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Robust Piezoelectric-Excited Millimeter-Sized Cantilever Sensors for Detecting Pathogens

David Maraldo1, Kishan Rijal1, and Raj Mutharasan2. (1) Chemical and Biological Engineering, Drexel University, 32nd and Chestnut St., Philadelphia, PA 19104, (2) Department of Chemical and Biological Engineering, Drexel University, 32nd and Chestnut St., Philadelphia, PA 19104

Purpose. To develop practical sensors for detecting waterborne pathogen E. coli O157:H7(EC) and others in small (1 mL) and in large volume samples (1 liter) without a sample preparation step. Method. Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors with recognition molecules immobilized on the surface was used for selective detection of the target pathogen. Upon binding of the antigenic target, the resonance frequency decreases and was monitored by an impedance analyzer. Two recognition strategies were used: (1) use of antibody specific to EC, and (2) use of identifying DNA sequence, Stx2 gene that is specific to EC O157:H7. Results. The sensors were first characterized and were found to exhibit 0.3 to 1.5 fg/Hz mass change sensitivity. Initial studies show that both strategies are successful, and limit of detection for EC is ~10 cells/mL with one mL sample of tap water or in proteinous complex fluids. Detection at 1 cell/mL in 1 liter sample was also successful. We use low-pH buffer release to confirm antibody-based detection. For Stx2-based detection, a simple sample prep that enabled extraction of genomic DNA in 30 minutes was used. Initial experiments show that 5,000 DNA copies of stx2 (which correspond to 5,000 cells) is easily detected without amplification. Dilution of the sample and subsequent hybridization shows detection at 300 cells is feasible with PEMC sensors. Conclusions. We conclude that antibody-based detection of EC can be accomplished in a practical format in less than 15 minutes at a detection limit of one cell. DNA-based method requires slightly longer time of ~45 minutes for 5,000 cells. These results suggest that detection of 50 cells with a simplified genomic extraction procedure might be feasible.