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Electrochemical methods offer great promise in meeting the demand for user-friendly on-site devices for Monitoring important parameters. The food industry often runs own lab procedures, for example, for mycotoxin analysis, but it is a major goal to simplify analysis, linking analytical methods with smart technologies. Enzyme-linked immunosorbent assays, with photometric detection of 3,3’,5,5’-tetramethylbenzidine (TMB),form a good basis for sensitive detection. To provide a straightforward approach for the miniaturization of the detectionstep, we have studied the pitfalls of the electrochemical TMB detection. By cyclic voltammetry it was found that the TMB electrochemistry is strongly dependent on the pH and the electrode material. A stable electrode response to TMB could be achieved at pH 1 on gold electrodes. We created a smartphonebased, electrochemical, immunomagnetic assay for the detection of ochratoxin A in real samples, providing a solid basis forsensing of further analytes.
Partikelbasierte Multianalyt- Fluoreszenzimmunoassays für die Unweltanalytik und Biotechnologie
(2021)
Zur Beurteilung der Wasserqualität können Markersubstanzen, wie Carbamazepin, Diclofenac, Koffein und Gallensäuren, verwendet werden. Erhöhte Konzentrationen dieser anthropogenen Marker weisen auf Einleitstellen von Abwasser hin und zeigen somit an, wo Gewässer verschmutzt werden. Diese Arbeit beschreibt deshalb die Etablierung, Optimierung und Validierung eines partikelbasierten Suspensionsarray Fluoreszenzimmunoassays (SAFIA), mit dem diese Substanzen gleichzeitig und parallel in vielen Proben quantitativ bestimmt werden können. Für SAFIA wurde das Format des kompetitiven indirekten Immunoassays gewählt. Als Plattform wurden fluoreszenzintensitätscodierte Polystyrol-Kern/Siliziumdioxid-Schale-Partikel, auf deren Oberfläche Haptene als kompetitive Bindungsstellen für Antikörper immobilisiert wurden, verwendet. Hier zeigte sich, dass eine selektive Erkennung der Haptenstruktur auf der Oberfläche der Partikel durch die Antikörper nur gegeben war, wenn die Partikel zusätzlich mit Polyethylenglykolgruppen funktionalisiert wurden. Zum Auslesen des Fluoreszenzsignals wurde ein Durchflusszytometer verwendet. Kompatibilität mit der Durchführung in Mikrotiterplatten wurde erreicht, indem eine Stopp-lösung für den SAFIA, basierend auf Formaldehyd, entwickelt wurde. Nach der Optimierung der Assayparameter können die oben genannten vier Analyten gleichzeitig in einer Probe mindestens bis zu einer Konzentration von 0.3 μg L-1 nachgewiesen werden. Der Assay kann ohne Wasch-schritte ausgeführt werden und ist somit einfacher als konventionelle Immunoassays, wie z. B. ELISA. Der SAFIA wurde hinsichtlich Interferenzen und Selektivität untersucht. Dabei zeigte sich, dass Matrixbestandteile, die in wässrigen Umweltproben vorkommen, keinen bzw. nur einen ge-ringen Einfluss auf den SAFIA hatten. Im simulierten Umweltscreening eines Flusses und mithilfe der Analyse von Abwasserproben wurde SAFIA validiert; hier zeigte SAFIA eine mit ELISA vergleich-bare Genauigkeit, bei gleichzeitiger Senkung der Analysenzeit und -kosten. Die gleichzeitige Detektion mehrerer anthropogener Marker erlaubte zudem Rückschlüsse auf die Art von Verschmutzungsquellen, im Gegensatz zu ELISA, mit dem nur ein Analyt bestimmt werden kann. Da hochaffine Antikörper für Immunoassays essenziell sind, wurde ein SAFIA zum Screening antikörperproduzierenden Zellen (Hybridomzellen) entwickelt und eingesetzt. Durch die Implementierung eines Sandwichimmunoassays zur IgG Bestimmung und durch Verwendung von homo- und heterologen Haptenstrukturen konnten aus einem Pool von Hybridomzellen diejenigen ausgewählt werden, deren sekretierter Antikörper die geringsten Nachweisgrenzen im Immunoassay erlauben. Damit konnte das Anwendungsspektrum des SAFIA maßgeblich vergrößert werden. Er vereinfacht als biotechnologisches Werkzeug stark sowohl die Produktion von monoklonalen Antikörpern als auch die Analyse von Umweltproben.
