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An electrochemical biosensor based on a polystyrene-gold nanocomposite was fabricated for the ultrasensitive sensing of clenbuterol. The PS-Au nanocomposite was synthesized by modifying polystyrene with triazine and aminothiazole groups, followed by the in-situ reduction of AuNPs. The PS-Au/SPCE exhibited a linear range (0.01–10 μM) with a limit of detection of 3.2 nM towards clenbuterol along with high selectivity and good stability. The sensor platform showcased its prowess by accurately quantifying clenbuterol in milk specimens fortified with known analyte concentrations, yielding recovery percentages within the range of 96.5–102.8 %, accompanied by relative standard deviations below 4.2 %. The superior performance of the PS-Au/SPCE is due to the synergistic effect of the high surface area and electrocatalytic activity of the AuNPs, the efficient adsorption of clenbuterol on the functionalized polystyrene matrix, and the enhanced electron transfer kinetics. [ABSTRACT FROM AUTHOR]

A rapid, simple but sensitive analytical method for the simultaneous monitoring of heavy metal ions and illegal additives in complex samples remains lacking. We reported a difunctional capture–detection surface-enhanced Raman scattering (SERS) immunochromatographic assay (ICA) for the direct detection of the heavy metal residue Cd ion (Cd2+) and illegal additive clenbuterol (CLE) with high sensitivity and specificity herein. A novel magnetic SERS tag (Fe@RAu) with a Raman reporter molecule–embedded raspberry-like Au shell was fabricated via a one-step reduction reaction that not only simplified the fabrication of common magnetic SERS nanomaterials but also improved SERS activity to increase detection sensitivity. The difunctional SERS–ICA enabled the fast magnetic enrichment of the target small molecules in complex specimens and ultrasensitive and quantitative determination of CLE/Cd2+ on the two test lines of the ICA system. The limits of detection of the developed method for CLE and Cd2+ were as low as 0.48 and 1.88 pg/mL, respectively, and about 2000 and 500 times lower than those of conventional AuNP-based ICA strips. The excellent practicability of Fe@RAu–ICA for real food samples analysis was also demonstrated, revealing the potential of the proposed assay for rapid and on-site screening of small-molecule pollutants. [Display omitted] • A Raman reporter molecule–embedded magnetic SERS tag was reported for the first time. • Raman reporter–embedded magnetic tag was used as capture/detection difunctional tool for small molecule detection. • Simultaneous monitoring of heavy metal ions and illegal additives on SERS–ICA was first realized. • The detection limits for CLE and Cd2+ were as low as 0.48 and 1.88 pg/mL, respectively. [ABSTRACT FROM AUTHOR]

Clenbuterol (CLB) is a bronchodilator for treating respiratory disorders. However, the irrational use of CLB in animal feeding can cause serious risks to human health. The development of selective and efficient adsorbent for CLB removal is of great significance. Herein, we report a convenient covalent grafting approach for the fabrication of molecularly imprinted covalent organic framework (COF) for selective adsorption and removal of CLB from environmental water and milk samples. By integrating the merits of both molecularly imprinted polymers (MIPs) and COFs, the molecularly imprinted COFs named BatBt-MIPs (2,5-bis(allyloxy)terephthalohydrazide, Bat; 1,3,5-benzenetricarboxaldehyde, Bt) presented the alluring traits of large surface area, tailored-made binding sites, uniform pore structure, and good selectivity for CLB. The adsorption equilibrium for CLB on BatBt-MIPs was quickly reached within 20 min and followed pseudo-second-order kinetic model. BatBt-MIPs showed a CLB adsorption capacity of 15.45 mg/g, which is about 3.7 times higher than that of non-imprinted COFs (BatBt-NIPs). Compared with BatBt-NIPs, BatBt-MIPs present improved adsorption selectivity for template CLB. Selective removal of CLB from complex environmental water and milk samples on BatBt-MIPs was also realized. This work provided a new covalent grafting approach to construct novel molecularly imprinted COFs and highlighted the great potential of COF-MIPs in contaminants elimination. [Display omitted] • Novel molecularly imprinted BatBt-MIPs was prepared via covalent graft strategy. • BatBt-MIPs exhibited good selectivity for the clenbuterol adsorption and removal. • BatBt-MIPs could effectively remove clenbuterol from water and milk sample. [ABSTRACT FROM AUTHOR]

[Display omitted] • The CuO@Ag composite was synthesized by metal self-reducing triggered green etching. • Flower-like morphology of the CuO@Ag improved its binding ability with antibodies. • The CuO@Ag has excellent photothermal conversion efficiency. • The dual-mode immunosensor can achieve high-sensitive detection of clenbuterol. Photothermal materials has aroused extensive attention for building multi-signal immunosensors. Herein, a metal self-reducing triggered green etching strategy was employed for the synthesis of CuO@Ag nanoparticles (NPs) with high photothermal conversion efficiency and better antibody coupling efficiency. The flower-like morphology of the CuO@Ag NPs has improved its binding ability with antibodies, which enables them to serve as the multifunctional signal label of the lateral flow immunoassay (LFIA). Then, a colorimetric-photothermal dual-mode LFIA (CuO@Ag-based LFIA) was designed for the highly sensitive detection of clenbuterol hydrochloride (CLE). After direct, the CuO@Ag NPs endowed T-line to be irradiated by an 808 nm laser to obtain photothermal signal owing to their strong photothermal conversion efficiency of CuO@Ag. Under optimal conditions, the detection limits of the CuO@Ag-based LFIA are 0.205 ng mL−1 and 0.131 ng mL−1 under colorimetric and photothermal modes, respectively, which are 13 and 20 times sensitive than that of the conventional AuNP-based LFIA. Additionally, the LFIA was assessed by measuring CLE in food samples with recovery rates ranging from 86.37 % to 103.72 % and coefficient of variations (CVs) ranging from 2.52 % to 9.64 %. Consequently, this work provides qualitative-quantitative point-of-care testing (POCT) approach and has important reference value for other facile, green, and low-cost synthesis of composite signal labels in the LFIA system. [ABSTRACT FROM AUTHOR]

The World Anti-Doping Agency (WADA) has prohibited the use of clenbuterol (CLN) because it induces anabolic muscle growth while potentially causing adverse effects such as palpitations, anxiety, and muscle tremors. Thus, it is vital to assess meat quality because, athletes might have positive test for CLN even after consuming very low quantity of CLN contaminated meat. Numerous materials applied for CLN monitoring faced potential challenges like sluggish ion transport, non-uniform ion/molecule movement, and inadequate electrode surface binding. To overcome these shortcomings, herein we engineered bimetallic zeolitic imidazole framework (BM-ZIF) derived N-doped porous carbon embedded Co nanoparticles (CN-CoNPs), dispersed on conductive cellulose acetate-polyaniline (CP) electrospun nanofibers for sensitive electrochemical monitoring of CLN. Interestingly, the smartly designed CN-CoNPs wrapped CP (CN-CoNPs-CP) electrospun nanofibers offers rapid diffusion of CLN molecules to the sensing interface through amine and imine groups of CP, thus minimizing the inhomogeneous ion transportation and inadequate electrode surface binding. Additionally, to synchronize experiments, machine learning (ML) algorithms were applied to optimize, predict, and validate voltametric current responses. The ML-trained sensor demonstrated high selectivity, even amidst interfering substances, with notable sensitivity (4.7527 μA/μM/cm2), a broad linear range (0.002–8 μM), and a low limit of detection (1.14 nM). Furthermore, the electrode exhibited robust stability, retaining 98.07% of its initial current over a 12-h period. This ML-powered sensing approach was successfully employed to evaluate meat quality in terms of CLN level. To the best of our knowledge, this is the first study of using ML powered system for electrochemical sensing of CLN. [ABSTRACT FROM AUTHOR]

