Source: PURDUE UNIVERSITY submitted to
SMALLSCALE METHODOLOGIES AND SENSING FOR DETECTION AND CHARACTERIZATION OF BIOENTITIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0206143
Grant No.
(N/A)
Project No.
IND010687
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Irudayaraj, J. M.
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Ag & Biological Engineering
Non Technical Summary
The overarching goal of this research is to develop nanotools and nanoscale methodologies for detection and mechanistic studies in biology. The specific focus of this effort is to develop and apply these novel technologies for food safety and bioremediation, two key issues relating to agriculture and environment. A wide variety of fresh or minimally processed fruits and vegetables and fruit juices have been linked to foodborne diseases (Beuchat, 1996; 1998). Data collected by the CDC indicate that the number of produce related outbreaks doubled between the periods 1973-1987 and 1988-1992. Despite the fact that America's food supply is one of the safest in the world, 76 million cases of food borne illnesses, 325,000 cases of hospitalization and 5000 deaths have been estimated every year according to the Center for Disease Control (CDC). The cost for the treatment and control is estimated to be between $1- 10 billion every year. While the development of sensor technologies is on the rise, a better understanding of the sensor materials in the context of biological interaction will help to develop sensors that are more specific and sensitive (Ravindranath et al., 2009). Such accuracy is critical because of the zero-tolerance mandate. Sensitivity using conventional biosensors is in the range between 103-104 colony forming units (CFU)/ml. Labeled PCR products of various genes from one food borne pathogen could be probed on one DNA micro array to create a Multi-Locus micro array subtyping scheme for various food borne pathogens. To detect at 1 CFU/ml sensitivity, to be able to answer questions at the molecular level, micro and nano-based technologies should be examined in conjunction with the existing methods. The overall objective of this study is to investigate biosensor and nanotechnology based methods and materials to further understand biomolecular interactions for improved detection and characterization of food pathogens. This problem in predicting and assessing bioremediation performance is compounded by the lack of fundamental knowledge of the molecular basis, regulatory mechanisms, and biochemistry enabling bacterial metal-reducing capabilities. As such, understanding the dynamics of microbially mediated reductive metal transformations will require novel, innovative, and ultrasensitive approaches capable of probing single cells at high resolution across cellular dimensions and under physiologically relevant conditions. Functionalized gold nanoprobes and native gold island-type structures grown in S. oneidensis offer excellent promise in developing single cell 3D chemical maps of metal reduction sites by enhanced raman spectroscopy. For the first time, we utilize the power of enhanced raman microscopy to obtain dynamic chemical maps of chromate reduction sites in living bacterial cells, thus paving the way to developing a general raman platform for single-cell chemical imaging. Upon completion, we will present the very first single cell chemical mapping platform to study the mechanism of metal reduction in situ.
Animal Health Component
(N/A)
Research Effort Categories
Basic
40%
Applied
50%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124099100040%
4047299202040%
1040199200020%
Knowledge Area
104 - Protect Soil from Harmful Effects of Natural Elements; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 404 - Instrumentation and Control Systems;

Subject Of Investigation
4099 - Microorganisms, general/other; 0199 - Soil and land, general; 7299 - Research equipment and methods, general/other;

