Progress 05/01/18 to 04/30/23
Outputs
Target Audience:The target audience we wish to reach through this proposal consists of fish/seafood consumers, suppliers, government regulators and the broad scientific community. During the five years, we have presented at multiple conferences including government agencies. Unfortunately, we have not made outreach to consumers and suppliers with the pandemic interruptions. We have published many papers, however, and will aim to publish our main paper showing widespread antibiotic contamination in US seafood this year. This will serve as our outreach mechanism to seafood consumers and suppliers. Changes/Problems:Like everyone else, we were severely impacted by the COVID-19 pandemic, which resulted in lab closure and delayed research pace during the 2020-2021 reporting period. However, because a one-year no-cost-extension was granted, we were able to complete and achieve all the goals listed in the original grant. Therefore, in light of everything, the team has succeeded in accomplishing the proposed objectives. One shortfall, however, is our efforts to reach out to seafood consumers and suppliers. We are still working on achieving this objective by publishing our main manuscript on the extent of antibiotic contamination in seafood. What opportunities for training and professional development has the project provided?The project has resulted in the training of many PhD students (per the FTE report) which have since left to pursue careers in industry or academia (post-doc position). How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community and government. We have probably failed in reaching out to seafood consumers and suppliers, but we still hope this goal will be achieved once our paper describing widespread antibiotic contamination in seafood gets published. What do you plan to do during the next reporting period to accomplish the goals?N/A as the grant ended in April of 2023.
Impacts
What was accomplished under these goals?
There were four primary objectives to this proposal. Aim 1 was to develop a field-deployable filter-nanofibrous membrane sensor chip for rapidly isolating and capturing 4 amphenicol antibiotics with a smart phone. Aim 2 was to test a novel surrogate standard-antibody approach for establishing quantitation. Aim 3 was to validate the new technology on standard reference material and compare it to standard FDA-approved methods. Aim 4 was to use the new sensor chip technology to quantify amphenicol exposure levels in off-the-shelf farmed and wild seafood products. We have successfully delivered on Aims 1 and 2. In Aim 1, we developed a nanofibrous membrane sensor that measured chloramphenicol (CAP), florfenicol (FF), thiamphenicol (TAP) and penicillin in spiked salmon with the naked eye, at a detection level of 0.3 ng/mL. This detection level was 33 times greater compared to a conventional ELISA, reflecting the utility of nanofibrous membranes for greatly enhancing sensitivity and improving antibiotic detection. Further optimization of the sensor by immobilizing the antibiotic antibody in larger fiber pore structures made of hydrophilic membranes, resulted in a further 200-fold increase in sensitivity. We also transferred the nanofibrous membrane inside a robotic microfluidics-enabled chamber which enables enhanced and regulated contact of the contaminated salmon extract with the nanofibrous membrane antibodies, resulting in increase binding probability of antibiotics to the antibibody on the membrane. With this method, we were able to quantify CAP at a low detection level of 0.1 ng/mL (100 times more sensitive than conventional ELISA). Importantly, precision ranged from 0.57 to 9.9%, and accuracy was within 90-113%. With regard to Aim 2, we realized that there was no need to use a surrogate standard because the extraction recovery of antibiotics was close to 100% wit our extraction protocol. The protocol involved homogenizing 1 gram of salmon with 3 mL PBS buffer, and filtering the extract through a Millipore filter to remove solid particles. the filtrate was then applied to the nanofibrous membrane. Aim 3 was a challenge. We were not able to compare the nanofiber membrane technology to the conventional mass-spec detection approach. This is because the mass-spec detection limits were 30-160 times greater than the nanofibrous sensor. Also, adding antibiotics to the sensor at levels matching the lower limits of detection of the mass-spec would result in sensor overloading beyond the standard curve. Therefore, direct comparison of the sensor to the mass-spec was not possible to achieve. In Aim 4 we used the mass-spec method to quantify antibiotic residues in 100+ seafood sample of farmed and wildtype origin, and from different countries including the US (i.e. imported versus produced within the US). We found widespread antibiotic contamination ranging from 1 to 70% in the samples assayed. Surprisingly, several antibiotics were more contaminated in wild fish compared to farmed, reflecting widespread environmental contamination. As expected, imported seafood had more antibiotics compared to US-produced seafood. This represents the largest survey of antibiotics in the US to date. The results show widespread antibiotic contamination that needs to be tackled immediately in order to reduce the spread of antibiotic drug resistance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhao C, Pan B, Wang M, Si Y, Taha AY, Liu G, Pan T, Sun G. Improving the Sensitivity of Nanofibrous Membrane-Based ELISA for On-Site Antibiotics Detection. ACS Sens. 2022 May 27;7(5):1458-1466. doi: 10.1021/acssensors.2c00208. Epub 2022 Apr 15.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Effects of carotenoid pigmentation in salmon on antibiotic extraction recovery, matrix effects and accuracy of quantification by ultrahigh performance liquid chromatography coupled to tandem mass spectrometry.
