Fluorescence Correlation Spectroscopy Studies of Dye Diffusion on Fresh and Aged Polyethylene Terephthalate.

Microplastics accumulate a wide variety of organic pollutants and thus may serve as efficient vectors for the transport of toxic substances. Much remains to be learned about how organic molecules interact with the surfaces of plastics and how these properties evolve as the microplastics are weathered. In this Letter, we report, for the first time, the application of confocal fluorescence correlation spectroscopy (FCS) to studies of organic molecules adsorbed from aqueous solution onto the surfaces of synthetic secondary microplastics. Both fresh and artificially aged poly(ethylene terephthalate) (PET) plastics are employed. The plastics are artificially aged in a UV-ozone chamber. Raman and infrared spectra confirm the composition of the PET microplastics. Water contact angle and surface roughness measurements reveal, respectively, an increase in wettability and a change in the nature of roughness with aging, consistent with surface oxidation. Rhodamine B (RhB) dye is used as a fluorescent probe in FCS studies and serves as an analogue for organic pollutants commonly found on microplastics. The FCS results reveal the accumulation of dye on the PET surfaces as they age. Dye motion is significantly slower on the plastics than in bulk aqueous solution and occurs by anomalous subdiffusion. The rate of diffusion becomes dramatically slower and more anomalous as the plastics are aged. Surface diffusion is likely slowed by either ionic interactions or hydrogen bonding between the dye and plastic. These results provide new insights critical to the understanding of how microplastics accumulate and transport organic pollutants as they weather in the environment.

Diffusion of hydrophilic to hydrophobic forms of Nile red in aqueous C12EO10 gels by variable area fluorescence correlation spectroscopy.

Solute diffusion within lyotropic liquid crystal gels prepared from a series of water and decaethylene glycol monododecyl ether (C12EO10) mixtures was explored by variable area fluorescence correlation spectroscopy. Aqueous C12EO10 gels were prepared in concentrations ranging from 55 : 45 to 70 : 30 wt% of surfactant and water. Small angle X-ray scattering revealed that these gels comprise hexagonal mesophases of cylindrical micelles. Micelle spacing was found to decrease with increasing C12EO10 concentration. Three different Nile red (NR) dyes were employed as model solutes and were separately doped into the gels at nanomolar levels. These include a hydrophilic form of NR incorporating an anionic sulfonate group (NRSO3-), a hydrophobic form incorporating a fourteen-carbon alkane tail (NRC14), and commercial NR as an intermediate case. FCS data acquired from the gels revealed that NRSO3- diffused primarily in 3D. Its diffusion coefficient exhibited a monotonic decrease with increasing gel concentration and micelle packing density, consistent with confinement of its motions by its exclusion from the micelle cores. NRC14 exhibited the smallest diffusion coefficient, most likely due to its larger size and enhanced interactions with the micelle cores. NR yielded an intermediate diffusion coefficient and the most anomalous behavior of the three dyes, attributable to its facile partitioning between core and corona regions, and greater participation by 1D diffusion. The results of these studies afford an improved understanding of molecular mass transport through soft-matter nanomaterials like those being developed for use in drug delivery and membrane based chemical separations.

Solvent-Induced Swelling Behaviors of Microphase-Separated Polystyrene-block-Poly(ethylene oxide) Thin Films Investigated Using In Situ Spectroscopic Ellipsometry and Single-Molecule Fluorescence Microscopy.

Block copolymers have attracted considerable interest in the fields of nanoscience and nanotechnology because these polymers afford well-defined nanostructures via self-assembly. An in-depth understanding of solvent effects on the physicochemical properties of these microdomains is crucial for their preparation and utilization. Herein, we employed in situ spectroscopic ellipsometry and single-molecule fluorescence techniques to gain detailed insights into microdomain properties in polystyrene-block-poly(ethylene oxide) (PS-b-PEO) films exposed to ethanol- and water-saturated N2. We observed a quick increase and a subsequent gradual decrease in the ellipsometric thickness of PS-b-PEO films upon exposure to ethanol-saturated N2. This observation was unexpected because ethanol-saturated N2 induced negligible thickness change for PS and PEO homopolymer films. The similarity in maximum thickness gain observed under ethanol- and water-saturated N2 implied the swelling of PEO microdomains. Ethanol vapor permeation through the PEO microdomains was supported by the redshift of the ensemble and single-molecule fluorescence emission of Nile red in PS-b-PEO films. Single-molecule tracking data showed the initial enhancement and subsequent reduction of the diffusion of hydrophilic sulforhodamine B molecules in PS-b-PEO films upon exposure to ethanol-saturated N2, consistent with the spectroscopic ellipsometry results. The higher ethanol susceptibility of the PEO microdomains was attributable to their amorphous nature, as shown by FTIR data.

