Every environment of the biosphere supports a particular mix of microorganisms called a microbiome. These diverse microbial communities play critical roles in the health of ecosystems and in higher organisms, including humans. Disruption or translocation of microbiomes may cause lethal infections, contaminate food and drug supplies, and adversely impact industrial activities. Microbiome detection and molecular characterization have emerged as priorities in many fields. Available methods cannot quickly and efficiently extract rare microorganisms in real specimens. Therefore, microbial detection and analysis require long incubation periods or the use of technically challenging molecular biotechnologies. These strategies are impractical in situations requiring immediate intervention. The intrinsic electric and dielectric properties of microbes permit their isolation by the phenomenon of dielectrophoresis in microfluidic devices. These microsystems have the potential to enhance microbial analysis but are plagued by low processing rates and the inability to interface with biological specimens containing high levels of interfering cells and debris. In this study, a method was created to discriminate between target microbes and undesired cells on the basis of their differential susceptibility to permeabilizing agents that altered cell dielectrophoretic responses. Fabrication techniques were developed to manufacture high-aspect ratio microfluidic channels that allowed the physical forces of gravity, diffusion and dielectrophoresis to be exploited to control cell positions over microscale distances normal to a Poiseuille flow gradient. Because the positioning effects were exploited in only one dimension, the other two dimensions of the channels could be scaled up to create large channel cross-sectional areas that supported rapid specimen processing rates while maintaining high separation efficiencies expected for the microscale effects. These strategies were applied in various ways to isolate microbes from whole blood, platelets, stool, saliva, and skin specimens. The dielectrophoretic extraction of microbes enabled by this approach was used to enable electrical impedance detection of ~100 bacteria in less than five hours. As a result, important technological barriers that have limited the applicability of dielectrophoresis in clinical and industrial settings were overcome by increasing throughput and addressing sample preparation requirements. These proof-of-concept data demonstrate the potential for accelerating microbial isolation and detection in diagnostics, screening, and microbiome research. / text

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Syphilis is a disease caused by the bacterium Treponema pallidum subsp. pallidum, which is generally transmitted through sexual contact, or vertically from a mother to her fetus. Syphilis is effectively treated with penicillin yet remains prevalent worldwide, due in part to the shortfalls of current diagnostic tests. Traditional serological testing algorithms screen with diagnostic tests specific for non-treponemal antibodies followed by subsequent screening of reactive samples for treponeme-specific antibodies. Limitations exist with both the sensitivity and specificity of non-treponemal and treponemal tests. Specific enzyme immunoassays, chemiluminescence assays and rapid point-of-care tests have been developed that contain the T. pallidum proteins TpN15 (Tp0171), TpN17 (Tp0435), TpN47 (Tp0574), and/or TpN44 (Tp0768; TmpA). These tests have also been shown to have suboptimal sensitivities, highlighting the need for identification of novel syphilis diagnostic candidates. In this study, soluble recombinant versions of two previously identified diagnostic candidates, Tp0326 and Tp0453, as well as a novel Tp0453-Tp0326 chimera were produced. The sensitivity of these recombinant proteins in enzyme-linked immunosorbant assays (ELISA) for diagnosis of syphilis was determined by screening characterized serum samples from primary, secondary, and latent stages of infection (n=169). The specificity was determined by screening uninfected individuals (n=13), false positives identified via the standard testing algorithm (n=19), and potentially cross-reactive infections caused by Leptospira, B. burgdorferi, H. pylori, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, and cytomegalovirus (n=38). The sensitivities for Tp0326, Tp0453, and the Tp0453-Tp0326 chimera were found to be 86%, 98% and 98%, respectively. The specificities for Tp0326, Tp0453, and the Tp0453-Tp0326 chimera were found to be 99%, 100% and 99%, respectively. These findings suggest that Tp0453 and the Tp0453-Tp0326 chimera show promise as novel syphilis-specific diagnostic candidates for accurate detection of all stages of infection and for future development into numerous diagnostic test formats including enzyme immunoassays, chemiluminescence assays, and rapid point-of-care tests. / Graduate

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Ability to diagnose cancer before it progresses into advanced stages is highly desirable for the best treatment outcome. A sensitive test to analyze complex samples for specific cancer biomarkers would provide with important prognostic information and help to select the best treatment regimen. A highly robust, ultra sensitive and cost-effective electronic chip platform was used to detect nucleic acid biomarkers in heterogeneous biological samples without any amplification or purification. Chronic myelogenous leukemia (CML) was chosen as a model disease due to its hallmark genetic abnormality. This disease state therefore has an ideal market to test the detection of the fusion transcripts in complex samples, such as blood. It was shown that the CML-related fusion can be detected from unpurified cell lysates and as low as 10 cells were needed for detection. Finally, patient samples were analyzed using the assay and the fusion transcripts were accurately identified in all of them.

Ability to diagnose cancer before it progresses into advanced stages is highly desirable for the best treatment outcome. A sensitive test to analyze complex samples for specific cancer biomarkers would provide with important prognostic information and help to select the best treatment regimen. A highly robust, ultra sensitive and cost-effective electronic chip platform was used to detect nucleic acid biomarkers in heterogeneous biological samples without any amplification or purification. Chronic myelogenous leukemia (CML) was chosen as a model disease due to its hallmark genetic abnormality. This disease state therefore has an ideal market to test the detection of the fusion transcripts in complex samples, such as blood. It was shown that the CML-related fusion can be detected from unpurified cell lysates and as low as 10 cells were needed for detection. Finally, patient samples were analyzed using the assay and the fusion transcripts were accurately identified in all of them.