Electrochemical Sensors and Biosensors Based on Self-Assembled

Downloaded by UNIV OF ARIZONA on November 27, 2012 | http://pubs.acs.org Publication Date (Web): November 26, 2012 | doi: 10.1021/bk-2012-1112.ch011

Chapter 11

Electrochemical Sensors and Biosensors Based on Self-Assembled Monolayers: Application of Nanoparticles for Analytical Signals Amplification Hanna Radecka,* Jerzy Radecki, Iwona Grabowska, and Katarzyna Kurzątkowska Laboratory of Bioelectroanalysis, Department of Biosensors, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland *E-mail: [email protected]. Phone: +48 89 523 46 36. Fax: +48 89 524 01 24

One of the most popular techniques to create well-defined functional surfaces is the formation of self-assembled monolayers (SAMs) based on the covalent interactions of thiols, disulphides, sulphides and other related molecules with the surfaces of noble metals, particularly gold, as well as platinum and mercury. This phenomenon is a base of sensors and biosensors presented. They can be divided into two main categories. The first type is based on ion-channel mimetic mode, where the analytical signals are governed by changing of accessibility of redox marker (present in the sample solution) to the electrode surface upon analyte recognition. This analytical approach was successfully applied for development of sensors and biosensors destined for determination of numerous analytes, relevant for medical diagnosis, such as: adenine nucleotides, ethene dicarboxylic acids, dopamine, viruses, His-tagged proteins and specific sequences of DNA. Biosensors based on ion-channel mimetic mode were also suitable for screening of interactions between amyloid β-peptide and potential drugs.

© 2012 American Chemical Society In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Another type of sensors and biosensors presented is based on redox active monolayers.The gold electrodes modified with (dipyrromenthene)2-Cu(II) and porphyrine-Fe (III) were applied for amperometric determination of paracetamol and L-histidine, respectively. The electrodes functionalized with dipyrromethe-Cu(II) SAM was suitable for oriented and stable immobilization of His-tagged proteins. This redox active monolayer was a base of biosensors destined for exploring the interactions between receptor protein and target species present in the aqueous solutions. The analytical signal amplification obtained through application of the different kind of nanoparticles such as colloidal gold, nanorods and carbon nanotubes for sensor construction will be disused. Keywords: Gold electrode; functionalization; self-assembling monolayers synthetic receptors; natural receptors; nanoparticles; voltammetry; electrochemical impedance spectroscopy

Introduction Nowadays the main streams of analytical research are directed to improve the efficiency of medical diagnosis, safety food production and environment screening. The development of all of these branches is not possible without suitable analytical methods. Such situation is a drive force for continuous work on the new trends in analytical chemistry clearly visible in the numerous recent published scientific literatures (1–8). The remarkable selectivity and sensitivity of molecular recognition processes occurring in the biological systems are going in water. This makes the inexhaustible source of inspiration for researchers dealing with supramolecular and analytical chemistry. Generally, intermolecular recognition processes are relied on the formation of hydrogen bonds, electrostatic interactions, Van der Waals interactions, π electron interactions, and Lewis acid – acid base interactions. The development of systems that can efficiently and selectively interact with target species in water is intrinsically difficult. Recent developments do however suggest that solutions can be found through a supramolecular approach which takes its inspiration from the natural world (9–11). Nature achieves high affinity and high selectivity towards targeted substrate in water through multiple weak, non covalent interactions between the functional groups on the binding partners. Many of synthetic and natural receptors have lack of water solubility, which prevents binding studies in aqueous solution. One way of solution of this problem is immobilization of receptors at the surface of solid electrodes. Such system allows for observation of intermolecular recognition processes at the interface 294 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


water / solid electrodes. Interface it is a place in which the most of biologically relevant recognition processes are going on. Thus, intermolecular recognition processes occurring at the border between the surface of electrode and water sample solution are the base for development of new electrochemical sensors and biosensors suitable for medical diagnosis, food analysis and environmental screening. Generally, sensor is a tool that integrates synthetic sensing element with physical transducer where the interactions between the sensing element and target molecules are directly converted into an electronic signal. Biosensors are sub-group of sensors that pose as a sensing element the biological molecules. Selectivity of sensors or biosensors is determined by selectivity of recognition process. On the other hands, sensitivity is governed by two parameters: sensitivity of intermolecular recognition and efficiency of transducer. In recent years, a continuous and fast increase of number of articles concerning the electrochemical sensors is clearly visible (12–14). Here we described the of electrochemical sensors and biosensors based on modified electrodes consist of receptors and nanomaterials designated for medical diagnosis, food analysis and environmental screening. According to mechanism of analytical signal generation, they can be divided into two general groups: • •

Sensors and biosensors based on ion-channel mimetic mode Sensors and biosensors based on bi-functional redox active layers, which serve as a transducer and analytically active element at the same time.

Sensors and Biosensors Based on Ion-Channel Mimetic Mode The ion-channel biomimetic sensor is one in which the kinetic of electron transfer reaction of the redox marker present in the sample solution is affected by analytes binding to the receptor. This system consist of two elements: electrode modified with receptor molecules and redox active marker in solution (Figure 1). This analytical approach was introduced by Umezawa and co – workers (15, 16). The mechanism of this sensing method relys on the control of the redox active marker accessibility to the electrode surface by the binding events between the analytes from solution and receptor molecules located on the surface of electrode. There are two sub-groups of ion channel sensors: •

•

Intermolecular type in which the receptor monolayer and analyte as results of recognition formed the charged supramolecular complex and access of marker to surface of electrode is controlled by electrostatic interactions (Figure 1A). Intramolecular type in which the access of marker to the surface of electrode is controlled by steric hindrance which appear at the surface of electrode as a consequence of recognition process between the analytes an receptor molecules located at the surface of electrode (Figure 1B). 295

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Figure 1. Working principle of ion-channel mimetic sensor: A - intermolecular type, B - intramolecular type. (see color insert)

The main advantage of ion-channel sensors is the possibility of determination with high sensitivity of the non electroactive analytes by using of amperometric techniques. Ion-channel sensors have the inherent possibility of signal amplification. The binding of only few numbers of analytes to the receptors monolayer can induce, or suppress oxidation and/or reduction of a greater numbers of markers (Figure 1A, B). Generally in the literature there are not so many examples of intramolecular ion channels sensors. One of this approach is the detection of redox-inactive and electrically neutral analytes by a cyclodextrin derivative monolayer (17). First examples intramolecular ion channels sensors developed in our laboratory are sensors based on gold electrodes modified with the macrocyclic polyamine as an analytically active element (18, 19). In these cases for incorporation of host molecules on the electrode surface we have applied two methods. The first one uses macrocyclic polyamine host molecules containing six SH groups responsible for covalent bonding to the surface of gold electrode (Figure 2A). The second method of modification employ macrocyclic polyamine host incorporated into a 1-dodecanethiol SAM immobilized onto the Au surface via hydrophobic and van der Waals forces (embedment modification) (Figure 2B). The electrodes modified in such manners were used for adenine nucleotides (18) and ethene dicarboxylic acids detection (19). The sensing of adenine nucleotides was done using both type of electrodes with two techniques, CV and OSWV. The measurements have been performed in the presence of borate buffer, because components of this buffer showed no influence on the voltammetric behaviour of gold electrodes coated with macrocyclic polyamine films. Two redox markers: [Ru(NH3)6]3+ and [IrCl6]2– showed quasi-irreversible CV curves in the absence of anionic guests. The interactions of adenine nucleotides with the polyamine hosts were examined with the both of markers (18). In the case of positive [Ru(NH3)6]3+ and negative [IrCl6]2-, the decrease of currents with increase of anionic guests concentration 296 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


were observed. The highest response of both types of electrodes, modified via covalent and embedment method was observed in the presence of ATP4–. The sensitivity sequence observed for both of redox markers was as follows:

The sensors prepared by the embedment method displayed two order of magnitude better detection limit (~1.0 × 10-7 M) for ATP4– than the sensors modified by the covalent technique. This enhanced detection limit suggests that flexibility of the receptors, and decreasing of free energy of ion transfer across the interface may play a crucial role in recognition of target analytes. Both types of electrodes are characterized by good durability and sensor–to–sensor repeatability.

