Integrated microfluidic system for electrochemical sensing of urinary proteins

This study reports a new microfluidic system integrated with a microfluidic control module and a micro electrochemical module for detection of urinary proteins. The integrated microsystem can automatically detect proteins in urine with a high sensitivity. The microfluidic control module consists of a new two-way, spiral-shape micropump which can transport the urine samples to the sensing regions. The net ionic charges of the protein samples can be detected while the samples flow through the sensing region of the micro electrochemical module. Two major urinary proteins including lysozyme and albumin are detected in a multiple-channel layout with little human intervention and are analyzed in a short period of time, while only consuming a 100-μl urine sample. The developed microfluidic system could lead to a convenient, yet crucial, platform for chemical and biological detection and diagnosis. Preliminary results of the current paper had been presented at the 1st Annual IEEE International Conference on Nano/Molecular Medicine and Engineering, August 6–9, 2007.     

Automatic microfluidic platform for cell separation and nucleus collection

This study reports a new biochip capable of cell separation and nucleus collection utilizing dielectrophoresis (DEP) forces in a microfluidic system comprising of micropumps and microvalves, operating in an automatic format. DEP forces operated at a low voltage (15 V_p–p) and at a specific frequency (16 MHz) can be used to separate cells in a continuous flow, which can be subsequently collected. In order to transport the cell samples continuously, a serpentine-shape (S-shape) pneumatic micropump device was constructed onto the chip device to drive the samples flow through the microchannel, which was activated by the pressurized air injection. The mixed cell samples were first injected into an inlet reservoir and driven through the DEP electrodes to separate specific samples. Finally, separated cell samples were collected individually in two outlet reservoirs controlled by microvalves. With the same operation principle, the nucleus of the specific cells can be collected after the cell lysis procedure. The pumping rate of the micropump was measured to be 39.8 μl/min at a pressure of 25 psi and a driving frequency of 28 Hz. For the cell separation process, the initial flow rate was 3 μl/min provided by the micropump. A throughput of 240 cells/min can be obtained by using the developed device. The DEP electrode array, microchannels, micropumps and microvalves are integrated on a microfluidic chip using micro-electro-mechanical-systems (MEMS) technology to perform several crucial procedures including cell transportation, separation and collection. The dimensions of the integrated chip device were measured to be 6 × 7 cm. By integrating an S-shape pump and pneumatic microvalves, different cells are automatically transported in the microchannel, separated by the DEP forces, and finally sorted to specific chambers. Experimental data show that viable and non-viable cells (human lung cancer cell, A549-luc-C8) can be successfully separated and collected using the developed microfluidic platform. The separation accuracy, depending on the DEP operating mode used, of the viable and non-viable cells are measured to be 84 and 81%, respectively. In addition, after cell lysis, the nucleus can be also collected using a similar scheme. The developed automatic microfluidic platform is useful for extracting nuclear proteins from living cells. The extracted nuclear proteins are ready for nuclear binding assays or the study of nuclear proteins.     

Biomedical microdevices synthesis of iron oxide nanoparticles using a microfluidic system

The preparation of nanoparticles is essential in the application of many nanotechnologies and various preparation methods have been explored in the previous decades. Among them, iron oxide nanoparticles have been widely investigated in applications ranging from bio-imaging to bio-sensing due to their unique magnetic properties. Recently, microfluidic systems have been utilized for synthesis of nanoparticles, which have the advantages of automation, well-controlled reactions, and a high particle uniformity. In this study, a new microfluidic system capable of mixing, transporting and reacting was developed for the synthesis of iron oxide nanoparticles. It allowed for a rapid and efficient approach to accelerate and automate the synthesis of the iron oxide nanoparticles as compared with traditional methods. The microfluidic system uses micro-electro-mechanical-system technologies to integrate a new double-loop micromixer, two micropumps, and a microvalve on a single chip. When compared with large-scale synthesis systems with commonly-observed particle aggregation issues, successful synthesis of dispersed and uniform iron oxide nanoparticles has been observed within a shorter period of time (15 min). It was found that the size distribution of these iron oxide nanoparticles is superior to that of the large-scale systems without requiring any extra additives or heating. The size distribution had a variation of 16%. This is much lower than a comparable large-scale system (34%). The development of this microfluidic system is promising for the synthesis of nanoparticles for many future biomedical applications.     

Improving the mixing performance of side channel type micromixers using an optimal voltage control model

Electroosmotic flow in microchannels is restricted to low Reynolds number regimes. Since the inertia forces are extremely weak in such regimes, turbulent conditions do not readily develop, and hence species mixing occurs primarily as a result of diffusion. Consequently, achieving a thorough species mixing generally relies upon the use of extended mixing channels. This paper aims to improve the mixing performance of conventional side channel type micromixers by specifying the optimal driving voltages to be applied to each channel. In the proposed approach, the driving voltages are identified by constructing a simple theoretical scheme based on a ‘flow-rate-ratio’ model and Kirchhoff’s law. The numerical and experimental results confirm that the optimal voltage control approach provides a better mixing performance than the use of a single driving voltage gradient.     

