Application of Ganglioside-Sensitized Liposomes in a Flow Injection

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Anal. Chem. 2007, 79, 246-250

Application of Ganglioside-Sensitized Liposomes in a Flow Injection Immunoanalytical System for the Determination of Cholera Toxin Ja-an Annie Ho,*,† Li-Chen Wu,‡ Ming-Ray Huang,‡ Yong-Jen Lin,† Antje J. Baeumner,§ and Richard A. Durst|

BioAnalytical Chemistry Laboratory, Department of Chemistry, National Tsing Hua University, Hsinchu, 300 Taiwan, Department of Applied Chemistry, National Chi-Nan University, Puli, Nantou, 545 Taiwan, Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14850, and Department of Food Science and Technology, Cornell University, Geneva, New York 14456

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 (GM1), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These GM1-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 GM1 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-1. The detection limit of this immunosensor was 6.6 × 10-17 g mL-1 in 200-µL samples (equivalent to 13 ag or 1.1 zmol). Increasing attention in the development of diagnostic testing paved the way for the application of alternative analytical devices for clinical diagnostics, environmental monitoring, on-site foodborne pathogen screening, and biodefense. Detection of bacterial toxins is of particular importance, because they represent a broad * To whom correspondence should be addressed. Fax: 886-3-571-1082. E-mail: [email protected]. † National Tsing Hua University. ‡ National Chi-Nan University. § Department of Biological and Environmental Engineering, Cornell University. | Department of Food Science and Technology, Cornell University.

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class of molecules that cause a variety of human diseases. The toxin secreted by the bacterium Clostridium botulinum is a neurotoxin that acts peripherally on the autonomic nervous system and interferes with acetylcholine signaling at the neuromuscular junction and can be used as a biological warfare agent. Enterotoxins produced by Escherichia coli or Salmonella typhimurium are common food contaminants that catalyze the ADP-ribosylation of host cell proteins and turn the synthesis of the metabolic regulator molecules cyclic AMP (cAMP) or cyclic GMP (cGMP) on and off in intestinal mucosal cells. High levels of cAMP and cGMP cause loss of electrolytes and water that results in diarrhea. Fumonisin, vomitoxin, and T-2 toxin are fungal toxins produced by several species of the genus Fusarium that appear in agricultural crops such as corn or peanuts, inhibit normal nucleic acid and protein synthesis, and cause cellular abnormalities. Waterborne infection and related morbidity and mortality are of major concern in many areas of Asia, Africa, and Latin America, where the incidences due to Vibrio cholerae, the etiological agent for the diarrheal disease cholera alone are estimated to cause more than 5 million cases per year.1 Cholera toxin (CT) is transmited to humans frequently by contaminated water and seafood and has been extensively studied over the years due to its profound effect in causing severe diarrhea, a devastating disease that causes water to flow from the blood through epithelial cells into the small intestine.2,3 CT, produced by the bacterium V. cholera, is an AB5 hexameric protein. It consists of five identical B subunits and a single A subunit. The A subunit surrounded by five B subunits is the pathogenic agent responsible for the symptoms of cholera and also the potent activator of adenylate cyclase.4,5 The B subunits are nontoxic binding components of the CT holotoxin that function in pentameric form to specifically recognize the ganglioside (GM1) presented on the surface of mucous cells.6 (1) Tauxe, R.; Seminario, L.; Tapia R.; Libel, M. The Latin American Epidemic. In Vibrio cholerae and Cholera; Wachsmuth, I. K., Blake, P. A., Olsvik, Ø., Eds.; American Society for Microbiology: Washington, DC, 1994; pp 321344. (2) Holmgren, J. Nature 1981, 292, 413-417. (3) Spangler, B. D. Microbiol. Rev. 1992, 56, 622-647. (4) Fishman, P. H. J. Membr. Biol. 1982, 69, 85-97. (5) King, C. A.; van Heyningen, W. E. J. Infect. Dis. 1973, 127, 639-647. (6) Sun, J. B.; Holmgren, J.; Czerkinsky, C. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10795-10799. 10.1021/ac060889n CCC: $37.00

