Electrochemical DNA sensors and aptasensors based on electropolymerized materials and polyelectrolyte complexes

Trends in Analytical Chemistry 79 (2016) 168–178

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Trends in Analytical Chemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c

Electrochemical DNA sensors and aptasensors based on electropolymerized materials and polyelectrolyte complexes Gennady Evtugyn a,b,*, Tibor Hianik b,c a b c

Analytical Chemistry Department, Kazan Federal University, 18 Kremlevskaya Street, Kazan 420008, Russian Federation OpenLab “DNA-Sensors”, Kazan Federal University, 18 Kremlevskaya Street, Kazan 420008, Russian Federation Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska dolina F1, 84248 Bratislava, Slovakia

A R T I C L E

I N F O

Keywords: Electropolymerization Layer-by-layer assembling DNA sensor Biosensor Hybridization detection Intercalator detection DNA damage

A B S T R A C T

DNA sensors based on oligonucleotides and aptamers immobilized using electropolymerization and layerby-layer assembling are reviewed. The conditions of electropolymerization and the role of electrosynthesized layers are considered for polyaniline, polypyrrole, polythiophene, polyphenazines and their derivatives with particular attention to immobilization of bioreceptors and signal detection principles. The performance of DNA sensors for hybridization detection and for the determination of low-molecular intercalators and DNA damaging factors is reviewed. Besides, the composition of polyelectrolyte complexes utilizing DNA receptors are considered depending on the analyte nature and functions of polyionic components and auxiliary reagents used for surface layer coatings. © 2016 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4.

Introduction ........................................................................................................................................................................................................................................................ Electropolymerized materials in DNA sensors and aptasensors ...................................................................................................................................................... 2.1. General characterization of electropolymerized materials and their inclusion in DNA sensor assembly ........................................................... 2.2. DNA sensors for hybridization detection .................................................................................................................................................................................... 2.3. Aptasensors based on electropolymerized materials ............................................................................................................................................................. Layer-by-layer assembling of biorecognition layers in DNA sensors .............................................................................................................................................. 3.1. Polyelectrolyte complex assembling ............................................................................................................................................................................................. 3.2. Electrochemical DNA sensors based on layer-by-layer assemblies ................................................................................................................................... Conclusion ........................................................................................................................................................................................................................................................... Acknowledgments ............................................................................................................................................................................................................................................ References ............................................................................................................................................................................................................................................................

1. Introduction The interest to the DNA sensors has been dramatically increased in the past decades due to great significance of their application including detection of hybridization events, DNA damage and antitumor drugs analysis [1,2]. Electrochemical transducers offer broad opportunities in DNA sensor design due to simple experiment protocols, inexpensive and mostly commercially available equipment. Together with well-developed theory of electrochemical phenomena, these advantages result in intensive progress in electrochemical DNA sensors development.

* Corresponding author. Tel.: +7 843 2337491; Fax: +7 843 2337416. E-mail address: [email protected] (G. Evtugyn). http://dx.doi.org/10.1016/j.trac.2015.11.025 0165-9936/© 2016 Elsevier B.V. All rights reserved.

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The use of electrochemical transduction principles has a natural limitation in rather low electrochemical activity of DNA molecules, which can be detected only with special measurement technique like differential pulse voltammentry (DPV) and adsorptive stripping voltammetry and chronoamperometry on mercury or carbon electrodes [3]. Recently, new approaches have been suggested to overcome this limitation by special design of appropriate transducer. Electropolymerized materials and polyelectrolyte complexes implementing DNA receptor elements are of special importance due to advantages they possess, i.e. variety of electrochemical characteristics, easy implementation of the DNA probes, preservation of native structure of DNA, high reproducibility of the biosensor characteristics and compatibility with different transducer types with no respect of their dimensions and shape. In this review, electrochemical DNA sensors based on electropolymerized materials and polyelectrolyte complexes are

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reviewed with examples of recent publications illustrating the advantages and limitations of these carriers within last decade. 2. Electropolymerized materials in DNA sensors and aptasensors 2.1. General characterization of electropolymerized materials and their inclusion in DNA sensor assembly Electropolymerization involves the formation of oligomeric or polymeric products by electrochemical oxidation of their monomers [4]. The products are insoluble and commonly deposited on the electrode surface. Although many different polymers obtained by electrolysis have been described in the DNA sensor assembly, only few of them are characterized to establish the mechanism of polymerization and product structure. The polymerization starts with the formation of a cation radical at high anodic potential. After that, headto-tail coupling leads to the dimer formation followed by its oxidation and addition of another monomer molecule. The appropriate schemes are presented for aniline, thiophene and pyrrole polymerization (1).

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simultaneous entrapment of auxiliary components, e.g., with chemical or electrochemical synthesis of metal nanoparticles, covalent binding of redox mediators etc. [6]. Electrochemical characteristics of the polymers including their ability of DNA wiring depend on the monomer nature and regularity of the polymer structure. It can be modified by implementation of appropriate functional groups in the side chain substituents. Thus, introduction of Methylene blue, a DNA intercalator, in the polythiohene structure (2) was applied for hybridization of complementary oligonucleotides which changed the redox activity of the phenothiazine dye [7]. Derived monomers applied for electropolymerization, both alone and in the mixture with unsubstituted analogs, are presented in Fig. 1 for aniline, pyrrole and thiophene. Chemical modification of the polymerization products is performed for two purposes, i.e. DNA immobilization and signal generation/transduction [8]. For polyaniline (PANI) derivatives, anionic substituents can also participate in electrostatic interaction with oxidized form of the polymer similarly to low-molecular counter anions. The formation of such “self-doped” PANI de-

(1)

(2)

The DNA addition to the reaction mixture affects polymerization due to coordination of the cationic intermediates to negatively charged phosphate residues in DNA structure [5]. The entrapment of DNA into the growing polymer film results in formation of polyelectrolyte complexes used for enhancement of redox activity or electroconductivity area and detection of specific DNA interactions especially those with small molecules, e.g., antitumor drugs. Alternatively, the DNA molecules can be immobilized onto the polymer layer by electrostatic interactions or covalent binding to the side groups. The electropolymerized film provides regular positioning of anchoring groups on the transducer surface. The accessibility of the oligonucleotides for bulky analytes in such coatings can be extended by introduction of appropriate spacers. The mechanisms of the DNA introduction described are compatible with

creases the pH dependence of its electroconductivity and redox activity. Introduction of DNA bearing large negative charge of phosphate residues plays similar role. This can be used for detection of hybridization events that increase the charge density of the double stranded (ds-) DNA helix against single DNA probe. Covalent immobilization assumes the reaction between the terminal functional groups of the substituents and those of DNA probe as shown for polythiohene derivative in Equation (3) [9]. Other examples of covalent DNA immobilization are given below in the description of appropriate biosensors. All the electropolymerization products can be subdivided into three groups in accordance with their electrochemical properties: (1) Electroconductive polymers that exert their own electron conductivity (PANI, polypyrrole (PPY), polythiophene and their structural

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(3)

derivatives) [8,10]. They exist in several redox forms depending on the redox potential and acidity of the environment. Commonly, oxidized (semi-oxidized in the case of PANI) form of the polymers exerts conductivity comparable to that of semiconductors. Reversible conversion of the reduced and oxidized centers can be monitored by optical methods, electrochemical impedance spectroscopy (EIS) or voltammetry in the presence of redox probes able to reversible electron transfer, e.g., ferricyanide ions or hydroquinone/benzoquinone pair. Regarding PANI (4) and to a less extent PPY, redox equilibria are accompanied with appropriate transfer of counter anions [11]. This results in significant changes of the own PANI volume similarly to that observed in swelling. This phenomenon has found application in some chemoresistors and DNA sensors based on detection of redox probe transfer through the polymer film. PANI is obtained mainly in the presence of strong inorganic acids stabilizing primary cation radical required for polymerization initiation. Its formation corresponds to irreversible anodic peak recorded on voltammograms at about 0.7–0.9 V. Decrease in the acidity of the polymerization mixture is achieved by substitution of sulfuric or hydrochloric acids with oxalic acid [12]. The AFM and SEM investigation showed the formation of rather spongiose film with DNA preserving ability to interact with small intercalator molecules. PPY and polythiophene derivatives are mainly synthesized in organic solvents. The polymerization of thiophene requires higher potential that its oxidation to non-conductive sulfone. For this reason,

Fig. 1. Functionalized pyrrole and thiophene derivatives applied in the electropolymerization step for the DNA/aptamer immobilization.

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dimers and trimers are applied as starting compounds instead of thiophene molecules for electropolymerization. (2) Electrochemically active polymers obtained by polymerization of phenazine dyes and some aromatic amines and phenols [13,14]. Their reactivity and equilibrium between reduced and oxidized form depends on the charge density and efficiency of intermolecular charge exchange. This can be recorded by direct current (DC) or differential pulse voltammetry (DPV). The appropriate signals decrease with the involvement of DNA probe in hybridization with complementary targets. Meanwhile, the changes in the surface charge and permeability of the film for diffusionally free redox probes are much lesser than those of electroconductive polymers. This prevents the use of EIS technique for signal detection; (3) Non-conductive polymers passivate electrodes and prevent measuring voltammetric signals. Nevertheless, they find limited application as supports for immobilization of DNA probes and auxiliary reagents in the surface layer. To some extent, such polymers can differentiate analytes and interferences in accordance with their size and hydrophobicity. This group of polymers is obtained by oxidation of some substituted phenols [15,16] or PPY (PANI) overoxidation [13]. The choice of the electropolymerization conditions depends on the protocol of the DNA immobilization. If the DNA probe is attached to the polymer film obtained before, there are no specific requirements. It can be performed in polar organic solvent (acetonitrile, dichloromethane) to improve solubility of the monomers (PPY or polythiophene derivatives). In other cases, biorecognition layer is assembled from aqueous solution of the monomer prior to or together with the DNA immobilization. It is preferable for the majority of the products due to simpler protocol, better biocompatibility of the product and stability of the film obtained and lower toxicity of the reagents required. The electrolysis regime plays crucial role in the establishment of necessary film properties, e.g. permeability and regularity of the structure, integrity of the layer and transducer, hydrophilicity/ hydrophobicity, roughness etc. Many of the parameters depend on the ratio of two processes, i.e. primary polymer nucleation and nuclei growth. The use of pulse techniques or sharp changes in the current density promote formation of well-dispersed branched polymer structures, i.e., nanofibers, nanoflacks, nanorods etc. In many DNA sensors, electropolymerization is performed by multiple potential cycling. Reversible redox conversion of the surface products results in rather low yield of target product. The film deposited is rather thin and well adhesive. The shape of the peaks on voltammograms recorded, their position and height specify electropolymerization mechanism and efficiency. The number of potential cycles required for electropolymerization is rather small. Full coverage of the electrode surface is achieved by 3rd–5th cycle. Meanwhile from 10 to 30 cycles are commonly preferred to reach higher durability of the coating and less influence of by-products leaching. Potentiostatic deposition provides rather high mass of the polymer which is more uniform in distribution of reduced and oxidized centers but can be contaminated with supporting electrolyte and by-products. Such impurities are removed by multiple cycling the potential or electrode polarization in the absence of the monomers. The same can be referred to galvanostatic electrolysis but the product formed contains more quantities of overoxidized or oxidatively damaged by-products. 2.2. DNA sensors for hybridization detection The hybridization assumes the formation of ds-DNA product between single-stranded (ss-) DNA and complementary target. The efficiency of detection system depends on its ability to distinguish

