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Cholera, an acute infectious disease associated with water and seafood contamination, is caused by the bacterium Vibrio cholerae, which lives and colonizes in the small intestine and secretes cholera toxin (CT), a causative agent for diarrhea in humans. Based on earlier lateral flow assays, a flow injection liposome immunoanalysis (FILIA) system with excellent sensitivity was developed in this study for the determination of CT at zeptomole levels. Ganglioside (GM]), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These G[M.sub.1]-sensitized, sulforhodamine B (SRB) dye-entrapping liposomes were used as probes in the FILIA system. Anti-CT antibodies were immobilized in its microcapillary. CT was detected by the formation of a sandwich complex between the immobilized antibody and G[M.sub.1] liposomes. During the assay, the sample was introduced first into the column, and then liposomes were injected to bind to all CT captured by the antibody in the microcapillary. Subsequently, the SRB dye molecules were released from the bound liposomes via the addition of the detergent octyl glucopyranoside. The released dye molecules were transported to a flow-through fluorescence detector for quantification. The FILIA system was optimized with respect to flow rate, antibody concentration, liposome concentration, and injected sample volume. The calibration curve for CT had a linear range of 10-16 to 10-14 g [mL.sup.-1]. The detection limit of this immunosensor was 6.6 x [10.sup.-7] g [mL.sup.-1] in 200-[micro]L samples (equivalent to 13 ag or 1.1 zmol).

An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes. Cholera is a diarrheal disease, often associated with water or seafood contamination. Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer. CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT. In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies. The intensity of the band could be visually estimated or measured by densitometry, using computer software. The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-[micro]L sample. The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays. The assay could be completed within 20 min. These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.

The use of immunospecific liposome migration offers many advantages for the extralaboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, and 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 polychlorinated biphenyls (PCBs). The liposome immunocompetition assay format measures the competitive reaction between analyte-tagged liposomes and the sample analyte for immobilized antibodies and can detect 0.4 nmol of PCB in less than 8 min. A more sensitive format, the liposome immunoaggregation (LIA) assay detects the inhibition of immunospecific liposome aggregation in solution and can detect 2.6 pmol of PCB in less than 23 min. Laser diffraction particle sizing has been used to study LIA-induced increases in liposome size over time and to determine optimal conditions for the application of this technique. 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.

Medium binding antibodies may be recycled to extend the utility of planar waveguide immunosensor for theophylline. Regeneration is done by flowing antigen-free buffer over the antigen-antibody complex for more than 10 half-lives of the dissociation rate constant. One sensor may be used for more than 15 cycles without serious degradation. The use of theophylline-tagged liposomes further enhances the utility of the immunoassay technique by amplifying its signals.

The development of automated flourometric immunoassay for the detection of Esherichia coli 0157:H7, using antibody directed liposomes encapsulating fluorophone as an analytical reagent is discussed. The result reveals the feasibility of using fluorophore-encapsulated immunoliposomes in a microtiter plate for the rapid and automated detection of molecules with multivalent antigenic sites.

To meet the technical challenge of accurately and rapidly detecting Cryptosporidium parvum oocysts in environmental water, the authors developed a single-use visual-strip assay. The first step in the overall assay procedure involves extracting C. parvum's mRNA coding for heat-shock protein hsp70, followed by amplification using nucleic acid sequence-based amplification (NASBA) methodology as described previously (Baeumner, A. J.; Humiston, M.; Montagna, R. A.; Durst, R. A. Anal Chem., in press). Subsequently, generated amplicons are hybridized with dye-entrapping liposomes bearing DNA oligonucleotides (reporter probes) and biotin on their surface. The liposome--amplicon complex is then allowed to migrate upward on a nitrocellulose membrane strip. On the nitrocellulose strip, antisense-reporter probes are immobilized in a capture zone and antibiotin antibodies are immobilized in a second zone above the capture zone. Depending on the presence or absence of amplicon in the sample, the liposomes will bind to the capture zone, or they will be caught via their biotin tag in the second zone. Visual detection or gray-scale densitometry allows the quantification of liposomes that are present in either zone. The detection limit of the assay was determined to be 80 fmol amplicon/test. High accuracy and an internal assay control is established using this competitive format, because the presence or absence of liposomes can be quantified in the two capture zones.

This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid-sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensities not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 [micro]L of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in [micro]-total analysis systems ([micro]-TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.

A reliable method using nucleic acid sequence based amplification (NASBA) with subsequent electrochemiluminescent detection for the specific and sensitive detection of viable oocysts of Cryptosporidium parvum in environmental samples was developed. The target molecule was a 121-nt sequence from the C. parvum heat shock protein hsp70 mRNA. Oocysts of C. parvum were isolated from environmental water via vortex flow filtration and immunomagnetic separation. A brief heat shock was applied to the oocysts and the nucleic acid purified using an optimized very simple but efficient nucleic acid extraction method. The nucleic acid was amplified in a water bath for 60-90 min with NASBA, an isothermal technique that specifically amplifies RNA molecules. Amplified RNA was hybridized with specific DNA probes and quantified with an electrochemiluminescence (ECL) detection system. We optimized the nucleic acid extraction and purification, the NASBA reaction, amplification, and detection probes. We were able to amplify and detect as few as 10 mRNA molecules. The NASBA primers as well as the ECL probes were highly specific for C. parvum in buffer and in environmental samples. Our detection limit was ~5 viable oocysts/sample for the assay procedure, including nucleic acid extraction, NASBA, and ECL detection. Nonviable oocysts were not detected.