Homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin employing Ru(bpy)(2)(dcbpy)NHS and carrier protein.

A highly sensitive homogeneous electrogenerated chemiluminescence (ECL) immunoassay for the determination of anti-digoxin antibody and digoxin hapten was developed employing Ru(bpy)(2)(dcbpy)NHS (bpy = 2,2'-bipyridyl; dcbpy = 2,2'-bipyridine-4,4'-dicarboxylic acid; NHS = N-hydroxysuccinimide ester) as an electrochemiluminescent label and bovine serum albumin (BSA) as a carrier protein. A digoxin hapten was indirectly heavily labelled with Ru(bpy)(2)(dcbpy)NHS through BSA to form Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate. The ECL intensity of the immunocomplex of the conjugate with anti-digoxin antibody markedly decreased when the immunoreaction between Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate and anti-digoxin antibody took place. Two formats, direct homogeneous immunoassay for anti-digoxin antibody and competitive immunoassay for digoxin, were developed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration in the range 7.6 x 10(-8)-7.6 x 10(-6) g/mL was determined by direct homogeneous format. Digoxin hapten was determined throughout the range 4.0 x 10(-10)-1.0 x 10(-7) g/mL with a detection limit of 1.0 x 10(-10) g/mL by competitive format. The relative standard derivation for 6.0 x 10(-9) g/mL was 4.3%. The method has been applied to assaying digoxin in control human serum.     

Determination of tetracycline, chlortetracycline and oxytetracycline by flow injection with inhibitory chemiluminescence detection using copper(II) as a probe ion.

This paper reports an indirect flow-injection (FI) method for the determination of the tetracycline drugs (TCs), tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC), using copper(II) as a probe ion. The method was based on the inhibition caused by these TCs to the copper(II)-catalysed chemiluminescence (CL) reaction between luminol and H(2)O(2). The CL reaction was induced on-line and injection of the sample produced negative peaks as a result of the copper(II) complexation or displacement by the analytes. The height of the peaks was proportional to the drug concentration in the sample. The choice of the catalyst ion, the concentration of luminol, H(2)O(2) and copper(II) are discussed. The linear range was 3.6 x 10(-8)-1.0 x 10(-5), 1.1 x 10(-7)-1.0 x 10(-5) and 1.9 x 10(-7)-1.0 x 10(-5) mol/L for TC, CTC and OTC, respectively. The detection limit was 5.0 x 10(-9) mol/L for TC, 1.0 x 10(-8) mol/L for CTC and 2.0 x 10(-8) mol/L for OTC (3sigma), respectively. The method was applied to the determination of TCs in pharmaceutical preparations and human urine with recoveries in the range 95-105%.     

Electrochemiluminescence determination of codeine or morphine with an organically modified silicate film immobilizing Ru(bpy)3(2+).

An ECL approach was developed for the determination of codeine or morphine based on tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+)) immobilized in organically modified silicates (ORMOSILs). Tetramethoxysilane (TMOS) and dimethyldimethoxysilane (DiMe-DiMOS) were selected as co-precursors for ORMOSILs, which were then immobilized on a surface of glassy carbon electrode (GCE) by a dip-coating process. Ru(bpy)(3)(2+) was immobilized in the ORMOSIL film via ion-association with poly(p-styrenesulphonate). The ORMOSIL-modified GCE presented good electrochemical and photochemical activities. In a flow system, the eluted codeine or morphine was oxidized on the modified GCE and reacted with immobilized Ru(bpy)(3)(2+) at a potential of +1.20 V (vs. Ag/AgCl). The modified electrode was used for the ECL determination of codeine or morphine and showed high sensitivity. The calibration curves were linear in the range 2 x 10(-8)-5 x 10(-5) mol/L for codeine and 1 x 10(-7)-3 x 10(-4) mol/L for morphine. The detection limit was 5 x 10(-9) mol/L for codeine and 3 x 10(-8) mol/L for morphine, at signal:noise ratio (S:N)=3. Both codeine and morphine showed reproducibility with RSD values <2.5% at 1.0 x 10(-6) mol/L. Furthermore, the modified electrode immobilized Ru(bpy)(3)(2+) was applied to the ECL determination of codeine or morphine in incitant samples.     

Determination of erythromycin in rat plasma with capillary electrophoresis-electrochemiluminescence detection of tris(2,2'-bipyridyl) ruthenium(II)

A fast and sensitive approach for determination of erythromycin in rat plasma was described. The method used capillary electrophoresis coupled with end-column electrochemiluminescence (ECL) detection of Ru(bpy)(3)(2+). The separation column used had an inner diameter of 75 microm. The running buffer was 15 mmol/L sodium phosphate (pH=7.5). The solution in the detection cell was 50 mmol/L sodium phosphate (pH=8.0) and 5 mmol/L Ru(bpy)(3)(2+). ECL intensity varied linearly with erythromycin concentration from 1.0 ng/mL to 10 microg/mL. The detection limit (S/N=3) was 0.35 ng/mL. The relative standard deviations, of ECL intensity and migration time for eight consecutive injections of 1.0 microg/mL erythromycin (n=8), were 1.3% and 1.8%, respectively. The method was successfully applied to erythromycin determination in rat plasma. The recovery ranged from 92.5 to 97.5%.     

Flow-injection electrogenerated chemiluminescence determination of fluoroquinolones based on its sensitizing effect.

A simple and sensitive flow-injection electrogenerated chemiluminescence (ECL) method for the determination of fluoroquinolones was developed. The method is based on the sensitizing effect of fluoroquinolones on the weak ECL signal of electrochemical oxidation of luminol on the surface of the platinum flake electrode in the medium of 0.1 mol/L Na2CO3-NaHCO3. At the optimum experimental conditions, the relative ECL intensity increased linearly with increasing fluoroquinolones concentration, in the ranges 1.0 x 10(-8)-2.0 x 10(-4) g/mL for norfloxacin, 5.0 x 10(-9)-6.0 x 10(-6) g/mL for oxfloxacin, 2.0 x 10(-8)-1.4 x 10(-5) g/mL for ciprofloxacin, 1.0 x 10(-8)-1.4 x 10(-5) g/mL for pefloxacin, and 1.0 x 10(-9)-1.0 x 10(-5) g/mL for enoxacin, with detection limits of 4.0 x 10(-9) g/mL, 2.0 x 10(-9) g/mL, 1.0 x 10(-8) g/mL, 8.0 x 10(-9) g/mL, and 8.0 x 10(-10) g/mL, respectively. The relative standard deviations were all less than 2.5% for the determination of 2.0 x 10(-6) g/mL fluoroquinolones (n = 11). The method was used to determine these medicines in pharmaceutical samples with satisfactory results.     

In-situ produced ascorbic acid as coreactant for an ultrasensitive solid-state tris(2,2'-bipyridyl) ruthenium(II) electrochemiluminescence aptasensor

Herein, an ultrasensitive solid-state tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL) aptasensor using in-situ produced ascorbic acid as coreactant was successfully constructed for detection of thrombin. Firstly, the composite of Ru(bpy)(3)(2+) and platinum nanoparticles (Ru-PtNPs) were immobilized onto Nafion coated glass carbon electrode, followed by successive adsorption of streptavidin-alkaine phosphatase conjugate (SA-ALP) and biotinylated anti-thrombin aptamer to successfully construct an ECL aptasensor for thrombin determination. In our design, Pt nanoparticles in Ru(bpy)(3)(2+)-Nafion film successfully inhibited the migration of Ru(bpy)(3)(2+) into the electrochemically hydrophobic region of Nafion and facilitated the electron transfer between Ru(bpy)(3)(2+) and electrode surface. Furthermore, ALP on the electrode surface could catalyze hydrolysis of ascorbic acid 2-phosphate to in-situ produce ascorbic acid, which co-reacted with Ru(bpy)(3)(2+) to obtain quite fast, stable and greatly amplified ECL signal. The experimental results indicated that the aptasensor exhibited good response for thrombin with excellent sensitivity, selectivity and stability. A linear range of 1 × 10(-15)-1 × 10(-8) M with an ultralow detection limit of 0.33 fM (S/N=3) was obtained. Thus, this procedure has great promise for detection of thrombin present at ultra-trace levels during early stage of diseases.     

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.     

Highly sensitive electrochemiluminescence determination of etamsylate using a low‐cost electrochemical flow‐through cell based on a tris(2, 2'‐bipyridyl)ruthenium(II)–Nafion‐modified carbon paste electrode

A simple and sensitive electrochemiluminescence (ECL) method for the determination of etamsylate has been developed by coupling an electrochemical flow‐through cell with a tris(2,2'‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)–Nafion‐modified carbon electrode. It is based on the oxidized Ru(bpy)₃²⁺ on the electrode surface reacting with etamsylate and producing an excellent ECL signal. Under optimized experimental conditions, the proposed method allows the measurement of etamsylate over the range of 8–1000 ng/mL with a correlation coefficient of r = 0.9997 (n = 7) and a limit of detection of 1.57 ng/mL (3σ), the relative standard deviation (RSD) for 1000 ng/mL etamsylate (n = 7) is 0.96%. The immobilized Ru(bpy)₃²⁺ carbon paste electrode shows good electrochemical and photochemical stability. This method is rapid, simple, sensitive and has good reproducibility. It has been successfully applied to the determination of the studied etamsylate in pharmaceutical preparations with satisfactory results. The possible ECL reaction mechanism has also been discussed. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrochemiluminescence detection of methamphetamine based on a Ru(bpy)32+‐doped silica nanoparticles/Nafion composite film modified electrode

An electrochemiluminescence (ECL) approach for methamphetamine determination was developed based on a glassy carbon electrode modified with a Ru(bpy)₃²⁺‐doped silica nanoparticles/Nafion composite film. The monodispersed nanoparticles, which were about 50 nm in size, were synthesized using the water‐in‐oil microemulsion method. The ECL results revealed that Ru(bpy)₃²⁺ doped in silica nanoparticles retained its original photo‐ and electrochemical properties. The ECL intensity was found to be proportional to methamphetamine concentration over the range from 1.0 × 10^?7 to 1.0 × 10^?5 mol L^?1, and the detection limit was found to be 2.6 × 10^?8 mol L^?1. The proposed ECL approach was used to analyze the methamphetamine content in drugs. Copyright © 2009 John Wiley & Sons, Ltd.     

Separation and determination of bupivacaine in plasma by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection.

A new, rapid, selective and sensitive method is described for determination of bupivacaine by capillary electrophoresis coupled with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)2+] electrochemiluminescence detection. The influence of parameters such as detection potential, Ru(bpy)(3)2+ concentration, buffer concentration and pH, injection time and separation voltage on separation efficiency and ECL peak intensity was systematically investigated. Under optimized conditions, the calibration curve was linear in the range 0.02-10 microg/mL. The RSD was 4.0% (n = 6). The detection limit was 3 ng/mL. The recoveries obtained were about 90%. This method was tested in the analysis of plasma samples taken from a rat after it had received bupivacaine injections.     

Flow injection chemiluminescent determination of tetracycline using a tris(2,2'-bipyridine)ruthenium(II)-cerium(IV) sulphate system.

A flow-injection chemiluminescence method for the determination of tetracycline was developed. The method is based on an enhancement by tetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine)ruthenium(II). In sulphuric acid medium, the chemiluminescence is generated by the continuous oxidation of tris(2,2'-bipyridine)ruthenium(II) by cerium (IV) sulphate. The light-emission intensity is greatly enhanced in the presence of tetracycline. Under the optimum conditions, the calibration curve is linear over the range 3.75 x 10(-8) g/mL-1.5 x 10(-5) g/mL for tetracycline with the linear equation: deltaINT = 205.898 x C - 20.442 (R2 = 0.9974). The detection limit is 3.27 x 10(-8) g/mL. The proposed method was also successfully used to determine tetracycline in pharmaceutical formulation (mean recovery of tetracycline, 100.7%).     

Determination of trimethylamine in fish by capillary electrophoresis with electrogenerated tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection.

A capillary electrophoresis with electrogenerated chemiluminescence (CE-ECL) method for the determination of trimethylamine (TMA) in fish was studied. In the presence of TMA, ECL from the reaction of analyte and in situ generated tris(2,2'-bipyridyl)ruthenium(III) [Ru(bpy)(3) (3+)] at electrode surface could be produced. The ECL detection was performed using a Pt working electrode biased at 1.23 V (vs. Ag/AgCl) potential in a 10 mmol/L sodium borate buffer solution, pH 9.2, containing 3 mmol/L Ru(bpy)(3) (2+). A linear calibration curve (correlation coefficient = 0.9996) was obtained in the range 8 x 10(-5)-4 x 10(-8) mol/L for TMA concentration. Recoveries obtained were in the range 98.78-101.46%. The method was successfully applied for the assay of TMA in fish, in combination with solid phase extraction (SPE) disks for sample clean-up and enrichment.     

A new sensitive flow-injection chemiluminescence method for the determination of H(2)-receptor antagonists.

Based on the chemiluminescence (CL) intensity generated from the potassium ferricyanide [K(3)Fe(CN)(6)]-rhodamine 6G system in sodium hydroxide (NaOH) medium, a new sensitive flow-injection chemiluminescence (FI-CL) method has been developed, validated and applied for the determination of three kinds of H(2)-receptor antagonists: cimetidine (CIMT), ranitidine (RANT) hydrochloride and famotidine (FAMT). Under the optimum conditions, the linear range for the determination was 1.0 x 10(-9)-7.0 x 10(-5) g/ml for CIMT, 1.0 x 10(-9)-5.0 x 10(-5) g/mL for RANT hydrochloride and 5.0 x 10(-9)-7.0 x 10(-5) g/mL for FAMT. During 11 repeated measurements of 1.0 x 10(-6) g/mL sample solutions, the relative standard deviations (RSDs) were all <5%. The detection limit was 8.56 x 10(-10) g/mL for CIMT, 8.69 x 10(-10) g/mL for RANT hydrochloride and 2.35 x 10(-9) g/mL for FAMT (S:N = 3). This method has been successfully implemented for the analysis of H(2)-receptor antagonists in pharmaceuticals.     

Protein analysis with terbium (III) and sodium dodecyl sulphonate by a second-order scattering technique.

This is the first report of terbium(III) as a probe of second-order scattering (SOS) for the determination of proteins in human serum at nanogram levels. A sensitive method has been developed using light scattering, based on the fact that the weak SOS of proteins can be enhanced in the presence of terbium(III) and sodium dodecyl sulphonate (SDS). With this method, 7.0 x 10(-9)-1.0 x 10(-5) g/mL human serum albumin (HSA) and 5.0 x 10(-9)-5.0 x 10(-6) g/mL gamma-globulin can be determined; the detection limits were 4.4 ng/mL for HSA and 3.1 ng/mL for gamma-globulin. The method has been applied to the detection of total proteins in human serum samples, and the results are consistent with those obtained by the Coomassie brilliant blue (CBB) G-250 assay.     

Capillary electrophoresis coupled with end‐column electrochemiluminescence for the determination of ephedrine in human urine,and a study of its interactions with three proteins

A tris(2,2‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10^–8–6.0 × 10^–6 g/mL. The detection limit was 4.5 × 10^–9 g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt‐C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt‐C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴ mol/L, 6.58 × 10³ mol/L and 1.59 × 10⁴ mol/L, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Fluorescence enhancement effect of the morin-Al3+ -sodium dodecyl benzene sulphonate-protein system and the determination of proteins.

The fluorescence intensity of the morin-Al(3+) complex was greatly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this, a new fluorimetric method for the determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence was in proportion to the concentration of proteins in the range 1.0 x 10(-8)-1.3 x 10(-5) g/mL for bovine serum albumin (BSA), 4.0 x 10(-8)-1.2 x 10(-5) g/mL for egg albumin (EA) and 5.0 x 10(-8)-1.2 x 10(-5) g/mL for human serum albumin (HSA). Their detection limits (S:N = 3) were 5.0 x 10(-9), 1.8 x 10(-8) and 1.6 x 10(-8) g/mL, respectively. The interaction mechanism was also studied.     

An assay for inorganic mercury(II) based on its post-catalytic enhancement effect on the potassium permanganate-luminol system.

