Progress 07/01/19 to 02/28/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:In performing our nanoplasmonic and electrochemical assays we found that the aptamer-target binding was less favorable than expected from the published results and our projected models. Consequently, detection of FDA relevant concentrations (<200 ppb OA) will be possible via analyte concentration. We believe a more thorough investigation of the aptamer-target binding conditions would be necessary to lower the current electrochemical limit of detection. This comprehensive testing would include exploring incubation temperatures, aptamer loading, buffer salt concentrations, electrode surface area, among other parameters to promote aptamer-target complexation. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Our results were communicated to marine toxin researchers and industry stakeholders during two conferences attended during the reporting period. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Using Giner's gold nanoparticle nanoplasmonic assay a rigorous study of published okadaic and domoic acid aptamers was performed. Based on the results it was determined that OA-34 was the best candidate to pursue electrode functionalization for electrochemical detection of okadaic acid. Giner successfully functionalized gold nanoparticle electrodes with the methylene blue modified OA-34 aptamer for use in toxin detection. Although immobilization of the aptamer onto the electrode surface reduced its functionality, we were able to detect OA at concentrations down to 500 parts per billion (ppb) equivalent to shellfish extracts. We believe that additional development of the assay procedures along with increasing the gold surface area would improve target detection levels and method consistency.
Publications
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
During Phase I Dr. Avni Argun (PI) attended the 235th Electrochemical Society conference in Dallas, TX to present Giners work on marine toxin sensors, Rapid and Low-cost Field Detection of Marine Toxins (2019). Additionally, Dr. Argun spoke at the 2020 Innovating for Coastal Resilience virtual conference hosted by the University of Florida, Envisioning Future Coastal Sensing Technologies (June 2020). During these talks Giner presented our sensor work on detection of okadaic and domoic acid in shellfish food stuffs. We had the opportunity to discuss these results with other academic and research professionals as well as shellfish industry stakeholders.
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Progress 07/01/20 to 02/28/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the first months of the program several experiments were performed to characterize the okadaic acid (OA) aptamerand target binding. Additionally, a procedure for functionalizing the screen printed electrodes (SPEs) with the aptamer wasdeveloped. The newly modified SPEs were also tested with samples of stock OA to establish a baseline sensitivity and limitof detection (LOD). Our current efforts are focused on investigating the assay methodology and improving the parameters ofthe electrochemical detection step for OA.I.A Aptamer-Target Binding ConfirmationThe initial Phase I work was performed using a an okadaic acid aptamer that had been previously published in researchliterature. The oligo sequence, shown in Table 1, was synthesized using a commercial vendor, Integrated DNA Technologies(Coralville, IA). Using this aptamer sequence we performed a novel colorimetric binding assay to confirm OA aptamer-targetbinding.Table 1. Okadaic acid aptamer OA-34 sequence and dissociation constant.Giner has developed a robust gold nanoplasmonic colorimetric procedure in house to characterize aptamer candidates.Briefly, the citrate-reduced gold nanoparticles (AuNP) possess a negative charge and the electrostatic repulsive forces givethem a characteristic red color (520 nm) in solution. In the presence of negatively charged aptamer and 150 mM NaCl, anegative charge cloud protects AuNPs from any salt-induced aggregation (Figure 1). When an aptamer binds to its target, itleaves the particle surface, inter-particle distance is reduced, and a salt-induced aggregation takes place, resulting in red-to-purple color transition to 700 nm in less than a minute. This simple mechanism allows us to obtain quantitative bindinginformation by monitoring the optical density at 520 nm (Figure 1 inset).Figure 2 shows the UV-Vis spectrometry data for the OA-34 aptamer and aptamer-target samples. The reduced 520 nmsignal indicates good binding of the target by the OA-34 aptamer. We are currently investigating several other aptamercandidates for both okadaic and domoic acid using this assay. Any resulting aptamers that are successfully screened canthen be functionalized onto Giner's electrodes and tested.I.B Aptamer Functionalized SPEsUsing the OA-34 aptamer a procedure was developed to functionalized gold nanoparticle carbon SPEs.
