Electrochimica Acta 51 (2006) 6217–6229 Review article Impedance spectroscopy: Ov electrochemical sensor op Nanoc Abstract There is c the detection surface. In f characterize of many typ charge trans processes ha the work tha of electroch © 2006 Else Keywords: Impedance spectroscopy; Material characterization; Membrane; Electrode; Electrochemical sensor; Potentiometric sensor; Biosensor; Interfacial kinetics Contents 1. Introd 2. Poten 2.1. 2.2. 3. Elect 3.1. 3.2. 3.3. 3.4. 4. Futur 5. Conc Ackn Refer 1. Introdu Signific have led to ∗ Correspon E-mail ad R.DeMarco@ 0013-4686/$ doi:10.1016/j uction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6217 tiometric sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6218 Glass and crystalline-based membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6219 Polymer-based membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6221 rochemical biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6222 Enzyme sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6222 Immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6223 DNA sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6224 Ion channel sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6225 e developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6226 lusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6226 owledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6227 ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6227 ction ant technological advances over the past few decades the development of numerous analytical devices in ding author. Fax: +61 8 9266 2300. dresses:
[email protected] (B. Pejcic), curtin.edu.au (R. De Marco). the monitoring of a wide range of analytes. The desire to mea- sure and understand every single variable in our environment has been the impetus behind the growth of many innovative tools. More specifically, electrochemical-based sensors are one group of analytical devices that have attracted considerable attention in recent times. The introduction of electrochemical sensors in the last century has revolutionized the way in which we lead our lives. This is not surprising considering that they play a – see front matter © 2006 Elsevier Ltd. All rights reserved. .electacta.2006.04.025 Bobby Pejcic, Roland De Marco ∗ hemistry Research Institute, Department of Applied Chemistry, Curtin University of Technology, GPO Box U 1987, Perth, WA 6845, Australia Received 23 February 2006; received in revised form 19 April 2006; accepted 20 April 2006 Available online 12 June 2006 onsiderable interest in the development of electroanalytical sensors (i.e., potentiometric, amperometric, electrochemical biosensors) for of a wide range of analytes. The success of many of these sensors is governed by the condition and stability of the membrane/electrode act, the response mechanism is dictated primarily by the surface structure and a considerable amount of work has been undertaken to the interfacial region. Consequently, electrochemical impedance spectroscopy (EIS) has played a pivotal role in the characterization es of sensors. EIS has been used to provide information on various fundamental processes (i.e., adsorption/film formation, rate of fer, ion exchange, diffusion, etc.) that occur at the electrode–electrolyte interface. Understanding and manipulating these interfacial s assisted in the development of membranes/electrodes with new and improved response characteristics. This paper reviews some of t has been undertaken using EIS over the past 35 years. More importantly, it evaluates the power of EIS in characterizing a wide range emical sensor systems. vier Ltd. All rights reserved. er 35 years of timization 6218 B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 crucial role in medical and clinical analysis, environmental and industrial monitoring [1–6]. The common feature binding all electrochem electrical p normally c potentiome excellent r recommend sensors is a due to the for the dete areas. A funda design lies between su sor materia pressing ne erties. Und sensor resp troanalytic ical stabili order to ac various ele or inside th ing of the adsorbs the or it could erties of th Sensor opti tion of an e with the int position, a be evaluate Understand mechanism developme response ch There ar sensor dev scanning e beyond the each techn brane/elect is designed reaction to however, i kinetic inf interface is trochemica steady-stat phenomena lies in its times over has been us the electric EIS has p corrosion r metals and metal-coated surfaces [13,14]. Nowadays, we are finding an increasing use of EIS to investigate the adsorption arge s. pur ation chem te th sor lized s on sens nov ubje ly re with in a g the d the enti o kn pass the ue co (i.e e ISE prim g pro e ion face ary port s un sorpt entio s tha nica ent ring tion [29– s of hout s is , bu amo that llen sed e nt s een iome on , wit ical sensors is that they rely on the detection of an roperty (i.e., potential, resistance, current), and are lassed according to the mode of measurement (i.e., tric, conductometric, amperometric). A number of eviews appear on the topic [7,8], and the authors them for further reading. Indeed, electrochemical large field that continues to evolve, and this is partly fact that the underlying electrochemical principles ction of analytes are highly relevant in many diverse mental challenge pertinent to electrochemical sensor in the molecular understanding of the relationship rface structure and reactivity. The fabrication of sen- ls with unique response characteristics has created a ed to understand their chemical and physical prop- erstanding the fundamental processes that govern onse in most cases leads to the development of elec- al devices with superior selectivity, excellent chem- ty, higher sensitivity, and lower detection limits. In hieve these objectives, it has been necessary to study ctrical processes that occur at the surface of the sensor e sensor membrane itself. This may involve tailor- chemistry of the surface layer so that it exclusively target molecule/ion in the presence of interferences, involve altering the bulk electrical conduction prop- e sensor by modifying the membrane composition. mization is one of the most crucial steps in the realiza- lectroanalytical device. Whether it involves tinkering erfacial properties or modifying the membrane com- suitable tool that allows the sensor performance to d under a range of conditions must be employed. ing how various parameters influence the response and interfacial reaction kinetics will assist with the nt of electrochemical sensors with new and improved aracteristics. e countless techniques available for electrochemical elopment/optimization (i.e., cyclic voltammetry, lectrochemical microscopy, etc.) [9–11], and it is scope of this review to discuss the relative merits of ique. A majority of these methods probe the mem- rolyte interface by using a large perturbation, which to provide mechanistic information by driving the a condition far from equilibrium. Another approach, s to apply a small perturbation to ensure that the ormation pertaining to the membrane/electrolyte at near zero current conditions. Subsequently, elec- l impedance spectroscopy (EIS) is a non-destructive e technique that is capable of probing the relaxation over a range of frequencies [12]. The power of EIS ability to provide in situ information on relaxation the frequency range 106 to 10−4 Hz. It is a tool that ed to identify and separate different contributions to and dielectric responses of a material. Traditionally, rovided a wealth of information pertaining to the ate and corrosion processes on a wide variety of and ch sensor The applic electro evalua ing sen specia focuse based If a in the s strong along to obta pinnin beyon 2. Pot Als encom cases, a uniq a solid and th that is bindin electiv the sur of prim ious re sensor chemi [28]. Pot sensor mecha instrum monito revolu levels billion throug sensor sample minute branes the cha been u differe have b potent review (ISEs) transfer processes of many types of electrochemical pose of this article is to review recent advances and s of EIS in unravelling the response mechanisms of ical-based sensors. Consequently, the authors will e effectiveness of EIS as a technique for investigat- response processes, and have aimed the article at a electrochemistry readership. Ultimately, the review the use of EIS in