| 摘要: | 微小核糖核酸 (microRNA, miRNA) 是一種長度約為21-25個核苷酸的非編碼RNA分子，可調控轉錄後基因的表現量，而miRNA的表達異常與人類的多種癌症密切相關，是現代醫療中備受矚目的癌症生物標記物。miRNA雖然具有良好的專一性，但miRNA對於目標基因並非只有直觀的一對一的關係，而是一對一、多對一以及網絡狀關係。因此檢測單一miRNA可能對於疾病診斷沒有實質性幫助，需檢測多種miRNA才能提升疾病診斷的準確率。
現今的癌症基因檢測是採取定量逆轉錄聚合酶鏈反應技術 (Quantitative reverse transcription polymerase chain reaction, RT-qPCR) ，該技術是目前核酸檢測技術的黃金標準 (gold standard) ，根據使用試劑及分析儀器不同，RT-qPCR的檢測極限範圍約在100 pM至100 fM 之間，通常會高於體內miRNA的濃度。因此，利用RT-qPCR檢測體內特定miRNA含量時往往會得到失真的數據。此外，RT-qPCR技術必須在實驗室經由專業人員才能進行操作，所用試劑以及檢測儀器都十分昂貴，且檢測流程往往需要耗時數小時。為克服RT-qPCR的這些限制，發展能夠檢測超低miRNA濃度，且操作簡便、成本低廉的檢測方法，對於提高癌症診斷的準確性具有重要意義。
本研究利用自製紙基萃取裝置提取出total RNA，再透過矽奈米線場效電晶體 (Silicon nanowire field-effect transistor, SiNW-FET) 定量檢測特定核酸序列之miRNA，以取代過往對於核酸萃取與檢測的繁複流程。由於紙質分析設備的成本價格低廉且具有毛細現象特性，不需額外的外部能源，讓紙質設備擁有巨大優勢。而 SiNW-FET 除了具有高靈敏度、免螢光標定、耗時短、即時檢測等優勢外，檢測極限可低至 1aM ，提供精確量測體內的特定 miRNA 濃度，因此被視為有力的檢測平台，有利於應用在定點照護檢驗 (Point-of-Care Testing, POCT) 。
於本研究的實驗流程中，首先在纖維素紙基表面上改質二氧化矽奈米顆粒，二氧化矽顆粒在酸性環境中水解聚縮合形成網狀結構，再滴於紙基並藉由高溫反應使二氧化矽與纖維紙上羥基機團發生脫水斷鍵反應，使得二氧化矽接枝於紙基，並藉由傅立葉轉換紅外光譜分析技術(Fourier-transform infrared spectroscopy, FTIR) 確認二氧化矽顆粒成功接之於纖維素紙上。在紙基萃取系統中，我們透過控制裝置閥門的開關，讓細胞中的核糖核酸在紙基上進行吸附、脱附與清洗，藉此來分離出細胞中的核酸，最後分別利用 RT-qPCR 與 SiNW-FET 檢測萃取出的核酸濃度並作比較。
本研究將此檢測系統應用於檢測人類直腸癌細胞株 (HCT116) 中的 miR-21 與 miR-155 的表現量。而為了檢測紙基裝置所萃取出的 total RNA ，我們事先利用定量標準品來建立 RT-qPCR 以及 SiNW-FET 的核酸濃度檢量線，並將萃取出的 total RNA 進行連續稀釋 (serial dilution) 後，分別利用 RT-qPCR 與 SiNW-FET 進行檢測。實驗結果顯示， RT-qPCR 檢測紙基裝置所萃取出的 miR-21及miR-155 透過已建立好的檢量線換算出來的核酸濃度分別為4.29×10-13 M以及4.65×10-13 M，而 SiNW-FET 檢測到的 miR-21、miR-155的表現量經過換算則是分別約為2.17 × 10-13 M、3.78 × 10-13 M，可得知 SiNW-FET 在檢測細胞中的 miRNA 濃度具有良好的相對準確性。
本研究成功開發出一套結合紙基核酸萃取與矽奈米線場效電晶體檢測的 miRNA 檢測系統，透過實驗驗證證實該系統相較於傳統的核酸萃取與分析，能夠快速、靈敏地檢測並區分出細胞中不同種類的超低濃度 miRNA ，得以更方便進行診斷、治療以及術後追蹤，為傳統癌症診斷方法提供了一種創新的替代方案。這項研究成果不僅為 miRNA 作為癌症生物標記物的研究提供了有力的技術支持，實踐 POCT 的理念。
;MicroRNAs (miRNAs), small non-coding RNA molecules about 21-25 nucleotides long, regulate gene expression post-transcriptionally. They are promising cancer biomarkers due to altered miRNA expression is highly associated with several types of human cancers. However, miRNAs interact with target genes in a variety of ways, such one-to-one, multiple-to-one, and network-like. As a result, detecting a single miRNA might not be enough to properly evaluate a disease; instead, multiple miRNAs are needed to detect.
