Electrochemical aptasensor for tetracycline detection

An electrochemical aptasensor was developed for the detection of tetracycline using ssDNA aptamer that selectively binds to tetracycline as recognition element. The aptamer was highly selective for tetracycline which distinguishes minor structural changes on other tetracycline derivatives. The biotinylated ssDNA aptamer was immobilized on a streptavidin-modified screen-printed gold electrode, and the binding of tetracycline to aptamer was analyzed by cyclic voltammetry and square wave voltammetry. Our results showed that the minimum detection limit of this sensor was 10 nM to micromolar range. The aptasensor showed high selectivity for tetracycline over the other structurally related tetracycline derivatives (oxytetracycline and doxycycline) in a mixture. The aptasensor developed in this study can potentially be used for detection of tetracycline in pharmaceutical preparations, contaminated food products, and drinking water.     

Chemiluminometric β-galactosidase detection as a basis for a tetracycline screening test in milk

The observation that tetracyclines inhibit the biosynthesis of β-galactosidase in Escherichia coli to a greater extent than other antibacterials was exploited for the development of a chemiluminometric method to detect residues of this class of antibiotics in milk. The procedure involves the incubation of a milk sample with 10⁷ CFU/ml of an E. coli strain in the presence of IPTG, an inducer of β-galactosidase, and of EGTA, a chelator of calcium ions, followed by a 1000-fold dilution and measurement of the residual enzymatic activity using the chemiluminogenic substrate Galacton. Chemiluminometry proved an essential tool in this procedure because the extensive dilution of the sample, necessary to avoid light quenching by turbidity, results in an insufficient level of β-galactosidase activity to be measurable by colorimetry. This tetracycline galactosidase (TG) test has been validated and compared in the field to existing commercial screening assays for antibiotics. Its detection limit for tetracyclines ranges between 40 and 65 μg/kg, which is below the European maximum residue limit (MRL = 100 μg/kg) in milk. No other antibacterials, at concentrations commonly expected in milk, were found to interfere with the TG test. Strategies to avoid false positive reactions possibly arising from very high somatic cell counts will be reported elsewhere. © 1998 John Wiley & Sons, Ltd.     

Further characterization of tunicate and tunichrome electrochemiluminescence

Electrophoresis, size exclusion chromatography, fluorescence, and electrochemiluminescence (ECL) data obtained from the cell-rich perivisceral fluid (‘blood’) of the tunicate, Molgula occidentalis, suggests that a yellowish protein or protein subunit of <6.5 kDa is probably responsible for the low level intrinsic ECL reported previously. Variable potential ECL scans and spectrofluorometric analyses directly indicated that chlorophylls from ingested phytoplankton were not significant contaminants of M. occidentalis blood samples. Chlorophylls were also examined indirectly from monocultures of dinoflagellate and diatom species to determine their ECL levels. While ECL was observed in these cultures, high concentrations (10⁶ organisms/mL) of dinoflagellates or diatoms were needed to produce ECL levels comparable to those observed for M. occidentalis blood. Additional work with two synthetic tunichrome isomers indicated 10-fold increases in ECL when the ‘2,3,4-tunichrome’ was reacted with Tl⁺ in 1:1 and 1:2 metal ion to ligand molar ratios. The ‘3,4,5-tunichrome’ isomer exhibited a titration curve with Hg²⁺ suggestive of the existence of at least two Hg²⁺ binding sites and a 9- to 10-fold increase in ECL output. © 1997 John Wiley & Sons, Ltd.     

The quenching of electrochemiluminescence upon oligonucleotide hybridization.

