Electrochemical impedance probing of transcriptional TATA binding protein based on TATA box site-specific binding

Electrochemical impedance probing of TATA binding protein (TBP) based on TATA box site-specific binding was described in this work. A sensitive detection of TBP was developed from TATA box DNA self assembly on the electrode and the impedance changes induced by TBP binding. Electrochemical impedance spectroscopy (EIS) probing of TBP had a sensitivity of 0.8 nM with excellent selectivity. Moreover, the interferences of triplex forming oligonucleotides (TFOs) and anticancer drug daunomycin on TBP binding to TATA box DNA were investigated by EIS. TFOs reduced the stability of TBP binding to TATA box, but daunomycin completely inhibited the TBP binding.     

The detection of DNA deamination by electrocatalysis at DNA-modified electrodes

The method of electrocatalysis based on using a methylene blue (MB) as an electrochemical indicator and ferricyanide ions [Fe(CN)6]3- as an electron acceptor was applied in screening DNA for lesions caused by deamination of nucleobases. The damaged DNA was modeled by short 18-mer oligonucleotides containing the different number of mismatched target bases (uracil instead of cytosine residues). The hybridization capacity of these oligomers with complementary probes (immobilized on gold electrodes or free) was investigated by both electrochemical methods and UV spectroscopy. We have shown that the amplitude of the reduction signal corresponding to ferricyanide ions considerably increases in the presence of MB. This electrocatalytic effect allowed us to detect the changes in electrochemical properties of DNA caused by dU.dG mismatches. Using differential pulse voltammetry and cyclic voltammetry, we showed that the electron transport from the electrode through the double-stranded DNA to MB and then to ferricyanide ions is suppressed by the mismatches in duplex structure. According to UV-monitored melting data, single or multiple wobble dU.dG base pairs destabilize 18-mer DNA duplex by 9-27 degrees C.     

Electrical impedance spectroscopy for detection of bacterial cells in suspensions using interdigitated microelectrodes

In this study, we present a new, simple and rapid impedance method to detect bacterial cells by making use of the impedance properties of bacterial cell suspensions using interdigitated microelectrodes. It was found that bacterial cell suspensions in deionized (DI) water with different cell concentrations could generate different electrical impedance spectral responses, whereas cell suspensions in phosphate buffered saline (PBS) solution could not produce any significant differences in impedance spectra in response to different cell concentrations. In DI water suspensions, impedance at 1 kHz decreased with the increasing cell concentrations in the suspensions. The impedance of cell suspensions in DI water was discussed and found that it was resulted from the cell wall charges and the release of ions or other osmolytes from the cells. A linear relationship between the impedance and the logarithmic value of the cell concentration was found in the cell concentration range from 10⁶ to 10¹⁰ cfu/ml, which can be expressed by a regression equation of Z (kΩ) = −2.06 log C (cells/20 μl) + 5.23 with R² = 0.98. The detection limit was calculated to be 3.45 × 10⁶ cfu/ml, which is comparable with many label-free immunosensors for detection of pathogenic bacteria reported in the literature. To achieve the selectivity of this method, we also demonstrated the feasibility of integrating magnetic separation to this impedance method. This study has demonstrated that bacterial cell concentration can be inferred by measuring the impedance of cell suspensions in DI water. This new detection mechanism could be an alternative to current impedance methods that have been reported for the detection of bacterial cells, e.g. impedance microbiology and electrical/electrochemical impedance biosensors.     

