Progress 12/15/13 to 12/14/17
Outputs
Target Audience:Regulatory agencies and officials overseeing trade in agricultural and biological materials, and food safety. Agricultural producers and processors. Food retailers. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Extension agents working on food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?-Application of industry standard engineering CAD software (SolidWorks) -Design and application of molecular diagnostic techniques like LAMP. -Design, servicing, maintenance, and application of equipment and techniques including plasma treatment, machine vision, spectroscopy. -Classic microbiological techniques including sterile culture and plate counting. -Collaboration and communication with industry professionals for commercial product manufacture. -Software development including for Android devices, embedded system control, and digital communications between devices. -Experimental design and data analysis. How have the results been disseminated to communities of interest?Results have been communicated to colleagues primarily through presentation at professional meetings and symposia, conference proceedings, and in peer-reviewed articles. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Executive Summary of Achievements in the Last Year of Project For the concluding year of this project, we were able to demonstrate the use of several portable and affordable technologies to rapidly enhance the sensitivity of detection of food-borne pathogenic bacteria in the field and in rudimentary lab settings. These technologies include the use of a fully automated "electroflotation" cartridge to isolate bacteria dispersed into large water samples (100s of ml), enabling at least two orders of magnitude improvement in detection limit using molecular tools. We also demonstrated the use of a handheld non-instrumented incubator to enrich Salmonella and E. coli, enabling detection of less than 1 CFU/g of these pathogens in food materials like spinach and chicken within an 8 hour shift. Notable achievements include: A complete diagnostic cycle for sensitive detection of food borne pathogens, including sample acquisition, concentration and detection of bacterial contaminants was demonstrated in less than 2 hours without a specialized laboratory facility or traditional enrichment methods. Overall the electroflotation system was capable of concentrating hundreds of mL containing 102 CFU/mL E. coli into 1 mL containing approximately 104-105 CFU/mL, enabling reliable detection using portable molecular tools. Development of comprehensive yet simple protocols that can be performed during field testing including DNA extraction (crude cell lysis) and removal of LAMP inhibitors. Optimization of surfactant (Pluronic F-68) and flocculant (chitosan) concentrations allowing us to reliably detect bacterial quantities of 102 CFU/mL at an average rate of 96.3%-100%. Demonstration of pathogen enrichment in field conditions without an instrumented incubator, enabling detection of pathogenic bacteria at 1 CFU/g levels in food within 8 hours. While the funded portion of this project has concluded, we are continuing to develop the technologies moving forward to improve performance, leading to potential commercialization of the technologies. Specific areas of focus include: Investigate the recovery and detection of other food-borne pathogens. Continued investigation of nanoparticles, adhesins, and other molecular tools to disrupt biofilms and enhance the recovery and detection of live bacterial pathogens from food materials. Investigation of effects of electroflotation on cell viability and vigor (to enable subsequent confirmation and/or signal enhancement by culture based methods) Investigation of fractionation effects to enhance the concentration effect of electroflotation and improve detection limits even further. Designs for improved manufacturability and facilitated assembly of electroflotation cartridges. Detailed research efforts Significant effort in the last project year involved extensive combinatorial experiments to optimize system conditions, development of standardized protocols, improved electroflotation-based recovery by the addition of chemical additives and validation testing/analysis using live cell suspensions. Extensive combinatorial experiments were done to determine conditions resulting in the most efficient recovery and concentration of dispersed microbes. During flotation, the "stirring effect" caused by rising clouds of bubbles can cause fluid to circulate which may positively impact the collection efficiency by increasing particle/bubble collisions and rate of mass transport. On the other hand, when bubble flux exceeds a critical limit, hydrodynamic forces due to turbulence in fluid circulation