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The techniques, protocols, and advancements revolving around printed circuit boards (PCBs) have been gaining sustained attention in the realm of micro-total analysis systems ( μTAS) as more and more efforts are devoted to searching for standardized, highly reliable, and industry-friendly solutions for point-of-care diagnostics. In this Perspective, we set out to identify the current state in which the field of μTAS finds itself, the challenges encountered by researchers in the implementation of these technologies, and the potential improvements that can be targeted to meet the current demands. We also line up some trending innovations, such as 3D printing and wearable devices, along with the development of lab-on-PCB to increase the possibility of multifunctional biosensing activities propelled by integrated microfluidic networks for a wider range of applications, anticipating to catalyze the full potential of μTAS.

Cell-membrane permeability to water (Lp) and cryoprotective agents (Ps) of a cell type is a crucial cellular information for achieving optimal cryopreservation in the biobanking industry. In this work, a new micro-vortex system was developed to virtually trap cells of interest in a flow-driven hydrodynamic circulation passively formed at the expansion region in a microfluidic channel. Trapped cells remain in suspension and flow along with the streamline of the localized vortex, thus, featuring no physical contact with the device structure, and furthermore supporting a pragmatic assumption of 100% sphericity and active surface area of cell membrane for estimating actual cell volume from two-dimensional images. Cell membrane permeability was therefore able to be accurately determined by directly visualizing, with automatic cell recognition from high-speed videos, the transient profile of cell-volume change in response to a sudden osmotic gradient instantaneously (within 2 seconds) applied between the intracellular and extracellular environments. An acute T-cell lymphoma cell line (Jurkat) was used as a model to examine the newly-adopted micro-vortex technology in this research, and our results indicate a moderately higher values (Lp = 0.34 μm min-1 atm-1 for the binary system, Lp = 0.16 μm min-1 atm-1 and Ps = 0.55 × 10-3 cm min-1 for the ternary system) than those in prior arts utilizing contact-based cell-trapping techniques, manifesting an significant influence of active surface area on the determination of cell-membrane permeability.

Periodontal disease is a chronic inflammatory disease. Given its high prevalence, especially in aging population, the detailed mechanisms about pathogenesis of periodontal disease are important issues for study. Neutrophil firstly infiltrates to periodontal disease-associated pathogen loci and amplifies the inflammatory response for host defense. However, excessive neutrophil-secreted neutrophil elastase (NE) damages the affected gingival. In lung and esophageal epithelium, NE had been proved to upregulate several growth factors including placenta growth factor (PGF). PGF is an angiogenic factor with proinflammatory properties, which mediates the progression of inflammatory disease. Therefore, we hypothesize excessive NE upregulates PGF and participates in the pathogenesis and progression of periodontal disease.In gingival epithelial cells (GEC), growth factors array demonstrated NE-increased growth factors and further be corroborated by Western blot assay and ELISA. The GEC inflammation was evaluated by ELISA. In mice, the immunohistochemistry results demonstrated ligature implantation-induced neutrophil infiltration and growth factor upregulation. By multiplex assay, the ligature-induced proinflammatory cytokines level in gingival crevicular fluid (GCF) were evaluated. Finally, alveolar bone absorption was analyzed by micro-CT images and Hamp; E staining.NE upregulated PGF expression and secretion in GEC. PGF promoted GEC to secret IL-1β, IL-6, and TNF-α in GCF In periodontal disease animal model, ligature implantation triggered NE infiltration and PGF expression. Blockade of PGF attenuated the ligature implantation-induced IL-1β, IL-6, TNF-α and MIP-2 secretion and ameliorated the alveolar bone loss in mice.In conclusion, the NE-induced PGF triggers gingival epithelium inflammation and promotes the pathogenesis and progression of periodontal disease.

