矽奈米線場效電晶體 (SiNW-FET) 現今已廣泛用做於生物感測器,應用在各種偵測上,像是去氧核醣核酸雜交 (DNA hybridization),蛋白質,病毒,或是小分子。由於SiNW-FET有高靈敏度,選擇性,免標記與及時偵測的優點,我們可以利用可重複使用的SiNW-FET去偵測小片段的核醣核酸。 後轉錄時期基因沉默 (post-transcriptional gene silencing;PTGS) 為植物抵抗外來病毒感染的一個策略,但是病毒會產生抑制蛋白,以防止這種攻擊病毒的-siRNA) 有很好的結合力。在本篇論文中,我們運用奈米科技而發展出修飾有P19的矽奈米線場效電晶體 (P19/SiNW-FET) 可以偵測基因沉默機制,番茄叢矮病毒 (Tombusvirus) 含有的抑制蛋白- P19 (分子量19 kDa),具有長度專一性的鑑別率,並與21 nt雙股小干擾核醣核酸 (double-stranded short-interfering RNA;dsds-siRNA。P19/SiNW-FET具有極高的靈敏度,不只可以偵測到500 pM的21 nt ds-siRNA,還可以分辨不同二級結構的ds-siRNA,像是不同長度或是未完全配對的ds-siRNA。再者, P19/SiNW-FET抓到的未知雙股核醣核酸 (dsRNA) ,在偵測實驗完後可以被沖提出來並回收,以進行下一步的鑑定,像是即時聚合酶連鎖反應 (real-time polymerase chain reaction;real time PCR)。從這些測量結果可以了解到SiNW-FET提供很好的平台去篩選多種蛋白質-核醣核酸之間的交互作用;另一個特點則是我們可以將這些小片段核醣核酸從P19/SiNW-FET給沖提出來,並且做進一步的定量分析而能了解其資訊。
Silicon nanowire field-effect transistors (SiNW-FETs) have been extensively used as biosensors in a variety of detections, ranging from DNA hybridization, proteins, virus, and small molecules. Taking advantages of the ultrahigh sensitivity, selectivity, label-free and real-time detection capabilities of a SiNW-FET, we have applied reusable SiNW-FETs for the study of protein-small RNA interactions. Post-transcriptional gene silencing (PTGS) is an antiviral strategy in plants to avoid the virus infection. However, the virus can produce a viral suppressor protein to suppress the PTGS in order to prevent the silencing machinery from attacking virus. P19 of Tombusvirus (with 19 kDa molecular weight) has been identified as a viral suppressor that can specifically bind 21 nucleotide (nt) double-strand short-interfering RNA (ds-siRNA) with high affinity and size selection. In this study, we reported the application of the nanotechnology to develop a highly sensitive SiNW-FET biosensor immobilized with P19 protein (referred to as P19/SiNW-FET) for the detection of interacting siRNAs. This P19/SiNW-FET biosensor is capable of not only detecting 21 nt ds-siRNAs to a 500 pM level, but also distinguishing the various secondary structures of ds-siRNAs, such as their size, and the mismatch between P19 and ds-siRNAs. Moreover, unknown ds-siRNAs captured by P19/SiNW-FET in the sensing measurement can be recovered after detection for the further identification with the polymerase chain reaction (PCR) technique. From these sensing measurements, we demonstrate that SiNW-FET biosensors provide an excellent platform for high-throughput screening protein-RNA interactions. In particular, the interacting siRNAs recorvered from a specific protein-modified SiNW-FET can be identified to reveal their profile.