NANOTECHNOLOGY AND BIOSENSORS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010505
• Multistate No.: NC-_old1194
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Project Director
Wu, MI.

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Biological & Environmental Engineering

Non Technical Summary
Water resources are threatened by the increasing occurrence of harmful algal blooms (HABs) when the sudden growth of certain species of cyanobacteria leads to intense blooms appearing on the water surface.The toxins associated with HABs deplete drinking water resource, endanger fish industries, and sustainability of all life forms in aquatic ecosystems.Just recently, Florida declared state of emergency because HABs have threatened drinking water resources in St Lucie and Martin Counties (http://www.flgov.com/2016/06/29/gov-scott-declares-state-of-emergency-in-st-lucie-and-martin-counties-following-algal-blooms/). Early 2016, Chilean salmon farmers were devastated by losses reported at 800 million in revenue due to HABs, which has had a lasting impact on the world fish market (March, 2016, http://www.reuters.com/article/us-chile-salmon-idUSKCN0WC0A2). Other notable HAB crises include the complete drinking water shutdown in Wuxi, China along Lake Taihuand Toledo, Ohio along Lake Erie (https://www.washingtonpost.com/news/capital-weather-gang/wp/2015/11/12/this-years-disgusting-green-algal-bloom-in-lake-erie-was-the-most-severe-on-record/).Despite the urgency of the problem, technologies used in HABs research are mostly large scale. They are typically large scale instrumentation, such as mass spectrometer, which are not suitable for field use. As such, it is difficult to predict the onset of HABs. The goal of this project is to develop a high throughput, smart phone based biosensor for detecting low level microcystins and/or microcystin-producing strains that are suitable for both field and lab studies. Microcystin is an broad spectrum enzyme that is responsible for producing toxins in cyanobacteria. The eventual goal of the proposed work is to establish a large data base and theoretical model for prediction and prevention of fresh water HABs.The proposed work support two objectives of the Multistate Research Project of Nanotechnology and Biosensors, which are:Objective 3: Develop devices and systems incorporating microfabrication and nanotechnology.Objective 4: Multidisciplinary experimentation and modeling methods at different scales will be used to fulfill the objectivesImpact: Together, these studies will advance both the state of art technology in HAB research as well as our basic understanding of how multiple environmental factors synergistically impact toxin production of cynobacteria.In the long term, the proposed project will serve as a stepping-stone for introducing biosensors into mechanistic understanding of the cause for HABs. This will allow general public (e. g. fish farmers, residents along the water way) to take control, and measure the toxins in the water, leading towards active HAB management. We note that although the proposed study is limited to fresh water and M. aeruginosa (a specific type of algae), we anticipate that tools and knowledge generated in this proposal will be adopted- by/inform studies focused on other photosynthetic microbes and marine ecosystems. These studies are timely because of the urgent need to protect water resources and maintain sustainable aquatic ecosystems.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
135	0210	1000	100%

Knowledge Area
135 - Aquatic and Terrestrial Wildlife;

Subject Of Investigation
0210 - Water resources;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

harmful algal blooms

biosensors

cyanobacteria

microfluidics

climate change

water safety

Goals / Objectives
Develop new technologies for characterizing fundamental nanoscale processes