Investigation of Molecular Diffusion at Block Copolymer Thin Films Using Maximum Entropy Method-Based Fluorescence Correlation Spectroscopy and Single Molecule Tracking.

Fluorescence correlation spectroscopy (FCS) has been widely used to investigate molecular diffusion behavior in various samples. The use of the maximum entropy method (MEM) for FCS data analysis provides a unique means to determine multiple distinct diffusion coefficients without a priori assumption of their number. Comparison of the MEM-based FCS method (MEM-FCS) with another method will reveal its utility and advantage as an analytical tool to investigate diffusion dynamics. Herein, we measured diffusion of fluorescent probes doped into nanostructured thin films using MEM-FCS, and validated the results with single molecule tracking (SMT) data. The efficacy of the MEM code employed was first demonstrated by analyzing simulated FCS data for systems incorporating one and two diffusion modes with broadly distributed diffusion coefficients. The MEM analysis accurately afforded the number of distinct diffusion modes and their mean diffusion coefficients. These results contrasted with those obtained by fitting the simulated data to conventional two-component and anomalous diffusion models, which yielded inaccurate estimates of the diffusion coefficients. Subsequently, the MEM analysis was applied to FCS data acquired from hydrophilic dye molecules incorporated into microphase-separated polystyrene-block-poly(ethylene oxide) (PS-b-PEO) thin films characterized under a water-saturated N2 atmosphere. The MEM analysis revealed distinct fast and slow diffusion components attributable to molecules diffusing on the film surface and inside the film, respectively. SMT studies of the same materials yielded trajectories for mobile molecules that appear to follow the curved PEO microdomains. Diffusion coefficients obtained from the SMT data were consistent with those obtained for the slow diffusion component detected by MEM-FCS. These results highlight the utility of MEM-FCS and SMT for gaining complementary information on molecular diffusion processes in heterogeneous material systems.

Single Molecule Spectroscopy Studies of Acid-Base Chemical Gradients Using Nile Red as a Probe of Local Surface Acidity.

Single molecule spectroscopy studies of local acidity along bifunctional acid-base gradients are reported. Gradients are prepared by directional vapor phase diffusion and subsequent reaction of 3-aminopropyl-trimethoxysilane with a uniform silica film. Gradient formation is confirmed by spectroscopic ellipsometry and by static water contact angle measurements. X-ray photoelectron spectroscopy is used to characterize the nitrogen content and degree of nitrogen protonation along the gradient. Nile Red is employed as the probe dye in single molecule spectroscopy studies of these gradients. While Nile Red is well-known for its solvent sensitivity, it is used here, for the first time, to sense the acid/base properties of the film in two-color wide-field fluorescence imaging experiments. The data reveal broad bimodal distributions of Nile Red emission spectra that vary along the gradient direction. The single molecule results are consistent with solution phase ensemble acid/base studies of the dye. The former reveal a gradual transition from a surface dominated by basic aminosilane sites at the high-amine end of the gradient to one dominated by acidic silanol sites at the low-amine end. The sub-diffraction-limited spatial resolution afforded by superlocalization of the single molecules reveals spatial correlations in the acid/base properties of the gradient over ∼200 nm distances. These studies provide data relevant to the use of aminosilane-modified silica in bifunctional, cooperative chemical catalysis.

Dysregulation of the AP2M1 phosphorylation cycle by LRRK2 impairs endocytosis and leads to dopaminergic neurodegeneration.