acting on suspended cells can break fragile flocs of cell aggregates causing shear stress or damage to cells. We observed that bubble flux and average bubble diameter were dependent on frequency and duty cycle applied to the electrodes. Lower frequencies and duty cycles, at the same current levels, generally resulted in smaller bubbles, less fluid circulation/ stirring and less mass flux, as individual bubbles stopped growing and were more likely to randomly detach from electrode surfaces during the longer "off" periods. To enhance viable cell recovery, variable concentrations of the non-ionic surfactant pluronic-F68was added to the prepared electroflotation bacterial cultures subjected to electroflotation. Results demonstrated improved detection rates at previously undetectable concentrations, illustrating potential protective effects of the additive used in the EF process. Chitosan is an inexpensive cationic polysaccharide and is particularly desirable to flocculate and aggregate negatively charged particles i.e. predominately negatively charged membrane of bacterial cells. To aggregate cells producing shear protected flocs, variable concentrations of agricultural grade chitosan oligosaccharide (0.001, 0.01, 0.1, 1 g L-1) was added to electroflotation bacterial cultures containing 102 CFU/mL E. coli and 0.1 g L-1 pluronic. When compared to corresponding treatments only containing pluronic, significant differences were observed for electroflotation treatments containing 0.01 and 0.1 g L-1 chitosan with average detection rates of 96.3% and 100% respectively. Previously we observed strong inhibitory effect of chitosan on downstream LAMP reactions. The amino group of chitosan is cationic below its pKa (~pH 9.5). In this state, it will bind through electrostatic interaction to negatively charged negatively charged bacterial cells i.e. E. coli and also anionic DNA. Chitosan binding to anionic DNA can also prevent LAMP primer binding and inhibit amplification. By raising the pH above the pKa, negatively charged particles i.e. DNA are released from chitosan. Following a simple methodology, appropriate volumes of NaOH were added to recovered 1mL samples containing chitosan following electroflotation to adjust sample pH. Chitosan present in concentrations of 0.01 and 0.1 g L-1 inhibited amplification of 0.2 ng of E. coli 25922. LAMP was not inhibited, however, for the same concentrations of chitosan (0.01, 0.1 g L-1) at pH 10. By adjusting the pH of the samples from pH 6 to pH 10, LAMP inhibition by chitosan was prevented and amplification of target was unaffected. We also demonstrated practical application of a handheld non-instrumented incubator (NI-incubator) to enable in-field incubation and rapid direct detection of pathogens from food samples. The sensitivity of nucleic acid based LAMP assay to detect pathogens in food can be greatly improved by subjecting target samples to short (<8 hours) enrichment periods using the NI-incubators described in this study to achieve cell densities above common LOD (103-104 CFU/mL) prior to pathogen screening. We were able to quantify bacterial growth rates for cultures of Salmonella typhimurium (ATCC 14028), Escherichia coli O157:H7 (C7927), Escherichia coli K12, and generic Escherichia coli (isolated from the environment) in non-instrumented incubators by growing them in an laboratory incubator programmed to replicate representative temperature profile. The empirical growth characteristics could be used to predict the minimum enrichment periods required to achieve reliable detection (>95% positive) of a given quantity of inoculum by loop mediated isothermal amplification (LAMP). Predictions were validated by inoculation of 5 g spinach with <1 CFU/mL of E.coli O157:H7, and 5 g ground chicken inoculated with <1 CFU/mL of Salmonella typhimurium. which could be reliably detected by LAMP after 6-8 hours of enrichment in non-instrumented incubators. The simple technology may be a useful tool for rudimentary labs and food processing facilities to enable detection of trace quantities (< 1 CFU / g) of E. coli and Salmonella on real food items within eight hours.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Diaz, L., 2017. Point-of-Care Electroflotation of Dispersed, Low Tolerance Pathogens Improves Detection Rates by Loop-mediated Isothermal Amplification. Department of Molecular Biosciences & Bioengineering, University of Hawaii at Manoa, Honolulu, HI.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Diaz, L., Kubota, R., Li, Y., and Jenkins, D.M. 2018. Enrichment of Escherichia coli O157:H7 and Salmonella typhimurium using a portable, point-of-care non-instrumented incubator increases the detection sensitivity of loop mediated isothermal amplification (LAMP). Journal of Food Protection.