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Fig. 2. Schematic outline of signal generation approaches applied in electrochemical DNA sensors for hybridization detection.

between fully complementary sequences and mismatches differed from them by one, two etc. bases. The analytical characteristics of some DNA sensors devoted to the hybridization detection are summarized in Table 1. As could be seen, biospecific interactions together with various amplification approaches make it possible to detect sub-nano and sub-picomolar concentrations of specific DNA sequences. General approaches to the detection of hybridization event are schematically outlined in Fig. 2. PANI and PPY contain positively charged areas able to accumulate DNA probes either in the bulk of the layer or on its surface. Hybridization increases density of negative charge which can be detected by EIS or DC voltammetry with ferricyanide ions (Fig. 2A). Instead of electrostatic accumulation, DNA probe can be attached to functional groups of the polymer by carbodiimide binding or avidin-biotin coupling. For this purpose, PPY or polythiophene derivatives with side chains bearing appropriate functional groups are described. Increased amounts of negatively charged species accumulated in the polymer layer also affect the equilibrium of reduced and oxidized centers of the polymer monitored by ratio of appropriate peak currents on voltammograms. The changes in the intrinsic redox activity of the polymers are quite attractive as measure of hybridization degree because they do not assume any additional manipulations with the DNA sequences and allow one-step measurement protocol. Redox activity of polythiophene derivatives is not applied for signal readout due to oxidative instability of the polymer. Another common approach to signal detection is based on the use of redox active intercalators that penetrate DNA helix and lose their activity (Fig. 2B). Methylene blue and ethidium bromide (4) belong to this class of redox probes. Methylene blue in oxidized cationic form is accumulated on the surface of ss-DNA probe prior to hybridization and this process increases its signal measured by DC

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or DPV whereas intercalation affects the signal in opposite direction. Besides, intercalation with acridone derivative (5) was employed for immobilization of ds-DNA on the co-polymer of pyrrole and pyrrolobutanoic acid [27].

As could be seen from short description of the detection principles, the structure of polymeric support and regular positioning of the DNA probe play significant role in sensitivity of the assay. Selection of current density and electrolysis regime makes it possible

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Labeled DNA sequences are used in competitive assay techniques. In these methods, DNA probe similar to the analyte is modified by redox molecule or enzyme which occurrence is easily detected. The hybridization event results in close attachment of the label to the electrode surface and in signal increasing with a labeled analyte concentration. In the presence of non-labeled target species the surface concentration of the label decays (Fig. 2C). In some approaches, general labeling techniques adapted to be used with many labels and signal detectors are applied. Thus, implementation of biotin as label makes it possible to attach various molecules conjugated with avidin or streptavidin to the ds-DNA (fluorescent label, enzyme, ferrocene derivative). Regarding enzymes, the signal recorded can be obtained by oxidation of appropriate product of enzyme conversion (Fig. 2D). Alkaline phosphatase commonly used in such biosensors shows high sensitivity of detection. Naphtylphosphate is hydrolyzed to α-naphtol involved in electrocatalytic redox cycle amplifying the hybridization response [23,24]. Glucose oxidase and horseradish peroxidase are the other examples of sensitive detection systems. Amplification of the signal is achieved in sandwich format with partially complementary DNA-probes. Target DNA interacts with capture probe on the transducer surface whereas second (signaling) probe, often labeled, is then attached to the non-hybridized fragment of target DNA sequence. This protocol can be used to form large ds-DNA sequences by multiple repetition of the binding of partially complementary DNA probes [48]. Signal amplification is attained due to increased accumulation of a larger amount of redox active intercalator in comparison to that possible for a single pair of capture and signaling DNA probes.

to control thickness, aggregation and permeability of the polymer layer. The use of functionalized monomers provides their regular distribution on the surface. For the same purpose, Au nanoparticles can be synthesized either chemically or electrochemically followed by immobilization of thiolated oligonucleotides. The implementation of Au nanoparticles is simplified by electrostatic attraction of negatively charged particles or AuCl4− anions as precursors on positively charged polymer. Interesting approach suggests simultaneous synthesis of both components of the surface layer by the pulses of anodic (polymerization) and cathodic (Au deposition) currents [41]. Most of the biosensors reported assume single use because of the problems with strong binding of hybridized sequences. Affine immobilization offers additional opportunities due to partial removal of the biosensing layer after measurement by reversible dissociation of the binding sites. Thus, chelate immobilization utilizes pyrrolo-nitrilotriacetic acid involved in electropolymerization together with unsubstituted pyrrole [30,31]. Soaking sensor in Cu2+ ions solution simplifies the inclusion of biochemicals bearing biotin or histidine tags (6). Removal of the central ion results in disintegration of the complex. After that, DNA probe can be attached again by repeated treatment with a new DNA probe in the presence of central metal ion. Other immobilization techniques utilize conventional binding methods with carbodiimides and glutaraldehyde frequently used in biosensors. Thiolated DNA probes are attached to Au nanoparticles distributed in the polymer layer. Aminated DNA probe can covalent bind chitosan added to the reaction media on the stage of aniline polymerization [18]. What is important for biosensor

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Table 1 Analytical characteristics of DNA sensors based on electropolymerized materials Analyte PANI 24-mer DNA oligonucleotide

DNA probe associated with breast cancer biomarker gene BRCA1 DNA probe associated with Neisseria gonorrhoeae DNA probe associated with Neisseria gonorrhoeae

DNA probe specific to Mycobacterium tuberculosis and genomic DNA 24-mer DNA oligonucleotide microRNA-16

DNA probe specific to CR/ABL fusion gene in chronic myelogenous leukemia PPY DNA probe specific to human adenoviruses HAdV 40/41

DNA probe specific to West Nile virus

Electropolymerization conditions

Signal measurement protocol, samples tested

LOD, linearity range

Ref.