Emami S, Taha AY. J Chromatogr B Analyt Technol Biomed Life Sci. 1216:123585. doi: 10.1016/j.jchromb.2022.123585. PMID: 36669255
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhao C, Si Y, Pan B, Taha AY, Pan T, Sun G. Design and fabrication of a highly sensitive and naked-eye distinguishable colorimetric biosensor for chloramphenicol detection by using ELISA on nanofibrous membranes. Talanta, Volume 217, 1 September 2020, 121054
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhao C, Si Y, Pan B, Shenghan Z, Bradley K, Taha AY, Pan T, Sun G. Diffusion of Protein Molecules through Microporous Nanofibrous Polyacrylonitrile Membranes. ACS Applied Polymer Materials, 3(3):1618-1627, 2021.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhou C, Fang Z, Zhao C, Mai X, Emami S, Taha AY, Sun G, Pan T. Sample-to-Answer Robotic ELISA. Anal Chem. 2021 Aug 24;93(33):11424-11432. doi: 10.1021/acs.analchem.1c01231.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Emami S, Lerno LA, Taha AY. Antibiotic standards stored as a mixture in water: methanol are unstable at various temperatures irrespective of pH and glass container silanization. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Jan;39(1):61-73. doi: 10.1080/19440049.2021.1982150. Epub 2021 Dec 2.
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:The target audience we wish to reach through this proposal consists of fish/seafood consumers, suppliers, government regulators and the broad scientific community. During the reporting year, we have presented to the USDA Food Safety and Inspection Service and to the Environmental Health Safety Center at UC Davis. All presentations were virtual. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two PhD students and one post-doc have been involved in this grant. The post-doc and the PhD student have recently transferred to academic positions. The other PhD student has recently started working as an analytical chemist at Charles River. How have the results been disseminated to communities of interest?The results have been disseminated to the public and government at the USDA Food Safety and Inspection Service, and to scientists at the Environmental Health Safety Core at UC Davis. What do you plan to do during the next reporting period to accomplish the goals?Over the next reporting period we aim to publish our work on antibiotic residue contamination in seafood samples. We also wish to extend the method to milk analysis if time and resources permit.
Impacts
What was accomplished under these goals?
The major goals of the proposed research are to (1) simplify the antibiotic extraction process by developing and validating a nanofibrous membrane sensor for on-site, rapid, inexpensive and simultaneous isolation and quantitaton of 4 amphenicol antibiotics commonly found in seafood imports, and 2) determine the extent of antibiotic contamination in imported seafood products. In prior years, we reported on the utility of the sensor for detecting amphenicol antibiotics in particular. This year, we reported on our efforts to improve on the sensitivity of the sensor by better controlling protein (or antibody) diffusion through the nanonfibrous membrane. Improved protein diffusivity was achieved by making controlled fabrication of the nanofibrous membrane and increasing its lipophilicity, so that proteins can easily go through pores and bind. More protein (or antibody binding) means easier detection of incoming antigens (i.e. antibiotics) from a sample. Another advance we made this year is the use of a miniaturized robotic ELISA that embeds the same nanofibrous technology to measure antibiotics. The robotic ELISA was also testedin salmon extracts spiked with antibiotics, and yielded consistent results with the spike amount. Finally, we reported on the storage, temperature and optimal container conditions to use when quantifing antibiotics with mass-spectrometry. It turns out that antibiotics stored in mixture (i.e. in a standard mixture with others) are unstable at various temperatures for more than a day, because they adsorb to the glass container used to store them. Glass silanization did not improve stability. This means that antibiotic standard mixtures must be prepared and run the same day of the mass-spec run in order accurately measure antibiotics in seafood. This year we completed the measurement of antibiotic contamination in 100+ seafood samples, learning from all the methodological work we did. We are working on compiling the data into a manuscript for publication.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhao C, Pan B, Wang M, Si Y, Taha AY, Liu G, Pan T, Sun G. Improving the Sensitivity of Nanofibrous Membrane-Based ELISA for On-Site Antibiotics Detection. ACS Sens. 2022 May 27;7(5):1458-1466. doi: 10.1021/acssensors.2c00208. Epub 2022 Apr 15.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhou C, Fang Z, Zhao C, Mai X, Emami S, Taha AY, Sun G, Pan T. Sample-to-Answer Robotic ELISA. Anal Chem. 2021 Aug 24;93(33):11424-11432. doi: 10.1021/acs.analchem.1c01231.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Emami S, Lerno LA, Taha AY. Antibiotic standards stored as a mixture in water: methanol are unstable at various temperatures irrespective of pH and glass container silanization. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Jan;39(1):61-73. doi: 10.1080/19440049.2021.1982150. Epub 2021 Dec 2.
