NANOTECHNOLOGY AND BIOSENSORS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0229641
• Project No.: NYC-123800
• Multistate No.: NC-_old1194
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2016

Project Director
Walter, MI, F..

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

Performing Department
Biological & Environmental Engineering

Non Technical Summary
The proposed research addresses the need of ensuring a safe food supply. The contamination of our foods with pathogens and toxins causes a real threat to the safety of the consumers and increases health care costs with multi-billion dollars associated to the treatment of foodborne illnesses. The CDC estimates that 1 in 6 people in the US experience foodborne illness per year resulting in 128,000 hospitalizations and 3000 annual deaths. The most troubling fact may be that the causative agents are not identified for the majority of these cases. The detection of foodborne pathogens and toxins is complex, time consuming and costly. As much as there is a need for point-of-care diagnostics in our health care to better serve patients and reduce health care costs; there is an urgent need for point-of-food diagnostics. These would enable rapid and inexpensive identification of contamination in foods before it is distributed; enable rapid identification in the case of an outbreak and therefore protect wide-spread foodborne illnesses from occurring. Public funding is essential so that emphasis can be put toward the development of inexpensive and simple point-of-food diagnostics to protect children, the elderly and immune-compromised who are the first to suffer severe illness from foodborne pathogens. Knowledge gained by nanotechnology research needs to be translated into prototypes of devices for real-world problems, so that subsequently industrial collaborators can develop commercially viable products, create jobs and protect consumers.

Animal Health Component

0%

Research Effort Categories

Basic

30%

Applied

30%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	5010	2020	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5010 - Food;

Field Of Science
2020 - Engineering;

Keywords

biosensors

nanotechnology

foodborne pathogens

detection

rapid diagnostics

on-site

in-field

food matrix

food safety

Goals / Objectives
Develop devices and systems incorporating microfabrication and nanotechnology

Project Methods
For sub-objective 1, an existing electrochemical microfluidic biosensor with embedded sample preparation of the Baeumner group will be transformed to enable electrochemiluminescence (ECL) detection. The biosensor is made from plastic to enable simple and rapid prototyping as well as inexpensive mass production in the future. Here, a new electrochemical 3-electrode set up will be developed driving the ECL reaction. The signals will be monitored by a simple photodiode. A small black housing will be designed to ensure portability. The molds required for microchannel designs and the electrodes will be fabricated using the Cornell University clean room facilities. Actual prototype fabrication can be carried out in a normal biochemical wet lab. The electrode design, microfluidic channels and the photodiode set up will be optimized to enable highly sensitive ECL detection using ruthenium bipyridyl complexes. For sub-objective 2, dendrimers tagged with the ruthenium complexes will be covalently coupled to anti-E. coli and anti-C. parvum antibodies. The organisms will be isolated from the food sample using immunomagnetic separation. The magnetic beads will be captured within the microfluidic device and cells will be quantified upon being bound by the reporter antibodies via the ECL reaction. Assay conditions will be optimized. For sub-objective 3, sensitive photopaper will be investigated as alternative to the photodiode. Photopaper will enable semi-quantitative detection of the ECL reaction that emits light at 620 nm. Assay conditions for signal development on the photopaper will be studied.

Progress 10/01/12 to 09/30/16

Outputs
Target Audience:The target audience are initially researchers interested in the development of sensors for pathogens in food and agricultural related applications. Ultimately, users are food production plants, farmers, authorities and health officials. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several graduate students were enabled to work on the project and learn about biosensor development, nanotechnology, independent research and issues relevant to pathogens and diseases in agricultural and food products. How have the results been disseminated to communities of interest?Through two publications and presentations at international conferences relevant findings have been distributed. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue efforts toward all three objectives. We will study improvements in electrochemiluminescence chemistry, nanofiber recognition and the specific detection of bacteria cells. We also plan on improving fabrication technology for the miniaturization of our microfluidic systems.

Impacts
What was accomplished under these goals? Objective 1: Investigations were carried out toward the development of a miniaturized dual detection system for the simultaneous detection of at least two food-borne pathogens. Various reports exist that use chemically modified electrochemiluminescence (ECL) markers to accomplish dual or multi-analyte detection. However, the molecules are not commercially available and require complex syntheses. Here, we therefore investigated the use of two commercially available luminescence molecules (a ruthenium complex and luminol). Specific standard curves for each analyte were prepared reaching limits of detection at 35 and 26 nM, respectively. In a dual detection approach, the simultaneous detection of the ECL components within a miniaturized measurement cell, an optical fiber and PIN detection system, limits of detection were reached that were at 0.7 and 1.7 microM. While higher than those obtained for the separate optimized standard curves, these values are excellent in comparison to other multi-analyte ECL approaches and will therefore be continued in further investigations. Objective 2 and 3: We also study the use of nanofibers for sample preparation purposes. Here, we investigate the isolation of E. coli cells from samples for concentration and subsequent detection. Preliminary data show a high isolation capability through electrostatic interactions resulting in concentration factors of > 10.000. Furthermore, we investigate the specific isolation of E. coli and subsequent detection using antibody-tagged nanofibers.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Reinholt, S., Baeumner, A.J. Microfluidic Nucleid Acid Purification Angewandte Chemie, International Edition (available online, DOI: 10.1002/anie.201309580) 2014
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Matlock-Colangelo, L., Baeumner, A.J. Biologically Inspired Nanofibers for Use in Translational Bioanalytical Systems Annu. Rev. Anal. Chem. 2014. 7:2342 (2014)

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience are initially researchers interested in the development of sensors for pathogens in food and agricultural related applications. Ultimately, users are food production plants, farmers, authorities and health officials. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The research was presented at international conferences such as the 11th BBMEC, Germany, the Gordon Conference on Nanoscale Science and Engineering in Agriculture and Food Systems. Also, Findings were published in a peer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue efforts toward all three objectives. We will study improvements in electrochemiluminescence chemistry, nanofiber recognition and the specific detection of bacteria cells. We also plan on improving fabrication technology for the miniaturization of our microfluidic systems.