Field Of Science
2020 - Engineering; 2000 - Chemistry; 1000 - Biochemistry and biophysics;
Goals / Objectives
1) Design and fabricate nanoprobes that are the most effective for enhanced sensing of pathogens. Gold (Au)-silver(Ag) nanostructures (i.e., Ag-tipped Au nanorods, Ag-Au nanocubes/nanoboxes and Ag-nanosphere/nanocube clusters) will be fabricated. Time domain finite difference (FDTD) simulation will be used to optimize the design of anisotropic, multi-compositional Au-Ag nanostructures. Suitable fabrication and biofunctionalization protocols (Swaminathan et al., 2004; Ravindranath et al., 2009) will be developed to create high-performance nanoprobes specifically targeting foodborne pathogens. Various (E. coli, Salmonella sp., Shigella sp., Listeria, Staphylococcus, Yersinia) foodborne and disease causing pathogens will be examined for specific vibrational fingerprints using surface enhanced Raman Spectroscopy (SERS) (Lee and Irudayaraj, 2009). The key goal is to develop strategies to detect as few as 10 CFU/ml or possibly a single CFU/ml. 2) Integrate nanomaterials with single molecule tracking strategies to detect and quantify cell surface receptors and to develop intracellular raman mapping strategies. In this aim we will use these nanomaterials with an appropriate targeting ligand (aptamers or antibodies) to target microbes to develop a highly sensitive spectroscopic biosensor. Second, we will extend our nanomaterial fabrication and Raman imaging techniques to develop a raman chemical and fluorescence imaging strategies for intracellular studies. We will develop single molecule methods for surface receptor quantification studies with a broader aim to integrate these efforts with surface and subcelular functionalities and mechanisms. Second we will develop intracellular imaging methods using nanomaterials using an application related to bioremediation. We will use nanomaterials functionalized with chromate [Cr(VI)] to detect chromate reduction sites in a single bacteria. Shewanella oneidensis - MR1 is used to reduce chromate (VI), a toxic contaminant of soils to chromate (III), as non toxic substance. This demonstration will use nanotools to develop an understanding of the reduction process at single cell level to develop effective bioremediation measures.
Project Methods
Objective 1 : In this project FDTD modeling will be used to optimize the design of nanostructures, and develop chemical procedures based on Ag-Au galvanic replacement reaction for effective synthesis. Silver nanocubes synthesized using well-established methods are subjected to galvanic replacement (Lu et al., 2007) reaction with hydrogen tetrachloroaurat. By adjusting the reaction conditions, porous and/or hollow Au-Ag alloy nanostructures with desired truncation on corners/edges or internal void can be fabricated. These multi-material, anisotropic SERS probes will display superior and consistent enhancement by SERS and functionalized appropriately for biological experiments (Yu et al., 2007; Yu and Irudayaraj, 2007). Culturing of pathogens (E. coli, Salmonella sp., Shigella sp., Listeria, Staphylococcus, Yersinia) are routinely done in Dr. Irudayaraj's laboratory. A Senterra (Bruker Inc.) confocal Raman spectrometer will be used and data analyzed using chemometrics (Kemsley, 1998). Methods for Objective 2 : This objective consists of two subaims: 1) development of single molecule strategies constituting Fluorescence correlation spectroscopy to quantify surface receptors so that effective detection methods can be developed and 2) Develop targeting nanoparticles to assess metal reduction sites using Raman chemical imaging and fluorescence lifetime imaging in single cells of Shewanella oneidensis. i) Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging techniques will be developed (Chen and Irudayaraj, 2009, 2010) for surface receptor quantification. ii) Nanoparticles to assess metal reduction sites in single cells of Shewanella oneidensis. by Raman chemical imaging and fluorescence lifetime imaging. The overall goal in this subaim is to demonstrate that nanoparticles can be used for mechanistic explorations. A robust single-cell monitoring platform for dynamic assessment of cellular and/or subcellular compartmentalization of chromate reduction sites in bacteria to address bioremediation mecahnisms will be studied. Tasks are to a) assess the impact of gold nanoparticle composition, geometry, and functionality on cell viability, growth, and efficacy of microbial chromate reduction using S. oneidensis MR-1 as a model system, b) Construct experiments to track the localization of chromate transformation at single-cell resolution using functionalized gold nanostructures as well as using intracellularly grown gold nanoislands by raman chemical imaging and lifetime imaging.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Target audience are Scientists, Industry professionals, postdoctoral scholars, and graduate and undergraduate students. Efforts included, presentation in national conferences as well in biannual meetings with the USDA collaborators. Lectures on biosensor technology development and laboratory demonstrations were also held for undergraduate students and peer scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have provided training to 6postdoctoral scholars, 5 graduate students, and 10 undergraduate students. Further, we have also provided opportunities to 3 visiting scholars. How have the results been disseminated to communities of interest?Conferences (Institute of Food Technologists, American Chemical Society, PittConn); Progress meetings - these are open to all students in the departments of Food Science, Agric and Biological Engineering, and other. 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) development of lateral flow assays to detect less than 100 cells/ml of pathogens 2) development of instrumentation to detect trancsripts in single cells 3) development of gold nanoparticle biosensors to monitor localization of mRNA in live cells 4) Biosensors for environment monitoring - detect nitrates, and organic contaminants. 5) Development of SERS technology to identify the localization of nanopartciles in metal reducing bacteria

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cho, I., Bhunia, A., Irudayaraj, J. 2015. Rapid pathogen detection by lateral-flow immunochromatographic assay with gold nanoparticle-assisted enzyme signal amplification. International Journal of Food Microbiology. 206: 60-66.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu, J., Hu, Y., Kumar, P., Cheng, G., and Irudayaraj, J. 2014. Enhanced Multi-Photon Emission from CdTe/ZnS Quantum Dots Decorated on Single Layer Graphene. J. Phys Chem. C. 119(1): 6331-6336.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Cho, I-H., Bhandari, P., Patil, P., and Irudayaraj, J. 2014. Membrane filter-assisted surface enhanced Raman spectroscopy for the rapid detection of E. coli O157:H7 in ground beef. Biosensors and Bioelectronics, 64: 171-176.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Craig, A., Franca, A., Oliveira, L., Irudayaraj, J., and Ileleji, K. 2014. Fourier transform infrared spectroscopy and near infrared spectroscopy for the quantification of defects in roasted coffee. Talanta, 03/2015; 134:379386. DOI: 10.1016/j.talanta.2014.11.038.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Craig, A., Franca, A., Oliveira, L., Irudayaraj, J., and Ileleji, K. 2014. Application of elastic net and infrared spectroscopy in the discrimination between defective and non-defective roasted coffees. Talanta 128, 393-400.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Cho, I-H., Mauer, L., and Irudayaraj, J. 2014. In-situ fluorescent immunomagnetic multiplex detection of foodborne pathogens in low numbers. Biosensors & Bioelectronics. 57, 143- 148.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kadam, U., Schulz, B., and Irudayaraj, J. 2014. Detection and quantification of alternative splice sites in Arabidopsis genes AtDCL2 and AtPTB2 with highly sensitive surface enhanced Raman spectroscopy (SERS) and gold nanoprobes. FEBS letters 588 (9), 1637-1643.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Cho, I., Mauer, L. and Irudayaraj, J. 2014. In-situ fluorescent immunomagnetic detection of foodborne pathogens. Biosensors and Bioelectronics. 57:143-148.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Cho, I., Radadia, A., Farrokhzad, K., Ximenes, E., Bae, E., Singh, A., Oliver, H., Ladisch, M., Bhunia, A., Applegate, B., Mauer, L., Bashir, R., and Irudayaraj, J. 2014. Nano/Micro and Spectroscopic Approaches to Food Pathogen Detection. Annual Review of Anal. Chemistry. 7(1):65-88.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Mura, S. Greppi, G., Roggero, P., Musu, E., Pittails, D., Carletti, A., Ghiglieri, G., and Irudayaraj, J. 2015. Functionalized gold nanoparticles for the detection of nitrates in water International. J. Environmental Science and Technology. 12(3):1021-1028.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target audience are students, and industry personnel. Students are taught the basic concepts of design so that they may develop better programs in the future. INdustry personnel.. implies that they can potentially be one of the end users. Changes/Problems: The goals and methods did not deviate in a significant manner. Minor alterations in sample processing and methods development was necessary. What opportunities for training and professional development has the project provided? We have graduated 3 Ph.D students, trainied 5 undergraduate interns, and 5 postdoctoral scholars How have the results been disseminated to communities of interest? We have presented in conferences and in professional society meetings; we have published several papers that are being cited fairly well in our field. What do you plan to do during the next reporting period to accomplish the goals? 1) We plan to extend the limit of detection of the pathogen sensors to < 10 CFU/ml 2) We plan to detect and differentiate live vs dead bacteria 3) we plan to develop a drug delivery system for commercial implementation

