Quantum dot (QD)-based probes for multiplexed determination of heavy metal ions.

Heavy metal contamination is a major global concern and additive toxicity resulting from the exposure to multiple heavy metal ions is more pronounced than that induced by a single metal species. Quantum dots (QDs) have demonstrated unique properties as sensing materials for heavy metal ions over the past two decades. With the rapid development and deep understanding on determination of single heavy metal ion using QD probes, this technology has been employed for sensing multiple metal ions. This review (with 97 refs.) summarizes the progress made in recent years in methods for multiplexed determination of heavy metal ions using QDs. Following an introduction into the importance of simultaneous quantitation of multiple heavy metal ions in environmentally relevant settings, the review discusses the applications of different types of QDs, i.e. chalcogenide, carbon, polymer and graphene in this field. Determination strategies based on fluorometric, colorimetric and electrochemical responses were reviewed including the testing mechanisms and differentiation between various metal ions. In addition, current state of the art sensor constructions, i.e. immobilization of QDs on solid substrate and sensor arrays have been highlighted. A concluding section describes the limitations, opportunities and future challenges of the QD probes. We also compiled a comprehensive table of currently available literature. The listed papers provided information in the following categories, i.e. type of QDs used, ligands or other components in the probe, metal ions tested, medium/substrate of the probe, transduction methods, discrimination mechanism, limit of detection (LOD) and concentration range. Graphic abstract.

Evaluation of free thyroxine determination based on one-step fluorometric immunoassay technique and the suboptimal concordance with two-step immunoassay.

Free thyroxine (FT4) quantification is continuing to be a concern. The purpose of the following study was to evaluate the analytical performance of Tosoh AIA900 based on a one-step technique and its comparison to Access 2 (two-step technique) over different clinical contexts (euthyroid, thyroid disorders, uncontrolled diabetes, renal failure and pregnancy). The protocol established by the French society of Clinical Biology was used to evaluate: imprecision, limit of detection, trueness, linearity, interferences and method comparisons. Within-run variation of 3.1%, 5.7% and 4.4% were found for the low, medium and high controls, respectively. Between-run was 5.8% for low control, 5.7% for medium control and 7.1% for high control. Common interferences did not affect one-step immunoassay FT4 results. The linearity was checked up to 86 pmol/L. The limit of detection was 5.5 pmol/L. The concordance correlation coefficient (CCC) showed a low agreement (0.6) between both methods. Bland-Altman plot revealed that AIA 900 one-step immunoassay technique provides a significant higher values ((+2.8 ±â€¯2.7 pmol/L;p < 0.0001). The Passing-Bablok regression demonstrated both proportional and systematic differences in comparison to Access 2. The lowest association was noted in subjects with impaired renal function (CCC = 0.27). At the time of the study, the results of on-step immunoassay are not directly comparable with Access 2.

Sensitivity test of derivative matrix isopotential synchronous fluorimetry and least squares fitting methods.

Determination of concentrations of spectrally overlapping compounds has special difficulties. Several methods are available to calculate the constituents' concentrations in moderately complex mixtures. A method which can provide information about spectrally hidden components in mixtures is very useful. Two methods powerful in resolving spectral components are compared in this paper. The first method tested is the Derivative Matrix Isopotential Synchronous Fluorimetry (DMISF). It is based on derivative analysis of MISF spectra, which are constructed using isopotential trajectories in the Excitation-Emission Matrix (EEM) of background solution. For DMISF method, a mathematical routine fitting the 3D data of EEMs was developed. The other method tested uses classical Least Squares Fitting (LSF) algorithm, wherein Rayleigh- and Raman-scattering bands may lead to complications. Both methods give excellent sensitivity and have advantages against each other. Detection limits of DMISF and LSF have been determined at very different concentration and noise levels.

Stopped-flow kinetic fluorimetric studies of the interaction of Ru(II) complex with DNA and its analytical application.

A simple, rapid, and sensitive stopped-flow kinetic fluorometric approach was established for the assay of DNA in synthetic samples and real samples by using the measures of initial reaction rate. The increased initial reaction rate is in proportion to the concentration of DNA in the range of 2.0x10(-8)M to 2.1x10(-6)M. The optimum conditions for various parameters on which the binding of Ru(phen)(2)(dppz)(2+) to DNA depends, were investigated. The influence of various surfactants on the interaction was discussed. Furthermore, stopped-flow techniques were employed to determine kinetic parameters of Ru(phen)(2)(dppz)(2+) binding to DNA under pseudo-first-order conditions. It was found that the interaction of Ru(phen)(2)(dppz)(2+) with DNA was very fast. A two-step reaction mechanism, a fast phase followed by a slow phase, was proposed.

