Tony Tien

In cellular electron microscopy, large macromolecular objects like filaments and ribosomes can be resolved. However, smaller macromolecules require additional contrast and are difficult to directly identify. The Ackerson group at Colorado State University is working on an approach to impart unique contrast to macromolecules that cannot otherwise be identified in cellular EM. The approach is based on a Glutathione Reductase-Like Metalloid Reductase (GRLMR) enzyme, which converts soluble selenium ions to high-contrast selenium nanoparticles. To localize the SeNPs to the enzyme (and protein of interest), the enzyme was genetically fused to a SeNP binding peptide. To better understand how the peptide can control the size of the enzymatically created SeNPs, we are varying the number of peptide repeats fused to the enzyme. Preliminary data has shown that size control characteristics appear to vary with different tandem repeat quantities. Ongoing efforts include more rigorous characterization of nanoparticle sizes formed by these variants by combining higher throughput TEM data collection via SerialEM with nanoparticle classing in software like CryoSPARC. Further investigation, particularly through refining higher throughput data collection workflows, would yield information about improving particle size tunability for easily trackable EM contrast markers.

Flash nanoprecipitation (FNP) is a rapid mixing technique that can be used to make nanoparticles of desired size and character given appropriate stream turbulence and composition. In collaboration with the Prud’homme group at Princeton University, the Cash Lab is working on the best methods to characterize FNP nanosensors designed to measure oxygen concentration. Ten oxygen nanosensor samples were formulated by the Prud’homme group using varying compositions of pegylated polystyrene particles (PS-PEG), 4-(4-Dihexadecylaminostyryl)-N-methylpyridinium iodide) (DiA), 5,10,15,20-(tetraphenyl)porphyrin (PtTPP), vitamin E or vitamin E acetate, and other inert core components. Fluorescence data were used to characterize the nanosensors using a combination of glucose/glucose oxidase tests (G+GOx) compared to ambient oxygen. In analyzing the fluorescence data, sample #5 was determined to have the best oxygenated to deoxygenated fluorescence ratio as well as the highest overall fluorescence for subsequent analysis. Additional characterization was performed with sample #5 to quantify oxygen response using direct gas bubbling with air and nitrogen; however, the nanosensors exhibited variations in the reference dye peak (DiA) compared to the oxygen-responsive peak (PtTPP) during bubbling, leading to inconclusive results regarding reliable oxygen detection. Future work aims to further refine fluorescence techniques and re-evaluate reference dyes for FNP oxygen detection.

Bacterial biofilms pose barriers to the current antibiotic selection strategies for treating affected clinical patients quickly. Pseudomonas aeruginosa, in particular, is a bacteria commonly found in the environment that poses risks of lung, blood, and other bodily infections with high contraction risk for people with compromised immune systems, open or recovering wounds, or exposure to contaminated medical devices. P. aeruginosa also colonizes the lungs of people with cystic fibrosis, providing a targeted interest for a type of biofilm that has proven difficult to treat. The exploration of a layer-by-layer growth technique for an artificial biofilm aims to mimic in vivo biofilm characteristics to improve the diagnosis and treatment of patients infected with biofilms. A layer-by-layer dipping technique involving a sequence of polymers, GFP-expressing P. aeruginosa PA01GFP, and water rinses provided a basis for the growth of biofilms on circular, glass microscope coverslips. Biofilm deposition and bacterial incorporation within the biofilm were then analyzed with fluorescence and confocal spectroscopy. A future implementation of these biofilms aims to take advantage of metabolite-sensitive nanosensors developed by the Cash Lab for the development of an effective antibiotic screening test for clinical biofilm treatment.

Current methods of measuring analyte concentrations, including molecular sensors and probes, are limited in their selectivity, sensitivity, and invasiveness for use inside biological systems. The creation of polymeric nanosensors for analyte concentration determination in vivo is designed to address the current limitations of molecular sensors and probes, focusing particularly on selective sensitivity to biological analyte ranges. The research at hand aims to investigate the role of varying nanosensor charges in the response characteristics that the nanosensors produce for target analytes. In particular, changes in the nanoparticle zeta potentials are indicative of the stability of these nanosensors, which can be used to match the coagulative interaction levels in solution. The lipid membrane charge ratio is varied with combinations of positive 18:0 TAP, negative 16:0 PG, and neutral DMG-PEG-2000 lipids. Nanosensor zeta potential is measured by zeta phase analysis light scattering (PALS), while nanosensor functionality is measured with fluorescence intensity in response to varying biological analytes of interest, including lithium, potassium, sodium, and calcium ions. With this information, improved methods for biological analyte concentration determination could be used for enhanced biological compatibility and in vivo monitoring capabilities desired by biomedical applications.

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