The residue of clenbuterol (CLB) in food poses a potential harm to human health. Herein, we presented an electrochemical aptasensor (E -A-CRISPR) based on employing an aptamer as a specific recognition element and asymmetric CRISPR-Cas12a as signal amplifiers for sensitive, and selective detection of CLB. In this E -A-CRISPR system, the target CLB bound to the aptamer and initiated cascade signal amplification through the DNase activity of CRISPR-Cas12a with two competitive gRNAs. Upon amplification, the active Cas12a cleaved the methylene blue-labeled hairpin probe on the electrode, reducing the peak current. Under optimal conditions, the E -A-CRISPR system showed a wide linear range (1 pM-100 nM) and a low detection limit (500 fM). This system could detect CLB in potable water, pig liver, and pork samples, showing significant potential for food safety monitoring. To our knowledge, this study is the first to use a CRISPR-Cas12a powered system for electrochemical sensing of CLB. • CRISPR-Cas12a coupled with an aptamer can achieve selective detection of clenbuterol (CLB). • Integrating asymmetric CRISPR-Cas12a with electrochemistry to enhance sensitivity • High selectivity and sensitivity detection of CLB in a linear range from 1 pM to 100 nM with a detection limit of 500 fM. • Reliable detection of CLB in potable water, pig liver, and pork samples was achieved. [ABSTRACT FROM AUTHOR]

To achieve high throughput detection of clenbuterol while reducing matrix interference, we developed a simple and integrated nano-inspection platform. Herein, based on the simplicity and convenience of paper chip, a multi-layer paper chip-chemiluminescence (CL) sensor was designed in the form of "48-well plate" type. The all-in-one paper chip-CL sensor has integrated the covalent organic frameworks (COFs) layer for sample matrix purification, high affinity nanobody layer for specific capture of target, and CL rapid response layer for the final determination. The signal readout was realized by the inhibitory effect of clenbuterol on the K 3 [Fe(CN) 6 ] CL system. Detection of clenbuterol in pork meat was verified by measuring the CL intensity and satisfactory recoveries (85.9 %–97.3 %) were achieved. 48 samples could be simultaneously detected on one-chip in one-test, favoring the high-throughput detection. Especially, the work explored a new pathway for design of portable analytical instruments to achieve immediate on-site detection of hazards. Multi-layer paper chip sensing platform was established by COF sample pretreatment, nanobody recognition, and luminescent sensing for high-throughput and rapid nano-inspection of food hazards. [Display omitted] • A new paper chip method offers low-cost, easy-to-use, efficient clenbuterol detection. • Novel COF layer contributed to matrix cleanup for complex samples. • High affinity nanobody layer ensured high selectivity in CL detection. [ABSTRACT FROM AUTHOR]

Skeletal muscle atrophy is a hallmark of severe spinal cord injury (SCI) that is precipitated by the neural insult and paralysis. Additionally, other factors may influence muscle loss, including systemic inflammation, low testosterone, low insulin-like growth factor (IGF)-1, and high-dose glucocorticoid treatment. The signaling cascades that drive SCI-induced muscle loss are common among most forms of disuse atrophy and include ubiquitin-proteasome signaling and others. However, differing magnitudes and patterns of atrophic signals exist after SCI versus other disuse conditions and are accompanied by endogenous inhibition of IGF-1/PI3K/Akt signaling, which combine to produce exceedingly rapid atrophy. Several well-established anabolic agents, including androgens and myostatin inhibitors, display diminished ability to prevent SCI-induced atrophy, while ursolic acid and β2-agonists more effectively attenuate muscle loss. Strategies combining physical rehabilitation regimens to reload the paralyzed limbs with drugs targeting the underlying molecular pathways hold the greatest potential to improve muscle recovery after severe SCI. [ABSTRACT FROM AUTHOR]

Background: Clenbuterol is a long-acting β-adrenergic agonist that is not Food and Drug Administration-approved for use in the United States, but may be obtained without a prescription from various unregulated sellers. It has seen increasing use as a performance-enhancing drug for sports. Literature on pediatric toxicity and treatment is limited.Case Report: We report a case of a 2-year-old female presenting after an exploratory ingestion of clenbuterol. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Use of performance-enhancing agents is increasing and physicians should be aware of the potential toxicity of intentional and unintentional ingestions of β-adrenergic agonists. Patients may exhibit nausea, vomiting, tremor, tachycardia, and hypotension, along with laboratory abnormalities, including hyperglycemia, hypophosphatemia, hypokalemia, and hyperglycemia. Hypotension might not respond to adrenergic agents and may require administration of β-adrenergic antagonists to maintain adequate perfusion. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel nanohybrid electrochemical sensor was fabricated by for the first time. • NPVMo/ZrO 2 nanocomposites may significantly amplify the electrochemical signal. • The nanohybrid exhibited excellent electrocatalytic activity to CLB and RAC. • The sensor was applied to the simultaneous measure of RAC and RAC in sample. Abstract A new electrochemical sensor based on isopolyacid anions (NH 4) 5 PV 8 Mo 4 O 40 (NPVMo) and transition metal zirconium dioxide (ZrO 2) nanocomposites was prepared in double signal amplification for the simultaneous detection of clenbuterol (CLB) and ractopamine (RAC). This is the first time that ZrO 2 was used as POM carrier to prepare electrochemical sensor. Scanning electron microscopic (SEM), nitrogen adsorption/desorption isotherms and X-ray diffraction (XRD) test showed that the synthesized ZrO 2 nanoparticles have a needlelike nanostructure and a larger surface area. The electrochemical measurements such as cyclic voltammetry (CV), amperometric and differential pulse voltammetry (DPV) indicated that the NPVMo/ZrO 2 /GCE exhibited much high stability, fast response, good conductivity and electrocatalytic actives, which enhanced peak current and negatively shifted the oxidation potentials of CLB and RAC. Due to the synergistic action between NPVMo and ZrO 2 nanoparticles, DPV results showed that the oxidation of CLB and RAC on NPVMo/ZrO 2 /GCE could be detected selectively and sensitively with low limits of detection, 5.03 × 10−9 mol L−1 for CLB and 9.3 × 10−7 mol L−1 for RAC, and a wide range for calibration curves of CLB and RAC from 0.1 to 1000 μM and 3.0 to 50 μM, respectively. In addition, the proposed sensor was applied to detect content of CLB and RAC in pork sample, and the recovery was over the range from 94.4 to 102.3%. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A unique hybrid reoxidized graphene oxide/Au nanoparticle (rGO/AuNPs) SERS substrates were prepared. • The substrate was stable within 180 days under 4 °C storage temperature • The mixture solvent (BEHP / methylene chloride, v/v, 1:800) was first reported as extraction reagent. • Melamine was selected as internal standard for quantitative calculation. • The total detection time was approximately 8 min/sample. Abstract A rapid, selective, and quantitative method to determine clenbuterol content in animal urine based on Surface-Enhanced Raman Spectroscopy (SERS) by aggregated re-oxidized graphene oxide/Au nanoparticle (roGO/AuNPs) hybrids and liquid-liquid extraction has been proposed in this study. To realize the quantitative calculation, melamine was optimized as the internal standard. The peak intensity of melamine observed at △ v = 709 cm−1 was first set to 1000 as basis, and the peak at △ v = 1474 cm−1 was selected as the characteristic peak of clenbuterol used for the quantitative calculation. Sufficient linearity was obtained in the range of 1–20 ng mL−1 (R 2 = 0.997). The limit of detection and quantification in urine matrix were as low as 0.5 and 1 ng mL−1, respectively. The recovery rates were 78.6–89.4% with coefficients of variation less than 9.8%, and the storage time of the substrates is approximately 180 days under 4℃. The detection results of the developed SERS detection method agree well with those determined by LC–MS/MS, while the cost time significantly reduced to 8 min/sample. This new protocol could be easily developed for the routine monitoring of the illicit use of clenbuterol in animal farming. [ABSTRACT FROM AUTHOR]