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
Diaz, L. and Jenkins, D.M. 2018. Concentrating E.coli 25922 with the Assistance of Flocculants and Surfactants by a Hand-held Electroflotation System Improves Detection Rates by Loop-mediated Isothermal Amplification. PLOS-Pathogens. (in preparation)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Diaz, L., Jenkins, D.M., Kubota, R., Walter, N., Yong, L., and McNealy, T. 2017. Electroflotation of Escherichia coli Improves Detection Rates by Loop-mediated Isothermal Amplification. Paper No. 1701064 Transactions of American Society for Agricultural and Biological Engineers.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Diaz, L., Jenkins, D.M., Li, Y., McNealy, T., and Tzeng, T. 2017. Electroflotation of Escheriscia coli improves detection rates by Loop Mediated Isothermal Amplification. Presentation/ paper no. 170164 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jenkins, D.M., Diaz, J.L., Li, Y., Kubota, R., and Obatake, J. 2017. Handheld non-instrumented incubator for field-based pathogen enrichment. Presentation / paper 1701434 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jenkins, D.M., Kubota, R., Berghorn, K., and Diaz, L. 2017. Clinical and Food-safety applications of point-of-care gene-based diagnostics. Presentation 1701430 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jenkins, D.M. and Reyes-de-Corcuera, J. 2017. Handheld, open-source potentiostat for high-performance electrochemical analysis in the field. Presentation 1701478 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jenkins, D.M. and Reyes-de-Corcuera, J. 2017. Open-source Android app for facilitating customized data acquisition, visualization, and control. Presentation 1701479 at 2017 International Meeting of American Society of Agricultural and Biological Engineers, Spokane, WA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Diaz, L., Li, Y., Jenkins, D.M., and McNealy, T. 2017. A novel application of electroflotation to concentrate disperse populations of low tolerance pathogens from environmental samples. Presentation at 2017 98th Annual Meeting of the American Association for the Advancement of Science, Waimea, HI.
- Type:
Other
Status:
Other
Year Published:
2017
Citation:
Diaz, L., Kubota, R., Yong, L., McNealy, T., and Jenkins, D. 2017. A novel application of electroflotation to physically separate and concentrate disperse populations of low tolerance pathogens from large volume environmental samples. Presentation at 2017 Student Research Symposium, University of Hawaii at Manoa.
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Progress 12/15/15 to 12/14/16
Outputs
Target Audience:Regulatory agencies and officials overseeing trade in agricultural and biological materials, and food safety. Agricultural producers and processors. Food retailers. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Extension agents working on food safety. Changes/Problems:The most significant challenges that we have faced in this project- i.e. to develop a planar electrode array capable of sustaining large electrolytic current densities over an extended period, and with surface characteristics enabling formation of bubble size distributions to promote efficient particulate recovery, have finally been resolved in the reported project year. Now that this challenge has been addressed, we anticipate that we will make significantly faster progress in process development for efficient bacterial concentration and recovery. Preliminary experiments have demonstrated successful concentration of viable bacteria, and we anticipate in the coming year todemonstration electroflotation as an effective technology for rapidly concentrating food-borne bacteria dispersed in/ on large samples, to facilitate rapid and highly sensitive detection of food-borne pathogens. What opportunities for training and professional development has the project provided?Students participating in this project have had opportunities for training in the following areas: -Application of industry standard engineering CAD software (SolidWorks) -Molecular diagnostic techniques like LAMP, and software tools for primer design and analysis. -Assembly, servicing, and use of analytical equipment and techniques including plasma treatment, CV imaging, spectroscopy, -Basic microbiological techniques including sterile culture, safe handling and manipulation, plating. -Collaboration and communication with industry professionals for execution of outsourced fabrication jobs. -Software development including in Android,embedded system control, and digital communications between systems. -Fundamentals of electrochemistry, as well as design and application of electrochemical techniques. -Experimental design and data analysis. How have the results been disseminated to communities of interest?To date results have primarily been communicated through presentations at national and regional conferences. Due to difficulties in obtaining reproducible results with previously developed hardware, we have not sought to publish results from these platforms, but with the demonstration of our new more stable operational platform we anticipate opportunities for publishing replicated data in peer-reviewed journals, as well as more opportunities for demonstrating our technologies at workshops and other events designed for technology transfer and promotion. What do you plan to do during the next reporting period to accomplish the goals?In this report year we have finally overcome many of the persistent challenges that we've encountered in previous years- notably in developing an easily serviceable, stable, planar electrode array capable of sustaining large electrolytic currents over a long period of time, consistently generating bubble morphologiesamenable to efficient particulate recovery from solution. With our new platform operating reproducibly, we will focus this last year of our project in optimizing electroflotation conditions to improve the speed and efficiency of recovery of live cells from suspension, and in demonstrating this technology in real samples inoculated with pathogenic bacteria (including steps to dislodge bacteria on food surfaces into suspension). The ultimate objective is to develop an electroflotation process to enable detection of trace contaminations (i.e. in the realm of 1 CFU / ml) of food-borne bacteria which can be easily automated to run very quickly (i.e. < 30 minutes). For direct testing on food we will also be evaluating comprehensive sample preparation protocols including surface treatments to release bacteria adhered on food surfaces into suspension that can then be concentrated/ recovered by electroflotation. To accelerate the electroflotation process we will also be experimenting with flocculents like chitosan in the media. Given previous observations that chitosan can inhibit molecular detection such as with LAMP, we will also develop simple protocols to separate nucleic acids (DNA and RNA) from recovered bacteria to enable detection.
