A microfluidic flow distributor generating stepwise concentrations for high-throughput biochemical processing

In this paper, we describe a microfluidic device in which solutions with stepwise concentrations can be accurately generated by continuously introducing two kinds of miscible liquids from each inlet, and biochemical processing can be conducted at the various conditions. Introduced liquid flows are geometrically divided into a number of downstream flows through multiple distribution channels, and each divided flow is then mixed with the divided flow of another liquid at a confluent point. The lengths of the precisely designed distribution channels determine the mixing ratio of the two liquids, without the influence of flow rate. In this study, a PDMS microfluidic device able to generate nine different concentrations was fabricated, and the performance of this device was estimated via colorimetric assay. As a biological application of this device, cell cultivation was performed under different concentration conditions. Due to its simplicity of operation, this microfluidic flow distributor will be applied to various kinds of biological analysis and screening systems.     

A method for dynamic system characterization using hydraulic series resistance

The pressure required to drive flow through a microfluidic device is an important characteristic of that device. We present a method to measure the flow rate through microfluidic components and systems, including micropumps and microvalves. The measurement platform is composed of two pressure sensors and a glass tube, which provides series resistance. The principle of the measurement is the fluid dynamical equivalent of Ohm's law, which defines the relationship between current, resistance, and voltage that are analogues to flow rate, hydraulic resistance, and pressure drop, respectively. Once the series resistance is known, it is possible to compute the flow rate through a device based on pressure alone. In addition, the dynamic system characteristics of the device-resistance and capacitance-can be computed. The benefits of this method are its simple configuration, capability of measuring flow rate accurately from the more easily measured pressure, and the ability to predict the dynamic response of microfluidic devices.     

An electrochemically driven poly(dimethylsiloxane) microfluidic actuator: oxygen sensing and programmable flows and pH gradients

We describe the fabrication and performance of an integrated microelectrochemical reactor-a design possessing utility for multiple applications that include electrochemical sensing, the generation and manipulation of in-channel microfluidic pH gradients, and fluid actuation and flow. The device architecture is based on a three-electrode electrochemical cell design that incorporates a Pt interdigitated array (IDA) working (WE), a Pt counter (CE), and Ag pseudo-reference (RE) electrodes within a microfluidic network in which the WE is fully immersed in a liquid electrolyte confined in the channels. The microchannels are made from a conventional poly(dimethylsiloxane)(PDMS) elastomer, which serves also as a thin gas-permeable membrane through which gaseous reactants in the external ambient environment are supplied to the working electrode by diffusion. Due to the high permeability of oxygen through PDMS, the microfluidic cell supports significantly (>order of magnitude) higher current densities in the oxygen reduction reaction (ORR) than those measured in conventional (quiescent) electrochemical cells for the same electrode areas. We demonstrate in this work that, when operated at constant potential under mass transport control, the device can be utilized as a membrane-covered oxygen sensor, the response of which can be tuned by varying the thickness of the PDMS membrane. Depending on the experimental conditions under which the electrochemical ORR is performed, the data establish that the device can be operated as both a programmable pH gradient generator and a microfluidic pump.     

Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing

Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic flow cytometer developed in this paper provides a practical platform that can be used for routine analysis in biological laboratories. Additionally, the 3D hydrodynamic focusing channel design can also be applied to various applications that can advance the lab on a chip research.     

Microfluidic Wheatstone bridge for rapid sample analysis

We developed a microfluidic analogue of the classic Wheatstone bridge circuit for automated, real-time sampling of solutions in a flow-through device format. We demonstrate precise control of flow rate and flow direction in the "bridge" microchannel using an on-chip membrane valve, which functions as an integrated "variable resistor". We implement an automated feedback control mechanism in order to dynamically adjust valve opening, thereby manipulating the pressure drop across the bridge and precisely controlling fluid flow in the bridge channel. At a critical valve opening, the flow in the bridge channel can be completely stopped by balancing the flow resistances in the Wheatstone bridge device, which facilitates rapid, on-demand fluid sampling in the bridge channel. In this article, we present the underlying mechanism for device operation and report key design parameters that determine device performance. Overall, the microfluidic Wheatstone bridge represents a new and versatile method for on-chip flow control and sample manipulation.     

An evaporation-based microfluidic sample concentration method

We present a method for sample concentration within microfluidic devices using evaporation-induced flow. Evaporation-induced flow is easy to incorporate into microfluidic designs and can be used to concentrate a wide variety of molecules. The practicality of this method was demonstrated with 0.2 microm fluorescent spheres and FITC-labeled BSA. Thirty two percent of the 0.6 microL fluorescent sphere suspension was concentrated into a well within a microfluidic device. In the same amount of time, 93% of the 0.6 microL FITC-labeled BSA solution was concentrated.     

Integrated three-dimensional filter separates nanoscale from microscale elements in a microfluidic chip

We report on the integration of a size-based three-dimensional filter, with micrometre-sized pores, in a commercial microfluidic chip. The filter is fabricated inside an already sealed microfluidic channel using the unique capabilities of two-photon polymerization. This direct-write technique enables integration of the filter by post-processing in a chip that has been fabricated by standard technologies. The filter is located at the intersection of two channels in order to control the amount of flow passing through the filter. Tests with a suspension of 3 μm polystyrene spheres in a Rhodamine 6G solution show that 100% of the spheres are stopped, while the fluorescent molecules are transmitted through the filter. We demonstrate operation up to a period of 25 minutes without any evidence of clogging. Preliminary validation of the device for plasma separation from whole blood is shown. Moreover, the filter can be cleaned and reused by reversing the flow.     

A microfluidic device for separating erythrocytes polluted by lead (II) from a continuous bloodstream flow

To sort and separate erythrocytes contaminated by lead (II) from whole bloodstream flow, the first step is to use a microchannel to transport the blood cells into a microdevice. Within the device, polluted erythrocytes can be separated from the bloodstream by applying local dielectrophoretic (DEP) forces. Exploiting the fact that Pb(2+) ions attach to the membranes of the erythrocytes, we utilize the microfluidic DEP device to perform property-based fractionation of the blood samples and to separate the polluted erythrocytes from the continuous bloodstream flow. Atomic absorption spectrometer analysis reveals that, to remove lead-polluted erythrocytes, the most effective driving velocity was less than 0.1 cm/s through our microfluidic DEP device, based on an applied power of 10 V(peak-peak) and a frequency of 15.5 MHz AC field. We were able to remove 80% of the polluted erythrocytes. Using gentle DEP manipulating techniques to efficiently sort unique cells within a complex biological sample may potentially allow biological sorting to be performed outside of hospitals, in facilities without biological analyzing equipment.     

Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

Characterization of a membrane-based gradient generator for use in cell-signaling studies

This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel. The high fluidic resistance of the membrane limits fluid flow caused by pressure differences in the system, but allows diffusive transport of a chemical species through the membrane and into the channel. The large volume sink region at the end of the microfluidic channel helps to maintain spatial and temporal stability of the gradient. The chemical gradient in a 0.5 mm region near the sink region experiences a maximum of 10 percent change between the 6 and 24 h data points. We present the theory, design, and characterization of this device and provide an example of neutrophil chemotaxis as proof of concept for future quantitative cell-signaling applications.     

Micro-droplet detection and characterization using thermal responses

We suggest a novel method to detect droplets and determine the protein content of droplets in microfluidic system using the 3ω method, which is a powerful tool to easily detect thermal response changes with a simple device. By measuring the thermal response of droplets and a carrying flow in real time, water droplets in an oleic acid carrying flow can be detected, and the concentration of bovine serum albumin in droplets can be estimated. This method is expected to increase the practicality and power of droplet-based microfluidic systems.     

Dielectrophoresis-based particle exchanger for the manipulation and surface functionalization of particles

We present a microfluidic device where micro- and nanoparticles can be continuously functionalized in flow. This device relies on an element called "particle exchanger", which allows for particles to be taken from one medium and exposed to some reagent while minimizing mixing of the two liquids. In the exchanger, two liquids are brought in contact and particles are pushed from one to the other by the application of a dielectrophoretic force. We determined the maximum flow velocity at which all the particles are exchanged for a range of particle sizes. We also present a simple theory that accounts for the behaviour of the device when the particle size is scaled. Diffusion mixing in the exchanger is also evaluated. Finally, we demonstrate particle functionalization within the microfluidic device by coupling a fluorescent tag to avidin-modified 880 nm particles. The concept presented in this paper has been developed for synthesis of modified particles but is also applicable to on-chip bead-based chemistry or cellular biology.     

Microfluidic add-on for standard electrophysiology chambers

We have developed a microfluidic brain slice device (microBSD) that marries an off-the shelf brain slice perfusion chamber with an array of microfluidic channels set into the bottom surface of the chamber substrate. As this device is created through rapid prototyping, once optimized, it is trivial to replicate and share the devices with other investigators. The device integrates seamlessly into standard physiology and imaging chambers and it is immediately available to the whole slice physiology community. With this technology we can address the flow of neurochemicals and any other soluble factors to precise locations in the brain slice with the temporal profile we choose. Dopamine (DA) was chosen as a model neurotransmitter and we have quantified delivery in brain tissue using cyclic voltammetry (CV) and fluorescence imaging.     

