16 research outputs found
Nonlinear Optical Methods for Noninvasive Analytics.
To date, many of the current tools and technologies used to explore biophysical processes and phenomena are limited in part by their inability to probe without external perturbation. Factors such as size restrictions, samples used for non-terminal studies, and small molecule dynamics cannot be addressed by these techniques. This has not only driven the field of noninvasive analytics, but has had a direct impact on shaping the next generation of biological and clinical assays. Highlights of my thesis work herein focus on the development of nonlinear optical spectroscopy and imaging modalities, sum frequency generation (SFG) spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy. Each has their own unique qualities that make them ideal as noninvasive tools and techniques.
Using these two techniques, my studies address fundamental questions about: (1) the orientation and behavior of chemically immobilized peptides on abiotic surfaces for the rational design of improved biosensors and bioactive textiles, (2) understanding the relations between cytosolic lipids, cellular energy homeostasis/consumption, and developmental biology in female reproductive cells, and (3) the ability to classify and measure male reproductive health as it relates to acrosome integrity.
Results from SFG, a surface-sensitive spectroscopy, demonstrate that surface tethering mechanisms govern both the orientation and activity of antimicrobial peptide MSI-78 on surfaces. Attachment of the n-terminus of this peptide results in an orientation perpendicular to the surface normal (lying down) and higher antimicrobial activity whereas c-terminus attachment leads to a parallel orientation (standing up) and lower activity. Other methods complementary to SFG including circular dichroism (CD) spectroscopy and coarse-grained simulation molecular dynamics simulations also support this conclusion.
CARS, a live cell noninvasive microscopy, is used to evaluate the contributions of lipid in oocyte growth, development, and for metabolic disease. Results show that lipid content fluctuates as oocytes progress through meiosis, indicated by an increase in content as the oocytes grow and a decrease as oocytes resume meiosis. Lipid content is also higher for oocytes from females who exhibit metabolic disease. As for male reproductive cells, CARS microscopy is beginning to be used for the identification of acrosome reaction, an important predictor of male infertility.PhDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/113659/1/jasensja_1.pd
Nano-bio interfaces probed by advanced optical spectroscopy: From model system studies to optical biosensors
Multireflection Sum Frequency Generation Vibrational Spectroscopy
We developed a multireflection data
collection method in order
to improve the signal-to-noise ratio (SNR) and sensitivity of sum
frequency generation (SFG) spectroscopy, which we refer to as multireflection
SFG, or MRSFG for short. To achieve MRSFG, a collinear laser beam
propagation geometry was adopted and trapezoidal Dove prisms were
used as sample substrates. An in-depth discussion on the signal and
SNR in MRSFG was performed. We showed experimentally, with “<i>m</i>” total internal reflections in a Dove prism, MRSFG
signal is ∼<i>m</i> times that of conventional SFG;
SNR of the SFG signal-to-background is improved by a factor of ><i>m</i><sup>1/2</sup> and <<i>m</i>. MRSFG also improved
the SFG sensitivity to resolve weak vibrational signals. Surface molecular
structures of adsorbed ethanol molecules, polymer films, and a lipid
monolayer were characterized using both MRSFG and conventional SFG.
