Biotinylation of quantum dots for application in fluoroimmunoassays with biotin-avidin amplification

A competitive microplate fluoroimmunoassay was developed for the determination of human serum albumin in urine. It is based on the use of biotinylated CdTe quantum dots (QDs) whose synthesis is optimised in terms of storage stability, purification, and signal-to-noise ratio. The bioconjugated QDs were characterised by gel chromatography and gel electrophoresis. Storage stability and quantum yield were investigated. The excitation/emission wavelengths are 360/620?nm. The immunoassay of human serum albumin in urine has a working range from 1.7 to 10?μg.mL^?1, and the limit of detection is 1.0?μg.mL^?1.

Determination of phenolic compounds in water samples by HPLC following ionic liquid dispersive liquid-liquid microextraction and cold-induced aggregation

We report on the determination of bisphenol A and 2-naphthol in water samples using ionic liquid cold-induced aggregation dispersive liquid-liquid microextraction combined with HPLC. Parameters governing the extraction efficiency (disperser solvent, volume of extraction and disperser solvent, pH, temperature, extraction time) were optimized and resulted in enrichment factors of 112 for bisphenol A and of 186 for 2-naphthol. The calibration curve was linear with correlation coefficients of 0.9995 and 0.9998, respectively, in the concentration range from 1.5 to 200?ng?mL^?1. The relative standard deviations are 2.3% and 4.1% (for n?=?5), the limits of detection are 0.58 and 0.86?ng?mL^?1, and relative recoveries in tap, lake and river water samples range between 100.1 and 108.1%, 99.4 and 106.2%, and 97.1 and 103.8%, respectively.

Visual detection and microplate assay for Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification

We have developed a specific method for the visual detection of Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification technology. A biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of a microplate via biotin-avidin binding. Then, the target bacteria (S. aureus), the biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and streptavidin-HRP were successively placed in the wells of the microplate. After adding TMB reagent and stop solution, the intensity of the yellow reaction product can be visually inspected or measured with a plate reader. Under optimized conditions, there is a linear relationship between absorbance at 450 nm and the concentration of S. aureus in the 10 to 10⁷ cfu mL^?1 concentration range (with an R² of 0.9976). The limit of detection is 8 cfu mL^?1.

Dispersive liquid-liquid microextraction of phenolic compounds using solidified floating organic droplets, and their determination by HPLC

We have developed a simple and efficient method for dispersive liquid-liquid microextraction of 4-nitrophenol, 2-naphthol and bisphenol A in real water samples. It is making use of solidified floating organic droplets of 1-dodecanol which has low density and a proper melting point. The type and volume of extraction solvent and dispersive solvent, the effect of salts, pH value and extraction time were optimized and resulted in enrichment factors of 84 for 4-nitrophenol, 123 for 2-naphthol, and 97 for bisphenol A. The limits of detection by HPLC are 1.50, 0.10 and 1.02 ng · mL^?1, respectively. Excellent linearity is observed in the concentration range from 10 to 800 ng · mL^?1, with coefficients of correlation ranging from 0.9988 to 0.9999. The relative standard deviations (for n?=?5) are from 3.2 to 5.3 %, and relative recoveries for the three phenols in tap, river and spring water range from 85.0 to 105.0 %, 98.3 to 110.0 %, and 98.6 to 109.0 %, respectively.

Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads

We report on a new scheme for the determination of the activity of caspase-3 using a specific peptide labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a chemiluminescent (CL) probe and on the development of magnetic separation technology. Firstly, the ABEI-labeled and biotinylated peptide was prepared and conjugated to streptavidin-coated magnetic beads (MBs) to form f-MBs (functionalized magnetic beads). The f-MBs contain a site (DEVD, Asp-Glu-Val-Asp) that is cleaved by caspase-3. Upon cleavage, the terminal residue attached to ABEI can dissociate from the f-MBs and can be used for CL detection. CL intensity is linearly related to the concentration of caspase-3 in the range 1.0 to 600 ng mL^?1, with a detection limit of 0.3 ng mL^?1. The relative standard deviation of the assay is 3.6 % at a level of 50 ng mL^?1 of caspase-3 (for n?=?11). The CL assay has been applied to the determination of caspase-3 in Jurkat cell extract with recoveries between 96.6 % and 106.1 % (n?=?5).