the development of electrochemical- ors and biosensing devices. ice to the EIS technique finds themselves interested ct matter of this fundamental review then the authors commend the superb EIS text by Macdonald [12], a compilation of excellent research articles [15–24], good grounding in the fundamental principles under- EIS technique, as this rudimentary information is scope of this specialized review paper. ometric sensors own as ion-selective electrodes (ISEs), these sensors a large subset of electrochemical sensors. In many potentiometric sensor comprises a membrane with mposition, noting that the membrane can be either ., glass, inorganic crystal) or a plasticized polymer, composition is chosen in order to impart a potential arily associated with the ion of interest via a selective cess at the membrane–electrolyte interface. A perms- -conducting membrane is used and the magnitude of potential is directly related to the activity or number ions in solution according to the Nernst equation. Var- s have been published suggesting that potentiometric dergo a selective response mechanism involving the ion [25–27] and/or phase-boundary potential model metric sensors are a popular class of analytical t generally possess long lifetimes and acceptable l stability. The main appeal lies in the simple ation, low cost, and their suitability for continuous . However, these sensors have recently undergone a in terms of lowering the detection limits to ultratrace 32]. Consequently, they are being used to perform measurements each year in virtually every hospital the world [2]. A major problem with potentiometric the leaching of the membrane components into the t this influence has been ameliorated by incorporating unts of electroactive ingredients in polymeric mem- have been used successfully in clinical analysers for ging analysis of whole blood. Nevertheless, EIS has xtensively to study the leaching mechanism of many ensor membranes. Since 1970, countless reports published in the literature on the characterization of tric sensors by EIS. Buck [33] published a seminal EIS in the investigation of ion-selective electrodes h 128 references, aimed at a more specific analytical B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 6219 chemistry readership. However, the early and pioneering research by a variety of groups helped to establish the founda- tions for im electrical p polymer-ba enabled the used to this of this foun [33–42] ha research ch potentiome focuses pri early and p excellent re 2.1. Glass By far, sensor is th ditioning o an importa subsequent Hwang and glass electr as a functio ion concen stant, whic stant at low interface [4 have also g the detectio and Boych may be use pioneering developme chtenberg membranes various me of impedan nism of the performed. formation o in the sensi the interfac on the Cu– ence of a s and the kine [53]. An ad when the m that the rea trol. Imped of membra stant shifte Cu(II) solu ing data wa A numb workers [54 genide glass ISEs. There is a general consensus that the potential generating process of a chalcogenide glass electrode is governed form er h e MS rane , mo ng/s SE i ave enso ises ns ( d tha arge is ge Fe3+ merc ated ties of t anc actio Nern e Hg surf effe strat pro liabl spe mic ntere envi usin ral w geni n sea ISE g2+ rds [ ion high pons tran eno erfo mon assi senc orpt o de anal rane e Fe by th stabl pedance spectroscopy, and used it to determine the roperties of a wide range of crystalline, glassy and sed ISEs [34–42]. These significant papers have development of many successful ISEs that are still day, and are a testimony of the excellence and impact dation research. Without a doubt, these early papers ve provided an excellent platform for subsequent EIS aracterizing the response mechanisms of various tric-based sensors. Accordingly, the present review marily on the follow-up work, as the majority of this ioneering research has already been captured in the view by Buck [33]. and crystalline-based membranes the most widely used and successful potentiometric e pH glass electrode [43]. It is well known that con- f the glass membrane in an aqueous electrolyte plays nt role on the formation of a hydrated gel layer and ly on the electrochemical properties of the pH sensor. Han [44] used EIS to show that the resistance of a pH ode, based on a lithium-silicate membrane, changes n of aqueous solution soaking time and hydronium tration. EIS detected both a high frequency time con- h was attributed to the glass matrix, and a time con- er frequencies that was assigned to the glass/solution 4]. ISEs comprising chalcogenide glass membranes ained acceptance as alternative chemical sensors for n of metal ions [45]. A recent review by Vassilev eva [46] has shown that chalcogenide glass sensors d in environmental and industrial monitoring. The work of Baker and Trachtenberg [47] led to the nt of an ISE based on a chalcogenide glass. Tra- and co-workers [47–51] were the first to use such as potentiometric sensors in the determination of tal ions in aqueous solution. However, the application ce spectroscopy to understand the response mecha- chalcogenide glass membrane has only recently been Koenig and Grabner [52] employed EIS to detect the f a modified surface layer and to determine its role ng mechanism. Similarly, Cali et al. [53] investigated ial properties of a copper(II) selective electrode based As–S alloy. The impedance spectra revealed the pres- ingle time constant due to a charge transfer reaction tics of charge transfer increased with elevated [Cu2+] ditional straight line was observed at low frequencies embrane was exposed to elevated [Cu2+] suggesting ction is under mixed charge transfer/diffusion con- ance measurements were also performed as a function ne soaking time, and it was found that the time con- d toward higher frequencies as the membrane aged in tion [53]. Unfortunately, no equivalent circuit model- s provided to rationalize the changes in time constant. er of articles have been published by Vlasov and co- –56] that discuss the response mechanism of chalco- by the this lay that th memb Indeed ditioni glass I [57] h Fe3+ s compr reactio showe the ch and th sive to based in elev proper ageing perform tion re in the that th during drastic demon mental that re The geoche icant i of the taken in natu chalco Hg2+ i Hg2+ wide H standa centrat much the res charge tion ph were p was de trode p the pre the ads used t electro memb the fre taken have e ation of a modified surface layer (MSL), noting that as the ability to undergo ion exchange. It is believed L is formed during conditioning of the chalcogenide in an electrolyte that contains the primary ion [52,55]. st potentiometric sensors need some type of con- urface preparation before use, and the chalcogenide s no exception. However, De Marco and co-workers shown that conditioning of the chalcogenide-based r (i.e., Fe2.5Se58.5Ge27.3Sb11.7) in an electrolyte that high levels of ferric ion leads to undesirable side i.e., membrane oxidation). Impedance spectroscopy t long-term ageing in ferric ion solution decreases transfer resistance of the chalcogenide membrane, nerates a sensor surface that is no longer respon- [52,57]. Similarly, conditioning of a chalcogenide- ury(II) sensor (i.e., Ag46.1Br25.2As9.7Si18.1Hg0.3I0.6) Hg2+ ion levels revealed changes in the interfacial with exposure time [58]. EIS showed that extended he sensor in Hg2+ electrolyte is detrimental to the e of the Hg2+ ISE, noting that the membrane oxida- n is facilitated and this is responsible for a change stian slope [58,59]. Impedance studies also revealed 2+ ISE undergoes a photochemical oxidation reaction ace/membrane exposure to light and this can have cts on the response mechanism [58]. These studies e that EIS is able to establish an appropriate experi- tocol for sensor conditioning/surface preparation so e potentiometric measurements can be made. ciation/distribution, along with the biological and al cycling of various elements in the ocean is of signif- st to organizations concerned with the preservation ronment. A great deal of research has been under- g electrochemical sensors to monitor trace metals aters. Recently, a mercury(II) ISE that employs a de membrane has been developed and used to detect water [60]. Potentiometric studies revealed that the yields a Nernstian slope (i.e., ∼29 mV/dec) over a concentration range using saline Hg(II) ion