Mutations in the kinase LRRK2 and impaired endocytic trafficking are both implicated in the pathogenesis of Parkinson's disease (PD). Expression of the PD-associated LRRK2 mutant in mouse dopaminergic neurons was shown to disrupt clathrin-mediated endocytic trafficking. Here, we explored the molecular mechanism linking LRRK2 to endocytosis and found that LRRK2 bound to and phosphorylated the µ2 subunit of the adaptor protein AP2 (AP2M1), a core component of the clathrin-mediated endocytic machinery. Analysis of human SH-SY5Y cells and mouse neurons and tissues revealed that loss of LRRK2 abundance or kinase function resulted in decreased phosphorylation of AP2M1, which is required for the initial formation of clathrin-coated vesicles (CCVs). In contrast, overexpression of LRRK2 or expression of a Parkinson's disease-associated gain-of-function mutant LRRK2 (G2019S) inhibited the uncoating of AP2M1 from CCVs at later stages and prevented new cycles of CCV formation. Thus, the abundance and activity of LRRK2 must be calibrated to ensure proper endocytosis. Dysregulated phosphorylation of AP2M1 from the brain but not thyroid tissues of LRRK2 knockout and G2019S-knockin mice suggests a tissue-specific regulatory mechanism of endocytosis. Furthermore, we found that LRRK2-dependent phosphorylation of AP2M1 mediated dopaminergic neurodegeneration in a Drosophila model of PD. Together, our findings provide a mechanistic link between LRRK2, AP2, and endocytosis in the pathogenesis of PD.

Fluorescence Microscopic Investigations of Molecular Dynamics in Self-Assembled Nanostructures.

Many analytical methods employ self-assembled nanostructured materials as chemical recognition media. Molecular permeation through these materials exhibits unique selectivity owing to nanoconfinement-induced enhancement of permeant-nanostructure interactions. This Personal Account introduces our efforts to investigate the detailed dynamics of single or a small number of molecules in nanostructured materials. We developed new experimental and analysis approaches built upon laser-based fluorescence microscopy to measure the detailed translational and orientational dynamics of molecules diffusing in horizontally-oriented, cylindrical nanostructures, including surfactant micelles, silica mesopores, block copolymer microdomains, and bolaamphiphile-based organic nanotubes. Our studies clarified nanoscale details on the structural/chemical heterogeneity of the nanostructures, and their impacts on molecular mass transport dynamics.

On the Importance of Silane Infusion Order on the Microscopic and Macroscopic Properties of Multifunctional Charge Gradients.

Four multicomponent charge gradients containing acidic and basic functionalities were prepared via sol-gel processes and the controlled-rate infusion (CRI) method to more clearly understand how preparation conditions influence macroscopic properties. CRI is used to form gradients by infusing reactive alkoxysilanes into a glass vial housing a vertically oriented modified silicon wafer. The concentration and time of infusion of the silane solutions were kept constant. Only the sequence of infusion of the silane solutions was changed. The first set of samples was prepared by initially infusing a solution containing 3-aminopropyltriethoxysilane (APTES) followed by a mercaptopropyltrimethoxysilane (MPTMS) solution. The individual gradients were formed either in an aligned or opposed fashion with respect to the initial gradient. The second set of samples was prepared by infusing the MPTMS solution first followed by the APTES solution, again in either an aligned or opposed fashion. To create charge gradients (NH3 +, SO3 -), the samples were immersed into H2O2. The extent of modification, the degree of protonation of the amine, and the thicknesses of the individual layers were examined by X-ray photoelectron spectroscopy (XPS) and spectroscopic ellipsometry. The wettability of the individual gradients was assessed via static contact angle measurements. The results demonstrate the importance of infusion order and how it influences the macroscopic and microscopic properties of gradient surfaces including the surface concentration, packing density, degree of protonation, and ultimately wettability. When the gradient materials are prepared via infusion of the APTES sol first, it results in increased deposition of both the amine and thiol groups as evidenced by XPS. Interestingly, the total thickness evaluated from ellipsometry was independent of the infusion order for the aligned gradients, indicative of significant differences in the film density. For the opposed gradients, however, the infusion of APTES first leads to a significantly thicker composite film. Furthermore, it also leads to a more pronounced gradient in the protonation of the amine, which introduces a very different surface wettability. The use of aminosilanes provides a viable approach to create gradient surfaces with different functional group distributions. These studies demonstrate that the controlled placement of functional groups on a surface can provide a new route to prepare gradient materials with improved performance.

