underlines the suitability of this assay for the AMX determination in these samples. Furthermore, cross-reactivities of structurally similar β-lactam antibiotics were examined and found to be low for ampicillin, penicillin G and penicillin V. In contrast, carbenicillin was recognized by the antibody even better than AMX indicating a positive effect of an additional carboxyl group on antibody binding. This is promising for further application of this assay, e.g. in the determination of the most relevant hydrolysis product of AMX, amoxicilloic acid.
Contamination of drinking water with pharmaceuticals is an emerging problem and a potential health threat. Immunochemical methods based on the binding of the analyte to specific antibodies enable fast & cost-effective on-site analyses. Magnetic bead-based immunoassays (MBBAs) allow for implementation into an immunosensor for online testing. Particles are prepared by either coupling the analyte molecule (diclofenac) or the respective antibody (amoxicillin) to the surface. For miniaturization, detection is performed electrochemically (chronoamperometry) on a microfluidic chip. The developed immunosensor will enable detection of pharmaceuticals directly in water supply pipes.
Increasing contamination of environmental waters with pharmaceuticals represents an emerging threat for the drinking water quality and safety. In this regard, fast and reliable analytical methods are required to allow quick countermeasures in case of contamination. Here, we report the development of a magnetic bead-based immunoassay (MBBA) for the fast and cost-effective determination of the analgesic diclofenac (DCF) in water samples, based on diclofenac-coupled magnetic beads and a robust monoclonal anti-DCF antibody. A novel synthetic strategy for preparation of the beads resulted in an assay that enabled for the determination of diclofenac with a significantly lower limit of detection (400 ng/L) than the respective enzyme-linked immunosorbent assay (ELISA). With shorter incubation times and only one manual washing step required, the assay demands for remarkably shorter time to result (< 45 min) and less equipment than ELISA. Evaluation of assay precision and accuracy with a series of spiked water samples yielded results with low to moderate intra- and inter-assay variations and in good agreement with LC–MS/MS reference analysis. The assay principle can be transferred to other, e.g., microfluidic, formats, as well as applied to other analytes and may replace ELISA as the standard immunochemical method.
Contamination of drinking water with pharmaceuticals is an emerging problem and a potential health threat. Immunochemical methods based on the binding of the analyte to specific antibodies enable fast & cost-effective on-site analyses. Magnetic bead-based immunoassays (MBBAs) allow for implementation into an immunosensor for online testing. Particles are prepared by either coupling the analyte molecule (diclofenac) or the respective antibody (amoxicillin) to the surface. For miniaturization, detection is performed electrochemically (chronoamperometry) on a microfluidic chip. The developed immunosensor will enable detection of pharmaceuticals directly in water supply pipes.
Immunanalytische Bestimmung von β-Lactam-Antibiotika und deren Hydrolyseprodukten im Trinkwasser
(2020)
Die speziellen Herausforderungen der immunanalytischen Bestimmung von β-Lactam-Antibiotika in (Trink-)Wasserproben werden anhand eines neu entwickelten ELISA (enzyme-linked immunosorbent assay) für das Breitbandantibiotikum Amoxicillin diskutiert. Es konnte gezeigt werden, dass die Hydrolyse des Analyten einen positiven Einfluss auf die Erkennung durch den Antikörper hat. Das entsprechende Hydrolyseprodukt, zu dem der Antikörper folglich eine höhere Affinität aufweist, wurde identifiziert und ein entsprechendes Verhalten wurde auch für andere β-Lactam-Antibiotika aus der Gruppe der Penicilline beobachtet. Eine Strategie für die Analyse von Wasserproben hinsichtlich Kontamination mit den hier betrachteten β-Lactamen und deren Hydrolyseprodukten wurde ausgearbeitet.
