Redox Tag Research Articles

Aptamer-Based Electrochemical Biosensor for Interferon Gamma Detection

In this paper, we describe the development of an electrochemical DNA aptamer-based biosensor for detection of interferon (IFN)-γ. A DNA hairpin containing IFN-γ-binding aptamer was thiolated, conjugated with methylene blue (MB) redox tag, and immobilized on a gold electrode by self-assembly. Binding of IFN-γ caused the aptamer hairpin to unfold, pushing MB redox molecules away from the electrode and decreasing electron-transfer efficiency. The change in redox current was quantified using square wave voltammetry (SWV) and was found to be highly sensitive to IFN-γ concentration. The limit of detection for optimized biosensor was 0.06 nM with linear response extending to 10 nM. This aptasensor was specific to IFN-γ in the presence of overabundant serum proteins. Importantly, the same aptasensor could be regenerated by disrupting aptamer-IFN-γ complex in urea buffer and reused multiple times. Unlike standard sandwich immunoassays, the aptasensor described here allowed one to detect IFN-γ binding directly without the need for multiple washing steps and reagents. An electrochemical biosensor for simple and sensitive detection of IFN-γ demonstrated in this paper will have future applications in immunology, cancer research, and infectious disease monitoring.

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.

Re-engineering aptamers to support reagentless, self-reporting electrochemical sensors

Electrochemical aptamer-based (E-AB) sensors have emerged as a promising and versatile new biosensor platform. Combining the generality and specificity of aptamer-ligand interactions with the selectivity and convenience of electrochemical readouts, this approach affords the detection of a wide variety of targets directly in complex, contaminant-ridden samples, such as whole blood, foodstuffs and crude soil extracts, without the need for exogenous reagents or washing steps. Signaling in this class of sensors is predicated on target-induced changes in the conformation of an electrode-bound probe aptamer that, in turn, changes the efficiency with which a covalently attached redox tag exchanges electrons with the interrogating electrode. Aptamer selection strategies, however, typically do not select for the conformation-switching architectures, and as such several approaches have been reported to date by which aptamers can be re-engineered such that they undergo the binding-induced switching required to support efficient E-AB signaling. Here, we systematically compare the merits of these re-engineering approaches using representative aptamers specific to the small molecule adenosine triphosphate and the protein human immunoglobulin E. We find that, while many aptamer architectures support E-AB signaling, the observed signal gain (relative change in signal upon target binding) varies by more than two orders of magnitude across the various constructs we have investigated (e.g., ranging from -10% to 200% for our ATP sensors). Optimization of the switching architecture is thus an important element in achieving maximum E-AB signal gain and we find that this optimal geometry is specific to the aptamer sequence upon which the sensor is built.

Exploiting Binding-Induced Changes in Probe Flexibility for the Optimization of Electrochemical Biosensors

Electrochemical sensors employing redox-tagged, electrode-bound oligonucleotides have emerged as a promising new platform for the reagentless detection of molecular analytes. Signal generation in these sensors is linked to specific, binding-induced changes in the efficiency with which an attached redox tag approaches and exchanges electrons with the interrogating electrode. We present here a straightforward means of optimizing the signal gain of these sensors that exploits this mechanism. Specifically, using square-wave voltammetry, which is exquisitely sensitive to electrode reaction rates, we can tune the frequency of the voltammetric measurements to preferentially enhance the signal associated with either the unbound or target-bound conformations of the probe. This allows us to control not only the magnitude of the signal gain associated with target binding but also the sign of the signal change, generating "signal-on" or "signal-off" sensors. This optimization parameter appears to be quite general: we show here that tuning the square-wave frequency can significantly enhance the gain of the sensors directed against specific oligonucleotide sequences, small molecules, proteins, and protein-small molecule interactions.

Comparing the Properties of Electrochemical-Based DNA Sensors Employing Different Redox Tags

Many electrochemical biosensor approaches developed in recent years utilize redox-labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported that employ a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers, and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum.

On the Signaling of Electrochemical Aptamer-Based Sensors: Collision- and Folding-Based Mechanisms.

Recent years have seen the emergence of a new class of electrochemical sensors predicated on target binding-induced folding of electrode-bound redox-modified aptamers and directed against targets ranging from small molecules to proteins. Previous studies of the relationship between gain and probe-density for these electrochemical, aptamer-based (E-AB) sensors suggest that signal transduction is linked to binding-induced changes in the efficiency with which the attached redox tag strikes the electrode. This, in turn, suggests that even well folded aptamers may support E-AB signaling if target binding sufficiently alters their flexibility. Here we investigate this using a thrombin-binding aptamer that undergoes binding-induced folding at low ionic strength but can be forced to adopt a folded conformation at higher ionic strength even in the absence of its protein target. We find that, under conditions in which the thrombin aptamer is fully folded prior to target binding, we still obtain a ca. 30% change in E-AB signal upon saturated target levels. In contrast, however, under conditions in which the aptamer is unfolded in the absence of target and thus undergoes binding-induced folding the observed signal change is twice as great. The ability of folded aptamers to support E-AB signaling, however, is not universal: a fully folded anti-IgE aptamer, for example, produces only an extremely small, ca. 2.5% signal change in the presence of target despite the larger steric bulk of this protein. Thus, while it appears that binding-induced changes in the dynamics in fully folded aptamers can support E-AB signaling, this signaling mechanism may not be general, and in order to ensure the design of high-gain sensors binding must be linked to a large-scale conformational change.

An Electrochemical Sensor for the Detection of Protein−Small Molecule Interactions Directly in Serum and Other Complex Matrices

Here we have demonstrated a general, sensitive, and selective approach for the detection of macromolecules that bind to specific small molecule recognition elements. Our electrochemical approach utilizes a redox-tagged DNA signaling scaffold that is conjugated to a small molecule recognition element and is covalently attached to an interrogating electrode. The binding of a protein to the small molecule recognition element alters the dynamics of the scaffold, increasing or decreasing the efficiency with which the redox tag collides with the electrode and thus altering the observed faradaic current. We optimized the scaffold using a biotin recognition element and streptavidin as a target to determine the variables that define sensor performance before then applying the approach to detection of anti-digoxigenin antibodies using the steroid as the recognition element. We generated streptavidin sensors exhibiting both signal-on (target binding increases the faradaic current) and signal-off behavior, of which only the signal-off approach was generalizable to the detection of antibodies. Sensors for both targets are sensitive (detection limits in the low nanomolar range), rapid (minutes), reusable, and selective enough to function directly in complex matrices including blood serum, soil, and foodstuffs.

Effects of Probe Length, Probe Geometry, and Redox-Tag Placement on the Performance of the Electrochemical E-DNA Sensor

Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architectures comprised of an electrode-immobilized, redox-tagged probe oligonucleotide. Recent studies suggest that E-DNA signaling is predicated on hybridization-linked changes in probe flexibility, which will alter the efficiency with which the terminal redox tag strikes the electrode. This, in turn, suggests that probe length, probe geometry, and redox-tag placement will affect E-DNA signaling. To test this we have characterized E-DNA sensors comprised of linear or stem-loop probes of various lengths and with redox tags placed either distal to the electrode or internally within the probe sequence (proximal). We find that linear probes produce larger signal changes upon target binding than equivalent stem-loop probes. Likewise, long probes exhibit greater signal changes than short probes provided that the redox tag is placed proximal to the electrode surface. In contrast to their improved signaling, the specificity of long probes is poorer than that of short probes, suggesting that sensor optimization represents a trade off between sensitivity and specificity. Finally, we find that sensor response time and selectivity are only minimally affected by probe geometry or length. The results of this comparative study will help guide future designs and applications of these sensors.

