Department of Bioengineering, University of California, Berkeley, California, USA.
PLoS One. 2013 Aug 1;8(8):e70266. doi: 10.1371/journal.pone.0070266. Print 2013.
The rapid detection and identification of infectious disease pathogens is a critical need for healthcare in both developed and developing countries. As we gain more insight into the genomic basis of pathogen infectivity and drug resistance, point-of-care nucleic acid testing will likely become an important tool for global health. In this paper, we present an inexpensive, handheld, battery-powered instrument designed to enable pathogen genotyping in the developing world. Our Microfluidic Biomolecular Amplification Reader (µBAR) represents the convergence of molecular biology, microfluidics, optics, and electronics technology. The µBAR is capable of carrying out isothermal nucleic acid amplification assays with real-time fluorescence readout at a fraction of the cost of conventional benchtop thermocyclers. Additionally, the µBAR features cell phone data connectivity and GPS sample geotagging which can enable epidemiological surveying and remote healthcare delivery. The µBAR controls assay temperature through an integrated resistive heater and monitors real-time fluorescence signals from 60 individual reaction chambers using LEDs and phototransistors. Assays are carried out on PDMS disposable microfluidic cartridges which require no external power for sample loading. We characterize the fluorescence detection limits, heater uniformity, and battery life of the instrument. As a proof-of-principle, we demonstrate the detection of the HIV-1 integrase gene with the µBAR using the Loop-Mediated Isothermal Amplification (LAMP) assay. Although we focus on the detection of purified DNA here, LAMP has previously been demonstrated with a range of clinical samples, and our eventual goal is to develop a microfluidic device which includes on-chip sample preparation from raw samples. The µBAR is based entirely around open source hardware and software, and in the accompanying online supplement we present a full set of schematics, bill of materials, PCB layouts, CAD drawings, and source code for the µBAR instrument with the goal of spurring further innovation toward low-cost genetic diagnostics.
快速检测和鉴定传染病病原体是发达国家和发展中国家医疗保健的迫切需要。随着我们对病原体感染性和耐药性的基因组基础有了更多的了解，即时核酸检测可能成为全球卫生的重要工具。在本文中，我们介绍了一种廉价、手持、电池供电的仪器，旨在使发展中国家能够进行病原体基因分型。我们的微流控生物分子扩增读取器（µBAR）代表了分子生物学、微流控、光学和电子技术的融合。µBAR 能够进行等温核酸扩增检测，并具有实时荧光读出功能，其成本仅为传统台式热循环仪的一小部分。此外，µBAR 还具有手机数据连接和 GPS 样本地理标记功能，可用于流行病学调查和远程医疗服务。µBAR 通过集成的电阻加热器控制检测温度，并使用 LED 和光电晶体管监测来自 60 个单独反应室的实时荧光信号。检测在 PDMS 一次性微流控卡上进行，无需外部电源即可进行样品加载。我们对仪器的荧光检测极限、加热器均匀性和电池寿命进行了表征。作为原理验证，我们使用 µBAR 通过环介导等温扩增（LAMP）检测了 HIV-1 整合酶基因。虽然我们这里主要关注纯化 DNA 的检测，但 LAMP 以前已经在各种临床样本中得到了证明，我们的最终目标是开发一种包括从原始样本中进行芯片上样品制备的微流控装置。µBAR 完全基于开源硬件和软件，在随附的在线补充材料中，我们提供了 µBAR 仪器的全套原理图、材料清单、PCB 布局、CAD 图纸和源代码，旨在推动低成本基因诊断的进一步创新。
