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As paper-based diagnostics has become predominantly driven by more advanced microfluidic technology, many of the research efforts are still focused on developing reliable and versatile fluidic control devices, apart from improving sensitivity and reproducibility. In this work, we introduce a novel and robust paper fluidic control system enabling versatile fluidic control. The system comprises a linear push-pull solenoid and an Arduino Uno micro controller. The precisely controlled pressure exerted on the paper stops the flow. We first determined the stroke distance of the solenoid to obtain a constant pressure while examining the fluidic time delay as a function of the pressure. Results showed that strips of grade 1 chromatography paper had superior reproducibility in fluid transport. Next, we characterized the reproducibility of the fluidic velocity which depends on the type and grade of paper used. As such, we were able to control the flow velocity on the paper and also achieve a complete stop of flow with a pressure over 2.0 MPa. Notably, after the actuation of the pressure driven valve (PDV), the previously pressed area regained its original flow properties. This means that, even on a previously pressed area, multiple valve operations can be successfully conducted. To the best of our knowledge, this is the first demonstration of an active and repetitive valve operation in paper microfluidics. As a proof of concept, we have chosen to perform a multistep detection system in the form of an enzyme-linked immunosorbent assay with mouse IgG as the target analyte. Funding Agencies|National Research Foundation of Korea (NRF) - Ministry of Science and ICT [NRF-2017M3A9E9072667, NRF-2015R1C1A1A01054292]

Electrical lysis of mammalian cells has been a preferred method in microfluidic platforms because of its simple implementation and rapid recovery of lysates without additional reagents. However, bacterial lysis typically requires at least a 10-fold higher electric field (∼10 kV/cm), resulting in various technical difficulties. Here, we present a novel, low-field-enabled electromechanical lysis mechanism of bacterial cells using electroconvective vortices near ion selective materials. The vortex-assisted lysis only requires a field strength of ∼100 V/cm, yet it efficiently recovers proteins and nucleic acids from a variety of pathogenic bacteria and operates in a continuous and ultrahigh-throughput (1 mL/min) manner. Therefore, we believe that the electromechanical lysis will not only facilitate microfluidic bacterial sensing and analysis but also various high-volume applications such as the energy-efficient recovery of valuable metabolites in biorefinery pharmaceutical industries and the disinfection of large-volume fluid for the water and food industries.

A controllable microchemostat can provide an ideal, powerful means to study the growth behavior of microorganisms by improving conventional macroscale chemostat. However, a challenge remains for implementing both continuous growth and active population control of microorganisms at the same time because they keep communicating with nearby culture environments by regulating their metabolism. Here, we present a novel microchemostat that enables reversible bacterial isolation, continuous chemical refreshment, and dynamic physicochemical stimulation. The microchemostat not only controls bacterial growth and subculture conditions in a completely automated and programmed manner but it also makes it possible to manipulate bacterial populations from a single bacterium to an ultrahigh density for long-term subculture periods with ultralow reagent consumption. Moreover, the microchemostat enables in situ measurement and feedback control of bacterial growth and population through various subculture programming modes that are sequentially performed using a single microchemostat over 720 h; to the best of our knowledge, this is the longest microchemostat culture of bacterial cells reported to date. Hence, we ensure that the microchemostat can be further applied to a wide range of microbial studies on a single chip, such as nutrient optimization, genetic induction, environmental selection, high-throughput screening, and evolutionary adaptation.

A compartmentalized microfluidic chamber array that offers not only separate cell culture environments but also independent control of the diffusion of small molecules provides an extremely useful platform for cell cultivations and versatile cellular assays. However, it is challenging to incorporate both cell compartmentalization and active diffusion control in real-time and precise manners. Here, we present a novel nanoscale hydrodynamic film (NHF) that is formed between a solid substrate and a polydimethylsiloxane (PDMS) surface. The thickness of the NHF can be adjusted by varying the pressure applied between them so that the mass transfer through the NHF can also be controlled. These novel phenomena are characterized and applied to develop a compartmentalized microchamber array with diffusion-tunable and solution-switchable chemostat-like versatile bacterial assays. The NHF-based compartmentalization technique is ideal for not only continuous bacterial cultivation by consistently refreshing various nutrient sources but also various diffusion-based microbial assays such as chemical induction of synthetically engineered bacterial cells and selective growth of a specific bacterial strain with respect to chemical environments. In addition, we show that tight compartmentalization protects cells in the chambers, while biofilm formation and nutrient contamination are eliminated by loading a lysis buffer, which typically hinders long-term continuous cultures and accurate microbial assays on a chip. Therefore, we ensure that the NHF-based compartmentalization platform proposed in this work will facilitate not only fundamental studies in microbiology but also various practical applications of microbes for production of valuable metabolites and byproducts in a high-throughput and highly efficient format.

