Papers

AVS2004 Session BI-ThA: Biosensors and Bio-Diagnostics

Thursday, November 18, 2004 2:00 PM in Room 210D

Thursday Afternoon

Start	Invited?	Item
2:00 PM		BI-ThA-1 Multi-analyte Immunoassays in Packed Microcolumns: Design and Analysis G.P. Lopez (University of New Mexico) This talk will present recent developments at the University of New Mexico in the development of microfluidic based biosensor systems and their use in analysis of biomolecular recognition. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column-format inside a microfluidic channel. The packed microcolumn format is (1) well suited for enhancing reaction times of analyte with immobilized receptors, (2) compatible with electro-osmotic pumping, and (3) allows detection of multiple analytes. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer (FRET). Several immunoassays have been developed that can potentially detect sub-femtomole quantities of antibody with high signal-to-noise ratio and a large dynamic range spanning nearly four orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. Kinetic and equilibrium constants for the reaction of this receptor-ligand pair are obtained through modelling of kinetic responses of the affinity microcolumn and are consistent with those obtained by flow cytometry. Because of the correlation between kinetic and equilibrium data obtained for the microcolumns, quantitative analysis can be done prior to the steady state endpoint of the recognition reaction. This method has the promise of combining the utility of affinity chromatography, with the advantage of direct, quantitative, and real-time analysis and the cost-effectiveness of microanalytical devices. The approach has the potential to be generalized for high sensitivity, high selectivity, rapid detection of a host of bioaffinity assay methods and analyte types.
2:40 PM		BI-ThA-3 Biosensing Based on Light Absorption of Immobilized Metal Nanostructures F. Frederix, K. Bonroy, G. Reekmans, C. Van Hoof (IMEC, Belgium); G. Maes (K.U.Leuven, Belgium) The "Transmission Plasmon Biosensor" is a novel, cheap and easy to handle biosensing technique. Surface plasmon resonance sensors are widely used for biosensing. These sensors are highly sensitive to the refraction index at the interface between the metal film deposited upon a prism and a sample upon this metal surface. This principle can also be applied to a dielectric planar surface coated with nanostructures. The plasmon absorption peak position and intensity is highly dependent on the size of these particles and on the close proximity of these particles immobilized onto a surface. This research compromises the synthesis of metal nanoparticles with different sizes and morphologies, which were covalently immobilized on transparent substrates, e.g. glass, quartz and polymers using a molecular glue of silane layers. Particle films were also realized using various evaporation strategies, e.g. thermal evaporation, e-beam evaporation, sputtering and electroless plating. The different strategies were evaluated using TEM, AFM and absorption spectroscopy. The resulting plasmon resonance and interband absorption bands in the visible and UV region were compared. Mixed SAMs were used to couple antibodies to the metal nanoparticle films. The change in absorbance properties of the nanoparticle films upon antibody-antigen binding was monitored in order to obtain quantitative information on the antibody-antigen interaction. Besides the localised plasmon resonance sensing, we observed a novel physical phenomenon namely the interband transition absorption enhanced sensing. Furthermore, the applied technique was identified to be a useful alternative for the most widely used clinical immunosensing technique, i.e. the ELISA technique. This promising alternative was applied onto modified microtitre plates, which allow the implementation into an array technology. The Transmission Plasmon Biosensor fulfils therefore the needs of an ideal, multi-analyte bio(nano)sensor.
3:00 PM		BI-ThA-4 2-D Array Biosensor using Waveguide Bragg Grating K.S. Choi, H.J. Lee, C.I. Jung, H.J. Park, N.W. Park (Chonnam National University, Korea) Bragg grating based planar integrated optical circuit technology is applied to biosensors. Varieties of detection mechanisms such as antigen-antibody are investigated and an appropriate structures of Bragg grating optical sensors circuit is demonstrated. Trenches are fabricated onto a planar waveguide substrate and polymer core material with high refractive index is deposited in these trenches.Bragg gratings are formed onto this polymer core and series of Bragg gratings with different periods are fabricated along these waveguides. These waveguides are repeated in serial such repetition makes 2-D array of bio sensors which might be vey effective in sensing of disease. We will introduce and demonstrate the Bragg grating based silica waveguide sensors in this paper and some theoretical designs will be considered.
3:20 PM		BI-ThA-5 Biological Sensors Based on Brownian Relaxation of Magnetic Nanoparticles S.-H. Chung, A. Hoffmann, S.D. Bader, L. Chen, C. Liu, B. Kay, L. Makowski (Argonne National Laboratory) We present a biological sensing platform that is based on a modification of the dynamic magnetic properties of ferromagnetic nanoparticles suspended in a liquid. For a narrow size range the ac magnetic susceptibility of the ferromagnetic nanoparticles is dominated by Brownian relaxation. By coating the nanoparticles with a suitable ligand the Brownian relaxation and thus the ac magnetic susceptibility can be modified through the binding to the corresponding bio-receptor. The size of the particles has to be large enough to avoid superparamagnetism and at the same time small enough to have a homogeneous single domain magnetization. We demonstrate a proof-of-principle of this concept by using avidin-coated Fe₃O₄ particles that are ~10 nm in diameter, which were investigated before and after binding to biotinylated S-protein and bacteriophage particles. The ac susceptibility measurements show that the magnetic relaxation occurs via a Brownian mechanism; the frequency shift for the peak in the imaginary part of the susceptibility after binding to the target indicates the increase of the hydrodynamic radius. We are currently developing magnetic phage viruses in order to further improve this bio-sensing platform. * Supported by DOE, BES under contract W-31-109-ENG-38, and DARPA under contract 8C67400.
