Pat Stayton U. Washington, Seattle...
24
Hybrid Biomaterials That Talk and Listen in Nanospace Pat Stayton U. Washington, Seattle [email protected]
Transcript of Pat Stayton U. Washington, Seattle...
Hybrid Biomaterials ThatTalk and Listen in Nanospace
Pat StaytonU. Washington, Seattle
Change in:Shape
MechanicsChemical Properties
ApplicationsDrug DeliveryBioanalytical
Biohybrid Smart Polymer-Biomolecule Materials
Polymer is Antennae and Actuator
++
+++ +
+
Protein Conjugate DNA Complex
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
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
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.
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
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
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
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
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
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
“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
“Smart” Streptavidin Conjugates
Poly (NIPAAm) synthesized by RAFTto control Mn polydispersity
-(CH2-CH)-C=O
NHCH
CH3CH3
“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
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
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
“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
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+
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
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
“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
1. bead suspension at RT
Doubly- modified bead
(100nm)
2. Increase temp. to 45ºC to cast net
∆T
Casting the Net
Inject Sample with Target Capture and Purify Target
- ∆T
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