Galvanostatic three-step electrolysis on GCE modified with reduced graphene oxide

DPV measurement of the changes in the PANI intrinsic redox activity DC measurement of ferricyanide redox current

LOD 3.25 × 10−13 M, conc. range 2.12 × 10−6–2.12 × 10−12 M

[17]

LOD 0.05 fM, conc. range 0.05–25 fM

[18]

DC measurement of decrease in the PANI redox activity

LOD 0.5 × 10−16 M, conc. range 1 × 10−6–1 × 10−16 M

[19]

DPV measurement of Methylene blue signal

LOD 1 × 10−17–1 × 10−19 M, conc. range 1 × 10−19– 1 × 10−6 M

[20]

DC or square-wave voltammetry (SWV) of Methylene blue as intercalator DPV measurement of Methylene blue signal Sandwich assay mode with alkaline phosphatase label and α-naphtol oxidation monitoring by DPV Sandwich assay mode with alkaline phosphatase label and α-naphtol oxidation monitoring by DPV

LOD 2.5 × 10−18 (DC) 0.125 × 10−18 (SWV) M, conc. range (2.5–15) × 10−18 M LOD 1 × 10−12 M, conc. range 2.25 × 10−12–2.25 × 10−10 M LOD 0.1 nM, conc. range 0.2–10 nM

[21]

LOD 2.11 pM, conc. range 10–1000 pM

[24]

Coulometric measurement of the charge passed

LOD 0.1 μM, conc. range 0.1 to 1.0 μM

[25]

Sandwich assay mode with biotinylated DNA sequence and avidin – glucose oxidase conjugate, amperometric detection of H2O2 formed in glucose oxidation Sandwich assay mode with biotinylated DNA sequence and avidin – glucose oxidase conjugate, amperometric detection of H2O2 formed in glucose oxidation Increase in the charge transfer resistance measured in the presence of [Fe(CN)6]3−/4− Increase in the charge transfer resistance measured in the presence of [Fe(CN)6]3−/4− Increase in the charge transfer resistance measurement in the presence of hydroquinone

LOD 1 fg/mL, conc. range 1 × 10−15–1 × 10−10 g/mL

[26]

LOD 1 pg/mL, conc. range 1 × 10−12–1 × 10−6 g/mL

[27]

Nanomolar concentrations detection

[28]

LOD 5 × 10−12 M, conc. range 1 × 10−11–1 × 10−7 M

[29]

LOD 1 × 10−15 M, conc. range 1 × 10−15–1 × 10−8 M

[30,31]

LODs 0.15 and 3.5 fM for 18- and 27-mer target DNA

[32]

LOD 1.82 × 10−21 M, conc. range 1 × 10−20–1 × 10−14 M

[33]

LOD 8.5 × 10−11 M, conc. range 1 × 10−10–1 × 10−8 M

[34]

LOD 2 nM, conc. range 5–18 nM

[35]

LODs of 1 and 0.01 nM on macro and microelectrodes, respectively

[36]

Potentiostatic electrolysis of aniline-chitosan mixture, DNA probe bonded to chitosan Galvanostatic tree-step electrolysis on ITO electrode followed by avidin immobilization via carbodiimide binding and biotinylated DNA probe attachment Galvanostatic electrolysis of the mixture of aniline, carboxylated multi-walled carbon nanotubes and Fe3O4 nanoprticles followed by DNA probe attachment by avidin-biotin or glutaraldehyde binding Galvanostatic electrolysis of aniline on Au film electrode followed by treatment with glutaraldehyde and DNA probe Galvanostatic three-step electrolysis on GCE followed by carbodiimide binding of DNA probe Multiple potential scanning in aniline solution followed by chemical synthesis of Au nanoparticles and thiolated DNA probe attachment Multiple potential scanning in aniline solution on the Au electrode modified with chitosan followed Au nanoparticles electrodeposition and thiolated DNA probe attachment Si nanoporous support doped with boron, PPY deposited from HF solution of pyrrole in potentiostatic regime, DNA probe electrostatically accumulated on PPY in potentiostatic mode Pt electrode with electrodeposited pyrrole anchoring N-succinimide group (multiple potential cycling) followed by covalent binding of aminated DNA probe

DNA probe specific to West Nile virus

Pt electrode with electrodeposited pyrrole anchoring N-succinimide group (multiple potential cycling) followed by covalent binding of aminated intercalator (acridone derivative) and ds DNA binding

DNA probe specific to Salmonella virulence invA gene 24-mer DNA oligonucleotide

Pt electrode covered with co-polymerized pyrrole and 4-(3-pyrrolyl)butanoic acid in potentiostatic regime, carbodiimide binding of DNA probe Multiple potential scanning on GCE in the dispersion of carboxylated MWCNTs and pyrrole followed by carbodiimide binding of DNA probe Pt electrode covered with poly(pyrrole-nitrilotriacetic acid) in potentiometric electrolysis in acetonitrile followed by soaking in CuCl2 solution and biotinylated DNA probe Pt electrode covered with PPY and polymer of 2,5bis(2-thienyl)-N-(3-phosphorylpropyl)pyrrole obtained in potentiostatic electrolysis followed by soaking in MgCl2 and DNA probe Pt electrode covered with PPY and polymer of 2,5bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole obtained in potentiostatic electrolysis followed by soaking in MgCl2 and DNA probe Multiple potential cycling of mixed solution of pyrrole and DNA probe on MWCNTs paste electrode

DNA probe specific to HIV

27-mer DNA probe

DNA probe specific to hepatitis C virus

32-mer DNA oligonucleotide

DNA probe specific to herpes virus

Multiple potential cycling of mixed solution of pyrrole and DNA probe on Au interdigitated electrode

25-mer DNA oligonucleoride

Potentiostatic electrolysis of 1-(phthalimidylbutanoate)-1′-(N-(3butylpyrrole)butanamide)ferrocene and pyrrole in acetonitrile on Au electrode followed by covalent binding of aminated DNA probe

DC measurements of the current related to chloride anions transfer in the inner layer DC measurements of the current related to chloride anions transfer in the inner layer Oxidation current of ethidium bromide as ds-DNA intercalator Increase in the charge transfer resistance measurement in the presence of [Fe(CN)6]3−/4− DC current of ferrocene redox pair

[22] [23]

(continued on next page)

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Table 1 (continued) Analyte

Electropolymerization conditions

Signal measurement protocol, samples tested

LOD, linearity range

Ref.

DNA probes specific to 35S promoter and NOS gene

Pyrrole polymerization in the presence of DNA probes and MWCNTs in potentiostatic regime or by potential cycling at Au microelectrode Potentiostatic electrolysis of pyrrole in the presence of gelatin on Pt integrated microelectrodes followed by DNA probe physical adsorption. Potentiostatic electrolysis of pyrrole in the presence of gelatin on interdigitated Pt electrodes followed by DNA probe binding to terminal groups of the polymer nanowires Independent synthesis of pyrrole and Au nanoparticles by applying recurrent potential pulses followed by treatment with thiolated carboxylic acid and carbodiimide binding of aminated DNA probe Co-polymerization of pyrrole and 3-pyrrolylacrylic acid by pulse electrolysis on GCE in the presence of polystyrol sulfonate (PSS) or LiClO4 followed by carbodiimide binding of aminated DNA probe

Increase in the charge transfer resistance measured in the presence of [Fe(CN)6]3−/4− Increase in the charge transfer resistance measured in the presence of [Fe(CN)6]3−/4− Increase in conductivity of the polymer layer

Conc.range 25–292 pM, direct assay of herbicide-resistance RR soybean LOD 10 pM, conc. range 10 pM–500 nM

[37]

LOD 0.1 nM, conc. range 2–11 nM

[39]

Increase in the charge transfer resistance measured in the presence of [Fe(CN)6]3−/4−

LOD 8.4 × 10−13 M, conc. range 2 × 10−13–2 × 10−6 M

[40]

Increase in the charge transfer resistance measured in the presence of hydroquinone or [Fe(CN)6]3−/4−

Conc. range 10−7–10−3 M

[41]

DPV measurement of daunorubicin signal as ds-DNA intercalator

C2nc. range 20–100 nM

[42]

Increase in the charge transfer resistance measured in the presence of hydroquinone or [Fe(CN)6]3−/4− Simultaneous increase of surface mass and charge transfer resistance DPV measurement of methylene blue signal as dsDNA intercalator

Testing in 4.03 μM solution of complementary DNA sequence

[43]

Testing in 1 μM solution of complementary DNA sequence

[44]

LOD 1.1 × 10−14 M, conc. range 1 × 10−13–1 × 10−7 M

[45]

Oxidation current related to intrinsic redox activity of the polymer matrix measured in DC mode DPV measurement of reduced redox activity of PEDOT layer

Testing in 0.1 mM solution of complementary DNA sequence

[46]

LOD 0.13 nM, conc. range 1–20 nM

[47]

DPV measurement of Ru phenanthroline complex signal measured after multiple hybridization of partially complementary probes DPV measurement of intrinsic redox activity of polythionine layer

LOD 4.03 × 10−14 M, conc. range 1 × 10−13–1 × 10−8 M

[48]

LOD 3.5 × 10−14 M, conc. range 1 × 10−13–1 × 10−8 M

[49]

LOD 1 × 10−13 M, conc. range 1 × 10−12–1 × 10−8 M

[50]

25-mer DNA oligonucleotide

DNA probes specific to Escherichia coli

DNA probe specific to the genus Lactococcus lactis

DNA probe specific to glycoforin A

Polythiophene derivatives 19-mer DNA oligonucleotide

18-mer DNA oligonucleotide

38-mer DNA oligonucleotide

DNA probe specific to colitoxin gene

DNA probe related to GMO related gene of Cauliflower mosaic virus DNA probe specific to hepatitus C virus

