In this work, a library-immobilization method was used to select DNA aptamers for ractopamine (RAC) and clenbuterol (CLB), two common "lean meat agents". Two RAC-binding aptamers, RAC-1 and RAC-5, exhibit dissociation constants (Kd) of 6.8 and 2.9 μM, respectively, based on isothermal titration calorimetry (ITC). Similar values were also obtained using a thioflavin T (ThT) fluorescence assay. A CLB aptamer named CLB-1 exhibited a Kd of 561 nM by the ThT assay and 885 nM by ITC. The RAC-5 aptamer showed a modest response to CLB, whereas CLB-1 exhibited strong specificity to CLB. Comparisons were made with previously published aptamers, and the aptamers reported here were shorter, had well-defined secondary structures, and were validated by rigorous binding assays. Furthermore, the ThT fluorescence responses of RAC-5 and CLB-1 were used to develop a label-free fluorescent sensor array to distinguish RAC and CLB from each other and from other structurally related molecules. These individual sensors had limits of detection of 1.2 μM for RAC and 0.16 μM for CLB. Finally, the label-free aptamer sensor was applied to spiked cat food and pork samples. These results demonstrate the potential of these aptamers for practical applications in food additive monitoring and safety assessment.

Abstract This study presents a novel electrochemical sensing platform based on a glassy carbon electrode modified with a mixed-metal metal–organic framework, Materials Institute of Lavoisier (MIL)-53(Al, Fe), for the highly sensitive detection of clenbuterol (CLE). The MIL-53(Al, Fe) material was synthesized and thoroughly characterized by x-ray diffraction, Fourier transform infrared spectroscopy, scanning and transmission electron microscopy, and nitrogen adsorption–desorption analysis, confirming the preservation of the MIL-53 framework and a high specific surface area, with numerous accessible active sites. For the first time, MIL-53(Al, Fe) is shown to act as an efficient electrocatalyst for CLE oxidation, significantly improving electron-transfer kinetics via a proton-coupled electron-transfer mechanism. Compared with the bare electrode, the modified electrode demonstrates a markedly increased current response, enhanced sensitivity, and a low detection limit of 0.11 μM within a linear range of 0.40–4.59 μM. The sensor also exhibits excellent repeatability, stability, and selectivity against common interfering species. Practical applicability was confirmed by the successful detection of CLE in pork samples, with satisfactory recoveries of 94.0%–104.1%. This work highlights the potential of mixed-metal MIL-53-based electrodes as a reliable strategy for on-site monitoring of CLE, thereby improving food safety control and regulatory surveillance.

Abstract The objective of this study is to detect residues of β-agonists, salbutamol (SAL), ractopamine (RCT), and clenbuterol (CLB), in meat and liver matrices simultaneously using high-performance liquid chromatography with a PDA detector. Evaluation of accuracy at three spiking levels (0.08, 0.5, and 1 μg/g for SAL and RCT; 0.0625, 0.125, and 0.25 μg/mL for CLB) produced recovery rates between 80.15±5.98% and 94.97±8.39%. Five calibration levels showed correlation coefficients (r) of 0.9996 to 0.9998 for SAL, RCT, and CLB. The limit of detection (LOD) of 0.1 ng/g (SAL), 0.2 ng/g (RCT), and 0.02 ng/g (CLB), and the limit of quantification (LOQ) of 0.4 ng/g, 0.85 ng/g, and 0.06 ng/g, respectively. These results are considerably lower than the established maximum residue limits (MRLs) for RCT and CLB, and they are adequate for SAL screening. An analysis of 60 samples revealed no SAL, RCT, or CLB in either meat or liver, except for two liver samples, which contained 2.92 μg/kg of SAL and 2.5 μg/kg of RCT. The modified extraction procedure and detection of the HPLC method were successfully developed for the simultaneous detection of β-agonists residues in beef and liver samples.

A Dopamine D1-Like Receptor Agonist Ameliorates Traumatic Brain Injury Through its Immunosuppressive Effects.

Rapid detection of clenbuterol and ractopamine in pork: Sulfonate functionalized ion exchangemagnetic solid phase extraction combined with visual protein microarray chip.

Beta-agonists are growth promoters sparking considerable interest in animal husbandry. However, their numerous negative effects on health have led to a number of restrictions on their presence in agricultural products, which differ depending on the type of preparation and food. In this regard, there is a demand for methods of their mass, rapid, and easy control with strict, focused selectivity. In this paper, a lateral flow immunoassay (LFIA) with clenbuterol (CLE), a priority β2-agonist for food safety, as the main target compound is proposed. The LFIA is based on indirect labeling of specific antibodies by gold nanoparticles via anti-species antibodies. The development of the LFIA involved optimizing the concentrations of immunoreagents and the composition of the reaction mixture (buffer type and pH, ionic strength, detergents, and additional components). In qualitative (visual) mode, the LFIA detects up to 1.0 ng/mL of CLE. In quantitative mode, the detection limit reaches 0.02 ng/mL, surpassing previously described colorimetric LFIAs. The selectivity of the obtained and used monoclonal antibodies allows for the group-specific detection of CLE and structurally close (the presence of a trimethyl residue, similar charge distribution in the benzene ring) common β2-agonists—salbutamol and mabuterol—distinguishing them from other β-agonists, including the widely used β1-agonist ractopamine, which differs in application and biological activity. The assay time is 15 min. The application of the LFIA for meat samples demonstrated that the CLE recovery ranged between 86% and 104%. The obtained results confirm the effectiveness and competitive potential of the developed assay for screening meat products outside of laboratories.

