基於指叉狀電極阻抗量測之適體結合動力學與親和力分析研究

Abstract

生物分子交互作用分析可謂是生醫研究中不可或缺的一環，其得以分析生物分子之間的親和力與動力學特性。雖然現今已發展出許多不同的即時分析技術，但大多數的分析儀器均受限於其量測原理而具有造價高昂、體積龐大的限制，有鑑於此，發展一可攜式且易於操作的技術有助於促進相關研究，如新藥與新型療法的開發。此研究以適體為例，固定於即時電化學阻抗分析平台表面以演示其與目標分子間的親和力與動力學特性分析。首先，製程與量測條件於標準金電極上進行調校與最適化，使用高離子強度的固定化環境（1 M磷酸鹽緩衝溶液）與較具彈性的間隔物（寡去氧胸苷）作為適體與電極間連接，可以提昇結合阻抗響應；進行阻抗量測時則需使用帶負電之氧化還原電子對（赤/黃血鹽）。以上的條件將進一步運用於發展之平台，此平台藉指叉狀電極將結合訊號轉換為電化學阻抗訊號，並能以終點感測、即時感測，共兩種不同量測模式進行分析。指叉狀電極晶片針對兩不同目標蛋白，包含凝血酶與黏蛋白-1 SEA單元，進行親和力分析。修飾有HTDQ29適體的晶片可選擇性地辨認其目標分子凝血酶，且其量測得之解離常數（18.4 nM）與以酵素酶聯寡核苷酸分析法量測得之解離常數（17.6 nM）十分相近；修飾有K1R4.2適體的晶片同樣也可選擇性地辨認其目標分子黏蛋白-1 SEA單元，相較之下，其量測得之解離常數（3.90 nM）與遠小於以表面電漿共振分析法量測得之解離常數（72.0 nM），可能是量測環境中流場的有無所導致。指叉狀電極微流體晶片則能達成親和力與動力學特性分析，在此，一固定的交流電頻率被選用以進行即時阻抗量測，在此頻率下所對應的阻抗大小與電荷轉移阻抗相似，同時其值不受到流率之影響，而流動式適體固定化也可在此晶片上達成，而即時阻抗分析的重複性、專一性及劑量依存性也被驗證。在動力學特性分析結果的部分，此平台上量測得凝血酶與HTDQ29適體的結合速率常數為2.40×105 M-1sec-1，而解離速率常數為8.12×10-3 sec-1；惟以此電化學法量測得之解離速率常數與表面電漿共振分析法量測得之解離速率常數有近十倍的差異，可能是由於表面電漿共振分析法所使用的CM5晶片的流道高度較低、清洗強度較強（CM5晶片：40 μm；指叉狀電極微流體晶片：100 μm）。而親和力分析結果則藉由不同濃度的凝血酶對應其穩態阻抗響應，量測得之解離常數為24.3 nM，略高於在指叉狀電極晶片的分析結果。本論文提出一微型化、易於操作之電化學分析法，未來可運用於各式不同分析物的交互作用分析，並與可攜式電化學分析儀整合，可望有助於生物物理與生物分析方法相關研究之推展。

Biomolecular interaction analysis (BIA) is one of the most essential parts in biomedical research, as the specific binding between biomolecules is substantial to biological processes. Although many real-time techniques have been currently used, most of the instrument is costly and bulky due to their detection modes. Therefore, a portable and easy-to-operate technique would boost the development of new drugs and therapies. In this thesis, an electrochemical impedance platform is built up, and aptamer is applied as recognition probe to demonstrate kinetics and affinity analysis. First of all, optimal fabrication and measurement conditions are tuned on standard gold electrode (SGE). With higher ionic strength of immobilization buffer (1 M phosphate buffer), using a flexible linkage between aptamer and electrode (oligo(dT) spacer), and negative-charged redox mediators (ferri/o-cyanide), its response signal can be improved. These conditions are further applied on the developed platform. This platform transduces the impedance signal with an interdigitated array (IDA) electrode and is able to be applied in two different measurement modes: endpoint detection via IDA electrode chip and real-time detection via IDA microfluidic chip. Affinity analysis is demonstrated on IDA electrode chip by two model proteins, thrombin and MUC1 SEA domain. The HTDQ29 aptamer-modified chip selectively detects thrombin; moreover, the measured KD (18.4 nM) is very closed to the KD of enzyme-linked oligonucleotide assay (ELONA) (17.6 nM). The K1R4.2 aptamer-modified chip also selectively detects MUC1 SEA domain. In contrast, the measured KD (3.90 nM) is far smaller than the KD of surface plasmon resonance (SPR) (72.0 nM), which could result from its flowing condition. Kinetics and affinity analysis are then demonstrated on IDA microfluidic chip. A fixed fAC whose impedance resembles Rct is chosen for impedance monitoring because of its independency on flow rate. In situ immobilization is achieved, and its repeatability, specificity, and dose-dependency are verified. The kinetics analysis of thrombin and HTDQ29 aptamer shows that the association rate constant (kon) is 2.40×105 M-1sec-1 and that the dissociation rate constant (koff) is 8.12×10-3 sec-1; nevertheless, there is 10-fold difference in koff between this electrochemical assay and SPR method. It could be attributed to the wash stringency caused by a lower channel height of CM5 chip (CM5 chip: 40 μm; IDA microfluidic chip: 100 μm). Affinity analysis can be done by correlating the steady-state response of different concentration of thrombin. The measured KD is 24.3 nM, which is slightly higher than the one measured on IDA electrode chip (18.4 nM). This thesis proposes miniaturize and simple-to-make electrochemical bioassays, which could be applied on various analytes and integrated with portable device in the future. It is believed to be beneficial to promote the research regarding to biophysics and bioanalytical methods.