Paper analytical devices for rapid, quantitative electrochemical detection of DNA and bacteria

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Brenes, Nicholas James

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In this thesis, two paper analytical devices (PADs) are described as proof of concept devices for point-of-care applications. The first PAD, termed the Esensor, was developed for quantitative detection of oligonucleotides. The detection component of the Esensor was based on DNA stem-loop probe hybridization with signal stranded DNA followed by transduction of an electrochemical signal via target-induced conformational switching. The electrochemical signal was produced by a redox label attached to the DNA stem-loop probe. The Esensor had a limit of detection of 30 nM for DNA, and the device-to-device reproducibility was better than 10%. Furthermore, the Esensor had a shelf life of at least 4 weeks and required only 20 µL of sample. The Esensor work presented in this thesis was published in Analytical Chemistry where the detection of DNA and thrombin was described.1 The Esensor work was completed in collaboration with Dr. Cunningham who is the primary author on the publication. This thesis focuses only on the detection of DNA by the Esensor, as I have made significant contributions to this portion of the work. The second PAD covered in this thesis was developed for the detection of whole-cell bacteria. The operation of the device involved a sandwich capture assay. Bacterial specificity was achieved using antibody-functionalized magnetic microbeads and silver nanoparticle (AgNP) labels. The AgNP labels allowed for electrochemical detection via anodic stripping voltammetry. In this sensor, there were two inherent forms of signal amplification: (1) magnetic concentration of microbeads complexed with bacteria at the working electrode surface and (2) electrochemical concentration of Ag+ ions at the working electrode surface. This PAD was nearly 100% specific for Escherichia coli (E. coli) in the presence of two additional bacterial species. The on-chip assay time was <4 min, the device fabrication was cost effective at $0.36 USD/device, and the limit of detection was 1.3 x 107 cells/mL. This device, termed the oSlipB, was similar to a PAD employed to detect DNA and proteins as previously reported;2–5 however, this new application of bacterial detection further exemplifies the versatility of this paper device.



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