AVS2001 Session BI-MoP: Biorecognition Poster Session
Monday, October 29, 2001 5:30 PM in Room 134/135
Monday Afternoon
Time Period MoP Sessions | Topic BI Sessions | Time Periods | Topics | AVS2001 Schedule
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BI-MoP-1 Silica-Elastin Like Polypeptide Smart Membranes-Switchable Molecular Filters
G.V. Rama Rao, S. Balamurugan, G.P. Lopez (The University of New Mexico); D. Meyer, A. Chilkoti (Duke University) Elastin-like polypetides (ELPs) are a class of synthetic polypetides comprising of Val-Pro-Gly-Xaa-Gly where Xaa is any amino acid with the exception of proline and exhibit inverse solubility temperature behavior in aqueous solutions. They undergo a transition from hydrophilic (extended conformation) to hydrophobic (compacted conformation) at the lower critical solution temperature (LCST). We demonstrate in this study that when ELPs are encapsulated in a silica matrix, the ELPs can act as molecular switches that control the selective permeability of the membranes. The pores resulting from the transition can selectively transport different molecular species depending on their size. Two different ELPs of molecular weights of 60 kDa (ELP1-150) and 13 kDa (ELP4-30) were used for the present study. Silica-ELP membranes were prepared by sol-gel processing on microcon centrifugal filter units with 30,000 -100,000 molecular weight cut-off membranes and on 1 inch diameter of ultrafiltration discs. The LCST of the membranes was established by permeation measurements and static contact angle measurements. Differential scanning calorimetric studies were employed to determine the LCST of bulk gels and found to be 34 and 44°C for ELP1-150 and ELP4-30 respectively. Cycling of the membranes between 25 and 40-45°C indicates that the membranes possessed reversible, variable permeability while maintaining good mechanical stability. Permeation experiments with various molecular weights of poly(ethylene glycol) (PEGs) on centrifugal filters and ultrafiltration membranes clearly demonstrated that these membranes are acting as a molecular switches by being impermeable below the LCST and permeating the lower molecular weights of PEGs and filtering out higher molecular weight PEGs above LCST. |
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BI-MoP-3 The Silanisation of Tantalum Pentoxide for Biosensor Realisation
W. Laureyn, F. Frederix, A. Campitelli (IMEC, Belgium); J.-J. Pireaux (FUNDP, LISE, Belgium); G. Maes (KULeuven, Belgium) Affinity biosensors allow the detection of affinity based interactions between bio-molecules, which occur e.g. in antibody-antigen recognition or DNA hybridisation. The presence of antigens in an analyte can be verified by the binding of these molecules to their complementary antibodies, immobilised onto a biosensor surface. Tantalum pentoxide is considered as a material with unique properties for biosensor realisation, being chemically very stable and attractive from an electronic point of view, owing to its high dielectric constant. In order to realise tailored bio-interfaces; bromoalkyltrichlorosilanes were deposited on Ta2O5. The use of chlorosilanes (as opposed to alkylethoxysilane derivatives) leads to the formation of reproducible and close-packed monolayers on the oxide surface. To allow antibody binding, the bromo-functionality was converted into a carboxyl-functionality, via a one step reaction with mercaptoacetic acid. XPS, FT-IR, contact angle goniometry, cyclic voltammetry and impedance measurements were applied for the characterisation of the cleaning of Ta2O5, its silanisation and of the demanded surface reaction. The results of this study indicate the success of our approach. Moreover, distinct differences are revealed for (mixed) silane monolayer formation with short or long-chain chlorosilanes, from the liquid or vapour phase. In future work, the use of bromoalkyltrichlorosilanes will be compared to the silanisation of Ta2O5 with allylalkyltrichlorosilanes, followed by an oxidation in order to generate carboxyl groups. This procedure will also allow the immobilisation of antibodies on Ta2O5. |
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BI-MoP-4 Organization of Multifunctional Co-polymers on Metal Oxide Surfaces for Optical Biosensing Applications
N.-P. Huang, I. Reviakine, S.M. De Paul, M. Textor, N.D. Spencer (ETH Zürich, Switzerland) A novel polymeric interface that combines the resistance to non-specific protein adsorption conferred to metal oxide surfaces by poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) with the high affinity of the well-known (strept)avidin-biotin system through the use of a biotinylated-PEG-containing copolymer (PLL-g-PEG/PEGbiotin) has recently been introduced by our group for use in biosensing applications. Biosensor chips with high sensitivity and selectivity can be designed with the above approach, making it an attractive surface technology. Optimization of this system for various biosensor applications depends critically on the knowledge of how the polymer film is organized at the metal oxide interface. Therefore, this work focuses on investigating the organization of the co-polymer film by atomic force microscopy (AFM), quartz crystal microbalance with dissipation (QCM-D) and optical waveguide lightmode spectroscopy (OWLS). This combination of spectroscopic and imaging techniques provides an insight into how the co-polymer mixture is organized at the surface and how this organization evolves with time. The effect of streptavidin addition was also investigated and biotinylated liposomes were used as a well-understood model analyte in AFM and QCM studies. |
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BI-MoP-5 Investigation of the Immobilization Process of Peptide Nucleic Acids
J.C. Feldner, M. Ostrop, O. Friedrichs, G. Gappa, D. Lipinsky, U. Gunst, S. Sohn, H.F. Arlinghaus (Universität Münster, Germany) In order to immobilize peptide nucleic acid (PNA) onto Au and Ag coated surfaces, a thiol linker (DTSP, 3,3'-Dithio-bis(propionic acid N-hydroxysuccinimide ester)) was used. The immobilization process of DTSP and PNA to these surfaces can be performed by either binding PNA to DTSP in a solution and then immobilizing it onto the surface or immobilizing DTSP onto the surface and then attaching PNA to it. In both methods, PNA binds to the reactive end group of DTSP and the thiol group of DTSP binds to the Au or Ag surface. The reactive end groups of the DTSP layer can be inactivated using primary amines after immobilization of PNA. Deprotenated (M-H)- signals of the different PNA bases as well as characteristic peaks of DTSP fragments could be used in TOF-SIMS and TP-SIMS (temperature programmed SIMS) measurements to study and optimize the different immobilization processes. A detailed investigation of the concentration of DTSP and its immobilization time on Au and Ag surfaces showed that the best result could be achieved at a concentration of 10 mM and an immobilization time of 24 hr. The binding of PNA to the DTSP layer takes significantly longer than attaching DTSP to the surface. TP-SIMS data, which are very sensitive to bonding strength, showed that characteristic ion signals of the bases start to decrease at a temperature of about 150°C, with differences in the point of onset for the different bases. From the obtained data it can be concluded that the second attachment method described above is preferable to the first one and also has the advantage of allowing to inactivate the complete Au or Ag surface for unspecific DNA attachment. |
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BI-MoP-6 Biotin-reactive Surfaces Based on Ω-substituted Alkanethiols on Au(111)
H. Tran, M. Chen, H. Lu, A. Neurauter, S. McManus-Munoz, D. Quincy, T. Langenbacher, P. Peluso, P. Kernen, S. Nock, P. Wagner (Zyomyx, Inc.) (Oligo)ethylene glycol-containing alkanethiols with ω-substituted N-hydroxysuccinimide-ester groups self-assemble in ordered monolayers on Au(111) surfaces (NHS-SAM) and form highly reproducible reactive interfaces for in-situ transformations and biomolecular immobilization. Surface sensitive spectroscopic techniques including FTIRRAS, and XPS, indicate that amino-biotin can be successfully coupled in-situ to the NHS-SAM. Such reactive surfaces have been tested for homogeneous surface coverage and selective binding of streptavidin- and biotin-conjugated dyes and proteins using fluorimetry, radiometry and surface plasmon resonance. These binding data on biotin-functionalized SAMs are compared with binding efficiencies of electrostatically adsorbed biotin derivatized poly(L-lysine)-grafted poly(ethylene glycol) layers on Au(111) surfaces. We incorporated these bioreactive interfaces into microfabricated three-dimensional structures in silicon and used these to test an immunoassay in a microarray format. |
