Browsing by Subject "Fluorescence quenching"
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Item Investigating molecular effects on membrane structure, dynamics and function(2019-05-01) Anderson, Cari Michelle; Webb, Lauren J.; Elber, Ron; Baiz, Carlos R; Eberlin, Livia S; Gordon, VernitaBiological membranes are heterogeneous structures with complex electrostatic profiles arising from lipids, sterols, membrane proteins, and water molecules. We investigated the effect of cholesterol and its derivative 6-ketocholestanol (6-kc) on membrane electrostatics by directly measuring the dipole electric field (F [arrow above F] [subscript d] ) within lipid bilayers containing cholesterol or 6-kc at concentrations of 0−40 mol% through the vibrational Stark effect (VSE). We found that adding low concentrations of cholesterol, up to ∼10 mol %, increases F [arrow above F] [subscript d], while adding more cholesterol up to 40 mol% lowers F [arrow above F] [subscript d]. In contrast, we measured a monotonic increase in F [arrow above F] [subscript d] as 6-kc concentration increased. We proposed that this membrane electric field is affected by multiple factors: the polarity of the sterol molecules, the reorientation of the phospholipid dipole due to sterol, and the impact of the sterol on hydrogen bonding with surface water. We used molecular dynamics simulations to examine the distribution of phospholipids, sterol, and helix in bilayers containing these sterols. At low concentrations, we observed clustering of sterols near the vibrational probe whereas at high concentrations, we observed spatial correlation between the positions of the sterol molecules. This work demonstrated how a one-atom difference in a sterol changes the physicochemical and electric field properties of the bilayer. Additionally, we set out to understand how a small molecule interacts with the lipid bilayer differently based on its charge. Our laboratory had previously reported that tryptophan permeated through a phosphatidylcholine lipid bilayer membrane at a faster rate when it was positively charged (Trp+) than when negatively charged (Trp−), which corresponded to a lower potential of mean force (PMF) barrier determined through simulations. In the work described here, we demonstrated that Trp+ partitions into the lipid bilayer membrane to a greater degree than Trp− by interacting with the ester linkage of a phosphatidylcholine lipid, where it is stabilized by the electron withdrawing glycerol functional group. These results are in agreement with tryptophan’s known role as an anchor for transmembrane proteins, though the tendency for binding of a positively charged tryptophan is surprising. We discussed the implications of our results on the mechanisms of unassisted permeation and penetration of small molecules within and across lipid bilayer membranes based on molecular charge, shape, and molecular interactions within the bilayer structure.Item Metal nanoparticles for signal amplification in biosensors and signal suppression in fluorescence measurements(2021-05-05) Kogan, Molly; Webb, Lauren J.; Elber, Ron; Baiz, Carlos R; Gordon, VernitaMetal nanoparticles are particles made up of many individual metal atoms ranging in size from one nanometer to hundreds of nanometers in diameter. Originally these particles were used in glazes to make lustrous patterns on pottery. It was not until Michael Faraday’s discovery of colloidal gold in 1856 that nanoparticles were characterized. Since then, nanoparticles of different compositions, sizes, and shapes have been implemented in fields such as catalysis and biotechnology. This dissertation will address two applications of nanoparticles: silver nanoparticles (AgNPs) to amplify signal in a paper-based fluidic device and gold nanoparticles (AuNPs) to quench signal in studies of cell penetrating peptides (CPPs). First, the paper-based device used concentrates a “sandwich-type” immunoassay, made of a capture antibody bound to a magnetic microbead and a detection antibody bound to a AgNP label, at a screen-printed carbon electrode by a magnet. The AgNP labels are oxidized to Ag ions, reduced, and stripped off the electrode in a technique known as anodic stripping voltammetry. Originally, this device could only detect AgNP concentrations as low as 2.1 pM. Scanning electron microscopy (SEM) was used to investigate the percentage of AgNPs oxidized in this detection method enabling optimization of the electrochemical parameters for higher signal output. This lowered the detection limit to 2.6 fM, which is now at a sensitivity comparable to a commercial pregnancy test. Second, this dissertation focuses on the use of AuNPs to suppress signal in fluorescence studies of cell penetrating peptides (CPPs), a class of peptides so effective in crossing cellular membranes that they can be used to carry cargo such as proteins or small molecules into a cell. The mechanism by which CPPs can permeate cell membranes is poorly understood. Here we use a model system composed of phospholipid vesicles and small fluorescent peptides to investigate the transport and mechanism of these peptides interacting with the lipid bilayer. While fluorescence signal can reveal details such as the amount of peptide that transports inside the vesicles, it cannot determine the depth that a peptide penetrates through the vesicles. By growing AuNPs inside these vesicles, however, we can use fluorescence quenching to determine the proximity of the peptide to the center of the vesicles, which will be important as we continue to study CPPs and their permeation through cell membranes.