Titanium-based dielectrophoresis devices for microfluidic applications

To date, materials selection in microfluidics has been restricted to conventional micromechanical materials systems such as silicon, glass, and various polymers. Metallic materials offer a number of potential advantages for microfluidic applications, including high fracture toughness, thermal stability, and solvent resistance. However, their exploitation in such applications has been limited. In this work, we present the application of recently developed titanium micromachining and multilayer lamination techniques for the fabrication of dielectrophoresis devices for microfluidic particle manipulation. Two device designs are presented, one with interdigitated planar electrodes defined on the floor of the flow channel, and the other with electrodes embedded within the channel wall. Using these devices, two-frequency particle separation and Z-dimensional flow visualization of the dielectrophoresis phenomena are demonstrated.     

Self-loading and cell culture in one layer microfluidic devices

We report on a simple method for self loading and culture of mammalian cells in microfluidic multi-chambers for high throughput screening. The device was obtained by using one layer soft lithography with polydimethylsiloxane (PDMS) and thermal bonding on a glass slide. Self loading of cell suspension could be possible after degassing of the PDMS device for 30 min. Both cell loading efficiency and cell proliferation behaviors have been analyzed with triangle chambers of different sizes, all connected to the main flow channels with small entrances. We found that the number of cells loaded into the micro-chamber increased with the side length of the triangle, showing well size dependence and that self loading at a single cell level was possible for small chambers. For large chambers, the cell area density after loading and proliferation is however quite heterogeneous. For demonstration, HeLa cell growth behavior has been followed for 11 days until the total area of the largest chambers was fully filled.     

A microfluidic cell culture platform for real-time cellular imaging

This study reports a new microfluidic cell culture platform for real-time, in vitro microscopic observation and evaluation of cellular functions. Microheaters, a micro temperature sensor, and micropumps are integrated into the system to achieve a self-contained, perfusion-based, cell culture microenvironment. The key feature of the platform includes a unique, ultra-thin, culture chamber with a depth of 180 μm, allowing for real-time, high-resolution cellular imaging by combining bright field and fluorescent optics to visualize nanoparticle-cell/organelle interactions. The cell plating, culturing, harvesting and replenishing processes are performed automatically. The developed platform also enables drug screening and real-time, in situ investigation of the cellular and sub-cellular delivery process of nano vectors. The mitotic activity and the interaction between cells and the nano drug carriers (conjugated quantum dots-epirubicin) are successfully monitored in this device. This developed system could be a promising platform for a wide variety of applications such as high-throughput, cell-based studies and as a diagnostic cellular imaging system.     

A microfluidic chip for formation and collection of emulsion droplets utilizing active pneumatic micro-choppers and micro-switches

The formation of emulsification droplets is crucial for many industrial applications. This paper reports a new microfluidic chip capable of formation and collection of micro-droplets in liquids for emulsion applications. This microfluidic chip comprising microchannels, a micro-chopper and a micro-switch was fabricated by using micro-electro-mechanical-systems (MEMS) technology. The microfluidic chip can generate uniform droplets with tunable sizes by using combination of flow-focusing and liquid-chopping techniques. The droplet size can be actively fine-tuned by controlling either the relative sheath/sample flow velocity ratios or the chopping frequency. The generated droplets can be then sorted to a specific collection area utilizing an active pneumatic micro-switch formed with three micro-valves. Experimental data showed that the olive oil and sodium-alginate (Na-alginate) droplets with diameters ranging from 3 mum to 70 mum with a variation less than 14% is successfully generated and collected. The development of this microfluidic system can be promising for emulsion, drug delivery and nano-medicine applications.     

The culture and differentiation of amniotic stem cells using a microfluidic system

Human mesenchymal stem cells (MSCs) have the potential to differentiate into multiple tissue lineages for cell therapy and, therefore, have attracted considerable interest recently. In this study, a new microfluidic system is presented which can culture and differentiate MSCs in situ. It is composed of several components, including stem cell culture areas, micropumps, microgates, seeding reservoirs, waste reservoirs and fluid microchannels; all fabricated by using micro-electro-mechanical-systems (MEMS) technology. The developed automated system allows for the long-term culture and differentiation of MSCs. Three methods, including Oil Red O staining for adipogenic cells, alkaline phosphatase staining and immunofluorescence staining are used to assess the differentiation of MSCs. Experimental results clearly demonstrate that the MSCs can be cultured for proliferation and different types of differentiation are possible in this microfluidic system, which can maintain a suitable and stable pH value over long time periods. This prototype microfluidic system has great potential as a powerful tool for future MSC studies.     

