Yinji Chen

A sensitive electrochemical aptasensor for multiplex antibiotics detection based on high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted cascade target recycling

A multiplex electrochemical aptasensor was developed for simultaneous detection of two antibiotics such as chloramphenicol (CAP) and oxytetracycline (OTC), and high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted target recycling was used to improve sensitivity. The cascade amplification process consists of the exonuclease-assisted target recycling amplification and metal ions encoded magnetic hollow porous nanoparticles (MHPs) to produce voltammetry signals. Upon the specific recognition of aptamers to targets (CAP and OTC), exonuclease I (Exo I) selectively digested the aptamers which were bound with CAP and OTC, then the released CAP and OTC participated new cycling to produce more single DNA, which can act as trigger strands to hybrid with nanotracers to generate further signal amplification. MHPs were used as carriers to load more amounts of metal ions and coupling with Exo I assisted cascade target recycling can amplify the signal for about 12 folds compared with silica based nanotracers. Owing to the dual signal amplification, the linear range between signals and the concentrations of CAP and OTC were obtained in the range of 0.0005-50 ng mL(-1). The detection limits of CAP and OTC were 0.15 and 0.10 ng mL(-1) (S/N=3) which is more than 2 orders lower than commercial enzyme-linked immunosorbent immunoassay (ELISA) method, respectively. The proposed method was successfully applied to simultaneously detection of CAP and OTC in milk samples. Besides, this aptasensor can be applied to other antibiotics detection by changing the corresponding aptamer. The whole scheme is facile, selective and sensitive enough for antibiotics screening in food safety.

An electrochemical aptasensor for multiplex antibiotics detection based on metal ions doped nanoscale MOFs as signal tracers and RecJf exonuclease-assisted targets recycling amplification

An ultrasensitive electrochemical aptasensor for simultaneous detection of oxytetracycline (OTC) and kanamycin (KAN) has been developed based on metal ions doped metal organic frame materials (MOFs) as signal tracers and RecJf exonuclease-catalyzed targets recycling amplification. The aptasensor consists of capture beads (the anti-single-stranded DNA Antibody, as anti-ssDNA Ab, labeled on Dynabeads) and nanoscale MOF (NMOF) based signal tracers (simplified as Apts-MNM, the NMOF labeled with metal ions and the aptamers). Particularly, the MOF (UiO-66-NH2), with large internal surface areas, ultrahigh porosity and abundant amine groups in the pores, was employed as substrates to carry plenty of metal ions (Pb2+ or Cd2+) and label aptamers of OTC or KAN. Thus, the aptasensor is formed by the specific recognition between anti-ssDNA Ab and aptamers. In the presence of targets (OTC and KAN), aptamers prefer to form targets-Apts-MNM complexes in lieu of anti-ssDNA Ab-aptamer complexes, which results in the dissociation of Apts-MNM from capture beads. With the employment of RecJf exonuclease, targets-Apts-MNM in supernatant was digested into mononucleotides and liberated the target, which can further participate in the next reaction cycling to produce more signal tracers. After magnetic separation, the enhanced square wave voltammetry (SWV) signals were produced from signal tracers. The aptasensor exhibited a linear correlation in the range from 0.5pM to 50nM, with detection limits of 0.18pM and 0.15pM (S/N=3) toward OTC and KAN respectively. This strategy provides specificity and sensitive approach for multiplex antibiotics detection and has promising applications in food analysis.

Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification

Novel label-free and multiplex aptasensors have been developed for simultaneous detection of several antibiotics based on a microchip electrophoresis (MCE) platform and target catalyzed hairpin assembly (CHA) for signal amplification. Kanamycin (Kana) and oxytetracycline (OTC) were employed as models for testing the system. These aptasensors contained six DNA strands termed as Kana aptamer-catalysis strand (Kana apt-C), Kana inhibit strand (Kana inh), OTC aptamer-catalysis strand (OTC apt-C), OTC inhibit strand (OTC inh), hairpin structures H1 and H2 which were partially complementary. Upon the addition of Kana or OTC, the binding event of aptamer and target triggered the self-assembly between H1 and H2, resulting in the formation of many H1-H2 complexes. They could show strong signals which represented the concentration of Kana or OTC respectively in the MCE system. With the help of the well-designed and high-quality CHA amplification, the assay could yield 300-fold amplified signal comparing that from non-amplified system. Under optimal conditions, this assay exhibited a linear correlation in the ranges from 0.001ngmL-1 to 10ngmL-1, with the detection limits of 0.7pgmL-1 and 0.9pgmL-1 (S/N=3) toward Kana and OTC, respectively. The platform has the following advantages: firstly, the aptamer probes can be fabricated easily without labeling signal tags for MCE detection; Secondly, the targets can just react with probes and produce the amplified signal in one-pot. Finally, the targets can be simultaneously detected within 10min in different channels, thus high-throughput measurement can be achieved. Based on this work, it is estimated that this detection platform will be universally served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.

Novel single-stranded DNA binding protein-assisted fluorescence aptamer switch based on FRET for homogeneous detection of antibiotics

Herein, a smart single-stranded DNA binding protein (SSB)-assisted fluorescence aptamer switch based on fluorescence resonance energy transfer (FRET) was designed. The FRET switch was synthesized by connecting SSB labeled quantum dots (QDs@SSB) as donor with aptamer (apt) labeled gold nanoparticles (AuNPs@apt) as acceptor, and it was employed for detecting chloramphenicol (CAP) in a homogenous solution. In the assay, the interaction between core-shell QDs@SSB and AuNPs@apt leads to a dramatic quenching (turning off). After adding CAP in the detection system, AuNPs@apt can bind the target specifically then separate QDs@SSB with AuNPs@apt-target, resulting in restoring the fluorescence intensity of QDs (turning on). Consequently, the fluorescence intensity recovers and the recovery extent can be used for detection of CAP in homogenous phase via optical responses. Under optimal conditions, the fluorescence intensity increased linearly with increasing concentrations of CAP from 0.005 to 100ngmL-1. The limit of this fluorescence aptamer switch was around 3pgmL-1 for CAP detection. When the analyte is changed, the assay can be applied to detect other targets only by changing relative aptamer in AuNPs@apt probe. Furthermore, it has potential to be served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.

Electro-deposited poly-luminol molecularly imprinted polymer coating on carboxyl graphene for stir bar sorptive extraction of estrogens in milk

Electrochemical polymerization of luminol molecularly imprinted polymer on carboxyl graphene (MIP/CG) was developed as stir bar sorptive extraction (SBSE) coating for selective pre-concentration and specific recognition of bisphenol A (BPA), hexoestrol and diethylstilbestrol in milk samples. Luminol was employed as monomer and BPA as the template to prepare MIP under 0-0.6V electro-polymerization. Carboxyl graphene was modified on pencil lead as the substrate to increase extraction capacity. The preparation and extraction conditions affecting the extraction efficiency were optimized. Under the optimized conditions, a good linearity of three estrogens was obtained in the range of 4-1000ngmL(-1). The average recoveries at the three spiked levels of the three estrogens ranged from 83.4% to 96.3% with the relative standard deviations (RSD)≤7.1%. The limits of detection were in the range of 0.36-1.09ngmL(-1). The developed method with low cost, high selectivity and good reproducibility can be potentially applied for determining trace estrogens in complex food samples.

A novel aptamer–quantum dot fluorescence probe for specific detection of antibiotic residues in milk

Herein, a facile, signal-on and homogenous fluorescence assay using novel aptamer-dsDNA antibody–quantum dot probes was designed for detecting antibiotic residues. In this assay, the fluorescent probes were fabricated by using a double-stranded DNA antibody (dsDNA Ab) covalently bonded on the surface of CdSe quantum dots. The antibody can bind double-stranded DNA (dsDNA) specifically. When dsDNA was added, the fluorescence could be efficiently quenched because the probes came into contact with each other with dsDNA as the bridge. The dsDNA was formed between an aptamer towards chloromycetin (CAP) and its complementary strand DNA (cDNA). When CAP was added into the reaction system, the aptamer reacted with CAP preferentially and the dsDNA was unwinded to single strand DNA (ssDNA) which cannot be recognized by the dsDNA antibody. Thus the probes were separated and fluorescence was recovered. Based on the scheme, a signal-on fluorescence assay for detecting antibiotics in a homogenous aqueous phase was developed. Under the optimized conditions, the assay shows a good liner range from 0.05 ng mL−1 to 100 ng mL−1 and the limit of detection (LOD) is 0.002 ng mL−1. If the analyte is changed, the assay can also be employed only by changing the corresponding aptamer and its cDNA. Whats more, the probes can be reused at least 10 times with the recovery above 90%. The fluorescence assay is facile and has promising application for detecting antibiotic contaminants in biological and environmental samples.

Three dimensional M × N type aptamer-functionalized solid-phase micro extraction fibers array for selectively sorptive extraction of multiple antibiotic residues in milk

In this study, a novel three dimensional (3D) M × N type aptamer-functionalized solid-phase micro extraction (SPME) fibers array (M represents the number of targets; N represents the number of samples) was developed for selective enrichment of multiplex antibiotic residues from milk samples, with three chloramphenicols (CAPs) as models. First, gold nanoparticles (AuNPs) were electrodeposited on a gold wire (Φ = 0.2 mm), which was wound around a conductive indium tin oxide (ITO) glassy fiber. Then, the fiber was immersed into thiol-functionalized aptamer solution, which could specifically recognize three CAPs, including chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF). The aptamer can be covalently immobilized on AuNPs through Au–S bonds. Thus, a 3D aptamer-functionalized fiber interface (3D-Apt@AuNPs@Au wire–ITO) was built and employed for specifically sorptive extraction of CAPs from the milk samples with a matrix complex based on the high affinity of the aptamer for the targets. The extraction capacities for CAP, TAP and FF were 887, 840 and 801 ng per fiber, respectively. The enrichment folds were more than 500 times, which is 3.1 folds higher than that of the 2D-Apt@AuNPs–ITO fiber and 6.6 folds higher than that of the 1D-Apt@Au wire–ITO fiber. Afterwards, several similar fibers were assembled together into an array for simultaneous enrichment of three CAPs from 12 samples in one run. Finally, the adsorbed targets were washed away using a pH 8.5 0.1 M Tris–HCl buffer and then detected by high performance liquid chromatography (HPLC)-Diode Array Detector (DAD). The parameters, including extraction temperature, shuttle rates, extraction and desorption pH, and extraction and desorption time, were investigated and discussed. Under the optimized conditions, the limits of detection (LOD) and quantitation (LOQ) were determined as 0.262–0.293 and 0.864–0.967 ng mL−1, respectively, for several CAPs. The fibers array can be applied in replicate batch-extraction for at least 60 extraction cycles with a recovery of over 80%. The SPME fibers assay coupled with HPLC detection possessed advantages of high-throughput, high selectivity and adsorption capacity in one run. Furthermore, the process is environmentally friendly without using organic solutions during the entire extraction process. Thus, the method is a universal platform that can be extended to the selective extraction of other organic pollutant residues if the modified aptamers are changed.

Simultaneous and specific enrichment of several amphenicol antibiotics residues in food based on novel aptamer functionalized magnetic adsorbents using HPLC-DAD

In this work, a novel aptamer functionalized magnetic adsorbent was developed and combined with magnetic dispersive solid phase extraction (MDSPE) for selective enrichment of several amphenicol antibiotics residues (chloramphenicol(CAP), thiamphenicol(TAP) and florphenicol(FF)) in foodstuff then determined by High Performance Liquid Chromatography (HPLC)-Diode array detector(DAD). Firstly, a magnetic silica-coated Fe3O4 microsphere(Fe3O4@SiO2) was synthetized by sol-gel method, then it was functionalized by amino groups through 3-Aminopropyltriethoxysilane (APTES) reagent to form Fe3O4@SiO2-NH2; Thirdly, the amino group on Fe3O4@SiO2-NH2 was transferred to carboxylic group via the succinic anhydride to form Fe3O4@SiO2-COOH. Finally a kind of DNA aptamer with amino group which can simultaneously recognize CAP, TAP, FF, was functionalized on Fe3O4@SiO2-COOH through adding the EDC/sulfo-NHS coupling reagent (Fe3O4@SiO2@Apt). Therefore, a very stable and useful adsorbent was synthesized for the detection of chloramphenicol series antibiotics. This adsorbent can specifically and simultaneously recognize and enrich CAP, TAP, and FF with high adsorption amount from some complicated food matrix, e.g. milk based on the high affinity of aptamer towards the analytes. The saturated extraction capacities for CAP, TAP and FF by the adsorbent were 2.82, 2.56, 2.72μg/g (mass of target/adsorbent)respectively and the enrichment folds were more than 100 times. Afterwards, the target analytes were washed away by pH 8.5 0.1M Tris-HCl buffer solution and detected by HPLC-DAD. The parameters including extraction temperature, extraction capacity, extraction & desorption pH, extraction & desorption time were investigated. With the optimized conditions, the limits of detection (LOD) and limits of quantitation (LOQ) were 0.12-0.17ng/ml and 0.40-0.55ng/ml for the amphenicols in milk. The adsorbent also has good reproducibility for extraction which can be reused at least for 60 cycles with the recovery over 80% (Fig. S2). The MDSPE combined with HPLC-DAD detection possessed the advantages of high selectivity, extraction capacity and very convenient for magnetic separation. In addition, this method is environment friendly and employed no organic solution in the period of pretreatment and extracting. It is a universal platform which can be extended to selective enrichment other organic pollutants residues if changing the modified aptamers.

Novel method for the rapid and specific extraction of multiple β2 -agonist residues in food by tailor-made Monolith-MIPs extraction disks and detection by gas chromatography with mass spectrometry.

A quick and specific pretreatment method based on a series of extraction clean-up disks, consisting of molecularly imprinted polymer monoliths and C18 adsorbent, was developed for the specific enrichment of salbutamol and clenbuterol residues in food. The molecularly imprinted monolithic polymer disk was synthesized using salbutamol as a template through a one-step synthesis process. It can simultaneously and specifically recognize salbutamol and clenbuterol. The monolithic polymer disk and series of C18 disks were assembled with a syringe to form a set of tailor-made devices for the extraction of target molecules. In a single run, salbutamol and clenbuterol can be specifically extracted, cleaned, and eluted by methanol/acetic acid/H2 O. The target molecules, after a silylation derivatization reaction were detected by gas chromatography-mass spectrometry. The parameters including solvent desorption, sample pH, and the cycles of reloading were investigated and discussed. Under the optimized extraction and clean-up conditions, the limits of detection and quantitation were determined as 0.018-0.022 and 0.042-0.049 ng/g for salbutamol and clenbuterol, respectively. The assay described was convenient, rapid, and specific; thereby potentially efficient in the high-throughput analysis of β2 -agonists residues in real food samples.

Application of a multifunctional magnetic mesoporous material for seafood sample clean-up prior to the determination of highly chlorinated polychlorinated biphenyls

In this study, a highly effective clean-up adsorbent was developed for eliminating matrix interferences, especially main organochlorine pesticide residues during the determination of highly chlorinated polychlorinated biphenyls in seafood. The multifunctional adsorbent was prepared by grafting carboxymethyl-β-cyclodextrin on the surface of amino functionalized mesoporous nanoparticles. The amino group functionalized mesoporous SiO2 can remove most of matrix interference in samples. Moreover, carboxymethyl-β-cyclodextrin has stronger host–guest complexation with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, 2,2-bis(p-chlorophenyl)-1,1-dichloro-ethylene, and 1,1-dichloro-2,2-bis(p-chloropheny)ethane. However, it showed weaker adsorption ability toward highly chlorinated polychlorinated biphenyls due to a steric hindrance effect. Based on this, a gas chromatography-mass spectrometry method coupled with the multifunctional adsorbent as a clean-up adsorbent for dispersive solid phase extraction was developed for the analysis of several highly chlorinated polychlorinated biphenyls in seafood samples. The results indicate that the multifunctional adsorbent as a purification material can easily and effectively remove matrix interferences in seafood samples within a short time. The recoveries for polychlorinated biphenyls were in the range of 88.4–103.2%, with relative standard deviations varying between 1.3 and 5.7%.