Fc Redox Research Articles

Redox-Functionalized Graphene Oxide Architecture for the Development of Amperometric Biosensing Platform

We describe the redox functionalization of graphene oxide (GO) and the development of versatile amperometric biosensing platforms for clinically important analytes such as cholesterol ester, uric acid and glucose. Ferrocene (Fc) redox units were covalently tethered onto the GO backbone using diamine sigma spacers of different chain lengths (C3-, C6-, and C9-diamines). The functionalized GO (Fc-GO) displays a pair of redox peak corresponding to Fc/Fc(+) redox couple at ~0.225 V. The surface coverage and heterogeneous electron transfer rate constant of Fc-GO depends on the length of sigma spacer. Amperometric biosensors for cholesterol (total), uric acid and glucose have been developed by integrating Fc-GO and the respective redox enzymes with screen printed electrode. Fc-GO efficiently mediates the bioelectrocatalytic oxidation of the substrates in the presence of the redox enzymes. The spacer length of Fc-GO controls the bioelectrocatalytic response of the biosensing platforms. The sensitivity of the biosensors based on C9 sigma spacer is significantly higher than the others. The detection limit (S/N = 3) of the biosensors for cholesterol and uric acid was 0.1 μM and for glucose it was 1 μM. Excellent stability, reproducibility, selectivity and fast response time were achieved. Biosensing of cholesterol, uric acid and glucose in human serum sample is successfully demonstrated with the biosensors, and the results are validated with the clinical laboratory measurement.

Kinetic and Thermodynamic Hysteresis Imposed by Intercalation of Proflavine in Ferrocene‐Modified Double‐Stranded DNA

Surface-confined immobilized redox species often do not show the expected zero peak separation in slow-scan cyclic voltammograms. This phenomenon is frequently associated to experimental drawbacks and hence neglected. However, a nonzero peak separation, which is common to many electrochemical systems with high structural flexibility, can be rationally assigned to a thermodynamic hysteresis. To study this phenomenon, a surface-confined redox species was used. Specifically, a DNA strand which is tagged with ferrocene (Fc) moieties at its 5' end and its complementary capture probe is thiolated at the 3' end was self-assembled in a monolayer at a Au electrode with the Fc moieties being located at the bottom plane of the double-stranded DNA (dsDNA). The DNA-bound Fc undergoes rapid electron transfer with the electrode surface as evaluated by fast scan cyclic voltammetry. The electron transfer is sensitive to the ion transport along the DNA strands, a phenomenon which is modulated upon specific intercalation of proflavine into surface-bound dsDNA. The electron transfer rate of the Fc(0/+) redox process is influenced by the cationic permselectivity of the DNA monolayer. In addition to the kinetic hindrance, a thermodynamic effect correlated with changes in the activity coefficients of the Fc(0/+) moieties near the gold-dsDNA interface is observed and discussed as source of the observed hysteresis causing the non-zero peak separation in the voltammograms.

Target-induced structure-switching DNA hairpins for sensitive electrochemical monitoring of mercury (II)

A simple, sensitive and reusable electrochemical sensor was designed for determination of mercury (II) (Hg2+) by coupling target-induced conformational switch of DNA hairpins with thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry. The hairpin probe consisted of a stem of 6 base pairs enclosing a 14 nucleotide (nt) loop and an additional 12 nt sticky end at the 3′ end. Each hairpin was labeled with ferrocene (Fc) redox tag in the middle of the loop, which was immobilized on the electrode via self-assembly of the terminal thiol moiety at the 5′ end. In the presence of target analyte, Hg2+-mediated base pairs induced the conformational change from the sticky end to open the hairpins, resulting in the ferrocene tags close to the electrode for the increasing redox current. The strong coordination reaction of T–Hg2+–T resulted in a good repeatability and intermediate precision down to 10%. The dynamic concentration range spanned from 5.0nM to 1.0μM Hg2+ with a detection limit of 2.5nM at the 3sblank level. The strategy afforded exquisite selectivity for Hg2+ against other environmentally related metal ions. Inspiringly, the developed sensor could be reused by introduction of iodide (I−).

Quantized Electron‐Transfer Pathways at Nanoparticle–Redox Centre Hybrids

Hexanethiolate gold monolayer-protected clusters (C6-MPCs) with an average core diameter of 1.8 nm and a capacitance of 0.6 aF are synthesised by a two-phase method. These clusters are functionalised with (6-ferrocenyl)-1-hexanethiol by a place exchange reaction at different molar ratios. The average number of ferrocene centres per cluster determined by (1)H NMR is ten, seven and four. Differential pulse voltammetry and cyclic voltammetry measurements for cluster solutions in 0.1 M TBAPF(6)/Tol:AN (2:1) clearly show the response of the Fc(+)/Fc redox couple and of quantized double layer (QDL) charging events of the gold core. A transition from single to multiple electron-transfer response for the redox couple is observed as the number of ferrocene units per cluster is increased. The distances between the redox moieties are estimated considering a homogeneous distribution of the redox sites on the nanoparticle ligand shell. In all the cases, the inter-ferrocene average separation is too large to observe self-exchange reactions and the most likely electron-transfer pathway is by fast rotational diffusion. The oxidation of the ferrocene groups results in an electrostatic switching-off of electron transfers between the electrode and the nanoparticle core.

Photoelectrochemical Behavior of Planar and Microwire‐Array Si|GaP Electrodes

AbstractGallium phosphide exhibits a short diffusion length relative to its optical absorption length, and is thus a candidate for use in wire array geometries that allow light absorption to be decoupled from minority carrier collection. Herein is reported the photoanodic performance of heteroepitaxially grown gallium phosphide on planar and microwire‐array Si substrates. The n‐GaP|n‐Si heterojunction results in a favorable conduction band alignment for electron collection in the silicon. A conformal electrochemical contact to the outer GaP layer is produced using the ferrocenium/ferrocene (Fc+/Fc) redox couple in acetonitrile. Photovoltages of ∼750 mV under 1 sun illumination are observed and are attributed to the barrier formed at the (Fc+/Fc)|n‐GaP junction. The short‐circuit current densities of the composite microwire‐arrays are similar to those observed using single‐crystal n‐GaP photoelectrodes. Spectral response measurements along with a finite‐difference‐time‐domain optical model indicate that the minority carrier diffusion length in the GaP is ∼80 nm. Solid‐state current–voltage measurements show that shunting occurs through thin GaP layers that are present near the base of the microwire‐arrays. The results provide guidance for further studies of 3D multi‐junction photoelectrochemical cells.

Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer spoilage bacteria Lactobacillus brevis

Attomole (10−18mol) levels of RNA and DNA isolated from beer spoilage bacterial cells Lactobacillus brevis have been detected by the electrochemical sandwich DNA hybridization assay exploiting enzymatic activity of lipase. DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design. The assay employs magnetic beads (MB) modified with a capture DNA sequence and a reporter DNA probe labeled with the enzyme, both made to be highly specific for L. brevis DNA. Lipase-labeled DNAs captured on MBs in the sandwich assay were collected on gold electrodes modified with a ferrocene (Fc)-terminated SAM formed by aliphatic esters. Lipase hydrolysis of the ester bond released a fraction of the Fc redox active groups from the electrode surface, decreasing the electrochemical signal from the surface-confined Fc. The assay, shown to be efficient for analysis of short synthetic DNA sequences, was ineffective with genomic double stranded bacterial DNA, but it allowed down to 16amole detection of 1563nts long RNA, isolated from bacterial ribosomes without the need for PCR amplification, and single DNA strands produced from ribosomal RNA. No interference from E. coli RNA was registered. The assay allowed analysis of 400 L. brevis cells isolated from 1L of beer, which fits the “alarm signal” range (from 1 to 100 cells per 100mL).

Ferrocene-Conjugated Copper(II) Complexes of l-Methionine and Phenanthroline Bases: Synthesis, Structure, and Photocytotoxic Activity

Ferrocene-conjugated reduced Schiff base (Fc-metH) copper(II) complexes of l-methionine and phenanthroline bases, namely, [Cu(Fc-met)(B)](NO3), where B is 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq in 2), dipyrido[3,2-a:2′,3′-c]phenazine (dppz in 3), and 2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (nip in 4), were prepared and characterized and their photocytotoxicity studied (Fc = ferrocenyl moiety). Complexes [Cu(Ph-met)(B)](NO3) of the reduced Schiff base from benzaldehyde and l-methionine (Ph-metH) and B (phen in 5, dppz in 6) were prepared and used as control species. Complexes 1 and 5 were structurally characterized by X-ray crystallography. Complex 1 as a discrete monomer has a CuN3OS core with the thiomethyl group as the axial ligand. Complex 5 has a polymeric structure with a CuN3O2 core in the solid state. Complexes 5 and 6 are formulated as [Cu(Ph-met)(B)(H2O)](NO3) in an aqueous phase based on the mass spectral data. Complexes 1–4 showed the Cu(II)–Cu(I) and Fc+–Fc redox couples at ∼0.0 and ∼0.5 V vs SCE, respectively, in DMF–0.1 M [Bun4N](ClO4). A Cu(II)-based weak d–d band near 600 nm and a relatively strong ferrocenyl band at ∼450 nm were observed in DMF–Tris-HCl buffer (1:4 v/v). The complexes bind to calf thymus DNA, exhibit moderate chemical nuclease activity forming •OH radical species, and are efficient photocleavers of pUC19 DNA in visible light of 454, 568, and 647 nm, forming •OH radical as the reactive oxygen species. They are cytotoxic in HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells, showing an enhancement of cytotoxicity upon visible light irradiation. Significant change in the nuclear morphology of the HeLa cells was observed with 3 in visible light compared to the nonirradiated sample. Confocal imaging using 4 showed its nuclear localization within the HeLa cells.

Tetrathia[7]helicene-Based Complexes of Ferrocene and (η5-Cyclohexadienyl)tricarbonylmanganese: Synthesis and Electrochemical Studies

Tetrathia[7]helicene ([7]TH)-based complexes substituted at the thienyl ring ends by a ferrocenyl group (Fc) or by a (η5-cyclohexadienyl)Mn(CO)3 derivative have been prepared by Sonogashira coupling reactions starting from the mono- or diiodo [7]TH compounds. The molecular structure of one of the diferrocenyl [7]TH complexes was established by X-ray analysis. Electrochemical investigation on the Fc-[7]TH systems show that the Fc groups are significantly electron poorer with respect to Fc (ΔE° ≈ 0.15 V), due to the effective conjugation of the Fc redox moiety with the triple bond + helicene system, as also confirmed by spectroscopic data. Potential cycling around the second oxidation peak, assigned to the thiahelicene moiety, affords fast, regular growth of electrodeposited conducting films, provided that one terminal α-thiophene position be available for coupling; on the other hand, long alkyl chains hamper film formation. The conducting films feature a broad oxidation wave resulting from the merging of several redox peaks, having its onset at the Fc oxidation. Since conducting films obtained by electrooligomerization of parent tetrathiahelicene have their onset potentials 0.45 V more positive than the Fc redox sites in this studied Fc-[7]TH conjugates, the above continuity could point to some coupling between Fc redox centers and conjugated π systems, favored by solid-state stacking.

Electrochemical “Signal‐On” Reporter for Amyloid Aggregates

The synthesis and characterization of four new Ferrocene (Fc) bioconjugates, bearing a podant (Lys)-Leu-Val-Phe-Phe motif, namely the hydrophobic sequence of amyloid-β-peptides (Aβ), is reported. The Fc-peptide conjugates are characterized by a reversible redox activity and the ability to undergo hydrophobic and hydrogen bonding interactions. Biomolecular interactions between Fc-bioconjugates with Aβ(12-28) fragments were studied by circular dichroism (CD), transmission electron microscopy (TEM), and electrochemistry. All four Fc-peptides interacted favourable with Aβ(12-28) and prevented fibril formation, the extent of which depended on the length of the peptide and the nature of the C-terminal group. The aggregates obtained for the Aβ(12-28)/Fc-peptide mixtures range from short fibrils to spherical aggregates. We demonstrated that in solution the peptide sequence and peptide charge affect the biomolecular interactions. Fc-peptide interactions with immobilized Aβ(12-28)-Cys films on Au surfaces were detected by measuring the current response of the Fc redox process. The formal redox potential, E(0), at ~440 (10) mV and i(pc)/i(pa) at 0.9 were observed characteristic for the monosubstituted Fc-derivative undergoing a one-electron redox process. On the surface, methyl ester-protected Fc-peptides (1 and 3) interacted only weakly with Aβ(12-28)-Cys films, giving rise to minimal redox activity. In contrast, charged Fc-peptides (2 and 4) gave a significant electrochemical readout following the interaction with Aβ(12-28)-Cys films. Interestingly, the Fc-peptide charge dictates the surface-assisted interactions, while hydrophobic and ionic effects contribute to the overall solution behaviour of the Fc-bioconjugates with Aβ(12-28).

