Smallscale Methodologies and Sensing for Detection and Characterization of Bioentities

<p>NON-TECHNICAL SUMMARY: <br/>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.

<p>APPROACH:<br/>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). <br/>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.

<p>PROGRESS: 2011/10 TO 2012/09
<p>OUTPUTS: <br/>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.

<p>PROGRESS: 2010/10/01 TO 2011/09/30
<p>OUTPUTS: <br/>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.

<p>PROGRESS: 2009/10/01 TO 2010/09/30
<p>OUTPUTS:<br/>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.
<p>PROGRESS: 2008/10/01 TO 2009/09/30
<p>OUTPUTS: <br/>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.

<p>PROGRESS: 2007/10/01 TO 2008/09/30
<p>OUTPUTS: <br/>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.

<p>PROGRESS: 2006/10/01 TO 2007/09/30

<p>OUTPUTS:<br/>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

<p>PROGRESS: 2005/10/01 TO 2006/09/30
<p>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.