This paper presents the combination of "bottom-up" layer-by-layer (LbL) nano self-assembly and "top-down" micromanufacturing techniques for MEMS and microelectronics applications. Two approaches, modified lift-off and metal mask, were utilized to pattern nano-assembled thin films effectively. With nano self-assembly and surface micromachining, highly flexible nanoparticle-based magnetic cantilever platform for micro sensing and actuation was self-assembled. Self-assembled magnetic thin films on polymeric tunneling sensors were functional as a polymer actuators for the actuation of highly sensitive sensors. For microelectronics devices, self-assembled silica nanoparticle thin film acted as the dielectric layer for field-effect transistors. Nanoparticle- and polymer-based field-effect transistors with embedded nano self-assembled films as dielectric and active layers were fabricated and characterized successfully as well.
Nanomedicine involves cell-by-cell regenerative medicine, either repairing cells one at a time or triggering apoptotic pathways in cells that are not repairable. Multilayered nanoparticle systems are being constructed for the targeted delivery of gene therapy to single cells. Cleavable shells containing targeting, biosensing, and gene therapeutic molecules are being constructed to direct nanoparticles to desired intracellular targets. Therapeutic gene sequences are controlled by biosensor-activated control switches to provide the proper amount of gene therapy on a single cell basis. The central idea is to set up gene therapy "nanofactories" inside single living cells. Molecular biosensors linked to these genes control their expression. Gene delivery is started in response to a biosensor detected problem; gene delivery is halted when the cell response indicates that more gene therapy is not needed.
Cell targeting of nanoparticles, both nanocrystals and nanocapsules, has been tested by a combination of fluorescent tracking dyes, fluorescence microscopy and flow cytometry. Intracellular targeting has been tested by confocal microscopy. Successful gene delivery has been visualized by use of GFP reporter sequences. DNA tethering techniques were used to increase the level of expression of these genes. Integrated nanomedical systems are being designed, constructed, and tested in-vitro, ex-vivo, and in small animals.
While still in its infancy, nanomedicine represents a paradigm shift in thinking-from destruction of injured cells by surgery, radiation, chemotherapy to cell-by-cell repair within an organ and destruction of non-repairable cells by natural apoptosis.
Layer-by-layer (LbL) nanoassembly in combination with traditional lithography and microfluidics was applied for the fabrication of ultrathin microcantilevers and for the modification of microchannel surface. Hundreds of cantilevers were fabricated on a silicon wafer simultaneously. The purpose is to develop chemical/biosensor arrays for parallel, massive data gathering. Microcantilever optical deflections were measured using a four-quadrant AFM head with integrated laser and position sensitive detector. In the second part, laminar flow fabrication of interpolyelectrolyte complexes was studied inside a microchannel. Polyelectrolyte micropatterns were studied using fluorescent confocal microscopy. Filament like, 15 μm, interpolyelectrolyte microstripes were formed at flow rate higher than 0.01 mL•min-1 and concentrations of the initial polyelectrolytes below 1 mg•mL-1. New, soft micropatterning technique for the anisotropic modification of polyelectrolyte nanocapsules was also demonstrated. The microchannel surface was made sensitive to pH by coating the surface of the channel with a pH sensitive dye using LbL assembly to control the reaction.
Extracellular enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP) from white rot fungus Phanerochaete chrysosoporium, have been shown to degrade various harmful organic compounds ranging from chlorinated compounds to polycyclic aromatic hydrocarbons (PAH) to polymeric dyes. The problems in using immobilized enzymes for biocatalysis/bioremediation are their loss of activity and long-term stability. To address these issues, adsorption by layer-by-layer assembly (LbL) using polyelectrolytes, entrapment using gelatin, and chmisorption using coupling reagents have been investigated. In order to increase surface area for catalysis, porous silicon, formed by electrochemical etching of silicon, has been considered. The efficacy of these extremely stable nanoassemblies towards degradation of model organic compounds-veratryl alcohol (VA and 2,6-dimethoxyphenol (DMP)-in aqueous and in a mixture of aqueous/acetone has already been demonstrated. In parallel, we are pursuing development of sensors using these immobilized enzymes. Experiments carried out in solution show that NO can reversibly bind Ferri-LiP to produce a diamagnetic complex with a distinct change in its optical spectrum. NO can be photolyzed off to produce the spectrum of native paramagnetic ferri-species. Preliminary data on the detection of NO by LiP, based on surface plasmon resonance (SPR) using fiber optic probe, are presented.