Flexible microfluidic devices supported by biodegradable insertion scaffolds for convection-enhanced neural drug delivery

Convection enhanced delivery (CED) can improve the spatial distribution of drugs delivered directly to the brain. In CED, drugs are infused locally into tissue through a needle or catheter inserted into brain parenchyma. Transport of the infused material is dominated by convection, which enhances drug penetration into tissue compared with diffusion mediated delivery. We have fabricated and characterized an implantable microfluidic device for chronic convection enhanced delivery protocols. The device consists of a flexible parylene-C microfluidic channel that is supported during its insertion into tissue by a biodegradable poly(DL-lactide-co-glycolide) scaffold. The scaffold is designed to enable tissue penetration and then erode over time, leaving only the flexible channel implanted in the tissue. The device was able to reproducibly inject fluid into neural tissue in acute experiments with final infusate distributions that closely approximate delivery from an ideal point source. This system shows promise as a tool for chronic CED protocols.     

A disposable lab-on-a-chip platform with embedded fluid actuators for active nanoliter liquid handling

In this work we present the development of a disposable liquid handling lab-on-a-chip (LOC) platform with embedded actuators for applications in analytical chemistry. The proposed platform for nanoliter liquid handling is based on a thermally responsive silicone elastomer composite, consisting of PDMS and expandable microspheres. In our LOC platform, we integrate active dosing, transportation and merging of nanoliter liquid volumes. The disposable platform successfully demonstrates precise sample volume control with smart microfluidic manipulation and on-chip active microfluidic components. It is entirely fabricated from low-cost materials using wafer-level processing. Moreover, an enzymatic reaction and real-time detection was successfully conducted to exemplify its applicability as an LOC.     

A microfluidic platform for sequential ligand labeling and cell binding analysis

Developing biochemical and cell biological assay for screening biomolecules, evaluating their characteristics in biological processes, and determining their pharmacological effects represents a key technology in biomedical research. A PDMS-based integrated microfluidic platform was fabricated and tested for facilitating the labeling of ligand on the nanogram scale and sequential cell binding analysis in a manner that saves both time and reagents. Within this microfluidic platform, ligand labeling, cell immobolization, and optical analysis are performed in a miniaturized, continuous and semi-automated manner. This microfluidic device for ligand labeling and cell analysis is composed of two functional modules: (i) a circular reaction loop for fluorophore-labeling of the ligand and (ii) four parallel-oriented incubation chambers for immobilization of cells, binding of ligand to different cell populations, and optical evaluation of interactions between the labeled ligand and its cell targets. Epidermal growth factor (EGF) as the ligand and different cell lines with various levels of EGF receptor expression have been utilized to test the feasiblity of this microfluidic platform. When compared to studies with traditional Petri dish handling of cells and tissues, or even microwell analyses, experiments with the microfluidic platform described here are much less time consuming, conserve reagents, and are programmable, which makes these platforms a very promising new tool for biological studies.     

A microfluidic device for continuous manipulation of biological cells using dielectrophoresis

The present study demonstrates the design, simulation, fabrication and testing of a label-free continuous manipulation and separation micro-device of particles/biological cells suspended on medium based on conventional dielectrophoresis. The current dielectrophoretic device uses three planner electrodes to generate non-uniform electric field and induces both p-DEP and n-DEP force simultaneously depending on the dielectric properties of the particles and thus influencing at least two types of particles at a time. Numerical simulations were performed to predict the distribution of non-uniform electric field, DEP force and particle trajectories. The device is fabricated utilizing the advantage of bonding between PDMS and SU8 polymer. The p-DEP particles move away from the center of the streamline, while the n-DEP particles will follow the central streamline along the channel length. Dielectrophoretic effects were initially tested using polystyrene beads followed by manipulation of HeLa cells. In the experiment, it was observed that polystyrene beads in DI water always response as n-DEP up to 1 MHz frequency, whereas HeLa cells in PBS medium response as n-DEP up to 400 kHz frequency and then it experiences p-DEP up to 1 MHz. Further, the microscopic observations of DEP responses of HeLa cells were verified by performing trapping experiment at static condition.     

A microfluidic platform for 3-dimensional cell culture and cell-based assays

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.     

A micro circulating PCR chip using a suction-type membrane for fluidic transport

A new micromachined circulating polymerase chain reaction (PCR) chip is reported in this study. A novel liquid transportation mechanism utilizing a suction-type membrane and three microvalves were used to create a new microfluidic control module to rapidly transport the DNA samples and PCR reagents around three bio-reactors operating at three different temperatures. When operating at a membrane actuation frequency of 14.29 Hz and a pressure of 5 psi, the sample flow rate in the microfluidic control module can be as high as 18 μL/s. In addition, an array-type microheater was adopted to improve the temperature uniformity in the reaction chambers. Open-type reaction chambers were designed to facilitate temperature calibration. Experimental data from infrared images showed that the percentage of area inside the reaction chamber with a thermal variation of less than 1°C was over 90% for a denaturing temperature of 94°C. Three array-type heaters and temperature sensors were integrated into this new circulating PCR chip to modulate three specific operating temperatures for the denaturing, annealing, and extension steps of a PCR process. With this approach, the cycle numbers and reaction times of the three separate reaction steps can be individually adjusted. To verify the performance of this circulating PCR chip, a PCR process to amplify a detection gene (150 base pairs) associated with the hepatitis C virus was performed. Experimental results showed that DNA samples with concentrations ranging from 10⁵ to 10²copies/μL can be successfully amplified. Therefore, this new circulating PCR chip may provide a useful platform for genetic identification and molecular diagnosis.     

Simulation and experimentation of a microfluidic device based on electrowetting on dielectric

Electrowetting on dielectric (EWOD) moving fluid by surface tension effects offers some advantages, including simplicity of fabrication, control of minute volumes, rapid mixing, low cost and others. This work presents a numerical model using a commercial software, CFD-ACE+, and an EWOD system including a microfluidic device, a microprocessor, electric circuits, a LCD module, a keypad, a power supply and a power amplifier. The EWOD model based on a reduced form of the mass conservation and momentum equations is adopted to simulate the fluid dynamics of the droplets. The EWOD device consists of the 2 x 2 mm bottom electrodes (Au/Cr), a dielectric layer of 3,000 A nitride, 500 A Teflon and a piece of indium tin oxide (ITO)-coated glass as the top electrode. The complete EWOD phenomenon is elucidated by comparing simulation with the experimental data on droplet transportation, cutting and creation. In transportation testing, the speed of the droplet is 6 mm/s at 40 V(dc). In addition, the droplet division process takes 0.12 s at 60 V(dc) in the current case. Finally, a 347 nl droplet is successfully created from an on-chip reservoir at 60 V(dc).     

Nanoporous membrane-sealed microfluidic devices for improved cell viability

Cell-laden microfluidic devices have broad potential in various biomedical applications, including tissue engineering and drug discovery. However, multiple difficulties encountered while culturing cells within devices affecting cell viability, proliferation, and behavior has complicated their use. While active perfusion systems have been used to overcome the diffusive limitations associated with nutrient delivery into microchannels to support longer culture times, these systems can result in non-uniform oxygen and nutrient delivery and subject cells to shear stresses, which can affect cell behavior. Additionally, histological analysis of cell cultures within devices is generally laborious and yields inconsistent results due to difficulties in delivering labeling agents in microchannels. Herein, we describe a simple, cost-effective approach to preserve cell viability and simplify labeling within microfluidic networks without the need for active perfusion. Instead of bonding a microfluidic network to glass, PDMS, or other solid substrate, the network is bonded to a semi-permeable nanoporous membrane. The membrane-sealed devices allow free exchange of proteins, nutrients, buffers, and labeling reagents between the microfluidic channels and culture media in static culture plates under sterile conditions. The use of the semi-permeable membrane dramatically simplifies microniche cell culturing while avoiding many of the complications which arise from perfusion systems.     

An integrated microfluidic chip for the analysis of biochemical reactions by MALDI mass spectrometry

Using an integrated microfluidic chip combined with mass spectrometry is an attractive method for parallel and multiple analyses because of its inherent simplicity, low sample consumption, and high sensitivity. To realize an effective microfluidic chip for the rapid analysis of biochemical reactions by matrix assisted laser desorption/ionization (MALDI)–mass spectrometry (MS), the basic operations on microfluids, namely loading, metering, cutting, transporting, mixing, and injecting, must be integrated. This study describes an integrated microfluidic chip with MALDI–MS that performs the on-chip analysis of biochemical reactions, such as enzymatic reactions. For on-chip multiple reactions, we present sequential fluidic manipulations with nanoliter-sized droplets, based on the precise control of wettability and the capillary pressure of a microchannel. The microfluidic chip we have developed successfully performed biochemical reactions and can dispense a droplet of a few hundred nanoliters on the MALDI target plate according to the designed multiple reaction procedure. Finally, the MS spectrum showed accurate and clear characteristic peaks for reaction products. Our investigations into reaction efficiency showed that the microfluidic chip could reduce the reaction time to one third, and the volume to one hundredth, of off-chip methods using conventional labware such as the micropipette and Eppendorf tube.