Miniaturized devices for isothermal DNA amplification addressing DNA diagnostics

Abstract

Microfluidics is an emerging technology enabling the development of Lab-on-a-chip (LOC) systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. Currently, available nucleic acid diagnostic tests take advantage of Polymerase Chain Reaction (PCR) that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. At the same time, isothermal methods for DNA amplification are being developed and are preferred for their simplified protocols and the elimination of thermocycling. Here, we present a low-cost and fast DNA amplification device for isothermal Helicase Dependent Amplification (HDA) implemented in the detection of mutations related to breast cancer as well as the detection of Salmonella pathogens. The device is fabricated by mass production amenable technologies on printed circuit board (PCB) substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zone necessary for isothermal amplification methods.

Combination of Immunomagnetic Separation and Real‐Time Recombinase Polymerase Amplification (IMS‐qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon Samples

“…Molecular methods have been demonstrated as reliable tools for the fast detection of foodborne pathogens, being just as specific as conventional methods, but capable of providing definitive results in a shorter period of time. Particularly, isothermal DNA amplification‐based methods, have attracted the interest of the scientific community in the last decade due to their advantages over other approaches, such as simplicity and reduced thermal budget (Kaprou et al., ). Likewise, nanotechnology has contributed to the development of faster analytical methods for foodborne pathogen detection, with IMS in combination with a DNA amplification technique as the most common combination.…”

“…Food samples. After IMS (M&M 2.3), the DNA was extracted from the bacteria captured by the beads following the protocol described by (Kawasaki et al, 2005). Briefly, the beads were directly resuspended in 200 µL of enzymatic solution (1 mg/mL lysozyme and 1 mg/mL achromopeptidase) and incubated with constant agitation (1,000 rpm) for 1 hr at 37°C.…”

The lab-on-PCB approach: tackling the μTAS commercial upscaling bottleneck

“…115,116 Furthermore, microfluidic devices for DNA amplification have been fabricated on 4-copper layer PCB substrates by using either CNC machining or photolithography of industrially used dry film photosensitive resists, for the formation of meandering microfluidic channels. 117,118 In the latter work also, copper facilitates the incorporation of on-chip resistive microheaters, serving both as thermal zones necessary for DNA amplification (Fig. 8A) and as temperature sensors 114 implemented for temperature regulation.…”

“…Furthermore, several academia/PCB industry partnerships have produced/are developing industrially manufactured PCB prototypes. 116,117,130,[137][138][139][156][157][158] Given the novelty of this approach and its commercial interest, there has been significant activity in patenting related innovations, ranging from PCB microfluidic processing to fuel cell microsystems. [159][160][161][162][163][164][165][166][167][168] In the past, a few collaborations have been established between academia/research groups and the PCB industry towards industrially manufactured (rigid or flexible) PCB prototypes for microfluidics and MEMS.…”

Combination of Microfluidic Loop-Mediated Isothermal Amplification with Gold Nanoparticles for Rapid Detection of Salmonella spp. in Food Samples

“…Such demands concerning accurate thermal control, limit the number of materials that can be used for the fabrication of miniaturized devices ( Jia et al, 2007 ), and consequently increase fabrication costs. Therefore, novel isothermal amplification techniques have emerged in recent years with the goal of providing an analytical solution to some of the drawbacks associated with PCR/qPCR, specially due to their simplicity and reduced thermal budget ( Kaprou et al, 2015 ). Among them, loop-mediated isothermal amplification (LAMP) has become the most popular technique ( Mori and Notomi, 2009 ; Fang et al, 2010b ; Rafati and Gill, 2015 ).…”