我們藉由在脫脂牛奶中偵測硫酸紫菌素 (Gentamycin)，建立了一個靈敏、簡易、平價並且快速的免疫分析平台。此偵測平台由奈米脂粒訊號放大系統、硫酸紫菌素微珠以及96孔過濾盤所組成；此競爭型免疫分析是藉由製成硫酸紫菌素微珠做為與分析物硫酸紫菌素競爭硫酸紫菌素抗體的競爭者。而所有的反應步驟皆於底部黏貼封膜的96孔過濾盤中操作，反應完成後清洗多餘未結合反應物的步驟則可輕易將大量清洗液通過已經除去封膜的96孔過濾盤而完成，無須任何反覆離心或者抽真空的步驟，而抗體將藉由與硫酸紫菌素微株的結合而滯留於96孔過濾盤中。接著加入接合蛋白質G並包有螢光物質的奈米脂粒，藉由蛋白質G與抗體之間的結合特性使滯留於96孔過濾盤中的硫酸紫菌素抗體與硫酸紫菌素微株複合物攜帶包有螢光物質的奈米脂粒，將多餘未結合的奈米脂粒洗除以後再將滯留於96孔過濾盤中的螢光訊號以介面活性劑將之自奈米脂粒釋放並收集在96孔盤中。此平台針對在Tris buffered saline緩衝液 (TBS) 以及脫脂牛奶中偵測硫酸紫菌素的偵測極限 (Limit of detection, LOD)分別為52.65 ng/mL 和14.16 ng/mL，皆足以偵測在Codex中法規所訂定的 200 ng/mL 最大殘留量濃度。在 TBS 以及脫脂牛奶中的偵測範圍為各自的偵測極限至100 μg/mL。最低可定量濃度個別為257.06 ng/mL 和 236.85 ng/mL。最後，此平台在 TBS 和脫脂牛奶中的50%抑制率濃度各自為199.66 ng/mL 和 360.81 ng/mL。亦藉由在脫脂牛奶中單獨偵測硫酸紫菌素與同時偵測硫酸紫菌素及另外5種抗生素的混合液來確認此偵測平的專一性。此偵測平台在60個不同樣本同時操作之下仍可在2小時內完成。而以上的結果皆可證明此新建立的生物感測法不但具有磁珠式分析法的優點，藉由96孔過濾盤分離法取代磁吸式分離法亦兼具節省成本以及操作簡易的優勢。

We developed a sensitive, simple, inexpensive and rapid immunoassay platform for the detection of Gentamycin in milk. This platform was composed of liposomal nanovesicle amplification system, Gentamycin sulfate beads and 96-well filtration plates. Gentamycin beads were constructed as a competitor of analyte for the recognition by antibody. The incubation for this competitive assay was conducted in a bottom-sealed 96-well filtration plate. By running washing buffers through the unsealed 96-well filtration plate with gravity but without any spin or vacuum force, the antibody-Gentamycin bead complexes were separated from the rest of the components in the solution. Fluorescent dye-loaded protein G-liposomal nanovesicles were added to specifically bind to antibodies on the beads retained in the sealed 96-well filtration plate. After washing unbound nanovesicles, millions of fluorescent dye molecules were released by adding a detergent solution to lyse liposomal nanovesicles. Results showed that the limit of detection (LOD) of this novel detection platform in TBS and in skim milk were 52.65 ng/mL and 14.16 ng/mL, which are both sufficient for detecting the 200 ng/mL Codex maximum residual level (MRL). The dynamic ranges were both from each of their LODs to 100μg/mL. The LOQs were 257.06 ng/mL and 236.85 ng/mL for detection in TBS and skim milk respectively. The 50% inhibition concentrations (IC50) in TBS and skim milk were 199.66 ng/mL and 360.81 ng/mL, respectively. We also demonstrated the good specificity of this platform by comparing detection results between pure Gentamycin solution and a mixture solution of Gentamycin with other 5 different antibiotics. The entire assay with 60 samples was conducted within 2 h. These results demonstrated that this novel biosensing platform not only fulfilled most benefits of magnetic bead-based assays, but also was inexpensive and convenient by replacing the magnetic separation with a 96-well filtration plate separation.

[1] Castilho Mde S, Laube T, Yamanaka H, Alegret S, Pividori MI, Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles. Anal Chem 83: 5570-5577, 2011, Jul 15.
[2] Tang D, Yu Y, Niessner R, Miro M, Knopp D, Magnetic bead-based fluorescence immunoassay for aflatoxin B1 in food using biofunctionalized rhodamine B-doped silica nanoparticles. Analyst 135: 2661-2667, 2010, Oct.
[3] Babu D, Muriana PM, Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of aflatoxin B(1). Journal of Microbiological Methods 86: 188-194, 2011, Aug.
[4] Chen CS, Baeumner AJ, Durst RA, Protein G-liposomal nanovesicles as universal reagents for immunoassays. Talanta 67: 205-211, 2005, Jul 15.
[5] Zacco E, Adrian J, Galve R, Marco MP, Alegret S, et al., Electrochemical magneto immunosensing of antibiotic residues in milk. Biosens Bioelectron 22: 2184-2191, 2007, Apr 15.
[6] Itoh S, Kariya M, Nagano K, Yokoyama S, Fukao T, et al., New rapid enzyme-linked immunosorbent assay to detect antibodies against bacterial surface antigens using filtration plates. Biological & Pharmaceutical Bulletin 25: 986-990, 2002, Aug.
[7] Sardinha JP, Gil MH, Mercader JV, Montoya A, Enzyme-linked immunofiltration assay used in the screening of solid supports and immunoreagents for the development of an azinphos-methyl flow immunosensor. J Immunol Methods 260: 173-182, 2002, Feb 1.
[8] Randall LP, Ridley AM, Cooles SW, Sharma M, Sayers AR, et al., Prevalence of multiple antibiotic resistance in 443 Campylobacter spp. isolated from humans and animals. J Antimicrob Chemother 52: 507-510, 2003, Sep.
[9] Bergwerff AA, Scherpenisse P, Determination of residues of malachite green in aquatic animals. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 788: 351-359, 2003, May 25.
[10] Reybroeck W, Schram E, Improved Filtration Method to Assess Bacteriological Quality of Raw-Milk Based on Bioluminescence of Adenosine-Triphosphate. Netherlands Milk and Dairy Journal 49: 1-14, 1995,
[11] Dewdney JM, Maes L, Raynaud JP, Blanc F, Scheid JP, et al., Risk assessment of antibiotic residues of beta-lactams and macrolides in food products with regard to their immuno-allergic potential. Food Chem Toxicol 29: 477-483, 1991, Jul.
[12] Dayan AD, Allergy to Antimicrobial Residues in Food - Assessment of the Risk to Man. Veterinary Microbiology 35: 213-226, 1993, Jun.
[13] Currie D, Lynas L, Kennedy DG, McCaughey WJ, Evaluation of a modified EC Four Plate Method to detect antimicrobial drugs. Food Addit Contam 15: 651-660, 1998, Aug-Sep.
[14] Berends BR, Van den Bogaard AEJM, Van Knapen F, Snijders JMA, Human health hazards associated with the administration of antimicrobials to slaughter animals - part II. An assessment of the risks of resistant bacteria in pigs and pork. Veterinary Quarterly 23: 10-21, 2001, Jan.
[15] Chen CS, Durst RA, 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, 2006, Mar 15.
[16] Zhang YL, Lu SX, Liu W, Zhao CB, Xi RM, Preparation of anti-tetracycline antibodies and development of an indirect heterologous competitive enzyme-linked immunosorbent assay to detect residues of tetracycline in milk. Journal of Agricultural and Food Chemistry 55: 211-218, 2007, Jan 24.
[17] Jeon M, Kim J, Paeng KJ, Park SW, Paeng IR, Biotin-avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk. Microchemical Journal 88: 26-31, 2008, Feb.
[18] Loomans EEMG, Van Wiltenburg J, Koets M, Van Amerongen A, Neamin as an immunogen for the development of a generic ELISA detecting gentamicin, kanamycin, and neomycin in milk. Journal of Agricultural and Food Chemistry 51: 587-593, 2003, Jan 29.