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Liposomes, microscopic spherical vesicles, are formed by the self-assembly of phospholipid molecules in an aqueous environment. They were discovered in the mid-1960s and were originally studied as cell membrane models.7 The coexistence of the hydrophilic and hydrophobic compartments in liposomes makes them a versatile carrier for a wide spectrum of amphipathic, watersoluble, and lipid-soluble molecules. Liposomes have been intensively used in our laboratories in developing immunodiagnostics,8-14 and more applications of liposomes have been reported including gene therapy,15 drug delivery,16,17 cosmetic skin conditioners,18,19 food and agriculture.20 Liposomes used in immunodetection systems not only occur as immunoliposomes (liposomes tagged with antibodies) but also as DNA- or analyte-tagged liposomes. Despite the fact that gangliosides (GM1) have high specificity and strong affinity for Shiga-like toxin comparable to anti-CT antibody, gangliosides (GM1) had not been used as recognition markers in liposomebased assays until very recently.21-34 Ganglioside-sensitized liposomes have advantages over immunoliposomes due to the (7) Bangham, A. D.; Standish, M. M.; Weissmann, G. J. Mol. Biol. 1965, 13, 253-259. (8) Ho, J-a. A.; Durst, R. A. Anal. Chim. Acta 2000, 414, 61-69. (9) Ho, J-a. A.; Hsu, H. W. Anal. Chem. 2003, 75, 4330-4334. (10) Ho, J-a. A.; Hsu, H. W.; Huang. M. R. Anal. Biochem. 2004, 330, 342-349. (11) Ho, J-a. A.; Huang, M. R. Anal. Chem. 2005, 77, 3431-3436. (12) Subramanian, V.; Wu, L. C.; Huang, M. R.; Ho, J-a. A. Anal. Chem. 2006, 78, 1115-1121. (13) Ho, J-a. A.; Zeng, S. C.; Huang, M. R.; Kuo, H. Y. Anal. Chim. Acta 2006, 556, 127-132. (14) Ho, J-a. A.; Wauchope, R. D. Anal. Chem. 2002, 74, 1493-1496. (15) Fletcher, S.; Ahmad, A.; Perouzel, E.; Heron, A.; Miller, A. D.; Jorgensen, M. R. J. Med. Chem. 2006, 49, 349-357. (16) Lurquin, P. F. In Liposome Technology: Entrapment of Drugs and Other Materials; Greroriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. II, Chapter 8. (17) Brandl, M.; Bachmann, D.; Drechsler, M.; Bauer, K. H. Drug Dev. Ind. Pharm. 1990, 16, 2167-2191. (18) Singh, A. K.; Carbonell, R. G. In Handbook of Nonmedical Applications of Liposomes; Lasic, D. D., Barenholz, Y., Eds.; CRC Press: Boca Raton, FL, 1996; pp 190-207. (19) Oh, Y. K.; Kim, M. Y.; Shin, J. Y.; Kim, T. W.; Yun, M. O.; Yang, S. J.; Choi, S. S.; Jung, W. W.; Kim, J. A.; Choi, H. G. J. Pharm. Pharmacol. 2006, 58 (2), 161-6. (20) Taylor, T. M.; Davidson, P. M.; Bruce, B. D.; Weiss, J. Crit. Rev. Food Sci. Nutr. 2005, 45 (7-8), 587-605. (21) Pan, J. J.; Charych, D. Langmuir 1997, 13, 1365-1367. (22) Singh, A. K.; Harrison, S. H.; Schoeniger, J. S. Anal. Chem. 2000, 72, 60196024. (23) Axelsson, B.; Ericksson, H.; Borrebaeck, C.; Mattiasson, B.; Sjogren, H. O. J. Immunol. Methods 1981, 41, 351-363. (24) Ahn-Yoon, S.; DeCory, T. R.; Baeumner, A. J.; Durst, R. A. Anal. Chem. 2003, 75, 2256-2261. (25) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 13611369. (26) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585-588. (27) Reicheret, A.; Nagy, J. O.; Spevak, W.; Charych, D. J. Am. Chem. Soc. 1995, 115, 1146-1147. (28) Charych, D.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113-120. (29) Song, X. D.; Nolan, J.; Swanson, B. I. J. Am. Chem. Soc. 1998, 120, 48734874. (30) Song, X. D.; Swanson, B. I. Anal. Chem. 1999, 71, 2097-2107. (31) Ahn-Yoon, S.; DeCory, T. R.; Durst, R. A. Anal. Bioanal. Chem. 2004, 378, 68-75. (32) Song, J.; Cheng, Q.; Zhu, S.; Stevens, R. C. Biomed. Microdevices 2002, 4 (3), 213-221. (33) Alfonta, L.; Willner, I. Anal. Chem. 2001, 73, 5287-5295. (34) Singh, A. K.; Harrison, S. H.; Schoeniger, J. S. Anal. Chem. 2000, 72, 60196024.

