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

Bio-mimetic vs Bio-inspired

inspired

mimetic

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

Acknowledgements

Financial Support: NIH R01 (NIDA, PECASE), NSF CAREER (CBET), DARPA,NSF CBET-1460922

Nitin ChopraUniv. Al

Mainak MajumderMonash Univ.

Collaborations:

Dali QianRodney AndrewsLeonidas BachasAudra Stinchcomb

Center for Nanoscale Science and Engineering UKy

Dr. Ji Wu, Georgia St