Author ORCID Identifier

https://orcid.org/0000-0001-6448-3505

Date Available

4-30-2025

Year of Publication

2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Engineering

Department/School/Program

Mechanical Engineering

Faculty

Scott M. Berry

Faculty

Jonathan F. Wenk

Abstract

Environmental Surveillance (ES) has emerged as a powerful tool to monitor the emergence and persistence of a pathogen within a facility or community using wastewater/water analysis. Over the past four years, implementation of ES has experienced an explosive acceleration, driven by efforts to monitor the spread of SARS-CoV-2, which has made ES a routinely used public health tool in many locations. We can now use ES to measure the emergence/presence/abundance of different viruses (e.g., SARS-CoV-2, influenza, mpox), bacteria (e.g., E. coli, V. cholerae, antibiotic-resistant strains), fungi (e.g., C. auris), and other biological markers indicative of infectious diseases. However, its broad implementation, especially in low-resource settings, is hindered by the need for specialized equipment, high consumable costs, complex workflows, and highly trained personnel. The goal of this dissertation is to find solutions for overcoming the technological barriers of ES. Our previous efforts led to a new method, termed exclusion-based sample preparation (ESP), that substantially simplifies nucleic acid extraction step.

In the first step, we developed an automated workflow based on a low-cost benchtop liquid handling robot. Our automated workflow takes 40 min from raw wastewater to purified RNA, which is several times faster than conventional methods. We believe that this technology is especially valuable in regions with basic infrastructure (e.g., electricity) but limited resources for rapid sample transport or centralized laboratories. In the next step, we focused on the concentration step and compared the performance of four inexpensive and simple concentration methods to detect SARS-CoV-2 in wastewater samples. All methods can be performed with basic laboratory equipment and minimal electricity usage which enables further application of ES in remote areas and low resource settings. Moreover, we present a novel Concentration-Extraction-Identification Device (CEID), a point-of-use platform that integrates nucleic acid concentration, extraction, and detection into an affordable and user-friendly chip. The CEID employs immiscible filtration assisted by surface tension (IFAST) for nucleic acid extraction and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for pathogen detection. This technology offers in-field sample processing, eliminating the need for complex centralized labs and mitigating challenges related to sample transportation.

Most of the technologies introduced in this dissertation serve as flexible platform technologies that can be readily adapted for a wide range of applications. We present the application of ESP in isolating specific subtype of extracellular vesicles (EVs). Studying specific subpopulations of cancer-derived EVs could help reveal their role in cancer progression. ESP is used for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line. Our results indicate that Redox EVs promote the growth of glioblastoma cells, and cause injury to normal astrocytes.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.89

Funding Information

This study was supported by the following:

  • National Institutes of Health (NIH) grants 1U01DA053903-01 and P30 ES026529, 2021-2023
  • Centers for Disease Control and Prevention (CDC) contract BAA 75D301-20-R-68024, 2021-2022
  • National Science Foundation (NSF) grants 2154934 and 2412446, 2022-2025
  • Lighthouse Beacon Foundation Graduate Scholarship, 2024-2025

Additional Files
RightsLink Printable License_LAMP Figure.pdf (157 kB)
LAMP Figure License
RightsLink Printable License - Redox EVs.pdf (155 kB)
Redox EVs License
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