New tools are enabling unprecedented advances in biology and medicine. This proposal describes an interdisciplinary research program that aims to create, test, and disseminate micro and nanofluidic technology for quantitative biological studies. Specifically, the behavior of individual bacterial cells will be investigated using nanofluidic electrochemical sensors in combination with traditional optical techniques. Initial experiments are focused on detection of quorum sensing (or auto-inducing) molecules which are small electroactive molecules that are excreted by many bacterial cells, including several involved in human diseases. The platform will be used to investigate effects of external stimuli, static heterogeneity, and the role of chemical communication in bacterial populations. This approach will utilize nanofluidic electrochemical sensors that are microfabricated on large silicon substrates. These nanofluidic sensors allow for real-time electrochemical detection of concentrations as low as 100 nM and can be easily integrated with larger fluidic systems. A microfluidic architecture, complete with control valves, will be aligned over the nanofluidic sensors on the silicon substrate to allow transport and trapping of individual bacterial cells near the sensors. The microfluidic architecture will be constructed using a photo-curable transparent polymer (polydimethylsiloxane) via soft lithography. The transparent nature of the microfluidic channels will allow for optical monitoring of the cells. The assembled system will allow the simultaneous real-time electrochemical and optical characterization of individual cells. Of particular interest are the physical and biochemical changes the cells will undergo when exposed to various external factors, such as pH, temperature, buffer concentration, surface modification, and drug molecules. Initial electrochemical testing will be conducted with known commercially available quorum sensing molecules (e.g. autoinducer-2, pyocyanin) and validation of the microfluidic architecture will utilize microscale beads to simulate bacterial cells. In the final stage of this project, bacterial cells (e.g. E. coli, P. aeruginosa) will be transported and trapped next to the nanofluidic sensing elements using a microfluidic platform. The generation of quorum sensing molecules by the cells will be monitored electrochemically while the cellular response to the molecules will be observed optically. The long term objective for this research is to provide an integrated chip-in-a-lab platform for systems biology experiments where researchers will be able to stimulate and monitor hundreds of individual cells simultaneously. Intellectual Merit: Few alternatives exist for monitoring single cells. The proposed work will investigate the feasibility and practicality of electrochemical detection as a technique for studying the behavior of individual cells in conjunction with existing optical techniques in a high-throughput manner. The proposal will also investigate microfluidic systems for handling of individual bacterial cells with diameters of 1-2 micrometers, which remains a difficult technological challenge. This broadly applicable platform will revolutionize the field of systems biology by providing label-free chemical information for potentially thousands of individual cells simultaneously. No such high-throughput electronic sensor technology is currently available. The fundamental questions being investigated will give insight to the behavior and interactions of bacterial cells that can be applied to biotechnology, medicine, and environmental research. Broader Impacts: The interdisciplinary nature of this research will foster collaborations between engineers and biologists and train researchers for emerging dynamic work environments. The proposed technology can be potentially employed for a large variety applications ranging from microbial fuel cells to drug screening to biomedical instrumentation to evolutionary biology. In general, a better fundamental understanding of the electrochemical and micro/nanoscale systems involved in this project will lead to the next generation of tools for researchers in the biosciences. The importance of this project and engineering in general will be disseminated to the public by actively engaging teachers and students ranging from the middle school to undergraduate levels. The PI will visit local inner city school to provide information about engineering career opportunities. Live and virtual laboratory tours will be offered. Hands on training in the laboratory will be provided for high school students and teachers from Boston inner city schools, such as Roxbury Preparatory Charter School. Middle and high school teachers participating in the training program will be partner with the research group to design kits and teaching modules to help educate students in junior high and high school science classes about nanobiotechonolgy. The developed material will initially be assessed at Pope John Paul II Catholic Grade School, a predominantly minority school on Chicago?s southwest side. At the college level, research experiences for undergraduates will be offered in the research group to reinforce engineering concepts learned in the classroom.

Project Report

The outcome of this work is an interdisciplinary research program that creates, tests, and disseminates micro and nanofluidic technology for quantitative biological studies. The program actively engages the public, as well as students from groups that have been traditionally underrepresented in engineering, by teaching them about the tools that are being developed and used to study bacterial behavior. Intellectual Merit: This project investigated the use of nanoscale electrochemical sensors to study the physiology of individual bacterial cells. This approach is particularly challenging as bacterial cells swim rapidly and can escape through constrictions with diameters below 1 micrometer. Several new insights have been garnered during the development and testing of the proposed technology, and new application areas have been ascertained as a result of these findings. One critical advancement was the development of a simple, stable, microscale pseudo-reference electrode for electrochemical measurements. This technology allows continuous monitoring of electrochemical activity for several days in nanoliter fluid volumes that is important for bacterial experiments as well as environmental and medical sensing. Several common bacterial species and quorum sensing molecules were evaluated using this technology. Pseudomonas aeruginosa was selected as the most promising bacterial species to investigate, as it produces and excretes an electroactive quorum sensing molecule, pyocyanin. Changes in the production rate of pyocyanin were examined by exposing the cells to amino acids that are precursor molecules in the metabolic pathway of pyocyanin synthesis. P. aeruginosa was also exposed to different antibiotics to access their effect on pyocyanin production. New approaches were developed for trapping individual cells near the miniaturized sensors. The developed technology can be utilized, as predicted, for high-throughput screening of bacterial species exposed to chemical stimuli, such as target drug compounds. Broader Impacts: As a broadening participation, research initiation grant in engineering, a central focus of the work was the engagement of groups that are historically underrepresented in engineering fields. Several initiatives were employed, as part of this project, to teach middle and high school students and teachers about the importance and challenges of studying bacteria. The PI visited his grade school to educate students about career opportunities in chemical and biological engineering. The PI hosted several high school students each summer for six or more weeks in his laboratory to teach them how to work with bacterial cultures and run electrochemical experiments. The training of two high school teachers from Boston area city schools was particularly successful, with a continuing collaboration established in the science department at Everett High School. The new insights and technology developed during this project are particularly useful for the development of sensors for monitoring bacterial infections. Technical findings from this work have been disseminated via presentations at conferences, publications in peer-reviewed journals, and articles in popular media outlets. To date, 30 presentations have been given at conferences and meetings, 5 papers have been published, and 3 patent applications have been filed. Of particular note, the results of this research were featured in April 2014 issue of The Rotarian magazine, online on the Fast Company magazine website, and in a book entitled "Material Innovation: Product Design" by Andrew Dent and Leslie Sherr.

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Northeastern University
United States
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