Design and characterisation of a thin-film electrode array with shared reference/counter electrodes for electrochemical detection

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In the current study, a novel electrode array and integrated microfluidics have been designed and characterised in order to create a sensor chip which is not only easy, rapid and cheaper to produce but also have a smaller imprint and good
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Design and characterisation of a thin- 󿬁 lm electrode array with sharedreference/counter electrodes for electrochemical detection Yildiz Uludag a, n , Zehra Olcer a,b , Mahmut Samil Sagiroglu a a UEKAE   –  BILGEM   –  The Scienti  󿬁 c and Technological Research Council of Turkey (TUBITAK), 41470 Gebze/Kocaeli, Turkey b Department of Chemistry, Gebze Institute of Technology, 41400 Gebze/Kocaeli, Turkey a r t i c l e i n f o  Article history: Received 27 November 2013Received in revised form16 January 2014Accepted 23 January 2014Available online 1 February 2014 Keywords: Elecrode designCyclic voltammetryAmperometryElectrochemical sensingBiosensor a b s t r a c t In the current study, a novel electrode array and integrated micro 󿬂 uidics have been designed andcharacterised in order to create a sensor chip which is not only easy, rapid and cheaper to produce butalso have a smaller imprint and good electrochemical sensing properties. The current study includes theassessment of the effects of an Au quasi-reference electrode and the use of shared reference/counterelectrodes for the array, in order to obtain a small array that can be produced using a  󿬁 ne metal mask. Inthe study, it is found that when Au is used as the quasi-reference electrode, the arrays with sharedreference and counter electrodes result in faster electron transfer kinetics and prevent the potentialchange with respect to scan rate, and hence is advantageous with respect to conventional electrodes. Inaddition, the resulting novel electrode array has been shown to result in higher current density(10.52  m A/cm 2 ; HRP detection assay) and measured diffusion coef  󿬁 cient (14.40  10  12 cm 2 /s; calculatedfrom the data of cyclic voltammetry with 1 mM potassium ferricyanide) with respect to conventionalelectrodes tested in the study. Using the new electrode arrays, the detection limits obtained from horseradish peroxidase (HRP) and bisphenol A assays were 12.5 ng/ml (2.84  10  10 M ) and 10 ng/ml(44  10  9 M), respectively. Performing the HRP detection assay in a  󿬂 ow injection system using arrayintegrated micro 󿬂 uidics provided 25 times lower detection limit (11.36  10  12 M), although Ti has beenused as electrode material instead of Au. In short, incorporation of this new electrode array to lab-on-a-chip or MEMs (micro-electro mechanic systems) technologies may pave the way for easy to useautomated biosensing devices that could be used for a variety of applications from diagnostics toenvironmental monitoring, and studies will continue to move forward in this direction. &  2014 Elsevier B.V. All rights reserved. 1. Introduction Biosensors have been envisioned to play a signi 󿬁 cant analyticalrole in diagnostics, bioprocesses, quality assurance in agriculture andfood industries, environmental monitoring and homeland security.All of the mentioned sectors have considerable market size. Oneexample is the global food safety testing market by contaminantswhich is estimated to grow at an annual growth rate of 10.46% to$2.5 billion in 2015 (MarketPublishers, 2011). Another importantapplication area is in vitro diagnostics (IVD) with a yearly worldwidemarket of $42 billion in 2007 (KaloramaInformation, 2008). There-fore, further research to develop new biosensor devices for foodsafety and diagnostics has a great socio-economic signi 󿬁 cance. Thedetection methodology of biosensors is quite varying and rangesfrom optical (e.g. ,  󿬂 uorescence detection, SPR, interferometry, opticalgrating) and piezoelectric to electrochemical (e.g., amperometric,impedimetric, voltammetric) transducers (Becker and Cooper, 2011;Cooper, 2002; Hintsche et al., 1994; Homola, 2006; Sheikh et al.,2008; Turner, 2000). High sensitivity, selectivity, rapid analysis, theability to operate in turbid solutions and the possibility of miniatur-isation enabled electrochemical biosensors to became the mostwidely used biosensors (Shah and Wilkins, 2003) (Newman and Turner, 2005). The design of an electrochemical biosensor involvescareful consideration of many parameters such as electrode design,sensor surface chemistry, recognition element immobilisation onelectrode surface, assay conditions and enzyme/mediator selection(Borgmann et al., 2011).Despite the numerous advantages of electrochemical sensorsand many years of scientists ’  efforts, important hurdles in theirdevelopment still remain. A key issue that needs to be addressed isthe development of electrode probes that can be fabricated intouseful arrays for multiplex detection (Drummond et al., 2003).Electrochemical measurements usually involve a three electrodesystem that consists of a working, counter and reference electrode.Contents lists available at ScienceDirect journal homepage: Biosensors and Bioelectronics  &  2014 Elsevier B.V. All rights reserved. n Corresponding author. Tel.:  þ 90 262 648 1910; fax:  þ 90 262 648 11 00. E-mail address: (Y. Uludag).Biosensors and Bioelectronics 57 (2014) 85 – 90  Author's personal copy Traditional electrochemical measurements involve the use of Ag/AgCl as the reference electrode. This functions as a redoxelectrode for the reaction between Ag metal and its salt, AgCl.This reference electrode is usually formed by a glass tube contain-ing a silver wire that is coated with a thin layer of silver chloride(Czaban and Rechnitz 1976). Although this reference electrodeprovides fast electrode kinetics, it is not practical when portable,smaller and single use electrochemical biosensors are required.Thus, a new type of electrochemical chip that comprises planarelectrodes in the form of either screen printed electrodes (SPEs) orthin  󿬁 lm electrodes has gained widespread use. SPEs are printedusing inks (carbon or metals together with a polymer matrix) onplastic or ceramic substrates (Hart and Wring, 1994) and thin  󿬁 lmelectrodes are deposited on glass or silicon wafers by means of evaporation or sputtering methods. Thin  󿬁 lm technologies enablethe manufacturing of electrodes with high precision and resolu-tion. If micron sized electrodes need to be produced a photolitho-graphy process is required (Wu et al.,1993); otherwise a  󿬁 ne metalmask is used to create the electrode patterns on substrates (Zhouet al., 2003). By a sequential process, different metal inks can beprinted on SPE; although carbon and gold inks are the most widelyused working or counter electrode materials, as pseudo-referenceelectrodes, Ag/AgCl ink is widely used. However, in the case of thin 󿬁 lm electrodes, it is not possible to evaporate or sputter Ag/AgCl toform a reference electrode. Therefore, for thin  󿬁 lm electrodes,as an alternative, application of quasi-reference electrodes in theform of Au or Pt has gained some use. While some studies existthat investigate the usability of Ag/AgCl pseudo electrodes, thestudies that examine the effects of quasi-reference electrodeon electrochemical measurements are rather limited (Kasem and Jones, 2008).A number of studies have been performed to assess the electro-chemical properties of microelectrodes with different geometriesand sizes (Guo and Lindner, 2009; Kurita et al., 2000). From thesestudies it was observed that, although microelectrodes do haveseveral advantages over macro-electrodes (Stett et al., 2003), suchas higher current density, smaller sensor footprint, and higherdiffusion coef  󿬁 cient, the most obvious disadvantage includes theirhigher impedance due to interfacial capacitance, which results invery low currents (within or below nano-ampere range) (Ordeiget al., 2008) and their need for expensive and time consumingfabrication involving photolithography (Fiaccabrino and Koudelka-Hep, 1998). Especially if single use sensors are considered forend user applications such as diagnostics, food or environmentaltesting, cheaper and less time consuming production proceduresneed to be considered. For this reason, in the current study a novelelectrode array has been designed and characterised in order tocreate a sensor chip which is not only easy, rapid and cheaper toproduce but also has a smaller imprint and good electrochemicalsensing properties. The current study includes the assessmentof the effects of an Au quasi-reference electrode and the use of shared reference/counter electrodes for the array. In addition, thisstudy investigates the electrochemical performance of new elec-trode arrays by means of an enzyme (horse radish peroxidase,HRP) and bisphenol A detection assays. 