Progress 07/01/18 to 06/30/23
Outputs
Target Audience:The primary audience for the sensor prototype development are researchers and companies focused on the engineeringand technology of sensors for agriculture and biomedical sectors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supportedtwo PhD students (one Materials Science and Engineering, and one Chemistry) and two post-doctoral researchers. All researchers and Principal Investigators attend a biweekly teleconference. The highly interdisciplinary nature of this project is providing the students with the basic language and understanding necessary to perform research at the intersection of animal health, agriculture, biochemistry, and electronics. 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?
Nothing Reported
Impacts
What was accomplished under these goals?
The potential to use assays to also measure IgM specific BPIV-3 antibodies responses as "early" infection markers has also been demonstrated and IgG and IgM antibody responses profiled sample sets from individual animals from birth through to vaccination. Six viral surface proteins were successfully expressed, purified and characterized for use as capture antigens in the sensor assay development.The recombinant proteins were expressed in baculovirus and E coli expression systems.Cluster Omega was used to compare the sequences of the wild-type version of the protein to the viral surface proteins. Affinity chromatography was used to purify and enrich the expressed viral surface proteins. Polyhistidine and streptavidin tags were bioengineered onto the N-and C termini of these proteins with the goal of downstream purification. These six viral surface proteins were successfully expressed, purified and characterized for use as capture antigens in the sensor assay development.The performance of the developed BPIV-3 HN immunoassay has been compared to commercial BPI-3 ELISA kits (e.g., SVANOVA) showing good comparison. The SPR was used to develop the multiplex sensor surface chemistry needed for the selective detection and quantification of the assay. To develop an electronic biosensor that is stable and selective, a bottomup approach is used to systematically optimize the sensor surface chemistry associated with these biosensors. This work highlights the importance of carefully selecting the structural properties of the surface linkers to enable the covalent immobilization of the surface linker whilst suppressing the drift associated with the spontaneous reorganization of these surface linkers in solution. To improve the selectivity and sensitivity of the assay, the sandwich ELISA, predominantly used in label-based approaches was translated for use in label-free technologies. To ensure the reliability and reproducibility of this approach, the fundamental differences in the sensing mechanism between the ELISA, a label-based approach and electronic biosensors, label-free approaches was elucidated. The sandwich ELISA was able to be successfully translated for use impedimetric biosensors. However, the utility of potentiometric biosensors were limited by the Debye screening effect. Lastly, this work utilizes SPR to elucidate the relationship between the analytic sensitivity of a biosensor and the biological buffer used. Additionally, the presence of the polyhistidine tags commonly used for protein enrichment and purification are hypothesized to impact the binding affinity of the antigenic protein for the target analyte.By utilizing this bottom up approach, this collaborative work has been able to express and purify viral surface proteins for use as capture antigens. Additionally, the development of stable and selective electronic biosensors is reliant on the elucidation and optimization of the fundamental properties of the sensor surface. This work utilizes a systematic approach to develop electronic biosensors that will become commercially available.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Robinson C., Juska V. B., ORiordan A., "Surface chemistry applications and development of immunosensors using electrochemical impedance spectroscopy: A comprehensive review," Environmental Research, 2023, 237, 116877
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Juska V. B., Moukri N., Estrela P., Maxwell G. D., Hendriks S., Santillan-Urquiza E., OBrien B., Patella B., Inguanta R., ORiordan A., "Biocompatible hydrogel electrodeposition enables the simultaneous preparation of multi-microinterfaces for ligand bioconjugation and multiplexed electrochemical detection," DOI: https://doi.org/10.1101/2023.08.17.553669
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Juska V.B., Maxwell G. D., Estrela P., Pemble M.E., ORiordan A., "Silicon microfabrication technologies for biology integrated advance devices and interfaces," Biosensors and Bioelectronics, 2023, 237, 115503
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Gezahagne H. F., Jin D. S., Vogel, E. M., "The Influence of Charge on the Translation of the Sandwich ELISA Approach to Electronic Biosensors," submitted to the Journal of Colloid And Interface Science
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Gezahagne H. F., Juska V. B., Mooney, M., ORiordan A., Vogel, E. M., "Effect of Buffers on the Selectivity of Biosensors," submitted to Biosensors and Bioelectronics.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Juska, V.B., "Silicon microtechnologies integrated ultramicro interfaces for electrochemical system development," A|nkara University, Chemistry, Ankara, Turkey, 07 July 2023.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Juska V. B., Maxwell G. D., ORiordan A., "Microfabrication of a multiplexed device for controlled deposition of miniaturised copper-structures for glucose electro-oxidation in biological and chemical matrices," Biosensors and Bioelectronics: X, 2023, 13, 100315, DOI: https://doi.org/10.1016/j.biosx.2023.100315
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Juska, V. B.; ORiordan A, "Silicon microtechnology based electrochemical biosensors," 17th International nanoscience & nanotechnology conference (NANO-TR), Izmir, Turkey, 27-29 August 2023,
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Juska, V. B.; ORiordan A, "Multiplexed miniaturised microfabricated silicon devices for electrochemical biosensing: from chemical to biological systems," 33rd International Symposium on Pharmaceutical and Biomedical Analysis (PBA 2023), Ankara, Turkey, 2-6 July 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Vogel. E., Gezahagne, H., Nzuma, R., Juska, V. B., Mooney, M., ORiordan, A., Development of electrochemical sensors for on-farm detection of bovine infections," CRWAD (Conference of Research Workers in Animal Diseases), Chicago, IL, 19-23 January 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Eric M. Vogel, Fundamentals of and Challenges with Electronic Biosensors, Electrochemical Society Meeting, Atlanta, GA, October 12th, 2022.