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BI-MoP-7 Reactivities and Biomolecular Immobilization on Self-assembled Alkanethiols with Ω-substituted N-hydroxysuccinimide-ester Groups on Au(111)
S. McManus-Munoz, D. Martin, C.E.J. Dentinger, R.L. Cicero, H. Tran, M. Chen, P. Kernen, P. Wagner (Zyomyx, Inc.) Alkanethiols with ω-substituted N-hydroxysuccinimide ester groups have been self-assembled on Au(111) surfaces. Stability and reactivity of the functional group were studied using reflection absorption infrared spectroscopy, contact angle, ellipsometry and radiometry measurements. Increased NHS-reactivity was observed for alkanethiols containing (oligo)ethylene glycol units. Effective NHS quenching combined with some effects on non-specific protein adsorption was found with various amino-containing compounds, e.g. glycine and amino-PEGs. Transformation of the NHS-group into a biotin exposing surface was obtained by in-situ coupling of amino-biotin. Specificity and homogeneity of streptavidin and biotin-conjugated protein binding was tested by spectroscopic and microscopic techniques and compared with the binding efficiencies on physisorbed polymer layers of biotin derivatized poly(L-lysine)-grafted poly(ethylene glycol) on metal oxides and on Au(111). |
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BI-MoP-8 Measuring Bound Water in Protein-resistant Coatings: A Combined OWLS and QCM-D Study of Poly(L-lysine)-g-poly(ethylene glycol)
S.M. De Paul, J. Vörös, I. Reviakine (ETH Zürich, Switzerland); C. Galli, M. Collaud Coen (University of Fribourg, Switzerland); M. Textor, N.D. Spencer (ETH Zürich, Switzerland) Metal oxide surfaces coated with poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) have been shown to resist non-specific adsorption of proteins. The hydrophilicity of the PEG chains is believed to play a crucial role in such behavior. In order to determine the amount of water coupled to PLL-g-PEG at aqueous metal oxide interfaces, we use results from two in situ methods: optical waveguide lightmode spectroscopy (OWLS), which detects the mass of polymer adsorbed at the surface, and quartz crystal microbalance with dissipation (QCM-D), which is sensitive to the mass of the polymer plus the mass of trapped or hydrodynamically coupled water. Complementary information about water content is provided by solid-state NMR measurements. We also examine how the amount of adsorbed polymer and its protein resistance vary with the choice of substrate (e.g., TiO2, SiO2) and with the surface topography as determined by atomic force microscopy (AFM). |
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BI-MoP-9 Examination of Bacterial and Protein Attachment and Release Using Tunable Poly(N-Isopropylacrylamide) as a Switchable Hydrophobic/Hydrophilic Substratum
L.K. Ista, S. Mendez, S. Balamurugan, G.P. Lopez (The University of New Mexico) Poly(N-isopropylacrylamide), or PNIPAAM, undergoes a critical solubility transition in response to temperature. Below 32°C, the polymer is freely water soluble, whereas above this temperature it is insoluble in water. When PNIPAAM is immobilized this property translates to an increase in surface hydrophobicity above the transition temperature (Tt). The process is fully reversible, with restoration of the original degree of solubility or hydrophilicity occurring upon cooling to temperatures below Tt. This switchable characteristic of grafted PNIPAAM has been exploited in the creation of fouling release surfaces. It has been discovered that when thin layers of PNIPAAM are grown from initiator-derivatized. alkanethiolate self-assembled monolayers (SAMs), the range of wettabilities observed above and below Tt can be tuned by altering the hydrophobicity of the underlying SAM. Because the hydrophobic/hydrophilic transition happens in situ, and rapidly over a short temperature range, tunable PNIPAAM substrata are ideal for examining the effect of changing surface hydrophobicity on the attachment and detachment of biofilm components, i.e. microbes and proteins. We report here the results of attachment and detachment studies on patterned tunable PNIPAAM surfaces using two bacteria which normally exhibit different and opposite responses to subtratum hydrophobicity (Halomonas marina and Staphylococcus epidermidis) as well as studies on adsorption and desorption of a variety of proteins known to play a role in biofilm formation. |