Other redox active polymers DNA probe specific to human papilloma virus

25-mer DNA oligonucleotide

23-mer hairpin locked nucleic acid (LNA) probe specific to BCR/ABL fusion gene

Potential cycling in EDOT solution on Pt microelectrode followed by potentiostatic polymerization of p-aminobenzoic acid and carbodiimide binding of DNA probe Potential cycling of 3-terthioheneacrylic acid solution on Pt electrode in dichloromethylene followed by covalent binding of aminated DNA probe Potentiostatic oxidation of 3-methylthiophene followed by deposition of Pt nanoparticles and thiolated DNA probe attachment Potential cycling in 2-thiophenesulfonyl chloride solution on GCE modified with submicroparticles of CoS2 followed by covalent attachment of aminated DNA probe Potentiostatic electrolysis of propylene dioxythiophene in ionic liquid solution on fluorenone doped tin oxide electrode followed by DNA probe electrostatic deposition Potential cycling in the solution of azidomethyl substituted ethylene dioxythiophene followed by covalent attachment of DNA probe with terminal acetylene gropu by click chemistry Potential cycling in the thionine solution in phosphate buffer on GCE modified with graphene/Au nanorods composite followed by thiolated DNA probe immobilization Potential cycling of thionine solution in phosphate buffer on GCE modified with graphene followed by potentiostatic deposition of Au nanoparticles and thiolated DNA probe attachment GCE modified with poly(eriochrome black T) by potential cycling followed by current pulse deposition of Au nanoparticles and attachment of thiolated hairpin LNA probe DPV measurement of

operation, the use of electropolymerized coatings diminishes the negative influence of non-specific sorption of oligonucleotides that limit the sensitivity of detection in conventional DNA assay techniques. Regarding application of Au nanoparticles as carriers, they are additionally covered with thiolated alcohols or acids to shield bare electrode surface and prevent deposition of serum proteins and other possible interferences present in the samples tested.

2.3. Aptasensors based on electropolymerized materials Aptamers are synthetic oligonucleotides obtained by combinatorial chemistry from random DNA/RNA library and selected against target analyte by affine chromatography [51]. Many of aptamers

DPV measurement of Methylene blue signal as dsDNA intercalator

[38]

show affinity toward target species comparable with that of antibodies. Besides, they are more stable and can be modified to simplify their entrapment in the biosensor assembly. The immobilization of aptamers is performed similarly to that of DNA probes. However, the anchoring terminal group involved in covalent immobilization is commonly separated from the binding area of the oligonucleotide by spacer consisting on methylene groups or short homonucleotide sequence. PANI has been described in the assembly of appropriate conductometric aptasensor [52] The polymer was grown in a microchannel between two Au electrodes. Interaction with immunoglobulin IgI changed the PANI conductivity. The aptasensor showed the LOD of 0.56 pg mL −1 with the concentration range from 1.0 pg mL−1 to 22.0 ng mL−1.

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Fig. 3. Self assembling of the layer from electropolymerized Neutral red and polycarboxylate derivative of calixarene followed by carbodiimide binding of redox label and an aptamer. Arrows indicate electron exchange responsible for the signal on aptamer – protein (mycotoxin) detection.

Sensitive aptasensor for aflatoxin M1 was developed on the base of interdigitated Au electrodes covered with PANI and dispersed Fe3O4 particles by cyclic voltammetry. The aptamers were immobilized by glutaraldehyde cross-linking. The signal referred to the changes in the conductivity of the PANI layer was recorded by SWV in the range from 6 to 60 ng mL−1 (LOD 1.98 ng mL−1) [53]. Human prion protein PrPC was detected with aptasensor based on Au electrode covered with electropolymerized copolymer of pyrrole and 3-(N-hydroxyphthalimidyl ester)pyrrole by cycling the potential in acetonitrile [54]. The polymer coating was consecutively modified with PAMAM dendrimer, ferrocene derivative and aminated aptamer. The signal was related to ferrocenyl group implemented between the dendrimer and aptamer layer and affected by prion protein binding. The biosensor detected from 1 pM to 1 nM of analyte (LOD 0.8 pM). Impedimetric biosensor for thrombin detection was assembled by galvanostatic deposition of poly(pyrrole-nitrilotriacetic acid) and chelate immobilization of aptamer as described above [55]. A linear quantification of thrombin based on detection of charge transfer resistance was obtained in the range from 4.7 × 10−12 to 5.0 × 10−10 M (LOD 4.4 × 10−12 M). Botulinum neurotoxin was detected with 16-pin Au array covered with PPY and streptavidin layer in sandwich assay format [56]. The modification was performed by loading the mixture of pyrrole and streptavidin modified DNA dendrimer on the electrode followed by square wave polarization. After target binding, fluorescein labeled signaling aptamer and anti-fluorescein – HRP conjugate were fixed at the surface. The amperometric signal was related to the reduction of tetramethylbenzidine, a HRP substrate. A family of aptasensors for the detection of thrombin and mycotoxins has been developed on the base of poly(Neutral red) and macrocyclic ligands bearing redox labels [57–60]. The assembly of the bioreceptor layer of the aptasensor is presented in Fig. 3. The assembling of aptasensors involved electrostatic accumulation of carboxylated carrier followed by carbodiimide binding of aminated aptamers and redox label (Neutral red or Ag nanoparticles). The signal toward target analytes was recorded with DC voltammetry or EIS. Binding of the analytes resulted in suppression of electron exchange between the redox labels. As a result, cathodic peak current of Neutral red decreased with increasing analyte concentration. Inclusion of analytes was confirmed by increased charge transfer resistance measured in the presence of ferricyanide ions. The biosensors made it possible to detect down to 0.05 nM of ochratoxin A, aflatoxin B1 and thrombin. As in the case of many other electropolymerized layers, no special measures against

non-specific binding of interferences are necessary. The aptasensors show high selectivity of the response and were tested on spiked samples of blood serum and foodstuffs.