Influences of Hydrogen Bonding-Based Stabilization of Bolaamphiphile Layers on Molecular Diffusion within Organic Nanotubes Having Inner Carboxyl Groups.

This paper reports molecular diffusion behavior in two bolaamphiphile-based organic nanotubes having inner carboxyl groups with different inner dimeters (10 and 20 nm) and wall structures, COOH-ONT10nm and COOH-ONT20nm, using imaging fluorescence correlation spectroscopy (imaging FCS). The results were compared to those previously obtained in a similar nanotube with inner amine groups (NH2-ONT10nm). COOH-ONT10nm, as with NH2-ONT10nm, were formed from a rolled bolaamphiphile layer incorporating triglycine moieties, whereas COOH-ONT20nm consisted of four stacks of triglycine-free bolaamphiphile layers. Imaging FCS measurements were carried out for anionic sulforhodamine B (SRB), zwitterionic/cationic rhodamine B (RB), and cationic rhodamine-123 (R123) diffusing within ONTs (1-9 µm long) at different pH (3.4-8.4) and ionic strengths (1.6-500 mM). Diffusion coefficients (D) of these dyes in the ONTs were very small (0.01-0.1 µm2/s), reflecting the significant contributions of molecule-nanotube interactions to diffusion. The D of SRB was larger at higher pH and ionic strength, indicating the essential role of electrostatic repulsion that was enhanced by the deprotonation of the inner carboxyl groups. Importantly, the D of SRB was virtually independent of nanotube inner diameter and wall structure, indicating the diffusion of the hydrophilic molecule was controlled by short time scale adsorption/desorption processes onto the inner surface. In contrast, pH effects on D were less clear for relatively hydrophobic R123 and RB, suggesting the significant contributions of non-Coulombic interactions. Interestingly, the diffusion of these molecules in COOH-ONT20nm was slower than in COOH-ONT10nm. Slower diffusion in COOH-ONT20nm was attributable to relatively efficient partitioning of the hydrophobic dyes into the bolaamphiphile layers, which was reduced in COOH-ONT10nm due to the stabilization of its layer by polyglycine-II-type hydrogen bonding networks. These results show that, by tuning the bolaamphiphile structures and their intermolecular interactions, unique environments can be created within the nanospaces for enhanced molecular separations and reactions.

Nanoconfinement and Mass Transport in Silica Mesopores: the Role of Charge at the Single Molecule and Single Pore Levels.

Polarization-dependent single molecule tracking was employed to simultaneously probe the translational and orientational diffusion of four perylene diimide (PDI) dyes, having different lengths and charges, within the one-dimensional (1D) nanoscale pores of surfactant-templated mesoporous silica films. The wide-field fluorescence videos acquired reveal that a significant fraction of the molecules follow 1D pathways and exhibit highly polarized fluorescence, consistent with their orientational confinement. Single-frame step size distributions prepared from these data were fit to a new model that accurately describes the distribution for 1D Fickian diffusion in the presence of finite localization precision. Average diffusion coefficients obtained from mean square displacement (DMSD) data were 20-100% larger for the two uncharged PDIs compared to the charged PDIs, reflecting electrostatic interactions of the latter with oppositely charged sites on the cationic surfactant headgroups and deprotonated silanol sites on the pore walls. Polarization-dependent tracking data show that the longest uncharged PDI was most strongly confined, while the three shorter dyes were less confined. The cationic PDI produced a wobbling angle distribution that was broader than the others, suggesting it explores more of the pore diameter. The results provide new knowledge on the mechanisms by which the dye molecules interact with the pore-filling medium and the pore surfaces, helping to elucidate the factors controlling the rate of mass transport.

