Polymer Pen Printing: A Tool for Studying 2D Enzymatic Lithography and Printing 3D Carbon Features

Polymer Pen Lithography (PPL) is a promising molecular printing approach which combines the advantages of both microcontact printing (low cost, high-throughput) and the dip pen lithography (DPN) (arbitrary writing, high-resolution) into one cohesive lithography method to create 2 dimensional (2-D) patterns with micro/nano-features on different substrates. The goal of this dissertation is to design and develop a new tool based upon PPL, which is not limited to forming 2D parallel patterns, but can also create 3D complex microstructures, finding applications in both biotechnology and Micro-Electro-Mechanical systems (MEMS) technology. This novel approach is named Polymer Pen Printing. Different from PPL using traditional dry-ink printing methods, an inking step is added to each printing repetition in the polymer pen printing process. Thus a wide range of ink materials with diverse viscosities can be transferred to substrates to create functional 2D and 3D microstructures. The polymer pen printing apparatus used in this thesis has been accomplished and introduced in Chapter 2. As a preliminary attempt, the single polymer pen printing approach was developed by simply attaching a solid polydimethylsiloxane (PDMS) pen tip to a multi-axis robot for small microarray fabrication. Compared to the single pen printing method, multi-pen printing can create large arrays of features. Therefore, an improved apparatus for polymer pen printing with high-throughput was discussed and built. Silicon molds, which consist of hundreds of uniform pyramidal openings, were photolithographically defined and etched using hydrofluoric acid (HF) followed by potassium hydroxide solution; after surface-modification with fluorosilane, these silicon molds were used to cast arrays of PDMS pyramidal pen tip. The cast PDMS pen array was mounted to a hollow holder with a 45° mirror inside. Therefore, each PDMS pen can be observed and monitored from the microscope on the side. To achieve prints less than 1 micron across, a Z axis stage with nanometer resolution was incorporated; and to control the compression of PDMS pen tips, a force gauge was also incorporated to detect 1 mg of applied force from the tips. The printing process for the multi-pen system is almost the same as single pen system. PDMS pens are coated with ink solution before each printing cycle by dipping into an inkwell and then brought into contact with the substrate surface. Thus multiple patterns, one from each tip, are created in parallel simultaneously. Furthermore, with control of the printing force, feature sizes could be controlled over the range submicron to tens of microns. Three ink candidates have been printed by polymer pen printing approach to fabricate 2D&3D microstructures. The first ink material is Barium Strontium Titanate (BST) nanocrystallites dispersed in a furfuryl alcohol (FA), which was printed by the single PDMS pen with 100 μm tip diameter (Chapter 3). After printing, samples were heated to crosslink FA monomers, forming a stable polymeric matrix with embedded BST nanocrystallites. Without shear-thinning properties, BST/FA ink cannot be used to build 3D posts, but it has the capability to create circular patterns with different thickness by the single or multi-tier deposition method. It was found that the thickness of film increased linearly with the number of deposits without changing the diameter significantly. This encouraging result could enable the formation of microcapacitors with multi-tiered structure. Moreover, the study of printing parameters, including printing height and ink pick-up position, shows that changes to the pen positions in the ink reservoir or substrate have essentially no impact on deposit thickness or diameter. Beyond that, the effect of surface chemistry of PDMS pen and silicon wafer have also been studied. The plasma treated hydrophilic PDMS pen can pen transfer more BST/FA than untreated one; and the larger diameters with smaller thickness were obtained on a hydrophilic silicon wafer. The second ink candidate is a dilute aqueous solution of enzyme Candia antartica lipase B (CALB), which is known to catalyze the decomposition of poly (ε-caprolactone) (PCL) films. By bringing enzymes into contact with pre-defined regions of a surface, a polymer film can be selectively degraded to form patterned features that are requited for applications in biotechnology and electronics. This so-called enzymatic lithography is an environmentally friendly process as it does not require any actinic radiation or synthetic chemicals to develop required features. But the need to restrict the mobility of the enzyme in order to maintain control of feature sizes poses a significant challenge. In Chapter 4, after writing 2D enzyme patterns onto a spin-cast PCL film by single pen printing, samples with CALB were incubated at 37 ℃ and 95% relative humidity (RH) for up to 7 days to develop features. The CALB selectively degraded the PCL film during incubation, forming openings through the film. The size of these features (10 to 50 μm diameter) is well suited for use as biocompatible micro-reactors. Previous study of patterning CALB by single polymer pen printing technique resulted in slow etch rates, low throughput and poor image quality. In Chapter 5, I present an improved enzymatic lithography approach, still based on enzyme CALB and PCL system, which can resolve fine-scale features (\u3c 1 μm across) in thick (0.1 - 2.0 μm) polymer films after 5 minutes to 2 hours of incubation at 37 ℃ and 87% RH. Immobilization of the enzyme on the polymer surface was monitored using fluorescence microscopy by labeling CALB with FITC. The crystallite size in the PCL films was systematically varied; small crystallites resulted in significantly faster etch rates (20 nm/min) and the ability to resolve smaller features (as fine as 1 μm). The effect of printing conditions and RH during incubation is also presented. Patterns formed in the PCL film were transferred to an underlying copper foil demonstrating a Green approach to the fabrication of printed circuit boards. In parallel, the third ink material is a mixture of 25 wt% graphite dispersed in a high viscosity phenolic resin n-methyl-2-pyrrolidone (NMP) solution, which can be converted into carbon/carbon composites after a pyrolysis process. The 3D polymeric posts were created by depositing multilayers of thixotropic phenolic ink on a silicon substrate by single polymer pen printing method with a 10 μm radius PDMS pen tip (Chapter 6). After pyrolysis at 1000 ℃ in a nitrogen (N2) atmosphere, the polymeric features were converted to the glassy carbon/graphite features with a high aspect ratio (\u3e2). These features may be used as microelectrodes. Last, arrays of needle-shaped glassy carbon have been developed by a drawing approach using multi-pen printing technique followed by simple pyrolysis process (Chapter 7). To build polymeric needles with ultra-high aspect ratio, the polymeric ink was prepared by dissolving phenolic resin in the high boiling point (204 ℃) solvent NMP without fillers to achieve good printability and suitable viscosity. By slowly lifting up the print head from substrate, liquid needle structures were formed and then solidified on silicon substrates or gold electrodes due to the solvent evaporation. In addition, suspended resin fibers connected to two electrodes have also been fabricated by precisely controlling the movement of the PDMS pen. After pyrolysis, these resin features were converted to glassy carbon and the 3D structures remained. The electrical characterization results showed that glassy carbon made by this method had relatively low resistivity (2.5 x 10-5 Ωm). Therefore the glassy carbon based microneedles are well-suited to be electrodes for electrochemical sensors for biological applications