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The current audience for the sensor prototype development are researchers and companies focused on the engineeringand technology of sensors for agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project is currently supporting two PhD students (one Materials Science and Engineering, one Biochemistry) and one post-doctoral researcher. All researchers and Principal Investigators attend a bi-monthly teleconference.The highly interdisciplinary nature of this project is providing the students the basic language and understanding necessary toperform research at the intersection of animal health, agriculture, biochemistry, and electronics. The students and post-docshave also attended a variety of training events including research integrity and team leadership training. How have the results been disseminated to communities of interest?To reach the broadest spread of stakeholders as possible, dissemination is being undertaken using a number ofdifferent approaches. For the scientific and technical audience,several papers have been submitted and conference presentations have been given. We have also engaged with regulatory control laboratories, governmental animal healthagencies and farms to access samples to aid development of test assays. Specifically, Queen's University has obtained calveserum/milk samples from the Agri-Food and Biosciences Institute, Belfast, Northern Ireland. Tyndally has obtained source ofinfection screened samples from Teagasc, Animal & Grassland Research Centre, Moorepark, Cork, Ireland. What do you plan to do during the next reporting period to accomplish the goals?QUB successfully developed a four-step protocol to purify and characterize disease-specific recombinant antigens. Consequently, surface plasmon resonance (SPR) will be used for the development of a sensor surface functionalization procedure. SPR will be used to quantify the amount of selective and non-selective attachment of the target analyte. Therefore, the first step of this sensor surface functionalization procedure involves optimizing and amplifying the response associated with the selective attachment of the target analyte. Protein blocking methods are being investigated in conjunction with the use of several surfactants with the goal of optimizing the response associated with the selectively bound target. Once the surface chemistry has been optimized, this protocol will be translated for use in electronic based biosensors.
Impacts
What was accomplished under these goals?
Impact The overall objective of this project is to develop and optimize an electronic sensing platform to simultaneously diagnose multiple infectious agents within minutes in on-farm settings focusing on viral diseases of key importance to bovine animal health and performance. If successful, this project will have a direct impact on the efficiency and sustainability of the agri-food sector which contributes $835 billion to the U.S. gross domestic product in 2014 and an estimated €26Bn to the Irish economy.The following is a brief description of the impact of our accomplishments this year: 1. The synthesis of recombinant forms of subunit viral proteins in sufficient quantities is key to the development of diagnostic assay formats using sensor-based approaches whose operation benefits from functionalization with enriched purer protein forms. The research work of the Queen's University Belfast this period has focused on the application of chromatographic techniques to sufficiently purify expressed proteins to be applied to electronic sensor test platforms. 2. One of the key challenges associated with commercializing electronic biosensors is the lack of stability and reproducibility associated with these techniques. Georgia Tech has demonstrated that the use of a polymer-based surface linker, ortho-aminobenzoic acid (o-ABA) results in significant improved drift of electrochemical biosensors. 3. An important starting point for the development of an emerging electronic biosensor is being able to reproducibly manufacture chips with high yield. Tyndall National Institute has developed a novel and reproducible method for fabricating electrochemical sensors based on an alkaline and acidic treatment of the gold sensor surface. Objectives and Detailed Accomplishments 1. Further develop and optimize the dual sensing platform to simultaneously diagnose multiple infectious agentswithin minutes focusing on viral diseases of key importance to bovine animal health and performance. 