Monitoring water quality regarding emerging pollutants, such as drug residues, demands for selective, high-throughput and multi-target analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored. On the other hand, estimation of removal efficiencies of pollutants, such as drug residues, is in the focus of industrial and public wastewater treatment. Immunoassays, such as ELISA, offer the possibility to be highly sensitive and selective due to the high target affinity and specificity of target recognition by antibodies. Batch-wise processing in microtiter plates allows for the necessary high-throughput, but only a single analyte can be determined within one measurement.
To overcome these disadvantages, we developed a four-plex Suspension Array Fluorescence Immunoassay (SAFIA), which is adaptable for the microtiter plate format. The modular and self-prepared bead support consists of polystyrene-core silica-shell microparticles. While the polystyrene core is used for encoding, by introducing different amounts of fluorescent dyes, the silica shell creates a solid-support for the immunoassay: the target analytes, three drugs, carbamazepine, diclofenac and caffeine, and the fecal marker isolithocholic acid are covalently coupled to amino groups on the surface via NHS chemistry. Specific recognition of these haptens is accomplished by further introduction of PEG moieties, suppressing non-specific binding. A competitive immunoassay is subsequently conducted in a simple mix-and-read procedure, eliminating the demand for laborious washing steps and decreasing time-to-result. An automated flow cytometer is used for simultaneous decoding and quantification.
After optimization, the SAFIA showed limits of detection for all analytes in the low µg L 1 range, meeting the sensitivity criteria for wastewater analysis. Then, applicability of the SAFIA was studied on real wastewater samples from three different wastewater treatment plants in Berlin. The results were in good comparability to LC-MS/MS indicating high matrix stability. Moreover, the accuracy of the assay exceeded that of the respective ELISA. Finally, we used SAFIA to assess the influent of treated and untreated wastewater on the Douro river estuary in Portugal. The obtained results of the analysis were comparable to ELISA. However, the measurements by SAFIA could be carried out in one quarter the time of analysis required for ELISA.
Acknowledgements: The authors acknowledge financial support from Deutscher Akademischer Austauschdienst and from Fundação das Universidades Portuguesas under the protocol CRUP-DAAD (Ações Integradas Luso-Alemãs nºE-20/16, DAAD Zuwendungsvertrag 57212899).
Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers.
Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater Treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension Array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer.
For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater Treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.
The development of an automated miniaturized analytical system that allows for the rapid monitoring of carbamazepine (CBZ) levels in serum and wastewater is proposed. Molecular recognition of CBZ was achieved through its selective interaction with microbeads carrying anti-CBZ antibodies. The proposed method combines the advantages of the micro-bead injection spectroscopy and of the flow-based platform lab-on-valve for implementation of automatic immunosorbent renewal, rendering a new recognition surface for each sample. The sequential (or simultaneous) perfusion of CBZ and the horseradish peroxidase-labelled CBZ through the microbeads is followed by real-time on-column Monitoring of substrate (3,30,5,50-tetramethylbenzidine) oxidation by colorimetry. The evaluation of the initial oxidation rate and also the absorbance value at a fixed time point provided a linear response versus the logarithm of the CBZ concentration. Under the selected assay conditions, a single analysis was completed after only 11 min, with a quantification range between 1.0 and 50 µg L⁻¹. Detection of CBZ levels in undiluted wastewater samples was feasible after a simple filtration step while good recoveries were attained for spiked certified human serum, analyzed without sample clean-up.
Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.
Commercial bead-based assays are commonly built upon polystyrene particles. The polymeric carrier can be encoded with organic dyes and has ideal material properties for cytometric applications such as low density and high refractive index. However, functional groups are conventionally integrated during polymerization and subsequent modification is limited to the reactivity of those groups. Additionally, polystyrene as the core material leads to many hydrophobic areas still being present on the beads’ surfaces even after functionalization, Rendering the particles prone to nonspecific adsorption during an application. The latter calls for several washing steps and the use of additives in (bio)analytical assays. In this contribution, we show how these limitations can be overcome by using monodisperse polystyrene (PS) core/silica (SiO2) shell particles (SiO2@PS). Two different hydrophobic BODIPY (boron−dipyrromethene) dyes were encapsulated inside a poly(vinylpyrrolidone) (PVP) -stabilized polystyrene core in different concentrations to create 5-plex arrays in two separate detection channels of a cytometer. A subsequent modification of the silica shell with an equimolar APTES/PEGS (aminopropyltriethoxysilane/polyethylene glycol silane) blend added multifunctional properties to the hybrid core/Shell microparticles in a single step: APTES provides amino groups for the attachment of a caffeine derivative (as a hapten) to create antigen-coupled microspheres; the PEG moiety effectively suppresses nonspecific binding of antibodies, endowing the surface with antifouling properties. The particles were applied in a competitive fluorescence immunoassay in suspension, and a highly selective wash-free assay for the detection of caffeine in beverages was developed as a proof of concept.
