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Multi Fluorescence Microsensors for Spatiotemporal Measurement of Culture Environment in a Microfluidic Chip 1 Keisuke Takagi, 1 Hisataka Maruyama, 1 Taisuke Masuda, 2 Osamu Suzuki, 1 Fumihito Arai 1 Department of Mechanical Science of Engineering, Nagoya University 2 Division of craniofacial function engineering, Tohoku University Spatiotemporal measurement of multi-parameters is important. Glass Inlet of fluorine Inlet of Cell Inlet of OCP Inlet of sensor materials Contact person： Keisuke Takagi E-mail: [email protected], URL: http://www.biorobotics.mech.nagoya-u.ac.jp/ TEL: 052-789-5220, FAX : 052-789-5027 Background Concept Fabrication of calibration of microfluidic chip Experiments Conclusions Bone biomineralization Environment measurement during conversion of OCP to hydroxy apatite (HA) Conventional approach Spatiotemporal measurement using multi fluorescence sensors in a chip PDMS Micro-channel Osteoblasts Fluorescence sensors OCP Parameter Ca 2+ pH Temp. Sensor material PEG-DA PEG-DA PEG-DA Fluorescence Fluo-3 FITC Lumidot 480 Diameter 20um 15um 10um Ex. 488 nm 488 nm 488 nm Em. 527 nm 510nm 515nm Characters of proposed method ・Simultaneous measurement of multi- parameters by single wavelength light ・No limitation of measurement parameter by arranging sensor shape and pattern Specification of fluorescence sensors On-chip measurement methods Temp. sensor pillar Ca 2+ sensor pillar pH sensor pillar 1. Mold fabrication 2. Replica molding 3. Bonding to glass 4. Making Ca 2+ sensor 5. Making pH sensor 6. Making Temp. sens Su-8 Si substrate PDMS Glass Mask UV Ca 2+ sensors pH sensors Temp. sensors Fabrication process of the chip and sensors Calibration of fluorescence sensors Ca 2+ calibration pH calibration Temp. calibration T pH f F FITC R ) ( ) ( R R F T pH f F ) ( ' Spatiotemporal measurement of variation of [Ca 2+ ] and pH during transformation of OCP to HA Reference: K. Takagi, H. Maruyama, T. Masuda, O. Suzuki, F. Arai, Multi Fluorescence Microsensors for Spatiotemporal Measurement of Culture Environment in a Microfluidic Chip Acknowledgement: This work was supported by Kakenhi (16H04301). Time course variation of [Ca 2+ ] and pH OCP Slit Ca 2+ sensor pH sensor 100 m Compensation of calibration with other sensors’ data FITC is sensitive to both pH and temp. Ca 2+ sensor pH sensor Temp. sensor OCP Culture area Fluorine 100 m 10 mm Spatiotemporal distribution of [Ca 2+ ] and pH was measured. Concentration of fluorine: 100 ppm Measurement parameter: [Ca 2+ ], pH Exposure time: 200 ms ・PO 4 2‐ emitted from OCP during conversion to HA is a trigger of differentiation of osteoblast cell. ・Environment surrounding OCP and differentiation of osteoblast cell have some relationship. Method Electric sensor MEMS sensor Fluorescence sensor Schema Ref. Zhiwei Zou, et al, Sens Actuators B,2008 N. Inomata, et al, Appl. Phys. Lett.(2012) L Basabe-Desmonts, et al, Anal Bioanal Chem, 2008, Integration of many sensors Possible Possible Possible Spatial resolution Low Low High Problems of multi-parameter measurement by conventional fluorescence method ・Limitation of number of parameters ・Low time resolution due to switching the excitation light On-chip spatiotemporal measurement of Ca 2+ and pH was succeeded using fluorescence multi-sensors. C1 C2 C3 C4 C5 C6 P1 P2 P3 P4 P5 P6

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Multi Fluorescence Microsensors for Spatiotemporal Measurement of Culture Environment in a Microfluidic Chip

1Keisuke Takagi, 1Hisataka Maruyama, 1Taisuke Masuda, 2Osamu Suzuki, 1Fumihito Arai1Department of Mechanical Science of Engineering, Nagoya University

2Division of craniofacial function engineering, Tohoku University

Spatiotemporal measurement of multi-parameters is important.

Glass

Inlet of fluorine

Inlet of Cell

Inlet of OCP

Inlet of sensor materials

Contact person： Keisuke TakagiE-mail: [email protected],URL: http://www.biorobotics.mech.nagoya-u.ac.jp/TEL: 052-789-5220, FAX : 052-789-5027

Background

Concept

Fabrication of calibration of microfluidic chip Experiments

Conclusions

Bone biomineralizationEnvironment measurement during conversion of OCP to hydroxy apatite (HA)

Conventional approachSpatiotemporal measurement using multi fluorescence sensors in a chip

PDMS Micro-channel Osteoblasts

Fluorescence sensors

OCP

Parameter Ca2+ pH Temp.Sensor material PEG-DA PEG-DA PEG-DAFluorescence Fluo-3 FITC Lumidot 480Diameter 20um 15um 10umEx. 488 nm 488 nm 488 nmEm. 527 nm 510nm 515nm

Characters of proposed method・Simultaneous measurement of multi-

parameters by single wavelength light・No limitation of measurement parameter

by arranging sensor shape and pattern

Specification of fluorescence sensors

On-chip measurement methods

Temp. sensor pillar

Ca2+ sensor pillarpH sensor pillar

1. Mold fabrication 2. Replica molding 3. Bonding to glass

4. Making Ca2+ sensor 5. Making pH sensor 6. Making Temp. sens

Su-8

Si substrate

PDMS

Glass

MaskUV

Ca2+ sensors pH sensors Temp. sensors

Fabrication process of the chip and sensors

Calibration of fluorescence sensors

Ca2+ calibration pH calibration Temp. calibration

TpHfF FITCR )()( RR FTpHfF )('

Spatiotemporal measurement of variation of [Ca2+] and pH during transformation of OCP to HA

Reference: K. Takagi, H. Maruyama, T. Masuda, O. Suzuki, F. Arai, Multi Fluorescence Microsensors for Spatiotemporal Measurement of Culture Environment in a Microfluidic Chip

Acknowledgement:This work was supported by Kakenhi (16H04301).

Time course variation of [Ca2+] and pH

OCPSlitCa2+sensorpH sensor

100 m

Compensation of calibration with other sensors’ data

FITC is sensitive to both pH and temp.

Ca2+ sensorpH sensorTemp. sensor

OCP

Culture area

Fluorine

100 m

10 mm

Spatiotemporal distribution of [Ca2+] and pH was measured.

Concentration of fluorine: 100 ppmMeasurement parameter: [Ca2+], pHExposure time: 200 ms

・PO42‐ emitted from OCP during conversion to HA is a trigger of differentiation of osteoblast cell.・Environment surrounding OCP and differentiation of osteoblast cell have some relationship.

Method Electric sensorMEMS sensor

Fluorescence sensor

Schema

Ref.Zhiwei Zou, et al, Sens Actuators B,2008

N. Inomata, et al, Appl. Phys. Lett.(2012)

L Basabe-Desmonts, et al, Anal Bioanal Chem, 2008,

Integration of many sensors

Possible Possible Possible

Spatial resolution Low Low High

Problems of multi-parameter measurement by conventional fluorescence method・Limitation of number of parameters・Low time resolution due to switching the excitation light

On-chip spatiotemporal measurement of Ca2+ and pH was succeeded using fluorescence multi-sensors.

C1

C2

C3

C4

C5

C6

P1

P2P3

P4

P5

P6