1.1. Capture protein design and synthesis. Target proteins with requisite dominant immunogenicity from each of the identified viruses of interest (i.e. BVDV NS3 / BHV-1 gB / BHV-1 gE / BRSV gF / BLV gp51) have been selected and amino acid sequences compiled from viral strains reported within repository databases and available literature. Homology analysis was performed to develop consensus sequences for each protein ensuring high degrees of compatibility of various viral strains to assays to be developed. Protein sequence truncation and additional tag / protein sequence incorporations were made. Amino acid sequences for the various subunit proteins have been successfully cloned into plasmid constructs and transformed into Sf9 / E. coli cellular systems, with target proteins successfully expressed. Optimum enrichment and purification of expressed capture antigens from protein-containing lysates has been established based on combinations of various chromatographic approaches including ion-exchange, hydrophobic interaction and size exclusion chromatography and ammonium sulphate precipitation. Immunogenicity of synthesized viral subunit proteins has been demonstrated and used to characterize viral specific IgG and IgM levels in sera within pre- and post-vaccination calves. 1.2 Sensor design and optimization. Georgia Tech has demonstrated that the use of a polymer-based surface linker, ortho-aminobenzoic acid (o-ABA) results in significant improved drift of electrochemical biosensors. The following provides a summary of the results; details can be found in: Hilena F. Gezahagne, Eleanor L. Brightbill, Decarle S. Jin, Siamalan Krishnathas, Billyde Brown, Mark Mooney, Alan O'Riordan, Niamh Creedon, Caoimhe Robinson and Eric M. Vogel6, "Suppression of Impedimetric Baseline Drift for Stable Biosensing," ECS Sensors Plus https://doi.org/10.1149/2754-2726/ac8fa1 (2022). Biosensors based on Electrochemical Impedance Spectroscopy (EIS) detect the binding of an analyte to a receptor functionalized electrode by measuring the subsequent change in the extracted charge-transfer resistance (RCT). Linker molecules, such as a carboxyl terminated thiol self-assembled monolayers on gold, are typically utilized to functionalize the sensing surface with a bioreceptor via amine reactive cross-linker chemistry. However, thiol-based SAMs have been shown to exhibit significant drift in the baseline RCT limiting their use in biosensing. In this work, the stability of a long chain alkanethiol, 16-mercaptohexadecanoic acid was compared to that of a polymer-based surface linker, ortho-aminobenzoic acid (o-ABA). The drift in RCT observed for the native SAM functionalized gold electrodes was observed to correlate to the drift in the subsequent receptor functionalized SAM. This indicates the importance of the gold-molecule interface for reliable biosensing. Additionally, the magnitude of the baseline drift correlated to the percentage of thiol molecules not properly bound to the gold electrode as evaluated using X-ray Photoelectron Spectroscopy (XPS). Alternatively, the o-ABA functionalized gold electrodes demonstrated negligible baseline RCT drift and reduced variability between electrodes prepared with identical procedures. Furthermore, these polymer functionalized gold electrodes do not require a stabilization period in the buffer solution prior to receptor functionalization. This allows for the practical application of o-ABA functionalized gold electrodes in EIS based Point of Care Technologies (PoCTs). 2. Translate the sensor platform from an advanced bench-based state to a field deployable format facilitating cost effective (~$5) on-farm testing and result reporting. Tyndall National Institute has developed a novel and reproducible method for fabricating electrochemical sensors. The following provides a summary of the results; details can be found in: Vuslat B. Juska, Graeme D. Maxwell, and Alan O'Riordan, "Controlled nano-foam-structured device enables rapid, sensitive detection for biological and chemical sensing," ChemRxiv DOI: 10.26434/chemrxiv-2022-pfkrb (2022). Advances in multiplexed sensing on a single chip device offer a range of advantages for the development of new biosensing platforms. The increasing number of reports on multiplexed sensing demonstrates the reliability and attractiveness of merging several closely packed sensing areas. However, the most important starting point for this emerging technology is being able to reproducibly manufacture chips with high yield. Microfabrication is arguably the most elegant approach to develop chips with sensing electrodes that have the highest reproducibility and design flexibility. With this approach, it is possible to manufacture devices at very small scales with any arrangement of the components on a single chip. Such devices are not only outstanding for sensing applications but also for building interfaces for electrochemistry with very high reproducibility at the micro and nanoscale, so called micro/nanoarchitectures. Here we show one route for a successful fabrication protocol of ultra-micro scaled sensing electrode chips. We demonstrate the effectiveness of alkaline and acidic treatments in terms of cleaning and reproducibility of electrochemical sensing on chip and on wafer. The alkaline treatment protocol established for these particular microfabricated chips will aid the development of highly reproducible biosensors. Herein, we clearly demonstrate the intimate link between the microfabrication and engineering micro-interfaces for high performance sensing tools in the fields of chemical and biological applications. 