食源性疾病是一項全球公共衛生問題，由被汙染的食物所引起的大範圍疾病。 已經有許多食源性病原體被公認會造成食源性疾病。O157:H7 型大腸桿菌 (E. coli O157:H7) 會產生志賀氏菌毒素 (STEC serotype)，引起的症狀較為嚴重且通常和食物中毒有關。已經有許多方法用以檢測 O157:H7 型大腸桿菌，但是這些方法相當耗時且需要技術人員及昂貴的儀器。因此，我們希望開發簡單、快速的標靶靈敏脂質體 (target-sensitive liposome) 以檢測 O157:H7 型大腸桿菌。本研究的目的是開發具有蛋白A標靶 (protein A-tagged)、包埋磺基羅丹明 B (SRB) 的標靶靈敏脂質體，進行單步驟檢測O157:H7 型大腸桿菌。利用棕櫚酸 N-羥基琥珀酸琥珀醯亞胺酯 (NHSP) 將蛋白 A 進行衍生化，後續將衍生化後的蛋白 A 作為配體綴合在脂質體表面上。當與抗體結合後，標靶靈敏脂質體會自發性不穩定並釋放出磺基羅丹明 B，後續使用ELISA讀值器檢測磺基羅丹明B的螢光訊號。對於IgG結合在標靶靈敏脂質體的能力，結果顯示出IgG結合能力與對照的比率約為2.65:1。另外，我們發現標靶靈敏脂質體結合IgG的最佳偵測溫度及時間分別是45 度和30 分鐘。O157:H7 型大腸桿菌免疫測定的檢測極限 (LOD) 約為4.36 x 106 CFU/mL， 動態範圍超過四個數量級從 106 到 109 CFU/mL，並在 107 CFU/mL 以上具有顯著增加。總而言之，標靶靈敏脂質體可成為一個具有實用性的載體，並單步驟檢測食源性病原體。

Foodborne disease is the global public health problem, and it includes a wild range of illness that caused by contaminated food. There are numerous foodborne pathogens that have been recognized for foodborne diseases. Escherichia coli O157:H7 (E. coli O157:H7), is the Shiga toxin-producing E. coli (STEC serotype). It has commonly associated with food poisoning outbreaks. There have been many methods to detect E. coli O157:H7. However, these methods are time-consuming. Therefore, it is ideal to develop easy and rapid target sensitive liposome to detect E. coli O157:H7. The aim of the present study is to develop a protein A-tagged, sulforhodamine B (SRB) loaded universal target-sensitive liposome for immunoassays of E. coli O157:H7 in one step. Derivatization of protein A with N-hydroxyl succinimide ester of palmitic acid (NHSP) which conjugated as a ligand on the liposome surface. After binding with antibody, target-sensitive liposome can spontaneously destabilize and release the entrapped SRB. The released SRB was assayed by measuring the intensity of fluorescence using ELISA reader. The result showed the ratio of IgG binding ability to control was approximately 2.65 to 1. Besides, we have found 45 ℃ and 30 min as the optimal conditions for lysing of target-sensitive liposome with IgG. And the limits of detection (LODs) of the immunoassay for E. coli O157:H7 was approximately 4.36 x 106 CFU/mL. The dynamic range of more than four orders of magnitude from 106 to 109 CFU/mL and greatly increased above 107 CFU/mL. In summary, this target-sensitive liposome can be a useful carrier to detect foodborne pathogens in one step.

Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S., Zarghami, N., Hanifehpour, Y., & Kouhi, M. (2013). Liposomes: Classification, preparation and applications. Nanoscale Res Lett 8 (1): 102. In: Springer.
Almeida, B., Nag, O. K., Rogers, K. E., & Delehanty, J. B. (2020). Recent Progress in Bioconjugation Strategies for Liposome-Mediated Drug Delivery. Molecules, 25(23), 5672.
Anderson, M., & Omri, A. (2004). The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations. Drug Deliv, 11(1), 33-39.
Barnadas-Rodrı́guez, R., & Sabés, M. (2001). Factors involved in the production of liposomes with a high-pressure homogenizer. International Journal of Pharmaceutics, 213(1-2), 175-186.
Brown, D. A., Allen, C. J., & Bignell, G. N. (1982). The use of a protein A conjugate in an indirect enzyme-linked immunosorbent assay (ELISA) of four closely related baculoviruses from Spodoptera species. Journal of General Virology, 62(2), 375-378.
