Nitric Oxide Sensor based on Silicon Planar TechnologyMarcelo B.A. Fontes§*,Lúcio Angnes‡, Koiti Araki‡,

Rogério Furlan§, Jorge J.Santiago-Avilés†

§Laboratório de Sistemas Integráveis (LSI), Universidade de São Paulo - Brasil -*bariatto@lsi.usp.br‡Instituto de Química da Universidade de São Paulo, †University of Pennsylvania -USA

AbstractThe small molecule nitric oxide (NO) mediates manyphysiological processes related to neurology ,immunology, and muscle cell action. In this study wemarried the technology of silicon planar with modifiedelectrodes to explore the electrochemical detection ofthis molecule using differential pulse voltammetry (DPV)measurements. Typical dimensions of the sensor are: alength of 10 mm and a width of 3 mm. It contains 8 golddetection microelectrodes (10 µm x 10 µm), a Ag/AgClreference electrode and a gold counter electrode. Theactivity of nitric oxide and nitride was detected in themicromolar range with and without covering theelectrodes with a well known cationic film Nafion®. Itwas observed the influence of two different DPVconditions in the sensor sensitivity. The detection limitand sensitivity obtained for the nitric oxide was10 µM and 7,02x10-4A/M respectively.

1. IntroductionMicroelectrode arrays based on silicon technology havebeen used with success on several applications, such asaction potentials1 and single neuron activity2 detection.Due to the small dimensions involved, microfabricationpresents many advantages including: batch fabrication,easy shape definition by computer designed masks, highreproducibility and low cost3-5.In this article we married the technology of siliconplanar with modified electrodes to explore theelectrochemical detection of the nitric oxide (NO)molecule using amperometric measurements (differentialpulse voltammetry - DPV)6,7. The NO molecule hasattracted attention in the recent years because the manyinteractions in the human body8,9. In the atmosphere it isa noxious chemical, but in the body, in small controlleddoses, it is extraordinarily beneficial. It helps to maintainblood pressure by dilating blood vessels, helps to killforeign invaders in the immune response, is a majorbiochemical mediator of penile erection and is probablya major biochemical component of long-term memory.NO measurement in biological systems is difficult due tothe small amounts present, usually in the nanomolarrange, and the rapid interaction with oxygen generatingnitride ion (NO2

-). The detection of the activity of suchmolecules in small concentrations is vital in manystudies in vitro and in vivo as well as in diagnostic andtherapy10,11.

We have developed a general purpose silicon basedelectrochemical sensor composed of an array ofmicroelectrodes, described in details in a previouswork12,14.The (DPV) technique was used in two differentconditions15,16 of applied potential to detect the activity ofboth NO and NO2

- in solution with and without coveringthe electrodes with Nafion® - a well known cationicexchange.

2. Sensor design and fabricationThe sensor is composed of a silver/silver chloridereference electrode, a gold counter electrode (2 mm x0.5 mm) and 8 gold detection microelectrodes (10 µm x10 µm). Typical dimensions of the sensor are: a lengthof 10 mm and a width of 3 mm. Silicon nitride film wasused as a protective material. In order to reduce theelectrolyte resistance in the electrochemicalmeasurements, the reference electrode was placed asclose as possible to the detection electrodes. The counterelectrode was placed relatively far from them, tominimize the interference of the chemical speciesgenerated on it. One feature that has to be pointed out isrelated to the counter electrode area. It has to be at least10 times larger than the detection electrodes12. In ourcase this factor is about 10,000.Figure 1 shows SEM images of the fabricated sensor,where the contact pads and the detection, reference andcounter electrodes can be seen.

Figure 1 - SEM image of the sensor front side and details.

Figure 2 shows schematically the sensor fabricationsequence.

Contacts PadsAu

0.5 x 0.5 mmCounter

Au 2 x 0.5 mm

Si3N4 ReferenceAg/AgCl

DetectionAu

10 x 10 µm

Lines20 µm

SENSOR3 x 10 mm

Figure 2 - Sensor fabrication sequence

The sensor was fabricated using the silicon planartechnology through the following sequence :a)Wet oxidation of 2" silicon wafers (p type, orientation:<100>, resistivity 1 - 10 Ωcm) to obtain at least 1 µm ofSiO2;b)NiCr e-gun evaporation (adhesive material);c)Gold film e-gun evaporation (300 nm);d)Gold and NiCr electrode lines (20 µm wide), contactpads (500 µm x 500 µm), reference (68,800 µm2) andcounter (2,000 µm x 500 µm) electrode areas, patterningwith mask 1;e)Si3N4 deposition by sputtering (~300 nm);f)Si3N4 plasma etching (CF4+O2) over contacts, detection,counter and reference areas, defined by mask 2, using anegative photoresist as a etching mask;g)Scribing to individuals sensors;h)Wiring bonding to a carrier;i)Covering of the bonding pads and the gold wires with apolyethylene resin;j)Selective silver electrodeposition and silver chlorideformation by chemical reaction, on the referenceelectrode.

After fabrication the sensors were physicallycharacterized by means of scanning electron microscopy(SEM) and atomic force microscopy (AFM). It was alsoperformed a electrochemical characterization of thesensors electrodes, as described in a previous work12,14.

3. Experimental procedureWe prepared the electrolyte solution by using a mixtureof 40 mM of K2HPO4 and 10 mM of KH2PO4 indionised water (DI, 18 MΩ) to achieve 50 mM ofphosphate buffered (PBS; pH = 7.4). The nitric oxidewas obtained by first purging the PBS with highly purenitrogen gas during 1 hour in a gas tight burette and sobubbling NO gas (White Martins,10 % in N2) duringanother one hour. We assume at this point that thesolution was saturated and the NO concentration wasabout 2 mM13. Our cell was filled with 75 ml of PBS andalso purged with N2 during 1 our. We achieved themicromolar range by adding microliters of the NOsaturated solution in the cell. For the nitride solution inthe micromolar range, several droppings of 75 ml of 10mM sodium nitride (NaNO2) were used. In order to

cover our electrodes with Nafion® (Aldrich, 1% inethanol), one drop of it was applied and let to air dry, soan uniform coverage was obtained. After each additionof analyte a stirring step was done.All experiments were performed with a fully computercontrolled three-electrode potentiostatic system (BAS -Bioanalytical System CV 50W) in a Faraday cage, figure3. Two different DPV conditions were used, as describedin the table 1 and illustrated by figure 4.

Figure 3 - Experimental setup for nitric oxide detection

Figure 4 - Illustration of the parameters of the appliedpotential in the DPV analysis.

Parameter Conditionno 1 no 2

Potential sweep (mV/s) 20 12.5Pulse amplitude (mV) 50 75Pulse width (mV) 50 80Pulse period (ms) 200 400Current sampling (ms) 17Potential range (mV) 400 - 900

Table 1 - DPV parameters

Gas tight burette

Electrochemical system

Faraday cage

Sensor assembly Reactionchamber

stirring

carrier

Pulse width

Potentialsweep

Pulse amplitude

Current sampling

Pulse period

4. Results and DiscussionThe response of one of the detection microelectrodes ofthe sensor for the nitride oxidation in concentrationsranging from 1 µm to 100 µm, is shown in figure 5, forboth conditions of applied potential. It can be seem forcondition no1, figure 5(a), that the oxidation potentialoccurs around 830 mV with a slight shift towards815 mV with the for increase of the nitrideconcentration. On the other hand for condition no2,figure 5(b), those values were observed for potential of805 mV and 800 mV, respectively. It is also observedthat the oxidation current increase by a factor ofapproximately 2 for the condition no2. This can beclearly seen in the figure 6 where the peak current isdisplayed against the nitride concentration. In both casesa linear relationship was observed in the measuredrange. Their slope are related to the sensor sensitivity.The sensitivity obtained were 3,92x10-5A/M and8,67x10-5 A/M for condition no1 and no2, respectively.The detection limit was around 5 µM for both cases.

-7

-6

-5

-4

-3

-2

-1

0400 450 500 550 600 650 700 750 800 850 900

100 µM

50 µM

10 µM

5 µM

1 µM

Potential (mV) Ag / AgCl

Cu

rre

nt (

nA)

-15

-12

-9

-6

-3

0

400 450 500 550 600 650 700 750 800 850 900

100 µM

50 µM

10 µM

5 µM

1 µM

Potential (mV) Ag / AgCl

Cu

rre

nt (

nA

)

Figure 5 - DPV response for one of the sensorsmicroelectrode with a increase of the nitride concentration,for both conditions of the applied potential: a) conditionno1 and b) condition no2.

4

6

8

10

12

14

Condition no 2

Condition no 1

- I p

ea

k (n

A)

Figure 6 - Response of the sensor for current versus nitrideconcentration for both conditions of the applied potential.

The Nafion® film, of used to reduce the activity of thenitride, is showed in the figure 7 covering the sensorsdetection microelectrodes.

Figure 7 - SEM images of the sensors microelectrodesmodified by a film of Nafion®

The response for the nitride in the one of the detectionmicroelectrodes, after being modified by the Nafion®, isshown in the figure 8. For both conditions of appliedpotential current obtained was strongly reduced forconcentrations below 60 µM. This is particularly true forcondition no1, where no peak could be defined.Comparing these results with the sensor withoutmodification, the peak current was reduced by a factorof 8, and the sensitivity was also reduced, by a factoraround 2. Clearly the nitride activity was reduced bymodification of the electrode with a film of Nafion®.

0

0.5

1.0

1.5

2.0

10 20 30 40 50 60 70 80 90 100

condition no 2

condition n o 1

[NO-1

2] (µM)

Cur

ren

t (n

A)

Figure 8 - Sensor response of one microelectrode modifiedwith a film of Nafion ® for increasing nitride concentrationfor both conditions of the applied potential: a) conditionno1 and b) condition no2.

The response of one of the sensors microelectrodes,modified with Nafion®, for the nitric oxide oxidation in amicromolar concentration, is showed in figure 9, forboth conditions of applied potential. It can be seem forcondition no1, figure 9(a), that the oxidation potentialoccurs around 790 mV with a slight shift towards770 mV for increasing nitric oxide concentration. Itappears that there are two oxidation reactions takingplace at electrode surface. The same behavior wasobserved for condition no2, figure 9(b), where thosevalues were observed for potential of 780 mV and755 mV, respectively. Again it was observed that theoxidation current increases by a factor of approximately2 for the condition no2.

Nafion

b)a)

Ο condition no2 condition no1

Ο condition no2 condition no1

-20

-16

-12

-8

-4

0

400 450 500 550 600 650 700 750 800 850 900

80 µM

70 µM

60 µM

40 µM

50 µM

30 µM

20 µM

10 µM

Potencial (mV) Ag / AgCl

Cor

rent

e (n

A)

-40

-32

-24

-16

-8

0

400 450 500 550 600 650 700 750 800 850 900

80 µM

70 µM

60 µM

50 µM

40 µM

30 µM

20 µM

10 µM

Potencial (mV) Ag / AgCl

Cor

rent

e (

nA)

Figure 8 - DPV response for one of the sensorsmicroelectrode modified with Nafion® with the increase ofnitric oxide concentration for both conditions of theapplied potential: a) condition no1 and b) condition no2

The peak current increases linearly with nitric oxideconcentration for both cases, figure 10, where theobtained sensor sensitivity presented two rangesdepending on the concentration. Values up to 50 µM,figure 9(a), presented sensitivity of 1,39x10-4 A/M and3,75x10-4 A/M for condition no1 and no2, respectively.For concentrations between 50 µM and 80 µM, figure9(b), these values were 3,91x10-4 A/M and 7,02x10-4

A/M for condition no1 and no2, respectively. Thedetection limit was around 10 µM for both cases.

0

2

4

6

8

10

12

14

15 20 25 30 35 40 45 50

[NO] (µM)

- I p

eak

(nA

)

0

8

16

24

32

40

55 60 65 70 75 80 85 90

[NO] (µM)

- I p

eak

(nA

)

Figure 10 - Sensor response of one microelectrode modifiedwith a film of Nafion ® for two ranges with the increase ofnitric oxide concentration for both conditions of theapplied potential: a) range up to 50 µM and b) from 50 µMto 80µM

A comparison of the sensors response for 40 µM ofnitric oxide and nitride with and without modifying thedetection microelectrodes with Nafion® is shown infigure 11, where condition no2 was applied due its highercurrent and sensitivity. It can be clearly seen the roleplayed of the nitride as a inteferent in a nitric oxidedetection, since their oxidation potential are very close.It is also shown the reduction of the nitride activity byusing Nafion®.

-10

-8

-6

-4

-2

0

400 450 500 550 600 650 700 750 800 850 900

NO2

- with Nafion

NO with Nafion

NO2

- without Nafion

Potential Ag / AgCl (mV)

Cu

rre

nt (

nA)

Figure 11 - Results for 40 µM of NO and NO2- with and

without Nafion®, using condition no2.

The reduction of the activity of the nitride is particularlyimportant for low nitric oxide concentrations. Figure 12presents the previous results comparing the variation ofsensors response in the micromolar range for nitric oxideand nitride with and without Nafion®.. For concentrationbelow 40 µM, the current detected by the sensor, withoutbeing modified, is higher for nitride than for nitric oxide.After the sensors modification, as described before, thecurrent due to nitride presented a reduction by a factor of8, indicating that its interference is satisfactorily reducedin the nitric oxide detection.

0

5

10

15

20

25

30

35

40

10 20 30 40 50 60 70 80 90

NO2

- with Nafion

NO2

- without Nafion

NO with Nafion

Concentration (µM)

- I

pea

k (n

A)

Figure 12 - Sensor response for nitric oxide and nitride in amicromolar range, with and without modification of itsmicroelectrodes with a film of Nafion®, using condition no2.

ConclusionsThis paper described the design and fabrication of anarray of microelectrodes based on silicon planartechnology, composing a general purposeelectrochemical sensor. It consists of 8 gold detectionmicroelectrodes, a Ag/AgCl reference electrode and agold counter electrode. The activity of nitric oxide andnitride was studied in the micromolar range with andwithout covering the electrodes with a well knowncationic film Nafion®.. It was also verified the influenceof the DPV parameters in the sensors response. Thecondition defined as no2, presented best results forsensors sensitivity and current. Its parameters are :potential range (400 mV to 900 mV), potential sweep

b)a)

b)a)

Ο condition no2 condition no1

Ο condition no2 condition no1

rate (12.5 mV/s), pulse amplitude (75 mV), pulse width(80 mV), pulse period (800 ms) and sample width(17 ms). Using this parameters, the measurements of thesensor for nitride, a interferent in the nitric oxidedetection, presents sensitivity of 8,67x10-5 A/M anddetection limit around 5 µM. It was observed that onedrop of Nafion® decrease the nitride activity by a factorof 8 and the sensor sensitivity was reduce by 2, for thisanalyte. Results in the nitric oxide detection, in the sameconditions, showed a concentration depend sensitivitywith values of 3,75x10-4 A/M up to 50 µM and7,02x10-4 A/M, for higher concentrations. The detectionlimit was about 10 µM. It was observed that nitridereduction of the activity by Nafion® is particularlyimportant for low nitric oxide concentrations, below40 µM, in our results.More research is on the way focusing to achievenanomolar concentration of the nitric oxide by electrodemodification and flow techniques.

AcknowledgmentsThe authors wish to acknowledge the support offered byFAPESP (contract 96/8274-5), LME/USP for the wirebonding process and the facilities of the Center forSensor Technology at UPENN and Institute ofChemistry at USP. The authors also wish to specialthank help offered by Mr. Vladimir Dominko atUPENN.

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