本研究為設計流體繞射晶片(Diffraction chip，DC)及聚苯乙烯球(Polystyrene，PS)偵測的鼠疫桿菌F1莢膜蛋白抗原(Yersinia pestis F1 capsular antigen)。基材上分別有柱型及線型兩種微結構，間距比率為1：1.5 的圖案化光阻矽晶圓。以此結構利用氧電漿機，進行表面電漿活化，矽烷類自組裝改質( (3-Aminoproyl)triethoxysilane，APTES )。接著利用APTES 末端有-NH2 官能基，透過EDC/NHS 結合 Protein G ，再利用生物親合法接枝鼠疫桿菌F1抗原之單株抗體(F1-4B5-3 MoAb)，進行F1抗原抓取。由於檢驗原理是用雷射會因基材表面規律的微結構產生光柵繞射，當基材表面結合到F1抗原標的時，微結構受到破壞的影響所產生的能量損失進行偵測，但抗原的三維尺寸較小，對微結構的影響有限，使的偵測受限，需額外建立三明治分析方式以增強訊號。三明治分析方式由聚苯乙烯微球為基材，改質上 Protein G與鼠疫桿菌F1抗原之多株抗體(Rabbit anti-F1 antibodies)後與被晶片抓取之抗原做結合，達到微結構破壞，增強光學訊號之目的。
接著對10 µg/ml ~ 12.5 pg/ml F1抗原濃度做靈敏度測試，並透過雷射共軛焦顯微鏡觀察螢光及高解析度場發射掃描式電子顯微鏡觀察表面形貌佐證，結果顯示當聚苯乙烯微球與晶片表面抗原結合時，雷射繞射強度呈現下降的趨勢，且下降的幅度與抗原濃度呈線性關係，其對F1抗原偵測極限濃度為25 pg/ml，說明利用雷射能量檢測F1抗原的靈敏度和其他文獻所提到的相比較高。而柱型及線型繞射晶片對於不同濃度的F1抗原經過光學訊號放大後所作出的線性回歸之相關係數值為0.9701及0.9792，非常有潛力作為即時檢測抗原的技術，並期望往後可以應用在檢測其他的病毒蛋白抗原。

This study designed a Diffraction chip (DC) and a Polystyrene microspheres (PS) to detect the Yersinia pestis F1 capsular antigen in the target. The substrate is a patterned photoresist silicon wafer with a pillar type and a line type with a pitch ratio of 1:1.5. With this structure, the oxygen plasma machine is used to perform surface plasma activation and silane self-assembly modification((3-Aminoproyl)triethoxysilane,APTES). Then, APTES, using the -NH2 functional group, bind Protein G through EDC/NHS, and then use the monoclonal antibody (F1-4B5-3 MoAb) to graft the F1 antigen of Yersinia pestis by bioaffinity to capture the F1 antigen. Because the principle of detection is that the laser has grating diffraction due to the regular microstructures on the surface, and compare it to the energy loss caused by the destruction of the microstructure. But the three-dimensional size of the antigen is small, and the damage to the microstructure is limited. Make detection unaccuracy. An additional sandwich analysis method is needed to enhance the signal. The sandwich analysis method uses polystyrene microspheres, modified with Protein G and multiclonal antibody (Rabbit anti-F1) of Yersinia pestis F1 antigen, and then combined with the antigen caught by the diffraction chip to achieve microstructure destruction and enhanced the optical signals.
Then, the sensitivity test was performed on the F1 antigen concentration of 10 µg/ml ~ 12.5 pg/ml, and the fluorescence was observed by Confocal Laser Scanning Microscope (CLSM) and the surface morphology was observed by Field-emission scanning electron microscope (FE-SEM). When the polystyrene microspheres are combined with the antigen on the surface of the chip, the laser diffraction intensity showed a linear decreasing trend, and the detection limit concentration was 25 pg/ml, which indicated that the sensitivity of using laser energy to detect F1 antigen was high. The pillar type and line type diffraction chips for different concentrations of F1 antigen after optical signal enhancing has correlation coefficient value of 0.9701 and 0.9792, which have great potential as a technology for rapid detection of antigens and are expected to be applied in the future. Detect other viral protein antigen signals.

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