結合銅奈米粒子與螢光檢測裝置應用於結核病分子診斷

The Development of Arduino-Based Fluorescent Detection Device and Copper Nanoparticles for Tuberculosis Molecular Diagnosis

李宗穎(Zong-Ying Li)

指導教授 ： 張建成

共同指導教授 ： 陳建甫

國立臺灣大學/工學院/應用力學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902565

摘要

無法簡單、快速的檢測是結核病傳播的主要原因。為此，本研究將銅奈米粒子之特性結合分子生物技術，利用其螢光性質取代傳統核酸擴增試驗修飾螢光標定物質之檢測方法，並應用於結核病快速檢測。除了聚合酶連鎖反應(Polymerase chain reaction, PCR)放大後，接著使用環滾式擴增法(Rolling circle amplification, RCA)對DNA片段進行二次放大，並以特定單股DNA做為模板，將銅離子還原形成螢光銅奈米粒子。在DNA擴增實驗中，調整分子倒位探針(Molecular inverse probes, MIP)循環數、RCA加熱時間及酵素使用量的參數測試，在生成銅奈米粒子的實驗中對不同濃度還原劑、銅離子溶液及反應時間做探討，選擇最適化的條件參數，最後利用真實檢體進行臨床試驗。研究結果顯示，本系統能有效率且精準地放大結核菌目標DNA片段之訊號，採用最適化條件參數製作R2值為0.9998之檢量線，DNA濃度檢測極限可達5 fg/μL，且臨床檢體的試驗結果顯示具有87.5 %的敏感度及100 %的特異度，證明此方法在實際臨床應用之可靠性。由以上實驗結果，本系統能在保有高靈敏度、精確度情形下，大幅降低檢測分子生物技術放大訊號之專業人力、金錢、時間成本，在檢測部分也開發可攜式螢光檢測平台，能夠在一分鐘內將以DNA為模板的銅奈米粒子螢光訊號轉為數值供使用者讀取，判斷DNA標準品濃度在20 fg/μL以上為陽性結果。期許未來結合可攜式核酸擴增裝置，將放大、檢測訊號步驟簡化且合併於系統化裝置中，使低資源地區的居民能更有效率檢測結核病，以實踐及早發現、及早治療的願景。

關鍵字

結核病 ； 生物感測器 ； 核酸擴增試驗 ； 螢光銅奈米粒子 ； Arduino系統

並列摘要

Detection without easily and rapidly testing is the primary reason for Tuberculosis spreading. For this purpose, the study combines molecular biological techniques and features of copper nanoparticles (CuNPs). The method of labeling fluorescent group for detection in Nucleic acid amplification techniques (NAAT) is substituted by optical properties of CuNPs and applied for rapid TB diagnosis. In addition to polymerase chain reaction (PCR), rolling circle amplification (RCA) is then performed to amplify the DNA fragmentation two times. And these specific single-stranded DNA (ssDNA) are used as templates for reduction of fluorescent CuNPs from Cu ions. In the part of DNA amplification experiment, the parameters of thermal cycles in Molecular inversion probes (MIP), heat time in RCA and unit of phi29 DNA polymerase were adjusted. In the other part experiment about the productivity of CuNPs, we discussed the various concentration of reductant and copper ion solution and reaction time to select optimized conditions and finally conducted clinical trials with real sample. The results show that we can efficiently and accurately amplify the signals of TB specific DNA fragment. We use the optimization parameters to construct the calibration curve with R2 value of 0.9998, LOD of standard DNA concentration 5 fg/μL, and the result of clinical tests shows detection rate with 87.5 % sensitivity and 100 % specificity, which demonstrates reliability of practical clinical applications. As mentioned above, the system keeps high sensitivity and specificity while greatly reducing professional labor, money and time cost of detecting signal amplificated by molecular biology. In term of detecting, we develop portable fluorescent detection platform converting optical signals from DNA-templated CuNPs to digital number for user reading in one minute and judge the concentration of DNA standard above 20 fg/μL as a positive result. In the future, we expect to combine this platform with portable NAAT device to simplify procedures and integrate amplification and detection DNA signal into a system device. And we look forward to helping the resident living in the low-resource area more efficiently detecting TB to realize the vision for early detection and treatment.

並列關鍵字

Tuberculosis ； biosensor ； NAAT ； fluorescent CuNPs ； Arduino system

參考文獻

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