3. Validate the developed diagnostic tests and sensor platform for on-farm testing using blood and milk as test matrices enabling rapid result reporting for beef and dairy sector herd management. Purified antigens have been delivered to GT to demonstrate sensor functionality.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Hilena L, Gezahagne, Eleanor L, Brightbill, Decarle S, Jin, Siamalan Krishnathas, Billyde Brown, and Eric M, Vogel, "Structural Properties of Alkanethiolates and Their Influence on The Stability and Reproducibility of Impedimetric Sensing," Electrochemical Society Meeting Abstract, MA2021-02, 1608 (2021).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Vuslat B. Juska, Graeme D. Maxwell, and Alan ORiordan, "Controlled nano-foam-structured device enables rapid, sensitive detection for biological and chemical sensing," ChemRxiv DOI: 10.26434/chemrxiv-2022-pfkrb (2022)
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2022
Citation:
Hilena F. Gezahagne, Eleanor L. Brightbill, Decarle S. Jin, Siamalan Krishnathas, Billyde Brown, Mark Mooney, Alan O'Riordan, Niamh Creedon, Caoimhe Robinson, and Eric M. Vogel, "Suppression of Impedimetric Baseline Drift for Stable Biosensing," ECS Sensors Plus, https://doi.org/10.1149/2754-2726/ac8fa1 (2022).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
V. B. Juska, G. D. Maxwell, and A. ORiordan, "Silicon microtechnologies based microsurface engineering for biosensing applications," 18th International Conference on Electroanalysis, Vilnius, Lithuania, 4-8 June 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
"Engineering micro and nano surfaces based on silicon microtechnologies for bio-sensing applications," 7th International Conference on Bio-Sensing Technology, Sitges, Spain, 22-25 May 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
V. B. Juska and A. O'Riordan, "Silicon microtechnologies driven biological sensing platforms," 7th International Congress on Biomaterials and Biosensors, Mugla, Turkey, 22-28 April 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Melissa Hardy, Gordon Allan, Eric Vogel, Alan ORiordan, Mark Mooney, "Development of Serological IgG and IgM Antibody Profiling Assays for Bovine Parainfluenza-3 Virus," European Society for Veterinary Virology (ESVV) - 12th International Congress for Veterinary Virology Conference, Ghent Belgium, September 20-23, 2022 .
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:The current audience for the sensor prototype development are researchers and companies focused on the engineering andtechnology of sensors. As the program progresses towardsvalidation and testing, the audience will move to the agri-foodsector including farmers and veterinarians. Changes/Problems:The pandemic has slightly slowed progress due to visa issues associated with the Tyndall researchers. We have also not been able to travel to the three sites as initially planned, which has slowed integration of the prototype. We intend to request a one-year no-cost extension and this should provide the necessary time to reach the overall objectives of the project. What opportunities for training and professional development has the project provided?The project is currently supporting three PhD students (one Materials Science and Engineering, one Biochemistry, andone Chemistry) and two post-doctoral researchers. All researchers and Principal Investigators attend a bi-monthlyteleconference. The highly interdisciplinary nature of this project is providing the students the basic language andunderstanding necessary to perform research at the intersection of animal health, agriculture, biochemistry, and electronics. The students and post-docs have also attended a variety of training events including research integrity and team leadership training. How have the results been disseminated to communities of interest?To reach the broadest spread of stakeholders as possible, dissemination is being undertaken using a number of differentapproaches. For the scientific and technical audience, two papers and a thesis have been published and additional papers will soon besubmitted. We have also engaged with regulatory control laboratories, governmental animal health agencies and farms to access samples to aid development of test assays. Specifically, Queen's University has obtained calve serum/milk samples from theAgri-Food and Biosciences Institute, Belfast, Northern Ireland. Tyndally has obtained source of infection screened samples fromTeagasc, Animal & Grassland Research Centre, Moorepark, Cork, Ireland. We will be working with these agencies to validateour sensor system using these samples. What do you plan to do during the next reporting period to accomplish the goals?The next period will continue expanding optimization of additionalproteins representative of other disease states. The individual portions of the system (microfluidics, potentiometric sensor, impedance sensor, associated surface chemistries) have been largely developed. The next period will involve integration of these into a final prototype followed by validation using blood and milk matrices.
Impacts
What was accomplished under these goals?
