bruce hinds univ. of washington, mse15_hinds... · separations, environmentalseparations,...
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Bio-inspired membrane nanoplatforms for engineering, biotechnology and medical applications
• Carbon nanotubes as a protein channel mimetic platform• Transdermal nicotine smart phone delivery demonstration• Active protein pump for bio-pharm separations
Protein Pump Adv. Funct. Mater. 2014 24(27) 4317-23 Electroosmosis Nature Nano 2012 7(2) 133-139TDD Animal study J. Pharm. Sciences 2012CNT flow mechanism ACS Nano 2011 5(5) 3867-3877EO device Proc. Nat. Acad. Sci. 2010 107(26) 11698-702Fast fluid flow Nature 2005, 438, 44
Bruce HindsUniv. of Washington, MSE
Membranes as Exciting Nano-Engineering Platforms:
Numerous applications including: water purification, energyseparations, environmental separations, agriculture, food processing, gas separations, fuel cells, batteries, health care, kidney dialysis …. Functional/dynamic chemistry of the pore entrance is key.
Key Challenges:Slow speedPrecise chemical selectivityFouling
Beating the upperbound:Perfect pore dist, facilitated
Nature:Perfect selectivityPerfect pumpingDefoulingRegeneration
How Nature does membranes … and far exceeds man-made performance
General cell wall structureSodium specific pump
Proton pumpsAquaporin water purifier3
α-type channels – α-helixes are a common ‘scaffold’ that gives proteins there form. They are fairly rigid and can be moved like levers. They can also open up the protein structure to form the channel. Known structures have 2-22 of these segments
β-barrel channels -- Sheet like H-bonded structure (not tight helix). They form a barrel of 0.6nm to 3nm in diameter for water channel. Polypeptide loops at the entrance give selectivity. These are associated with simple organisms. The larger channels 2.5-3nm are activated to kill cells (apoptosis).
Protein Channels Basics
Passive Channel – rely on concentration gradient to transport across the channel. Selectivity is given by chemistry at pore entrance
Active Channels – Can pump against chemical gradient. Typically use ATP to push alpha helical lever and supply the energy.
Nature appears to ‘squeeze toothpaste’
T.M. Devlin ‘Textbook of Biochemistry 6th ed’
2n+m=3q metalic
The CNT as Bio-Inspired Platform
3-key attributes unique to CNT membranes:* Atomically flat hydrophobic core* Functional chemistry by necessity at cut entrances* CNTs are conductive
Applications in:Chemical separations Catalysis Bio-materialsWater Purification FO power generation Energy storageProgrammable drug delivery
The ultimate membranes are found in the walls of every, living cell
Ion Channel Conduction for Bio-Sensing
R RR R
Analyte
Ru(bpy)32+
Bound streptavidin blocks ion pore. The process is reversible with desthiobiotin. General scheme of complementary biological pairs blocking an ion channel is demonstrated.
Time hr
P. Nednoor, N. Chopra, V. Gavalas, L. G. Bachas, B. J. Hinds Chem. Mater. ‘05Hinds Group
0
10
20
30
40
50
60
0 5 10 15 20 25 30
DSB-X Funct. CNTM
Strept. Bound DSB-CNT
After Biotin Release
Flux
nM
Ru(
bpy)
32+
Gas Permeability of CNT Membrane
0.0E+00
1.0E-07
2.0E-07
3.0E-07
4.0E-07
5.0E-07
Hydrogen Methane Nitrogen Air Oxygen Argon
Perm
eabi
lity
(mol
es/m
2-se
c-Pa
)
0
20
40
60
80
100
Enha
ncem
ent F
acto
r
Permeability Enhancement Factor
>1 order of magnitude enhancement of flow for most gases
>2 order observed by Holt et al. in ~ 2 nm SWNT and DWNT membranes (ScienceMay 2006)
Porosity estimated from KCl Diffusion measurements
Enhancement factor = Experimental permeability/Calculated Knudsen permeability
Specular Reflection at Smooth Surfaces, G. Arya et al. PRL, 91(2) , 2003, 26102-1
vs.
Slip Length and the Nature of the Solvents
0
10
20
30
40
50
60
water EtOH IPA Hexane Decane
Slip
leng
th (m
icro
ns)
Slip lengths in Carbon nanotubes decrease with increasing hydrophobicity of the solvents (more interaction with CNT)
Majumder et al (Hinds group) Nature 2005, 438, 44Hinds Group
Chemical modification of CNT core to ruin perfect slip conditions
(a)
CNT Surface
i ii,iii iv
SO3-
SO3-
NNNNNN
OH
SO3-
NH -O3S
HN
OC
2
O
Chemical Functionalization
Flow Velocity normalized at 1 bar
(cm/s)
Enhancementover Newtonian flow
Normalized Diffusive Flux
As made (plasma-oxidized)
10.9(±5.1) 4.6 (±2.1) x 104 1
Spacer-dye(tip region)
4.7 (±0.7) x 10-2 2 (±0.3) x 102 0.93
Spacer-dye(core and tip)
< 1.26 x 10-3 < 5.3 1.03
Membranes showed dramatic loss of flow enhancement with chemical functionality but similar ionic diffusive flux. Confirms slip boundary condition. Conclusive evidence against cracks. Electrochemical grafting only on CNTs
Grand intellectual Challenge:High flow and selectivity, Nature does it all the time
Majumder , Chopra, Hinds ACS Nano 2011 5(5) 3867-3877
The next step: active membrane structures
Electrostatically modulated gate keeper ligands at entrance to CNT pore
Figure from NSF CAREER proposal 2002
SO3H
SO3H
N N N N N N
HO
HO3S
HNSO3H
O
NH
CNT
2
Positive flow of inert fluid during electrochemical diazonium grafting
N N
O
HO
+
v*t > √D*t20cm
CNT-SG
CNT-FG
“Voltage Gated Carbon Nanotube Membranes” Majumder, M.; Zhan, X; Andrews, R; Hinds, B.J * Langmuir 2007 23, 8624. Hinds Group
Electro-osmotic flow through DWCNT membraneBias study on double-walled CNT membrane
Dye modified and unmodified (PECVD) 5mM MV2+ and 5mM caffeine in 0.01 MKCl, trans membrane bias
0.02.04.06.08.0
10.012.014.016.018.020.022.024.026.028.030.0
-800 -600 -400 -200 0 200 400 600 800applied bias in mV
nmol
es/h
r
MV unmodified
Caffein unmodified
MV modified
Caffein modified
0 0
• 10 fold electro-osmotic flow (caffeine) over diffusion• 20 fold electro-phoretic flow (MV2+)
V
RCS Nanoscale 2011 3(8) 3321-28Hinds Group
Observed EO in 1nm SWCNT membranes
With electroosmosis giving a ‘plug flow’ and ions having bulk mobility in the moving frame of reference, dramatic electroosmotic velocities are seen.
EO velocity1nm SWCNT 3 cm/s-V~2nm DWCNT 0.18 cm/s-V7nm id MWCNT 0.16 cm/s-VAnionic AAO 0.0004 cm/s-V
4 orders of magnitude EO enhancement seen in small SWCNTs without counter ion current.Anion repulsion is ‘screened’ above 20 mM salt concentrationWu et al (Hinds) Nat Nanotech Feb 2012
The Dopamine Cascade: Origin of learning and addiction
• Learning (neural networking) is reinforced by dopamine cascades that give pleasure: “LOVE OF LEARNING” is real
• Chemical addiction gives strong dopamine cascades that reduce natural dopamine receptors thereby inhibiting learning
• Success of counseling for addiction is that positive ‘thoughts’ are reinforced to compete with chemical cascades.
• Nicotine patch is only 9% effective compared to highly expensive personal counseling ~40%
• For large populations, better to use remote counseling/algorithms combined with timed replacement therapy
• This can be achieved with blue-tooth enabled device with actively pumping membrane
Dopamine MRI imaging of cocaine cues, Nora Volkow et al
Transdermal Nicotine Delivery
15
Cum
ulat
ive
Nic
otin
e R
elea
sed
(µm
oles
)
-600 mV0 mV 0 mV
c
b
a
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0.0
0.5
1.0
1.5
2.0
2.5
0 10 20 30 40 50 60 70 80 90 1000
1
2
3
4
5
6
Cumulative nicotine drug release from threeCNTs/Gel/Human Skin design using 0 and -600 mV biases. Different CNT membranesand skin samples were used for eachexperiment (a-c) with solid curves assimulated data and solid points asexperimental data.
Wu et al, Proc. Nat. Acad. Sci. 2010 107(26) 11698-11702Hinds Group
Programmed Nicotine delivery to Hairless Guinea Pigs
16
• Observe programmed delivery in-vivo blood stream• Quantify time lag on skin structure• Demonstrate mechanical safety on free moving animal
0
5
10
15
20
0 10 20 30 40 50Cotin
ine
conc
(ng/
ml)
Time (h)
OFF/ON (n=3) OFF (n=2)
Plasma concentration time profiles of cotinine in hairless guinea pigs after application of a patch containing CNT membrane and nicotine gel. For ON/OFF study, the patch was switched off for the first 24 h and then switched on until 48 h. For control or OFF study, the patch was switched off for 48 h
J. Pharm. Sci. 2012 in pressHinds Group
Wireless ‘bluetooth’ control of nicotene delivery through CNT membrane
17
Demonstration of wireless control of nicotine delivery based on survey response• Using ‘App builder and Bluetooth’ kit technologies• Watch alarm wire connected to membrane
Hinds Group
Bio-Pharmaceutical Separations
• Our ability to modify microorganisms to produce or ‘express’ human proteins and enzymes is a foundational technology of the biotech revolution.
• However, we must extract ppm expressed human proteins from all the proteins necessary for organism to live.
• For instance urokinase treatment is ~$100k/yr with 80% of cost in separations.
• Ideal is to have an active membrane continuously extracting human proteins from media. Potential regulatory benefits in quality control of process.
Actively Pumping Bio-Inspired Membrane Systems
Electrophoretic pumping flux of BSA before, after his-tagged GFP blocking and imidazolereleasing
Schematic of AAO membrane blocked by his-tagged GFP
Dynamic Electrochemical Membranes for Continuous Affinity Protein SeparationZ. Chen, T. Chen, X. Sun, B.J. HindsAdv. Funct. Mater. 24(27) 4317-23 2014
Pulse Electrophoretic Pumping Process
1. Binding cycle: the imidazole at the feed pore is repelled and His-tagged GFP is selectivelycaptured by the nickel receptor thereby blocking the pores as a gatekeeper.2. Release and pumping cycle: Cationic Imidazole from the permeate solution is pumped tothe feed solution to release the His-tagged GFP. The anionic His-tagged GFP is selectivelytransported to the permeate solution by electrophoresis.
Dynamic Electrochemical Membranes for Continuous Affinity Protein SeparationZ. Chen, T. Chen, X. Sun, B.J. Hinds Adv. Funct. Mater. 24(27) 4317-23 2014
2
+
Pulsed Electrophoretic Pumping Success in Separations
Fluorescence spectra showing the emission intensity of the feed and permeate solutions after pulse electrophoresis.The release and pumping cycle time is 14s, the biding cycle time is 1s. The concentration of imidazole in thepermeate solution is 10 mMThe trade off challenge between flux and selectivity is addressed with the pulse electrophoretic pumping. The selectivity achieved was as high as 16.
Chen Z … Hinds BJ Adv. Funct. Mater. Adv. Funct. Mater. 24(27) 4317-23 2014
480 500 520 540 620 640 660 680 700
0.1ug/ml
0.1ug/ml
Permeate solution2.56 ug/ml
Permeate solution
Feed solution 7ug/ml(Texas Red conjugated BSA)Feed solution 7ug/ml
(Fluoresecence green protein)
Wavelength (nm)
Nor
mal
ized
Em
issi
on (A
.U.)
White Blood Cell Electroporation System: Membrane Electrode-Microfluidic System for Cellular
Electroporation
High cell viability with low voltage electroporation High throughput with continuous flowing electroporation process Low cost with electrophoretic pumping of needed transfection reagents
Conclusions
•CNT’s offer idealistic bio-inspired protein channel• Dramatic flow rates rivaling aquaporine seen with atomically smooth cores • Platform for ‘selective gatekeeper’ binding• Highly efficient electroosmotic pumping demonstrated
• Neuro-biology impact of membrane•Interactions with neurons aren’t just electrical. Chemical cascades can be induced with active membranes•Smart-phone devices offer a way to interact with complex psycology
•Industrial impact of membranes• Membranes are one of the ‘original’ nanotech. Touch many areas including the Water/Energy/Food Nexus•Opportunity to transform membrane as bio-inspired pumps
Hinds Group