amphiphilicity of the gangliosides. Gangliosides are composed of a hydrophobic ceramide, which can be easily incorporated into a lipid bilayer structure, while immunoliposome preparation requires multistep operations for covalent conjugation. Liposomes, encapsulating detectable molecules, such as visible or fluorescent dyes and electroactive markers, provide instantaneous enhancement rather than time-dependent enhancement as seen in enzyme immunoassays. Several studies have utilized the affinity binding of GM1 to CT and have developed detection schemes for the toxin. Since the CT could be recognized by GM1-containing lipid membranes, colorimetric detection of the CT was demonstrated with poly(diacetylene)-functionalized liposomes that included GM1,32 optical sensors for CT that involved resonance energy transfer and self-quenching,33 and fluorescence immunoassays for CT by ganglioside-sensitized liposomes were also reported.24,33 The use of molecular tools for detection and characterization of Vibrio isolates is another approach, which are either based on PCR35 or the extensions of PCR as amplified fragment length polymorphism fingerprinting.36-38 In this study, an immunodetection system that combines flow injection analysis with a sandwich immunoassay for the detection of cholera toxin is described. The microcapillary flow injection liposome immunoanalysis (µ-FILIA) system, designed for the determination of cholera toxin, utilizes a microcapillary with antiCT antibodies immobilized on its inner surface and GM1 liposomes containing SRB as the signal amplifier. CT is first captured by the antibodies on the column and is subsequently bound by GM1 expressed at the outside of the liposomes, followed by the measurement of fluorescence intensity of released SRB dye after the rupture of the liposomes. The FILIA format demonstrated improved sensitivity comparing to that reported by Ahn-Yoon et al.24 and Subramanian et al.12 EXPERIMENTAL SECTION Safety note: Organic solvents used in this study are suspected carcinogens and should be handled with extreme care. Reagents and Materials. Monosialoganglioside (GM1), cholera toxin B subunit (CT), sodium azide, sodium borohydride, sodium hydroxide, cholesterol, Sephadex G-50, Trizma base (tris(hydroxymethyl)aminomethane), sodium chloride, protein A, glutaraldehyde, ethanolamine, γ-aminopropyltriethoxysilane, triethylamine, n-octyl β-D-glucopyranoside (OG), chloroform, hydrochloric acid, and methanol (MeOH) were purchased from Sigma (St. Louis, MO). Isopropyl ether was purchased from Acros. Dipalmitoylphosphatidylcholine (DPPC), Dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylglycerol (DPPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Sulforhodamine B (SRB) were purchased from Molecular Probes (Eugene, OR). Mouse monoclonal antibodies to the B subunit of cholera toxin were obtained from Biodesign International (Saco, ME). Dimethyl pimelimidate (DMP) was purchased from Pierce Chemical Co. (Rockford, IL). Dialysis bag, MWCO 12-14 kDa, (35) Shirai, H.; Nishibuchi, M.; Ramamurthy, T.; Bhattacharya, S. K.; Pal, S. C.; Takeda, Y. J. Clin. Microbiol. 1991, 29, 2517-2521. (36) Jiang, S. C.; Louis, V.; Choopun, N.; Sharma, A.; Huq, A.; Colwell, R. R. Appl. Environ. Microbiol. 2000, 66, 140-147. (37) Purohit, H. J.; Kapley, A.; Khanna, P. Ind. J. Clin. Biochem. 1997, 12, 111114. (38) Kapley, A.; Purohit, H. J. Med. Sci. Monit. 2001, 7 (2), 242-245.

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Figure 1. Schematic diagram of the FILIA. TBS, 10 mM Trisbuffered saline (pH 7.0); OG, n-octyl β-D-glucopyranoside (30 mM); MeOH, 30% methanol solution.

was purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA). Undeactivatated fused-silica capillary was ordered from Alltech Associates (Deerfield, IL). FILIA Apparatus. A schematic diagram of the FILIA system is shown in Figure 1. The sequence of injections and the data collection are computer-programmed, providing precise control of timing in the system. Commercially available PEEK tubing (0.020-in. i.d.) and standard fingertight fittings were purchased from Upchurch Scientific Inc. (Oak Harbor, WA). The Rheodyne (model 7725i) injector with 1-mL sample loop (Rainin, Emeryville, CA) was used for injection of sample and liposome solutions. Three different mobile phases were used, the carrier (20 mM TrisHCl, containing 0.15 M NaCl, 0.15 M sucrose, and 0.01% sodium azide, pH 7), detergent solution (30 mM OG), and regeneration buffer (30% MeOH), all of which were kept in pressurized reservoir bottles. Two three-way, 12-V solenoid pinch valves (Bio-Chem Valve, East Hanover, NJ) were used to switch to the proper mobile phase. An LC pump (model LC-203) at the inlet of the FILIA was used to maintain a flow rate of 0.2 mL/min and was obtained from Everseiko (Tokyo, Japan). A MacIntegrator I Data Analysis Package, used for valve operation and data collection, was purchased from Rainin (Emeryville, CA). Final signal integration was performed using the MacIntegrator I, version 1.4, software system running on a Macintosh Performa 6214 CD Power PC computer. The fluorescence detector was obtained from Jasco Corp. (Tokyo, Japan) Preparation of GM1 Liposomes. Liposomes were prepared using the film hydration method.39,40 The lipid mixture consisted of a 10:10:1:0.4 molar ratio of DPPC, cholesterol, DPPG, and GM1. The total lipid mixture was dissolved in 4 mL of a solvent mixture consisting of chloroform, isopropyl ether, and methanol (volume ratio 6:6:1), followed by a 1-min sonication at 45 °C under N2. One milliliter of SRB solution (110 mM) was added to the lipid mixture. After sonication of the solution for 3 min more, the organic solvent was removed under vacuum on a rotary evaporator, leaving a dark purple, gel-like suspension of liposomes. An additional 1 mL of SRB was added, followed by another 3 min of sonication. The liposome preparation was incubated in a 45 °C water bath for 35 min before passing through a 0.2-µm polycarbonate filter 20 times to produce a homogeneous suspension of uniform size. Any (39) Alving, C. R.; Swartz, J. G. In Liposome Technology. Preparation of liposomes for use as drug carriers in the treatment of leishmaniasis; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. II, pp 55-68. (40) Nasseau, M.; Boublik, Y.; Meier, W.; Winterhalter, M.; Fournier, D. Biotechnol. Bioeng. 2001, 75, 615-618.

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unencapsulated dye or trace organic solvent was removed from the liposome preparation by gel filtration on a 1.5 × 25 cm Sephadex G-50 column at room temperature, followed by dialysis in 10 mM TBS buffer (MWCO, 12-14 kDa) at 4 °C in the dark. Preparation of the Capillary Immunoreactor Column. Immobilization of antibody molecules on the surface of the solid support is a very important procedure in developing a heterogeneous assay. It is essential to develop a suitable antibody immobilization strategy to avoid the loss of antibody bioactivity caused by the randomly oriented immobilization of antibodies.13 Antibody binding site-directed immobilized procedures, described previously for better reproducibility of the immunoassay, were adapted in current study.10,11,13,41,42 In short, the modification procedures were initiated by the treatment with 1 M NaOH overnight, followed by the silyation with 5% methanolic γ-aminopropyltriethoxysilane. Protein A, having high affinity for the constant region of IgG, was subsequently immobilized on the inner wall of the microcapillary by glutaraldehyde, a homobifunctional cross-linker. This was followed by the attachment of anti-CT antibody molecules. DMP was then used to covalently convert the protein A-antibody complex to a more permanent immunoaffinity matrix. The capillary, filled with TBS, was then stored at 4 °C until use. Assay Procedure. The CT sample (200 µL) was introduced into the protein A-antibody-coated capillary immunoreactor by manual injection (at time 0:00 min). This was followed by manual injection of 60 µL of GM1 liposomes into the column. The sandwich complexes (immobilized antibody-CT-GM1 liposomes) were thus formed. Unbound liposomes were eluted, and a detergent solution (30 mM OG) was passed through the immunoreactor (at time 18:00 min) to lyse the bound liposomes. The fluorescence signal generated by the released SRB was measured at an excitation wavelength of 544 nm and emission wavelength of 596 nm. Finally, the bound CT was dissociated from the antibody binding sites using a 30% methanol solution (at time 21: 00 min), and the column was reconditioned for subsequent analyses by returning the mobile phase to the carrier buffer (at time 26:00 min). An analysis was completed within 40 min. Each assay was run at a flow rate of 0.2 mL/min using a 1 psi N2 head pressure on each of the mobile-phase reservoirs in conjunction with the LC pump. Stability Study and Characterization of Liposomes. Liposomes were used as probes in the proposed immunodetection system; therefore, it is important to monitor the stability of liposomes, with respect to the maintenance of their integrity. The intactness of liposomes can be determined by measuring fluorescence intensity before and after lysis. When a high concentration of SRB dye solution (110 mM SRB dye solution was used in this study) was entrapped inside of the liposomes, light could hardly pass through the sample to cause excitation; thus, very low fluorescence was found with a high concentration of SRB (this is called concentration quenching). The fluorescence measurement before and after lysing the liposomes relies on dequenching of the putatively self-quenched SRB. For these fluorescence tests, the SRB dye was excited at 544 nm and the fluorescent emission intensity was measured at wavelength of 596 nm. Temperature (41) Larsson, P. O. Methods Enzymol. 1984, 104, 212-223. (42) de-Frutos, M.; Paliwal, S. K.; Regnier, F. E. Anal. Chem. 1993, 65, 21592163.

