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Yeo W.-H.,University of Washington | Yeo W.-H.,University of Illinois at Urbana - Champaign | Lee H.-B.,University of Washington | Kim J.-H.,University of Washington | And 2 more authors.
Nanotechnology | Year: 2013

Rapid and sensitive detection of low-abundance viral particles is strongly demanded in health care, environmental control, military defense, and homeland security. Current detection methods, however, lack either assay speed or sensitivity, mainly due to the nanosized viral particles. In this paper, we compare a dendritic, multi-terminal nanotip ('dendritic nanotip') with a single terminal nanotip ('single nanotip') for dielectrophoretic (DEP) concentration of viral particles. The numerical computation studies the concentration efficiency of viral particles ranging from 25 to 100 nm in radius for both nanotips. With DEP and Brownian motion considered, when the particle radius decreases by two times, the concentration time for both nanotips increases by 4-5 times. In the computational study, a dendritic nanotip shows about 1.5 times faster concentration than a single nanotip for the viral particles because the dendritic structure increases the DEP-effective area to overcome the Brownian motion. For the qualitative support of the numerical results, the comparison experiment of a dendritic nanotip and a single nanotip is conducted. Under 1 min of concentration time, a dendritic nanotip shows a higher sensitivity than a single nanotip. When the concentration time is 5 min, the sensitivity of a dendritic nanotip for T7 phage is 104 particles ml-1. The dendritic nanotip-based concentrator has the potential for rapid identification of viral particles. © 2013 IOP Publishing Ltd.


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 | Yeo W.-H.,University of Washington | Shu Z.,University of Washington | Soelberg S.D.,University of Washington | And 11 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2012

A rapid, accurate tuberculosis diagnostic tool that is compatible with the needs of tuberculosis-endemic settings is a long-sought goal. An immunofluorescence microtip sensor is described that detects Mycobacterium tuberculosis complex cells in sputum in 25 minutes. Concentration mechanisms based on flow circulation and electric field are combined at different scales to concentrate target bacteria in 1 mL samples onto the surfaces of microscale tips. Specificity is conferred by genus-specific antibodies on the microtip surface. Immunofluorescence is then used to detect the captured cells on the microtip. The detection limit in sputum is 200 CFU mL -1 with a success rate of 96%, which is comparable to PCR. © The Royal Society of Chemistry.


Kim J.-H.,University of Washington | Inoue S.,University of Washington | Cangelosi G.A.,University of Washington | Lee K.-H.,Nanofacture, Inc. | Chung J.-H.,University of Washington
Journal of Micromechanics and Microengineering | Year: 2014

A long-sought goal for infectious disease care is a rapid and accurate diagnostic tool that is compatible with the needs of low-resource settings. To identify target biomarkers of infectious diseases, immunoassays utilizing the binding affinity between antigen and antibody have been widely used. In immunoassays, the interaction between antigen and antibody on sensor surfaces should be precisely controlled for specific identification of targets. This paper studies the specific capturing mechanisms of target bacteria onto sensor surfaces through investigation of combined effects of capillary action and binding affinity. As a model system, cells of both Escherichia coli and the Bacillus Calmette-Guérin strain of Mycobacterium bovis were used to study specific and nonspecific capturing mechanisms onto a microtip sensor. The capillary action was observed to arrange the concentrated cells onto the two-dimensional sensor surface. Due to the capillary-induced organization of target cells on the antibody-functionalized sensor surface, the number of the captured target cells was three times greater than that of the non-targeted cells. The capturing and detection capabilities varied with the width of a microtip. The specific capturing mechanism can be used to enhance the sensitivity and specificity of an immunoassay. © 2014 IOP Publishing Ltd.


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.


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.


Kim J.-H.,University of Washington | Shen A.Q.,University of Washington | Lee K.-H.,Nanofacture, Inc. | Cangelosi G.A.,University of Washington | Chung J.-H.,University of Washington
Analyst | Year: 2014

Immunoassays analyzing interactions between antigens and antibodies can be affected by capillary action together with binding affinity. This paper studies contact-angle changes of bacterial suspensions on antibody immobilized surfaces. The capillary action and the dried pattern of the bacterial suspensions are analyzed and correlated with specific- and nonspecific bindings between bacteria and antibodies. © 2014 The Royal Society of Chemistry.


Yeo W.-H.,University of Washington | Chou F.-L.,University of Washington | Fotouhi G.,University of Washington | Oh K.,University of Washington | And 6 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2010

Rapid, low cost screening of tuberculosis requires an effective enrichment method of Mycobacterium tuberculosis (MTB) cells. Currently, microfiltration and centrifugation steps are frequently used for sample preparation, which are cumbersome and time-consuming. In this study, the size-selective capturing mechanism of a microtip-sensor is presented to directly enrich MTB cells from a sample mixture. When a microtip is withdrawn from a spherical suspension in the radial direction, the cells that are concentrated by AC electroosmosis are selectively enriched to the tip due to capillary- and viscous forces. The size-selectivity is characterized by using polystyrene microspheres, which is then applied to size-selective capture of MTB from a sample mixture. Our approach yields a detection limit of 800 cells mL-1, one of the highest-sensitivity immunosensors to date. © 2010 The Royal Society of Chemistry.


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: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2012

DESCRIPTION (provided by applicant): The current SBIR Phase I proposal addresses a need for the rapid and simple sample preparation of genomic DNA from human saliva and blood through a novel technology: microscale tip enrichment probes that employ electric fields and capillary action. With the action of a microtip-installed device, DNA contained in a given saliva sample can be selectively captured without centrifugation on the proposed microtip, and stored as a dried form for long- term preservation and delivery. The preserved DNA on microtips is released into a desired medium for PCR analysis without additional purification steps. Therefore, this innovative technology enables the one-step concentration of DNA from human saliva and blood as a purified, durable form. This application describes a feasibility study of the proposed method and system to explore and optimize the protocol of centrifuge-free preparation of PCR-ready DNA. PUBLIC HEALTH RELEVANCE: This proposal aims to conduct a feasibilitystudy for a portable device capable of concentrating and purifying human genomic DNA from saliva without centrifuge. The success of the project would provide a convincing rationale and form a technical basis for the proposed methodology for rapid and simple preparation of PCR-ready DNA from various sample specimens with a high throughput at a low cost.

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