Browsing by Author "Kisley, Lydia"
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Item Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation(American Chemical Society, 2016) Dominguez-Medina, Sergio; Kisley, Lydia; Tauzin, Lawrence J.; Hoggard, Anneli; Shuang, Bo; Indrasekara, A. Swarnapali D.S.; Chen, Sishan; Wang, Lin-Yung; Derry, Paul J.; Liopo, Anton; Zubarev, Eugene R.; Landes, Christy F.; Link, StephanThe response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.Item Charge-Dependent Transport Switching of Single Molecular Ions in a Weak Polyelectrolyte Multilayer(American Chemical Society, 2014) Tauzin, Lawrence J.; Shuang, Bo; Kisley, Lydia; Mansur, Andrea P.; Chen, Jixin; de Leon, Al; Advincula, Rigoberto C.; Landes, Christy F.The tunable nature of weak polyelectrolyte multilayers makes them ideal candidates for drug loading and delivery, water filtration, and separations, yet the lateral transport of charged molecules in these systems remains largely unexplored at the single molecule level. We report the direct measurement of the charge-dependent, pH-tunable, multimodal interaction of single charged molecules with a weak polyelectrolyte multilayer thin film, a 10 bilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy and single-molecule tracking, two modes of interaction were detected: (1) adsorption, characterized by the molecule remaining immobilized in a subresolution region and (2) diffusion trajectories characteristic of hopping (D ∼ 10–9 cm2/s). Radius of gyration evolution analysis and comparison with simulated trajectories confirmed the coexistence of the two transport modes in the same single molecule trajectories. A mechanistic explanation for the probe and condition mediated dynamics is proposed based on a combination of electrostatics and a reversible, pH-induced alteration of the nanoscopic structure of the film. Our results are in good agreement with ensemble studies conducted on similar films, confirm a previously-unobserved hopping mechanism for charged molecules in polyelectrolyte multilayers, and demonstrate that single molecule spectroscopy can offer mechanistic insight into the role of electrostatics and nanoscale tunability of transport in weak polyelectrolyte multilayers.Item Extending single molecule fluorescence observation time by amplitude-modulated excitation(IOP Publishing, 2013) Kisley, Lydia; Chang, Wei-Shun; Cooper, David; Mansur, Andrea P.; Landes, Christy F.We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors.Item Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis(American Chemical Society, 2014) Shuang, Bo; Cooper, David; Taylor, J. Nick; Kisley, Lydia; Chen, Jixin; Wang, Wenxiao; Li, Chun Biu; Komatsuzaki, Tamiki; Landes, Christy F.; Rice Quantum InstituteWe introduce a step transition and state identification (STaSI) method for piecewise constant single-molecule data with a newly derived minimum description length equation as the objective function. We detect the step transitions using the Student’s t test and group the segments into states by hierarchical clustering. The optimum number of states is determined based on the minimum description length equation. This method provides comprehensive, objective analysis of multiple traces requiring few user inputs about the underlying physical models and is faster and more precise in determining the number of states than established and cutting-edge methods for single-molecule data analysis. Perhaps most importantly, the method does not require either time-tagged photon counting or photon counting in general and thus can be applied to a broad range of experimental setups and analytes.Item Fluorescence Correlation Spectroscopy Study of Protein Transport and Dynamic Interactions with Clustered-Charge Peptide Adsorbents(Wiley-Blackwell, 2012-08) Daniels, Charlisa R.; Kisley, Lydia; Kim, Hannah; Chen, Wen-Hsiang; Poongavanam, Mohan- Vivekanandan; Reznik, Carmen; Kourentzi, Katerina; Willson, Richard C.; Landes, Christy F.Ion-exchange chromatography (IEX) relies on electrostatic interactions between the adsorbent and the adsorbate, and is used extensively in protein purification. Conventional IEX utilizes ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clusteredcharge adsorbent surfaces using Fluorescence Correlation Spectroscopy. We can resolve protein free diffusion, hindered diffusion and association with bare glass, agarose-coated, and agaroseclustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered charge adsorbent and other surface interactions at the molecular scale.Item High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study(Elsevier, 2014) Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Dominguez-Medina, Sergio; Kulla, Eliona; Kang, Marci; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of ?-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.Item Improved Analysis for Determining Diffusion Coefficients from Short, Single-Molecule Trajectories with Photoblinking(American Chemical Society, 2013) Shuang, Bo; Byers, Chad P.; Kisley, Lydia; Wang, Lin-Yung; Zhao, Julia; Morimura, Hiroyuki; Link, Stephan; Landes, Christy F.; Rice Quantum InstituteTwo Maximum Likelihood Estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular,short, single-molecule trajectories with photoblinking were studied, and our method was compared with existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars.Item Molecular Approaches to Chromatography Using Single Molecule Spectroscopy(American Chemical Society, 2015) Kisley, Lydia; Landes, Christy F.; Rice Quantum InstituteItem Single Molecule Studies of Ion-Exchange Chromatography(2015-04-20) Kisley, Lydia; Landes, Christy F; Weisman, R. Bruce; Pasquali, MatteoAs the pharmeceutical industry moves away from traditional small organic molecules towards biologically-based treatments, ion-exchange separation methods must be investigated to improve the cost and time required for protein purification. Several new single molecule, super-resolution techniques are presented to offer a mechanistic experimental understanding of chromatography unachievable through traditional ensemble-averaged methods. Super-resolution analysis visualizes single protein adsorption kinetics to single, super-resolved ligands, allowing for the first experimental validation of the statistical mechanical stochastic theory of chromatography. Imperative results on the spatial charge-distribution of ligands, reduction of heterogeneity by ionic strength, and tuning of protein/stationary phase interfacial interactions by pH are observed. A common finding that the sterics of the agarose support induces separation heterogeneity leads to super-resolution imaging of the agarose structure and diffusion properties. Finally, the single molecule techniques are applied to several applications beyond protein chromatography to demonstrate the potential for future materials research. Overall, we have shown that single molecule spectroscopy can aid in the mechanistic experimental and theoretical understanding of the ion-exchange chromatographic separation of proteins.Item Super-Resolution mbPAINT for Optical Localization of Single-Stranded DNA(American Chemical Society, 2013) Chen, Jixin; Bremauntz, Alberto; Kisley, Lydia; Shuang, Bo; Landes, Christy F.We demonstrate the application of superlocalization microscopy to identify sequence-specific portions of single-stranded DNA (ssDNA) with sequence resolution of 50 nucleotides, corresponding to a spatial resolution of 30 nm. Super-resolution imaging was achieved using a variation of a single-molecule localization method, termed as ﾓmotion blurﾔ point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate. Short, dye-labeled, and complementary ssDNA molecules stochastically bound to the target ssDNA, with repeated binding events allowing super-resolution. Sequence specificity was demonstrated via the use of a control, noncomplementary probe. The results support the possibility of employing relatively inexpensive short ssDNAs to identify gene sequence specificity with improved resolution in comparison to the existing methods.Item Transforming Separation Science with Single-Molecule Methods(American Chemical Society, 2020) Calabrase, William; Bishop, Logan D.C.; Dutta, Chayan; Misiura, Anastasiia; Landes, Christy F.; Kisley, Lydia; Smalley-Curl InstituteEmpirical optimization of the multiscale parameters underlying chromatographic and membrane separations leads to enormous resource waste and production costs. A bottom-up approach to understand the physical phenomena underlying challenges in separations is possible with single-molecule observations of solute–stationary phase interactions. We outline single-molecule fluorescence techniques that can identify key interactions under ambient conditions. Next, we describe how studying increasingly complex samples heightens the relevance of single-molecule results to industrial applications. Finally, we illustrate how separation methods that have not been studied at the single-molecule scale can be advanced, using chiral chromatography as an example case. We hope new research directions based on a molecular approach to separations will emerge based on the ideas, technologies, and open scientific questions presented in this Perspective.Item Troika of single particle tracking programing: SNR enhancement, particle identification, and mapping(Royal Society of Chemistry, 2014) Shuang, Bo; Chen, Jixin; Kisley, Lydia; Landes, Christy F.Single particle tracking (SPT) techniques provide a microscopic approach to probe in vivo and in vitro structure and reactions. Automatic analysis of SPT data with high efficiency and accuracy spurs the development of SPT algorithms. In this perspective, we review a range of available techniques used in SPT analysis programs. In addition, we present an example SPT program step-by-step to provide a guide so that researchers can use, modify, and/or write a SPT program for their own purposes.