College of Information Science and Electronic Engineering, Zhejiang University, Hangzhou, 310027, PR China; International Joint Innovation Center, Zhejiang University, Haining, 314400, PR China.
College of Information Science and Electronic Engineering, Zhejiang University, Hangzhou, 310027, PR China.
Biosens Bioelectron. 2022 Oct 1;213:114499. doi: 10.1016/j.bios.2022.114499. Epub 2022 Jun 23.
In this paper, we report on an integrated microfluidic digital PCR system for rapid and high-performance absolute quantification of DNA at a single-molecular level. Microchannel plate (MCP), a highly porous glass membrane conventionally used as a particle multiplier in detectors, is demonstrated here as an ideal platform for the sample partition and high-fidelity DNA detection. The density of the microreactors reaches up to 1563 mm, with a total number of 25,000 chambers each in 100 pL volumes embedded in 4 × 4 mm MCP. This glass membrane consisting of highly-regular, densely-packed and high-aspect ratio microchannels is batch-fabricated through fiber-draw technology that requires no cumbersome lithography or etching process. The sidewalls, having a slight tilting angle with respect to the surface normal, can be functionalized with ZnO nanorods through one-step shadowing-based deposition without resorting to complicated solution-process or lithography. In this way, the required thermal cycling time for end-point detection has been reduced from 40 min to 25 min through plasmonic fluorescence enhancement compared to bare MCP without nanostructures. The digital PCR performance of our system has been validated by using bacteriophage λDNA and PLAU genes. The dynamic range achieved is noted as 10 ranging from 1 × 10 to 1 × 10 copies/μL and the measurement deviation is less than 5%. The detection limit is determined to be 1.4 copies/μL. The density of microchambers could be easily scaled for extensive applications and detection ranges by fabricating various MCP matrix structures. Given this high performance and a straightforward fabrication process of MCP, the device is expected to replace conventional PCR equipment for high fidelity and wide dynamic range single-molecule counting.
在本文中，我们报告了一种集成的微流控数字 PCR 系统，用于在单分子水平上快速、高性能地对 DNA 进行绝对定量。微孔板（MCP）是一种传统上用作探测器中的粒子倍增器的高多孔玻璃膜，在这里被证明是用于样品分离和高保真 DNA 检测的理想平台。微反应器的密度高达 1563mm，每个 MCP 中嵌入了 4×4mm 的 25000 个腔室，每个腔室的体积为 100μL。这种玻璃膜由高度规则、密集排列和高纵横比的微通道组成，通过光纤拉制技术批量制造，不需要繁琐的光刻或蚀刻工艺。侧壁相对于表面法线有一个轻微的倾斜角，可以通过一步阴影沉积技术在不使用复杂溶液处理或光刻的情况下功能化 ZnO 纳米棒。通过等离子体荧光增强，与没有纳米结构的裸 MCP 相比，用于终点检测的所需热循环时间从 40 分钟减少到 25 分钟。通过使用噬菌体 λDNA 和 PLAU 基因对我们系统的数字 PCR 性能进行了验证。所达到的动态范围为 10，范围从 1×10 到 1×10 拷贝/μL，测量偏差小于 5%。检测限确定为 1.4 拷贝/μL。通过制造各种 MCP 基质结构，可以轻松扩展微室的密度以实现广泛的应用和检测范围。鉴于这种高性能和 MCP 的简单制造工艺，该设备有望取代传统的 PCR 设备，用于高保真度和宽动态范围的单分子计数。