Many genomic assays rely on a distance-dependent interaction between luminescent labels, such as luminescence quenching or resonance energy transfer. We studied the interaction between electrochemically excited Ru(bpy)(3) (2+) and Cy5 in a hybridization assay on a chip. The 3' end of an oligonucleotide was labelled with Ru(bpy)(3) (2+) and the 5' end of a complementary strand with Cy5. Upon the hybridization, the electrochemiluminescence (ECL) of Ru(bpy)(3) (2+) was efficiently quenched by Cy5 with a sensitivity down to 30 nmol/L of the Cy5-labelled complementary strand. The quenching efficiency is calculated to be 78%. A similar phenomenon was observed in a comparative study using laser-excitation of Ru(bpy)(3) (2+). The hybridization with the non-labelled complementary or labelled non-complementary strand did not change the intensity of the ECL signal. Resonance energy transfer, electron transfer and static quenching mechanisms are discussed. Our results suggest that static quenching and/or electron transfer are the most likely quenching mechanisms.     

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.     

Enhanced electrochemiluminescence of Ru(bpy)32+ by flavone compounds

Flavones such as morin, rutin, quercitrin, quercetin and wogonin were found to be able to strongly enhance the electrochemiluminescence (ECL) of the Ru(bpy)₃²⁺ system. Based on this, a novel ECL method with good stability and reproducibility could be developed for determination of flavones. Under the optimum conditions, the enhanced ECL intensity was linear with the flavones concentration in a wide range. The detection limits (defined as S:N = 3) for morin, rutin, quercitrin, quercetin and wogonin were 3.2 × 10^–7 mol/L, 4.3 × 10^–7, 1.8 × 10^–7, 8.0 × 10^–8 and 1.0 × 10^–7 mol/L, respectively. In addition, the possible mechanism for the Ru(bpy)₃²⁺ ECL system in the presence of flavones is also discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

A label-free electrochemiluminescence aptasensor for thrombin based on novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles

In the work, a label-free electrochemiluminescence (ECL) aptasensor for the sensitive and selective detection of thrombin was constructed based on target-induced direct ECL signal change by virtue of a novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles (luminol-AuNPs). It is the first label-free ECL biosensor based on luminol and its analogs functionalized AuNPs. Streptavidin AuNPs coated with biotinylated DNA capture probe 1 (AuNPs-probe 1) were firstly assembled onto an gold electrode through 1,3-propanedithiol. Then luminol-AuNPs co-loaded with thiolated DNA capture probe 2 and thiolated thrombin binding aptamer (TBA) (luminol-AuNPs-probe 2/TBA) were assembled onto AuNPs-probe 1 modified electrode through the hybridization between capture probes 1 and 2. The luminol-AuNPs-probe 2/TBA acted as both molecule recognition probe and sensing interface. An Au/AuNPs/ds-DNA/luminol-AuNPs/TBA multilayer architecture was obtained. In the presence of target thrombin, TBA on the luminol-AuNPs could capture the thrombin onto the electrode surface, which produced a barrier for electro-transfer and influenced the electro-oxidation reaction of luminol, leading to a decrease in ECL intensity. The change of ECL intensity indirectly reflected the concentration of thrombin. Thus, the approach showed a high sensitivity and a wider linearity for the detection of thrombin in the range of 0.005-50nM with a detection limit of 1.7pM. This work reveals that luminol-AuNPs are ideal platform for label-free ECL bioassays.     

Preliminary electrochemiluminescence studies of metal ion–bacterial diazoluminomelanin (DALM) interactions

Electrochemiluminescence (ECL) studies of the chemiluminescent (CL) polymer diazoluminomelanin (DALM) biosynthesized in nitrate reductase transfected Escherichia coli JM109 bacteria revealed noteworthy anodic ECL and even more intense cathodic ECL. Bacterial DALM (BD) ECL was also assessed in the presence of 100 ppm of 33 different metal and non-metal ions which revealed specific anodic, but not cathodic, enhancements of BD ECL with Ag⁺, Hg²⁺ and Ru³⁺. The precursors and intermediate polymers which comprise DALM, such as luminol, 3-amino-L -tyrosine (3-AT), aminomelanin (AM) and diazomelanin (DM) were screened for ECL enhancement against the same set of elemental ions. Significant anodic ECL enhancements were observed for luminol with Hg²⁺ in the presence of tripropylamine (TPA), but not for any other DALM component in combination with other elemental ions, either anodically or cathodically. Comparison of BD with luminol in the presence and absence of TPA and Hg²⁺ revealed very different ECL activity patterns and suggested different mechanisms for BD and luminol ECL. © 1998 John Wiley & Sons, Ltd.     