Impacts
What was accomplished under these goals?
Significant accomplishments made this project year involved improved system hardware, advanced user control interface, expanded and continued collaboration with Clemson University. At the University of Hawaii, significant challenges were overcome and primary objectives were met from the previous project year. Previously a major challenge was identifying a feasible, conductive material that could protect and support custom fabricated printed circuit board electrode array from corrosion during sustained periods of anodic current. In previous years we attempted to develop planar, corrosion resistant electrode arrays by screen printing carbon pastes and other conductive materials onto commercially produced customized printed circuit boards. However, while some of these tested approaches were demonstrated to lengthen the servicable life of the electrode arrays and result in surface characteristics promoting bubble morphology for efficient particulate (i.e. bacterial cell) recovery, in every case our material coatings failed to protect underlying metal layers in the long term, with the result that we could not obtain reproducible electroflotation data. In the latest iteration of electrode array design we used an entirely different approach to address corrosion issues. We designed individual solid electrode patterns, configured into an array interfaced to an underlying circuit board through a waterproof elastomeric material. Fabrication of each of these custom-designed components was outsourced to commercial manufacturers. Electrodes were machined in solid grade-1 titanium and plated with a 5-micron layer of platinum to impart corrosion resistance (anodization of the platinum results in a passivated conductive metal oxide that is still capable of supporting electrolytic reactions at the surface). To date, we have operated these electrode arrays for over 30 hours with anodic current densities exceeding 1 kA / m2 with no observable corrosion or change in electrode performance. The custom elasomeric seal for these electrode arrays and underlying circuit board have also dramatically reduced the amount of hardware and time required for system assembly and maintenance, facilitating rapid sterilization between electroflotation treatments. Our initial experimentation with the new hardware configuration has involved optimizing system parameters affecting the efficiency of recovery and concentration of dispersed microbes. Simulated environmental samples were prepared by inoculating 380 ml of sterilized phosphate buffer (0.1 M, pH 6.6) with serial dilutions ranging from 101 to 106 CFU/ml of Escherichia coli (E. coli) ATCC_25922. Samples were subjected to 10 and 15 minute durations of electroflotation treatment with a sustained anodic current of 500 mA (density of about 1 kA / m2) using the current control feature in our Android app that interfaces to the hardware. Importantly, the current control feature allows a user defined current to be maintained irrespective of the buffer type, conductivity or concentration of dissolved ions. To evaluate the effects of EF treatment on detection limit, we used a LAMP assay using primers targeting a single-copy gene (glycerate kinase) in generic E. coli. While the primer set was taken from published literature, using the software Primer Explorer we chose a generated 6 sets of primer candidates to make the assay more robust. Primer tests were conducted on 4 different strains of E. coli, and successfully improved the sensitivity and robustness of the assay. LAMP detection limits for untreated samples were observed to be 104 CFU/ml, yielding zero positive detection in 81 tests each at concentrations of 102 and 103 CFU/ml (3 experiments with 27 assays for each experiment). In contrast, E. coli inoculated into samples at 102 CFU / ml could be detected in the displaced volume recovered from 10 or 15 minutes of electroflotation about 25% of the time. This has demonstrated that the basic electroflotation process can be used to automate the concentration and recovery of bacteria from a simple suspension to improve diagnostic detection limits. We did not observe any inhibitory effects of electroflotation using our new hardware on downstream LAMP reactions, and similarly demonstrated that candidate nanoparticles to be used to help disrupt biofilms to disperse bacteria into suspensions that could be recovered with electroflotation did not inhibit LAMP reactions. However we did observe strong inhibitory effects of chitosan, which we are proposing to use to flocculate suspended bacteria to accelerate recovery and concentration, on detection using LAMP. Data from Clemson using the new electroflotation system have demonstrated that greater than 50% of E. coli cells inoculated into a sample at about 102 CFU / ml and subjected to our electroflotation process continue to be viable after the process. This contrasts sharply to results we observed using our old electrode array systems, in which virtually no viable cells could ever be recovered from any fraction of the electroflotation media. In summary, the primary achievements for the project in this report year (December 15, 2015 - December 14, 2016) are as follows: Development of a highly stable, reusable electrode array to enable long-term sustained electrolysis and reproducible experimentation with electroflotation conditions. Redesign