Selective spectrofluorimetric determination of glutathione in clinical and biological samples using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene

This work reports the development of a selective, sensitive and rapid spectrofluorimetric method for the determination of reduced glutathione (GSH) in the presence of relatively high levels of cysteine (Cys) in clinical and biological samples using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M). The fluorescence from TMPAB-o-M is strongly quenched by its maleimide moiety, but after reaction with thiol, the fluorescence is restored with a 350-fold intensity increase (fluorescence quantum yield from 0.002 to 0.73). In H₃Cit-Na₂HPO₄ buffer (pH 7.40), the derivatization is completed in just 5 min under 37 °C. The linear range is 0.005-0.2 μmol L⁻¹, with detection limit of 1.1 × 10⁻¹⁰ mol L⁻¹ (signal-to-noise ratio = 3). Almost all amino acids, including Cys, impose no interference even if present at relatively high concentrations (amino acids:GSH = 100:1, Cys:GSH = 1:1, molar ratio, C_GSH = 3 × 10⁻⁷ mol L⁻¹). The sample can be used directly without further treatment after the protein is removed. The developed method is precise with a relative standard deviation (R.S.D.) lower than 5.0% (n = 6) and has been applied to the determination of GSH in human blood and pig’s liver with recoveries between 94.4 and 105.6%.     

Highly sensitive and selective detection of biothiols using graphene oxide-based “molecular beacon”-like fluorescent probe

A fluorometric method for quantity analysis of biothiols was developed using a graphene oxide (GO)-based “molecular beacon”-like probe, which consisted of FITC labeled thymine (T)-rich single-stranded DNA (ssDNA), GO and Hg²⁺ ions. The labeled ssDNA containing T–T mismatches would self-hybridize to duplex in the presence of Hg²⁺, which can avoid its adsorption on GO and the fluorescence of this GO-based probe was recovered. The fluorescence of the probe quenched after the addition of biothiols such as glutathione (GSH) and cysteine (Cys) owing to thiol groups can selectively competitive ligation of Hg²⁺ ions with T–T mismatches. In the present work, the GO-based probe was used for the determination of GSH and Cys. Under the optimal conditions, a linear correlation was established between fluorescence intensity ratio I₀/I and the concentration of GSH in the range of 2.0 × 10⁻⁹–5.0 × 10⁻⁷ mol L⁻¹ with a detection limit of 1.0 × 10⁻⁹ mol L⁻¹. The linear range for Cys is from 5.0 × 10⁻⁹ to 4.5 × 10⁻⁷ mol L⁻¹ with a detection limit of 2.0 × 10⁻⁹ mol L⁻¹. The proposed method was applied to the determination of GSH in human serum and cell extract samples with satisfactory results.     

Determination of adrenaline by using inhibited Ru(bpy)3 electrochemiluminescence

Adrenaline was found to inhibit strongly the electrochemiluminescence (ECL) from the Ru(bpy)₃²⁺/tripropylamine system when a working Pt electrode was maintained at 1.05 V (versus Ag/AgCl) in pH 8.0 phosphate buffer. On this basis, a flow injection (FI) procedure with inhibited electrochemiluminescence detection has been developed for determination of adrenaline. The method exhibited a good reproducibility, sensitivity, and stability with a detection limit (signal-to-noise ratio = 3) of 7.0×10⁻⁹ mol l⁻¹ and dynamic concentration range of 2×10⁻⁸ to 1×10⁻⁴ mol l⁻¹. The relative standard deviation was 2.2% for 1.0×10⁻⁶ mol l⁻¹ adrenaline (n=11). The method was successfully applied to the determination of adrenaline in pharmaceutical samples. Moreover, ECL emission spectra, UV-Vis absorption spectra and cyclic voltammograms of Ru(bpy)₃²⁺/tripropylamine/adrenaline were studied. The inhibition mechanism has been proposed as the interaction of electrogenerated Ru(bpy)₃^2+* and the o-benzoquinone derivatives, adrenochrome and adrenalinequinone, at the electrode surface.     

