<OL> <LI> 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. <LI> Develop fully automated flow-injection immunoanalysis systems, microfluidic systems and simple extra-laboratory assay devices (e.g., biosensors and lateral-flow devices).
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.
APPROACH: 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. <P>
PROGRESS: 2003/10 TO 2008/09 <BR>
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.
IMPACT: 2003/10 TO 2008/09<BR>
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.