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BI-MoP-10 Novel Immunosensor Interfaces Based on Mixed Self-assembled Monolayers of Thiols
F. Frederix, W. Laureyn, K. Bonroy (IMEC, Belgium); W. Dehaen, G. Maes (KULeuven, Belgium) An ideal biosensor is characterized by its stability, reproducibility sensitivity and specificity towards a desired analyt. However, reduction of the size of the transducer and thus of the active area, requires an optimization of the sensing area. Our research is therefore also focussing on the biological recognition layer, which is based on an optimized covalent coupling of the antibodies to mixed self-assembled monolayers of thiols on gold. Since cleanliness and structural properties of the gold are of the utmost importance for perfect SAM formation, we have evaluated different cleaning procedures and induced the gold(111) structure. Characterization was performed with XRD and STM. The stability of the SAMs on gold with various properties was evaluated. To attach antibodies and/or avoid non-specific adsorption, novel thiols were synthesized. For standard covalent coupling procedures mercaptoethanol and ethanolamine are normally used as blocking molecules. We have synthesized blocking molecules based on ethylene oxide groups which show enhanced properties towards non-specific adsorption. The mixed monolayer formation was characterized using contact angle, cyclic voltammetry, impedance spectroscopy, XPS and GA-FTIR. The advantages of an orientated immobilization of chemically modified antibodies are demonstrated using SPR. We have evaluated the random amino and streptavidine-biotin coupling in comparison with the orientated aldehyde and thiol coupling procedures. Also the chemical modifications of these antibodies were optimized towards an increased sensitivity. Finally, we compared the sensitivity and selectivity with commercially available biological recognition layers, illustrating their enormous potential for further sensing applications. |
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BI-MoP-11 Piezoresistive Microcantilever Sensors for the Detection of Biological Molecules
T.L. Porter, M.P. Eastman, D.L. Pace, T.R. Dillingham (Northern Arizona University) Microsensors capable of recognizing single biological molecules have been fabricated using piezoresistive microcantilever technology. Using 25 base single strand DNA layers as the active sensing material, small piezoresistive microcantilevers in contact with this surface were able to recognize the presence of the complimentary strand, while ingoring the presence of strands differing by 2-5 base units. The analyte recognition is by means of a simple resistance change in the microcantilever, meaning only simple, inexpensive electronics are required for this device. Several sensors may be grouped together to form small bio-sensing arrays. |
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BI-MoP-12 Protein Template-Imprinting to Enhance Specific Protein Adsorption and Cell Adhesion
J. Wang, X.H. Cheng, J. Schwartz, B.D. Ratner (University of Washington) Molecularly imprinted polymers (MIPs) are synthetic materials that possess specific recognition properties, and have found applications in enantiomer separation, enzyme mimics, and biomimetic biosensors. Among the common methods for molecular imprinting, the self-assembly approach is important for its resemblance to antibody-antigen, substrate-receptor, and enzyme-inhibitor interactions.1 The key for MIPs to perform recognition is that both the template and imprint are complementary in size, shape, and chemical functionality at the binding site. Nevertheless the self-assembly approach to molecular imprinting has been primarily done in organic solutions, and is less successful in directly imprinting larger biomolecules (proteins). Shi et al.2 developed an alternative approach to imprint protein molecules with disaccharide molecules and polymeric thin films. The protein imprints can preferentially recognize the original template protein, which is mainly attributed to cooperative non-covalent interactions including hydrogen bonds, hydrophobic interactions and van der Waals forces. We applied a similar imprint process with optimized conditions to create highly reproducible albumin (Alb) and fibronectin (FN) imprint surfaces. Surface analyses including ESCA, SIMS, and AFM were performed to characterize the variation in chemical composition after each imprint step. BAE cell adhesion studies demonstrated that the FN imprint surface could enhance cell attachment. Results from 125I labeled binary protein competitive adsorption also showed that the FN imprint surface could preferentially adsorb higher amount of FN in a binary protein solution with Alb as the competing protein. |
