3 research outputs found
Atomic Force Microscopy-Infrared Spectroscopy of Individual Atmospheric Aerosol Particles: Subdiffraction Limit Vibrational Spectroscopy and Morphological Analysis
Chemical
analysis of atmospheric aerosols is an analytical challenge,
as aerosol particles are complex chemical mixtures that can contain
hundreds to thousands of species in attoliter volumes at the most
abundant sizes in the atmosphere (∼100 nm). These particles
have global impacts on climate and health, but there are few methods
available that combine imaging and the detailed molecular information
from vibrational spectroscopy for individual particles <500 nm.
Herein, we show the first application of atomic force microscopy with
infrared spectroscopy (AFM-IR) to detect trace organic and inorganic
species and probe intraparticle chemical variation in individual particles
down to 150 nm. By detecting photothermal expansion at frequencies
where particle species absorb IR photons from a tunable laser, AFM-IR
can study particles smaller than the optical diffraction limit. Combining
strengths of AFM (ambient pressure, height, morphology, and phase
measurements) with photothermal IR spectroscopy, the potential of
AFM-IR is shown for a diverse set of single-component particles, liquid–liquid
phase separated particles (core–shell morphology), and ambient
atmospheric particles. The spectra from atmospheric model systems
(ammonium sulfate, sodium nitrate, succinic acid, and sucrose) had
clearly identifiable features that correlate with absorption frequencies
for infrared-active modes. Additionally, molecular information was
obtained with <100 nm spatial resolution for phase separated particles
with a ∼150 nm shell and 300 nm core. The subdiffraction limit
capability of AFM-IR has the potential to advance understanding of
particle impacts on climate and health by improving analytical capabilities
to study water uptake, heterogeneous reactivity, and viscosity
Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein
Polymer–ligand conjugates
are designed to bind proteins
for applications as drugs, imaging agents, and transport scaffolds.
In this work, we demonstrate a folic acid (FA)-triggered exosite binding
of a generation five polyÂ(amidoamine) (G5 PAMAM) dendrimer scaffold
to bovine folate binding protein (bFBP). The protein exosite is a
secondary binding site on the protein surface, separate from the FA
binding pocket, to which the dendrimer binds. Exosite binding is required
to achieve the greatly enhanced binding constants and protein structural
change observed in this study. The G5<sub>Ac</sub>-COG-FA<sub>1.0</sub> conjugate bound tightly to bFBP, was not displaced by a 28-fold
excess of FA, and quenched roughly 80% of the initial fluorescence.
Two-step binding kinetics were measured using the intrinsic fluorescence
of the FBP tryptophan residues to give a <i>K</i><sub>D</sub> in the low nanomolar range for formation of the initial G5<sub>Ac</sub>-COG-FA<sub>1.0</sub>/FBP* complex, and a slow conversion to the
tight complex formed between the dendrimer and the FBP exosite. The
extent of quenching was sensitive to the choice of FA-dendrimer linker
chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in
roughly 50% fluorescence quenching of the FBP. The G5<sub>Ac</sub>-COG-FA, which has a longer linker containing a 1,2,3-triazole ring,
exhibited an ∼80% fluorescence quenching. The binding of the
G5<sub>Ac</sub>-COG-FA<sub>1.0</sub> conjugate was compared to polyÂ(ethylene
glycol) (PEG) conjugates of FA (PEG<sub><i>n</i></sub>-FA).
PEG<sub>2k</sub>-FA had a binding strength similar to that of FA,
whereas other PEG conjugates with higher molecular weight showed weaker
binding. However, no PEG conjugates gave an increased degree of total
fluorescence quenching
Quantitative Measurement of Cationic Polymer Vector and Polymer–pDNA Polyplex Intercalation into the Cell Plasma Membrane
Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30–50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials