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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.

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.