This paper reports the first demonstration of a fluorescence resonance energy transfer based glucose sensor, wherein a competitive binding (CB) assay is encapsulated into polyelectrolyte microcapsules. The work supports the concept that microcapsules are superior to hydrogel systems or other matrixes for competitive-binding-based system, as they provide free movement of the sensing elements within the capsule interior while constant total sensing assay concentration is maintained. The transduction approach employed in these preliminary experiments is also a novel CB system based on a model apoenzyme, apo-glucose oxidase (AG), which is highly specific to beta-d-glucose, as the model target-binding protein. The glucose sensitivity of the fluorescein isothiocyanate (FITC)-dextran and tetramethylrhodamine isothiocyanate-AG encapsulated in microcapsules showed 5 times greater specificity for beta-D-glucose over other sugars, with sensitivity (change in intensity ratio) in the range of 2-6%/mM. It was observed that the sensitivity and range of the response can be tailored by controlling the assay concentration using different FITC-dextran molecular weight and total capsule concentration. The findings support the concepts of using microcapsules to encapsulate CB assays for reversible and stable sensors and the use of apoenzymes as specific molecular recognition elements in CB assays. Further, characterization results for microcapsule glucose sensors demonstrate their suitability for monitoring physiological glucose levels.

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.

Systems for glucose monitoring based on resonance energy transfer (RET) and competitive binding using Concanavalin A (Con A) are problematic as a result of problems of toxicity, aggregation, and irreversible binding. This paper presents an improved RET assay wherein Con A was replaced by apo-glucose oxidase (apo-GOx). The basic principle for transduction is identical to that used in assays based on Con A-dextran: a reduction in RET from fluorescein isothiocyanate (FITC) to tetramethyl rhodamine isothiocyanate (TRITC) occurs when FITCdextran (donor) is displaced from TRITC-apo-GOx (acceptor) as a result of the competition of glucose. Fluorescence measurements confirm that the apo-GOx/dextran complexes are highly sensitive to glucose, measured as an increase in the donor peak relative to acceptor due to stepwise addition of glucose. The solution-phase assay showed strong signals and excellent repeatability, with a sensitivity of 0.0163 (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.

Fluorescent sensing systems offer the potential for noninvasive monitoring with implantable devices, but they require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility while maintaining adequate response characteristics. A recent development towards this goal is a highly specific and sensitive competitive binding assay for glucose using apo-glucose oxidase (apo-GOx) as the recognition element and dextran as the competing ligand; this has been demonstrated as a glucose sensor system by encapsulating the competitive binding assay in semipermeable microcapsule carriers. This paper describes the extension of this sensor design to longer wavelengths in an attempt to increase the applicability to in vivo monitoring. The glucose sensitivity of the tetramethylrhodamine isothiocyanate-dextran (TD) and cyanine Cy5-apo-GOx (CAG) complexes showed five to 10 times greater specificity for beta-D-glucose over other sugars. Microcapsules loaded with TD/CAG complexes exhibited a linear, totally reversible response in the range of 0-720 mg/dL, with a sensitivity (percent change in intensity ratio) of 0.06%/(mg/dL). The decrease in sensitivity observed with the use of longer-wavelength dyes is most likely to be compensated with the deeper penetration of light and reduced tissue scattering. These findings imply that the encapsulation of sensing assay elements in microcapsules is a simple and translatable method for the fabrication of stable biosensors, and optimization of resonance energy transfer pairs and assay component preparation will further improve the response to approach clinically relevant performance.

Recommended by Igor MedintzFluorescence-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.