| dc.description.abstract | 由於矽奈米線場效電晶體 (Silicon-nanowire field effect transistors, SiNW-FETs)具備高靈敏性、無須生物/螢光標記、設備成本低廉、即時檢測等優勢，近年來已被視為極具發展潛力的檢測平台。
以往， SiNW-FETs檢測核酸雜交的機制，是透過核酸雜交後表面電荷密度的改變，誘發電晶體元件通道內載子數量的變化，以此得到不同電流-電壓曲線圖(I-V curve)來當作檢測方法。以n-type FETs為例，若檢測之目標分子帶負電，其產生的電場將會排斥通道內的載子(e-)，造成通道內載子密度下降從而降低源汲電流；反之，若目標分子帶正電，其產生的電場將會吸引額外的載子至通道內，造成通道內載子密度上升從而增加源汲電流。
然而，真實表面現象更為複雜且超出原本想像。根據實驗結果，緩衝液中的反離子(Counter-ion)除了會受到雙股螺旋核酸上帶負電的磷酸根所吸引外，也會因為閘極電壓施加的關係而蓄積於閘極氧化層表面上。這些反離子產生的電場會與雙股核酸所產生的電場發生競爭進而影響通道內載子可以有不同變化。
為此，本研究旨在探討表面經最適化矽烷處理後，分析不同鹽離子濃度環境對於SiNW-FETs檢測表面探針與微核醣核酸雜交後造成之電訊號影響，並嘗試找出最適合檢測核酸雜交的鹽離子濃度環境，作為產品設計之建議。首先，我們將Mixed-SAMs (silane-PEG-NH2：OH=1：3(mM/mM))與戊二醛 (Glutaraldehyde, GA)修飾於元件表面，接著透過還原胺化反應將 miR-21 去氧核醣核酸探針與戊二醛反應。待表面改質完成後，會利用光電子能譜儀與原子力顯微鏡觀察表面特徵以確保Mixed-SAMs於表面修飾之最適化條件，並確認miR-21探針成功接枝於表面。另外，為了確認雜交後使用不同鹽濃度環境對於核酸雜交穩定性與雜交量的影響，本研究利用穿透式與反射式小角度散射系統 (Grazing Incident Small-angle X-ray Scattering, GISAXS)與雙晶片(Charge Coupled Device, CCD)全自動倒立螢光顯微鏡進行觀察。從小角度散射的結果顯示，即使改變不同鹽濃度環境依然能維持雙股螺旋結構，並從螢光顯微鏡的結果得知，隨著鹽離子濃度上升，探針與目標物核酸之間的雜交量呈現上升的趨勢。最後，我們利用COB (Chip on Board)檢測系統來檢測不同鹽離子濃度環境下核酸雜交之電訊號變化。
根據檢測結果可以得知，在三種鹽濃度環境中，我們找出50 mM BTP在檢測核酸雜交能得到最大電訊號變化，並利用Liquid gate FETs之相等電路圖中，電容的概念來描述核酸雜交之複雜變化。接著，我們研究了兩種不同反離子大小的緩衝溶液1,3-雙(三(羥甲基)甲氨基)丙烷 (Bis-tris propane, BTP)和磷酸鹽緩衝生理鹽水 (Phosphate buffer saline, PBS)對於檢測核酸雜交的影響，發現到緩衝溶液中的BTP因離子半徑大小大於PBS中的鈉離子，能有效減少反離子蓄積於閘極氧化層的電荷密度從而提升電晶體元件靈敏性。最後，在最適化鹽離子濃度的環境下，我們研究了檢測不同目標物核酸的電訊號變化，並將實驗結果帶入Langmuir isotherm理論計算中，得出不同目標物核酸下的Ka值。計算結果顯示，DNA探針與目標DNA具有較大的Ka值，代表著其之間的結合親和力越大，能夠讓感測器檢測到越低濃度的DNA。 | zh_TW |
| dc.description.abstract | Due to the advantages of high sensitivity, label-free operation, low device cost, and real-time detection, silicon-nanowire field effect transistors (SiNW-FETs) have been widely considered as a highly promising detection platform in recent years.
Traditionally, the detection mechanism of SiNW-FETs for detecting nucleic acid hybridization relies on the change in surface charge density after nucleic acid hybridization, thereby inducing variations in the number of carriers within the electron channel, which are represented by different current-voltage (I-V) curves. For n-type field effect transistors, if the target molecule is negatively charged, the electric field it generates repels carriers (e-) in the channel, decreasing carrier density and reducing source-drain current, and vice versa.
However, the real surface phenomena are more complex and beyond the original assumptions. Based on experiment result, counter-ions in the buffer solution were not only drawn towards the negatively charged phosphate groups on the double-stranded nucleic acid but also drived by the gate voltage and thereby accumulated on the gate oxide surface. The electric field generated by these counter-ions competes with the electric field generated by the double-stranded nucleic acid, thereby affecting the variation of carriers within the channel.
Therefore, this study aims to investigate the effects of different salt ion concentrations on the electric fields caused by surface probe and microRNA hybridization after optimal silanization treatment on the surface, and to explore the most suitable salt ion concentration environment for nucleic acid detection as a product design recommendation. Initially, we modify the device surface with mixed self-assembled (silane-PEG-NH2：OH = 1：3 (mM/mM)) and glutaraldehyde. Then, DNA probes were reacted with glutaraldehyde through reductive amination. After surface modification, AFM and (ESCA) are used to observe surface characteristics to ensure the optimized conditions of Mixed-SAMs on the surface and the successful grafting of miR-21 probes.
Furthermore, in order to investigate the effects of different salt concentrations on the stability and amount of nucleic acid hybridization, we used (GISAXS) and IX83 Inverted Microscope for observation. Results from GISAXS demonstrate that the double-stranded helical structure remained stable despite changes in salt concentration, and fluorescence microscopy results reveal that hybridization amount between probes and target miRNA and DNA increased with higher salt concentrations.
Next, we used the Chip on Board (COB) detection system to detect changes in electrical signals during nucleic acid hybridization under different salt ion concentrations. From the COB detection results, it is observed that among the three salt ion concentration environments, 50mM Bis-tris propane (BTP) yields the most significant variation in electrical signals. Additionally, the concept of capacitance is applied to describe the complex variations in nucleic acid hybridization using the equivalent circuit of liquid gate FETs. Subsequently, we investigate the effects of two different buffer solutions with two different counter-ion sizes, 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP) and Phosphate-buffered saline (PBS), on nucleic acid hybridization detection. We found that BTP, with larger ion radii compared to the sodium ions in PBS, effectively reduced counter-ion accumulation on the gate oxide surface, enhancing the sensitivity of the transistor device.
Finally, under the optimized salt ion concentration environment, we studied the electrical signal changes in detecting different target nucleic acids. The experimental results were applied to the Langmuir isotherm theory to calculate the Ka values for different target nucleic acids. The results showed that DNA probes and target DNA had higher Ka values, indicating stronger binding affinity and allowing the sensor to detect lower concentrations of DNA. | en_US |