Wir berichten hier über die Entwicklung plattenbasierter Magnetpartikel-Immunoassays für die Bestimmung des Breitbandantibiotikums Amoxicillin und des nicht-steroidalen Antirheumatikums Diclofenac im Trinkwasser. Für die beiden Analyten wurden unterschiedliche Ansätze entwickelt, mit partikelgebundenen Antikörpern für AMX und Analyt-gekoppelten Partikeln für DCF, die die simultane Detektion beider Kontaminanten im nanomolaren Konzentrationsbereich erlauben.
Der Bedarf nach schnellen und kostengünstigen Analysemethoden in der (Trink-)Wasseranalytik steigt mit dem zunehmenden Eintrag pharmazeutisch aktiver Substanzen in die Umwelt.
Insbesondere die Kontamination mit Antibiotika erscheint neben ökotoxikologischen Effekten vor allem in Hinblick auf die Evolution resistenter Keime im Wasser besorgniserregend.
Wir berichten hier über die Entwicklung eines magnetpartikelbasierten Immunoassays (MBBA) zur Detektion des Breitbandantibiotikums Amoxicillin (AMX) in Wasserproben, der im Vergleich zum entsprechenden Enzyme-linked Immunosorbent Assay (ELISA) eine wesentlich kürzere Analysendauer sowie einen breiteren Messbereich mit niedrigerer Nachweisgrenze aufweist. Das angewendete Assayprinzip ist dabei leicht auf weitere Analyten übertragbar und ermöglicht die Implementierung in einen Immunosensor.
The demand for quick and reliable online sensors is rising not only in the field of environmental analysis with new and diversifying sources of pollution. A major aspect of this is the contamination of (drinking) water with pharmaceuticals representing a potential health threat and requiring timely countermeasures in case of contamination.
In this regard, antibody-based methods can be helpful as they enable fast and cost-effective on-site and potentially online analyses. The transition from the conventional plate-based formats like ELISA (enzyme-linked immunosorbent assay) to an immunosensor fit for online sensing can be achieved by using magnetic microparticles as a mobile sensing platform.
Beads are prepared by either coupling the antibody or the analyte to the surface. Both these strategies are presented using the example of two relevant contaminants in water: diclofenac (DCF) & amoxicillin (AMX), and can be transferred to other analytes.
The developed magnetic bead-based assays (MBBAs) exhibit lower LODs, enhanced sensitivity as well as shorter time of analysis than the respective ELISAs and can be integrated into an immunosensor with electrochemical detection on a microfluidic chip.
Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β-lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.
We present the development of magnetic bead-based immunoassays for two potential contaminants of drinking water: the nonsteroidal anti-inflammatory drug diclofenac and the broad-spectrum antibiotic amoxicillin. For these analytes, different assay formats were developed which can generally be transferred to other analytes. The use of magnetic beads not only enables implementation of the assays into an online system since the beads can be reversibly immobilized by applying a magnetic field. Furthermore, our MBBAs hold several advantages over the conventional ELISAs (enzyme-linked immunosorbent assays) for both analytes, such as lower limits of quantification, shorter assay duration and less washing steps required.
Strategies for the setup and miniaturization of an online immunosensor including electrochemical readout on a microfluidic chip will be discussed.
Die zunehmende Kontamination von (Trink)-Wasser mit pharmazeutischen Rückständen stellt ein potenzielles Gesundheitsrisiko dar und verlangt nach schnellen und zuverlässigen Analysemethoden. Hierbei können antikörper-basierte Methoden helfen, da sie schnelle und kostengünstige Vor-Ort-Messungen sowie Onlineanalysen ermöglichen.
Der Übergang von konventionellen plattenbasierten Formaten, wie ELISA (Enzyme-linked Immunosorbent Assay), zu einem Immunosensor für Online-Messungen wird durch den Einsatz magnetischer Mikropartikel als mobile Messplattform ermöglicht.
