Hope T. Beier

Nanofluidic Biosensing for β-Amyloid Detection Using Surface Enhanced Raman Spectroscopy

Trace detection of the conformational transition of beta-amyloid peptide (Abeta) from a predominantly alpha-helical structure to beta-sheet could have a large impact in understanding and diagnosing Alzheimers disease. We demonstrate how a novel nanofluidic biosensor using a controlled, reproducible surface enhanced Raman spectroscopy active site was developed to observe Abeta in different conformational states during the Abeta self-assembly process as well as to distinguish Abeta from confounder proteins commonly found in cerebral spinal fluid.

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.

Bright emission from a random Raman laser

Random lasers are a developing class of light sources that utilize a highly disordered gain medium as opposed to a conventional optical cavity. Although traditional random lasers often have a relatively broad emission spectrum, a random laser that utilizes vibration transitions via Raman scattering allows for an extremely narrow bandwidth, on the order of 10 cm−1. Here we demonstrate the first experimental evidence of lasing via a Raman interaction in a bulk three-dimensional random medium, with conversion efficiencies on the order of a few percent. Furthermore, Monte Carlo simulations are used to study the complex spatial and temporal dynamics of nonlinear processes in turbid media. In addition to providing a large signal, characteristic of the Raman medium, the random Raman laser offers us an entirely new tool for studying the dynamics of gain in a turbid medium.

Stimulated Raman scattering using a single femtosecond oscillator with flexibility for imaging and spectral applications.

Stimulated Raman scattering (SRS) is a powerful tool for obtaining background-free chemical information about a material without extrinsic labeling. Background-free spectra are particularly important in the fingerprint region (~800 and 1800 cm(-1)) where peaks are narrow, closely-spaced, and may be in abundance for a particular chemical. We demonstrate a method for obtaining SRS spectra using a single femtosecond laser oscillator. A photonic crystal fiber is used to create a supercontinuum to provide a range of Stokes shifts from ~300 to 3400 cm(-1). This SRS approach provides for collection capabilities that are easily modified between obtaining broadband spectra and single-frequency images.

Activation of intracellular phosphoinositide signaling after a single 600 nanosecond electric pulse

Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways--phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2 kV/cm, 600 ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.

Resolving the spatial kinetics of electric pulse-induced ion release

Exposure of cells to nanosecond pulsed electric fields (nsPEF) causes a rapid increase in intracellular calcium. The mechanism(s) responsible for this calcium burst remains unknown, but is hypothesized to be from direct influx through nanopores, the activation of specific ion channels, or direct disruption of organelles. It is likely, however, that several mechanisms are involved/activated, thereby resulting in a complex chain of events that are difficult to separate by slow imaging methods. In this letter, we describe a novel high-speed imaging system capable of determining the spatial location of calcium bursts within a single cell following nsPEF exposure. Preliminary data in rodent neuroblastoma cells are presented, demonstrating the ability of this system to track the location of calcium bursts in vitro within milliseconds of exposure. These data reveal that calcium ions enter the cell from the plasma membrane regions closest to the electrodes (poles), and that intracellular calcium release occurs in the absence of extracellular calcium. We believe that this novel technique will allow us to temporally and spatially separate various nsPEF-induced effects, leading to powerful insights into the mechanism(s) of interaction between electric fields and cellular membranes.

Whispering Gallery Mode Biosensors Consisting of Quantum Dot-Embedded Microspheres

New methods of biological analyte sensing are needed for development of miniature biosensors that are highly sensitive and require minimal sample preparation. One technique employs optical resonances, known as whispering gallery modes (WGMs), in spherical or cylindrical microstructures. The spectral positions of these resonant modes are very sensitive to the local refractive index and spectral shifts may be used to sense changes in the index. To excite these WGMs and enable remote excitation, quantum dots are embedded in polystyrene microspheres to serve as local light sources. Using a simple continuous wave excitation optical system, these sensors are demonstrated by monitoring the wavelength shift of multiple resonant modes as bulk index of refraction is changed in ethanol–water mixtures. The potential for targeted biosensing is explored through addition of a protein that adsorbs to the microsphere surface, thrombin, and one that does not, bovine serum albumin (BSA). The thrombin produced a spectral shift that was much larger than that due to the bulk index change. The BSA produced a significantly smaller shift that was slightly larger than the expected shift due to bulk index change. Most likely due to the thin, high index layer of quantum dots, microsensor response in all cases demonstrated increased sensitivity over theoretical predictions.

Assessment of tissue heating under tunable near-infrared radiation

Abstract. The time-temperature effects of laser radiation exposure are investigated as a function of wavelength. Here, we report the thermal response of bulk tissue as a function of wavelength from 700 to 1064 nm. Additionally, Monte Carlo simulations were used to verify the thermal response measured and predict damage thresholds based on the response.

Microporated PEG Spheres for Fluorescent Analyte Detection

Poly(ethylene glycol) (PEG) hydrogels have been used to encapsulate fluorescently labeled molecules in order to detect a variety of analytes. The hydrogels are designed with a mesh size that will retain the sensing elements while allowing for efficient diffusion of small analytes. Some sensing assays, however, require a conformational change or binding of large macromolecules, which may be sterically prohibited in a dense polymer matrix. A process of hydrogel microporation has been developed to create cavities within PEG microspheres to contain the assay components in solution. This arrangement provides improved motility for large sensing elements, while limiting leaching and increasing sensor lifetime. Three hydrogel compositions, 100% PEG, 50% PEG, and microporated 100% PEG, were used to create pH-sensitive microspheres that were tested for response time and stability. In order to assess motility, a second, more complex sensor, namely a FITC-dextran/TRITC-Con A glucose-specific assay was encapsulated within the microspheres.

600 ns pulse electric field-induced phosphatidylinositol4,5-bisphosphate depletion

The interaction between nsPEF-induced Ca(2+) release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca(2+) the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca(2+) (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca(2+) is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca(2+) leads to a whole cell response. The reported observations of Ca(2+) dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane.