Source: N Y AGRICULTURAL EXPT STATION submitted to NRP
LIPOSOME-AMPLIFIED BIOANALYSIS OF TOXIC CHEMICALS AND PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0162162
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
GENEVA - FOOD SCIENCE & TECH
Non Technical Summary
Rapid and highly specific techniques are needed to detect toxins and pathogens in the field. The development of commercialized bioanalytical devices and sensors based on these studies will eventually lead to simple, rapid assays that can be used in extra-laboratory venues (on-site) for the real-time detection of toxic chemicals and pathogenic organisms in foods and the environment.
Animal Health Component
40%
Research Effort Categories
Basic
10%
Applied
40%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7115010100050%
7125010100050%
Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5010 - Food;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
Determine optimal methods using nucleic acid-, natural receptor-, analyte- or antibody-tagged liposomes containing detectable markers (e.g., dyes, quantum dots) for the instantaneous amplification of the competitive binding or sandwich hybridization reaction between sample analyte molecules or organisms and liposomes. Develop fully automated flow-injection immunoanalysis systems, microfluidic systems and simple extra-laboratory assay devices (e.g., biosensors and lateral-flow devices).
Project Methods
A variety of encapsulated markers will be studied (optical, electrochemical, and enzymatic) to evaluate the characteristics of each in terms of several operational parameters including stability, sensitivity and specificity. Highly specific antibodies, natural receptors and nucleic acid probes for the analytes of interest will be developed. Field- and laboratory-testable devices will be developed. Antibodies and other types of receptors will be tested for cross reactivity with metabolites and relevant compounds of similar structure. Food commodities, and surface and ground water samples will be analyzed in the field and lab in validation studies. NASBA will be used for nucleic acid amplification in the development of sandwich assays.

Progress 10/01/03 to 09/30/08

Outputs
Research was focussed on the development of sensitive and specific bioanalytical assays based on liposomal amplification strategies. The assay platforms fall primarily under two formats: (1) automated, computer-controlled Flow-Injection Liposome ImmunoAnalysis (FILIA) or Nucleic-acid Analysis (FILNA) systems and (2) rapid, simple lateral flow assays (LFA). Considerable success has been made with both approaches. With the FILIA/NA systems, assays have been completed for the determination of the herbicides imazethapyr and alachlor, the pathogens Escherichia coli and Listeria monocytogenes, and the mycotoxin fumonisin B1. With the LFA approach, assays have been completed for the detection of the pathogens Escherichia coli, Cryptosporidium parvum, Salmonella spp. and Listeria monocytogenes, for the pesticide alachlor, for the natural glycoalkaloidal toxins solanine and chaconine, for Shiga toxins I and II, and for the peanut allergen Ara h1. Overall, in both approaches the methodologies and components have been refined and improved. Furthermore, the lateral-flow assays for C. parvum, E. coli and Shiga toxins have been performed as nucleic acid-probe (RNA gene-probe) assays which not only provide detection data but also the viability status of the microorganisms. The assays are additionally being incorporated into simple microfluidic devices using microfabrication approaches with the possibility for either electrochemical or optical detection. E. coli has also been determined by a fluorescence tube assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces). This work has been extended by the use of immunomagnetic bead separation and concentration of the E. coli prior to detection with the immunoliposomes. Also, extremely sensitive and specific assays have been developed for cholera and botulinum toxins using a hybrid recognition LFA approach: ganglioside-liposomes and capture antibodies. Finally, several projects have been completed: the detection of the principal peanut allergen, Ara h1 in chocolate; E. coli using 'universal' immunoliposomes prepared with protein G conjugated to the liposome surface; a nucleic-acid LFA for Streptococcus pyogenes; a LFA based on nucleic-acid detection of C. parvum and an antibody immunoassay for Erwinia amylovora, the organism causing fire blight in fruit.

Impacts
The lateral-flow nucleic-acid based assay for Cryptosporidium parvum is currently undergoing field testing. If these tests are successful, several collaborating companies will be adding new fabrication facilities and personnel for production, commercialization and marketing. Subsequently, several of the other assays are expected to be commercialized using similar technology. These simple, inexpensive, single-use tests will be further developed by the use of microfluidics and should improve food safety, homeland security and environmental quality.

Publications

  • Wen, H.-W., Borejsza_Wysocki, DeCory, T.R and Durst, R.A. 2006. Investigation of NeutrAvidin-Tagged Liposomal Nanovesicles as Universal Detection Reagents for Bioanalytical Assays, Talanta 68(4): 1264-1272
  • Chen, C.-S. and Durst, R.A. 2006. Simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes with an array-based immunosorbent assay using universal protein G-liposomal nanovesicles, Talanta 69: 232-238.
  • Borejsza-Wysocki, W., Aldwinckle., H. and Durst, R.A. 2006. Immunoliposomes for early detection of Erwinia amylovora. Acta Horticulturae 704: 63-68.


Progress 01/01/05 to 12/31/05

Outputs
Research is focussed on the development of bioanalytical assays based on liposomal amplification strategies. The assay platforms fall primarily under two formats: (1) an automated, computer-controlled Flow-Injection Liposome ImmunoAnalysis (FILIA) or Nucleic-acid Analysis (FILNA) system and (2) a rapid, simple lateral flow assay (LFA). Considerable success has been made with both approaches. With the FILIA/NA systems, assays have been completed for the determination of the herbicides imazethapyr and alachlor, the pathogen Listeria monocytogenes, and the mycotoxin fumonisin B1. With the LFA approach, assays have been completed, or are ongoing, for the detection of the pathogens Escherichia coli, Cryptosporidium parvum, Salmonella spp. and Listeria monocytogenes, for the pesticide alachlor, for the natural glycoalkaloidal toxins solanine and chaconine, for Shiga toxins I and II, and for the peanut allergen Ara h1. Overall, in both approaches the methodologies and components have been, and continue to be, refined and improved. Furthermore, the lateral-flow assays for C. parvum, E. coli and Shiga toxins have been performed as nucleic acid-probe (gene-probe) assays which not only provide detection data but also the viability status of the microorganisms. The assays are additionally being incorporated into simple microfluidic devices using microfabrication approaches with the possibility for either electrochemical or optical detection. E. coli has also been determined by a fluorescence tube assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces). This work has been extended by the use of immunomagnetic bead separation and concentration of the E. coli prior to detection with the immunoliposomes. Also, extremely sensitive and specific assays have been developed for cholera and botulinum toxins using a hybrid recognition LFA approach: ganglioside-liposomes and capture antibodies. Finally, several projects have been completed: the detection of the principal peanut allergen, Ara h1, in chocolate; E. coli using 'universal' immunoliposomes prepared with protein G conjugated to the liposome surface; a nucleic-acid LFA for Streptococcus pyogenes; and LFAs based on nucleic-acid detection of C. parvum and an antibody immunoassay for Erwinia amylovora, the organism causing fire blight in fruit are currently in progress.

Impacts
The lateral-flow nucleic-acid based assay for Cryptosporidium parvum is currently undergoing field testing by an independent laboratory. If these tests are successful, several collaborating companies will be adding new fabrication facilities and personnel for commercialization and marketing. Subsequently, several of the other assays are expected to be commercialized using similar technology. These simple, inexpensive, single-use tests will be further developed by the use of microfluidics and should improve food and homeland security and environmental safety.

Publications

  • DeCory, T., Durst, R.A., Zimmerman, S., Garringer, L., Paluca, G. and Montagna, R.A. 2005. Development of an Immunomagnetic Bead-Immunoliposome Fluorescence Assay for Rapid Detection of E. coli O157:H7 in Aqueous Samples and Comparison of the Assay with a Standard Microbiological Method in Appl. Environ. Microbiol. 71(4): 1856-1864
  • Wen, H.-W., Borejsza-Wysocki, W., DeCory, T.R., and Durst, R.A. 2005. Development of a Competitive Liposome-Based Lateral Flow Assay for the Rapid Detection of the Allergenic Peanut Protein Ara h1 in Anal. Bioanal. Chem. 382(5): 1217-1226
  • Chen, C.-S., Baeumner, A.J. and Durst, R.A. 2005. Protein G-Liposomal Nanovesicles as Universal Reagents for Immunoassays in Talanta 67(1): 205-211
  • Wen, H.-W., Borejsza_Wysocki, DeCory, T.R and Durst, R.A. 2005. A Novel Extraction Method for Peanut Allergenic Proteins in Chocolate and Their Detection by a Liposome-based Lateral Flow Assay in European Food Res. Tech. 221: 564-569


Progress 01/01/04 to 12/31/04

Outputs
Research is focussed on the development of bioanalytical assays based on liposomal amplification strategies. The assay platforms fall primarily under two formats: (1) an automated, computer-controlled Flow-Injection Liposome ImmunoAnalysis (FILIA) or Nucleic-acid Analysis (FILNA) system and (2) a rapid, simple lateral flow assay (LFA). Considerable success has been made with both approaches. With the FILIA/NA systems, assays have been completed for the determination of the herbicides imazethapyr and alachlor, the pathogen Listeria monocytogenes, and the mycotoxin fumonisin B1. With the LFA approach, assays have been completed, or are ongoing, for the detection of the pathogens Escherichia coli, Cryptosporidium parvum and Listeria monocytogenes, for the pesticide alachlor, for the natural glycoalkaloidal toxins solanine and chaconine, and for Shiga toxins I and II. Overall, in both approaches the methodologies and components have been, and continue to be, refined and improved. Furthermore, the lateral-flow assays for C. parvum, E. coli and Shiga toxins have been performed as nucleic acid-probe (gene-probe) assays which not only provide detection data but also the viability status of the microorganisms. The assays are additionally being incorporated into simple microfluidic devices using microfabrication approaches with the possibility for either electrochemical or optical detection. E. coli has also been determined by a fluorescence tube assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces). This work is being extended by the use of immunomagnetic bead separation and concentration of the E. coli prior to detection with the immunoliposomes. Also, extremely sensitive and specific assays have been developed for cholera and botulinum toxins using a hybrid recognition LFA approach: ganglioside-liposomes and capture antibodies. Finally, good progress has been made on several new projects: the detection of the principal peanut allergen, Ara h1, in chocolate; histamine for the detection of seafood spoilage; E. coli using 'universal' immunoliposomes prepared with protein G conjugated to the liposome surface; a nucleic-acid LFA for Streptococcus pyogenes; and LFAs based on nucleic-acid detection and an antibody immunoassay for Erwinia amylovora, the organism causing fire blight in fruit.

Impacts
Several of these assays are in the final stages of commercialization and prototype devices for Cryptosporidium parvum and E. coli O157:H7 are expected to be tested in 2005. If, as expected, these tests are successful, several collaborating companies will be adding new fabrication facilities and personnel. Subsequently, several of the other assays are expected to be commercialized. These simple, inexpensive, single-use tests should improve food and homeland security and environmental safety.

Publications

  • Kim, M., Oh, S. and Durst, R.A. 2003. Detection of Escherichia coli O157:H7 using Combined Procedure of Immunomagnetic Separation and Test Strip Liposome immunoassay. J. Microbiol. Biotech. 13(4):509-516.
  • Kim, M., Lee, J., Shin, W.S. and Durst, R.A. 2003. Flow-Injection Liposome Immunoanalysis System for Potato Glycoalkaloids. Food Sci. Biotech. 12(4):430-434.
  • Park, S., Oh, S. and Durst, R.A. 2004. Immunoliposome Sandwich Fluorometric assay (ILSF) for Detection of Escherichia coli O157:H7, J. Food Science 69(6):M151-M156.
  • Ahn-Yoon, S., DeCory, T.R. and Durst, R.A. 2004. Ganglioside-Liposome Immunoassay for the Detection of Botulinum Toxin. Anal. Bioanal. Chem. 378:68-75.
  • Park, S. and Durst, R.A. 2004. Modified Immunoliposome Sandwich Assay for the Detection of Escherichia coli O157:H7 in Apple Cider. J. Food Prot. 67(8):1568-1573.
  • Baeumner, A.J. and Durst, R.A. 2004. Liposome-Amplified Bioanalytical Techniques. LabPlus Intl. 2004:21-25.


Progress 01/01/03 to 12/31/03

Outputs
Research is focussed on the development of bioanalytical assays based on liposomal amplification strategies. The assay platforms fall primarily under two formats: (1) an automated, computer-controlled Flow-Injection Liposome ImmunoAnalysis (FILIA) or Nucleic-acid Analysis (FILNA) system and (2) a rapid, simple immunomigration (lateral-flow) assay. Considerable success has been made with both approaches. With the FILIA/NA systems, assays have been completed for the determination of the herbicides, imazethapyr and alachlor, the pathogen, Listeria monocytogenes, and the mycotoxin, fumonisin B1. With the immunomigration approach, assays have been completed, or are ongoing, for the detection of the pathogens, Escherichia coli, Cryptosporidium parvum and Listeria monocytogenes, for the pesticide, alachlor, for the glycoalkaloids, solanine and chaconine, which are natural toxins found in potatoes, and for Shiga toxins I and II. Overall. In both approaches, the methodologies and components have been, and continue to be, refined and improved. Furthermore, the lateral-flow assays for C. parvum, E. coli and Shiga toxins have been performed as nucleic acid-probe (gene-probe) assays which not only provide detection data but also the viability status of the microorganisms. The assays are additionally being incorporated into simple cassette devices with the possibility for either electrochemical or optical detection. Microminiaturization of the assay is also being attempted using nanofabrication approaches. E. coli has also been determined by an immunomigration sandwich-assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces). This work is being extended by the use of immunomagnetic bead separation and concentration of the E. coli prior to detection with the immunoliposomes. During the current period, extremely sensitive and specific assays have been developed for cholera and botulinum toxins using a hybrid recognition approach: ganglioside-liposomes and capture antibodies. Finally, new projects have been initiated for the detection of the peanut allergen, Ara h1, histamine for the detection of seafood spoilage, for E. coli using "universal" immunoliposomes containing quantum dot markers, for a nucleic-acid LFA for Streptococcus pyogenes, and for a nucleic-acid LFA for Erwinia amylovora, the organism causing fire blight in fruit.

Impacts
Several of these assays are in the final stages of commercialization and prototype devices for Cryptosporidium parvum, E. coli O157:H7 and histamine are expected to be beta tested in 2004. If, as expected, these test are successful, several companies will be adding new fabrication facilities and personnel. Subsequently, several of the other assays will be commercialized. These simple, inexpensive, single-use tests should improve food security and environmental safety.

Publications

  • Ho, J.-A. and Durst, R.A. 2003. Detection of Fumonisin B1: Comparison of Flow-Injection Liposome Immunoanalysis (FILIA) with HPLC, Anal. Biochem. 312, 7-13.
  • Ahn-Yoon, S., DeCory, T. Baeumner, A.J. and Durst, R.A. 2003. Ganglioside-Liposome Immunoassay for the Ultrasensitive Detection of Cholera Toxin, Anal. Chem. 75, 2256-2261.


Progress 01/01/02 to 12/31/02

Outputs
Research is continuing on the development of bioanalytical assays and biosensors based on liposomal amplification strategies. The assays fall basically under two formats: automated, computer-controlled Flow-Injection ImmunoAnalysis (FILIA) systems and rapid, simple sensitive and specific migration assay devices. Considerable success has been made with both approaches. With the FILIA system, assays have been completed on the use of a nucleic acid-probe FILIA for the determination of Listeria monocytogenes in food products. Using the immunomigration approach, assays have been completed, or are ongoing, for the detection of the pathogenic organisms, Escherichia coli, Cryptosporidium parvum and Listeria monocytogenes, and for Shiga toxin-producing E. coli. In both approaches, the methodologies and components have been refined and improved. Furthermore, the migration assays for C. parvum, E. coli and Shiga toxin-producing E. coli are being performed as nucleic acid probe (gene-probe) assays which not only provide detection data but also the viability status of the microorganisms. The assays are additionally being incorporated into a simple cassette devices with the possibility for either electrochemical or optical detection. E. coli has also been determined by a new immunomigration sandwich-assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces). This work is being extended by the use of immunomagnetic bead separation and concentration of the E. coli prior to detection with the immunoliposomes containing fluorescent markers. The lastest bioassay is a hybrid technique using natural receptors (gangliosides) for cholera and botulinum toxins combined with antibodies for the same toxins. Preliminary results in an optical migration-strip, sandwich-assay format showed extremely high sensitivity and excellent specificity.

Impacts
These studies will result in simple, rapid, sensitive and specific bioassays that can be used in extralaboratory venues (on-site) for the real-time detection of toxic chemicals and pathogenic organisms in foods and the environment.

Publications

  • No publications reported this period


Progress 01/01/01 to 12/31/01

Outputs
Research is continuing on the development of bioanalytical assays based on liposomal amplification strategies. The assays fall basically under two formats: an automated, computer-controlled Flow-Injection ImmunoAnalysis (FILIA) system and a rapid, simple immunomigration assay. Considerable success has been made with both approaches. With the FILIA system, assays have been completed for the determination of the mycotoxin, fumonisin B1, and for the simultaneous determination of the glycoalkaloid, solanine, and the hericide, alachlor. An ongoing project is focussed on the use of a nucleic acid-probe FILIA for the determination of Listeria monocytogenes in food products. Using the immunomigration approach, assays have been completed, or are ongoing, for the detection of the pathogenic organisms, Escherichia coli, Cryptosporidium parvum and Listeria monocytogenes, and for Shiga-like toxin-producing E. coli. In both approaches, the methodologies and components have been refined and improved. Furthermore, the migration assays for C. parvum, E. coli and Shiga toxin-producing E. coli are being performed as nucleic acid probe (gene-probe) assays which not only provide detection data but also the viability status of the microorganisms. The assays are additionally being incorporated into a simple cassette devices with the possibility for either electrochemical or optical detection. Microminiaturization of the assay for C. parvum has also been achieved using nanofabrication approaches. E. coli has also been determined by a new immunomigration sandwich-assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces). This work is being extended by the use of immunomagnetic bead separation and concentration of the E. coli prior to detection with the immunoliposomes.

Impacts
These studies will eventually lead to simple, rapid assays that can be used in extralaboratory venues (on-site) for the real-time detection of toxic chemicals and pathogenic organisms in foods and the environment.

Publications

  • Baeumner, A.J., Humiston, M.C., Montagna, R.A. and Durst, R.A., 2001. Detection of Viable Oocysts of Cryptosporidium parvum Following Nucleic Acid Sequence Based Amplification, Anal. Chem. 73, 1176-1180.
  • Park, S., Worobo, R.W., and Durst, R.A., 2001. Escherichia coli O157:H7 as an Emerging Foodborne Pathogen: A Literature Review, Crit. Revs. in Biotechnol. 21, 27-48.
  • Esch, M.B., Locascio, L.E., Tarlov, M.J. and Durst, R.A., 2001. Detection of Viable Cryptosporidium parvum using DNA-Modified Liposomes in a Microfluidic Chip, Anal. Chem. 73, 2952-2958.
  • Esch, M.B., Baeumner, A.J. and Durst, R.A., 2001. Detection of Viable Cryptosporidium parvum on using Oligonucleotide-tagged Liposomes in a Competitive Assay Format, Anal. Chem. 73, 31-62-3167.


Progress 01/01/00 to 12/31/00

Outputs
Research is continuing on the development of bioanalytical assays based on liposomal amplification strategies. The assays fall basically under two formats: an automated, computer-controlled Flow-Injection ImmunoAnalysis (FILIA) system and a rapid, simple immunomigration assay. Considerable success has been made with both approaches. With the FILIA system, assays have been completed for the determination of the herbicides, imazethapyr and alachlor, and for the mycotoxin, fumonisin B1. Overall, seven papers have been published on this work and another is in press. An ongoing project is focussed on the use of FILIA for the determination of Listeria monocytogenes in food products. With the immunomigration approach, assays have been completed, or are ongoing, for the detection of the pathogenic organisms, Escherichia coli, Cryptosporidium parvum and Listeria monocytogenes, for the pesticide alachlor, for the glycoalkaloids, solanine and chaconine, which are natural toxins found in potatoes, and for Shiga-like Toxins I and II. Overall, nine papers have been published on this work, three are in press, eight are in preparation. In both approaches, the methodologies and components have been refined and improved. Furthermore, the migration assays for C. parvum, E. coli and Shiga-like toxins are being performed as a nucleic acid probe (gene-probe) assay which not only provides detection data but also the viability status of the microorganisms. The assays are additionally being incorporated into a simple cassette devices with the possibility for either electrochemical or optical detection. Microminiaturization of the assay is also being attempted using nanofabrication approaches. E. coli has also been determined by a new immunomigration sandwich-assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces). This work is being extended by the use of immunomagnetic bead separation and concentration of the E. coli prior to detection with the immunoliposomes.

Impacts
These studies will eventually lead to simple, rapid assays that can be used in extralaboratory venues (on-site) for the real-time detection of toxic chemicals and pathogenic organisms in foods and the environment.

Publications

  • Lee, M., Durst, R.A., Spittler, T.D. and Forney, D.R. 2000. FILIA Determination of Imazethapyr Herbicide in Water, Agrochemical Fate and Movement: Perspective and Scale of Study, Steinheimer, T.R., Ross, L.J. and Spittler, T.D., eds., ACS Symposium Series 751, Amer. Chem. Soc., Washington, DC.
  • Ho, J.-A. and Durst, R.A. 2000. Preparation of Reagents for the Determination of Fumonisin B1 by Flow-Injection Liposome ImmunoAnalysis, Anal. Chim. Acta 414:51-60.
  • Ho, J.-A. and Durst, R.A. 2000. Development of a Flow-Injection Liposome ImmunoAnalysis System for Fumonisin B1, Anal. Chim. Acta 414:61-69.
  • Glorio, P. and Durst, R.A. 2000. Determination of Potato Glycoalkaloids Using a Liposome Immunomigration, Liquid-Phase Competition Immunoassay, J. Agric. Food Chem. 48:1678-1683.
  • Park, S. and Durst, R.A. 2000. Immunoliposome Sandwich Assay for the Detection of Escherichia coli O157:H7, Anal. Biochem. 280:151-158.
  • Martorell, D., S.T.A. Siebert and Durst, R.A. 1999. Liposome Dehydration on Nitrocellulose and its Application in a Biotin Immunoassay, Anal. Biochem. 271:177-185.
  • Park, S., Worobo, R.W., and Durst, R.A. 1999. Escherichia coli O157:H7 as an Emerging Foodborne Pathogen: A Literature Review, Critical Revs. in Food Science and Nutrition 39:481-502.


Progress 01/01/99 to 12/31/99

Outputs
Research is continuing on the development of bioanalytical assays based on liposomal amplification strategies. The assays fall basically under two formats: an automated, computer-controlled Flow-Injection ImmunoAnalysis (FILIA) system and a rapid, simple immunomigration assay. Considerable success has been made with both approaches. With the FILIA system, assays have been completed, or are ongoing, for the determination of the herbicides, imazethapyr and alachlor, and for the mycotoxin, fumonisin B1. With the immunomigration approach, assays have been completed, or are ongoing, for the detection of the pathogenic organisms, Escherichia coli, Cryptosporidium parvum and Listeria monocytogenes, for the pesticide alachlor, and for the glycoalkaloids, solanine and chaconine, which are natural toxins found in potatoes. Two papers have been published on this work, two are in press, two are in preparation. In both approaches, the methodologies and components have been refined and improved. Furthermore, the migration assay for C. parvum is being performed as a nucleic acid probe (gene-probe) assay which not only provides detection data but also the viability status of the microorganism. The assay is additionally being incorporated into a simple cassette device with the possibility for either electrochemical or optical detection. E. coli is also being determined by a new sandwich-assay approach using immunoliposomes (liposomes with antibodies conjugated to their surfaces).

Impacts
(N/A)

Publications

  • Lee, M. and Durst, R.A., Determination of Imazethapyr using Capillary-Column Flow Injection Liposome Immunoanalysis, J. Agric. Food Chem. 44, 4032-4036 (1996)
  • Rule, G.S., Montagna, R.A. and Durst, R.A., Characteristics of DNA-Tagged Liposomes Allowing Their Use in Capillary-Migration, Sandwich-Hybridization Assays, Anal. Biochem. 244, 260-269 (1997).
  • Lee, M., Durst, R.A., and Wong, R.B., Comparison of Liposome Amplification and Fluorophor Detection in Flow-Injection Immunoanalysis, Anal. Chim. Acta 354, 23-28 (1997)
  • Lee, M., Durst, R.A., and Wong, R.B., Development of Flow-Injection Liposome Immunoanalysis (FILIA) for Imazethapyr, Talanta 46, 851-859 (1998).


Progress 01/01/98 to 12/31/98

Outputs
An automated, computer-controlled Flow-Injection Liposome ImmunoAnalysis (FILIA) system has been modified from a previous design, using specific environmental contaminants, the herbicide imazethapyr and the mycotoxin fumonisin B1, as model analytes. Signal amplification has been achieved by means of analyte-tagged liposomes containing either fluorescent or electrochemical markers to provide high detection sensitivity. The relationship between antibody concentration and assay speed and sensitivity has been studied, and the possibility of using the system for determination of multiple analytes is under investigation. The use of immunospecific liposome migration offers many advantages for the extra-laboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, robust, and respond to the presence of analyte at low-parts-per-billion concentrations. Investigations have been carried out that determine optimal interactions for key components of these assays, and techniques are presented for the development of generalized liposome immunoassays. Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of several analytes: alachlor, glycoalkaloids and polychlorinated biphenyls. Both formats utilize capillary action to transport liposome-containing solutions along strips of nitrocellulose. Measurement of color intensity is then carried out visually or with a desktop scanner. More recently, these latter approaches are being applied to the detection of pathogenic organisms, including bovine herpesvirus, Cryptosporidium parvum, and E. coli O157:H7, using either antibodies or nucleic acid probes for biorecognition. In addition, the use of electrochemical detection on the test strips is being investigated using interdigitated ultramicroelectrode arrays for enhanced sensitivity. Patent No. 5,753,519. May 19, 1998. Patent No. 5,756,362. May 26, 1998. Patent No. 5,789,154. Aug. 4, 1998.

Impacts
(N/A)

Publications

  • Lee, M., Durst, R.A. and Wong, R.B. 1998. "Development of Flow-Injection Liposome Immunoanalysis (FILIA) for Imazethapyr", Talanta 46, 851-859.
  • Mathai, M., Siebert, K.J., Siebert, S.A., Durst, R.A. and Reeves, S.G. 1998. "Development of a Signal Measurement Technique for Application in Multi-analyte Liposome Immunomigration Assays", Food Agric. Immunol. 10, 195-202.
  • Rule, G. S., Montagna, R. A. and Durst, R. A. 1997. "Characteristics of DNA-Tagged Liposomes Allowing Their Use in Capillary-Migration." Sandwich-Hybridization Assays, Anal. Biochem, 244, 260-269. in Biosciences, T. T. Ngo and D. Narinesingh, eds., Plenum Press.
  • Lee, M., Durst, R.A. and Wong, R.B. 1997. "Comparison of Liposome Amplification and Fuorophor Detection in Flow-Injection Immunoanalysis", Anal. Chim. Acta 354, 23-28.
  • Lee, M. and Durst, R. A. 1996. "Determination of Imazethapyr Using Capillary-Column FILIA (Flow-Injection Liposome Immunoanalysis). J. Agric. Food Chem. 44, 4032-4036.


Progress 01/01/97 to 12/31/97

Outputs
An automated Flow-Injection Liposome ImmunoAnalysis (FILIA) system has been modified from a previous design, using specific environmental contaminants, the the mycotoxin fumonisin and the herbicides alachlor and imazethapyr, as model analytes. Signal amplification has been achieved by means of analyte-tagged liposomes containing either fluorescent or electrochemical markers to provide high detection sensitivity. The computer controlled system is composed of commercially available components, with the exception of the column packing material, which has to be prepared for each specific analyte to be determined. The relationship between antibody concentration and assay speed and sensitivity has been studied, and the possibilities of using the system for determination of multiple analytes is under investigation. The use of immunospecific liposome migration offers many advantages for the extra-laboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, robust, and respond to the presence of analyte at low-parts-per-billion concentrations. Investigations have been carried out that determine optimal interactions for key components of these assays, and techniques are presented for the development of generalized liposome immunoassays. Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of several analytes: alachlor, glycoalkaloids, E. coli and polychlorinated biphenyls. Both formats utilize capillary action to transport liposome-containing solutions along strips of nitrocellulose. Measurement of color intensity is then carried out visually or with a desktop scanner.

Impacts
(N/A)

Publications

  • Lee, M. and Durst, R. A. 1996. "Determination of Imazethapyr Using Capillary-Column FILIA (Flow-Injection Liposome Immunoanalysis). J. Agric. Food Chem. 44, 4032-4036.
  • Rule, G. S., Montagna, R. A. and Durst, R. A. 1997. "Characteristics of DNA-Tagged Liposomes Allowing Their Use in Capillary-Migration." Sandwich-Hybridization Assays, Anal. Biochem, 244, 260-269.
  • Lee, M. and Durst, R. A. 1997. "Comparison of Liposome Amplification and Fluorophore Detection in Flow-Injection Immunoanalyses." Anal. Chim. Acta 354, 23-28.
  • Locascio-Brown, L., Choquette, S., Reeves, S. G. and Durst, R. A. 1998. "Liposome ImmunoAnalysis (FILIA)." Flow Injection Analysis Applications in Biosciences, T. T. Ngo and D. Narinesingh, eds., Plenum Press.
  • Roberts, M. A., Reeves, S. G., Siebert, S. T. A. and Durst, R. A. 1998. "Liposome-Enhanced Immunomigration Strips for Field Screening of Toxic Chemicals. Field Screening Methods for Hazardous Wastes and Toxic Chemicals.(in press)


Progress 01/01/96 to 12/30/96

Outputs
An automated Flow-Injection Liposome ImmunoAnalysis (FILIA) system has been modified from a previous design, using specific environmental contaminants, the herbicides alachlor and Pursuit, as model analytes. Signal amplification has been achieved by means of analyte-tagged liposomes containing either fluorescent or electrochemical markers to provide high detection sensitivity. The computer controlled system is composed of commercially available components, with the exception of the column packing material. The relationship between antibody concentration and assay speed and sensitivity has been studied, plus using the system for determination of multiple analytes is under investigation. The use of immunospecific liposome migration offers many advantages for the extra-laboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, robust, and respond to the presence of analyte at low-parts-per-billion concentrations. Investigations have been carried out that determine optimal interactions for key components of these assays, and techniques are presented for the development of generalized liposome immunoassays. Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of several analytes: alachlor, glycoalkaloids and polychlorinated biphenyls. Both formats utilize capillary action to transport liposome-containing solutions along strips of nitrocellulose. Measurement of color intensity is then carried out.

Impacts
(N/A)

Publications

  • Reeves, S. G., Siebert, S. T. A. and Durst, R. A. 1996. Liposome-Amplified Immunodetermination... (17) Vol. IV: From Gene Delivery and Diagnostics to Ecology, D. D. Lasic and Barenholz, Y., eds. CRC Press, Boca Raton, FL, 247-256.
  • Rule, G. S., Montagna, R. A. and Durst, R. A. 1996. Rapid Method for Visual Identification of Specific DNA Sequences Based on DNA-Tagged Liposomes. Clin. Chem. 42 (8), 1206-1209.
  • Roberts, M. A., Locascio-Brown, L., MacCrehan, W. A. and Durst, R. A. 1996. Liposome Behavior in Capillary Electrophoresis. Anal. Chem. 68 (19), 3434-3440.
  • Rule, G. S., Montagna, R. A. and Durst, R. A. 1997. Characteristics of DNA-Tagged Liposomes Allowing Their Use in Capillary-Migration. Sandwich-Hybridization Assays, Anal. Biochem, 244.
  • Locascio-Brown, L., Choquette, S., Reeves, S. G. and Durst, R. A. 1997. Liposome ImmunoAnalysis (FILIA). Flow Injection Analysis Applications in Biosciences, T. T. Ngo and D. Narinesingh, eds., Plenum Press. (in press).
  • Roberts, M. A., Reeves, S. G., Siebert, S. T. A. and Durst, R. A. 1997. Liposome-Enhanced Immunomigration Strips for Field Screening of Toxic Chemicals.Field Screening Methods for Hazardous Wastes and Toxic Chemicals. (in press).
  • Lee, M. and Durst, R. A. 1997. Determination of Imazethapyr Using Capillary-Column FILIA (Flow-Injection Liposome Immunoanalysis). J. Agric. Food Chem. (in press).


Progress 01/01/95 to 12/30/95

Outputs
An automated Flow-Injection Liposome ImmunoAnalysis (FILIA) system has been modified from a previous design, using specific environmental contaminants, the herbicides alachlor and Pursuit, as model analytes. Signal amplification has been achieved by means of analyte-tagged liposomes containing either fluorescent or electrochemical markers to provide high detection sensitivity. The computer controlled system is composed of commercially available components, with the exception of the column packing material, which has to be prepared for each specific analyte to be determined. The relationships between antibody concentration and assay speed and sensitivity has been studied, and the possibilities of using the system for determination of multiple analytes is under investigation. The use of immunospecific liposome migration offers many advantages for the extra-laboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, robust, and respond to the presence of analyte at low-parts-per-billion concentrations. Investigations have been carried out that determine optimal interactions for key components of these assays, and techniques are presented for the development of generalized liposome immunoassays. Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of several analytes: alachlor, glycoalkaloids and polychlorinated biphenyls. Both formats utilize capillary action to transport liposome-containing solu.

Impacts
(N/A)

Publications

  • ROBERTS, M. A. and DURST, R. A. 1995. Investigation of liposome-based immunomigration sensors for the detection of polychlorinated biphenyls. Anal. Chem. 67:482-491.
  • RULE, G. S., PALMER, D. A., REEVES, S. G., and DURST, R. A. 1994. Use of protein A in a liposome-enhanced flow-injection immunoassay. Anal. Proc. 31:339-340.
  • EDWARDS A. J. and DURST, R. A. 1995. Flow-injection liposome immunoAnalysis (FILIA) with electrochemical detection. Electroanalysis 7:838-845.
  • SIEBERT, S. T. A., REEVES, S. G., ROBERTS, M. A., and DURST, R. A. 1995. Improved liposome immunomigration strip assay for alachlor determination. Anal. Chim. Acta 311:309-318.
  • REEVES, S. G., SIEBERT, S. T. A., and DURST, R. A. 1995. Liposome- amplified immunoanalysis for pesticides, Chapter 15 in Immunoanalysis of Agrochemicals: Emerging Technologies, J.O. Nelson, A.E. Karu and R.B. Wong, eds., ACS, Washington, D
  • REEVES, S. G., SIEBERT, S. T. A., and DURST, R. A. 1995. Liposome immunosensing devices for environmental contaminant screening. Trends Anal. Chem. 14:351-355.


Progress 01/01/94 to 12/30/94

Outputs
An automated Flow-Injection Liposome ImmunoAnalysis (FILIA) system has been modified from a previous design, using a specific environmental contaminant, the herbicide alachlor, as a model analyte. Signal amplification has been achieved by means of analyte-tagged liposomes containing either fluorescent or electrochemical markers to provide high detection sensitivity. The computer controlled system is composed of commercially available components, with the exception of the column packing material, which has to be prepared for each specific analyte to be determined. The relationships between antibody concentration and assay speed and sensitivity has been studied, and the possibilities of using the system for determination of multiple analytes is under investigation. The use of immunospecific liposome migration offers many advantages for the extra-laboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, robust, and respond to the presence of analyte at low-parts- per-billion concentrations. Investigations have been carried out that determine optimal interactions for key components of these assays, and techniques are presented for the development of generalized liposome immunoassays. Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of alachlor and polychlorinated biphenyls.

Impacts
(N/A)

Publications