Dai, Hai-Lung

Gonella, Grazia

Elsevier - Publisher Connector 

Tuesday, February 5, 2013 501aThe method involves using antibody functionalized magnetic beads to target
CD3, a subunit of the TCR complex to induce TCR clustering. using magnetic
tweezers, we move the magnetic beads through the cell membrane and simul-
taneously measure the trafficking of co-receptors and involved proteins. One of
these is CD6 co-receptor, which is considered a co-stimulator for cell activa-
tion. We study CD6 during cluster formation.
We introduce aCD3 coated magnetic beads of sizes 1mm and 4.5mm respec-
tively to CD6-GFP transfected Jurkat T-Cells. using confocal fluorescence mi-
croscopy, we measure recruitment of CD6 and co-localization with proteins
involved in cell activation pathway as the bead is pulled through the membrane.
First experiments suggest that while CD6 is not physically associated with TCR
complex, it becomes actively recruited into the contact site mimicking the IS,
and moves along with the IS as it translocates.
We will downscale this method by using functionalized magnetic nanoparticles
in order to address individual T-cell receptors. using magnetic fields this allows
to actively induce nanoscale TCR clusters of predefined size. This will allow us
to study the importance of TCR cluster size on cell activation.
2562-Pos Board B581
Near-Field Optical Immobilization of Antibodies for Novel Fluorescent
Bioassays
Xavier Serey, Pilgyu Kang Dakota O’Dell, David Erickson.
Cornell University, Ithaca, NY, USA.
Optical manipulation techniques have enabled the study of single molecules in
biophysics. Optical tweezers have allowed force spectroscopic measurements
of large biopolymers bond to microspheres. A newer generation of optical
traps, based on near field optics, allow trapping of smaller molecules with
the hope to manipulate directly the biomolecules rather than through micro-
spheres. We previously developed a photonic crystal trap able to capture small
biomolecules, such as Wilson disease molecule, while observing them with
fluorescent microscopy. The technique has been shown to be compatible
with biophysical experiments as it can be performed in the biological buffer
without significant temperature increase. Although these techniques have
been developed with the idea of trapping single molecules, in this presentation,
we steer away from the single molecule focus. A novel bioassay platform is
presented that takes advantage of the optical forces exerted on molecules
near the photonic crystal. The aim of this method is to replace the chemical
immobilization on a surface by optical immobilization because molecules op-
tically attracted to the optically active surface can freely rotate on it and sample
more orientations than molecules bond to a surface. On this platform, using
fluorescent microscopy, we observe the aggregation of antibodies in the optical
trap and measure their binding rates to antigens at various temperatures.
Because of the optical and hydrodynamic forces at play, we expect to observe
higher binding rates than in mass-transport limited equivalent systems.
2563-Pos Board B582
Singlet Oxygen Triplet Energy Transfer (STET) Based Imaging
Technologies for Mapping Protein-Protein Proximity and Interactions in
Intact Cells
Tsz-Leung To, Xiaokun Shu.
University of California San Francisco, San Francisco, CA, USA.
Forster resonance energy transfer (FRET) has been widely used for the detec-
tion of protein-protein interactions but its detection distance is <10 nm. To
overcome the short detection range of FRET, we have developed a new concept
for molecular imaging: Singlet oxygen Triplet Energy Transfer (STET). We
have engineered genetically encoded singlet oxygen generator (SOG) and sin-
glet oxygen sensor (SOS). The proximity of protein X to protein Y can be as-
sayed by fusing X to SOS and Y to SOG. Upon illumination, SOG is excited
and converts molecular oxygen into singlet oxygen through triplet energy trans-
fer. The singlet oxygen transfers to SOS through diffusion and chemically re-
acts with the sensor. The closer X and Y are, the faster SOS will respond to
illumination of SOG. STET has a distance range (~50-100 nm) an order of mag-
nitude greater than that of FRET and does not depend on the orientation of
chromophores. The utility of STET in mapping proximity is illustrated in
a three-membrane-spanning protein complex that physically tethers ER and mi-
tochondria in yeast. In fixed yeast cells, STET can detect any pair-wise protein
proximity spanning ER, mitochondrial outer and inner membranes, and mito-
chondrial matrix (i.e. a detection range of >40nm). The strength of STET ro-
bustly reflects the relative distances between the protein pairs. In a separate set
of experiments, we have demonstrated that STET can monitor the sub-second
time course of the association of two proteins upon ligand induction inside live
cells, highlighting the temporal resolution and single-molecule sensitivity of
STET. Taken together, STET will find broad applications in (1) estimating
the topology of a stable protein complex and (2) identifying and monitoring
transient protein-protein interactions.2564-Pos Board B583
Characterization of Nanoparticles for Therapeutics, Diagnostics and
