On-chip three-dimensional tumor spheroid formation and pump-less perfusion culture using gravity-driven cell aggregation and balanced droplet dispensing

This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.     

Laser-based patterning for fluidic devices in nitrocellulose

In this report, we demonstrate a simple and low cost method that can be reproducibly used for fabrication of microfluidic devices in nitrocellulose. The fluidic patterns are created via a laser-based direct-write technique that induces polymerisation of a photo-polymer previously impregnated in the nitrocellulose. The resulting structures form hydrophobic barriers that extend through the thickness of the nitrocellulose and define an interconnected hydrophilic fluidic-flow pattern. Our experimental results show that using this method it is possible to achieve microfluidic channels with lateral dimensions of ∼100 μm using hydrophobic barriers that form the channel walls with dimensions of ∼60 μm; both of these values are considerably smaller than those that can be achieved with other current techniques used in the fabrication of nitrocellulose-based fluidic devices. A simple grid patterned nitrocellulose device was then used for the detection of C-reactive protein via a sandwich enzyme-linked immunosorbent assay, which served as a useful proof-of-principle experiment.     

A hybrid microfluidic platform for cell-based assays via diffusive and convective trans-membrane perfusion

We present a novel 3D hybrid assembly of a polymer microfluidic chip with polycarbonate track-etched membrane (PCTEM) enabling membrane-supported cell culture. Two chip designs have been developed to establish either diffusive or convective reagent delivery using the integrated PCTEM. While it is well suited to a range of cell-based assays, we specifically employ this platform for the screening of a common antitumor chemotoxic agent (mitomycin C – MMC) on the HL60 myeloid leukemia cell line. The toxic activity of MMC is based on the generation of severe DNA damage in the cells. Using either mode of operation, the HL60 cells were cultured on-chip before, during, and after exposure to MMC at concentrations ranging from 0 to 50 μM. Cell viability was analysed off-chip by the trypan blue dye exclusion assay. The results of the on-chip viability assay were found to be consistent with those obtained off-chip and indicated ca. 40% cell survival at MMC concentration of 50 μM. The catalogue of capabilities of the here described cell assay platform comprises of (i) the culturing of cells either under shear-free conditions or under induced through-membrane flows, (ii) the tight time control of the reagent exposure, (iii) the straightforward assembly of devices, (iv) the flexibility on the choice of the membrane, and, prospectively, (v) the amenability for large-scale parallelization.     

Gold coated optical fibers as three-dimensional electrodes for microfluidic enzymatic biofuel cells: Toward geometrically enhanced performance

For the first time, we report on the preliminary evaluation of gold coated optical fibers (GCOFs) as three-dimensional (3D) electrodes for a membraneless glucose/O₂ enzymatic biofuel cell. Two off-the-shelf 125 μm diameter GCOFs were integrated into a 3D microfluidic chip fabricated via rapid prototyping. Using soluble enzymes and a 10 mM glucose solution flowing at an average velocity of 16 mm s⁻¹ along 3 mm long GCOFs, the maximum power density reached 30.0 ± 0.1 μW cm⁻² at a current density of 160.6 ± 0.3 μA cm⁻². Bundles composed of multiple GCOFs could further enhance these first results while serving as substrates for enzyme immobilization.     

Hand-powered microfluidics: A membrane pump with a patient-to-chip syringe interface

In this paper, we present an on-chip hand-powered membrane pump using a robust patient-to-chip syringe interface. This approach enables safe sample collection, sample containment, integrated sharps disposal, high sample volume capacity, and controlled downstream flow with no electrical power requirements. Sample is manually injected into the device via a syringe and needle. The membrane pump inflates upon injection and subsequently deflates, delivering fluid to downstream components in a controlled manner. The device is fabricated from poly(methyl methacrylate) (PMMA) and silicone, using CO₂ laser micromachining, with a total material cost of ∼0.20 USD/device. We experimentally demonstrate pump performance for both deionized (DI) water and undiluted, anticoagulated mouse whole blood, and characterize the behavior with reference to a resistor-capacitor electrical circuit analogy. Downstream output of the membrane pump is regulated, and scaled, by connecting multiple pumps in parallel. In contrast to existing on-chip pumping mechanisms that typically have low volume capacity (∼5 μL) and sample volume throughput (∼1–10 μl/min), the membrane pump offers high volume capacity (up to 240 μl) and sample volume throughput (up to 125 μl/min).     