Figure 2. Schematic illustration of receptor immobilization on the surface of gold electrodes by: A - covalent bonds, B - embedment method, C - using nanoparticles. (see color insert)

As it was mentioned above we have observed that upon increasing concentration of anionic analytes, the current of redox reaction decreased, for both types of applied markers and macrocyclic polyamine modified gold electrodes. 297 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


A possible explanation of this phenomenon might be as follows. In this particular case, the molecular layer packing factor induced by host–guest complexation, which may cause a decrease in permeability of the cationic marker, seems to dominate over the effect of reduction of charge repulsion (16, 17). The results obtained indicated that macrocyclic hexapolyamines incorporated into SAMs immobilized onto gold surfaces form rather intramolecular ion channel sensors. In such type of sensors, the interaction between host and guest control the permeation of marker ions through the intramolecular cavity of polyamines. With the increase of adenine nucleotides or ethene dicarboxylic acids concentration, the decrease of peak currents and their shifts to the negative potentials were observed. It appears that macrocyclic polyamines form on the surface of gold the intramolecular channel type sensors sensitive towards anionic guests (18, 19). The sensor for determination of dopamine is an example of intermolecular ion channel sensor developed in our laboratory (20). Dopamine (DA) is one of the most significant neurotransmitters because of its role in the functioning of the cardiovascular, renal, hormonal, and central nervous system. The majority of electrochemical sensors for dopamine determination exploit its ease of oxidation. However, the oxidative approaches suffer from interferences caused by other electroactive substances existing in the physiological samples. One of the main interferences is ascorbic acid (AA). The concentration of DA is extremely low (0.01-1 μM) while that of AA is as high as 0.1 mM in biological systems (21). Moreover, at almost all electrodes materials, DA and AA are oxidized at nearly the same potential, which results in overlapped voltammetric response. In order to solve this problem, we decided to apply the different approach for the detection of DA based on an intermolecular ion-channel mimetic sensor. In such type of sensor the binding of analytes to receptors immobilized on electrode surfaces facilitates or suppresses the access of an anionic (cationic) marker ion to the modified surface due to electrostatic attraction or repulsion of the marker and/or distortion of the modification layer arrangement. This leads to the changes of the electron transfer rate between the marker and electrode surface through the modification layer. This type of ion-channel sensor is a subject of numerous scientific literatures (15, 16, 22–24). It has been already reported that corrole derivatives form complexes with dihydroxybenzene derivatives through a NH· · ·OH hydrogen bond (25). Thus, this compound has been selected as a suitable receptor for dopamine. Figure 1A illustrates the working principle of the sensor proposed. The corrole host molecules have been covalently attached on the surface of gold electrodes through Au-S bonds. In the measuring condition (pH 7.0), corrole exists as an uncharged molecule (25). The monolayer created on the electrode surface is quasi permeable for a redox marker existing in the aqueous solution. Upon addition of dopamine, the corrole-DA complex is formed and the monolayer gained the positive charge, which repealed the positive charged redox marker. This leads to the decreasing of the electron transfer rate between the marker and electrode surface through the modification layer. The electroanalytical signals generated based on the corrole-DA complex formed at the electrode surface were explored using Osteryoung square-wave voltammetry (OSWV) and electrochemical impedance spectroscopy (EIS) in the presence of [Ru(NH3)6 ]+3 298 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


as an electroactive marker, which does not interfere with the oxidation of the DA or AA. The proposed sensor is effective regarding the following parameters: very good sensitivity toward DA (detection limit in 10-12 M range by using both EIS and OSWV techniques), very good selectivity (human plasma components at 80-fold dilution have no influence on dopamine determination), simple, and quick procedure of electrode preparation. Therefore, the gold electrodes modified with mixed corrole-mercaptohexanol SAM work as intermolecular “ion-channel mimetic sensors” in the presence of [Ru(NH3)6]Cl3 as the redox marker and could be applied for the determination of dopamine in clinical analysis (20). The similar working manner was applied for the sensor designed for determination of acrylic acid in food samples based on gold electrode modified with tetralactam (26). The recent example of ion-channel mimetic sensor electrochemically developed in our laboratories is genosensor with covalently attached HS-ss DNA probe destined to detection of specific DNA sequence of avian influenza virus H5N1 (27).The hybridization processes with target DNA was monitored with OSWV in the presence of [Fe(CN)6]3-/4- redox probe. The genosensor displayed good sensitivity, namely detection limits of 2.2 × 10-11 M and 2.4 × 10-11 M for complementary 20-mer ssDNA and double stranded 181-bp DNA containing 20 nucleotides complementary to the probe. This genosensor was also able to discriminate different positions (at 3’-end, middle and 5’-end) of the complementary part in the PCR products.

Electrochemical Sensors and Biosensors Incorporating Nanoparticles Nanotechnology has recently become one of the most exiting fields in analytical chemistry. A wide variety of nanomaterials, especially gold nanoparticles have been applied in broad area (28). They allow increase in surface area of modified electrodes. This leads to increase of biomolecules numbers in analytically active layers and improving the sensitivity of analytical devices. Nanoparticles (in the size range of 1-100 nm) exhibit unique chemical, physical and electronic properties that are different from those of bulk materials, and can be used to construct electrochemical sensors and biosensors with improving sensing properties (28). The most popular metal nanoparticles are colloidal gold (29–31) and gold nanorods (32–35). The isotropic nature of spherical colloidal gold nanoparticles prevents the selective binding of molecules on surfaces. In contrast, the anisotropic features of Au nanorods allow their assembly in various orientations (35). Both of them, having excellent conductivity and biocompatibility are widely used in electrochemical sensors for analytical signal amplification (29, 31, 33, 36–40). The dynamic assembly and disassembly of double–strand DNA modified Au nanorods at different temperature was a base of plasmonic circular dichroism response towards ssDNA at nM level (41). 299 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The type of nanoparticles suitable electrochemical sensor design are carbon nanotubes (42–44) and more recently graphene (6–8, 45, 46). The one of the crucial role of carbon nanotubes, graphene and gold nanoparticles in senor or biosensor construction is the enhancement of electron transfer from redox centers to the electrode surface (8, 28). The graphene is a flat monolayer of carbon atoms, tightly packed into a two–dimensional (2D) honeycomb lattice (46). Whereas, single-walled carbon nanotube (SWCNTs) can be interpreted as the graphene sheets rolled up to form hollow tubes with diameters ranging between 0.4 and 2 nm (1D structure) (42). The electronic properties of the tubes are strongly affected by both the rolling angle of the graphene sheets and their final diameter. These carbon nanostructures are both one atom thick, and their electronic properties are extremely sensitive to adsorption of chemical species on their surface. The carbohydrate - functionalized carbon nanotubes as well as graphene could be employed for the rapid identification of bacteria in samples consisting of water systems, soils, or human specimens (45). Multilayered graphene – Al2O3 nanopore sensors showed high sensitivity for detection of DNA and DNA-protein complexes (47). Urea, clinically important analyte, was successfully detected with ITO electrode functionalized with multilayered graphene (46). The self-assembly of gold nanoparticles on single–walled carbon nanotubes is the recent excellent example of complex nano-structure successfully applied for sensing of different gasses (48). Taking into account the properties of the gold, as well as carbon nanostructures, we have applied them for fabrication of sensors and biosensors working in ion-channel bio-mimetic mode. Two main strategies were employed for receptor immobilization on the nanoparticle surface: one is based on electrostatic interactions (Figure 3A) and another one is based on covalent bonds between Au–S atoms (Figure 3B).

Figure 3. Schematic illustration of sensors based on nanoparticles. Receptor immobilization : A - via electrostatic interactions; B - via covalent bonds. (see color insert)

300 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


One of the example of the application of colloidal gold particles is ion-channel mimetic biosensor destined for the determination of association constant of β amyloid with selected alkaloids (49, 50). The main therapeutic strategy in Alzheimer’s disease relays on use the inhibitors which should bind or stabilized the α-helical or early formed β-sheet structures (51, 52). The compounds used as the inhibitors of amyloidosis are: peptides (53), antibodies (54), isoflavones (52), nicotine and its metabolites (51, 55), just to name a few. Using the electrochemical biosensor based on impedance spectroscopy (EIS), the association constants of (−)cotinine and (−)nicotine ditartrate and selected alkaloids with Aβ (1–40) peptide were determined (49, 50). Such attempt mimics well the physiological processes occurring at the interface. The crucial step of proposed method is the suitable immobilization of Aβ (1–40) peptide on the electrode surface. It is known that, adsorption of proteins onto bulk metal surfaces leads to their denaturation and loss of their bioactivity (56). The additional modification of gold electrodes with colloidal gold nanoparticles is one of the solution of the above problem. It has been recently reported that proteins adsorbed onto colloidal Au retain their bioactivity (56, 57). In the work presented, the gold electrodes were modified with Au nano-particles by self-assembly with using 1,6-hexanedithiol as cross-linkers. So, the Aβ (1–40) peptide was adsorbed directly on the surface of colloidal Au (Figure 3A). The interactions between Aβ (1–40) peptide and with two potent drug molecules (−)nicotine ditartrate and (−)cotinine (49) and selected alkaloids (50) were monitored with using electrochemical impedance technique (EIS). It might be concluded that the electrochemical impedance spectroscopy, together with electrode modified with self-assembled layer of colloidal Au is very suitable technique for screening the interactions between Aβ-peptides and compounds, which could be applied as the potential drugs. This measuring system is relatively simple in the comparison to other spectroscopic one and allows to determine not only thermodynamic of the interfacial interactions, but their kinetics as well (57). The immunosensors incorporated antibodies as a sensitive element mainly work according to mechanism of intramolecular ion-channel. This type of biosensors are promising tools for detection of pathogens since antibodies are natural receptors responsible for binding of antigens harmful for the organism. Thus their binding selectivity and efficiency are naturally high. The complex between antigen and specific antibody adsorbing on a surface of an electrode forms an insulating layer. This phenomenon can be monitored by electrochemical impedance spectroscopy (58). In order to improve immunosensors sensitivity the colloidal gold and gold nanorods were applied for their fabrication (59, 60) (Figure 3A). The successive electrode modification steps: (1) 1,6-hexanedithiol SAM formation on the Au electrode, (2) gold nanoparticles layer formation through Au-S covalent bonds, (3) deposition of antibody, (4) blocking the remaining spaces on gold layer with BSA, were controlled using CV and EIS. 301 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Both type of gold nanoparticles facilitate the electron transfer between the redox marker and the electrode surface, and at the same time create the suitable environment for biomolecule deposition assuring keeping their biological activity (49, 50, 59, 60). The immobilization of antibody on the gold nanoparticle layer, forming the insulating layer, decreased the accessibility of [Fe(CN)6 ]-3/-4. Further decreasing of the faradaic current was observed upon immobilization of BSA onto the remaining space on the gold layer. The electron interfacial resistance increased linearly with increasing concentration of specific antigen His6-rSPI2 in the range between 10pg/ml and 1 ng/ml. Immunosensors using gold nanorods showed 40% better analytical response then this incorporating colloidal gold (59). Nanorods, unlike more symmetrically shape nanoparticles have the ability to assemble aligned configuration. This property make them suitable for electrochemical devices. The immunosensors for His-tagged proteins determination were very selective. The presence of culture medium has no influence on immunosensors performance (59, 60). The similar strategy was applied for the immunosensor fabrication destined for determination of Plum pox virus virus PPV (61). The formation of specific complex between antibody attached to the gold nanoparticles covalently attached to the electrode surface and viruses present in the sample solution caused the increase of interfacial electron transfer resistance dependent. The quantitative assessment of sensitivity of PPV detection was performed using EIS with a series of dilutions of purified virions. The virus was suspended (and then diluted) either in 0.1 M PBS buffer pH 6.0, or in healthy plant (plum or tobacco) leaf extracts. The immunosensor proposed is not sensitive to the matrix of healthy leave extract. Only a very small increase of electron transfer resistance was observed between the value recorded for the pure PBS buffer and the value recorded for healthy plum leave extract (61). The immunosensor displayed very good analytical parameters: a very low detection limit (10 pg PPV) and very good dynamic range from 10 pg PPV/mL to 200 pg/mL. The immunosensors based on electrochemical impedance spectroscopy incorporating gold nanaoparticles are promising devices for fast, reliable and simple PPV detection and His-tagged proteins control (59–61). The determination of nucleic acid sequences humans, animals, bacteria and viruses constitute the starting point to solve different problems such as: food and water contamination caused by microorganisms, as well as detection of genetic disorders. The electrochemical genosensors could be very interesting analytical tools for solving these problems (27, 62). Among DNA sensors, two main groups can be distinguished according to the different protocols, based on labeling DNA target or using a label-free approach. Regarding the first approach, common label used for hybridization detection can be fluorescent dyes, redox active enzymes, magnetic particles or different kinds of nanoparticles. An indirect labeling scheme consist of the use of redox couple indicators such as metal complexes in solution, which generate electrochemical analytical signal (63). 302 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


A polylysine/single-walled carbon nanotubes modified electrode was used for the impedimetric detection of transgenic plants gene fragment with an estimated detection limit around 0.1 pM (64). Bonanni et al. (65) employed screen-printed electrodes modified with carboxyl functionalized multi-walled carbon nanotubes as platforms for impedimetric genosensing of oligonucleotide sequences specific for transgenic insect resistant Bt maize. Amino-modified DNA probe was covalently immobilized by EDC-NHS chemistry. A similar platform, consisting of carboxylic acid functionalized single-walled carbon nanotubes modified graphite sensors was employed by Caliskan et al. (66) for electrochemical monitoring of DNA hybridization related to specific sequence of Hepatitis B virus. The presence of carbon nanotubes enhanced the electrochemical (voltammetric and impedimetric) signals in comparison to bare graphite. A novel biosensing platform was introduced by Nebel et al. (67) by combining a geometrically controlled DNA bonding using vertically aligned diamond nano-wires, a superior transducer material. Ultra-hard vertically aligned diamond nano-wires were electrochemically modified to bond phenyl linker molecules to their tips which provide mesospacing for DNA molecules on the transducer. Electro- and bio-chemical sensor properties were investigated using cyclic and differential pulse voltammetry as well as impedance spectroscopy with Fe(CN)63−/4− as redox markers, which provided sensitivities of 2 pM on 3 mm2 sensor areas and superior DNA bonding stability over 30 hybridization/denaturation cycles. Yang et al. (68) deposited a poly-2,6-pyridinedicarboxylic acid film (PDC) on a glassy carbon electrode (GCE). Then gold nanoparticles (NG) were added to the platform to prepare NG/PDC/GCE. After that, the ssDNA probe was immobilized on the NG/PDC/GCE by the interaction of NG with DNA. The electron transfer resistance (Ret) of the electrode surface in [Fe(CN)6]3−/4− solution increased after the immobilization of the DNA probe on the NG/PDC/GCE. The hybridization of the DNA probe with cDNA made Ret increase further. The NG modified on the PDC dramatically enhanced the immobilization amount of the DNA probe and greatly improved the sensitivity of the label-free detection of the sequence-specific DNA related to PAT gene in the transgenic plants with detection limit of 2.4 × 10−11 mol L−1. It might be concluded that the electrochemical sensors and biosensors working according to ion channel mimetic mechanism which are relatively simple measuring systems could be successfully applied as analytical devices with very high sensitivity as well as selectivity. The main advantages of ion -channel mimetic sensors are as follow: • •

•

Offering capability of signal amplification Suitability for detecting large, hydrophilic ionic species that have high charge number such as peptides, proteins, polysacharides, oligonucleotides as well as uncharged molecules Unique signal transduction - gating of the immobilized receptor membrane’s permeability to redox marker ions in response to charged and uncharged analytes


•

• •

Signal transduction does not have to be changed or tailored for each analyte–receptor combination, in contrast with many conventional chemical or biosensors Permeability changes for redox markers are the only means for signal transduction for ion-channel sensors Incorporation of nanoparticles for ion-channel mimetic sensors improve their sensitivity


Electrochemical Sensors and Biosensors Based on Redox Active Monolayers One of the most popular techniques to create well-defined functional surfaces of electrodes for their future application in electrochemical sensors and biosensors is formation of self-assembled monolayers (SAMs) based on the covalent interactions of thiols, disulfides, sulfides, and other related molecules with the surfaces of noble metals, particularly gold, as well as platinum and mercury (69, 70). Electrodes with such way modified surface could be applied as a place of intermolecular recognition process in ion channel type sensors. Despite of the promising properties of SAMs, they also have some weak points. The most important is the presence of pinholes and defects, which effect on the faradaic response of blocking monolayers. In consequence, the ion channel type sensors based on such surfaces could loss their sensitivity and selectivity. Attaching a redox centers to SAMs is a possible solution of the above problem (70, 71). Such prepared surface could play a double role as an analytically active element of sensor as well as transducer. The possible way of electrode functionalization with redox active receptors, via covalent Au-S bonds and via embedment strategy are illustrated in Figure 4 A,B,C. In electroactive monolayers, all redox centres could be located on the outer surface of the SAM (Figure 4A, C) or could be buried into SAM (Figure 4B). The different locations of redox centre might have influence on their properties. Because of this, they display many advantages in comparison to monolayers without redox centres: •