An optimal cooling rate is a critical factor affecting the survival of biological cells during cryopreservation. In this paper, a system for on-board cooling-rate-controlled cryopreservation under low-temperature (-80 °C) environments is developed with disposable, biocompatible polydimethylsiloxane (PDMS) storage chambers on top of localized heaters on printed circuit boards. The assembly allows the storage chambers to be removed from the temperature-controlled board and transferred from a −80 °C freezer to a liquid nitrogen tank for long-term cryopreservation. The use of PDMS enables the insertion of a syringe needle for loading samples, and during freezing, seeding extracellular ice formation. The PDMS storage chambers were fabricated using a polymethyl methacrylate mold made using a laser cutting machine. For each device, a copper thin film was deposited on a fiberglass epoxy substrate using electroless plating, and patterned using photolithography techniques into a micro-serpentine shape. The copper film functioned simultaneously as a resistive heating element and a temperature sensor with a proportional-integral-derivative feedback control program embedded in a microcontroller to semi-actively control the transient temperature profiles during the freezing process for multiple samples with different cooling rate requirements. The results show that the proposed devices are able to maintain a stable cooling rate down to 1 °C/min, which covers the optimal range for some mammalian cell types with low cell membrane permeability, for which low cooling rates are required. A heat transfer simulation was established to model transient and spatial temperature profiles of the device during freezing. Preliminary biological tests on yeast cells and their survival rates after on-board cryopreservation suggest that the prototype device can be a low-cost, reliable, and convenient tool for laboratory use in cryopreservation.

A compact biosensing system is presented that contains an array of devices composed of three-microelectrode electrochemical sensors and resistive heaters. The devices are intended for employment in quantitative polymerase chain reactions in multiple chambers that can be controlled via microcontroller. Arrays of sensors and heaters were developed using an inexpensive advanced printed circuit board technology featuring three different metals. The three-microelectrode sensors were fabricated by a new series of photolithographic and electroless plating processes. The surface morphology of the microelectrodes was characterized by several imaging techniques, including scanning electron microscopy and atomic force microscopy. The electrochemical properties of the microelectrodes were studied by cyclic voltammetry in order to estimate the active electrochemical surface area by solving the Randles–Sevcik equation. The on-board thermal cyclers were realized by feedback control embedded in a portable microcontroller. Quantitative polymerase chain reactions with methylene blue as the redox indicator were carried out as an example of biosensing with the proposed devices, and the results indicate that the prototype array is able to serve as an inexpensive, practical platform for mass production of portable point-of-care instrumentation containing arrays of addressable heaters and sensors.

Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs. To obtain these unknown parameters, membrane transport properties of the human UT-7/TPO megakaryocytic cell line were investigated using a microfluidic perfusion system. UT-7/TPO cells were immobilized in a microfluidic system on poly-D-lysine-coated glass substrate and perfused with various hyper-osmotic salt and CPA solutions at suprazero and subzero temperatures. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and the information was processed and analyzed using the Kedem-Katchalsky model to determine the membrane transport properties. The osmotically inactive cell volume (V(b)=0.15), the permeability coefficient to water (Lp) at 37°C, 22°C, 12°C, 0°C, -5°C, -10°C, and -20°C, and dimethyl sulfoxide (DMSO; Ps) at 22, 12, 0, -10, -20, as well as associated activation energies of water and DMSO at different temperature regions were obtained. We found that MKs have relatively higher membrane permeability to water (Lp=2.62 μm/min/atm at 22°C) and DMSO (Ps=1.8×10(-3) cm/min at 22°C) than most other common mammalian cell types, such as lymphocytes (Lp=0.46 μm/min/atm at 25°C). This information could suggest a higher optimal cooling rate for MKs cryopreservation. The discontinuity effect was also found on activation energy at 0°C-12°C in the Arrhenius plots of membrane permeability by evaluating the slope of linear regression at each temperature region. This phenomenon may imply the occurrence of cell membrane lipid phase transition.

