利用金奈米粒子-免疫球蛋白鍵結物配合電熱式原子吸收光譜法檢測尿液中金黃色葡萄球菌

Determination of Staphylococcus aureus in Urine by Electrothermal Atomic Absorption Spectrometry Based on Gold Nanoparticle/Immunoglobulin Conjugates

蔡東昇(Tong-Shung Tsay)

指導教授 ： 黃友利

高雄醫學大學/健康科學院/生物醫學檢驗學研究所/碩士(2007年)

摘要

蛋白質A是金黃色葡萄球菌細胞壁上的成分之ㄧ，可和人類免疫球蛋白以抗原抗體方式進行結合；金奈米粒子則可依據靜電作用力與人類免疫球蛋白進行結合，以形成金黃色葡萄球菌-人類免疫球蛋白-金奈米粒子之複合物，於是即可根據金黃色葡萄球菌所結合上金奈米粒子的含量，用以測定尿中金黃色葡萄球菌之菌液濃度。在分析尿中金黃色葡萄球菌前，須先以pH 8及pH 6緩衝液清洗數次，並控制其菌液酸鹼值為6，接著加入100 μL之250 μg/mL人類免疫球蛋白於4℃及室溫下進行反應各5分鐘，再藉由pH 6緩衝液去除未結合的人類免疫球蛋白後，添加800 μL 100 μg/L金奈米粒子溶液與金黃色葡萄球菌表面的人類免疫球蛋白進行反應25分鐘，待反應結束後，以2500 rpm的轉速進行離心3分鐘，用以分離未結合金奈米粒子，並利用原子吸收光譜法測定其含量，最後即可依據添加金奈米粒子的總量，計算金黃色葡萄球菌所結合上金奈米粒子的含量，以求得尿液中金黃色葡萄球菌之菌液濃度，本分析方法最低可測得尿中金黃色葡萄球菌之菌液濃度為2.7 × 104 cfu/mL，且於尿中添加金黃色葡萄球菌兩種濃度之回收率均分別為100.0 ± 5.4% 與114.8 ± 3.1%，顯示分析方法之可靠性，最後將此分析方法實際應用於臨床尿液檢測中，其分析臨床尿液檢體中含有4件金黃色葡萄球菌之尿液與433件尿液檢體不含金黃色葡萄球菌之尿液，並將本分析方法所測得結果與臨床結果進行比對，顯示本分析方法之靈敏度與特異性分別為100% 及94.3%，表示本研究所開發測定尿中金黃色葡萄球菌之分析技術，確實可應用於臨床尿液檢體之檢測。

關鍵字

金奈米 ； 金黃色葡萄球菌 ； 電熱式原子吸收光譜法

並列摘要

A novel method for quantification of Staphylococcus aureus (S. aureus) in urine using electrothermal atomic absorption spectrometry (ETAAS) has been developed. Protein A is a cell wall constituent of S. aureus, which reacts directly with human immunoglobulin G (IgG) through antibody-antigen interaction. The Citrated gold nanoparticles (GNPs) with negative charge surfaces can bind with IgG according to the electrostatic interaction. The S. aureus of urine were washed by pH 8 and pH 6 washing buffer and reacted with 100 μL of 250 μg/mL IgG. The unbound IgG were washed by pH 6 washing buffer, and reacted with 800 μL of 100 μg/L GNPs. The samples were centrifuged at 2500 rpm for 3 mins, and the supernatants were analyzed by ETAAS for the determination of unbound GNPs. The bound GNPs were obtained from the difference of unbound GNPs and the total GNPs. According to the concentration of bound GNPs, the concentrations of S. aureus can be calculated. The recoveries of two concentrations of S. aureus in urine are 100.0 ± 5.4% and 114.8 ± 3.1%, respectively. The lowest concentration of S. aureus we detected in urine sample was 2.7×104 cfu/mL based on the analytical procedures. To show the usefulness of this assay, the sensitivity and specificity in this assay were 100% and 94.5% for 4 S. aureus strain and 433 non-S. aureus strains in urine sample from patients, respectively. The use of a bioassay for determination of S. aureus in urine by ETAAS based on gold nanoparticles/immunoglobulin conjugates.

並列關鍵字

Gold nanoparticle ； Staphylococcus aureus ； electrothermal atomic absorption spectrometry

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