Malkiat S. Johal

Multiphasic DNA Adsorption to Silica Surfaces under Varying Buffer, pH, and Ionic Strength Conditions

Reversible interactions between DNA and silica are utilized in the solid phase extraction and purification of DNA from complex samples. Chaotropic salts commonly drive DNA binding to silica but inhibit DNA polymerase amplification. We studied DNA adsorption to silica using conditions with or without chaotropic salts through bulk depletion and quartz crystal microbalance (QCM) experiments. While more DNA adsorbed to silica using chaotropic salts, certain buffer conditions without chaotropic salts yielded a similar amount of eluted DNA. QCM results indicate that under stronger adsorbing conditions the adsorbed DNA layer is initially rigid but becomes viscoelastic within minutes. These results qualitatively agreed with a mathematical model for a multiphasic adsorption process. Buffer conditions that do not require chaotropic salts can simplify protocols for nucleic acid sample preparation. Understanding how DNA adsorbs to silica can help optimize nucleic acid sample preparation for clinical diagnostic and research applications.

Tuning the optical properties of a water-soluble cationic poly(p-phenylenevinylene): surfactant complexation with a conjugated polyelectrolyte.

In this work, we investigate the emission and absorbance properties of the novel water-soluble cationic conjugated polymer poly{2,5-bis[3-(N,N,N-triethylammonium bromide)-1-oxapropyl]-1,4-phenylenevinylene}, denoted here as P2, in the presence of varying amounts of the anionic surfactant sodium dodecylsulfate (SDS). We show that the absolute photoluminescence quantum efficiency (PLQE), the absorption wavelength, and the emission wavelength of an aqueous solution of P2 can be adjusted according to the surfactant/polymer ratio in aqueous solution. In particular, we show that the addition of SDS to P2 increases the polyelectrolytes PLQE to approximately 40%. An observed red shift in the emission spectra upon addition of the surfactant is attributed to the reduction in electrostatic repulsive interactions between side chains that minimize the benzene ring twisting along the backbone structure. At the surfactants critical micelle concentration, the P2 chains wrap around the outer surface of the SDS micelles. This work has implications on the development of new stable poly(p-phenylenevinylene)-based photovoltaic and electroluminescent materials with tunable optical properties.

Quantification of the Layer of Hydration of a Supported Lipid Bilayer

Dual polarization interferometry (DPI) and quartz-crystal microgravimetry (QCM-D) were used to investigate the adsorption of DOPC vesicles to a solid hydrophilic surface. The layer of hydration formed between a self-assembled DOPC bilayer and a silica solid support was probed in assemblies constructed using H(2)O and D(2)O buffers. We used QCM-D to measure the mass of the bilayer, including the mass contribution of the coupled solvent that resides between the membrane-solid interface. The mass of only the DOPC in the bilayer was resolved using DPI. By comparing these two measurements, and also accounting for the bulk phase effects on mass, we have been able to determine the mass of water below the bilayer. The thickness of this hydration layer, calculated by relating its mass to the density of the layer, was determined to be 10.46 A +/- 0.15 A for trapped D(2)O and 10.21 A +/- 0.40 A for trapped H(2)O, in agreement with measurements obtained by other methods. This work establishes the feasibility of concurrently using DPI and QCM-D to gauge the extent of hydration in thin films.

Dual-beam polarization interferometry resolves mechanistic aspects of polyelectrolyte adsorption.

The electrostatically driven binding dynamics of a polyelectrolyte multilayer (PEMU) film was investigated in real-time using dual-beam polarization interferometry (DPI) and independently supported by quartz crystal microbalance with dissipation monitoring (QCM-D) studies. Multilayer assemblies of the polyanions poly[1-[4[(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl sodium salt] (PAZO) and poly(styrene sulfonate) (PSS) were respectively constructed with the polycation poly(ethylenimine) (PEI) on anionic functionalized substrates using the layer-by-layer electrostatic self-assembly method. DPI measurements indicate that polyelectrolyte adsorption occurs in three distinct stages. In the first stage, for approximately 5 s, coil-like segments of polyanion partially tether to the surface of the oppositely charged PEI. In the second stage, these coils unfurl over a period of approximately 10 s to cover the surface resulting in an increase in average density of the film. During the final adsorption step, the surface-bound polyelectrolyte diffuses into the multilayer assembly, exposing the surface to further deposition. This last step occurs over a much longer time period and results in a highly interpenetrated film containing a charge-overcompensated region at the film surface.

Polymer–surfactant complexation in polyelectrolyte multilayer assemblies

Layer-by-layer self-assembly can be used to incorporate amphiphilic molecules into multilayered polyelectrolyte architectures. This review examines equilibrium LbL assemblies constructed by direct adsorption from aqueous solution. LbL systems have not only provided fundamental insight into the nature of polyion-surfactant complexation, but have also yielded functional materials with useful surface, optical, and electronic properties.

Controlling tyrosinase activity on charged polyelectrolyte surfaces: a QCM-D analysis.

