Quantum dot-based nuclease-resistant molecular beacons for visualizing the Coxsackievirus replication in living cells via TAT peptide delivery

論文發表人: 葉曉芸(加州大學河濱分校化學工程學研究所)
http://portal.acs.org/portal/acs/corg/content?_nfpb=true&_pageLabel=PP_TRANSITIONMAIN&node_id=859&use_sec=false&sec_url_var=region1

 

分子信標技術是基於螢光共振能量轉移現象和鹼基互補配對原則建立，於顯微鏡下直接觀測與新合成病毒RNA結合而產生的螢光，方法簡單且不需探針分離的步驟。進行活體細胞即時研究時，細胞酶會導致分子信標水解進而破壞其結構,有機螢光分子也易受光降解的影響。我們發展抗核酸酶分子信標結合無機螢光共振能量轉移對 - 量子點與金奈米粒子。TAT胺基酸於15分鐘內能成功將信標組送入活體細胞裡。BGMK細胞以10倍病毒連續稀釋感染,於顯微鏡下即時監控螢光的密度並觀察完整的病毒繁殖週期。病毒劑量與螢光細胞的數量呈正相關。與傳統的48小時病毒斑檢驗法比較,本研究提供一更為迅速且同等準確的檢驗方法。

 

Molecular beacons (MB), which produce fluorescence upon target binding, provide a simple and separation-free detection scheme. By directly visualizing the fluorescent hybrids with newly synthesized viral RNA, MB can be used to provide a rapid and sensitive detection of infectious viruses. For real-time studies in living cells, however, the durability of MBs is affected due to nuclease degradation and the rapid photodegradation of the organic fluorophore. In this study, we developed nuclease-resistant MBs composed of CdSe/ZnS quantum dots as donors and gold nanoparticles as quenchers for the in vivo detection of infectious viruses. For intracellular delivery, a hexahistidine-appended cell-penetrating TAT peptide was self-assembled onto the quantum dot surface via metal-affinity interactions. Presence of the TAT peptide allowed nearly 100% intracellular delivery within 15 min. Confluent buffalo green monkey kidney (BGMK) monolayers were infected with virus dilutions and the fluorescence intensity was monitored in real time. Fluorescence microscope was used to directly visualize infected cells and to subsequently follow virus spread among cells in vivo. The number of fluorescent cells increased in a dose-responsive manner and enabled the direct quantification of infectious viral doses. Using the 2-h infection window, the validation of the MB-based fluorescence assay for viral quantification was demonstrated by comparing with the traditional 48-h plaque assay.