TY - GEN A1 - Wilke, Marco A1 - Röder, Bettina A1 - Paul, Martin A1 - Weller, Michael G. T1 - Sintered Glass Monoliths as New Supports for Affinity Columns T2 - Preprints N2 - A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run. KW - Affinity Chromatography KW - Glass KW - Purification KW - Antibodies KW - Solid support KW - HPLC KW - FPLC KW - Separation PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529117 DO - https://doi.org/10.20944/preprints202103.0298.v1 SP - 1 PB - MDPI CY - Basel AN - OPUS4-52911 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Paul, Martin A1 - Tannenberg, Robert A1 - Tscheuschner, Georg A1 - Wilke, Marco A1 - Weller, Michael G. T1 - Cocaine Detection by a Laser-induced Immunofluorometric Biosensor T2 - Preprints N2 - The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement. KW - Drug search KW - Customs KW - Confiscation KW - Border surveillance KW - Narcotics KW - International drug trade KW - Drug trafficking KW - Illicit drug KW - Immunosensor KW - Antibodies KW - Detection PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529951 DO - https://doi.org/10.20944/preprints202107.0521.v1 SP - 1 PB - MDPI CY - Basel AN - OPUS4-52995 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - THES A1 - Ecke, Alexander T1 - Integrated Diagnostics of Pharmaceutical Contaminants in Water Supply and Management Systems N2 - 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. N2 - Die Kontamination von Trinkwasser mit Arzneimitteln stellt eine ernste Gesundheitsgefahr dar. Um die Trinkwasserqualität kontinuierlich überwachen und im Falle einer Verunreinigung zeitnah reagieren zu können, sind neuartige Sensoren erforderlich. Hier können immunanalytische Methoden, die auf der Bindung des Analyten an hochselektive Antikörper beruhen, hilfreich sein. In dieser Arbeit wurden magnetpartikelbasierte Immunoassays (MBBAs) für zwei relevante Kontaminanten des Trinkwassers entwickelt: Diclofenac (DCF) und Amoxicillin (AMX). Bei letzterem erwiesen sich neben der Ausgangsverbindung auch dessen Hydrolyseprodukte (HPs) als relevant für die Gefährdungsbeurteilung. In einer umfassenden Studie wurde der Einfluss von externen Faktoren und intrinsischen Eigenschaften des Wassers auf die Hydrolysegeschwindigkeit untersucht. Da die Hydrolyse von AMX auch die Erkennung durch den Antikörper beeinflusst, wurde eine Strategie zur Analyse von Proben mit unbekanntem Hydrolysegrad von AMX unter Verwendung des Enzyms β-Lactamase in der Probenvorbereitung entwickelt. Für beide Analyten ermöglichen die MBBAs eine schnelle Quantifizierung mit Ergebnissen in weniger als einer Stunde, was eine wesentliche Verbesserung gegenüber herkömmlichen Immunoassays wie dem Enzyme-linked Immunosorbent Assay (ELISA) darstellt. Im Vergleich zu den entsprechenden ELISAs mit denselben Antikörpern weisen die MBBAs zudem verbesserte analytische Parameter auf, wie einen breiteren Messbereich und niedrigere Nachweisgrenzen. Aufgrund der magnetischen Eigenschaften der Partikel, die als Plattform für die Assays dienen, eignen sie sich für den mobilen und automatisierten Einsatz vor Ort. Ein integriertes Diagnosesystem, bei dem die elektrochemische Detektion mittels Chronoamperometrie auf einem mikrofluidischen Chip eine weitere Miniaturisierung des Systems ermöglicht, wurde entworfen, um die Überwachung der Trinkwasserqualität online in Wasserwerken zu ermöglichen. KW - Antibiotics KW - Immunoassay KW - Amoxicillin KW - Diclofenac KW - Biosensor KW - Antibodies PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-591901 DO - https://doi.org/10.18452/25853 SP - 1 EP - 129 CY - Berlin AN - OPUS4-59190 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -