| Autor: |
Pillai RG; National Research Council Canada- Quantum and Nanotechnologies Research Center, 11421 Saskatchewan Drive, Edmonton T6G 2M9, AB, Canada. abebaw.jemere@nrc-cnrc.gc.ca., Azyat K; National Research Council Canada- Quantum and Nanotechnologies Research Center, 11421 Saskatchewan Drive, Edmonton T6G 2M9, AB, Canada. abebaw.jemere@nrc-cnrc.gc.ca., Chan NWC; Defence Research and Development Canada - Suffield Research Centre, Medicine Hat T1A 8K6, AB, Canada., Jemere AB; National Research Council Canada- Quantum and Nanotechnologies Research Center, 11421 Saskatchewan Drive, Edmonton T6G 2M9, AB, Canada. abebaw.jemere@nrc-cnrc.gc.ca.; Department of Chemistry, Queen's University, Kingston K7L 3N6, ON, Canada.; Department of Chemistry, University of Waterloo, Waterloo N2L 3G1, ON, Canada. |
| Abstrakt: |
Herein, we describe a rapid and facile fabrication of electrochemical sensors utilizing two different toll-like receptor (TLR) proteins as biorecognition elements to detect bacterial pathogen associated molecular patterns (PAMPs). Using potential-assisted self-assembly, binary mixtures of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MCH), or MUA and an in-house synthesized zwitterionic sulfobetaine thiol (DPS) were assembled on a gold working electrode within 5 minutes, which is >200 times shorter than other TLR sensors' preparation time. Electrochemical methods and X-ray photoelectron microscopy were used to characterize the SAM layers. SAMs composed of the betaine terminated thiol exhibited superior resistance to nonspecific interactions, and were used to develop the TLR sensors. Biosensors containing two individually immobilized TLRs (TLR4 and TLR9) were fabricated on separate MUA-DPS SAM modified Au electrodes (MUA-DPS/Au) and tested for their response towards their respective PAMPs. The changes to electron transfer resistance in EIS of the TLR4/MUA-DPS/Au sensor showed a detection limit of 4 ng mL -1 for E. coli 0157:H7 endotoxin (lipopolysaccharide, LPS) and a dynamic range of up to 1000 ng mL -1 . The TLR4-based sensor showed negligible response when tested with LPS spiked human plasma samples, showing no interference from the plasma matrix. The TLR9/MUA-DPS/Au sensor responded linearly up to 350 μg mL -1 bacterial DNA, with a detection limit of 7 μg mL -1 . The rapid assembly of the TLR sensors, excellent antifouling properties of the mixed SAM assembly, small size and ease of operation of EIS hold great promise for the development of a portable and automated broad-spectrum pathogen detection and classification tool. |
