Peter Yim

Quantitative Characterization of Quantum Dot-Labeled Lambda Phage for Escherichia coli Detection

We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (λ) phage, and Escherichia coli are examined by several cross‐validated methods. Flow and image‐based cytometry clarify fluorescent labeling of bacteria, with image‐based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image‐based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4–17 QDs) and show that λ phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal‐biological conjugates. Biotechnol. Bioeng. 2009;104: 1059–1067. Published 2009 Wiley Periodicals, Inc.

The polymerization of actin: Thermodynamics near the polymerization line

Studies of the dependence of actin polymerization on thermodynamic parameters are important for understanding processes in living systems, where actin polymerization and depolymerization are crucial to cell structure and movement. We report measurements of the extent of polymerization, Φ, of rabbit muscle actin as a function of temperature [T=(0–35) °C], initial G-actin concentration [[G0]=(1–3) mg/ml], and initiating salt concentration [[KCl]=(5–15) mmol/l with bound Ca2+], in H2O and D2O buffers and in the presence of adenosine triphosphate (ATP). A preliminary account of the data and analysis for H2O buffers has appeared previously [P. S. Niranjan, J. G. Forbes, S. C. Greer, J. Dudowicz, K. F. Freed, and J. F. Douglas, J. Chem. Phys. 114, 10573 (2001)]. We describe the details of the studies for H2O buffers, together with new data and analysis for D2O buffers. The measurements show a maximum in Φ(T) for H2O buffers and D2O buffers. For H2O buffers, Tp decreases as either [G0] or [KCl] increases. For D2...

Structural analysis of soft multicomponent nanoparticle clusters

Quantitative techniques are essential to analyze the structure of soft multicomponent nanobioclusters. Here, we combine electrospray differential mobility analysis (ES-DMA), which rapidly measures the size of the entire cluster, with transmission electron microscopy (TEM), which detects the hard components, to determine the presence and composition of the softer components. Coupling analysis of TEM and ES-DMA derived data requires the creation and use of analytical models to determine the size and number of constituents in nanoparticle complexes from the difference between the two measurements. Previous ES-DMA analyses have been limited to clusters of identical spherical particles. Here, we dramatically extend the ES-DMA analysis framework to accommodate more challenging geometries, including protein corona-coated nanorods, clusters composed of heterogeneously sized nanospheres, and nanobioclusters composed of both nanospheres and nanorods. The latter is critical to determining the number of quantum dots attached to lambda (λ) phage, a key element of a rapid method to detect bacterial pathogens in environmental and clinical samples.

The polymerization of actin: extent of polymerization under pressure, volume change of polymerization, and relaxation after temperature jumps.

The protein actin can polymerize from monomeric globular G-actin to polymeric filamentary F-actin, under the regulation of thermodynamic variables such as temperature, pressure, and compositions of G-actin and salts. We present here new measurements of the extent of polymerization (phi) of actin under pressure (P), for rabbit skeletal muscle actin in H2O buffer in the presence of adenosine triposphate and calcium ions and at low (5-15 mM) KCl concentrations. We measured phi using pyrene-labeled actin, as a function of time (t) and temperature (T), for samples of fixed concentrations of initial G-actin and KCl and at fixed pressure. The phi(T,P) measurements at equilibrium have the same form as reported previously at 1 atm: low levels of polymerization at low temperatures, representing dimerization of the actin; an increase in phi at the polymerization temperature (Tp); a maximum in phi(T) above Tp) with a decrease in phi(T) beyond the maximum, indicating a depolymerization at higher T. From phi(T,P) at temperatures below Tp, we estimate the change in volume for the dimerization of actin, DeltaVdim, to be -307+/-10 ml/mol at 279 K. The change of Tp with pressure dTp/dP=(0.3015+/-0.0009) K/MPa=(30.15+/-0.09) mK/atm. The phi(T,P) data at higher T indicate the change in volume on propagation, DeltaVprop, to be +401+/-48 ml/mol at 301 K. The phi(t) measurements yield initial relaxation times rp(T) that reflect the behavior of phi(T) and support the presence of a depolymerization temperature. We also measured the density of polymerizing actin with a vibrating tube density meter, the results of which confirm that the data from this instrument are affected by viscosity changes and can be erroneous.

Fluorescence intermittency and spectral shifts of single bio-conjugated nanocrystals studied by single molecule confocal fluorescence microscopy and spectroscopy

We have fabricated a combined measurement system capable of confocal microscopy and fluorescence spectroscopy to simultaneously evaluate multiple optical characteristics of single fluorescent nanocrystals. The single particle detection sensitivity is demonstrated by simultaneously measuring the dynamic excitation-time-dependent fluorescence intermittency and the emission spectrum of single cadmium selenide/zinc sulfide (CdSe/ZnS) nanocrystals (quantum dots, QDs). Using this system, we are currently investigating the optical characteristics of single QDs, the surface of which are conjugated with different ligands, such as trioctylphosphine oxide (TOPO), mercaptoundecanoicacid (MDA), and amine modified DNA (AMDNA). In this paper, we present the progress of our measurements of the time-dependent optical characteristics (fluorescence intermittency, photostability, and spectral diffusion) of single MDA-QDs and AMDNA-MDA-QDs in air in an effort to understand the effects of surface-conjugated biomolecules on the optical characteristics at single QD sensitivities.

Thermal properties of gold nanoshells in lipid vesicles studied by single particle tracking measurements

Photothermal therapy employing nanomaterials is a promising approach to selectively treat targeted tissues with abnormal characteristics such as tumors. While vital research has focused on the use of these materials in biomedical applications, net effects of these materials in biological environments are still not well understood. For reliable biomedical applications, it is crucial to quantitatively evaluate thermal properties of these materials in biological and physiological environments. To this end, we have developed a highly integrated measurement platform and examined local thermal properties of single gold shell nanocrystals in biomimetic environments. These nanoshells consist of a silica core with an outer gold coating. For quantitative measurement of the local thermal profile of gold nanoshells, we monitor lipid phase transitions triggered by gold nanoshell thermal excitation. Dried lipid layers with adsorbed gold nanoshells were placed in an aqueous environment. Photothermal excitation of the gold nanoshells induced localized liposome budding as the lipids were raised above their transition temperature. Single particle tracking of gold nanoshells in solution and within liposomes revealed larger diffusion rates for the confined nanoparticles, likely due to a raised local temperature.