Diffusion Behavior of Differently Charged Molecules in Self-Assembled Organic Nanotubes Studied Using Imaging Fluorescence Correlation Spectroscopy.

The diffusion behavior of fluorescent molecules within bolaamphiphile-based organic nanotubes (ONTs) was systematically investigated using imaging fluorescence correlation spectroscopy (imaging FCS). Anionic sulforhodamine B, zwitterionic/cationic rhodamine B, or cationic rhodamine 123 was loaded into ONTs having cylindrical hollow structures (ca. 10 nm in inner diameter) with amine and glucose groups on the inner and outer surfaces, respectively. Wide-field fluorescence video microscopy was used to acquire imaging FCS data for dye-doped ONTs in aqueous solutions of different ionic strengths (1-500 mM) at different pH (3.4-8.4). The diffusion behavior of these dyes was discussed on the basis of their apparent diffusion coefficients ( D) that were determined by autocorrelating the time transient of fluorescence intensity at each pixel on an ONT. Molecular diffusion in the ONTs was significantly slowed by the molecule-nanotube interactions, as shown by the very small D (10-1 to 10-2 µm2/s). The pH dependence of D revealed that dye diffusion was basically controlled by electrostatic interactions associated with the protonation of the amine groups on the ONT inner surface. The pH-dependent change in D was observed over a wide pH range, possibly because of electrostatically induced variations in the p Ka of the densely packed ammonium ions on the ONT inner surface. On the other hand, the influence of ionic strength on D was relatively unclear, suggesting the involvement of non-Coulombic interactions with the ONTs in molecular diffusion. Importantly, individual ONTs of different lengths (1-5 µm) afforded similar diffusion coefficients for each type of dye at each solution condition, implying that the properties of the ONTs were uniform in terms of solute loading and release. These results highlight the characteristics of the molecular diffusion behavior within the ONTs and will help in the design of ONTs better suited for use as drug vehicles and contaminant adsorbents.

Vapor-Phase Plotting of Organosilane Chemical Gradients.

Vapor-phase plotting of organosilane-based self-assembled monolayer (SAM) gradients is demonstrated for the first time. Patterned SAMs are formed by delivering gas-phase organotrichlorosilane precursors to a reactive silica surface using a heated glass capillary. The capillary is attached via a short flexible tube to a reservoir containing the precursor dissolved in toluene. The proximal end of the capillary is positioned at an experimentally optimized distance of 30 µm above the substrate during film deposition. The capillary is mounted to a stepper-motor-driven X, Y plotter for raster scanning above the surface. Two different organotrichlorosilane precursors are employed in this initial demonstration: n-octyltrichlorosilane and 3-cyanopropyltrichlorosilane. The dependence of SAM deposition on ambient relative humidity, capillary-substrate separation, raster-scanning speed, and solvent viscosity and volatility is explored and optimum deposition conditions are identified. The optimized procedures are used to plot uniformly modified square "pads" and gradients of the silanes. Film formation is verified and the gradient profiles are obtained by sessile drop water contact angle measurements, spectroscopic ellipsometry measurements of film thickness, and X-ray photoelectron spectroscopy mapping. The resolution of the plotting process is currently in the millimeter range and depends on capillary diameter and distance from the substrate surface. Vapor-phase plotting affords a unique direct-write method for producing patterned and chemically graded SAMS that may find applications in microfluidic devices, planar chromatography, and optical and electronic devices.

Probing the Local Dielectric Constant of Plasmid DNA in Solution and Adsorbed on Chemically Graded Aminosilane Surfaces.

Nile Red dye was used to determine the dielectric constant, ε, of nonpolar microenvironments in double stranded DNA (ds-DNA) both in aqueous buffer solution and when adsorbed on amine-modified surfaces. The value of ε within the DNA decreased with increasing buffer concentration. Values of ε ∼ 6.75 and ∼3.00 were obtained in 0.1 mM phosphate buffered saline (PBS) and in 10 mM PBS, respectively. Similar effects were observed upon adsorption to chemically graded amine-modified silica surfaces. Under 1 mM buffer, ε was measured to be ∼2.84 and ∼1.90 at the low amine (high silica) and high amine (low silica) ends of the gradient, respectively. An increase in the buffer concentration again led to a decrease in ε, but only at the low amine end. It is concluded that high buffer concentrations and binding to an amine surface cause a condensation of the DNA, forming less polar microenvironments. These results provide important knowledge of the factors governing the polarity of DNA microenvironments to which intercalators bind.

pH and Surface Charge Switchability on Bifunctional Charge Gradients.

