微小流路による微小管集団運動の制御
Control of Microtubules Collective Motion by Microflow Channel

Daisuke Inoue

CytoArchitec Lab, Faculty of Design, Kyushu University

Introduction

Micro-biosensor device activated by ATP

In vitro gliding assay＋ATP+ATP (Chemical energy)

Micro-volume analytes(e.g. virus RNA, rare earth) + ATP

MTs/kineisn

25 nm
20 μm

Kinesin motor protein propels MTs
by converting the chemical energyof ATP into mechanical work.
Microtubule(MT) Kinesin 1

AnalyteCondensation of analyte

Sample-loading area

ATP driven micro transportation devices

Collective motion

MT/kinesin system can transportand organize material at their size
scales at high energy efficiency.

T. Fischer, et al.,Nat. Nanotechnol. 2009

Highly-dense gliding MTs autonomously organize
directed flow and increase their local density.
Aim of project

Microflow channel can guidemoving direction of MTs.

To develop a microdevice to concentrate analytes
by controlling microtubule collective motion.

50 μm

M. G. L., van den Heuvel, et al., Science 2006

D. Inoue, et al., Nanoscale 2015

Materials & Methods
Photomask (Toppan)

Concentration and detection of diluted target
analytes by MT local accumulation induced bycollective motion

Experiment 1: Poly(N,N-dimethylacrylamide) gel
Microfabrication

Cheap LED UV light (Jaxaman)

OHOH

GlassVinylsilane
CH2

Wavelength: 365 nm
Radiance: 1600 mW/sr/m2

Photomask

Available resins to make microflow channel
Materials

UV

Descriptions
Nonionic, neutral hydrogelLess interaction with proteins.
Low toxicity unlike acrylamide.
Weak bond with glass substrate.
UV responsive epoxy resin
Less steps for preparation
Strong bond with glass substrate
Non-specific interaction with proteins.

N,N-dimethylacrylamide(DMAAm)

Norland Optical Adhesive(NOA61)

100 μm

4M DMAAm mix†
LAP*β-ME,LAP

MT gliding assay in PDMAAm microflow channel

UV(365nm),
1 sec

30’

0 sec

60’

135’

UV, 1 minPDMAAm gel

In vitro Gliding assay

Taxol-stabilized
ATTO565-MTs

Wash

Methylcellulose+ATP

MTs stuck in the softhydrogel wall.
→Gel is not suitable
Material to guide MTs.

PDMAAm gel

Wash†4M DMAAm, 4mol% MBAA,

SNAP-kinesin1

1% BSACH2

0.05mol% LAP, 53.6% Glycerol*LAP (lithium phenyl-2,4,6trimethylbenzoylphosphinate)→ Water-soluble photoinitiator tostart radical polymerization of the gel

Glass substrate

MTskinesin

Experiment 2: Norland Optical Adhesive 61 (NOA61)
Microfabrication

Dense bundle formation of MTs in microflow channel

Photomask

NOA610 sec

300 sec

600 sec

UV,1sec
20 μm80oC(SU8 developer, IPA)
100 μm

MT gliding assay in NOA microflow channel
100

Probability (%)

NOA

GuidingGlass

100 μmGuidingkinesin

80

MTs
Width of channel (μm)

Climbing up

Follow
Glimbing up

60
40

Climbingup

NOA61

200GlassNOA

90% MTs climb up NOA wall where kinesin is absorbed→ NOA can’t guide MTs.

Passivation of NOA surface by using PEG & Pluronic F127
PEG modification
CH2NOA61

CH2mPEG-SH, 10k+

NOA61LAP
Thiol–ene click reaction

GlassNOA+PEG

Normalized densityof microtubules*2 mg/mL Pluronic F127 was deposited onto NOA

10.8
0.6
0.4
0.2

NOA+PEG+PluronicF127

80Follow

PEG

Glimbing up

60Guiding40

Pluronic
B

NOA61

200+Pluronic

NOA+Pluronic

50100+PEG+PEG+Pluronic

150200250

11.6 μm
50100150200

26.2 μm
50100150200250

Fusion of MT bundles at thecentre line of the channel
Distance(um)Intensity inchannel: Iin

41.4 μm

50100150200

250

Intensity out ofDistance(um)channel: Iout

50.8 μm50

100

150200250

76.4 μm50

100

150200250

MT intensity in channel (a.u.)

Dispersion of MTs
in the channel
I
Concentration = inIoutefficiency

1.5
1.4
1.3
Distance(um)

1.2
1.1
1

2600

Distance(um)

250

52

20 μm
20 μm

WideDistance(um)

5.6

11.6

26.2

41.4

50.8

76.4 NOA only(24.5)
Width of channel (μm)

Width of channel
Width of smallerthan(μm50) μm is
optimal for concentrating MTs.
Distance(um)SummaryProbability (%)

20 μmGlass523926
1300

Quantification of MTs bundle formation
Narrow+PEG+Pluronic*
10004228
14005.6 μm78

MT gliding assay in PEG-PluronicNOA microflow channel

UV, 1min

63001260027
18
900

Concentration
efficiency

Wash+PEG,Pluronic

PEG & Pluronic modification ofPEG + PluronicF127 modification minimizes
NOA enables to guide MTs at 100%.
kinesin-MTs absorption onto NOA.

・ PDMAAm gel was not available to guide gliding MTs.
・ Modification of NOA61 with PEG and PluronicF127 improved
guiding probability of MTs from 10% to 100%.
・ Microflow channel with a certain width (<50 μm) increased thedensity of MTs and assemble dense MT bundles.

Next plan: Demonstration of molecular sensing of small volume analytes
Acknowledgement:This work is supported by Q-pit Support Program for Young Researchers and Doctoral Students. , JSPS
LEDER program, JSPS Grant-in-Aid for Early-Career Scientists, Nikki-Saneyoshi Scholarship Foundation
Grant, QR project, Wakaba-challenge, Grant-in-Aid for Transformative Research Areas, (A).