Converting steady laminar flow to oscillatory flow through a hydroelasticity approach at microscales

We report a hydroelasticity-based microfluidic oscillator that converts otherwise steady laminar flow to oscillatory flow. It incorporates an elastic diaphragm to enhance nonlinearity of the flow. Negative differential flow resistance is observed. High-frequency oscillatory flow is produced passively through interactions among hydrodynamic, elastic and inertial forces, without resorting to external actuators and control equipment. Driven by fluid flow and pressure, this device can operate in either steady laminar flow or oscillatory flow states, or work as a valve. Its applications for flow control and operation, and mixing enhancement are demonstrated.     

An active bubble trap and debubbler for microfluidic systems

We present a novel, fully integrated microfluidic bubble trap and debubbler. The 2-layer structure, based on a PDMS valve design, utilizes a featured membrane to stop bubble progression through the device. A pneumatic chamber directly above the trap is evacuated, and the bubble is pulled out through the gas-permeable PDMS membrane. Normal device operation, including continuous flow at atmospheric pressure, is maintained during the entire trapping and debubbling process. We present a range of trap sizes, from 2 to 10 mm diameter, and can trap and remove bubbles up to 25 muL in under 3 h.     

Quantitative modeling of the behaviour of microfluidic autoregulatory devices

We develop a theoretical model for a fluidic current source consisting of a via, a detour channel, and a push-up type micro-valve. The model accurately describes the non-linear behaviour of this type of device, which has been previously measured experimentally. We show how various structural parameters and material properties of the device influence the saturated flow rate and the minimum driving pressure required for the device to function as a current source. Conversely, the model can be used to design a fluidic current source with a desired saturated flow rate and low operational pressure. The present model can be straightforwardly applied to microfluidic circuits composed of many functional autoregulatory devices.     

Continuous separation of particles using a microfluidic device equipped with flow rate control valves

We propose herein an improved microfluidic system for continuous and precise particle separation. We have previously proposed a method for particle separation called "pinched flow fractionation." Using the previously reported method, particles can be continuously separated according to differences in their diameters, simply by introducing liquid flows with and without particles into a specific microchannel structure. In this study, we incorporated PDMS membrane microvalves for flow rate control into the microfluidic device to improve the separation accuracy. By adjusting the flow rates distributed to each outlet, target particles could be precisely collected from the desired outlet. We succeeded in separating micron and submicron-size polymer particles. This method can be used widely for continuous and precise separation of various kinds of particles, and can function as an important part of microfluidic systems.     

An inertia enhanced passive pumping mechanism for fluid flow in microfluidic devices

We describe and characterize a pumping mechanism that leverages the momentum present in small droplets ejected from a micro-nozzle to drive flow in an open microfluidic device. This approach allows driving flow in a microfluidic device in a regime that offers unique features different to those achievable with typical passive pumping or syringe-pump driven flow. Two flow regimes with specific flow characteristics are described: inertia enhanced passive pumping, in which fluid exchange times in the channel are significantly reduced, and inertia actuated flow, in which it is possible to initiate flow in an empty channel or against natural pressure gradients. Momentum is leveraged to create rapid fluid exchanges, instantaneous flow reversal, filling and mixing inside the microfluidic device.     

A hydrogel-based microfluidic device for the studies of directed cell migration

We have developed a hydrogel-based microfluidic device that is capable of generating a steady and long term linear chemical concentration gradient with no through flow in a microfluidic channel. Using this device, we successfully monitored the chemotactic responses of wildtype Escherichia coli (suspension cells) to alpha-methyl-DL-aspartate (attractant) and differentiated HL-60 cells (a human neutrophil-like cell line that is adherent) to formyl-Met-Leu-Phe (f-MLP, attractant). This device advances the current state of the art in microchemotaxis devices in that (1) it demonstrates the validity of using hydrogels as the building material for a microchemotaxis device; (2) it demonstrates the potential of the hydrogel based microfluidic device in biological experiments since most of the proteins and nutrients essential for cell survival are readily diffusible in hydrogel; (3) it is capable of applying chemical stimuli independently of mechanical stimuli; (4) it is straightforward to make, and requires very basic tools that are commonly available in biological labs. This device will also be useful in controlling the chemical and mechanical environment during the formation of tissue engineered constructs.     

A parallel diffusion-based microfluidic device for bacterial chemotaxis analysis

We developed a multiple-channel microfluidic device for bacterial chemotaxis detection. Some characteristics such as easy operation, parallel sample adding design and fast result readout make this device convenient for most biology labs. The characteristic feature of the design is the agarose gel channels, which serve as a semi-permeable membrane. They can stop the fluid flow and prevent bacteria getting across, but permit the diffusion of small molecules. In the device fabrication process a novel thermal-based method was used to control the shape of agarose gel in the microfluidic channel. The chemical gradient is established by diffusion which can be precisely controlled and measured. Combined with an 8-channel pipette, different attractants, repellent chemicals or different bacteria were analyzed by a two step operation with a readout time of one hour. This device may be useful in the high throughput detection of chemotaxis related molecules and genes.