Device Engineering for Infectious Disease Diagnosis using Isothermal DNA Amplification and Lateral Flow Detection

Technologies that enable infectious diseases diagnosis in low-resource settings could greatly facilitate effective treatment and containment of such diseases. Nucleic acid amplification testing can be used to identify pathogens, but typically requires highly-trained personnel and large, expensive lab equipment, neither of which is available in low-resource settings. Our overall goal is to develop a portable diagnostic system that utilizes a low-cost, disposable, mesofluidic cartridge and a handheld electronics unit to perform fully-integrated nucleic acid testing at the point of care in low-resource settings. As a first step toward this goal, we developed a subunit to execute isothermal nucleic acid amplification coupled with lateral flow detection, in parallel with the development of a sample preparation subunit by our collaborators at Claremont BioSolutions. Fluid handling inside the amplification and detection cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard, scalable manufacturing techniques, such as injection molding. Using an initial prototype system, we demonstrated detection of purified Mycobacterium tuberculosis genomic DNA. We then developed a refined amplification and detection cartridge in conjunction with an improved portable instrument, which automates pumping, heating, and timing, using a design format compatible with eventual integration with the sample preparation subunit. This refined cartridge incorporates a novel, inexpensive, stand-alone, passive valve, smaller, integrated pump components, a more complex injection molded polycarbonate cartridge core piece, and enhanced lateral flow chambers to improve visual detection. The independent valve component can be tailored for a variety of fluidic systems. We demonstrated appropriate fluidic and thermal control, and successful isothermal nucleic acid amplification within this refined amplification and detection subunit. We have developed a separate fluidic module for master-mix reagent storage and reconstitution that is designed to act as the interface between the amplification and detection subunit and the upstream sample preparation subunit. We envision that the merger of these two subunits into a fully-integrated cartridge will enable user-friendly, automated sample-in to answer-out diagnosis of infectious diseases in primary care settings of low-resource countries with high disease burden

Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

<div><p>Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of <i>Mycobacterium tuberculosis</i> (<i>M.tb</i>) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.</p></div

Thermal control within the cartridge.

<p>(a) Measured temperature of the fluid inside the reaction pouch of a cartridge attached to the heater as a function of time. Filling the pump pouch with liquid improves the thermal transfer from the heater to the reaction fluid, which reaches a stable temperature of 62±0.1°C within approximately ten minutes. Thermal paste applied between the heater surface and pump pouch provides no further improvements. (b) and (c) Thermal simulations (Comsol Multiphysics) of the cartridge on top of the heater, showing horizontal cross-sections within the center of the reaction fluid layer of the cartridge. (b) Uniform heating to the desired temperature (63°C) is observed using a model with ideal thermal contact between cartridge and heater, with no air gap. (c) Introducing a 150 µm thick thermally-resistive layer between cartridge and heater leads to a lower temperature and less uniform heating within the reaction pouch. The cross-hatches in the middle of the pump pouches indicate the dimensions of the circular reaction pouches. An air bubble was intentionally introduced into the outlet port of the reaction chamber to simulate trapped air in the reaction pouches after fluid is inserted.</p

Isothermal DNA amplification coupled to NALF detection.

<p>Conceptual depiction for (a) LAMP, and (b) EXPAR. (i) amplified master-mix applied to the conjugate pad enables amplicons to interact with colored polystyrene microspheres functionalized with appropriate capture moieties. (ii) After migrating along the nitrocellulose membrane, microspheres carrying amplicons are captured at the test line. (iii) At the control line, microspheres are captured irrespective of the presence of amplicon. (c) LAMP based detection of <i>M.tb</i> genomic DNA performed in the cartridge on the instrument: NALF strips of two representative cartridges, after 10 minutes of isothermal amplification, followed by 10 min for lateral flow detection. (d) LAMP master-mix amplified in the cartridge on the heater, analyzed via gel electrophoresis (Lanes 1 and 2), compared to amplification performed in reaction tubes on a standard heat block (Lanes 3 and 4). Lane 5: DNA molecular weight markers. For (c) and (d), positive (+) reactions show LAMP product starting from 3000 copies of <i>M.tb</i> DNA, and negative (−) reactions show no product since no <i>M.tb</i> DNA was added to the reaction. (e) EXPAR based detection of <i>M.tb</i> genomic DNA performed in the cartridge on the instrument: NALF strips of two representative cartridges, after 60 minutes of isothermal amplification, followed by 10 min for lateral flow detection. Positive (+) reactions contained 6×10⁵ copies <i>M.tb</i> DNA, and negative (−) reactions contained no <i>M.tb</i> DNA.</p

System components.

<p>(a) Top side of cartridge: lateral flow strips are sealed inside the grooves forming the lateral flow strip pouches, and electrolytic chambers are press-fit in place. (b) Heater with cartridge secured in place, prior to test execution. (a) and (b) The red line on the NALF strips represents the colored microspheres immobilized on the treated conjugate pad.</p

Cartridge concept.

<p>(a) Cartridge viewed from the top, showing access ports to the reaction and pump pouches on the underside of the cartridge (indicated in gray outline), and the position of lateral flow strips in anti-parallel orientation. (b) Cartridge viewed from the bottom, showing reaction pouches with overlaid pump pouches. (c) and (d) Side view of the cartridge on the heater (not to scale), illustrating the operating principle. (c) Master-mix is injected through the septum inlet into the reaction pouch, and targeted DNA sequences are isothermally amplified on top of the temperature-controlled heater surface. (d) Applying current to the electrolytic pump pushes fluid into the pump pouch. The pump pouch expands, thereby compressing the reaction pouch against the dome-shaped recess in the cartridge, forcing fluid out of the reaction pouch through the one-way check valve and into the lateral flow strip pouch.</p