of the electroflotation cartridge to facilitate assembly / maintenance / sterilization. New Android software to facilitate control and automate different conditions and durations of the electroflotation process, for both the concentration and recovery phases of the process. Demonstration of reproducible electroflotation conditions, and recovery / concentration of E. coli from a simple media. For the last year of the project, we will be doing more combinatorial experiments to determine conditions resulting in most efficient recovery of bacteria, including the use of additives to flocculate cells, and protect them from stresses such as those occurring at gas/ liquid interfaces in the media. Pending successful demonstration of concentration and recovery from even more dilute samples, we will also investigate options for further research and/or commercialization.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Diaz, L.M. and Jenkins, D.M. 2016. Recovery and concentration of microbial pathogens using a portable electroflotation system. Presentation 162461482 at 2016 Annual International Meeting of the American Society of Biological and Agricultural Engineers, Orlando, FL, July 18, 2016.
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Progress 12/15/14 to 12/14/15
Outputs
Target Audience:Regulatory agencies and officials overseeing trade in agricultural and biological materials, and food safety. Agricultural producers and processors. Food retailers. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Extension agents working on food safety. Changes/Problems:As described in earlier sections, we've had significant difficulties in making planar corrosion-resistant electrode arrays with high surface densities of conductors and small inter-electrode spacings, and also encountered difficulties in actually recovering live cells from the electroflotation process. We are therefore behind in much of the experimental phase of this project- but otherwise we believe that we are on track to move forward to address the stated project objectives without any substantive changes in approach. Given that we are behind schedule, however, we will likely request a one year no cost extension to allow us to complete the experimental phase of the project. What opportunities for training and professional development has the project provided?To date this project has mostly provided opportunities for training of students in instrumentation and experimental design, including: - model platforms for training students in instrumentation design and system fabrication, for both undergraduates and graduates - use of powerful analytical equipment and techniques (i.e. zeta potential analysis and various forms of spectroscopy including dynamic light scattering, scanning electron, and Electron Dispersive) - design of protocols and experiments to evaluate the effects of nanoparticles and electroflotation conditions on bacterial recovery - studying fundamental physics and biochemistry underlying redox processes and fundamental biomolecular interactions - safely handling and manipulating pathogenic organisms (i.e. plating, streaking, culturing, decontamination etc). - software engineering for data acquisition and control (primarily in C, Arduino, Android) How have the results been disseminated to communities of interest?To date results have primarily been communicated through presentations at national and regional conferences. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we expect the PI from Clemson to conduct an 8 week sabbatical in Honolulu so we can begin putting the two parts of the project together in one. We are currently working to make sure that we can build out new designs of the electroflotation cartridge with new electrode array designs (ensuring good quality coverage of corrosion resistant coatings) and controller configuration are available when the sabbatical starts (late April 2016), so that both groups can conduct practical electroflotation tests- coupled with pre-treatments of contaminated food surfaces to disperse biofilm associated pathogens into suspension. We are also hoping to possibly investigate the development of more sophisticated electroflotation based systems to enable not just the automated ejection of floated material from the cell but electrolysis mediated automation of downstream processes like DNA purification. We believe that this will be a busy year catching up with the experimental phases proposed for electroflotation (i.e. testing/ optimizing conditions for recovering pathogens dispersed in a suspension)- especially given challenges we've already observed in preventing cell shear at the gas liquid interface (resulting in cell lysis and inability to recover live cells or DNA), balanced with the need for bubbles to contact cell particulates in solution to enable the electroflotation process. While we've been able to prevent cell death (in an earlier year of the project) by use of additives to reduce the hydrophobicity of structures on the cell surface, these additives have the effect of preventing adhesion of cells to bubbles. In the coming year all of the collaborators will be working closely to formulate molecular conditions in the suspension to improve the recovery of cells without damaging them (for example, through the use of nanomaterials and other additives to promote flocculation or cell aggregation in the suspension).