Multifunctionalized pH-sensitive silica gradients containing acidic and basic functional groups have been prepared to evaluate how the spatial arrangement of active sites on a surface influences the surface charge and pH switchability. The gradient surfaces were prepared using controlled rate infusion in such a manner that the individual gradients in the strong acid (sulfonic acid) and in the weak base (propylamine) align, whereas a gradient in the weakly acidic silanol groups opposes them. The relative amounts of the three species were varied by controlling the composition of the deposition solution, whereas the hydrophobicity of the underlying surface was set by using base layer-coated substrates prepared from either tetramethoxysilane or tetramethoxysilane/octyltrimethoxysilane mixtures. Results from X-ray photoelectron spectroscopy confirm that aligned gradients are formed in both amine and sulfonic acid groups, and the relative amounts bound to the surface follow that expected from the solution composition. Water contact angle measurements show a 40°-50° change across the length of the gradient, the exact values being dependent on the hydrophobicity of the base layer. Zeta potential measurements on gradient mimics reveal that there is a pH where the net charge on the gradient surface is predicted to have a constant but nonzero value. Static contact angle measurements and modeling confirm this prediction. At a pH acidic of this value, the gradient in charge runs in one direction, whereas at a pH basic of this value, the gradient in charge runs in the other direction. This point can be strategically moved from acidic values to basic values by changing the relative amounts of acidic and basic functionalities on the surface. The origin of this unique pH switchability can be found in acid-base chemistry. By modeling the charge along the gradient surface using a simple equilibrium model, a distribution of pKa values were noted in these materials.

Organosilane Chemical Gradients: Progress, Properties, and Promise.

Chemical gradients play an important role in nature, driving many different phenomena critical to life, including the transport of chemical species across membranes and the transport, attachment, and assembly of cells. Taking a cue from these natural processes, scientists and engineers are now working to develop synthetic chemical gradients for use in a broad range of applications, such as in high-throughput investigations of surface properties, as means to guide the motions and/or assembly of liquid droplets, vesicles, nanoparticles, and cells and as new media for stationary-phase-gradient chemical separations. Our groups have been working to develop new methods for preparing chemical gradients from organoalkoxysilane and organochlorosilane precursors and to obtain a better understanding of their properties on macroscopic to microscopic length scales. This review highlights our recent work on the development of controlled-rate infusion and infusion-withdrawal dip-coating methods for the preparation of gradients on planar glass and silicon substrates, on thin-layer chromatography plates, and in capillaries and monoliths for liquid chromatography. We also cover the new knowledge gained from the characterization of our gradients using sessile drop and Wilhelmy plate dynamic water contact angle measurements, X-ray photoelectron spectroscopy mapping, and single-molecule tracking and spectroscopy. Our studies reveal important evidence of phase separation and cooperative interactions occurring along multicomponent gradients. Emerging concepts and new directions in the preparation and characterization of organosilane-based chemical gradients are also discussed.

Spectroscopic imaging studies of nanoscale polarity and mass transport phenomena in self-assembled organic nanotubes.

