takehiko kitamori the university of tokyo -...
TRANSCRIPT
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Takehiko Kitamori
The University of Tokyo
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Micro Unit Operations (MUO) Continuous Flow Chemical Processing
(CFCP)
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Condensation Distillation
Column Membrane
L/L
G/L
L/S
Others
Phase contact
Extraction
Phase separation
Mixing & reaction
Reaction Extraction
Concentration
Babble sep.
Adsorp. or
reac.
Heating Cell culture
Phase cont. Phase sep.
Micro Unit Operation and Continuous Flow Chemical Processing
MUO & CFCP
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Sample
Aq./Org. (shaking)
Colorimetry
Organic solution
Unit operation MUO Experimental procedure
Reagent
Aq. Org.
Aq. Org.
Aq. Org.
HCl
NaOH
Aq. Org.
Water
Extraction
Phase separation
Detection
Mixing & reaction
Phase contact
Phase contact
Phase contact
Phase contact
Phase separation
Phase separation
Phase separation
・Continuous Flow Chemical Processing (CFCP)
100mm
Design Protocol for Unit Operation Based Microfluidics Example: Wet Analysis for Trace Metal
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Stabilization of Parallel Microflows
Org.
Aq.
Aq.
Aq.
Org.
Aq.
Org.
Aq.
500 µm
70 mm
30 mm
140 µm
Stabilization of multiphase microflow
Aq. Org.
① Structure of channel
② Surface Modification
③ Flow condition
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Format Change between Parallel-Droplet Microfluidics
By Gucha-Gucha Chip
500 µm 500 µm 500 µm Hydrophilic surface
Hydrophobic surface
Phase separation Droplet formation
Water
Oil
Hydrophilic surface
Droplet-to-plug
Hydrophilic surface
Hydrophobic surface
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Our Concept of Micro Integration of Chemical & Biological Process
Micro Unit Operation
50mm
Separation Confluence
Mixing, reaction Extraction
Continuous flow chemical processing (CFCP)
Tokeshi (Kitamori Lab.), Anal. Chem., 2002
CPU
Chemical CPU
Chemical CPU
Chemical instruments
etc., etc., almost 20 kinds
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Microchips Installed Chemical Instruments
m-Extraction chip and
environmental analysis system m-ELISA chip and serum
immunoassay system
Compact
Rapid
Simple m-Gas extraction chip and clean room gas monitor
7,500 parallel CPUs
Gel particle plant
(30t/year)
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Institute of Microchemical Technology
IMT Products
Micro Chemical Chip Peripheral Devices & Accessories
Detectors: TLM Systems
www.i-mt.co.jp
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1 ~Å nm µm mm
10 100 1 10 100 1 100 10 1
Micro chemical chip
(Device)
System
Nanotech
Quantum effect
Object Molecule,
Nano molecule
Field
Principle
CNT, Nano pore Method
Extended-nano space
Nano space
Micro chemical chip
Microspace
Continuous fluid
Micro chemistry
Classical dynamics
Bulk space
ガラス器具
Components: Micro unit operations
Circuit: Continuous flow chemical processing (CFCP)
Micro unit operations
Macro unit operations (MUO)
Mixing Confluence Extraction Separation
100 µm
10cm
Divided by 500
100nm
100nm
Extended-nano space
50 mm
UV-Vis light
Background: Micro and Extended-Nano Fluidics
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Olympic Game in Analysis
Sample volume: Smaller
Concentration: Lower
Separation: Finer
Duration: Faster
Throughput: Higher
Cost: Lower
Why extended-nano fluidics?
Single Molecule Immunoassay at Femto-Liter
on Extended–nano Fluidic Device:
A Crazy ELISA (ITP 2013 Plenary)
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Strategy of Micro/E-nano Fluidics for Analysis
Our world
fluidics
Micro
fluidics
Extended-nano
fluidics
Micro
fluidics
Our world
fluidics
mm/nm
interface
nm/mm
interface
Cell process Molecule process
Detection
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MENU
Fabrication
Detection
Separation 1 Chromatography
Separation 2 Immunochemical
Separation 3 L/L extraction
Sampling interface
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Nanofabrication (yellow room in Kitamori Lab.)
Plasma etching Glass bonding @ RT
Electron beam lithography
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Substrate
EB
Development of channel fabrication method
Top-down fabrication by electron beam (EB) lithography and plasma etching
Plasma
Resist
Channel
EB & plasma etching
Optimization of EB
exposure time (0.4 ms)
100 nm
100 nm square
Extended-nano channels EB spot
100 nm
100 nm
Extended
nanochannel
Top view
Roughness: 3 nm
100 nm
Cross-section view
Depth:100 nm
Width:106 nm
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Bonding
・ Weaker bonding than thermal one ・ Good for fnctionalization
Low temperature (25-100℃), several hours Pressure 1000-5000 N
Low-Temperature Bonding Giving Chemical Function to EN Channel
Thermal fusion bonding Low-temperature bonding
Catalyst
Electrode
Biomolecules
1060℃, 6 hours
Thermal fusion Burned out
・ Strong and clean bonding ・ Difficult to functionalize
Catalyst
Electrode
Biomolecule
Chemically-
functionalized
Activation by O2 plasma
Hydrophobidized by Fluorine
Surface treatment
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MENU
Fabrication
Detection
Separation 1 Chromatography
Separation 2 Immunochemical
Separation 3 L/L extraction
Sampling interface
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Fluorescence
Detection in Extended-Nano Channels (DIC: Differential Interference Contrast TLM: Thermal Lens Microscope)
Excitation beam
EN channel
d=100nm
Probe beam
phase
Phase change
EN channel
d=100nm
Change of RI
LIF DIC-TLM
For fluorescent analyte For non-fluorescent analyte
Heat
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Peak a
rea[a
.u.]
0 5 10 15 20 25
0
100
200
300
400
500
600
0 5 10 15
Concentration [pM]
Absolute number [molecules]
Peak area plot
Results: Calibration Curve
0
2
4
6
8
10
12
14
16
18
0 30 60 90 120
Time [sec]
Therm
al le
ns s
ignal [m
V]
Blank
2.7 pM
5.3 pM
11 pM
Raw data
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MENU
Fabrication
Detection
Separation 1 Chromatography
Separation 2 Immunochemical
Separation 3 L/L extraction
Sampling interface
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Atto-Litter Chromatography
910 nm
470 nm
1600 mm
loading channel
separation channels
depth : 200 nm
pressure controller
valve
fluorescence microscope
Glass microchip vial
Schematic image of nanochannel
7 cm
3 cm
nanochannels
hole
microchannel
Glass or quartz microchip
15 cm
Anal Chem (2010), Small (2012), J. Chro. A (2011)( 2012)
550 aL
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Normal phase
Time [s]
強度
[a
.u.]
0
0.4
0.8
1.2
0 1 2 3 4 5
Reversed phase
0
0.4
0.8
1.2
0 4 8 12 Time [s]
Hydrophilic interaction
0 1 2 3 4 5
0
0.4
0.8
1.2
Time [s]
Various Separation Modes by Surface Modification
Time [s] 0 500 400 300 200 100
0.4
0.8
1.2
0
Column HPLC
強度
[a
.u.]