Pipetting techniques play a crucial role in obtaining reproducible and reliable results, especially when seeding cells on small target areas, such as on microarrays, biochips or microfabricated cell culture systems. For very rare cells, such as human primary skeletal muscle cells (skMCs), manual (freehand) cell seeding techniques invariably result in nonuniform cell spreading and heterogeneous cell densities, giving rise to undesirable variations in myogenesis and differentiation. To prevent such technique-dependent variation, we have designed and fabricated a simple, low-cost pipet guidance device (PGD), and holder that works with hand-held pipettes. This work validates the accuracy and reproducibility of the PGD platform and compares its effectiveness with manual and robotic seeding techniques. The PGD system ensures reproducibility of cell seeding, comparable to that of more expensive robotic dispensing systems, resulting in a high degree of cell uniformity and homogeneous cell densities, while also enabling cell community studies. As compared to freehand pipetting, PGD-assisted seeding of C2C12 mouse myoblasts showed 5.3 times more myotube formation and likewise myotubes derived from PGD-seeded human primary skMCs were 3.6 times thicker and 2.2 times longer. These results show that this novel, yet simple PGD-assisted pipetting technique provides precise cell seeding on small targets, ensuring reproducible and reliable high-throughput cell assays.

We present a diffusion-driven and long-range concentration gradient generator that uses hydrogel as a porous membrane to prevent convection flows but allow the diffusion of cell signaling molecules for the study of bacterial chemotaxis in a microfluidic device. Using this device, we characterized the critical concentrations associated with the chemotactic responses of cells that initially created a population band and then migrated in bands in the presence of multiconcentration gradients. In addition, this device can be used to study the preferential chemotaxis of bacterial cells toward different carbon sources: glucose, galactose, and mannose were preferred over arabinose and xylose, in this order. This was possible since the device is able to simultaneously produce long-range concentration gradients of different chemicals as well. The method presented in this study is easy to perform and the device is cheap to fabricate, so that we believe that these characteristics not only make this device a very useful tool to study the chemotaxis of various, motile microorganisms but also permit parallel experimentation and reduce the time and effort needed in characterizing bacterial responses to various chemicals.

To precisely purify and study aged (senescent) cells, we have designed, fabricated, and demonstrated a novel diamond-structure (DS) microfluidic filter. Nonuniform flow velocities within the microfilter channel can compromise microfluidic filter performance, but with this new diamond structure, further optimized via simulation, we achieve a uniform microfilter flow field, improving the throughput of size-based separation of senescent cells, as obtained by 39-passaged human dermal fibroblasts. After separating these aged cells into two groups, consisting of large- and small-sized cells, we assessed senescence by measuring lipofuscin accumulation and β-galactosidase activity. Our results reveal that even though these senescent cells had been equivalently passaged in culture, a high degree of size distribution and senescent phenotype heterogeneity was observed. In particular, the smaller-sized cells tended to express a younger phenotype while the larger aged cells demonstrated an older phenotype. We suggest that size-based separation of senescent cells, subtyped into small- and large-sized cohorts, offers an alternative method to purify such aged cells, thereby enabling more precise study of the mechanisms of aging, autophagy impairment, and rejuvenation.

Recent advances in controlling the cracking phenomena established a novel unconventional fabrication technique to generate mixed-scale patterns/structures with resolution and accuracy comparable to conventional nanofabrication techniques. Here, we adapt our previous cracking-assisted nanofabrication technique (called "crack-photolithography") relying on only the standard photolithography to develop micro/nanofluidic devices with greatly reduced time and cost. The crack-photolithography makes it possible not only to simultaneously produce micropatterns and nanopatterns with various dimensions but also to replicate both of the mixed-scale patterns in a high-throughput manner. Therefore, a microfluidic channel network can easily be fabricated with a nanochannel array that can function as a nanoporous membrane wherever necessary, which basically plays a key role in diffusion-allowed but convection-suppressed microfluidic devices. In addition, the nanochannel array can manipulate the transport of small molecules by adjusting its dimension and/or number at will, so that nanochannel-array-integrated micro/nanofluidic devices prove even more robust and accurate in diffusion control than conventional membrane-integrated microfluidic devices. As an application of such micro/nanofluidic devices, we employed synthetic bacterial cells and found that their genetic induction and expression are dominated by extracellular diffusive microenvironments that were completely engineered using the nanochannel array. Hence, the crack-photolithography could provide innovative fabrication techniques for unprecedented micro/nanofluidic devices that show substantial potential for a wide range of biological and chemical applications.

Full automation with high purity for circulating tumor cell (CTC) isolation has been regarded as a key goal to make CTC analysis a "bench-to-bedside" technology. Here, we have developed a novel centrifugal microfluidic platform that can isolate the rare cells from a large volume of whole blood. To isolate CTCs from whole blood, we introduce a disc device having the biggest sample capacity as well as manipulating blood cells for the first time. The fully automated disc platform could handle 5 mL of blood by designing the blood chamber having a triangular obstacle structure (TOS) with lateral direction. To guarantee high purity that enables molecular analysis with the rare cells, CTCs were bound to the microbeads covered with anti-EpCAM to discriminate density between CTCs and blood cells and the CTCs being heavier than blood cells were only settled under a density gradient medium (DGM) layer. To understand the movement of CTCs under centrifugal force, we performed computational fluid dynamics simulation and found that their major trajectories were the boundary walls of the DGM chamber, thereby optimizing the chamber design. After whole blood was inserted into the blood chamber of the disc platform, size- and density-amplified cancer cells were isolated within 78 min, with minimal contamination as much as approximately 12 leukocytes per milliliter. As a model of molecular analysis toward personalized cancer treatment, we performed epidermal growth factor receptor (EGFR) mutation analysis with HCC827 lung cancer cells and the isolated cells were then successfully detected for the mutation by PCR clamping and direct sequencing.

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.