3. Layer-by-layer assembling of biorecognition layers in DNA sensors 3.1. Polyelectrolyte complex assembling The protocol of layer-by-layer assembling is based on electrostatic interactions between negatively and positively charged polyelectrolytes. Oppositely charged reactants are consecutively added to the solid support with intermediate washing to remove unbounded molecules. Self-assembling results in formation of regular reproducible structures with alternating areas of positive and negative charge. The structure and properties of such polyelectrolyte complexes depend on the charge distribution and flexibility of the chains bearing charged/ionized groups. Small counter ions are commonly squeezed out during the complex formation. Some their quantities remain in the layer and diminish the electrostatic attraction of polyelectrolytes. The counter ions participate in ion exchange with supporting electrolyte and affect the stability of the assemblies especially in electrolyte solutions. The polyelectrolyte complexes can also entrap low-molecular compounds and nanoparticles, e.g., metal complexes, CNTs, noble metals etc. that play significant role in the electron transduction and generation of the biosensor signal. The DNA molecules are easily implemented in the polyelectrolyte complexes due to high density of negative charge of the phosphate residues. Poly(dimethyldiallylammonium chloride) (PDDA), poly-L-lysine (PLL), polyvinylpyrrolidone (PPV), polyethylene imine (PEI), poly(allylamine hydrochloride) (PAH) and chitosan are mainly described among cationic parts of polyelectrolyte complexes. Besides DNA, PSS, nafion and carbon nanotubes can be implemented in the layers as anionic species. Chemical structures and schematic outline of polyelectrolyte complex assembling is outlined in Fig. 4. The polyelectrolytes mentioned above can be modified to extend the performance of appropriate biosensors. The examples of covalent attachment of redox labels to the polyelectrolye complexes as well as the conditions of their redox activity measurement are given in review [61]. They involve the implementation of Os and Ru bipyridine complexes and ferrocene units as redox labels for detection of hybridization events and oxidative DNA damage.

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Fig. 4. Polyelectrolytes applied in layer-by-layer deposition of surface coatings in DNA sensors and schematic outline of self-assembling process.

3.2. Electrochemical DNA sensors based on layer-by-layer assemblies Self-assembling of DNA-polyelectrolyte layers is now rarely used for the detection of hybridization due to shielding of the DNA probe by non-covalent interactions within the layer. The multi-point interaction with complementary sequence is possible if DNA is positioned in outer layer facing the solution. However, such an assembly is less stable and needs to be strengthen by implementation of Au nanoparticles or CNTs. Thus, EIS detection of hybridization was described with multilayered DNA sensors with self-doped PANI and Fe2O3 microspheres deposited on the carbon paste electrode [62]. The PEPCase gene specific DNA sequence was electrostatically deposited on the surface and increase in the charge transfer resistance recorded in the presence of ferricyanide ions. The DNA sensor makes it possible to determine from 1.0 × 10−13 to 1.0 × 10−7 M of DNA sequence with the LOD of 2 × 10−14 M. Au-PLL complex was applied for the detection of hybridization by the electric charge passed in the presence of [Ru(NH3)6]3+ probe [63]. This signal linearly depends on the concentration of complementary DNA sequence in the range from 1.0 × 10−13 to 1.0 × 10−10 M (LOD 3.5 × 10−14 M). The formation of bioreceptor layers with polyelectrolytes is easily combined with electropolymerization technique described above. Thus, PDDA – poly(2,6-pyridine dicarboxylic acid) – single walled carbon nanotubes (SWCNTs) composite has been employed for the detection of biomarkers of GMA products, i.e. PAT and NOS gene specific sequences [64]. First the polymer was deposited from the monomer suspension in the presence of SWCNTs and then PDDA and DNA probe were consecutively added and accumulated in the surface layer by electrostatic interactions. The hybridization was monitored by EIS and DC voltammetry in the presence of ferricyanide ions. The calibration graph was linear in the range from 1.0 × 10−11 to 1.0 × 10−6 M with the LOD of 2.6 × 10−12 M. Similarly to hybridization detection, layer-by-layer complexes have been proposed to use for detection of the aptamer – protein binding [65]. Multilayer coating was obtained by consecutive GCE treatment with PEI modified with ferrocene units and MWCNTs suspension. Thrombin or lysozyme-binding aptamers were electrostatically attached to the outer PEI layer. Changes in the ferrocene DPV signal were proportional to the protein concentration in the ranges 0.3–165 ng mL−1 for thrombin (LOD 0.14 ng mL−1) and 0.2 ng mL−1–1.66 μg mL−1 for lysozyme (LOD 0.17 ng mL−1). More frequently, polyelectrolyte complexes are used for detection of target DNA interactions with small molecules, i.e., intercalation of ds-DNA molecules or their oxidative damage. Thus, a number of

multilayered coatings consisting of DNA, PAH and PSS have been assembled on glassy carbon electrodes modified with electropolymerized poly(Methylene blue) and poly(Methylene green) [66]. The behavior of three- and four-layer coatings was tested with Fenton reagent as the source of reactive oxygen species. As was shown by EIS and DC voltammetry, maximal increase of the charge transfer resistance was observed for the coatings including poly(Methylene green) and direct contact of the DNA and polyphenothiazine. Meanwhile the effect of DNA introduction was more pronounced for the coatings based on poly(Methylene blue). The difference in the behavior of the polyelectrolyte complexes can be used for differentiation of the influence on the film permeability related to the DNA damage and changes in the redox status of polyphenothiazines. PLL and ds-DNA were alternatively adsorbed on SWCNTs modified carbon paste electrode to form 1–3 bilayers. The charge transfer resistance and DPV signal of redox indicator methylene violet (7) were recorded to estimate damaging influence of Cd2+ ions [67]. The influence of damaging agent provoked released of the bonded dye from multilayer coating followed by its increased signal on voltammogram.

