Droplet based microfluidic fabrication of designer microparticles for encapsulation applications

Developing carriers of active ingredients with pre-determined release kinetics is a main challenge in the field of controlled release. In this work, we fabricate designer microparticles as carriers of active ingredients using droplet microfluidics. We show that monodisperse droplet templates do not necessarily produce monodisperse particles. Magnetic stirring, which is often used to enhance the droplet solidification rate, can promote breakup of the resultant microparticles into fragments; with an increase in the stirring time, microparticles become smaller in average size and more irregular in shape. Thus, the droplet solidification conditions affect the size, size distribution and morphology of the fabricated particles, and these attributes of the microparticles strongly influence their release kinetics. The smaller the average size of the microparticles is, the higher the initial release rate is. The release kinetics of drug carriers is strongly related to their characteristics. The understanding of this relationship enables the fabrication of tailor-designed carriers with a specified release rate, and even programmed release to meet the needs of applications that require a complex release profile of the active ingredients.     

Polydimethylsiloxane microfluidic chip with integrated microheater and thermal sensor

A microheater and a thermal sensor were fabricated inside elastomeric polydimethylsiloxane microchannels by injecting silver paint (or other conductive materials) into the channels. With a high-precision control scheme, microheaters can be used for rapid heating, with precise temperature control and uniform thermal distribution. Using such a microheater and feedback system, a polymerase chain reaction experiment was carried out whereas the DNA was successfully amplified in 25 cycles, with 1 min per cycle.     

Integrated electrical concentration and lysis of cells in a microfluidic chip

Lysing cells is an important step in the analysis of intracellular contents. Concentrating cells is often required in order to acquire adequate cells for lysis. This work presents an integrated concentration and lysis of mammalian cells in a constriction microchannel using dc-biased ac electric fields. By adjusting the dc component, the electrokinetic cell motion can be precisely controlled, leading to an easy switch between concentration and lysis of red blood cells in the channel constriction. These two operations are also used in conjunction to demonstrate a continuous concentration and separation of leukemia cells from red blood cells in the same microchannel. The observed cell behaviors agree reasonably with the simulation results.     

A microfluidic chip for highly efficient cell capturing and pairing

This paper examined the feasibility of a microfluidics chip for cell capturing and pairing with a high efficiency. The chip was fabricated by the polydimethylsiloxane-based soft-lithography technique and contained two suction duct arrays set in parallel on both sides of a main microchannel. Cells were captured and paired by activating two sets of suction ducts one by one with the help of syringe pumps along with switching the cell suspensions inside the main microchannel correspondingly. The effects of suction flow rate and the dimensions of suction channels on the cell capturing and pairing efficiency were characterized. The present chip was capable of creating 1024 pairs of two different cell populations in parallel. The preliminary experimental results showed that the cell capturing efficiency was 100% and the pairing one was 88% with an optimal suction rate of 5 μl/min in the chip in the 2 μm-sized suction duct chip. The cell viability after capture inside the microfluidic device was 90.0 ± 5.3%. With this cell capturing and pairing chip, interaction between cells in a single pair mode can be studied. The ability to create cell pairs has a number of biological applications for cell fusion, cell-cell interaction studies, and cell toxicity screening.     

Sub-nanomolar detection of thrombin activity on a microfluidic chip

Bioluminescence resonance energy transfer (BRET) is a form of Förster resonance energy transfer. BRET has been shown to support lower limits of detection than fluorescence resonance energy transfer (FRET) but, unlike FRET, has not been widely implemented on microfluidic devices for bioanalytical sensing. We recently reported a microscope-based microfluidic system for BRET-based biosensing, using a hybrid, high quantum-efficiency, form of BRET chemistry. This paper reports the first optical fiber-based system for BRET detection on a microfluidic chip, capable of quantifying photon emissions from the low quantum-efficiency BRET² system. We investigated the effects of varying core diameter and numerical aperture of optical fibers, as well as varying microfluidic channel design and measurement conditions. We optimized the set-up in order to maximize photon counts and minimize the response time. The optimized conditions supported measurement of thrombin activity, with a limit of detection of 20 pM, which is lower than the microscope-based system and more than 20 times lower than concentrations reported to occur in plasma clots.     