Electrochemical analysis of HIV-1 reverse transcriptase serum level: Exploiting protein binding to a functionalized nanostructured surface

This manuscript describes an electrochemical approach to the detection of the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT) in serum exploiting an organometallic peptide conjugate that is chemically linked to a nanostructured gold surface. The assay format is based on the formation of a thin film of a ferrocene-labeled lipoic acid (Fc-LA) onto a gold nanoparticles-functionalized screen-printed carbon electrode (GNPs-SPCE). Time-of-Flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy were employed to confirm the binding of the Fc-LA to the electrode surface via formation of a gold–thiol bond. The RT biosensor was developed by covalent attachment of the peptide VEAIIRILQQLLFIH to the carboxylic acid group of Fc-LA. Square wave voltammetry offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. This allowed a linear quantification of the target RT in the range of 1–500pgmL−1 equivalent to 0.9–427fM, with a detection limit of 0.8pgmL−1 (0.7fM) with a short response time.

Electrochemical Considerations for Determining Absolute Frontier Orbital Energy Levels of Conjugated Polymers for Solar Cell Applications

Narrow bandgap conjugated polymers in combination with fullerene acceptors are under intense investigation in the field of organic photovoltaics (OPVs). The open circuit voltage, and thereby the power conversion efficiency, of the devices is related to the offset of the frontier orbital energy levels of the donor and acceptor components, which are widely determined by cyclic voltammetry. Inconsistencies have appeared in the use of the ferrocenium/ferrocene (Fc + /Fc) redox couple, as well as the values used for the absolute potentials of standard electrodes, which can complicate the comparison of materials properties and determination of structure/property relationships.

A bioorganometallic approach for rapid electrochemical analysis of human immunodeficiency virus type-1 reverse transcriptase in serum

Determination of low-abundance human immunodeficiency virus (HIV) enzymes is essential for early detection of the viral infection. Designed for the rapid detection of the virus in human serum, a rapid and ultrasensitive electrochemical assay for HIV-1 reverse transcriptase (HIV-1 RT) is presented in this article. The assay format is based on the formation of a self-assembled monolayer (SAM) of a synthesized ferrocene (Fc)-labeled lipoic acid onto a gold electrode. X-ray photoelectron spectroscopy (XPS) and Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) were employed to confirm the binding of the Fc-labeled lipoic acid to the gold via the Au–S bond. A short RT-specific peptide, VEAIIRILQQLLFIH, was covalently attached to the Fc-labeled lipoic acid. Square wave voltammetry (SWV) offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. This allowed a linear quantification of the target RT in the range of 100–500pgmL−1, equivalent to 85.5–427.4fM, with a detection limit of 50pgmL−1 (42.7fM). The developed biosensor is inexpensive, easy to prepare and operate, and allows a highly selective detection of RT in 20s.

Proton Medium Effect Based on the Ferrocinium Assumption in 80 Mass-% Propylene Carbonate + 20 Mass-% p-Xylene Medium

Conventional electrochemical studies on the ferrocinium–ferrocene (Fc+–Fc) redox system in 80 mass-% propylene carbonate (PC) + 20 mass-% p-xylene (PX) medium are reported, leading to a calculation of the proton medium effect. The dissociation constant of ferrocinium picrate in this medium was determined by conductivity measurements. The value of \(E_{\mathrm{Fe^{+} /Fe}}^{\mathrm{o}}\) against the Hg/HgCl2(s) reference electrode in 80 mass-% PC + 20 mass-% PX was determined potentiometrically in conjunction with conductivity data, and found to be 0.133±0.001 V versus the SHE at 25 °C. The \(E_{\mathrm{Fe^{+} /Fe}}^{\mathrm{o}}\) value has also been calculated using cyclic voltammetric data obtained in 80 mass-% PC + 20 mass-% PX at a micro platinum working electrode against the Ag/AgCl (nonaq.) reference electrode, and found to be comparable with the value obtained by potentiometry. The proton medium effect for transfer from water to the present mixed solvent medium was calculated using the value of \(E_{\mathrm{Fe^{+}/Fe}}^{\mathrm{o}}\) versus SHE at 25 °C and found to be 4.5. Based on values of proton medium effect reported in the literature, it is inferred that the present mixed solvent medium is slightly more basic than pure PC.

