根據美國疾病與預防控制中心之國家院內感染監視系統及流行病原監控的統計資料，顯示院內血管通路的微生物感染最常見為金黃色葡萄球菌(S. aureus)，佔有13％的高比率。又因感染得到菌血症誘發之敗血症常是多重症加護病房病患最主要的死因之一，所以臨床檢驗上，快與準即為能救命與否的關鍵。傳統的檢測技術為酵素連結免疫分析(ELISA)，然此法在檢體前處理與測定步驟相當繁瑣，且需耗費大量的人力與時間。基於此，本研究以傳統三明治模型ELISA為雛型做改良，將粒徑約20 nm的金奈米膠體溶液 (5.4×1011 particles/mL)，與抗體免疫球蛋白G (80 μg/ml)於pH值為6.5(抗體之等電位點, pI=7.4)的磷酸鹽緩衝溶液中以等體積混合，再予以離心純化。其後把表面被覆予特異性抗體的奈米金粒(IgG-AuNP)和具獨特指紋區的拉曼活性分子探針5,5’-二硫基-雙(2-硝基苯甲酸) (DTNB)，形成在1340 cm-1具有很強的拉曼訊號之奈米金粒(IgG-AuNP-DTNB)後，因AuNP之間DTNB的表面增顯散射(SERS)作用，而可達訊號放大的目的。以常引起菌血症之S. aureus細胞壁所特有的蛋白質Protein A作為抗原，讓其在鍍金基盤上先物理性吸附有IgG層行親和性作用後，清洗後投入IgG-AuNP-DTNB，期以SERS作用中DTNB的1340 cm-1強度來呈現Protein A的濃度，因而取代原本帶有酵素的二次抗體之ELISA技術。我們將Protein A調整在10-6至10-13 g/ml之間進行偵測，結果顯示此量測方式之檢測極限值可達10-11 g/ml，相較於傳統免疫分析技術僅在10-8至10-9 g/ml，靈敏度約提高了2到3個級數。本研究進行之際也感受到，若(1)再擇取更佳的分子探針，並(2)改良具SERS效能功能性之奈米金粒，(3)金基盤的粗糙度，以及(4)將激發雷射改用較短波長(如532 nm之Nd:YAG雷射)，若許可將檢測極限再往下推進2-4個級數。

According to the statistics of National Nosocomial Infection Surveillance System in Centers for Disease Control and Prevention and Surveillance and Control of Pathogens of Epidemiologic Importance, Staphylococcus aureus was the most common infectant of nosocomial bloodstream microbial infection detected. It accounts for 13 percentages at the high rate. Sepsis induced by bacteremia is one of the main causes of death of critically ill patients in intensive care unit. In clinical tests, the most traditional medical examination technique is enzyme-linked immunosorbent assay (ELISA). However, this method in the sample pretreament and measurement steps is rather cumbersome, and would entail a lot of manpower and time.For this reason, this study is modified from traditional sandwich ELISA model as prototype. We mixed gold colloidal solution (20 nm, 5.4×1011 particles/mL) and Immunoglobulin G (IgG) (80 μg/ml) in phosphate buffer solution (pH=6.5) at pH below isoelectric point (pI=7.4) of antibodies isovolumically. After centrifugation, 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) can be employed as Raman-active molecular probes to modify on IgG-coated gold nanoparticles. This molecule has strong Raman signals at 1340 cm-1, and the gold nanoparticle can amplify the Raman scattering of molecular probe. The enzyme-linked secondary antibodies in traditional immunoassay are replaced with the immunogold molecular probes we fabricated. Protein A is the specific antigen of Staphylococcus aureus, will be used as a model system. The antigen concentrations were adjusted to be 10-6~10-13 g/ml, and detected by surface-enhanced Raman scattering (SERS). As a result, it showed that the detection of limit could reach to 10-11 g/ml. Comparing with traditional immunoassay only to 10-8~10-9 g/ml, the sensitivity could promote 2~3 order of magnitude higher. In addition to improve the sensitivity at low concentration, this method may also provide us Raman spectra and information of biological samples. We may (1) choose better molecular probes, and (2) change type of nanoparticles with higher SERS efficiency (3) increase roughness of substrate, and (4) switch to shorter LASER excitation wavelength (e.g. 532 nm Nd : YAG LASER), to lower the detection limit.

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