0
0.4
0.8
1.2
Time [s] 0 100 200 300 400 500
Column HPLC Extended-nano
Time [s] 500 400 200 0
0.4
0.8
1.2
0 300 100
Column HPLC Extended-nano Extended-nano
Separation performance
Column HPLC :
45,000 plates/m
Hydrophilic interaction
Nonpolar
Stationary
Stationary
Mobile
OH OH OH OH OH OH
Polar
OH OH OH OH OH OH
Polar
Hydrophilic interaction
Polar
OH OH OH OH OH OH
OH OH OH OH OH OH
Polar
Polar
Hydrophobic interaction
Polar
CH3 CH3 CH3 CH3 CH3 CH3
Nonpolar
CH3 CH3 CH3 CH3 CH3 CH3
Nonpolar
Separation performance
Extended-nano :
440,000 plates/m
Separation performance
Column HPLC :
43,000 plates/m
Separation performance
Column HPLC :
44,000 plates/m
Separation performance
Extended-nano :
350,000 plates/m
Separation performance
Extended-nano :
910,000 plates/m
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H = A + B/u + C・u
A
B 200 nm
C 200 nm
200 nm
No vortic diffusion
No particle
H = A + B/u + C・u
Small space
100 mm
Plate height:H
(Term A、B、C : factors to decrease separation efficiency)
Conventional HPLC Extended-nano chromatography
Vortic diffusion
Diffusion in flow direction
Diffusion in depth direction
Flow rate:u
Theoretical Discussion about Innovative Performance
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MENU
Fabrication
Detection
Separation 1 Chromatography
Separation 2 Immunochemical
Separation 3 L/L extraction
Sampling interface
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Single molecule detection and counting T
LM
sig
na
l [m
V]
Time [sec]
Detection point :1.5 mm downstream
Concept of Single Molecular Detection in Extended-nano ELISA
200nm
2mm
Antibody-modified region: 3 mm 1.5 mm
Injection
Antibody-modified region
Non-specific adsorption
of HRP-antibody
Spot size
1.1mm
DIC-TLM Analyte in proximity Isolated analyte
Non-specific adsorption
of HRP-antibody
Diffusion length
±400 mm
1 molecule
2 molecules
Perfect capture
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Results of Single Molecule ELISA
5
4
3
2
1
02520151050
secTime [sec]
TLM
sig
na
l[m
V]
Measurement 1
Measurement 2
Measurement 3
Results
Inlet Outlet
Signal from antibody-immobilized region
5 molecules
1 molecule Non-specific
Design and fabricated device
Extended-nano ELISA chamber
Inlet Outlet
Antibody
140 mm
3.3 mm
200 nm
Antigen
~100 fL
200 nm
depth
Diffuse in
~2 sec
Reaction field volume : = 100 fL
Concentration : = 10 pM (1 molecule/100 fL)
Reaction time : = 2 sec (traveling time)
Collision frequency
37000 times
DIC-TLM
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MENU
Fabrication
Detection
Separation 1 Chromatography
Separation 2 Immunochemical
Separation 3 L/L extraction
Sampling interface
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Concept of Unit Operation in Micro/Extended-nano Fluidics
Microchemistry (10-100 mm)
Integration concept using multiphase flow
50mm
Micro unit operations (MUO)
Separation Confluence
Mixing, reaction Extraction
Continuous flow chemical processing (CFCP)
Various complicated chemical process
Environmental analysis, synthesis etc.
Tokeshi (Kitamori Lab.), Anal. Chem., 2002
Aq. Org.
10-1000 nm
Extended-nano chemistry (10-1000 nm)
1/500
Novel functional devices:
Volume: aL-fL 100kPa
g : surface tension
d : diameter d
g
PLap=
Glass
Partial surface modification
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MENU
Fabrication
Detection
Separation 1 Chromatography
Separation 2 Immunochemical
Separation 3 L/L extraction
Sampling interface
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Advantage
Proposal of Micro/Extended-nano Interface by Lipid fusion
Extended-nano channel
20 mm
900 nm
Lipid bilayer
Cell
900 nm
Lipid bilayer
Lipid fusion
(molecular interaction)
(1) Formed ~ 102 nm hole on cell membrane
Realizing micro/extended-nano sampling interface by lipid fusion
(3) Tight junction ( no leakage) by lipid fusion (2) Keeping viability of cell due to the small size
10 mm Pressure
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Evaluation of Sampling Volume
L: 50 ± 0.4 μm
20 μm
50 ± 0.4 μm
Nano channel volume: 39 fL ( 978 nm × 826 nm × 50 μm )
Evaluation of sampling volume
Observation after bonding
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Why Extended-nano Fluidics?
Molecular behavior restriction
Smaller than unit diffusion distance of single molecule
Scientific uniqueness
Property change
Solution properties
Fluid characteristics
Transport phenomena
Photonic properties
High viscosity (x5) dielectric cons. (x1/4)
Surface slipping
High proton mobility (x20)
Blue-shifted optical near field
Engineered uniqueness
Molecular position control
Nano fabrication and nano surface modification
= Guiding target molecule to designed position
virus Cell
femto liter ( 1 mm)3
Smallness
nano liter (100 mm)3 atto liter (100 nm)3
pico liter ( 10 mm)3 zept liter ( 10 nm)3 Bacterium
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fL
200nm
Microtiter plate
Positioning in microtiter plate & extended-nano fluidic device
Extended-nano fluidic device
I am here. Where shall I be caught?
mL
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1H-1/T1
Intermolecular
Intramolecular
1H experiment
1H-1/T1 OH / D
850nm
750nm
850nm
950nm
DE
Restriction of
molecular mortion
Activation energy of
proton
Proton localization Proton transfer
NMR Measurement of Water in Extended Nano-Channels
2 µm
SEM image
Anal. Chem. (2002)
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Field of nanochemistry and nanofluidics
Nanopore: ~2000 papers Nanochannel: ~350 papers
Year (-) Year (-)
Num
ber
of
paper
(-)
Hibara, Kitamori, et al
Anal. Chem. (2002)
Size-regulated fluidic channel Unregulated nanospace
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Si O Si O Si O Si O
Si O Si O Si O Si O
50 nm
Wall
H+
H+
H+
H+
H+
H+ H+
H+
Fluid dynamics in extended nanospace
Extended nano physical chemistry
Stokes-Einstein Hopping
2 26
B B HS H
k T k TD
r z F
m
• NMR
• Streaming potential
• Capillary action
• Electric
• nano-PIV
• STED
• RAMAN
Measurement
• viscosity (×4) • dielectric constant (×1/4) • proton mobility (×20) • conductivity (×80) • condensation T (120ºC)
• chemical equilibrium shift
• Surface Slipping flow
• High E near field
Properties
Publications • web-site of Kitamori-Lab (#>30)
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EN-channel
Priventing H2,O2 mixing
O2
EN--channel (hydrophilic)
H2
H2/O2 generation and separation by light illumination
H2 O2
e-
H+ H2O
H+ transfer
H+
Nanochannel
20X Higher H+ mobility
H+ H+ Glass
Gas-liquid separation
H2
Hydrophobic
Laplace pressure to gas Laplace pressure to water
Pt cathode Nano-structured photocatalyst (photoanode)
Hydrophobic Hydrophobic
Design of Fuel (H2/O2)) Supplier Device
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Automatic H2/O2 generation and separation
Pt cathode
Nanochannels
Results
500 mm 4x
Stoichiometric H2/O2 generation
WO3/BiVO4 photocatalyst
1. Observation of H2/O2 generation
2. Analysis of generated gas by GC-MS
H2 O2
0
50
100
H2 O2 Genera
tion r
ate
[nL/m
in] Generation rate of O2 : H2≒1:2
Microscope
Experimental set up
photoanode Pt cathode
Electrolyte: 0.5 M NaClO4 Bias : 0.25 V vs Ag/AgCl ref. electrode
Water separation and H2/O2 generation by Vis light illumination
Fuel (H2/O2)) Supplier Device
500nm
WO3/BiVO4 Photocatalyst
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Solar Light Driven μ-Fuel Cell Device
Introduction Microchannel
(w: 600 um, d: 6 um)
PA:
WO3/BiVO4
C: Pt
Fuel Supplier (FS)
Solar Light
e-
Fuel Cell (FC)
Device Design
H2O Circulation
Nanochannels
(φ: 200 nm)
Extended-nano channels
(φ: 200 nm)
Hydrophobic channel
(w: 400 um, d: 0.5 um)
PA-photoanode; C-cathode: A-Anode
C: Pt
A: Pt/Pd
Fuel generation & separation
H2
O2 e- H2O H+
H2
O2
FC electricity generation
H2
O2
e-
H2O
H+
Nanochanel for H2O circulation
H2O
Resistance~1200MΩ: (Electrically disconnected)
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Kitamori Lab 2015
http://park.itc.u-tokyo.ac.jp/kitamori/
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