Source: UNIV OF MASSACHUSETTS submitted to
DEVELOPMENT OF A LABEL-FREE SERS MAPPING BASED PLATFORM FOR MULTI-BACTERIAL DETE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005211
Grant No.
2015-67021-22993
Project No.
MASW-2014-03641
Proposal No.
2014-03641
Multistate No.
(N/A)
Program Code
A1511
Project Start Date
Feb 1, 2015
Project End Date
Jan 31, 2019
Grant Year
2015
Project Director
He, L.
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Food Science
Non Technical Summary
The increased incidence of food pathogen outbreaks placed a new emphasis on the requirement of a rapid, sensitive, and reliable detection method for pathogens in food samples. The overall goal of this project is to develop a detection platform for pathogens in foods that is superior to the current detection methods, in terms of analytical time, sensitivity, and accuracy. The platform is based on a technique called Surface enhanced Raman spectroscopy (SERS). It is a combination of Raman microscopic and nano-techniques. Raman microscopy can identify bacterial cells based on their biochemical signatures within a few seconds. The use of a nanosubstrate can enhance the Raman signals tremendously, reaching a single cell detection limit. In this proposal, a novel SERS platform is developed and evaluated for rapid detection of a mixture of two important food pathogens, Salmonella enterica and Listeria monocytogenes, in milk and ground beef. After some simple steps of food sample pretreatment, bacteria cells will be captured and concentrated by the capturers conjugated onto the nanosubstrate. The identification of will be based on the Raman biochemical signatures of the cells. The quantification of will be based on the mapping technique. With the developed SERS platform, we expect to concentrate, identify and quantify multiple bacterial cells from food samples before them being distributed further. This will improve the long-range sustainability of U.S. agriculture and food systems by reducing the economic loss due to the product recalls and minimizing the health risk caused by food pathogens.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240102000100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
2000 - Chemistry;
Goals / Objectives
The overall goal of this project is to develop a platform for pathogen detection in foods that is superior to the current detection methods, in terms of analytical time, sensitivity, and accuracy, which is based ona novel label-free surface enhanced Raman scattering (SERS) mapping technique. With the developed SERS platform, we expect to concentrate, identify and quantify multiple bacterial cells from food samples before them being distributed further. This will improve the long-range sustainability of U.S. agriculture and food systems by reducing the economic loss due to the product recalls and minimizing the health risk caused by food pathogens.Objectives:Determine the best capturers for bacteria concentration and SERS identification.Optimize the SERS mapping method for cell quantification.Evaluate and optimize sample pretreatments for bacterial capture and SERS identification.Fabricate SERS slides for multi-bacterial detection in food samples.
Project Methods
Determine the best capturers for bacteria concentration and SERS identification. Salmonella enterica and Listeria monocytogenes will be used as the targets. Selective capturers (antibodies and aptamers) and nonselective capturers (antibiotics and antimicrobial peptides) will be conjugated onto silver dendrites and their performance will be evaluated. Capture capacity will be tested using the traditional plate count methods. SERS identification capacity will be evaluated by principal component analysis. Both live and dead cells (thermal inactivated) will be tested for SERS identification. The best capturers will be determined for each bacterium and bacterial mixture.Optimize the SERS mapping method for cell quantification. Different parameters of mapping will be applied and optimized for rapid and accurate quantification.Evaluate and optimize sample pretreatments for bacterial capture and SERS identification. Milk (an example of liquid food) and ground beef (an example of solid food) will be tested in this study. The sample pretreatments (purification and/or enrichment) will be evaluated and optimized for the best detection.Fabricate SERS slides for multi-bacterial detection in food samples. SERS slides with capturer functionalized silver dendrites will be fabricated and evaluated for detection of a mixture of Salmonella and Listeria from food samples after the optimized pretreatments.

Progress 02/01/15 to 01/31/19

Outputs
Target Audience:food industry, private labs, governmental agencies who want to have a rapid detection method for pathogens in food; researchers and students who are interested in learning and applications of nanotechnology in agriculture and food systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students and postdocs are trained through research activities and participation in conferences and short courses. The work on the smartphone detection of bacteria also ledto the initiative of establishing a start-up company (in progress) where students engaged had opportunities for the entrepreneurtraining program on campus. How have the results been disseminated to communities of interest?We published total of 6 papers and two master thesesand presented in more than 10 conferences, anticipate to have two phd dissertations from this project this summer. The work on the smartphone detection of bacteria has been reported by more than 10 media. We also did a Raman/SERS short course open to the public to teach some basics of Raman/SERS and their applications to study chemicals,microbes, and engineered nanomaterials in environmental and food matrices. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We developed several platforms for detecting bacteria in environmental, agricultureand food matrices. 1. a SERS based sandwich assay for total bacteria detection in a well plate 2. two SERS based mapping methods for detecting total and specific bacteria on a filter membrane, respectively. 3. a SERS based method for detecting bacteria on/in plant leaves in situ 4.an optical method to detect bacteria on a chip

Publications

  • Type: Other Status: Published Year Published: 2018 Citation: Invited talk. Lili He. Rapid detection of bacteria using a smartphone. USDA-Food Safety and Inspection Service (FSIS) seminar. June 27, 2018.
  • Type: Other Status: Published Year Published: 2018 Citation: Inivited talk. Lili He. Rapid detection of bacteria using a smartphone. Partnership for Food Safety Education meeting. May 15, 2018.
  • Type: Other Status: Published Year Published: 2018 Citation: Inivted talk. Lili He. Rapid detection of bacteria using a smartphone. Information & Communication Technology Summit. March 23, 2018. UMass Amherst
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Michael Hickey and Lili He. "Raman Surveillance of Bacterial Communities in situ on plant surfaces". Eastern analytical symposium. Nov. 2018. NJ.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Madeline Tucker. Development of methodology for rapid bacteria detection in complex matrices using SERS.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Gao, Siyue, Brooke Pearson, and Lili He*. Mapping bacteria on filter membranes, an innovative SERS approach. Journal of microbiological methods 147 (2018): 69-75.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Siyue Gao and Lili He. Poster presentation: Mapping Salmonella on a filter membrane using SERS imaging. IAFP annual meeting. July 2018. Salt Lake City, UT.
  • Type: Other Status: Published Year Published: 2019 Citation: Invited talk. Lili He. Rapid detection of bacteria using a smartphone. Connecticut Department of Public Health monthly meeting. Feb 4, 2019.


Progress 02/01/17 to 01/31/18

Outputs
Target Audience:food industry, private labs, governmental agencies who want to have a rapid detection method for pathogens in food; researchers and students who are interested in learning and applications of nanotechnology in agriculture and food systems. Changes/Problems:We requested a no-cost extension to finish up the aptamer based detection work. What opportunities for training and professional development has the project provided?Three graduate students, two undergraduates have been involved and trained through this project. How have the results been disseminated to communities of interest?We published two papers and one master thesis, presented in IFT conference and thermo fisher webinar. What do you plan to do during the next reporting period to accomplish the goals?We are currently working on using aptamer as the binding agent to selective detect food pathogens on chips and filter membranes.

Impacts
What was accomplished under these goals? We developed a sensitive and reliable surface-enhanced Raman spectroscopy (SERS) sandwich assay based on 3-mercaptophenylboronic acid (3-MBPA) as a capturer and indicator molecule was developed for rapid bacteria detection. Both optical and chemical (SERS mapping) imaging were used as mechanisms for bacterial detection and quantification. Using Salmonella enterica and Listeria monocytogenes as the model bacteria, we have identified a unique bacterial SERS signal upon the interaction between the captured bacteria, 3-MBPA and AgNPs, which was used as the base for reliable detection of bacteria using SERS mapping. The non-specific assay also possesses unique optical properties allowing for the enhanced visualization of bacteria at low microscope magnifications (10 and 20x objective lenses). Using 3-MBPA owe achieved sensitive detection and quantification of as low as 102 CFU/mL and a capture efficiency of 92.1% for nonselective detection of Salmonella. Further,we explored the advantages and constraints of this assay over the conventional aerobic plate count (APC) method and further developed methods for detection in real environmental and food matrices. The SERS sandwich assay was able to detect environmental bacteria in pond water and on spinach leaves at higher levels than the APC method. In addition, the SERS assay appeared to have higher sensitivity to quantify bacteria in the stationary phase. On the other hand, the APC method was more sensitive to cell viability. Finally, a method to detect bacteria in a challenging high-sugar juice matrix was developed to enhance bacteria capture. This study advanced the SERS technique for real applications in environment and food matrices.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Brooke Pearson, Panxue Wang, Alexander Mills, Shintaro Pang, Lynne McLandsborough, Lili He*. Innovative sandwich assay with dual optical and SERS sensing mechanisms for bacterial detection. Analytical Methods, 2017, 9, 4732-4739.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Brooke Pearson, Alexander Mills, Madeline Tucker, Siyue Gao, Lynne McLandsborough, Lili He*. Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matrices.Food Microbiology. 2017. Accepted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Alexander Mills and Lili He. 2017. Colorimetric Detection and Inactivation of Escherichia Coli based on Gold Nanoparticles and Sodium Hydroxide. IFT undergraduate research competition. Las Vegas, NV.
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Brooke Pearson. "DEVELOPMENT OF A SERS SANDWICH ASSAY PLATFORM FOR RAPID DETECTION OF BACTERIA".
  • Type: Other Status: Published Year Published: 2017 Citation: Lili He. Invited Talk. Surface-enhanced Raman spectroscopy: One tool for multiple food contaminants. Thermo Fisher Scientific Webinar. June. 2017.