Impacts
What was accomplished under these goals?
Most of the accomplishments this project year have involved refinements in protocols and improvements in the systems engineering to enable economically feasible achievement of the objectives. The two institutions collaborating on this proposal have mostly pursued distinct but complementary objectives- at Clemson University work has focused on the use of nanoparticles to disrupt biofilms to facilitate recovery of pathogenic organisms from food surfaces, and at the University of Hawaii work has continued on developing materials, hardware, and software systems to implement a reliable and stable electroflotation process. At Clemson University protocols havebeen developed for EPS isolation from mature biofilms, and interactions of these with nanoparticles have been studied using dynamic light scattering and zeta potential analysis, in an effort to better understand and exploit the mechanisms of biofilm disruption. We have completed the characterization of the candidate functionalized nanoparticles with early (1 hr) and mature (24 hr) biofilms of E. Coli, and are preparing to conduct similar experiments with Salmonella. Work at the University of Hawaii has focused on overcoming some of the difficulties encountered in the previous year of the project. Notably, one of the primary objectives of this project is to develop an affordableplanar electrode array with high density of conductive surface where electrolysis can be supported, and a relatively small interelectrode spacing to reduce energy losses and ohmic heating in the electrolyte. A major challenge was identifying materials that could resist anodic corrosion, support large current densities, and still be patterned easily and affordably into the desired geometries. Previously we used expensive "dimensionally stable anode" ribbon and wire assembled by hand into a serpentine array, which was a time-consuming process that also made it difficult to engineer seals and simple "plug-and-play" connectors to engage the array to the controller. Efforts to passivate othermetal electrodesby coating with graphene were similarly difficult, and not effective for imparting long-term corrosion resistance to the underlying metals. As a result, a significant amount of effort was spent this year focusing on materials engineering to develop affordable materials that could be used to impart better corrosion resistance to planar electrode arrays and printing these materials onto arrays without bridging across the electrode contacts. For the materials engineering, we focused on two different coatings including a commercial silver filled conductive epoxy coating (MasterBond EP21TDCS), and a various formulations of cured conductive carbon pastes. While the silver epoxy coating was a risky choice (since silver is easily oxidized/ corroded), we experimented with this material based on several potential benefits derived from the properties of the silver/ silver chloride redox reaction: 1) the anodic reaction occurs at a very low potential, so that electrolytic hydrogen production might be supported at extremely small applied potential (saving energy in a portable battery powered system); 2) the anodic reaction is easily reversible, so that corroded anodic surfaces could easily be restored by reversing the current through the cell, or from a separate counter electrode in a regeneration reaction between electroflotation cycles, and; 3) the scavenging of chloride ions by elemental silver at the anode(especially at low applied potentials) might prevent generation of reactive chlorine gas and other highly oxidativespecies at the anode in the presence of chloride, which could result in lysing of bacterial cells and makeit extremely difficult to recover live bacteria and/or nucleic acids for downstream detection. We conducted several electrochemical tests on electrodes coated in the conductive silver epoxy to determine their suitability as a "stable" anode material. We were able to demonstrate oxidation of silver in the coating to silver chloride using Scanning Electron Microscopy (SEM) and Electron Dispersive Spectroscopy (EDS), and that the conductor material could easily be restored to elemental silver by applying a reducing current. Unfortunately, the anodic current from this material rapidly dropped off to a small fraction of it's initial value within a minute of the onset of electrolysis, indicating that the oxidized surface could not sustain an electrolysis reaction for the 10s of minutes we believe will be required for an electroflotation process to have a significant recovery/ concentration cells in suspension. The second material we've been working on for coating electrode arrays is a curable carbon paste. We started with a commercial formulation (DuPont CCP7102), and subsequently been customizing a formulation for improved conductivity and cohesion. For fabrication of the array, we have used industry-standard techniques to develop an electrode array pattern on the surface of a custom printed circuit board (with gold plated surface conductors), and working on screen printing the carbon material onto the array surfaces. Preliminary tests have illustrated that the material is indeed very stable. Electrode arrays prepared using this technique have sustained stable anodic currents for hours. While the edges of some anodic contacts