3:40 PM		BI-ThA-6 G-Protein Coupled Receptor Biosensors - New Opportunities and Applications E.J. McMurchie, W.R. Leifert (CSIRO Health Sciences and Nutrition, Australia); L. Wieczorek, B. Raguse (CSIRO Telecommunications and Industrial Physics, Australia) Future diagnostic and biosensor platforms will require development of cell-free, high-throughput, microarray formats with bioengineered sensors mimicking the specific interactions between ligand and cell membrane receptors. For bio-diagnostic technologies, G-protein coupled receptors (GPCRs) are likely to have application as biosensors reporting on ligands influencing physiological and pathophysiological functions. GPCRs are a large and ubiquitous class of membrane-associated receptors activated by a wide range of extracellular ligands, (biogenic amines, amino acids, ions, peptides, and bioactive lipids) which act as hormones, neurotransmitters, chemokines etc. Signalling through these receptors regulates responses such as neurotransmission, chemotaxis, inflammation, cell proliferation, muscle contractility, and visual and chemosensory perception. GPCRs signal to numerous down stream cellular effectors via a set of heterotrimeric G-proteins through GTP dependant processes. GPCRs are the target for >50% of current therapeutic drugs with drug discovery programs relying on high throughput screening technologies. The future development of microarray technologies for GPCRs is relevant for the development of highly specific ligands in drug discovery and for utilising GPCRs as potential biosensors. Present assays for ligand screening against GPCRs can be classified into two major categories; whole cell assays with cell-associated, down-stream signalling systems for detecting activated receptors, and homogeneous, cell-free assays consisting of membrane fragments containing (usually cloned) GPCRs. For the latter, some form of signalling/reporting system must be added if functional assays, as opposed to ligand binding, are to be used. Our strategic objective is the construction of a cell-free system to enable reconstitution and nanoconstruction onto appropriate surfaces for future adaptation to microarray formats suitable for high throughput, multiplex screening.
4:20 PM		BI-ThA-8 Detection of Human Immunodeficiency Virus-1 Using Micro-Cantilever Deflection Biosensors Y. Lam, N. Abu-Lail, M. Alam, S. Zauscher (Duke University) Having a simple, efficient, and sensitive technique for the diagnosis of human immunodeficiency virus-1 (HIV-1) is extremely important due to the increasing trend in HIV-1 cases, and the current lack of a rapid and simple method to detect the disease. We show that surface modified micro-cantilevers, decorated with monoclonal antibodies (mAb) A32, deflect upon specific binding of mAb A32 to HIV-1 envelope glycoprotein gp120 (HIV-1 Env gp120). This deflection of the micro-cantilever is a direct result of the surface stress induced by molecular recognition mediated protein binding. The specific binding between the two proteins was confirmed through force spectroscopy measurements between mAb 17b tethered to a surface and HIV-1 Env gp120 immobilized on the cantilever; HIV-1 gp120 will only bind mAb 17b if the former has bound to mAb A32. Our results show that micro-cantilever deflection can effectively be used for the sensitive detection of molecular recognition events, encouraging the further development of this technique as a rapid response biosensor for disease diagnosis. Work on determining the detection limits of the micro-cantilever deflection method and its extension to whole virus detection is in progress.
4:40 PM		BI-ThA-9 Realization and Characterization of Porous Gold for Increased Protein Coverage for Biosensor Applications K. Bonroy, J.-M. Friedt, F. Frederix, R. De Palma (IMEC, Belgium); M. Sàra (Center for NanoBiotechnology, Austria); B. Goddeeris (KULeuven, Belgium); G. Borghs (IMEC, Belgium); P. Declerck (KULeuven, Belgium) In recent years, there has been an increasing need for the detection of biochemical substances with low molecular weight. Biosensors could be an alternative to conventional analytical methods for monitoring these substances. However, generally applied biosensor systems are often not sensitive enough for direct detection of these compounds. Therefore, our research focuses on the development of biosensors with improved transducer capabilities and biological interfaces. We chose gold surfaces in combination with SAMs of thiols as platform for the immobilization of biomolecules because of its compatibility with existing biosensors. In previous research, the use of SAMs of thiols on flat gold surfaces showed several advantages concerning specificity and reproducibility for final biosensor applications. However, the main disadvantage of this approach is the 2D aspect of these layers compared to 3D surfaces (e.g. polymers). 3D surfaces, such as porous gold, would allow for the immobilization of a large number of molecules per surface area, facilitating higher biosensor responses. The presented research describes the analysis of the different parameters, which define the electrochemical growth of porous gold, starting from flat gold. QCM-D technique was used for online monitoring of the porous gold deposition. The resulting surfaces were characterized using SEM, cyclic voltammetry and contact angle measurements. Applied potentials of -0.5V were found to be the most adequate conditions to grow porous gold, resulting in a 16x increase in surface area. In addition, we evaluated the immobilization degree of S-layer and IgG proteins on these porous surfaces. The optimized deposition conditions for realizing porous gold substrates, lead to 3x increase of S-layer adsorption and 5x increase of anti-IgG recognition using QCM-D as biological transducer. We can conclude that the high specific area of the porous gold amplifies the final sensitivity of the original flat surface device.