Production of a matrix certified reference material for measurement and risk monitoring of clenbuterol in mutton.

A novel matrix certified reference material (CRM) for clenbuterol in mutton (GBW 10216) was developed to assist measurement and risk monitoring of clenbuterol in mutton. The candidate CRM raw samples were obtained by oral administration of clenbuterol and investigating the pharmacokinetics of clenbuterol in sheep. A high-precision isotope dilution coupled with liquid chromatography tandem mass spectrometry (LC-ID-MS/MS) method was established and assigned the value of clenbuterol in mutton powder through combined detection of nine inter-laboratories. The certified value with expanded uncertainty was 21.1 ± 2.2 µg/kg (k = 2, 95% confidence) for clenbuterol in mutton. The prepared matrix CRM was sufficiently homogeneous between and within bottles. The long-term stability of clenbuterol in mutton powder was evaluated for 12 months at -20℃ and short-term stability for 7 days at 4℃ and 50℃. The uncertainties originating from characterization, homogeneity, and stability were systematically analyzed and evaluated. The prepared matrix CRM can be applied for proficiency testing and nationwide risk monitoring programs to guarantee the accuracy and comparability of clenbuterol measurement results in mutton.

Determination of ß2-Agonist Residues in Fermented Ham Using UHPLC-MS/MS after Enzymatic Digestion and Sulfonic Resin Solid Phase Purification.

ß2-agonists are a class of synthetic sympathomimetic drugs with acute poisoning effects if consumed as residues in foods. To improve the efficiency of sample preparation and to overcome matrix-dependent signal suppression in the quantitative analysis of four ß2-agonists (clenbuterol, ractopamine, salbutamol, and terbutaline) residues in fermented ham, an enzyme digestion coupled cation exchange purification method for sample preparation was established using ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). Enzymatic digests were subject to cleanup treatment on three different solid phase extraction (SPE) columns and a polymer-based strong cation resin (SCR) cartridge containing sulfonic resin was found to be optimal compared with silica-based sulfonic acid and polymer sulfonic acid resins based SPEs. The analytes were investigated over the linear range of 0.5 to 10.0 µg/kg with recovery rates of 76.0-102.0%, and a relative standard deviation of 1.8-13.3% (n = 6). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/kg and 0.3 µg/kg, respectively. This newly developed method was applied to the detection of ß2-agonist residues in 50 commercial ham products and only one sample was found to contain ß2-agonist residues (clenbuterol at 15.2 µg/kg).

Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

BACKGROUND: Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the ß2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties. METHODS AND RESULTS: Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20 weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles. CONCLUSION: The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression.

Electromembrane extraction of clenbuterol from swine urine for monitoring illegal use in livestock.

The illegal use of clenbuterol seriously endangers food safety and human health. Accurate monitoring of the illegal use of clenbuterol in livestock can efficiently prevent the clenbuterol residue pork products from entering the consumer market. Thus, in this study, a simple, rapid, and sensitive method for the determination of clenbuterol in swine urine was developed using electromembrane extraction combined with liquid chromatography-tandem mass spectrometry. It should be noted that the electromembrane extraction method presented many advantages of simple operation, fast mass transfer rate, good sample clean-up capability, and less organic solvent consumption. The effect of important factors on the extraction efficiency of clenbuterol was investigated. Under the optimal conditions, good linearity was achieved for clenbuterol over the range of 1-1000 ng/ml (linear correlation [R2 ] = 0.9996). The recoveries of clenbuterol in swine urine at three spiked levels ranged from 83.7% to 110.0% with relative standard deviation values lower than 9.7% (n = 4). The limits of detection and quantification for clenbuterol were 0.07 and 0.25 ng/ml, respectively. These results suggested that the proposed method has great potential for the extraction and determination of trace analyte in a complex sample matrix for monitoring illegal use in livestock.

A new potentiometric platform: Antibody cross-linked graphene oxide potentiometric immunosensor for clenbuterol determination.

