Impedance spectra analysis to characterize interdigitated electrodes as electrochemical sensors

Electrochimica Acta 56 (2011) 8559–8563

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Impedance spectra analysis to characterize interdigitated electrodes as electrochemical sensors Sunil Rana ∗ , Robin H Page, Calum J McNeil Diagnostic and Therapeutic Technologies, Institute of Cellular Medicine, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE24HH, UK

a r t i c l e

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Article history: Received 12 May 2011 Received in revised form 12 July 2011 Accepted 13 July 2011 Available online 23 July 2011 Keywords: Electrochemical impedance spectroscopy Interdigitated electrodes Geometric capacitance Solution resistance Solution conductivity

a b s t r a c t Interdigitated electrode (IDE) arrays have been used widely for electrochemical impedance spectroscopy (EIS). Here, we present an in-depth analysis of interdigitated electrode impedance spectra. Such an analysis is necessary for the identification of the contribution of each interdigitated electrode circuit element to the impedance change. We also discuss the importance of the solution conductivity in impedance spectroscopy showing that the use of low conductivity solutions is advantageous when inter-digit solution resistance and geometric capacitance need to be monitored. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Impedance sensors are a sub-category of electrochemical sensors which use electrochemical impedance spectroscopy (EIS) as a method of signal transduction. Considerable interest has been shown in using EIS with interdigitated electrode (IDE) arrays for biological sensing [1–6] as these arrays show promise for realization of low cost point-of-care clinical test systems, for e.g., cancer diagnosis [2] and heart disease [7] due to their ease of miniaturization, batch fabrication and automation. The micro-fabricated arrays can easily be coupled with microfluidic sample preparation platforms to implement point-of-care micro-total analysis systems (␮TAS). Moreover, the possibility of label free detection makes EIS an attractive technique for biosensing [8]. Although IDE based sensors have been reported by several groups, much of the literature is based on molecular coverage of electrode surface leading to a change in Faradaic impedance (charge transfer resistance) or interfacial double layer capacitance [9,10]. This approach typically requires the use of redox ions (usually [Fe(CN)6 ](3−/4−) ) in the solution. Fig. 1(I) shows an equivalent circuit model for an IDE array in a solution containing redox ions. Here CP , CG and CDL represent the parasitic capacitance (introduced by the sensor substrate and electrical cables), the geometric capacitance between the electrode digits (with aqueous solution as the dielectric medium) and the double layer capacitance at the electrode–solution interface

∗ Corresponding author. Tel.: +44 01912227991. E-mail address: [email protected] (S. Rana). 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.07.055

respectively. RSOL , RCT and RO represent the solution resistance, charge transfer resistance (as a result of oxidation–reduction reactions at electrode surface) and the ohmic resistance in the wiring and electrode tracking respectively. A major drawback of redox reaction-based sensing is that it is difficult to obtain a stable uniform coverage of the electrode surface. Any pinholes in the molecular layer would permit reduction–oxidation reactions at the electrode surface which would in turn significantly suppress the impedance signal [11]. Moreover, extra reagents in form of electrochemical probes are required which may be photosensitive and degrade over time [12]. Another approach to impedance based sensing uses the interdigit geometric capacitance and/or the solution resistance as the transduction parameters [6]. Molecules immobilized on or between the digits alter both the geometric capacitance (CG ) as well as the solution resistance (RSOL ). It has been demonstrated that the interdigit space can be selectively functionalized using a microfabrication compatible processes [13]. This method involving the use of CG and/or RSOL is more robust and reliable as an incomplete molecular coverage of either the electrodes or the inter-digit space does not introduce noise as in the case of Faradaic impedance based method [11]. Also, in the absence of redox species in the solution, the equivalent circuit model gets simplified as the Faradaic and the Warburg impedances can be ignored. A simplified equivalent circuit model for IDEs in a redox ion free electrolytic solution is shown in Fig. 1(II). Here CP is included in CG (assuming CG  CP ) and the series combination of two double layer capacitors is represented by CDL . The term RO can be ignored as gold IDEs (presented here) have a low resistance. The above approach of using

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2. Experimental 2.1. Theory (CDL , RSOL and CG ) The impedances for RSOL (ZR ), CDL (ZCdl ) and CG (ZCg ) are RSOL , 1/jωCDL and 1/jωCG respectively where ω represents angular frequency. When these values are input in the IDE equivalent circuit (Fig. 1(II)), the total impedance can be expressed as follows: ZT = Re(ZT ) + Im(ZT )