Impacts
What was accomplished under these goals? 1) We have redesigned the biosensors and have adopted a lateral flow concept to improve the limit of detection by over 1000-fold compared to the conventional method. For bacteria, we are able to detect at 50 cfu/ml in food matrices in an onsite setting. 2) We have achieved the intracellular detection and mapping of chromate reduction sites in single bacterium. We have also mapped the chromate reduction profiles in shewanella oneidensis in an in situ condition. 3) We have developed techniques to map the presence of mRNA's in single cells.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Quantification of abortive transcription of TWD1 gene in Arabidopsis thaliana, using Surface-enhanced Raman spectroscopy (SERS). Plant Biotechnology. 12(5):568-577
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: . Functionalized gold nanoparticles for the detection of nitrates in water International. J. Environmental Science and Technology. DOI 10.1007/s13762-013-0494-7.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: . Detection and quantification of alternative splice sites in Arabidopsis genes AtDCL2 and AtPTB2 with highly sensitive surface enhanced Raman spectroscopy (SERS) and gold nanoprobes. FEBS letters 588 (9), 1637-1643.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Targeted in vivo photodynamic therapy with epidermal growth factor receptor-specific peptide linked nanoparticles. International Journal of Pharmaceutics. 471(1-2):421-9.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: In-situ fluorescent immunomagnetic multiplex detection of foodborne pathogens in very low numbers. Biosensors and Bioelectronics 57, 143-148.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools. Analytical biochemistry 458, 58-60
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Application of elastic net and infrared spectroscopy in the discrimination between defective and non-defective roasted coffees. Talanta 128, 393-400.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Vibrational Spectroscopy for food quality and safety screening. In: Arun K. Bhunia; Moon S. Kim; Chris R. Taitt. High throughput screening for food safety assessment: biosensor technologies, hyperspectral imaging and practical applications


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Target audience are scientists, students, and industry personnel. We expect scientists to adopt our protocols for their applications. Students are trained in cross-disciplinary fields. Our methods can be adopted by industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project was instrumental in training postdoctoral researchers and graduate students, in addition to undergraduate students. The students involved in the project receive multidisciplinary training so that they are well-prepared to handle complex projects, How have the results been disseminated to communities of interest? We have published in highy reputed journals and presented in various international conferences. What do you plan to do during the next reporting period to accomplish the goals? Our goal in developing pathogen sensors is to further develop these technologies for simultaneous detection of multiple pathogens in food matrices in an onsite setting in an economically feasible manner. The simplicity of the detection platform to detect at this high level of sensitivity could be the first step to developing a highly sensitive portable optical sensor that can be deployed onsite for routine monitoring of food safety. Our parallel development of ELISA and fluorescence biosensors will be tailored to specific industry needs so that a high level of detection is possible for routine monitoring of pathogens. In addition we will incorporate a live and dead cell detection module in the biosensor, which is of critical importance in the industry sectors. Our Raman platform to study bioremediation mechanisms in single cells will have far reaching impact in understanding how bacteria reduces metals, which so far has not been possible at single cell level. Once developed we expect that our technologies could be used to study bioremediation mechanisms of Shewanella under a variety of conditions. Other efforts to integrate nanoparticle-based sensing for disease targeting and therapy is also in progress. Preliminary proof has shown that nanoparticle-based targeted therapeutics could be developed for treatment. \]e have also developed microscopic methods for detecting epigenetic marks, which we expect to provide fundamental insights on gene expression.

Impacts
What was accomplished under these goals? Multiplex approaches based on immune biosensors were designed and developed for the simultaneous detection of three key pathogens: E. coli O157:H7; Salmonella enteritidis, and Listeria monocytogenes. Detection limit was estimated to be about 10-100 cells/ml. These methods are being expanded to more sensitive lateral flow methods for onsite detection In addition detection of live vs dead cells are also being pursued that are simple and easy to use on a routine basis. Parallel efforts using Raman spectroscopy is also directed towards studying the metal reduction sites in bioremediating bacteria, Shewanella Oneidensis using gold nanoislands grown inside cells that can act as molecular beacons to enhance the signal of target molecule. Using surface-enhanced Raman spectroscopy we have developed a protocol to assess the metal reduction sites in single cells of Shewanella. Our single cell assay was validated by population studies. High resolution microscopic techniques were developed for single cell phenotyping as well as to detect key epigenetic marks in single cells. These are expected to have a significant effect in understanding basic biology.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Chen, J., Miller, A., Kirchmaier, A., and Irudayaraj, J. 2012. Single Molecule Tools Elucidate H2A.Z Nucleosome Composition. J. Cell Science. 125(12):2954-2964.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Wang, Y. and Irudayaraj, J. 2013. Surface-enhanced Raman spectroscopy at single molecule scale and its implication in biology. Philosophical Transactions B. 368(1611): 20120026.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Mura, S., Greppi., G., Innocenzi, P., Piccinini, M., Figus, C., Marongiu, L., Guo, C., and Irudayaraj. 2013. Nanostructured thin films as surface-enhanced Raman scattering substrates. J. Raman Spectroscopy. 44:35-40.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Jae-Sung, K., Ravindranath, S., Aloke, K., Irudayaraj, J. and Wereley, S. 2012. Opto- electrokinetic manipulation technique for high-performance on-chip biological assay. Lab on Chip. 12(23):4955-4959.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Damayanti, N., Parker, L., Irudayaraj, J. 2013. Monitoring phosphorylation in live cells by lifetime imaging. Angew Chemie. 52(14): 3931-3934.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Cho, I. and Irudayaraj, J. 2013. In-situ immunogold nanoparticle network-based ELISA for biosensor for highly sensitive pathogen detection. Intl. J. Microbiology. 164(1): 70-75.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Cho, I and Irudayaraj, J. 2013. Lateral-flow immune concentration for detection of Listeria monocytogenes. Analytical Bioanalytical Chemistry. 405(10: 3313-3319.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Lee, K. and Irudayaraj, J. 2013. Correct Spectral Conversion between Surface-enhanced Raman and Plasmon Resonance Scattering from Nanoparticle Dimers for Single Molecule Detection. Small J. 9(7): 1106-1115.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Multiplex approaches based on Raman biosensors were designed and developed for the simultaneous detection of several pathogens. Surface enhanced Raman spectroscopy (SERS) was used to detect three key foodborne pathogens: E. coli O157:H7; Salmonella enteritidis, and Listeria monocytogenes. Detection limit was estimated to be about 100 cells/ml. These methods are being expanded to more sensitive biosensors based on hybrid ELISA and fluorimetry-based techniques that are simple and easy to use on a routine basis. Parallel efforts using Raman spectroscopy is also directed towards studying the metal reduction sites in bioremediating bacteria, Shewanella Oneidensis using gold nanoislands grown inside cells that can act as molecular beacons to enhance the signal of target molecule. Using surface-enhanced Raman spectroscopy we have developed a protocol to assess the metal reduction sites in single cells of Shewanella. Our single cell assay was validated by population studies. Further we have also expanded our SERS approach to classifying up to 15 soil bacteria using appropriate statistical tools. Biosensors for detection of metal contamination is also being developed. PARTICIPANTS: 1) Stefania Mura from University of Sassari (Italy) TARGET AUDIENCES: Sensors are developed for implementaion in an industry setting. Target audience are industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our goal in developing pathogen sensors is to further develop these technologies for simultaneous detection of multiple pathogens in food matrices in an onsite setting in an economically feasible manner. The simplicity of the detection platform to detect at this high level of sensitivity could be the first step to developing a highly sensitive portable optical sensor that can be deployed onsite for routine monitoring of food safety. Our parallel development of ELISA and fluorescence biosensors will be tailored to specific industry needs so that a high level of detection is possible for routine monitoring of pathogens. Our Raman platform to study bioremediation mechanisms in single cells will have far reaching impact in understanding how bacteria reduces metals, which so far has not been possible at single cell level. Once developed we expect that our technologies could be used to study bioremediation mechanisms of Shewanella under a variety of conditions. Other efforts to integrate nanoparticle-based sensing for disease targeting and therapy is also in progress. Preliminary proof has shown that nanoparticle-based targeted therapeutics could be developed for treatment.