Molecular orientation information on lipid molecules with a 9% composition
in a mixed monolayer was measured using MRSFG, which showed a good
agreement with that derived from 100% lipid surface coverage using
conventional SFG. MRSFG can both improve the spectral quality and
detection limit of SFG spectroscopy and is expected to have important
applications in surface science for studying structures of molecules
with a low surface coverage or less ordered molecular moieties
Molecular Orientation Analysis of Alkyl Methylene Groups from Quantitative Coherent Anti-Stokes Raman Scattering Spectroscopy
Quantitative data analysis in coherent
anti-Stokes Raman scattering
(CARS) spectroscopy is important for extracting molecular structural
information. We developed a method to derive molecular tilt angle
with respect to the surface normal based on quantitative CARS spectral
analysis. We showed that the tilt angle of methylene alkyl chains
on a surface can be directly obtained from the CH<sub>2</sub> symmetric/asymmetric
peak ratio in a CARS spectrum. The lipid alkyl chain tilt angle from
a lipid monolayer was measured to be ∼0° and was verified
by sum frequency generation spectroscopy, which probes the orientations
of the lipid methyl end groups. The tilt angle of a silane monolayer
alkyl chain was derived to be ∼35°, which agrees with
the theoretical prediction. This method is submonolayer sensitive
and can also be used to interpret polarization-dependent signals in
CARS microscopy. It can be applied to elucidate detailed molecular
structure from CARS spectroscopic and microscopic measurements
Quantitative Spectral Analysis of Coherent Anti-Stokes Raman Scattering Signals: C–H Stretching Modes of the Methyl Group
Coherent
anti-Stokes Raman scattering (CARS) vibrational spectroscopy
has been extensively developed into a powerful analytical technique
to study various molecules. Quantitative interpretation of CARS spectra
can help to improve CARS for chemical analysis and extend its analytical
applications. In this work, we quantitatively analyzed CARS signals
originating from the methyl groups in poly(dimethylsiloxane) (PDMS),
with the help of the bond additivity method. Experimentally, a home-built
CARS spectrometer modified from a commercial sum frequency generation
spectrometer was used to collect CARS spectra from a PDMS film. Theoretically,
we successfully reproduced the peak intensity ratio of C–H
symmetric and asymmetric stretching modes of the PDMS methyl group
in different polarization combinations based on bond additivity method
and Raman depolarization ratio. This research shows that bond additivity
theory can help to obtain the third-order nonlinear susceptibility
tensor properties probed by different polarization combinations used
in CARS spectroscopy. The method developed in this work could also
be applied to CARS vibrational stretching analysis of other functional
groups, providing quantitative understanding of CARS spectrum for
applications in spectroscopy
Monitoring Antimicrobial Mechanisms of Surface-Immobilized Peptides in Situ
Antimicrobial peptides
(AMPs) in free solution can kill bacteria
by disrupting bacterial cell membranes. Their modes of action have
been extensively studied, and various models ranging from pore formation
to carpet-like mechanisms were proposed. Surface-immobilized AMPs
have been used as coatings to kill bacteria and as sensors to capture
bacteria, but the interaction mechanisms of surface-immobilized AMPs
and bacteria are not fully understood. In this research, an analytical
platform, sum frequency generation (SFG) microscope, which is composed
of an SFG vibrational spectrometer and a fluorescence microscope,
was used to probe molecular interactions between surface-immobilized
AMPs and bacteria in situ in real time at the solid/liquid interface.
SFG probed the molecular structure of surface-immobilized AMPs while
interacting with bacteria, and fluorescence images of dead bacteria
were monitored as a function of time during the peptide–bacteria
interaction. It was believed that upon bacteria contact, the surface-immobilized
peptides changed their orientation and killed bacteria. This research
demonstrated that the SFG microscope platform can examine the structure
and function (bacterial killing) at the same time in the same sample
environment, providing in-depth understanding on the structure–activity
relationships of surface-immobilized AMPs
Effect of Solvent on Surface Ordering of Poly(3-hexylthiophene) Thin Films
Enhancement
of charge transport in organic polymer semiconductors
is a crucial step in developing optimized devices. A variety of sample
preparation conditions, such as film fabrication method, solvent species,
and annealing, were found to influence the hole mobility of organic
polymers. Despite the fact that many factors can influence their performance,
it is believed that polymer surface ordering plays a key role in determining
organic polymer function. Here, sum frequency generation (SFG) vibrational
spectroscopy was used to nondestructively map the surface/interfacial
ordering of poly(3-hexylthiophene) (P3HT) films prepared using different
solvents; we believe that solvent interactions determine the degree
of surface/interfacial ordering. Both X-ray diffraction (XRD) spectroscopy
and scanning electron microscopy (SEM) were used to supplement SFG
to systematically study bulk crystallinity and surface morphology.