Project Methods
Efforts: The short term goal of this project is to develop a high throughput, smart phone based biosensor for detecting low level microcystins and/or microcystin-producing strains that are suitable for both field and lab studies. The eventual goal of the proposed work is to establish a large data base and theoretical model for prediction and prevention of fresh water HABs.The proposed work support two objectives of the Multistate Research Project of Nanotechnology and Biosensors, which are:Objective 3: Develop devices and systems incorporating microfabrication and nanotechnologyObjective 4: Multidisciplinary experimentation and modeling methods at different scales will be used to fulfill the objectivesWater resource issues are involved in agricultural and food development due to the product and waste disposal to water systems. Development of sensing technology based on molecular phenomena is the core subject in the Multistate Research Project. Our collaboration with field specialists in New York and North Carolina will meet the experimentation at different states.Under Objective 3, we will develop a smart phone based biosensor to detect toxin, microcystin, in fresh water samples. The bio-recognition element will be an oligonucleotide primer responsible for mRNA of microcystin synthetase (mcyA or mcyB ) along with a fluorescent tag. We will use the standard fluorescence in situ hybridization (FISH) method to quantify the microcystin synthetase activites within the cyanobacteria. We expect a high detection sensitivity since our analyte is inside a cell in contrast to methods for detecting extracellular toxin.For hybridization and washing steps, we will utilize a newly developed microfluidic device (Kim et al., Lab on a Chip, 2015) for establishing a controlled environment for the samples as well as providing an imaging platform compatible with smart phone camera. This biosensor will be validated using a standard HPLC system.Under Objective 4, we will apply the biosensor to detect toxin in water samples from field in collaboration with Dr. Hairston in New York state (Honeoye Lake), Dr. Paerls in North Carolina state (Carolina Rivers in North Carolina and also Lake Taihu in China).In parallel to this, we will explore roles of microenvironment (nutrient gradients, temperature, light) in toxin kinetics using a model microcystis strain. These in vitro data along with data from field will be integrated to establish a data driven model for prediction and prevention of HABs. Evaluations We will use traditional sensing technology such as Mass spectrometer, or immuno assay to validate the measurements from newly developed biosensor.MilestonesYear 1 An understanding of enzymes responsible for toxin productionYear 2 Develop the biosnsor that detects toxins associated with HABs.Year 3 Validate and evaluate the biosensor, integrate analysis and readoutwith iphone and test it in fields.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Local community that cares about water resources, this includes New York State HABs consortium and citizen scientists in the Finger Lakes region. Scientists around the world who works on the problem of harmful algal bloom. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunity to train the following students in microfluidics, imaging, mathematical modeling and computation. (1) Graduate student, Fengchen Liu, who started graduate program with our group in Jan. 2018. (2) Postdoctoral researcher, Nicole Wagner, who was trained in basic microfluidics and imaging. Nicole came from a biological science background. (3) Visiting Graduate student from Cambridge University, Kasia Warburton, for working with us on the project over the summer of 2018. (4) Undergraduate student, Muhammad Moughal, for working on the project during the entire funding period. How have the results been disseminated to communities of interest?Regionally, we disseminated our research results to the New York State Harmful Algal Bloom Consortium. My students presented posters at the Cornell Biological and Environmental Engineering department annual symposium, Cornell Nanofabrication Facility annual meeting. Nationally, my student presented our results at the American Society of Agricultural and Biological Engineers (ASABE) meeting in Michigan, in July, 2018. Internationally, Wu gave a public lecture (part of the presentation involves research from this project) at Cambridge University in England in December, 2018. What do you plan to do during the next reporting period to accomplish the goals?Our ultimate goal for this multi-state grant is to use nanotechnology to gain an understanding of how nutrient conditions impact the outbreak of harmful algal blooms, and its relation to toxin production. In the next funding period, we will focus on the development of toxin detection technology. This will lay a solid foundation for the work proposed to do in the remaining two year of this multistate grant, which is the relation between the nutrient condition and the toxin production.

