發展早期診斷新生兒巨細胞病毒感染之表面電漿共振生醫感測器

Abstract

Human cytomegalovirus (HCMV) 人類巨細胞病毒又稱人類皰疹病毒第五型，是一種雙股DNA病毒，有非常強的傳染力。如果該病毒感染健康人時，通常不會觀察到明顯症狀，但若感染免疫不全的個體 (如:器官移植病人、AIDS病人和新生兒)，就可能會危及生命。另外，母體若於懷孕時期感染HCMV，病毒有很高的機率藉由胎盤、乳汁及唾液等途徑傳染給胎兒，造成新生兒出現呼吸困難、意識運動障礙、智力遲鈍、肝脾腫大、耳聾和中樞神經系統異常等嚴重的臨床症狀。目前診斷HCMV的方式之一為定量血液樣品中IgG及IgM的濃度；若兩者皆呈陽性反應，則過兩周後須再抽血定量一次。若血中IgG的量是前一次的四倍以上則表示已被感染HCMV。此方法雖為臨床常用的檢驗方法，但診斷結果無法即時得知。由於過去文獻曾指出，HCMV病毒帶有多個微小核酸片段分子(microRNAs)，並可在被感染的病人血液中被偵測出，因此設計一套表面電漿共振生醫感測系統，可以快速且準確檢測 HCMV感染，進而輔助醫生早期診斷，並適時給予適當藥物以預防母嬰垂直感染即為本研究的主要實驗目的。在這實驗中想利用表面電漿共振生醫感測器(surface plasmon resonance, SPR)來偵測血清中HCMV的microRNA，藉由磁珠抓住目標microRNA，再利用Poly A polymerase來將目標microRNA加上200~300 bases poly A tail ，利用Adenine對金有較強的吸附力，吸附在表面電漿共振系統偵測金表面上，造成表面電漿共振訊號改變來偵測是否有感染HCMV。目前實驗已經證明Poly A polymerase確實可以延長目標microRNA，而藉由表面電漿共振訊號圖，也可以證明經過延長的目標microRNA可以吸附在金表面上並且訊號有隨濃度的變化而提高。目前整個系統的偵測極限是在250pM。

Human cytomegalovirus (HCMV), known as human herpesvirus-5 (HHV-5), is a double strand DNA viruses. Vertical transmission of HCMV occurs frequently in newborns, then leads to congenital abnormalities. MicroRNAs (miRNAs) are short (18~23 nucleotide), non-coding nucleotides that play an important role in human diseases. The abundant expression of multiple microRNAs in blood that are encoded by HCMV, have been discovered after infection. In this study, we aimed to develop a surface plasmon resonance biosensing platform for the rapid detection of HCMV viral microRNA that can be used in the prediction of possible vertical transmission from mother to newborns. The utilization of the capture probe-modified magnetic nanoparticle enabled us to extract our target microRNA via biotin−streptavidin interaction. The function of later-added polymerase was used for elongation of our target microRNA. Finally the analytical measurement was performed on a surface plasmon resonance biosensing system to determine the amount of elongated nucleic acid sequences. The total assay time for our SPR sensing platform is less than 2 hours, and detection limit is calculated as 250 pM. This newly-developed sensing technology holds great promise in assisting physicians to facilitate treatment decision making, thus preventing timely congenital abnormalities in newborns.