Nanomolar detection of p-nitrophenol via in situ generation of p-aminophenol at nanostructured microelectrodes

A new method and a new buffer medium, expected to be practical for miniaturized electrochemical immunoassays, were developed for rapid detection of nanomolar levels of p-nitrophenol (p-NP). The method exploits rapid reduction of p-NP to p-aminophenol (p-AP) by fast scan cyclic voltammetry at 500 V s(-1) at nanostructured carbon fiber microdisk electrodes (～7 μm dia.) fabricated from polyacrylonitrile (PAN) fibers. Large surfaces of the nanostructured microdisks facilitate the rapid reduction of p-NP to p-aminophenol (p-AP), as confirmed by the overlap with the analytical signals of the standards, which is then rapidly detected by fast scan cyclic voltammetry. A new 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris)-HAc buffer medium was developed in order to allow adaptation of this detection strategy to alkaline phosphatase (ALP)-based immunoassays. Tris-HAc is a stable medium, while the traditional Tris-HCl buffer medium produces large residual faradaic currents attributed to chloride oxidation. Addition of sodium acetate to a Tris-HAc buffer medium allows sensitivity enhancement by a factor of 2 to 0.85 nA μM(-1), similar to the best sensitivity reported at the nanostructured PAN carbon fiber microdisk sensors.     

Frequency-dependent electrical detection of protein binding events

Frequency-dependent electrochemical impedance spectroscopy has been used to characterize the changes in electrical response that accompany specific binding of a protein to its substrate, using the biotin-avidin system as a model. Our results show that avidin, at concentrations in the nanomolar range, can be detected electrically in a completely label-free manner under conditions of zero average current flow and without the use of any auxiliary redox agents. Impedance measurements performed on biotin-modified surfaces of gold, glassy carbon, and silicon were obtained over a wide frequency range, from 5 mHz to 1 MHz. On each biotin-modified surface, binding of avidin is most easily detected at low frequencies, <1 Hz. Electrical circuit modeling of the interface was used to relate the frequency-dependent electrical response to the physical structure of the interface before and after avidin binding. Electrical measurements were correlated with measurements of protein binding using fluorescently labeled avidin.     

Diffusion coefficients at rotated microelectrodes

The limiting current for several electrode reactions at a rotated microelectrode is observed to vary with the two-thirds power of the diffusion coefficient at infinite dilution in accordance with the theories of TACHI, EISBNBERG, LIN, and others. However, the uncertainties involved in estimating diffusion coefficients at given ionic strength require, that for accurate work, the limiting currents for different electrode reactions be compared on the basis of electrode sensivity, k_ion= i₁/C, and without regard to the role of diffusion in the transport process. The method of LAITINEN AND KOLTHOFF using an electrode reaction with only linear diffusion is recommended for measuring diffusion coefficients at given ionic strength under conditions similar to those extant in voltammetry.     

Insight of electrolyte-free voltammetry at microelectrodes

Distance (w)-changeable Pt wire electrodes and the resistivity of water when voltage over 1 V was applied to the two electrodes.

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Voltammetric determination of mifepristone at a DNA-modified carbon paste electrode

A new strategy for the preparation of a DNA-modified carbon paste electrode is developed. It is found that the anodic response of mifepristone is greatly enhanced at the dsDNA-modified carbon paste electrode comparing with that obtained at the bare electrode, while the response at a ssDNA-modified electrode is similar to bare electrode. So the dsDNA-modified electrode is employed as a sensitive biosensor for the detection of mifepristone. A linear dependence of the peak currents on the concentration is observed in the range 2.0 x 10(-7) approximately 2.0 x 10(6) mol/L, with a detection limit of 1.0 x 10(-7) mol/L. The relative standard deviation is 4.3% for six successive determinations of 1.0 x 10(6) mol/L mifepristone. The determination of mifepristone tablets is carried out and satisfactory results are obtained.     

Voltammetric study of the ion-exchange binding of non-electroactive metal cations to DNA-modified surfaces

We describe a simple electrochemical protocol for studying the ion-exchange binding of non-electroactive ions, specifically mono- and divalent metal cations of biological relevance (Mg(2+), Ca(2+), and K(+)), to DNA-modified surfaces. After incubation in a dilute solution of multiply charged transition metal complex (5.0 microM [Ru(NH(3))(6)]Cl(3)), gold electrodes modified with thiolate-DNA monolayers respond to the presence of these non-electroactive metal cations by producing significant changes in the cyclic voltammograms (i.e., decrease of the integrated charge and shift of formal potential) of the surface-bound redox complex ([Ru(NH(3))(6)](3+)). The divalent cations (particularly Mg(2+)) can be detected at very low concentrations (<10 microM), while the on-set value for K(+) is substantially higher (50 mM). The equilibrium binding constants for Mg(2+) and Ca(2+) to DNA-modified surfaces were calculated.     