The illegal use of β-agonists like clenbuterol (CLB) and ractopamine (RAC) as livestock growth promoters poses severe threats to human health. To address this, we developed a novel electrochemical sensor based on violet phosphorene (VP) and carboxylated multi-walled carbon nanotubes (COOH-MWCNTs) with Nafion-isopropanol mixed solution for simultaneous voltammetric detection of CLB and RAC in beef. The sensing platform was optimized through response surface methodology coupled with particle swarm optimization, enabling multi-objective parameter (VP concentration, COOH-MWCNTs concentration, and pH value) refinement that enhanced sensitivity while minimizing experimental trials. This design leveraged the synergistic properties of VP (high carrier mobility) and COOH-MWCNTs (electrocatalytic activity), yielding a sensor with ultra-low detection limits of 5.8nM for CLB and 6.4nM for RAC, excellent selectivity with negligible interference from biological matrices, and high stability. Support vector machine (SVM) nonlinear models achieved robust quantification (R2 > 0.9948 and RPD > 7.09 for both analytes), validated by high recoveries (98.4-103.1% for CLB and 96.4-103.7% for RAC) in beef samples. This work establishes a rapid, cost-effective platform for monitoring β-agonist residues, advancing food safety surveillance.

In this study, rice straw-based carbon dots (RSCD) synthesized via a hydrothermal method were effectively utilized for modifying glassy carbon electrodes in differential pulse voltammetry to determine ractopamine (RTP) and clenbuterol (CLB) simultaneously. The structural, optical, and elemental properties of RSCD were characterized using X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, tunneling electron microscopy, UV–vis, photoluminescence spectra, and energy-dispersive X-ray elemental mapping. The RSCD-modified glassy carbon electrode (RSCD/GCE) demonstrated enhanced electrochemical outcome in cyclic voltammetry tests and differential pulse voltammetry tests, compared to bare GCE. The RSCD/GCE showed distinct oxidation peaks for RTP (+0.88 V) and CLB (+1.88 V), indicating high selectivity. The method stated a broad linear concentration range of 0.1–92 μM with limits of detection of 0.09 μM for RTP and 0.03 μM for CLB. Interference studies revealed negligible impact from common ions and organic substances. The RSCD/GCE demonstrated good repeatability, reproducibility, and stability over a week. This study underscores the potential of RSCD/GCE as a working electrode for reliable and sensitive DPV determination of β-agonists residue in food safety applications.

In the present study, the carbon dots (CD) were extracted from rice straw (RS) and denoted as RSCD. The obtained material was examined by using techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM), and nitrogen adsorption/desorption isotherms. The RSCD material was employed as an electrode modifier to improve the effectiveness of an electrochemical sensor for determining clenbuterol (CLB). RSCD showed fabulous oxidation towards clenbuterol. The modest detection limit could confirm an LOD value of 0.094 µM within a linear range between 0.1 and 1.8 µM under suitable conditions and the proposed technique –– the differential pulse voltammetry (DPV). The developed sensor exhibited several benefits: simplicity, low cost, effortless generation, fabulous selectivity, and good reproducibility.

Asymmetric CRISPR-Cas12a powered electrochemical aptasensor for clenbuterol detection based on competitive gRNA mediated cascade signal amplification

A clenbuterol detection method based on magnetic separation up-conversion fluorescent probe

Sports doping remains a significant challenge in competitive sports. Given that urine analysis is the standard for detecting doping, developing rapid, sensitive, accurate, and high-throughput methods for stimulant detection in urine is crucial. Surface-enhanced Raman scattering (SERS) tag-based immunoassays have emerged as powerful analytical tools known for their high sensitivity and specificity, holding particular promise for stimulant detection in urine samples. However, both the Raman signals of typical SERS tags and sample matrices are within the Raman fingerprint region (<1800 cm-1), which could lead to spectrum overlap, potentially reducing detection accuracy and sensitivity. By recognizing this, we designed a competitive immunoassay that integrates two types of zero-background SERS tags and magnetic separation. These innovative SERS tags exhibit distinctive Raman peaks within the Raman-silent region (1800-2800 cm-1), effectively mitigating potential spectrum overlap with background sample signals. Moreover, magnetic separation not only enhances operational simplicity but also improves the system's anti-interference capability. Using clenbuterol (CL) and higenamine (HM) as model targets, the SERS-based competitive immunoassay demonstrated sensitive detection of individual CL or HM standards, with limits of detection (LODs) of 0.87 and 0.71 pg/mL, respectively. In multiplex mode, CL and HM can be simultaneously detected with LODs of 1.0 and 0.81 pg/mL, respectively. Furthermore, the recovery rates in urine samples ranged from 83 to 116% (relative standard deviation, RSD ≤ 6.4%) for CL and from 82 to 103% (RSD ≤ 5.1%) for HM, further confirming the reliability of the SERS-based immunoassay for practical applications.

Half-antibody spur one-to-one recognition mechanism can function with chemical-staining technology for next-generation lateral flow immunoassay

Fabrication of Raman reporter molecule–embedded magnetic SERS tag for ultrasensitive immunochromatographic monitoring of Cd ions and clenbuterol in complex samples

Core–satellite-structured magnetic nanozyme enables the ultrasensitive colorimetric detection of multiple drug residues on lateral flow immunoassay

Conventional lateral flow immunoassay (LFIA) usually suffers from poor antimatrix interference, unsatisfactory sensitivity, and lack of quantitative ability for target analyte detection in food matrices. In response to these limits, here, multifunctional nanomaterial ZnFe2O4 nanoparticles (ZFOs) were developed and integrated into LFIA for powerful magnetic separation/enrichment and colorimetric/photothermal target sensing. Under optimum conditions, the detection for clenbuterol (CL) with magnetic enrichment achieves 9-fold higher sensitivity compared to that without enrichment and 162-fold higher sensitivity compared to that based on traditional colloidal golds. Attributing the improved performances of ZFOs, CL can be detected at ultralow levels in pork and milk with 10 min of immunoreaction time. The vLODs were 0.01 μg kg-1 for two modes, and the cutoff values of CL were about 5 and 3 μg kg-1, respectively. More importantly, the enrichment ZFO-mediated LFIA (ZE-LFIA) exhibits a similar limit of detection (LOD) in both buffer solution and food matrix, demonstrating a universal resistance to the food matrix. The multitudinous performance merits of this ZE-LFIA with high sensitivity, matrix tolerance, accuracy, and specificity have ensured a broad application potential for target detection of clenbuterol and can serve as an experience for other veterinary drug residues' detection.

A high-accuracy measurement procedure for salbutamol, ractopamine, and clenbuterol in pork by isotope dilution-liquid chromatography/tandem mass spectrometry

We aimed to determine associations between experimentally impaired uterine clearance or treatment with ecbolic drugs on luteal development in estrous mares after insemination. In a crossover design, eight mares were treated with saline (CON), clenbuterol (CLEN), oxytocin (OXY) and carbetocin (CARB) from the day of first insemination until 2 days after ovulation. Between treatments, the mares rested for one cycle. Estrous mares were examined for the presence of free intrauterine fluid by transrectal ultrasound. Endometrial swabs for cytology and bacteriology were collected on days 1 and 14. Blood samples were collected daily before AI until day 14 after ovulation for determination of progesterone and PGF2α metabolites (PGFM). Differences between treatments were compared by a general linear model for repeated measures (SPSS 29). One mare was excluded because of a uterine infection in the control cycle. In all other mares, only minor amounts of free intrauterine fluid were present after insemination and decreased over time (P<0.05) with no treatment x time interaction. There was no effect of treatment on polymorphonucleated cells (PMN) in endometrial cytology after ovulation or PGFM secretion. Progesterone release from day 1–14 as well as pregnancy rate and conceptus size on day 14 was not influenced by treatment. In conclusion, treatment with clenbuterol does not impair uterine clearance in estrous mares resistant to endometritis. Repeated injection of the oxytocin analogue carbetocin during the early postovulatory period is not detrimental to corpus luteum function and can be recommended to enhance uterine clearance.

Machine learning powered CN-coordinated cobalt nanoparticles embedded cellulosic nanofibers to assess meat quality via clenbuterol monitoring

Clenbuterol (CLB) as an illegal feed additive may cause a great security risk to food safety. However, convenient and efficient detection means for CLB in practical application remain a formidable challenge. Herein, a stable Eu-based organic framework {[H2N(CH3)2]2[Eu2(ttca)2]·H2O}n (compound 1) (H4ttca = [1,1':2',1″-terphenyl]-4,4',4″,5'-tetracarboxylic acid) has been harvested, exhibiting excellent chemical stability and thermal stability. Luminescence investigation reveals that compound 1 can sensitively and selectively detect CLB without being affected by different components from simulated serum and urine (limit detection: 22.7 nM). Furthermore, sensor 1 can also be applicable to CLB recognition in real swine feeds, presenting excellent anti-interference performance. The good cyclicity of compound 1 endows CLB determination with many advantages: low cost, high stability, and simplicity. Importantly, in view of the indication of the luminescence color (red to blue), test membranes were fabricated and employed for convenient and fast CLB detection, providing a valuable scheme for the visual monitoring of CLB in meat products. This work enriches rare earth metal compounds and luminescence sensor portfolios and breaks the concentration record (nM) for detecting CLB compared with reported complex materials, providing an effective monitoring platform for CLB visually.