5 research outputs found
Temperature Mapping in Hydrogel Matrices Using Unmodified Digital Camera
We report a simple,
generally applicable, and noninvasive fluorescent
method for mapping thermal fluctuations in hydrogel matrices using
an unmodified commercially available digital single-lens reflex camera
(DSLR). The nanothermometer is based on the complexation of short
conjugated polyelectrolytes, polyĀ(phenylene ethynylene) carboxylate,
with an amphiphilic polymer, polyvinylpyrrolidone, which is in turn
trapped within the porous network of a gel matrix. Changes in the
temperature lead to a fluorescent ratiometric response with a maximum
relative sensitivity of 2.0% and 1.9% at 45.0 Ā°C for 0.5% agarose
and agar, respectively. The response was reversible with no observed
hysteresis when samples were cycled between 20 and 40 Ā°C. As
a proof of concept, the change in fluorescent signal/color was captured
using a digital camera. The images were then dissected into their
red-green-blue (RGB) components using a Matlab routine. A linear correlation
was observed between the hydrogel temperature and the green and blue
intensity channels. The reported sensor has the potential to provide
a wealth of information when thermal fluctuations mapped in soft gels
matrices are correlated with chemical or physical processes
Site-Specific Fluorescent Labeling and Oriented Immobilization of a Triple Mutant of CYP3A4 via C64
The generation of site-specific bioconjugates of proteins
is highly
desired for a number of biophysical and nanotechnological applications.
To this end, many strategies have been developed that allow the specific
modification of certain canonical amino acids and, more recently,
noncanonical functional groups. P450 enzymes are heme-dependent monooxygenases
involved in xenobiotic metabolism and in the biosynthesis of a variety
of secondary metabolites. We became interested in the site-specific
modification of these enzymes, CYP3A4 in particular, through our studies
of their <i>in vitro</i> biocatalytic properties and our
desire to exploit their remarkable ability to oxidize unactivated
CāH bonds in a regio- and stereospecific manner. Obtained via
a partial cysteine-depletion approach, a functional triple mutant
of CYP3A4 (C98S/C239S/C468G) is reported here which is singly modified
at C64 by maleimide-containing groups. While cysteine-labeling of
the wild-type enzyme abolished >90% of its enzymatic activity,
this
mutant retained ā„75% of the activity of the unmodified wild-type
enzyme with 9 of the 18 maleimides that were tested. These included
both fluorescent and solid-supported maleimides. The loss of activity
observed after labeling with some maleimides is attributed to direct
enzyme inhibition rather than to steric effects. We also demonstrate
the functional immobilization of this mutant on maleimide-functionalized
agarose resin and silica microspheres
Bacterial Recognition of Silicon Nanowire Arrays
Understanding how living cells interact
with nanostructures is
integral to a better understanding of the fundamental principles of
biology and the development of next-generation biomedical/bioenergy
devices. Recent studies have demonstrated that mammalian cells can
recognize nanoscale topographies and respond to these structures.
From this perspective, there is a growing recognition that nanostructures,
along with their specific physicochemical properties, can also be
used to regulate the responses and motions of bacterial cells. Here,
by utilizing a well-defined silicon nanowire array platform and single-cell
imaging, we present direct evidence that <i>Shewanella oneidensis</i> MR-1 can recognize nanoscale structures and that their swimming
patterns and initial attachment locations are strongly influenced
by the presence of nanowires on a surface. Analyses of bacterial trajectories
revealed that MR-1 cells exhibited a confined diffusion mode in the
presence of nanowires and showed preferential attachment to the nanowires,
whereas a superdiffusion mode was observed in the absence of nanowires.
These results demonstrate that nanoscale topography can affect bacterial
movement and attachment and play an important role during the early
stages of biofilm formation
Interaction of Anionic Phenylene Ethynylene Polymers with Lipids: From Membrane Embedding to Liposome Fusion
Here
we report spectroscopic studies on the interaction of negatively
charged, amphiphilic polyphenylene ethynylene (PPE) polymers with
liposomes prepared either from negative, positive or zwitterionic
lipids. Emission spectra of PPEs of 7 and 49 average repeat units
bearing carboxylate terminated side chains showed that the polymer
embeds within positively charged lipids where it exists as free chains.
No interaction was observed between PPEs and negatively charged lipids.
Here the polymer remained aggregated giving rise to broad emission
spectra characteristic of the aggregate species. In zwitterionic lipids,
we observed that the majority of the polymer remained aggregated yet
a small fraction readily embedded within the membrane. Titration experiments
revealed that saturation of zwitterionic lipids with polymer typically
occurred at a polymer repeat unit to lipid mole ratio close to 0.05.
No further membrane embedding was observed above that point. For liposomes
prepared from positively charged lipids, saturation was observed at
a PPE repeat unit to lipid mole ratio of ā¼0.1 and liposome
precipitation was observed above this point. FRET studies showed that
precipitation was preceded by lipid mixing and liposome fusion induced
by the PPEs. This behavior was prominent for the longer polymer and
negligible for the shorter polymer at a repeat unit to lipid mole
ratio of 0.05. We postulate that fusion is the consequence of membrane
destabilization whereby the longer polymer gives rise to more extensive
membrane deformation than the shorter polymer
Postmetalated Zirconium Metal Organic Frameworks as a Highly Potent Bactericide
Metalāorganic frameworks (MOFs)
have emerged as an important class of hybrid organicāinorganic
materials. One of the reasons they have gained remarkable attention
is attributed to the possibility of altering them by postsynthetic
modification, thereby providing access to new and novel advanced materials.
MOFs have been applied in catalysis, gas storage, gas separation,
chemical sensing, and drug delivery. However, their bactericidal use
has rarely been explored. Herein, we developed a two-step process
for the synthesis of zirconium-based MOFs metalated with silver cations
as a potent antibacterial agent. The obtained products were thoroughly
characterized by powder X-ray diffraction, scanning electron microscopy,
UVāvisible, IR, thermogravimetric, and BrunauerāEmmettāTeller
analyses. Their potency was evaluated against <i>E. coli</i> with a reported minimal inhibitory concentration and minimal bactericidal
concentration of as low as 6.5 Ī¼g/mL of silver content. Besides
the novelty of the system, the advantage of this strategy is that
the MOFs could be potentially regenerated and remetalated after each
antibacterial test, unlike previously reported frameworks, which involved
the destruction of the framework