Calin, M., Stan, D., Schlesinger, M., Simion, V., Deleanu, M., Constantinescu, C. A., . . . Simionescu, M. (2015). VCAM-1 directed target-sensitive liposomes carrying CCR2 antagonists bind to activated endothelium and reduce adhesion and transmigration of monocytes. European Journal of Pharmaceutics and Biopharmaceutics, 89, 18-29.
Castellana, E. T., & Cremer, P. S. (2006). Solid supported lipid bilayers: From biophysical studies to sensor design. Surface Science Reports, 61(10), 429-444.
Cevc, G. (1988). Effect of lipid headgroups and (nonelectrolyte) solution on the structural and phase properties of bilayer membranes. Berichte der Bunsengesellschaft für physikalische Chemie, 92(9), 953A-961.
Chen, C.-S., & 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(1), 232-238.
Chen, C. S., Baeumner, A. J., & Durst, R. A. (2005). Protein G-liposomal nanovesicles as universal reagents for immunoassays. Talanta, 67(1), 205-211.
Crommelin, D., Fransen, G., & Salemink, P. (1986). Stability of liposomes on storage. In Targeting of Drugs With Synthetic Systems (pp. 277-287): Springer.

Deamer, D., & Bangham, A. (1976). Large volume liposomes by an ether vaporization method. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis, 443(3), 629-634.
Ding, W., Palaiokostas, M., Wang, W., & Orsi, M. (2015). Effects of lipid composition on bilayer membranes quantified by all-atom molecular dynamics. The journal of physical chemistry B, 119(49), 15263-15274.
Drescher, S., & van Hoogevest, P. (2020). The Phospholipid Research Center: Current Research in Phospholipids and Their Use in Drug Delivery. Pharmaceutics, 12(12), 1235.
Ellens, H., Bentz, J., & Szoka, F. C. (1984). pH-induced destabilization of phosphatidylethanolamine-containing liposomes: role of bilayer contact. Biochemistry, 23(7), 1532-1538.
Fatouros, D., Gortzi, O., Klepetsanis, P., Antimisiaris, S., Stuart, M., Brisson, A., & Ioannou, P. (2001). Preparation and properties of arsonolipid containing liposomes. Chemistry and Physics of Lipids, 109(1), 75-89.
Finger, J., Baroni, W., Maffei, D. F., Bastos, D. H. M., & Pinto, U. M. (2019). Overview of Foodborne Disease Outbreaks in Brazil from 2000 to 2018. Foods, 8(10).
Fu, Z., Rogelj, S., & Kieft, T. L. (2005). Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR. International Journal of Food Microbiology, 99(1), 47-57.
Gehring, A. G., Brewster, J. D., Irwin, P. L., Tu, S.-I., & Van Houten, L. J. (1999). 1-Naphthyl phosphate as an enzymatic substrate for enzyme-linked immunomagnetic electrochemistry. Journal of Electroanalytical Chemistry, 469(1), 27-33.
Hafez, I. M., Ansell, S., & Cullis, P. R. (2000). Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids. Biophysical Journal, 79(3), 1438-1446.
Hashimoto, Y., Endoh, H., & Sugawara, M. (1984). Chemical methods for the modification of liposomes with proteins or antibodies. Liposome T~~ h~ o~ ogy Volume III.
Heath, T. D., & Martin, F. J. (1986). The development and application of protein-liposome conjugation techniques. Chemistry and Physics of Lipids, 40(2-4), 347-358.
Hernández‐Caselles, T., Villalain, J., & Gómez‐Fernández, J. (1990). Stability of liposomes on long term storage. Journal of Pharmacy and Pharmacology, 42(6), 397-400.
Ho, R. J., Rouse, B. T., & Huang, L. (1986a). Destabilization of target-sensitive immunoliposomes by antigen binding--a rapid assay for virus. Biochemical and Biophysical Research Communications, 138(2), 931-937.
Ho, R. J., Rouse, B. T., & Huang, L. (1986b). Target-sensitive immunoliposomes: preparation and characterization. Biochemistry, 25(19), 5500-5506.
Ho, R. J., Rouse, B. T., & Huang, L. (1986). Target-sensitive immunoliposomes: preparation and characterization. Biochemistry, 25(19), 5500-5506.
Ho, R. J., Rouse, B. T., & Huang, L. (1987a). Interactions of target-sensitive immunoliposomes with herpes simplex virus. The foundation of a sensitive immunoliposome assay for the virus. Journal of Biological Chemistry, 262(29), 13979-13984.
Ho, R. J., Rouse, B. T., & Huang, L. (1987b). Target-sensitive immunoliposomes as an efficient drug carrier for antiviral activity. Journal of Biological Chemistry, 262(29), 13973-13978.