2. Materials and instrumentation Phosphate buffered saline (PBS, 0.01 M phosphate buffer,0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4)tablets, mercaptoethanol, mercaptoundecanoic acid (MUDA), ethano-lamine, spectrophotometric grade ethanol, horse radish peroxidase(HRP), TMB ready to use reagent (containing H 2 O 2 ), bisphenol A andpotassium ferricyanide were purchased from Sigma-Aldrich (Poole,UK). Potassium chloride (KCl) was purchased from Fisher Scienti 󿬁 c(Loughborough, UK). Oxygen free argon was purchased from Habas( İ stanbul, Turkey). Ultrapure water (18 M Ω /cm) was obtained from aMilli-Q water system (Millipore Corp., Tokyo, Japan). 3. Methods  3.1. Transducer fabrication Three electrode arrays were designed.  Design 1  consists of eightAu electrodes of different sizes (diameter sizes: 1.5, 2, 3, and 4 mm)and each has its own Au counter and quasi-reference electrode. Design 2  consists of eight Au electrodes of different sizes (diametersizes: 1.5, 2, 3, and 4 mm) and all share the same Au counter andquasi-reference electrodes.  Design 3  consists of eight Au electrodes of 1.5 mm diameter and all share the same Au counter and quasi-reference electrodes. An electronbeam evaporatordevicewas usedtodeposit Ti and Au metals on the glass slides. The designs of theelectrodes were formed on the glass slides by means of Fine MetalMasks made of a laser cut patterned stainless steel. Before theapplication of Au (200 nm), a 20 nm Ti layer is applied on to the  󿬁 neglass slides as an intermediary adhesive layer to increase theadhesion between Au and glass slide. A  󿬂 ow cell was designed andfabricated using PMMA to be used for the electrode arrays.  3.2. Electrochemical analysis Cyclic voltammetry measurements were performed with aDropsens MicroStat 8000 Electrochemical Analyzer with the gen-eral purpose electrochemical software Dropview 1.4 (Dropsens,Astuias, Spain). The electrochemical analyzer and the purposebuilt shielded cables enable simultaneous electrochemical mea-surements of eight electrodes. Cyclic voltammetry (CV) tests wereperformed using 1 mM potassium ferricyanide solution in 1 M KCl.  3.3. Assay Bisphenol Adetection assay was performed by injecting bisphenolA (in PBS)at varying concentrations on tothe plasma cleaned bare Auelectrode array. After bisphenol A injection, the  󿬂 ow stopped and0.5 V potential has been applied to the electrode arrays for 60 s andthe current vs. time plot has been obtained. The current value at the60th second of the test has been recorded as sensor response. For thehorse radish peroxidase (HRP) assays, initially plasma cleaned bareAu electrode arrays were immersed in ethanolic solution of 2 mMmercaptoundecanoic acid (80%) and 2 mM mercaptoethanol (20%)mixture for overnight. Later the electrode arrays were rinsed withethanol and water.Afterdrying with nitrogen stream,the arrays werestored at  þ 4  1 C till use. Enzyme assays were performed by mixingHRP and TMB reagent, then injecting on to the  󿬂 ow cell containingelectrode arrays (Fig.1). The chronoamperometric responses obtainedat   0.1 V potential at 60 s of the measurements were used as assayresponse.Three data points were used to obtain the mean and standarddeviation of the results. The limit of detection (LOD) was calcu-lated as the signal obtained from the assays that is equivalent tothree times the standard deviation of the signals obtained fromthe blank standards. 4. Results and discussion 4.1. Cyclic voltammetry To investigate the electrochemical behaviour of the designedelectrode arrays, a cyclic voltammetry technique has been utilised Y. Uludag et al. / Biosensors and Bioelectronics 57 (2014) 85 – 90 86  Author's personal copy and voltammograms were recorded for a model electroactivespecies, potassium ferricyanide. Typical voltammograms for thethree designs of electrode arrays are presented in Fig. 2. Au thin 󿬁 lm electrodes with Au quasi-reference electrodes had shownoxidation and reduction peaks for potassium ferricyanide similarto those generated when Ag/AgCl was used as a referenceelectrode (Fig. 2(A)). The  design 3  electrode array consists of eightworking electrodes with shared counter and reference electrodes.For the array to be potentially used as a multianalyte detectionsensor, each electrode within the array has to result in the sameelectrochemical response irrespective of their position in the array.Therefore, as can be seen from Fig. 2(C), all eight electrodes withinthe same array displayed the same voltammogram, indicating thesuitability of the array for multiplexed measurements. 