We conclude that SFG is a powerful tool to elucidate the surface/interfacial
structural information on polymer semiconducting films. We demonstrate
that the solvent composition used to prepare P3HT thin films influences
the resulting film surface morphology, surface/interfacial ordering,
and bulk crystallinity
Capsaicin-Inspired Thiol–Ene Terpolymer Networks Designed for Antibiofouling Coatings
Novel
photocurable ternary polymer networks were prepared by incorporating <i>N</i>-(4-hydroxy-3-methoxybenzyl)-acrylamide (HMBA)
into a cross-linked thiol–ene network based on poly(ethylene
glycol)diacrylate (PEGDA) and (mercaptopropyl)methylsiloxane
homopolymers (MSHP). The ternary network materials displayed bactericidal
activity against <i>Escherichia coli</i> and <i>Staphylococcus
aureus</i> and reduced the attachment of marine organism <i>Phaeodactylum tricornutum</i>. Extensive soaking of the polymer
networks in aqueous solution indicated that no active antibacterial
component leached out of the materials, and thus the ternary thiol–ene
coating killed the bacteria by surface contact. The surface structures
of the polymer networks with varied content ratios were studied by
sum frequency generation (SFG) vibrational spectroscopy. The results
demonstrated that the PDMS Si-CH<sub>3</sub> groups and mimic-capsaicine
groups are predominantly present at the polymer–air interface
of the coatings. Surface reorganization was apparent after polymers
were placed in contact with D<sub>2</sub>O: the hydrophobic PDMS Si-CH<sub>3</sub> groups left the surface and returned to the bulk of the polymer
networks, and the hydrophilic PEG chains cover the polymer surfaces
in D<sub>2</sub>O. The capasaicine methoxy groups are able to segregate
to the surface in an aqueous environment, depending upon the ratio
of HMBA/PEGDA. SFG measurements in situ showed that the antibacterial
HMBA chains, rather than the nonfouling PEG, played a dominant role
in mediating the antibiofouling performance in this particular polymer
system
Observing a Model Ion Channel Gating Action in Model Cell Membranes in Real Time in Situ: Membrane Potential Change Induced Alamethicin Orientation Change
Ion channels play crucial roles in transport and regulatory
functions
of living cells. Understanding the gating mechanisms of these channels
is important to understanding and treating diseases that have been
linked to ion channels. One potential model peptide for studying the
mechanism of ion channel gating is alamethicin, which adopts a split
α/3<sub>10</sub>-helix structure and responds to changes in
electric potential. In this study, sum frequency generation vibrational
spectroscopy (SFG-VS), supplemented by attenuated total reflectance
Fourier transform infrared spectroscopy (ATR-FTIR), has been applied
to characterize interactions between alamethicin (a model for larger
channel proteins) and 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine
(POPC) lipid bilayers in the presence of an electric potential across
the membrane. The membrane potential difference was controlled by
changing the pH of the solution in contact with the bilayer and was
measured using fluorescence spectroscopy. The orientation angle of
alamethicin in POPC lipid bilayers was then determined at different
pH values using polarized SFG amide I spectra. Assuming that all molecules
adopt the same orientation (a δ distribution), at pH = 6.7 the
α-helix at the N-terminus and the 3<sub>10</sub>-helix at the
C-terminus tilt at about 72° (θ<sub>1</sub>) and 50°
(θ<sub>2</sub>) versus the surface normal, respectively. When
pH increases to 11.9, θ<sub>1</sub> and θ<sub>2</sub> decrease
to 56.5° and 45°, respectively. The δ distribution
assumption was verified using a combination of SFG and ATR-FTIR measurements,
which showed a quite narrow distribution in the angle of θ<sub>1</sub> for both pH conditions. This indicates that all alamethicin
molecules at the surface adopt a nearly identical orientation in POPC
lipid bilayers. The localized pH change in proximity to the bilayer
modulates the membrane potential and thus induces a decrease in both
the tilt and the bend angles of the two helices in alamethicin. This
is the first reported application of SFG to the study of model ion
channel gating mechanisms in model cell membranes