Junichi Miwa

Adhesion-Based Cell Sorter With Antibody-Coated Amino-Functionalized-Parylene Surface

An adhesion-based cell-separation device is developed for the extraction of rare cells from a cell mixture. The cell-separation principle mimics leukocyte recruitment from blood vessels in our body, where leukocytes are decelerated by antigen-antibody interaction at the sites of inflammation or injury. Separation of cell mixture can be accomplished by simply introducing the sample plug through an antibody-immobilized microchannel without any pre- or postprocessing. A new class of amino-functionalized parylene (diX AM) is employed in order to provide amino group on the channel-wall surface. The amount of immobilized biomolecules on diX AM surface is characterized through quartz-crystal-microbalance measurements. The number density of immobilized biotin is as large as , which indicates an amount of amino group enough to immobilize biotin and other biomolecules in a closely packed state. It is shown by the measurement of cell velocity in the CD31-coated diX AM microchannel that the flowing velocity of human endothelial cells are reduced by up to 70% due to specific adhesion of CD31 antigens and antibodies. The results are further analyzed by using a 2-D membrane-peeling model, with which the cell velocity can be estimated under different conditions of antibody number density and bulk mean velocity. Based on the experimental results, a cell-separation device for treating a 1- cell-mixture plug is designed and microfabricated. A mixture of human endothelial cells and leukocytes is successfully separated into plugs of each cell type within a shorter period of time as compared to conventional cell-separation methods which require sample preprocessing.

Label-free continuous cell sorter with specifically adhesive oblique micro-grooves

We report the development of a label-free continuous cell sorting method based on specific adhesivity between cells and surface-immobilized adhesion molecules. The separation of cells is induced by cross-flow adhesive force on micron-sized stripes with adhesion molecules immobilized on the surface. In order to accurately form the adhesive stripes on a microchannel wall, 1 µm wide micro-grooves are fabricated at a certain angle with respect to the flow direction using direct electron-beam lithography. Amino-functionalized parylene is used as the groove surface material, and streptavidin is immobilized on the entire surface, resulting in a surface with periodic adhesive patterns. The effectiveness of the proposed cell sorting principle is verified by flow-through experiments using functionalized particles as model cells. Measurements of the motion of biotin-coated microparticles show that the particles decelerated by specific adhesivity are displaced in the cross-flow direction. The observed cross-flow displacement is around 0.8% of the streamwise travelling distance. It is also shown that the rate of cross-flow displacement is independent of the flow rate or the stripe angle. Finally, it is demonstrated that a mixture of streptavidin- and biotin-coated microparticles can be completely separated after flowing over a 20 mm long patterned surface. The proposed label-free continuous lateral separation scheme has a wide range of potential applications for separation of cells which could not be distinguished by size or separated using dielectric forces.

Development of a high-speed scanning micro PIV system using a rotating disc

A novel high-speed scanning micro particle image velocimetry system has been developed in order to provide time-resolved, pseudo-three-dimensional flow-field information. The focal plane is moved rapidly in the out-of-plane direction by changing the optical length using a rotating disc having glass windows with different thicknesses. In the present prototype system, the extent of the scanning depth is 50 µm and the maximum scanning frequency is 100 Hz. Through a series of experiments in a two-dimensional microchannel, it was found that the velocity data are in good agreement with the analytical solution. The fluctuation in the flow rate delivered by a syringe pump is successfully measured with the present system. This demonstrates the applicability of the present scanning system for unsteady flow measurements.

Active Continuous-Flow Micromixer Using an External Braille Pin Actuator Array

We present a continuous-flow active micromixer based on channel-wall deflection in a polydimethylsiloxane (PDMS) chip for volume flows in the range up to 2 μL s −1 which is intended as a novel unit operation for the microfluidic Braille pin actuated platform. The chip design comprises a main microchannel connected to a series of side channels with dead ends aligned on the Braille pins. Computer-controlled deflection of the side-channel walls induces chaotic advection in the main-channel, which substantially accelerates mixing in low-Reynolds number flow. Sufficient mixing (mixing index MI below 0.1) of volume flows up to 0.5 μL s −1 could be achieved within residence times ~500 ms in the micromixer. As an application, continuous dilution of a yeast cell sample by a ratio down to 1:10 was successfully demonstrated. The mixer is intended to serve as a component of bio-analytical devices or as a unit operation in the microfluidic Braille pin actuated platform.

A Novel Microfluidic Platform for Continuous DNA Extraction and Purification using Laminar Flow Magnetophoresis

We present a novel microfluidic platform using laminar-flow magnetophoresis for combined continuous extraction and purification of DNA. All essential unit operations (DNA binding, sample washing and DNA elution) are integrated on one single chip. The key function is the motion of magnetic beads given by the interplay of laminar flow and time-varying magnetic field. The time for extraction was 1 minute. The device is a central part of a complete biochemical system for continuous monitoring of cell-growth in bioreactors. The novel platform allows continuous purification of DNA, but is also applicable to purification of RNA, proteins or cells, including their subsequent real-time analysis in general.

Adhesion-based cell sorter with antibody-immobilized functionalized-parylene surface

We report the development of a micro cell sorter for extraction of rare cells from a limited amount of sample. The separation principle is based on specific adhesion with the aid of surface-immobilized antibodies. Antibody-immobilized channel walls are fabricated with a versatile technique using functionalized parylene. Performance of the cell sorter is characterized through cell velocity measurements, in which deceleration of positive cells up to 60% is observed. Cell separation with a mixture containing positive and negative cells is also demonstrated.

Label-Free Continuous Micro Cell Sorter with Antibody-Immobilized Oblique Grooves

We report development of a novel label-free continuous cell separation method for the extraction of rare cells from a limited amount of sample. The separation method is based on specific adhesion between target cells and antibody immobilized on oblique micro grooves etched into the channel wall. Due to asymmetric adhesive force, cross-flow displacement of the target cells is induced. Label-free lateral separation of micro beads as the cell model has been successfully achieved. Cross-flow displacement of human umbilical-vein endothelial cells (HUVEC) with the CD31 antibody is also demonstrated.

Evaluation of Cell Velocity Regulation in a Microfabricated Adhesion-Based Cell Separation Device

This paper reports the characterization of adhesion-based cell velocity regulation in a prototype microfabricated cell separation device for regenerative medicine. The principle of cell sorting is based on immunoreaction for accurate recognition of target stem cells. Target-cell specific antibody is immobilized on the micro channel wall to form a selectively adhesive surface, where a new class of functionalized parylene is used as the surface material for antibody immobilization. The flowing velocity of sample cells in a prototype microfabricated cell separation column is examined under the microscope. The measurement results show that the cell velocity is reduced by 40% due to the effect of antigen/antibody interaction

High-density antibody-immobilized surface for adhesion-based cell separation

We develop a versatile biomolecule immobilization method using amino-functionalized parylene, and examine the surface density of antibody immobilized on the parylene surface by using quartz crystal microbalance (QCM). We also demonstrate label-free cell sorting for the extraction of rare cells from a limited amount of sample. The sorting principle is based on cell-type specific adhesion with the aid of high-density surface-immobilized antibodies. Continuous lateral separation is also successfully achieved with antibody-immobilized oblique micro grooves in a micro channel.