Grantee Research Project Results
2024 Progress Report: Open-Source Droplet Digital PCR (ddPCR) System for the Rapid and Accurate Detection of Bacteria from Environmental Water Samples
EPA Grant Number: SU840868Title: Open-Source Droplet Digital PCR (ddPCR) System for the Rapid and Accurate Detection of Bacteria from Environmental Water Samples
Investigators: Li, Yiyan , Chatterley, Christie
Institution: Fort Lewis College
EPA Project Officer: Cunniff, Sydney
Phase: I
Project Period: January 1, 2024 through April 25, 2025
Project Period Covered by this Report: January 1, 2024 through December 31,2024
Project Amount: $75,000
RFA: 20th Annual P3 Awards: A National Student Design Competition Focusing on People, Prosperity and the Planet Request for Applications (RFA) (2023) RFA Text | Recipients Lists
Research Category: Urban Air Toxics , Heavy Metal Contamination of Soil/Water , P3 Awards , P3 Challenge Area - Safe and Sustainable Water Resources
Objective:
The overall goal of this project is to develop and validate an open-source Droplet Digital Polymerase Chain Reaction (ddPCR) system to rapidly detect specific waterborne bacteria from environmental water samples. To achieve this goal, the following objectives are proposed: (1) develop the ddPCR assays to detect bacteria from environmental water samples; (2) develop a new sampling and filtering procedure to boost the bacteria recovery rate from environmental water samples; (3) develop an integrated and open-source ddPCR system for field workers and environmental engineers; (4) collaborate with environmental non-profit organizations, high schools, and Native American tribes in the four-corners area on community outreach regarding the importance of water resource protection and the technologies that support environmental conservation.
Progress Summary:
Our team has made substantial progress across multiple technical domains critical for developing an open-source, low-cost ddPCR system. In collaboration with the Mountain Studies Institute, we established standardized protocols for environmental water sampling from Colorado's Animas River, achieving 34-66% bacterial recovery rates using InnovaPrep™ concentration technology. The developed fluorescence detection system demonstrates precise measurement capabilities, utilizing a 473nm laser path with calcium fluoride optics and a 545nm-tuned PMT detector, validated with 10-106μm fluorescent particles showing <0% error at low concentrations. Significant advancements include the creation of a custom neural network (7 convolutional layers, TensorFlow implementation) that classifies optical signals with 80/20 train/validation split accuracy. Parallel development of our open-source thermocycler features a 56-well aluminum block with four Peltier modules, achieving precise thermal control through a Raspberry Pi interface and VNH5019 current drivers. While initial challenges with droplet generation oil were resolved, current efforts focus on optimizing signal quantification for bacterial detection. With all core systems now operational, we are preparing for final environmental testing this summer. This work represents pioneering efforts to create the first accessible ddPCR platform for sparse DNA target analysis, with publication of results anticipated following completion of validation studies. The integrated system shows particular promise for field-deployable water quality monitoring applications.
Future Activities:
The team has successfully developed the optical detector, PCR machine, and ddPCR protocol for this project. Encouraging results have emerged from these advancements, and the system is now prepared for validation using environmental water and bacterial samples. The primary focus is on enhancing droplet quality and collecting data to confirm the accuracy of the ddPCR system. After several years of development, the ddPCR system is nearly ready for real-time environmental water monitoring.
Supplemental Keywords:
ddPCR, optics, filtration, waterborne bacteria detectionProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.