Determination of cadmium(II) using carbon paste electrode modified with a Cd-ion imprinted polymer

We have synthesized cadmium(II) ion-imprinted polymers (IIP) and non-imprinted polymers (NIP) using 1-(2-pyridylazo)-2-naphthol as a ligand. The materials were used to prepare a carbon paste electrode for the determination of Cd(II). Polymerization was performed with (a) methacrylic acid as a functional monomer, (b) ethyleneglycol dimethacrylate as the crosslinking monomer, and (c) 2,2′-azobis(isobutyronitrile) as the initiator. Imprinted cadmium ion was removed from the polymeric matrix using nitric acid. The measurements were carried out in an closed circuit after accumulation at ?1.2?V, this followed by electrolysis of the accumulated Cd(II) by voltammetric scanning from ?1.0 to ?0.6?V. The parameters governing the response of the electrode were studied. Under optimized conditions, the response of the electrode is linear in the range from 2.0 to 200?ng?mL^?1. The detection limit is 0.31?ng?mL^?1. The relative standard deviations are ±3.4 and ±2.1?% for 7 successive determinations of 20.0 and 50.0?ng?mL-1 of Cd(II), respectively. The method was applied to the determination of cadmium (II) in water and food samples.

Electromagnetic induction-assisted heating as a new method for on-line cloud point extraction of cadmium from water samples

We report on a novel method for on-line cloud point extraction (CPE) for preconcentration of cadmium ions. It is based on electromagnetic induction-assisted heating (EMIH) of iron particles in a packed bed contained in a quartz tube that acts as an on-line CPE enrichment column. The cadmium complex of 1-(2-pyridylazo)-2-naphthol is quantitatively retained by the column under the cloud point temperature with the help of EMIH. The column was then eluted with alcoholic borax buffer at room temperature and on-line coupled to FAAS. Under optimum conditions, the limit of detection (3 s_b/b) and limit of quantification (10 s_b/b) are 0.21 μg?L^?1 and 0.70 μg?L^?1 of Cd(II), respectively, and the relative standard deviation is 3.8 % (for n?=?8; at 20 ng?mL^?1). An enhancement factor of 76 is typically achieved. The correlation coefficient of the calibration graph using the present method was 0.9986. The method was successfully applied to determine Cd(II) in water samples

Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10⁷ CFU mL^?1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

Determination of the rare earth elements La, Eu, and Yb using solidified floating organic drop microextraction and electrothermal vaporization ICP-MS

We have developed a method for the determination of trace levels of the rare earth elements La, Eu, and Yb in biological and environmental samples. It is based on solidified floating organic drop microextraction using 1-(2-pyridylazo)-2-naphthol (PAN) as a chelator, followed by electrothermal vaporization (ETV) and quantification by inductively coupled plasma mass spectrometry. PAN also acts as a modifier in ETV. The effects of pH, amount of PAN, extraction time, stirring rate, volume of sample solution, and temperature program were examined. Under optimized conditions, the detection limits are 2.1, 0.65 and 0.91 pg mL^?1 for the elements La, Eu and Yb, respectively. The relative standard deviations are <6.0 % (c?=?0.1 ng mL^?1, n?=?9). When applied to the analysis of (spiked) natural water samples, the recoveries range from 92 to 105 %. The accuracy was validated with certified reference materials (combined sample of branch and leaf of shrub: GBW 07603 and human hair: GBW 07601), and the results were in good agreement with the certified values.

Double layer enzyme modified carbon nanotubes as label for sandwich-type immunoassay of tumor markers

An immunosensor was prepared for the determination of carcinoembryonic antigen (CEA). It is based on the use of multiwalled carbon nanotubes (MWCNTs) along with horseradish peroxidase-labeled antibody. The enzyme was assembled onto MWCNTs templates using the layer-by-layer technique and then conjugated to carcinoembryonic secondary antibodies (Ab₂) as the enzyme label. The resulting assembly results in a largely amplified sensitivity. The response is linear in the range of 0.05 to 45?ng?mL^-1, with a detection limit of 16.0?pg?mL^-1. The immunosensor possesses good stability and good reproducibility.

Single-drop microextraction for the determination of manganese in seafood and water samples

We describe a method for single drop microextraction of manganese from fish, mollusk, and from natural waters using the reagent 1-(2-pyridylazo)-2-naphthol as the complexing agent and chloroform as the fluid extractor. After extraction, the analyte was directly submitted to graphite furnace electrothermal atomic absorption spectrometry. Once optimized, the method has a detection limit of 30 ng L^?1, a limit of quantification of 100 ng L^?1, and an enrichment factor of 16. Its accuracy was verified by applying the procedure to the following certified reference materials: apple leaves, spinach leaves, bovine liver, and mussel tissue. The procedure was also successfully applied to the determination of manganese in seafood and natural waters.

Highly sensitive determination of doxorubicin and daunorubicin based on their effect on the resonance light scattering signals of the ethidium-DNA complex

We have developed a resonance light scattering (RLS) quenching assay for the highly sensitive determination of doxorubicin (DOX) and daunorubicin (DAU). It is based on the reduction of the intensity of the shoulder of the RLS spectra at 443?nm. The intensity of the RLS of the ethidium-DNA system decrease linearly on addition of trace quantities of DOX or DAU within the concentration range of 0.008 to 12.0???g?mL^?1 for DOX, and of 0.010 to 21.0???g?mL^?1 for DAU. The detection limits are 3.0 and 5.0?ng?mL^?1, respectively. The assay was successfully applied to the determination of DAU in synthetic and serum samples. Compared to the reported methods for anthracyclines, this assay displays higher sensitivity, lower detection limits, and a wider linear range.

Lead ion sensor with electrodes modified by imidazole-functionalized polyaniline

Glassy carbon electrodes (GCE) and carbon paste electrodes (CPE) were modified with imidazole functionalized polyaniline with the aim to develop a sensor for lead (II) in both acidic and basic aqueous solution. The electrodes were characterized by cyclic voltammetry and differential pulse adsorptive stripping voltammetry. The limit of detections obtained with glassy carbon electrode and carbon paste electrode are 20?ng?mL^-1 and 2?ng?mL^-1 of lead ion, respectively. An interference study was carried out with Cd(II), As(III), Hg(II) and Co(II) ions. Cd(II) ions interfere significantly (peak overlap) and As(III) has a depressing effect on the lead signal. The influence of pH was investigated indicating that bare and modified GCE and CPE show optimum response at pH?4.0 ± 0.05.

Analysis of dextromethorphan and pseudoephedrine in human plasma and urine samples using hollow fiber-based liquid–liquid–liquid microextraction and corona discharge ion mobility spectrometry

We report on the use of hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) followed by corona discharge ion mobility spectrometry for the determination of dextromethorphan and pseudoephedrine in urine and plasma samples. The effects of pH of the donor phase, stirring rate, ionic strength and extraction time on HF-LLLME were optimized. Under the optimized conditions, the linear range of the calibration curves for dextromethorphan in plasma and urine, respectively, are from 1.5 to 150 and from 1 to 100 ng mL^?1. The ranges for pseudoephedrine, in turn, are from 30 to 300 and from 20 to 200 ng mL^?1. Correlation coefficients are better than 0.9903. The limits of detection are 0.6 and 0.3 ng mL^?1 for dextromethorphan, and 8.6 and 4.2 ng mL^?1 for pseudoephedrine in plasma and urine samples, respectively. The relative standard deviations range from 6 to 8%.

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Micro-solid phase extraction of ochratoxin A, and its determination in urine using capillary electrophoresis

We describe a simple, environmentally friendly and selective technique for the determination of ochratoxin A (OTA) in urine. It involves (a) the use of a molecularly imprinted polymer as a sorbent in micro-solid-phase extraction in which the sorbent is contained in a propylene membrane envelope, and (b) separation and detection by capillary electrophoresis (CE). Under optimized conditions, response is linear in the range between 50 and 300 ng mL^?1 (with a correlation coefficient of 0.9989), relative standard deviations range from 4 to 8 %, the detection limit for OTA in urine is 11.2 ng mL^?1 (with a quantification limits of 32.5 ng mL^?1) which is lower than those of previously reported methods for solid-phase extraction combined with CE. The recoveries of OTA from urine spiked at levels of 50, 150 and 300 ng mL^?1 ranged from 93 to 97 %.

Comparison of three different configurations of dual ultramicroelectrodes for the decomposition of S-Nitroso-L-glutathione and the direct detection of nitric oxide

Podoplanin (PDP) is a small transmembrane protein and widely present in various specialized cells throughout the human body. It is a specific marker for identification of lymphatic vessel and a candidate marker for cancer stem cells in squamous cell carcinoma of the lung. We report on method for the highly selective determination of PDP by using a surface plasmon resonance imaging (SPRI) that exploits the highly selective interaction between PDP and anti-human PDP monoclonal antibody (IgG). The sensor has a dynamic range between 0.25 and 1.0?ng?mL^?1, and a detection limit of 15?pg?mL^?1. It was applied to the determination of PDP in blood plasma and tissue homogenates from paired normal and lung tumor tissue.

Immunoassay for serum alpha-fetoprotein using silver nanoparticles and detection via resonance light scattering

The assay for alpha-fetoprotein (AFP) is based on the use of immobilized anti-AFP labeled with silver nanoparticles (AgNPs). The immunoreaction between the labeled antibody against AFP and free AFP takes place in pH 6.0 solution and leads to the formation of the respective immunocomplex which displays enhanced resonance light scattering (RLS) intensity at 480 nm. Under the optimal conditions, the intensity of the enhanced RLS is proportional to the concentration of AFP in the range from 0.10 to 50 ng mL^?1, with a detection limit of 40 pg mL^?1. The characteristics of RLS, the immunocomplex, the immuno response, and the optimum conditions of the immunoreaction have been investigated. The concentration of AFP in 20 serum specimens was determined by the new assay, and results are consistent with those obtained with a commercially available ELISA kit.

In vitro monitoring of picogram levels of risperidone in human urine via luminollysozyme flow injection chemiluminescence

The anti-schizophrenic drug risperidone (RSP) exerts an inhibitory effect on the chemiluminescence (CL) of the luminol-lysozyme system. This finding forms the basis for a sensitive flow injection method for its determination at picogram levels. RSP binds to Trp62 in the lysozyme, and this leads to a conformational change upon which the CL of the system is quenched. The decrease in CL is proportional to the logarithm of the concentration of RSP, and the calibration graph is linear in the range from 0.1 pg?mL^?1 to 1.0 ng?mL^?1, with relative standard deviations of <5.0%, and a detection limit of 0.05 pg?mL^?1 (3σ). At a flow rate of 2.0 mL?min^?1, the whole process including sampling and washing is completed within 20 s. The method was successfully applied to monitoring RSP in human urine after incorporation of 2 mg of RSP, with a total excretion of 16.6% within 8.5 h.

Ultrasensitive electrochemiluminescent immunoassay for morphine using a gold electrode modified with CdS quantum dots, polyamidoamine, and gold nanoparticles

We report on a novel electrochemiluminescent (ECL) immunoassay for the ultrasensitive determination of morphine by making use of a gold electrode which was modified with a nanocomposite film containing self-assembled polyamidoamine (PAMAM) CdS quantum dots and electrodeposited gold nanoparticles (Au-NPs). The highly uniform and well-dispersed quantum dots were capped with PAMAM dendrimers. Due to the synergistic effect of the modified quantum dots and the electrodeposited Au-NPs, the ECL response is dramatically enhanced. Under optimal experimental conditions, the immunoreaction between morphine and anti-morphine antibody resulted in a decrease of the ECL signal because of steric hindrance. The calibration plot is linear in the morphine concentration range from 0.2 to 180 ng?mL^?1, with a detection limit as low as 67 pg?mL^?1. The sensor was successfully applied to the determination of morphine in blood plasma. This kind of assay is expected to pave new avenues in label-free drug assays.