Swetha Chinnayelka

Glucose-sensitive nanoassemblies comprising affinity-binding complexes trapped in fuzzy microshells.

A new design for glucose monitoring with “smart” materials based on self assembly, competitive binding, and resonance energy transfer (RET) is presented. The basic transduction principle is changing RET efficiency from fluorescein isothiocyanate (FITC) to tetramethylrhodamine isothiocyanate (TRITC), as FITC-dextran is displaced from TRITC-Concanavalin A (Con A) with the addition of glucose. Nanoscale fabrication by self-assembly of Con A/dextran into multilayer films, followed by polymer multilayers. The advantages of this approach include physical localization and separation of sensing molecules from the environment via entrapment of the biosensor elements in a semi-permeable polymeric shell, and only functional molecules are included in the sensors. To realize these nanostructures, dissolvable resin microparticles were coated with FITC-dextran+TRITC-Con A multilayers, followed by polyelectrolyte multilayers, and the core particles were then dissolved to yield hollow capsules. The nanoassembly process was studied using microbalance mass measurements, fluorescence spectroscopy, confocal fluorescence microscopy, and zeta-potential measurements. The key findings are that the specific binding between Con A and dextran can be used to deposit ultrathin multilayer films, and these exhibit changing RET in response to glucose. Fluorescence spectra of a microcapsules exhibited a linear, glucose-specific, 27% increase in the relative fluorescence of FITC over the 0–1800 mg/dL range. These findings demonstrate the feasibility of using self-assembled microcapsules as optical glucose sensors, and serve as a basis for work toward better understanding the properties of these novel materials.

Near-Infrared Resonance Energy Transfer Glucose Biosensors in Hybrid Microcapsule Carriers

Fluorescence-based sensing systems offer potential for noninvasive monitoring with implantable devices, but require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility. Recent developments towards this goal include a competitive binding assay for glucose that has been encapsulated in semipermeable microcapsule carriers. This paper describes an extension of this work to increase the applicability to in vivo monitoring, wherein two significant developments are described: (1) a near-infrared resonance energy transfer system for transducing glucose concentration, and (2) novel hybrid organic-inorganic crosslinked microcapsules as carriers. The quenching-based assay is a competitive binding (CB) system based on apo-glucose oxidase (AG) as the receptor and dextran as the competitive ligand. The encapsulated quencher-labeled dextran and near infrared donor-labeled glucose receptor showed a stable and reversible response with tunable sensitivity of 1–5%/mM over the physiological range, making these transducers attractive for continuous monitoring for biomedical applications.

Microfabricated interferometer and integrated fluidic channel for infrared spectroscopy of aqueous samples

Components of a microspectrometer for operation in the IR range has been designed, fabricated, and characterized. An adjustable Fabry-Perot interferometer is used to select the resonant frequency of the system through electrostatic actuation, allowing tuning for certain optical frequencies to pass. Silicon microfabrication techniques are employed for the fabrication of the device. The intended use of the device is for spectroscopic study of liquids in biomedical and environmental applications; therefore, a sample containment chamber has been integrated into the device. The device was designed using finite element modeling to determine the stress distribution on the silicon nitride membrane due to deflection and the voltage required for the suitable displacement of the membrane to which one mirror is attached. The devices have been fabricated using a combination of processing steps to sputter gold mirrors on nitride membranes, to deposit electrodes and spacers using evaporation and photosensitive polyimide, to etch channels and sacrificial layers, and to bond chips to obtain a resonant cavity. Optical characterization was performed with an FTIR spectrometer. Initial results presented here support the feasibility of the approach in developing standalone microspectrometers for analysis of aqueous samples including biological fluids.