Progress 02/01/16 to 01/31/17

Outputs
Target Audience:food industry, private labs, governmental agencies who want to have a rapid detection method for pathogens in food; researchers and students who are interested in learning and applications of nanotechnology in agriculture and food systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students, one undergraduate,and one postdoc have been involved and trained through this project. How have the results been disseminated to communities of interest?We have publish one paper, presented in 6 seminars, conferences, and meetings during this time. What do you plan to do during the next reporting period to accomplish the goals?1. we are currently working on an optical and SERS dual sensing sandwich assay for capturing and detecting bacteria on a slide. 2. we are also working on the integration of filteration techniquewith SERS mapping for detection bacteria cells captured on a membrane.

Impacts
What was accomplished under these goals? A novel method was developed to rapidly concentrate, detect, and differentiate bacteria in skimmed milk using surface enhanced Raman scattering (SERS) mapping on 4-mercaptophenylboronic acid functionalized silver dendrites. The 4-mercaptophenylboronic acid (4-MPBA) functionalized silver (Ag) dendritic SERS substrate was used to capture the bacterial cells and enhance the bacterial signal. Salmonella, a significantly important food pathogen was used as the representative strain to optimize and evaluate the developed method. The capture efficiency for Salmonella enterica subsp enterica BAA1045 (SE1045) was 84.92 ± 3.25% at 106 CFU/mL and as high as 99.65 ± 3.58% at 103 CFU/mL. Four different strains, two gram-negative and two gram-positive can be clearly distinguished by their SERS spectra using principle component analysis. A mapping technique was utilized to automatically collect 400 spectra over an area of 60 μm × 60 μm to construct a visual image for a sensitive and statistically reliable detection within 30 min. Using this method, we were able to detect as low as 103 CFU/mL bacterial cells in 50 mM NH4HCO3 solution and 102 CFU/mL cells in both 1% casein and skimmed milk. Our results demonstrate the feasibility of using SERS mapping method coupled with 4-MPBA functionalized Ag dendrites for rapid and sensitive bacteria detection in complex liquid samples.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Panxue Wang, Shintaro Pang, Brooke Pearson, Yayoi Chujo, Lynne McLandsborough, Mingtao Fan, Lili He*. Rapid concentration detection and differentiation of bacteria in skimmed milk using surface enhanced Raman scattering mapping on 4-mercaptophenylboronic acid functionalized silver dendrites. Analytical and Bioanalytical Chemistry. DOI 10.1007/s00216-016-0167-8
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lili He. Invited Talk.  Surface enhanced Raman Spectroscopy: one tool for all food contaminants. ACS C&EN Talented 12 symposium. Aug 2016.
  • Type: Other Status: Other Year Published: 2016 Citation: Lili He. Invited talk.  Development and applications of surface enhanced Raman spectroscopy in food safety. College of Chemistry and Chemical Engineering. Xiamen University. March, 2016.
  • Type: Other Status: Other Year Published: 2016 Citation: Lili He. Invited talk. Development of Surface Enhanced Raman Spectroscopy for Food Safety applications. Stockbridge of Agriculture seminar. Feb.22. 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lili He. oral and poster presentation. Label-free mapping bacterial cells using surface enhanced Raman microscopy. NIFA nanotechnology program director annual meeting. June 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Brooke Pearson, Lili He. Oral presentation. Innovative SERS chemical sandwich assay for rapid, sensitive, and reliable detection of bacteria. Eastern analytical syposium. Somerset, New Jersey. Nov. 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Siyue Gao, Lili He. Oral presentation. Mapping bacteria on filter membranes, an innovative SERS approach. Eastern analytical syposium. Somerset, New Jersey. Nov. 2016