[The use of the fluorimetric method for studying DNA integrity exemplified by an assessment of dioxidine genotoxicity]. / Primenenie fliuorimetricheskogo metoda issledovaniia tselostnosti DNK na primere otsenki genotoksichnosti dioksidina.

The genotoxic effect of dioxidine (300, 100, 30, and 10 mg/kg, i.p.) on the liver cells of outbred male rats was studied by fluorometric analysis of DNA unwinding (FADU) using the cell DNA samples taken from animals sacrificed 1, 6, 18, and 24 h after drug introduction. At a dose of 10 mg/kg, dioxidine produced no statistically significant increase in the amount of DNA damage relative to control. Increase in the dioxidine dose to 30 mg/kg resulted in a significant DNA damage 1 h after drug administration, while a dose of 100 or 300 mg/kg reliably caused damage at all times of exposure.

[Analytical control over 4,4'-bis-maleinimide diphenylmethane]. / Analiticheskii kontrol' za 4,4'-bismaleinimidadifenilmetanom.

The authors studied detection of 4,4-bis-maleinimid-diphenylmethane in aethylacetate by means of fluorimetry and direct spectrophotometry. Detection of the individual substance is highly sensitive, reproducible and express. Limit of 4,4-bis-maleinimid-diphenylmethane detection through spectrophotometry is 0.1 mg/ml and that through fluorimetry is 0.03 mg/ml. Studies covered interference of the synthesis' initial components on 4,4-bis-maleinimid-diphenylmethane detection in aethylacetate. Multicomponent mixtures of bis-maleinimids were analyzed according to Firordt additivity rule. The techniques elaborated are useful for sanitary and hygienic control over 4,4-bis-maleinimid-diphenylmethane in the air of workplace.

Plasma, urinary, and erythrocyte concentrations of guanidino compounds in patients with chronic renal failure.

Guanidino compounds are among the most likely candidates for uremic toxins. We determined the plasma, erythrocyte, and urinary concentration of guanidino compounds in 30 hemodialysis patients and 15 patients with chronic renal failure who had not undergone hemodialysis. Guanidino compounds were measured by high-performance liquid chromatography. Plasma levels of taurocyamine, guanidinosuccinic acid, alpha-N-acetyl-L-arginine, creatine, guanidinobutyric acid, guanidine, and methylguanidine were significantly increased in patients with chronic renal failure with or without hemodialysis. In contrast, plasma guanidinoacetic acid concentrations were significantly decreased. Erythrocyte concentrations of creatinine, guanidinosuccinic acid, guanidine and methylguanidine were also markedly elevated. No correlation was observed between plasma creatinine concentration and erythrocyte concentration of guanidinosuccinic acid or methylguanidine. However, there was a significant correlation between plasma and erythrocyte methylguanidine, and between plasma and erythrocyte guanidinosuccinic acid.

A fluorescent microplate assay for diarrheic shellfish toxins.

A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples.

A descriptive model for the kinetics of a homogeneous fluorometric immunoassay.

A descriptive mathematical model was chosen to fit the antigen-antibody association kinetics of a new homogeneous immunometric assay for prolactin, involving time-resolved fluorescence detection (TRACE technology, Time Resolved Amplified Cryptate Emission). We paid special attention to the methodology and criteria applied, to yield a convenient and statistically valid model, designed to allow potential exploitation of kinetic information in the data processing of the assay. We compared specific parameterizations of an hyperbolic model, the Gompertz, and the monomolecular models on the basis of morphological considerations, a statistical analysis of fit, and an assessment of the parameters estimation quality, over a wide range of antigen concentrations. The monomolecular model gave the best fit, and the most precise and stable estimation of its parameters. The study of parameter properties confirmed this choice.

Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase activity.

OBJECTIVE: To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity. METHODS: The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer. RESULTS: The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay. CONCLUSIONS: The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

A specific fluorometric assay for hexosamines in glycosaminoglycans, based on deaminative cleavage with nitrous acid.

Glycosaminoglycan (GAG) hexosamines were measured after deacetylation (2 h acid hydrolysis), deaminative cleavage by nitrous acid, and coupling of the 2,5-anhydro sugars thus produced with 3,5-diaminobenzoic acid (DABA) to give a fluorescent product. Analyses were performed after the addition of an aliquot of potassium acetate solution to the acid hydrolysates, to adjust the pH. Results on a series of GAGs were compared with an Elson-Morgan (E-M) procedure. Our method is faster, more convenient, 10-20 times more sensitive, and always gave higher figures for hexosamine content. In particular it avoids long, destructive hydrolysis in concentrated strong acid and the complications of the Moggridge-Neuberger effect. Results agreed with calculations and titrimetric data. Using internal standards, our method is tolerant of cetylpyridinium chloride, peptides, and salts. A simple fluorometer easily handled less than 1 microgram GAG per sample in 1.0 ml volume. Results are available in 4-5 h. The method is suitable for automation. A strongly polycationic environment, as in chitosan, markedly slowed nitrous acid deamination, possibly because of Donnan-type exclusion from the domain of the polycation of the cationic nitrosonium species. Whereas all other alpha- and beta-hexosaminides gave yield-hydrolysis time profiles that peaked in approximately 1 h in the DABA method, the E-M profiles, particularly for 2-sulfamato-alpha-hexosaminides, took longer and/or achieved lower optimal yields. All the usually encountered forms of the 2-amino-2-deoxy group (free, acetylated, sulfamato) are readily assayed using the same reagents, with appropriate prefluorometric stages.