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BI-MoP-13 Surface Technologies to Optimize Osteopontin-immobilized Surfaces for Healing Biomaterials
S.M. Martin, R. Ganapathy, T. Kim, L.A. Martinson, D. Leach-Scampavia, S.L. Golledge, C. Giachelli, B.D. Ratner (University of Washington) Our efforts to develop biomaterial surfaces that modulate the healing and inflammatory response focus on immobilizing specific biological triggers of healing onto a bland, relatively non-protein adsorptive surface. This study illustrates the use of surface analysis tools to characterize such surfaces. Osteopontin (OPN) is a protein known to regulate inflammatory responses. Although its precise role is not fully understood, it has been implicated in wound-healing processes. We have thus chosen OPN as the protein to immobilize in these model experiments. ESCA data in our laboratory demonstrated that poly(2-hydroxyethyl methacrylate) (pHEMA) shows low protein adsorption, making it a suitable material for our immobilization studies. In the present study, a technique using carbonyldiimidazole (CDI) was used to immobilize OPN to the polyHEMA surface. We employed several techniques to verify presence of protein on the polyHEMA surface (ESCA, TOF-SIMS, FTIR) and quantified the amount immobilized (ELISA, radioiodination of OPN). ESCA high-resolution N 1s spectra indicated existence of OPN on the surface, and this data was confirmed by TOF-SIMS. Furthermore, data using I-125-labeled OPN showed a dose-response corresponding to the varying amounts of OPN used for the immobilization experiment. Though less accurate than the radiolabel data, the ELISA showed protein amounts in a similar range as well. These findings represent an important first step toward the creation of novel healing materials for biomedical applications and to using modern surface analytical tools to verify the surface engineering of a biomaterial. Studies funded by UWEB, EEC9529161. |
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BI-MoP-14 Competitive Oligonucleotide Adsorption Equilibria at a Silane-Water Interface
A.D. Suseno, R.S. Gascon, J.L. DelosReyes, J.E. Forman (Zyomyx, Inc.) Adsorption of complimentary oligonucleotide sequences to surface bound oligonucleotide probes can produce surface bound duplex structures that are more prone to dissociation than their solution phase counterparts. The stability (as indicated by the observed melting temperature or Tm) can be altered by a number of factors, including probe orientation on the surface and surface bound probe density. A bound orientation that does not allow the probe to fully interact with its complimentary sequence can result in a non-optimal (and thus lower stability) duplex structure. Such effects are expected when immobilized probes are bound through exo-cyclic amines or crosslinking of thymidine residues to the surface. For surface bound probe densities, crowded surfaces that limit the amount of bound target through unfavorable steric and/or electrostatic interactions serve to destabilize the surface bound duplexes. Likewise, surfaces that interact with the immobilized probe, will compete with adsorption of complimentary sequences and can ultimately reduce duplex stability. Yet, despite these potential hindrances to duplex formation, surface bound probes can adsorb complimentary sequences from solutions in which those sequences are present in double stranded form (where the second strand is non-complimentary to the surface bound probes). We will present results from experiments in which covalently immobilized oligonucleotide probes on silanated substrates interact with both single- and double-stranded oligonucleotide target sequences to illustrate consequences of surface immobilization strategy, as well as what effect the introduction of a competing solution phase duplex formation equilibria has on adsortion to the surface bound probes. |