Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests

Abstract

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges (‘chips’) that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed.

Lab-on-a-Chip Technologies for the Single Cell Level: Separation, Analysis, and Diagnostics

“…Some salient examples thereof are circulating tumor cells (CTC) of various types of cancer [1,[36][37][38], rare cells (e.g., sickle-cell variants of red blood cells) [39,40], parasites, like Plasmodium falciparum [1,7,10,11,[13][14][15][21][22][23]36,[41][42][43][44][45][46][47][48][49][50] and Trypanosoma spp. [8,44,[51][52][53][54][55][56][57] and even plant pathogens [26,58], as well as-after cells have been lysed-subcellular infection markers (e.g., DNA, RNA fragments) [10,11,22,43,[59][60][61][62]. Given the vast adaptability of microfluidics to any kind of single or multi-cellular assay [63], the ability to combine it with various light microscopy techniques…”

“…An alternative technique for diagnosing sub-cellular targets is loop-mediated isothermal amplification (LAMP), which can run at a single temperature of around 65 • C and inside a single tube where the cell lysate is mixed with DNA polymerase and a set of four (or more) specifically designed primers [214]. The fact that it needs less equipment to be run, and that it can even be more portable [55], or fully non-instrumented [48], makes LAMP and its derivatives very promising for use in the field for resource-scarce settings [11,22,47,48,55,61].…”

“…Fourth, by heating the reaction volume to 65 • C, the amplification loop is started. In some cases, the heater also melts away an encapsulation between the (lyophilized) enzymes and reagents and the water [61]. Finally, after the amplification has run for the desired amount of time, the reaction mixture is excited with a light source and the fluorescent response of the reaction mixture is measured to assess the presence and concentration of the target [61].…”

“…In some cases, the heater also melts away an encapsulation between the (lyophilized) enzymes and reagents and the water [61]. Finally, after the amplification has run for the desired amount of time, the reaction mixture is excited with a light source and the fluorescent response of the reaction mixture is measured to assess the presence and concentration of the target [61]. Chip has one or more flow-through chambers for isothermal amplification and includes a filter-like, flow-porous nucleic acid binding phase (e.g.…”

An integrated biosensor platform for extraction and detection of nucleic acids

“…Among these, solid‐phase extraction (SPE) is one of the most effective extraction method based on electrostatic interactions between NA molecules and SiO 2 surfaces (Berensmeier, 2006). It has been implemented in several formats, such as silicon pillars (Petralia, Sciuto, & Conoci, 2017), filter membranes (Mauk, Song, Liu, & Bau, 2018), and magnetic beads (MagaZorb DNA Mini‐Prep Kit Technical Bulletin, 2011). When magnetic beads are used, the silica surface covers microparticles' magnetic core, making the NA–SiO 2 electrostatic interactions tunable by using proper washing and elution buffers to obtain purified and concentrated NA samples.…”