Recent Advances in Electrochemical Aptamer-Based Sensors

Aptamers are with their selectivity and affinity for specific target ligands ideal biorecognition units for any biosensor development. The development of improved techniques for in vitro selection of aptamers with high affinities (comparable to antibodies) for target molecules has transformed aptamers from costly and restrictedly accessible species into readily synthetically available biorecognition material for almost all possible targets - from small molecules to proteins and whole cells. Unsurprisingly, the new potential of aptamers has also been applied for development of novel aptamer-based electrochemical biosensors. In this paper we overview general tendencies and recent advances in this field, exemplified by impedimetric and electrochemical conformational aptamer-based biosensors, electrochemical aptamer-based biosensors exploiting redox indicators, catalytic and quantum dot labels, and inherent redox activity of DNA, and aptamer-modified field-effect transistors. Keywords: Apatmers, DNA, RNA, electrochemical aptamer-based biosensors, aptasensors, SELEX, adenosine monophosphate (AMP), neomycin-modified electrode, neomycin B, methylene blue (MB), aptamer-thrombin, ferrocene (Fc) redox, platelet-derived growth factor (PGDF), cocaine biosensor

Towards an early diagnosis of HIV infection: an electrochemical approach for detection ofHIV-1 reverse transcriptase enzyme

Oriented for the rapid diagnosis of HIV infection, highly sensitive and facile electrochemical assays for HIV-1 reverse transcriptase (HIV1-RT) are presented in this article. A non-labeled and a labeled assay format were based on the formation of self-assembled monolayers (SAMs) of either lipoic acid active ester or a newly synthesized ferrocene (Fc)-labeled cystamine derivative on electrode surfaces, respectively. A short RT-specific peptide, VEAIIRILQQLLFIH, was covalently attached to the surface of the formed SAMs. Electrochemical impedance spectroscopy (EIS) allowed a sensitive interrogation of RT in the non-labeled assay format. Furthermore, square wave voltammetry (SWV) offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. These techniques allowed a linear quantification of the target RT in the range of 75 to 750 pg mL(-1), with a limit of detection of 50 pg mL(-1). Furthermore, the developed biosensors showed a good specificity and allowed a proper discrimination between RT and other HIV enzymes.

Impact of metal on the DNA photocleavage activity and cytotoxicity of ferrocenyl terpyridine 3d metal complexes

Ferrocenyl terpyridine 3d metal complexes and their analogues, viz. [M(Fc-tpy)(2)](ClO(4))(2) (1-4), [Zn(Ph-tpy)(2)](ClO(4))(2) (5) and [Zn(Fc-dpa)(2)]X(2) (X = ClO(4), 6; PF(6), 6a), where M = Fe(II) in 1, Co(II) in 2, Cu(II) in 3 and Zn(II) in 4, Fc-tpy is 4'-ferrocenyl-2,2':6',2''-terpyridine, Ph-tpy is 4'-phenyl-2,2':6',2''-terpyridine and Fc-dpa is ferrocenyl-N,N-dipicolylmethanamine, are prepared and their DNA binding and photocleavage activity in visible light studied. Complexes 2, 4, 5 and 6a that are structurally characterized by X-ray crystallography show distorted octahedral geometry with the terpyridyl ligands binding to the metal in a meridional fashion, with Fc-dpa in 6a showing a facial binding mode. The Fc-tpy complexes display a charge transfer band in the visible region. The ferrocenyl (Fc) complexes show a quasi-reversible Fc(+)-Fc redox couple within 0.48 to 0.66 V vs. SCE in DMF-0.1 M TBAP. The DNA binding constants of the complexes are ∼10(4) M(-1). Thermal denaturation and viscometric data suggest DNA surface binding through electrostatic interaction by the positively charged complexes. Barring the Cu(II) complex 3, the complexes do not show any chemical nuclease activity in the presence of glutathione. Complexes 1-4 exhibit significant plasmid DNA photocleavage activity in visible light via a photoredox pathway. Complex 5, without the Fc moiety, does not show any DNA photocleavage activity. The Zn(II) complex 4 shows a significant PDT effect in HeLa cancer cells giving an IC(50) value of 7.5 μM in visible light, while being less toxic in the dark (IC(50) = 49 μM).

