Progress 07/01/12 to 06/30/16
Outputs
Target Audience:1. People attended the 2014 Rocky Mountain Food Safety Conference and the 118th Association of Food and Drug Officials (AFDO) Annual Educational Conference, including food safety professionals, food service managers, retail managers, students etc. 2. People attend 2015 Pittcon including scientist, educator and students related to food and agriculture, chemistry, biology. 3. People attended International Conference & Expo on Smart Materials and Structures, including students, scientists and educators in material science, food science, environmental and engineering. 4. people read the research papers published on scientific journals. Changes/Problems:The original plan of this project includes detecting the ELISA product (a colored compound) using optical absorption spectrometry with a 2 meters liquid core waveguide (LCW) as a sample cell, which if successed can increase the sensitivity of ELISA by more than100 times. However, it was discovered that ELISA reaction results in a blank signal, which is also amplified by the LCW spectrometric method. The concentration of the colored compound in a blank sample of ELISA is very low and is not a problem in traditional ELISA test using plate reader or traditional UV/Vis spectrometer. However, when the LCW is used in the detection, the colored compound in the blank solution absorbs almost all the light which causes a high noise signal in measuring light absorption signal. The PI switched to fiber optic immuno-separation with on fiber laser indueced fluorescence technqiue and achieved the detection of 1.0*10e4 molecules, which is about three orders of magnitude lower than that achieved with traditional ELISA techniques. What opportunities for training and professional development has the project provided?1. Four undergraduate students from West Texas A&M University (WTAMU) and University of Texas involved in the research activity of this project. The PI provided training on bioanalytical technologies and food safety test techniques to these students. The PI also provide traning on how to do scientific research to these students. These training significantly helped students in thier career development. One of the students involved in this project has graduated from WTAMU and was employed as a scientist by a federal agent (Department of Energy) funded research institute. A second student is graduating from WTAMU and has been enrolled in an engineering PhD program in a research university. The student from University of Texas has been enrolled in a medical school. The fourth student graduated from WTAMU and has been enployed as a teacher in a regional high school. 2. Three undergraduate students who signed up research courses with the PI has been involved technology development related to this project. One of the students graudauted from WTAMU and has been enrolled in a PhD program in another high education institute in Texas. The other two students are applying for medical schools. How have the results been disseminated to communities of interest?The research discoveries were disseminated to communities by presenting in related conferences/events, publishing papers on peer-reviewed scientific journals. One of the papers was published on an open-access journal (Nano Research & Applications) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Developed an IMS technique for successfully separating Salmonella bacteria from raw egg samples. 2. Integrated IMS separation with ELISA for detecting Salmonella bacteria in raw egg samples and achieved a detection limit of 7.0*10e4 Salmonella cell/mL sample solution. 3. Developed a new immuno-separation technique using antiboby-coated optical fibers as supporting material for separating food-borne pathogenic bacteria. 4. Developed fiber optic fluorescence tecniques for detecting fluorescence compound used to label pathogenic bacteria in immuno fluorescence assay and achieved the detection of 1.0*10e4 fluorescence molecules trapped on optical fiber surface.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
C. A. Guyer, M. Alex and S. Tao, Magnetic micro-particle immune separation combined with enzyme linked immunosorbent assay for quick detection of Salmonella typhimurium in raw eggs , Nano Research & Applications, 2, 13:1-13:6 (2016).
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
J. C. Ray, M. S. Almas and S. Tao, Exciting fluorescence compounds on an optical fibers side surface with a liquid core waveguide , Optics Letters, 41, 100-103 (2016).
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
C. A. Guyer and S. Tao, Total internal reflection fluorescence spectrometry using a dual optical fiber sample cell , Applied Spectroscopy, 69, 608-612 (2015).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
C. A. Guyer, S. Tao and M. Alex, "Immunomagnetic separation combined with enzyme linked immunosorbent assay for quick detection of Salmonella typhimurium in raw eggs", 2014 Rocky Mountain Food Safety Conference and the 118th Association of Food and Drug Officials
(AFDO) Annual Educational Conference
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
S. Tao, C. A. Guyer and M. Alex, "Immunomagnetic Separation Combined with Enzyme Linked Immunosorbent Assay for Quick Detection of Salmonella Typhimurium in Raw Eggs", 2015 PittCon Abstract# 21008, 2015 Pittcon Conference & Expo, March 8-12, 2015, New Orleans, Louisiana
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
C. A. Guyer, S. Tao and M. Alex, "Immunomagnetic separation combined with enzyme linked immunosorbent assay for quick detection of Salmonella in raw eggs", International Conference and Expo on Smart Materials and Structures, June 15-17, 2015 Las Vegas, NV.
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Progress 07/01/14 to 06/30/15
Outputs
Target Audience:scientists in field of agriculture and material related research. Changes/Problems:It was found that immunoseparation using magnetic particles as antibody carrier for separating pathogenic bacteria from raw egg has many problems, including loss of magnetic particles to sample, non-specific adsorption of proteins to magnetic particle surface, and long reaction/washing time needed. In addition, the existence of magnetic particles in solution interfere the detection of pathogens using any optical spectroscopic method, and therefore, must be separated before optical spectroscopic detection. A major change of technical approach is to use an optical fiber as an antibody carrier for immunoseparation. It is much easier to separate pathogen-captured optical fibers from food sample and wash the optical fibers. In addition, an optical fiber is an excellent optical cell for optical spectroscopic detection. There is no need of separating antibody carrier before optical spectroscopic detection of captured pathogens. The detection limit of the fiber optic fluorescence technique I developed is also much lower than detection limit of present ELISA techniques. What opportunities for training and professional development has the project provided?Three undergraduate students have participated in the research activity of this project during the reporting period. The students had hand-on experience in developing fiber optic immunofluorescence spectroscopic techniques, and gained broad spectrum of knowledge about food safety issue, biotechnologies, laser spectroscopy. How have the results been disseminated to communities of interest?The discovery of research results has been published or submitted for publication on peer-reviewed scientific journals and presented in international scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?The fiber optic fluorescence technique for detecting fluorescence molecules absorbed on fiber surface developed from the work of this reporting period will be furhter modified for detecting pathogenic bacteria using bioaffinity optical fibers for trapping target bacteria from food samples to fiber surface. Chemical/biochemical techniwques will be developed for coating antibody of pathogenic bacteria on surface of optical fibers. The conditions for using the obtained bioaffinity optical fiber for separating pathogenic bacteria from food samples will be investigated. A fluorescence conjugated antibody will be used to label the bacteria trapped on optical fiber surface. The fiber optic fluorescence technique developed from this past reporting period will be tested for detecting bacteria trapped on optical fiber surface.
Impacts
What was accomplished under these goals?
A new fiber optic fluorescence spectroscopic method using a liquid core waveguide (LCW) as an excitation element has been developed for detecting a fluorescence compound absorbed on optical fiber surface. A laser light beam was coupled into a multimode silica optical fiber by using a focus lens. The distal end of the fiber was inserted into an LCW. The diverging light emerged from the fiber's distal end was collected and guided within the LCW. A tapered optical fiber was inserted into the LCW from the other side. Laser light traveling in the LCW evenly illuminates the tapered fiber surface and excites fluorescence molecules absorbed on the fiber's surface. Fluorescence light emitted from the molecules on tapered fiber surface was collected with the tapered fiber, and delivered through the fiber to an optical fiber compatible spectrometer for detection. This new technique provides an efficient way for evenly exciting fluorescence compounds absorbed on an optical fiber's surface. A chemical process was developed for coating a single molecular layer of ImmunoPure NeutrAvidin on the surface of tapered optical fibers. A fluorescence reagent conjugated biotin (Atto 550-biotin) was used as a sample in this work. Preliminary test results indicate that this newly developed fiber optic fluorescence technique can detect 1.0*104 Atto 550-biotin molecules absorbed on a tapered optical fiber surface through avidin-biotin reaction.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Cole A. Guyer and Shiquan Tao, "Total Internal Reflection Fluorescence Spectrometry Using a Dual Optical Fiber Sample Cell", Applied Spectroscopy, 69, 608-612 (2015)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Jason C. Ray, Muhammad S. Almas and Shiquan Tao, "Exciting fluorescence compounds on an optical fibers side surface with a liquid core waveguide", submitted to Optics Letters.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
S. Tao, M. Alex and C. Guyer, "Immunomagnetic Separation Combined with Enzyme Linked Immunosorbent Assay for Quick Detection of Salmonella Typhimurium in Raw Eggs", Abstract# 2100-8, 2015 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 8-12, 2015 New Orleans, LA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
S. Tao, C. Guyer and M. Alex "Immunomagnetic Separation Combined with Enzyme Linked Immunosorbent Assay for Quick Detection of Salmonella Typhimurium in Raw Eggs", Proceedings of International Conference on Smart Materials & Structures, June 15-17, 2015, Las Vegas
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Cole A. Guyer and Shiquan Tao, "Total Internal Reflection Fluorescence Spectrometry Using a Dual Optical Fiber Sample Cell", Abstract has been accepted for publication on The International Chemical Congress of Pacific Basin Societies 2015.
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Progress 07/01/13 to 06/30/14
Outputs
Target Audience: People attended the 2014 Rocky Mountain Food Safety Conference and the 118th Association of Food and Drug Officials (AFDO) Annual Educational Conference, including food safety professionals, food service managers, retail managers, students etc. Changes/Problems: I am starting developing a fiber optic immunoseparation technique for separating Salmonella bacteria from raw egg samples. A major problem I found out from the work of the past reporting period is that it is difficult to recover all the magnetic microparticles added to a sample such as a raw egg, which contains high concentration of macrobiomolecules and is high viscosity. The loss of magnetic microparticles to sample matrix will cause false negative results. I plan to develop a technique to covalently bind anti-Salmonella antibogy on surface of silica optical fibers or organic polymer optical fibers, using these antibody coated optical fibers for immunoseparating Salmonella bacteria from raw egg samples. The bacteria-trapped optical fibers can be easily separated from sample matrix. The DOF-TIRF technique developed from my work during the past reporting period will be used for detecting the bacteria trapped on optical fibers after labeling with a fluorescence compound. What opportunities for training and professional development has the project provided? Three West Texas A&M University (WTAMU) students involved in this research project. I trained the students to do research on immunomagnetic separation, ELIZA test, as well as to use a high-power (class IV) laser and fiber optic spectroscopic instruments. 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? My laboratory research work for this project during next reporting period will be focused on: 1. develop a technique for coating anti-Salmonella antibody on surface of silica optica fibers. 2. investigate the feasibility of using such an antibody coated optical fiber for immuno separation of Salmonella bacteria from raw egg samples 3. develop a new ELISA technique, fiber optic ELISA, for detecting Salmonella bacteria trapped on an optical fiber. The substrate of the labeling enzyme for the ELISA test will be dissolved into a polymer solution. The substrate-containing polymer is then coated on the surface of bacteria-trapped optical fiber. The enzyme catalyzed reaction product(s) will be continuously monitored by using fiber optic spectroscopic techniques. 3. develop a fiber optic sandwitch fluorescence immunoassay method using DOF-TIRF as the fluorescence detection technique for detecting Salmonella bacteria trapped on optical fiber surface. I'll continue to train WTAMU undergraduate students involved in this research project to develop scientific research capability. I plan to present two oral presentations related to work of this project in 2015 Pittsburgh conference on analytical chemistry and analytical spectroscopy. I also plan to present one oral presentation related to work of this project in Pacifichem 2015 conference. I plan to submit one to two papers reporting discovery made from the work of thisw project to peer-reviewed journals for publication.
Impacts
What was accomplished under these goals?
During this reporting period my work is focused on developing a highly sensitive fiber optic fluorescence spectroscopic technique:dual optical fiber total internal reflection fluorescence (DOF-TIRF) spectroscopy. TIRF is ad advanced fluorescence technique , in which fluorescence molecules on the surface of an optical waveguide are excited by the evanescence wave (EW) of excitation light guided inside the optical waveguide. The excitation light and the fluorescence photons are spatially separated. Therefore, a lens used to collect the fluorescence photons will not pick up scattered excitation light, which is the major noise signal when an intensive light source, such as a laser, is used for excitation. In TIRF, the diffuse reflection caused noise is negligible as long as a high quality waveguide (a prism or a microscope slide) is used as an excitation element. Due to its extremely low noise level, TIRF has achieved single molecular detection (SMD). Planar waveguide and prism have been used in TIRF as optical waveguides in reported TIR fluorescence microspectroscopy. An optical fiber is a cylindrical optical waveguide. Light is guided inside an optical fiber through TIRs. If a fluorescence molecule exists on the surface of an optical fiber core the molecule can be excited by the EW of light guided within the fiber core to emit fluorescence photons. Theoretically, this optical fiber TIRF phenomenon can be used for detecting the fluorescence compound. However, with traditional fluorescence detection methods, such as using a large lens to collect the optical fiber TIRF fluorescence photons, the lens will collect fluorescence photons from only a small area where the fiber is located, but also pick up noise photons from a large area where there is no optical fiber. In this paper, we present a special method for efficiently collecting the optical fiber TIRF photons. The new technique developed from my work uses a liquid core waveguide (LCW), which is a special optical fiber, to collect the TIRF photons. The efficiency of LCW for collecting optical fiber TIRF photons is much higher than that can be achieved with a traditional method using an optical lens. The excitation optical fiber is deployed inside an LCW, and emitted fluorescence photons are collected by the LCW and delivered to a highly sensitive photon detector. Because the LCW collects only the fluorescence photons, a very low noise fluorescence spectroscopic detection can be achieved. This new fluorescence technique will be integrated with an optical fiber immunoseparation method, which I plan to develop in the near future for detecting food pathogens.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Immunomagnetic separation combined with enzyme linked immunosorbent assay for quick detection of Salmonella typhimurium in raw eggs
Cole Guyer, Shiquan Tao*
Department of Mathematics, Chemistry and Physics, West Texas A&M University,
Canyon, TX 79016, USA (* correspondence author e-mail: stao@wtamu.edu)
Maya Alex
School of Medicine, University of Texas at El Paso, El Paso, TX 79968, USA
Abstract: An immunomagnetic separation (IMS) method was developed for separating Salmonella typhimurium bacteria from large-volume samples of raw eggs. An egg was homogenized with a blender. The homogenized egg material was diluted with a 0.05% Triton X-100 solution to make a 200 mL sample mixture. Anti-Salmonella typhimurium antibody coated magnetic micro particles (MMP) were used to trap the Salmonella bacteria in the sample mixture. The Salmonella-trapped MMP were separated from the egg sample mixture by using a large magnet. An enzyme-linked immunosorbent assay method was adopted and revised for detecting the bacteria trapped onto the MMP. A horseradish peroxidase (HRP) labeled anti-Salmonella typhimurium antibody was used to label the trapped bacteria, and a SureblueTM solution was used as a substrate. The color compound resulted from HRP-catalyzed reaction was detected with UV/Vis absorption spectrometry using a 1 cm sample cell. This simple method can detect 1.4*107 Salmonella typhimurium cells in one raw egg (7.0*104 Salmonella typhimurium cells/mL in a sample mixture) without any pre-enrichment. The results presented in this paper demonstrate the feasibility of using IMS for separating bacteria from large volume complex samples, which could be adopted for detecting bacteria in other type samples in food safety inspection.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Immunomagnetic separation combined with enzyme linked immunosorbent assay for quick detection of Salmonella in raw eggs
Cole Guyer1, Maya Alex2, Shiquan Tao1*
1 Department of Mathematics, Chemistry and Physics, West Texas A&M University,
Canyon, TX 79016, USA
2 School of Medicine, University of Texas at El Paso, El Paso, TX 79968, USA
Abstract: An immunomagnetic separation (IMS) method was developed for separating Salmonella bacteria from large-volume samples of raw eggs. An egg was homogenized with a blender. The homogenized egg material was diluted with a 0.05% Triton X-100 solution to make a 200 mL sample mixture. Anti-Salmonella typhimurium antibody coated magnetic micro particles (MMP) were used to trap Salmonella typhimurium bacteria in the sample mixture. The Salmonella-trapped MMP were separated from the egg sample mixture by using a large magnet. An enzyme-linked immunosorbent assay method was adopted and revised for detecting the bacteria trapped onto the MMP. Horseradish peroxidase labeled anti-Salmonella typhimurium antibody was used to label the trapped bacteria, and a SureblueTM solution was used as a substrate. The color compound resulted from horseradish peroxidase catalyzed reaction was detected with UV/Vis absorption spectrometry using a 1 cm sample cell. This simple method can detect 1.4*107 Salmonella typhimurium cells in one raw egg (7.0*104 Salmonella cells/mL in a sample mixture) without any pre-enrichment. The method is quick, can obtain test results within 5 hours. The results presented in this poster demonstrate the feasibility of using IMS for separating bacteria from large volume complex samples, which could be adopted for detecting bacteria in other type samples in food safety inspection.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Total Internal Reflection Fluorescence Spectrometry Using a Dual Optical Fiber Sample Cell
Cole A. Guyer and Shiquan Tao* (e-mail: stao@wtamu.edu)
Department of Mathematics, Chemistry & Physics, West Texas A&M University, WTAMU Box 60787, Canyon, TX 79016
Abstract: A novel total internal reflection fluorescence (TIRF) spectrometric technique using a dual optical fiber sample cell was developed. A conventional silica optical fiber was used for exciting fluorescence compounds in its evanescence wave (EW) field. A liquid core waveguide (LCW) was used to collecting the fluorescence photons emitted from fluorescence compounds existing in excitation fibers EW field. The collected fluorescence photons were guided through the LCW and sent to a fluorescence spectrometer for detection. The spatially separation of excitation light and fluorescence light reduces the excitation light related optical noise signal, which is the major factor limiting fluorescence techniques from achieving lower detection limit. Preliminary results obtained from this work indicate that the optical fiber TIRF system of this work can detect 4.6*10-18 mole rhodamine 6G (2.7*106 molecules) existing in the EW field of the excitation optical fiber.
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Progress 07/01/12 to 06/30/13
Outputs
Target Audience: 1. Students at West Texas A&M University who taking PI's classes. 2. Students and faculty of West Texas A&M University who attanded PI's seminar presentation in department of mathematics, chemistry and physics at West Texas A&M University. Changes/Problems: A major change is the method of separating Salmonella-trapped micromagnet particles (MMP) from raw egg sample matrix. In my original design, the Salmonella-trapped MMP in raw egg sample will be flowed through a tube, which has a small open in one side of the wall. A large magnet is attached to the wall of the tube to pull the MMP out of the egg sample. It was found from experiment that this method doesn’t work. The new method I developed is placing a 1” diameter N50 magnet disc inside a plastic zip bag, and deploying the magnet in the zip bag to an egg sample mixture inside a beaker to pull up the MMP. What opportunities for training and professional development has the project provided? 1. One undergraduate student in West Texas A&M University has been hired to work on this project. Thisprovides the student an opportunity to develop his skills in scientific research. 2. ELISA has been taught as part of PI's upper level and graduate courses in West Texas A&M University (total student enrollment: 36) 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? My work in next reporting period will be focused on: 1. Developing more sensitive detection technologies, such as liquid core waveguide optical spectrometry, fiber optic laser induced fluorescence spectrometry, fiber optic chemiluminescence, for detecting Salmonella separated from raw egg samples. 2. Build up liquid core waveguide fiber optic spectrometric device as a detector for my IMS-ELISA method. 3. Improving the IMS-ELISA method. 4. Test the IMS-ELISA method with new detecting device for field applications
Impacts
What was accomplished under these goals?