Highlights • Selenium nanoparticle (SeNp) of 40.78 nm was synthesized successfully with SDS and PEG. • The SeNp was spherical form and uniform dispersion. • SeNp can be used for clenbuterol detection by lateral flow immunoassay (LFIA). • The SeNP-based LFIA can be stored at 37 °C for 60 days. Abstract Lateral flow immunoassays (LFIAs) have been applied widely in clinical diagnosis, food safety, drug abuse, environmental monitoring and other fields due to their characteristics of rapidity, sensitivity, stability and low cost. Choosing the proper labels is key to the preparation of LFIAs. An ideal probe requires a simple synthesis, has stable performance and is economical. This study aimed to establish a simple, rapid and convenient method for the preparation of selenium nanoparticles (SeNPs) that can be used as labels in LFIAs. Appropriate SeNPs were successfully prepared by reducing 2 mM H 2 SeO 3 with 16 mM l -ascorbic acid (Vc), 5 mM sodium dodecyl sulfate (SDS) and 100 mM polyethylene glycol (PEG) as stabilizing agents. The spherically formed and uniformly dispersed nanoparticles were applied to the preparation of an LFIA. In this study, the preparation of the SeNPs was economical and was accomplished at room temperature. We used clenbuterol (CLE) as an analyte in an LFIA; the limit of detection (LOD) of CLE in pig urine was found to be 3 ng/mL, which meets the industry standard and has the same sensitivity as commercially available flow assays. In addition, the SeNP-based LFIA can be stored at 37 °C for 60 days. [ABSTRACT FROM AUTHOR]

• A sample collection component was designed to detect clenbuterol. • The problem of poor reproducibility of SERS detection was solved. • The optimal particle size of gold colloid for detection of clenbuterol was found. • This method can be used for the rapid crude screening of clenbuterol in pork. Clenbuterol is often used as a feed additive to increase the percentage of lean meat in livestock. Meat containing clenbuterol can cause many illnesses and even death for people. In this paper, the particle growth method was used to prepare gold colloids of different sizes, and the enhanced effectiveness of gold colloids of different sizes on clenbuterol in pork was investigated. The results showed that the gold colloid with the best enhanced effectiveness for clenbuterol had a particle size of approximately 90 nm. Second, a sample collection component was designed to detect clenbuterol from bottom to top, solving the problem of poor reproducibility of Surface-enhanced Raman scattering (SERS) detection caused by different droplet sizes and shapes. Then, the influence of different volumes of samples and concentrations of aggregating compounds on the enhanced effectiveness was optimized. The results showed that, based on the sample collection components designed in this article, 5 µL of enhanced substrate, 7.5 µL of clenbuterol and 3 µL of 1 mol/L mixed detection of NaCl solution had the best enhanced performance. Finally, 88 pork samples (0.5, 1, 1.5,..., 10, 12, 14 µg/g) with different concentrations were divided into correction sets and prediction sets in a ratio of 3:1. Unary linear regression models were established between the concentration of clenbuterol residue in the pork and the intensity of the bands at 390, 648, 1259, 1472, and 1601 cm−1. The results showed that the unary linear regression models at 390, 648, and 1259 cm−1 had lower root mean square errors than those at 1472 and 1601 cm−1. The intensity of the three bands and the concentration of clenbuterol residue in the pork were selected to establish a multiple linear regression model, and the concentration of clenbuterol residue in the pork was predicted. The results showed that the determination coefficients (R2) of the correction set and the prediction set were 0.99 and 0.99, respectively. The root mean square errors (RMSE) of the correction set and the prediction set were 0.169 and 0.184, respectively. The detection limit of clenbuterol in pork by this method is 42 ng/g, which can realize the crude screening of pork containing clenbuterol in the market. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fe 3 O 4 has enzyme-like activities, can be used as an alternative for natural enzymes. • Fe 3 O 4 @mSiO 2 @MMT-SDS nanoparticles were prepared by modifying Fe 3 O 4 @mSiO 2 with MMT and SDS to impart negative charges to the particle surfaces. • Fe 3 O 4 @mSiO 2 @MMT-SDS nanoparticles exhibited excellent catalytic activity for the catalytic oxidation of clenbuterol (CLB) by H 2 O 2 , turning the solution blue in the presence of the 3,3ʹ, 5,5ʹ-tetramethylbenzidine chromogen.. • A novel colorimetric method for clenbuterol detection were demonstrated based on the peroxidase-like activity of Fe 3 O 4 @mSiO 2 @MMT-SDS. This study demonstrates a novel colorimetric method for clenbuterol (CLB) detection based on the peroxidase-like activity of SiO 2 -coated Fe 3 O 4 nanoparticles (Fe 3 O 4 @mSiO 2) modified with montmorillonite (MMT) and sodium dodecyl sulfate (SDS), namely, Fe 3 O 4 @mSiO 2 @MMT-SDS. Fe 3 O 4 @mSiO 2 @MMT-SDS nanoparticles were prepared by modifying Fe 3 O 4 @mSiO 2 with MMT and SDS to impart negative charges to the particle surfaces. The structural properties, steady-state kinetics, catalytic performance, and reaction conditions of the as-prepared nanoparticles were analyzed. Fe 3 O 4 @mSiO 2 @MMT-SDS nanoparticles exhibited excellent dispersion and enzyme-like activity, which improved the affinity with substrate (TMB and H 2 O 2) for the catalytic oxidation of CLB by H 2 O 2 , turning the solution blue in the presence of the 3,3ʹ, 5,5ʹ-tetramethylbenzidine chromogen. Under optimal reaction conditions, a strong linear correlation between the absorbance of solution and CLB concentration (R2 = 0.9983) was observed. Here, the limit of detection, spike recovery, and relative standard deviation were 9.6 × 10-9 mol·L-1, 94.2 %–102.2 %, and 2.2 %–4.4 %, respectively. The proposed method is a simple, fast, cost-efficient, and sensitive assay, which can be used to rapidly and accurately screen CLB in pork and animal feeds. [ABSTRACT FROM AUTHOR]

[Display omitted] • Misuse of veterinary drug, Clenbuterol as growth promoters in humans and livestock. • A comprehensive discussion on usage of nanosensors to detect clenbuterol in food samples. • The application of nano-sensors for real sample analysis has been discussed. • The limit of detection and recovery percentage values of each nanosensor has been compared. Veterinary drugs which are primarily meant for livestock treatment have now been categorised under potential food contaminant due to its unregulated usage and abuse. Their over usage by animal workers lead to production of contaminated animal-based food products which contain veterinary drug residues. These drugs are also misused as growth promoters to enhance the muscle to fat ratio in human body. This review highlights the misuse of such a veterinary drug; Clenbuterol. In this review, we have comprehensively discussed the usage of nanosensors to detect clenbuterol in food samples. Colorimetric, fluorescent, electrochemical, SERS and electrochemiluminescence are major categories of nanosensors that have been utilized for this purpose. The mechanism through which these nanosensors detect clenbuterol have been discussed in detail. The limit of detection and recovery percentage values of each nanosensor have been compared. This review will impart significant information on various nanosensors for clenbuterol detection in real samples. [ABSTRACT FROM AUTHOR]

Abstract A multiplex lateral flow immunoassay sensor based on highly luminescent green-emitting Au nanoclusters (AuNCs-MLFIA sensor) was successfully established for the simultaneous and quantitative determination of clenbuterol (Clen) and ractopamine (RAC) in swine urine. The antigens of Clen and RAC were dispersed on a nitrocellulose membrane as two test lines, and the Au nanoclusters were synthesized from 6-aza-2-thiothymine and l -arginine to obtain highly green luminescence and ultra-small nanoparticles (Arg/ATT/AuNCs). Free carboxyl groups on Arg/ATT/AuNCs enabled conjugation with biomolecules to afford an indicator for the biosensor. The AuNCs-MLFIA sensor is based on the indirect competition assay and could successfully detect samples within 18 min without sample pretreatment, qualitative results can be obtained by visual inspection under a UV lamp. The limits of detection of Clen and RAC by the naked eye were both 0.25 μg L−1. In addition, the AuNCs-MLFIA sensor allowed quantitative detection combined with a portable fluorescence reader. The half-maximal inhibitory concentrations of Clen and RAC were 0.06 and 0.32 μg L−1, respectively, with detection limits of 0.003 and 0.023 μg L−1. Thirty blind-spiked swine urine samples were analyzed by the AuNCs-MLFIA sensor and liquid chromatography–tandem mass spectrometry, and the results of the two methods showed a significant correlation. The newly developed AuNCs-MLFIA sensor overcomes several limitations of conventional LFIA sensors, including their low sensitivity, limitation to quantify analytes, and single-analyte detection. Graphical abstract Multiplex LFIA sensor based on highly luminescent green-emitting gold nanoclusters for simultaneous and quantitative determination of Clen and RAC in swine urine. Image 1 Highlights • Highly luminescent green-emitting AuNCs were applied to LFIA for the first time. • AuNCs-MLFIA sensor was developed for simultaneous and quantitative determination of Clen and RAC in swine urine. • The developed AuNCs-MLFIA sensor revealed superior in sensitivity than other LFIA sensors. [ABSTRACT FROM AUTHOR]

Graphical abstract With large size Prussian blue nanoparticles (PBNPs) as the labeling substrate to reduce the labeled quantity of antibodies and improve visible signal, a lateral flow biosensor was developed for sensitive and cost-effective detection of clenbuterol. Highlights • MOFs of PBNPs were firstly introduced as novel labels in lateral flow assays (LFAs). • Assay cost was reduced because a fewer antibodies were needed in the PBNP-mAb probe. • Assay performance of the PBNP-based LFA was 5-fold improved for clenbuerol detection. • The work opens new possibilities to choose MOFs as labeling matrices in immunoassays. Abstract Prussian blue nanoparticles (PBNPs), one kind of metal organic frameworks with the general formula of Fe 4 [Fe(CN) 6 ] 3 ·nH 2 O, are increasing attractive due to their excellent properties. Here, with PBNPs as labels, a PBNP-based competitive lateral flow assay (LFA) for efficient and sensitive detection of clenbuterol was developed. With unique control structure, PBNPs are quite larger than most of the commonly used labels, meaning much fewer antibodies are needed in the probe to produce clearly visible results, which will bring a more sensitive dose-response relation between the analyte concentration and signal intensity. Under optimal conditions, the PBNP-based LFA showed a great linear relationship over the range of 0.5–5 ng mL−1 for clenbuterol. The visual limit of detection was 1.0 ng mL−1, 5-fold improvement in sensitivity compared with that of the traditional gold nanoparticles based LFA. In our perception, our work opens new possibilities to choose metal organic frameworks as labeling matrices in immunoassays. [ABSTRACT FROM AUTHOR]

Immunochromatographic assays (ICAs) are most frequently used for on-site rapid screening of clenbuterol. To improve sensitivity, a novel probe with bacteria as signal carriers was developed. Bacteria can load a great deal of gold nanoparticles (AuNPs) on their surface, meaning much fewer antibodies are needed to produce clearly visible results, although low concentrations of antibody could also trigger fierce competition between free analyte and the immobilized antigen. Thus, a limited number of antibodies was key to significantly improved sensitivity. Analytical conditions, including bacterial species, coupling method, and concentration, were optimized. The visual detection limit (VDL) for clenbuterol was 0.1 ng/mL, a 20-fold improvement in sensitivity compared with traditional strips. This work has opened up a new route for signal amplification and improved performance of ICAs. Furthermore, inactivated bacteria could also be environment-friendly and robust signal carriers for other biosensors. [ABSTRACT FROM AUTHOR]

Equine hair is becoming an increasingly popular biological matrix for doping control of horse sports; one of the reasons for this is the significantly longer detection window hair can offer. Hair analysis opens up the opportunity for longitudinal monitoring of drug exposure which would otherwise not be possible with the more traditional and common biological matrices, such as urine and blood. As such, there is a need for more multi-target screening methods covering a broad range of prohibited substances in equine hair at the required sensitivities for equine doping control. This paper describes a sensitive ultra-high performance liquid chromatography – tandem mass spectrometry (UHPLC–MS/MS) method for the detection of 121 drugs and/or their metabolites in equine hair covering ten classes of prohibited substances with estimated limits of detection between 0.1 and 10 pg/mg. To our knowledge, this is the first report of a screening method in equine hair which can cover such a broad range and well over one hundred prohibited substances in a single analytical run. This method has been validated for its specificity, precision and extraction recovery. Applicability of this method has been demonstrated by: (i) the successful identification of clenbuterol, 2-(1-hydroxyethyl) promazine sulfoxide, acepromazine and tetrahydrozoline in genuine equine mane samples; as well as (ii) the detection of drugs from artificially incurred mane hair samples which have been prepared by soaking blank hair samples in solutions of drug targets. [ABSTRACT FROM AUTHOR]

This study proposed the filtration method for removal of inhibitors from real urine samples for immunoassay without centrifuge. Although the inhibitors could not be removed by the physical filtration, the carboxyl group terminated silica effectively removed the inhibitors. In a low pH, antibody formed aggregation due to the protonation. We propose to adjust pH of the sample solution by adding a phosphate buffer solution (pH 7.5). As a result of pretreatment, the SPR immunosensing achieved the SPR signal of 45 mdeg and a low limit of detection with 100 ppq (100 fg mL −1 ). [ABSTRACT FROM AUTHOR]

A novel immunochromatographic assay (ICA) using colored silica nanoparticles (colored SiNPs) as optical directly-detected label was developed for point-of-care detection of clenbuterol (CLE). To our knowledge, this is the first report that using colored SiNPs as nanolabel to develop an ICA. CLE was selected as the model analyte for competitive ICA. The coloration of monodispersed silica nanoparticles (SiNPs) was preceded in water with the presence of aminosilanes and C.I Reactive Violet 5. The purple SiNPs were introduced into a pre-hydrolyzed Stöber system to regrow a silica shell, forming core-shell purple SiNPs. The silica shell prevented dye on SiNPs from leaking. Under appropriate conditions, the core-shell purple SiNPs based ICA was developed to screen for CLE detection by naked eyes at cut-off values of 3 ng/mL, 6 ng/mL and 5 ng/mL in phosphate buffered saline, urine and pork, respectively. The core-shell purple SiNPs based immunoassay system has no cross activity with other nonspecific structural analogues. The results showed that colored SiNPs are promising to be alternative visible labels for the ICA strip. [ABSTRACT FROM AUTHOR]

In this study, a paper-based microfluidic enzyme-linked immunosorbent assay (ELISA) was developed as a screening system for rapid detection of clenbuterol, which is illegally used as a growth promoter for food-producing animals. The microfluidic paper-based analytical device (μPAD) was combined with ELISA and the intrinsic properties of paper allowed the entrapment of antibody through cellulosic fibres, validating to be an alternative to 96-well ELISA microplate for food safety monitoring. Detection of clenbuterol in milk was achieved by measuring the intensity of colour change that was proportional to the analyte concentration with a detection limit of 0.2 ppb. The μPAD effectively reduces the cost, volume of reagents, and time required to run ELISA for food sample testing. [ABSTRACT FROM AUTHOR]

A simple, efficient and sensitive molecularly imprinted electrochemiluminescence sensor (MIECLS) based on reduced graphene oxide (rGO) and upconversion nanoparticles (UCNPs) was developed for determination of clenbuterol (CLB). In this study, rGO generated by electrodeposition of graphene oxide not only acted as carrier for immobilizing UCNPs, but also had a significant impact in boosting electrochemiluminescence (ECL) response of UCNPs thanks to its high conductivity, superior electron transport rate and large specific surface area. UCNPs as an advanced ECL emitter possessed wonderful ECL performance. Furthermore, the introduction of molecularly imprinted polymers (MIP) endowed the ECL sensor a new character of specifically identifying analyte CLB. Under the optimal experimental conditions, the ECL signal was proportional to the logarithm of CLB concentration in the range of 10 nM to 100 µM with a low detection limit of 6.3 nM. The proposed MIECLS combining the advantages of UCNPs-ECL and MIP exhibited good sensitivity, desirable selectivity and favorable stability, indicating enormous potential in the future of food safety detection. [ABSTRACT FROM AUTHOR]