Clinical, environmental, and food analysis, require efficient and accurate quantitative analysis. Immunoassays take advantage of highly selective and affine binding of an antibody towards an antigen, being easy-to-use and allowing for high sample throughput. However, common immunoassays, e. g. ELISA are not capable of multiplexed analysis, thus limiting possible applications. On the other hand, multi-analyte methods, e.g. liquid chromatographytandem mass spectrometry requires expensive equipment, trained analysts and the time of analysis usually limits sample throughput. This bottleneck can be overcome combining the suspension array technology, where particles are encoded to allow multiplexed detection, with fluorescence immunoassays to create suspension array fluorescence immunoassays (SAFIA).
Polystyrene core/silica shell microparticles serve as platform for SAFIA. While an encoding dye is introduced in the polystyrene core, the silica shell is used for immobilization: For competitive hapten immunoassays small molecules are immobilized on the surface, serving as a competitive binding site for selective antibodies. For quantification of proteins, antibodies can be immobilized on the surface and the sandwich immunoassay format can be employed. All mentioned immunoassays can be executed wash-free and highly parallelized, allowing up to 500 determinations within three hours. In contrast to ELISA, short incubation times, a wash-free mix-and-read procedure and automated flow-cytometric read-out makes SAFIA applicable for even non-trained personal.
SAFIA showed excellent performance in studies on the origin and fate of drug residues in waste water, which is important to assess clearance rates of wastewater treatment plants. Furthermore, SAFIA can be employed in environmental screenings, detecting pollution of natural river waters by tracking anthropogenic marker substances. It was successfully employed in clinical and food analysis applications, e.g. the quantification of the anti-inflammatory drug diclofenac in breast milk or the quantification of caffeine in beverages. Due to multiplexing capacities, SAFIA can significantly improve biotechnological processes, as in the screening of hybridoma cells for selective and efficient antibody production. For small molecule analytes, limits of detection down to 4 ng/L and for proteins below 25 ng/mL (IgG) were observed, which makes SAFIA applicable to all addressed analytical issues. Moreover, in the analysis of real-world samples, SAFIA shows higher accuracy in contrast to ELISA, indicating higher matrix stability and thus higher robustness in analysis.
Spheriplex multiplexed immunoassays: A practical tool for environmental, food and bioanalysis
(2019)
Clinical, environmental, and food analysis, require efficient and accurate quantitative analysis. Immunoassays take advantage of highly selective and affine binding of an antibody towards an antigen, being easy-to-use and allowing for high sample throughput. However, common immunoassays, e. g. ELISA are not capable of multiplexed analysis, thus limiting possible applications. On the other hand, multi-analyte methods, e.g. liquid chromatographytandem mass spectrometry requires expensive equipment, trained analysts and the time of analysis usually limits sample throughput. This bottleneck can be overcome combining the suspension array technology, where particles are encoded to allow multiplexed detection, with fluorescence immunoassays to create suspension array fluorescence immunoassays (SAFIA).
Polystyrene core/silica shell microparticles serve as platform for SAFIA. While an encoding dye is introduced in the polystyrene core, the silica shell is used for immobilization: For competitive hapten immunoassays small molecules are immobilized on the surface, serving as a competitive binding site for selective antibodies. For quantification of proteins, antibodies can be immobilized on the surface and the sandwich immunoassay format can be employed. All mentioned immunoassays can be executed wash-free and highly parallelized, allowing up to 500 determinations within three hours. In contrast to ELISA, short incubation times, a wash-free mix-and-read procedure and automated flow-cytometric read-out makes SAFIA applicable for even non-trained personal.
SAFIA showed excellent performance in studies on the origin and fate of drug residues in waste water, which is important to assess clearance rates of wastewater treatment plants. Furthermore, SAFIA can be employed in environmental screenings, detecting pollution of natural river waters by tracking anthropogenic marker substances. It was successfully employed in clinical and food analysis applications, e.g. the quantification of the anti-inflammatory drug diclofenac in breast milk or the quantification of caffeine in beverages. Due to multiplexing capacities, SAFIA can significantly improve biotechnological processes, as in the screening of hybridoma cells for selective and efficient antibody production. For small molecule analytes, limits of detection down to 4 ng/L and for proteins below 25 ng/mL (IgG) were observed, which makes SAFIA applicable to all addressed analytical issues. Moreover, in the analysis of real-world samples, SAFIA shows higher accuracy in contrast to ELISA, indicating higher matrix stability and thus higher robustness in analysis.