Hirokazu Sugino

On-chip microfluidic sorting with fluorescence spectrum detection and multiway separation

The microfluidic platform is an important tool for diagnosis and biomedical studies because it enables us to handle precious cells and infectious materials safely. We have developed an on-chip microfluidic sorter with fluorescence spectrum detection and multiway separation. The fluorescence spectrum of specimens (495-685 nm) in the microchannels was obtained every 2 ms using a 1 x 16 arrayed photomultiplier tube. The specimen was identified by its spectrum and collected into the corresponding channel based on our previously reported thermoreversible gelation polymer technique (Y. Shirasaki, J. Tanaka, H. Makazu, K. Tashiro, S. Shoji, S. Tsukita and T. Funatsu, Anal. Chem., 2006, 78, 695-701). Four kinds of fluorescence microspheres and three kinds of Escherichia coli cells, expressing different fluorescent proteins, were successfully separated with accuracy and purity better than 90% at a throughput of about one particle per second.

Integration in a multilayer microfluidic chip of 8 parallel cell sorters with flow control by sol–gel transition of thermoreversible gelation polymer

Microfluidic systems have significant implications in the field of cell separation since they could provide platforms with inexpensive, disposable and sterile structures. Here, we present a novel strategy to integrate microfluidic sorters into a single chip for high throughput sorting. Our parallel sorter consists of a microfluidic chip with a three-dimensional channel network that utilizes flow switching by a heat-induced sol-gel transition of thermoreversible gelation polymer. The 8 parallel sheathed sample flows were realized by injecting sample and buffer solutions into only 2 inlets. The sheathed flows enabled disposal of unwanted sample waste without laser irradiation, and collection of wanted sample upon irradiation. As an application of the sorter, two kinds of fluorescent microspheres were separated with recovery ratio and purity of 70% or 90% at throughputs of about 100 or 20 particles per second, respectively. Next, Escherichia coli cells expressing green fluorescent protein were separated from those expressing DsRed with recovery ratio and purity of 90% at a throughput of about 20 cells per second.

High-speed particles and biomolecules sorting microsystem using thermosensitive hydrogel

Two types of high-speed particle and biomolecule sheath flow sorters were realized using thermosensitive hydrogel. One is all hydrogel sheath flow and the other is two-phase sheath flow that consists of a water flow including samples and two hydrogel carrier flows; these have been utilized instead of the air and water droplet two-phase flow used in the conventional commercialized cell sorting system. Flow switching is performed by the sol–gel transfer of the thermosensitive hydrogel generated by focused infrared (IR) laser irradiation. High-speed sorting less than 5.0 ms and no error sorting (120 000 counts) in the all hydrogel sheath flow system while 20 ms in the two-phase sheath flow was realized. Since sorting was performed in simple PDMS-glass microchannels without any electric stimulation and mechanical valve structures, the proposed system is suitable for many biochemical applications.

On-Chip Cell Sorting System Using Thermoreversible Gelation Polymer

We have developed a microfabricated fluorescence-activated cell sorter system using laminar flow of thermoreversible gelation polymer (TGP). The glass sorter chip consists of microchannels with two inlets for sample and buffer solutions, and two outlets for collection and waste of the specimen. A biological specimen containing fluorescently labeled cells, is mixed with a solution containing a TGP. The laminar flow of the mixed solution and buffer solution are then introduced into the sorter chip. The fluorescently labeled target cells were detected with sensitive fluorescence microscopy. In the absence of a fluorescence signal, the laminar flow of the specimen is directed into the waste channel. Upon detection of a fluorescence signal from the target cells, the sol-gel transformation was locally induced by site-directed infrared laser irradiation for the flow switching and for allowing the fluorescent cells to be channeled into the collection reservoir. The flow switching time of 100 ms was achieved. Using this system, we have demonstrated the sorting of Escherichia coli cells expressing fluorescent proteins. These cells were found to be viable after extraction from the sorting system, indicating no damage to the cells

High Through-Put Parallel Biomolecules Sorting Microsystem with Three Dimensional PDMS Stack

High through-put parallel biomolecules sorter was fabricated with the three dimensional PDMS stack structure. Three dimensional channel structure was realized by using PDMS dry etching. High speed switching of 10 ms order in each channel was realized with four and eight parallel type sorter. Sorting rate is about 20 samples/sec in the case of the four parallel type sorter. High through-put sorting of about 20 times higher than that of the previous single channel sorter was obtained in actual E. coli cells separation.

High Speed Particles and Biomolecules Sorting Microsystem Using Thermosensitive Hydrogel and Water Two-Phase Flow

High speed particles and biomolecules sorter was realized using thermosensitive hydrogel and water two-phase sheath flow system. The two phase sheath flow consists of a water flow including samples and two hydrogel carrier flows is utilized instead of the air and water droplet two-phase flow used in the conventional commercialized cell sorter. Flow switching is performed by the sol-gel transfer of the thermosensitive hydrogel generated by the focused IR laser irradiation. High speed sorting less than 5.0 msec and no error sorting (3000 counts) was realized. Since sorting performed in simple PDMS-glass microchannels without any electric stimulation, the proposed system is suitable for bio-applications.