Preliminary studies of the enhanced electrochemiluminescence of 2,3-diaminonaphthalene in the presence of specific metal ions

Electrochemiluminescence (ECL) of 200 ppm 2,3-diaminonaphthalene (2,3-DAN) was studied alone and in conjunction with 100 ppm of 34 different metal and non-metal ions and revealed three relatively intense ECL responses from interactions of 2,3-DAN with Au⁺, Fe⁺³ and V⁺⁵. ECL responses from Cr⁺⁶ or Ru⁺³ with 2,3-DAN were less intense, but noteworthy, as was the coloured fluorescent product of the non-metal ion Se⁺⁴ interaction with 2,3-DAN. Several intense 2,3-DAN–metal ion ECL reactions were studied in greater detail and revealed various titration curves with ionic detection limits in the low ppm range, using a fixed level (200 ppm) of 2,3-DAN. © 1998 John Wiley & Sons, Ltd.     

Multi-channel electrochemiluminescence of luminol at a copper electrode.

Multi-channel electrochemiluminescence (ECL) of luminol at a copper electrode has been studied under conventional cyclic voltammetric (CV) conditions. Compared with the ECL of luminol at other electrodes, three ECL peaks were observed at 0.30, -0.24 and -0.65 V (vs. SCE), respectively, which was also imaged by a CCD camera. The effects of potential scan direction, anodic reverse potential, the presence of N2 and O2 of the solution, the pH of the solution, the NaNO3 concentration and the potential scan rate were examined. The effect of n-alkanethiol self-assembled monolayers on copper electrodes and 20 L-amino acids, dopamine, adrenaline and noradrenaline on the ECL of luminol were also investigated. The emission spectra of various ECL peaks at different potentials demonstrated that all ECL peaks were related to the luminol reaction. The results show that the oxygen dissolved in solution and copper oxide covered on the surface of the electrode play an important role in the luminol ECL process at a copper electrode. It has been proposed that three ECL channels of luminol at a copper electrode resulted from the reactions of luminol or luminol radical electrooxidized by luminol with various electrogenerated oxygen-containing species, such as O2, OOH- and copper oxides at different potentials.     

Cathodic electrochemiluminescence of the peroxydisulphate–ciprofloxacin system and its analytical applications

The cathodic electrochemiluminescence (ECL) of peroxydisulphate (S₂O₈^2?)–ciprofloxacin (CPF) system at a wax‐impregnated graphite electrode was studied. When CPF was absent, S₂O₈^2? was electrochemically reduced to sulphate free radical (SO₄^??), and dissolved oxygen absorbed on the electrode surface was reduced to protonated superoxide anion radical (HO₂^?). The HO₂^? was oxidized by SO₄^?? to produce molecular oxygen in both singlet and triplet states. Some of the singlet molecular oxygen (¹O₂) further combined through collision to be an energy‐rich precursor singlet molecular oxygen pair (¹O₂)₂. A weak ECL was produced when ¹O₂ or (¹O₂)₂ was converted to ground‐state molecular oxygen (³O₂). When CPF was present, a stronger ECL was produced, which originated from two emitting species. The main emitting species was excited state CPF (CPF*), which was produced by accepting energy from (¹O₂)₂. The other emitting species was excited singlet molecular oxygen pair [(¹O₂)₂*], which originated from the chemical oxidation of CPF by SO₄^?? and dissolved oxygen. Based on the stronger ECL phenomenon, an ECL method for the determination of either S₂O₈^2? or CPF was proposed. The proposed ECL method has been applied to the determination of CPF in pharmaceutical preparations. Copyright © 2011 John Wiley & Sons, Ltd.     