Diffusion-controlled current at elliptically deformed microelectrodes

Diameters of invisible microelectrodes have been estimated from steady-state diffusion-controlled currents of a known concentration of redox species on the assumption of a disk form. However, geometry of the disk is often deformed by polishing the electrode surface obliquely against a polishing pad, by malleability of metal, and/or by distortion of metal wire. Then, the exposed surface is close to an ellipse with rough circumference. The diameter estimated from the steady-state current should be an average value among a major radius, a minor radius, and circumference length. In order to obtain a way of the average, we obtained here voltammetric steady-state currents at elliptic electrodes which were fabricated by polishing glass-coated platinum wire obliquely. Values of the diffusion-controlled currents at the elliptic electrode with smooth edge agreed with the theoretical values with 4% error. The steady-state current at a deformed electrode was approximately proportional to the square root of the area of the electrode rather than the length of the edge, as opposed to the conventional concept of the edge effect on the current. Even if electrode geometry is uncertain, the diameter evaluated from the steady-state current corresponds to the square root of the area.     

Diffusion-controlled current at elliptically deformed microelectrodes

Diameters of invisible microelectrodes have been estimated from steady-state diffusion-controlled currents of a known concentration of redox species on the assumption of a disk form. However, geometry of the disk is often deformed by polishing the electrode surface obliquely against a polishing pad, by malleability of metal, and/or by distortion of metal wire. Then, the exposed surface is close to an ellipse with rough circumference. The diameter estimated from the steady-state current should be an average value among a major radius, a minor radius, and circumference length. In order to obtain a way of the average, we obtained here voltammetric steady-state currents at elliptic electrodes which were fabricated by polishing glass-coated platinum wire obliquely. Values of the diffusion-controlled currents at the elliptic electrode with smooth edge agreed with the theoretical values with 4% error. The steady-state current at a deformed electrode was approximately proportional to the square root of the area of the electrode rather than the length of the edge, as opposed to the conventional concept of the edge effect on the current. Even if electrode geometry is uncertain, the diameter evaluated from the steady-state current corresponds to the square root of the area.

     

Label-free detection of protein binding with multisine SPR microchips

Label-free techniques such as surface plasmon resonance (SPR) have used a step-response excitation method to characterize the binding of two biochemical entities. A major drawback of the step response technique is its high susceptibility to thermal drifts and noise which directly determine the minimum detectable binding mass. In this paper we present a new frequency-domain method based on the use of multisine chemical excitation that is much less sensitive to these disturbances. The multisine method was implemented in a PDMS microfluidic chip using a dual channel, dual multiplug chemical signal generator connected to functionalized and reference SPR binding spots. Kinetic constants for the reaction are extracted from the characteristics of the sense spot response versus frequency. The feasibility of the technique was tested using a model system of Carbonic Anhydrase-II analyte and amino-benzenesulfonamide ligand. The experimental signal to noise ratio (SNR) for the multisine measurement is about 32 dB; 7 dB higher than that observed with the single step-response method, while the overall measurement time is twice as long as the step method.     