buffered 60]. Although it was shown that the free Hg2+ con- in seawater, as determined by the Hg(II) ISE, is er than expected [60,61]. EIS studies revealed that e mechanism of the Hg2+ ISE is underpinned by a sfer process and the membrane undergoes a passiva- menon in seawater [60]. Further impedance studies rmed to understand the fouling mechanism, and it strated that chloride is partly responsible for elec- vation/interference effect in seawater [58]. However, e of natural organic matter in seawater suppresses ion of chloride [58,62], and this information is being velop an appropriate measurement protocol for the ysis of Hg2+. The development of a chalcogenide (i.e., Fe2.5Se58.5Ge27.3Sb11.7) to selectively probe 3+ concentration in seawater has also been under- e group of De Marco [63,64]. Various researchers ished that iron plays a major role in regulating phy- 6220 B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 toplankton growth in seawater [65–67], and the development of a portable sensor for the real-time detection of iron has impor- tant ramific essential tr were one o anism of t conditions. anism of F revealed th reactions in provided im developme the membr time are im measureme work is sti Fe(II) ISE seawater. O future mem Crystall materials th which unde interface. T powder, (b together by aqueous so oride (LaF group of M field effect were collec LaF3 thick was used t contrast, Si ion exchan selective e cations in s current wa potentiome EIS can als brane selec importance sor based o in potential the membr [79] emplo a gallium n in the pres ClO4−). Im electron-tra [ClO4−] [7 There h ature on th pressed pel formation o leads to de erties for t fact, EIS w jalpaite membrane as a function of rotation speed, seawater age- ing, copper(II), chloride, and humic acid concentrations [86]. ork gov rane/ ntro inter I) at er an AgC func ize t odyn is) a he m ting chan sible thod s we iron ive g elop ate obal analy e d 88] d ate o een wire De ele tran osph ogen yer o s tha s m t of a lysi entio omo of th nd th ratur rese nd th rial s. Co teriz sen ]. In of th ditio comp s of ations for understanding the biological cycling of this ace element. De Marco and co-workers [57,68–72] f the first to use EIS to scrutinize the response mech- he Fe2.5Se58.5Ge27.3Sb11.7 membrane under saline EIS was employed to understand the leaching mech- e2.5Se58.5Ge27.3Sb11.7 and this comprehensive study at the chalcogenide membrane undergoes oxidation seawater, which are under kinetic control. EIS has portant mechanistic information pertaining to the nt of an iron(III) sensor, noting that surface treatment, ane composition, and membrane stability/immersion portant considerations when making potentiometric nts in seawater [69,71,72]. Notwithstanding, further ll needed to improve the chemical durability of the before reliable Fe3+ measurements can be made in bviously, EIS will continue to play a pivotal role in brane optimization studies. ine-based ISEs are another large group of inorganic at act as ionic conductors at room temperature, and rgo ion exchange reactions at the membrane/solution hese sensor membranes can be classed as: (a) pressed ) single crystal, and (c) powdered salts that are held an inert binder [43]. The detection of fluoride in lutions employing a single crystal of lanthanum flu- 3) membrane is well established. Consequently, the oritz [73,74] used EIS to develop an ion-selective transistor (ISFET) sensor based on LaF3. EIS spectra ted as a function of surface preparation/composition, ness, and fluoride concentration, and the information o understand the quality of the ISFET device. By ebert and co-workers [75–77] used EIS to probe the ge reactions occurring on a NASICON-based sodium lectrode. The exchange current densities of various olution were measured by EIS, and the ionic exchange s related directly to the selectivity as determined via try [77]. More importantly, this work has shown that o be used to provide invaluable information on mem- tivity. Bohnke and Fourquet [78] demonstrated the of using EIS to understand the response of a pH sen- n a lithium lanthanum titanate material. The changes as a function of pH were linked to grain boundaries in ane as detected by EIS. More recently, Alifragis et al. yed EIS to study the interfacial reactions occurring on itride crystalline membrane anion-selective electrode ence of various levels of anions (i.e., Cl−, HPO42−, pedance spectra revealed significant changes in the nsfer kinetics as a function of [Cl−], [HPO42−] and 9]. as been a great deal of work reported in the liter- e analysis of copper(II) in various systems using a let of CuS–Ag2S [30,80–83]. It was shown that the f a ternary sulfide called jalpaite (i.e., Ag1.5Cu0.5S) sirable electrochemical and electroanalytical prop- he detection of Cu(II) in natural waters [84,85]. In as used to investigate the interfacial kinetics of the This w ISE is memb sion co ligand of Cu( seawat tion of it non- minim a hydr analys onto t allevia the me respon sor me It i the env excess the dev phosph this gl based real-tim et al. [ phosph have b cobalt ies by of this charge and ph dihydr face la proces aqueou opmen the ana Pot the aut Many tures, a tempe active nism a a mate sensor charac ric gas [93–96 istics of con brane in term revealed that the response mechanism of the Cu(II) erned by a Cu2+/Cu+ charge transfer process at the electrolyte interface, and the reaction is under diffu- l [86]. Furthermore, EIS demonstrated that the weak ference effect associated with organic complexation the electrode diffusion layer poses no problems for alyses [86]. However, it was shown that the forma- l on the surface of the jalpaite Cu(II) ISE can render tional and various strategies have been developed to he chloride interference [87]. Reports suggest that amic electrode rotation or flow regime (e.g., flow long with the adsorption of seawater organic ligands embrane surface, suppress the formation of AgCl, the chloride interference [31,85–87]. Consequently, istic information gathered from EIS has been partly for the development of a successful and reliable sen- for the determination of copper in natural waters. ll established that phosphate is a major culprit in mental problem of eutrophication that results in the rowth of algae in natural waters. It is viewed that ment of an analytical device for the measurement of is an important step in developing strategies to negate problem. There is no doubt that a potentiometric- tical tool, which can be deployed in the field for the etection of phosphate, will be extremely useful. Chen emonstrated that a cobalt wire ISE is able to monitor ver a wide concentration range. Various mechanisms proposed to explain the response behaviour of the electrode towards phosphate [89,90], and EIS stud- Marco et al. [91] clarified the response mechanism ctrode [89,90] by showing that the kinetics of the sfer process is dependent on both the hydrogen ion ate concentrations. It has been rationalized that the phosphate species dissolves the cobalt oxide sur- f the cobalt wire electrode, facilitating the corrosion t controls the response of the metallic electrode in edia [91]. This mechanistic work has led to the devel- cobalt wire flow injection potentiometric method for s of phosphate in fertilizers and wastewaters [91,92]. metric gas sensors have played a significant role in tive industry for the monitoring of exhaust emissions. ese gas sensors are employed at elevated tempera- e development of new materials with lower operating es and improved selectivity/sensitivity is an area of arch. Similarly, understanding the response mecha- e diffusion properties of ions/charge carriers within are important considerations when developing gas nsequently, a number of groups have used EIS to e the electrical properties of various potentiomet- sors based on an oxide-semiconductor membrane most of these studies, the impedance character- e oxide membranes were measured under a range ns (i.e., gas atmospheres, temperatures, and mem- ositions), and the observed changes were discussed gas adsorption, ionic transfer and diffusion processes. B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 6221 Ramamoorthy et al. [96] also determined the time constants of several electrodes, and showed that the time constant varied as a function of indicative o 2.2. Polym In recen developme sensors. A ISEs has fo tivities and published r area [97,98 to characte ion-selectiv influence o on the elec only a sing impedance tivity were structure, m [99]. Anoth the impeda PVC-based and anion changes we it was sugg is desirable eral time c electrode a dielectric p anionic sit determined correlated s Similarly, M cial exchan various ele metric sele fast interfac independen [103]. Sim between th exchange c the polarize surfactant- physicoche interface. T of a thiocy thiocyanate membrane istics. Legi eral plastic tridodecylm trolyte (ET membrane tivity varie Arrhenius equation. A variety of kinetic parameters (i.e., activa- tion energy, pre-exponential factor) were used to rationalize the of d mem -prin as be ed a eome t ele re h ersta mem 110] in) o xcha IS r io o nisti VC-b nge o plica cantl s an ties o on t izer an ties o of su rfact of ad trati [11 uen sens elec ion ion [ d th eri bser y, B orpt ce. T t of bran st of es th ateri mpr in a Imp acr ve se and etics ercu temperature. The variations in the time constant are f changes in the charge transfer mechanism. er-based membranes t years, there has been tremendous growth in the nt of polymeric ionophore-based potentiometric ion majority of the work reported on polymeric-based cussed on improving the electrode sensitivities, selec- detection limits. A number of articles have been eviewing some of the major advances made in this ]. Similarly, many papers have emerged using EIS rize the interfacial properties of various PVC-based e electrodes. Schwake et al. [99] investigated the f several quaternary ammonium based ion exchangers trical properties of a PVC membrane and observed le time constant attributed to the bulk membrane . However, variations in the bulk membrane conduc- observed as a function of quaternary ammonium salt embrane ageing and the nature of the plasticizer er paper by the same authors revealed differences in nce properties of Ca2+-selective and Mg2+-selective membranes in the presence of a combined cation exchanger [100]. More importantly, the impedance re linked to variations in membrane selectivity and ested that a combined cation and anion exchanger [100]. Zhang and Spichiger [101] observed sev- onstants for a polymer membrane Mg2+-selective nd showed that the bulk membrane resistance and roperties depended on the presence of lipophilic es. In addition, the charge transfer resistance was in electrolytes containing different cations, and it trongly with potentiometric selectivity trends [101]. ikhelson et al. [102] used EIS to obtain the interfa- ge current densities of a lithium selective electrode in ctrolytes observing a relationship between potentio- ctivity and current density, and also demonstrated a ial charge transfer for primary and discriminated ions t of whether the ISE response was Nernstian or not ilarly, Xie et al. [104] used EIS to show a correlation e selectivity of a polymer ISE and the concomitant urrent density at the electrolyte/electrode interface of d electrode. Gabrielli et al. [105] developed an ionic selective electrode, and used EIS to understand the mical processes occurring at the membrane/solution he group of Yuan [106] measured the impedance anate-selective PVC-based sensor as a function of concentration, and established a link between the impedance and its potentiometric response character- n et al. [107,108] employed EIS to characterize sev- ized PVC membranes with different concentrations of ethylammonium chloride and lipophilic inert elec- H 500). A single time constant due to the bulk resistance was observed, and the membrane conduc- d as a function of temperature in accordance with the degree in the screen acid h observ bulk g contac The to und based Zhou [ album Ca2+ e ever, E the rat mecha able P of a ra the ap signifi factant proper things plastic EIS in proper levels that su extent concen Pretsch the infl based Ca2+-s in the centrat reveale a polym were o recentl the ads interfa opmen a mem Mo involv mer m that co ether) [117]. of the m selecti cations fer kin that m issociation of tridodecylmethylammonium chloride brane. The electrical behaviour of an all-solid-state ted PVC-modified potentiometric sensor for ascorbic en investigated by EIS [109]. Veltsistas et al. [109] single high frequency time constant attributed to the tric impedance; however, the impedance of the solid ctrode varied with and without a redox mediator. as also been a great deal of interest in using EIS nd the adsorption and fouling mechanism of PVC- branes. EIS studies carried out by Covington and showed that protein adsorption (i.e., bovine serum nto a calcium ion-selective PVC membrane inhibits nge across the membrane–solution interface. How- evealed that adsorption was less problematic when f ligand to Ca2+ in the membrane was high. This c information has enabled the development of reli- ased potentiometric sensors for the clinical analysis f ions in a complex matrix such as blood [2]. Indeed, tion of polymeric-based sensors can be broadened y by chemically modifying the membranes with sur- d proteins. It has been demonstrated that the surface f plasticized PVC are highly dependent among other he preparation method, the type and proportion of [111,112]. The group of Horvai [112,113] have used attempt to understand the adsorption and blocking f PVC-based membranes in the presence of various rfactants and proteins. Impedance spectra revealed ants (sugar alkyl ester type) adsorb strongly, and the sorption depended on the molecular structure and the on of the surfactant [113]. Similarly, Muslinkina and 4,115] showed the importance of EIS in evaluating ce of various surfactants on the response of a PVC- or. Non-ionic lipophilic surfactants were added to a tive DOS/PVC membrane, and EIS detected changes transfer resistance as a function of surfactant con- 115]. Studies involving stearyl-�-d-glucopyranoside e formation of a self-organized layer at the surface of c membrane and changes in the interfacial properties ved after the addition of concanavalin A [114]. More akker and co-workers [116] used EIS to investigate ion of the neutral surfactant Brij-35 at a liquid–liquid his work has important consequences for the devel- sensors for the detection of ionic transference across e/solution interface during a biorecognition event. the work reported in the literature on polymer ISEs e use of plasticized PVC; however, alternative poly- als have also been studied. A nanocomposite material ises a macrocyclic ionophore (i.e., polythiacrown- n organopolysiloxane network has been developed edance spectroscopy was used to understand the role ocyclic ligand in the selective response of a silver ion- nsor [117]. The membrane was exposed to various the impedance spectra revealed that the charge trans- was most favourable for silver, although it appears ry (Hg2+) exerts an interference effect [117]. Con- 6222 B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 ducting polymers are attractive materials for the fabrication of electrochemical sensors because they display some degree of ion selectiv tivity. This directly int been under of these ma transducers substrate–m workers [1 conducting line, etc.) a electrodes under vario ness, memb compositio anism. Thi polymers w application 3. Electro Biosens is not surpr the monito diagnoses of diabetes the develop [128]. A bi biological with a sign for the targ are present a biologica acts with th detected by blies on va the basis o survey has in the liter (i.e., ampe ric) [5,130 the relative [5,131,132 A numb EIS in the [133,134] troscopy to at conducti al. [135] re ous enzym recently, an the power screening. more speci Most of optimizing cial steps in the realization of electrochemical-based biosensing devices is the deposition of a biomolecule film or coating onto a semi l, it i d ir stry a eacti imiz a k enso pas -asse orta cal s prov y me sista 39–1 our ondu scop surf n a w solid l too s, an sor d rface nzym se s yme erom of g on o nium dete sor r sor d nsor tion omet mole tion/ e. A pol 56]. omet din-l dea viro are ed r emi lectr n of ity, and have a mixed ionic and electronic conduc- feature allows the ionic response to be transformed o an electrical signal, and a great deal of work has taken to improve the selectivity and redox sensitivity terials. The success of polymer-based ion-to-electron in solid-state ISEs is primarily determined by the embrane interface. Consequently, Ivaska and co- 18–127] have thoroughly studied a wide range of polymers (i.e., polypyrrole, polythiophene, polyani- s solid-state contacts in several different ion-selective using EIS to investigate the response mechanism us conditions. It was shown that the membrane thick- rane composition, membrane ageing, and electrolyte n play an important role on the sensor response mech- s work has resulted in the fabrication of conducting ith desirable properties for potentiometric sensor s. chemical biosensors ors have received a lot of attention lately, and this ising considering that they play a significant role in ring of a wide range of diseases and pathogens. The and management of the worldwide health problem has made life much easier for many patients since ment of the electrochemical-based glucose biosensor osensor is an analytical device, which incorporates a recognition element in close proximity or integrated al transducer to provide a sensing system specific et analyte [129]. Many different types of biosensors ly available; however, all of them basically comprise l recognition element or bioreceptor, which inter- e analyte and responds in some manner that can be a transducer. The construction of biological assem- rious conductive and semiconductive surfaces forms f most electrochemical-based biosensors. A recent revealed that more than half of the biosensors reported ature involve the electrochemical detection method rometric; potentiometric; capacitive; conductomet- ]. Several reviews have been published discussing merits of various electrochemical-based biosensors ]. er of investigators have reviewed the application of development of biosensors. Katz and co-workers outlined the importance of using impedance spec- detect and understand the biomolecular interactions ve and semiconductive surfaces. Similarly, Guan et ported on the application of EIS to characterize vari- e-based and microorganism-based biosensors. More article by K’Owino and Sadik [136] demonstrated of EIS in cell culture monitoring and bimolecular These previously published reviews were aimed at a fic biosensor readership. the past research on biosensors has focussed on the surface structure. In fact, one of the most cru- metal/ cessfu attache chemi high r as min tion is a bios ing the as self an imp chemi EIS to tion b and re [137,1 behavi onto c spectro sensor lored i to the an idea action biosen ing, su 3.1. E The an enz in amp dation detecti ammo for the biosen of sen of a se adsorp amper of bio adsorp surfac with a ing [1 amper an avi a great and en trodes improv sonoch microe ablatio conductor surface. For a biosensor to be highly suc- s essential that the biorecognition molecule remains reversibly to the transducer. Controlling the surface nd coverage is of paramount importance in assuring vity, stability, orientation, and accessibility, as well ing non-specific adsorption processes. Immobiliza- ey step in determining the overall performance of r, and considerable work has been carried out dur- t few decades in forming ordered organic films such mbled monolayers (SAMS) [137,138]. SAMs play nt role in the selectivity and sensitivity of electro- ensors. Subsequently, a number of groups have used ide important mechanistic information about adsorp- asuring the changes in the interfacial capacitance nce of SAMs and bilayer lipid membrane surfaces 52]. Similarly, the immobilization and adsorption of various biomolecules (i.e., proteins, protamine) ctive surfaces has been investigated by impedance y [153–155]. Evidently, the interface between the ace and the chemical/biological system must be tai- ay that ensures that the receptor molecules attached support retain their activity. Consequently, EIS is l for observing the dynamics of biomolecular inter- d it has been used to predict important aspects of evelopment such as rates of reaction, surface load- reactivity/stability, and binding constants. e sensors ensors are based primarily on the immobilization of onto an electrode, either a metallic electrode used etry (e.g., detection of the enzyme catalysed oxi- lucose) or an ISE employed in potentiometry (e.g., f the enzyme catalysed liberation of hydronium or ions). The development of enzyme-based sensors ction of glucose in blood represents a major area of esearch. Materials selection is an important aspect evelopment as it allows the response characteristics to be altered in a way that minimizes non-specific by other molecules. The fabrication of a long-term ric enzyme biosensor for the real-time monitoring cules in blood demands a device that inhibits the reaction of interfering components with the sensor recent study revealed that covering the membrane ymer is a useful route in overcoming sensor foul- EIS was used to evaluate the performance of an ric enzyme biosensor, which had been coated with oaded conductive polymer [156]. Similarly, there is l of interest in microelectrode sensors for biomedical nmental applications [157], noting that microelec- capable of providing lower limits of detection and esponse times [9]. Recently, Barton et al. [158] used cal ablation to fabricate an enzyme-based glucose ode array. It was demonstrated that sonochemical a thin insulating polymer film on an electrode surface B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 6223 exposes localized areas which act as a microelectrode and col- lectively as a microelectrode array [158]. More importantly, EIS was used to the sensor, glucose co activity of have shown glucose bio Glucose ox Au nanopa modified g ous stages A major ometric tra al. [160] di ever, there transducers tion of ana evaluate th ricated a co of hydroge cal propert lization an [162] deve enzyme sen action betw nase, and th Impedance modificatio tance incre [162]. Tak based enzy the interfac centration. at various constant ch structure/co EIS to mon sensor for observed a surface coa 3.2. Immu The app range of an trol is well e point of car tive to exist In most imm a conductiv the interfac antigen of antibodies sor device. along with are import (i.e., selectivity, sensitivity, stability, response time, etc.) of the immunosensor. The formation of an antigen–antibody complex ondu ce, a to stu suri this gro mam ation on cific S is an im dy–a vale need Cui yer goat odifi r res /biot own osen ino y of lay e., H rev levat extr rope ate of be s tion is c ance ed in ect E by e (al cial E. c t en sed ignifi osen ped mon nine kinet ctrop rnat oper tive characterize the electrochemical redox behaviour of and variations in the impedance as a function of the ncentration were rationalized in terms of enzymatic glucose oxidase [158]. Studies by Zhang et al. [159] that the charge transfer process of a gold-modified sensor is under both kinetic and diffusion control. idase was covalently attached to a cystamine-coated rticle followed by immobilization onto the dithiol old electrode, and EIS was able to confirm the vari- of immobilization [159]. ity of enzyme-based biosensors employ the amper- nsduction method, and a review by Habermuller et scusses various electron-transfer mechanisms. How- is also interest in the development of alternative (i.e., conductometric, potentiometric) for the detec- lytes other than glucose, and EIS has been used to e performance of sensors. Sergeyeva et al. [161] fab- nductimetric-based enzyme sensor for the detection n peroxide, and used EIS to investigate the electri- ies of the membrane at various stages of immobi- d solution treatment. By contrast, Kharitonov et al. loped an ion-sensitive field effect transistor (ISFET) sor for lactate. EIS was used to probe in situ the inter- een NAD+-dependent enzyme, lactate dehydroge- e NAD+-functionalized Au-electrode surface [162]. spectra were collected at different stages of electrode n, and it was shown that the electron-transfer resis- ases with elevated levels of lactate dehydrogenase histov [163] fabricated a nano-patterned alumina- me biosensor for penicillin, and detected changes in ial impedance as a function of the penicillin con- EIS also revealed differences in the phase angle stages of biosensor fabrication [163], and the time anges are consistent with variations in the surface mposition. More recently, Zucolotto et al. [164] used itor the capacitance changes of a gold-based enzyme catechol. Significant variations in capacitance were s a function of catechol concentration and the gold ting [164]. nosensors lication of immunosensors for the detection of a wide alytes in clinical diagnostics and