Capillary electrophoresis coupled with electrochemiluminescence for determination of atomoxetine hydrochloride and the study on its interactions with three proteins

A simple, rapid and sensitive method for the determination of atomoxetine hydrochloride (AH) by capillary electrophoresis with electrochemiluminescence detection (CE‐ECL) using tris(2,2′‐bipyridyl) ruthenium (II) was developed. Under optimized conditions, the determinations of AH in capsules and rat plasmas and the study on its interactions with three plasma proteins, including bovine serum albumin, cytochrome c and myoglobin were performed successfully. Relative to some previous studies, in this paper the methodologies for the determination of AH in aqueous solution and spiked plasma systems were established, respectively. By comparing the difference between the two work curves of two systems, the matrix effect in plasma samples on the determination of AH by the CE‐ECL method was discussed in detail. The results indicated that the effect of the matrix in plasma samples should not be ignored even if no obvious interference was found in the electropherograms and the establishment of method validation in complex samples by the CE‐ECL method was necessary. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of Cryptosporidium parvum oocyst recovery efficiencies from various filtration cartridges by electrochemiluminescence assays

AIMS: To evaluate four types of filtration cartridges for their capacities, efficiency for capture and release of Cryptosporidium parvum oocysts for detection. METHODS AND RESULTS: Filtration cartridges included in this evaluation were IDEXX Filta-Max, Gelman Envirochek HV, Corning CrypTest, and Filterite Sigma+. Various dosages of C. parvum oocysts were spiked into water samples with a wide range of turbidity (10-50 NTU). Electrochemiluminescence assays were employed to enumerate viable or total number of C. parvum oocysts in these eluates. Among the cartridges tested, Filta-Max consistently showed higher oocyst recovery efficiency, especially with large volume, highly turbid water samples. CONCLUSIONS: Filta-Max filter is the best performer because of its higher oocyst recovery efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall sensitivities of various C. parvum oocyst detection assays in water samples can be improved if highly efficient oocyst recovery filtration cartridges such as Filta-Max are incorporated in sample preparation.     

An interdigital array microelectrode aptasensor based on multi-walled carbon nanotubes for detection of tetracycline

Bioprocess and Biosystems Engineering - In this study an impedance aptasensor was designed for sensitive, selective, and fast detection of tetracycline (TET) based on an interdigital array...     

Indirect electrochemiluminescence detection of lysine and histidine separated by capillary electrophoresis based on charge displacement

Indirect electrochemiluminescence (ECL) detection was applied for the analysis of lysine (Lys) and histidine (His) separated by capillary electrophoresis (CE). With the most effective electrophoretic buffer system, which contained 15 mM phosphate buffer (pH = 5.8) and 0.5 mM Tripropylamine (TPA), fast separation of the two basic amino acids could be performed within 7 min. The linear ranges were 10–35 μM, 35–150 μM for Lys; and 5–35 μM, 35–150 μM for His. The detection limits (S/N = 3) were 0.3 μM for Lys and 1.0 μM for His, respectively. The proposed method was also successfully used for the determination of Lys in the oral pharmaceutical formulations. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of arecoline in areca nut based on field amplification in capillary electrophoresis coupled with electrochemiluminescence detection

A sensitive capillary electrophoresis–electrochemiluminescence (CE–ECL) assay with an ionic liquid (IL) was developed for the determination of arecoline in areca nut. The IL, 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMImBF₄), was an effective additive improved not only the separation selectivity but also the detection sensitivity of the analyte. BMImBF₄ in the separation electrolyte made the resistance of the separation buffer much lower than that of the sample solution, which resulted in an enhanced field amplified electrokinetic injection CE. ECL intensity of arecoline is about two times higher than that of the analyte with phosphate–IL buffer system. Resolution between arecoline and other unknown compounds in real samples was improved. Under the optimized conditions (ECL detection at 1.2 V, 16 kV separation voltage, 20 mmol/L phosphate with 10 mmol/L BMImBF₄ buffer at pH 7.50, 5 mmol/L Ru(bpy)₃²⁺ and 50 mmol/L phosphate buffer in the detection reservoir), a detection limit of 5 × 10^–9 mol/L for arecoline was obtained. Relative standard deviations of the ECL intensity and the migration time were 4.51% and 0.72% for arecoline. This method was successfully applied to determination of the amount of arecoline in areca nut within 450 s. Copyright © 2012 John Wiley & Sons, Ltd.     

Flow injection analysis of thiamazole based on strong Ru(bpy)32+ co‐reactant electrochemiluminescence

Based on the strong electrochemiluminescence (ECL) reaction between thiamazole and tris(2,2′‐bipyridine)ruthenium(II) (Ru(bpy)₃²⁺), a sensitive, simple and rapid flow injection analysis method for the determination of thiamazole was developed. When a Pt working electrode was maintained at a potential of +1.50 V (vs Ag/AgCl) in pH 12.0 H₃PO₄–NaOH solution containing thiamazole and Ru(bpy)₃²⁺ at a flow rate of 1.0 mL/min, a linear range of 2.0 × 10⁻⁷–1.0 × 10⁻⁴ mol/L with a detection limit of 5.0 × 10⁻⁸ mol/L was obtained for the detection of thiamazole. The method showed good reproducibility with a relative standard deviation (RSD) of 0.75%. The method has been successfully applied to the determination of thiamazole in spiked animal feeds. In addition, a co‐reactant ECL mechanism was proposed for the thiamazole–Ru(bpy)₃²⁺ system. Copyright © 2014 John Wiley & Sons, Ltd.     

Sensitive determination of carbidopa through the electrochemiluminescence of luminol at graphene‐modified electrodes

Using the concept of electrogenerated chemiluminescence (ECL), a sensitive analytical method for the determination of carbidopa is described. Electro‐oxidation of carbidopa on the surface of a graphene oxide (GO)‐modified gold electrode (GE) leads to enhancement of the weak emission of oxidized luminol. Under optimum experimental conditions, the ECL signal increases linearly with increasing carbidopa concentrations over a range of 1.0 × 10^‐9–1.7 × 10^‐7 M, with a detection limit of 7.4 × 10^‐10 M. The proposed ECL method was successfully used for the determination of carbidopa in urine samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Generation of free radicals and electrochemiluminescence from simple aromatic molecules in aqueous solutions.

Oxide-covered aluminium electrodes were used to demonstrate that aromatic compounds, such as the simple derivatives of benzene, can be electrochemically excited at cathodically pulse-polarized conductor/insulator/electrolyte (C/I/E) tunnel junction electrodes (e.g. oxide-covered aluminium electrodes). The primary cathodic process at these electrodes was a tunnel emission of hot electrons into an aqueous electrolyte solution. Fluorescence (FL) and electrochemiluminescence (ECL) spectra were compared and the dependence of the electrochemiluminescence on the concentrations of benzene, toluene, phenol, p-cresol and aniline were measured and detailed mechanisms for the present electrochemiluminescence are proposed.     

Determination of metformin hydrochloride using precolumn derivatization with acetaldehyde and capillary electrophoresis coupled with electrochemiluminescence

A novel method was developed using capillary electrophoresis (CE) coupled with tris(2,2′‐bipyridyl)ruthenium(II) electrogenerated chemiluminescence (ECL) for highly sensitive detection of metformin hydrochloride (MH) derivatizatized with acetaldehyde. The precolumn derivatization of MH with acetaldehyde was performed in phosphate buffer solution (0.3 mol/L, pH 7.5) at room temperature for 120 min. The effects of acetaldehyde concentration, buffer pH, electrokinetic voltage and injection time were investigated. Under optimized detection conditions, the MH ECL detection sensitivity was more than 120 times that without derivatization. The linear concentration range for MH was 0.001–15.00 μg/mL (with a correlation coefficient of 0.9992). The detection limit was 0.31 ng/mL with a signal:noise ratio of 3. The recoveries of MH in human urine were in the range 98.50–99.72%. Copyright © 2011 John Wiley & Sons, Ltd.