Abstract
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.
Highlights
Integrated microfluidic diagnostic systems present an unprecedented opportunity to facilitate high-quality, low-cost healthcare in remote and resource-limited settings [1]
As a proof-ofprinciple, we have shown that the HIV integrase gene can be detected using the Loop-Mediated Isothermal Amplification (LAMP) assay on the Microfluidic Biomolecular Amplification Reader (mBAR) platform
The LAMP assay has been demonstrated for detecting drug resistance in TB from sputum samples [27] as well as detecting HIV [28] and malaria [29] in blood samples, and these assays can be readily adapted to the mBAR platform
Summary
Integrated microfluidic diagnostic systems present an unprecedented opportunity to facilitate high-quality, low-cost healthcare in remote and resource-limited settings [1]. These systems show promise for both acute infection discovery and chronic disease monitoring and management [2]. A wide array of nucleic acid tests (NATs) are emerging to identify pathogen species as well as specific clinically-relevant characteristics such as pathogenicity, origin, and drug susceptibility [3], [4]. Recent efforts have focused on automating standard sample preparation and PCR techniques; the significant assay cost and relatively low throughput remain prohibitive barriers for wide-scale adoption [7]
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