Publications

  • 1. Ravindranath, S., Kadam, U., and Irudayaraj, J. 2012. Intracellularly grown gold nanoislands as SERS substrates for monitoring chromate, sulfate and nitrate localization sites in remediating bacteria biofilms by Raman chemical imaging. Analytica Chimica Acta. 745:1-9. (Cover article)
  • 2. Stephen, K., Nakatsu, C., and Irudayaraj, J. 2012. Surface Enhanced Raman Spectroscopy (SERS) for the discrimination of Arthrobacter strains based on variations in cell surface composition. Analyst. 137: 4280-4286. 3. Dong, C. and Irudayaraj, J. 2012. Hydrodynamic Size-Dependent Cellular Uptake of Aqueous QDs Probed by Fluorescence Correlation Spectroscopy. J. Physical Chem. C. 116(40):12125-32.
  • 4. Wang, Y., Book, B., and Irudayaraj, J. 2012. Folic acid protected doxorubicin coated silver nanocarriers for targeted drug delivery. J. Biomedical Nanotechnology. 8(5):751-759.
  • 5. Book, B. Wang, Y., and Irudayaraj, J. 2012. Multifunctional gold nanorods for targeted drug delivery using 2-photon excitation. Euro Journal of Medicinal Chemistry. 48:330-337.
  • 6. Mura, S., Greppi, G., Marongiu, M., Roggero, P., Ravindranath, S., Mauer, L., Schibeci, N., Perria, N., Piccinini, M., Innocenzi, P., and Irudayaraj, J. 2012. FTIR nanobiosensors for Escherichia coli detection. 2012. Beilstein J. Nanotechnology. 3:885-92.
  • 7. Wang, Y., Chen, J., and Irudayaraj, J. 2011. Nuclear targeting dynamics of gold nanoclusters for enhanced therapy of HER2+ breast cancer. ACS NANO. 5(12):9718-9725.
  • 8. Panchapakesan, B., Book, B., Seth R., and Irudayaraj, J. 2011. Gold nanoprobes for theragnostics and diagnostics. Journal of Nanomedicine, 6(10):1-7.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Multiplex approaches based on Raman biosensors were designed and developed for the simultaneous detection of several pathogens. Surface enhanced Raman spectroscopy (SERS) was used to detect three key pathogens: E. coli O157:H7; Salmonella enteritidis, and Listeria monocytogenes via a cross-platform detection strategy. Detection limit was estimated to be about 100 cells/ml. These methods are being expanded to more sensitive biosensors to detect one cell/ml. In parallel we have also developed techniques for strain level differentiation of pathogens using Raman spectroscopy. Parallel efforts using Raman spectroscopy is also directed towards studying the metal reduction sites in bioremediating bacteria, Shewanella Oneidensis using gold nanoislands grown inside cells that can act as molecular beacons to enhance the signal of target molecule. Using surface-enhanced Raman spectroscopy we have developed a protocol to assess the metal reduction sites in single cells of Shewanella. Our single cell assay was validated by population studies. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Technologies for food security and bioremediation are targeted to secure the safety of foods and to remediate the lands. Knowledge gained from these efforts will be beneficial for the Industries and government agencies to implement these techniques. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our goal in developing pathogen sensors is to further develop these technologies for detection of pathogens in food matrices in an onsite setting. The simplicity of the detection platform to detect at this high level of sensitivity could be the first step to developing a highly sensitive portable optical sensor that can be deployed onsite for routine monitoring of food safety. Our parallel development of ELISA biosensors will be tailored to specific industry needs so that a high level of detection is possible for routine monitoring of pathogens. Our Raman platform to study bioremediation mechanisms in single cells will have far reaching impact in understanding how bacteria reduces metals, which so far has not been possible at single cell level. Once developed we expect that our technologies could be used to study bioremediation mechanisms of Shewanella under a variety of conditions

Publications

  • Lee, K., Drachev, V., and Irudayaraj, J. 2011. DNA-Gold nanoparticle networks grown at cell surface marker sites: Diagnostics of cancer stem cells. ACS NANO. 5(3):2109-2117.
  • Wang, Y. and Irudayaraj, J. 2011. A SERS DNAzyme Biosensor for Lead Ion Detection. Chemical Communications. 47:4394-4396.
  • Ravindranath, S., Henne, T., Thompson, D., and Irudayaraj, J. 2011. Intracellular Bioreduction of Chromate-Decorated Gold Nanoparticles by Shewanella oneidensis MR-1 by Raman Chemical Imaging. ACS NANO. 5(6):4729-36.
  • Guo, C. and Irudayaraj, J. 2011. Fluorescent Ag Clusters via a Protein-directed Approach as a Hg(II) Ion Sensor. Analytical Chemistry. 83(8):2883-9.
  • Ravindranath, S., Wang, Y., Irudayaraj, J. 2011. SERS driven Cross-platform based multiplex pathogen detection. Sensors & Actuators: B. Chemical. 152: 183-190.
  • Ravindranath, S., Henne, K., Thompson, D., and Irudayaraj, J. 2011. Surface-enhanced Raman Imaging of Intracellular Bioreduction of Chromate in Shewanella oneidensis. PLoS ONE. 6(2): e16634 (10 pages).


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: We have developed nanoparticles to detect foodborne pathogens at a high level of sensitivity. We have shown that we can detect as low as 10 cells per ml. Silver nanospheres with roughened surfaces have been fabricated and these novel particles have been used to demonstrate detection at this level of sensitivity. In parallel we have also developed techniques for strain level differentiation of pathogens using Raman spectroscopy. Parallel efforts using Raman spectroscopy is also directed towards studying the metal reduction sites in bioremediating bacteria, Shewanella Oneidensis. Using surface-enhanced Raman spectroscopy we have developed a protocol to assess the metal reduction sites in single cells of Shewanella. Chromate coated 3.5 and 13 nm particles were used to illustrate this concept. Our single cell assay was validated by population studies. PARTICIPANTS: Dr. Chobi Debroy, The Pennsylvania State University (Collaborator) Dr. Lisa Mauer, Purdue University (Collaborator) TARGET AUDIENCES: Food companies and Bioremediation inspectors. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our goal in developing pathogen sensors is to further develop these technologies for detection in food matrices and in the field. Since Raman spectroscopy has a portable feature, we expect that the benchtop studies can be further developed to detect in field conditions. The simplicity of the detection platform to detect at this high level of sensitivity could be the first step to incorporate a highly sensitive portable optical sensor. Our Raman platform to study bioremediation mechanisms in single cells will have far reaching impact in understanding how bacteria reduces metals, which so far has not been possible at single cell level. Once developed we expect that our Raman approach could be used to study bioremediation mechanisms of Shewanella under a variety of conditions. We have also optimized single molecule detection techniques to detect protein interactions in living cells. This approach will be used to quantify receptor interactions in living cells.

