Electrochimica Acta 51 (2005) 938–942

Electrochemical nitric oxide microsensors based on two-dimensionalcross-linked polymeric LB films of oligo(dimethylsiloxane) copolymer

Dai Katoa, Masashi Kunitakeb, Matsuhiko Nishizawac,Tomokazu Matsuec, Fumio Mizutania,∗

a National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-higashi, Sapporo 062-8517, Japanb Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan

c Graduated School of Engineering, Tohoku University, Sendai 980-8579, Japan

Received 22 October 2004; received in revised form 14 March 2005; accepted 25 April 2005Available online 15 August 2005

Abstract

Two-dimensional cross-linked polysiloxane Langmuir–Blodgett (LB) films were prepared and applied to nitric oxide (NO)-permselectivem micro-disce nsitivity toN electrodet size ands©

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embranes in order to block other electroactive interfering species. The cross-linked siloxane LB films deposited on platinumlectrodes (10�m in diameter) offered revealing high performances as a permselective membrane for NO sensor such as high seO (detection limit, 40 nM) and high selectivity (e.g., the ratio of current response for acetaminophen or uric acid on the modified

o that on the bare electrode, less than 10−3). Furthermore, the permselective membrane could be easily deposited irrespective of thehape of electrode.2005 Elsevier Ltd. All rights reserved.

eywords: Microsensor; polysiloxane; Langmuir–Blodgett film; Nitric oxide; Scanning electrochemical microscopy

. Introduction

A sensitive and rapid measurement method for in situ nitricxide (NO) monitoring is a field of great interest since NO haseen found to be a molecular messenger in biological systems

1–3]. Amperometric determination of NO can be accom-lished by the utilization of electrochemical oxidation at aetal or catalyst-attached electrode[4–10]. However, such

ystems generally suffer from electrochemical interferencey other oxidizable species such as nitrite, acetaminophen,ric acid andl-ascorbic acid, resulting in a positive error in

he current response[11]. The use of the coating layer aimedt ensuring to impermeability to the other electroactive inter-

ering species. Moreover, we need to downsize the electrodes,hich can be used for measurement of NO released from

he biological samples. For example, a micro NO-sensinglectrode equipped with a scanning electrochemical micro-

∗ Corresponding author. Tel.: +81 11 857 8920; fax: +81 11 857 8915.E-mail address: mizutani.fumio@aist.go.jp (F. Mizutani).

scopic (SECM) system[12–16] would be a useful tool foin vivo NO monitoring[17,18]. Consequently, it is necesary to reduce the thickness of the permselective memaccording to the size of the electrode. In order to achsuch a NO-microelectrode sensing system, the developof microelectrode coated with an appropriate, ultrathin mbrane as a permselective layer is of importance.

We have investigated homogeneous two-dimensionalcross-linked Langmuir–Blodgett (LB) films of oligo(dmethyl-siloxane) copolymer prepared by polymer–polycross-linking on an air–water interface[19–21]. The crosslinked LB films showed high permselectivity for NO eventhe monolayer thickness, whereas the LB films without crlinking revealed almost no restriction for interfering specThese results clearly show that the cross-linking of thefilms played an important role in permselectivity. In adtion, the combination of the 2D cross-linked network wthe flexible side chains gave a higher mechanical strengthe practical usage of ultrathin films on a platinum electwith a millimeter size[20,21]. Thus, the cross-linked silo

013-4686/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.

oi:10.1016/j.electacta.2005.04.072

D. Kato et al. / Electrochimica Acta 51 (2005) 938–942 939

ane LB films could be applied for the fabricating the sensorswith micro size. This paper describes the fabrication of theNO-selective microelectrode modified with the permselec-tive films based on the cross-linked siloxane LB films and itsproperties as a NO microsensor.

2. Experimental

2.1. Microsensor fabrication

We used a platinum disc microelectrode (diameter: 10�m,outer diameter including the insulating glass part; 4.0 mm,Bioanalytical Systems, West Lafyette, IN). The microelec-torode was initially cleaned by a wet chemical treatment in afreshly prepared piranha solution (3:1 v/v mixture of H2SO4and 30% H2O2) at 80◦C for 1 h followed by an extensiverinse with ultraclean water (Millipore)[22]. After blowingthe microelectrode surface dry with nitrogen, trimethylsily-lation of the glass portion surrounding the Pt electrode wasperformed by immersing the microelectrodes for 4 h in a1,1,1,3,3,3-hexamethyldisilazane (HMDS, Aldrich) solution(HMDS/chloroform = 1:1 v/v) at 40◦C [23]. The HMDS-modified microelectrode was washed with chloroform andacetone and dried.

yer,a db n-d1 tain-i no rriedo s.T lu-t ledw ched

F matici

where the L film is the state of the limited occupied area (ca.1.2 nm2/siloxane chain unit). The L film was cross-linked bythe injection of a polyallylamine (PAA,Mw: 100,000; NittoBoseki Co., Japan) aqueous solution into the trough (finalconcentration of PAA, ca. 10 mMunit). The 2D cross-linkedmonolayers were found to be microscopically homogeneousby Brewster angle microscopy. Finally, the cross-linked Lfilms were deposited onto the HMDS-treated microelectrodeby a downward stroke of horizontal deposition method. TheLB films-modified microelectrode in this way is schemati-cally illustrated inFig. 1(b). Furthermore, a microelectrodefor SECM (diameter: 10�m, outer diameter; ca. 30�m,Hokuto Denko, Tokyo) was also fabricated in the same man-ner as described above.

2.2. Amperometric measurements

A potentiostat (HA-150; Hokuto Denko, Tokyo) was usedin a three-electrode configuration; the LB films modifiedelectrode, an Ag/AgCl electrode (saturated with NaCl,Bioanalytical Systems) and a platinum wire were employedas the working, reference and auxiliary electrodes, respec-tively. The electrodes were placed in a stirred 0.1 M sodiumphosphate buffer solution (pH 7.0, 20 mL) at 25± 0.2◦Cunder argon atmosphere. The magnetic stirrer and the testsolution were placed in the Faraday cage to minimizee ciess .85 Vv no moS dera no mM[

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ntot elec-t eri-m enti t theh e LBfi sub-s obice r andt rode(

tedl ode( (in0 cal

In order to form the homogenous siloxane monolan amphiphilic siloxane copolymer (Fig. 1(a)) was preparey the copolymerization of a vinyl monomer with peant oligosiloxane side-groups (SILAPLANE FM0711,Mw:000; Chisso Co., Japan) and glycidyl methacrylate, con

ng epoxy groups for covalent cross-linking[20]. Preparatiof the 2D cross-linked polysiloxane monolayers was caut as previously reported[20]. The procedure is as followo form a Langmuir monolayer (L film), a benzene soion of the copolymer was spread into a LB trough filith pure water, and compressed until a point is rea

ig. 1. The chemical structure of siloxane copolymer (a) and a schellustration of the cross-linked LB films-based microelectrode (b).

lectric noises. Then, either NO or interfering speolutions were added and the current responses at +0ersus Ag/AgCl were recorded[24,25]. A standard solutiof NO was prepared by bubbling it (99.7%, Sumitoeika Chemicals, Tokyo) into pure water, which was unrgon atmosphere, at 25.0± 0.2◦C. The NO concentratiof the saturated solution was reported to be 1.88

26].

. Results and discussion

.1. Effect of the hydrophobic treatment of the glassurface of the microeletrode

Before the deposition of the cross-linked LB films ohe microelectrode, the outer glass part of the base microrode was modified with HMDS as indicated in the expental section. It is well known that the HMDS treatm

s widely used as the surface modified reagents to geydrophobic surfaces of the solid substrates. The siloxanlms demonstrate superior deposition onto hydrophobictrates rather than hydrophilic ones, owing to the hydrophffect between the oligosiloxane groups of the copolyme

he trimethylsilyl groups on the glass surface of the electseeFig. 1(b)) [27].

Fig. 2 shows cyclic voltammograms of bare Pt (dotine) and the HMDS-treated Pt (solid line) microelectrØ-10�m) in 5 mM potassium ferrocyanide solution.1 M KCl). Both voltammograms indicated the typi

940 D. Kato et al. / Electrochimica Acta 51 (2005) 938–942

Fig. 2. Cyclic voltammograms of bare Pt (dashed line) and the HMDS-treated Pt (solid line) microelectrode (Ø-10�m) in 5 mM potassium ferro-cyanide solution (in 0.1 M KCl), at the rate of 20 mV s−1.

sigmoidal-shape cyclic voltammetric responses. There wereno significant differences in voltammograms between thebare and the HMDS-modified electrode. These resultssuggest that the electron transfer of ferrocyanide remainednearly unaffected by the trimethylsilyl groups on the glasssurface of the electrode. In fact, in both electrodes systems,the equivalent oxidation currents to electroactive speciessuch as NO and the other interfering substances wereobserved. These results indicated that the trimethylsilylgroups did not interfere with the oxidation of the elec-troactive species; the treatment was effective for enhancingonly the adhesion of the cross-linked LB films to the basemicroelectrode.

In the case of using the LB films-modified microelectrodewithout the hydrophobic treatment, the obtained responsesshowed significant sample-to-sample variability (i.e., theobtained current varies from 10 to 40�A cm−2 for NO),whereas the modified electrode with the hydrophobic treat-ment exhibited the reproducible results (ca. 15.0�A cm−2).Moreover, the electrode without the hydrophobic treatmentshowed a significant increase in the current response forthe electroactive species during the course of electrolysis.This suggests that LB films at the platinum/hydrophilicglass boundary were gradually removed or destroyed,with the result that electroactive species diffused easilytoward the platinum surface (to undergo the electrochemicalr withH edL ndr medw byu

3

e (a)a e tof ,a , the

Fig. 3. Current–time curves for the bare (a) and the cross-linked siloxaneLB films-modified (b) microelectrode (Ø-10�m) exposed to (1) NO, (2)l-ascorbic acid, (3) acetaminophen and (4) uric acid. The concentration ofeach species was 20�M. Electrode potential, +0.85 V (vs. Ag/AgCl).

current increased immediately after the addition of samplesand reached a maximum current response within 10 s(Fig. 3(a)). The cross-linked LB films-modified electrodealso exhibited a discernible response with the injection ofNO (16.9�A cm−2). In contrast, the responses for the otherinterfering species were almost negligible on the modifiedmicroelectrode (Fig. 3(b). In the case ofl-ascorbic acid,the current response was decreased from 102�A cm−2 fora bare electrode to 0.4�A cm−2 for the cross-linked LBfilms-modified electrode. The responses of acetaminophenand uric acid for bare electrode were 49.0 and 13.4,respectively, but no responses were observed (less than0.1�A cm−2).

Thus, the cross-linked LB films have proved to be effec-tive in the elimination of the interfering responses. The ratiosof current response on the cross-linked LB films-modifiedelectrode to that on the bare electrode for a variety of elec-troactive species were plotted against their molecular weight,as shown inFig. 4. The ratio obtained is clearly relative tothe permeability of the cross-linked LB films. The perme-ability of the solute into the cross-linked layer was obviouslydependent on the molecular weight; the ratio was around0.1 for the electroactive species having a molecular weight<100 and drastically decreased with increase in the molec-ular weight over 100. Mizutani et al. have reported that apolyion complex layer consisting of polycation and polyan-i ith ac tr withm ver,t gh itir er-i antlyo ers.

eaction). Thus, the modification of the glass surfaceMDS proved to be useful for obtaining cross-linkB films-based microelectrode with high selectivity aeproducibility. Subsequent experiments were perforith the cross-linked LB films-based system preparedsing the HMDS-modified electrode.

.2. Permselectivity of the cross-linked siloxane LB films

Fig. 3 shows the current response curves for the barnd the cross-linked LB films-modified (b) microelectrod

our kinds of the electroactive species; NO,l-ascorbic acidcetaminophen and uric acid. As for the bare electrode

on showed permselectivity based on the solute size wut-off molecular weight of ca. 100[28–30]. The presenesults also demonstrated a similar size exclusion effectonolayer thickness. As for hydrogen peroxide, howe

he ratio was much smaller than that of NO, even thous about the same molecular weight as NO (Fig. 4(2)). Theesult indicated that the elimination of hydrophilic interfng species such as hydrogen peroxide would be dominriginated from defect-free hydrophobic polysiloxane lay

D. Kato et al. / Electrochimica Acta 51 (2005) 938–942 941

Fig. 4. Effect of the molecular weight of the oxidizable species on the ratioof current increase after the addition of the species for the cross-linked LBfilms-based microelectrode to that for a bare microelectrode. The speciesused are: (1) NO, (2) hydrogen peroxide, (3) nitrite, (4) hydroquinone,(5) acetaminophen, (6) dopamine, (7) uric acid, (8) ascorbic acid and (9)adrenalin. The concentration of each species was 20�M and the potentialsapplied to the electrodes were +0.85 V vs. Ag/AgCl. Each ratio was averagedover three measurements.

3.3. Performance of the cross-linked LB films-modifiedmicroelectrode for SECM

Fig. 5 shows the current response curves on the cross-linked LB films-modified microelectrode for SECM. Thismicroelectrode can be used for the anodic detection of NOwithout serious error from such interfering species as muchas the case of the micro-disk electrode. As a NO microsensor,this electrodes gave a linear current response up to 100�MNO. The lower detection limit of NO was 40 nM (signal-to-noise ratio; ca. 2). The permselectivity was remained in

F films-m Thec s.A

consistent performance for at least half a year, because therewas no essential change of current responses for NO and theother interfering species, which is proving for the practicalreuse.

We attempted to monitor NO released from bovine aortalendothelial cells upon stimulation with bradykinin, using theSECM system as described in the previous papers[13–15].The tip of the cross-linked LB films-modified microelectrodefor SECM was set at 100�m distance from the cell surface.The modified microelectrode showed almost no responsesfor the interfering substances such as nitrite andl-ascorbicacid, and demonstrated the high stability even in the useof the cell experiments. However, the current response toNO was extremely low (a few pA). The small responsefor NO from the endothelial cells is attributable to the lowefficiency for collecting NO on the electrode. The tip–celldistance (100�m) was too large to collect the NO generatedfrom the cell by the tip with the small size. Furthermore,the NO generated would be partly degraded through thereaction with O2 and/or other biological molecules in theair-saturated cell-containing medium[25] during the courseof the diffusion from the cells to the tip. Therefore, theminimization of tip–cell distance is necessary to monitorNO released from the cells. We are now constructing ameasuring system with the tip–cell distance being severalmicrons.

4

rea Thec heh lingh sen-s rode.F ouldb rada-t rodew t tot itorN icro-e ary.F vityi

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ig. 5. Current–time curves response for the cross-linked siloxane LBodified microelectrode for SECM exposed to NO (a) and nitrite (b).

oncentration of each species was 20�M. Electrode potential, +0.85 V (vg/AgCl).

. Conclusion

The 2D cross-linked polysiloxane LB films wepplied to the permselective films for NO-microsensor.ross-linked siloxane LB films had applicability to tydrophobic-treated microelectrode and offered reveaigh performances as a permselective membrane for NOor, irrespective of the size and the shape of the electurther, the modified electrodes were very stable and ce used repeatedly for more than half a year without deg

ion as sensors. The cross-linked LB films-modified electith high stability is the great advantage with respec

he continuous monitoring. We are beginning to monO released from the cells using the NO-sensing mlectrode/SECM system while the data are still preliminurther investigation for detecting NO with higher sensiti

s in progress.

cknowledgment

We are very grateful to Chisso Co. for supply of the sine monomer.

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