Magdalena Gębala

Controlled Orientation of DNA in a Binary SAM as a Key for the Successful Determination of DNA Hybridization by Means of Electrochemical Impedance Spectroscopy

Determination of DNA hybridization at electrode surfaces modified with thiol-tethered single-stranded DNA (ssDNA) capture probes and co-assembled with short-chain thiol derivatives using electrochemical impedance spectroscopy requires a careful design of the electrode/electrolyte interface as well as an in-depth understanding of the processes at the interface during DNA hybridization. The influence of the electrode potential, the ssDNA coverage, the ionic strength, the nature of the thiol derivative and especially the Debye length are shown to have a significant impact on the impedance spectra. A mixed monolayer comprising--in addition to the ssDNA capture probe--both mercaptohexanol (MCH) and mercaptopropionic acid (MPA) is suggested as an interface design which allows a high efficiency of the DNA hybridization concomitantly with a reliable modulation of the charge-transfer resistance of the electrode upon hybridization.

A single-electrode, dual-potential ferrocene-PNA biosensor for the detection of DNA.

A Fc–PNA biosensor (Fc: ferrocenyl, C10H9Fe) was designed by using two electrochemically distinguishable recognition elements with different molecular information at a single electrode. Two Fc–PNA capture probes were therefore synthesized by N‐terminal labeling different dodecamer PNA sequences with different ferrocene derivatives by click chemistry. Each of the two strands was thereby tethered with one specific ferrocene derivative. The two capture probes revealed quasi‐reversible redox processes of the Fc0/+ redox couple with a significant difference in their electrochemical half‐wave potentials of ΔE1/2=160 mV. A carefully designed biosensor interface, consisting of a ternary self‐assembled monolayer (SAM) of the two C‐terminal cysteine‐tethered Fc–PNA capture probes and 6‐mercaptohexanol, was electrochemically investigated by square wave (SWV) and cyclic voltammetry (CV). The biosensor properties of this interface were analyzed by studying the interaction with DNA sequences that were complementary to either of the two capture probes by SWV. Based on distinct changes in both peak current and potential, a parallel identification of these two DNA sequences was successful with one interface design. Moreover, the primary electrochemical response could be converted by a simple mathematical analysis into a clear‐cut electrochemical signal about the hybridization event. The discrimination of single‐nucleotide polymorphism (SNP) was proven with a chosen single‐mismatch DNA sequence. Furthermore, experiments with crude bacterial RNA confirm the principal suitability of this dual‐potential sensor under real‐life conditions.

Mechanistic Studies of Fc‐PNA(⋅DNA) Surface Dynamics Based on the Kinetics of Electron‐Transfer Processes

N-Terminally ferrocenylated and C-terminally gold-surface-grafted peptide nucleic acid (PNA) strands were exploited as unique tools for the electrochemical investigation of the strand dynamics of short PNA(⋅DNA) duplexes. On the basis of the quantitative analysis of the kinetics and the diffusional characteristics of the electron-transfer process, a nanoscopic view of the Fc-PNA(⋅DNA) surface dynamics was obtained. Loosely packed, surface-confined Fc-PNA single strands were found to render the charge-transfer process of the tethered Fc moiety diffusion-limited, whereas surfaces modified with Fc-PNA⋅DNA duplexes exhibited a charge-transfer process with characteristics between the two extremes of diffusion and surface limitation. The interplay between the inherent strand elasticity and effects exerted by the electric field are supposed to dictate the probability of a sufficient approach of the Fc head group to the electrode surface, as reflected in the measured values of the electron-transfer rate constant, k(0). An in-depth understanding of the dynamics of surface-bound PNA and PNA⋅DNA strands is of utmost importance for the development of DNA biosensors using (Fc-)PNA recognition layers.

Optimization of an electrochemical DNA assay by using a 48-electrode array and redox amplification studies by means of scanning electrochemical microscopy.

Sensible DNA: An electrochemical DNA assay based on specific Salmonella spp. capture probes and enzyme labeling with alkaline phosphatase was optimized by using a 48‐electrode microarray and scanning electrochemical microscopy (SECM). SECM was further used to evaluate potential amplification strategies due to redox cycling.

Amplified detection of DNA hybridization using post-labelling with a biotin-modified intercalator

A 32-electrode microelectrode array modified with a self-assembled monolayer of a thiolated DNA capture strand and 11-mercapto-l1-undecanol was used for the detection of multi-resistant Staphylococcus aureus (MRSA) upon hybridization of the complementary target DNA. In the proposed assay strategy the obtained double-stranded DNA (dsDNA) is at first non-covalently labeled by intercalation of a proflavine derivative which is functionalized via a flexible spacer with biotin moieties. Subsequent to this epost-labelling a avidin/alkaline phosphatase conjugate is bound to the biotin moieties thus introducing a reporter group at sites bearing dsDNA. Hybridization and hence the presence of MRSA DNA is detected via oxidation ofp-aminophenol enzymatically generated from p-aminophenylphosphate. The assay strategy was carefully evaluated using ferrocene-modified target strands. An increase in sensitivity of the proposed label-free DNA assays based on a careful design of the sensing interface and the implemented enzymatic amplification was achieved.