Synthetic organic nanotubes self-assembled from bolaamphiphile surfactants are now being explored for use as drug delivery vehicles. In this work, several factors important to their implementation in drug delivery are explored. All experiments are performed with the nanotubes immersed in ethanol. First, Nile Red (NR) and a hydroxylated Nile Red derivative (NR-OH) are loaded into the nanotubes and spectroscopic fluorescence imaging methods are used to determine the apparent dielectric constant of their local environment. Both are found in relatively nonpolar environments, with the NR-OH molecules preferring regions of relatively higher dielectric constant compared to NR. Unique two-color imaging fluorescence correlation spectroscopy (imaging FCS) measurements are then used along with the spectroscopic imaging results to deduce the dielectric properties of the environments sensed by mobile and immobile populations of probe molecules. The results reveal that mobile NR molecules pass through less polar regions, likely within the nanotube walls, while immobile NR molecules are found in more polar regions, possibly near the nanotube surfaces. In contrast, mobile and immobile NR-OH molecules are found to locate in environments of similar polarity. The imaging FCS results also provide quantitative data on the apparent diffusion coefficient for each dye. The mean diffusion coefficient for the NR dye was approximately two-fold larger than that of NR-OH. Slower diffusion by the latter could result from its additional hydrogen bonding interactions with polar triglycine, amine, and glucose moieties near the nanotube surfaces. The knowledge gained in these studies will allow for the development of nanotubes that are better engineered for applications in the controlled transport and release of uncharged, dipolar drug molecules.

Single Molecule Catch and Release: Potential-Dependent Plasmid DNA Adsorption along Chemically Graded Electrode Surfaces.

Single molecule detection methods were employed to study the potential dependent adsorption and desorption of dye labeled plasmid DNA along chemical gradients prepared on indium tin oxide (ITO) electrodes. Gradients were formed over silica-base-layer-coated ITO surfaces by exposing them in a directional fashion to aminopropyltrimethoxysilane from the vapor phase. Sessile drop water contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to verify that a gradient was formed and to characterize its wettability, thickness, and composition as a function of position. The gradient-coated ITO electrode served as both the working electrode and a window into the electrochemical cell used to manipulate DNA adsorption. For single molecule studies, the electrochemical cell was filled with buffer solution containing YOYO-1-labeled plasmid DNA. Fluorescence videos acquired along the gradients depicted clear position-, potential-, and pH-dependent variations in DNA adsorption and desorption. The results demonstrate that DNA adsorption was largely independent of applied potential and irreversible at high amine coverage (i.e., multilayers), under pH ∼ 6 buffer. DNA adsorption became more reversible as the amine coverage decreased and the solution pH increased. Potential dependent control over DNA adsorption and desorption was best achieved at monolayer-to-submonolayer aminosilane coverage under pH ∼ 8 buffer. The knowledge gained in these studies will aid in the development of electrochemical methods for the capture and release of DNA and other polyelectrolytes at electrode surfaces.

Base Layer Influence on Protonated Aminosilane Gradient Wettability.

Protonated amine gradients have been prepared on silicon wafers via programmed controlled rate infusion (CRI) with varying degrees of hydrophobicity and characterized by X-ray photoelectron spectroscopy (XPS) and static and Wilhelmy plate dynamic contact angle measurements. Initially, base layers were spin coated from sols containing tetramethoxysilane (TMOS) and either phenyltrimethoxysilane (PTMOS), dimethyldimethoxysilane (DMDMOS), or octyltrimethoxysilane (OTMOS, C8). Amine gradients were then prepared from 3-aminopropyltriethoxysilane (APTEOS) via CRI. Gradients were exposed to concentrated HCl vapor for amine protonation. XPS showed that NH2 functional groups were distributed in a gradient fashion as a result of CRI controlling the time of exposure to APTEOS. Interestingly, the overall extent of N modification depended on the type of base layer used for gradient formation. The C8-derived base layer had about half the amount of nitrogen on the surface as compared to those prepared from TMOS, which was attributed to a reduction in the number and accessibility of surface silanol groups. The wettability and contact angle (CA) hysteresis were also dependent on the base layer and varied along the length of the gradient. The greatest CA change across the length of the gradient was observed on the gradient formed on the C8-derived base layer. Likewise, the CA hysteresis was approximately 2 times larger on the C8-modified surfaces, indicative of greater chemical inhomogeneity. In contrast to uniformly modified substrates, Wilhelmy plate CA analysis that involves the immersion of samples gave a unique S-shaped CA distance curve for the gradients. The three curve segments correspond to hydrophilic, hydrophobic, and a middle connecting region. Importantly, these curves give precise CAs along the gradient that reflect the surface chemistry and coverage defined by programmed CRI processing.