environmental con- stablished. The development of hand held devices for e measurements is a promising and attractive alterna- ing laboratory-based immunochemical assays [131]. unosensor reports, the antibody is immobilized onto e support (i.e., gold), and the electrical properties of e are modified when the antibody reacts with the interest. The surface organization and assembly of is a critical step in the fabrication of an immunosen- Characterizing the immobilization of the antibody, its stability/activity and interaction with the antigen ant steps in optimizing the analytical performance on a c interfa sively by mea panies The human inform itance) the spe that EI ity of antibo that co face is [166]. multila body ( acid m transfe avidin has sh immun of 4-am efficac surface gen (i. studies with e EIS is face p phosph sitivity times genera catalys imped modifi to det plished enzym interfa els of withou [172] u time s immun develo and de the thio under Ele an alte tric pr conduc ctive support alters the impedance features of the nd impedance spectroscopy has been used exten- dy the antigen–antibody molecular recognition event ng the resistive and/or capacitive change that accom- reaction. up of Jie [165] developed an immunosensor for mary tumour, and used EIS to collect mechanistic (i.e., charge transfer resistance, double layer capac- antibody adsorption onto gold and interaction with antigen. Studies by Cony et al. [166] have shown a powerful method for predicting the surface activ- munosensor. The impact of immobilization on the ntigen binding event was studied, and it revealed nt attachment of an antibody to the electrode sur- ed for a successful impedance-based immunosensor et al. [167] used EIS to characterize the growth of a film that comprised avidin and a biotin-labeled anti- anti-hIgG antibody) formed on a mercaptopropionic ed gold electrode. It was observed that the electron- istance increases proportionally with the number of in–antibody layers [167]. Likewise, Wang et al. [168] that it is possible to improve the sensitivity of an sor by using a combined self-assembled monolayer thiophenol and Au colloidal particles to increase the antibody immobilization. The assembly of various ers along with the effect of incubation time and anti- epatitis B virus) were evaluated by EIS [168]. These ealed that the electron-transfer resistance increases ed antigen concentrations [168]. Others showed that emely useful in monitoring the immunosensor sur- rties before and after antibody immobilization in a buffer solution [169]. There is no doubt that the sen- EIS in characterizing interfacial phenomena can at ystem dependent; however, it was shown that the of a precipitate onto the electrode surface via bio- an lead to a substantial increase in the interfacial [170]. The group of Li [170,171] used an antibody- dium tin oxide electrode in conjunction with EIS scherichia coli. Signal amplification was accom- using a combined redox probe and antibody labeled kaline phosphatase), and it was demonstrated that the electron-transfer resistance increases at elevated lev- oli [170]. Similar impedance trends were observed zymatic amplification [171]. Recently, Tang et al. EIS to show that the pH, temperature and incubation cantly influenced the analytical performance of an sor for Hepatitis B. The same group of authors [173] an amperometric immunosensor for �-fetoprotein, strated using EIS that the electron-transfer process of -entrapped Nafion-coated gold-modified electrode is ic control. olymerization of electrically conducting polymers is ive method of controlling the thickness and dielec- ties of the immunosensor surface. As the use of electroactive polymers in the development of elec- 6224 B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 trochemical immunosensors continues to grow, there is a need to understand the electrochemistry of antibody-modified con- ducting po labeled con elucidate it of Sadik [1 an antibod a polypyrro reaction is r polymer. It charged an give rise to also investi entrapment a silane m troscopy (i terize the s also electro bilized an coupling re low freque suggesting modified [1 impedance lizing an an G onto a po that the ele fetoprotein number of enced the s [179]. In most tion has be is catalysed system (i.e ever, there of direct o been used f [180,181] h EIS as a pl ducting po and EIS sp electrode t this strateg remains to complex m metric tran sera. Imped surface tre system [18 that furthe detection c work of Ra based imm coli. A mic in the fabr the frequen impedance changes as a function of the concentration of E. coli [183]. ost de o s int ble ped pro at a semb Au carbo nsdu imm he d ies. Z tigen ckne tly, B ed o yed E face NA s use g an t cas ngle posu in a t of stran ing E 92] arbo A h ases e pre , alo ese f a inves carb ange 4] ex ry el ature ric l stud nd o s [19 ctron treat ncen d an urre lymer electrodes. Some workers used a polyaniline- ductometric immunosensor, and employed EIS to s response mechanism [174]. By contrast, the group 75,176] studied the charge transfer mechanism of y (i.e., anti-human serum albumin) immobilized on le electrode, and revealed that the antibody–antigen esponsible for varying the capacitive behaviour of the is suggested that interactions between the negatively tibody and the positively charged polypyrrole chain variations in the capacitance [175]. Sadik et al. [177] gated several antibody immobilization methods (i.e., in a polypyrrole layer and covalent attachment with olecule) and developed a form of impedance spec- .e., differential impedance spectroscopy) to charac- urface density and antibody–antigen reaction. Others deposited polypyrrole onto a gold surface and immo- antibody (i.e., anti-human IgG) via an avidin–biotin action [178]. EIS detected impedance changes in the ncy region as a function of antigen concentration, that the surface layer of the immunosensor becomes 78]. Recently, Miao and Guan [179] developed an -based immunosensor for �-fetoprotein by immobi- ti-human monoclonal�-fetoprotein immunoglobulin lyaniline modified carbon electrode. EIS confirmed ctron-transfer resistance increases with elevated �- levels in the range 200–800 ng/ml, and both the polymerization cycles and the applied potential influ- ensitivity of the immunosensor towards�-fetoprotein studies involving EIS, the antibody–antigen interac- en monitored via a reduction/oxidation reaction that by an enzyme or by the use of an electroactive/redox ., [Fe(CN)6]4−/[Fe(CN)6]3−) [167–170,179]. How- has been a great deal of interest in the development r reagentless sensor formats, noting that EIS has or this purpose. Subsequently, Lillie and co-workers ave developed a simple immunosensor that employs atform for reagentless sensing. An electrically con- lymer (polypyrrole) was loaded with an antibody, ectra were collected before and after exposure of the o the luteinising hormone [180,181]. It appears that y can be used to detect various analytes; however, it be seen if this method works well for analytes in a atrix such as blood. Diniz et al. [182] used the impedi- sduction platform to detect Chagas disease in blood ance spectra were collected at a range of potentials, atments and in the presence and absence of a redox 2]. Despite poor reproducibility, it was concluded r mechanistic work is needed before impedimetric an be used in clinical diagnosis. By contrast, the dke and Alocilja [183] revealed that an impedimetric- unosensor shows great promise for the detection of E. roelectrochemical system technology was employed ication of the sensor, and EIS spectra collected in cy range of 13 MHz to 10 Hz revealed significant In m are ma variou or dou develo of the event self-as talline using ric tra many est in t strateg itor an the thi Recen sor bas emplo ent sur 3.3. D The growin In mos of a si and ex results opmen single- out us [190–1 ious c of DN acid b surfac tration that th ment o [193] glassy the ch al. [19 mercu of den dielect Others face, a proces the ele trode and co Gol carry c EIS studies reported in the literature, measurements ver a wide frequency range in order to obtain the erfacial parameters (i.e., charge transfer resistance layer capacitance). However, Dijksma et al. [184] an electrochemical immunosensor for the detection tein interferon-� by monitoring the biorecognition single frequency. The immunosensor comprised a led monolayer of cysteine attached to a polycrys- surface, and