Progress 02/01/15 to 01/31/16

Outputs
Target Audience:food industry, private labs, governmental agencies who want to have a rapid detection method for pathogens in food; researchers and students who are interested in learning and applications of nanotechnology in agriculture and food systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students and one visiting scholar have been involved and trained through this project. How have the results been disseminated to communities of interest?We published two manuscripts and presented two posters and one oral in professional conferences. journal publication Panxue Wang, Shintaro Pang, Hua Zhang, Mingtao Fan, Lili He. 2015. Surface enhanced Raman scattering characterization of Lactococcus lactis responding to ampicillin and ciprofloxacin. Analytical and Bioanalytical Chemistry. doi:10.1007/s00216-015-9184-2. Panxue Wang,Shintaro Pang,Juhong Chen,LA McLandsborough,Sam Rasmussen Nugen,Mingtao FanandLili He.Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy. Analyst.DOI:10.1039/C5AN02175H conference publication Hua Zhang, Dillon Murray, Lynne A. McLandsborough,Lili He. Oral presentation: "Characterization of outer membrane of Salmonella responding to different inactivation treatments using surface enhanced Raman spectroscopy". IAFP. July 2015 Panxue Wang, Mingtao Fan andLili He. poster presentation: "Characterization of biochemical profiles of Lactococcus lactis cell wall responding to ciprofloxacin using surface-enhanced Raman spectroscopy". IFT annual conference. Chicago, IL. July 2015. Panxue Wang, Lynne McLandsborough,Lili He. Poster presentation: "Identification and quantification of a bacterial mixture using surface enhanced Raman spectroscopy". Gordon conference. Bentley, MA, June 2015 What do you plan to do during the next reporting period to accomplish the goals?we will testthe capacity ofdifferent capturers for the SERS detection of bacterial cells. we will optimize the sample pretreatment of real food products (milk and ground beef) for detection of bacterial cells.

Impacts
What was accomplished under these goals? The increased incidence of food pathogen outbreaks placed a new emphasis on the requirement of a rapid, sensitive, and reliable detection method for pathogens in food samples. Here we developed a label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy (SERS) mapping.The results show the developed method is able to detect single bacterial cells adsorbed on the silver dendrites with a limit of detection as low as 104 CFU/mL.Moreover, the developed SERS mapping method can realize simultaneous detection and identification of Salmonella enterica and Escherichia coli from a mixture sample. Our results demonstrate the great potential of the label-free SERS mapping method to detect, identify and quantify bacteria and bacterial mixtures simultaneously. Decades of antibiotic use or misuse have resulted in antibiotic resistance in lactic acid bacteria (LAB), a group of common culture starters and probiotic microorganisms. This urges researchers to study how LAB respond to antibiotics, in order to have a better strategy to identify and predict the antibiotic resistant bacteria. Here we characterizedthe biochemical profiles of Lactococcus lactis responding to antibiotics using SERS. Results show different antibiotics induced different spectral changes and these changes revealed the detailed biochemical information of cellular responses. This study demonstrated that the developed SERS method could not only sense the changes in the bacterial cell wall, but also reveal the details on the biochemical profiles, which help us to understand further on how LAB respond to antibiotics, as well as to set a base for the detection of antibiotics susceptibility of bacteria using SERS.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Hua Zhang, Dillon Murray, Lynne A. McLandsborough, Lili He. Oral presentation: "Characterization of outer membrane of Salmonella responding to different inactivation treatments using surface enhanced Raman spectroscopy". IAFP. July 2015
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Panxue Wang , Shintaro Pang , Juhong Chen , LA McLandsborough , Sam Rasmussen Nugen , Mingtao Fan and Lili He. Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy. Analyst. DOI: 10.1039/C5AN02175H.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Panxue Wang, Shintaro Pang, Hua Zhang, Mingtao Fan, Lili He. 2015. Surface enhanced Raman scattering characterization of Lactococcus lactis responding to ampicillin and ciprofloxacin. Analytical and Bioanalytical Chemistry. doi:10.1007/s00216-015-9184-2.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Panxue Wang, Lynne McLandsborough, Lili He. Poster presentation: "Identification and quantification of a bacterial mixture using surface enhanced Raman spectroscopy". Gordon conference. Bentley, MA, June 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Panxue Wang, Mingtao Fan and Lili He. poster presentation: "Characterization of biochemical profiles of Lactococcus lactis cell wall responding to ciprofloxacin using surface-enhanced Raman spectroscopy". IFT annual conference. Chicago, IL. July 2015.