Table 1. Characteristics of the Liposomes mean diameter (nm) volume of liposome (µL) liposome concentration (number/mL) SRB concentration (mM) SRB molecules per liposome GM1 molecules on the liposome surface

210 4.7 × 10-12 1.3 × 1013 110 2.8 × 105 4200

effects on liposome stability were conducted by adding 10 µL of liposome solution to 3 mL of osmotically balanced TBS, pH 7.0, in a test tube preheated to 30-70 °C in a water bath. The liposomes were incubated for 5 min before measurement of fluorescence intensity. The diameter of the liposomes was measured with a Coulter N4 particle size analyzer (Coulter Corp., Miami, FL) according to the manufacturer’s instructions. RESULTS AND DISCUSSION Characterization of Liposomes. A homogeneous GM1 liposome population with respect to size is important for any diagnostic application in order to obtain high reproducibility of the assay. Extrusion of the liposome preparation was used as a means to decrease the size distribution and generate liposomes of a size found suitable for FILIA applications.8,10,11 GM1 liposomes used in this study remained intact at the temperature ranging from 30 to 70 °C, showing a good heat stability. Their mean diameter of 210 nm with a standard deviation of 19 was determined by analysis with a Coulter N4 particle size analyzer. With liposomes of 210-nm diameter, it is possible to calculate that the average volume of a single liposome is 4.7 × 10-12 µL and the volume entrapped (assuming a bilayer thickness of 4 nm) is 4.2 × 10-12 µL. Assuming the SRB concentration inside of the liposomes is equal to the original solution used (110 mM), and comparing the fluorescence of lysed liposomes to that of standard SRB solutions, it is possible to calculate that there were ∼1.3 × 1013 liposomes/ mL and that each liposome contained ∼2.8 × 105 molecules of SRB. The amount of GM1 in the resulting liposomes was measured by the dimethyl methylene blue dye method,43 and it was found that ∼47% of the GM1 in the lipid mixture was successfully incorporated in the formation of the GM1 liposome. Therefore it was estimated that ∼4200 molecules of GM1 were on the outer surface of each liposome. The characteristics of the liposomes are summaried in Table 1. Optimization of Assay Parameters. A one-factor-at-a-time optimization approach was taken in this study for system optimization. A series of experiments was performed to establish the conditions with maximum signal intensity. The effects of flow rate, antibody concentration, dilution factor of GM1 liposome solution, injected volume of liposome solution, and injected volume of sample cholera toxin solution were investigated. The optimization of the flow rate is an important step in the development of an efficient flow-based immunoliposome assay. In general, low flow rates will result in more time for the immunorecognition reaction to occur; however, low flow rate will also compromise the sampling rate. In this study, flow rates between 0.2 and 0.5 mL/min were investigated, and the highest signal-to-noise ratios were found for (43) De Lucca, A. J.; Jacks, T. J.; Brogden, K. A. Mol. Cell. Biochem. 1995, 151, 141-148.

Figure 2. Effect of various injected volumes and dilutions of GM1 liposomes on the generation of the fluorescence signal. Values are means ( SD (n ) 3); error bars represent ( 1 SD. *p < 0.1.

