Real-Time Measurement of Polymer Brush Dynamics Using Silicon Photonic Microring Resonators: Analyte Partitioning and Interior Brush Kinetics.

Polymer brushes are found in biomedical and industrial technologies, where they exhibit functionalities considerably dependent on polymer brush-solvent-analyte interactions. It remains a difficult challenge to quickly analyze solvent-swollen polymer brushes, both at the solvent-polymer brush interface and in the brush interior, as well as to monitor the kinetics of interaction of solvent-swollen brushes with key analytes. Here, we demonstrate the novel use of silicon photonic microring resonators to characterize in situ swollen polymer brush-analyte interactions. By monitoring resonant wavelength shifts, we find that brush-solvent-analyte interaction parameters can be extracted from a single set of data or from successive analyte introductions using a single brush-coated sensor. The partition coefficient of three industrially relevant plasticizers into hydrophobic and hydrophilic brushes was determined and found to be in agreement with known solubility trends. We found that the diffusion coefficient of the plasticizer into the brush decreases as brush thickness increases, supporting a model of a dense inner brush layer and diffuse outer layer. pKa's of pH-sensitive brushes were determined on the microring resonator platform; upon increasing the dry brush thickness, the pKa for poly(2-dimethylamino ethyl methacrylate) decreased from 8.5 to approach the bulk material pKa of 7.3 and showed dependence on the presence and concentration of salt. These proof-of-concept experiments show how the surface-sensitive nature of the microring resonator detection platform provides valuable information about the interaction of the polymer brushes with the solvents and analytes, not easily accessed by other techniques.

Acid-Triggered, Acid-Generating, and Self-Amplifying Degradable Polymers.

We describe the 3-iodopropyl acetal moiety as a simple cleavable unit that undergoes acid catalyzed hydrolysis to liberate HI (p Ka ∼ -10) and acrolein stoichiometrically. Integrating this unit into linear and network polymers gives a class of macromolecules that undergo a new mechanism of degradation with an acid amplified, sigmoidal rate. This trigger-responsive self-amplified degradable polymer undergoes accelerated rate of degradation and agent release.

Soft Three-Dimensional Microscale Vibratory Platforms for Characterization of Nano-Thin Polymer Films.

Vibrational resonances of microelectromechanical systems (MEMS) can serve as means for assessing physical properties of ultrathin coatings in sensors and analytical platforms. Most such technologies exist in largely two-dimensional configurations with a limited total number of accessible vibration modes and modal displacements, thereby placing constraints on design options and operational capabilities. This study presents a set of concepts in three-dimensional (3D) microscale platforms with vibrational resonances excited by Lorentz-force actuation for purposes of measuring properties of thin-film coatings. Nanoscale films including photodefinable epoxy, cresol novolak resin, and polymer brush with thicknesses as small as 270 nm serve as the test vehicles for demonstrating the advantages of these 3D MEMS for detection of multiple physical properties, such as modulus and density, within a single polymer sample. The stability and reusability of the structure are demonstrated through multiple measurements of polymer samples using a single platform, and via integration with thermal actuators, the temperature-dependent physical properties of polymer films are assessed. Numerical modeling also suggests the potential for characterization of anisotropic mechanical properties in single or multilayer films. The findings establish unusual opportunities for interrogation of the physical properties of polymers through advanced MEMS design.

Soluble Zwitterionic Poly(sulfobetaine) Destabilizes Proteins.

The widespread interest in neutral, water-soluble polymers such as poly(ethylene glycol) (PEG) and poly(zwitterions) such as poly(sulfobetaine) (pSB) for biomedical applications is due to their widely assumed low protein binding. Here we demonstrate that pSB chains in solution can interact with proteins directly. Moreover, pSB can reduce the thermal stability and increase the protein folding cooperativity relative to proteins in buffer or in PEG solutions. Polymer-dependent changes in the tryptophan fluorescence spectra of three structurally-distinct proteins reveal that soluble, 100 kDa pSB interacts directly with all three proteins and changes both the local polarity near tryptophan residues and the protein conformation. Thermal denaturation studies show that the protein melting temperatures decrease by as much as ∼1.9 °C per weight percent of polymer and that protein folding cooperativity increases by as much as ∼130 J mol-1 K-1 per weight percent of polymer. The exact extent of the changes is protein-dependent, as some proteins exhibit increased stability, whereas others experience decreased stability at high soluble pSB concentrations. These results suggest that pSB is not universally protein-repellent and that its efficacy in biotechnological applications will depend on the specific proteins used.

Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels.

We apply fast relaxation imaging (FReI) as a novel technique for investigating the folding stability and dynamics of proteins within polyacrylamide hydrogels, which have diverse and widespread uses in biotechnology. FReI detects protein unfolding in situ by imaging changes in fluorescence resonance energy transfer (FRET) after temperature jump perturbations. Unlike bulk measurements, diffraction-limited epifluorescence imaging combined with fast temperature perturbations reveals the impact of local environment effects on protein-biomaterial compatibility. Our experiments investigated a crowding sensor protein (CrH2) and phosphoglycerate kinase (PGK), which undergoes cooperative unfolding. The crowding sensor quantifies the confinement effect of the cross-linked hydrogel: the 4% polyacrylamide hydrogel is similar to aqueous solution (no confinement), while the 10% hydrogel is strongly confining. FRAP measurements and protein concentration gradients in the 4% and 10% hydrogels further support this observation. PGK reveals that noncovalent interactions of the protein with the polymer surface are more important than confinement for determining protein properties in the gel: the mere presence of hydrogel increases protein stability, speeds up folding relaxation, and promotes irreversible binding to the polymer even at the solution-gel interface, whereas the difference between the 4% and the 10% hydrogels is negligible despite their large difference in confinement. The imaging capabilities of FReI, demonstrated to be diffraction limited, further revealed spatially homogeneous protein unfolding across the hydrogels at 500 nm length scales and revealed differences in protein properties at the gel-solution boundary.

Developing bivalent ligands to target CUG triplet repeats, the causative agent of myotonic dystrophy type 1.

An expanded CUG repeat transcript (CUG(exp)) is the causative agent of myotonic dystrophy type 1 (DM1) by sequestering muscleblind-like 1 protein (MBNL1), a regulator of alternative splicing. On the basis of a ligand (1) that was previously reported to be active in an in vitro assay, we present the synthesis of a small library containing 10 dimeric ligands (4-13) that differ in length, composition, and attachment point of the linking chain. The oligoamino linkers gave a greater gain in affinity for CUG RNA and were more effective when compared to oligoether linkers. The most potent in vitro ligand (9) was shown to be aqueous-soluble and both cell- and nucleus-permeable, displaying almost complete dispersion of MBNL1 ribonuclear foci in a DM1 cell model. Direct evidence for the bioactivity of 9 was observed in its ability to disperse ribonuclear foci in individual live DM1 model cells using time-lapse confocal fluorescence microscopy.

A novel CUG(exp)·MBNL1 inhibitor with therapeutic potential for myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is caused by an expanded CUG repeat (CUG(exp)) that sequesters muscleblind-like 1 protein (MBNL1), a protein that regulates alternative splicing. CUG(exp) RNA is a validated drug target for this currently untreatable disease. Herein, we develop a bioactive small molecule (1) that targets CUG(exp) RNA and is able to inhibit the CUG(exp)·MBNL1 interaction in cells that model DM1. The core of this small molecule is based on ligand 2, which was previously reported to be active in an in vitro assay. A polyamine-derivative side chain was conjugated to this core to make it aqueous-soluble and cell-penetrable. In a DM1 cell model this conjugate was found to disperse CUG(exp) ribonuclear foci, release MBNL1, and partially reverse the mis-splicing of the insulin receptor pre-mRNA. Direct evidence for ribonuclear foci dispersion by this ligand was obtained in a live DM1 cell model using time-lapse confocal microscopy.