Publications

  • Wang, Y., Lee, K., and Irudayaraj, J. 2010. SERS aptasensor from nanorod-nanopaticle junction for protein detection. Chem. Communications. 46(6):613-615.
  • Chen, J. and Irudayaraj, J. 2010. Fluorescence lifetime cross correlation spectroscopy resolves EGFR and antagonist interaction in live cells. Analytical Chemistry, 82(15):6415-6421.
  • Wang, Y., Seebald, J., Szeto, D., and Irudayaraj, J. 2010. Biocompatibility and biodistribution of SERS nanoprobes in zebrafish embryos: in vivo and multiplex imaging. ACS Nano. 4(7):4039-4053.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Our efforts explore the use of nanoparticles to detect pathogens and protein interactions for specific use in eludidating mechanisms related to food, plant, and environment. We have developed nanoprobes to detect multiple foodborne pathogens in selected food samples using uv-visble spectrometer as well as Raman spectrometer. We have developed multifunctional probes to target cancer cells. The same probes can also be used in conjunction with single molecule spectroscopy to detect protein interactions in serum. PARTICIPANTS: Ann Kirchmaier. Chobi Debroy, Pennsylvania State University. TARGET AUDIENCES: Target audience include students, peer researchers (scientists and engineers), and industry personnel. The message was deliveres during special lectures, conferences, and workshops. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The multifunctional nanoparticles developed can be used to separate and detect pathogens directly from a food matrix. This technology could be used for onsite detection of threat agents. The uv-vis-NIR based multiple pathogen detector can detect foodborne pathogens at 100 - 10 cells/ml sensitivity. The simplicity of the detection platform to detect at this high level of sensitivity is the first step to incorporate a highly sensitive portable optical sensor. We have optimized single molecule detection techniques to detect protein interactions in living cells.

Publications

  • Sun, L. and Irudayaraj, J. 2009. PCR-free quantitation of splice variants in Cancer Gene using Surface Enhanced Raman Spectroscopy. J. Phys. Chem. 113(4):14021-5.
  • Sun, L. and Irudayaraj, J. 2009. Quantitative Surface Enhanced Raman for gene expression estimation, Biophysical Journal. 96:4609-16.
  • Wang, C. Chen, J. Talavange, T. and Irudayaraj, J. 2009. Gold nanorod/Fe3O4 nanoparticle Nano-Pearl-Necklaces for simultaneous targeting, dual-mode imaging, and photothermal ablation of cancer cells. Angewandte Chemie. 121(15):2797-2801.
  • Chen, J., Wang, C., and Irudayaraj, J. 2009. Ultrasensitive on-step immunoassay in blood serum using gold nanoparticle probes by single molecule spectroscopy. Biomedical Optics Letters. 14(4):040501-3
  • Vidi, P., Chen, J., Irudayaraj, J. and Watts, V. 2008. Higher-order Adenosine A-2A receptor oligomers revealed by BiFC-FRET and FLIM. FEBS Letters. 582(29):3985-3990.
  • Yu, C., Jacob, R., Ritchie, K., Prasad, N., Irudayaraj, J. 2008. Receptor Overexpression or Inhibition Alters Cell Surface Dynamics of EGF-EGFR interaction: New Insights from Real-time Single Molecule Analysis. Biochemical and Biophysical Research Communication. 378(3):376-382.
  • Lee, K. and Irudayaraj, J. 2009. Periodic and dynamic 3-D gold nanoparticle-DNA network structures for surface-enhanced raman spectroscopy-based quantification. J. Phys. Chem. C. 113:5980-5983.
  • Ravindranath, S., Mauer, L., Chitrita, D., and Irudayaraj, J. 2009. Biofunctionalized magnetic nanoparticle integrated mid-infrared pathogen sensors for food matrices. Analytical Chemistry. 81(8):2840-2846.
  • Bakker, R., Yuan, H., Liu, Z., Pedersen, R., Boltasseva, A., Chen, J., Irudayaraj, J., Kildishev, A., Drachev, V., and Shalaev, V. 2008. Nanoantenna induced fluorescence enhancement: dipolar coupling and reduced lifetimes. New Journal of Physics. 10:125022.
  • Sun, L., Yu, C. and Irudayaraj, J. 2008. Raman multiplexers for alternative gene splicing. Analytical Chemistry. 80(9):3342-9.
  • Wang, C. and Irudayaraj, J. 2008. Gold nanorod probes detects multiple pathogens. Small. 4(12):2204-2208.
  • Shamsaie, A., Heim, J. and Irudayaraj, J. 2008. Quantification of MBA in living cell by surface enhanced raman spectroscopy. Chemical Physics Letters. 461:131-135
  • Varghese, L., Sinha, R., and Irudayaraj, J. 2008. Single molecule kinetic investigations of protein association and dissociation using fluorescence cross-correlation spectroscopy. Analytica Chimica Acta. 625(1):103-109.
  • Tapasree Sarkar. Ph.D. Dissertation. Biophysical methods for understanding signalling cascades. Fall 2008.
  • Lan Sun. Ph.D. Multiplex quantification of genetic material by surface enhanced Raman spectroscopy. Spring 2009.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: The overall effort is to develop nanoparticles for pathogen detection or to study protein interactions for specific use in eludidating mechanisms related to food, plant, and environment. We have developed a methodology to combine iron nanoparticles and a portable mid-infrared spectrometer to detect foodborne pathogens in selected food samples. A procedure to calculate protein interactions and monitor multiple protein binding kinetics using uv-vis-NIR spectrometer was developed using gold nanorods of different aspect ratios. Novel gold nanorods tagged with iron nanoparticles were fabricated to detect food borne pathogens as well as to kill the targeted pathogens by laser irradiation was developed. PARTICIPANTS: Key individuals who worked on this project include: Lan Sun, Chenxu Yu, Ali Shamsaie, Soo-Jin Jun. Individuals from other institutions include: Ali Demirci (The Pennsylvania State University) Chobi Debroy (The Pennsylvania State University) Sreevatsan (University of Minnesota) TARGET AUDIENCES: Scientists and Engineers in academic units, Industry personnel - quality and safety control departments, federal and regularory agencies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The portable mid-infrared iron nanoparticle immunoassay is capable of detecting pathogens in real food matrix. This technology could be used for onsite detection of threat agents. The uv-vis-NIR based multiple pathogen detector can detect foodborne pathogens at 100 - 10 cells/ml sensitivity. The simplicity of the detection platform to detect at this high level of sensitivity is the first step to incorporate a highly sensitive portable optical sensor. The magnetic particle bearing gold nanorods is a novel nanomaterial possessing optical and magnetic properties. This multimaterial is extremely valuable because of its multifunctional use: Targeting, deteting, and photokilling.

