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Chemical sensors utilizing immobilized enzymes and proteins are important for monitoring chemical processes and biological systems. In this study, calcium-cross-linked alginate hydrogel microspheres were fabricated as enzyme carriers by an emulsification technique. Glucose oxidase (GOx) was encapsulated in alginate microspheres using three different methods:  physical entrapment (emulsion), chemical conjugation (conjugation), and a combination of physical entrapment and chemical conjugation (emulsion−conjugation). Nano-organized coatings were applied on alginate/GOx microspheres using the layer-by-layer self-assembly technique in order to stabilize the hydrogel/enzyme system under biological environment. The encapsulation of GOx and formation of nanofilm coating on alginate microspheres were verified with FTIR spectral analysis, ζ-potential analysis, and confocal laser scanning microscopy. To compare both the immobilization properties of enzyme encapsulation techniques and the influence of nanofilms with uncoated microspheres, the relationship between enzyme loading, release, and effective GOx activity (enzyme activity per unit protein loading) were studied over a period of four weeks. The results produced four key findings:  (1) the emulsion−conjugation technique improved the stability of GOx in alginate microspheres compared to the emulsion technique, reducing the GOx leaching from microsphere from 50% to 17%; (2) the polyelectrolyte nanofilm coatings increased the GOx stability over time, but also reduced the effective GOx activity; (3) the effective GOx activity for the emulsion−conjugation technique (about 3.5 × 10^-5 AU μg^-1 s^-1) was higher than that for other methods, and did not change significantly over four weeks; and (4) the GOx concentration, when compared after one week for microspheres with three bilayers of poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) ({PAH/PSS}) coating, was highest for the emulsion−conjugation technique. As a result, the comparison of these three techniques showed the emulsion−conjugation technique to be a potentially effective and practical way to fabricate alginate/GOx microspheres for implantable glucose biosensor application.

A simple and high-efficiency approach to loading macromolecules into microscale carriers is presented. Calcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique and then used as negatively charged templates to form polyelectrolyte multilayer coatings. A calcium ion chelator, EDTA, was used to free the Ca²⁺-cross-linked alginate hydrogel within {poly(allylamine hydrochloride)/poly (styrene sulfonate)}4 ({PAH/PSS}4) coating, allowing partial release of alginate. The retention of alginate in {PAH/PSS}4 microcapsule was confirmed by FTIR spectroscopy and confocal microscopy. Real-time confocal microscopy was used to investigate the loading process of positively charged macromolecules (dextran-amino, and peroxidase) into alginate-templated microcapsules, which showed the loading occurred in <2 min for dextran-amino and <10 min for peroxidase, respectively. A high loading efficiency of 25 μg peroxidase in ~1.0 × 10⁷ microcapsules (2.5 pg POx/capsule) was achieved with a low concentration of peroxidase loading solution (10 μg/mL). This spontaneous loading technique for encapsulating positively charged molecules in alginate-templated polyelectrolyte microcapsules shows strong potential for biosensor and drug delivery applications.

Inorganic colloids are usually prepared via precipitation reactions, which involve two sequential steps:  nucleation and growth of the nuclei. On the basis of this theory, preformed manganese carbonate nanoseeds were prepared by mixing diluted ammonium hydrogen carbonate with manganese sulfate solution to serve as nuclei for production of monodisperse manganese carbonate microparticles. It was found that the average particle diameter can be finely tuned from 1 to 10 μm by adding a different volume of nanoseed solution. It was further verified that the manganese carbonate microparticles can be used as templates for efficient formation of uniform, smooth multilayer polyelectrolyte microcapsules, which are promising systems for biomedical and other applications, such as biosensors, bioreactors, and drug-delivery devices.

A stable enzyme encapsulation technique based on the conversion of weak interactions between diazo resin/poly(styrene sulfonate) to covalent bonds was explored. Photosensitive diazoresin-based polyelectrolyte microcapsules were prepared via layer-by-layer electrostatic self-assembly of poly(styrene sulfonate) and diazoresin on MnCO3 templates. UV−vis and ζ-potential measurements confirmed the alternate deposition of {PSS/DAR} multilayers on the micrometer-sized dissolvable templates. The DAR-based microcapsules were demonstrated to be permeable to enzymes prior to UV irradiation, while the permeability of the multilayer wall was changed substantially after photo-cross-linking. Encapsulated molecules were stably entrapped after UV irradiation, as shown by confocal microscopy and atomic force microscopy images. Activity assays revealed that encapsulated glucose oxidase possessed 52.8% of the catalytic activity exhibited by the same amount of free enzyme, proving the preservation of native conformation and accessibility of substrate. This encapsulation technique is promising for many biomedical and biotechnological applications, particularly enzyme biosensors, which require stable immobilization of functional components while allowing sufficient transport rates for substrate molecules.

Methods for producing protein patterns with defined spatial arrangement and micro- and nanoscale features are important for studying cellular-level interactions, including basic cell−cell communications, cell signaling, and mechanisms of drug action. Toward this end, a straightforward, versatile procedure for fabricating micropatterns of bioactive nanofilm coatings as multifunctional biological testbeds is demonstrated. The method, based on a combination of photolithography and layer-by-layer self-assembly (LbL), allows for precise construction of nanocomposite films of potentially complex architecture, and patterning of these films on substrates using a modified lift-off (LO) procedure. As a first step in evaluating nanostructures made with this process, “comparison chips,” comprising two coexisting regions of square patterns with relevant proteins/polypeptides on a single substrate, were fabricated with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Using neuronal cells as a model biological system, comparison chips were produced with secreted phospholipase A2 (sPLA2), a known membrane-active enzyme for neurons, for direct comparison with gelatin, poly-l-lysine (PLL), or bovine serum albumin (BSA). Fluorescence microscopy, surface profilometry, and atomic force microscopy techniques were used to evaluate the structural properties of the patterns on these chips and show that the patterning technique was successful. Preliminary cell culture studies show that neurons respond and bind specifically to the sPLA2 enzyme embedded in the polyelectrolyte thin films and present as the outermost layer. These findings point to the potential for this method to be applied in developing test substrates for a broad array of studies aimed at identifying important biological structure−function relationships.

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 FITC−dextran (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.

Organized multilayers of nanoparticles (9-, 20-, and 45-nm-diameter silica or 12-nm magnetite) and glucose oxidase (GOx) were assembled in alternation with oppositely charged polyelectrolytes on 420-nm latex particles. Stepwise growth of the multilayer films on latex was confirmed by microelectrophoresis and transmission electron microscopy. The inclusion of silica layers on latex yields a higher surface area, resulting in greater GOx adsorption and thereby increasing the catalytic activity of the bioreactor. The bioactivity was proportional to the core surface area and also to the number of GOx layers in the shells. Also, the presence of magnetic nanoparticles allows self-stirring of the nanoreactors with a rotating magnetic field and enhances its productivity. The ensemble of GOx and fluorescent dyes in the shells demonstrated the correlation between Ru-bpy fluorescence and glucose concentration in solution.