Impacts
What was accomplished under these goals? In this funding period, we focused on the roles of environmental parameters, Nitrogen (N) and phosphrous (P), on the growth of cyanobacteria. For doing that, we developed a microfluidic platform that can provide dual gradients N and P. This allows us to investigate not only the roles of each individual nutrients on cell growth but also synergistic roles of N and P in cell growth. This device has been calibrated, and tested using a model cyanobacteria, Chlamydomonas reinhardtii. We also carried out experiments learning about an important cyanobacteria, microcystis aeruginosa. We have carried out experiments on how P and N influences its growth using a conventional 96 well plate. This is to set the stage for our next year's work.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Fangchen Liu, Beum Jun Kim, Nicole D. Wagner, Jason Zarate, Beth A. Ahner, S. C. Winans, and Mingming Wu, Microhabitat Platform to Study Harmful Algal Blooms, ASABE 2018 Annual International Meeting. July 29-Aug 01. Detroit, Michigan.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Nicole Wagner, Jane Kwon, Beum Jun Kim, Fangchen Liu, Jason Zarate, Beth Ahner, Stephen Winans and Mingming Wu, Cell to cell communication in halmful algal blooms, Nanoscale Science and Engineering for Agriculture and Food Systems Gordon Research Conference, June 3-8, 2018, Mount Holyoke College in South Hadley, MA.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Researchers in nanobiotechnogy and biosensors, bioengineers and environmental engineers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project provided opportunities to train two undergraduate students along with a new postdoc in the field of micro- nano- systems engineering. One undergraduate student, Sam Reffsin received Engineering Initiative Research fellowships twice from Cornell. Another undergraduate student, Sebastian gave an excellent talk at the ASABE meeting representing all the people in the project. Both students finished an honor's thesis on the subject (see citation). Starting August 2017, a new postdoc, Nicole Wagner, was hired into the program. Nicole's background is ecology, and this project provided her an opportunity for an interdisciplinary training in both microsystems engineering and basic biology. How have the results been disseminated to communities of interest?We have presented our results at the American Society of Agricultural and Biological Engineers (ASABE) meeting in Spokan Washington. We also have one paper published in peer reviewed journal (See citation part). What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan to spend about 6 months to confirm about the roles of quorum sensing in the formation of cell aggregates, write a paper about this result. In the remaining time, we will then investigate the correlation of cell aggregate formation and toxin production. Our hypothesis is that cell aggregation is correlated with the toxin production.

Impacts
What was accomplished under these goals? In this funding period, we focused on roles of quorum sensing in the production of toxins by algal blooms. More specifically, we used a model cynobacteria, microcystis aeruginosa, and a microfluidic platform to ask the question whether cell-cell communication regulates the formation of algal blooms, or whether cells need to talk to each other to form algal blooms. Towards this front, we have (1) developed a microfluidic platform that is suitable for studying roles of environmental cues in the formation of algal blooms, and subsequently, toxin production. (2) We have evidence that cell-cell communication molecules are present in the supernatant of the cell culture. (3) Cells clusters earlier or at a lower cell concentration (a necessary condition for algal blooms) when synthetic quorum sensing or cell-cell communication molecules are added to the cell culture. (3) Cells are chemotactic to the synthetic quorum sensing molecule, OOHL. These results are still at a preliminary stage, and need to be repeated and verified.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Sebastian Jusuf, Motility analysis of aeruginosa under chemotactic influences, Undergraduate Honor's thesis, Biological and Environmental Engineering, Cornell University, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sebastian Jusuf, Sam Reffsin, Lubna Richter, Beth Ahner, Steve Winans and Mingming Wu, The roles of cell-cell communication in the formation of algal blooms. ASABE conference, Spokane, Washington, July 18, 2017.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Sam Reffsin, ROLE OF QUORUM SENSING IN CELL-CELL COMMUNICATION AND AGGREGATION OF THE CYANOBACTERIA M.AERUGINOSA, Undergraduate Honors Thesis, Biological and Environmental Engineering, Cornell University, 2017.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Matthew R. Gellert, Beum Jun Kim, Samuel E. Reffsin, Sebastian E. Jusuf, Nicole D. Wagner, Stephen C. Winans, and Mingming Wu, Nanobiotechnology for the Environment: Innovative Solutions for the Management of Harmful Algal Blooms, Journal of Agricultural and Food Chemistry, Article ASAP. DOI: 10.1021/acs.jafc.7b04271 (2017).