An immunosensor is based on the signal measurement obtained upon the reaction of an antibody antigen complex. It plays a significant role in various fields such as environmental analysis, production monitoring, drug detection or screening, veterinary medicine, and animal food. In this study, an antibody crosslinked graphen oxide (GO)-based potentiometric sensor has been developed for fast, simple, and economical detection of clenbuterol. In this context, the photosensitive amino acid bound GO platform is synthesized and used for the preparation of electrode material. Then, polymeric structure is characterized by infrared spectroscopy, and the performance of immunonano platform prepared by potentiometric sensor is evaluated. The effect of pH, response time, selectivity, and sensitivity is investigated. Under the optimized conditions, a simple and rapid method for the determination of clenbuterol from milk sample is established by immuno-potentiometric sensor. The detection limit has found to be 0.87 × 10-9 mmol L-1 for this immuno-potentiometric sensor. This immuno-potentiometric sensor has optimum pH at 7.0, a wide linear response (1.0 × 10-2 to 1.0 × 10-9 mmol L-1 ), rapid response time (2 Min) and 36 weeks operational lifetime.

In situ synthesis of graphene oxide/gold nanocomposites as ultrasensitive surface-enhanced Raman scattering substrates for clenbuterol detection.

A highly sensitive approach to detect trace amount of clenbuterol (CB) based on graphene oxide/gold nanoparticles (GO/Au NPs) by surface-enhanced Raman spectroscopy (SERS) was presented. To be specific, the GO/Au nanocomposites were formed by depositing Au NPs onto the surface of GO through an in situ reduction process, where a high density of inherent hot spots was created between Au NPs. By optimizing the depositing density of Au NPs, the strongest electromagnetic coupling effect originating from highly dense hot spots was obtained. The optimized GO/Au was demonstrated to enhance the Raman signals of CB by 4.8 times more than that of CB enhanced by Au NPs. Moreover, GO/Au nanocomposites exhibit good biocompatibility and accessible surface for high adsorption of target molecules through the pi-pi stacking with graphene oxide. Hence, the proposed GO/Au nanocomposites were utilized to capture aromatic molecules like CB and served as excellent sensitive SERS-active substrates for sensing of it, which exhibited an excellent linear performance in the range of 5 × 10-8 to 1 × 10-6 mol/L with a limit of detection (LOD) of 3.34 × 10-8 mol/L (S/N = 3). Due to high-density hot spots with easy operation, this proposed GO/Au nanocomposite-based SERS technique holds great potential in the application of food safety analysis and biomedical science.

A molecularly imprinted electrochemiluminescence nanoprobe based on complexes consisting of CdTe and multiwall carbon nanotube for sensitive determination of clenbuterol.

An electrochemiluminescence (ECL) nanoprobe was fabricated for the determination of clenbuterol (CLB). A molecularly imprinted polymer (MIP) film was coated on the surface of the glassy carbon electrode modified with CdTe-doped multiwall carbon nanotubes. The MIP film with CLB as the template molecule improves the selectivity of the nanoprobe, CdTe is used as ECL signal amplifier, and MWCNT works as the carrier. The ECL intensity is altered by elution and reabsorption of CLB. The possible reaction mechanism and experimental parameters of the nanoprobe are discussed. Under optimized conditions, the quenched ECL intensity and the CLB concentration have a linear relationship in the range 2.3 × 10-9 to 1.5 × 10-5 mol·L-1, and the detection limit is 1.0 × 10-9 mol·L-1 (S/N = 3). The nanoprobe was successfully applied to the determination of CLB in pork samples. Graphical abstract Schematic representation of the molecularly imprinted electrochemiluminescence nanoprobe based on complexes consisting of CdTe and multiwall carbon nanotube used to determinate clenbuterol.

Integrated immunochromatographic assay for qualitative and quantitative detection of clenbuterol.