Automatic sequential fluid handling with multilayer microfluidic sample isolated pumping

To sequentially handle fluids is of great significance in quantitative biology, analytical chemistry, and bioassays. However, the technological options are limited when building such microfluidic sequential processing systems, and one of the encountered challenges is the need for reliable, efficient, and mass-production available microfluidic pumping methods. Herein, we present a bubble-free and pumping-control unified liquid handling method that is compatible with large-scale manufacture, termed multilayer microfluidic sample isolated pumping (mμSIP). The core part of the mμSIP is the selective permeable membrane that isolates the fluidic layer from the pneumatic layer. The air diffusion from the fluidic channel network into the degassing pneumatic channel network leads to fluidic channel pressure variation, which further results in consistent bubble-free liquid pumping into the channels and the dead-end chambers. We characterize the mμSIP by comparing the fluidic actuation processes with different parameters and a flow rate range of 0.013 μl/s to 0.097 μl/s is observed in the experiments. As the proof of concept, we demonstrate an automatic sequential fluid handling system aiming at digital assays and immunoassays, which further proves the unified pumping-control and suggests that the mμSIP is suitable for functional microfluidic assays with minimal operations. We believe that the mμSIP technology and demonstrated automatic sequential fluid handling system would enrich the microfluidic toolbox and benefit further inventions.     

Ion concentration polarization on paper-based microfluidic devices and its application to preconcentrate dilute sample solutions

Microfluidic paper-based analytical devices (μPADs) are a promising solution for a wide range of point-of-care applications. The feasibility of inducing ion concentration polarization (ICP) on μPADs has thus far attracted little attention. Accordingly, this study commences by demonstrating the ICP phenomenon in a μPAD with a Nafion ion-selective membrane. We are the first to measure the current-voltage curve on a Nafion-coated μPAD in order to indicate that the ion depletion occurs and the ICP is triggered when the current reaches the limiting current. The ICP effect is then exploited to preconcentrate fluorescein on μPADs incorporating straight and convergent channels. By an optimal geometric design, it is shown that the convergent channel results in a greater preconcentration effect than the straight channel. Specifically, a 20-fold enhancement in the sample concentration is achieved after 130 s given an initial concentration of 10^?5 M and an external potential of 50 V. By contrast, the straight channel yields only a 10-fold improvement in the concentration after 180 s. Further, the practical feasibility of the proposed convergent-channel μPAD is demonstrated using fluorescein isothiocyanate labeled bovine serum albumin. The experimental results show that a 15-fold enhancement of the initial sample concentration (10^?5 M) is obtained after 120 s given an external potential of 50 V.     

A microfluidic chip for direct and rapid trapping of white blood cells from whole blood

Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 10³ from 3.3 × 10³ WBCs.     

A capillary dielectrophoretic chip for real-time blood cell separation from a drop of whole blood

This study proposes a capillary dielectrophoretic chip to separate blood cells from a drop of whole blood (approximately 1 μl) sample using negative dielectrophoretic force. The separating efficiency was evaluated by analyzing the image before and after dielectrophoretic force manipulation. Blood samples with various hematocrits (10%–60%) were tested with varied separating voltages and chip designs. In this study, a chip with 50 μm gap design achieved a separation efficiency of approximately 90% within 30 s when the hematocrit was in the range of 10%–50%. Furthermore, glucose concentration was electrochemically measured by separating electrodes following manipulation. The current response increased significantly (8.8-fold) after blood cell separation, which was attributed not only to the blood cell separation but also to sample disturbance by the dielectrophoretic force.     