A fully automated spectrophotometric method based on flow-batch analysis has been developed for the determination of clenbuterol including an on-line solid phase extraction using a molecularly imprinted polymer (MIP) as the sorbent. The molecularly imprinted solid phase extraction (MISPE) procedure allowed analyte extraction from complex matrices at low concentration levels and with high selectivity towards the analyte. The MISPE procedure was performed using a commercial MIP cartridge that was introduced into a guard column holder and integrated in the analyzer system. Optimized parameters included the volume of the sample, the type and volume of the conditioning and washing solutions, and the type and volume of the eluent. Quantification of clenbuterol was carried out by spectrophotometry after in-system post-elution analyte derivatization based on azo-coupling using N- (1-Naphthyl) ethylenediamine as the coupling agent to yield a red-colored compound with maximum absorbance at 500 nm. Both the chromogenic reaction and spectrophotometric detection were performed in a lab-made flow-batch mixing chamber that replaced the cuvette holder of the spectrophotometer. The calibration curve was linear in the 0.075–0.500 mg L −1 range with a correlation coefficient of 0.998. The precision of the proposed method was evaluated in terms of the relative standard deviation obtaining 1.1% and 3.0% for intra-day precision and inter-day precision, respectively. The detection limit was 0.021 mg L −1 and the sample throughput for the entire process was 3.4 h −1 . The proposed method was applied for the determination of CLB in human urine and milk substitute samples obtaining recoveries values within a range of 94.0–100.0%. [ABSTRACT FROM AUTHOR]

A molecularly imprinted electrochemiluminescence sensor (MIECLS) is constructed to selectively detect clenbuterol (CLB) based on boron nitride quantum dots@gold nanoflowers/silver nanowires (BNQDs@AuNFs/AgNWs). The abundant amino and hydroxyl groups on the surface of the BNQDs generate an electrostatic self-assembly effect with the multi-tipped spatial structure of AuNFs, constituting a novel nanoscale co-reaction accelerator (NCRA) with high activity and large load capacity. An NCRA embedded in the network structure of the AgNW luminophores significantly promotes the reduction of peroxydisulfate (S 2 O 8 2−) to sulfate anion radicals (SO 4 −•) through the catalysis of amino groups and boron radicals (B•) and the electron acceleration of AuNFs while also reducing the reaction distance between SO 4 −• and AgNWs−•, realizing the multiple synergistic amplification of the electrochemiluminescence (ECL) signal. Imprinted cavities in the molecularly imprinted polymers (MIPs) prepared by electropolymerization can generate a "blocking-effect" by recognizing CLB, realizing ECL signal quenching. Analytical results indicate that the established MIECLS detects CLB in a line concentration range of 0.5–50000 nM and detection limit of 0.00693 nM. The spiked recoveries are 85.90%–97.77%, with the relative standard deviations (RSD) under 5.1%, consistent with those of high-performance liquid chromatography (HPLC). This work demonstrates that an efficient NCRA can significantly enhance the output of the ECL signal in collaboration with the original luminophore, providing a new method to realize the ultra-detection of targeted substances by MIECLS. [Display omitted] • AgNWs with excellent conductivity and transparency were used as ECL luminophores. • Green BNQDs can be electrostatically assembled onto surface of multi-tipped AuNFs. • Novel NCRA based on BNQDs@AuNFs had multiply synergistic catalyses of S 2 O 8 2−. • The MIIECLS showed selective "blocking-effect" detection strategy for clenbuterol. • The sensitive MIECLS had been successfully applied to the detection of real samples. [ABSTRACT FROM AUTHOR]

• Two enantiomers of clenbuterol in pig tissues are separated and quantified. • No reciprocal transformation of two enantiomers during pretreatment is observed. • Chirality distributions of clenbuterol in tissues of pigs are investigated. To establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R -(-)-clenbuterol & S -(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs. A LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid–liquid extraction using tert -butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated. R -(-)-clenbuterol and S -(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5–500 ng/g. Accuracies ranged from −11.9–13.0% for R -(-)-clenbuterol and −10.2–13.2% for S -(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R -(-)-clenbuterol and 1.6–5.9% for S -(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1. The analytical method has good specificity and robustness in determination of R -(-)-clenbuterol and S -(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation. [ABSTRACT FROM AUTHOR]

This study uses media reports to analyze food safety incidents in South Korea that occurred between January 1998 and March 2016. The total number of incidents during this period was 975, with an average of 51.3 per year and 4.3 per month. The top five types of food involved were fruits and vegetables, fish and fish products, meat and meat products, beverages, and confectionery. Of the 975 incidents, 406 (41.6%) were related to chemical hazards—such as food additives and pesticides—221 (22.7%) were due to biological hazards, and 199 (20.4%) were due to physical hazards. Through analysis, this study determines at what stage a breakdown in food safety is likely to occur: primary production is the most common stage with 615 (63.1%) incidents, followed by incidents during the handling and distribution stages, at 240 (24.6%) and 65 (6.7%), respectively. The results of this study can be used as data for risk analysis or food safety strategies. [ABSTRACT FROM AUTHOR]

Clenbuterol is banned for sports use and strictly regulated in livestock industry. In this study, a procedure for the determination of clenbuterol residues in water and urine has been developed using selective supports combined with ion mobility spectrometry (IMS) determination. A molecular imprinted polymer (MIP) was used for efficient clean-up and preconcentration of clenbuterol followed to a dispersive liquid-liquid microextraction (DLLME) in order to improve selectivity and sensitivity of IMS determinations. Quantitative recoveries from 81 to 99% were obtained for water and spiked urine samples using MIP-DLLME-IMS method. A precision of 4.5%, established as the relative standard deviation of analysis at a concentration level of 5 μg L − 1 , was obtained, being the limit of quantification (LOQ) of 0.2 μg L − 1 . A certified reference and several blind-spiked urine samples were analyzed by the proposed methodology and results were compared to a reference ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS-MS) procedure, being statistically comparable. Thus, the proposed methodology is an attractive strategy for the sensitive and selective analysis of clenbuterol residues in water and urine samples. [ABSTRACT FROM AUTHOR]

A fluffy porous ethylenediamine-connected graphene/carbon nanotube composite (EGC), prepared by a simple and time-saving one-pot synthesis, was successfully applied as an adsorbent in pipette-tip solid-phase extraction (PT-SPE) for the rapid extraction and determination of clenbuterol (CLB) from pork. In the one-pot synthesis, carbon nanotubes were inserted into graphene sheets and then connected with ethylenediamine through chemical modification to form a three-dimensional framework structure to prevent agglomeration of the graphene sheets. Under the optimum conditions for extraction and determination, good linearity was achieved for CLB in the range of 15.0–1000.0 ng g −1 ( r = 0.9998) and the recoveries at three spiked levels were in the range of 92.2–96.2% with relative standard deviation ≤9.2% ( n = 3). In comparison with other adsorbents, including silica, NH 2 , C 18 , and Al 2 O 3 , EGC showed higher extraction and purification efficiency for CLB from pork samples. This analytical method combines excellent adsorption performance of EGC and high extraction efficiency of PT-SPE. [ABSTRACT FROM AUTHOR]

As an ideal biomimetic antibody, molecularly imprinted polymer (MIP) has shown great promise in immunoassays. Here, we developed a covalent imprinting method to prepare MIP artificial antibody towards clenbuterol on the well surface of a microtiter plate. Template molecule (clenbuterol) was splited by hydrolysis with functional monomer and removed by methanol/acetic acid solution, and then three-dimensional cavities were fabricated in the MIP, which can rebind template molecule via hydrogen bond interaction. Using this artificial antibody, we developed a novel biomimetic enzyme-linked immunosorbent assay (ELISA) with excellent sensitivity and specificity. The linear range for clenbuterol was 10 −5 to 1000 μg L −1 , with a detection limit of 10 −7 μg L −1 . Cross reactivity of this MIP artificial antibody to other four structural analogs was less than 0.93%. This method was applied to determine clenbuterol in real samples with satisfactory result, suggesting a promising application of the biomimetic ELISA in food and environmental analysis. [ABSTRACT FROM AUTHOR]