Impact The overall objective of this project is to develop and optimize an electronic sensing platform to simultaneously diagnose multiple infectious agents within minutes in on-farm settings focusing on viral diseases of key importance to bovine animal health and performance. If successful, this project will have a direct impact on the efficiency and sustainability of the agri-food sector which contributes $835 billion to the U.S. gross domestic product in 2014 and an estimated €26Bn to the Irish economy. The following is a brief description of the impact of our accomplishments this year: Expression and purification of capture proteins for detection of BLV. Almost all cattle infected with BLV have detectable antibodies to the viral protein gp51. Queen's University at Belfast has developed new expression and purification techniques to produce the BLV gp51 protein so that it can be applied to our biosensor to capture its associated antibody and detect BLV. Development of a technique to prevent sensor degradation in ambient storage conditions. Our sensors employ carboxylic-acid-terminated self-assembled monolayers (COOH-SAMs) on the gold sensor surface to permit covalent attachment of our protein receptor molecules. Although oxidation of these COOH-SAMs is known to decrease biosensing reliability, techniques to limit their oxidation have not previously been established. Georgia Tech has demonstrated a new gelatin preblocking technique that allows subsequent receptor functionalization for selective biosensing with degradation prevention in ambient conditions that outperforms storage in nitrogen. Modeling, design and fabrication of microfluidics and sensor electrodes for a farm deployable biosensor with fast response time. Tyndall National Institute has used detailed models of hydrodynamic flow to design microfluidics and sensor electrodes. The initial prototype was fabricated and showed results consistent with the models. Objectives and Detailed Accomplishments 1. Further develop and optimize the dual sensing platform to simultaneously diagnose multiple infectious agents within minutes focusing on viral diseases of key importance to bovine animal health and performance. 1.1. Capture protein design and synthesis. To develop a suitable sequence for expression of a recombinant form of the BLV gp51 protein amenable to application in developed sensor assays, a consensus sequence constructed from a collection of 256 sequences of BLV env genes deposited on NCBI was initially considered. Cluster Omega was used to compare this sequence with BLV gp51 sequences from around the world randomly selected from Genbank. The coincidence degree of the consensus sequence with the known sequence for comparison is more than 95%, and the few amino acids not completely overlapping are also the same as the corresponding amino acids in most sequences. Therefore, the consensus sequence was adopted to express recombinant gp51. The DNA sequence (839 bp in length) of the translated BLV gp51 protein consensus sequence optimized by Genscript for expression in E. coli was obtained. The pET-32(a+) plasmid vector was selected for expression of gp51 recombinant protein. The cloned pET32a(+)-gp51 plasmid construct was then transformed into Mach1-T1 E. coli cells, and the purified plasmid DNA was digested with EcoRl-HF and EcoRl-HF double enzymes. A band that is slightly larger than 800 bp on the gel, which matches the size of the gp51 insert, confirms that the expected gp51 protein gene was successfully cloned between EcoRl-HF and Ncol-HF restriction sites. Purified pET32a (+)-gp51 plasmid was transformed into BL21(DE3) E. coli expression host cells. The transformed BL21(DE3) cells were induced by IPTG to produce a BLV gp51 protein band of 47 kDa in the total protein fraction. Large scale purification of expressed gp51 protein was performed by His-tag affinity Talon beads using optimized conditions. Protein expression and purification was shown to produce sufficient concentrations of protein, at low cost and simple operation and expressed gp51 protein can be applied to biosensor assay methods requiring pure and concentrated capture antigens. 1.2 Sensor design and optimization. Georgia Tech has developed a new technique, gelatin pre-blocking, that allows these sensors to be viable on-farm devices. The following provides a summary of the results; details can be found in: Eleanor L. Brightbill, Hilena F. Gezahagne, Decarle S. Jin, Billyde Brown, and Eric M. Vogel, "Protein blocking inhibits ambient degradation of self-assembled monolayers for affinity biosensing," Applied Surface Science 557, 149843 (2021). The use of a simple protein blocking procedure prior to receptor functionalization is shown to inhibit the oxidation of carboxylic-acid-terminated thiolate self-assembled monolayers (COOH-SAMs) in ambient conditions. This finding has direct implications for the engineering and design of point-of-care and field affinity biosensors. While an abundance of label-free sensor systems utilizing COOH-SAM functional surfaces have been demonstrated, these layers degrade quickly in ambient conditions, severely limiting their shelf life. With gelatin blocking, we have demonstrated no surface degradation (oxidation) is observed via X-ray photoelectron spectroscopy nor reductive desorption for 21 days, compared to observable degradation in only one day for an unblocked COOH-SAM. Additionally, unlike a traditional postblocking method that requires the sensor to first be functionalized with active receptor, this blocking procedure occurs directly after COOH-SAM formation and does not prevent the subsequent functionalization of the biosensor. These blocked sensors are shown to maintain sensing performance for at least one week. Additionally, the blocking technique for degradation prevention is shown to even outperform COOH-SAM storage in N2. 2. Translate the sensor platform from an advanced bench-based state to a field deployable format facilitating cost-effective (~$5) on-farm testing and result reporting. Tyndall National Institute has performed simulations to explore molecular diffusion to microband sensor electrodes. Ferrocene Monocarbolxylic Acid (FCA) was used as a redox probe. A simulation model was designed to explore the oxidation and reduction of a redox molecule at the microband electrodes, using the FEA software COMSOL Multiphysics 5.3. These simulations were undertaken to investigate the diffusional mass transport and consequent current signal obtained at a range of electrode widths and arrays in two dimensions. The modeled diffusion profile of a 3-electrode array under quiescent conditions showed diffusional overlap between the electrodes. This large profile means the analyte is greatly dispersed throughout the system, relying on diffusion only to be delivered to the electrode surface. The diffusional overlap observed also reduces the sensitivity of the array, with each electrode no longer behaving independently. In contrast, once a laminar flow of 400 um/s is introduced to the system, the diffusional profile is greatly reduced as well as the degree of overlap. This clearly indicates that the analyte has been more successfully delivered to the electrode surface, since more than one mode of mass transport is now being employed, using convection as well as the pre-existing diffusion. Initial experimental results for the designed microfluidic system corroborate the simulations produced to date. An increase in the limiting current clearly indicates the increased sensitivity of detection as the flowrate is increased, as expected from the simulations. 3. Validate the developed diagnostic tests and sensor platform for on-farm testing using blood and milk as test matrices enabling rapid result reporting for beef and dairy sector herd management. Blood and milk matrices have been collected by Tyndall and Queen's Universityfor future testing of the fully developed system.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Eleanor L. Brightbill, Hilena F. Gezahagne, Decarle S. Jin, Billyde Brown, and Eric M. Vogel, Protein blocking inhibits ambient degradation of self-assembled monolayers for affinity biosensing, Applied Surface Science 557, 149843 (2021).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Eleanor L. Brightbill, Katherine T. Young, Hilena F. Gezahagne, Decarle S. Jin, Bryce Hitchcock and Eric M. Vogel, Protein interactions with chemical vapor deposited graphene modified by substrate, 2D Materials 8, 025015 (2021).