Activated Carbon Fabric Mask Reduces Lead Absorption and Improves the Heme Biosynthesis and Hematological Parameters of Battery Manufacturing Workers

Activated carbon fabrics (ACF) mask prevents the absorption of lead and reduce its adverse effects of human health. Aim of this study to know the blood lead level and its effects on heme biosynthesis and hematological parameters after using 2 months activated carbon fabric mask of battery manufacturing workers (BMW). Blood lead level, heme biosynthesis and hematological parameters were measured by using standard method. Blood lead level (P < 0.001, − 13.5%) was significantly decreased, activated δ-aminolevulinic acid dehydratase (P < 0.001, 11.97%) and non-activated δ- aminolevulinic acid dehydratase (P < 0.001, 23.17%) enzyme activity were significantly increased, however, the ratio of activated to Non-activated δ- ALAD (P < 0.001, − 10.13%) was significantly decreased, urinary excretion of δ- aminolevulinic acid (P < 0.001, − 10.49%) and porphobilinogen (P < 0.001, − 7.38%) were significantly decreased after using 2 months ACF mask as compared to before using mask of BMW. Hematological parameters i.e Hb (P < 0.05, 13.42%), PCV (P < 0.05, 7.23%), MCV (P < 0.05, 1.9%) were significantly increased and total WBC count (P < 0.05, − 5.18%) was significantly decreased after using 2 months ACF mask as compared to before using mask of BMW. Two months using ACF mask reduces the blood lead level and improves the δ-ALDH activity and hematological parameters, decreases the urinary excretion of δ-ALA, PBG of battery manufacturing workers. Therefore, the regular using of ACF mask is beneficial to prevent the lead absorption and its adverse effects on human health.     

Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip

While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 10¹ to 5 × 10⁶ copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs.     

Formation of lipid bilayer membrane in a poly(dimethylsiloxane) microchip integrated with a stacked polycarbonate membrane support and an on-site nanoinjector

This paper describes a new and facile approach for the formation of pore-spanning bilayer lipid membranes (BLMs) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Commercially, readily available polycarbonate (PC) membranes are employed for the support of BLMs. PC sheets with 5 μm, 2 μm, and 0.4 μm pore diameters, respectively, are thermally bonded into a multilayer-stack, reducing the pore density of 0.4 μm-pore PC by a factor of 200. The BLMs on this support are considerably stable (a mean lifetime: 17 h). This multilayer-stack PC (MSPC) membrane is integrated into the PDMS chip by an epoxy bonding method developed to secure durable bonding under the use of organic solvents. The microchip has a special channel for guiding a micropipette in the proximity of the MSPC support. With this on-site injection technique, tens to hundreds of nanoliters of solutions can be directly dispensed to the support. Incorporating gramicidin ion channels into BLMs on the MSPC support has confirmed the formation of single BLMs, which is based on the observation from current signals of 20 pS conductance that is typical to single channel opening. Based on the bilayer capacitance (1.4 pF), about 15% of through pores across the MSPC membrane are estimated to be covered with BLMs.     

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.     

Ultraviolet-assisted microfluidic generation of ferroelectric composite particles

We report on the feasible fabrication of microfluidic devices for ferroelectric polymers'' synthesis in a rapid and stable fashion. Utilizing micro-mixing and flow-focusing in microchannels, poly(vinylidene fluoride-trifluoroethylene) and copper phthalocyanine are uniformly dispersed in one hydrogel particle, which are then demonstrated to immediate and complete on-chip steady polymerization by moderate ultraviolet treatment. The advantage of our droplet-based microfluidic devices is generating versatile particles from simple spheres to disks or rods, and the lengths of particles can be precisely tuned from 30 to 400 μm through adjusting the flow rates of both disperse and oil phases. In addition, this mixed technique allows for the continuous production of dielectric microparticles with controlled dielectric properties between 10 and 160. Such a microfluidic device offers a flexible platform for multiferroic applications.     

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.     