Die Partikel werden dazu entweder mit dem Antikörper oder dem Analyten gekoppelt. Diese beiden Strategien werden am Beispiel zweier relevanter Kontaminanten des Wassers gezeigt: Diclofenac (DCF) & Amoxicillin (AMX), und sind leicht auf andere Analyten übertragbar. Die magnetpartikelbasierten Assays (MBBAs) weisen niedrigere Nachweisgrenzen, breitere Messbereiche sowie kürzere Analysezeiten als die entsprechenden ELISAs auf und können in einen Immunosensor mit elektrochemischer Detektion auf einem mikrofluidischen Chip integriert werden.
Magnetpartikelbasierte Immunoassays für die Bestimmung von Amoxicillin und Diclofenac im Trinkwasser
(2021)
Wir berichten hier über die Entwicklung von magnetpartikelbasierten Immunoassays für die Bestimmung zweier relevanter Kontaminanten des Trinkwassers: Diclofenac (DCF) und Amoxicillin (AMX). Für die beiden Analyten wurden die Assays nach unterschiedlichen Prinzipien entwickelt, die sich auch auf weitere Analyten übertragen lassen. Im Vergleich zum herkömmlichen ELISA weisen die hier entwickelten magnetpartikelbasierten Assays einen größeren Messbereich mit niedrigeren Bestimmungsgrenzen bei gleichzeitig deutlich reduzierter Analysendauer auf. Darüber hinaus können die Immunoassays in ein automatisierbares Onlinesystem zur Analyse in der Trinkwasserversorgung implementiert werden. Strategien hierfür werden erläutert.
Contamination of drinking water with pharmaceuticals is an emerging problem and a potential health threat. Immunochemical methods based on the binding of the analyte to specific antibodies enable fast & cost-effective on-site analyses. Magnetic bead-based immunoassays allow for implementation into an immunosensor for online testing. Particles are prepared by either coupling the analyte molecule (diclofenac) or the respective antibody (amoxicillin) to the surface. For miniaturization, detection is performed electrochemically (chronoamperometry) on a microfluidic chip. The developed immunosensor will enable detection of pharmaceuticals directly in water supply pipes.
Contamination of drinking water with pharmaceuticals is an emerging problem and a potential health threat. Immunochemical methods based on the binding of the analyte to specific antibodies enable fast and cost-effective on-site analyses. Magnetic bead-based immunoassays allow for implementation into an immunosensor for online testing. These particles are prepared by either coupling the analyte molecule (diclofenac) or the respective antibody (amoxicillin) to the surface. For miniaturization, detection is performed electrochemically on a microfluidic chip. The developed immunosensor will enable detection of pharmaceuticals directly in water supply pipes.
Herein we present the data for a newly developed immunoassay for the determination of amoxicillin (AMX) in water samples using a commercially available monoclonal anti-AMX antibody. During optimization and testing of the assay, we observed a significant decrease of the C-value (IC50) of the ELISA calibration curves with aged standard solutions. Running the assay with standard solutions of synthesized AMX hydrolysis products revealed that this decreased C-value could be attributed mainly to the hydrolysis product amoxicilloic acid. Therefore, the limit of detection of the ELISA for AMX determination in water could be enhanced by either hydrolyzing the samples or testing for amoxicilloic acid as marker substance.
The environmental fate of the frequently used broad-spectrum β-lactam antibiotic amoxicillin (AMX) is of high concern regarding the potential evolution of antimicrobial resistance (AMR). Moreover, it is known that AMX is prone to hydrolysis, yielding a variety of hydrolysis products (HPs) with yet unknown effects. Studies to identify those HPs and investigate their formation mechanisms have been reported but a long-term study on their stability in real water samples was missing. In this regard, we investigated the hydrolysis of AMX at two concentration levels in four distinct water types under three different storage conditions over two months. Concentrations of AMX and four relevant HPs were monitored by an LC-MS/MS method revealing pronounced differences in the hydrolysis rate of AMX in tap water and mineral water on the one hand (fast) and surface water on the other(slow). In this context, the occurrence, relative intensities, and stability of certain HPs are more dependent on the water type than on the storage condition. As clarified by ICP-MS, the main difference between the water types was the content of the metals copper and zinc which are supposed to catalyze AMX hydrolysis demonstrating an effective method to degrade AMX at ambient conditions.
