A paper-based platform for detection of viral RNA.
A paper-based platform for detection of viral RNA.
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用于检测病毒 RNA 的纸质平台。
DOI：
10.1039/c6an02452a
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Deo,SapnaK
中科院分区：
文献类型：
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作者：
Zhang,Daohong;Broyles,David;Hunt,EricA;Dikici,Emre;Daunert,Sylvia;Deo,SapnaK
即使是最敏感的分析技术，病毒检测也面临着一系列挑战，而常见检测平台的复杂性通常会阻碍其便携性。考虑到这些考虑，我们设计了一种纸质微区平板病毒检测系统，用于检测病毒遗传物质，可以使用简单的仪器进行。该传感系统可以利用生物素标记的捕获探针和Alexa Fluor®647标记的报告探针来检测从总RNA提取中反转录的病毒cDNA。生物素化捕获探针通过物理吸附在纸张上的NeudAvidin®连接到纸张表面。在依次加入逆转录样品和报告探针后，逆转录靶标捕获报告探针并以桥接的形式将其与捕获探针捆绑在一起。使用Western印迹成像系统对荧光强度进行成像，Alexa Fluor®647的发射强度增加，可以看到更高的目标浓度。通过利用纸张，该检测装置还可以作为一种蒸发样品浓缩方法，在需要时可以显著降低检测限。该检测平台利用EB病毒(EBV)RNA作为概念验证，通过检测逆转录产生的cDNA，可进一步扩展为对其他病原体的通用检测方法。EBV是一种众所周知的人类肿瘤病毒，最近也被认为与宫颈癌的发展有关。该方法在两小时内完成，包括室温下提取RNA和逆转录步骤。此外，本文的微区平板平台设计坚固，界面方便，便于携带，具有潜在的应用前景，可用于POC检测试剂盒或设备的开发。实验可以在每一步之后停止，并在以后的时间继续。改进的纸板设置的保质期至少为3个月，信号没有明显变化，从第一天开始的结果可以在3个月内读取-这两个标准都是POC分析测试工具的重要标准，特别是在资源匮乏的环境中。所有所需的分析步骤都可以在没有任何重要设备的情况下使用廉价的纸质微区板进行，这将是进一步开发POC测试设备的理想选择。尽管该平台不处于可直接用于护理点设置的阶段，但它确实具有诸如稳定的平台、简单的检测方法以及与POC系统紧密对准的相对常见的试剂等基本特征。
Viral detection presents a host of challenges for even the most sensitive analytical techniques, and the complexity of common detection platforms typically preclude portability. With these considerations in mind, we designed a paper microzone plate-based virus detection system for the detection of viral genetic material that can be performed with simple instruments. The sensing system can detect viral cDNA reverse-transcribed from total RNA extraction by utilizing a biotinylated capture probe and an Alexa Fluor® 647-labeled reporter probe. The biotinylated capture probe was linked to the paper surface via NeutrAvidin® that was physically adsorbed on the paper. After addition of reverse-transcribed sample and reporter probe in sequence, the reverse-transcribed target captured the reporter probe and tethered it to the capture probe in a bridged format. Fluorescence intensity was imaged using a Western blot imaging system, and higher target concentration was visible by the increased emission intensity from Alexa Fluor® 647. By utilizing paper, this detection setup could also serve as a sample concentration method via evaporation, which could remarkably lower the detection limit if needed. This detection platform used Epstein-Barr virus (EBV) RNA as a proof-of-concept by sensing cDNA resulting from reverse transcription and can be further expanded as a general method for other pathogens. EBV is a well-known human tumor virus, which has also recently been linked to the development of cervical cancer. The assay was accomplished within two hours including the room-temperature RNA extraction and reverse transcription steps. Also, this paper microzone plate-based platform can potentially be applicable for the development of point-of-care (POC) detection kits or devices due to its robust design, convenient interface, and easy portability. The experiment could be stopped after each step, and continued at a later time. The shelf-life of the modified paper plate setup was at least 3 months without a discernible change in signal, and the result from day 1 could be read at 3 months – both of which are important criteria for POC analytical testing tools, especially in resource-poor settings. All of the required assay steps could potentially be performed without any significant equipment using inexpensive paper microzone plates, which will be ideal for further development of POC testing devices. Although, this platform is not at the stage where it can be directly used in a point-of-care setting, it does have fundamental characteristics such as a stable platform, a simple detection method, and relatively common reagents that align closely with a POC system.