the antibody was covalently bonded diimide/succinimide chemistry [184]. Amperomet- ction is a common detection method employed in unosensors; however, there also appears to be inter- evelopment of other electrochemical-based detection ayats et al. [185] fabricated an ISFET device to mon- –antibody binding processes, and used EIS to follow ss of various films formed on the ISFET surface. etty et al. [186] fabricated a capacitive immunosen- n electrolyte-insulator porous silicon structures, and IS to characterize the interfacial region after differ- treatments. ensors of nucleic acid recognition layers represents a rapidly d an exciting area of biosensor research [187–189]. es, the detection of DNA relies on the immobilization -stranded oligonucleotide onto a transducer surface, re of the sensor to a sample containing the target hybridization event. A crucial step in the devel- DNA biosensors involves the immobilization of a ded DNA, and a great deal of work has been carried IS to understand this process. Brett and co-workers employed impedance spectroscopy to evaluate var- n-based electrodes as transducers for the detection ybridization. The adsorption of DNA and nucleic onto glassy carbon was studied as a function of paration, electrode potential, nucleic acid concen- ng with electrolyte conditioning, and it was revealed parameters play an important role in the develop- DNA biosensor [190,191]. Likewise, Zhao and Ju tigated the interaction of various redox probes on a on DNA-modified electrode, and used EIS to verify s in the interfacial structure. By contrast, Strasak et amined the adsorption mechanism of DNA onto a ectrode surface, and concluded that the desorption d single-stranded DNA is accompanied by higher osses than the desorption of double stranded DNA. ied DNA adsorption/hybridization onto a gold sur- bserved a single time constant attributed to the redox 5–197]. More importantly, EIS detected changes in -transfer resistance with immobilization time, elec- ment/preparation, oligonucleotide sequence length, tration [195–197]. d carbon are the most common materials used to nt/charge during DNA sensing [198]. However, there B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 6225 has been considerable interest in the development of biosen- sors that use alternative substrates. A silicon-based platform for the detecti the basis o and microp to a silicon tive and pr Hamers an interfacial after expos EIS spectra that the hig hybridizati tion, spectr produced a stranded D able to det wise, the g lization of EIS. The p silane, and a function workers ha for amplify was emplo hybridizati sulfide (Cd covalently was demon nanoparticl oligonucleo electron-tra group of au ties (i.e., in role/carbon More recen chemical D used EIS to centration i rate consta ness plays a The use of for immob research. C response m up new pos Most re the probe D has been so Vagin and c sor that is b It was show modified d structural d Zhao et al. gemini surf surements a By contrast, Guo et al. [208] showed that the polyanionic DNA molecule can be electrostatically adsorbed onto a gold electrode, had b e) c reme proc ox cial n-tra A [2 acit ant r any ctroa act activ peda mpe redo indi . Ho detec s pl asso s in and ade pi-E orma micr . Th le, a ingl e in as at les thod e col and in u the ed w as in so cial n ch de etic sor that etic arma dev of E estig on of DNA is highly attractive since silicon forms f most microelectronic devices such as amplifiers rocessors. It has been shown that DNA attachment substrate generates a surface that is highly selec- ovides good stability [199,200]. More importantly, d co-workers [201] employed EIS to investigate the properties and stability of a silicon (1 1 1) substrate ure to solutions comprising various DNA molecules. collected at various stages of hybridization revealed h frequency impedance (i.e., >1 kHz) increases upon on with the complementary sequence [201]. In addi- a collected on a non-complementary oligonucleotide similar impedance response compared to the single- NA-modified silicon surface, confirming that EIS is ect changes due to DNA hybridization [201]. Like- roup of Lawrence [202] investigated the immobi- a peptide nucleic acid onto a silicon surface with eptide nucleic acid was covalently attached via a the impedance properties of the interface varied as of potential and DNA concentration [202]. Other ve shown that quantum dots can provide a means ing the signal during DNA detection, noting that EIS yed in conjunction with a redox marker to detect the on reaction [203]. Xu et al. [203] coated cadmium S) nanoparticles with carboxyl compounds, and then bonded these with an amine-modified ssDNA. It strated that the target DNA labeled with the CdS e was more sensitive compared to non-CdS tagged tide, noting that EIS detected an increase in the nsfer resistance after hybridization [203]. The same thors [204] also investigated the impedance proper- terfacial capacitance and resistance) of a polypyr- nanotube modified electrode for DNA detection. tly, Peng et al. [205] developed a label-free electro- NA sensor coated with a conducting copolymer, and show that the complementary oligonucleotide con- nfluences the heterogenous standard charge transfer nt. Similarly, it was demonstrated that the film thick- n important role on the sensitivity of the sensor [205]. materials other than gold or carbon as a substrate ilizing DNA presents an exciting area of biosensor onsequently, EIS is an ideal tool for probing the echanism of new biosensor platforms, and this opens sibilities in DNA research. ports in the literature have covalently immobilized NA molecule onto a metal substrate. However, there me interest in alternative immobilization procedures. o-workers [206] used EIS to develop a DNA biosen- ased on a bilayer comprising the surfactant Brij-52. n that the interfacial capacitance and resistance are uring hybridization, and this was attributed to some isordering of the surfactant bilayer [206]. Recently, [207] adsorbed a multilayer comprising DNA and a actant onto a gold surface, and performed EIS mea- s a function of surfactant concentration and structure. which chlorid measu cation the red interfa electro of DN the cap import In m an ele ate the electro for im (i.e., a use of hybrid strands ble to EIS ha anism change before were m Guisep helix f itated digits) molecu nated s increas this w molecu this me tra wer Higson of EIS fish on modifi DNA w DNA interfa 3.4. Io The biomim cal sen shown biomim cal, ph for the the use for inv een modified with a poly(diallyldimethylammonium oated multi-walled carbon nanotube (MWNT). EIS nts were performed at various stages of the modifi- ess, and it was revealed that the MWNT improves properties of the gold electrode by increasing the surface area [208]. It was also observed that the nsfer resistance increases at elevated concentrations 08]. This work shows that nano-sized materials have y to provide a much larger surface area, and this has amifications for improved sensor sensitivity. EIS studies that involve DNA sensors, the use of ctive probe/indicator has been employed to evalu- ivity of the electrode. The advantage of using an e probe is that it can serve as a reference point nce studies. Electrochemical-based DNA biosensors rometric, impedimetric, etc.) normally involve the x labels, soluble redox mediators, and redox-active cators that bind more strongly to hybrids than single wever, various studies have shown that it is possi- t DNA hybridization without the use of labels, and ayed an important part in understanding the mech- ciated with transduction. Hleli et al. [209] revealed the low frequency time constant of a gold electrode after DNA hybridization, noting that measurements at the open circuit potential. Recently, Hang and lie [210] used EIS to investigate the DNA double tion on a microlithographically fabricated interdig- osensor (comprising glass and platinum electrode e glass surface was modified with an organosilane nd this was covalently coupled to a 5′-amine termi- e-stranded oligonucleotide (30-mer) probe [210]. An the impedance was observed after hybridization, and tributed to a reduction in the concentration of water between the double helix [210]. It was revealed that is reasonably sensitive considering that the EIS spec- lected at the open circuit potential. Similarly, work by co-workers [211] have demonstrated the importance