Imaging by Means of Second Harmonic Scattering
Grazia Gonella, Hai-Lung Dai.
Department of Chemistry - Temple University, Philadelphia, PA, USA.
A new field of study, called nano-bio interactions, investigates how nanomate-
rials interact with biological systems. This is an important issue because, in or-
der to be able to engineer nanostructures capable of traveling in the body and
interacting with cells, we need to develop an understanding of how the physico-
chemical properties of nanoparticles (NPs) affect the way these objects interact
with biological systems. For instance, when considering a cancer nanomedicine
different parameters have to be taken into consideration [1]. An understanding
of how the NP size, shape, composition, structure, charge and surface chemistry
influence transport to the tumor site [2] can help to maximize the efficiency of
delivery of the drugs or contrast agents for imaging as well as the effectiveness
of photothermal therapy [3].
Second harmonic scattering (SHS) is a coherent second-order optical technique
that is specifically surface sensitive and can be performed in-situ [4]. It has also
been recently shown to be sensitive to size, shape and composition of metallic
and dielectric nano and microparticles with or without adsorbed molecular
monolayers [5], presenting a huge potential in the characterization of nano-
bio interactions as well as for use in imaging and photothermal therapy.
[1] A. Albanese, et al. Annu. Rev. Biomed. Eng. 2012, 14, 1.
[2] M. Ferrari. Trends Biotechnol. 2010, 28, 181.
[3] R. Bardhan, et al. Adv. Func. Mater. (2009) 19, 3901.
[4] S. Roke and G. Gonella. Annu. Rev. Phys. Chem. 2012, 63, 353.
[5] S.-H. Jen, et al. J. Phys. Chem. A 2009, 113, 4758; S.-H. Jen, et al. J. Phys.
Chem. C 2010, 114, 4302; G.Gonella and H.-L. Dai. Phys. Rev. B 2011, 84,
121402; G.Gonella, et al. J. Phys. Chem. Lett. 2012, 3, 2877.
2565-Pos Board B584
Spherical and Cubic Magnetic Colloids to Decipher the Mechanics of the
Cytoskeleton
Thomas Pujol, Joe Tavacoli, Pierre Bauer, Olivia du Roure,
Julien Heuvingh.
PMMH ESPCI, Paris, France.
We developed a new method of mechanical measurement, based on the dipolar
attraction between magnetic colloids. A spherical gel formed around magnetic
colloids is progressively deformed between two surfaces as the dipolar attrac-
tion between the colloids increase. Applied force is controlled by the external
magnetic field, while the gel deformation is monitored by video-microscopy.
Elastic modulus of the gel material is extracted by the fit of the force-
deformation curve. This method was used on in vitro dense branched networks
formed by the Arp2/3-complex nucleator, which are crucial for cell migration.
The high throughput of this method allowed the probing of thousands of gels,
orders of magnitude more than previous experiments done with demanding
techniques such as atomic force microscopy. By changing the concentration
of proteins used during the growth of the gel, we changed the proportion of
branches and the length of the filaments, and conducted the first systematic
study linking the architecture of the actin networks to their architecture. A lim-
itation of this technique is the requirement of sphericity to extract the elastic
modulus from force-deformation curves. We solved this issue by developing
new magnetic colloids of arbitrary shapes (cubes, cylinders, disks, etc.). This
allows the deformation of objects between two flat surfaces while retaining
the high throughput of this method. The technique now permits the measure-
ment of non-linear elasticity, viscous modulus and speed of polymerization ver-
sus applied force for dense branched actin gels. Micro-objects of different
shapes can be probed, and development is underway to use this technique for
the measurement of adherent cells’ mechanical properties. This technique
could then be used as a diagnosis tool by probing metastatic cancer cells
with a great throughput.
2566-Pos Board B585
Novel Approaches for Identifying Phosphorus Species in Terrestrial and
Aquatic Ecosystems with 31P NMR
Johan E. Vestergren1, Andrea G. Vincent2, Per Persson1, Mats Jansson3,
Ulrik Ilstedt2, Reiner Giesler3, Jurgen Schleucher4, Gerhard Gro¨bner1.
1Department of Chemistry, Umea˚, Sweden, 2Swedish agricultural University,
Umea˚, Sweden, 3Dept. of Ecology and Environmental Sciences, Umea˚,
Sweden, 4Medical Biophysics and Biochemistry Dept., Umea˚, Sweden.
Phosphorus (P) limits plant growth especially where agriculture is performed
on older soils. Predictions indicate that rock-P mining peaks within 35 years
with severe impacts on global food production1. Several studies have been
made to completely understand fate and cycle of P in soils but knowledge in
this field remains limited. Therefore we aim in our physicochemical/ecological


Characterization of Nanoparticles for Therapeutics, Diagnostics and Imaging by Means of Second Harmonic Scattering 

file:///data/core-remote/dit/data/elsevier/pdf/096/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUxMjA0MDEyeA==.pdf

Characterization of Nanoparticles for Therapeutics, Diagnostics and Imaging by Means of Second Harmonic Scattering

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector