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Use of Enzymatic and Chemical Fragment Polymorphism by Electrochemical Analysis to Differentiate Meat Species, Animal Origins and Pathological Bacterial Strains
peptide/amino acid analysis
|摘要:||本計劃之主要目的在利用合作團隊所開發之專利奈米銅網版印刷電極(CunSPE)，進一步探討其在肉品安全衛生、病原菌區鑑定及法醫學領域(動物種別鑑定)之應用潛力。本團隊以光照技術製造出粒徑10-30 nm之奈米銅電極，特別對於小分子有機酸具有高選擇性及敏感度，顯著降低分析生體樣本時之蛋白質干擾，提高了分析之解析度及偵測極限，提供了以胺基酸與小胜肽為標的物之研究者一項新工具，在生命科學、診斷醫學、藥學、食品公共衞生及蛋白質體學等領城皆具實際應用潛力。本研究前二年旨在引進限制酵素片段長度多型性（restriction-enzyme fragment length polymorphism, RFLP）的概念，配合CunSPE之電化學特性，開發酵素及化學片段多型性（enzymatic and chemical fragment polymorphism, ECFP）的方法，建立層析譜資料庫並予以電腦化管理，發展出可與分子生物學技術媲美但更快速的鑑定系統。此方法之開發在公共衛生方面預期還可應用於評估肉品的等級及新鮮度而在法醫學方面則可以實際利用肌肉、血液或毛髮之層析資料庫來建立常規鑑定動物種別的輔助工具。我們已發表可初步區別食用肉品種別的電化學分析方法並獲得化學網誌專文報導(SeparationNOW.com)。本計劃期望能進一步確認用以區別種別之特異性成分之結構以及肉品蛋白質降解過程所衍生之胜肽/胺基酸動力學，強化此一研究之理論基礎。第三年，將嘗試以CunSPE建立以分析有機酸代謝產物及內、外毒素為基礎的細菌性病原鑑定系統，用以區別產毒株與非產毒株之不同。目前已初步完成多株巴東氏菌(Bartonella spp)之電化學層析譜分析。我們期望本計劃之完成能延續本團隊在CunSPE上6項次專利及7篇科學文獻發表之基礎再生產至少2項專利及3篇以上的科學發表。以化學層析方法而非以聚合酶鏈鎖反應(PCR)測試胜肽片段多型性以及病原鑑定可謂分析學上一創新的思維和嘗試，若能成功將成為化學分析在此一方面應用之首例。|
The main purposes of the study are to further explore the potential applications of the developed, patented cupper nano-particle screen-plated electrode (CunSPE) in the areas of public health, bacteriology and forensic medicine; more specifically, the food-meat safety and the differentiation of pathogenic bacteria and animal species. The CunSPE produced by photo-electrochemical deposition method exhibited Cu particle sizes ranged from 10-30 nm in diameter, and was selective to small organic acids with high sensitivity. The major advantage of CunSPE detection is its ability to directly detect peptides and amino acids/peptides that exhibit little or no chromophoric or fluorescent properties. Under appropriate chromatographic conditions, reliable detection is feasible without sample pretreatment, making it a promising tool for studies targeting amino acid/peptides in multiple disciplines including biomedical sciences, diagnostic medicines, pharmacology, food safety and proteomics. The goals of this research in the first two years are to employ the concept of restriction-enzyme fragment length polymorphism (RFLP) to be applied to electrochemical analysis using CunSPE to create a new “enzymatic and chemical fragment polymorphism (ECFP)＂system that utilizes enzyme and chemical digestion to produce specific chromatographic product patterns for species identification. A computerized recognition system using data collected from electrochemical analysis will be developed for fast, economic and marketable identification of meats from multiple species and to utilize this system for simultaneous evaluation of meat freshness and meat grades. The applications also extend to the identification of endangered animal species and carry forensic significances in which chromatographic patterns of EC signals from blood and hair samples of human remains will also be studied and recorded. A preliminary result of this initial idea has been published demonstrating the first HPLC-EC method that is suitable for differentiation of meat species in a regular analytical lab. Further identification of the structures of the species-specific components and the establishment of a computerized fast recognition system are necessary steps to solidify the scientific evidence behind the theory. Our publication has drawn attention from a professional publisher of analytical chemistry (SeparationsNOW.com) and a special report has been dedicated to the introduction of our novel method. The goal of the third year is to differentiate pathological bacterial strains from the non-pathological strains based on the metabolic profiles of organic acids of the bacteria and the exo-toxins they secreted. A preliminary study was completed with successful demonstration of chromatographical differences among various strains of Bartonells spp. It is our ultimate goal to generate 2 new patents and at least 3 scientific publications from the 3-year proposal in addition to the 6 patents and 7 publications associated with CunSPE we have accomplished previously. The idea of ECFP and direct chromatographical analysis of bacterial pathogen proposed here are to our knowledge, the first of its kind.
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