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Bellevue, WA, United States

Kim S.,University of Hawaii at Manoa | Lu L.,University of Hawaii at Manoa | Chung J.-H.,University of Washington | Lee K.,Nanofacture, Inc. | And 2 more authors.
Innovative Food Science and Emerging Technologies | Year: 2011

A rapid immunofluorescence method for foodborne pathogens in food systems using microwire sensor coupled with high frequency alternating current was developed. The method was intended to enrich and quantify E. coli cells internalized in baby spinach leaves. The targeted bacterial cells in the sample solution were captured on microwires in a diameter of 25 μm, and were bound to fluorescein isothiocyanate (FITC) labeled polyclonal E. coli antibodies. Fluorescent images of the FITC antibodies were obtained using a fluorescence microscope equipped with a charge-coupled-device (CCD) camera, and the fluorescent intensity (FI) was quantified through image processing. The capture of E. coli K-12 in PBS buffer was optimized when the electric field was generated at the frequency of 3 MHz and 20 Vpp with bacterial concentration of 107 CFU/mL. The detection limit of our sensing device was determined to be 103 CFU/mL. Field emission scanning electron microscopy (FESEM) was used to validate and visualize E. coli cells captured on the tip surface. The sensitivity and specificity of the developed sensor has been successfully validated by testing E. coli internalized in baby spinach leaves. The immunofluorescence detection has been completed within 15 min. Moreover, it was found that the enrichment process of E. coli cells using the dielectrophoretic force was rarely affected by food particles, which proved the sensing selectivity. The developed sensor is expected to meet the steady demand for a simple, rapid, highly sensitive detection approach to quantify the targeted microbes in food systems. Industrial Relevance: There has been an increase in the number of foodborne illnesses linked to the consumption of fresh and minimally processed fruits and vegetables. Some E.coli strains such as E.coli O 157:H7, can cause a variety of diseases, including diarrhea, urinary tract infections, respiratory diseases, meningitis and more. In general, consumers wash the fresh produces under cold running tap water to remove any lingering dirt on the surface of the produces before eating or preparing. However, how do the consumers know if there is any possible pathogen hiding inside of the fresh produce after rinsing? It was reported from many researchers that, the E.coli internalization, which may occur when fresh produces intake E. coli containing water or manure from the soil, would be a main cause of the foodborne illness outbreak. To ensure the safety of drinking water, E.coli concentration cannot be higher than 1 CFU/mL. How can we detect such a low level of E.coli in an easy yet efficient way? To our knowledge, none of the traditional detecting approaches such as cultural based method, polymerase chain reaction(PCR), surface plasmon resonance (SPR) biosensor, and Latex Agglutination, has performed perfectly. Hence, a rapid and accurate technique for detecting foodborne pathogens in fresh produce is urgently needed in order to secure the food safety. To overcome this issue, a simple detection method for foodborne pathogens in food systems using the microwire sensor coupled with high frequency alternative current was developed. The sensitivity and specificity of the developed sensor have been successfully validated by testing with E. coli internalized inside baby spinach leaves. It was found that spinach particles rarely affect the performance of our sensing device, which shows a promising prospect of its application in food industries.© 2011 Elsevier Ltd. All rights reserved.


Grant
Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase II | Award Amount: 500.00K | Year: 2010

This Small Business Technology Transfer Research (STTR) Phase II project is to develop a prototype biosensor array system for rapid surveillance of Methicillin-Resistant Staphylococcus aureus (MRSA) operated by minimally-trained personnel. MRSA, one of the major bacterial pathogens for healthcare acquired infections (HAI), afflicts overcrowded and understaffed US hospitals. Thus, an urgent need exists for a more rapid, reliable yet affordable testing method for HAI screening. The proposed tip sensor?s novel sample concentration mechanism enables rapid screening of whole cells followed by confirmation of genetic signatures. The project implements a proprietary sample concentration mechanism for highly efficient capture and detection of bacterial pathogens in a size-exclusive manner. The novelty of the proposed work involves studying DNA reaction kinetics enhanced by a high-frequency electric field on a high aspect ratio tip. The transformative nature of the proposed biosensing technology enables screening for pathogens and nanoparticles without culture and amplification. The broader impact/commercial potential of this project is to establish a solid fabrication and detection method for a high-throughput biosensor. The tip sensors offer a specific concentration of whole bacterial cells (screening) and an accelerated DNA detection (confirmation). The proposed method will pave the way to high-throughput screening of pathogens through the specific detection in terms of target-geometry, electric properties, and affinity chemistry. The operation cost and time can be minimized through superior concentration performance. Considering the concentration and detection mechanisms, the tip sensor works as a universal platform for low cost detection of various pathogenic analytes including bacteria and viruses, proteins and nucleic acids in clinical samples. The societal impact of this biosensor platform will fulfill an unmet need to save healthcare costs associated with specific pathogens. The technology would eventually be deployed in resource-limited settings including individual uses, for the detection of various pathogens. Thus, this technology will directly impact the fields of micro/nanochip fabrication, biomedical sensors, and low-cost diagnostics.


Grant
Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase I | Award Amount: 65.00K | Year: 2008

This STTR Phase I research proposal will demonstrate the feasibility of a nanoscale needle (nano-needle) probe biosensor assaying low-abundance nucleic acids without target-/signal amplification. The unique point in the proposed work lies in the combined use of dielectrophoresis and capillary action to fabricate a high-aspect ratio nano-needle made of a hybrid nanomaterial and to operate the nano-needle as a biosensor, achieving high sensitivity through size-exclusive sample concentration. The company has already succeeded in nonspecifically sampling/assaying intercalator-treated lambda-DNA spiked into a buffer or cell solution in a quantitative manner. The sensitivity of this achievement was around 10pg/mL (0.3fM) which is comparable to the concentration of naturally occurring DNA species and sufficient for screening/detection of circulating DNA in blood (sub-50ng/mL). The method will pave the way to high throughput fabrication of high aspect ratio nano-needle structures and the application to the biosensing platform. The proposed nano-needle biosensor device will enable simple, rapid, yet sensitive detection without target-/signal amplification and minimize the operation in terms of the physical size, corresponding energy consumption, sample size, and sample preparation time as a field-deployable device. The biosensor is aimed for point-of-care-testing using minimally treated or raw samples. Eventually, the device will be applicable to nucleic acid testing (NAT) for rapid screening of diseases (e.g. cancer), which is enabled through minimally treated samples.


Shu Z.,University of Washington | Weigel K.M.,Seattle Biomedical Research Institute | Soelberg S.D.,University of Washington | Lakey A.,Seattle Biomedical Research Institute | And 4 more authors.
Journal of Clinical Microbiology | Year: 2012

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live"cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Kim J.-H.,University of Washington | Hiraiwa M.,University of Washington | Lee H.-B.,University of Washington | Lee K.-H.,Nanofacture, Inc. | And 2 more authors.
RSC Advances | Year: 2013

Electric detection using a nanocomponent may lead to platforms for rapid and simple biosensing. Sensors composed of nanotips or nanodots have been described for highly sensitive amperometry enabled by confined geometry. However, both fabrication and use of nanostructured sensors remain challenging. This paper describes a dendritic nanotip used as an amperometric biosensor for highly sensitive detection of target bacteria. A dendritic nanotip is structured by Si nanowires coated with single-walled carbon nanotubes (SWCNTs) for generation of a high electric field. For reliable measurements using the dendritic structure, Si nanowires were uniformly fabricated by ultraviolet (UV) lithography and etching. The dendritic structure effectively increased the electric current density near the terminal end of the nanotip according to numerical computation. The electrical characteristics of a dendritic nanotip with additional protein layers was studied by cyclic voltammetry and I-V measurement in deionized (DI) water. When the target bacteria dielectrophoretically captured onto a nanotip were bound with fluorescence antibodies, the electric current through DI water decreased. Measurement results were consistent with fluorescence- and electron microscopy. The sensitivity of the amperometry was 10 cfu/sample volume (103 cfu mL-1), which was equivalent to the more laborious fluorescence measurement method. The simple configuration of a dendritic nanotip can potentially offer an electrolyte-free detection platform for sensitive and rapid biosensors. This journal is © The Royal Society of Chemistry 2013.

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