Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.

Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer-target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics.

Aptamer-modified biosensors to visualize neurotransmitter flux.

Chemical biosensors with the capacity to continuously monitor various neurotransmitter dynamics can be powerful tools to understand complex signaling pathways in the brain. However, in vivo detection of neurochemicals is challenging for many reasons such as the rapid release and clearance of neurotransmitters in the extracellular space, or the low target analyte concentrations in a sea of interfering biomolecules. Biosensing platforms with adequate spatiotemporal resolution coupled to specific and selective receptors termed aptamers, demonstrate high potential to tackle such challenges. Herein, we review existing literature in this field. We first discuss nanoparticle-based systems, which have a simple in vitro implementation and easily interpretable results. We then examine methods employing near-infrared detection for deeper tissue imaging, hence easier translation to in vivo implementation. We conclude by reviewing live cell imaging of neurotransmitter release via aptamer-modified platforms. For each of these sensors, we discuss the associated challenges for translation to real-time in vivo neurochemical imaging. Realization of in vivo biosensors for neurotransmitters will drive future development of early prevention strategies, treatments, and therapeutics for psychiatric and neurodegenerative diseases.

Biosynthesis of Glycoconjugate Virus-like Particles (VLPs).

The outermost surface of bacterial pathogens consists primarily of complex carbohydrate structures-polysaccharides, glycolipids, and glycoproteins. To raise a long-lasting and effective immune response against carbohydrate antigens, they generally require covalent attachment to an immunogenic carrier protein-a so-called glycoconjugate vaccine. One hurdle to the development of glycoconjugate vaccines is that carbohydrate antigens remain inaccessible to recombinant production. Thus, the carbohydrate antigen is typically purified from the pathogen and then chemically conjugated to an immunogenic protein. Recent developments in the field of bacterial glycoengineering have opened the opportunity for total recombinant production of glycoconjugate vaccines. In this method, we describe the production of proteinaceous, virus-like particles (VLPs) bearing the conserved N-glycan of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumoniae.