**3. Challenges of EIS-based biosensors**

The detection of contaminates in water is very important since high pollution (heavy metals, pesticide and antibiotic residues, etc.) or the presence of pathogens (infectious microorganisms like viruses, bacteria, and fungi) can seriously endanger human health.

Several technical challenges hinder the development and construction of EISbased biosensors: limitations to detect small molecules, reusability, and sufficient stability for repetitive measurements [37, 38]. However, the most crucial problem is whether the impedance biosensors have sufficient selectivity for their application in real samples, which typically contain an unknown amount of nontarget molecules.

There are two main types of impedimetric biosensors—with or without a specific biorecognition element [30]. The most common biorecognition elements used are specific antibodies [39, 40]. The key to the successful performance of EIS-based biosensors is how to decrease the *non-specific bindings* and increase the *selectivity*. Selectivity is particularly important in real samples where the analyte concentration can be much smaller than the concentration of nontarget molecules. Non-specific binding is typically ascribed to proteins contained in a sample matrix attaching to the sensor interface through an unwanted process not involving the bimolecular recognition [41]. One option to decrease non-specific binding is to use blocking agents like bovine serum albumin (BSA), cysteine, or ethanolamine [42–44]. The choice of a blocking agent depends on the particular system. For example, Puttharugsa and Kamolpach used BSA for prevention of non-specific binding on gold electrodes, and the selectivity of the constructed biosensors was testes toward *Escherichia coli* K12 (*E. coli* K12) as a model with EIS [45]. When BSA is adsorbed physically onto the surface, the penetration of redox probe was reduced resulting in the increase of the semicircle Nyquist curve proving that BSA prevents the adsorption of bacteria onto the blocked surface by delaying the interfacial electrontransfer kinetics and increasing the electron-transfer resistance. Riquelme et al. studied several blocking agents (mercaptoundecanol, polyethylene glycol, BSA, and chicken serum albumin) to study the effect of biomolecule size and hydrophilic properties on blocking capacity on gold electrodes [43]. Higher impedance change was observed with lower molecular weight blocking agents, due to higher molecular packing on gold electrode. In addition lower blocking agent concentrations may be required if the electrode surface has already been bio-functionalized.

In addition to blocking agents, antifouling agents can be used to prevent target depletion via non-specific bindings [46]. Blocking agent reduces the non-specific binding by blocking the active functional groups on the surface and can stabilize the biomolecule bound to the surface [47]. Antifouling (or non-fouling) agent is

**57**

*Challenges and Applications of Impedance-Based Biosensors in Water Analysis*

a compound that has the capability to ensure the resistivity to the non-specific adsorption of proteins, cells, or other biological species [48]. Ortiz-Aguayo and Valle tried to decrease the non-specific adsorption to the graphite-epoxy composite electrode surface of an EIS-based wine aptasensor using polyethylene glycol [46]. Even though the aptasensor showed good sensitivity, the blocking did not work so

One of the main challenges is the *sensitivity*, which depends on the thickness of the sensing layer [49–53]. If the sensing layer is too thin, the electrode surface may be exposed, which would decrease the signal to noise ratio and decrease the sensitivity. If the sensing layer is too thick, the detected AC impedance current reduces meaning that the electron transfer between layers is hindered and the sensitivity is decreased. For example, Groß et al. studied the effect of the thickness on the base resistance in the range 30 to 150 μm and found that the sensitivity decreased along with the sensitive layer getting thicker [49]. They also found that there is a trade-off between wide linear range and high sensitivity. In addition, the sensor signal became slower as the thickness of the sensitive layer increased [49]. Functionalization of the electrodes with high-affinity biomolecules enhances besides selectivity and also the sensitivity of the system. Therefore, EIS is very often combined with different nanostructured interfaces in order to increase the amount of biorecognition material on the surface and therefore to improve the sensor sensitivity and extend its linear working [54–59]. This improvement is associated with the dimensions of nanomaterials, which endows them with a large surface/volume ratio and high specific area enabling to immobilize bigger amount of biomolecules

*Reusability* of the antibody-based biosensors can be problematic because of the strength and irreversibility of antibody-antigen binding, and the regeneration of these surfaces without damaging the antibody layer can be complicated due to harsh conditions [61]. For impedance biosensors, extreme pH values of strong acids or bases may not be compatible with the chemistries employed for the protein immobilization, meaning that the reusability of a biosensor can be problematic. Radhakrishnan et al. studied the regeneration of antibody-based Si electrodes [62]. Even though they could regenerate the surfaces for 15 days, the impedance spec-

trum gradually degraded during these multiday regeneration trials.