A Single‐Electrode, Dual‐Potential Ferrocene–PNA Biosensor for the Detection of DNA

A Fc-PNA biosensor (Fc: ferrocenyl, C(10)H(9)Fe) 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 Fc(0/+) redox couple with a significant difference in their electrochemical half-wave potentials of Delta E(1/2)=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.

Touching Surface-Attached Molecules with a Microelectrode: Mapping the Distribution of Redox-Labeled Macromolecules by Electrochemical-Atomic Force Microscopy

We report on the development of a mediator-free electrochemical-atomic force microscopy (AFM-SECM) technique designed for high-resolution imaging of molecular layers of nanometer-sized redox-labeled (macro)molecules immobilized onto electrode surfaces. This new AFM-SECM imaging technique, we call molecule touching atomic force electrochemical microscopy (Mt/AFM-SECM), is based on the direct contact between surface-anchored molecules and an incoming microelectrode (tip). To validate the working-principle of this microscopy, we consider a model system consisting of a monolayer of nanometer long, flexible, polyethylene glycol (PEG) chains covalently attached by one extremity to a gold surface and bearing at their free end a ferrocene (Fc) redox tag. Using Mt/AFM-SECM in tapping mode, i.e., by oscillating the tip so that it comes in intermittent contact with the grafted chains, we show that the substrate topography and the distribution of the redox-tagged PEG chains immobilized on the gold surface can be simultaneously and independently imaged at the sub-100 nm scale. This novel type of SECM imaging may be found useful for characterizing the surface of advanced biosensors which use electrode-grafted, redox-tagged, linear biochains, such as peptides or DNA chains, as sensing elements. In principle, Mt/AFM-SECM should also permit in situ imaging of the distribution of any kind of macromolecules immobilized on electrode surfaces or simply conducting surfaces, provided they are labeled by a suitable redox tag.

Synthesis and Application of a Triazene−Ferrocene Modifier for Immobilization and Characterization of Oligonucleotides at Electrodes

A new DNA modifier containing triazene, ferrocene, and activated ester functionalities was synthesized and applied for electrochemical grafting and characterization of DNA at glassy carbon (GC) and gold electrodes. The modifier was synthesized from ferrocenecarboxylic acid by attaching a phenyltriazene derivative to one of the ferrocene Cp rings, while the other Cp ring containing the carboxylic acid was converted to an activated ester. The modifier was conjugated to an amine-modified DNA sequence. For immobilization of the conjugate at Au or GC electrodes, the triazene was activated by dimethyl sulfate for release of the diazonium salt. The salt was reductively converted to the aryl radical which was readily immobilized at the surface. DNA grafted onto electrodes exhibited remarkable hybridization properties, as detected through a reversible shift in the redox potential of the Fc redox label upon repeated hybridization/denaturation procedures with a complementary target DNA sequence. By using a methylene blue (MB) labeled target DNA sequence the hybridization could also be followed through the MB redox potential. Electrochemical studies demonstrated that grafting through the triazene modifier can successfully compete with existing protocols for DNA immobilization through the commonly used alkanethiol linkers and diazonium salts. Furthermore, the triazene modifier provides a practical one-step immobilization procedure.

Effect of Serum on an RNA Aptamer-Based Electrochemical Sensor for Theophylline

Electrochemical performance of the ferrocene (Fc) redox-labeled RNA aptamer based sensor for theophylline (Th) is essentially inhibited in serum, but is restored in serum-free buffer solutions. This phenomenon is inconsistent with the data on methylene-blue-labeled aptamer beacon systems, which operational potential window is more negative compared to the Fc redox label. Electrochemical studies with a ferricyanide redox probe, having redox potential close to the Fc redox couple, and interfacial capacitance measurements unambiguously demonstrate that it is adsorption of serum proteins at positively charged electrode surface that slows down the kinetics of the electrode reactions in serum and interferes with the biosensor performance. In filtered serum solutions, in the absence of serum proteins, the Fc-labeled aptamer-based biosensor performed similarly to the pure buffer solutions, ad the signal for Th could be linearly calibrated versus Th concentration. These results on interfacial effects of serum are of particular importance for future research and development of the beacon-type biosensors for in vivo applications.