Impacts
What was accomplished under these goals? Objective 1: Investigations were carried out toward the development of a miniaturized dual detection system for the simultaneous detection of at least two food-borne pathogens. Various reports exist that use chemically modified electrochemiluminescence (ECL) markers to accomplish dual or multi-analyte detection. However, the molecules are not commercially available and require complex syntheses. Here, we therefore investigated the use of two commercially available luminescence molecules (a ruthenium complex and luminol). Previously, we had optimized the ECL reactions both for the individual markers as well as for combined ones. Current effort focused on the development of liposomes for signal amplification entrapping the ruthenium complexes. Liposomes were synthesized, optimized and investigated for stability. Initial tests were done for the detection of RNA sequences of Cryptosporidium parvum. Further investigations are needed in order to compare the ECL liposomes to other ECL-amplification systems, as well as generating luminol-entrapping liposomes. Microfabrication platforms were investigated for the realization of a dual-detection system within microfluidic channels. Hot embossing using glass, steel and SU-8 templates were considered. Further optimization of the fabrication process is necessary. Objective 2 and 3: We also study the use of nanofibers for sample preparation purposes. Here, we investigate the isolation of E. coli cells from samples for concentration and subsequent detection. Here, we were able to demonstrate the E. coli cells can be concentrated using non-specific interactions based on electrostatic attraction, as well as antibody-derived specific interaction.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Matlock-Colangelo, L., Coon, Barbara, Pitner, C.L:, Frey, M.W., Baeumner, A.J. Funcationalized electrospun poly(vinyl alcohol) nanofibers for on-chip concentration of E. coli cells Analytical and Bioanalytical Chemistry, DOI 10.1007/s00216-015-9112-5

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Research scientists focusing on the development of bioanalytical methods for food safety and related analytical areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several graduate students were enabled to work related to the project and learn about biosensor development, nanotechnology, independent research and issues relevant to pathogens and diseases in agricultural and food products. How have the results been disseminated to communities of interest? Yes. Through two publications and presentations at international conferences relevant findings have been distributed. What do you plan to do during the next reporting period to accomplish the goals? We plan to continue efforts toward all three objectives. We will study improvements in electrochemiluminescence chemistry, nanofiber recognition and the specific detection of bacteria cells. We also plan on improving fabrication technology for the miniaturization of our microfluidic systems.

Impacts
What was accomplished under these goals? Objective 1: Investigations were carried out toward the development of a miniaturized dual detection system for the simultaneous detection of at least two food-borne pathogens. Various reports exist that use chemically modified electrochemiluminescence (ECL) markers to accomplish dual or multi-analyte detection. However, the molecules are not commercially available and require complex syntheses. Here, we therefore investigated the use of two commercially available luminescence molecules (a ruthenium complex and luminol). Specific standard curves for each analyte were prepared reaching limits of detection at 35 and 26 nM, respectively. In a dual detection approach, the simultaneous detection of the ECL components within a miniaturized measurement cell, an optical fiber and PIN detection system, limits of detection were reached that were at 0.7 and 1.7 microM. While higher than those obtained for the separate optimized standard curves, these values are excellent in comparison to other multi-analyte ECL approaches and will therefore be continued in further investigations. Objective 2 and 3: We also study the use of nanofibers for sample preparation purposes. Here, we investigate the isolation of E. coli cells from samples for concentration and subsequent detection. Preliminary data show a high isolation capability through electrostatic interactions resulting in concentration factors of > 10.000. Furthermore, we investigate the specific isolation of E. coli and subsequent detection using antibody-tagged nanofibers.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Reinholt, S., Baeumner, A.J. Microfluidic Nucleid Acid Purification Angewandte Chemie, International Edition (available online, DOI: 10.1002/anie.201309580) 2014
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Matlock-Colangelo, L., Baeumner, A.J. Biologically Inspired Nanofibers for Use in Translational Bioanalytical Systems Annu. Rev. Anal. Chem. 2014. 7:2342 (2014)

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? The developed microfluidic set-up will be used to integrate an electrochemiluminescence detection into the chip and detect nucleic acid sequences from pathogens.

Impacts
What was accomplished under these goals? The main objective of the proposed research is the development of biosensors that can rapidly detect foodborne pathogens with limits of detection required to identify on-site contamination of food products. Based on previous biosensors that have demonstrated applicability to food-related detection due to included sample preparation, focus here will be on a novel sensing technology enabling extremely high sensitivity coupled with little hardware requirements and simple user handling. In the first year of the project we focused on developing a new platform of a microfluidic sensor that will facilitate the integration of the highly sensitive detection strategy of electrochemiluminescence. Here, focus was on the fabrication of the microfluidic device, flow rate requirements for a superparamagnetic bead based assay approach, buffer compositions and overall analytical conditions. Initial experiments were done using fluorescently labeled liposomes as well as electrochemically active liposomes. Conditions were established that allowed the fabrication of a stable 3-electrode system with an on-chip reference electrode. Also, linear-flow velocities were determined that yielded in efficient superparamagnetic bead capture as well as avoiding non-specific settling of the beads in the channel system.

Publications