nderstanding and discriminating different species of basis of DNA. A screen-printed carbon electrode ith various polymers and containing single-stranded exposed to complementary and non-complementary lution, and EIS revealed significant changes in the capacitive properties [211]. annel sensors velopment of biosensing techniques based on ion channels is a growing area of electrochemi- research. The review by Buhlmann et al. [212] has chemically modified membranes, which incorporate ion channels, play an important role in the biomedi- ceutical and bioanalytical fields. A unique approach elopment of biomimetic ion channel technology is IS. In fact, it has been shown that EIS is an ideal tool ating the formation of tethered bilayer membrane ion 6226 B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 channels [140,149,213]. Impedance measurements were made over a range of ionic species, ionic concentrations, reservoir chemistries More impo in terms of the ionic d group of S through Gr strates. Imp interfacial preparation centration [ to study th nanotube, w lane and ex (DBS) [21 DBS) acts bic [216]. is a reliable currents an 4. Future EIS cha behaviour i sive at diff measureme anywhere b an EIS spe processes/s range chose since the sy typical EIS ment time Park and co possible to rationalized chronoamp by Fourier signals in t formations method; ho capable of chemical b biosensor s the develop ments to be for future k Evident obtained us Most of th reported in conduction vided no in bution over electrochem interrogatin reported on the use of this technique in electrochemical sensor research. However, LEIS has been used extensively in corro- tudie cre oub ction er, f surf loca mpo s som icro ew d copi derst rs [2 forc t wor EIS cal p to u IS st pme nclu inc rial a und alyti brac f ele gat chem nt w tiniz stem our. te th cial cap view ting an imp rich lude text do n ons any prete nt te an be ed th d th ity is , tether structures, spacer molecules and potentials. rtantly, the changes in capacitance were interpreted various models (i.e., Stern, etc.), which account for istribution on a metal surface [140]. Similarly, the teinem [214,215] used EIS to evaluate ion transport amicidin channels on various lipid bilayer solid sub- edance spectra revealed significant differences in the capacitance and resistance as a function of surface , immobilization, ionic composition, and cation con- 214,215]. By contrast, Kohli et al. [216] utilized EIS e conduction mechanism of an alumina membrane hich had been modified with octadecyltrimethoxysi- posed to various levels of 1-dodecanesulfonic acid 6]. It was shown that the surfactant molecule (i.e., as a switch by making the surface less hydropho- It is clearly evident from the above studies that EIS and sensitive tool for detecting the transmembrane d interfacial changes on various modified surfaces. developments racterization relies on a system whose electrical s dependent on various processes, which are respon- erent rates or frequencies. It is well known that EIS nts normally take a long time, i.e., it can take from etween ∼10 min to more than several hours to collect ctrum. Obviously, this will depend on the relaxation tability of the system under study, and the frequency n. Unfortunately, this can lead to interpretation errors stem may have changed during the time frame of a study. Clearly, it would be desirable if the measure- could be significantly reduced, and recent work by -workers [217,218] has demonstrated that it may be make rapid impedance measurements. It has been that a derivative signal can be obtained from a erometric signal at a given bias potential followed transform of the derivative signal to a series of ac he frequency domain [218]. Kramers–Kronig trans- validated the impedance response obtained using this wever, it still remains to be seen if the technique is shedding new mechanistic insights into the electro- ehaviour of the potentiometric and electrochemical ystems discussed in this review [219]. In any event, ment of an instrument that will allow EIS measure- made much faster is without doubt an attractive tool inetic studies. ly, this review has discussed some interesting results ing EIS on various electrochemical sensor systems. e impedance spectroscopy research that has been the literature has mainly addressed the macroscopic properties of the sensors. Such measurements pro- formation on defects, their properties, and their distri- the surface. Although it is well known that localized ical impedance spectroscopy (LEIS) is capable of g the microscopic processes, limited work has been sion s pit and is no d condu howev scopic films, ous co defect these m itate n micros for un worke atomic Recen to use chemi needed that E develo 5. Co The indust need to tant an has em range o mation electro excelle to scru sor sy behavi separa interfa that is This re evalua for new It is mation to exc the con papers data. C before is inter transie data c Provid ory, an causal s, noting that various corrosion mechanisms (i.e., vice corrosion) occur on a microscale [220]. There t that EIS is an invaluable tool for elucidating the properties of surface-modified electrodes/sensors; urther information could be gleaned if the micro- ace defects were probed (i.e., impurities/defects in lized oxidation, galvanic interactions between vari- nents on a membrane surface). Microscopic surface etimes lead to sensor failure, and a tool that allows scale processes to be investigated will certainly facil- evelopments in this area. Coupling EIS with various c-based techniques offers a powerful combination anding nano/microscale processes. Teeters and co- 21] demonstrated that EIS can be combined with an e microscope, and used to probe nanoscale processes. k by Pemkopf et al. [222] has shown that it is possible in conjunction with a microelectrode to map electro- rocesses on a microscale, although future work is still nderstand the impedance changes. There is no doubt udies on a nano/microscale will help foster future nts in miniaturized electrochemical sensors. sions reasing use of electrochemical sensors in medical, nd environmental applications has created a pressing erstand the bulk and surface properties of such impor- cal systems. It appears that the scientific community ed impedance spectroscopy for characterizing a wide ctrochemical sensor systems. The mechanistic infor- hered from this technique has been used to fabricate ical sensors with desirable properties. Indeed, some ork has appeared in the literature on the use of EIS e the fundamental electrochemical processes of sen- s, and provide new mechanistic insights into their Not many techniques are available that can easily e bulk membrane charge transport processes from the reactions; however, EIS is one of very few techniques able of providing this information simultaneously. has shown that EIS is a powerful diagnostic tool for sensor stability and performance, and paves the way d exciting possibilities in electroanalysis. ortant to note that EIS spectra are intrinsically infor- ; however, they need to be analyzed with caution artefacts and to ensure that the data are analyzed in of the most appropriate model. Unfortunately, many ot question the validity and reliability of their fitted equently, the system needs to be well characterized of the data extracted from equivalent circuit modeling d. An important advantage of EIS over other classical chniques is that the validity of the experimental EIS checked using Kramers–Kronig transforms [223]. at the system complies with the linear systems the- at the interface is stable over the time of sampling, obeyed, and the transfer function may be identi- B. Pejcic, R. De Marco / Electrochimica Acta 51 (2006) 6217–6229 6227 fied as impedance. Regrettably, a majority of studies that use EIS to explore the response mechanism of electrochemical sen- sors seldom experiment to display a this is a po examinatio etc.) other interpretati still play a importance characteriz Acknowled The fina (ARC) and Centre (AB Reference [1] J. Wan [2] E. Bakk (1999) [3] R.I. Ste 366 (20 [4] S. Don [5] P. D’O [6] S. And [7] E. Bak [8] E. Bak [9] J. Wan 2000. [10] J.O.M. chemis Academ [11] A.J. Ba Applic [12] J.R. Ma rials & [13] F. Man [14] D.D. M [15] D.D. M (1998) [16] R.P. Bu [17] D.C. S [18] M.V. T trochem [19] B.A. B [20] S. Fletc [21] G. Fafi [22] D.D. 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