**1. Introduction**

Currently, Biosensor technology has provided a number of benefits to detect both biological and chemical molecules. Abiosensor is a promising device, which is combination of sensitivi‐ ty of electrochemistry and specificity of biological recognition, enables to detect any kind of molecules in a short time with selectively and sensitively. Likewise many analytical methods, it has also limitations, such as high oxidation potentials lead to detection of non-target mole‐ cules, furthermore non-electroactive species cannot show electroactive signal for measure‐ ment or some biomolecules cannot be transformed by enzymes, even if they can be transformed, they require secondary molecules such as mediators, coenzymes or labels. In or‐ der to detect molecules without electrochemical reaction, electrochemical impedance spectro‐ scopy (EIS) can be employed as a measurement technique "to see electrode surface modifications just by looking impedance curves". As it is known, electrochemical impedance spectroscopy is an electrochemical technique that provides the examination of electrical prop‐ erties of electrode surface and binding kinetics of molecules between electrolyte and elec‐ trode surface. Therefore it can be used for biomolecular recognition, biomolecular bindings and biomolecular interactions between molecules such as DNA-DNA, DNA-protein, Receptor-Li‐ gand, Protein-Ligand, Antibody-Antigen, and Ion Channels-Ligands. As a consequence of this affinity provides label-free detection without chemical transformation and this binding prop‐ erty can be monitored by electrochemical impedance spectroscopy expeditiously. In this chap‐ ter, the information will answer a number of questions about the development of impedimetric biosensors. In fact that is focused on the usage of impedance for biosensor technology, and to demonstrate impedance curves and the meaning of electrical elements for obtained Nyquist Plots especially faradaic impedance. Employed biorecognition receptors and not yet em‐ ployed biorecognition receptors, which have different chemical residues, are discussed.

© 2013 ErtuğruL and Uygun; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 ErtuğruL and Uygun; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Theory of electrochemical impedance spectroscopy for biosensors**

Electrochemical impedance spectroscopy and the method of impedance are widely used for corrosion, batteries, bioelectrochemistry and electrochemistry. EIS provide electrochemical examination of electrical properties of electrode surface; on the other hand it can be called as electrochemical surface characterization. Therefore it is possible to realize the differentiation of electrode surface alterations easily. In biosensor technology it is used for monitoring bio‐ sensor modifications, layer formation on electrode surface and binding kinetics between molecules such as DNAs, receptors, antibodies, antigens, proteins, ions etc. This advantage provides examination binding kinetics of molecules, just by using obtained impedance spec‐ trums for binding kinetics of molecules leads to label-free detection. As it is known, for en‐ zyme based biosensors, a molecule needs to be transformed into another molecule by enzyme for obtain electroactive molecules or electrons for gain an electroactive signal elec‐ troactive signal can be disturbed by other molecules, which oxidation reduction potentials are same as analyte molecule. Electrochemical impedance spectroscopy overcomes this problem and provides non-electroactive detection of molecules. There is only one condition, which is the most important handicap, is to find a most specific biorecognition receptor for analyte. Likewise all electrochemical measurement techniques, by employing EIS for biosen‐ sor technology has the same fundamentals, which are composed of electrical circuits in or‐ der to determine electrochemical measurement. This electrical circuit is affected by AC current, which is generally used for impedimetric experiments. Alternative current (AC) is a wave shaped and has a frequency; therefore both potential and current oscillate (Fig. 1). This oscillation causes differentiation in time because AC excitation signal and sinusoidal current response are both based on Ohm law. As it is known the Ohm law includes; a potential, a current and a resistance for ideal DC circuits. However, for AC, some mathematical units must be added because of the frequency of AC.

**Figure 1.** Alternative current; Et and It.

As it can be seen in figure 1, the sinusoidal fluctuation of both current and potential show a difference, this difference, Φ, is determined as impedance which is an alternative current system resistance. Mathematical equationof this system is transformed into this equation 1 (Z; impedance, Et ; potential in a time, It ; current in a time, E0; potential at zero point, I0; cur‐ rent at zero point, ω; frequency, t; time)

**2. Theory of electrochemical impedance spectroscopy for biosensors**

must be added because of the frequency of AC.

180 State of the Art in Biosensors - General Aspects

**Figure 1.** Alternative current; Et and It.

Electrochemical impedance spectroscopy and the method of impedance are widely used for corrosion, batteries, bioelectrochemistry and electrochemistry. EIS provide electrochemical examination of electrical properties of electrode surface; on the other hand it can be called as electrochemical surface characterization. Therefore it is possible to realize the differentiation of electrode surface alterations easily. In biosensor technology it is used for monitoring bio‐ sensor modifications, layer formation on electrode surface and binding kinetics between molecules such as DNAs, receptors, antibodies, antigens, proteins, ions etc. This advantage provides examination binding kinetics of molecules, just by using obtained impedance spec‐ trums for binding kinetics of molecules leads to label-free detection. As it is known, for en‐ zyme based biosensors, a molecule needs to be transformed into another molecule by enzyme for obtain electroactive molecules or electrons for gain an electroactive signal elec‐ troactive signal can be disturbed by other molecules, which oxidation reduction potentials are same as analyte molecule. Electrochemical impedance spectroscopy overcomes this problem and provides non-electroactive detection of molecules. There is only one condition, which is the most important handicap, is to find a most specific biorecognition receptor for analyte. Likewise all electrochemical measurement techniques, by employing EIS for biosen‐ sor technology has the same fundamentals, which are composed of electrical circuits in or‐ der to determine electrochemical measurement. This electrical circuit is affected by AC current, which is generally used for impedimetric experiments. Alternative current (AC) is a wave shaped and has a frequency; therefore both potential and current oscillate (Fig. 1). This oscillation causes differentiation in time because AC excitation signal and sinusoidal current response are both based on Ohm law. As it is known the Ohm law includes; a potential, a current and a resistance for ideal DC circuits. However, for AC, some mathematical units