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:The target audience we wish to reach through this proposal consists of fish/seafood consumers, suppliers, government regulators and the broad scientific community. During the reporting year, we did not reach any of our target audience due to restrictions imposed by COVID-19. We hope to ramp up these efforts during the 2021-2022 reporting year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has so far supported two PhD trainees who are mentored the by the 2 PIs on the project (Taha and Sun). The research is highly interdisciplinary and it is projected that it will further the professional development of trainees involved. 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?As outlined in the grant proposal, our goal over the next year is to finish testing how the sensor output compares to UPLC-MS/MS, having validated the UPLC-MS/MS method. Our second goal is to probe antibiotic levels in imported versus domestic seafood, to determine the extent of antibiotic contamination.
Impacts
What was accomplished under these goals?
The major goals of the proposed research are to (1) simplify the antibiotic extraction process by developing and validating a hand-held seonsor for on-site, rapid, inexpensive and simultaneous isolation and quantitaton of 4 amphenicol antibiotics commonly found in seafood imports, and 2) determine the extent of antibiotic contamination in imported seafood products. To date, we have developed a hand-held sensor that incorporates antibodies embedded onto porous nanofibrous membranes to detects amphenicols. Progress has been stalled for at least 6 months due to COVID-19 restrictions. However, we have made progress on several fronts. First, we continued to explore the properties of the nanofibrous membrane sensor using surrogate antibodies, as reported in our recent publication in ACS Applied Polymer Materials. We found that the membrane pore size must be at least 1000-fold greater than the protein size in order to diffusion to be consistent across the membrane and not variable. This is key for efficiently and evenly immobobilizing antibiotic antibodies in the nanofibrous membrane sensor. We will test the pore size ratio in follow-up studies using amphenicol antibodies to improve the sensitivity of the sensor. Parallel to this, we've developed and optimized the UPLC-MS/MS measurement. As mentioned in last year's progress report, we discovered that analytical standards need to be prepared fresh before running them on UPLC-MS/MS; this is because they adhere to the sides of the glass, irrespective of the degree of salinization. Upon overcoming this hurdle, we established LODs, LOQs and standard spike recoveries for the antibiotic UPLC-MS/MS method. Finally, we initiated an experiment to compare the sensor to the UPLC-MS/MS method.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhao C, Si Y, Pan B, Shenghan Z, Bradley K, Taha AY, Pan T, Sun G. Diffusion of Protein Molecules through Microporous Nanofibrous Polyacrylonitrile Membranes. ACS Applied Polymer Materials, 3(3):1618-1627, 2021.
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Progress 05/01/19 to 04/30/20
Outputs
Target Audience:The target audience we wish to reach through this proposal consists of fish/seafood consumers, suppliers, government regulators and the scientific community. I have communicated our findings of our preliminary surveying of antibiotic residues in seafood, to program directors from the USDA and FDA. We have published a paper in Talenta, communicating our advancements in the antibiotic sensor development. Changes/Problems:In year 1, we discovered that other antibiotics (besides amphenicols) were detected in off-the-shelf supermarket seafood products. Hence, we optimized the UPLC-MS/MS method to probe for approximately 30 antibiotics used in aquaculture. However, as mentioned above, we experienced problems with the standard stability and determined that they had to be freshly prepared in order to obtain reproducible data. None of this affects the budget or stated objectives in the grant proposal. What opportunities for training and professional development has the project provided?The project has so far supported two PhD trainees who are mentored the by the 3 PIs on the project (Taha, Sun and Pan). The research is highly interdisciplinary and it is projected that it will further the professional development of trainees involved. 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?As outlined in the grant proposal, our goal over the next year is to test how the sensor output compares to UPLC-MS/MS data. To do so, we need to first validate the UPLC-MS/MS method. Now that we know that the analytical standards need to be freshly prepared in order to ensure data reproducibility, we will test the extent of matrix effects and standard recoveries from spiked seafood samples, establish the LOD and LOQ, and determine the inter- and intra-day variability. Once the UPLC-MS/MS method is validated, we will compare it to the sensor data.