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Eleanor L. Brightbill, "Optimization of Surface-Protein Interactions for Next Generation Biosensors," Doctoral Dissertation, Georgia Institute of Technology, School of Materials Science and Engineering (2021).
|
Progress 07/01/19 to 06/30/20
Outputs
Target Audience:The audience for the sensor prototype development will be researchers and companies focused on the engineering andtechnology of sensors. As the program progresses towards validation and testing, the audience will move to the agri-foodsector including farmers and veterinarians. Changes/Problems:The pandemic significantly slowed progress this year. The laboratories at each institution were closed for approximately 4 months (March-June 2020) resulting in significant experimental delays. Also, Tyndall National Institute was delayed in the hiring of a key post-doctoral researcher for the project. Finally, the Georgia Tech team had planned on visiting Ireland this summer to begin sensor validation there. This trip had to be cancelled. With the laboratories now open, experimental work has re-started andthe Tyndall post-doc is now on-site. Instead of visiting Ireland to perform sensor validation, the sensors will now be validated by Queen's University sending the developed proteins to both Tyndall and Georgia Tech. What opportunities for training and professional development has the project provided?The project is currently supporting three PhD students (one Materials Science and Engineering, one Biochemistry, and oneChemistry) and two post-doctoral researchers. All researchers and Principal Investigators attend a monthly teleconference.The highly interdisciplinary nature of this project is providing the students the basic language and understanding necessary toperform research at the intersection of animal health, agriculture, biochemistry, and electronics. 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 next period will focus on optimization of protein expression / purification to produce sufficient capture antigen to enable extensive sensor validation and field deployment activities to proceed. Large scale expression and purification via chromatographic approaches (e.g. ion exchange, size exclusion) will be employed using produced Baculovirus expressed material (lysates / supernatants). Production of capture antigens for BLV assays (e.g. gp51) will also be initiated using an E coli protein expression system. These proteins, developed by Queen's University in Belfast, will be provided to Georgia Tech and Tyndall National Institute to perform extensive sensor validation.
Impacts
What was accomplished under these goals?
