AVS2011 Session BI-ThP: Biomaterial Interfaces Poster Session

We present a new technique called surface initiated enzymatic polymerization (SIEP), which uses terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer. We utilized TdT’s ability to polymerize a long DNA chain while incorporating non-natural chemically reactive dNTPs to create new functional materials and to generate signal amplification. Previously, we have shown that fluorescent-dNTP can be directly polymerized by TdT. In this work, we will show the incorporation of amine- and aldehyde-modified dNTPs to impart reactive moieties into the polymerized DNA chain. We quantified the number of reactive dNTPs and their effect on the polymerization efficiency. We then further investigated the reactivity of the functional group for subsequent reactions, which include fluorescent dye conjugation for signal amplification, selective DNA metallization, and oligonucleotide conjugation to create a branched structure. We found that multiple reactive groups can be incorporated and they are active for subsequent reactions. For characterization of SIEP, we utilized a quartz crystal microbalance with dissipation monitoring (QCM-D) to monitor increase in surface mass during DNA polymerization in real-time. The mass increase versus concentrations of dNTP allows us to determine the reaction constant, which reflects the growth kinetics of DNA polymerization on the surface. In addition, we examined the effect of grafting density on the polymerization reaction and the conformation of DNA brushes.

The ability to predict and manipulate biomolecule behavior at the biointerface determines the success of biomaterials in applications ranging from biosensing to medical devices and therapeutic products. However, precise biointerface engineering will remain elusive until the roles of physical and chemical properties of surfaces on abiotic and biotic interfacial interactions are well understood. We have shown that plasma treatment of polymers generates chemically reactive surfaces for successful silanization and biomolecule immobilization [1, 2]. The focus of this work is to investigate the influences of surface chemistry and surface morphology on biomolecule attachment. We fine-tune our plasma system to favor the production of specific functional groups that promote subsequent biomolecule attachment. The effects of surface morphology on biomolecule immobilization are also assessed. Plasma diagnostics and modeling allows us to elucidate the effects of plasma parameters (plasma density, electron temperature and the resulting kinetic ion energy) on the polymer surface modifications. The work was supported by the Office of Naval Research.

References:

1) E. H. Lock, J. Wojciechowski, S. H. North, S. G. Walton, C. R. Taitt, “Exploring the mechanism of biomolecule immobilization on plasma treated polymer substrate”, to be published NATO Science for Peace and Security Series by Springer.

2) S. H. North, E. H. Lock, C. J. Cooper, J. B. Franek, C. R. Taitt, S. G. Walton, “Plasma-based surface modification of polystyrene microtitre plates for covalent immobilization of biomolecules”, ACS Appl. Mater.& Interfaces 2, 2884-91 (2010).

Atomic layer deposited (ALD) Al₂O₃ has been widely used as encapsulation material for organic LEDs and solar cells due to its low water vapor transmission rate (WVTR) (~5×10^-6 g-H₂O/m²-day). However, its coating performance for implantable devices still needs investigation. Parylene has been commonly applied as encapsulation for implantable devices, such as Utah Electrode Arrays (UEAs). The idea of combing Al₂O₃ and parylene is based on the concept that Al₂O₃ works as moisture barrier and parylene as ion barrier. In this paper, Al₂O₃ was deposited by both thermal and plasma-enhanced ALD on interdigitated electrodes (IDEs) for comparison. AFM micrographs (Fig. 1) show that Al₂O₃ films deposited on silica substrate (RMS surface roughness of 0.17 nm) by thermal and plasma-enhanced ALD have RMS surface roughness of 0.51 nm and 0.48 nm, respectively. XPS shows that ALD films had an oxygen to aluminum ratio of 1.2 while thermal ALD Al₂O₃ is 1.09, indicating that the former is closer to Al₂O₃. A 6-µm thick parylene-C layer was deposited by CVD using Gorman process on top of Al₂O₃ and saline A-174 (Momentive Performance Materials) was used used as adhesion promoter. The samples were soaked in 1× PBS at 37 °C and 57 °C for accelerated lifetime test. Electrochemical impedance spectroscopy (EIS) and chronoamperometry were used to evaluate the performance of the encapsulation. Preliminary data shows that the leakage current (Fig. 2) remained very small (~ 7 pA) and the electrochemical impedance (Fig. 3) was consistently high (~ 3 MΩ at 1 kHz) after 5 days of soaking test at 57 °C (equivalent to at least 20 days of soaking test at 37 °C). Comparing with parylene and Al₂O₃ control samples, the Al₂O₃-parylene coated sample showed lower leakage current ( Fig. 2). The impedance for three different types of samples was almost the same. However, the phase of parylene-C coated sample slightly declined after 5 days of soaking test (Fig. 3), suggesting that sample coated with Al₂O₃ had lower WVTR. No obvious difference has been observed yet for samples soaked at different temperatures since the soaking period is relatively short. Parylene came off after one day of soaking test for one sample, which might be caused by the poor adhesion between Al₂O₃ and parylene. In conclusion, preliminary results shows that the Al₂O₃-parylene bi-layer encapsulation scheme is promising encapsulation in terms of leakage current and electrochemical impedance. Long-term soaking tests are being performed to further investigate the functionality of this novel encapsulation scheme.