Highly Specific and Sensitive Electrochemical Genotyping via Gap Ligation Reaction and Surface Hybridization Detection

This paper developed a novel electrochemical genotyping strategy based on gap ligation reaction with surface hybridization detection. This strategy utilized homogeneous enzymatic reactions to generate molecular beacon-structured allele-specific products that could be cooperatively annealed to capture probes stably immobilized on the surface via disulfide anchors, thus allowing ultrasensitive surface hybridization detection of the allele-specific products through redox tags in close proximity to the electrode. Such a unique biphasic architecture provided a universal methodology for incorporating enzymatic discrimination reactions in electrochemical genotyping with desirable reproducibility, high efficiency and no interferences from interficial steric hindrance. The developed technique was demonstrated to show intrinsic high sensitivity for direct genomic analysis, and excellent specificity with discriminativity of single nucleotide variations.

Reagentless, electrochemical approach for the specific detection of double- and single-stranded DNA binding proteins.

Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.

Optimization of a Reusable, DNA Pseudoknot-Based Electrochemical Sensor for Sequence-Specific DNA Detection in Blood Serum

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem's loop forms part of the second stem, is modified with a methylene blue redox tag at its 3' terminus and covalently attached to a gold electrode via the 5' terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density ( approximately 1.8 x 10(13) molecules/cm(2) apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3' stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3' loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.

Cymantrene Radical Cation Family: Spectral and Structural Characterization of the Half-Sandwich Analogues of Ferrocenium Ion

The anodic one-electron oxidation of three members of the half-sandwich family of piano-stool compounds MnCp (gamma)(CO) 3, where Cp (gamma) is a generic cyclopentadienyl ligand, has been studied in a CH 2Cl 2/[NBu 4][TFAB] electrolyte (TFAB = [B(C6F5) 4] (-)). The long-sought 17 e (-) radical cation of the parent complex MnCp(CO) 3 (cymantrene, 1, E 1/2 = 0.92 V vs ferrocene) has been shown to be persistent in solutions that use weakly coordinating anions in place of more nucleophilic traditional electrolyte anions. Spectroscopically characterized for the first time, 1 (+) was shown to absorb in the visible (530 nm), near-IR (2066 nm), and IR (2118, 1934 cm (-1)) regions. It was ESR-active at low temperatures (g parallel = 2.213, g perpendicular = 2.079, A parallel (Mn) = 79.2 G, A perpendicular (Mn) = 50 G) and NMR active at room temperature (delta = 22.4 vs TMS). The radical cations of the Cp-functionalized analogues, Mn(eta (5)-C5H 4NH2)(CO) 3, 2, E 1/2 = 0.62 V, and MnCp*(CO) 3 (Cp*= eta (5)-C 5Me 5, 3), E 1/2 = 0.64 V, were generated electrochemically as well by the chemical oxidant [ReCp(CO) 3] (+). The structures of 2 (+) and 3 (+) were determined by X-ray crystallographic studies of their TFAB salts. Compared to the structures of the corresponding neutral compounds, the cations showed elongated Mn-C(O) bonds and shortened C-O bonds, displaying the effect of diminished metal-to-CO backbonding. The bond-length changes in the Mn(CO) 3 moiety were much larger in 3 (+) (avg changes, Mn-C(O) = + 0.142 A, C-O = -0.063 A) than in 2 (+) (avg changes, Mn-C(O) = + 0.006 A, C-O = -0.003 A). Although there were only minor changes in the metal-to-center ring distances upon oxidation of either 2 or 3, there was decidedly less bending of the C(N) atom out of the cyclopentadienyl plane in 2 (+) compared to 2. The optical, vibrational, and magnetic resonance spectra of radicals 2 (+) and 3 (+) were also observed. The spectral data argue for the SOMOs of the 17-electron species being largely located on the Mn(CO) 3 moiety, having 40-50% Mn d-orbital character, with the ground states of the radicals, most likely (2)A'', lying close in energy (within about 6000 cm (-1)) to excited states that are responsible for their rapid electronic relaxations. The cymantrenyl moiety is proposed as an anodic redox tag (or label) having physical and chemical properties that are significantly different from those of its ferrocenyl analogue.

A New Electrocatalytic [2+2] Cross-Coupling Reaction Supported by a "Redox Tag" for Anodic EC-backward-E Process

not Available.

Ultrasensitive label-free aptamer-based electronic detection.

