FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

Dielectric Nanoparticles Coated upon Silver Hollow Nanosphere as an Integrated Design to Reinforce SERS Detection of Trace Ampicillin in Milk Solution

Surface-enhanced Raman scattering (SERS) technique is competent to trace detection of target species, down to the single molecule level. The detection sensitivity is presumably degraded by the presence of non-specific binding molecules that occupy a SERS-active site (or hot spot) on the substrate surface. In this study, a silver hollow nano-sphere (Ag HNS) with cavity has been particularly designed, followed by depositing dielectric nanoparticles (Di NPs) upon Ag HNS. In the integrated nanostructures, Di NPs/Ag HNS were furthermore confirmed by cutting through the cross sections using the Focused Ion Beam (FIB) technique, which provides the Scanning Electron Microscope (SEM) with Energy-dispersive Spectroscope (EDS) mode for identifying the distribution of Di NPs upon Ag HNS. The results have indicated that Di NPs/Ag HNS exhibits small diameter of cavity, and among Di NPs in this study, Al2O3 with lower dielectric constant provides a much higher SERS enhancement factor (e.g., ~6.2 × 107). In this study, to detect trace amounts (e.g., 0.01 ppm) of Ampicillin in water or milk solution, Al2O3 NPs/Ag HNS was found to be more efficient and less influenced by non-specific binding molecules in milk. A substrate with integrated plasmonic and dielectric components was designed to increase the adsorption of target species and to repulse non-specific binding molecules from SERS-active sites.

Microfluidic Device for Screening for Target Cell-Specific Binding Molecules by Using Adherent Cells

This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on the surface of target cells, washing away unbound molecules, and collecting target cell-specific binding molecules from target cells. These functions were effectively implemented by using our previously developed micro pillar arrays for cell homogeneous dispersion and pneumatic microvalves for tall microchannels. The device was also equipped with serially connected filter chambers in which non-target cells were cultured to reduce the molecules binding to non-target cells as much as possible. We evaluated the performance of the device using cancer cell lines (N87 cells as target cells and HeLa cells as non-target cells) and two fluorescent dye-labeled antibodies: Anti-human epidermal growth factor receptor 2 (anti-HER2) antibody that binds to target cells and anti-integrin antibody that binds to non-target cells. The results showed that the device could reduce anti-integrin antibodies to the detection limit of fluorescent measurement and collect anti-HER2 antibodies from the target cells.

Beyond antibodies ? lessons from bacterial ?immunity?

Isolation and production of highly specific protein-based binding molecules are crucial to the ever expanding diagnostics, therapeutics and protein array fields. Traditionally, such reagents have been sourced from vertebrate immune systems, where antibodies have evolved over millennia into highly effective molecules of immune surveillance capable of targeting a huge range of targets in response to infection and disease. Now, a growing number of alternative protein scaffolds are being investigated as specific binding molecules incorporating a diverse and powerful range of binding and recognition interfaces. These are being sourced from human proteins, from alternative immune molecules present in evolutionarily old vertebrates, and from highly evolved binding proteins in prokaryotic systems.

Affinity Capillary Electrophoresis for the Screening of Novel Antimicrobial Targets

The increasing number of multiantibiotic-resistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), requires the development of novel chemotherapies that are structurally distinct and exempt from current resistance mechanisms. Bioinformatics data mining of microbial genomes has revealed numerous previously unexploited essential open reading frames (ORFs) of unknown biochemical function. The potential of these proteins as screening targets is not readily apparent because most screening technologies rely on knowledge of biological function. To address this problem, the authors employed affinity capillary electrophoresis (ACE) to identify antimicrobial compounds that bound the novel target YihA. Screening a small-molecule library of 44,000 compounds initially identified 115 binders, of which 76% were confirmed. Furthermore, the ACE assay distinguished diverse compounds that possessed drug-like properties and antimicrobial activity against drug-resistant clinical isolates. These data validate ACE as a valuable tool for the fast, efficient detection of specific binding molecules that possess biological activity.