A strong chemiluminescence (CL) response is observed when potassium permanganate solution is injected into basic luminol solution. When the CL reaction terminates, subsequent injection of Hg2+ solution into the reaction mixture results in a new CL reaction. Based on the post-catalytic enhancement effect of Hg2+ on the potassium permanganate-luminol system in basic media, a fast and simple CL-coupled flow injection analysis for the determination of Hg2+ was developed. In optimum conditions, CL intensity is proportional to Hg2+ concentration over the range 1.0 x 10(-8)-1.0 x 10(-5) g/mL, with a detection limit of 2.0 x 10(-9) g/mL. The relative standard deviation (RSD) is 3.6% for 1.0 x 10(-7) g/mL Hg2+ (n = 11). After pretreatment with sulphydryl cotton fibre, environmental water samples were analysed by the proposed method for total mercury determination with satisfactory results. The results were in good agreement with those given by hydride generation-cold vapour atomic absorption spectrometry (HG-CVAAS).     

Chemiluminescence of synephrine based on the cerium(IV)-rhodamine B system.

A new chemiluminescence (CL) method is described for the determination of synephrine. It is based on the reaction between synephrine and Ce(IV) in a nitric acid medium and measurement of the CL intensity produced by rhodamine B used as a luminophore, similar to luminol or lucigenin in basic media, instead of as a sensitizer. In the optimum conditions, the increase of CL intensity was correlated with synephrine concentration over the range 5.0 x 10(-9)-1.0 x 10(-6) g/mL with a detection limit of 1.0 x 10(-9) g/mL. The relative standard deviation (RSD) was 2.9% for 1.0 x 10(-7) g/mL synephrine (n = 11). The method was applied to the determination of a drug in herbal products, citrus fruit and biological samples, with satisfactory results. The results given by the proposed method are in good agreement with those given by HPLC-UV and UV spectrophotometry.     

Electrochemiluminescence of CdSe quantum dots for immunosensing of human prealbumin

We describe a non-labeled electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) for the detection of human prealbumin (PAB, antigen). The immunosensor was fabricated by layer by layer coupled with nanoparticle-amplification techniques. After two gold nanoparticle layers were self-assembled onto the gold electrode surface through cysteamine, anti-PAB (antibody) were conjugated with -COOH groups of both the CdSe QDs and cysteine, which were linked to the gold nanoparticle-modified electrode. The principle of ECL detection was that the immunocomplex inhibited the ECL reaction between CdSe QDs and K(2)S(2)O(8), which resulted in the decrease of ECL intensity. On the one hand, the immunocomplex increased the steric hindrance. On the other hand, the immunocomplex maybe inhibit the transfer of K(2)S(2)O(8) to the surface of the CdSe QD-electrode. The PAB concentration was determined in the range of 5.0 x 10(-10) to 1.0 x 10(-6) g mL(-1), and the detection limit was 1.0 x 10(-11) g mL(-1). The developed CdSe QD-based ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which could be applied in more bioanalytical systems.     

Determination of ketotifen fumarate by capillary electrophoresis with tris(2,2′‐bipyridyl) ruthenium(II) electrochemiluminescence detection

On the basis of an europium (III)‐doped Prussian blue analog film modifying platinum electrode as the working electrode, a Ru(bpy)₃²⁺‐based electrochemiluminescence (ECL) assay coupled with capillary electrophoresis has been first established for the determination of ketotifen fumarate (KTF). Analytes were injected onto a separation capillary of 50 cm length (50 μm i.d., 360 μm o.d.) by electrokinetic injection for 10 s at 10 kV. Parameters related to the separation and detection were discussed and optimized. It was proved that 15 mm phosphate buffer at pH 8.0 could achieve the most favorable resolution, and the highest sensitivity of detection was obtained using the detection potential at 1.25 V and 5 mm Ru(bpy)₃²⁺ in 100 mm phosphate buffer at pH 8.0 in the detection reservoir. Under the optimized conditions, the ECL intensity was in proportion to KTF concentration over the range from 3.0 × 10^?8 to 5.0 × 10^?6 g mL^?1 with a detection limit of 2.1 × 10^?8 g mL^?1 (3σ). The relative standard deviations of the ECL intensity and the migration time were 0.95 and 0.26%, respectively. The developed method was successfully applied to determine KTF contents in pharmaceuticals and human urine with recoveries between 99.5 and 107.0%. Copyright © 2010 John Wiley & Sons, Ltd.