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer \u27epicuticular\u27 layer as well as the blend consisting of the cutin biopolymer and \u27intracuticular\u27 waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Schäffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties. Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-μm length scale. The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment. 1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer \u27epicuticular\u27 layer as well as the blend consisting of the cutin biopolymer and \u27intracuticular\u27 waxes. 2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato ( Solanum lycopersicum ) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Schäffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle. 3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13 C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13 C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH 2 O) chemical moieties

Patterned Enzymatic Degradation of Poly(ε-caprolactone) by High-Affinity Microcontact Printing and Polymer Pen Lithography

This paper reports deposition of Candida antarctica Lipase B (CALB) on relatively thick poly­(ε-caprolactone) (PCL) films (300–500 nm) to create well-defined patterns using two different writing techniques: high-affinity microcontact (HA-μCL) and polymer pen (PPL) lithography. For both, an aqueous CALB ink is absorbed onto a polydimethylsiloxane (PDMS) writing implement (PDMS stamp or a PDMS pen tip), which is transferred to a spun-cast PCL film. HA-μCL experiments demonstrated the importance of applied pressure to obtain high-resolution patterns since uniform contact is needed between raised 20 μm parallel line regions of the PDMS stamp and the surface. AFM imaging shows pattern formation evolves gradually over incubation time only in areas stamped with CALB cutting through spherulites without apparent influence by grain boundaries. Strong binding of CALB to PCL is postulated as the mechanism by which lateral diffusion is limited. PPL enables formation of an arbitrary image by appropriate programming of the robot. The PDMS pen tips were coated with an aqueous CALB solution and then brought into contact with the PCL film to transfer CALB onto the surface. By repeating the ink transfer step multiple times where pen tips are brought into contact with the PCL film at a different locations, a pattern of dots is formed. After printing, patterns were developed at 37 °C and 95% RH. Over a 7-day period, CALB progressively etched the PCL down to the silicon wafer on which it was spun (350 nm) giving round holes with diameters about 10 μm. AFM images show the formation of steep PCL walls indicating CALB degraded the PCL film in areas to which it was applied. This work demonstrates that high-resolution patterns can be achieved without immobilizing the enzyme on the surface of polymeric stamps that limits the depth of features obtained as well as the throughput of the process

