結合石英晶體微天平法與奈米金球構建登革熱病毒檢測之核酸生物感測平台

Abstract

本研究中結合oligonucleotides-functionalized 之奈米金球 (gold nanoparticles; AuNPs)應用於循環迴流石英晶體微天平中以架構一登革熱病毒核酸檢測平台。在此石英晶體微天平系統中，oligonucleotides-functionalized AuNPs (AuNPs-probes) 利用層疊方式雜交於晶片上以達到大幅放大偵測訊號的效果。首先將人工合成之probe (DENV2-P1; 30 mer外加12-dT)以自組裝方式固定化於晶片表面，由於DENV2-P1可與登革熱病毒膜套蛋白序列互補，因此當檢測物中含有人工合成的登革熱病毒膜套蛋白的DNA序列 (60 mer) 或以不對稱聚合酶連鎖反應所合成的產物 (130 mer) 皆會導致表面重量增加進而改變震盪頻率 (ΔF)。此外，表面修飾probe (DENV2-P2; 30 mer 附加 12-dT) 的奈米金球亦被運用在此系統中並扮演著target捕捉以及序列確認的角色，同時也具有增大頻率的變化的功能。為了能充分利用循環迴流系統中的target，DENV2-P1修飾於奈米金球上並隨後應用於此系統中，藉由未雜交的target作為架橋層疊於晶片上進行第二次的訊號放大。此實驗中並比較不同粒徑大小的奈米金球 (直徑5, 13, 20, and 50 nm) 的撘配層疊對於訊號放大的效果，結果發現13 nm AuNPs-DENV2-P2 (第一層) 與13 nm AuNPs-DENV2-P1 (第二層) 具有最適的訊號放大效果。此外，在血液中當病毒數量在2 × 100 to 2 × 106 PFU/mL間其感測器所測的的頻率變化(ΔF)方面亦與病毒的濃度呈現正線性相關(R2 = 0.987)，此系統可偵測極限為2 PFU/mL 微量的登革熱病毒。

In this study, a circulating-flow quartz crystal microbalance (QCM) sensor combining oligonucleotide-functionalized gold nanoparticles (AuNPs) used to detect dengue virus (DENV) has been established. In the QCM system, two specific oligonucleotides-functionalized AuNPs (i.e., AuNPs-probes) were layer-by-layer hybridized onto the QCM chip to significantly amplify the detection signal. A synthesized probe (DENV2-P1; 30 mer with an additional 12-dT) specific to complement the target sequences, the gene sequences encoding DENV envelope protein (E), was first immobilized onto the surface of QCM chip by self-assembly. Furthermore, hybridization was performed by exposing the immobilized probe to the target sequences, synthesized target oligonucleotides (60 mer) or a DNA fragment (130 bp) amplified by asymmetric polymerase chain reaction, resulting in mass increase and consequently oscillatory frequency change (ΔF) of the QCM sensor. A second probe (DENV2-P2; 30 mer with 12-dT) conjugated to AuNPs was then applied onto the QCM chip as a “target capturer” and “sequence verifier” to amplify the ΔF of QCM sensor. In order to make the target sequences could been sufficiently hybridized in the circulating-flow QCM system, the DENV2-P1 was also conjugated with AuNPs and applied onto the QCM chip by using the surplus target sequences as a bridge for layer-by-layer hybridization and enhancing the detection signal. The effect of four AuNPs-probes sizes (5, 13, 20, and 50 nm in diameter) on the layer-by-layer hybridization has been evaluated and the results indicated that 13 nm AuNPs-DENV2-P2 (1st layer) collocated with 13 nm AuNPs-DENV2-P1 (2nd layer) showed the best hybridization efficiency. As low as 2 plaque forming unit (PFU)/mL of DENV serotype 2 could be specifically detected by the established DNA-QCM sensor. Additionally, a linear correlation (R2 = 0.987) of ΔF vs. virus titration from 2 × 100 to 2 × 106 PFU/mL was found.