Microfluidic size separation of cells and particles using a swinging bucket centrifuge

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.     

Manufacturing and wetting low-cost microfluidic cell separation devices

Deterministic lateral displacement (DLD) is a microfluidic size-based particle separation or filter technology with applications in cell separation and enrichment. Currently, there are no cost-effective manufacturing methods for this promising microfluidic technology. In this fabrication paper, however, we develop a simple, yet robust protocol for thermoplastic DLD devices using regulatory-approved materials and biocompatible methods. The final standalone device allowed for volumetric flow rates of 660 μl min⁻¹ while reducing the manufacturing time to <1 h. Optical profilometry and image analysis were employed to assess manufacturing accuracy and precision; the average replicated post height was 0.48% less than the average post height on the master mold and the average replicated array pitch was 1.1% less than the original design with replicated posts heights of 62.1 ± 5.1 μm (mean ± 6 standard deviations) and replicated array pitches of 35.6 ± 0.31 μm.     

Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chip

We describe a scalable artificial bilayer lipid membrane platform for rapid electrophysiological screening of ion channels and transporters. A passive pumping method is used to flow microliter volumes of ligand solution across a suspended bilayer within a microfluidic chip. Bilayers are stable at flow rates up to ∼0.5 μl/min. Phospholipid bilayers are formed across a photolithographically defined aperture made in a dry film resist within the microfluidic chip. Bilayers are stable for many days and the low shunt capacitance of the thin film support gives low-noise high-quality single ion channel recording. Dose-dependent transient blocking of α-hemolysin with β-cyclodextrin (β-CD) and polyethylene glycol is demonstrated and dose-dependent blocking studies of the KcsA potassium channel with tetraethylammonium show the potential for determining IC₅₀ values. The assays are fast (30 min for a complete IC₅₀ curve) and simple and require very small amounts of compounds (100 μg in 15 μl). The technology can be scaled so that multiple bilayers can be addressed, providing a screening platform for ion channels, transporters, and nanopores.     

Surface protein gradients generated in sealed microchannels using spatially varying helium microplasma

Spatially varied surface treatment of a fluorescently labeled Bovine Serum Albumin (BSA) protein, on the walls of a closed (sealed) microchannel is achieved via a well-defined gradient in plasma intensity. The microchips comprised a microchannel positioned in-between two microelectrodes (embedded in the chip) with a variable electrode separation along the length of the channel. The channel and electrodes were 50 μm and 100 μm wide, respectively, 50 μm deep, and adjacent to the channel for a length of 18 mm. The electrode separation distance was varied linearly from 50 μm at one end of the channel to a maximum distance of 150, 300, 500, or 1000 μm to generate a gradient in helium plasma intensity. Plasma ignition was achieved at a helium flow rate of 2.5 ml/min, 8.5 kV_pk-pk, and 10 kHz. It is shown that the plasma intensity decreases with increasing electrode separation and is directly related to the residual amount of BSA left after the treatment. The plasma intensity and surface protein gradient, for the different electrode gradients studied, collapse onto master curves when plotted against electrode separation. This precise spatial control is expected to enable the surface protein gradient to be tuned for a range of applications, including high-throughput screening and cell-biomolecule-biomaterial interactions.     

A chitosan coated monolith for nucleic acid capture in a thermoplastic microfluidic chip

A technique for microfluidic, pH modulated DNA capture and purification using chitosan functionalized glycidyl methacrylate monoliths is presented. Highly porous polymer monoliths are formed and subsequently functionalized off-chip in a batch process before insertion into thermoplastic microchannels prior to solvent bonding, simplifying the overall fabrication process by eliminating the need for on-chip surface modifications. The monolith anchoring method allows for the use of large cross-section monoliths enabling high flowrates and high DNA capture capacity with a minimum of added design complexity. Using monolith capture elements requiring less than 1 mm² of chip surface area, loading levels above 100 ng are demonstrated, with DNA capture and elution efficiency of 54.2% ± 14.2% achieved.