The performance of the developed BPIV-3 HN immunoassay has been compared to commercial BPI-3 ELISA kits (e.g. SVANOVA) showing good comparison. The potential to use assays to also measure IgM specific BPIV-3 antibodies responses as "early" infection markers has also been demonstrated and IgG and IgM antibody responses profiled sample sets from individual animals from birth through to vaccination. Alternative capture antigen immobilisation procedures have been developed with a view to enhancing the levels of protein specifically functionalised to SPR sensor chip surfaces. This approach seeks to utilise the interaction of His-tag regions of expressed proteins with cobalt ions present on sensor surfaces to form through oxidative processes a covalently bound capture antigen layer. To minimise potential for assay interference with cross-reactivity to non-viral related circulating antibodies, the glycosylation profile of expression proteins (gF and NS3) has been modified by expression in SF9 cells rather than originally utilised Hi-5 cells.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Decarle Sibi Jin, Xingyuan Zhu, Eleanor L Brightbill, Billyde Brown, Eric M Vogel,"Chemical and Biological Sensor Capsules for Real-Time Measurement of Cell Properties in Bioreactors," 237th ECS Meeting with the 18th International Meeting on Chemical Sensors, May 10-14, 2020
|
Progress 07/01/18 to 06/30/19
Outputs
Target Audience:The audience for the sensor prototype development will be researchers and companies focused on the engineering and technology of sensors.As the program progresses towards validation and testing, the audience will move to the agri-food sector including farmers andveterinarians. Changes/Problems:The Ireland (Tyndall) and Northern Ireland (Queen's University Belfast) portions of the project have been somewhat delayed due to delays in recruiting students and post-docs to the project. However, these positions are now filled and progress will accelerate. What opportunities for training and professional development has the project provided?The project is currently supporting threePhD students (one Materials Science and Engineering, one Biochemistry, and one Chemistry) and one post-doctoral researcher. All researchers and Principal Investigators attend a monthly teleconference. The highly interdisciplinary nature of this project is providing the students the basic language and understanding necessary to perform research at the intersection of animal health, agriculture, biochemistry, and electronics. How have the results been disseminated to communities of interest?To reach the broadest spread of stakeholders as possible, dissemination is being undertaken using a number of different approaches.For the scientific and technical audience, two papers have been published and additionalpapers will soon be submitted to international conferences including IEEE sensors 2019, ACM Nanocom 2019 and European Conference On Precision Livestock Farming 2019.Concerning education and public engagement a toy farm comprising fully integrated sensors, water features and pumps has been built using Lego and other toy components.This farm has proven very successful in engaging children and their parents and has been used successfully to introduce the concept of precision farming, on-farm disease diagnostics and environmental monitoring; see figure 3.At a recent state visit to Ireland (12th-14thJune 2019) the smart farm was also presented to the King and Queen of the Netherlands. What do you plan to do during the next reporting period to accomplish the goals?In the next report period, work will intensify in the following areas: Optimising immunoassay - work will focus on developing a robust surface attachment chemistry for the immobilisation of antibodies at gold sensor surfaces while also reducing non-specific binding.A number of conducting and non-conducting polymers will be assessed and benchmarked against ELISA. New Chip Design for "on-farm" testing- a new chip is being designed to enable up to eight multiplexed immunoassays to be undertaken simultaneously.A number of changes have been incorporated includinga new edge connector pinout with 20 electrical connections, and integrationwith on-chip microfluidics. Initial testing using blood and milk test matrices will begin.
Impacts
What was accomplished under these goals?
Impact One of the most common methods for identification of viral, bacterial and parasitic infections in animals is based on detection of circulating antibodies to these infections within blood, milk, and tissue samples using lab-based techniques such as enzyme linked immuno-sorbant assays (ELISA). However, ELISA-based testing is limited, as it typically relies on the detection of a single virus within an individual test sample (~$5 per sample test) - this means screening for each disease under investigation must be performed separately. Consequently, the costs associated with the performance of multiple ELISA testing for pathogens in animal populations can be prohibitively expensive and many animals do not undergo frequent testing compromising the effectiveness of disease control, prevention and management procedures. Additionally, ELISA does not facilitate on-farm testing as samples must be collected and transported to a central laboratory, further increasing the cost.This project will have a direct impact on the efficiency and sustainability of the agri-food sector which contributes $835 billion to the U.S. gross domestic product in 2014 (with ~$88Bn for farm gate receipts in cattle and calves) and an estimated €26Bn to the Irish economy (generating 10% of Ireland's exports, with beef and dairy accounting for 58% of agricultural output). In 2014, there were 39 million cattle in the U.S. with ~$7Bn of beef export and 6.3 million cattle in Ireland, with exports of €1.8Bn for beef, and €2.9Bn for milk products. Objectives and Accomplishments 1.Further develop and optimize the dual sensing platform to simultaneously diagnose multiple infectious agents within minutes focusing on viral diseases of key importance to bovine animal health and performance - specifically Bovine Viral Diarrhoea (BVDV), Bovine Parainfluenza Virus-3 (BPIV-3), Bovine Respiratory Syncytial Virus (BRSV), Infectious Bovine Rhinotracheitis (IBR) and Bovine Leukemia Virus (BLV). 