Efficient Optosensing of Hippuric Acid in the Undiluted Human Urine with Hydrophilic "Turn-On"-Type Fluorescent Hollow Molecularly Imprinted Polymer Microparticles.

The development of complex biological sample-compatible fluorescent molecularly imprinted polymers (MIPs) with improved performances is highly important for their real-world bioanalytical and biomedical applications. Herein, we report on the first hydrophilic "turn-on"-type fluorescent hollow MIP microparticles capable of directly, highly selectively, and rapidly optosensing hippuric acid (HA) in the undiluted human urine samples. These fluorescent hollow MIP microparticles were readily obtained through first the synthesis of core-shell-corona-structured nitrobenzoxadiazole (NBD)-labeled hydrophilic fluorescent MIP microspheres by performing one-pot surface-initiated atom transfer radical polymerization on the preformed "living" silica particles and subsequent removal of their silica core via hydrofluoric acid etching. They showed "turn-on" fluorescence and high optosensing selectivity and sensitivity toward HA in the artificial urine (the limit of detection = 0.097 µM) as well as outstanding photostability and reusability. Particularly, they exhibited much more stable aqueous dispersion ability, significantly faster optosensing kinetics, and higher optosensing sensitivity than their solid counterparts. They were also directly used for quantifying HA in the undiluted human urine with good recoveries (96.0%-102.0%) and high accuracy (RSD ≤ 4.0%), even in the presence of several analogues of HA. Such fluorescent hollow MIP microparticles hold much promise for rapid and accurate HA detection in the clinical diagnostic field.

Uricase based fluorometric determination of uric acid based on the use of graphene quantum dot@silver core-shell nanocomposites.

The authors describe a fluorescent "turn-on" assay for detection of uric acid (UA) based on the use of graphene quantum dots coated with a shell of silver (GQD@Ag). The fluorescence of the GQDs is quenched by the silver shell. However, if the silver shell was removed via etching with H2O2 (which is produced by uricase catalyzed oxidation of UA), the fluorescence of the GQDs is restored. The resulting increase in fluorescence at 466 nm depends directly on the concentration of H2O2, which, in turn, depends on the concentration of UA. The method allows UA to be quantitated with a 2 µM detection limit. It was applied to the analysis of human urine samples and exhibited satisfactory results. The method is cost-effective, sensitive and selective for UA. In our perception, it provides a useful tool in clinical analysis and may be extended to other assays based on the use of oxidases. Graphical abstract Schematic of the reduction of Ag(I) and the growth of a silver shell on the surface of graphene quantum dots (GQDs) to form a GQD@Ag nanocomposite whose fluorescence is quenched. Uricase catalyzes the oxidation of uric acid (UA) to produce allantoin and H2O2 which etches the silver shell. This results in the release of GQDs and increased fluorescence, allowing quantitative analysis of UA.

Determination of urinary carnitine levels as a potential indicator of uterine fibroids caused by nonylphenol exposure.

Our previous studies have shown that uterine fibroids are associated with nonylphenol (NP) exposure, and the changes of carnitines in critical reproductive tissues and body fluids could be used to indicate the female reproductive toxicity caused by NP exposure. In this work, on the basis of further clarifying the correlation between NP exposure level and uterine fibroids, the possibility of the urinary carnitine levels as a potential indicator of uterine fibroids caused by NP exposure was discussed. The urine samples were collected from 84 female volunteers: the control group of 34 healthy women without gynecological disease and 50 uterine fibroids patients, respectively. Methods were respectively established for the determination of NP and eight carnitines in human urine samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the NP level of uterine fibroids group was significantly higher than that of control group (P = 0.002), indicating that NP exposure was an important environmental factor in the occurrence of uterine fibroids. It was further found that in urine samples of the uterine fibroids group, the levels of L-Carnitine (C0), L-Acetyl-carnitine (C2), L-Octanoyl-carnitine (C8), Tetradecanoyl-carnitine (C14), Oleoyl-carnitine (C18:1) and Linoleoyl-carnitine (C18:2) had obviously increased compared with those in the control group (P < 0.001; < 0.001; < 0.001; = 0.003; < 0.001; = 0.010). The concentrations of L-Hexanoyl-carnitine (C6) and L-Palmitoyl-carnitine (C16) in the uterine fibroids group were also higher than those in the control group, although the difference was not statistically significant (P > 0.05). The results suggested that the changes in urinary carnitine levels might be a potential indicator to help to warn of the risk of uterine fibroids caused by NP exposure at the early stage.

The use of high-performance liquid chromatography with diode array detector for the determination of sulfide ions in human urine samples using pyrylium salts.

Hydrogen sulfide is a toxic gas involved in the regulation of some essential biological processes. A novel, precise, accurate and rapid method based on high-performance liquid chromatography with diode array detection for the determination of sulfide ions in human urine sample is proposed. The method involves the derivatization of sulfide with pyrylium salts - (2,4,6-triphenylpyrylium hydrogensulfate(VI) (L1) and 4-[p-(N,N-dimethylamino)phenyl]-2,6-diphenylpyrylium chlorate(VII) (LN1). The separation occurs on InfinityLab Poroshell 120 EC C18 column using acetonitrile and phosphate buffer as a mobile phase. The detectors utilized a wavelength of 371 or 580 nm. The calibration curves were linear in the range of 2-150 µmol L-1 and 1-50 µmol L-1 for L1 and LN1 derivatives, respectively. The samples were found to be stable from sample collection to final analysis. The method was successfully applied to samples from apparently healthy volunteers.

Development of extremely stable dual functionalized gold nanoparticles for effective colorimetric detection of clenbuterol and ractopamine in human urine samples.

New glutamic acid (Glu) and polyethylenimine (PE) functionalized ultra-stable gold nanoparticles (PE-Glu-AuNPs) were developed via a simple NaBH4 reduction method. The low toxicity and biocompatibility of PE-Glu-AuNPs were confirmed via an MTT assay in Raw 264.7 cells. Excitingly, PE-Glu-AuNPs were found to be extremely stable at room temperature up to six months and were utilized in an effective colorimetric naked eye assay of clenbuterol (CLB) and ractopamine (RCT) at pH 5. It was found that the selective assay of CLB and RCT is not affected by any other interferences (such as alanine, phenylalanine, NaCl, CaCl2, threonine, cysteine, glycine, glucose, urea and salbutamol). Furthermore, the detection of these ß-agonists can be visually accomplished through change color from wine red to purple blue. Notably, the aggregation induced detection of CLB and RCT was well confirmed through transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies. DLS investigations, clearly showed, that in the presence of CLB and RCT, the initial size of PE-Glu-AuNPs (12.8 ±â€¯8.6 nm) was changed to 84.8 ±â€¯52.3 and 79.5 ±â€¯47.8 nm, respectively, via aggregation. Furthermore, the colorimetric assays of CLB and RCT with PE-Glu-AuNPs were effective starting from CLB and RCT concentrations of 200 nM and 400 nM, respectively, and could be visualized using the naked eyes. Remarkably, UV-vis titrations of PE-Glu-AuNPs with CLB and RCT could be used to well estimate their sub nanomolar detection limits (LODs) via standard deviation and linear fittings. The contribution of surface functional groups that support the analyte recognition was confirmed by fourier-transform infrared spectroscopy (FTIR) analysis. Moreover, the CLB and RCT assays with PE-Glu-AuNPs were supported by examination of human urine samples.

Hydrophilic interaction chromatography combined with dispersive liquid-liquid microextraction as a preconcentration tool for the simultaneous determination of the panel of underivatized neurotransmitters in human urine samples.

A simple and sensitive method using dispersive liquid-liquid microextraction (DLLME) followed by liquid chromatography coupled to mass spectrometry (LC-MS) with a hydrophilic interaction chromatography (HILIC) column was developed for the simultaneous determination of 13 compounds of different polarities, comprising monoamine neurotransmitters (dopamine, norepinephrine, epinephrine and serotonin) along with their respective precursors and metabolites, in human urine samples. The microextraction procedure was based on the fast injection of a mixture of ethanol (disperser solvent) and dichloromethane (extraction solvent) into a human urine sample, forming a cloudy solution in the Eppendorf tube. After centrifugation, the sedimented phase was collected and subsequently analyzed by LC-HILIC-MS in about 12min without a derivatization step. The separation was performed on an XBridge Amide™ BEH column 3.0×100mm, 3.5mm and the mobile phase consisted of phase A: 10mM ammonium formate buffer in water pH 3.0 and phase B: 10 mM ammonium formate buffer in acetonitrile, under gradient program elution. Tyrosine, tryptophan, 5-hydroxytryptophan, dopamine, epinephrine, norepinephrine, serotonin, 3-methoxytyramine, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxy-l-phenylalanine and norvaline (internal standard) were detected in the positive ionization mode. While vanillylmandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxybenzylamine (internal standard) were detected in the negative ionization mode. Parameters influencing DLLME and LC-HILIC-MS were investigated. Under the optimum conditions, the proposed method exhibited a low detection limit (5-10ngmL(-1)), and good linearity with R between 0.9991 and 0.9998. The recoveries in human urine samples were 99.0%±3.6%. for the 13 studied biogenic amines with intra- and inter-day RSDs of 0.24-9.55% and 0.31-10.0%, respectively. The developed DLLME-LC-MS method could be successfully applied for the determination of trace amounts of polar endogenous compounds, such as neurotransmitters, in human urine samples, including samples with a reduced volume obtained from pediatric patients.

A sensitive electrochemical sensor for rapid determination of methadone in biological fluids using carbon paste electrode modified with gold nanofilm.

A novel and effective electrochemical sensor for the determination of methadone (MET) at pH 9.0 using gold nanoparticles, electrodeposited on a multi-walled carbon nanotube modified carbon paste electrode (GNPs/MWCPE), is introduced. The voltammetric behavior of MET at this modified electrode was studied using cyclic and square wave voltammetric techniques and the results were compared with those obtained at the multi-walled carbon nanotube modified carbon paste electrode (MWCPE). The oxidation of MET was irreversible and exhibited an adsorption controlled process at the GNPs/MWCPE and a diffusion controlled process at the MWCPE. The effect of various experimental parameters including pH, scan rate, and accumulation potential and time on the voltammetric response of MET was investigated. At the optimum conditions, the concentration of MET was determined using square wave voltammetry (SWV) in a linear range of 0.1-500.0 µmol L(-1) with a correlation coefficient of 0.9901 at the GNPs/MWCPE, and 0.5-300.0 µmol L(-1) with a correlation coefficient of 0.993 at the MWCPE and the detection limits were found to be 0.005 and 0.3 µmol L(-1), respectively. The proposed electrode was successfully applied to the determination of MET in a pharmaceutical dosage form, urine and saliva samples. The effects of common interferences, namely some of different cations and anions, on the current response of MET were investigated. This revealed that the GNPs/MWCPE shows excellent analytical performance for the determination of MET in terms of a very low detection limit, high sensitivity, very good repeatability and reproducibility.