(2dr) A Split Enzyme-Based Self Amplification System for Ultrasensitive Detection of Proteins and Small Molecules at the Point of Care

My research interests involve the development of novel technologies for disease detection that will expand access to healthcare in under-served populations. Specifically, I plan to bridge the gap between new methods in synthetic biology for nucleic acid, protein, and small molecule biosensing and clinical implementation of diagnostic devices incorporating those methods. My academic and research experiences have provided an excellent background for obtaining this goal.

My graduate training under the mentorship of Dr. Rebecca Richards-Kortum focused my research interests on the development of point-of-care technologies to detect maternal and neonatal bacterial infections in low-resource settings. I developed The LeukoScope, a portable device for measuring WBC and neutrophil counts at the point of care. I designed a one-step blood drop sample processing cartridge and a small fluorescence microscopy-based measurement device; additionally, I developed software for device control, sample image processing and analysis, and user interface and performed two separate clinical trials to assess device accuracy, including a 300-patient trial in Blantyre, Malawi. I was first author on an article describing The LeukoScope’s design and initial clinical testing performed in Houston, TX and on a second paper describing the clinical validation performed in a low-resource setting in Blantyre, Malawi. I also developed and optimized lateral flow strips for the semi-quantitative detection of two protein biomarkers of neonatal and maternal sepsis; I will be co-first author on a forthcoming paper describing the design and pilot clinical testing of these test strips.

For my postdoctoral training with Dr. Keith Tyo, I am developing novel pathogen detection techniques by introducing methods of protein engineering and in vitro protein signaling network design for ultrasensitive biomarker sensing, bridging the gap between the sensitivity of nucleic acid diagnostic tests (e.g., PCR) and the speed of point-of-care antigen tests (e.g., lateral flow assays). The detection assay leverages split enzyme engineering and a novel autocatalytic feedback loop made up of engineered protein components with cooperative protein-protein interactions that results in rapid, bistable, ultrasensitive in vitro sensing of antigen biomarkers of disease, a diagnostic development that has yet to be realized in point-of-care testing. This assay would bypass many of the time and infrastructure barriers preventing the implementation of molecular diagnostics in low-resource settings while providing ultrasensitive detection of protein, small molecule, and nucleic acid biomarkers of disease in a simple to measure fluorometric output, expanding access to potentially life-saving disease detection worldwide. I will be first author on a paper currently in preparation describing the in vitro sensing of analytes via split adenylate cyclase with fluorescence readout; I will also be first author on a planned paper describing the autocatalytic feedback loop and bistable switch-like behavior of the novel in vitro protein signaling network. My postdoctoral work and results to date are described in detail below.

As an independent investigator, I plan to build a research program at the cross-section of my previous and current work: the engineering of novel biosensing pathways via protein engineering that can be implemented in vitro and the development and initial clinical translation of these pathways into diagnostics for use at the point of care. Specifically, I plan to build on my postdoctoral work developing novel assays via split enzyme and protein engineering for analyte detection. I plan to optimize the split adenylate assay I am currently developing for deployment into a point-of-care settings via component protein lyophilization and the development of instrumentation for field-ready fluorescence measurement. I will leverage my current network of global health clinicians from my PhD and the Northwestern Institute for Global Health to perform pilot studies on assay accuracy for both Hepatitis C Core Antigen and p24 detection for HIV screening. I will also expand my research into several new areas for novel antigen detection, including the incorporation of other detection readout modalities stimulated by upstream enzyme activation more suited for point-of-care implementation than fluorescence measurement, including colorimetric (e.g., β galactosidase activated by cAMP accumulation) and electrical (e.g., glucose dehydrogenase activated by cAMP accumulation) measurement. In all cases, assay development will be carried out in collaborative efforts with protein engineers and global health experts to ensure downstream translation of devices into deployable diagnostics.

Teaching Interests:

As a teacher, I strive to empower students to develop both the technical and non-technical skills that are vital to their success as chemical and biological engineers. In addition to course technical content, learning objectives center on how to synthesize ideas across scales, how to critically consider and analyze problems, how to clearly communicate ideas, roadblocks, and innovations, and how to work well within teams. To achieve these learning goals with my students, my approach to teaching is focused on maximizing student engagement within the classroom. While lecturing time can be necessary to communicate and explain new material, I minimize lecture time in my classes to incorporate teaching methods that involve more student participation, allowing students to actively engage with course material, rather than solely absorbing it through passive notetaking and reading. Additionally, I believe it is vital for engineering students, as both scientists and engineers, to understand the impact of their work. In the Biotechnology and Global Health course that I designed, the second half of the course is largely driven by student interests. Each student group proposes an unmet need in global health and new technology advancement in the field to drive the discussion portion of the class on their assigned day. This approach gives students the opportunity to shape the course to their interests and inspires more students to engage critically with issues within the biological engineering and global health fields. Finally, I foster an equitable learning environment in my classroom, ensuring that all students can participate and engage with material throughout the course. At the beginning of each term, I anonymously survey students to assess background knowledge and learning preferences to ensure I am appropriately teaching to the students in my classroom. This survey, along with an emphasis on student engagement in office hours and midterm evaluations, allows me to personalize my teaching to the needs of students with diverse educational backgrounds. Overall, it is my goal for all students, regardless of past academic experiences, to have the opportunity for success in my courses. As I move into my career as an independent researcher and faculty member, I am enthusiastic to engage in student learning through both teaching courses and research-based learning experiences for students. I look forward to continuing to grow as a classroom educator throughout my career as I engage with students, their learning goals, and their feedback for me as an educator.

Detailed Description of Postdoctoral Research (Abstract):

Rapid, inexpensive detection of biomarkers at the point of care is vital for many clinical purposes. However, limitations in current detection platforms have prevented the sensitive detection of many protein and small molecule biomarkers; specifically, there has been a lack of innovation for methods to amplify signal from the detection of low concentration proteins and small molecules at the point of care. Biology, on the other hand, has evolved intricate mechanisms for rapidly amplifying protein signals in vivo. Towards the goal of developing rapid, ultrasensitive diagnostics, we have developed a novel sensing platform that leverages split enzyme engineering and a novel autocatalytic feedback loop made up of engineered protein components that results in rapid, bistable, ultrasensitive in vitro sensing of biomarkers. The detection platform uses split adenylate cyclase to detect the presence of an analyte of interest. The analyte mediates interactions of the two enzyme component halves fused to binding domains (i.e., a sandwich assay in solution), resulting in cAMP accumulation. Proof of concept demonstration has been performed by detecting rapamycin, a small molecule, via the well characterized FKBP12-rapamycin-FRB complex. We engineered protein constructs with split halves of adenylate cyclase (nAC and cAC) fused to rapamycin binding domains (FRB and FKBP12) and showed that the system transitions from an OFF state to an ON state between rapamycin concentrations of 10 and 100nM. Final detection of cAMP is achieved via a FRET-based cAMP biosensor that shifts fluorescence output in the presence of cAMP. Additionally, we have designed a feed-forward system composed of split adenylate cyclase fused to cAMP receptor protein (CRP) and small DNA fragments that act as binding scaffolds that further reconstitutes split adenylate cycles in the presence of cAMP, driving further cAMP generation. Computational modeling of the full system demonstrates ultrasensitive, bistable sensing of biomarkers that is tunable over several orders of magnitude. This system will be broadly applicable for protein and small molecule detection and could be used to detect a wide range of clinical target analytes with known binding domains.