This Is AuburnElectronic Theses and Dissertations

Low-Cost, Rapid, Sensitive Detection of Pathogenic Bacteria Using Phage-Based Magnetoelastic Biosensors




Horikawa, Shin

Type of Degree



Materials Engineering


As part of the ongoing efforts to secure food safety as well as to guard against possible bioterrorism, the role of pathogen detection technologies has become vital. However, conventional and standard detection methods, including culture-, immunology-, and polymerase chain reaction-based methods, are generally expensive, time-consuming, and labor-intensive. Hence, there is a need for new detection technologies that outperform the conventional methods and enable the rapid, on-site detection of pathogenic substances. Although label-free biosensors have proven to be among the most promising methods, meeting various performance criteria (e.g., sensitivity, selectivity, assay time, thermal stability, and longevity) simultaneously still remains a challenge. Hence, further research and development are essential before biosensors become a reliable, alternative solution. Phage-based magnetoelastic (ME) biosensors, a novel class of wireless, mass-sensitive biosensors, are among potential candidates that could overcome the above performance challenge. These biosensors are not only thermally robust, but their wireless nature of detection offers great flexibility in design and use, which facilitates on-site pathogen detection. In addition, the sensitivity of ME biosensors can be improved by reducing their dimensions, and the fabrication cost per sensor can be reduced via batch fabrication. Hence, this dissertation presents investigations into the performance improvement of phage-based ME biosensors, in terms of cost-effectiveness, rapidness, and sensitivity, and into the enhanced detection of pathogenic bacteria, Salmonella Typhimurium and Bacillus anthracis spores, for food safety and biosecurity. To enhance both cost-effectiveness and sensitivity, micron- to millimeter-scale ME biosensors were batch-fabricated and used. In this way, the fabrication cost per sensor was reduced to a fraction of a cent. In addition, the following two methodologies were employed to dramatically shorten assay time: (1) direct detection of S. Typhimurium on fresh spinach leaves and (2) detection of B. anthracis spores with the aid of a designed microfluidic flow cell, which ensures efficient physical contact between a biosensor and flowing spores. By using these methodologies with low-cost, miniature ME biosensors, (1) S. Typhimurium cells on the order of 10^4 cells/cm^2 were detected with 150-μm long sensors in 45 min, and (2) down to 106 B. anthracis spores were detected with 200-μm long sensors in 10 min. Additionally, to further enhance the detection capabilities of phage-based ME biosensors, the following effects were studied: (1) the effects of mass position on the sensitivity of ME biosensors and (2) the effects of surface functionalization on surface phage coverage. The mass sensitivity of ME biosensors was found to be largely dependent on the dimensions of the sensors as well as on the position of attached masses. From numerical simulation results, a formula that predicts the mass-position-dependent sensor response for a single localized mass was also derived. In addition, surface phage coverage on bare and surface-functionalized ME biosensors was quantified by atomic force microscopy. The results showed that activated carboxyl-based covalent attachment produced a surface phage coverage of ∼ 50%, which is comparable to that obtained through physical adsorption, the traditional method of phage immobilization. By contrast, much lower surface phage coverages (∼ 5%) were obtained for aldehyde- and methyl-terminated sensor surfaces. These differences in surface phage coverage was also found to affect the quantity of a subsequently captured analyte. Hence, by properly functionalizing the sensor surface, both surface phage coverage and the quantity of the captured analyte can be controlled. Finally, with the results of the mass-position-dependence of sensor response, a concept of phage layer patterning was introduced. Phage may be patterned onto desired parts of the sensor surface to further enhance the detection capabilities of ME biosensors.