In spite of the urgent need for better tools, automated, in situ methods for the determination of the presence, abundance and activity of marine microbes are still in the development stages. The Capillary Waveguide Biosensor (CWB) will potentially make possible long-term, in situ, unattended detection of microorganisms and monitoring of microbial processes. This is a renewal of the PI?s EAGER proposal. They were able to complete two of the three tasks originally proposed. With this addition funding they will complete the third task, which was to optimize hybridization protocols and achieve a dynamic range satisfactory for use in real-world studies.
Broader Impacts:
The potential for training students at graduate, undergraduate and high school levels in this project is particularly interesting and exciting, because it will offer students the opportunities to cross-train in microbial ecology and engineering. Also, education and training of minorities and exposure of the general public to science are two other likely benefits of this project. Participation of C-MORE students is great. If molecular techniques are going to be used more frequently in the future, students are going to have to get used to the technology.
In situ methods for unattended collection of real-time information about microbial abundance and population dynamics, biomass production, and gene expression in the marine environment will allow a better understanding of processes mediated by microorganisms and their responses to stresses such as climate change or hypoxia. They can also allow detection and quantification of microbial contaminants which are important to monitor for estuary management, shellfish aquaculture, and water quality. Additionally, the ability to monitor environments that may be only intermittently accessed by ordinary means would be a major advance. This project continued the development of a sensor system, the Capillary Waveguide Biosensor (CWB), for in situ monitoring of microorganisms in the ocean. The CWB employs nucleic acid hybridization for detection and quantification of specific microorganisms in conjunction with a large volume water sample collection system, the Monterey Bay Aquarium Research Institute (MBARI)-Environmental Sample Processor (ESP). The ESP can be deployed for periods of up to three months and programmed to regularly collect water, concentrate microorganisms, extract their nucleic acids, and pass them to sensor modules, such as the CWB, where target microorganisms would be detected. Data would then be stored for remote retrieval or transmitted in real time. The CWB interfaces with the ESP through a daughter microfluidic block platform, the MFB. Over the last two years, design of a temperature microcontroller and electronic and optical data acquisition system were perfected allowing for an efficient merging of the CWB with the MFB. The heart of the CWB is a capillary with an interior coating of capture probe molecules designed to bind targeted RNA or DNA. Coating protocols were developed to ensure that the probe layer was uniform and effective. This allowed for major improvements in the magnitude and uniformity of the fluorescent signal, and guaranteed that the probes remained firmly attached to the capillary interior and could be regenerated for repeated use. Our work to date is essential to making the CWB a reliable platform to work with environmental samples, where concentrations of microorganisms can be variable and the target organism may be only a small proportion of the total microbial numbers. Because nucleic acid concentration ultimately relies both on target organism abundance and on the water filtration capacity of the ESP (which can range from a few hundred mililiters to tens of liters depending on water quality/conditions), optimizing detection capabilities has been a priority. Although we have been unable in this funding period to achieve our ultimate goal of detecting target RNA from natural communities at low concentrations, we have overcome many methodological challenges and are encouraged by our advances. Human activities are dramatically affecting ecosystems at the Earth surface and driving major global environmental changes. A large number of these anthropogenic activities primarily affect coastal waters. Continuing to work on sensor technology improves our ability to evaluate the impacts and predict future consequence of anthropogenic forcing on complex coastal zone systems where the majority of the world’s population reside whose resources must be monitored and protected.
