Implantable Fluorogenic DNA Biosensor for Stress Detection.

Implantable sensors that can monitor analytes related to cognitive and physiological status have gained significant focus in recent years. We have developed an implantable biosensor to detect dehydroepiandrosterone sulfate (DHEA-S), a biomarker related to stress. The biosensor strategy was based on the principle of forced intercalation (FIT) aptamers designed to detect subtle intramolecular changes during aptamer-target binding events.

Preparation of Multifunctional Silk-Based Microcapsules loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing β-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions.

Improving the Activity of DNA-Encoded Sensing Elements through Confinement in Silk Microcapsules.

Assembling synthetic bioparts into simplified artificial cells holds tremendous promise for advancing studies into the synthesis, biosensing, and delivery of biomolecules. Currently, the most successful techniques for encapsulation of the transcription-translation machinery exploit compartmentalization in liposomal vesicles. However, improvements to these methods may increase permeability to polar molecules, functionalization of the membrane with biologically active elements, and encapsulation efficiency.

Immobilization of Recombinant Cells in a Bacterial Cellulose-Silk Composite Matrix To Preserve Biological Function.

Strategies for the encapsulation of cells for the design of cell-based sensors require efficient immobilization procedures while preserving biological activity of the reporter cells. Here, we introduce an immobilization technique that relies upon the symbiotic relationship between two bacterial strains: cellulose-producing cells; and recombinant cells harboring recombinase-based dual-color synthetic riboswitch (RS), as a model for cell-based sensor. Following sequential coculturing of recombinant cells in the cellulose matrix, final immobilization of cells was completed after reconstituted silk fibroin (SF) protein was added to a "living membrane" generating the composite bacterial cellulose-silk fibroin (BC-SF) scaffold.

Printed Dual Cell Arrays for Multiplexed Sensing.

We demonstrated inkjet printing of large-scale dual-type encapsulated bacterial cell arrays for prospective multiplexing sensing. The dual cell arrays were constructed on the basis of two types of bioengineered cells hosting fluorescent reporters (green-GFPa1 and red-turboRFP) capable of detecting different target chemicals. The versatility of inkjet printing allows for the fabrication of uniform multilayered confined structures composed of silk ionomers that served as nests for in-printing different cells.

Silk macromolecules with amino acid-poly(ethylene glycol) grafts for controlling layer-by-layer encapsulation and aggregation of recombinant bacterial cells.

This study introduces double-brush designs of functionalized silk polyelectrolytes based upon regenerated silk fibroin (SF), which is modified with poly-L-lysine (SF-PLL), poly-L-glutamic acid (SF-PGA), and poly(ethylene glycol) (PEG) side chains with different grafting architecture and variable amino acid-PEG graft composition for cell encapsulation. The molecular weight of poly amino acids (length of side chains), molecular weight and degree of PEG grafting (D) were varied in order to assess the formation of cytocompatible and robust layer-by-layer (LbL) shells on two types of bacterial cells (Gram-negative and Gram-positive bacteria). We observed that shells assembled with charged polycationic amino acids adversely effected the properties of microbial cells while promoting the formation of large cell aggregates.

Inkjet printing of silk nest arrays for cell hosting.

An inkjet printing approach is presented for the facile fabrication of microscopic arrays of biocompatible silk "nests" capable of hosting live cells for prospective biosensors. The patterning of silk fibroin nests were constructed by the layer-by-layer (LbL) assembly of silk polyelectrolytes chemically modified with poly-(l-lysine) and poly-(l-glutamic acid) side chains. The inkjet-printed silk circular regions with a characteristic "nest" shape had diameters of 70-100 μm and a thickness several hundred nanometers were stabilized by ionic pairing and by the formation of the silk II crystalline secondary structure.

Cell surface engineering with edible protein nanoshells.

Natural protein (silk fibroin) nanoshells are assembled on the surface of Saccharomyces cerevisiae yeast cells without compromising their viability. The nanoshells facilitate initial protection of the cells and allow them to function in encapsulated state for some time period, afterwards being completely biodegraded and consumed by the cells. In contrast to a traditional methanol treatment, the gentle ionic treatment suggested here stabilizes the shell silk fibroin structure but does not compromise the viability of the cells, as indicated by the fast response of the encapsulated cells, with an immediate activation by the inducer molecules.

Permeability and micromechanical properties of silk ionomer microcapsules.

We studied the pH-responsive behavior of layer-by-layer (LbL) microcapsules fabricated from silk fibroin chemically modified with different poly amino acid side chains: cationic (silk-poly L-lysine, SF-PL) or anionic (silk-poly-L-glutamic acid, SF-PG). We observed that stable ultrathin shell microcapsules can be assembled with a dramatic increase in swelling, thickness, and microroughness at extremely acidic (pH < 2.5) and basic (pH > 11.

pH-responsive layer-by-layer nanoshells for direct regulation of cell activity.

Saccharomyces cerevisiae yeast cells encapsulated with pH-responsive synthetic nanoshells from lightly cross-linked polymethacrylic acid showed a high viability rate of around 90%, an indication of high biocompatibility of synthetic pH-responsive shells. We demonstrated that increasing pH above the isoelectric point of the polymer shell leads to a delay in growth rate; however, it does not affect the expression of enhanced green fluorescent protein. We suggest that progressive ionization and charge accumulation within the synthetic shells evoke a structural change in the outer shells which affect the membrane transport.

Robust and responsive silk ionomer microcapsules.

We demonstrate the assembly of extremely robust and pH-responsive thin shell LbL microcapsules from silk fibroin counterparts modified with poly(lysine) and poly(glutamic) acid, which are based on biocompatible silk ionomer materials in contrast with usually exploited synthetic polyelectrolytes. The microcapsules are extremely stable in an unusually wide pH range from 1.5 to 12.

Direct probing of micromechanical properties of hydrogen-bonded layer-by-layer microcapsule shells with different chemical compositions.

The mechanical properties of hydrogen-bonded layer-by-layer (LbL) microcapsule shells constructed from tannic acid (TA) and poly(vinylpyrrolidone) (PVPON) components have been studied in both the dry and swollen states. In the dry state, the value of the elastic modulus was measured to be within 0.6-0.

Truly nonionic polymer shells for the encapsulation of living cells.

Engineering surfaces of living cells with natural or synthetic compounds can mediate intercellular communication and provide a protective barrier from hostile agents. We report on truly nonionic hydrogen-bonded LbL coatings for cell surface engineering. These ultrathin, highly permeable polymer membranes are constructed on living cells without the cationic component typically employed to increase the stability of LbL coatings.