Ho, R. J., Ting-Beall, H. P., Rouse, B. T., & Huang, L. (1988). Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus. Biochemistry, 27(1), 500-506.
Horn, R. G., & Israelachvili, J. (1980). Direct measurement of forces due to solvent structure. Chemical Physics Letters, 71(2), 192-194.
Huang, L. (1990). Target-sensitive immunoliposomes-preparation and characterization. In: Google Patents.
Jin, T., Tiwari, D. K., Tanaka, S.-i., Inouye, Y., Yoshizawa, K., & Watanabe, T. M. (2010). Antibody–ProteinA conjugated quantum dots for multiplexed imaging of surface receptors in living cells. Molecular Biosystems, 6(11), 2325-2331.
Laouini, A., Jaafar-Maalej, C., Limayem-Blouza, I., Sfar, S., Charcosset, C., & Fessi, H. (2012). Preparation, characterization and applications of liposomes: state of the art. Journal of colloid Science and Biotechnology, 1(2), 147-168.
Lee, Y., & Thompson, D. (2017). Stimuli‐responsive liposomes for drug delivery. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 9(5), e1450.
Lekowska-Kochaniak, A., Czajkowska, D., & Popowski, J. (2002). Detection of Escherichia coli O157:H7 in raw meat by immunomagnetic separation and multiplex PCR. Acta Microbiologica Polonica, 51(4), 327-337.
Leserman, L. D., Machy, P., & Barbet, J. (2018). Covalent coupling of monoclonal antibodies and protein A to liposomes: specific interaction with cells in vitro and in vivo. Liposome Technology Volume III.
Li, J., Wang, X., Zhang, T., Wang, C., Huang, Z., Luo, X., & Deng, Y. (2015). A review on phospholipids and their main applications in drug delivery systems. Asian journal of pharmaceutical sciences, 10(2), 81-98.
Lian, T., & Ho, R. J. (2001). Trends and developments in liposome drug delivery systems. Journal of Pharmaceutical Sciences, 90(6), 667-680.
Lin, C., Guo, Y., Zhao, M., Sun, M., Luo, F., Guo, L., . . . Chen, G. (2017). Highly sensitive colorimetric immunosensor for influenza virus H5N1 based on enzyme-encapsulated liposome. Analytica Chimica Acta, 963, 112-118.
Lin, C., Zheng, H., Sun, M., Guo, Y., Luo, F., Guo, L., . . . Chen, G. (2018). Highly sensitive colorimetric aptasensor for ochratoxin A detection based on enzyme-encapsulated liposome. Analytica Chimica Acta, 1002, 90-96.
Mackay, S. M., Myint, D. M. A., Easingwood, R. A., Hegh, D. Y., Wickens, J. R., Hyland, B. I., . . . Tan, E. W. (2019). Dynamic control of neurochemical release with ultrasonically-sensitive nanoshell-tethered liposomes. Communications Chemistry, 2(1), 1-10.
Maherani, B., & Arab-Tehrany, E. (2011). R Mozafari M, Gaiani C, Linder M. Liposomes: a review of manufacturing techniques and targeting strategies. CurrNanosci, 7(3), 436-452.
Marčelja, S., & Radić, N. (1976). Repulsion of interfaces due to boundary water. Chemical Physics Letters, 42(1), 129-130.
March, S. B., & Ratnam, S. (1986). Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. Journal of Clinical Microbiology, 23(5), 869-872.
Milsmann, M. H., Schwendener, R. A., & Weder, H.-G. (1978). The preparation of large single bilayer liposomes by a fast and controlled dialysis. Biochimica et Biophysica Acta (BBA)-Biomembranes, 512(1), 147-155.
Mui, B., Chow, L., & Hope, M. J. (2003). Extrusion technique to generate liposomes of defined size. Methods in Enzymology, 367, 3-14.
Newman, G. C., & Huang, C.-H. (1975). Structural studies on phosphatidylcholine-cholesterol mixed vesicles. Biochemistry, 14(15), 3363-3370.
Ng, K., Zhao, L., Liu, Y., & Mahapatro, M. (2000). The effects of polyethyleneglycol (PEG)-derived lipid on the activity of target-sensitive immunoliposome. International Journal of Pharmaceutics, 193(2), 157-166.
Niculescu, A.-G., Chircov, C., Bîrcă, A. C., & Grumezescu, A. M. (2021). Nanomaterials Synthesis through Microfluidic Methods: An Updated Overview. Nanomaterials, 11(4), 864.