Depending on the reagents and instruments used, the detection limit of RT-qPCR typically ranges from 100 pM to 100 fM, which is higher than the concentration of miRNA in the human body. Because of this, RT-qPCR often provides inaccurate results when detecting specific miRNA levels in biological samples. Furthermore, the reagents and equipment needed for RT-qPCR are costly, and the procedure must be carried out in a lab by experienced technicians. Additionally, it might take numerous hours to accomplish the entire procedure. It is essential to develop detection techniques that can quantify ultra-low miRNA concentrations with less difficulty and cost to overcome these limitations. These developments would greatly increase the accuracy of cancer diagnosis.
In this study, total RNA was extracted using a self-designed paper-based extraction method. A silicon nanowire field-effect transistor (SiNW-FET) was next used to detect and quantify specific nucleic acid sequences. The conventional complex procedures for nucleic acid extraction and detection will be replaced by this approach. The low cost and capillary mechanism of the paper-based analytical device, which doesn′t require an external power source, provide significant advantages. SiNW-FET provides real-time analysis, label-free detection, high sensitivity, and rapid process times. It is a potential platform for Point-of-Care Testing (POCT) since it enables the accurate determination of specific miRNA concentrations in biological samples with a detection limit as low as 1 aM.
In this study, we started by coating silica particles on the cellulose paper′s surface. The silica particles hydrolyzed and polycondensed to form a network structure in an acidic environment. Following the drop of this solution onto the paper substrate, a high-temperature reaction was employed that caused the silica to dehydrate and break its bond to the hydroxyl groups on the cellulose fibers, allowing the silica to graft onto the paper. RNA may be adsorbed, desorbed, and washed on the paper substrate by adjusting the valve switches on the device, enabling the extraction of nucleic acids.
The human colorectal cancer cell line (HCT116) was used in this study to evaluate the expression levels of miR-21 and miR-155 with the detection approach. We first developed calibration curves for nucleic acid concentration using RT-qPCR and SiNW-FET with quantitative standards aiming to verify the total RNA extracted by the paper-based device. The extracted total RNA was then subjected to serial dilution and analyzed separately using RT-qPCR and SiNW-FET. According to RT-qPCR based on the calibration curve, the experimental results indicated that the nucleic acid concentrations of miR-21 and miR-155 extracted by the paper-based device were 4.29× 10⁻¹³ M and 4.65 × 10⁻¹³ M, respectively. On the contrary one, upon conversion, the concentrations of miR-21 and miR-155 detected by SiNW-FET were roughly 2.17 × 10⁻¹3 M and 3.78 × 10⁻¹3 M.
This research successfully developed a miRNA detection system that combines SiNW-FET detection with paper-based nucleic acid extraction, offering a novel alternative for conventional cancer detection methods. In comparison with conventional nucleic acid extraction and analysis approaches, experimental validation demonstrated that this approach enables for the rapid and highly specific detection of miRNAs at ultra-low concentrations. This allows for more practical uses in postoperative monitoring, diagnosis, and treatment. Despite providing solid scientific support for the investigation of miRNAs as cancer biomarkers, the results establish a basis for the creation of quick and easy-to-use cancer diagnostic instruments that are consistent with the POCT concept. |
