Membrane-based on-line optical analysis system for rapid detection of bacteria and spores.

We report here the adaptation of our electronic microchip technology towards the development of a new method for detecting and enumerating bacterial cells and spores. This new approach is based on the immuno-localization of bacterial spores captured on a membrane filter microchip placed within a flow cell. A combination of microfluidic, optical, and software components enables the integration of staining of the bacterial species with fully automated assays. The quantitation of the analyte signal is achieved through the measurement of a collective response or alternatively through the identification and counting of individual spores and particles. This new instrument displays outstanding analytical characteristics, and presents a limit of detection of approximately 500 spores when tested with Bacillus globigii (Bg), a commonly used simulant for Bacillus anthracis (Ba), with a total analysis time of only 5 min. Additionally, the system performed well when tested with real postal dust samples spiked with Bg in the presence of other common contaminants. This new approach is highly customizable towards a large number of relevant toxic chemicals, environmental factors, and analytes of relevance to clinical chemistry applications.

[1]  Ihab Abdel-Hamid,et al.  Highly sensitive flow-injection immunoassay system for rapid detection of bacteria , 1999 .

[2]  Dean P. Neikirk,et al.  Solution-based analysis of multiple analytes by a sensor array: toward the development of an electronic tongue , 1998, Other Conferences.

[3]  A. Gehring,et al.  Immunoelectrochemical assays for bacteria: use of epifluorescence microscopy and rapid-scan electrochemical techniques in development of an assay for Salmonella. , 1996, Analytical chemistry.

[4]  Angela Gennaccaro,et al.  Risk and control of waterborne cryptosporidiosis. , 2002, FEMS microbiology reviews.

[5]  D. J. Mason,et al.  The ability of membrane potential dyes and calcafluor white to distinguish between viable and non-viable bacteria. , 1995, The Journal of applied bacteriology.

[6]  Michael Krihak,et al.  An integrated, stacked microlaboratory for biological agent detection with DNA and immunoassays. , 2002, Biosensors & bioelectronics.

[7]  Joseph Berkson,et al.  THE ERROR OF ESTIMATE OF THE BLOOD CELL COUNT AS MADE WITH THE HEMOCYTOMETER , 1939 .

[8]  H. Leclerc,et al.  Microbiological safety of natural mineral water. , 2002, FEMS microbiology reviews.

[9]  M. Szczęsna-Antczak,et al.  Bacillus subtilis cells immobilised in PVA-cryogels. , 2001, Biomolecular engineering.

[10]  Dmitri Ivnitski,et al.  Biosensors for detection of pathogenic bacteria , 1999 .

[11]  Michael Steinert,et al.  Legionella pneumophila: an aquatic microbe goes astray. , 2002, FEMS microbiology reviews.

[12]  J B Shear,et al.  Characterization of multicomponent monosaccharide solutions using an enzyme-based sensor array. , 2001, Analytical biochemistry.

[13]  George P. Anderson,et al.  Automated Fiber Optic Biosensor for Multiplexed Immunoassays , 2000 .

[14]  John T McDevitt,et al.  A microchip-based multianalyte assay system for the assessment of cardiac risk. , 2002, Analytical chemistry.

[15]  Tanja Popovic,et al.  Bacillus anthracis Aerosolization Associated with a Contaminated Mail Sorting Machine , 2002, Emerging infectious diseases.

[16]  J. Brewster,et al.  Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157:H7. , 1998, Journal of immunological methods.

[17]  Raymond C. Stevens,et al.  “Smart” Materials for Biosensing Devices: Cell-Mimicking Supramolecular Assemblies and Colorimetric Detection of Pathogenic Agents , 2002 .

[18]  J. Watkins,et al.  A simple modified membrane filtration medium for the enumeration of aerobic spore-bearing bacilli in water. , 2001, Water research.

[19]  H M Davey,et al.  Variable selection and multivariate methods for the identification of microorganisms by flow cytometry. , 1999, Cytometry.

[20]  George M. Whitesides,et al.  Polymeric Thin Films That Resist the Adsorption of Proteins and the Adhesion of Bacteria , 2001 .

[21]  A. M. Paton,et al.  The observation and enumeration of micro-organisms in fluids using membrane filtration and incident fluorescence microscopy. , 1975, The Journal of applied bacteriology.

[22]  H. Yu,et al.  Comparative studies of magnetic particle-based solid phase fluorogenic and electrochemiluminescent immunoassay. , 1998, Journal of immunological methods.

[23]  D. Kell,et al.  Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms. , 1997, Cytometry.

[24]  G L Andersen,et al.  Sequence-specific identification of 18 pathogenic microorganisms using microarray technology. , 2002, Molecular and cellular probes.

[25]  P Belgrader,et al.  A battery-powered notebook thermal cycler for rapid multiplex real-time PCR analysis. , 2001, Analytical chemistry.

[26]  M. Tabacco,et al.  Optical sensors for detection of bacteria. 1. General concepts and initial development. , 2001, Analytical chemistry.

[27]  Gary L Andersen,et al.  Development of a high‐volume aerosol collection system for the identification of air‐borne micro‐organisms , 2002, Letters in applied microbiology.

[28]  A. Bennett,et al.  Enzyme-linked immunosorbent assay for the detection of airborne microorganisms used in biotechnology , 1997 .

[29]  A Maquieira,et al.  A comparative study by the enzyme-linked immunofiltration assay of solid phases used in the development of flow immunosensors. , 1997, Journal of immunological methods.

[30]  Douglas B. Kell,et al.  Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry , 1992 .

[31]  Kazutaka Yamada,et al.  Optimization of a rapid and sensitive identification system for Salmonella enteritidis by capillary electrophoresis with laser-induced fluorescence. , 2002, FEMS microbiology letters.

[32]  H. Cheun,et al.  Detection of anthrax spores from the air by real‐time PCR , 2001, Letters in applied microbiology.

[33]  Yung-sung Cheng,et al.  Detection of bioaerosols using multiwavelength UV fluorescence spectroscopy , 1999 .

[34]  J B Shear,et al.  Development of multianalyte sensor arrays composed of chemically derivatized polymeric microspheres localized in micromachined cavities. , 2001, Journal of the American Chemical Society.

[35]  Wei Wang,et al.  Antigen detection using microelectrode array microchips , 2001 .

[36]  J. Brewster,et al.  Filtration capture immunoassay for bacteria: optimization and potential for urinalysis. , 1999, Journal of microbiological methods.

[37]  P. Belgrader,et al.  PCR Detection of Bacteria in Seven Minutes , 1999, Science.

[38]  U. Szewzyk,et al.  Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis. , 2002, Journal of microbiological methods.

[39]  H. Cheun,et al.  A simple and sensitive detection system for Bacillus anthracis in meat and tissue , 2001, Journal of applied microbiology.

[40]  K. Klose,et al.  Vibrio cholerae and cholera: out of the water and into the host. , 2002, FEMS microbiology reviews.

[41]  D A Stahl,et al.  Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach. , 2001, Environmental microbiology.

[42]  A. Fox.  Report of the "Bioterrorism Workshop." Duke University Thomas Center on April 2-4, 2002, organized by US Army Research Office. , 2002, Journal of microbiological methods.

[43]  Steve B. Brown,et al.  Autonomous detection of aerosolized Bacillus anthracis and Yersinia pestis. , 2003, Analytical chemistry.

[44]  D. Kell,et al.  Tinopal AN as a selective agent for the differentiation of phytopathogenic and saprophytic Pseudomonas species , 1985 .

[45]  L M Tender,et al.  Array biosensor for simultaneous identification of bacterial, viral, and protein analytes. , 1999, Analytical chemistry.

[46]  T. Sugo,et al.  Capture of microbial cells on brush-type polymeric materials bearing different functional groups. , 1997, Biotechnology and bioengineering.

[47]  B. V. Bronk,et al.  A review of molecular recognition technologies for detection of biological threat agents. , 2000, Biosensors & bioelectronics.

[48]  P Atanasov,et al.  Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7. , 1999, Biosensors & bioelectronics.

[49]  B. Dunn,et al.  A MEMS based amperometric detector for E. coli bacteria using self-assembled monolayers. , 2001, Biosensors & bioelectronics.

[50]  B. C. Weimer,et al.  Solid-Phase Capture of Proteins, Spores, and Bacteria , 2001, Applied and Environmental Microbiology.

[51]  Bill W Colston,et al.  A reusable flow-through polymerase chain reaction instrument for the continuous monitoring of infectious biological agents. , 2003, Analytical chemistry.

[52]  D. Stahl,et al.  Distribution and abundance of Gram-positive bacteria in the environment: development of a group-specific probe. , 2001, Journal of microbiological methods.

[53]  H. Yu.  Enhancing immunoelectrochemiluminescence (IECL) for sensitive bacterial detection. , 1996, Journal of immunological methods.

[54]  K. Martin,et al.  Differentiation between spores of Bacillus anthracis and Bacillus cereus by a quantitative immunofluorescence technique , 1983, Journal of clinical microbiology.

[55]  K Dill,et al.  Rapid, sensitive and specific detection of whole cells and spores using the light-addressable potentiometric sensor. , 1997, Journal of biochemical and biophysical methods.

[56]  G. Blomquist,et al.  Application of PCR and probe hybridization techniques in detection of airborne fungal spores in environmental samples. , 2002, Journal of environmental monitoring : JEM.