•

Close packing prevents motion of the redox centres toward the electrode, toward a pinhole or a defect. Therefore, the faradaic current due to redox reaction at (or near) pinholes and defects becomes a negligible component of the total faradaic current. Because the electroactive centers are combined with the electrode surface, the diffusion does not have any influence on cyclic voltammetric responses. Thus, the double-layer correction for the surface concentration versus the bulk concentration is not necessary.

On the other hand, the new double-layer effect becomes important due to the changing oxidation state of the surface confined redox centres. Voltammetric waves frequently deviate from the ideal behavior, depending on such factors as the 304 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


dielectric constants of film and solution, the concentration of electroactive species, supporting electrolyte composition and the film thickness (70, 71).

Figure 4. Schematic illustration of redox active centers location in SAM deposited on the electrode surface: A - outer, B - inner, C - using embedment method. (see color insert) The distortions caused by double-layer effects are less significant when the distances between redox active units are greater and when the electrolyte concentration is high. Thus, the formation of mixed SAMs with a relatively low concentration of redox sites and working in concentrated electrolyte is highly recommended. We have developed the method for creation of electroactive monolayers based on the two step complexation reaction of Cu(II) performing on the surface of a gold substrate, previously modified with dipyrromethene derivative (72, 73). As the example of transition metal ions, Cu(II) was selected. The obtained electroactive SAMs were characterized by wettability contact angle measurements, cyclic voltammetry, and atomic force microscopy. The better reversibility of the redox processes proceeding on the electrode surface was observed for dipyrromentene SAMs with the Cu(II) redox centers located closer to the electrode surface. On the other hand, the electron transfer rate constants were very similar for both systems studied, 0.74 ± 0.21 and 0.44 ± 0.15 s−1 for dodecanethiol-dipyrromethene SAM and ethanethiol–dipyrromethene–Cu(II), respectively. This indicates that the electron transfer rate constant depends not only on the distance of the redox centers from the electrode surface but also on the presence 305 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


of hetero atoms such as nitrogen or oxygen. Their presence changed the dielectric properties of the system. The proposed method of formation of electroactive SAMs might be useful for derivatization of gold surfaces with functional groups such as COOH, NH2, or OH. The possibility of the attachment of biomolecules to dipyrromethene−Cu(II) SAMs with controlled orientation offers an excellent framework for the study of interactions occurring in interfacial environments between biomolecules of the type protein−protein, protein−hormone, or ssDNA−drugs. Having surface confined Cu(II) centres, the system proposed might respond toward interfacial molecular recognition events without the redox markers in the solution. This is particularly beneficial, because some of them might cause the loss of biomolecule activity. By performing the transition metal coordination on the electrode surface by dipyrromethene derivatives, many electroactive monolayers could be designed and used to define paths of electron transfer into and out of the biomolecules. They could work also as the transducing layer of biosensors for exploring the interfacial molecular recognition processes. This research is currently in progress in our laboratory One example of application of electroactive SAM with Cu(II)dipyrromethene concerns the determination of paracetamol, a common analgesic and antipyretic drug. The Cu(II)-dipyrromethene molecules were immobilized on gold electrode surfaces, previously modified with a dodecanethiol monolayer via hydrophobic and van der Waals interactions (embedment technique) (Figure 4C) (74). The dipyrromethene complex with Cu(II), selected as a host molecule for voltammetric sensor for paracetamol determination shows the reduction/oxidation peaks within the potential windows in which the paracetamol molecules remain electrochemically inactive. To our knowledge, this is the first report on paracetamol voltammetric sensors based on host–guest recognition and electroactive layer which plays a role of transducer. The interaction between dipyrromethene-Cu(II) and paracetamol changed the redox properties of Cu(II) centers in quantitative relation with paracetamol concentration in human plasma. Fe(III)-porphyrin was selected by us for determination of L-histidine (75). This host molecule, immobilized on the surface of Au electrode by the embedment method, (Figure 4C) played also transducer role. Taking into account the simply way of preparation, low detection of 4.9×10-10 M limit and wide dynamic range, the sensor based on Fe(III)-porphyrin is superior to those already reported. The proposed mechanism of the recognition between the porphyrin host and L-histidine guest molecules might be associated to the Fe(III) centre coordination ability. It is well known that iron porphyrins display a selective affinity towards imidasol group in histidine due to strong coordination ability of Fe(III) centre to nitrogen atoms (76). The idea working principle of sensors based on monolayers incorporated redox centres is illustrated in Figure 5. The mechanism of electrochemical signal generation by biosensor based on redox active SAM upon stimulation of specific analyte is not fully understood. 306 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The recognition reaction might cause a change in microenvironment surrounding the receptor molecule, which influences the double layer structure (77). The model and potential distribution of the electroactive film was provided by Ohtani (78, 79). In the algorithm to compute the voltammetric responses of quasi-reversible system for a surface-tethered redox species, the double layer effect on the electron transfer kinetics and ion-pair formation between surface confined redox species and electrolyte ions were considered. The effect of the position of the redox centres immobilized onto electrode surfaces, as well as the effect of the ions present in the electrolyte on the thermodynamics of redox active monolayers were reported by Rawe and Creager (80, 81). They discovered that the distance of ferrocene units from the electrode surface as well as the presence of different anions in the basic electrolyte strongly influence the voltammetric response of redox active monolayer.

