Biosensors and Bioelectronics 20 (2005) 1559–1565

Simultaneous detection of the release of glutamate andnitric oxide from adherently growing cells using an array of

glutamate and nitric oxide selective electrodes

Jaime Castilloa, Sonnur Isikb, Andrea Blochlc, Nazare Pereira-Rodriguesd,Fethi Bediouid, Elizabeth Csoregia, Wolfgang Schuhmannb, Joshua Onib,∗

a Department of Biotechnology, Lund University, S-22100 Lund, Swedenb Anal. Chem., Elektroanalytik and Sensorik, Ruhr-Universit¨at Bochum, Universit¨atsstr. 150, D-44780 Bochum, Germanyc Lehrstuhl fur Molekulare Neurobiochemie, Ruhr-Universit¨at Bochum, Universit¨atsstr. 150, D-44780 Bochum, Germany

d Ecole Nationale Sup´erieure de Chimie de Paris, Laboratoire de Pharmacologie Chimique et G´enetique, Fre Cnrs Enscp 2463 + U Inserm 266,11 rue Pierre et Marie Curie, 75231 Paris cedex 05, France

Received 21 May 2004; received in revised form 24 July 2004; accepted 4 August 2004Available online 27 September 2004

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individuale hydrogel-b ly charged Nip ectrode bya ntly growingC ses recordeda uale ighbouringe©

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bstract

The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which thelectrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redoxased glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positiveorphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working elbienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adhere6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current respont each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individlectrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the nelectrode or other analytes present in the test mixture2004 Elsevier B.V. All rights reserved.

eywords: Electrode array; Biosensor; Glutamate oxidase; Nitric oxide; Modified electrodes; Transmitter release

. Introduction

The simultaneous detection of chemical substances re-eased by adherently growing cells is not only of utmost im-ortance for the elucidation of signal transduction pathways

n vivo but also for drug testing and the possible replace-ent of animal tests by cell-based assays. Of equal signifi-

ance is the simultaneous detection of the release of multipleransmitter substances by living cells upon stimulation withhe appropriate agents. Neurotransmitters are chemical sub-tances that aid in transmitting impulses between nerve cells

∗ Corresponding author. Tel.: +49 234 322 5464; fax: +49 234 321 4683.E-mail address:joshua.oni@ruhr-uni-bochum.de (J. Oni).

or between a nerve cell and a muscle (Dox et al., 1979).Thus the highly sophisticated mechanism of communtion between populations of neurons is largely kept oational by neurotransmitters. A complex interplay betwthe variations in the concentration levels of several trmitter substances is one of the many factors that canto the manifestation of neurological and psychiatric diders (Bradford, 1986; Marsden and Fahn, 1982; Bird andIversen, 1974). The ability to carry out simultaneous dettion of neurotransmitters will go a long way in shedding mlight onto the complex mechanisms involved in the deopment of neuropsychiatric disorders and considerablyprove the procedure of diagnosis and management ofdiseases.

956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2004.08.021

1560 J. Castillo et al. / Biosensors and Bioelectronics 20 (2005) 1559–1565

The development of sensors that can display high sensitiv-ity and selectivity towards the analysis of various neurotrans-mitters but at the same time being insensitive to interferencespresent in the complex biological media is the major hurdlethat has to be crossed in order to make the simultaneous detec-tion of neurotransmitters released by cells a reality. An arrayof electrodes in which the individual electrodes making upthe array are separately addressable and permits the tailoringof the sensitivity and selectivity of each electrode towardsa particular analyte, by functionalisation with the appropri-ate sensing chemistry, provides an easy access to the devel-opment of sensors for the simultaneous detection of multi-ple analytes. The feasibility of the modification of individ-ual electrodes in an array with different sensing chemistriesfor the detection of nitric oxide released by endothelial cellswas recently demonstrated (Oni et al., 2004). The concept ishereby extended to the simultaneous detection of glutamateand nitric oxide from adherently growing C6 cells.

Glutamate is an important excitatory transmitter in themammalian central nervous system (Fagg and Foster, 1983)believed to be responsible for certain fundamental processessuch as learning, memory, neuro-development and synapticplasticity (Martin et al., 2000; Johnston and Rogers, 1998;Urbanska et al., 1998; Riedel and Reymann, 1996). Excessivestimulation of glutamatergic receptors can cause excitotoxicn Ca2+

l ies.O ducet k loopw ts.T ancenJ ei d/orm

itsi um-b tho-l andH

ell-e ely dev ;C( -a ion ofg ntlyg

2

2

Cl,N r

(all from Riedel de-Haen, Seelze, Germany). Arginine andl-glutamic acid was from Sigma (Steinheim, Germany).Glutamate oxidase (EC 1.4.3.11, Cat No. 7804) from Ya-masa Corporation (Tokyo, Japan) and horseradish peroxi-dase (EC 1.11.1.7, code HRP3C) from Biozyme Laborato-ries (Blaenavon, Wales, UK) were obtained as lyophilisedpowders having activities of 8.7 and 150 U mg-1 solid, re-spectively. Poly(vinylimidazole) complexed with [Os(4,4′dimethylbipyridine)2Cl] (PVI-dme-Os) was synthesised ac-cording to a well established procedure (Ohara et al., 1994).Poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) usedfor cross-linking glutamate oxidase, horseradish peroxidaseand the Os-modified redox polymer was obtained from Poly-science, Warrington, PA, USA. 4-(N-tetramethyl)pyridyl por-phyrin (H2TmPyP) free base and bradykinin were purchasedfrom Aldrich (Steinheim, Germany). The metallation of theporphyrin free base was accomplished by adopting the pro-cedure previously described (Adler et al., 1970) to obtainNi(4-N-tetramethyl)pyridyl porphyrin (NiTmPyP). Acrylicacid, butyl acrylate and di-tert-butyl peroxide used for thesynthesis of electrodeposition paint (EDP) (Isik et al., 2004;Neugebauer et al., 2003) were purchased from Fluka (Seelze,Germany).

2.2. Electrode preparation

ucedb witha RL,H ei m-i Ptfi( rlyd er onP wasm mgN5 ctro-c tialso sA gc n theapmfw f itsp least2

2

glem um,2 an

eurodegeneration by extensive increase of intracellularevels followed by the formation of reactive oxygen specn the other hand, several misfunctions, like ischemia, in

he release of glutamate and thereby enforce a feedbachich will facilitate the development of excitotoxic evenherefore, glutamatergic transmitter release might enheurodegeneration after stroke or hypoglycaemia (Stone andavid, 1983; Lodge, 1988). A hypofunctioning of glutamatn the central nervous system may result in psychotic an

emory disturbances.The interest in the detection of nitric oxide arises from

dentification as a bio-regulatory molecule that plays a ner of important roles in several physiological and pa

ogical processes (Dawson and Dawson, 1996; Moncadaiggs, 1995; Durante et al., 1988).In this report, we describe the incorporation of w

stablished sensing chemistries that have been separateloped for the detection of glutamate (Belay et al., 1999ollins et al., 2001; Mikeladze et al., 2002) and nitric oxide

Oni et al., 2004; Isik et al., 2004) into an array of individully addressable electrodes for the simultaneous detectlutamate and nitric oxide from a population of adhererowing C6 cells.

. Materials and methods

.1. Chemicals

Hank’s buffer (pH 7.4) was prepared from NaCl, KaH2PO4, MgCl2, CaCl2, NaHCO3, glucose, HEPES buffe

-

Planar electrodes arrays (1 mm diameter) were prody sputter deposition of gold onto a polycarbonate basen intermediate titanium layer as adhesion layer by Ceathrow, UK (see alsoOni et al., 2004). The electrod

ntended for modification with nitric oxide sensing chestry was first platinised by reductive deposition of thinlms from an oxygen-free aqueous solution of 2 mM H2PtCl6Schuhmann, 1998a,b) because earlier investigations cleaemonstrated that the sensing chemistry performs bettt than on Au. The resulting platinised gold electrodeodified by electrodeposition of a mixture containing 1iTmPyP dissolved in 500�L triply distilled water and00�L EDP polymer suspension in a three-electrode elehemical cell using pulse amperometry with pulse potenf 2200 mV (versus Ag|AgCl) for 0.2 s and 0 mV (versug|AgCl) for 5 s (Isik et al., 2004). The glutamate sensinhemistry was imparted onto the gold electrode surface irray by carefully dropping a premixed hydrogel (2�L) pre-ared by mixing horseradish peroxidase (5 mg mL-1), gluta-ate oxidase (10 mg mL-1), PVI-dme-Os (2.5 mg mL-1) and

reshly prepared PEGDGE (2.5 mg mL-1) in doubly distilledater. The PEGDGE solution was used within 15 min oreparation. The hydrogel was allowed to set in air for at0 h before use.

.3. C6 cell culture

C6-glioma were grown in Dulbecco’s modified eaedium (DMEM) supplemented with 10% foetal calf sermM glutamine and 100 U penicillin/streptomycin in

J. Castillo et al. / Biosensors and Bioelectronics 20 (2005) 1559–1565 1561

atmosphere of 10% CO2. The cells were routinely splittedin a ratio of 1:3 every three days using phosphate bufferedsaline (PBS) with 2 mM EDTA for detachment. On the daybefore experiments, cells were seeded in a density of 106 per10 mm diameter tissue culture inserts with nanopore mem-brane. For measurements, the insert was transferred into awell of a 24 well microtiter plate containing the modifiedelectrodes at its bottom and incubated with modified Hank’sBuffer.

2.4. Instrumentation

A CHI 1030 eight-fold potentiostat (CH Instruments,Austin, USA) was used for the electrodeposition of the por-phyrin paint mixture onto platinised gold electrode surfaces

Fte

in a conventional three electrode electrochemical cell set-up.The simultaneous amperometric detection of nitric oxide andglutamate was carried out with a Biometra EP-30 bipotentio-stat (Biometra, Gottingen, Germany). A potential of−50 mVwas applied to the electrode modified with glutamate sensingchemistry while a potential of +750 mV was applied to theelectrode modified with nitric oxide sensing chemistry.

3. Results and discussion

3.1. Sequence of electrode surface modification

A representation of the design of the electrode array at thebottom of a well used in this investigation is shown inFig. 1.

ig. 1. (a) Representation of the array of electrode employed in this study:he reference electrode. (b) Schematic representation and photograph of thlectrodes on the bottom.

i, ii and iii are working electrodes; iv, v and vi are counter electrodes; and vii ise well plate with and without the membrane insert positioned over the integrated

1562 J. Castillo et al. / Biosensors and Bioelectronics 20 (2005) 1559–1565

The array of electrodes consists of three working electrodes(i, ii, iii), three counter electrodes (iv, v, vi) with one counterelectrode close to each working electrode and a shared pseudoAg/AgCl reference electrode (vii).

Two of the three working electrodes in the array werefunctionalised with the appropriate sensing chemistry for thedetection of NO and glutamate, while the third electrode wasnot used. One major factor that determines the success of theuse of an array of electrodes, whose individual componentsare to be modified with different sensing chemistries, is thesequence of the electrode modification step. This has to becarried out in such a way that the properties of the modifieron an already modified electrode surface are not perturbedor altered by the modification process of the neighbouringelectrode. The two sensing chemistries were considered anda sequence of imparting them on the electrode surface wasfashioned out based on the volume of reagents involved indepositing them on the electrode and an evaluation of therisk of the “contamination” of a sensing chemistry alreadyin place on a neighbouring electrode. The NO sensing chem-istry needed to be deposited from a “relatively large” volumeof reagents (≈1 mL that covered the entire base of the elec-trochemical cell containing all the electrodes) as comparedto the glutamate oxidase/redox hydrogel-based biosensor ar-chitecture (≈2�L, confined to the surface of the individuale NOs ge ofd n oft ateb NOs mates o airab TheN thise elec-t ando n ofN t ando shlym

3

im-p ce ofe hichi rvesd , ob-t NOa NOo earc ationo t thee others it is

intended to be employed. In particular, in the present study,beside other potential interferences such as ascorbic acid, ni-trite, nitrate the electrode fashioned as NO sensor should notbe sensitive to glutamate and vice versa. Therefore, at firstonly glutamate was deliberately added to the test solutionleading to the expected response at the glutamate-selectiveelectrode but no visible effect at the NO sensor. In the sameway, the current signals of glutamate were not affected by theaddition of NO to the test mixture. These results clearly sug-gest that the two electrodes modified with sensing chemistriesfor the selective detection of glutamate and NO, respectively,can be employed simultaneously for the detection of NO andglutamate without any expected chemical crosstalk betweenthem.

3.3. Choice of cell culture for the simultaneousdetection of NO and glutamate

To detect the simultaneous release of NO and glutamate,a cell line is required that is capable of secreting both NOand glutamate. After a careful consideration of different celllines, C6-glioma cells were selected. C6-glioma cells are ob-tained from a glial tumour in the rat brain. They are knownto express bradykinin receptors (Brismar, 1995) as well asglutamatergic receptors of the metabotropic (Martin et al.,1s neu-r ns do( it-a luta-m torst withK ofg

3i

ellsw e fore d onC thenc elec-t e ofb -i ctc gede sur-f (e

mVv ena se ofNl oni-t n of

lectrode desired using a microsyringe). Therefore, theensing chemistry was deposited first. Another advantaepositing the NO sensing chemistry first is the preventio

he contact of its reagents with the hydrogel of the glutamiosensor; thereby avoiding a possible adsorption of theensing chemistry reagent on the surface of the glutaensing chemistry. A setting period of 20 h of exposure tt room temperature or in the refrigerator at 4◦C is requiredy the hydrogel before it can be employed for analysis.O sensing chemistry is sufficiently stable to withstandxposure to ambient air for 20 h. The performance of anrode, modified with NO sensing chemistry and left to stn the laboratory bench for 24 h, towards the oxidatioO, measured by the magnitude of the oxidation currenxidation potential, was about the same as that of a freodified electrode (not shown).

.2. Selectivity of modified electrodes

When the appropriate sensing chemistry has beenarted onto the surface of both electrodes, the performanach of the modified electrodes towards the analyte for w

ts sensitivity was tailored was evaluated. Calibration cueveloped from plots of the magnitude of current signals

ained by constant potential amperometry at +750 mV fornd−50 mV for glutamate, against the concentration ofr glutamate were linear. It is not only important that linorrelations should be obtained between the concentrf analyte in solution and the current signals obtained alectrodes, the electrode should also be insensitive topecies that could be present in the test mixture in which

998) and the NMDA subtypes (Amano et al., 1992). Be-ides, it has been verified that this cell line releasesotransmitter substances in a manner similar as neuroBugnard et al., 2002). C6-glioma cells were considered suble for use in the simultaneous detection of NO and gate since it is possible to activate its bradykinin recep

o release NO while the depolarisation of the membraneCl induces an influx of Ca2+ which stimulates the releaselutamate.

.4. Detection of the release of NO and glutamatendividually

The release of NO and glutamate from C6-glioma cas detected using the appropriate modified electrodach of the analytes. Typically, the cells were seedeellagen® disc, whose diameter is 10 mm. The disc wasarefully inserted into the chamber that constituted therochemical cell around the electrodes. The total volumuffer solution in the electrochemical cell was 500�L. Seed

ng the cells on Cellagen® discs is important to avoid direontact of the cells with the electrode surface. A prolonxposure of cells to an applied potential on an electrodeace has been shown to lead to the death of the cellsOnit al., 2004).

For the detection of NO, a constant potential of +750ersus Ag|AgCl was applied to the working electrode. Whstable background current was established, the releaO was stimulated by the addition 20�L of bradykinin so-

ution (80 nM) and the changes in current signals were mored. A representative current–time plot for the detectio

J. Castillo et al. / Biosensors and Bioelectronics 20 (2005) 1559–1565 1563

Fig. 2. Constant potential amperometric curves for the detection of (a) NOat +750 mV vs. Ag|AgCl and (b) glutamate at +50 mV vs. Ag|AgCl fromC6-glial cells. The release of NO was stimulated with bradykinin whileglutamate release was stimulated with KCl solution.

NO released from C6-glioma cells is shown inFig. 2a. Thegradual rise in the background current is due to the oxidationof NO that has diffused to the electrode surface.

Glutamate was detected at a constant potential of−50 mV.Its release from C6-glioma cells was induced by the additionof 20�L of KCl solution (1 M) to the buffer solution closeto the cells. A representative current–time trace obtained forglutamate release is shown inFig. 2b.

3.5. Effect of addition of KCl on the signal of the NOsensor and the effect of bradykinin on the signal of theglutamate-selective biosensor

Bradykinin and KCl are required to elicit the release ofNO and glutamate, respectively, from C6-glioma cells. It isnecessary to investigate the effect the addition of KCl so-lution could have on the current signals of the NO sensorand the effect the addition of bradykinin could have on thecurrent signals of the glutamate biosensor. The release ofNO from C6-glioma cells was repetitively invoked by meansof bradykinin addition (Fig. 3a, points (ii) and (iii)). 20�Lbradykinin solution (80 nM) subsequent to a first stimulation

Fig. 3. (a) Current–time plot recorded to demonstrate that the addition ofbradykinin has no effect on the current signal of glutamate, points (ii) and(iii) and (b) the addition of KCl solution has no effect on the current signalof NO, point (iii).

of glutamate release by means of KCl addition (20�L 1 MKCl solution,Fig. 3a, point (i)) and the current at the gluta-mate sensor was recorded.

There was no significant change observed in the initialcurrent signal, which was due to glutamate release from thecells, upon the subsequent addition of bradykinin. Similarly,after invoking the release of NO from C6-glioma cells uponstimulation with bradykinin, (Fig. 3b, points (i) and (ii)), KClsolution was added (point (iii)) and the current at the NO sen-sor was monitored. Although the addition of KCl solution re-sulted in a sharp drop in the “baseline” current due to the localchange in ionic strength of the electrolyte solution concomi-tant with the need to re-establish the electrical double layer,the previous background current was attained. The observa-tions clearly demonstrate that bradykinin can be used safelyin the vicinity of the glutamate biosensor to stimulate NOrelease from C6 glioma cells without affecting the responseof the glutamate biosensor. Although effects of variationsof the ionic strength in the vicinity of the NO sensor seemto lead to intermediate changes in the background current

1564 J. Castillo et al. / Biosensors and Bioelectronics 20 (2005) 1559–1565

Fig. 4. Constant potential amperometric curves showing the simultaneousdetection of (a) NO at +750 mV vs. Ag|AgCl and (b) glutamate at−50 mVvs. Ag|AgCl released from C6-glioma cells after simultaneous stimulationof the cells with a mixture of bradykinin and KCl.

of the NO sensor, NO determination is again possible af-ter waiting for the re-establishment of the initial backgroundcurrent.

3.6. Simultaneous detection of the release of NO andglutamate from C6-glioma cells

NO and glutamate were detected at two different poten-tials. A constant potential of +750 mV was applied to the NOsensor while−50 mV was applied to the glutamate sensor.Thus, a bipotentiostat was necessary to allow for the applica-tion of two different potential values to two electrodes at thesame time. A Cellagen® disc containing a population of C6-glioma cells was carefully inserted into the electrochemicalchamber around the electrodes. After the establishment of astable background current at both electrodes, 20�L of a mix-ture containing 10�L (160 nM) bradykinin and 10�L KClsolution (2 M) was carefully injected into the buffer solutionaround the cells, and changes in current signals at both sensorwere monitored. The result is shown inFig. 4. The current sig-nals due to the detection of NO (curve a) and glutamate (curveb) released from the cells can be clearly seen. This demon-strates the feasibility of a simultaneous detection of chemicalsubstances released from cells, provided the appropriate sensing chemistries are imparted to the individual electrodes ande

4

singc lec-t thes dher-e erK eache ual

electrode in the array to detect the analyte towards which itssensitivity and selectivity were targeted without interferencefrom the neighbouring electrode or other analytes present inthe test mixture. Future development will aim on the appli-cation of the developed system for drug screening and eval-uation of complex signal transduction pathways.

Acknowledgements

Financial support from the European Union in the frame-work of the projects “Cellsens” (QLK-CT-2001-00244) and“INAS” (INTAS 01-2065) is acknowledged. The authors aregrateful to Dr. H. Kusakabe (Yamasa, Tokyo, Japan) for thegift of glutamate oxidase.

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