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An Electrically Heated Au Electrode for ElectrochemicalDetection in Microchip System

Di Wu, Jian Wu, Yu-Hua Zhu, Jing-Juan Xu,* Hong-Yuan Chen

The Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing210093, P. R. China*e-mail: xujj@nju.edu.cn

Received: October 25, 2009Accepted: January 8, 2010

AbstractAn electrically heated Au electrode which can be used for microchip electrophoresis has been constructed andevaluated. The Au electrode is heated by direct current (DC) getting across the copper wire enlacing the Au electrode.Control and measurement of the temperature at the surface of the heated Au electrode can be easily achieved.Electrochemical characteristics of the heated electrode are investigated. Separation and determination of dopamine(DA) and catechol (CA) on poly(dimethylsiloxane) (PDMS) microchip are performed to evaluate the feasibility andreliability of this heated electrode detection system.

Keywords: Heated electrode, Amperometric detection, Electrophoresis, Microchip, Dopamines, Catechol

DOI: 10.1002/elan.200900512

1. Introduction

The performance of the amperometric detection is stronglyinfluenced by the working electrode where the reaction ofinterest occurs. The current response of the electrode isinfluenced by temperature for its impact on free energy,diffusion coefficients, and kinetic parameters such as theelectron-transfer rate at the electrode surface. Heatingelectrode to improve sensitivity has become a new tendencysince temperature can influence every parameter in electro-chemistry. Heated electrode offers several advantages forelectroanalysis. Firstly, higher temperature on surface ofelectrode can enhance the thermal convection in the vicinityof the electrode surface. Secondly, some reactions inert atroom temperature are accelerated at increased temperature.Many electrochemical reactions are kinetically sluggish,while occur at considerable rates at a higher temperature.

Microchip electrophoresis is becoming a powerful tech-nique in separation science due to its short analysis time, lowsample consumption, and cheap instrumentation. Amper-ometry is the most widely reported EC detection method formicrochip electrophoresis. Heated electrode can be used toimprove the response of the amperometry. Heated elec-trode which can be used as a detection electrode inmicrochip should meet two requirements. Firstly, theelectrode should be small enough to suit the microchip.The working area of the electrode should be the approx-imate size of the separation channel section. Secondly, theelectrode should be thermal insulated from the cell of themicrochip electrophoresis. The whole buffer in the cell couldbe heated easily if the electrode was heated without anythermal insulator.

Two kinds of techniques for heating can be used to elevatethe temperature of the electrode; direct heating and indirectheating. Direct heating means that the electrode body itselfis heated [1 – 2]. In 1966 Ducret and Cornet first described adirect electrode heating [3]. Gr�ndler [4 – 7] have designedsymmetrical hot-wire electrodes which can avoid undesireddistortion caused by alternating current. A series of researchwas done by Wang and Gr�ndler [8 – 10] and hot wireelectrodes were first proposed to be used in electroanalyt-ical flow systems, developing a new promising thermalmodulation amperometric approach for monitoring flowingschemes. Sun [11, 12] fabricated a new heated graphitecylinder electrode, and applied an electrically heated carbonpaste electrode to capillary electrophoresis [13, 14]. Bar-anski [15, 16] introduced a hot-disk microelectrode heatedby using an alternating potential of very high frequency(0.1 – 2 GHz) and high amplitude (up to 2.8 V rms). How-ever it is difficult to make them suit the microchip system,because symmetrical structure which is required for thesehot wire electrodes to avoid AC distortion limits theirfurther miniaturization. The smaller the electrode is, themore asymmetric resulting from fabrication technology willbe, which will lead to large interference.

On the contrary, indirect heating is increasing the temper-ature of the electrode by heater with an electrical isolationbetween electrode and heater. An indirectly electricallyheated gold film electrode was developed by Harima et al. in1976 [17]. Gr�ndler and co-workers designed indirectlyelectrically heated electrodes based on Low TemperatureCo-fired Ceramic (LTCC) technology and did a lot ofresearch [18 – 22]. An array of indirectly heated Pt micro-electrodes was presented by Yang et al. [23]. Capillary

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electrophoresis with microwave-enhanced electrochemicaldetection [24] was presented, inducing strong localizedthermal activation to enhance sensitivity. Indirect heatingelectrodes which are getting rid of the distortion of AC aremuch easier to be miniaturized than the direct heating oneand there is an opportunity to use the indirect heatingelectrodes in microchip electrophoresis.

In this paper, we apply a unique indirect heating approachby copper wire to heat Au electrode in microchip electro-phoresis for the first time which is a relatively unexploredarea of chemistry. Separation and determination of dop-amine(DA) and catechol(CA) on poly(dimethylsiloxane)(PDMS) microchip are performed to evaluate the feasibilityand reliability of this heated electrode detection system.

2. Experimental

2.1. Reagents and Solutions

Sylgard 184 (PDMS) was from Dow Corning (Midland, MI,USA). DA and CAwere purchased from Sigma-Aldrich. K3

[Fe(CN)6], K4[Fe(CN)6], KCL, Na2HPO4, KH2PO4 andperchloric acid were purchased from Nanjing ChemicalReagents Factory (Nanjing, China). Phosphate buffersolution (PBS) as the running buffer was prepared withNa2HPO4 and KH2PO4. Before use, all samples and buffersolutions were filtered through 0.22 mm cellulose acetatefilter (Xinya Purification Factory, Shanghai, China). Thestock solutions of samples (10.0 mM) were prepared bydissolving analytes in 0.1 M perchloric acid. Prior to use,they were diluted with corresponding running buffer. Allother chemicals were of analytical grade and used without

further purification. All solutions were prepared in deion-ized water by a Milli-Q water purification system (Millipore,Milford, MA, USA).

2.2. Working Electrode Construction

The construction of heated Au electrode is shown schemati-cally in Figures 1A – C. A 200 mm gold wire (Fig. 1A) wascoiled by a lacquered wire (diameter¼ 130 mm) twice inadverse direction (Fig. 1B) avoiding induction voltage. Asshown in Figure 1C, gold wire twisted by copper wire wasspread by epoxy resin. Then the whole thing fabricated inthe former step was covered by a plastic shell. The room waskept between epoxy resin and plastic shell for thermalinsulation. The section of the spun gold was used as theworking electrode and the lacquered wire was the heater.Before use, the working area of Au electrode was carefullypolished with shammy and rinsed with pure water andcorresponding buffer. The electrode can be used carefullyfor a long time avoiding heating to an extremely hightemperature.

2.3. Instruments and Procedure

PDMS/PDMS microchip was fixed on a Plexiglas holderwith a precisely three-dimensional adjustor (Shanghai LianYi Instrument Factory of Optical Fiber and Laser, China). Atraditional three-electrode system was used with the heatedAu electrode as a working electrode, a platinum wire as anauxiliary and an Ag/AgCl as a reference one. The end of theheated Au electrode was placed ca. 40 mm far from the end

Fig. 1. A – C Schematic diagram of the heated Au electrode. The construction of working electrode. a: gold wire; b: lacquered wire; c:epoxy resin; d: plastic shell; e: working area of the electrode. D – E The Image of the microchip electrophoresis coupled with heated Auelectrode; D: schematic diagram of the heated system; E: infrared thermal image of the heated system reaching the stable temperature of35 8C. (E is transformed from a colorful picture. The real color at electrode part is gray with the temperature of 35 8C, while thesurrounding color is changed from white to red with the temperature from 17.2 8C to 14 8C.)

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of the separation channel. The structure of the chip and theheating electrode is shown in Figure 1D. Electrochemicaldetection was performed using “amperometric i – t curve”mode with a CHI 630A electrochemical workstation (CHICo., Shanghai, China). A homemade high-voltage power(0 – 5000 V) was used to supply high voltage. A directcurrent (DC) power supply RXN-303A (Shengzhen ZhaoXin Electronic Equipments and Instruments Producer,Shengzhen, China) was connected to the lacquered wire ofthe heated Au electrode to provide steady current forheating. Infrared Thermal Imager SC3000 (FLIR System,USA) was used to examine the temperature of the solution.

Samples were injected into the separation channel using asimple crossing mode. During the separation procedure, thesampling and waste buffer reservoirs were kept floating. Theseparation current could be monitored in real time.

3. Results and Discussion

3.1. Control and Measurement of the Temperature at theSurface of the Heated Au Electrode

As mentioned earlier, the thermal insulation of the elec-trode is very important, so an infrared thermal imager wasused to evaluate the effect of thermal insulation of theelectrode .The solution temperature in the vicinity of theelectrode can be observed through an infrared thermalimager. Figure 1D is the schematic diagram of the heatedsystem, pane in the diagram is the area of the electrode.Figure 1E displayed the final steady heating state; differenttemperature was expressed in different colors. In Figure 1E,the temperature of the solution in a small distance of theelectrode surface increased 1 – 2 8C while the whole bulksolution remained unchanged.

Control and measurement of the electrode temperature isalso important for the investigation of the electrochemicaland microchip electrophoresis signals acquired with heatedAu electrode. Indirect temperature calibration by open

circuit potentiometry [5] was reported because directmeasurement of the temperature was not practicallyfeasible. For calibration, a heated Au electrode and a Ptelectrode were placed into a solution of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] in 1 M KCl. The potential of the heatedelectrode changes due to the change in temperature, leadingto change of open circuit potential of a reversible redoxcouple. The exact electrode temperature can be calculatedusing the temperature coefficient (1.56 mV/K for K3

[Fe(CN)6]/K4[Fe(CN)6]) of the electrode potential. Thenthe relationship between the electrode potential with theheating current (A) was also determined. The relationshipbetween the heating current and the temperature ofelectrode surface could be deduced as shown in Figure 2.

3.2. Electrochemical Characteristics of the Heated AuElectrode

As shown in inset of Figure 3, a constant potential (0.7 V vs.Ag/AgCl) was supplied to the working electrode in 20 mMPBS. The current increased as the increase of the temper-ature at the electrode surface in accordance with Gr�n-dler�s report [5]. No obvious noise augment was observedat high temperature. So the analytical signal of the analytescould be enhanced without noise enhancement when thetemperature of the electrode surface increased. Thecurrent could reach a stable value rapidly when thetemperature altered indicating that the control of temper-ature was sensitive.

The voltammetric behavior of the heated Au electrodewas tested using K3[Fe(CN)6] system. Figure 3 showed thecyclic voltammograms of the heated Au electrode at differ-ent temperatures. Increasing the electrode surface temper-ature resulted in the increase of the peak current. Increasing

Fig. 2. Relationship between the heating voltage and the tem-perature.

Fig. 3. CV of K3[Fe(CN)6] on heated Au electrode in KClsolution at different temperature values: K3[Fe(CN)6], 5 mM; KCl,1 M; scan rate 50 mV/s; a :20 8C; b : 30 8C; c : 40 8C; d :47; e :53 8C.Inset: Current on the heated CPE at different temperature. (1)17 8C; (2) 22 8C; (3) 28 8C; (4) 35 8C; (5) 41 8C; (6) 54 8C.

Electrically Heated Au Electrode

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the temperature of electrode could greatly speed updiffusion and also led to convection of redox couple at athin layer near the surface of the electrode [14]. At lowertemperature, the electrode showed the macroelectrodebehavior with peak-shaped signals due to diffusion. How-ever, at higher temperature limiting current for both redoxcouples can be obtained and the electrode showed thesteady state behavior. The limiting current could beattributed to enhanced convection and diffusion at theelectrode surface. Temperature gradient would also existresulting in enhanced convection. So, the diffusion andconvection would be enhanced and the steady statebehavior would exist as a result. With the increase of thecurrent, the analytical signal of the analytes would beenhanced and we could profit from it.

In the vicinity of the electrode, when the temperature ofthe electrode surface increased, the viscosity of the solutionthere would decrease causing a larger diffusion coefficientdue to the Stoke – Einstein equation.

D¼ kT/6prih (1)

where D is the diffusion coefficient, k is Boltzmann�sconstant, T is the absolute temperature, ri is the radius of thediffusing species, and h is the viscosity of the solution. It wasreported [7] that the temperature dependence of thelimiting currents shows an Arrhenius-type behavior as isexpected based on the temperature dependence of thediffusion coefficient

Ilim¼ Ilim,1 exp(�EA/RT) (2)

Ilim(T)¼ zFAcbulkD(T)/ri (3)

where Ilim is the limiting current at temperature T, z is thenumber of electrons transferred per molecule, F is Faraday�sconstant, A is the electrode area, cbulk is the bulk concen-

tration. Apparently, the limiting current will increase if thetemperature increases.

3.3. Analysis of DA and CA

DA and CA were employed here as a model system todemonstrate the temperature effects. The cyclic voltam-metric measurements of 50 mm DA and CA at cold and hotconditions were obtained in 100 mM phosphate bufferpH 7.0. The effect of the temperature increase was demon-strated in Figure 4. A significant rise of the current and amore reversible wave shape were observed in accordancewith literature [24]. The current increased with the rise of thetemperature and the degree of DA and CA signal enhance-ment was almost the same.

The separation and determination of DA and CA wereperformed to evaluate the feasibility and reliability of thisdetection system. There are many factors that affect micro-chip electrophoresis separation and determination, such asthe pH and concentration of the buffer solutions, thepotential applied to the working electrode, separation andsample injection voltages and so on. In our experiment, theconditions of separation and detection of DA and CA wasoptimized as that the separation buffer solution was pH 7.0,20 mM PBS, the injection voltage was 500 V, the injectiontime was 3 s, the separation voltage was 1000 V, thedetection potential was 0.8 V. Under these conditions, DAand CA were baseline separated within 70 s in a 2.8 cmseparation channel. Good reproducibility was gained bothat room temperature and under elevated temperature.RSDs of DA and CA for run to run, day to day, chip to chip atcold and hot conditions were all less than 1.6 (n¼ 3).

What we care most was the improvement of the signal ofDA and CA under elevated temperature. The peak currentwas greatly enhanced and the noise was not changed toomuch as shown in Figure 5A. The following equation can be

Fig. 4. CV of 50 mM DA(A) and CA(B) on heated Au electrode at 17, 25, 35, 45, 55 8C (from inner to outer) in 0.1 M PBS with a scanrate of 50 mV/s.

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used to express the enhancement of peak current underheating electrode:

R¼ Iheating/I0 (4)

where Iheating is the peak current when the electrode washeated, and I0 is the peak current without heating. As isshown in Figure 5B, R increased with the rise of thetemperature. In addition, no obvious augment of noise wasobserved compared with the obvious enhancement of peakcurrent in the range of 17 – 40 8C. Noise can not be ignoredwould exist at a temperature higher than 40 8C. So, asignificant improvement in sensitivity and detection limitdue to the improvement of the signal-to-noise ratio (S/N)was achieved. The linear ranges for DA and CA at cold andhot conditions were all from 50 to 500 mM with thecorrelation coefficients (r) above 0.99 (Figure 6). Theelectropherograms of 5 mM DA and CA is shown in theInset of Figure 6. The detection limits at high temperaturewere much lower than the results obtained at the unheatedelectrode, which was consistent with the trend of R. Thedetection limit was 3.0 mM (DA) and 7.4 mM (CA) at 17 8Cwhile 0.9 mM (DA) and 2.8 mM (CA) at 40 8C. The detectionfor DA and CA indicated that the present microchipelectrophoresis coupled with heated Au electrode providedhigh sensitivity and low detection limit.

4. Conclusions

An electrically heated Au electrode which can be used formicrochip electrophoresis has been constructed and firstused in microchip system. The Au electrode is heated by DCgetting across copper wire enlacing the Au electrode. Thetemperature at the surface of the heated Au electrode can bedetected by measurement of open circuit potential. Electro-chemical characteristics of the heated electrode are inves-

tigated. Separation and determination of dopamine(DA)and catechol(CA) on poly(dimethylsiloxane) (PDMS) mi-crochip are performed to evaluate the feasibility andreliability of this heated electrode detection system. Theelectrode has been tested by separation and detection of DAand CA, and the results indicated that the present microchipelectrophoresis detection system equipped with an electri-cally heated Au electrode provides lower detection limitthan the unheated one. The preliminary results indicatedthat the new microchip electrophoresis -heated electrodesystem is a promising approach with strong potentialapplications. Different substances can be distinguished bytheir different characters at different temperature. Thesystem described here opens up a way to couple the heated

Fig. 5. (A) Electropherograms of 50 mM DA and CA at different temperature. 1):17 8C; 2): 20 8C; 3):25 8C; 4): 30 8C; 5):40 8C. (B)Influence of different Te on the enhancement of peak currents. Conditions: running buffer, 20 mM PBS (pH 7.0); injection voltage, atþ500 V for 3 s; separation voltage, at þ1000 V; detection potential, at 0.8 V; the peaks were DA and CA in the migration time order.

Fig. 6. The calibration curve of DA and CA at 40 8C (a) and17 8C (b). Inset: Electropherograms of 5 mM DA and CA at 40 8C(a) and 17 8C (b). Measuring condition are the same as those inFig. 5.

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electrode technique with microchip electrophoresis andfurther research needs to be done.

Acknowledgements

This work was supported by the National Natural ScienceFoundation (No. 20890021), the National Natural ScienceFunds for Creative Research Groups (20821063), and the 973Program (2007CB936404, 2007CB714501) of China. Wethank Prof. J. J. Sun for his help in fabrication of heatedelectrodes.

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