A microfluidic chip for controlled release of drugs from microcapsules

A new microfluidic device with liquid-droplet merging and droplet storage functions for the controlled release of drugs from microcapsules is reported. A switching channel is designed and integrated within the microfluidic device, facilitating the generation and capturing of uniform droplets by the storage chambers. The drug model is the MnCO₃ microparticle, which is encapsulated by a microcapsule and fabricated using a simple layer-by-layer nanoassembly process. The merging function is used for dynamically adding the control solution into the droplets, which contain drugs within the microcapsules (DWμCs) and water. The storage chambers are used for collecting DWμCs-laden droplets so that the controlled-drug release in specific droplets can be monitored for an extended period of time, which has been experimentally implemented successfully. This technology could offer a promising technical platform for the long-term observation and studies of drug effects on specific cells in a controlled manner, which is especially useful for single cell analysis.     

Integrated microfluidic chip for rapid DNA digestion and time-resolved capillary electrophoresis analysis

An integrated microfluidic chip is proposed for rapid DNA digestion and time-resolved capillary electrophoresis (CE) analysis. The chip comprises two gel-filled chambers for DNA enrichment and purification, respectively, a T-form micromixer for DNA/restriction enzyme mixing, a serpentine channel for DNA digestion reaction, and a CE channel for on-line capillary electrophoresis analysis. The DNA and restriction enzyme are mixed electroomostically using a pinched-switching DC field. The experimental and numerical results show that a mixing performance of 97% is achieved within a distance of 1 mm from the T-junction when a driving voltage of 90 V/cm and a switching frequency of 4 Hz are applied. Successive mixing digestion and capillary electrophoresis operation clearly present the changes on digesting φx-174 DNA in different CE runs. The time-resolved electropherograms show that the proposed device enables a φx-174 DNA sample comprising 11 fragments to be concentrated and analyzed within 24 min. Overall, the results presented in this study show that the proposed microfluidic chip provides a rapid and effective tool for DNA digestion and CE analysis applications.     

On-chip antibody immobilization for on-demand and rapid immunoassay on a microfluidic chip

Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.     

Construction and operation of a microrobot based on magnetotactic bacteria in a microfluidic chip

Magnetotactic bacteria (MTB) are capable of swimming along magnetic field lines. This unique feature renders them suitable in the development of magnetic-guided, auto-propelled microrobots to serve in target molecule separation and detection, drug delivery, or target cell screening in a microfluidic chip. The biotechnology to couple these bacteria with functional loads to form microrobots is the critical point in its application. Although an immunoreaction approach to attach functional loads to intact MTB was suggested, details on its realization were hardly mentioned. In the current paper, MTB-microrobots were constructed by attaching 2 μm diameter microbeads to marine magnetotactic ovoid MO-1 cells through immunoreactions. These microrobots were controlled using a special control and tracking system. Experimental results prove that the attachment efficiency can be improved to ∼30% via an immunoreaction. The motility of the bacteria attached with different number of loads was also assessed. The results show that MTB can transport one load at a velocity of ∼21 μm/s and still move and survive for over 30 min. The control and tracking system is fully capable of directing and monitoring the movement of the MTB-microrobots. The rotating magnetic fields can stop the microrobots by trapping them as they swim within a circular field with a controllable size. The system has potential use in chemical analyses and medical diagnoses using biochips as well as in nano/microscale transport.     

Red blood cell recognition and posture estimation in microfluidic chip based on lensless imaging

On the one hand, lensless imaging technology has become one of the key technologies to achieve point-of-care testing; on the other hand, microfluidic technology has shown great application potential in the field of biological detection. Using mainstream lensless imaging technology to achieve biological cell imaging in microfluidic chips has technical limitations. In particular, it is more difficult to achieve lensless imaging for non-spherical cells in microfluidic chips such as red blood cells. Achieving red blood cell recognition and posture estimation in a microfluidic chip under the lensless imaging, combined with mainstream lensless imaging technology, can provide more effective red blood cell morphological parameters for medical diagnosis. In this paper, the method for red blood cell recognition and posture estimation in microfluidic chips based on lensless imaging is given. First, the relevant theoretical basis is introduced. Then, the models of red blood cell recognition and posture estimation in microfluidic chips based on lensless imaging are given. The effect of red blood cell flipping on lensless imaging is analyzed in the modeling process. Finally, the effectiveness of the proposed method is verified by experiments. Experiments show that the proposed method can well achieve red blood cell recognition and posture estimation through the shape characteristics of red blood cells.     

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.     

A practical guide for the fabrication of microfluidic devices using glass and silicon

This paper describes the main protocols that are used for fabricating microfluidic devices from glass and silicon. Methods for micropatterning glass and silicon are surveyed, and their limitations are discussed. Bonding methods that can be used for joining these materials are summarized and key process parameters are indicated. The paper also outlines techniques for forming electrical connections between microfluidic devices and external circuits. A framework is proposed for the synthesis of a complete glass/silicon device fabrication flow.     

Phaseguide-assisted blood separation microfluidic device for point-of-care applications

We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 μl of whole blood but yielding approximately 0.38 μl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.     

Biomolecule storage on non-modified thermoplastic microfluidic chip by ink-jet printing of ionogels

This paper reports an innovative technique for reagents storage in microfluidic devices by means of a one-step UV-photoprintable ionogel-based microarray on non-modified polymeric substrates. Although the ionogel and the ink-jet printing technology are well published, this is the first study where both are used for long-term reagent storage in lab-on-a-chip devices. This technology for reagent storage is perfectly compatible with mass production fabrication processes since pre-treatment of the device substrate is not necessary and inkjet printing allows for an efficient reagent deposition process. The functionality of this microarray is demonstrated by testing the release of biotin-647 after being stored for 1 month at room temperature. Analysis of the fluorescence of the ionogel-based microarray that contains biotin-647 demonstrated that 90% of the biotin-647 present was released from the ionogel-based microarray after pumping PBS 0.1% Tween at 37 °C. Moreover, the activity of biotin-647 after being released from the ionogel-based microarray was investigated trough the binding capability of this biotin to a microcontact printed chip surface with avidin. These findings pave the way for a novel, one-step, cheap and mass production on-chip reagents storage method applicable to other reagents such as antibodies and proteins and enzymes.     

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.     

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.     

High-throughput particle manipulation by hydrodynamic,electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.     

Advances in three-dimensional rapid prototyping of microfluidic devices for biological applications

The capability of 3D printing technologies for direct production of complex 3D structures in a single step has recently attracted an ever increasing interest within the field of microfluidics. Recently, ultrafast lasers have also allowed developing new methods for production of internal microfluidic channels within the bulk of glass and polymer materials by direct internal 3D laser writing. This review critically summarizes the latest advances in the production of microfluidic 3D structures by using 3D printing technologies and direct internal 3D laser writing fabrication methods. Current applications of these rapid prototyped microfluidic platforms in biology will be also discussed. These include imaging of cells and living organisms, electrochemical detection of viruses and neurotransmitters, and studies in drug transport and induced-release of adenosine triphosphate from erythrocytes.     

Cell chip array for microfluidic proteomics enabling rapid in situ assessment of intracellular protein phosphorylation

We discuss the ability to perform fluorescent immunocytochemistry, following cell fixation, using a microfluidic array of primary, nonadherent, single CD34+ stem cells. The technique requires small cell samples and proceeds with no cell loss, making it well-suited to monitoring these rare patient-derived cells. The chip allows us to correlate live cell dynamics across arrays of individual cells with post-translational modifications of intracellular proteins, following their exposure to drug treatments. Results also show that due to the microfluidic environment, the time scale of cell fixation was significantly reduced compared to conventional methods, leading to greater confidence in the status of the protein modifications studied.     

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.