MICRO VALVE SYSTEM FOR THE SINGLE BIO-MORECULE DETECTION

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					                            MICRO VALVE SYSTEM
                  FOR THE SINGLE BIO-MORECULE DETECTION

                                          1            1            2                        2
                    Takahiro Arakawa , Yukiko Sato , Taro Ueno , Tomoya Sameshima ,
                                                  2                 1
                                   Takashi Funatsu and Shuichi Shoji

                        1; Graduate School of Science and Engineering, Waseda University,
                                            Tokyo, JAPAN 169-8555
                    2; Graduate School of Pharmaceutical Sciences, University of Tokyo,
                                              Tokyo, 113-0033, Japan
                                    e-mail; arakawa@shoji.comm.waseda.ac.jp



We present a multi-reagent rapid exchange flow system integrated with Total Internal
Reflection Fluorescent Microscopy (TIRFM). The flow system consists of four PDMS
microvalves were integrated with an optical detector of single fluorescent dye molecules.
Since switching time of the leakage free PDMS microvalves are 100 msec in Open-to-Close
mode and 120 msec in Close-Open mode, a reagent exchange time within 200 msec was
obtained. A single cell or biomolecular level observation capability was confirmed in the
fabricated TIRFM microflow system.

Fig.1 is the cross-sectional view of this system which is consists of a glass substrate having
pinhole-free Pyrex glass channels for fluid flow and an observation area, and a part of PDMS
having pneumatic membranes and air pressure channels for active microvalves. The flat glass
surface for TIRFM was realized by the mix acid of HF-H2SO4. The cross-sectional view of
the monolithically fabricated in-channel valve with the smooth surface glass microchannel is
shown in Fig.2.We measured response time of the microvalve using high speed CCD camera
by applying stepped air pressure. The response time of the Close-Open mode was 120 msec
while that of the Open-Close mode was 100 msec. The longer response found in the Close-
Open mode is caused by the sticking of PDMS membrane to the channel wall.

Firstly, we evaluated the reagent exchange behavior of the flow system. Fig. 3 shows the top
views around the cross point of the four microvalve system observed by CCD camera. Three
of microvalves are used for introduction of Rhodamine-B (V1), DI water (V2) and methylene-
blue (V3). One microvalve (V4) is connected to the observation area. Cyclic exchanges of the
reagents are performed within 200 msec under each flow rate of 1.0µl/min. The imaging of
carboxylated fluorescent polystyrene beads of 1µm in diameter as the test material also was
performed to evaluate the capability of single molecular level detection. Very low
background noise image was obtained at the etched glass surface. This automatic flow control
system is going to apply for dynamics observations of the chaperonin (GroEL) - cochaperonin
(GroES) interactions.
                                                                                         V3
                                                                                   Methylene
                                                                  Pyrex glass
     Sample inlet          Sample outlet                                           blue
                                                                                 DI water
         Microvalves         PDMS
                                                              PDMS membrane

                                                                                 V2              Observation
    Pyrex glass                                                                                  area V4
                       Observation area                                 PDMS
                                                                        30µm            V1       Rhodamine B
                     YAG laser
                                              Fig.2 Cross-sectional SEM view
 Fig.1 Cross-sectional view of the in-        of the PDMS and Pyrex glass       Fig.3 Top view of the
 channel microvalve system for TIRFM          microvalve system for TIRFM       microvalve system for TIRFM

				
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