AVS1997 Session BI-WeA: Biosensor-Biology Interface

Wednesday, October 22, 1997 2:00 PM in Room F

Wednesday Afternoon

Start	Invited?	Item
2:00 PM	Invited	BI-WeA-1 The Neuron-Silicon Junction and Beyond. P. Fromherz (Max-Planck-Institute for Biochemistry) Nerve cells are coupled directly to silicon devices by electrical induction¹. At first the stimulation and recording of electrical activity in neurons of the leech is reviewed²^,³^,⁴. In a second part a representative circuit of the junction is presented as determined by AC-measurements⁵^,⁶. Then the structure of cell adhesion is studied by fluorescence-interference contrast (FLIC) microscopy⁷^,⁸ and by closely packed arrays of transistors⁹. The neuron-silicon junction is described as a planar two-dimensional core-coat conductor. In the fourth part a nonlinear switching of membrane conductance is discussed which is caused by adhesion¹⁰^,¹¹. Finally progress and problems are considered with respect to the application of large-scale integration, to the coupling of vertebrate neurons and to the development of hybrid neuronal nets. ¹P.Fromherz, Ber.Bunsenges.Phys.Chem. 100 (1996) 1093-1102. ²P.Fromherz, A.Offenhaeusser, T.Vetter, J.Weis, Science 252 (1991) 1290-1293. ³P.Fromherz, A.Stett, Phys.Rev.Lett. 75 (1995) 1670-1673. ⁴A.Stett, B.Mueller, R.Weis, Phys.Rev.E 55 (1997) 1779-1782. ⁵P.Fromherz, C.Mueller, R.Weis, Phys.Rev.Lett. 71 (1993) 4079-4082. ⁶R.Weis, P.Fromherz, Phys.Rev.E 55 (1997) 877-889. ⁷A.Lambacher, P.Fromherz, Appl.Phys.A 63 (1996) 207-216. ⁸D.Braun, P.Fromherz, Appl.Phys.A submitted ⁹R.Weis, B.Mueller, P.Fromherz, Phys.Rev.Lett. 76 (1996) 327-330. ¹⁰M.Jenkner, P.Fromherz, Phys.Rev. submitted ¹¹P.Fromherz, Phys.Rev.Lett. (1997) in press
2:40 PM		BI-WeA-3 Reagentless Biosensors Based on Molecular Recognition and Transduction at Self-Assembled Monolayers G.P. Lopez (University of New Mexico) Several model systems based on multicomponent self-assembled monolayers (SAMs) are presented that accomplish reagentless sensing of biological molecules in aqueous solution. The SAMs are designed to display affinity elements (e.g., receptors and ligands) within a surface that incorporates high densities of oligo(ethylene glycol) moieties that prevent nonspecific adsorption of proteins and nonspecific cellular attachment. In some cases the SAMs are also designed to incorporate fluorophores that can be used for transduction (i.e., to detect the binding of the target analyte to the affinity element.) Changes in fluorescence intensity and lifetime can be used to detect analyte binding. In other cases, established reagentless techniques such as surface plasmon resonance (SPR) can be used to detect binding. Established techniques such as SPR also provide a good calibration technique to examine the effects of specific and nonspecific binding to fluorescent SAMs. Model analytes include antibodies, biotinylated molecules and pollutants and model receptors include proteins and peptides. This talk will examine several aspects of the structure-property relationships of multicomponent SAMs as model biosensor platforms.
3:00 PM		BI-WeA-4 Reversible Blocking of Ligand Binding to Streptavidin with Stimuli-Responsive Polymers. P. Stayton, V. Bulmus, Y. Hayashi, Z. Ding, C. Long, A. Hoffman (University of Washington) Stimuli-responsive polymers exhibit reversible phase changes in water in response to small changes in temperature, pH or other physical and chemical stimuli. We have previously reported that the binding of biotin to streptavidin can be gated by the collapse of a polyN-isopropyl acrylamide (pNIPAAm) oligomer that is conjugated to the protein near the biotin binding site at amino acid position 49. We have now extended these studies to include a study of the effect of molecular weight on the ability of pNIPAAm to gate biotin binding, a study of a pH sensitive copolymer of NIPAAm and acrylic acid (coP[NIPAAm/AAc]), and an investigation of a new conjugation site at position 116. This new position was chosen due to its proximity to tryptophan 120 which plays a critical energetic role in biotin binding. pNIPAAm oligomers with molecular weights ranging from 500 - 9000 were conjugated to the engineered cysteine side-chain at position 116 and the percent biotin block was observed to be dependent on molecular weight over this range. The pH-dependent coP[NIPAAm/AAc] oligomers are expanded coils at pH 7.4 and collapsed at pH 4.0 at 37 degrees celsius. 90% of biotin binding was blocked at pH 4.0 relative to pH 7.4 at this temperature when the coP[NIPAAm/AAc] was conjugated to cysteine 116. The ligand binding affinity of streptavidin can thus be controlled by either temperature or pH by conjugating the pNIPAAm or coP[NIPAAm/AAC] polymers at position 116. The environmentally-triggered "gating" and "switching" capabilities of these polymer-protein conjugates could allow eluate-free recovery of affinity ligands and regeneration of "fouled" affinity biosensor surfaces.
3:20 PM		BI-WeA-5 A New Method for Patterning Antibodies onto a Surface R.A. Brizzolara (NSWC - Carderock Division) The patterning of multiple antibodies (each to a different antigen) on a single substrate is an important step in the development of a biosensors with greatly improved chemical sensing capability. The envisioned devices would require patterning antibodies onto a substrate with micron spatial resolution, with adjacent pixels on the substrate containing antibodies against different antigens. In this paper, a new method for forming patterns of antibodies on a surface is described. This method has been used to form a rudimentary pattern of a single antibody on a polystyrene surface. This substrate was then exposed to an antigen labelled with 10 nm gold particles. X-ray photoelectron microscopy (XPS) and atomic force microscopy (AFM) were used to detect the spatial distribution of gold on the surface. The XPS and AFM results showed that there was more gold on the areas of the surface where antibody had been adsorbed. This data demonstrates that a single antibody can be patterned onto a surface using this technique. Additionally, data will be presented that indicates the possibility of patterning multiple antibodies onto a substrate using this technique. The NSWC Intra-Laboratory Independent Research Program provided funding support for this work.
3:40 PM		BI-WeA-6 Grafting Polyethylene Glycol on Surfaces to Create Biocompatible Coatings N.A. Alcantar, J.N. Israelachvili, E.S. Aydil (University of California, Santa Barbara) Surfaces covered with polyethylene glycol, (i.e., PEG, HO-(CH₂-CH₂-O)_n-H) have been shown to be biocompatible because PEG's properties enhance nonimmunogenicity, nonantigenicity, and protein rejection. In order to produce a biocompatible surface coating, we have developed a direct method for grafting PEG onto amorphous activated silica films. We deposit an amorphous silica film by plasma enhanced chemical vapor deposition (PECVD) from SiH₄ and O₂ gases, which gives the flexibility to coat diverse materials with different shapes. These silica films are activated by exposing them to water plasma, thus increasing the number of hydroxyl groups on the surface. The silanols (Si-OH) on the resulting surface chemically react with the hydroxyl end of the PEG chain and form an ester bond, Si-O-C. The surface reactions were monitored using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The vibrational absorption band for the C-O and for the -CH₂ bonds increase with time and saturate indicating that PEG is adsorbed onto the surface. Simultaneously with this increase, the Si-OH absorption band decreases showing that the surface silanols react with PEG and are depleted. The PEG-covered surfaces have been characterized by atomic force microscopy (AFM), Auger electron spectroscopy, ellipsometry, and contact angle measurements. These characterization techniques give solid evidence for the existence of bonded PEG on the surface. For instance, AFM images show that the PEG covered silica surfaces are smoother than as deposited silica surfaces. Auger electron spectra show that atomic species of PEG remain on the surface even after washing the surface with chloroform and water. The advancing contact angle values of water on the PEG covered surfaces are lower for the bare silica surfaces showing that they are more hydrophilic.
4:00 PM	Invited	BI-WeA-7 Diffusion in Microfluidic Systems P. Yager, J.P. Brody (University of Washington) Two prominent physical effects dominate microfluidic devices, diffusion and viscous fluid dynamics. A number of interesting devices can be constructed for chemical sensing, extraction, and mixing based on these two effects.
4:40 PM		BI-WeA-9 Stabilization of Self-Assembled Monolayers on Glass and Silicon D. Kelly, T.W. Schneider, H. Schessler, J.J. Hickman (Science Applications International Corporation) We have previously demonstrated the ability of self-assembled monolayers (SAMs) on glass and silicon surfaces to present a surface chemistry template favorable to biotechnology applications. However, the longevity of surface modifications afforded through SAMs is generally dependent upon the conditions of exposure of the modified substrates. We have undertaken XPS investigations aimed at elucidating new methods through which the longevity of adsorbed SAMs may be controlled. By employing methods of both substrate stabilization and SAM protection through the coadsorption of proteins, we are able to control the time frame during which the integrity of a SAM remains intact. We report the stability, as a function of time, of two SAMs important in biotechnological applications, namely (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethyltrichlorosilane (13F) and trimethoxysilylpropyldiethylenetriamine (DETA), under biologically relevant conditions. XPS results indicate that approximately 90% of a self-assembled monolayer of DETA on substrate-stabilized glass or silicon remains intact after 1 week in phosphate buffered saline (PBS), compared with 60% for the case of an unstabilized system. In the case of 13F adsorbed on glass or silicon, approximately 85% of the SAM is intact on stabilized substrates after 10 days in PBS, compared with 65% stability afforded by SAM protection by coadsorbed bovine serum albumin, and 45% for an unstabilized or unprotected system over the same time period. Results of longer term (on the order of several weeks) stability experiments and the potential impact of SAM stabilization to a number of applications will also be discussed.
5:00 PM		BI-WeA-10 In situ Two-Color Surface Plasmon Resonance Spectroscopy of Biopolymer Film Formation at Solid/Liquid Interfaces K.P. Peterlinz, R.M. Georgiadis (Boston University) The adsorption and self assembly of biopolymers is important for a number of processes, including separations, chromatography, and the fabrication of biosensors. In this work we present results of in situ two-color surface plasmon resonance spectroscopy measurements. With this method, we achieve unprecendented accuracy and precision for the determination of the average coverage, thickness, and density of biopolymer films adsorbed on gold surface in the presence of buffer solution. From these in-situ data and detailed studies of formation kinetics, we develop detailed kinetic models and discuss probable mechanisms for adsorption and self assembly. We also examined the effects of molecular size and film density on the interpenetration of small functionalized alkane and biopolymer molecules into these films. The films studied include DNA oligomers, thiol functionalized DNA, and acidic polysaccharide films.