Bovine serum albumin (BSA) is a widely studied globular protein that contains ca. 68% of alpha-helices and <2% of beta-sheets in its native conformation. The well characterized secondary structure of BSA is commonly used as a benchmark for electronic (ECD) and vibrational (VCD) studies of proteins in solution. Both ECD and VCD indicated a substantial loss of helical structure accompanied by an increase of beta-sheet character in BSA thermally denatured in solution. In surface adsorption experiments, hydroxyapatite microspheres were incubated in solutions with low (1.0 mg/mL) or high (50.0 mg/mL) concentrations of BSA for one hour, then triple rinsed and re-suspended in buffer for analysis. The ECD spectra were similar for BSA adsorbed from low and high concentration solutions, both showing a sizeable increase in beta-sheet character upon adsorption, while being dominated by alpha-helical features. The VCD spectra also exhibited stronger peaks in the beta-sheet region upon adsorption of BSA on hydroxyapatite. VCD signal enhancement, however, was observed upon adsorption from the high concentration BSA solution, indicating the formation of macroscopic chiral structures. The analysis of proteins adsorbed on surfaces thus can be enhanced by taking advantage of the complementary sensitivities of ECD and VCD spectroscopies to the secondary structures of biomolecules.

PTEN is a phosphatidylinositolphosphate (PIP) phosphatase frequently mutated in human cancer [1]. By lowering PI(3,4,5)P₃ levels in the plasma membrane, it functions as an antagonist to PI3-kinase in the regulatory circuit that controls cell proliferation and survival. wt PTEN has only weak affinity to zwitterionic phosphatidylcholine (PC) membranes but a strong interaction with anionic lipids. Its C2 domain was shown to bind in a Ca²⁺ independent manner to phosphatidylserine (PS) and phosphatidylglycerol (PG), whereas a short N-terminal domain binds specifically to PI(4,5)P₂ [2,3]. H93R PTEN is an autism related mutant which has decreased phosphatase activity [4].

Using Surface Plasmon Resonance (SPR), we characterized the affinity of wt and H93R PTEN to tethered bilayer lipid membranes (tBLMs) that contain PC and PS, PC and PI(4,5)P₂, and PC, PS and PI(4,5)P₂. As compared with wt PTEN, we find that the H93R mutation is sufficient to cause significant increases in the protein's association with lipid membranes containing PS. PI(4,5)P₂ enhances the apparent binding constant for both proteins and leads to intriguing binding kinetics of the protein to the membrane. The binding of either protein to membranes containing both PS and PI(4,5)P₂ shows a biphasic behavior, suggesting two independent binding sites. This supports the hypothesis of non-competitive binding of the protein to PS and PI(4,5)P₂ [5]. We also performed neutron reflectivity experiments to determine the structure and orientation of PTEN bound to the membrane.

1. R. J. Shaw, L. C. Cantley, Nature 441 (2006), 424-430.

2. J. O. Lee, et al., Cell 99 (1999), 323-334.

3. S. Das, J. E. Dixon, W. Cho, Proc. Natl. Acad. Sci. USA 100 (2003), 7491-7496.

4. R. E. Redfern, et al., Protein Sci. 19 (2010), 1948-1956.

5. R. E. Redfern, et al., Biochemistry 47 (2008), 2162-2171.

The cytoplasmic domain of the T-cell receptor zeta subunit, ζ_cyt, a cell surface protein complex responsible for binding peptide fragments of foreign antigens bound to major histocompatibility complex (MHC) proteins, is sufficient to couple receptor ligation to intracellular signaling cascades [1]. These domains carry immunoreceptor tyrosine-based activation motifs (ITAMs), i.e. signaling motifs that are phosphorylated by tyrosine kinases following receptor crosslinking. The phosphorylation of ITAMs is a first and obligatory step in signal transduction. ζ_cyt has been shown to be unstructured in aqueous solution and to assume a helical conformation in the presence of anionic lipid vesicles [2,3]. Membrane binding and membrane-induced conformational changes likely plays an important role in signal transduction, but no direct structural information on these functionally important lipid-bound states was available so far. Using a synthetic membrane model, i.e., fluid lipid bilayers tethered to planar solid supports [4,5], we report surface plasmon resonance (SPR) results on the binding kinetics and neutron reflectivity investigations of the association of the disordered ζ_cyt with membranes. We determine the extent to which the protein penetrates into the bilayer and discuss structural details of the ζ_cyt–lipid interaction.

1. M. E. Call, K. W. Wucherpfennig, Annu. Rev. Immunol. 23 (2005), 101-125.

2. A. Sigalov, Semin. Immunol. 17 (2005), 51-64.

3. D. Aivazian, L. J. Stern, Nat. Struc. Biol. 7 (2000), 1023-1026.

4. D. J. McGillivray, et al., Biointerphases 2 (2007), 21-33.

Biofouling, the undesired growth of marine organisms on submerged surfaces, is a global problem with both economic and environmental consequences. The attachment of bacteria to surfaces is an important step in the biofouling process, and the development of ways to attenuate microbial attachment or to achieve their easy removal is desirable. We built a microfuidic system and applied it to determine the adhesion strength of bacterial biofilms. Marine bacteria, e.g. Cobetia marina are cultivated in this microfluidic device on the surfaces of interest. The bacterial detachment is caused by a hydrodynamic shear flow which is continuously increased and the removal is recorded via video microscopy. The adhesion strength is determined as the shear stress needed to detach 50% of the bacteria. The parameters incubation time, medium and increase of shear stress were varied in order to find the optimal conditions to carry out the biological assays. The applicability of the technique is demonstrated using self assembled monolayers with a different ability to bind water. Well hydrated surfaces lead to decreased adhesion strength while bacteria stick stronger to less hydrated surfaces. Based on these findings, a range of polysaccharide hydrogels were tested towards their potential to reduce adhesion strength.