本研究是針對困難梭菌產生的雙毒素進行定量檢測。困難梭菌透過口腔進入人體釋放出腸毒素（Toxin A）及細胞毒素（Toxin B），導致患者腸道黏膜引發炎症反應，甚至造成腹膜炎而死亡。由於艱難梭菌具有傳染之風險，且複發率和死亡率高，故檢測毒素有利於及時採取隔離措施和後續追蹤，但困難梭菌為多株菌體，可能同時含有兩種毒素，或是只有一種，甚至沒毒素，易造成偽陰性結果，因此同時檢測雙毒素是非常關鍵的指標。
本實驗先以微影製程製作矽晶片繞射模具，透過奈米壓印取得PET繞射薄膜，其表面有解析度500奈米之一維線型及二維柱型奈米陣列圖案，可使雷射光產生不同型態的繞射。在此結構修飾(3-氨基丙基)三乙氧基矽烷(APTES)單分子自組裝及EDC/NHS活化劑耦合反應，成功接枝上蛋白質G(Protein G)，利用生物親合法接枝Toxin A單株抗體(Toxin A mAb)與Toxin B單株抗體(Toxin B mAb)，後續以PET - Toxin A mAb、PET - Toxin B mAb表示改質完的PET，接著進行Toxin A、Toxin B抗原抓取。但當毒素抗原結合於基材上，因毒素尺寸較小，對微結構的影響有限，無法對雷射繞射強度產生高靈敏性而限制了偵測的效能，故採用抗體-抗原-抗體(聚苯乙烯球)三明治結構，用2μm聚苯乙烯微米球為基材，改質上 Protein G 、Toxin A、Toxin B多株抗體 (Toxin A pAbs、Toxin B pAbs)及牛血清白蛋白(BSA)，後續以PS@pAb表示改質完成的聚苯乙烯微米球。PS@ pAb與被晶片抓取之抗原做結合，達到破壞規則圖案，進而放大雷射繞射強度變化量。
探討不同奈米尺寸高度基材mAb-L250、mAb -L500、mAb -L750、mAb -L1000、mAb -L1250、mAb -L1500、mAb -P250、mAb -P500、mAb -P750、mAb -P1000、mAb -P1250、mAb -P1500 (L表示線型，P表示柱型，後面數字代表高度nm尺寸)，捕捉100pg/m、10ng/ml Toxin A抗原後，再流入PS@pAbA，比較繞射基材在未捕捉Toxin A、捕捉Toxin A與PS@pAbA流入後之雷射損失能量百分比，最後選定mAb-L750為雷射損失百分比最大的尺寸進行後續實驗。
接著對0 pg/ml, 25 pg/ml, 50 pg/ml, 100 pg/ml, 500 pg/ml, 1 ng/ml, 10 ng/ml Toxin A、Toxin B抗原濃度做靈敏度測試，毒素抗原溶液透過注射幫浦以流速1.5 ml/ hr流入微流體繞射晶片，之後以PBST溶液流速6 ml/ hr進行清洗，再流入PSPS@pAbA、PS@pAbB固定流速為3ml/ hr，並以流速6 ml/ hr PBST溶液清洗，最後計算mAb-750和PS@pAbA與PS@pAbB結合L750- mAb上的toxin A、toxin B抗原後的雷射損失能量百分比，並將雷射損失能量百分比與抗原濃度的對數做線性回歸訂定出雙毒素之檢量線，結果顯示toxin A相關係數值為0.964；toxin B為0.983，皆為高度正相關，依截斷值判斷雙毒素之偵測極限濃度約為50 pg/ml，優於現行醫院採用酵素免疫分析法的偵測毒素極限濃度500pg/ml，並期望往後可以應用在困難梭菌感染之臨床應用。

This work is focused on the quantitative determination of Toxin A and Toxin B that generated by Clostridium difficile. Clostridium difficile always infects the human body through gastrointestinal tract to release two toxins including Toxin A and Toxin B, leading to inflammation of the intestinal mucosa in patients, and in severe cases. In that case, patients may result peritonitis, even death without early stage diagnosis. Because of contagious of Clostridium difficile, high recurrence and mortality rates, toxin detection is essential for early stage diagnosis and follow-up monitoring. However, Clostridium difficile is a polymorphic bacterium that simultaneously generates both toxins. Therefore, detection of these two kinds of toxins with high sensitivity and selectivity at early stage is a crucial.
In this work, silicon chip diffraction molds were initially fabricated using photolithography. Sequentially, PET diffraction films were obtained through nanoimprint, featuring one-dimensional and two-dimensional periodic relief grating with a resolution of 500 nm. The heights of these patterns are varied to investigate their intensity, denoted as L250、L500、L750、L1000、L1250、L1500、P250、P500、P750、P1000、P1250、P1500 (Item L and P represent line and pillar structure, the later number represents the height. )The surfaces of these chips were modified with APTES and Protein G through EDC/NHS activation coupling reaction. The Toxin A monoclonal antibodies (Toxin A mAb) and Toxin B monoclonal antibodies (Toxin B mAb) could be immobilized on the chips, denoted as ab-L250、ab-L500、ab-L750、ab-L1000、ab-L1250、ab-L1500、ab-P250、ab-P500、ab-P750、ab-P1000、ab-P1250、ab-P1500, respectively, to perform the detection of Toxin A and Toxin B antigens. However, when toxin antigens bound to the substrate, their smaller size limited their impact on the nanostructures, resulted the low sensitivity in laser diffraction intensity and thus restricting the detection efficiency. Therefore, a sandwich structure of antibody-antigen-antibody (polystyrene spheres) was employed to amplify the loss of diffraction intensity. 2 μm-polystyrene microspheres employed as the amplifier that modified with Protein G, polyclonal antibodies (Toxin A pAbs, Toxin B pAbs), and bovine serum albumin (BSA), reduced the non-specific binding, denote as PS@pAbA and PS@pAbB. Both PS@Abs were then bound to the captured antigens on the chip, disrupting the regular patterns and thereby amplifying the change in laser diffraction intensity.
The mAb-L250、mAb -L500、mAb -L750、mAb -L1000、mAb -L1250、mAb -L1500、mAb -P250、mAb -P500、mAb -P750、mAb -P1000、mAb -P1250、mAb -P1500 were used to investigate the impact of different nano-sized substrates on laser loss energy percentages at 100 pg/ml and 10 ng/ml of Toxin A antigen. Sequentially, PS@pAbA were introduced through the chip for amplification. The ab-L750 exhibited the highest laser energy loss percentage, selected for further experiments. Sensitivity were conducted for toxin A and toxin B antigen concentrations of 0 pg/ml, 25 pg/ml, 50 pg/ml, 100 pg/ml, 500 pg/ml, 1 ng/ml, and 10 ng/ml. Toxin antigen solutions were delivered into the microfluidic diffraction chip at a flow rate of 1.5 ml/hr using an injection pump. Subsequently, the PS@pAbA and PS@pAbB solutions were introduced at a fixed flow rate of 3 ml/hr. Laser energy loss percentages were plotted against logarithms of antigen concentrations to establish the calibration curves for both toxins. The results revealed a correlation coefficient of 0.964 for toxin A and 0.983 for toxin B, indicating a highly positive correlation. Based on the cutoff values, the limit of detections (LOD) for both toxins were approximately 50 pg/ml. This LOD of this methodology surpasses 10 times of the sensitivity of ELISA (500 pg/ml) for toxin concentration. This methodology provides a rapid and low cost of determination of toxins in practice, which can diagnose Clostridium difficile infections at early stage.

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