Formulation of Reactive Nanostructured Adhesive Microbial Ink-Jet Inks for Miniature Biosensors and Biocatalysis

Reactive adhesive microbial inks can be formulated from aqueous latex emulsions for drop on demand (DOD) piezoelectric deposition. Ink-jet printing of a high density of living microorganisms many be useful to generate microstructures for micro-biosensors, as biocatalytic coatings in micro-fluidic devices, or micro-channel bioreactors. Microbial inks are viable wet cell paste mixed with aqueous (organic solvent-free) emulsions of adhesive polymer particles that dry rapidly with arrested coalescence as thin, adhesive, nanoporous coatings. Latex ink viscosity (∼1.5 to 3 cP) is semi-shear rate dependent. Nanoporosity is essential for the embedded microorganisms to retain viability and reactivity. The nanoporous latex inks in this study contain glycerol and sucrose added to a low Tg acrylate/vinyl acetate latex emulsion (particle dia. ∼280nm, Tg ∼10°C). The glycerol and sucrose arrest polymer particle coalescence during film formation (porogens) and also act as osmoprotectants. Viability and reactivity is measured by bioluminescence following deposition, drying, and rehydration in nitrogen-limited (nongrowth) media. As a model system, the reactivity of Escherichia coli (<1 μm by ∼2 μm, 5 x 10 4 cfu/nl ink) containing a mercury (Hg +2 )-inducible promoter-lux fusion was studied by printing patches, microwells and multi-layer dot arrays onto polyester using office ink-jet printers and a piezoelectric nano-plotter. E. coli can be printed at a density of ∼1.5 cells/μm 2 . Reactivity (luminescence resulting from luxCDABE expression) is a function of drying conditions (temperature, relative humidity), nanoporosity following rehydration, Hg +2 concentration, and volume printed. Print resolution is determined by piezo tip aperture diameter (25 μm, ∼0.2 nl/droplet; 50 μm, ∼0.5 nl/droplet) and drying rate. Profilometry of dry latex microstructures indicated significant surface-tension driven flow prior to ink drying. Viable Kluyveromyces fragilis (∼12 μm diameter) have also been printed as a larger model microbe for developing ink-jet methods for bio-patterning of surfaces with microcolonies.