A layer-by-layer nanoassembly (LbL) allows production of ultrathin films with a precision of 1-2 nm and needed composition across the multilayer. It was used in combination with traditional lithography to develop micropatterns in ordered nanoparticle multilayers. A selective nanoparticle film growth was also demonstrated for microchannel silicon chips. Microfluidic properties of nanoorganized polymer microcapsules were studied with the microchannel device. Nanoorganized microcapsules production: A LbL-assembly of 20-nm thick
poly(styrenesulfonate) / poly(allylamine) shell on microtemplates and loading such hollow polyion shells with enzymes allowed fabrication of catalytic "bioreactors," as it was demonstrated for glucose oxidase, hemoglobin, and myoglobin ensembles.
A microfluidic system was designed, fabricated and implemented to study the behavior of polyelectrolyte capsules flowing in microscale channels. The silicon component of the system contains microchannels that leads into constrictions, which were fabricated using lithography techniques. Polyelectrolyte microcapsules were also fabricated with well-known layer-by-layer assembly technique, on a spherical template. Once the template was removed, the resulting hollow capsules were introduced into the system. The behavior of the capsules at the constrictions was visualized and the properties of the capsules were investigated. Capsules recovered from the system appear to have a undergone a plastic deformation.
Hollow microshells are being fabricated for potential use as versatile sensors for real-time measurements of biochemicals. These shells are assembled using Layer-by-Layer (LBL) assembly of polyions onto colloidal polymer template particles. The latex particles are subsequently dissolved leaving behind stable, hollow shells into which analyte-sensitive and reference dyes are introduced. Fundamental studies have been performed to determine optical and chemical characteristics of these shells. Tests have been performed to determine the integrity of shells with respect to leaching and structural stability and robustness. The microshells were then loaded with an assay composed of a sodium sensitive fluorophore (SBFI) or a potassium sensitive fluorophore (PBFI), along with an analyte insensitive reference dye. It has been shown that the dye-loaded capsules retain their sensitivity to ion concentration with fluorescence characteristics similar to those of the dyes in liquid-phase, and that the sensor response to increasing ion concentration is linear over a physiologically significant range. In addition, preliminary results have demonstrated the ability of these capsules to be loaded with other sensor chemistry, including Ruthenium/Glucose-oxidase, and a FITC-dextran/TRITC-ConA competitive binding assay for glucose sensing based on fluorescent resonance energy transfer (FRET) methods.
Self-assembled thin films containing embedded enzymes and fluorescent indicators are being developed for use as highly specific glucose biosensors. The sensors are fabricated using electrostatic Layer-by-Layer (LBL) adsorption to create oxygen-sensitive (Ruthenium-based) layers, the fluorescent intensity of which responds to changes in local oxygen levels. Oxygen is consumed locally by the reaction between glucose oxidase (GOx) molecules and glucose. Latex particles serve as the templates for our sensors and fabrication is carried out through the alternate adsorption of multiple levels of {GOx/polycation} and {Ruthenium-polycation/polyanion} bilayers. Additional fluorescence layers as well as fluorescent latex are being considered as internal intensity references to allow ratiometric monitoring. Films adsorbed to the nanoparticle templates are being studied to understand the fundamental chemical and optical properties, including enzymatic activity, spectral shape and emission intensity. Enzymatic activity is retained and stability is improved after adsorption, and increased surface area afforded by the particles allows use of increased numbers of molecules. Fluorescence is also maintained, though blue shifts are observed in emission spectra, and indicator activity remains. In vitro characterization studies demonstrate the feasibility of the particles as glucose biosensors, and future work will aim to optimize the response for neural monitoring.
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