Finally, it can be challenging to detect *small molecules* like heavy metals, pesticides, or antibiotic residues with EIS due to the exponential increase of the charge-transfer resistance through the polymer-protein layer to the underlying electrode surface [41, 63]. Small molecules (less than kDa) alone usually induce very small detectable response, which can be very difficult to measure especially in real samples where the concentration of the target molecule can be very low [41]. One possibility to improve the detection of small molecules is to conjugate these via a functional group to a larger carrier molecule (i.e., a protein) or with electrochemically bright metal and semiconductor nanomaterials, as changes due to binding of large molecules can be detected more easily detected. For example, Radhakrishnan et al. used impedance-based biosensor to detect two endocrine-disrupting chemicals (EDC) [41], which are small compounds found in various materials such as pesticides, additives, or contaminants in food [64]. It was found that for detecting small molecules, impedance biosensors can be operated at only one or a few frequencies that are most sensitive to analyte binding, and the sensitivity improved when attained with analyte conjugation. Gold nanoparticles (Au-NPs) have been used due to their electrochemically active surface; in particular, Au-NPs bound to the electrode digits disrupt the formation of the double layer around the electrodes, thus changing the double-layer capacitance [65–67]. For example, de Macedo et al. used Au-NPs for signal amplification, and comparing the results of free and

*DOI: http://dx.doi.org/10.5772/intechopen.89334*

onto biosensor surface [60].

efficiently; and the recovery was approximately 77%.

#### *Challenges and Applications of Impedance-Based Biosensors in Water Analysis DOI: http://dx.doi.org/10.5772/intechopen.89334*

a compound that has the capability to ensure the resistivity to the non-specific adsorption of proteins, cells, or other biological species [48]. Ortiz-Aguayo and Valle tried to decrease the non-specific adsorption to the graphite-epoxy composite electrode surface of an EIS-based wine aptasensor using polyethylene glycol [46]. Even though the aptasensor showed good sensitivity, the blocking did not work so efficiently; and the recovery was approximately 77%.

One of the main challenges is the *sensitivity*, which depends on the thickness of the sensing layer [49–53]. If the sensing layer is too thin, the electrode surface may be exposed, which would decrease the signal to noise ratio and decrease the sensitivity. If the sensing layer is too thick, the detected AC impedance current reduces meaning that the electron transfer between layers is hindered and the sensitivity is decreased. For example, Groß et al. studied the effect of the thickness on the base resistance in the range 30 to 150 μm and found that the sensitivity decreased along with the sensitive layer getting thicker [49]. They also found that there is a trade-off between wide linear range and high sensitivity. In addition, the sensor signal became slower as the thickness of the sensitive layer increased [49]. Functionalization of the electrodes with high-affinity biomolecules enhances besides selectivity and also the sensitivity of the system. Therefore, EIS is very often combined with different nanostructured interfaces in order to increase the amount of biorecognition material on the surface and therefore to improve the sensor sensitivity and extend its linear working [54–59]. This improvement is associated with the dimensions of nanomaterials, which endows them with a large surface/volume ratio and high specific area enabling to immobilize bigger amount of biomolecules onto biosensor surface [60].

*Reusability* of the antibody-based biosensors can be problematic because of the strength and irreversibility of antibody-antigen binding, and the regeneration of these surfaces without damaging the antibody layer can be complicated due to harsh conditions [61]. For impedance biosensors, extreme pH values of strong acids or bases may not be compatible with the chemistries employed for the protein immobilization, meaning that the reusability of a biosensor can be problematic. Radhakrishnan et al. studied the regeneration of antibody-based Si electrodes [62]. Even though they could regenerate the surfaces for 15 days, the impedance spectrum gradually degraded during these multiday regeneration trials.