Publications

  • Krishnamurthy, K., S. Jun, J. Irudayaraj, and A. Demirci. 2007. Efficacy of infrared heat treatment for inactivation of staphylococcus aureus in buffer and milk. Journal of Food Process Engineering 31(6):1-6.
  • Kouassi, G., P. Wang, J. Irudayaraj, and S. Sreevatsan. 2007. Detection of prion protein on streptavidin-aptamer mediated magnetic and gold-coated magnetic nanoparticles. Biotechnology Progress (23): 1239-1244
  • Jun, S., H. Khurana, K. Krishnamurthy, A. Demirci, and J. Irudayaraj. 2007. Infrared heating in food processing: An Overview. Comprehensive reviews in Food Science and Food Safety 7(1):2-13.
  • Veale, E., J. Irudayaraj, and A. Demirci. 2007. An on-line approach to monitor ethanol fermentation using FTIR spectroscopy. Biotechnology Progress Online publication 02/21/07.
  • Yu, C. and J. Irudayaraj. 2007. A multiplex biosensor using gold nanorods. Analytical Chemistry 79(2): 572-579.
  • Mizrach, A., Z. Schmilovitch, R. Korotic, J. Irudayaraj, and R. Shapira. 2007. Yeast detection in apple juice using raman spectroscopy. Transactions of ASABE 50(6):1-8
  • Yu, C. L. Varghese, and J. Irudayaraj. 2007. Surface modification of CTAB capped gold nanorods to make molecular probes. Langmuir 23:9114-9119.
  • Yu, C., and J. Irudayaraj. 2007. Selectivity and sensitivity kinetics of nanorod biosensors. Biophysical Journal, 93:3684-3692.
  • Yu, C., H. Nakshatri, and J. Irudayaraj. 2007. Single cell Identity profiling using multiplex nanorods. Nano Letters 7(8):2300-2306.
  • Lan, S., C. Yu, and J. Irudayaraj. 2007. Surface enhanced raman scattering based nonfluorescent probes for multiplex DNA detection. Analytical Chemistry 79(11):3981-3988
  • Shamsaie, A., J. Sturgisand, P. Robinson, and J. Irudayaraj. 2007. Intracellularly Grown Gold Nanoparticles (IGAuN) as Potential SERS Probes. Journal of Biomedical Optics 12 (2), 020502: 1-3.


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Experiments to build a library of fourier transform and raman spectroscopic fingerprints for detecting foodborne pathogens have been initiated and well in progress. Similarly raman spectroscopic fingerprints are also being accumulated in the library. This work is being done in collaboration with Penn State University. Various different nanoparticles have been fabricated. These include gold, iron, and gold coated iron particles. At present most of these particles are being used as sensors. Efforts to incorporate these in bioremediation is in progress. A laboratory for single molecule tracking in live cells is being established. This will allow the monitoring of receptor interactions and drug aggregation in living cells. TARGET AUDIENCES: Scientists interested in biosenses for detecting food pathogens

Impacts
Nanoparticle DNA detection technology for detecting bacterial genes. This technology will help to detect DNA at femto molar sensitivity. Spectroscopic detection technology for probing structural changes in food systems. This will help in rapid assessment of the quality of irradiated food. Spectroscopic methods for detecting foodborne pathogens. This will lead to the development of a portable system for detecting foodborne pathogens.

