pat stayton u. washington, seattle...
TRANSCRIPT
![Page 1: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/1.jpg)
Hybrid Biomaterials ThatTalk and Listen in Nanospace
Pat StaytonU. Washington, Seattle
![Page 2: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/2.jpg)
Change in:Shape
MechanicsChemical Properties
ApplicationsDrug DeliveryBioanalytical
Biohybrid Smart Polymer-Biomolecule Materials
Polymer is Antennae and Actuator
++
+++ +
+
Protein Conjugate DNA Complex
![Page 3: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/3.jpg)
How Do We “Talk” To Molecular Componentryin Bioanalytical Devices?
Micro Total Analytical SystemsRapid Diagnostic DevicesChips, Arrays
DNA Antibodies Enzymes Streptavidin Beads
Currently Make Large, Nonspecific Changes in T, pH, salt, etc.
Nanogen
Burns Lab
![Page 4: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/4.jpg)
ON(hydrated)
OFF(collapsed)
“Smart” Biohybrid MaterialsThat Talk and Listen
Smart Polymer is Antennae & Actuator
Endoglucanase12A
Nature 378, 472 Bioconj. Chem. 12, 314Nature, 411, 59 PNAS, 99, 16592
![Page 5: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/5.jpg)
CH 2 CH
O
S
O
CH 2 CH 2 CH 2 CH 2O S
NCH 3CH 3
N N
CH 2 CHC O
CH 2 CHC O
[ ]m n
][
Vinyl sulfone group for conjugation to
unique, engineered thiols
R
= O (DMAA) = NH (DMAAm)
R
Dimethyl acrylamide (DMA)
Photo-Responsive Polymers
Photo-sensitive azobenzene group
Endoglucanase 12AGenencor Int.
![Page 6: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/6.jpg)
LCST Behavior of DMAAm
0
20
40
60
80
100
Rel
ativ
e A
bsor
banc
e (%
)
25 30 35 40 45 50 55 60
Temperature (°C)
UV
VIS
0
20
40
60
80
100
Rel
ativ
e A
bsor
banc
e (%
)25 30 35 40 45 50 55 60
Temperature (°C)
UV
VIS
DMAAm
DMAA
DMAAm 5.9% Mw 18 kD
Coil
CollapsedCoil
Aggregate
![Page 7: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/7.jpg)
Magneticbead
1. Immobilized Conjugate
Application Formats
D pH, hn, T D pH, hn, T
2. Free Conjugate
Nanoparticle
Protein
Polymer
Enzyme
Polymer
Nature 378, 472
Nature, 411, 59
PNAS, 99, 16592
Bioconj. Chem. 12, 314
![Page 8: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/8.jpg)
Thermo- and Photo-Responsive Activity SwitchThermo- and Photo-Responsive Activity Switch
Thermo-Cycling (under VIS) Photo-Cycling (42oC)
0
25
50
75
100
o-n
itro
ph
enol
(m
M)
0 5 10 15 20 25 30 35 40
Time (min)
32 °C 32 °C 52 °C52 °C
N55C-DMAA
N55C-DMAAm
0
50
100
150
200
o-n
itro
ph
eno
l (
mM
)
0 5 10 15 20 25 30 35 40
Time (min)
N55C-DMAA
N55C-DMAAm
VIS VISUV UV
![Page 9: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/9.jpg)
Molecular Molecular ““DimmersDimmers””
0
5
10
15
20
25
Cata
lytic
Act
ivity
(ONP
mM/m
in)
30 35 40 45 50 55
Temperature (°C)
N55C-DMAAm (UV)
N55C-DMAAm (VIS)
N55C (UV)
N55C (VIS)
LCST
LCST
Temperature (oC)
Enzyme Alone
![Page 10: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/10.jpg)
Switch Can Be Optimized To Match/ManipulateEnzyme-Substrate Properties
• Conjugation Position vs. Polymer Mn,
e.g. more distant needs larger Mn
• Substrate Size vs. Polymer Mn, e.g. larger substrate needs smaller Mn
![Page 11: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/11.jpg)
Effect of Conjugation Position onEffect of Conjugation Position onThermal Switching ActivityThermal Switching Activity
(in 50 mM AB pH5.5, at 45 (in 50 mM AB pH5.5, at 45 °°C)C)
Catalytic Activity Relative toN55C (%)
N55C-DMAAm
S25C-DMAAm
WT-DMAAm
0
20
40
60
80
100
32 52 32 52 32 52
ONPC
![Page 12: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/12.jpg)
“Smart” Molecular Matchmakers
D pH, hn, T D pH, hn, T
Particle “On”
D pH, hn, T D pH, hn, T
Particle “Off”
Protein/DNA Species Brought Together On CommandMolecular Orientation in Particle Determines Activity
![Page 13: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/13.jpg)
“Smart” Streptavidin Conjugates
Poly (NIPAAm) synthesized by RAFTto control Mn polydispersity
-(CH2-CH)-C=O
NHCH
CH3CH3
![Page 14: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/14.jpg)
“Smart” Particle Stability
0
500
1000
1500
2000
2500
1 10 100 1000Time (min)
Hyd
rod
ynam
ic d
iam
eter
(n
m)
PNIPAAm (14.9 kDa) Free Polymer Grows
Temperature = 35 oC, 3 mM
PNIPAAm (14.9 kDa)-Streptavidin Conjugate
Conjugate Growth Capped
![Page 15: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/15.jpg)
Particle Size Controlled By Conjugate Concentration
0
200
400
600
800
1000
0 1 2 3 4 5 6 7 8 9 10
Concentration (uM)
Hy
dro
dy
na
mic
ra
diu
s (
nm
)
14.9 kDa
25.9 kDa
Temperature = 42 C
![Page 16: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/16.jpg)
Effect of Polymer Molecular WeightOn Particle Size
FWHM’s ca. 50 nm
Conjugates at 3 uM and 42 oC0
200
400
600
800
1000
1200
0 5 10 15 20 25 30
Molecular weight (kDa)
Ave
rag
e h
ydro
dyn
amic
dia
met
er (
nm
)
PNIPAAm 4.8 kDaconjugate
PNIPAAm 14.9 kDaconjugate
PNIPAAm 25.9 kDaconjugate
PNIPAAm 2.9 kDaconjugate
![Page 17: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/17.jpg)
“Smart” Meso- and Nano-Scale Particles
• Polymer-Protein/-DNA conjugates reversibly form particles
• Particles have controllable sizes ranging from 100nm to 1 mm
• Purification of diagnostic targets using reversible size or diffusion properties of particles
• Development of new bioassays – traditional and microfluidic format
![Page 18: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/18.jpg)
Comonomer Content (wt. %)
LCST (°C)
Polymer ResponsivenessTemp. ResponsivepH ResponsiveLight ResponsiveIonic Strength Responsive
Signals Are Molecular Species Specific:Some Multiplexing Possible
Multiple Transition PropertiesWithin Each Class+
![Page 19: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/19.jpg)
Matchmaker Technology
On
Talking to molecules in complex mixtures: species specific & resettable
OffOff
Off
Off
OffOn
OffOn
D T
DD T
D pH
D hn
Flow
Signal 1
Signal 2
Signal 3
![Page 20: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/20.jpg)
SmartFishingNet 1
Collect Target 1Collect Target 2
SmartFishingNet 2
Bioanalytical Sample Processing
“Smart” Microfluidic Components:Dissolvable “Fishing” Nets
Analytical Chemistry (2003) 75, 2943
Heater Heater
![Page 21: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/21.jpg)
“Smart” Beads for Microfluidic Separations
surface of doubly- modified bead
(100nm)
PNIPAAm b-PEG
NCH CH2 Sn H
C O
NH
CH
CH3 CH3
CH2 CH2 C O
O
O
O
SC
NHNH
CH2 OCH2 C NO
O
O
O
n
NH
CCH2CH2CH2CH2
O
O
NCH CH2 Sn H
C O
NH
CH
CH3 CH3
CH2 CH2 C O
O
O
O
SC
NHNH
CH2 OCH2 C NO
O
O
O
n
NH
CCH2CH2CH2CH2
O
O
NHS-PNIPAAm
NHS-PEG-biotin
![Page 22: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/22.jpg)
1. bead suspension at RT
Doubly- modified bead
(100nm)
2. Increase temp. to 45ºC to cast net
∆T
Casting the Net
![Page 23: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/23.jpg)
Inject Sample with Target Capture and Purify Target
- ∆T
![Page 24: Pat Stayton U. Washington, Seattle stayton@u.washingtonenglish.fsao.eng.osaka-u.ac.jp/summer_prog/UW2006/... · U. Washington, Seattle stayton@u.washington.edu. Change in: Shape Mechanics](https://reader034.vdocuments.us/reader034/viewer/2022052015/602d5131227580574638ce03/html5/thumbnails/24.jpg)
Immunoassay Format
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 200 400 600 800 1000
Volume Eluted (microliters)
Fra
ctio
nal
flu
ore
scen
ce
0
5
10
15
20
25
30
35
40
Tem
per
atu
re S
et P
oin
t (d
egre
es C
) 0 uM
1.28 uM
2.56 uM
12.8 uM
Temperature
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 200 400 600 800 1000
Volume Eluted (microliters)
Fra
ctio
nal
flu
ore
scen
ce
0
5
10
15
20
25
30
35
40
Tem
per
atu
re S
et P
oin
t (d
egre
es C
) 0 uM
1.28 uM
2.56 uM
12.8 uM
Temperature
Malmstadt et al., Lab on a Chip, in press
Streptavidin
PEG-biotinPNIPAAm
Nanobead