0.2 mL/min. Thus, a flow rate of 0.2 mL/min was chosen as optimum for subsequent experiments. The amount of antibody immobilized in the capillary immunoreactor column is also a key parameter to the performance of this sensing system. In this study, the sensor response increased when the concentration of antibody loaded on the microcapillary inner surface was changed from 0.5 to 2 mg/mL. With further increase in the antibody concentration to 3 mg/mL, the increase of the sensor response was not observed. Next, the amount of liposomes and volume of liposomes per assay was optimized. First, various volumes of diluted liposome solutions were investigated (Figure 2) where each experiment contained the same total number of liposomes. The highest fluorescence signals were obtained when the liposome preparation was diluted 5 and 10 times with an injection volume of 20 and 40 µL, respectively. Thus, it was found that lower concentrations paired with larger volumes exhibited a better condition for binding to the CT immobilized in the column. Since liposomes are in general more stable at higher concentrations, the 5 times diluted condition was chosen as optimal and used in subsequent assays. Second, the overall volume of the GM1 liposome solution was investigated, i.e., resulting in varying amounts of liposomes used per analysis (Figure 3). The highest fluorescence signals were obtained when the liposome preparation was diluted 5 times with an injection volume of 60 µL. Thus, each analysis can be done within 40 min. Combined with an autosampler, the FILIA system is therefore an excellent choice for routine medium-throughput CT analysis at very low concentration levels. Assay Performance. For each analysis, 200 µL of CT was injected into the immunoreactor. The number of CT molecules bound to the antibodies occupied only a fraction of the total number of antibody binding sites in proportion to its concentration. A 60-µL aliquot of liposomes was subsequently injected into the column, where they bound to the captured CT. A sandwich immunocomplex (immobilized antibodies-CT-GM1 liposome) is thus formed. For an immunoreactor prepared with 2.0 mg/mL antibody, the calibration curve generated using CT standard is shown in Figure 4. The curve was fitted by Origin scientific graphing and analysis software, version 6.0 (Originlab Corp., Northampton, MA) to the data plot using a logistical equation. Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

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Figure 3. Effect of various injected volumes of GM1 liposomes on the generation of the fluorescence signal. Values are means ( SD (n ) 3); error bars represent ( 1 SD. **p < 0.01.

Figure 4. Calibration curve for cholera toxin (each point represents the mean of five measurements; error bars represent ( 1 SD).

The calibration curve for the CT standard solutions has a working range of 10 ag/mL-10 fg/mL. The largest value for the coefficient of variation for triplicate measurements was 5.8%, indicating that the reproducibility of this proposed immunodetection system is very good. Various factors may limit the linear dynamic range of the calibration curve to 3 orders of magnitude in comparison to the 5 orders of magnitude reported previously using a lateral flow assay,24 for example, system flow rate, programmed assay time sequence, immobilized antibody concentration, and injected immunoliposome volume and concentration which will be further investigated in the future. According to the definition by the IUPAC, the detection limit in this study was calculated as the concentration corresponding to a signal 3 SD above the mean for a calibrator that is free of

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CT, i.e., a blank sample. Therefore, the limit of detection (LOD) was determined to be ∼1.1 zmol (equivalent to 200-µL injection of 66 ag/mL) of CT with a 99.7% level of confidence. The elution strategy for efficient regeneration of antibody activity was also investigated. The regeneration of the immunoreactor column was performed by flowing 30∼40% MeOH through the system for 5 min. It was considered a successful elution of the column if the fluorescence intensity of the analytical peak for the sample containing the same amount of analyte CT (10-14 g/mL) showed peak area values similar to those previously obtained. The most efficient regeneration of immobilized antibody activity was obtained using 30% MeOH. With this treatment, the immunoreactor could be used for at least 50 sample injections without any loss of reactivity. CONCLUSIONS In this study, the successful immobilization of anti-CT antibodies in a microcapillary column via protein A and its use as an immunoreactor/immunoseparator in a sandwich assay format for detecting CT were demonstrated. The proposed microcapillary flow injection liposome immunoanalysis system permits the detection of ∼1.1 zmol of CT. The sensitivity of FILIA for CT is improved by more than 7-fold compared to that of the liposomebased lateral flow assay (LOD 8.0 zmol)24 and the immunoelectrochemical assay that we reported previously (LOD 8.3 zmol).12 We assume that this significant improvement in the LOD is due to the sequential injection of analyte CT and GM1-liposomal probe, providing more opportunity for sample CT to bind with the antiCT antibodies modified on the inner wall of microcapillary immunoreactor. The FILIA is able to detect CT at room temperature, and a single assay can be performed within 40 min. Considering its low detection limit and reasonable assay speed, the FILIA may be employed in the monitoring and surveillance of CT in water and food supplies to ensure the health of humans. Future studies will focus on the detection of CT in actual sample matrices. ACKNOWLEDGMENT This work was supported by the National Science Council in Taiwan, ROC, under NSC 93-2113-M-007-051; NSC 94-2113-M-007043; NSC 42122F.

Received for review May 15, 2006. Accepted October 11, 2006. AC060889N