1.1.Capture protein design and synthesis Baculovirus expression systems have been used to design and producerecombinant BPI-3hemagglutinin-neuraminidase (HN),BVDV non-structural protein 3 (NS3) and BRSV fusion (gF) proteins as capture antigens for application within immuno-based analyses.Baculovirus-mediated expression of a recombinant BVDV non-structural protein 3 (NS3) complete with a C-terminus hexahistidine tag has been performed with the protein successfully expressed and secreted into the growth media. A 684 amino acid consensus sequence of the most commonly occurring sequence was generated for BVDV-NS3 following multiple sequence alignment of 227 previously published sequences available from online databases. The consensus sequence shares over 60% identity with each of the aligned NS3 sequences illustrating the highly conserved nature of the protein. Baculovirus-mediated expression of a truncated recombinant BVDV NS3 protein complete with a C-terminus hexahistidine tag has been performed with the protein intracellularly expressed and secreted into the growth media. The protein was found to be expressed in a soluble form with a determined molecular weight of approximately 80 kDa. Native Western Blot analysis using hyperimmune and known pos/neg serum samples from differentially exposed respiratory infections have shown specificity to BVDV with no cross-reactivity to BRSV/PI-3/BHV-1.It was estimated that recombinant NS3 is present in high abundance (approx. 46% of total protein content in cell culture media of NS3 baculovirus-infected cells) and hence initial assays have been carried out using the NS3 containing media to develop indirect ELISA assays with a monoclonal antibody conjugate confirm the BVDV antigenicity of expressed NS3. Investigation of performance of developed NS3 ELISA for analysis of BVDV positive and negative serum samples has revealed binding of serum to components within cell media. Baculovirus-mediated expression of a recombinant BRSV fusion (gF) protein complete with a C-terminus hexahistidine tag has similarly been performed with the protein successfully expressed and secreted into growth media.Ongoing work is focused on further purification of expressed NS3 and gF to a level which is amenable for application to immune-based analyses. 1.?2.Sensor design and optimization The detection principle of virtually all biosensors requires a reaction of analytes in a solution with receptors on the sensor surface, which is then transduced into a measurable signal.However, as analyte concentrations become increasingly low, the number of molecules in a solution decreases and the transport of the analyte to the sensor surface becomes progressively more difficult, often being the factor determining the limit of detectionof biosensors. Understanding the time scales of binding is critical for sensor optimization. Georgia Tech has developed a general model for mass transport to planar and nanowire biosensors under flow that is applicable over a wide range of variables [Decarle S. Jin, Eleanor L. Brightbill, and Eric M. Vogel,Journal of Applied Physics125, 114502 (2019)]. The model approximated experimental binding data under a wide variety of conditions quite well and is being used to design and optimize the biosensors. Carboxylterminated thiol self-assembled monolayers (COOH-SAMs) on Au are utilized to functionalize the sensing surface with a bioreceptor via amine coupling. However, commonly used COOH-SAM preparation methods result in large defect densities due to cooperative hydrogen bonding between carboxylic acid end groups, which in turn leads to large nonselective adsorption (NSA) of proteins to hydrophobic surfaces exposed by these defects. Georgia Tech has developed a procedure where the surface of COOH-SAMs is pretreated by a protein (e.g. gelatin) orthogonal to the immunological system of interest to cover hydrophobic, nonselective sites on the sensor surface while still leaving carboxylic acid head groups available for covalent functionalization [Eleanor L. Brightbill, Bryce Hitchcock, Meng-Yen Tsai, Adam Verga, and Eric. M. Vogel,The Journal of Physical Chemistry C123, 16778 (2019)]. This preblocking step has been shown to shield the COOH-SAM from oxidation, improve baseline stability, and prevent receptor denaturation. This method performs similarly to conventional postblocking, while allowing the more expensive, functional protein receptor to be conserved. 2.Translate the sensor platform from an advanced bench-based state to a field deployable format facilitating cost-effective (~$5) on-farm testing and result reporting. Using commercial off-the-shelf electronics components along with an ADI CM350 chip potentiostat and Bluetooth communications, thesensor platform has been translated from an advanced bench-based state to a field deployable format.A new metal stack was developed to improve coupling between the on-chip pin out and a commercial SD type edge connector.This approach greatly reduced poor electrical connections arising from the edge connector scratching through the on-chip metallisation and permitted up to 50 mating's without any degradation in sensor performance.In addition, work has begun on developing bespoke electronics system to operate the sensor cartridge in the field.Using commercial off the shelf electronics components along with an ADI CM350 chip potientiostat and Bluetooth communications, a bespoke prototype system has been developed to enable operation and measurement of the sensor devices.An app has also been written to enable control of this systems using a mobile phone.At present the system is capable of undertaking a variety of different voltametric and amperometric analytical methods remotely in the field including Cyclic voltammetry, Square wave voltammetry, and Generator Collector voltammetry.Work is now focusing on expanding the capability to this system to enable impedance measurements to be undertaken remotely.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Decarle S. Jin, Eleanor L. Brightbill, and Eric M. Vogel, "General model for mass transport to planar and nanowire biosensor surfaces," Journal of Applied Physics, vol. 125, art. no. 114502 (2019)
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Eleanor L. Brightbill, Bryce Hitchcock, Meng-Yen Tsai, Adam Verga, and Eric M. Vogel, "Preblocking Procedure to Mitigate Nonselective Protein Adsorption for Carboxyl-SAMs Used in Biosensing," The Journal of Physical Chemistry C, vol. 123, pp. 16778-16786 (2019)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Eric M. Vogel, Nano-material Potentiometric Biochemical Sensors for Diagnostic Applications, Biomedical Engineering Society Meeting, Atlanta, GA, Oct. 20, 2018.
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