蛍光酸素センサを用いたオンチップ単一卵子酸素計測

蛍光酸素センサを用いたオンチップ単一卵子酸素計測
○鬼頭雅伸1，丸山央峰2，新井史人2
１名古屋大学大学院工学研究科機械理工学専攻
2名古屋大学大学院工学研究科マイクロ・ナノシステム工学専攻
Concept
Background
Evaluation of oocyte quality
Mouse oocyte
100 m
Indicator of quality evaluation
・Morphological evaluation
・Mechanical characteristics
・Oxygen consumption rate（OCR）
Scan
OCR fmol/s
15
New
Method
Oocyte
Comparison of OCR of mamarian oocyte
20
Y. Kumasako et.al. Ova research. 2013
SARAH E. HARRIS, Molecular reproduction
and development (2009) , etc.
Amount of consumption depends on each mammalian （1 fmol/s
～20 fmol/s)
10
5
Oocyte
Problems: Damage to Oocyte, Probe is easily broken
Time‐Consuming (Several Minutes)
0
Low
Oxygen concentration
High
High
Fluorescence intensity
Low
Non‐contact measurement Measurement time : several seconds
Oxygen sensor
mouse porcine bovine
rat
Oxygen
diffusion
Culture Medium
Scanning electrochemical microscope
（SECM）
Tris(2,2′‐bipyridine) dichloro‐ruthenium(II) hexahydrate
(Ru(bpy)3Cl2) +PEGDA + Pure water
(ex. 561 nm, em. 620 nm)
Photo-bleaching is
less than 0.1％ for 32s
High sensitive oxygen measurement method is required!
Stripe shaped → Spherical diffusion theorty
is applicable !
Glass
Sensor chip fabrication
Fabrication process
4. Inject ruthenium solution
Cross section
6. Bond chamber layer
1
0.8
I/I0
Oxygen Consumption Rate
Culture medium
dC(rs )
dr
r  (rs t)2 (x)2
OCR= F  4rs2  D
Value
0.21 mol/m3
75 μm
Oxygen consumption rate
1.0×10‐15 mol/s
Oxygen diffusion coefficient in solution
2.18×10‐9 m2/s
Oxygen diffusion coefficient in PDMS
2.18×10‐9 m2/s
Oxygen diffusion coefficient in sensor
3.4×10‐11 m2/s
B
C
D
E
F
G
H
I
1.2
PDMS
1st try
2nd try
C
(b) Stripe type (pitch: 5 um)
200
I: Fluorescent intensity (measured value)
I0: Fluorescent intensity (saturated value)
Comparison of sensor sensitivity
Fluorescent intensity ratio 150
Oxygen concentration [μmol/l]
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
D
100 um
(b) Fluorescence img.
with oocyte
210
200
190
180
Sample 1 Direction: (A ,B ,C ,D )
170
rs
160
150
50
60
100
200
Experimantal condition:
110
120
OCR distribution of each oocyte
Sample
G
100 um
H
I
(c) Fluorescence img.
without oocyte
Regulating concentration of saturated solution : Na2SO3
100 um
(d) Img. of Fluorescence
ratio ((b)/(c))
1.
2.
OCR at each
Average Divergence direction fmol/s
OCR of OCR A
B
C
D
fmol/s
fmol/s
1
0.58
0.56
0.60
0.64
0.59
0.03
2
0.53
0.23
0.66
0.48
0.47
0.18
3
0.21
0.34
0.31
0.39
0.31
0.07
4
0.07
0.29
0.22
0.11
0.19
0.10
F
Laser: 561nm
Contact person： Masanobu Kito
E‐mail: [email protected]‐u.ac.jp
URL: http://www.biorobotics.mech.nagoya‐u.ac.jp/
Furo‐cho, Chikusa‐ku, Nagoya 464‐8603, Japan
Dept. of Mechanical Science Engineering, Nagoya University
TEL: 052‐789‐5220, FAX : 052‐789‐5027
100
Profile of oxygen concentration at each direction
Conclusions
Temp.: 37 oC, CO2 : 5 %
90
B
E
0
80
A
D
Deviation of oxygen concentration: 9.7 μmol/l
70
Distance from center of oocyte (side view) r [μm]
C
Oxygen concentration [μmol/l]
SECM (radius of Our sensor (by
electrode=5μm) using 16 bit CCD)
0.01 μmol/l
0.016 μmol/l
B
(a) Image of oocyte
50μm
0.2
0
220
100 um
3rd try
C: Oxygen concentration [mol/m3]
r: Distance from center of cytoplasm [m]
rs: Radius of cytoplasm [m]
t: Distance between sensor and cytoplasm [m]
x: Distance from center of cytoplasm on image [m]
PDMS
(a) Planar type
A
Liniarization
100
x2
Experiments
1.4
50
x1
PDMS
Culture medium
Bulk oxygen concentration
Diameter of oocyte
A
r2
r1
t=5μm
PDMS
FEM analysis
I
 1.2 10 3  [O2 ]  1.23
I0
rs
Steady state analysis
Calibration
0
Oocyte
Planar sensor
Stripe sensor
Parameters
0.4
rs=75μm
t
30 m
10 mm
2. Put on aluminum jig 5. Cut out PDMS
on the substrate
0.6
Culture medium
Oocyte
t=5μm
Fabricated chip
3. Make PDMS mold
Culture medium
A’
Oxygen concentration[μmol/l]
1. Make SU8 pattern
A
A’
A
OCR can be calculated based on spherical diffusion equation
This method achieve measurement of two-dimensional distribution of OCR
(sensitivity : 0.016μmol/l) in culture by acquiring the fluorescent image
(Average OCR: 0.39 ± 0.20 fmol/s, Divergence of OCR: 0.1 ±0.08 fmol/s)
Acknowledgements：
本研究は科学研究費（2468602）の支援をうけて行われたものです．
Reference: 鬼頭雅伸, 丸山央峰, 新井史人, “蛍光酸素センサを用いたオンチップ単一
卵子酸素消費量計測”, 化学とマイクロ・ナノシステム学会第28回研究会
講演要旨集 1P07, 2013

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