REVIEW The use of electrochemical impedance spectroscopy for biosensing F. Lisdat & D. Sch��fer Received: 28 November 2007 /Revised: 8 February 2008 /Accepted: 9 February 2008 /Published online: 16 April 2008 # Springer-Verlag 2008 Abstract This review introduces the basic concepts and terms associated with impedance and techniques of measur- ing impedance. The focus of this review is on the application of this transduction method for sensing purposes. Examples of its use in combination with enzymes, antibodies, DNA and with cells will be described. Important fields of application include immune and nucleic acid analysis. Special attention is devoted to the various electrode design and amplification schemes developed for sensitivity enhancement. Electrolyte insulator semiconductor (EIS) structures will be treated separately. Keywords Impedance . Sensor . DNA . Antibodies . Cells . Enzymes Introduction Impedance spectroscopy is a powerful method of analysing the complex electrical resistance of a system and is sen- sitive to surface phenomena and changes of bulk properties. Thus, it is a valuable method in electrochemical research, where an entire conference series is devoted to progress in and applications of this technique (the EIS Symposium Series, the most recent of which took place in 2007 in Argeles sur Mer, France). It has been intensively used, for example, for the elucidation of corrosion mechanisms, the characterisation of charge transport across membranes and membrane/solution interfaces, and the optimisation of bat- teries. In the field of biosensors, it is particularly well-suited to the detection of binding events on the transducer surface. First examples of its use were reported at the end of the 1980s however, the method has found increasing application in recent years due to advances made in instrumentation. Besides the detection of biorecognition processes, it is a valuable tool for characterising surface modifications, such as those that occur during the immobilisation of biomolecules on the transducer. In this review, a short introduction to the measuring principles of electrochemical impedance spectros- copy will be provided, followed by an overview of the usage of the technique in the area of biosensors. The technique has the inherent potential for label-free detection, which is of special interest in bioanalysis since this circumvents the need to modify biomolecules with fluorescence dyes, enzymes, redox or radioactive labels. Several examples of its applica- tion to the detection of DNA, antigens or antibodies have been demonstrated. However, an amplification step is often necessary to achieve a defined response with very low analyte concentrations. The different approaches used to obtain signal enhancement will be also treated within this review. Basic principles and terms in impedance spectroscopy The impedance Z of a system is generally determined by applying a voltage perturbation with a small amplitude and detecting the current response. From this definition, the impedance Z is the quotient of the voltage���time function V(t) and the resulting current���time function I(t): Z �� V t�� �� I t�� �� �� V0 sin 2�� f t�� �� I0 sin 2�� f t �� ���� �� �� 1 Y ��1�� where V0 and I0 are the maximum voltage and current signals, f is the frequency, t the time, �� the phase shift Anal Bioanal Chem (2008) 391:1555���1567 DOI 10.1007/s00216-008-1970-7 F. Lisdat (*) : D. Sch��fer Biosystems Technology, Wildau University of Applied Sciences, 15745 Wildau, Germany e-mail: flisdat@igw.tfh-wildau.de

between the voltage���time and current���time functions, and Y is the complex conductance or admittance. The impedance is a complex value, since the current can differ not only in terms of the amplitude but it can also show a phase shift �� compared to the voltage���time function. Thus, the value can be described either by the modulus |Z| and the phase shift �� or alternatively by the real part ZR and the imaginary part ZI of the impedance. This is illustrated in Fig. 1. Therefore the results of an impedance measurement can be illustrated in two different ways: using a Bode plot which plots log|Z| and �� as a function of logf, or using a Nyquist plot which plots ZR and ZI. The name impedance ���spectroscopy��� is derived from the fact that the impedance is generally determined at different frequencies rather than just one. Thus, an impedance spectrum is obtained that allows the characterisation of surfaces, layers or membranes as well as exchange and diffusion processes. To achieve this, the impedance spectrum is often analysed using an equivalent circuit. This circuit, which commonly consists of resistances and capacitances, represents the different physicochemical properties of the system under investigation [1���3]. Alternatively, the system can be described based on transfer functions derived from the basic laws of the processes involved, such as electro- kinetics, diffusion, partition, etc. However, it is not only possible to describe a system of interest, but the technique can also be used for analytical purpose. In this case, the change of one impedance element��� a resistance or a capacitance���as a function of the solution composition is evaluated. In some cases it is also possible to correlate the overall impedance to a change in concentration. This can simplify measurements, since it is often sufficient to determine the impedance at just one selected frequency or within a limited frequency window (where the relative changes are largest) in such cases. In electrochemical impedance spectroscopy, where the electrolyte solution is one component of the system to be investigated, four elements are usually used to describe the impedance behaviour: ohmic resistance, capacitance, constant- phase element and Warburg impedance. These elements and their definitions are summarised in Table 1. Equivalent circuits are used in order to approximate the experimental impedance data with these ideal or distributed impedance elements arranged in series and/or in parallel. Many electrochemical systems have been analysed according to this procedure. One can probably find a model for describing the impedance behavior of any particular system in the literature. This can be used at least as a starting point for analysis. For the situation of an electrode in contact with an electrolyte, the so-called Randles circuit is used (as shown in Fig. 2), comprising the solution resistance Rs, the charge transfer resistance Rct, the double layer capacitance Cdl and the Warburg impedance W. In the Nyquist plot shown in Fig. 2, the values for Rs and Rct can be easily determined. The double layer capacitance can be calculated from the frequency at the maximum of the semicircle (��=2��f=1/RctCdl). The product of Rct and Cdl is often termed the time constant �� of the electrochem- ical process. The 45�� line indicating Warburg-limited behaviour can be extrapolated to the real axis. The intercept is equal to Rs + Rct ��� 2��Cdl, from which �� and subsequently diffusion coefficients can be calculated (see Table 1). For analytical applications, however, the equivalent circuit is often simplified by neglecting the Warburg impedance. This can be done by choosing a frequency range where no 45�� line is observed in the Nyquist plot and the interfacial or bulk impedance is dominant. In order to characterise a biological material, e.g. anti- bodies or cells, electrodes must be introduced into the system, thus forming an electrochemical cell. Upon applying an ac voltage perturbation, the current is coerced to flow through all components of the system���the working elec- trode, the biological material, the solution and the counter electrode. The measured impedance is the sum of all of the individual contributions. Figure 3 provides a simplified equivalent circuit for this situation, neglecting the potential Fig. 1 Impedance is a complex value that is defined as the quotient of the voltage(time) and current(time) functions. It can be expressed as the modulus ���Z��� and the phase angle ��, or it can be specified by the real (ZR) and the imaginary (ZI) parts of the impedance Table 1 Definitions, frequency dependences and phase shifts of the impedance elements most often used to describe (bio)electrochemical systems Impedance element Definition Phase angle Frequency dependence R Z=R 0�� No C ZC �� 1 jwC 90�� Yes CPE ZCPE �� 1 A jw��a �� 0���90�� Yes W (infinite)a ZW �� s��������� w p 1 j�� �� 45�� Yes s �� RTp������ n2F2 2 1 D0c0 p������������ �� DR p���������������1 cR a When the diffusion region is limited a different behaviour will be observed. A description of the two borderline cases with blocked and nonblocked transport at the end of the diffusion layer is given in [3]) 1556 Anal Bioanal Chem (2008) 391:1555���1567