As it can be seen in figure 1, the sinusoidal fluctuation of both current and potential show a difference, this difference, Φ, is determined as impedance which is an alternative current

$$\dot{Z} = \frac{E\_t}{I\_t} = \frac{E\_0 \text{Sin(\omega t)}}{I\_0 \text{(Sin(\omega t + \Phi))}} = Z\_0 \frac{\text{Sin(\omega t)}}{\text{Sin(\omega t + \Phi)}} \tag{1}$$

In this equation, impedance is represented as Z, and Z is a phase shift of AC, furthermore this phase shift is angle of impedance curve of Nyquist plot.

This theory has been performed for biosensor technology for a long time, its aim is examina‐ tion of electrical characteristics of electrode surface for every layer formation and every in‐ teraction between molecules, and the obtained signal variations. In fact that charged groups of molecules have effect on impedance curves, layer has influence on electrical characteristic of electrode, this causes distribution of electrode surface charge, subsequently capacitive current varies, hence electrical circuit of the system keeps it balance and impedance increas‐ es or decreases [1].

EIS has an advantage over the other electrical measurement technique, which is an opportu‐ nity to design electrical circuit, according to obtained Nyquist plot curve. For figure 2, there is an electrical circuit model for obtained impedance curve. As you can see there are resis‐ tances and capacitance, in figure 2 there are both parallel capacitance and resistance, which they represent electrode surface, and a resistance is serial over this circuit, capacitance repre‐ sents electrical double layer of electrode, R2 represents resistance of the electrode, and R1 represents resistance of the solution in cell which is located electrodes inside of it. The Ny‐ quist plot of this electrode starts not zero point which means that the solution in the cell shows a resistance (If it started at zero point, that means the resistance of solution(R1) does not exist); therefore a resistance element(R1) is added in circuit model. The rest of the curve shows a characteristic sinusoidal impedance curve, which means only a resistance occurs, and R2 is added on circuit, and capacitance always occurs as a function of capacitive current, which represents in homogenity on electrode surface, because electrical double layer occurs.

**Figure 2.** Non-faradaic impedance curve, R2 electrode surface resistance and capacitance, and R1cell surface resistance.

Variation on impedance curve changes the electrical circuit model, an alteration especially on Nyquist curve an circuit element is added after R2 circuit element as serial [2].

**Figure 3.** Faradaic impedance curve, R2; electrode surface resistance, W; Warburg impedance and C; capacitance, and R1; cell solution resistance.

For figure 3, there is an additional circuit element, Warburg impedance(W) which repre‐ sents mass transfer to electrode surface. This resistance occurs when an interaction forma‐ tion, which is formed by electrical interaction, adsorption e.g., between electrode surface and solution a mass transfer occurs towards electrode surface, this transfer cannot be calcu‐ lated as diffusion because there is an accelerated mass transfer by affection. Therefore Ny‐ quist curve varies and becomes linear; this linearity represents Warburg impedance, which means mass transfer resistance. The interaction between electrode surface and solution, keep the balance between Warburg impedance and electrode surface resistance(R1), this balance can be unbalanced by mass transfer and after any increase on mass transfer the Warburg impedance shows dominancy on electrode surface resistance (R1) [3]. This domination shields resistance and sometimes resistance doesn't occur. A study was performed by Uy‐ gun and Sezgintürk, in this study gold film modified glassy carbon electrode was modi‐ fied by SAM of Cysteamine layer and positively charged Cysteamine attracted negatively charged redox probe(Fe(CN)6 -3/Fe(CN)6 -4), this mass transfer and this attraction shad‐ owed no resistance and Warburg impedance showed dominancy on circuit [4]. On the oth‐ er hand a redox probe as [Ru(NH3)6] 3+ will be repelled by positively charged modification. As you can see there are a number of conditions for impedance to design a circuit model, which surface of electrode, content of solution, characteristics of redox probe are impor‐ tant on electrical circuit modelling.

**Figure 4.** A schematic representation of electrode surface and redox probe interaction and their impedance spectrums.

For electrochemical impedance spectroscopy based biosensor systems, frequency scanning between two frequencies were chosen according to the solution, on the other hand electrical conductivity of solution is very important for choosing frequencies, in higher electrical con‐ duction ability of solution, which means the solution is highly concentrated by ions, lowest frequencies can be chosen especially in the presence of redox probe (lower than 0.1 Hz). A potential must be applied to gain a proper signal, this potential is called as AC excitation signal [2]. Its magnitude depends on the solution of measurement system's cell. When the solution includes redox probe such as ferricyanide, osmium complexes or ferrocene, accord‐ ing to the oxidation or reduction potentials of these materials, the beginning of the electro‐ chemical transformation potential is chosen. An unknown solution or unknown potential can be measured by cyclic voltammetry to find out the beginning oxidation/reduction po‐ tential of the electrolyte solution.