Oliver, S. P., & Murinda, S. E. (2012). Antimicrobial resistance of mastitis pathogens. Veterinary Clinics of North America: Food Animal Practice, 28(2), 165-185.
Olson, F., Hunt, C., Szoka, F., Vail, W., & Papahadjopoulos, D. (1979). Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochimica et Biophysica Acta (BBA)-Biomembranes, 557(1), 9-23.
Park, C. H., Vandel, N. M., & Hixon, D. L. (1996). Rapid Immunoassay for detection of Escherichia coli O157 directly from stool specimens. Journal of Clinical Microbiology, 34(4), 988-990.
Patil, Y. P., & Jadhav, S. (2014). Novel methods for liposome preparation. Chemistry and Physics of Lipids, 177, 8-18.
Pinnaduwage, P., & Huang, L. (1988). β-Galactosidase-induced destabilization of liposome composed of phosphatidylethanolamine and ganglioside GM1. Biochimica et Biophysica Acta (BBA)-Biomembranes, 939(2), 375-382.
Pinnaduwage, P., & Huang, L. (1992). Stable target-sensitive immunoliposomes. Biochemistry, 31(11), 2850-2855.
Rangel, J. M., Sparling, P. H., Crowe, C., Griffin, P. M., & Swerdlow, D. L. (2005). Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerging Infectious Diseases, 11(4), 603-609.
Rhys, N. H., Duffy, I. B., Sowden, C. L., Lorenz, C. D., & McLain, S. E. (2019). On the hydration of DOPE in solution. The Journal of chemical physics, 150(11), 115104.
Schmidt, H., Geitz, C., Tarr, P. I., Frosch, M., & Karch, H. (1999). Non-O157:H7 pathogenic Shiga toxin-producing Escherichia coli: phenotypic and genetic profiling of virulence traits and evidence for clonality. Journal of Infectious Diseases, 179(1), 115-123.
Sen, A., & Hui, S.-W. (1988). Direct measurement of headgroup hydration of polar lipids in inverted micelles. Chemistry and Physics of Lipids, 49(3), 179-184.
Shukla, S., Haldorai, Y., Hwang, S. K., Bajpai, V. K., Huh, Y. S., & Han, Y. K. (2017). Current Demands for Food-Approved Liposome Nanoparticles in Food and Safety Sector. Frontiers in Microbiology, 8, 2398.
Silk, T. M., & Donnelly, C. W. (1997). Increased detection of acid-injured Escherichia coli O157: H7 in autoclaved apple cider by using nonselective repair on trypticase soy agar. Journal of Food Protection, 60(12), 1483-1486.
Todd, E. (2014). Foodborne diseases: Overview of biological hazards and foodborne diseases. Encyclopedia of Food Safety, 221.
Torchilin, V. P. (2018). Immobilization of specific proteins on liposome surface: systems for drug targeting. Liposome Technology, 3.
Uyttendaele, M., Van Boxstael, S., & Debevere, J. (1999). PCR assay for detection of the E. coli O157: H7 eae-gene and effect of the sample preparation method on PCR detection of heat-killed E. coli O157: H7 in ground beef. International Journal of Food Microbiology, 52(1-2), 85-95.
Vaidya, B., Nayak, M. K., Dash, D., Agrawal, G. P., & Vyas, S. P. (2011). Development and characterization of site specific target sensitive liposomes for the delivery of thrombolytic agents. International Journal of Pharmaceutics, 403(1-2), 254-261.
Vaidya, B., Nayak, M. K., Dash, D., Agrawal, G. P., & Vyas, S. P. (2016). Development and characterization of highly selective target-sensitive liposomes for the delivery of streptokinase: in vitro/in vivo studies. Drug Deliv, 23(3), 801-807.
Velusamy, V., Arshak, K., Korostynska, O., Oliwa, K., & Adley, C. (2010). An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnology Advances, 28(2), 232-254.
Wu, W., Zhang, J., Zheng, M., Zhong, Y., Yang, J., Zhao, Y., . . . Wang, Q. (2012). An aptamer-based biosensor for colorimetric detection of Escherichia coli O157: H7. PloS One, 7(11), e48999.
Yu, L., Reed, S., & Golden, M. (2002). Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157: H7 in apple cider. Journal of Microbiological Methods, 49(1), 63-68.
Zhao, Y., Zeng, D., Yan, C., Chen, W., Ren, J., Jiang, Y., . . . Tang, F. (2020). Rapid and accurate detection of Escherichia coli O157: H7 in beef using microfluidic wax-printed paper-based ELISA. Analyst, 145(8), 3106-3115.