| 摘要: | microRNAs，是一種長度約為21-25個核苷酸的非編碼小片段RNA，它會通過辨識mRNA並與其結合，抑制後續蛋白質的轉譯，藉此來調控基因的表現。某些microRNA在各個發育階段的全部細胞中都有表達，某些microRNA在不同組織、不同發育階段中的表達水平也有著顯著的差異，因此，目前已經有越來越多的研究將miRNAs作為診斷、治療和預後的重要生物標記物。然而，microRNA和目標基因之間雖然有一定的專一性，但卻不是一對一的關係，一種microRNA可以結合一種以上的目標mRNA，從而對多種基因進行調控，也就是說，一種基因的表達可能是多種microRNA造成的，這是一種動態與複雜的系統。
檢測miRNAs的方法有許多種，而在眾多檢測方法中，由於矽奈米線場效應電晶體 (Silicon nanowire field-effect transistors, SiNWFETs)具有高靈敏度、免螢光標定、耗時短等特性使其在未來醫療的診斷方面具有很大的應用潛力。通常對於SiNWFETs的生物分子固定化方式大多採用整片式固定法( all area modification )，這使得SiNWFETs只能對單一一種microRNA進行檢測，但是，若是只透過檢測單一種microRNA來對癌症進行診斷及追蹤，其可靠度可能還是偏低，因此，本研究將嘗試將整片SiNWFETs晶片分為兩部分固定上兩種生物探針對miR-21和miR-155兩種microRNA同時進行檢測，以驗證SiNWFETs的多重感測(multiplexing)的可行性。
在使用COB (Chip on board)系統對本研究所使用的多重生物感測器(multiplex biosensor)所產生的電訊號進行研究前，我們會使用Cyanine3 (Cy3) 染料修飾的miR-21，透過螢光顯微鏡來觀察其在表面經過不同條件的清洗方式所剩餘的螢光的量的多少，以此來篩選出最佳的清洗條件。最後我們發現，先使用70℃熱水沖洗，再經過8M之尿素5分鐘的浸泡，最後再用70℃熱水沖洗為最佳的清洗方式。
對於multiplex biosensor的製作，本研究會先對整片SiNWFETs晶片使用Mixed-SAMs (Silane-PEG-NH2：Silane-PEG-OH=1：3(mM/mM))以及戊二醛 (Glutaraldehyde)對元件進行表面改質，之後將一片晶片分為兩個部分分別修飾上與miR-21和miR-155具有專一性的兩種不同的DNA生物探針，以此來完成multiplex biosensor的改質。
接下來我們會對multiplex biosensor的專一性、可重複性、靈敏度以及抗干擾能力來對其進行研究。對於miR-21和miR-155的專一性，我們分別使用1fM之miR-21和miR-155作為target並於multiplex biosensor上使用COB系統對其進行檢測，最後發現，兩者都只會在在與自身具專一性的生物探針上產生較大的訊號變化，說明miR-21與miR-155皆只會和與自身具有專一性的生物探針雜交，具有較高的專一性；同時我們也將上述實驗重複進行了三次，結果三次實驗所得到的電訊號值大小皆差不多，這說明該biosensor具有良好的的可重複性。
對於其靈敏度，我們使用上述multiplex biosensor分別對單一miR-21 target和單一miR-155 target進行檢測，之後再對同樣線性範圍濃度之miR-21和miR-155的混合物進行檢測。單一 target 之檢測極限 (Limit of detection)分別為 4.73 aM 和 7.27 aM ，對於其混合物，檢測極限則變為 5.58 aM 和 7.32 aM ，線性範圍 (Linear range)則皆為 10 aM-100 fM。若進行對比，會發現對混合物檢測結果所得到之電訊號雖然相比於單一target檢測得到之電訊號有些許減少，電訊號隨濃度變化之斜率也有降低，但其檢測極限並不會受到明顯的影響。
對於其抗干擾能力，首先我們使用了1 pM 的miR-155作為干擾物分別與不同線性範圍濃度內的miR-21混合後使用SiNWFETs進行檢測，經過計算，此時之檢測極限為 5.97 aM ; 接著使用1 pM 的miR-21作為干擾物分別與不同線性範圍濃度內的miR-155混合後使用SiNWFETs進行檢測，經過計算，此時之檢測極限為 7.62 aM，該結果表明非專一性之microRNA並不會對檢測極限以及線性範圍造成影響。再來我們使用了SiNWFETs對1fM miR-210，1fM miR-21與miR-155之混合物，1fM miR-210、miR-21與miR-155之混合物進行檢測，該結果表明，miR-210對於兩種目標物之檢測幾乎不會造成影響，說明該multiplex biosensor具有較高的抗干擾能力。
最後，將 miR-21 以及 miR-155 之混合物 spike in 人類血清中進行檢測，這時， miR-21 之檢測極限為 0.42 fM ，而miR-155 之檢測極限則是 0.48 fM。
;MicroRNAs are small, non-coding RNA molecules containing 21-25 nucleotides. Multiple microRNAs regulate gene expression, which is a dynamic and complex process. Targeting the complex and dynamic process by monitoring the microRNA profiling facilitates the usage of a silicon nanowire field-effect transistor (SiNWFET), which has the characteristics of real-time, label-free, high sensitivity, and the possibility of multiplexing. This study aims to verify the feasibility of multiplexing SiNWFETs by simultaneously detecting two microRNAs, miR-21 and miR-155, with high sensitivity and specificity requirements.
Before using the COB (Chip on board) system to study the electrical signals generated by the multiplex biosensor, we used a fluorescence microscope to observe the amount of remaining Cyanine3 (Cy3) dye-modified miR-21 on the surface to obtain an optimized cleaning process for detection. Finally, we found that the best cleaning procedure is first to rinse the surface with 80°C water, then immerse in 8M urea for 5 minutes, and finally rinse with 80°C water.
For the specificity of miR-21 and miR-155, we used the COB system to detect 1fM of miR-21 and miR-155 on the multiplex biosensor. Finally, we found that only probes that are specific to themselves generated significant signal changes, indicating that both miR-21 and miR-155 have high specificity. We also repeated the above experiment three times. As a result, the electrical signals obtained in the three experiments were all similar, which shows that the biosensor has good repeatability.
For the sensitivity, we used the multiplex biosensor to detect miR-21, miR-155, and the mixture of miR-21 and miR-155, respectively. The detection limit of single target are 4.73 aM for miR-21 and 7.27 aM for miR-155. For mixture, the detection limit of miR-21 is 5.58 aM, the detection limit of miR-155 is 7.32 aM ,the linear range are all 10 aM-100 fM. If compared, we can find that although other non-specific microRNA has little influence on the electrical signal and slope, it has almost no influence on the detection limit and linear range.
For anti-interference ability, we used 1 pM miR-155 as interference to detect miR-21 in different concentrations and compared the calibration line obtained from this result with the calibration line obtained from the detection of single miR-21 target, we found that non-specific microRNA has almost no influence on the detection limit and linear range. We can see the same result if we use 1 pM miR-21 as interference to detect miR-155. Finally, we used SiNWFETs to detect 1fM miR-210, the mixture of 1fM miR-21 and miR-155, and the mixture of 1fM miR-210, miR-21 and miR-155. The results show that miR-210 has almost no influence on the detection of target, indicating that our multiplex biosensor has high anti-interference ability.
Finally, we detected the mixture of miR-21 and miR-155 which was spiked in human serum . The detection limit of miR-21 was 0.42 fM, while the detection limit of miR-155 was 0.48 fM. |