For the rapid and robust detection of both parent clenbuterol (CLB) and its metabolites in swine urine samples, a novel quartz crystal microbalance (QCM) sensor array for CLB detection based on molecularly imprinted polymers (MIPs) was developed in this investigation. At first, clenbuterol and the structural analogs of its metabolites, 4-Aminohippuric acid (AHA) and 4-hydroxymandelic acid (HMA), were chosen as molecular templates. Through computational molecular modeling, the optimum ratio between the functional monomer and molecular template was selected. The surface imprinting method was applied to modify QCM electrode surface to graft a thin MIP film. The grafting polymer was characterized by Fourier-transformed infrared spectrometry (FTIR) and atomic force microscopy (AFM), respectively. After then, an array system composed of three sensors was employed to test the responses with different solutions and the principal component analysis (PCA) was adopted to analyze the corresponding data. As a result, for the designed sensor to clenbuterol, a linear equation y=100.07x-722.96 (R 2 =0.9928) was found between the sensor frequency shift ΔF and negative logarithm of clenbuterol concentration (-lgC). The limitation of detection (LOD) was 3.0 ng/mL, which is lower than the Codex Alimentarius Commission regulations residue limit 10 μg/L. The corresponding data of the three template solutions were analyzed by PCA, obtaining 100% recognition. The result demonstrated the feasibility that the developed method could be applied to detect whether the livestock was feed with CLB nutrient redistribution agent by checking the urine samples. [ABSTRACT FROM AUTHOR]

Clenbuterol (CLB), a kind of β 2 -adrenergic agonists, could disrupt cardiovascular and central nervous system. In this work, a new electrochemical non-enzyme sensor for detecting Clenbuterol (CLB) was fabricated based on MoS 2 -Au-PEI-hemin layered nanocomposites. The two-dimensional (2-D) MoS 2 nanosheets were first in-situ assembled with Au nanoparticles, and polyethylenimine (PEI), then hemin molecules were immobilized onto the MoS 2 -Au-PEI film-modified glassy carbon electrode (GCE) via amide bond. Scanning electron microscopy (SEM) and Zeta potential measurements were employed to characterize the MoS 2 -based nanomaterials. Cyclic voltammetry (CV) was used to investigate electrochemical activity of the immobilized hemin on the modified electrode. Upon the optimum conditions, the proposed electrochemical sensor showed an excellent response for CLB including a wide linear ranging from 10 ng/mL to 2 μg/mL and a detection limit (LOD) of 1.92 ng/mL CLB (S/N=3) with favorable reproducibility and stability. Furthermore, this presented method could be feasible for determining CLB in the real pork samples. [ABSTRACT FROM AUTHOR]

Lanthanide ions, the excellent fluorescence markers, have the potential to simplify the performance of multi-analytes analysis. In this work, a powerful time-resolved fluoroimmunoassay (TRFIA) based on dual-labeled format were performed for detecting clenbuterol and salbutamol simultaneously in water and swine feed samples. Europium (Eu 3+ )-labeled goat anti-mouse IgG and Samarium (Sm 3+ )-labeled goat anti-rabbit IgG were prepared and used as fluorescent probes. Under optimal conditions, the half-maximal inhibition concentration (IC 50 ) and the limit of detection (LOD, IC 10 ) of the TRFIA were 0.263 and 0.007 ng/mL for clenbuterol, and 0.179 and 0.014 ng/mL for salbutamol, respectively. The cross-reactivities of the TRFIA between these two β 2 -agonists and with their analogues were negligible, thus indicated high specificity and promoted the simultaneous determination. The spiked recoveries were 72.2%–102.2% with relative standard deviations (RSDs) of 3.4–13.8% for clenbuterol and salbutamol in water and swine feed samples. Furthermore, the results of TRFIA correlated well with those obtained by HPLC. The proposed dual-labeled TRFIA was a satisfactory tool for simultaneous, sensitive, efficient and labour-saving detection of clenbuterol and salbutamol in water and swine feed samples. [ABSTRACT FROM AUTHOR]

The potential of a commercially available imidazolium-based ionic liquid (C 2 C 1 imC 1 SO 4 ) to selectively separate two model emerging contaminants, ethynylestradiol and clenbuterol, from aqueous solutions containing non-ionic surfactants (Triton X-100 and Triton X-102) was proved. After a first stage where the binodal curves and tie-lines of the systems were obtained, all the experimental data were suitably correlated with different empirical equations. Then, a 2-step sequential extraction of ethynylestradiol and clenbuterol was investigated by means of a factorial design. In this way, while ethynylestradiol completely partitions to the light layer no matter the operating conditions, clenbuterol extraction can be tuned by a cautious selection of the ionic liquid concentration. Then, while ionic liquid concentrations higher than 70% led to about 4% of clenbuterol extraction in the upper phase, the operation at concentration levels lower than 18% entailed up to 96% of removal. The confirmation of this behavior with synthetic urine proved the relevance of the present data for the implementation of a separation process allowing the selective removal of clenbuterol and ethynylestradiol from aqueous streams, which poses undoubted significant environmental and analytical interest. [ABSTRACT FROM AUTHOR]

A novel method coupling molecular imprinted monolithic column with two-dimensional liquid chromatography was developed and validated for the analysis of clenbuterol in pork liver and swine urine samples. The polymers were characterized by using Fourier transform infrared spectroscopy, nitrogen adsorption desorption analyses, frontal analysis and the adsorption of selectivity. The results indicated that the imprinted columns were well prepared and possessed high selectivity adsorption capacity. Subsequently, the MIMC-2D-LC (molecular imprinted monolithic column-two dimensional liquid chromatography) method was developed for the selective analysis of clenbuterol in practical samples. The accuracy ranged from 94.3% to 99.7% and from 93.7% to 99.6% for liver and urine, respectively. The relative standard deviation (RSD) of repeatability was lower than 8.6% for both analyses. The limit of detections was 16 ng·mL −1 for liver and 25 ng·mL −1 for urine, respectively. Compared with the reported methods, the disturbance of endogenous impurity could be avoided by the 2D-LC method. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, and currently there is no effective cure for this devastating disease. Decreases in the levels of β 2 -adrenoceptor (β 2 -AR) and norepinephrine have been reported in several regions of AD brains. The activation of β 2 AR can prevent the amyloid β (Aβ)-mediated inhibition of LTP (Long-term potentiation), but the mechanism is not fully understood. Here, we used APP/PS1 mice to study whether the activation of β 2 AR could remodel synaptic and/or dendritic plasticity. We found that the activation of β 2 AR by Clenbuterol (Clen) ameliorated memory deficits and promoted dendrite ramification and spine generation in hippocampal CA1 neurons, which was accompanied by the upregulation of postsynaptic density protein 95 (PSD95), synapsin 1 and synaptophysin. Conversely, the inhibition of β 2 AR by a siRNA blocked the Clen-induced increase in dendrite ramification and dendritic spines in primary hippocampal neurons. Furthermore, the activation of β 2 AR decreased cerebral amyloid plaques through the up-regulation of α-secretase activity and by decreasing the phosphorylation of APP at Thr668. Based on the roles of β 2 AR in dendrite ramification and spine generation, memory deficits and AD pathogenesis, compounds designed to activate β 2 AR might shed light on the cure of AD. [ABSTRACT FROM AUTHOR]

[Display omitted] • The simultaneous detection of RAC and CLB has been achieved by HPMo/MoS 2 composite. • HPMo nanoparticles are well distributed on the MoS 2 nanofibers of GCE surface. • The sensor was applied to the simultaneous detection of RAC and CLB in pork sample. A novel electrochemical sensor based on MoS 2 nanofibers and polyoxometalate H 3 PMo 12 O 40 (HPMo) composite was used for the highly selective and sensitive simultaneous detection of ractopamine (RAC) and clenbuterol (CLB). The electrochemical sensor was prepared firstly by MoS 2 electrospun nanofibers as polyoxometalate (POM) carrier. Then, the morphology structure and physicochemical properties of the nanofiber composites modified electrode were characterized by X-ray diffraction (XRD), scanning electron microscopic (SEM), nitrogen adsorption/desorption isotherms, X-ray photoelectron spectroscopy (XPS), Fourier Transform Infrared Spectroscopy (FTIR) and electrochemical technologies. The results showed that HPMo decorated MoS 2 electrospun nanofibers as modified electrodes can not only promote electron transfer but also enhance electrochemical sensing ability towards RAC and CLB. Differential pulse voltammetry (DPV) results proved that the proposed sensor exhibits good stability, high sensitivity and selectivity in terms of a wide range of 1–70 μM for RAC and 1–350 μM for CLB and a low detection limit of 0.056 μM for RAC and 0.085 μM for CLB, respectively. Furthermore, the proposed sensors were applied to real pork sample analysis, which the recovery of RAC and CLB were 98.5 % − 105.6 % and 98.9 % − 103.1 %, respectively. The results indicate that the electrochemical sensors have a wide application prospect in the detection of β-agonists in animal food sample. [ABSTRACT FROM AUTHOR]

There is a lack of data on the effects of chronic exposure to common drugs and stimulants on the developing nervous system. Freshwater planarians have emerged as a useful invertebrate model amenable to high-throughput behavioral phenotyping to assay chemical safety in adult and developing brains. Here, we leverage the unique strength of the system to test in parallel for effects on the adult and developing nervous system, by screening ten common drugs and stimulants (forskolin, clenbuterol, LRE-1, MDL-12,330A, adenosine, caffeine, histamine, mianserin, fluoxetine and sertraline) using the asexual freshwater planarian Dugesia japonica. The compounds were tested up to 100 μM nominal concentration for their effects on planarian morphology and behavior. Quantitative phenotypic assessments were performed on days 7 and 12 of exposure using an automated screening platform. The antidepressants sertraline and fluoxetine were the most potent to induce lethality, with significant lethality observed at 10 μM. All ten compounds caused sublethal morphological and/or behavioral effects, with the most effects, in terms of potency and breadth of endpoints affected, seen with mianserin and fluoxetine. Four of the compounds (forskolin, clenbuterol, mianserin, and fluoxetine) were developmentally selective, causing effects at lower concentrations in regenerating planarians. Of these, fluoxetine showed the greatest differences between the two developmental stages, inducing many behavioral endpoints in regenerating planarians but only a few in adult planarians. While some of these behavioral effects may be due to neuroefficacy, these results substantiate the need for better evaluation of the safety of these common drugs on the developing nervous system. • Multidimensional planarian screening reveals differential neuronal effects of drugs. • Mianserin caused many morphological and behavioral effects in planarians. • Fluoxetine exhibited developmentally selective behavioral effects. [ABSTRACT FROM AUTHOR]

• Bismuth sulfide nanoparticles (Bi 2 S 3 NPs) were used for the first time as a signal substrate in LFIA. • Bi 2 S 3 NPs can connect to the anti-clenbuterol monoclonal antibody (mAb) through hydrogen bonding force. • As a label tag, more mAbs are bound by Bi 2 S 3 NPs, and Bi 2 S 3 NPs-mAb has binding stability. • The analysis results of milk, beef, and pork, show that Bi 2 S 3 NPs-LFIA has excellent practicability in food samples. Improving detection sensitivity is still a major research emphasis for lateral flow immunoassay (LFIA). Increasing the binding efficiency and stability of the probe is an achievable and effective solution. In this work, we developed a highly sensitive lateral flow immunoassay for clenbuterol detection by using bismuth sulfide nanoparticle (Bi 2 S 3) nanoparticles (NPs) as a novel marker. Here, Bi 2 S 3 NPs can link with the antibody by hydrogen bonding to improve the performance of the probe, e.g., stability and sensitivity. Benefiting from the direct hydrogen bonding between Bi 2 S 3 NPs and the monoclonal antibody (mAb), high sensitivity is obtained by the proposed LFIA with a lower visible detection limit of 0.1 ng mL−1 and a cut-off value of 4 ng·mL−1 for CLE detection, which is 5-fold and 7.5-fold improved than the conventional Au NPs based LFIA. In addition, the encouraging practical application results in milk, pork, and beef show that the bismuth sulfide nanoparticle has a great popularizing potential in the performance promotion of LFIAs for food safety monitoring. [ABSTRACT FROM AUTHOR]

• Some chemical dyes were studied to improve the catalytic activity of HRP. • Chemical staining strategy for enhancing performance of ELISA was firstly proposed. • Sensitivity and detection range of proposed ELISA were enhanced simultaneously. Exploring a novel strategy for strengthening the catalytic activity of enzyme facilitates the development of a sensitive enzyme-linked immunosorbent assay (ELISA). Herein, a chemical staining (CS) strategy was firstly discovered to possess the ability to directly improve the catalytic activity of horseradish peroxidase. Based on this discovery, coomassie brilliant blue was introduced into ELISA to establish a CS enhanced ELISA (CS-ELISA) to detect clenbuterol (CL) by simply staining monoclonal antibodies. Satisfactorily, the most important analytical parameters of CS-ELISA, including sensitivity (0.074 ng mL−1) and linear range (0.2–2 ng mL−1) were all improving 2-folds compared with conventional ELISA. Moreover, the CS-ELISA shows good applicability in the detection of CL in pork tenderloin samples. The proposed CS-ELISA shows various advantages, such as cost-effective, easily accessible, enhanced catalytic activity of enzyme, higher sensitivity, and broader linear range, providing a new insight into enhanced ELISA for food safety. [ABSTRACT FROM AUTHOR]

Immunochromatographic test strips (ITSs) have gained great practicability in point-of-care testing (POCT). Developing novel labels of ITSs with an additional signal to carry out quantitative analysis favors the development of sensitive POCT. Herein, tannic acid (TA) assisted stripping WS 2 nanosheets (TA-WS 2 NSs) based on colorimetric/photothermal ITS (TWITS) for clenbuterol (CLE) monitoring. After WS 2 NSs loaded TA, it retained excellent photothermal performance and an intact surface structure for monoclonal antibody (mAb) labeling, at the same time, the stability of WS 2 NSs and the activity of mAb were improved effectively. Satisfactory, the visual limit of detection (vLOD) of the photothermal signal was 0.3 ng/mL, which was enhanced 7-fold at least compared with traditional gold-based nanoparticles ITS (GNPs ITS). Importantly, this work can offer a new referenced method for other small molecule pollutants. • Developed a sensitive colorimetric/photothermal sensor for clenbuterol detecting in food. • Tannic acid improved effectively the stability of WS 2 NSs and the activity of monoclonal antibody. • The result of quantitative analysis effectively improve the visual limit of detection (vLOD) of the strip. [ABSTRACT FROM AUTHOR]

We have developed a fast, highly sensitive and low-cost biosensing system for the detection of clenbuterol (CLB), using a homemade mobile electrochemical device with an electric field-driven acceleration strategy. This system consists of an embedded circuit in smartphone for signal processing and a screen-printed carbon electrode (SPCE) modified with multi-walled carbon nanotubes (MWNTs) and goat anti mouse-immunoglobulin G (IgG) sensing layer (MWNTs-I-layer). CLB monoclonal antibody was assembled through its binding to the surface-confined antibody. Such modified electrodes were used for rapid and sensitive amperometric immunosensing detection of CLB. Horseradish peroxidase-coupled CLB (CLB–HRP) competed with free CLB in the samples to bind the monoclonal antibody. By using this mobile system, we could detect CLB ranging from 0.3 ng ⋅ mL − 1 to 100 ng ⋅ mL − 1 with the detection limit of 0.076 ng ⋅ mL − 1 . The whole competitive-type detection process was finished within 6 min. We expect this device can meet the requirements for field detection of various food security-related species. [ABSTRACT FROM AUTHOR]

In this work, a highly sensitive electrochemiluminescence (ECL) sensor for the detection of clenbuterol (CBT) in pork is reported. The fabrication of the sensor was carried out by modification of a glassy carbon electrode (GCE) with platinum nanoparticles (PtNPs)/silica nanoparticles doped with [Ru(bpy) 3 ] 2 + (RuSiNPs)/Nafion composite. The morphology, composition and electrochemical behavior of the PtNPs/RuSiNPs were characterized by scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy and electrochemical impedance spectroscopy, respectively. It was found that all the PtNPs are formed on the surface of RuSiNPs and the electron transfer resistance was obviously reduced on the PtNPs/RuSiNPs GCE. Taking the advantage of the Si-O-Si grid of RuSiNPs, the [Ru(bpy) 3 ] 2 + luminophore leaching from the modified electrode could largely be prevented, benefiting the ECL signal stability. Moreover, due to the enhanced electron transfer rate and electrocatalytical property of the PtNPs, the sensitivity of the PtNPs/RuSiNPs-based sensor was greatly improved. Under optimized conditions, the ECL peak intensity was linear with the concentration of CBT in the range of 5–100 ng mL − 1 , with a detection limit as low as 0.8 ng mL − 1 . The recoveries of the CBT obtained from spiked pork samples at PtNPs/RuSiNPs/Nafion GCE were between 89.5% and 106.3% and the RSDs were lower than 6.21%. The proposed ECL sensor based on PtNPs/RuSiNPs composite presented good characteristics for CBT determination in terms of rapid, high sensitivity and good stability, promising the applicability of this sensor in real samples. [ABSTRACT FROM AUTHOR]

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, aβ2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy. [ABSTRACT FROM AUTHOR]

Increased activity of the noradrenergic system in the amygdala has been suggested to contribute to the hyperarousal symptoms associated with post-traumatic stress disorder (PTSD). However, only two studies have examined the content of noradrenaline or its metabolites in the amygdala of rats previously exposed to traumatic stress showing inconsistent results. The aim of this study was to investigate the effects of an inescapable foot shock (IFS) procedure (1) on reactivity to novelty in an open-field (as an index of hyperarousal), and (2) on noradrenaline release in the amygdala during an acute stress. To test the role of noradrenaline in amygdala, we also investigated the effects of microinjections of propranolol, a β-adrenoreceptor antagonist, and clenbuterol, a β-adrenoreceptor agonist, into the amygdala of IFS and control animals. Finally, we evaluated the expression of mRNA levels of β-adrenoreceptors (β1 and β2) in the amygdala, the hippocampus and the prefrontal cortex. Male Wistar rats (3 months) were stereotaxically implanted with bilateral guide cannulae. After recovering from surgery, animals were exposed to IFS (10 shocks, 0.86 mA, and 6 s per shock) and seven days later either microdialysis or microinjections were performed in amygdala. Animals exposed to IFS showed a reduced locomotion compared to non-shocked animals during the first 5 min in the open-field. In the amygdala, IFS animals showed an enhanced increase of noradrenaline induced by stress compared to control animals. Bilateral microinjections of propranolol (0.5 μg) into the amygdala one hour before testing in the open-field normalized the decreased locomotion observed in IFS animals. On the other hand, bilateral microinjections of clenbuterol (30 ng) into the amygdala of control animals did not change the exploratory activity induced by novelty in the open field. IFS modified the mRNA expression of β1 and β2 adrenoreceptors in the prefrontal cortex and the hippocampus. These results suggest that an increased noradrenergic activity in the amygdala contributes to the expression of hyperarousal in an animal model of PTSD. [ABSTRACT FROM AUTHOR]

Clenbuterol is a β-adrenergic agonist used as additive to increase the muscle mass of meat-producing animals. Previous studies were limited to evaluations of animal growth performance and determination of the residues. Several studies have focused on urine samples. Little information about the underlying molecular mechanisms that can explain Clenbuterol metabolism and promote energy repartition in animal muscle and fatty tissue is available. Therefore, this research aims to detect the metabolite variations in muscle and fatty tissue acquired from Chinese pigs fed with Clenbuterol using gas chromatography/mass spectrometry (GC/MS). Ten two-month old Enshi black pigs were fed under the same condition; five of which were fed with basic ration containing Clenbuterol for one month, whereas the other five pigs were fed only with basic ration. Muscle and fatty tissue were subjected to metabolomics analysis using GC/MS. Differences in metabolomic profiles between the two groups were characterized by multivariate statistical analysis. The muscle samples showed that 15 metabolites were significantly different in the Clenbuterol-treated group compared with the control group; 13 potential biomarkers were found in the fatty tissue. Most of the metabolites were associated with fatty acid metabolism and amino acid metabolism. Glycerol, phenylalanine, and leucine were the common metabolites between the muscle and fatty tissue. These metabolites may provide a new clue that contributes to the understanding of the energy reassignment induced by Clenbuterol. [ABSTRACT FROM AUTHOR]

Electrochemical immunosensors based PEDOT/MWCNT hybrid composite for detection of clenbuterol antibiotic has been developed. It shows promising properties as biosensors platform with combination of high conductive properties of poly(3,4-ethylenedioxythiophene) (PEDOT) and high surface area of carbon nanotube (CNT). Based on cyclic voltammetry (CV) measurement in potassium ferricyanide (K 3 [Fe(CN) 6 ]), lower peak potential separation (90.33 mV) and higher peak current (0.197 mA) were obtained compared with unmodified electrode (114.75 mV and 0.163 mA).This immunosensor, which was based on direct competitive method, was examined with standard clenbuterol hydrochloride for detection of clenbuterol through chronoamperometry at -0.1 V. [ABSTRACT FROM AUTHOR]

β-agonists are phenylethanolamines with different substituent groups on the aromatic ring and the terminal amino group which have the effect of nutrition redistribution and can accumulate in body tissues causing acute or chronic poisoning when consumed. Therefore, it is very important to establish a fast screening method for the detection of several kinds of β-agonists in food safety control. In this study, the aptamer-agonists (AP-Ago) has screened out by Isothermal Titration Calorimetric method. AP-Ago was a single-strand DNA with 22 base-pairs. The dissociation constant ( K d ) to phenylethanolamine (PHL) was 3.34×10 −5 mol L −1 . The AP-Ago based electrode was constructed by self-assembling on gold electrode. A label-free electrochemical aptasensor was then developed with AP-Ago-based gold electrode, which was sensitive to phenylethanolamine(PHL), clenbuterol (CLB), ractopamine (RAC), salbutamol (SAL) and procaterol (PRO). The detection limits were 0.04 ng/mL (RAC), 0.35 pg/mL (CLB), 1.0 pg/mL (PHL), 0.53 pg/mL (SAL) and 1.73 pg/mL(PRO), respectively, The detection time was 15 min. The reproductivity of the mentioned aptasensor is good with RSD of 2.09%. Comparing with ELISA and HPLC on β-agonists detection in actual sample, this aptasensor is advantage of fewer steps and fast screen-detection of these five β-agonists or their mixtures. This study suggests that the aptasensor can be developed to a rapid screening means with multi-β-agonists (may be one or more) in sample. [ABSTRACT FROM AUTHOR]

A novel multiplexed immunochromatographic assay (ICA) based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of β-agonists, by utilizing ractopamine (RAC) and clenbuterol (CLE) as the models. Different from conventional multiplexed ICA methods which usually require two or more test lines, this strategy was developed for detection of two β-agonists by using only one test line on the nitrocellulose membrane. In this study, horseradish peroxidase and alkaline phosphatase were used as the signal probes to label RAC antibody and CLE antibody, respectively. The two CL reactions with flash type and glow type kinetics characteristics were triggered simultaneously by injecting the coreactants, then the signals for RAC and CLE detections were recorded at 3 s and 300 s after coreactants injection, respectively. Owing to the utilization of CL detection, this protocol showed ideal sensitivity for quantitation. Under the optimal conditions, the detection limits for RAC and CLE were 0.17 ng mL −1 and 0.067 ng mL −1 (S/N = 3), respectively. The whole assay process can be accomplished within 20 min without complicated sample pretreatment. The proposed method was successfully applied for the detection of RAC and CLE in spiked swine urine. It opens up a new pathway for designing a low cost, time-efficiency and multiplexed strategy for rapid screening and field assay. [ABSTRACT FROM AUTHOR]