Figure 5. Working principle of sensor based on electroactive SAM. (see color insert)

A decrease in peak current of the Ni(II)-phenanthroline complex deposited onto glassy carbon electrode was observed after interactions with dsDNA (82). The authors concluded that binding of dsDNA to the redox active Ni(II)-phenantroline significantly blocked the electron transfer process. A similar phenomenon was observed for a system consisting of dipyrromethene-Cu(II) complex deposited on the surface of a gold electrode used for exploring the interactions between His-Tagged V domain of Receptor for Advanced Glycation End Products (RAGE) and Aβ peptide (83). 307 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

The main advantages of electrochemical sensors and biosensors based on redox active monolayers are as follow:

• •


•

No need of using redox marker in the sample solution. Lack of influence of pinholes in the monolayers carring the receptors on sensors performance Redox active centers can simultaneously act as host as well as a transducer of electrochemical signals generated upon analyte recognition

The incorporation of gold as well as carbon nanoparticles into fabrication of sensors and biosensors based on redox active monolayers might be very beneficial. Their presence facilitating the electron transfer from redox centers to the electrode surface might improve sensors performance (1, 8, 84, 85). There is still much room for scientific research and application of nanostructures. This strategy is currently developing also in our laboratories. The transition metals complexex with porphyrines, dipyromethenes, terpyrydines deposited onto gold nanoparticles as well as carbon nanostructures such as single-walled carbon nanorods and graphene, will be applied for proper receptor proteins and ssDNA probe immobilization. The biosensors prepared in such way will be tested for sensing of target analytes important for medical diagnosis as well as for environmental monitoring.

Conclusions The two main types of electrochemical sensors: based on ion-channel mimetic mode and based on redox active layers were presented. The main advantage of ion-channel mimetic sensors, apart from high sensitivity and selectivity, is the possibility for application in the investigations of recognition processes occurring at the water/solid interface. It is very important from biological as well as medical point of view. The main disadvantage of this type of sensors, in particularly from analytical point of view, is the necessity of using the redox markers in the solutions, which may have toxic effect on biomolecules tested. The electrochemical sensors based on redox active layer are relatively new direction in sensing devices development. Their main advantage is the lack of the necessity of using the external redox marker. The application of redox centres in sensor giving very interesting possibilities. They can simultaneously act as host molecules and as well as transducers. The incorporation of nanoparticles into ion-channel mimetic sensors and also in sensors based on redox active monolayers improve their analytical parameters, in particular sensitivity. It might be concluded that they are relatively cheap analytical tools suitable from medical diagnosis and environment and food quality control. 308 In Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 1; Hepel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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