Ultrasensitive label-free aptamer-based electronic detection.

(Allenylidene)ruthenium Complexes with Redox‐Active Substituents and Ligands

AbstractWe describe the allenylidene complexes [TpL2Ru=C=C=CPhR]+ SbF6− [Tp = HB(pz)3−, L2 = 2 PPh3 or 1,1′‐bis(diphenylphosphanyl)ferrocene (dppf), R = Ph or ferrocenyl] and their spectroscopic and electrochemical characteristics. Three of these compounds possess redox‐active, ferrocene‐based substituents or ligands − that are oxidized at lower potentials than the ruthenium center itself − attached either to the terminal carbon atom of the allenylidene ligand or to the ruthenium atom. The Fc/Ph‐substituted complexes 3a and 3b provide unique examples of hindered rotation of the allenylidene substituent around the M=C bond. For 3a (L2 = 2 PPh3), two isomers differing in the orientation of the vertically aligned, unsymmetrically substituted allenylidene ligand are discernible even at 388 K. The dppf‐substituted congener 3b has the allenylidene ligand in a horizontal orientation and exhibits a rotational barrier, as determined by dynamic 31P NMR spectroscopy, of ΔG≠ = 47 kJ/mol at TC = 238 K. The changes in the spectroscopic and electrochemical properties upon replacement of the PPh3 by a dppf ligand and the Ph by an Fc moiety can be explained in terms of the bonding within these systems. These effects are attenuated when the ferrocene‐based redox tags are oxidized, as shown by IR and UV/Vis spectroelectrochemistry. Thus, infrared spectroelectrochemistry reveals a blue shift of the allenylidene stretch upon oxidation of the dppf ligand, while oxidation of the ferrocene covalently linked to the unsaturated C3 ligand has the opposite effect. Oxidation of the ruthenium atom influences the bonding within the unsaturated ligand more profoundly. Results from IR spectroelectrochemistry point to a vinylidene structure in the RuIII state. Reduction enhances the contribution of alkynyl‐type resonance forms to the overall bonding description, as also shown by IR spectroelectrochemistry. For the ferrocenyl‐substituted allenylidene complexes, oxidation and reduction result in bleaching of the intense optical low‐energy absorption band attributed to a ferrocenyl‐to‐allenylidene charge‐transfer process. The EPR spectra of the paramagnetic reduced forms of these complexes also indicate spin delocalization into the aryl substituents attached to the allenylidene ligand. The complexes Tp(dppf)RuCl and [Tp(dppf)Ru=C=C=CPh2]+ SbF6− were also characterized by X‐ray crystallography. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Microchip-based amperometric immunoassays using redox tracers.

A new chip-based electrochemical immunoassay protocol, based on the use of a ferrocene redox label, is described. Two reaction formats, based on direct (noncompetitive) and competitive modes of operation, were employed for illustrating the use of redox tracers in chip-based electrochemical immunoassays. The direct assay consisted of mixing the ferrocene-tagged antibody and the antigen analyte, a rapid electrophoretic separation of labeled free antibody and the labeled antigen/antibody complex, and a downstream anodic detection of the ferrocene tracer at gold-plated carbon screen-printed electrode detector. The competitive assay integrates precolumn reactions of the labeled antigen and the target antigen with the antibody with electrophoretic separation of the free and bound labeled antigens, along with amperometric detection of the redox tag. An internal standard has been used to normalize the peak area for the construction of calibration plots. Fundamental operating variables are examined and optimized. The use of a redox tracer offers the advantages of simplified protocol, wider linear range, higher stability, and higher separation efficiency compared to an analogous use of enzyme tags. The direct mouse-immunoglobulin G (IgG) assay and the competitive 3,3',5-triiodo-L-thyronine (T(3)) one were accomplished within less than 150 and 130 s (with field strengths of 256 and 192 V/cm), and offer minimum detectable concentrations of 2.5 x 10(-12) and 1 x1 0(-6) g/mL, respectively. Such use of redox labels for chip-based amperometric immunoassay protocols offers considerable promise for decentralized clinical or environmental testing.