appear to have slight discoloration and swelling suggesting corrosion, most of the surfaces are corrosion free suggesting that it is simply a quality control problem in the printing process to ensure complete coverage and adhesion to the underlying metal surfaces. We are already redesigning the geometry of the system slightly, and our cleaning protocols, to ensure complete corrosion resistance in subsequent iterations. Importantly, we have tested buffers subjected to hours of electrolysis with these electrodes, and have observed no inhibition in the diagnostic reactions we use for pathogen detection- indicating that these materials are a viable option for use in a pre-treatment process to facilitate detection. Finally, we have spent considerable effort this year in refining our system design to facilitate operation (i.e. "plug-and-play" approach where an electroflotation cartridge with electrode array can be easily plugged into a small control module, and subsequently detached for easy cleaning and reuse). We have a working prototype where the control module is a small circuit board that plugs directly into the side of the cartridge, and communicates to a custom Android app with a graphical user interface to set the process conditions and record the conditions of the process. We are currently redesigning this circuit board to fix one problem identified with anodic current leakage to non specific electrode arrays on our electrolysis board, and also to enable current to be reversed in the cells which may improve our ability to control bubble morphology to improve pathogen recovery.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Jwanowski, K., Fanning, B., Jenkins, B., Jenkins, D., and McNealy, TL. 2015 October. Nanoparticle Induced Biofilm Dispersal of Escherichia coli O157:H7. SC Branch ASM Meeting, Columbia SC.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Fanning, B., Mefford, T., McNealy TL. 2015 May. DLS Spectroscopy and Zeta Potential Analysis of Interactions between Nanoparticles and Biofilm Components. American Society of Microbiology General Meeting. New Orleans, LA.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Jenkins, D. M., McNealy, T. L., Kubota, R., Rodriguez, R., Horwitz, E. 2015. Rapid Recovery and Concentration of Bacterial Pathogens Using Portable Electroflotation Cartridge. 2015 Annual International Meeting of American Society of Biological and Agricultural Engineers. New Orleans, LA.
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Progress 12/15/13 to 12/14/14
Outputs
Target Audience: Regulatory agencies and officials overseeing trade in agricultural and biological materials, and food safety. Agricultural producers and processors. Food retailers. Basic experimental biologists. Analytical Chemists. Farmers, and agricultural diagnostic service providers. Extension agents working on food safety. Changes/Problems: In our first experiments with live bacteria electroflotation has been observed to be very unsuccessful at concentrating bacteria. We are currently investigating the mechanisms of this, with two operating hypotheses (we have eliminated the possibility of generation of reactive chlorine species by elimination of all chloride in our test media): - cell death is occurring due to large levels of cell membrane shear/ tension occurring by the adhesion forces to the generated gas bubbles - cell death is occurring due to exposure of large electric fields and or currents resulting in cell electroporation We are currently revising our electroflotation cell design to include a large cylindrical "reference" electrode throughout the electroflotation column, to ensure that the bulk solution is at a single isopotential with zero current, and will use numerical methods to predict the maximum local field strengths and currents in the cell. We will also evaluate different additives to help stabilize the cell membranes to shear, and to help aggregate microbial cells through flocculation providing additional protection against cell shear. Approaches to make corrosion resistant electrodes using graphene coatings were only partially successful. For fabrication of inexpensive and corrosion resistant planar electrode arrays for use in a disposable or reusable electroflotation cell, we are investigating different approaches such as graphene composites and conductive polymers. What opportunities for training and professional development has the project provided? To date this project has mostly provided opportunities for training of students in instrumentation and experimental design, including: - model platforms for training students in instrumentation design and system fabrication, for both undergraduates and graduates - design of protocols and experiments to evaluate the effects of nanoparticles and electroflotation conditions on bacterial recovery How have the results been disseminated to communities of interest? To date results have primarily been communicated through presentations at national and regional conferences. What do you plan to do during the next reporting period to accomplish the goals? For the next reporting period we will continue with the evaluation of the different technologies already underway, with modifications designed to address the limitations already observed (see "project changes" in next section), and to expand the testing to include Salmonella.
