Browsing by Subject "Electrochemical detection"

Now showing 1 - 2 of 2

Paper analytical devices for rapid, quantitative electrochemical detection of DNA and bacteria
(2016-12) Brenes, Nicholas James; Crooks, Richard M. (Richard McConnell)
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.
Synthesis and application of electrochemically active oligonucleotides
(2017-05) Lim, Byung Joon (Ph. D. in chemistry); Sessler, Jonathan L.; Ellington, Andrew D.; Anslyn, Eric V; Liu, Hung-wen; Russell, Rick
Modified oligonucleotides with redox-active functional groups could emerge as attractive tools for sensor development. In principle, changes in oligonucleotide hybridization or conformation may be read out as a change in an electrochemical signal. Monitoring this signal might allow for a direct interface between biology and electronics. This dissertation describes efforts devoted to creating redox active oligonucleotide derivatives designed to allow these application goals to be pursued. The focus is primarily on the synthesis, characterization, and application of oligonucleotides bearing on of two electroactive moieties, namely ferrocene and methylene blue. Chapter 1 provides a brief overview of electrochemically modified oligonucleotides and is designed to provide an historical perspective. Synthetic methodology, fabrication of electrode system, and current applications are introduced. Chapter 2 describes the synthesis of a ferrocene-modified oligonucleotide and its use as a multiplexing signal probe. Included in this chapter are syntheses of a ferrocene subunit bearing alkynes, as well as modified nucleoside phosphoramidites and the oligonucleotide syntheses they permit. A synopsis of electrochemical studies are also provided. Chapter 3 describes a ratiometric electrochemical DNA sensor (a so-called E-Sensor) based on the ferrocene-modified oligonucleotide described in Chapter 2 and its used in the detection of specific genes with greatly improved reproducibility. Oligonucleotide syntheses achieved through enzyme ligation, the fabrication of an E-sensor, and the results of electrochemical assays are provided in this chapter. Chapter 4 describes the design and fabrication of possible wearable devices with the modified electrochemically active oligonucleotides toward real diagnostic applications. This work is being done in collaboration with Dr. Nanshu Lu’s group in the Dept. of Aerospace Engineering and Engineering Mechanics at the University of Texas at Austin. Chapter 5 the details the synthetic procedures, provides characterization of all new products, and contains electrochemical analytical data discussed in this dissertation.