High Throughput, High Resolution Enzymatic Lithography Process: Effect of Crystallite Size, Moisture, and Enzyme Concentration

By bringing enzymes into contact with predefined regions of a surface, a polymer film can be selectively degraded to form desired patterns that find a variety of applications in biotechnology and electronics. This so-called “enzymatic lithography” is an environmentally friendly process as it does not require actinic radiation or synthetic chemicals to develop the patterns. A significant challenge to using enzymatic lithography has been the need to restrict the mobility of the enzyme in order to maintain control of feature sizes. Previous approaches have resulted in low throughput and were limited to polymer films only a few nanometers thick. In this paper, we demonstrate an enzymatic lithography system based on Candida antartica lipase B (CALB) and poly­(ε-caprolactone) (PCL) that can resolve fine-scale features, (<1 μm across) in thick (0.1–2.0 μm) polymer films. A Polymer Pen Lithography (PPL) tool was developed to deposit an aqueous solution of CALB onto a spin-cast PCL film. Immobilization of the enzyme on the polymer surface was monitored using fluorescence microscopy by labeling CALB with FITC. The crystallite size in the PCL films was systematically varied; small crystallites resulted in significantly faster etch rates (20 nm/min) and the ability to resolve smaller features (as fine as 1 μm). The effect of printing conditions and relative humidity during incubation is also presented. Patterns formed in the PCL film were transferred to an underlying copper foil demonstrating a “Green” approach to the fabrication of printed circuit boards

A supramolecular hybrid material constructed from graphene oxide and a pillar[6]arene-based host-guest complex as an ultrasound and photoacoustic signal nanoamplifier

Photoacoustic imaging combines the merits of ultrasound imaging and optical imaging, and allows a fascinating imaging paradigm with deeper tissue penetration than optical imaging and higher spatial resolution than ultrasound imaging. Herein, we develop a supramolecular hybrid material composed of graphene oxide (GO) and a pillar[6] arene-based host-guest complex (CP6 superset of PyN), which can be used as an ultrasound (US) and photoacoustic (PA) signal nanoamplifier. Triggered by the near-infrared (NIR) light mediated photothermal effect, CO2 nanobubbles are generated on the surface of GO@CP6 superset of PyN due to the decomposition of the bicarbonate counterions, thus strongly amplifying its US and PA performances. Our study, for the first time, demonstrates enhanced US and PA activity in a supramolecular hybrid material on the basis of host-guest chemistry as a photoacoustic nanoplatform

Development of a Scalable, Chromatography-Free Synthesis of <i>t</i>‑Bu-SMS-Phos and Application to the Synthesis of an Important Chiral CF₃‑Alcohol Derivative with High Enantioselectivity Using Rh-Catalyzed Asymmetric Hydrogenation

A chromatography-free, asymmetric synthesis of the C2-symmetric P-chiral diphosphine <i>t</i>-Bu-SMS-Phos was developed using a chiral auxiliary-based approach in five steps from the chiral auxiliary in 36% overall yield. Separtion and recovery of the auxiliary were achieved with good yield (97%) to enable recycling of the chiral auxiliary. An air-stable crystalline form of the final ligand was identified to enable isolation of the final ligand by crystallization to avoid chromatography. This synthetic route was applied to prepare up to 4 kg of the final ligand. The utility of this material was demonstrated in the asymmetric hydrogenation of trifluoromethyl vinyl acetate at 0.1 mol % Rh loading to access a surrogate for the pharmaceutically relavent chiral trifluoroisopropanol fragment in excellent yield and enantiomeric excess (98.6%)