Ultrafast, low-power, PCB manufacturable, continuous-flow microdevice for DNA amplification.

The design and fabrication of a continuous-flow µPCR device with very short amplification time and low power consumption are presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9-m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the µPCR device in the established PCB industry. The µPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering the presented µPCR one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Detailed numerical calculations of the DNA residence time distributions, within an acceptable temperature range for denaturation, annealing, and extension, performed for the first time in the literature, provide useful information regarding the actual on-chip PCR protocol and justify the maximum volumetric flow rate for successful DNA amplification. The calculations indicate that the shortest amplification time is achieved when the device is operated at its enzyme kinetic limit (i.e., extension rate). Graphical abstract.

Isothermal Recombinase Polymerase Amplification (RPA) of E. coli gDNA in Commercially Fabricated PCB-Based Microfluidic Platforms.

Printed circuit board (PCB) technology has been recently proposed as a convenient platform for seamlessly integrating electronics and microfluidics in the same substrate, thus facilitating the introduction of integrated and low-cost microfluidic devices to the market, thanks to the inherent upscaling potential of the PCB industry. Herein, a microfluidic chip, encompassing on PCB both a meandering microchannel and microheaters to accommodate recombinase polymerase amplification (RPA), is designed and commercially fabricated for the first time on PCB. The developed microchip is validated for RPA-based amplification of two E. coli target genes compared to a conventional thermocycler. The RPA performance of the PCB microchip was found to be well-comparable to that of a thermocycler yet with a remarkably lower power consumption (0.6 W). This microchip is intended for seamless integration with biosensors in the same PCB substrate for the development of a point-of-care (POC) molecular diagnostics platform.

Rapid Methods for Antimicrobial Resistance Diagnostics.

Antimicrobial resistance (AMR) is one of the most challenging threats in public health; thus, there is a growing demand for methods and technologies that enable rapid antimicrobial susceptibility testing (AST). The conventional methods and technologies addressing AMR diagnostics and AST employed in clinical microbiology are tedious, with high turnaround times (TAT), and are usually expensive. As a result, empirical antimicrobial therapies are prescribed leading to AMR spread, which in turn causes higher mortality rates and increased healthcare costs. This review describes the developments in current cutting-edge methods and technologies, organized by key enabling research domains, towards fighting the looming AMR menace by employing recent advances in AMR diagnostic tools. First, we summarize the conventional methods addressing AMR detection, surveillance, and AST. Thereafter, we examine more recent non-conventional methods and the advancements in each field, including whole genome sequencing (WGS), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometry, Fourier transform infrared (FTIR) spectroscopy, and microfluidics technology. Following, we provide examples of commercially available diagnostic platforms for AST. Finally, perspectives on the implementation of emerging concepts towards developing paradigm-changing technologies and methodologies for AMR diagnostics are discussed.

Towards PCB-Based Miniaturized Thermocyclers for DNA Amplification.

In recent years, printed circuit board (PCB)-based microfluidics have been explored as a means to achieve standardization, seamless integration, and large-scale manufacturing of microfluidics, thus paving the way for widespread commercialization of developed prototypes. In this work, static micro polymerase chain reaction (microPCR) devices comprising resistive microheaters integrated on PCBs are introduced as miniaturized thermocyclers for efficient DNA amplification. Their performance is compared to that of conventional thermocyclers, in terms of amplification efficiency, power consumption and duration. Exhibiting similar efficiency to conventional thermocyclers, PCB-based miniaturized thermocycling achieves faster DNA amplification, with significantly smaller power consumption. Simulations guide the design of such devices and propose means for further improvement of their performance.