| 摘要: | 據世界衛生組織統計，2019年僅歐洲地區因心血管疾病死亡的人數已高達約420萬人，佔該地區總死亡人數42.5%，而全球範圍內每年更有約1億人患病。當心肌因急性心肌梗塞而受損時，會在血液中釋放眾多心血管疾病之相關生物辨識物。其中，心肌肌鈣蛋白I (cardiac troponin I, cTnI) 得益於其對急性心肌梗塞極高的專一性，以及患病初期之血液內高釋放速率，因而被視為檢測急性心肌梗塞之黃金標準。
為了縮短診斷時長，以期能在抵達急診室前進行檢測及預判病程，從而降低病患術後死亡率，定點照護檢驗 (point of care testing, POCT) 設備已成為現今技術發展之重點。然而，由於市售手持設備靈敏性之限制，雖然對於納入 (rule-in) 病患有較高準確性，然而對於排除 (rule-out) 病患仍然有其不足。因此，近年來基於矽奈米線場效應電晶體 (silicon nanowire field effect transistors, SiNW FETs) 之檢測平台因具有高靈敏性、即時檢測及容易攜帶等優勢，而被視為極具潛力的檢測平台。
本研究利用對cTnI具專一性之單鏈變異區片段 (single-chain variable fragment, scFv) (PDB：4P48) 抗體探針發展了基於多晶矽奈米線場效應電晶體檢測cTnI之平台的表面改質與最適化測量條件之研究。scFv抗體探針相對於傳統完整IgG抗體探針，有著更小的物理尺寸，在SiNW FETs的測量過程中可較大程度地規避德拜遮蔽效應 (Debye shielding effect) 所帶來的檢測靈敏度低下問題，達成提升檢測極限之目的。
本研究利用由Silane-PEG-NH2：Silane-PEG-OH = 1：10所形成的混合自組裝單層膜 (mixed Self-assembled monolayers, mixed-SAMs) 及戊二醛 (glutaraldehyde, GA) 對於SiNW FETs表面進行化學改質，並以共價鍵結將anti-cTnI scFv探針固定於表面，從而實現高靈敏性測量。
本研究驗證anti-cTnI scFv與cTnI在矽晶片上之結合能力探討經由ELISA所得之不同離子強度環境溶液之KD值而得。實驗結果表明其皆在一個數量級內，說明該單鏈變異區片段抗體探針與cTnI抗原間的結合鮮少受到溶液離子強度之影響。再者，COB (chip on board) 檢測系統所得實驗結果可知，對於分別測定三種離子強度溶液 (10, 50, 150 mM bis-tris propane, BTP) 作為檢測溶液環境之下，以10 mM BTP作為最適化之離子強度溶液，可獲得更顯著之訊號變化量，並且cTnI與電訊號之間亦有更高的相關性。
此外，為了驗證探針於表面化學改質的成功與否，本研究利用原子力顯微鏡進行表面形貌及粗糙度分析，隨後利用光電子能譜儀進行表面化學鍵能分析，從而交叉確認了生物探針於矽晶片上的功能化。
研究中亦比較了scFv抗體探針與完整IgG抗體片段探針在SiNW FETs檢測平台上的檢測表現，結果說明分子量與物理尺寸皆較小的scFv探針對比完整抗體在電訊號檢測上較不受德拜遮蔽效應影響，從而較容易達成高靈敏度檢測之需求。此外，使用最佳化條件之離子強度溶液對人類血清環境下結合cTnI，所得之LOD約為0.5 pg/mL，低於市售手持檢測平台，並可充分滿足臨床上排除病患之標準，證實其臨床應用上之研究潛力。;According to statistical data from the World Health Organization, cardiovascular diseases caused approximately 4.2 million deaths in Europe alone in 2019, accounting for 42.5% of the total deaths in the region. Globally, around 100 million people are affected by cardiovascular diseases each year. When the myocardium is damaged due to acute myocardial infarction (AMI), various biomarkers associated with cardiovascular diseases are released into the bloodstream. Among these, cardiac troponin I (cTnI) is regarded as the gold standard for AMI detection due to its exceptionally high specificity and rapid release rate in the early stages of the disease.
To reduce diagnostic time and facilitate early detection before hospital admission, thereby improving patient outcomes and lowering postoperative mortality, point-of-care testing (POCT) devices have become a crucial topic in recent years of medical development. However, due to the sensitivity limitations of commercially available handheld devices, while they exhibit high accuracy in rule-in diagnosis, they still remain insufficient for rule-out diagnosis. Consequently, detection platforms based on silicon nanowire field-effect transistors (SiNW FETss) have recently garnered attention as promising platform due to their high sensitivity, real-time detection capability, and portability.
This study develops a SiNW FETs-based detection platform for cTnI by employing a single-chain variable fragment (scFv) antibody probe (PDB: 4P48) with specificity for cTnI. The study focuses on optimizing the surface functionalization and measurement conditions of the platform. Compared to conventional full-length IgG antibody probes, scFv antibody probes possess a smaller physical size, which effectively mitigates the Debye shielding effect that typically reduces detection sensitivity in SiNW FETs measurements, thereby improving the detection sensitivity.
To functionalize the SiNW FETs surface, a mixed self-assembled monolayer (mixed-SAM) composed of silane-PEG-NH₂ and silane-PEG-OH in a 1:10 ratio was utilized, followed by chemical modification using glutaraldehyde (GA). The anti-cTnI scFv probe was then immobilized onto the surface via covalent bonding to achieve high-sensitivity detection.
The binding affinity of anti-cTnI scFv to cTnI on a silicon oxide surface was evaluated using ELISA under different ionic strength conditions, yielding dissociation constant (KD) values within the same order of magnitude. These results indicate that the binding affinity between the scFv antibody probe and the cTnI antigen is minimally affected by variations in ionic strength of sensing environment. Furthermore, results from the chip-on-board (COB) detection system revealed that among three different ionic strength solutions (10, 50, and 150 mM bis-tris propane, BTP), the 10 mM BTP solution provided the most significant signal variation and demonstrated a higher resolution between cTnI concentration and electrical signal response.
Additionally, to confirm the success of surface modification, atomic force microscopy (AFM) was employed for surface morphology and roughness analysis, while X-ray photoelectron spectroscopy (XPS) was used for surface chemical bonding analysis. These methods cross-verified the effective functionalization of the bioprobe on the silicon oxide wafer.
A comparative study between scFv antibody probes and full-length IgG antibody probes on the SiNW FETs detection platform demonstrated that a smaller molecular weight and physical size of the scFv probe resulted in improved sensitivity, thereby facilitating high-sensitivity detection. Moreover, when using the optimized ionic strength solution in human serum samples, the achieved limit of detection (LOD) for cTnI was 0.5 pg/mL, which is lower than that of commercially available handheld detection platforms. This high sensitive biosensor meets clinical standards for rule-out diagnosis, highlighting the potential clinical applicability of this detection platform. |
