Research Program
Nanoscale Materials

Participating Faculty:             Carl A. Batt, Geoffrey W. Coates, and Christopher K. Ober

NBTC Students/Postdocs:     Nuttawee Niamsiri

Other Students/Postdocs:     Soazig C. Delamarre, Esha Mathew

Objectives

1. • Understand the mechanisms of enzymatic surface-initiated polymerization (ESIP) of polyhydroxybutyrate (PHB) and enhance the growth of PHB on the solid surfaces

   • Create PHB microstructures in situ inside microfluidic channels and evaluate their performance as microfluidic valves and barriers

   • Investigate the interaction between mammalian cells and PHB derivatized surfaces and evaluate their performance for mammalian cell patterning.

Methods
Enhance the growth of PHB on solid surfaces and inside microfluidic channels

PHB is a biodegradable and biocompatible aliphatic polyester produced by a variety of microorganisms as a reserve energy source. Here, site-specific attachment of the key catalytic enzyme, PHB synthase, on lithographic fabricated surfaces and subsequent addition of 3-hydroxybutyryl-CoA substrates (3HBCoA), allowed us to create spatially ordered PHB polymeric micro-/nano-structures on patterned surfaces via in situ ESIP. We studied the effect of various additives, such as detergents, phospholipids, and other proteins, to enhance the in situ growth of PHB structures under physiological conditions. Various physical and chemical characterizations of surface synthesized PHB via fluorescence, AFM, and FTIR analysis were used to gain a better understanding of its surface growth mechanisms. In addition, PHB microstructures were in situ synthesized inside microfluidic channels as an imbedded polymeric valve and/or barrier for microfluidic mixing device.

Fabrication and analysis of PHB microstructures for special control of mammalian cell adhesion

Various gold patterned substrates used for our surface polymerization and cells studies were fabricated using standard lithography techniques housed within NBTC & CNF at Duffield Hall. We also collaborated with Professor Jun-Lin Guan ( College of Veterinary Medicine , Cornell University ) to study the interaction between mouse embryonic fibroblasts and fabricated PHB patterned surfaces.

Summary
In this study, the effect of BSA protein on stabilizing the immobilized enzyme and the PHB polymeric structure was found to be the most important factor for a successful surface polymerization in order to form thick PHB films up to 1 μm in height with full surface coverage (Figure 1). Fluorescence immunodection and surface contact angle analysis showed that when PHB synthesized in the presence of BSA, BSA was bound to the PHB polymer matrix and, thus reduced the overall hydrophobicity of the polymeric surface. The resulting PHB surface structure obtained via BSA-stabilizing process is, to the best of our knowledge, the highest surface polymeric structure synthesized to date compared to other techniques using biological catalysts. We believe that the use of this novel biofabrication approach offers many practical applications in different areas. For example, PHB microstructures could be formed in situ inside microfluidic channels on designated areas and potentially used as either microfluidic valves and/or passive micromixers. The model devices were shown in Figure 2. In additon, the modification of solid surfaces with PHB could potentially yield a novel biocompatible polymer-coated implant for tissue engineering. Cell studies performed with mouse embryonic fibroblasts showed a substantially increased cell attachment and proliferation on the fabricated PHB surface as compared to the unmodified surface. Moreover, we demonstrated that micropatterning of PHB structures created through a combination of parylene lift-off and in situ biofabrication of PHB could be used for mammalian cell patterning (Figure 3). As a result, we believe that our ESIP of PHB strategy will be a versatile tool for surface modification.