Publications
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Progress 07/01/19 to 06/30/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Giner needs more time due to Covid in order to reach our goals set out and have requested an extension. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Program Progress During the first months of the program several experiments were performed to characterize the okadaic acid (OA) aptamer and target binding. Additionally, a procedure for functionalizing the screen printed electrodes (SPEs) with the aptamer was developed. The newly modified SPEs were also tested with samples of stock OA to establish a baseline sensitivity and limit of detection (LOD). Our current efforts are focused on investigating the assay methodology and improving the parameters of the electrochemical detection step for OA. I.A Aptamer-Target Binding Confirmation The initial Phase I work was performed using a an okadaic acid aptamer that had been previously published in research literature. The oligo sequence, shown in Table 1, was synthesized using a commercial vendor, Integrated DNA Technologies (Coralville, IA). Using this aptamer sequence we performed a novel colorimetric binding assay to confirm OA aptamer-target binding. Table 1. Okadaic acid aptamer OA-34 sequence and dissociation constant. Giner has developed a robust gold nanoplasmonic colorimetric procedure in house to characterize aptamer candidates. Briefly, the citrate-reduced gold nanoparticles (AuNP) possess a negative charge and the electrostatic repulsive forces give them a characteristic red color (520 nm) in solution. In the presence of negatively charged aptamer and 150 mM NaCl, a negative charge cloud protects AuNPs from any salt-induced aggregation (Figure 1). When an aptamer binds to its target, it leaves the particle surface, inter-particle distance is reduced, and a salt-induced aggregation takes place, resulting in red-to-purple color transition to 700 nm in less than a minute. This simple mechanism allows us to obtain quantitative binding information by monitoring the optical density at 520 nm (Figure 1 inset). Figure 2 shows the UV-Vis spectrometry data for the OA-34 aptamer and aptamer-target samples. The reduced 520 nm signal indicates good binding of the target by the OA-34 aptamer. We are currently investigating several other aptamer candidates for both okadaic and domoic acid using this assay. Any resulting aptamers that are successfully screened can then be functionalized onto Giner's electrodes and tested. I.B Aptamer Functionalized SPEs Using the OA-34 aptamer a procedure was developed to functionalized gold nanoparticle carbon SPEs.
Publications
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Progress 07/01/20 to 09/30/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the first months of the program several experiments were performed to characterize the okadaic acid (OA) aptamerand target binding. Additionally, a procedure for functionalizing the screen printed electrodes (SPEs) with the aptamer wasdeveloped. The newly modified SPEs were also tested with samples of stock OA to establish a baseline sensitivity and limitof detection (LOD). Our current efforts are focused on investigating the assay methodology and improving the parameters ofthe electrochemical detection step for OA.I.A Aptamer-Target Binding ConfirmationThe initial Phase I work was performed using a an okadaic acid aptamer that had been previously published in researchliterature. The oligo sequence, shown in Table 1, was synthesized using a commercial vendor, Integrated DNA Technologies(Coralville, IA). Using this aptamer sequence we performed a novel colorimetric binding assay to confirm OA aptamer-targetbinding.Table 1. Okadaic acid aptamer OA-34 sequence and dissociation constant.Giner has developed a robust gold nanoplasmonic colorimetric procedure in house to characterize aptamer candidates.Briefly, the citrate-reduced gold nanoparticles (AuNP) possess a negative charge and the electrostatic repulsive forces givethem a characteristic red color (520 nm) in solution. In the presence of negatively charged aptamer and 150 mM NaCl, anegative charge cloud protects AuNPs from any salt-induced aggregation (Figure 1). When an aptamer binds to its target, itleaves the particle surface, inter-particle distance is reduced, and a salt-induced aggregation takes place, resulting in red-to-purple color transition to 700 nm in less than a minute. This simple mechanism allows us to obtain quantitative bindinginformation by monitoring the optical density at 520 nm (Figure 1 inset).Figure 2 shows the UV-Vis spectrometry data for the OA-34 aptamer and aptamer-target samples. The reduced 520 nmsignal indicates good binding of the target by the OA-34 aptamer. We are currently investigating several other aptamercandidates for both okadaic and domoic acid using this assay. Any resulting aptamers that are successfully screened canthen be functionalized onto Giner's electrodes and tested.I.B Aptamer Functionalized SPEsUsing the OA-34 aptamer a procedure was developed to functionalized gold nanoparticle carbon SPEs.
Publications
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