In this study, colloidal gold (CG) and time-resolved fluorescent nanobead (TRFN) probes were used to establish an integrated immunochromatographic assay (ICA) to qualitatively and quantitatively detect clenbuterol (CLE). The best experimental conditions for the two probes in separate ICAs were obtained by optimizing the antibody labeling concentration, the amount of antigen, and the concentration of probe. When the CG and TRFN probes co-existed in the ICA, the latter had no effect on the sensitivity of qualitative detection of the CG probe-based ICA. However, the CG probe optimized the linear range of quantitative detection in the TRFN probe-based ICA. The integrated test strip can be used for qualitative and quantitative detection of CLE in one step. When the amount of antigen reached 0.4 mg/mL, the CG probe concentration reached 1.2 µg/mL, and the TRFN probe concentration reached 0.68 µg/mL. The qualitative sensitivity of the integrated ICA was 0.5 ng/mL and its quantitative limit of detection was 0.04 ng/mL with a detection range of 0.1-2.7 ng/mL. This developed method is of great significance for large-scale samples screening and positive monitoring in the field of food safety testing.

A novel murine antibody and an open sandwich immunoassay for the detection of clenbuterol.

Clenbuterol (CLEN) is a sympathomimetic amine used as a decongestant and bronchodilator while treating breathing disorders. It is also used in food-producing animals as it improves the rate of red meat production. However, it is prohibited in many countries nowadays due to human health and safety concerns. Unfortunately, the illegal use of CLEN is still rampant. Thus, monitoring it in food and livestock is important. Here, we report a novel murine antibody and an open sandwich enzyme linked immunosorbent assay (OS-ELISA) to detect CLEN based on antigen-antibody reactions. The genes of antibody variable regions in mice immunized with CLEN conjugated with bovine serum albumin were cloned into a phagemid (pDong1/Fab) to construct a phage-display antibody library, from which a novel antibody, A12, was selected. Then, an OS-ELISA was developed to detect CLEN using separated variable regions of the A12 antibody. The limit of detection of the assay was found to be 8 ng/mL, which was useful for monitoring CLEN usage.

Phosphorene nanocomposite with high environmental stability and antifouling capability for simultaneous sensing of clenbuterol and ractopamine.

A series of phosphorene (BP) nanocomposites was prepared to realize simultaneous electrochemical determination of clenbuterol (CLB) and ractopamine (RAC). CLB and RAC are the most commonly used ß-agonists in animal-derived food. The BP nanohybrid was obtained by co-decoration with both mono(6-mercapto-6-deoxy)-ß-cyclodextrin and poly(3,4-ethylenedioxythiophene) nanoparticles. It displays high stability, antifouling capability, a large electrochemical active surface and good electrochemical response. The electrochemical assisted antifouling strategy was selected by further eliminating the fouling of the electrode surface using continuous cyclic voltammetry. The electrode was employed for electrochemical sensing of CLB and RAC at typical peak voltages of 0.8 and 1.0 V (vs. SCE). Responses are linear in the 0.3-90 µM concentration range for CLB, and from 0.3 to 9.4 µM for RAC under optimal conditions. The limit of detection are 0.14 and 0.12 µM, respectively. The sensor was employed for simultaneous determination of CLB and RAC in (spiked) beef, feed and bovine serum samples with acceptable recoveries. Graphical abstractAn electrochemically assisted anti-fouling method for simultaneous voltammetric nanosensing of clenbuterol (CLB) and ractopamine (RAC) in edible cattle product samples using high-stable and anti-foul phosphorene (BP) co-decorated with mono(6-mercapto-6-deoxy)-ß-cyclodextrin (S-ß-CD) and poly(3,4-ethylenedioxythiophene) (PEDOTNPs).

A fluorometric clenbuterol immunoassay using sulfur and nitrogen doped carbon quantum dots.