Self-assembly of polymer/nanoparticle films for fabrication of fiber optic sensors based on SPR

Surface Plasmon Resonance (SPR) is an optical phenomenon which can be used for the sensitive detection of macromolecular interactions at a sensor surface by detecting small changes in refractive index resulting from adsorbed species. Previous work toward fiber-optic SPR sensors has employed metal films sputtered or evaporated onto waveguides. In this work, a novel nanofabrication approach using a combination of self-assembled monolayers (SAMs) and electrostatic layer-by-layer (LbL) self assembly was investigated toward precise deposition of metal nanomaterials onto fibers, which could enable excellent control over surface properties as well as provide an enhanced plasmon signal due to the roughened metal surfaces. Furthermore, nanoassembly allows production of nanocomposite materials that may possess attractive optical properties. To study this possibility, ultrathin films with architecture {Au/polymer}n (n=1-10) were deposited on flat silica substrates, then on optical fibers. Physical measurements of deposited mass were performed with quartz crystal microbalance (QCM). The influence of particle size, number of layers, and distance from surface on the magnitude of optical signals was investigated by measuring the absorption spectrum for each configuration. In addition, sequential and simultaneous bimetallic (Au/Ag) film layering and testing was also completed to assess the effect of nanocomposite metal films on SPR signals. Fluorescent anti-Immunoglobulin G (IgG) antibody was deposited on the outside surface, which was then exposed to IgG for observation of shifting resonance peak due to target binding. The results show that nanoassembly is a promising approach to precise yet cost-effective fabrication of optical biosensors.

RET nanobiosensors using affinity of an apo-enzyme toward its substrate

Fluorescent biosensors can be highly specific and sensitive, and may be engineered as implantable devices for metabolic monitoring. Commonly-used systems for glucose monitoring based on resonance energy transfer (RET) and competitive binding involve Concanavalin A (Con A), which is known to be toxic, and has problems of aggregation and irreversible binding. This work presents an improved RET assay wherein Con A was replaced by apo-glucose oxidase (apo-GOx). Fluorescence measurements confirm that the apo-GOx/dextran complexes are highly sensitive to glucose, observed as an increase in the donor peak relative to acceptor with the stepwise addition of glucose solution. The assay showed strong signals and excellent repeatability, with a sensitivity of 9/spl times/10/sup -4/ (ratio units)/mM over the range of 0-90 mM glucose. If properly encapsulated, these sensors can potentially be used for in vivo sensing without the concern of toxicity associated with Con A.

Competitive binding assays in microcapsules as "smart tattoo" biosensors

This paper demonstrates a novel fluorescence resonance energy transfer (RET) sensor system, wherein a competitive binding (CB) assay is encapsulated into microcapsules. For CB approaches intended as smart tattoos, microcapsules are superior to hydrogel microparticle systems, as they provide space in the capsule interior for the free movement of the sensing elements while maintaining constant sensing assay concentration. Previously-developed CB glucose sensors suffer from toxicity, nonspecificity, lack of efficient encapsulation technology, and poor reversibility. To overcome some of these limitations, apo-glucose-oxidase (AG) was used as a glucose binding protein and was entrapped in polyelectrolyte microcapsules. The glucose-sensitive change in RET of the fluorescein isothiocyanate (FITC)-dextran and tetramethylrhodamine isothio-cyanate (TRITC)-AG entrapped in microcapsules showed 5times more specificity towards glucose over other sugars, with a sensitivity of 0.035units/mM in the range of 0-40mM. These response characteristics appear to be suitable for glucose monitoring in diabetic patients

Modeling and fabrication of mirrors used in micro-spectrometers for infrared analysis of biofluids

The spectrometers in general are used to detect the chemical composition of a sample by measuring its absorption or emission spectra. The intended application of the device is for doing the qualitative and quantitative analysis of liquids. The design, fabrication and characterization of micro-mirrors used in the Fabry-Perot interferometer (FPI) operating in the infrared (IR) region are discussed. The miniaturized spectrometer basically comprises three components: infrared emitter, FPI and a photo-detector. The FPI is critical, as it is the wavelength-selecting component. Mirrors in the FPI should have low loss, high and broadband reflectance in the infrared region to operate the FPI in ER region. Mirrors were modeled and optical properties were observed for varying mirror thickness. The mirrors were microfabricated and tested using FTIR. Experimental results of mirror transmittance closely agree with the modeling results.