Membrane-activated microfluidic rotary devices for pumping and mixing

Microfluidic devices are operated at a low-Reynolds-number flow regime such that the transportation and mixing of fluids are naturally challenging. There is still a great need to integrate fluid control systems such as pumps, valves and mixers with other functional microfluidic devices to form a micro-total-analysis-system. This study presents a new pneumatic microfluidic rotary device capable of transporting and mixing two different kinds of samples in an annular microchannel by using MEMS (Micro-electro-mechanical-systems) technology. Pumping and mixing can be achieved using a single device with different operation modes. The micropump has four membranes with an annular layout and is compact in size. The new device has a maximum pumping rate of 165.7 μL/min at a driving frequency of 17 Hz and an air pressure of 30 psi. Experimental data show that the pumping rate increases as higher air pressure and driving frequency are applied. In addition, not only can the microfluidic rotary device work as a peristaltic pumping device, but it also is an effective mixing device. The performance of the micromixer is extensively characterized. Experimental data indicate that a mixing index as high as 96.3% can be achieved. The developed microfluidic rotary device can be easily integrated with other microfluidic devices due to its simple and reliable PDMS fabrication process. The development of the microfluidic rotary device can be promising for micro-total-analysis-systems.     

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.     

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.     

Magnetic-based microfluidic platform for biomolecular separation

A novel microfluidic platform for manipulation of micro/nano magnetic particles was designed, fabricated and tested for applications dealing with biomolecular separation. Recently, magnetic immunomagnetic cell separation has attracted a noticeable attention due to the high selectivity of such separation methods. Strong magnetic field gradients can be developed along the entire wire, and the miniaturized size of these current-carrying conductors strongly enhances the magnetic field gradient and therefore produces large, tunable and localized magnetic forces that can be applied on magnetic particles and confine them in very small spots. Further increases in the values of the generated magnetic field gradients can be achieved by employing miniaturized ferromagnetic structures (pillars) which can be magnetized by an external magnetic field or by micro-coils on the same chip. In this study, we demonstrate magnetic beads trapping, concentration, transportation and sensing in a liquid sample under continuous flow by employing high magnetic field gradients generated by novel multi-functional magnetic micro-devices. Each individual magnetic micro-device consists of the following components: 1. Cu micro-coils array embedded in the silicon substrate with high aspect ratio conductors for efficient magnetic field generation 2. Magnetic pillar(s) made of the magnetic alloy NiCoP for magnetic field focusing and magnetic field gradient enhancement. Each pillar is magnetized by its corresponding coil 3. Integrated sensing coil for magnetic beads detection 4. Microfluidic chamber containing all the previous components. Magnetic fields of about 0.1 T and field gradients of around 300 T/cm have been achieved, which allowed to develop a magnetic force of 3 x 10(-9) N on a magnetic particle with radius of 1 mum. This force is large enough to trap/move this particle as the required force to affect such particles in a liquid sample is on the order of approximately pN. Trapping rates of up to 80% were achieved. Furthermore, different micro-coil designs were realized which allowed various movement modes and with different step-sizes.These results demonstrate that such devices incorporated within a microfluidic system can provide significantly improved spatial resolution and force magnitude for quick, efficient and highly selective magnetic trapping, separation and transportation, and as such they are an excellent solution for miniaturized mu-total analysis systems.     

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 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.     

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 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.     

DC-Dielectrophoretic separation of biological cells by size

DC-Dielectrophoresis (DC-DEP), the induced motion of the dielectric particles in a spatially non-uniform DC electric field, is applied to separate biological cells by size. The locally non-uniform electric field is generated by an insulating hurdle fabricated within a PDMS microchannel. The cells experience a negative DEP (accordingly a repulsive) force at the corners of the hurdle where the gradient of local electric-field strength is the strongest. The DC-DEP force acting on the cells is proportional to the cells’ size. Thus the moving cells deviate from the streamlines and the degree of deviation is dependent on the cell size. In this paper, we demonstrated by using this method that, combined with the electroosmotic flow, mixed biological cells of a few to tens of micrometers difference in diameter can be continuously separated into different collecting wells. For separating target cells of a specific size, all that is required is to adjust the voltage outputs of the electrodes.     

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.     

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.     

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies

The current state-of-art in 3D microfluidic chemotaxis device (μFCD) is limited by the inherent coupling of the fluid flow and chemical concentration gradients. Here, we present an agarose-based 3D μFCD that decouples these two important parameters, in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled by the transport property of the agarose gel, which—in contrast to the conventional microfabrication material such as polydimethylsiloxane (PDMS)—provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion. We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient buffer) before adding the 3D cell-containing matrix, and the dextran (10 kDa) concentration gradients can be re-established within 10 min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic response of murine dendritic cells to a gradient of CCL19, an 8.8 kDa lymphoid chemokine, within a type I collagen matrix. This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly migrating cells like mature dendritic cells and neutrophils. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ulrike Haessler and Yevgeniy Kalinin have equal contribution.     

A high throughput perfusion-based microbioreactor platform integrated with pneumatic micropumps for three-dimensional cell culture

This study reports a new perfusion-based, micro three-dimensional (3-D) cell culture platform for high-throughput cell culture using enabling microfluidic technologies. In this work, the micro 3-D cell culture platform is fabricated based on SU-8 lithography and polydimethylsiloxane replication processes. The micro cell culture platform can maintain homogenous and stable culture environments, as well as provide pumping of multiple mediums and efficient cell/agarose (scaffold) loading functions, which allows realization of more precise and high-throughput cell culture-based assays. In this study, the design of a high-throughput medium pumping mechanism was especially highlighted. A new serpentine-shaped pneumatic micropump was used to provide the required medium pumping mechanism. Pneumatic microchannels with a varied length and U-shape bending corners were designed to connect three rectangular pneumatic chambers such that one can fine-tune the pumping rate of the S-shape micropump by using the fluidic resistance. To achieve a high-throughput medium pumping function, a pneumatic tank was designed to simultaneously activate all of the 30 pneumatic micropumps with a uniform pumping rate. Results show that the pumping rates of the 30 integrated micropumps were statistically uniform with a flow rate ranging from 8.5 to 185.1 μl h^-1, indicating the present multiple medium pumping mechanism is feasible for high-throughput medium delivery purposes. Furthermore, as a demonstration case study, 3-D culture of oral cancer cell was successfully performed, showing that the cell viability remained as high as 95% - 98% during the 48 h cell culture. As the result of miniaturization, this perfusion-based 3-D cell culture platform not only provides a well-defined and stable culture condition, but also greatly reduces the sample/reagent consumption and the need for human intervention. Moreover, due to the integrated capability for multiple medium pumping, high-throughput research work can be achieved. These traits are found particularly useful for high-precision and high-throughput, 3-D cell culture-based assay.     

Dielectrophoretic separation and enrichment of CD34+cell subpopulation from bone marrow and peripheral blood stem cells

Dielectrophoresis has been used to enrich selected cell subpopulations in a mixed cell population by exploiting differential dielectric properties. Six-fold enrichment of stem cells expressing the CD34+ antigen has been achieved for bone marrow samples and peripheral blood, without the requirment for initial chemical treatment associated with immunoadsorption techniques.     

Lectin-modified microchannels for mammalian cell capture and purification

There is a need for cell purification strategies suitable for handling small cell populations (tens to thousands of cells). We present here a novel strategy for small scale cell purifications using lectin-modified microchannels. Mammalian cells can be selectively captured in lectin-modified channels and eluted with a solution of the lectin’s inhibiting sugar. As a proof-of-principle demonstration, two cell lines with different binding affinities to the lectin Sambucus nigra agglutinin (SNA) were combined at various ratios and introduced into SNA-modified microchannels. After incubation and washing, enrichments of approximately ten-fold were obtained for the SNA-binding cell type. The results demonstrate the potential of this miniaturized device for manipulation and purification of limited quantities of cells.     

A cell culturing system that integrates the cell loading function on a single platform and evaluation of the pulsatile pumping effect on cells

In this paper, we present a novel microfluidic system with pulsatile cell storing, cell-delivering and cell culturing functions on a single PDMS platform. For this purpose, we have integrated two reservoirs, a pulsatile pumping system containing two soft check valves, which were fabricated by in situ photopolymerization, six switch valves, and three cell culture chambers all developed through a simple and rapid fabrication process. The sample volume delivered per stroke was 120 nl and the transported volume was linearly related to the pumping frequency. We have investigated the effect of the pulsatile pneumatic micropumping on the cells during transport. For this purpose, we pumped two types of cell suspensions, one containing human breast adenocarcinoma cells (MCF-7) and the other mesenchymal stem cells (hMSCs) derived from bone marrow. The effect of pulsatile pumping on both cell types was examined by short and long-term culture experiments. Our results showed that the characteristics of both cells were maintained; they were not damaged by the pumping system. Evaluations were carried out by morphological inspection, viability assay and immunophenotyping analysis. The delivered MCF-7 cells and hMSCs spread and proliferated onto the gelatin coated cell culture chamber. This total micro cell culture system can be applied to cell-based high throughput screening and for co-culture of different cells with different volume.     

Lab-on-CD microfluidic platform for rapid separation and mixing of plasma from whole blood

Traditional clinical methods for separating whole blood into blood cells and cell-free plasma are labor intensive and time consuming. Accordingly, the present study proposes a simple compact disk (CD) microfluidic platform for the rapid separation of plasma from whole human blood and the subsequent mixing of the plasma with a suitable reagent. The performance of three CD microfluidic platforms incorporating square-wave mixing channels with different widths is evaluated both numerically and experimentally. The results show that given an appropriate specification of the microchannel geometry and a CD rotation speed of 2000 rpm, a separation efficiency of 95 % can be achieved within 5?~?6 s given a diluted blood sample with a hematocrit concentration of 6 %. Moreover, a mixing efficiency of more than 96 % can be obtained within 5 s given a CD rotation speed of 2200 rpm. The practical feasibility of the proposed device is demonstrated by performing a prothrombin time (PT) test. It is shown that while the time required to perform the PT test using a conventional bench top system is around 15 min, the proposed CD microfluidic platform allows the test to be completed within 1 min.     

Microfluidic device for cell capture and impedance measurement

This work presents a microfluidic device to capture physically single cells within microstructures inside a channel and to measure the impedance of a single HeLa cell (human cervical epithelioid carcinoma) using impedance spectroscopy. The device includes a glass substrate with electrodes and a PDMS channel with micro pillars. The commercial software CFD–ACE+ is used to study the flow of the microstructures in the channel. According to simulation results, the probability of cell capture by three micro pillars is about 10%. An equivalent circuit model of the device is established and fits closely to the experimental results. The circuit can be modeled electrically as cell impedance in parallel with dielectric capacitance and in series with a pair of electrode resistors. The system is operated at low frequency between 1 and 100 kHz. In this study, experiments show that the HeLa cell is successfully captured by the micro pillars and its impedance is measured by impedance spectroscopy. The magnitude of the HeLa cell impedance declines at all operation voltages with frequency because the HeLa cell is capacitive. Additionally, increasing the operation voltage reduces the magnitude of the HeLa cell because a strong electric field may promote the exchange of ions between the cytoplasm and the isotonic solution. Below an operating voltage of 0.9 V, the system impedance response is characteristic of a parallel circuit at under 30 kHz and of a series circuit at between 30 and 100 kHz. The phase of the HeLa cell impedance is characteristic of a series circuit when the operation voltage exceeds 0.8 V because the cell impedance becomes significant.