A fluorometric clenbuterol immunoassay is described that uses S- and N-co-doped carbon quantum dots as the fluorescent probe. Strongly fluorescent S/N-doped carbon quantum dots (S/N-CDs) were synthesized by hydrothermal method using fructose as the carbon precursor and L-cysteine as S/N sources. The S/N-CDs were characterized by transmission electron microscopy, energy dispersive spectroscopy and Fourier transform infrared spectroscopy (FTIR). Under 350 nm photoexcitation, they display strong purple fluorescence with an emission peak at 405 nm. In pH 4.0 solution, the amino groups (confirmed by FTIR) on the carbon quantum dots were coupled to clenbuterol antibody (Ab) by amine-amine coupling reaction to quench the fluorescence. If clenbuterol (Clen) is added, it binds to the Ab to generate a stable Ab-Clen immunocomplex and free S/N-CD. This causes the fluorescence of nanoprobe to be restored. The fluorescence of the system increases linearly in the 0.07-1.7 ng·mL-1 Clen concentration range. The probe of type S/N4-CD displays the best sensitivity. The detection limit is 23 pg·mL-1. Graphical abstract Schematic presentation of clenbuterol fluorometric immunoassay using sulfur and nitrogen doped carbon quantum dots.

A silica nanoparticle based 2-color immunochromatographic assay for simultaneous determination of clenbuterol and ractopamine.

An immunochromatographic assay (ICA) is presented that can be applied to simultaneous detection of clenbuterol (CLE) and ractopamine (RAC). It is making use of two red and blue silica nanoparticles (SiNPs) that act as labels for encoding the antibodies. This design permits multiplexed analysis in a single test line and does not require an external source for photoexcitation. Anti-CLE was labeled with red SiNPs, and anti-RAC with blue SiNPs. The capture antigens CLE-BSA and RAC-BSA were placed onto the conjugate pad and the test line of the test strip, respectively. Under bare eye examination, no cross-colored lines or nonspecific bioconjugate adsorption were observed, and the visible limit of detections for CLE (red) and RAC (blue) are 3 and 2 ng‧mL-1, respectively. This design allows for multiplexed detection with reduced device dimensions and costs, and with easy integration and manufacturing. Conceivably, the method may be extended to simultaneous determination of numerous other analytes. Graphic abstract The principle of qualitative detection strategy of multiplex immunochromatographic assay for clenbuterol (CLE) and ractopamine (RAC) is schematically illustrated. Depending on the type and ratio of organic dyes, the color of colored silica nanoparticle can be tuned from red to purple and even to black (lower right corner).

A metal organic framework-functionalized monolithic column for enantioseparation of six basic chiral drugs by capillary electrochromatography.

Poly(glycidyl methacrylate)-co-(ethylene dimethacrylate) [poly(GMA-co-EDMA)] monoliths were used as a support to grow a zeolitic imidazolate framework-8 (ZIF-8) via layer-by-layer self-assembly. Pepsin, acting as as chiral selector, was covalently linked to the surface of the amino-modified ZIF-8 through the Schiff base method. The material was characterized by scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, Fourier transform infrared spectroscopy and elemental analysis. The pepsin-ZIF-8-poly(GMA-co-EDMA) column was utilized to the enantioseparation of the racemic forms of hydroxychloroquine (HCQ), chloroquine (CHQ), hydroxyzine (HXY), nefopam (NEF), clenbuterol (CLE) and amlodipine (AML). In comparison with a pepsin-poly(GMA-co-EDMA) monolithic column (without self-assembled ZIF-8 nanoparticles), the resolution is strongly enhanced (HCQ: 0.34 → 2.50; CHQ: 0.45 → 1.97; HXY: 0.39 → 1.43; NEF: 0.27 → 0.81; CLE: 0 → 0.81; AML: 0.16 → 0.72). Effects of self-assembly layers of ZIF-8, pepsin concentration, buffer pH values and applied voltage were investigated with hydroxychloroquine as the model analyte. The reproducibility of run-to-run, day-to-day and column-to-column were explored, and found to be satisfactory. Graphical abstractSchematic representation of capillary electrochromatography (CEC) systems with a pepsin-zeolitic imidazolate framework-8 (ZIF-8) modified poly(glycidyl methacrylate)-co-(ethylene dimethacrylate) [poly(GMA-co-EDMA)] monolithic column as stationary phases for separation of basic racemic drugs. ZIF-8 modified column was prepared via layer-by-layer self-assembly.

Visualization and colorimetric determination of clenbuterol in pork by using magnetic beads modified with aptamer and complementary DNA as capture probes, and G-quadruplex/hemin and DNA antibody on the metal-organic framework MIL-101(Fe) acting as a peroxidase mimic.

A visualization strategy is described for the detection of clenbuterol (CLB). It is using of antibody against dsDNA and G-quadruplex/hemin labeled on a metal organic framework of type MIL-101(Fe) (G-quadruplex/hemin-anti-DNA/MIL-101) acting as a peroxidase mimetic, and magnetic beads modified with aptamer and complementary DNA (MB/Apt-cDNA) as capture probes. The detection reagent was prepared via the reactions between the double stranded DNA (Apt-cDNA) in capture probes and anti-DNA in peroxidase mimetic. In the presence of CLB, the aptamer on the magnetic beads preferentially binds CLB, and the peroxidase mimetic is released to the supernatant after magnetic separation. The released peroxidase mimetic can catalyze the TMB/H2O2 chromogenic system under mild conditions. This leads to the development of a blue-green coloration whose absorbance is measured at 650 nm. The detection limit is as low as 34 fM of CLB. The method was applied to the determination of CLB in pork samples and gave results that were consistent with data obtained with an ELISA kit. Graphical abstract A visualization strategy is described for the detection of clenbuterol. The selectivity of detection system for clenbuterol is excellent compared with other interferents. The method was applied to the determination of CLB in pork samples.

A signal-on electrochemiluminescence sensor for clenbuterol detection based on zinc-based metal-organic framework-reduced graphene oxide-CdTe quantum dot hybrids.

Clenbuterol (CLB) is harmful to human health when used long term, and it has been listed by the World Anti-Doping Agency (WADA). In this work, a novel zinc-based metal-organic frameworks-reduced graphene oxide-CdTe quantum dots (ZnMOF-RGO-CdTe QDs) hybrid was used to construct an electrochemiluminescence (ECL) sensor for detecting CLB. CdTe QDs, loaded by RGO, exhibited an enhanced ECL signal. In addition, the ZnMOFs catalyzed OH• generation by coreactant H2O2, which further strengthened the ECL signal of the CdTe QDs. The integration of ZnMOFs and RGO-CdTe QDs endowed the sensor with high sensitivity for CLB detection. The intensity of the ECL signal increased as the concentration of CLB increased. The linear range of CLB detection was 3.0 × 10-13 M to 6.0 × 10-10 M, and the detection limit was estimated to be 1.0 × 10-13 M. Furthermore, the sensor displayed a good repeatability and stability. The ZnMOF-RGO-CdTe QD hybrids described in this study provide a foundation for the development of new methods of detecting CLB. Graphical abstract ᅟ.

A fluorometric clenbuterol immunoassay based on the use of organic/inorganic hybrid nanoflowers modified with gold nanoclusters and artificial antigen.

Organic/inorganic hybrid nanoflowers were synthesized from calcium phosphate and protein modified fluorescent gold nanoclusters and antigens. These nanoflowers are shown to be well suited labels for bioassay because they fulfill the functions of biological recognition and signal output. A fluorometric immunoassay was developed that was combined with immunomagnetic separation. In the detection system, the red fluorescence of the supernatant (measured at excitation/emission wavelengths of 360/640 nm) is found to be proportional to the clenbuterol (Clen) concentration after two immunomagnetic separations. The assay has a linear response in the 0.5 µg L-1 to 40 µg L-1 Clen concentration range, and 0.167 µg L-1 limit of detection. This makes it well suited for food safety monitoring. The average recoveries from spiked samples range from 92.7 to 109.1% (intra-assay) and 101.2 to 125.7% (inter-assay) with relative standard deviations of <11.6%. Spiked swine urine samples were analyzed by this method, and the results correlated well with data obtained by LC-MS/MS. Graphical abstract Fluorescent hybrid nanoflowers were fabricated with gold nanoclusters (BSA-AuNCs) and antigens. A fluorometric immunoassay based on the use of such nanoflowers and based on immunomagnetic separation was developed to detect clenbuterol residues in swine urine with satisfactory recoveries and acceptable accuracy.

A highly stable black phosphorene nanocomposite for voltammetric detection of clenbuterol.

A nanocomposite was prepared from graphene-like two-dimensional black phosphorene (BP, an allotrope of phosphorus) and nafion (Nf) treated with isopropanol (IP). A glassy carbon electrode (GCE) modified with this nanocomposite was found to be a viable sensor for voltammetric determination of clenbuterol (CLB). Unlike previously reported pure BP, the BP nanocomposite was stable towards water and oxygen. Its morphology, structure, electrochemically active surface area and electrochemical stability were investigated. The BP-Nf (IP) modified GCE displayed good electrochemical stability and electrocatalytic capacity with a low working potential of 0.94 V (vs. SCE), excellent peak current response for CLB in a linear concentration range of 0.06-24 µM with a detection limit of 3.7 nM (3σ/m) and a sensitivity of 0.14 µA·µM-1·cm-2 under optimal conditions. A sensing mechanism for the electro-oxidation of CLB was suggested and verified by density functional theory calculations under imitation of aqueous solution conditions. The sensor was successfully applied to the determination of CLB in bovine meat and bovine serum samples. Graphical abstract Highly-stable black phosphorene (BP) nanocomposite based on Nafion (Nf) was used to modify a glassy carbon electrode (GCE). It is shonw to be a viable electrochemical platform for sensitive voltammetric determination of trace clenbuterol (CLB) in bovine beef and bovine serum.

A voltammetric immunosensor for clenbuterol based on the use of a MoS2-AuPt nanocomposite.

An ultrasensitive immunosensor for the direct detection of the illegally used livestock feed clebuterol (CLB) is described. It is based on the use of a glassy carbon electrode modified with an MoS2-AuPt nanocomposite and on biotin-streptavidin interaction. The use of MoS2-AuPt accelerates electron transfer, and this leads to a sharp increase in the electrochemical signal for the electrochemical probe hydrogen peroxide. Differential pulse voltammetry was used to record the current signal at a peak potential of -0.18 V (vs SCE). Under optimal conditions, the electrode has a linear response in the 10 pg·mL-1 to 100 ng·mL-1 CLB concentration range and a 6.9 pg·mL-1 detection limit (based on the 3σ criterium). This immunosensor is sensitive, highly specific and acceptably reproducible, and thus represents a valuable tool for the determination of CLB in pork. Graphical abstract Schematic of a voltammetric immunosensor for the determination of clenbuterol (CLB) based on the use of a nanocomposite prepared from molybdenum disulfide and a gold-platinum alloy (MoS2-AuPt), and making use of the biotin-streptavidin system.

Development of Polyclonal Antibody against Clenbuterol for Immunoassay Application.

Development of an immunoassay for clenbuterol (CLB) detection required an anti-CLB antibody as an important bioreceptor. In this study, we report our work on production and purification of a rabbit-derived polyclonal anti-CLB antibody. The antibody was then purified by nProtein A Sepharose affinity column and the antibody purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The activities of purified antibody were evaluated based on high antibody titer determined from enzyme-linked immunosorbent assay (ELISA). The sensitivity and selectivity of this antibody was evaluated and exhibits negligible cross-reactivity to antibiotics other than ß-agonist families. Evaluation of the antibody as bioreceptor in immunoassay was performed using direct competitive ELISA and exhibited linear calibration plot (R² = 0.9484). The antibody was used to detect the content of CLB in spiked milk samples and the recovery of more than 92% indicating significant performance as bioreceptor for the development of a rapid and simple immunoassay.

Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor.

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5'-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33⁻97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.