Contamination of the environment with antibiotics is of great concern as it promotes the evolution of antimicrobial resistances. In case of amoxicillin (AMX) in the aquatic environment, further risk arises from hydrolysis products (HPs) which can cause allergy. To assess these risks, a comprehensive investigation and understanding of the degradation of AMX is necessary. We investigated the hydrolysis rate of AMX in different types of water as well as the influence of temperature and irradiation. The content of the heavy metal ions copper and zinc was found to be crucial for the hydrolysis rate of AMX and stability of HPs. Eventually, a new degradation pathway for AMX could be elaborated and confirmed by tandem mass spectrometry (LC-MS/MS).
Contamination of drinking water with pharmaceuticals is an emerging problem and a potential health threat. Immunochemical methods based on the binding of the analyte to specific antibodies enable fast & cost-effective on-site analyses. Magnetic bead-based immunoassays (MBBAs) allow for implementation into an immunosensor for online testing. Particles are prepared by either coupling the analyte molecule (diclofenac) or the respective antibody (amoxicillin) to the surface. For miniaturization, detection is performed electrochemically (chronoamperometry) on a microfluidic chip. The developed immunosensor will enable detection of pharmaceuticals directly in water supply pipes.
Immunoassays, based on the recognition and capture of analytes by highly selective antibodies, are now used extensively in all areas of diagnostics, but the challenge is to further integrate them into online sensors. To improve the transition from laboratory immunoassays to immunosensors, we have developed a complete flow system, based on a microfluidic core flow cell to enable automated detection of one of the most commonly used immunoassay substrates, TMB, by chronoamperometry. The architecture and fluidic optimisation of the system showed that a specially designed 3D flow cell allows higher flow rates (500 μL min−1) than a standard enlarged microfluidic channel (50 μL min−1) resulting in a significantly shorter detection time of 30 seconds per sample and making the system more robust against interferences due to bubble formation in the chip. The electrochemical measurements showed an improved signal-to-noise ratio (SNR) and thus higher sensitivity for a model immunoassay for diclofenac (SNR = 59), compared to the analytical performance of a conventional laboratory microplate-based assay with optical detection (SNR = 19). In general, this system facilitates the conversion of any conventional immunoassay into an immunosensor with automatic and continuous detection.
The contamination of drinking water with pharmaceuticals represents a severe health risk. In order to monitor the drinking water quality continuously and enable quick countermeasures in case of contamination, novel sensors are required. Here, immunoanalytical methods based on the binding of the analyte to highly selective antibodies can be helpful. In this work, magnetic bead-based immunoassays (MBBAs) have been developed for the detection of two relevant contaminants of drinking water: diclofenac (DCF) and amoxicillin (AMX). In case of the latter, not only the parent drug is of interest in the risk assessment but also its hydrolysis products (HPs). In a comprehensive study, the influence of external factors and intrinsic properties of the water on the rate of hydrolysis was investigated. As the hydrolysis of AMX further impacts the recognition by the antibody, a strategy to analyze samples with unknown hydrolysis degree of AMX was established employing the enzyme β-lactamase in sample preparation. For both analytes, the MBBAs enable the fast quantification with results obtained in less than one hour which represents a major improvement over conventional immunoassays like the enzyme-linked immunosorbent assay (ELISA). Compared to the respective ELISAs with the same antibodies, the MBBAs further exhibit improved analytical parameters such as a broader measurement range and lower limits of detection. Due to the magnetic properties of the beads that serve as a platform for the assays, they are suitable for the mobile and automated detection at the point-of-care. An integrated diagnostic system was designed in which electrochemical detection with chronoamperometry on a microfluidic chip allows for further miniaturization of the system to enable monitoring of the drinking water quality online in water supply pipes at waterworks.