Development of a sensitive enzyme immunoassay for the detection of phenyl-β-d-thioglucuronide in human urine

Immunoassays for the measurement of glucuronides in human urine can be a helpful tool for the assessment of human exposure to toxic chemicals. Therefore an enzyme immunoassay (EIA) for the specific detection of phenyl-β-d-thioglucuronide was developed. The immunoconjugate was formed by coupling p-aminophenyl-β-d-thioglucuronide to the carrier protein thyroglobulin leaving an exposed glucuronic acid. The hapten-protein conjugate was adsorbed to gold colloids in order to enhance the immunogenic effect. Rabbits were injected with the immunogold conjugates to raise polyclonal antibodies. The resulting competitive assay showed an inhibition by phenyl-β-d-thioglucuronide at sample concentrations of 23.0 ± 1.3 ng/mL (50% B/B₀) and a high cross-reactivity to p-aminophenyl-β-D-thioglucuronide (120%). Little cross-reactivities (< 2%) were observed for potential urinary cross reactants. In addition human urine samples were incubated with β-glucuronidase in order to investigate the EIA for specific matrix effects. An integration of high-performance liquid chromatography (HPLC) and EIA was developed in an attempt to decrease the matrix effects and increase the sensitivity of the overall method. The hyphenated technique HPLC-EIA may be used to monitor human exposure to toxic thiophenol which is excreted by mammals as urinary phenyl thioglucuronide.     

Study of a toxin–alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4–15 ng mL⁻¹ with a limit of detection (LOD) of 2 ng mL⁻¹ (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2–50 ng mL⁻¹ and the LOD was1 ng mL⁻¹.     

Washing-free chemiluminescence immunoassay for rapid detection of cardiac troponin Ⅰ in whole blood samples

Chemiluminescence immunoassay(CLⅠA) has always been a great challenge in detecting cardiac troponin Ⅰ(c Tn Ⅰ) in whole blood samples without centrifugation because of the interference of red blood cells and low sensitivity. Ⅰn this study, the antigens and erythrocytes in the blood were captured by the antibodies immobilized on the magnetic particles, recognized by another biotinconjugated c Tn Ⅰ antibody and detected by streptavidin/acridine aster-conjugated polychloromethylstyrene microspheres(...     

X-ray photoelectron spectroscopy for detection of the different Si–O bonding states of silicon

X-ray photoelectron spectroscopy (XPS) was used to detect the bonding between a silica particle surface and attached silanes. In addition to the commonly recorded Si 2p spectrum, the Si 1 s level is also accessible when monochromatic Ag Lalpha X-rays are applied. Furthermore, the spectrum of the Si 1 s level shows a fine structure. After spectrum deconvolution, we assigned the fitted spectral peaks to Si-C bonds of the silanes and to the Si-O bonds of the silica network. The recorded Si 1 s spectra were deconvoluted into peaks originating from Si-C bonds and the Si-O-Si silica network. To check the results of spectrum deconvolution, several differently functionalized silanes containing stoichiometric amounts of heteroatoms were applied for silica surface modification. We conclude that spectra deconvolution of the Si 1 s signal is an appropriate means for quantification of surface attached silane molecules.     

A highly sensitive and specific enzyme immunoassay for detection of β-amanitin in biological fluids

Polyclonal antisera to -amanitin were generated in sheep and used to construct a competitive ELISA for measurement of the toxin in human serum and urine. The assay had a detection limit of about 80 pg mL^–1, a dynamic range of 80–2,000 pg mL^–1, a cross reactivity of 22% with -amanitin, and no cross reactivities with cyclic peptides from algal sources. Assay responses in buffer, serum, and urine were remarkably similar. Coupling of the toxin to carrier proteins was carried out by previously unreported methods. The key step that allowed the construction of the highly sensitive assay was the introduction of a novel heterologous hapten derivative made of -amanitin-cyanuric chloride derivative. The new derivative overcame the problems of bridge binding that was, in this case, particularly serious with the homologous hapten derivative. The study demonstrated that the developed antiserum and ELISA procedure can be used to detect -amanitin and related toxins from Amanita phalloides in human serum and urine samples from suspected poison cases and enable early treatment to be administered.     

Magnetic nanomaterial–based electrochemical biosensors for the detection of diverse circulating cancer biomarkers

Electrochemical biosensors are highly compatible with modern advancements in magnetic nanomaterials. In particular, the versatile nature of magnetic nanomaterials as a universal platform for selective isolation of diverse forms of cancer biomarkers in body circulation, is highly synergistic with electrochemical biosensors for elevating biosensing performance to unprecedented levels. Such diverse circulating target biomolecules include cell surface proteins of circulating tumor cells and extracellular vesicles (EVs), as well as circulating tumor nucleic acids (i.e. ctDNA/ctRNA). This focussed review serves to discuss the latest work in the fields of magnetic nanomaterials and electrochemistry to tackle existing analysis challenges of diverse circulating biomarkers in cancer.     

Nanogold–polyaniline–nanogold microspheres-functionalized molecular tags for sensitive electrochemical immunoassay of thyroid-stimulating hormone

Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold–polyaniline–nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab₂) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0 mmol L⁻¹ H₂O₂ by using GPG-labeled HRP-Ab₂ as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab₂. Several labeling protocols comprising HRP-Ab₂, nanogold particle-labeled HRP-Ab₂, and polyaniline nanospheres-labeled HRP-Ab₂, were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab₂. With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab₂ to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20 μIU mL⁻¹ with a detection limit (LOD) of 0.005 μIU mL⁻¹ TSH at the 3s_B criterion. Significantly, no significant differences at the 0.05 significance level were encountered in the analysis of 15 spiking serum samples between the developed electrochemical immunoassay and the commercially available enzyme-linked immunosorbent assay (ELISA) method for determination of TSH.     

Graphene–Au composite sensor for electrochemical detection of para-nitrophenol

A novel electrochemical sensor for para-nitrophenol (p-NP) was constructed with graphene–Au composite containing 10 % Au (G–Au 10 %). In the composite, Au nanoparticles with the size of ca. 11 nm were regularly scattered on graphene sheet without aggregation, which offers dramatically higher electrocatalytic activity on the redox of K₃[Fe(CN)₆]/K₄[Fe(CN)₆] couple than sole Au nanoparticles. Compared to sole Au nanoparticles, the G–Au 10 % also exhibited dramatically improved electrocatalytic activity on the reduction of p-NP. Amperometric detection of p-NP at G–Au 10 % modified electrode displayed a wide linear range of 0.47–10.75 mM with detection limit of 0.47 μM and a high sensitivity of 52.85 μA/mM. Considering the thrifty in utilization of noble Au, the G–Au 10 % can be successfully applied as a low-cost and powerful sensing material for trace detection of p-NP.     

Spun films of perylene diimide derivative for the detection of organic vapors with host–guest principle

The optical characterization and chemical vapor sensing properties of 1,7-dibromo-N,N′-(bicyclohexyl)-3,4:9,10-perylene diimide thin film against to organic vapors were discussed in this study by using spin coating, UV–Vis spectroscopy, atomic force microscopy, surface plasmon resonance (SPR) and Quartz Crystal Microbalance (QCM) techniques. The perylene diimide thin films were fabricated with a refractive index values from 1.55 to 1.60 and thicknesses in the range between 15.80 and 26.32 nm using different spin speeds from 1000 to 5000 rpm. In this study, perylene diimide thin film sensor was exposed to dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran and ethyl acetate vapors by using both SPR and QCM techniques. Also, the swelling behaviors of the perylene diimide thin films prepared at different spin speeds were investigated with respect to dichloromethane vapor at the room temperature by using SPR data. Diffusion coefficients were found to be 11.34?×?10^?17 (1000 rpm), 2.56?×?10^?17 (3000 rpm) and 0.38?×?10^?17 cm² s^?1 (5000 rpm) for dichloromethane vapor by using the Fick’s law of diffusion. It might be proposed that perylene diimide thin film optical chemical sensor element has a good sensitivity and selectivity for the dichloromethane vapor at room temperature.     

A rational design of the multiwalled carbon nanotube–7,7,8,8-tetracyanoquinodimethan sensor for sensitive detection of acetylcholinesterase inhibitors

A new, simple and effective amperometric acetylcholinesterase biosensor was developed using screen-printed carbon electrodes modified with carbon nanotubes (MWCNTs)–7,7,8,8-tetracyanoquinodimethane (TCNQ). The design of the biosensor was based on the supramolecular arrangement resulted from the interaction of MWCNTs and TCNQ. This arrangement was confirmed by spectroscopic and electrochemical techniques. Two different supramolecular arrangements were proposed based on different MWCNTs:TCNQ ratios. The synergistic effect of MWCNTs and TCNQ was, for the first time, exploited for detection of thiocholine at low potential with high sensitivity. The biosensor developed by immobilization of acetylcholinesterase (AChE) in sol–gel allowed the detection of two reference AChE inhibitors, paraoxon-methyl and chlorpyrifos with detection limits of 30 pM (7 ppt) and 0.4 nM (0.1 ppb), respectively. Efficient enzyme reactivation was obtained by using obidoxime.     

Quantum dot–NBD–liposome luminescent probes for monitoring phospholipase A2 activity

In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot–fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A_2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C₆-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A_2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A₂ as low as 0.0075 U mL^?1 and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A₂ inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).     

The calculation of the detection efficiency in the calibration of gross alpha–beta systems

This paper presents a method for efficiency calibration of a measuring alpha–beta system PROTEAN ORTEC, MPC-2000-DP, using standard radioactive sources. The system is used to measure gross alpha–beta activity concentrations in environmental samples. The calculated efficiencies of detection were subsequently introduced in the system for two working geometries: measuring geometry—gross alpha–beta $ \varepsilon_{\alpha }^{g} $  = 31,37 ± 0.25 (%)—the alpha efficiency and $ \varepsilon_{\beta }^{g} $  = 44.94 ± 0.69 (%)—the beta efficiency, where the spillover factor is $ X_{\text{talk}}^{g} $  = 25.59 ± 0.50 (%) and measuring geometry up alpha–beta $ \varepsilon_{\alpha }^{u} $  = 36.23 ± 0.29 (%)—the alpha efficiency and $ \varepsilon_{\beta }^{u} $  = 48.53 ± 0.74 (%)—the beta efficiency, where the spillover factor is $ X_{\text{talk}}^{u} $  = 31.08 ± 0.60 (%).     

Analytical techniques for the detection of α-amino-β-methylaminopropionic acid

The non-protein amino acid L-α-amino-β-methylaminopropionic acid (BMAA) has been linked to several neurodegenerative diseases. Its presence in trace amounts in complex sample such as bacterial, plant and mammalian tissue extracts and hydrolyzates makes analysis a complicated process requiring good analytical technique. There are conflicting reports in the literature regarding the presence or absence of BMAA in key samples, but the absence of standardized or validated methods makes comparison of the disparate findings difficult to compare. This critical review will summarize the historic and recent literature, and provide suggestions for improving the methods currently in practice.     

N-Dansyl-N′-ethylthiourea for the fluorometric detection of heavy metal ions

N-Dansyl-N-ethylthiourea (DET) forms fluorescent chelates with a large number of heavy metal ions. The influence of the pH-value on the luminescence of DET and its metal chelates was investigated. The addition of Cu(II) to DET causes a bathochromic shift of the emission maximum, which is linearly dependent on the Cu(II) concentration. Low detection limits and a wide linear range of detection are achieved by HPLC using RP 18 columns as stationary phase. Also presented are first measurements of fluorescence decay times of the ligand as well as some complexes.     

CuS–MWCNT based electrochemical sensor for sensitive detection of bisphenol A

The rapid and simple detection of bisphenol A is very important for the safety and reproduction of organisms. Here, a sensitive and reliable electrochemical sensor was established for bisphenol A detection based on the high amplification effect of copper sulfide-multi-walled carbon nanotube (CuS–MWCNT) nanocomposites. The flower-like CuS–MWCNT were successfully synthesized by a simple hydrothermal method accompanied by polyvinylpyrrolidone (PVP). Compared with bare glassy carbon electrode (GCE), CuS–MWCNT modified GCE could amplify the electrochemical signals in about ten times, which was attributed to the synergistic effect of CuS and MWCNT. The MWCNT could increase the specific surface area of electrodes and improve the electrode activity. The integration of CuS could further enhance the electrode conductivity as well as accelerate the electron transfer rate. Raman spectra and transmission electron microscope (TEM) were used to characterize the successful fabrication of CuS–MWCNT nanocomposites and its uniform and monodispersed morphology. Under optimizing conditions, the oxidation currents of bisphenol A via the differential pulse voltammetric (DPV) showed a good linear relationship with its concentration in a wide range of 0.5–100 μM, with a detection limit of 50 nM. This electrochemical sensor of bisphenol A provided a convenient and economical platform with high sensitivity and reproducibility, which had great potential in environmental monitoring.     

Quantitative LC–MS/MS method in urine for the detection of drugs used to reverse the effects of chemical castration

The chemical castration law, which targets child molesters with recidivism, was introduced in Korea in 2011. For this, leuprolide, a gonadotropin-releasing hormone agonist, is used to decrease testosterone production and suppress libido. In order to achieve efficient law enforcement, it is necessary to monitor intentional ingestion of drugs that antagonize the effect of leuprolide. Therefore, an analytical method for the simultaneous detection of mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, icariin, alprostadil, and yohimbine, which are the major impotence treatment drugs, legitimately or otherwise, in Korea, as well as their selected metabolites, in human urine was established and validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). First, different sample preparation methods, two solid-phase extractions with different cartridges and protein precipitation, were compared and protein precipitation was chosen for the entire study because it showed better matrix effects and recoveries. Thus, the drugs and metabolites in urine were extracted by protein precipitation and then filtered and analyzed by LC–MS/MS with polarity switching electrospray ionization. The validation results of selectivity, matrix effect, recovery, linearity, intra- and inter-assay precision and accuracy were satisfactory. The limits of detection ranged from 0.25 to 10 ng/mL, and the limits of quantification were 2.5 to 50 ng/mL. The drugs and metabolites in urine did not show any degradation under storage for 7 and 15 days at 4 and ?20 °C as well as after three freeze–thaw cycles. The developed method will be very useful for monitoring the illegal use of impotence treatment drugs.     

Liquid chromatography–tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions

A new LC–MS/MS method for the separation and detection of the most prominent marine lipophilic toxin groups comprising okadaic acid, dinophysistoxins, yessotoxins, azaspiracids, pectenotoxins, spirolides and some okadaic acid fatty acid esters has been developed. With this method 28 different marine lipophilic biotoxins can be analysed in a single run. Separation was achieved with an acetonitrile/water gradient containing ammonium hydroxide (pH 11). All toxins were stable under these basic conditions. Compared to chromatography using an acidic gradient, the limit of detection (LODs) for okadaic acid, yessotoxin, gymnodimine and 13-desmethyl spirolide C were improved two- to three-fold, mainly due to better peak shapes. The azaspiracids and pectenotoxins-2 showed comparable LODs under acidic and basic conditions. A major advantage of the developed method is that toxins can be clustered in retention time windows separated for positively and negatively ionized molecular ions. Therefore, there is no need for rapid polarity switching or two separate runs for one sample. The new method showed good repeatability and reproducibility and is an important step in the development of alternatives to the animal tests currently in use for shellfish toxin analysis.     

Advancement and future challenges of metal–organic coordination polymers: A case study of optical sensor for the detection of the environmental contaminants

Fast industrialization, increasing population, rapid urbanization, and the greediness of creamy layer in the society without the issues of caring the sustainability of ecosystem are the main out of many reasons behind the environmental catastrophe. The ecological balance is disturbed with the noxious materials generated from the uncontrolled use of modern science and technology and also unscientific and unsystematic societal growths. To save the modern civilization, a fast-track task is the monitoring of the toxic materials for the maintenance of sustainable ecological health and their utilization in a circular economy. Accordingly, optical methods are employed for the detection and estimation of environmental contaminants for immediate monitoring. The stability and structural tunability of the coordination polymeric (CPs) materials make them promising optical sensors for easy, low cost, reliable, selective, and sensitive detection of toxic ions/molecules. The crystallinity, thermal, mechanical, magnetic, and optical stability and flexibility of the CPs play a beneficial role for the accurate and stress-free recognition of the toxicants. In this review, the recent developments as well as the outlook on the sensing of pollutants at a very low concentration level using CPs as selective and specific sensors by spectro-fluorometric method are summarized. The progresses and challenges in the fabrication of hybrid materials and understanding their structure property correlations are described with an aim to motivate and facilitate the future researchers to perform research in the area.