Nafion-CNT coated carbon-fiber microelectrodes for enhanced detection of adenosine

Adenosine is a neuromodulator that regulates neurotransmission. Adenosine can be monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes and ATP is a possible interferent in vivo because the electroactive moiety, adenine, is the same for both molecules. In this study, we investigated carbon-fiber microelectrodes coated with Nafion and carbon nanotubes (CNTs) to enhance the sensitivity of adenosine and decrease interference by ATP. Electrodes coated in 0.05 mg mL(-1) CNTs in Nafion had a 4.2 ± 0.2 fold increase in current for adenosine, twice as large as for Nafion alone. Nafion-CNT electrodes were 6 times more sensitive to adenosine than ATP. The Nafion-CNT coating did not slow the temporal response of the electrode. Comparing different purine bases shows that the presence of an amine group enhances sensitivity and that purines with carbonyl groups, such as guanine and hypoxanthine, do not have as great an enhancement after Nafion-CNT coating. The ribose group provides additional sensitivity enhancement for adenosine over adenine. The Nafion-CNT modified electrodes exhibited significantly more current for adenosine than ATP in brain slices. Therefore, Nafion-CNT modified electrodes are useful for sensitive, selective detection of adenosine in biological samples.     

DNA-modified electrodes. Part 6. A new method for the determination of binding constants and binding site sizes

A new method was developed to obtain the binding constant (K) and binding site size (n) for the interaction of DNA with other molecules using DNA-modified gold electrodes. This method is simple and microsample-consuming compared with conventional solution methods. A Q-basic program was designed to calculate K and n values. The results show that the K and n values obtained by the new method approach those obtained by conventional electrochemical methods, indicating the suitability of this method.     

High frequency faradaic rectification voltammetry at microelectrodes

An experimental setup for carrying out faradaic rectification measurements at micrometer-sized electrodes under potential control is described. A new method of data analysis is proposed that allows the determination of the standard rate constant and the electron-transfer coefficient of a fast charge transfer process without knowing the impedance of the microelectrode. This method is based on the frequency dependence of the shape of the faradaic rectification voltammograms (i.e., the average width of the peaks and the ratio of the peak heights) rather than on the magnitude of the faradaic rectification signal. The method was tested in the determination of heterogeneous electron transfer kinetics of Fe(CN)6(3-/4-) and Ru(NH3)6(2+/3+) in aqueous solutions on a platinum microelectrode (12.5 microm in radius) and ferrocene/ferrocinum redox couple in a dimethylformamide solution on a gold microelectrode (12.5 microm in radius).     

DNA-modified electrodes. Part 6. A new method for the determination of binding constants and binding site sizes

A new method was developed to obtain the binding constant (K) and binding site size (n) for the interaction of DNA with other molecules using DNA-modified gold electrodes. This method is simple and microsample-consuming compared with conventional solution methods. A Q-basic program was designed to calculate K and n values. The results show that the K and n values obtained by the new method approach those obtained by conventional electrochemical methods, indicating the suitability of this method. Received: 22 July 1998 / Revised: 24 November 1998 / Accepted: 27 November 1998     

Magnetic focusing of redox molecules at ferromagnetic microelectrodes

The electrochemical behavior of Fe and Ni disk-shaped microelectrodes (25 to 250 μm radius) in a uniform magnetic field (1 T) is reported. Magnetization of the ferromagnetic microelectrode generates a magnetic field gradient across the depletion layer that can be used to focus paramagnetic molecules toward the electrode surface. The magnetic force acting on the depletion layer is given by F_∇B=(χ/μ)B·∇B, where χ and μ are the volume magnetic susceptibility and magnetic permeability of the depletion layer, respectively, and B is the magnetic field. Magnetic field focusing of the nitrobenzene radical anion at Fe and Ni microelectrodes is demonstrated.     

A supercoiled DNA-modified mercury electrode-based biosensor for the detection of DNA strand cleaving agents

DNA-damaging agents in the environment represent a serious danger to human health. We use a supercoiled DNA-modified mercury electrode as a fast-response biosensor for the detection of DNA strand cleaving agents. The sensor is based on a strong difference between the a.c. voltammetric responses of covalently closed circular (supercoiled) and of open circular (nicked) plasmid DNA. We show that the sensor can detect hydroxyl radicals in laboratory-prepared solutions and in various natural and industrial water samples. The sensor is also capable of detecting unknown DNA-damaging agents in industrial waters.