4.2. Scan rate dependence of peak current  The observed peak current on the forward potential scan of thecyclic voltammetry measurements is given for the case of rever-sible electron transfer by the Randles – Sevcik equation (at 25  1 C) I  p ¼ 0 : 4463 nFAC  nF  ν DRT    1 = 2 ð 1 Þ where  I  p  is peak current,  n  is number of electrons transferredin the redox event,  A  is electrode area,  F   is Faraday constant, D  is diffusion coef  󿬁 cient,  C   is concentration,  ν  is scan rate, and T  ¼ 298 K.The Randles – Sevcik equation de 󿬁 nes a linear correlationbetween the peak current and the square root of the scan ratefor a reversible electron transfer (Wang, 2006). Thus, plots of   I  p and  ν 1/2 are a very convenient tool to characterise the electro-chemical reversibility of a given redox system using a selectedelectrode system. The irreversible reactions result in slow reactionkinetics which means the equilibria are not reached rapidly withrespect to the voltage scan rate, and this causes the shift in themaximum current as the voltage scan rate is changed. Threeparameters are generally derived from cyclic voltammetry mea-surements in order to characterise a reversible process:   the peak potential separation  Δ E  p ¼ E  pc  E  pa ¼ 59/n mV at allscan rates at 25  1 C;   the peak current ratio is  I  pa / I  pc ¼ 1 at all scan rates;   the peak current function  I  p / ν 1/2 is independent of   ν  .The cyclic voltammograms obtained from the use of threedifferent electrode array designs have been analysed to assessthe effect of electrode design and the reversibility of the reactionon the electrode surface in light of these three parameters.The peak potential separation of   designs 1 ,  2  and  3  electrodeswas calculated to be 91 mV, 56 mV and 87 mV, respectively. For allthe designs  I  pa  and  I  pc  ratio was found as 1, as expected from areversible reaction. For all the electrode array designs, the increaseof cyclic voltammetry scan rate resulted in an increase of peakcurrent obtained from the measurment, as expected from areversible reaction. However, when the scan rate was increased,a shift in the peak potential was observed for  design 1  (Fig. 3(A)).No shift in peak potential was observed for other designs (Fig. 3).The electrode array designs have also been compared in termsof measured diffusion coef  󿬁 cient that has been calculated usingEq. 1. As seen from Table 1, electrode arrays of   design 3  with its1.5 mm diameter electrodes resulted in the highest measureddiffusion coef  󿬁 cient (14.40  10  12 cm 2 /s), indicating that thisdesign can be preferred over others for electrochemical detection. 4.3. Au quasi-reference electrode In the current study, a shorter/quicker and a lower-cost proto-col has been utilised for electrode array fabrication by using theFine Mask Method instead of photolithography. In addition to this,a new electrode array has been designed in which sharedreference and counter electrodes have been used, minimising thesize of the sensor. This has eliminated the main disadvantage of  Fig.1.  (A)  Design 1  electrode array consists of eight Au electrodes of different sizes(diameter sizes: 1.5, 2, 3, 4 mm) and each has its own Au counter and quasi-reference electrode. (B) Enzyme assays were performed by mixing HRP and TMBreagent, and then injecting to the  󿬂 ow cell containing electrode arrays. Fig. 2.  Cyclic voltammetry ( CV  ) has been performed with 1 mM K 4 [Fe(CN) 6 ]/KCl at 100 mV/s scan rate, using (A) conventional screen printed Au electrode with Ag/AgClpseudo-reference electrode, (B)  design 1  and (C)  design 2  arrays containing eight Au electrodes with diameters 1.5, 2, 3 and 4 mm; and (D)  design 3  array containing eightAu electrodes with diameter 1.5 mm. Y. Uludag et al. / Biosensors and Bioelectronics 57 (2014) 85 – 90  87  Author's personal copy larger macroelectrodes. The important factors for this miniaturisa-tion are the effect of shared reference and counter electrodes onthe electrochemical response obtained from the sensors and theuse of an Au quasi-reference electrode. The experiments show thatmeasured diffusion coef  󿬁 cient is higher when smaller electrodesare utilised. Here the signi 󿬁 cance is that different designs (indivi-dual [ design 1 ] and shared electrode design [ design 2 ]) behavedsimilarly in terms of measured diffusion coef  󿬁 cient. The arrayswith shared reference and counter electrodes ( designs 2  and  3 )have been found more advantageous with respect to  design 1 electrode arrays, since  designs 2  and  3  do not cause a potentialpeak shift when the scan rate is increased. Although we are notsure about the reasons of this result, one interpretation could bethe use of Au quasi-reference electrodes for testing in  design 1 ,as suggested by Prakash and colleagues (Prakash et al. 2008).However, as we change the electrode array to  ‘ design 2  or  3 ’  withshared Au quasi-reference electrode, this problem disappears.This indicates that use of shared Au quasi-reference electrodeprovides better potential control during the reaction between theworking and reference electrodes. From these results we mayconclude that, when Au is used as quasi-reference electrode, thearrays with shared reference and counter electrodes ( design 2 )result in faster electron transfer kinetics and hence can bepreferred with respect to  design 1  (more conventional electrodes)to be used as electrochemical sensors. Within  design 2  array,electrodes with 1.5 mm diameter have shown the highest currentdensity and measured diffusion coef  󿬁 cient, which provided thebasis for  design 3  electrodes.  Design 3  array provides a smallersensor chip imprint that would be particularly useful when a  󿬂 owcell is designed for sensing which allows the implementation of micro 󿬂 uidics for sample and reagent transportation to the sensorsurface for automated, easy to use biosensing applications. 4.4. Assay performance4.4.1. Bisphenol A detection Bisphenol A (a potential endocrine disrupter) has been widelyused as a major component in the production of polycarbonateand epoxy resins that are used to manufacture plastic food/watercontainers and cans. However, there is a risk of contamination of food due to the migration of bisphenol A from packaging to foodand beverages. As a result, under EU regulations the maximumallowed concentration of bisphenol A in drinking water is 500 ng/ml (Commission of the European Communities,1980). The currentbisphenol A detection involves the use of gas chromatography (GC)and HPLC techniques. Both of these techniques are expensive, timeconsuming, require an expert user and a laboratory environment.An electrochemical sensor, on the contrary, may provide a quick,cheap and portable method for bisphenol A testing (Portaccioet al., 2010; Ruana et al., 1993). Amperometric detection is basedon measuring the current generated by the oxidation or reductionof an electroactive species at a working electrode while thepotential is kept constant. As bisphenol A is an electroactivesubstance, it is possible to detect its presence by means of amperometric measurements (Hiroi et al., 1999). Therefore, toassess the performance of electrode arrays developed and to Fig. 3.  The  CV   of   design 1  ( d ¼ 3 mm) (A) and  design 3  ( d ¼ 1.5 mm), (B) arrays at varying scan rates: 50,100,150, 200, 250 mV/s (inner to outer  CV   traces, respectively). Theresults of cyclic voltammograms at varying scan rates have been used to obtain scan rate 1/2 vs. oxidation/reduction current (C), and scan rate vs. oxidation/reductionpotential (D).  Table 1 The measured diffusion coef  󿬁 cient for three electrode array designs.Electrode type Measured diffusion coef  󿬁 cient (cm 2 /s)Design 2  –  1.5 mm 9.30  10  12 Design 3  –  1.5 mm 14.40   10  12 Design 1  –  2 mm 6.90  10  12 Design 2  –  2 mm 5.00  10  12 Design 1  –  3 mm 3.05  10  12 Design 2  –  3 mm 6.45  10  12 Design 2  –  4 mm 5.21  10  12 Y. Uludag et al. / Biosensors and Bioelectronics 57 (2014) 85 – 90 88
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