Impacts
What was accomplished under these goals?
The major goals of the proposed research are to (1) revolutionize the antibiotic extraction process by developing and validating a hand-held seonsor for on-site, rapid, inexpensive and simultaneous isolation and quantitaton of 4 amphenicol antibiotics commonly found in seafood imports, and 2) determine the extent of antibiotic contamination in imported seafood products. To date, we have developed a hand-held sensor that incorporates antibodies embedded onto porous nanofibrous membranes to detects amphenicols. Over the past year, we substantially improved the sensitivity of the sensor and showed that it is at least 10-fold higher than conventional ELISA assays. We also confirmed that the sensor can detect chloramphenicol, in spiked seafood residues. Lastly, we determined that quantitation is feasible at very low concentrations, using an external standard curve. Parallel to these efforts, we also initiated the validation process of antibiotics measurements on UPLC-MS/MS. Our current analytical method can detect 30 antibiotics (including amphenicols). However, matrix effects and standard recoveries, which are important aspects of the quantitative analysis, have not been determined. In our attempt to assess these, it was realized that the signal intensity from the analytical standards was not reproducible. Further experimentation revealed that the stock standard solutions were unstable and needed to be freshly prepared prior to running them on UPLC-MS/MS. We are preparing a manuscript to disseminate this important technical finding. We will continue to develop and improve the UPLC-MS/MS quantitative method, so we can validate our new nanofibrous membrane sensor method.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhao C, Si Y, Pan B, Taha AY, Pan T, Sun G. Design and fabrication of a highly sensitive and naked-eye distinguishable colorimetric biosensor for chloramphenicol detection by using ELISA on nanofibrous membranes. Talanta, Volume 217, 1 September 2020, 121054
|
Progress 05/01/18 to 04/30/19
Outputs
Target Audience:The target audience we wish to reach through this proposal consists of fish/seafood consumers, suppliers and government regulators. In our preliminary survey of antibiotic residues in seafood, we met and presented our findings to the USDA and FDA. We have not reached out to a broad audience as of yet because our findings are preliminary and need further replication (ongoing). Changes/Problems:In view of our discovery that other antibiotics, besides amphenicols were detected in off-the-shelf supermarket seafood products, we will optimize our extraction and analytical UPLC-MS/MS methods to probe for approximately 30 antibiotics used in aquaculture. This should not affect our budget. What opportunities for training and professional development has the project provided?The project has so far supported two PhD trainees that are mentored the by the 3 PIs on the project (Taha, Sun and Pan). The research is highly interdisciplinary and it is projected that it will further the professional development of trainees involved. How have the results been disseminated to communities of interest?Too preliminary to report at this point, without a conclusion. We did, however, report our progress to date with program directors from the USDA and FDA. What do you plan to do during the next reporting period to accomplish the goals?When we submitted the grant, our focus was amphenicols. However, due to the sensitivity and broad coverage of UPLC-MS/MS techniques, we were able to detect 'other' types of antibiotics in seafood (e.g. azithromycin). We also detected florfenicol. While these findings are preliminary in nature, we will continue to probe for amphenicols and other antibiotic classes with UPLC-MS/MS. Currently, we are optimizing the extraction method of at least 30 antibiotics, in order to improve their recovery and analytical detectability with UPLC-MS/MS. The sensor work will continue as planned, focusing on validating amphenicol levels it on seafood samples. To date, the sensor has been validated using standards only.
Impacts
What was accomplished under these goals?
The major goals of the proposed research are to revolutionize the antibiotic extraction process by developing and validating a hand-held lab-on-a-chip device for on-site, rapid, inexpensive and simultaneous isolation and quantitaton of 4 amphenicol antibiotics commonly found in seafood imports, and 2) determine the extent of antibiotic contamination in imported seafood products. To date, we have developed the hand-held device to incorporate the 4 amphenicols of interest. We have confirmed using standards that the hand-held sensor can detect them. Our next step is to test the device on real seafood samples. Parralel to this, we have developed a UPLC-MS/MS method for measuring 32 antibiotics (including amphenicols) in seafood. We started by surveying a few seafood samples (6 only) but realized that we need to optimize the extraction process further to improve antibiotic yield (particularly for amphenicols). Ultimately, we hope to tackle the tissue / matrix effects so we can validate the sensor on them using our UPLC-MS/MS methods.
Publications
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