開發高靈敏度之液晶感測器以應用於癌症生物標記CA-125之無標記免疫偵測

Abstract

現行之傳統免疫檢測皆須使用螢光或酵素標記之抗體進行待測物之辨識，其檢測過程相當耗時，且用於標記之試劑有危害環境的風險。相較之下，液晶生物感測器具無標記、快速、低汙染之優點，然而其感測之穩定度與靈敏度仍略顯不足。本論文提出改善液晶免疫檢測技術之方法，開發出超高靈敏度之液晶免疫感測器。由實驗結果顯示，相較於液晶5CB，以高折射率液晶做為感測材料，於偵測相同濃度之待測物時，能造成較顯著的光學紋理。此外，本研究透過紫外光修飾感測器之配向層，使待測物更易吸附於配向層表面，進而提高液晶免疫檢測之再現性與靈敏度。我們將此項液晶免疫檢測技術實際應用於癌症生物標記CA-125之篩檢，透過液晶材料的選擇與感測器表面的修飾，可降低CA-125偵測極限至10 pg/ml。此外，我們亦對此免疫檢測技術的專一性進行測試，結果顯示在非專一性抗體的作用下，CA-125免疫複合體無法形成，故液晶分子維持在未被擾動的垂直配向。最後，本研究比較了螢光免疫檢測與液晶免疫檢測之靈敏度，螢光免疫檢測之偵測極限為4–5 μg/ml，較本研究所開發之液晶免疫偵測極限高。我們預期未來可將此項技術推廣到其他種類癌症生物標記的免疫偵測，實際應用於臨床癌症篩檢。

Conventional label-based immunoassay is time-consuming, and the labeling reagent may result in environmental pollution. In comparison, liquid crystal (LC)-based biosensor is rapid, label-free, and poses lower environmental risk, but its stability and sensitivity remains to be improved. In this work, we proposed two approaches to enhance the sensitivity of LC-based immunodetection. Using liquid crystals of high birefringence as the sensing element, a more significant phase retardation, and thus higher sensitivity, in the presence of biomolecules was observed under a crossed polarized microscope compared to 5CB, a type of liquid crystal commonly studied in LC-based immunoassay. In addition, we applied UV irradiation to the alignment layer to introduce hydrophilic functional groups, which allowed for more stable binding of the analyte. With a liquid crystal of high birefringence and modified alignment layer, the detection limit of LC-based immunoassay for the cancer biomarker CA-125 was lowered to 0.01 ng/ml. In the presence of non-specific antibodies, which were unable to complex with the CA125 antigen and disrupt LC alignment significantly, the orientation of LCs remained homeotropic, suggesting that LC-based immunodetection is specific and sensitive to the formation of immunocomplexes. Finally, we compared the sensitivity of LC-based immunoassay with an array-based fluorescence immunoassay, which has a detection limit of 4-5 μg/ml CA-125, significantly higher than that of LC-based immunoassay. It is expected that the highly sensitive LC-based immunodetection can be applied in the detection of a wider range of biomarker proteins, with the potential of replacing conventional immunoassays used in clinical cancer screening.