Covalent immobilization of DNA and hybridization on microchips by microsecond electric field pulses
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Covalent immobilization of DNA and hybridization on microchips by microsecond electric field pulses F. Fixe1,2, H.M.Branz1,4, D.M.F. Prazeres2, V. Chu1 and J.P. Conde1,3 INESC Microsistemas e Nanotecnologias, Lisbon, Portugal 2 Center for Biological & Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal 3 Department of Materials Engineering, Instituto Superior Técnico, Lisbon, Portugal 4 National Renewable Energy Laboratory, Golden, CO, USA 1
ABSTRACT Single square voltage pulses were used to enhance by 7 and 9 orders of magnitude the rate of covalent immobilization and hybridization, respectively, of single stranded DNA probes on a chemically functionalized thin film surface (silicon dioxide) using 2 mm size electrodes. These electrodes were scaled down to 20 µm. Photolithography was used to define the electrode voltage line, ground line, and functionalized thin-film area on a plastic substrate (polyimide). At all electrode dimensions, electric field-assisted DNA immobilization and hybridization can be achieved in the microsecond time scale, far faster than the 2 hr or 16 hr needed for immobilization and hybridization, respectively, without the electric field. Pulse conditions optimized with the large-size electrodes (2 mm) were used in the microelectrodes. INTRODUCTION DNA microarrays are powerful devices that integrate the specificity and selectivity of biological molecules with electronic control and parallel processing of information. This combination will potentially increase the speed and reliability of biological analysis [1]. The capability of analyzing mRNA, cDNA, proteins and other biological samples with high throughput has opened new frontiers in genetic analysis, pathogen identification and expression analysis [1-3]. However, the widespread application of DNA chips requires that the chips be inexpensive to fabricate and that diagnostics are simple and reliable. The technologies presently used to produce arrays of DNA probes are expensive and their use is therefore limited to very specialized applications [4]. To date, DNA probes are normally attached to inorganic substrates (silicon and glass are the most widely used [5-8]) with immobilization achieved passively (by covalent bonding or adsorption) [9-11]. Nanogen has developed the concept of electric field assisted DNA immobilization [4, 12-13]. The chip developed by Nanogen (NanoChip) uses an affinity-based DNA immobilization (between the surface and the capture probes) and the DNA is addressed to selected pixels by applying a dc electric field for 5 minutes, based on an electrophoresis mechanism. Other groups used electrochemistry to enhance the kinetics of DNA probe immobilization and hybridization [14]. All these electrical addressing schemes have drawbacks, including unwanted electrochemical reactions that damage the DNA molecules. Previously, we have reported the use of a single square voltage pulse to enhance by 7 to 9 orders of magnitude the rate of covalent immobilization and hybridization of single stranded DNA probes
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