Simultaneous determination of N-acetyl-p-aminophenol and p-aminophenol with poly(3,4-ethylenedioxythiophene) modified glassy carbon electrode

A sensitive and selective method was developed for the determination of N-acetyl-p-aminophenol (APAP) and p-aminophenol (PAP) using poly(3,4-ethylenedioxythiophene) (PEDOT)-modified glassy carbon electrode (GCE). Cyclic voltammetry and differential pulse voltammetry were used to investigate the electrochemical reaction of APAP and PAP at the modified electrode. Both APAP and PAP showed quasireversible redox reactions with formal potentials of 367 mV and 101 mV (vs. Ag/AgCl), respectively, in phosphate buffer solution of pH 7.0. The significant peak potential difference (266 mV) between APAP and PAP enabled the simultaneous determination both species based on differential pulse voltammetry. The voltammetric responses gave linear ranges of 1.0 × 10⁻⁶-1.0 × 10⁻⁴ mol L⁻¹ and 4.0 × 10⁻⁶-3.2 × 10⁻⁴ mol L⁻¹, with detection limits of 4.0 × 10⁻⁷ mol L⁻¹ and 1.2 × 10⁻⁶ mol L⁻¹ for APAP and PAP, respectively. The method was successfully applied for the determination of APAP and PAP in pharmaceutical formulations and biological samples.     

Localized surface plasmon resonance sensing detection of glucose in the serum samples of diabetes sufferers based on the redox reaction of chlorauric acid

In this paper, the formation of gold nanoparticles (Au NPs) as a result of the thermo-active redox reaction of chlorauric acid (HAuCl₄) and glucose in alkaline medium was identified by measuring the plasmon resonance absorption, localized surface plasmon resonance (LSPR), and transmission electron microscopy (TEM) images, for the formation of Au NPs displays characteristic plasmon resonance absorption bands and corresponding LSPR signals. It was found that the resulted LSPR signals could be easily detected with a common spectrofluorometer. With increasing glucose concentration, the LSPR intensity displays linear response with the glucose content over the range from 2.0 to 250.0 μmol l⁻¹. Thus, a novel assay of glucose was established with the limits of determination (3σ) being 0.21 μmol l⁻¹, and the detection of glucose could be made easily in the serum samples of diabetes sufferers. Mechanism investigations showed that the activation energy and molar ratio of the reaction were 34.8 kJ mol⁻¹ and 3:2, respectively.     

Ultrasensitive and selective determination of trace amounts of nitrite ion with a novel fluorescence probe mono[6-N(2-carboxy-phenyl)]-β-cyclodextrin

A novel fluorescence probe, mono[6-N(2-carboxy-phenyl)]-β-cyclodextrin (OACCD), has been developed for the determination of trace nitrite, In dilute HCl medium, the fluorescence intensity of the newly synthesized fluorescence probe OACCD was quenched in presence of trace nitrite at room temperature. Based on this, a simple, sensitive, and selective method for rapid determination of nitrite was described. Furthermore, common ions do not interfere the determination of trace amounts of nitrite. The fluorescence quenching intensity was linear over a nitrite concentration of 0.02-1.7 μmol l⁻¹ with a detection limit of 0.2 nmol l⁻¹ (S/N = 3). The method was applied to the determination of nitrite in different water samples, soil samples, and food samples with satisfactory results.     

Sensitized extraction spectrophotometric determination of Hg(II) with dithizone after its flotation as ion-associate using iodide and ferroin

This paper describes a simple and highly selective method for the separation, preconcentration and spectrophotometric determination of extremely low concentration of mercury. The method is based on the flotation of an ion-associate of HgI₄²⁻ and ferroin between aqueous and n-heptane interface at pH 5. The ion-associate was then separated and treated with ammonia and dithizone solutions to extract only the mercury chelate with CH₂Cl₂. The measurement is feasible when the volume of the water sample containing Hg(II) was varied over 50-800 ml. Beer's law was obeyed over the concentration range of 8 × 10⁻⁹ to 1.6 × 10⁻⁷ mol l⁻¹ with an apparent molar absorptivity of 6.53 × 10⁶ l mol⁻¹ cm⁻¹ for a 500 ml aliquot of the water sample. The detection limit (n = 7) was 5.0 × 10⁻¹⁰ mol l⁻¹ and the R.S.D. (n = 5) for 8.0 × 10⁻⁷ mol l⁻¹ of Hg(II) was 3.7%. A notable advantage of the method is that the determination of Hg(II) is free from the interference of almost all cations and anions found in the environmental and waste water samples. The determination of Hg(II) in tap, synthetic sea water and human hair samples was carried out by the present method and cold vapor atomic absorption spectrometry (CV-AAS). The results were satisfactorily comparable so that the applicability of the proposed method was confirmed to the real samples.     

Flow-injection chemiluminescence determination of ascorbic acid by use of the cerium(IV)-Rhodamine B system

A highly sensitive flow-injection (FI) method with chemiluminescence (CL) detection is used for the determination of l-ascorbic acid. The method is based on the CL reaction of Rhodamine B with cerium(IV) in sulfuric acid media. l-Ascorbic acid is suggested to be a catalyst utilized in the energy-transferred excitation process. The proposed procedure allows quantitation of l-ascorbic acid in the range 3.8×10⁻¹³ to 1.0×10⁻¹⁰ mol l⁻¹ with a correlation coefficient of 0.9998 (n=5) and relative standard deviation (R.S.D.) of 0.92% (n=11) at 1.0×10⁻¹¹ mol l⁻¹. The detection limit (3×blank) was 1.0×10⁻¹³ mol l⁻¹. The method is successfully used to determine l-ascorbic acid in fresh vegetables. The possible mechanism of the chemiluminescence in the system is discussed.     

Flow-injection chemiluminescence determination of fluoroquinolones by enhancement of weak chemiluminescence from peroxynitrous acid

A flow-injection chemiluminescence (CL) method is described for the determination of fluoroquinolones including ciprofloxacin, norfloxacin and ofloxacin. The method is based on the enhancement by these compounds of the weak CL from peroxynitrous acid. The linear ranges are 1.0×10⁻⁷ to 1.0×10⁻⁵ mol l⁻¹ for ciprofloxacin and norfloxacin, and 3.0×10⁻⁷ to 3.0×10⁻⁵ mol l⁻¹ for ofloxacin, respectively. The detection limits (S/N=3) are 4.5×10⁻⁸ mol l⁻¹ ciprofloxacin, 5.9×10⁻⁸ mol l⁻¹ norfloxacin and 1.1×10⁻⁷ mol l⁻¹ ofloxacin, respectively. The proposed method was applied to the determination of fluoroquinolones in pharmaceutical preparations.     

Simultaneous determination of total homocysteine, cysteine and methionine in hypothyroid patients’ plasma by liquid chromatography using platinum/poly(methyl violet) modified electrode

The fabrication and application of a novel electrochemical detection (ED) system with the platinum/poly(methyl violet) (Pt/MV) chemically modified electrode (CME) for high performance liquid chromatography (HPLC) were described. The Pt particles deposited on the poly-MV film were characterized by atomic force microscope (AFM). It was found that the Pt/MV CME exhibited efficiently electrocatalytic effect on the current responses of cysteine (Cys), homocysteine (Hcy) and methionine (Met) with relatively high sensitivity, stability and long-life of activity. In HPLC-ED, these three amino acids had good and stable current responses at the CME and their linear ranges were over three orders of magnitude (R ≥ 0.9996) with the detection limits being 7.5 × 10⁻⁸ mol L⁻¹ for Cys, 1.0 × 10⁻⁷ mol L⁻¹ for Hcy, 5.0 × 10⁻⁷ mol L⁻¹ for Met. The application of this method coupled with microdialysis sampling for the determination of Cys, Hcy and Met in plasma from patients with hypothyroidism was satisfactory.     

Spectrophotometric catalytic determination of Fe(III) in estuarine waters using a flow-batch system

A flow-batch system was developed for the determination of Fe(III) in estuarine waters with high variability in salinity. The method is based on the catalytic effect of iron(III) on the oxidation rate of N,N-dimethyl-p-phenylenediammonium dichloride (DmPD) by hydrogen peroxide and the formed product is spectrophotometrically monitored at 554 nm. A controlled addition of sodium chloride to every assayed sample is accomplished for in-line individual salinity matching.The proposed system processes about 30 samples h⁻¹ and yields reproducible results. Relative standard deviations were estimated as <1.5% after 10 injections of typical samples (10.0-50.0 μg l⁻¹ Fe; ca. 0.5 mol l⁻¹ Cl). Synthetic samples (15.0 μg l⁻¹ Fe; 0.25-1.0 mol l⁻¹ NaCl) were efficiently processed, and no significant differences in results were found at a probability level of 99.7%. The method works for the full range of salinities. Only 120 μg DmPD are consumed per determination. The analytical curve is linear up to about 60 μg l⁻¹ Fe (r>0.999; n=5) and the detection limit is 5 μg l⁻¹ Fe. Results are in agreement with graphite furnace atomic absorption spectrometry.     

A novel histidine assay using tetraphenylporphyrin manganese (III) chloride as a molecular recognition probe by resonance light scattering technique

A novel histidine-selective method has been developed for the determination of histidine in aqueous solutions by resonance light scattering (RLS) technique. At pH 8.0, the weak RLS intensity of tetraphenylporphyrin manganese (III) chloride [MnTPPCl] was greatly enhanced by the addition of histidine with the maximum peak located at 483 nm. Under the optimum conditions, it was found that the enhanced RLS intensity was in proportion to the concentration of histidine in the range 7.8 × 10⁻⁷-2.4 × 10⁻⁵ mol l⁻¹. Low detection limit of 9.2 × 10^-8 mol l⁻¹ has been achieved. The histidine concentrations in synthetic samples and real samples were determined with satisfactory results. The sensitivity and selectivity of this method are high enough to permit the determination of trace amounts of histidine without any significant interference from high levels of other components such as common anions and especially, other amino acids.     

Sensitive detection of biothiols and histidine based on the recovered fluorescence of the carbon quantum dots–Hg(II) system

In this paper, we presented a novel, rapid and highly sensitive sensor for glutathione (GSH), cysteine (Cys) and histidine (His) based on the recovered fluorescence of the carbon quantum dots (CQDs)–Hg(II) system. The CQDs were synthesized by microwave-assisted approach in one pot according to our previous report. The fluorescence of CQDs could be quenched in the presence of Hg(II) due to the coordination occurring between Hg(II) and functional groups on the surface of CQDs. Subsequently, the fluorescence of the CQDs–Hg(II) system was recovered gradually with the addition of GSH, Cys or His due to their stronger affinity with Hg(II). A good linear relationship was obtained from 0.10 to 20 μmol L⁻¹ for GSH, from 0.20 to 45 μmol L⁻¹ for Cys and from 0.50 to 60 μmol L⁻¹ for His, respectively. This method has been successfully applied to the trace detection of GSH, Cys or His in human serum samples with satisfactory results. The proposed method was simple in design and fast in operation, which demonstrated great potential in bio-sensing fields.     

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples

In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λ_ext/λ_em = 340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L⁻¹ CH₃COONH₄ (pH = 2.5)/CH₃CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25–5.0 μmol L⁻¹ and the LOD was 19 nmol L⁻¹. No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.     

Chemiluminometric photo-induced determination of Strychnine in a Multicommutation flow assembly

This paper deals on the determination of Strychnine, a potent and dangerous pesticide and the analytical procedure is based on the photo-induced chemiluminescence of the pesticide by means of the Multicommutation continuous-flow methodology. Small segments of the pesticide solution were sequentially alternated with segments of the solution for adjusting the suitable medium for the photodegradation. The required time of UV irradiation was obtained by stopped-flow during 150 s; then, the resulting solution formed alternated segments with the oxidizing solution containing 5 × 10⁻³ mol l⁻¹ Ce(IV) in 0.6 mol l⁻¹ nitric acid. The calibration range, from 2 μg l⁻¹ to 50 mg l⁻¹, resulted in a linear behaviour over the range 25 μg l⁻¹ to 20 mg l⁻¹ and fitting the equation: I = 4706x + 624 with a correlation coefficient of 0.9955. The limit of detection was 2 μg l⁻¹ and the sample throughput 15 h⁻¹. After testing the influence of a large series of potential interferents, the method was applied to different kinds of samples.     

Glucose biosensors prepared by electropolymerization of p-chlorophenylamine with and without Nafion

Two different glucose biosensors for the amperometric determination of glucose, based on poly(p-chlorophenylamide) (PCPA) and bilayer film of PCPA and Nafion (PCPA/Nafion), are successfully developed. These two biosensors show linear amperometric responses to glucose ranging from 2.0×10⁻⁴ to 3.5×10⁻² mol l⁻¹ and 5.0×10⁻⁴ to 7.5×10⁻² mol l⁻¹, respectively, with the same correlation coefficient of 0.9988. Effects of polymerization potential and polymerization time on the performance of enzyme sensors are studied. It is found that PCPA, as a non-conducting polymer, can largely reduce the influence of electroactive interferents. Introduction of inner Nafion membrane not only further eliminates the influence of ascorbic acid on the sensor response but also increases electrode stability.     

p-Dimethylaminobenzaldehyde thiosemicarbazone: a simple novel selective and sensitive fluorescent sensor for mercury(II) in aqueous solution

A novel and simple fluorophore, p-dimethylaminobenzaldehyde thiosemicarbazone (DMABTS), was prepared in order to find available fluorescent chemosensor for mercuric ion in aquesous solution. DMABTS emitted fluorescence at 448 nm in aqueous solution and its fluorescence intensity was completely quenched upon interaction with Hg²⁺ ions, which should be attributed to the 1:1 complex formation between DMABTS and Hg²⁺. The binding constant of the complex was determined as 7.48 × 10⁶ mol l⁻¹. The linear range of quantitative detection of 0 to 5.77 × 10⁻⁶ mol l⁻¹ and the detection limit of 7.7 × 10⁻⁷ mol l⁻¹ for Hg²⁺ in the 6.3 × 10⁻⁶ mol l⁻¹ DMABTS aqueous solution were obtained from a calibration curve. The coexistence of several transition metal ions and anions did interfere the fluorometric titration of Hg²⁺ ion by less than 4% in the emission change.     

Synchronous fluorescence determination of DNA based on the interaction between methylene blue and DNA

The fluorescence intensity of methylene blue (MB) quenched by DNA in the pH range of 6.5-8.0 was studied with synchronous fluorescence technology. A novel method for detecting single-stranded and double-stranded DNA was developed. The decreased fluorescence intensity at 664 nm is in proportion to the concentration of DNA in the range of 0.28-11.0 μmol L⁻¹ for ctDNA, 0.14-8.25 μmol L⁻¹ for thermally denatured ctDNA and 0.28-8.25 μmol L⁻¹ for hsDNA. The detection limits (S/N = 3) are 0.11, 0.04 and 0.04 μmol L⁻¹, respectively. The method is rapid, selective, and the reagents are lower toxic. It has been used for the determination of DNA in synthetic samples with good satisfaction. In addition, the interaction modes between MB and ctDNA and the mechanism of the fluorescence quenching were also discussed in detail. The experimental results from absorption spectra and fluorescence polarization indicate that the possible interaction modes between MB and DNA are the electrostatic binding and the intercalation binding.     

Ultrasensitive cysteine sensing using citrate-capped CdS quantum dots

The importance of cysteine (Cys) in biological systems has stimulated a great deal of efforts in the development of analytical methods for the determination of this amino acid. In this work, a novel fluorescent probe for Cys based on citrate (Cit)-capped CdS quantum dots (QDs) is reported. The Cit-capped CdS QDs fluorescent probe offers good sensitivity and selectivity for detecting Cys. A good linear relationship was obtained from 1.0 × 10⁻⁸ mol L⁻¹ to 5.0 × 10⁻⁵ mol L⁻¹ for Cys. The detection limit was calculated as 5.4 × 10⁻⁹ mol L⁻¹. The proposed method was applied to detect Cys in human urine samples, which showed satisfactory results. This assay is based on both the lability of Cit and the strong affinity of thiols to the surface of CdS QDs. The addition of Cys improved the passivation of the surface traps of CdS QDs and enhanced the fluorescence intensity.     

A multicommuted flow system for sequential spectrophotometric determination of hydrosoluble vitamins in pharmaceutical preparations

A multicommuted flow system is proposed for spectrophotometric determination of hydrosoluble vitamins (ascorbic acid, thiamine, riboflavine and pyridoxine) in pharmaceutical preparations. The flow manifold was designed with computer-controlled three-way solenoid valves for independent handling of sample and reagent solutions and a multi-channel spectrophotometer was employed for signal measurements. Periodic re-calibration as well as the standard addition method was implemented by using a single reference solution. Linear responses (r=0.999) were obtained for 0.500-10.0 mg l⁻¹ ascorbic acid, 2.00-50.0 mg l⁻¹ thiamine, 5.00-50.0 mg l⁻¹ riboflavine and 0.500-8.00 mg l⁻¹ pyridoxine. Detection limits were estimated as 0.08 mg l⁻¹ (0.5 μmol l⁻¹) ascorbic acid, 0.8 mg l⁻¹ (2 μmol l⁻¹) thiamine, 0.2 mg l⁻¹ (0.5 μmol l⁻¹) riboflavine and 0.1 mg l⁻¹ (0.9 μmol l⁻¹) pyridoxine at 99.7% confidence level. A mean sampling rate of 60 determinations per hour was achieved and coefficients of variation of 1% (n=20) were estimated for all species. The mean reagent consumption was 25-fold lower in relation to flow-based procedures with continuous reagent addition. Average recoveries between 95.6 and 100% were obtained for commercial pharmaceutical preparations. Results agreed with those obtained by reference methods at 95% confidence level. The flow system is suitable for application in quality control processes and in dissolution studies of vitamin tablets.