Biosensing with Functionalized Single Asymmetric Polymer Nanochannels

Communication

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Biosensing with Functionalized SingleAsymmetric Polymer Nanochannels

Mubarak Ali,* Birgitta Schiedt, Reinhard Neumann, Wolfgang Ensinger

In this work, we describe the direct covalent attachment of protein recognition elements(biotin) with the carboxyl groups present on the walls of polyimide nanochannels. Sub-sequently, these biotinylated channels were used for the bio-specific sensing of proteinanalytes. Moreover, surface charge of these asymmetric nanochannels was reversed fromnegative to positive via the conversion of carboxylgroups into terminated amino groups. The nega-tively charge (carboxylated) and positivelycharged (aminated) channels were further usedfor the electrochemical sensing of bovine serumalbumin (BSA, pI¼ 4.7). These biorecognitionevents were assessed from the changes in theionic current flowing through the nanochannel.

Introduction

Solid-statenanochannels fabricated in ion-trackedpolymer

membranes have a great range of applications in bio-

technology,[1] where they are suitable for sensing bio-

molecules,[2] and act as stimuli-responsive devices,[3] and

molecular filters of high selectivity[4] as well as nanofluidic

diodes.[5] Therefore, for all these applications, it is highly

desirable to control the channel-surface properties, i.e. to

functionalize the surface in order to match specific

requirements concerning hydrophobicity, selectivity, and

to achieve desired interactions with molecules of interest.

It has been proven, that the asymmetric nanochannels

in polymer membranes rectify the ionic current[6] (i.e.

preferential transports of cations (anions) from the narrow

M. Ali, W. EnsingerTechnische Universitat Darmstadt, Fachbereich Material-u.Geowissenschaften, Fachgebiet Chemische Analytik,Petersenstraße 23, D-64287 Darmstadt, GermanyE-mail: [email protected]. SchiedtMPI, Universite Val d’Essonne, 91025 Evry Cedex, FranceR. NeumannGSI Helmholtzzentrum fur Schwerionenforschung GmbH,Planckstr. 1, D-64291 Darmstadt, Germany

Macromol. Biosci. 2010, 10, 28–32

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

entrance towards the wide opening of the channel) similar

tovoltage-gatedbiological ion channels. The rectificationof

ionic current occurs due to asymmetry in the electro-

chemical potential inside the asymmetric nanochannel

havingfixed surface charges. Previously,Wei et al. havealso

reported the rectification of ionic current at the nanopipet

electrodesystemsand investigatedthat thecurrent-voltage

(I-V) behavior depends sensitively on the size of electrodes

as well as on the concentration and pH value of the

electrolyte solution.[6e]

Single asymmetric nanochannels in polyimide (PI)

membranes have been used for the detection of DNA[7a]

and porphyrin molecules,[7b] which cause the blockage of

ionic current during their translocation through the

channel. Recently, based on the rectification behavior,

these nanochannels were successfully used for the sensing

of organic analytes (crown ether)[7c] and drugmolecules[7d]

in theelectrolyte solutionused formeasuring the I-V curves.

Previously, Siwy et al. have reported the protein sensing[8]

with gold coated asymmetric nanotubes, where the

incorporation of molecular recognition element was

achievedvia thechemisorptionof thiolmolecules. Recently,

we have demonstrated the protein sensing[9] by incorpor-

ating an electrostatic self-assembly of bifunctional macro-

molecule (biotinylated poly(allylamine hydrochloride)

having biorecognition moieties in its backbone. I-V curves

DOI: 10.1002/mabi.200900198

Biosensing with Functionalized Single Asymmetric . . .

reflect the polarity of the channel surface, and can also be

able to detect or sense any foreign analyte in the electrolyte

via bio-specific or electrostatic interactionwith the channel

surface functionalities. Within this framework, here we

describe a very simple and facile strategy for the direct

covalent attachment of bio-recognition elements with the

walls of PI nanochannels (Figure 1).

Experimental Part

Materials

Polyimide (PI) (Kapton 50 HN, DuPont) membranes of 12mm

thickness were irradiated at the linear accelerator UNILAC (GSI,

Darmstadt) with single swift heavy ions (Pb, U, and Au) of energy

11.4MeV per nucleon. N-(3-dimethylaminopropyl)-N’-ethylcarbo-

diimide hydrochloride (EDC, 98%, Fluka), pentafluorophenol (PFP,

99þ%, Aldrich), ethylenediamine (EDA, 99þ%, Merck, Germany)

and biotin-PEO3-amine (Pierce) were used as received for the

chemical modification. Albumin bovine serum (BSA, 99%, Sigma,

Germany), phosphate-buffered saline (PBS, pH¼7.6, Sigma),

Figure 1. Schematic representation of a single asymmetric nanochannenoncovalent binding of streptavidin analyte.



potassium chloride (Merck, Germany) and fluorescein (FITC)-

conjugated streptavidin (Pierce) were used as received.

Asymmetric Nanochannel Fabrication

In order to obtain asymmetric nanochannels, the swift heavy ion

irradiated foilswereetched fromonesideonly inaconductivitycell

in which it served as a dividing wall between the two compart-

ments.[10,11] Sodium hypochlorite (13% active chlorine content),

was used as the etching solution, while a stopping solution (1M KI)

was filled on the other side of the membrane. The etching process

was carried out at 50 8C. In order to monitor the etching process, a

voltage of �1V was applied across the membrane. Initially, the

current flowing across themembranewas remained zero and after

the break-through, continuous increase of ionic current was

observed. The etching process was terminated when the quantity

of current flowing through the nascent channel reached a certain

value. Shortly after breakthrough the channels were washed with

stopping solution, followed by deionized water.

After etching, the diameter of the large opening (D) of the channel

was measured by field emission scanning electron microscopy

l functionalized with biotin-PEO3-amine and subsequent bio-specific

www.mbs-journal.de 29

M. Ali, B. Schiedt, R. Neumann, W. Ensinger

30

(FESEM)usingaPI samplecontaining107 channelscm�2whichwas

etched simultaneously with the single channel under the same

conditions. The diameter of the small opening (d) was estimated

from its conductivity by the following relation:[10]

d ¼ 4LI=pDkV

where L represents the length of the channel, k the specific

conductivity of the electrolyte, V the voltage applied across the

membrane and I indicates the measured current.

Functionalization with Ethylenediamine and

Biotinamine

All the surface functionalization reactions were carried out in the

same cell used for the etching process. The carboxyl groups on the

channel surface were first activated by derivatizing into penta-

fluorophenyl esters.[16] For activation, an ethanolic solution

containing 0.1M EDC and 0.2M PFP was placed on both sides of

the track-etched PI membrane with single nanochannel. The

activation was carried out for 1 h at room temperature. After

washingwith ethanol, the solutionwas replacedwith an ethanolic

solution of ethylenediamine (0.100M) or biotin-PEO3-amine

(0.050M) on both sides of the membrane and allowed to react

overnight. Then, the chemically modified membranes having

terminus amino and biotin functionalities were thoroughly

washed first with ethanol followed by distilled water.

Current-Voltage (I-V) Measurements

The membrane containing the single asymmetrical nanochannel

wasmounted between the two halves of the conductivity cell, and

both half of the cell were filledwith 0.01M phosphate buffer saline

(0.138M NaCl; 0.0027M KCl, pH¼ 7.6) prepared in 1M KCl solution.

ThepHof theelectrolytewasadjustedwith0.1MNaOHor0.1MHCl.

AAg/AgCl electrodewasplaced into eachhalf-cell solution, and the

Keithley 6487picoammeter/voltage source (Keithley Instruments,

Cleveland, OH) was used to apply the desired transmembrane

potential in order to measure the resulting ion current flowing

Figure 2. (a) Current-voltage curves for single asymmetric PI nanochannand (b) I-V curves corresponding to a biotin functionalized asymmetricand with adding 10�7 M of (*) lysozyme, (*) BSA, and (") streptavi



through the nanochannel by applying a scanning triangle voltage

from �2 toþ 2V on the tip side while the base side of the channel

remain connected to the ground electrode.

Results and Discussion

Single asymmetric nanochannelswere fabricated in PI foils

of 12mm thickness, irradiated with single swift heavy ions

from the UNILAC linear accelerator (GSI, Darmstadt) by an

asymmetric track-etching technique.[10] During the track-

etching process, carboxyl (�COOH) groups were generated

on the channel surface. These carboxyl groups were first

converted into amine-reactive pentafluorophenyl esters by

reacting with an ethanol solution containing a mixture of

EDC and PFP. Subsequently, these PFP esters were further

coupled with either EDA or biotin-PEO3-amine, leading to

the terminated�NH2 andbiotinmoieties, respectively. The

characterization of the functionalized nanochannels was

done by measuring the I-V curves, which originate from

their charged surfaces. Here, we also describe the bio-

specific/electrochemical sensing of protein analytes using

functionalized nanochannels.

Figure 2 (a) describe that before functionalization, the

single asymmetric nanochannel rectified the ionic current

atpH¼ 7.6,asexpected fornegativesurfaces,whileatacidic

pH¼ 2.0, the carboxyl groups were protonated (neutral

surface), leading to the linear I-V curve.

After functionalization with bio-recognition elements, a

significant change in the I-V curves was observed,

attributed to the diminution of the channel surface charge

due to the conversion of carboxyl groups into terminated

biotinmoieties. This leads to the loss of rectifying behavior

of the channels (Figure 2b). The biofunctionalized channels

were further used for the sensing of bio-specific protein

analytes. When the biotinylated channel was exposed to

electrolyte containing either lysozyme or bovine serum

el (carboxylated) having small opening diameter (d¼9 nm) in 1 M KClnanochannel in 1 M KCl at pH¼ 7.6, (&) without any protein analyte,din, respectively.

DOI: 10.1002/mabi.200900198

Biosensing with Functionalized Single Asymmetric . . .

albumin (BSA),wedidnotobserveanysignificant change in

the ionic current. Because both lysozyme and BSA are

nonspecific, i.e., do not biorecognize biotin moieties. When

the I-V curves were measured in the presence of strepta-

vidin (SVn), a significant decrease in ionic current was

observed (Figure 2b) due to the noncovalent binding of

streptavidin with the biotin groups. This suppression of

ion current have also been observed in biotin-tagged

alamethicin and gramicidin A channels.[12] As is well-

known the biotin/SVn system shows a very stable and

strong interaction[13] having a dissociation constant of

KD¼ 4� 10�14. Therefore, this additional decrease in ionic

current is likely to be caused by the partial blockage of

the channel by the streptavidin molecule. The above

experimental results demonstrate that the covalently

functionalized biotin groups inside the channel can only

bio-specifically recognize SVn but not lysozyme or BSA

proteinanalytes, and that thesebiorecognitioneventswere

manifested via the changes in the ionic current flowing

through the nanochannel.

In addition to bio-specific interaction, carboxylated

(negatively charged), and aminated (positively charged)

channels were also studied for the electrostatic interaction

of BSA (pI¼ 4.7) protein. The overall charge on the BSA

molecules was tuned with the change of pH value of the

solution. At neutral and basic pH values, the BSA is

negatively charged while it becomes positively charged at

lower acidic pH.[14]

At neutral pH, when the channel was exposed to BSA in

the electrolyte solution, we did not observe any change in

the I-V curve, measured before the addition of BSA analyte

(Figure 3a). At this pH, an electrostatic repulsion exists

between the both negatively charged channel and BSA

molecules.[16] At acidic pH¼ 3.0, the PI nanochannel still

rectifies the ionic current but to a lower degree, indicating

the presence of some ionized �COO� groups along with

protonated�COOHgroups. ByaddingBSA in theelectrolyte

Figure 3. I-V characteristics of a single asymmetric carboxylated cha(b) acidic pH¼ 3.0, (&) prior and (*) after the addition of BSA (10�



solution, the ionic current flowing through the channelwas

drastically reduced (Figure 3b) due to the strong electro-

static interactions between the oppositely charged analyte

molecules and channel surface. We have already reported

the electrochemical interaction of BSA analytes with

the surface of nanochannels, fabricated in ion-tracked

poly(ethylene terephthalate) (PET) membranes.[16] In con-

trast to PI nanochannels, at pH¼ 3.5 the carboxylate groups

present on the PET nanochannels were completely proto-

nated and the rectification vanishes. Furthermore, the

hydrophobic adsorption of positively charged BSA analytes

led to the inversionof rectification. But in thecaseofPI, even

at lower acidic pH¼ 3.0, the nanochannel still showed the

rectification of ionic current. This means that the PI

nanochannels were still negatively charged at pH¼ 3.0 as

compared to PET nanochannels. The novelty in the present

work is that here we have investigated an additional effect

of strong electrostatic interactions between BSA analytes

and PI nanochannel at low pH values. From the above

discussion, it is evident that the properties of the bulk

material as well as the surface charge density also play a

significant role for the interaction of BSA analytemolecules

with the channel surface.

When the carboxyl groups were modified with EDA, the

overall charge on the channel surface is nowpositive due to

the protonation of terminated amino groups into ammo-

nium ions. This leads to the inversion of rectification as

expected for a positively charged channel.[5b,15] Figure 4a

shows that at acidic pH, the addition of BSA did not induce

any significant change in the I-V curve because both

(channel and BSA) are positively charged and electrostatic

repulsion occurs between them. At neutral pH, the

negatively charged BSA molecules electrostatically shield

the positively charged ionized amino groups. This results in

a linear I-V curve (Figure 4b) which represents that the

positively charged channel surface was neutralized by

negatively charged BSA molecules.

nnel (d¼ 10 nm) measured in 1 M KCl solution at (a) neutral pH and7 M) in the electrolyte, respectively.

www.mbs-journal.de 31

M. Ali, B. Schiedt, R. Neumann, W. Ensinger

Figure 4. I-V characteristics of a single aminated channel (d¼ 12 nm) measured in 1 M KCl solution at (a) acidic pH¼ 3.0 and (b) neutral pH,(&) prior and (*) after the addition of BSA (10�8 M) in the electrolyte, respectively.

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Conclusion

In conclusion, here we demonstrated the covalent attach-

ment of biological ligands (biotin) and subsequent sensing

of protein analytes with the biofunctionalized nanochan-

nels. In addition, it was also shown that protein (BSA)

analyte interact with the charged channel surface by

simply tuning the pH of the external environment.

These results explore the possibility to biofunctionalize

single asymmetric nanochannels in polyimide foils, which

are especially chemically robust and have very stable ion

current signals. This opens theway to use nanochannels in

this material as a biosensor for the sensing of protein

analytes bio-specifically or electrochemically.

Acknowledgements: M. A. thanks M. Nawaz Tahir (MainzUniversity) for the fruitful and enlightening discussions aboutthe use of biomolecules.

Received: June 4, 2009; Accepted: June 10, 2009; Published online:August 14, 2009; DOI: 10.1002/mabi.200900198

Keywords: functionalization of polymers; ion channels; mem-branes; nanotechnology; polyamides; sensors

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DOI: 10.1002/mabi.200900198

Biosensing with Functionalized Single Asymmetric Polymer Nanochannels

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