The quartz crystal microbalance (QCM) was used to monitor the immobilization of tyrosinase on polycationic and polyanionic precursor assemblies in situ and in real-time. The resulting enzymatic surfaces were then exposed to various flavonoids, and the degree of binding was measured using QCM. We show that enzyme activity is retained when immobilized on polycationic films (flavonoid binding observed), while the active site is blocked when assembled on a polyanionic film (no flavonoid binding to the enzyme). We rationalize these observations by considering a combination of interlayer interpenetration and strong electrostatic interactions between the polyelectrolyte and tyrosinases dicopper 2(+) center. Ion-pair formation between anionic moieties of the polyanion and the metal-coordinated active site is suggested as the dominant mechanism leading to the deactivation of tyrosinase. We are currently working to expand this research to achieve a more general theory of how various metal-coordinated enzymes react with polyelectrolyte surfaces of varying structural morphology, charge density, and chemical composition.

Structural Changes in a Polyelectrolyte Multilayer Assembly Investigated by Reflection Absorption Infrared Spectroscopy and Sum Frequency Generation Spectroscopy

The structure of polyelectrolyte multilayer films adsorbed onto either a per-protonated or per-deuterated 11-mercaptoundecanoic acid (h-MUA/d-MUA) self assembled monolayer (SAM) on gold was investigated in air using two surface vibrational spectroscopy techniques, namely, reflection absorption infrared spectroscopy (RAIRS) and sum frequency generation (SFG) spectroscopy. Determination of film masses and dissipation values were made using a quartz crystal microbalance with dissipation monitoring (QCM-D). The films, containing alternating layers of the polyanion poly[1-[4-(3-carboxy-4-hydroxyphenylazo) benzenesulfonamido]-1,2-ethanediyl, sodium salt] (PAZO) and the polycation poly(ethylenimine) (PEI) built on the MUA SAM, were formed using the layer-by-layer electrostatic self-assembly method. The SFG spectrum of the SAM itself comprised strong methylene resonances, indicating the presence of gauche defects in the alkyl chains of the acid. The RAIRS spectrum of the SAM also contained strong methylene bands, indicating a degree of orientation of the methylene groups parallel to the surface normal. Changes in the SFG and RAIRS spectra when a PEI layer was adsorbed on the MUA monolayer showed that the expected electrostatic interaction between the polymer and the SAM, probably involving interpenetration of the PEI into the MUA monolayer, caused a straightening of the alkyl chains of the MUA and, consequently, a decrease in the number of gauche defects. When a layer of PAZO was subsequently deposited on the MUA/PEI film, further spectral changes occurred that can be explained by the formation of a complex PEI/PAZO interpenetrated layer. A per-deuterated MUA SAM was used to determine the relative contributions from the adsorbed polyelectrolytes and the MUA monolayer to the RAIRS and SFG spectra. Spectroscopic and adsorbed mass measurements combined showed that as further bilayers were constructed the interpenetration of PAZO into preadsorbed PEI layers was repeated, up to the formation of at least five PEI/PAZO bilayers.

DNA Adsorption to and Elution from Silica Surfaces: Influence of Amino Acid Buffers

Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers. Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction.

Desolvation of BSA-ligand complexes measured using the quartz crystal microbalance and dual polarization interferometer.

By taking advantage of their unique difference in hydration sensitivity, we have shown that dual polarization interferometer (DPI) and quartz-crystal microbalance with dissipation monitoring (QCM-D) measurements can be used together to explore the degree of desolvation involved in the binding of small drug molecules to an immobilized bovine serum albumin film in real time. Results with DPI and QCM-D show significantly different mass values for three ligands of varying hydrophobicities that may be attributed to changes in the degree of hydration of the ligand-protein complexes in accordance with the physicochemical properties of the ligands. Furthermore, our data suggest that masses measured by QCM-D can be overwhelmed by changes in water content of ligand-protein, binary complexes, which has important consequences for future studies using mechanical resonators to study protein-binding events.

Monitoring N3 Dye Adsorption and Desorption on TiO2 Surfaces: A Combined QCM-D and XPS Study

Understanding the kinetics of dye adsorption and desorption on semiconductors is crucial for optimizing the performance of dye-sensitized solar cells (DSSCs). Quartz crystal microbalance with dissipation monitoring (QCM-D) measures adsorbed mass in real time, allowing determination of binding kinetics. In this work, we characterize adsorption of the common RuBipy dye N3 to the native oxide layer of a planar, sputter-coated titanium surface, simulating the TiO2 substrate of a DSSC. We report adsorption equilibrium constants consistent with prior optical measurements of N3 adsorption. Dye binding and surface integrity were also verified by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy (XPS). We further study desorption of the dye from the native oxide layer on the QCM sensors using tetrabutylammonium hydroxide (TBAOH), a commonly used industrial desorbant. We find that using TBAOH as a desorbant does not fully regenerate the surface, though little ruthenium or nitrogen is observed by XPS after desorption, suggesting that carboxyl moieties of N3 remain bound. We demonstrate the native oxide layer of a titanium sensor as a valid and readily available planar TiO2 morphology to study dye adsorption and desorption and begin to investigate the mechanism of dye desorption in DSSCs, a system that requires further study.