Impacts
What was accomplished under these goals?
A prototype electroflotation cell was developed to evaluate the concentration of different bacteria under different electrolysis conditions. The cell includes a planar electrode array using "dimensionally stable anode" materials (corrosion resistant electrodes with coatings of conductive metal oxides), and two compartments configured concentrically to enable a concentration flotation step in the inner compartment, and then ejection of the concentrated material by electrolysis driven displacement from the outer compartment. The ability of the electroflotation device to concentrate pathogens was tested using an environmental isolate of E. coli as a test organism. E. Coli samples at concentrations from 10-2 cfu/mL to 105 cfu/mL were prepared in a variety of systems, including tap water, 0.1 sodium bicarbonate, and a 0.1 M, pH 7 phosphate buffer. In each case recovery of live bacteria using plate counts was significantly (i.e., orders of magnitude) lower in the electroflotation treated "concentrated" samples than in the untreated samples. We are currently investigating the mechanisms of this cellular damage using the proposed treatment, whether recovery of detectable nucleic acids from the treated samples is similarly impaired, and the potential for design modifications or system amendments to protect cells and enhance their recovery from large samples (see next section, "project changes"). Several approaches were investigated for the fabrication of inexpensive and corrosion resistant electrodes for use in electroflotation cells. These include direct transfer of single layer graphene onto copper electrodes, and laser based photopatterning of graphene in suspensions of graphite oxide coated onto a substrate. Both approaches were successful in fabricating corrosion resistant graphene based electrode surfaces, but defects at the electrode edges in the first instance resulted in undercutting corrosion of those electrodes starting from the edges, and photopatterned electrode arrays were too thin to support substantial currents required in the electroflotation application. We are currently investigating alternative approaches such as composite graphite or conductive polymer electrodes. For studying the bubble size distributions from different electrode materials/ configurations and electrolysis conditions, we are also finalizing the development of a laser diffraction method. The approach uses a laser of a known wavelength significantly smaller than the size of the particle, and the sizes the passing particles based on interference fringes on a projected image. The experimental values required to determine the particle size include: the aperture and focal plane of the lense, the index of refraction of the buffer solution, the distance between the lense and the particles, and the distance between the particles and the projected plane. By placing a sensor at the projection plane, the size of the particle can be determined by the frequency distribution of the detected image. An estimation of the number of particles can be derived from the energy of the image. Segmentation analysis is an alternative method to count the number of particles, which would require a high speed camera and a powerful processor. This instrument will be used to optimize the size and number of microbubbles generated. By varying the distance between electrodes, as well as the voltage and current output of the electrodes, the size and quantity of microbubbles can be adjusted and optimized. For evaluating the effectiveness of biofilm disruption and recovery of bacteria into suspension, we have established a standard protocol for E.coli biofilm growth and imaging, and tested three varieties of AuNPs for biofilm dispersal. We are currently analyzing the interactions of nanoparticles with various biofilm matrix components.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Fanning, B., Mefford, O.T., and McNealy, T.L. 2014. DLS spectroscopy and zeta potential analysis of interactions between nanoparticles and biofilm components. SC Branch American Society for Microbiology Meeting Fall 2014, Columbia SC.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Jwanowski, K., Jenkins, B., Jenkins, D., and McNealy, T. 2014. Nanoparticles Induce Biofilm Dispersal in Escherichia coli O157:H7 and Salmonella typhimurium LT2. International Conference on the Environmental Effects of Nanoparticles and Nanomaterials. Fall 2014. Columbia, SC.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Jenkins, D.M., Li, Y., Kubota, R., Garmire, D., and McNealy, T. 2014. Nanoparticle assisted biofilm disruption for rapid recovery and detection of bacterial pathogens. 2014. IEEE-NEMS Paper #328. IEEE Nano/ micro Engineered and Molecular Systems Conference, Waikiki, HI.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Kubota, R. and Jenkins, D.M. 2014. Portable diagnostic platform for real-time LAMP based typing of Ralstonia solanacearum in the field. (Presentation only, # 1899698). ASABE Annual International Meeting, Montreal, Canada.
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