A fluorometric assay for glycosylasparaginase activity and detection of aspartylglycosaminuria.

Recent experimental work on the mechanism of action of glycosylasparaginase suggests that the enzyme specifically reacts toward the L-asparagine or L-aspartic acid moiety of its substrates. Based on this, a new sensitive assay for glycosylasparaginase activity has been developed using L-aspartic acid beta-(7-amido-4-methylcoumarin) as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. At pH 7.5, Km = 93 microM, and as little as 1 ng of glycosylasparaginase could be detected with the assay. Hydrolysis of the substrate was inhibited by diazo-oxonorvaline, a specific inhibitor of glycosylasparaginase. In biological samples, the fluorometric assay is 40-100 times more sensitive than other published methods for glycosylasparaginase. This new assay enables a rapid enzymatic diagnosis of aspartylglycosaminuria--a genetic deficiency of glycosylasparaginase activity--with leukocyte and fibroblast samples.

An enzymatic fluorometric assay for adenylate cyclase activity.

Adenylate cyclase, the catalytic protein that converts ATP to cAMP, plays a fundamental role in adrenergic signal transduction. Adenylate cyclase activity (pmol cAMP/mg/min) is generally assayed by measuring radiolabeled cAMP generated from [alpha-32P]ATP. Although sensitive, the radioactive approach is costly and time consuming. Given safety and environmental concerns, we developed a highly sensitive fluorometric assay for adenylate cyclase activity. This assay depends upon the breakdown of cAMP by phosphodiesterase to AMP, and the subsequent stimulation by AMP of glycogen phosphorylase a. Radioactive and fluorescence methods were compared using the same ventricular membrane preparations from five different rabbit hearts. Theophylline, used in the fluorometric assay, increased basal adenylate cyclase activity. However, adenylate cyclase kinetics, the dose response to isoproterenol, and the "fold" stimulation (agonist stimulated/basal adenylate cyclase activity) after isoproterenol (10(-6)M), guanylyl-5'-imidodiphosphate (GppNHp) (10(-4)M), and NaF (10(-2)M) were nearly identical with both methods. Adenylate cyclase activity can be measured with the fluorometric assay in samples as small as 10 micrograms of membrane protein. In summary, this new fluorometric assay is highly sensitive, safer, less costly, and less time consuming than radioactive assays for adenylate cyclase activity.

Tissue viability measurement by in situ fluorometry.

A prototype laser-fiber optic based sensor for in situ monitoring of nicotinamide adenine dinucleotide (NADH) has been developed. This system is based on a compact neodymium-yttrium-aluminum-garnet (Nd:YAG) laser with associated harmonic generators. Light distribution to and from tissue is handled by a fiber optic network, including a long optical fiber to be inserted into the target tissue. Immobilizing the enzyme lactate dehydrogenase on the fiber tip converts the monitoring channel into a lactate sensor. A new dual beam reflection approach for blood volume artifact compensation is tested. Detection sensitivity of free NADH in the micromolar region is achieved. The method and system configuration appear feasible for continuous in situ monitoring of two important indices of ischemia and hypoxia in a clinical setting.

A sensitive method for the assay of serum prolyl endopeptidase.

A new fluorimetric assay for the determination of prolyl endopeptidase (EC 3.4.21.26) was developed. The synthetic substrate Z-glycyl-prolyl-4-methylcoumarinyl-7-amide (0.2 mmol/l; 5 microliters), K-phosphate buffer (100 mmol/l, pH 7.5; 100 microliters), and serum (10 microliters) are incubated for 120 min at 37 degrees C. The reaction is stopped with acetic acid (1.5 mol/l; 500 microliters) and the released 7-amino-4-methylcoumarin is measured fluorimetrically. The mean value of prolyl endopeptidase catalytic activity concentration in serum for 120 healthy volunteers was 0.455 (SD = 0.092) mumol of 7-amino-4-methylcoumarin released per litre of serum per minute. The proposed procedure is sensitive, robust, and economical.