Detection Of SEB Gene By Bilayer Lipid Membrane Nucleic Acid Biosensor Supported By Patch-clamp Pipette Electrode

Staphylococcus aureus entoxin B (SEB) encoded by SEB gene was a crucial toxin arousing food-poisoning and also one of the important biological warfare agents. This paper was focused on a new kind of nucleic acid biosensor for fast, sensitive, and specific detection of SEB gene. The bilayer lipid membranes (BLMs) played the role as the sensitive molecular recognized element which fixed on oligonucleotide probe specific to SEB gene. The biosensor was fabricated by utilizing patch-clamp pipette technology, which characterized in sensitive changes of ionic current though physiological cellular membranes. The main research contents are listed as follows:1. Components and proportion of the membrane forming solution were optimized. A new kind of BLMs supported by patch-clamp pipette was developed. Patch-clamp pipette amplifier recorded the I-V plot of BLMs formation on the top of patch-clamp pipette electrode. The scene of BLMs formation could be observed by optics microscope (10x40). The authenticity of the BLMs' formation was validated by the way of embedding ionic channel protein,i.e. Gramicidin on it . The preparation was simple and reproductive.2. The oligonucleotide probe which specific to SEB gene was designed by means of biological informatics and was modified at the 5' end with an unbranched dodecane hydrophobic C₁₂ tail (C₁₂-ssDNA) which could improve the ablity of ssDNA's fixation. 3.Currents responses of fixation on BLMs by different sequences of ssDNA probes and that of the different concentrations of the 18bp C12-SSDNA, respectively. It showed that currents of C₁₂-ssDNA were more stable than that of the unmodified. The difference of electrochemical currents versus the different concentration of ssDNA probe appeared linear positive correlation by 50mV electrostatic potential. The ssDNA probe (273.65ng/mL) fixed on BLMs as the biological sensitive element concentration of the BLMs nucleic acid biosensor. Relationship between detective currents and the imposing step voltages was favorably linear correlation.