Major activity completed The work of this project in this reporting period is focused on the development of an immunomagnetic separation (IMS) method for directly separating Salmonella bacteria from raw egg.Experiments have been done to investigate reaction of anti-Salmonella antibody coated on micro magnetic particles (MMP) with Salmonella in raw egg samples.A method for separating Salmonella-trapped MMP from raw egg sample matrix has also been developed. An enzyme linked immunosorbent assay (ELISA) method with UV/Vis optical absorption spectrometry has been developed for detecting Salmonella bacteria trapped on MMP which have been separated from raw egg samples. Specific objectives met The developed IMS-ELISA method can be used to directly separate Salmonella bacteria from raw egg (diluted with a surfactant solution).The whole IMS-ELISA process takes less than 5 hours.With standard UV/Vis optical absorption spectrometry as a detection method, the developed IMS-ELISA can detect 1.1*107 Salmonella cells in one raw egg. Significant results achieved A: directly separate Salmonella bacterial from raw egg. B: The developed IMS-ELISA method achieved detecting Salmonella in raw eggs in 5 hours. Key outcomes or other accomplishments realized Key outcome: A method has been developed for using anti-Salmonella antibody coated MMP to directly separate Salmonella from raw egg samples. An ELISA method has been developed for detecting the separated Salmonella cells. The developed IMS-ELISA method can be used for directly quick detection (<5 hours) of Salmonella in raw eggs. Problems and further work: With traditional UV/Vis optical absorption spectrometry as a detection method, the developed IMS-ELISA achieved a detection limit of 1.1*107 Salmonella cells/egg, which is still not sensitive enough for food inspection program. Our work in next reporting period will be focused on developing more sensitive detection technologies, such as liquid core waveguide optical spectrometry, fiber optic laser induced fluorescence spectrometry, fiber optic chemiluminescence, for detecting Salmonella separated from raw egg samples.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Title: Immunomagnetic separation combined with enzyme linked immunosorbent assay for quick detection of Salmonella in raw eggs
Abstract: An immunomagnetic separation (IMS) method was developed for separating Salmonella bacteria from large-volume samples of raw eggs. The yolk and white from a raw egg was diluted with a 0.05% Triton X-100 solution to make a 200 mL sample mixture. Anti-Salmonella antibody coated magnetic micro particles (MMP) were used to trap Salmonella bacteria in the sample mixture. The Salmonella-trapped MMP were separated from the egg sample mixture by using a large magnet. An enzyme-linked immunosorbent assay method was adopted and revised for detecting the bacteria trapped onto the MMP. Horseradish peroxidase labeled anti-Salmonella antibody was used to label the trapped bacteria, and a SureblueTM solution was used as a substrate. The color compound resulted from horseradish peroxidase catalyzed reaction was detected with UV/Vis absorption spectrometry using a 1 cm sample cell. This simple method can detect 1.1*107 Salmonella cells in one raw egg (5.5*104 Salmonella cells/mL in a sample mixture) without any pre-enrichment. The results presented in this paper demonstrate the feasibility of using IMS for separating bacteria from large volume complex samples, which could be adopted for detecting bacteria in other type samples in food safety inspection.
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