Electrochemical detection or analysis of bio-molecules is an effective technique in laboratories due to its simplicity over conventional optical approaches. The technique is usually performed using three-electrode voltammetry composed of a working, counter and reference electrode that are made of noble metals. Graphite electrodes are major alternatives for resource-limiting experimental conditions such as point-of-care diagnostics and rapid screening, which would require disposable electrodes. To further reduce the cost of graphite-based electrodes for personal hormone-level monitoring in which several sampling activities are needed each day in months, pencil leads are proposed as an electrode material in this paper. Competitive-based enzyme immunoassay (EIA) is a method for the quantification of hormones, which is performed in a 96-well plate where the plate surface is coated with a capturing antibody in charge of hormone binding. Target hormones in the sample and artificial hormones conjugated with a peroxidase compete for limited binding sites on the plate surface. Peroxidase is an enzyme catalyzing the oxidation by hydrogen peroxide of 3, 3', 5, 5'-Tetramethylbenzidine (TMB), an electro-active redox dye indicator. The low-level hormone in the sample would lead to a high signal of oxidized TMB due to the large presence of the conjugated peroxidase, and vice versa. In this paper, the use of pencil leads allows a direct dip-in process to measure electrochemical signals from tested solution in a 96-well plate. Our work presents a portable enzyme immunoassay system that contains an electrochemical voltammetry composed of a three-electrode sensor module, potentiostat and data acquisition. The devices are intended for generic employment in the quantification of cortisol, a steroid hormone and indicator of human stress level. The three-electrode sensors were developed based on mechanical pencil-leads. We also report the preparation of Ag/AgCl reference electrodes, for the first time, fabricated by directly casting and chemically modifying silver epoxy on pencil-lead electrodes to form Ag/AgCl layers. The electrochemical properties of the redox indicator, TMB, were studied by cyclic voltammetry. The enzymatic activity of peroxidase was evaluated to indicate its linear characteristics in terms of time. The amperometric responses of varying concentration of cortisol samples (3200, 800, 400, 200, and 0 pg/ml) were obtained by our system. The results are consistent to the mechanism of competitive-based EIA in which a low amount of oxidized TMB should be measured due to a low amount of conjugated peroxidase molecules captured by the antibody is in presence if the cortisol level of a test sample is high. The non-linear relationship between the peak current values and cortisol concentration of samples at low and high concentration range was demonstrated indicating diffusion-limited reactions. The electrochemical enzyme immunoassay was for the first time performed using the pencil-lead based sensors as presented in this paper. The results promise that the proposed electrochemical assay is able to quantify hormone levels and serve as an ultra-low-cost biosensing system for point-of-care diagnostics and personal health monitoring. Figure 1

This paper presents a new fabrication technique to achieve ultra high-aspect ratio artificial cilia micro-patterned from flexible highly magnetic rare earth nanoparticle-doped polymers. We have developed a simple, inexpensive and scalable fabrication method to create cilia structures that can be actuated by miniature electromagnets, that are suitable to be used for lab-on-a chip (LOC) and micro-total-analysis-system (μ-TAS) applications such as mixers and flow-control elements. The magnetic cilia are fabricated and magnetically polarized directly in microfluidic channels or reaction chambers, allowing for easy integration with complex microfluidic systems. These cilia structures can be combined on a single chip with other microfluidic components employing the same permanently magnetic nano-composite polymer (MNCP), such as valves or pumps. Rare earth permanent magnetic powder, (Nd0.7Ce0.3)10.5Fe83.9B5.6, is used to dope polydimethylsiloxane (PDMS), resulting in a highly flexible M-NCP of much higher magnetization and remanence [1] than ferromagnetic polymers typically employed in magnetic microfluidics. Sacrificial poly(ethylene-glycol) (PEG) is used to mold the highly magnetic polymer into ultra high-aspect ratio artificial cilia. Cilia structures with aspect ratio exceeding 8:0.13 can be easily fabricated using this technique and are actuated using miniature electromagnets to achieve a high range of motion/vibration.

Rapid, low cost screening of tuberculosis requires an effective enrichment method of Mycobacterium tuberculosis (MTB) cells. Currently, microfiltration and centrifugation steps are frequently used for sample preparation, which are cumbersome and time-consuming. In this study, the size-selective capturing mechanism of a microtip-sensor is presented to directly enrich MTB cells from a sample mixture. When a microtip is withdrawn from a spherical suspension in the radial direction, the cells that are concentrated by AC electroosmosis are selectively enriched to the tip due to capillary- and viscous forces. The size-selectivity is characterized by using polystyrene microspheres, which is then applied to size-selective capture of MTB from a sample mixture. Our approach yields a detection limit of 800 cells mL(-1), one of the highest-sensitivity immunosensors to date.