Publications

  • Kizil, R. and Irudayaraj, J. 2006. Discrimination of irradiated starch gels using FT-Raman spectroscopy and chemometrics. J. Agric. and Food Chemistry, 54(1):13-18
  • Kizil, R. and Irudayaraj, J. 2007. Rapid evaluation and discrimination of gamma-irradiated carbohydrates using FT-Raman spectroscopy and canonical discriminant analysis. J. Science of Food and Agriculture, 87(7):1244-1251.
  • Ganjoo, A., H. Jain, C. Yu., R.Song, J.V. Ryan, J. Irudayaraj, Y.J. Ding and C.G. Pantano. 2006. Planar Chalcogenide Glass Waveguides for IR Evanescent Wave Sensors, J. Non-crystalline Solids 352:584-588.
  • Rudnitskaya, A., Kirsanov, D., Legin, A., Beullens, K., Lammertyn, J., Nicolai, B., Irudayaraj, J. 2006. Analysis of apples varieties - comparison of electronic tongue with different analytical techniques. Sensors and Actuators, B: Chemical, v 116, n 1-2, Jul 28, 2006, p 23-28.
  • Kouassi, G. and Irudayaraj, J. 2006. Magnetic and gold-coated magnetic particles as a DNA sensor. Analytical Chemistry. 78(10):3234-3241.
  • Yu, C., Gestl, E., Eckert, K., Allara, D., Irudayaraj, J. 2006. Confocal Raman based differentiation and biomarker identification in human breast epithelial cells. Cancer Detection and Prevention. 30(6):515-522.
  • Veale, E., Irudayaraj, J., Demirci, A. 2007. An on-line approach to monitor ethanol fermentation using FTIR spectroscopy. Biotechnology Progress. Online publication 02/21/07.
  • Krishnamurthy, K., Demirci, A., and Irudayaraj, J. 2007. Inactivation of Staphylococcus aureus in Milk Using Flow-through Pulsed UV-Light Treatment System, 72(7):M233-M239.


Progress 10/01/05 to 09/30/06

Outputs
Routine identification of pathogenic microorganisms predominantly based on nutritional and biochemical tests is a time-consuming process; the delay may lead to fatal consequences at times. When developing diagnostic methods for food safety or clinical purposes, the food or clinical sample could potentially contain more than one microbial species. A key question on detecting food borne pathogens in microbial cocktail is addressed via spectroscopic fingerprinting. To identify a particular microbial species in a microbial cocktail, it is therefore necessary to identify the key features that are characteristic to the species in question and to establish a database. A Fourier transform infrared spectroscopy based approach was developed to identify the presence of five possible pathogenic bacteria in ten different microorganism mixtures with each cocktail containing three different species. The average prediction accuracy was 98%. A parallel effort to combine a biosensor-based detection and spectroscopic fingerprinting was addressed via novel infrared sensitive flims. New functionalization strategies were developed and the specificity and sensitivity of the films were demonstrated. Standardization of the assays were accomplished using surface Plasmon resonance biosensors. To detect ultra small volumes a nanoparticle-based approach was also adopted. Nanomaterials used were iron and gold coated iron particles. Preliminary protocols to tether biomolecules to iron nanoparticles and gold coated iron nanoparticles were demonstrated as a first step towards using magnetic and optical fields in detection and diagnostics for food safety.

Impacts
a) The first experiment and analysis procedure for spectroscopic detection of a specific food pathogen in mixed culture was developed and successfully validated. b) Spectroscopic assays for the detection of food pathogens was also successfully tested and validated in food matrices.. c) A biosensor and spectroscopy based assay was designed to capitalize on the bioaffinity and spectroscopic modes of detection to increase detection specificity.

Publications

  • Yu, C. and J. Irudayaraj, J. 2006. Detection and identification of pathogenic microorganisms in mixed cultures by FTIR spectroscopy. Trans of ASABE. 49(5):1-10.
  • Kouassi, G. and Irudayaraj, J. 2006. A nanoparticle based immobilization assay for prion protein kinetics study. Journal of Nanobiotechnology. 4:8: 1-10.
  • Swaminathan, A. and Irudayaraj, J. 2006. Surface plasmon resonance based immunosensing of E. coli O157:H7 in apple juice. Transactions of ASABE. 49(5): 1257-1262.
  • Yu, C., Ganjoo, A., Jain, H., Pantano, C., and Irudayaraj, J. 2006. Towards a mid-IR biosensor: Detection and fingerprinting of pathogens on chalcogenide films. Analytical Chemistry, 78(8):2500-2506.
  • Gupta, M., Irudayaraj, J., Schmilovitch, Z., Mizrach, A. 2006. Identification and quantification of foodborne pathogens in different food matrices using FTIR and artificial neural networks. Trans of ASAE 49(5):1249-1256.
  • Waswa, J., Debroy, C., and Irudayaraj, J. 2006. Rapid detection of Salmonella enteritidis and Escherichia coli using the BiacoreTM Surface Plasmon Resonance biosensor. J. Food Process Engineering, 29: 373-385.