(7)

{PDDA/DNA}4 coating was obtained by layer-by-layer deposition on glassy carbon electrode for the detection of NO2· radicals generated in HRP catalyzed oxidation of nitrite ions [68] (8).

H2O2 + NO2− ⎯HRP ⎯⎯ → OH− + NO•2

(8)

The influence of free radicals was monitored by the DPV signal of Ru bipyridine complex which decreased with the degree of damage. The results were compared with those obtained with similar biosensors containing HRP in the polyelectrolyte complex. Besides similarity of the changes in the redox indicator signal, HRP immobilization made it possible to observe protecting effect of catalase inserted in the surface layer to prevent an access of hydrogen peroxide to the HRP active site and hence to exclude formation of NO2· species. Pencil graphite electrode was modified by DNA by means of two positively charged polyelectrolytes, i.e., PDDA and chitosan, with dispersed MWCNTs and TiO2 nanoparticles by consecutive dipping into appropriate dispersion and intermediate drying of the layers adsorbed [69]. The stability of immobilized DNA was checked by DPV signals of direct oxidation of guanine and adenine residues recorded prior to and after the contact of the DNA sensor with Methylene blue. The DNA sensor was applied for quantification of immobilized DNA (LOD 10.43 μg mL−1) and comparison of the stability of the multilayer coating. As was shown, the use of TiO2 dispersed in positively charged polymers increased the lifetime of the biosensor to a higher extent than MWCTs. 4. Conclusion Immobilization of DNA probes and aptamers is one of the most important steps of biosensors development because it determines their lifetime and sensitivity of the signal toward various analytes. Selection and optimization of immobilization protocols is often complicated by high density of negative charge of phosphate residues and rather strict requirements to the spatial accessibility of the binding sites to rather bulky complementary oligonucleotides and proteins specifically binding to DNA. Regarding determination of low-

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molecular species, e.g., anticancer drugs, intercalators and reactive oxygen species, immobilization should provide reliable and sensitive detection of minimal changes in a bioreceptor resulted in its interaction with a target which size and effect on biopolymer structure are about negligible. To meet such contradictory requirements, modern DNA sensors often utilize Au support, both as compact electrode and nanoparticles that specifically bind to thiolated terminal groups of bioreceptors. This technology is well developed but shows drawbacks related to non-specific chemisorption of interferences on bare metal surface and limited electron transfer in the biolayer. Electropolymerized materials and layer-by-layer immobilization are alternative approaches directed to use self-association and self-assembling processes to adapt the structure and properties of the immobilization matrices to the specific requirements of DNA probes mentioned above. Simple protocol with one-step attachment of oligonucleotides and renewable polyelectrolyte complexes deposition offer new opportunities to the increase of the sensitivity and stability of appropriate biosensors with minimal expenses on their operation. It should be mentioned that many of the protocols described in the review have analogs with chemically synthesized polymers which show some advantages when deposited on non-conductive particles. Nevertheless, electrochemistry provides a convenient way to establish the mechanism of signal generation and surface layer optimization for such materials, too. Started from proof-of-concept with model DNA probes and analytes, the DNA sensors utilize now more and more sophisticated matrices involving both electropolymerized and polyionic components often combined with electroconductive nanoparticles providing mechanical stability and electrical wiring of the binding sites. Many of the biosensors described were tested with real PCR products or natural DNA fragments which size exceeds that of conventional DNA probes used in standard assay techniques. It can be expected that the electropolymerization tools will incorporate some other modern approaches, e.g., sol-gel technologies and molecular imprinting concepts to increase the sensitivity and specificity of the response especially to pharmaceuticals and cancer biomarkers. Acknowledgments Financial support of the Scientific Grant Agency VEGA (project No. 1/0152/15), Slovak Research and Development Agency (Project No. APVV-14-0267) and Russian Foundation for Basic Research (grant No. 14-03-00409) is gratefully acknowledged. The work has been also performed according to the Russian Government Program of Competitive Growth of Kazan Federal University. References [1] R. Rosario, R. Mutharasan, Nucleic acid electrochemical and electromechanical biosensors: a review of techniques and developments, Rev. Anal. Chem. 33 (2014) 213–230. [2] A. Sassolas, B.D. Leca-Bouvier, L.J. Blum, DNA biosensors and microarrays, Chem. Rev. 108 (2008) 109–139. [3] E. Palecˇek, M. Bartošík, Electrochemistry of nucleic acids, Chem. Rev. 112 (2012) 3427–3481. [4] T. Ahuja, I.A. Mir, D. Kumar, Rajesh, Biomolecular immobilization on conducting polymers for biosensing applications, Biomaterials 28 (2007) 791–805. [5] J.P. Tosar, J.L. Holmes, S.D. Collyer, F. Davis, J. Laíz, S.P.J. Higson, Template and catalytic effects of DNA in the construction of polypyrrole/DNA composite macro and microelectrodes, Biosens. Bioelectron. 41 (2013) 294–301. [6] A. Chen, S. Chatterjee, Nanomaterials based electrochemical sensors for biomedical applications, Chem. Soc. Rev. 42 (2013) 5425–5438. [7] M. Liu, C. Luo, H. Peng, Electrochemical DNA sensor based on methylene blue functionalized polythiophene as a hybridization indicator, Talanta 88 (2012) 216–221. [8] G. Inzelt, Rise and rise of conducting polymers, J. Solid State Electrochem. 15 (2011) 1711–1718.

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