Flow-Based CL-SMIA for the Quantification of Protein Biomarkers from Nasal Secretions in Comparison with Sandwich ELISA.

Protein biomarkers in nasal secretions can be used as a measure to differentiate between allergies, airway diseases and infections for non-invasive diagnostics. The point-of-care quantification of biomarker levels using flow-based microarray facilitates precise and rapid diagnosis and displays the potential for targeted and effective treatment. For the first time, we developed a flow-based chemiluminescence sandwich microarray immunoassay (CL-SMIA) for the quantification of nasal interferon-beta (IFN-ß) on the Microarray Chip Reader-Research (MCR-R). Polycarbonate foils are used as a cost-effective surface for immobilizing capture antibodies. By using a commercially available set of anti-human IFN-ß antibodies, the CL-SMIA can be compared directly to an enzyme-linked immunosorbent assay (ELISA) performed in microtiter plates concerning the bioanalytical performance and economic issues. Pre-incubation of the sample with detection antibodies facilitates the lower consumption of detection antibodies, as this allows for a longer interaction time between the antibody and the biomarker. The direct injection of pre-incubated samples into the microarray chips eliminates the adsorption of proteins in the tubing as well as the contamination of the tubing and valves of the MCR-R with clinical samples. The small flow cell allows for a low sample volume of 50 µL. The limit of detection of 4.53 pg mL-1 was slightly increased compared to a sandwich ELISA performed on microtiter plates which were 1.60 pg mL-1. The possibility to perform the CL-SMIA in a multiplexed mode makes it a promising assay for the rapid and cost-effective non-invasive detection of biomarkers in nasal secretions.

Macroporous Epoxy-Based Monoliths Functionalized with Anti-CD63 Nanobodies for Effective Isolation of Extracellular Vesicles in Urine.

Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.

Flow-Based Chemiluminescence Microarrays as Screening Platform for Affinity Binders to Capture and Elute Bacteria.

Affinity describes the non-covalent but selective interaction between an affinity binder (e.g., proteins, antibiotics, or antibodies) and its counterpart (e.g., bacteria). These affinity binders can serve to detect bacteria and respond to the need for selective concentration via affinity chromatography for trace analysis. By changing the pH value or salt and protein contents, affinity bindings can be reversed, and bacteria can be recovered for characterisation. Analytical microarrays use multiple affinity binders immobilised on the surface in a distinct pattern, which immensely reduces screening time for the discovery of superior binding motifs. Here, flow-based microarray systems can inform not only about binding, but also about desorption. In this work, we pioneer a screening assay for affinity binders against both gram-positive and negative bacteria based on an automated flow-based chemiluminescence (CL) microarray. Biotinylation of model organisms E. coli and E. faecalis enabled labelling with horseradish-peroxidase-coupled streptavidin, and detection with CL. Polymyxin B, an antibiotic against gram-negative bacteria, was found to bind both E. coli and E. faecalis. Simultaneous screening for desorption methods unexpectedly revealed methyl alpha-D-mannopyranoside as a promising buffer for desorption from Polymyxin B. This proof-of-principle study shows that our new platform greatly facilitates the screening of new affinity binders against bacteria, with promise for future automation.