Micromixers are crucial components within micro biomedical systems. This article presents a comprehensive review of the main state-of-the art applications of micromixers in biomedical systems over the past ten years. The article commences by reviewing the fundamental fluidic behaviors involved in microfluidic mixing. The biomedical applications of micromixers are then described in regard to their use particularly for (1) sample concentration, (2) chemical synthesis, (3) chemical reaction, (4) polymerization, (5) extraction and purification, (6) biological analysis, and (7) droplet/emulsion processes and others. The review concludes with a brief statistical analysis of the published literature in the micromixer field and an overview of the relative advantages of passive micromixers compared to active micromixers.

Recent advances and applications of micromixers

Commercialization of lab-on-a-chip devices is currently the "holy grail" within the μTAS research community. While a wide variety of highly sophisticated chips which could potentially revolutionize healthcare, biology, chemistry and all related disciplines are increasingly being demonstrated, very few chips are or can be adopted by the market and reach the end-users. The major inhibition factor lies in the lack of an established commercial manufacturing technology. The lab-on-printed circuit board (lab-on-PCB) approach, while suggested many years ago, only recently has re-emerged as a very strong candidate, owing to its inherent upscaling potential: the PCB industry is well established all around the world, with standardized fabrication facilities and processes, but commercially exploited currently only for electronics. Owing to these characteristics, complex μTASs integrating microfluidics, sensors, and electronics on the same PCB platform can easily be upscaled, provided more processes and prototypes adapted to the PCB industry are proposed. In this article, we will be reviewing for the first time the PCB-based prototypes presented in the literature to date, highlighting the upscaling potential of this technology. The authors believe that further evolution of this technology has the potential to become a much sought-after standardized industrial fabrication technology for low-cost μTASs, which could in turn trigger the projected exponential market growth of μTASs, in a fashion analogous to the revolution of Si microchips via the CMOS industry establishment.

/pdf/the-lab-on-pcb-approach-tackling-the-mtas-commercial-3vffptnpp6.pdf

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

Recent progress of microfluidic reactors for biomedical applications

https://www.mdpi.com/2079-6374/6/3/41/pdf

Microfluidic devices for forensic DNA analysis: A review

Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100% as well as the positive and negative predictive values.

/pdf/combination-of-microfluidic-loop-mediated-isothermal-1vatj0cl60.pdf

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

A passive micromixer for enzymatic digestion of DNA

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).

/pdf/ultrafast-low-power-pcb-manufacturable-continuous-flow-29w9b3ljdg.pdf

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

Microfluidics is an emerging technology enabling the development of lab-on-a-chip 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 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 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 substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zone necessary for isothermal amplification methods.

Miniaturized devices for isothermal DNA amplification addressing DNA diagnostics

A planar split and merge (SAM) passive micromixer with labyrinthine microchannels is proposed to efficiently mix biomolecular solutions by combining several advantages of existing micromixer designs in one realization. First, to demonstrate the labyrinth-SAM micromixer advantages, it is compared with three passive micromixers of different geometries, i.e., a zigzag, a spiral, and a linear one, with respect to their mixing efficiency, by means of a computational study. The geometrical specifications are imposed from flexible printed circuit (FPC) technology which is used for their fabrication and the diffusion coefficient from the applications to be implemented, i.e., the mixing of biochemical reagents. The computations include the numerical solution of continuity, Navier–Stokes, and mass conservation equations in 3d. It is demonstrated that the labyrinth-SAM micromixer exhibits the highest mixing efficiency. Specifically, compared to a linear micromixer, which shows a mixing efficiency of 0.328, the spiral micromixer improves the mixing efficiency by 8 %, the zigzag by 11 %, and the SAM by 92 %; the diffusion coefficient of the biomolecules is 10−10 m2/s, the Reynolds number is 0.5, and the volume of each micromixer is 2.54 μl. Second, the proposed SAM micromixer is realized simply and inexpensively, with a small footprint, implementing FPC technology, commonly available in the production lines of printed circuit board manufacturers. Finally, its mixing efficiency is experimentally evaluated by means of fluorescence microscopy, while it is further validated for enzymatic digestion of DNA. The latter is achieved even within 30 s of sufficient mixing of DNA and enzyme solutions through the SAM. Despite the numerous works on micromixers, the labyrinth-SAM is a novel design of an efficient passive micromixer. The efficiency together with its simplicity, which is manifested by (a) the planar (and not complex three-dimensional) geometry, (b) the two-inlet, instead of multiple-inlet, configuration, (c) the small number of fabrication steps, and (d) the compatibility with mass production, makes the proposed micromixer a good candidate for integration in bioanalytical miniaturized platforms.

A labyrinth split and merge micromixer for bioanalytical applications

Two types of micro-PCR (polymerase chain reaction) devices, a continuous-flow and a static-chamber device, with specifications imposed from flexible printed circuit technology, are compared through a computational study. Models for laminar flow, heat transfer in both solid and fluid, mass conservation of species, Joule heating, PCR kinetics, and temperature control feedback are coupled. The comparison is performed under identical conditions: the same material stack, i.e., flexible thin polymeric films with metal layers for integration of microheaters, the same volume of PCR mixture, and the same PCR protocol. Performance is quantified in terms of DNA amplification, energy consumption, and total operating time. The calculations show that the efficiency of DNA amplification is almost the same in both devices. However, contrary to what is generally believed, the static-chamber device fabricated on thin substrates, despite the necessary temperature ramping up within each thermal cycle, requires (2–4 times) lower energy consumption compared to the continuous-flow device. The constant energy losses to the ambient continuously during the operation of the continuous-flow device overcome the energy required for the thermal cycling of the static-chamber device. However, this result is reversed when the substrate thickness increases above 1000 μm. Concerning the speed, the total time required for the static-chamber device is only 1.1–2 times greater than that of the continuous-flow device due to relatively rapid heating and cooling rates for such thin substrates. These advantages for static-chamber micro-PCR devices arise as a result of the small thickness of the substrate where microchannels and microheaters are closely spaced. Both the low energy consumption and the inherent protocol flexibility indicate an attractive potential for static-chamber micro-PCR devices realized on flexible thin substrates with integrated microheaters.

I. Kefala

Papers

A passive micromixer for enzymatic digestion of DNA

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

Miniaturized devices for isothermal DNA amplification addressing DNA diagnostics

A labyrinth split and merge micromixer for bioanalytical applications

Comparison of continuous-flow and static-chamber μPCR devices through a computational study: the potential of flexible polymeric substrates