Impact of single basepair mismatches on electron-transfer processes at Fc-PNA⋅DNA modified gold surfaces.

Gold-surface grafted peptide nucleic acid (PNA) strands, which carry a redox-active ferrocene tag, present unique tools to electrochemically investigate their mechanical bending elasticity based on the kinetics of electron-transfer (ET) processes. A comparative study of the mechanical bending properties and the thermodynamic stability of a series of 12-mer Fc-PNA⋅DNA duplexes was carried out. A single basepair mismatch was integrated at all possible strand positions to provide nanoscopic insights into the physicochemical changes provoked by the presence of a single basepair mismatch with regard to its position within the strand. The ET processes at single mismatch Fc-PNA⋅DNA modified surfaces were found to proceed with increasing diffusion limitation and decreasing standard ET rate constants k(0) when the single basepair mismatch was dislocated along the strand towards its free-dangling Fc-modified end. The observed ET characteristics are considered to be due to a punctual increase in the strand elasticity at the mismatch position. The kinetic mismatch discrimination with respect to the fully-complementary duplex presents a basis for an electrochemical DNA sensing strategy based on the Fc-PNA⋅DNA bending dynamics for loosely packed monolayers. In a general sense, the strand elasticity presents a further physicochemical property which is affected by a single basepair mismatch which may possibly be used as a basis for future DNA sensing concepts for the specific detection of single basepair mismatches.

Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed.

Amperometric sensing — Bioelectroanalysis

It seems quite timely that Analytical and Bioanalytical Chemistry (ABC) is devoting a special issue to sensing. Less obvious is that the issue is limited to electrochemical sensing, even less so to amperometric sensing, bioelectrosensing included. Actually, electroanalysis is not the most widely applied analytical technique. Optical methods definitely overwhelm the electrochemical ones in popularity; once mass spectrometry, chromatography, and the relevant hyphenated techniques, are taken into account together with the optical ones, only a restricted room is seemingly left to electrochemical methods of analysis. However, especially in the field of biosensors, this general description does not alway hold and electroanalytical techniques are presently dominating successful commercial applications of biosensors. A big change in electroanalysis has occurred in the last 30 years, caused by the invention of modified electrodes. The modification of electrode surfaces has also led to improved anchoring of highly specific biological recognition elements, allowing unprecedented performance in electrocatalytic biosensing. Electrode systems have attained increasing complexity and a large variety of electrode modifiers have been proposed, such as synthetic and natural macromolecules, different types of nanoparticles and conducting polymers or clays. Among the novel materials developed in the last 20 years, only a limited number have been tested and used in an electrochemical context, and an even much lower number have been considered for electroanalysis. This encourages analytical chemists to closely follow developments in the field of material chemistry, basic studies in electrochemistry and electrochemical applications to potentially transfer this knowledge to effective electroanalytical systems. Concomitantly with the development of novel electrode materials, optimization of the overall system structure has become more complex, requiring its characterization using a large number of spectroscopic, microscopic and electrochemical techniques. Consequently, interdisciplinarity has become a must for the effective development of novel modified electrodes for electroanalysis. Only combinations of analytical techniques provide sufficient insight into the in-depth functioning of modified electrodes for electroanalysis and the interplay between the different components ultimately composing the complex electrode architecture. Complete knowledge, in the widest sense, is necessary to achieve the best performance of an envisaged sensor as a whole. A lot of time has passed since the years in which ‘bare’ electrodes were used in molecular electrochemistry studies, which were subsequently transferred to electroanalysis. More or less complex electrode mechanisms were studied only by electrochemical techniques that allowed the electrode mechanism to be fully elucidated from a qualitative and even quantitative point of view. Such a ‘simple’, though rigorous approach is not possible with more complex modified electrode systems any more, owing to the enormous variety of possible structures, depending on a number of possible compositions and the conditions under which the system has been realized. A danger lies in the tendency to separate the field into two ‘different kinds’ of scientists doing electroanalysis: those strictly following an ‘it works!’ approach describing often useful effects which cannot be given an in-depth account, owing to the too great complexity of the system, Published in the topical collections Amperometric Sensing with guest editors Renato Seeber, Fabio Terzi, and Chiara Zanardi and Bioelectroanalysis with guest editors Nicolas Plumere, Magdalena Gebala, and Wolfgang Schuhmann. R. Seeber (*) : F. Terzi :C. Zanardi Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via G. Campi, 183, 41100 Modena, Italy e-mail: renato.seeber@unimore.it