Finally, it can be challenging to detect *small molecules* like heavy metals, pesticides, or antibiotic residues with EIS due to the exponential increase of the charge-transfer resistance through the polymer-protein layer to the underlying electrode surface [41, 63]. Small molecules (less than kDa) alone usually induce very small detectable response, which can be very difficult to measure especially in real samples where the concentration of the target molecule can be very low [41]. One possibility to improve the detection of small molecules is to conjugate these via a functional group to a larger carrier molecule (i.e., a protein) or with electrochemically bright metal and semiconductor nanomaterials, as changes due to binding of large molecules can be detected more easily detected. For example, Radhakrishnan et al. used impedance-based biosensor to detect two endocrine-disrupting chemicals (EDC) [41], which are small compounds found in various materials such as pesticides, additives, or contaminants in food [64]. It was found that for detecting small molecules, impedance biosensors can be operated at only one or a few frequencies that are most sensitive to analyte binding, and the sensitivity improved when attained with analyte conjugation. Gold nanoparticles (Au-NPs) have been used due to their electrochemically active surface; in particular, Au-NPs bound to the electrode digits disrupt the formation of the double layer around the electrodes, thus changing the double-layer capacitance [65–67]. For example, de Macedo et al. used Au-NPs for signal amplification, and comparing the results of free and

*Biosensors for Environmental Monitoring*

transfer resistance (*R*ct).

than π/2 [29, 36].

ger human health.

phase element (CPE) having capacitive-like properties in parallel with the charge-

layer capacitance (*C* dl), which introduces the constant phase shift of -π/2 rad (−90°) at all the frequencies. In reality, the CPE introduces the phase shift φ less

The faradaic sensor model includes the Warburg impedance (*W*), which describes diffusion phenomenon taking place due to chemical redox processes. The ideal Warburg impedance introduces the phase shift of π/4. Values of the charge transfer *Rct* and *W* depend on physicochemical parameters of a system. In real systems, impedance spectra are usually more complicated, and, therefore, the Randle's

The detection of contaminates in water is very important since high pollution (heavy metals, pesticide and antibiotic residues, etc.) or the presence of pathogens (infectious microorganisms like viruses, bacteria, and fungi) can seriously endan-

Several technical challenges hinder the development and construction of EISbased biosensors: limitations to detect small molecules, reusability, and sufficient stability for repetitive measurements [37, 38]. However, the most crucial problem is whether the impedance biosensors have sufficient selectivity for their application in real samples, which typically contain an unknown amount of nontarget molecules. There are two main types of impedimetric biosensors—with or without a specific biorecognition element [30]. The most common biorecognition elements used are specific antibodies [39, 40]. The key to the successful performance of EIS-based biosensors is how to decrease the *non-specific bindings* and increase the *selectivity*. Selectivity is particularly important in real samples where the analyte concentration can be much smaller than the concentration of nontarget molecules. Non-specific binding is typically ascribed to proteins contained in a sample matrix attaching to the sensor interface through an unwanted process not involving the bimolecular recognition [41]. One option to decrease non-specific binding is to use blocking agents like bovine serum albumin (BSA), cysteine, or ethanolamine [42–44]. The choice of a blocking agent depends on the particular system. For example, Puttharugsa and Kamolpach used BSA for prevention of non-specific binding on gold electrodes, and the selectivity of the constructed biosensors was testes toward *Escherichia coli* K12 (*E. coli* K12) as a model with EIS [45]. When BSA is adsorbed physically onto the surface, the penetration of redox probe was reduced resulting in the increase of the semicircle Nyquist curve proving that BSA prevents the adsorption of bacteria onto the blocked surface by delaying the interfacial electrontransfer kinetics and increasing the electron-transfer resistance. Riquelme et al. studied several blocking agents (mercaptoundecanol, polyethylene glycol, BSA, and chicken serum albumin) to study the effect of biomolecule size and hydrophilic properties on blocking capacity on gold electrodes [43]. Higher impedance change was observed with lower molecular weight blocking agents, due to higher molecular packing on gold electrode. In addition lower blocking agent concentrations may be

circuit with a corresponding plot may not give proper results [31].

required if the electrode surface has already been bio-functionalized.

In addition to blocking agents, antifouling agents can be used to prevent target depletion via non-specific bindings [46]. Blocking agent reduces the non-specific binding by blocking the active functional groups on the surface and can stabilize the biomolecule bound to the surface [47]. Antifouling (or non-fouling) agent is

**3. Challenges of EIS-based biosensors**

Sometimes simplifications are introduced, and the CPE is replaced by a double-

**56**

conjugated protein, the latter generated a measured signal 40–50 times higher and the limit of detection 64 times lower [68]. MacKay et al. also used Au-NPs to evaluate the sensing ability of biosensor chips using impedance measurements and found that the adsorption of Au-NPs to the surface binding sites increased the impedance through double-layer capacitance and higher sensitivity is gained using single frequent measurement [65].