(1)

where Re(ZT ) =

RSOL CDL 2 (CG + CDL )2 + (ωRSOL CG CDL )2

(2)

and Im(ZT ) = j

Fig. 1. (I) Equivalent circuit model for IDEs in solution containing redox ions and (II) simplified circuit model for IDEs in a solution lacking redox ions. Nodes A and B represent adjacent electrode digits. The arrows represent frequency dependent preferential paths for current.

redox-ion free solutions for sensing has been reported by several groups [3,6] although, to best of our knowledge, an in-depth analysis of the IDE impedance spectrum, specifying the transduction parameter of interest, has not been provided. Such an analysis is important because different regions of the impedance spectrum are dominated by different circuit elements. Without a detailed analysis of the impedance spectrum it is difficult to determine which sections of the spectrum provide the useful signal. Such an analysis would also help to quantify the impedance shift (whether CG or RSOL ) as a result of molecular immobilization. In this paper we present fabrication and characterization of gold IDEs on a fused silica substrate. We analyse the impedance spectra obtained from probes (that lack geometric capacitance) and use the results to analyse the IDE impedance spectra detailing the regions dominated by CG , RSOL and CDL . Such an analysis would help quantify the change in the parameter(s) (CG or RSOL ) used for transduction.

CG + CDL + CG (ωRSOL CDL )2 ω[(CG + CDL )2 + (ωRSOL CG CDL )2 ]

(3)

A simulated response of individual impedances (with RSOL = 500 , CDL = 1 × 10−6 F and CG = 1 × 10−8 F) and the impedance of Fig. 1(II) circuit (using Eqs. (2) and (3)), over the 10 Hz–1 MHz frequency range, is shown in Fig. 2. The value of CDL is taken higher than CG because, in practice, the double layer capacitance of electrode digits exceeds the geometric capacitance between the electrodes. This was also noticed consistently in experiments (data is presented in Section 3). On a Bode plot, a purely resistive impedance gives a flat line, while a purely capacitive response forms a line with a slope of −45◦ . It is evident from the frequency response of the IDE equivalent circuit that the total impedance (|ZT | in Fig. 2) is dominated by different circuit elements at different frequencies. Since ZR > ZCdl at high frequencies, the R − CDL path (Fig. 1(II)) is shunted by ZCg at high frequencies (region I in Fig. 2). However, as the frequency decreases, ZCg increases, surpassing ZR and the CG path gets shunted by ZR . Since ZCdl < ZCg , for a finite frequency range the current in the R − CDL path is only limited by ZR (region II in Fig. 2). For low frequencies ZCdl surpasses ZR and starts actively limiting the current (region III in Fig. 2). 2.2. Materials and methods The interdigitated electrodes were fabricated on fused silica substrates which, due to their lower relative permittivity, reduced

Fig. 2. Simulated data: left – impedances of CG (|ZCg |), CDL (|ZCdl |) and RSOL (|ZR |) individually and total impedance of IDE equivalent circuit (|ZT |). Right – phase response of ZT . Pure capacitive phase response would be at 90◦ while pure resistive at 0◦ throughout the frequency range.

S. Rana et al. / Electrochimica Acta 56 (2011) 8559–8563

Fig. 3. Fabrication process flow: (a) metal deposition – Cr/Au (15/200 nm) (b) photoresist spin-on and pattern, (c) ion beam etch of the exposed regions, (d) photoresist removal and clean.

the substrate parasitic capacitance in comparison to silicon substrates. The electrodes, including the inter-digit space, occupied an area of 1 mm2 . Each digit was 3 ␮m wide with a 3 ␮m inter-digit space. The fabrication details are listed in Fig. 3. A set of IDEs are shown in Fig. 4. Impedance measurements were first carried out in air in order to determine the system CG in air (CG path). This value of CG would include any parasitic capacitances in parallel, for example, due to the cables connecting the IDEs to the frequency response analyzer. This was followed by measurements in aqueous solutions of KCl of increasing conductivity (2 ␮S–10 mS). The KCl solution introduced additional impedances in the system (R − CDL path). The solution conductivity was measured using a conductivity meter (MettlerToledo International Inc.).

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Fig. 4. A set of interdigitated electrodes.

2.3.1. Probes Tungsten needle probes, mounted on a set of micromanipulators (Wentworth Labs Inc.), were used to investigate the double layer capacitance in KCl solution. The needles were positioned 20 mm apart in order to ensure there was negligible geometric capacitance between them. The needles were connected to the FRA module using shielded cables. The needle probes were dipped to a depth of 5 mm in the solution. 2.3.2. IDEs Sets of IDEs were mounted on custom made PCBs and gold wire bonded to contact pads on the PCBs. All contact pads were subsequently covered with an epoxy adhesive to electrically isolate them. Electrical connections from the FRA module to the PCB were established using single-in-line sockets. 3. Results and discussion

2.3. Impedance measurement setup 3.1. Impedance response – probes A frequency response analyser (FRA) module, on a Solartron Modulab system (AMETEK Inc.), was used for the impedance measurement experiments. Frequency scans were performed over the range 100 Hz–500 KHz with a signal amplitude of 50 mV. No DC bias was applied.

The impedance response of probes in air was purely capacitive (Fig. 5). This impedance, however, was a result of the parasitic capacitances from the connection cables/instrument because when the probes were dipped in 2 ␮S KCl solution, the impedance profile

Fig. 5. Left – impedance response of probes in air and KCl solutions of increasing conductivities; right – phase response of probes in air and KCl solutions of increasing conductivities.

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Fig. 6. Left – impedance response of IDEs in air and KCl solutions of increasing conductivities; right – phase response of IDEs in air and KCl solutions of increasing conductivities.

followed the air impedance for most of the frequency range. The geometric capacitance between the probes was negligible, otherwise it would have increased in aqueous solution (due to higher permittivity), thus lowering the total impedance. For solution conductivities in the range of 2 ␮S–1 mS a saturation in impedance value was observed as the frequency was scanned to lower values. This saturation occurred at progressively lower impedance values and at progressively higher frequencies as the solution conductivity was increased. This flat profile in the impedance response indicated a resistive impedance in parallel to the parasitic capacitance (RSOL in Fig. 1(II)). However, at very high solution conductivity (10 mS) an increase in impedance was observed at low frequencies. This indicated a capacitive impedance (i.e. CDL in Fig. 1(II)) in series with RSOL . The resistance, R, in a solid conductor is given by R = .L/A where  is the material resistivity, L the conductor length and A the cross sectional area. Similarly, the resistance between two conductors in an electrolyte can be approximated by Eq. (4) where A is the probe surface area interfacing with the solution and ˛ accounts for the probe separation and additional electrolytic effects such as electrode polarization.

ZR = ˛.

 A

(4)

The capacitance between two parallel plates is given by C = εo εr .A/d where ε0 is the electrical permittivity of free space, εr is the relative permittivity of the medium between the plates, A is the plate surface area and d is the separation between them. Double layer capacitance is similar to parallel plate capacitance where d comprises of the width of a few electrolyte molecules. However, the effect of decreasing ionic charge concentration into the solution needs to be taken into account [14]. Considering the above CDL , and hence ZCdl , can be expressed as follows

CDL =

ˇA 2 ⇒ |ZCdl | = 2 ωˇA

(5)

where ˇ accounts for ε0 , εr , d and the effects of varying charge distribution at electrode surface. A denominator of 2 in Eq. (5) is due to the fact that CDL is composed of two double layer capacitors (each originating from an area, A) in series. Using the simplifications

in Eqs. (4) and (5) it can be shown that the ratio of ZR to ZCdl is independent of the electrode surface area. ZR ˛ˇ · ω = ZCdl 2

(6)

The importance of ZR /ZCdl lies in the fact that it determines the knee point on Bode plot where ZCdl starts dominating ZR . In impedance based sensors (such as interdigitated electrodes) that use CG and/or RSOL as the transduction parameters, CDL contributes to noise. Therefore, as long as ZR /ZCdl > 1 the effect of CDL can be excluded from the impedance profile. It is evident from Eq. (6) that for a given geometry (i.e. for fixed electrode separation) the easiest way to eliminate the effect of CDL is to increase solution resistivity (higher ) or to perform impedance measurements at higher frequencies (higher ω). Alternatively, ZR could be increased by increasing the electrode separation, however, in case of interdigitated electrodes this would result in a decrease in of CG – an undesired effect if CG is of interest. 3.2. Impedance response – IDEs As in the case of the probes, the frequency response of the IDE impedance in air gave a straight line on Bode plot (Fig. 6). This impedance includes the effect of parallel parasitic capacitances due to substrate as well as cables/instrument. When the impedance was measured in a 2 ␮S KCl solution, the response was capacitive in the low as well as the high frequency regions while resistive in the mid-frequency range. The high frequency capacitive region was dominated by CG while the low frequency region was dominated by CDL . The impedance profile at high frequency (80 KHz–500 KHz) was parallel to that of air impedance, however, the impedance was significantly decreased. This decrease was due to the increase in IDE CG as a result of the increase in the electrical permittivity of the medium. With an increase in solution conductivity, the high frequency capacitive response (due to CG ) disappeared. This was also evident from the phase response – as the solution conductivity increased, the resistive region moved towards higher frequencies. This indicated that RSOL started shunting CG and the current started preferring the R − CDL path. With the increase in solution conductivity the ZR − ZCdl knee point moved towards higher frequencies and at a conductivity of 10 mS CDL dominated nearly the entire frequency range.

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For impedance sensing based purely on CG , both RSOL and CDL would contribute to noise. In order to utilize CG as the transduction parameter, only the impedance values right of the ZCg − ZR knee point can be used. Thus, a frequency range for which ZCg /ZR < 1 needs to be selected. 4. Conclusions and future work In this paper we have provided an insight into the operation of IDEs in aqueous solutions, in the absence of redox ions. Nyquist plots are generally used to visualize the frequency response of IDEs [5,6]. When quantification of transduction parameters is of essence, however, the use of Bode plots provides better representation. The use of Bode plots makes it easier to identify the contribution of different circuit parameters to the frequency response of IDEs. We used the frequency response of a pair of tungsten probes to demonstrate that ZCdl is higher than ZR only in low frequency range (the frequency range in turn depends upon solution conductivity). Therefore, when RSOL is used as transduction parameter, quantification can be achieved in the frequency region where ZR /ZCdl < 1 < ZR /ZCg . Similarly, when CG is used as transduction parameter, quantification can be achieved in the frequency region where ZR /ZCg > 1. The work presented here focuses on the quantification of RSOL and CG in the IDE equivalent circuit. It is important from the point of view of realization of a portable, point-of-care biosensing platform. Frequency scans such as the ones presented in Section 3 are routinely performed in laboratories using commercially available frequency response analyzers. These analyzer units are often quite bulky and cannot be used as part of a portable sensor. It is easier to construct a smaller module that can target one or two given frequencies and monitor the impedance at these frequencies. In order to realise such a module it is essential that the frequencies, where a quantifiable measurement of the chosen transduction parameter can be made, are known. The analysis presented in Section 3 indicates that the solution conductivity plays an important role

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in determining the frequency range over which a certain circuit element will dominate. A low conductivity solution provides an advantage that both the ZR /ZCdl and ZR /ZCg knee points are moved to lower frequency values thus permitting the ZR and ZCg measurements to be performed at relatively lower frequencies. The impedance sensing work presented here is ongoing. The interdigitated electrodes presented here will be used as the sensing element in a point-of-care biosensing platform for coeliac disease. Coeliac disease specific antibodies will be trapped in the inter-digit space and RSOL and CG will be used as the transduction parameters. Acknowledgements This work has been carried out with financial support from the Commission of the European Communities, Framework 7 programme “Coeliac Disease Management Monitoring and Diagnosis” using Biosensors and an Integrated Chip System, CD MEDICS [FP72007-ICT-1 IST-2007-2-216031]. References [1] C.J. McNeil, D. Athey, M. Ball, W.O. Ho, S. Krause, R.D. Armstrong, J.D. Wright, K. Rawson, Anal. Chem. 67 (1995) 3928. [2] C. Fernández-Sánchez, C.J. McNeil, K. Rawson, O. Nilsson, Anal. Chem. 76 (2004) 5649. [3] C. Fernández-Sánchez, A.M. Gallardo-Soto, K. Rawson, O. Nilsson, C.J. McNeil, Electrochem. Commun. 6 (2004) 138. [4] R.H. Page, Site-specific modification of surfaces for electrochemical immunoassay. PhD Thesis, Newcastle University, U.K., (2007). [5] R. Rica, C. Fernández-Sánchez, A. Baldi, Electrochem. Commun. 8 (2006) 1239. [6] A. Bratov, J. Ramón-Azcón, N. Abramova, A. Merlos, J. Adrian, F. Sánchez-Baeza, M. Marco, C. Domínguez, Biosens. Bioelectron. 24 (2008) 729. [7] A.M. Gallardo-Soto, K. Rawson, C.J. McNeil, The Chem. Eng. 734 (2002) 30. [8] J.S. Daniels, N. Pourmand, Electroanalysis 19 (2007) 1239. [9] W. Laureyn, D. Nelis, P. Van Gerwen, K. Baert, L. Hermans, R. Magnéeb, J.J. Pireaux, G. Maes, Sens. Actuators B 68 (2000) 360. [10] T. Balkenhohl, F. Lisdat, The Analyst 132 (2007) 314. [11] E. Katz, I. Willner, Electroanalysis 15 (2003) 913. [12] A.P. Grigin, A.D. Davydov, Russ. J. Electrochem. 36 (2000) 286. [13] G. Suárez, N. Keegan, J.A. Spoors, P.M. Ortiz, R.J. Jackson, J. Hedley, X. Borrisé, C.J. McNeil, Langmuir 26 (2010) 6071. [14] R.L. Spyker, R.M. Nelms, IEEE Trans. Aerosp. Electron. Syst. 36 (2000) 829.