Anping Deng

Core-shell structured Ag@C for direct electrochemistry and hydrogen peroxide biosensor applications

Ag@C core-shell nano-composites have been prepared by a simple one-step hydrothermal method and are further explored for protein immobilization and bio-sensing. The electrochemical behavior of immobilized horseradish peroxidase (HRP) on Ag@C modified indium-tin-oxide (ITO) electrode and its application as H₂O₂ sensor are investigated. Electrochemical and UV-vis spectroscopic measurements demonstrated that Ag@C nano-composites provide excellent matrixes for the adsorption of HRP and the entrapped HRP retains its bioactivities. It is found that on the HRP-Ag@C/ITO electrode, HRP exhibited a fast electron transfer process and good electrocatalytic reduction toward H₂O₂. Under optimum experimental conditions the biosensor linearly responds to H₂O₂ concentration in the range of 5.0×10⁻⁷-1.4×10⁻⁴ M with a detection limit of 2.0×10⁻⁷ M (S/N=3). The apparent Michaelis-Menten constant (K(app)(M)) of the biosensor is calculated to be 3.75×10⁻⁵ M, suggesting high enzymatic activity and affinity toward H₂O₂. In addition, the HRP-Ag@C/ITO bio-electrode shows good reproducibility and long-term stability. Thus, the core-shell structured Ag@C is an attractive material for application in the fabrication of biosensors due to its direct electrochemistry and functionalized surface for efficient immobilization of bio-molecules.

Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC–MS/MS)

Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist illegally used as feed additives for growth promotion. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of PA in tissue and feed samples was developed and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). By reduction of nitryl group to amino group, the PA derivative was synthesized and coupled to carrier proteins with diazobenzidine method. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All antisera displayed high sensitivity with IC50 values lower than 0.48 ng mL(-1). The most sensitive ELISA was established with IC50 and limit of detection (LOD) values of 0.049 ng mL(-1) and 0.003 ng mL(-1), respectively. The cross-reactivity (CR) values of the antisera with three frequently used β-adrenergic agonists (clenbuterol, salbutamol and ractopamine) were lesser than 0.39%; there was no CR of the antisera with other six compounds including two structurally related substances (isoproterenol, phenylephrine). To investigate the accuracy and precision of the assay, swine kidney, liver, meat and feed samples were fortified with PA at different content and analyzed by ELISA. Acceptable recovery rates of 92.2-113.7% and intra-assay coefficients of variation of 3.8-10.9% (n=3) were achieved. Seven spiked samples were simultaneously analyzed by ELISA and LC-MS/MS. There was a high correlation coefficient of 0.9956 (n=7) between the two methods. The proposed ELISA proven to be a feasible quantitative/screening method for PA analysis in tissue and feed samples with the properties of high sensitivity and specificity, high sample throughput and low expensive.

Sensitive electrochemical sensor of tryptophan based on Ag@C core-shell nanocomposite modified glassy carbon electrode.

We here reported a simple electrochemical method for the detection of tryptophan (Trp) based on the Ag@C modified glassy carbon (Ag@C/GC) electrode. The Ag@C core-shell structured nanoparticles were synthesized using one-pot hydrothermal method and characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), and Fourier transform-infrared spectroscopy (FTIR). The electrochemical behaviors of Trp on Ag@C/GC electrode were investigated and exhibited a direct electrochemical process. The favorable electrochemical properties of Ag@C/GC electrode were attributed to the synergistic effect of the Ag core and carbon shell. The carbon shell cannot only protect Ag core but also contribute to the enhanced substrate accessibility and Trp-substrate interactions, while nano-Ag core can display good electrocatalytic activity to Trp at the same time. Under the optimum experimental conditions the oxidation peak current was linearly dependent on the Trp concentration in the range of 1.0×10(-7) to 1.0×10(-4) M with a detection limit of 4.0×10(-8) M (S/N=3). In addition, the proposed electrode was applied for the determination of Trp concentration in real samples and satisfactory results were obtained. The technique offers enhanced sensitivity and may trigger the possibilities of the Ag@C nanocomposite towards diverse applications in biosensor and electroanalysis.

Sensitivity and specificity enhanced enzyme-linked immunosorbent assay by rational hapten modification and heterogeneous antibody/coating antigen combinations for the detection of melamine in milk, milk powder and feed samples

The adulteration of food products with melamine has led to an urgent requirement for sensitive, specific, rapid and reliable quantitative/screening methods. To enhance the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the detection of melamine in milk, milk powder and feed samples, rational hapten modification and heterogeneous antibody/coating antigen combinations were adopted. Three melamine derivatives with different length of carboxylic spacer at the end were synthesized and linked to carrier proteins for the production of immunogens and coating antigens. Monoclonal antibody against melamine was produced by hybridoma technology. Under optimal experimental conditions, the standard curves of the ELISAs for melamine were constructed in range of 0.1-100 ng mL(-1). The sensitivity was 10-300 times enhanced compared to those in the published literatures. The cross-reactivity values of the ELISAs also demonstrated the assays exhibited high specificity. Five samples were spiked with melamine at different concentrations and detected by the ELISA. The recovery rates of 72.8-123.0% and intra-assay coefficients of variation of 0.8-18.9% (n=3) were obtained. The ELISA for milk sample was confirmed by high-performance liquid chromatography with a high correlation coefficient of 0.9902 (n=6). The proposed ELISA was proven to be a feasible quantitative/screening method for melamine analysis.

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol.

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH=9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H2O2 product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL(-1) to 1000 ng mL(-1), and a low detection limit was 0.02 ng mL(-1). The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.

Highly sensitive electrochemiluminescent immunosensor based on gold nanoparticles-functionalized zinc oxide nanorod and poly(amidoamine)-graphene for detecting brombuterol

β-adrenergic agonists (β-agonists) recognized as a growth promoter will reflect the health of human. Sensitive detection of β-agonists in foodstuff is valuable for the health of animals and human. A novel ultrasensitive competition-type electrochemiluminescent (ECL) immunosensor was developed for detecting brombuterol (Brom) based on CdTe Quantum dot (QDs) and polyamidoamine dendrimer (PAMAM, G2) modified graphene oxide (GO) (CdTe QDs-PAMAM-GO composite) as bioprobe for the first time. The surface of glassy carbon electrode (GCE) was coated with AuNPs-ZnO NRs composite film as the platform, which facilitated the electronic transmission rate to enhance the ECL intensity and provide enough active sites for capturing antibody. The resulting ECL immunosensor enabled the real samples detection of Brom with a lower detection limit of 0.3pgmL(-1) (S/N=3) and a wider linear range from 0.001 to 500ngmL(-1). The proposed immunosensor coupled with the excellent advantages of CdTe QDs-PAMAM-GO and AuNPs-ZnO NRs composite displayed high sensitivity and long-term stability, and provided an approach for determining other important biomarkers.

Development of an immunoaffinity chromatography column for selective extraction of a new agonist phenylethylamine A from feed, meat and liver samples

Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist that has been illegally used as an animal feed additive for growth promotion in China. In this study, an immunoaffinity chromatography (IAC) column for selective extraction of PA from swine feed, meat and liver samples was developed. The IAC column was constructed by covalently coupling specific polyclonal antibody (Ab) against PA to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, selectivity, extraction recovery and stability. The maximum capacity of the ICA for PA extraction was found to be 239.4ng. For selectivity testing, 100ng of other three β-adrenergic agonists (clenbuterol, ractopamine and salbutamol) was separately loaded onto the column, and it was observed that the tested compounds could not be captured on the column, e.g. the column could only selectively recognize PA. The recovery of the IAC for PA extraction was found within 96.47-101.98% when 10, 50 and 100ng PA were separately loaded onto IAC column. The IAC column was also applied to real sample extraction. Swine feed, meat and liver samples were collected and spiked with PA in range of 1.0-20ngg(-1). The spiked and unspiked samples were extracted by IAC column and measured by high performance liquid chromatography (HPLC). It was found that there was no detectable PA in the blank samples, and the extraction recoveries of the IAC for PA from the spiked samples were within 89.48-104.89%. The stability of the column was also tested. It was showed that after 35 times repeated usage, 60% of the maximum capacity was still remained. The proposed IAC was proven to be a feasible extraction method for PA from different matrices with the properties of high maximum capacity, selectivity, extraction efficiency and stability.

A novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering for the sensitive and quantitative determination of clenbuterol

In this study, we present a novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for the sensitive and quantitative determination of clenbuterol in urine samples. The principle of this new ICA is similar to that based on colloidal gold particles, but using AuMBA@Ag–Ab [e.g. the polyclonal antibody of clenbuterol labelled Au@Ag core–shell nanoparticles (NPs) sandwiched with a Raman reporter (4-mercaptobenzoic acid, MBA)] as a probe. The clenbuterol in solution and the clenbuterol–ovalbumin conjugate previously coated on the test line of the ICA strip competed for limited antibody binding sites on the probe. The specific Raman scattering intensity of MBA on the test line was measured for the quantitative detection of clenbuterol. This proposed assay was completed within 15 min. The IC50 and limit of detection (LOD) values of the assay for clenbuterol were found to be 0.02 ng mL−1 and 0.24 pg mL−1, respectively. The relative standard deviations (RSD) of the signal obtained from 10 points along the middle part of the test line were within 4.3–8.7%. There was no cross-reactivity (CR) of the assay with ractopamine or phenylethanolamine A, and only 19% CR with salbutamol. The recoveries of clenbuterol from spiked swine urine samples were in the range of 93.8–112.4% with RSD values in range of 5.2–11.5% (n = 9). The proposed SERS-based ICA was demonstrated to be a rapid, simple and ultrasensitive analytical method for detecting clenbuterol in urine samples and could also be applied for the determination of other target analytes in different matrices.

Sensitive and specific detection of a new β-agonist brombuterol in tissue and feed samples by a competitive polyclonal antibody based ELISA

Brombuterol, a new β-adrenergic agonist which can enhance the lean meat-to-fat ratio, is forbidden as an additive in animal feeds for livestock production due to its adverse effects on consumers. In this study, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for the detection of brombuterol in tissue and feed samples was developed. The immunogen and coating antigen were prepared by directly linking brombuterol to carrier proteins using the diazobenzidine method. The antisera against brombuterol were obtained from immunized rabbits. The IC50 and limit of detection (LOD) values of the superior ELISA were 0.165 ng mL−1 and 0.007 ng mL−1, respectively. The cross-reactivity (CR) values of ELISA with clenbuterol, terbutaline and cimbuterol were less than 6.2%; there was no CR of ELISA with nine other compounds. To investigate the accuracy and precision of the assay, liver, meat and feed samples were spiked with brombuterol at different concentrations and analyzed by ELISA. Acceptable recovery rates of 91.9–115.4% and intra-assay coefficients of variation of 1.5–9.5% (n = 3) were achieved. The ELISA was also validated by HPLC. As revealed, both methods were highly correlated (R2 = 0.9927–0.9994, n = 6). The proposed ELISA was proven to be a feasible method for quantitative analysis of brombuterol in tissue and feed samples.

Development of a sensitive monoclonal antibody-based ELISA for the determination of a β-adrenergic agonist brombuterol in swine meat, liver and feed samples

A sensitive enzyme-linked immunosorbent assay (ELISA) based on a newly prepared monoclonal antibody (mAb) for the determination of a β-adrenergic agonist brombuterol in swine meat, liver and feed samples was developed. The immunogen and coating antigen were synthesized by directly linking brombuterol to carrier proteins using the diazobenzidine method. Mice immunized with the brombuterol–bovine serum albumin (BSA) conjugate were utilized for mAb generation whereas brombuterol–ovalbumin (OVA) was used as the coating antigen for the development of ELISA. Two hybridoma clones (13D12C1 and 4D11C1) specifically secreting antibodies against brombuterol were successfully isolated. Under optimal conditions, the IC50 and LOD values of the ELISA for brombuterol based on the mAb secreted by the 13D12C1 clone were found to be 0.56 ng mL−1 and 0.047 ng mL−1, respectively. The ELISA displayed no cross-reactivity with structurally related β-agonists (ractopamine, salbutamol, phenylethanolamine A, phenylephrine and isoproterenol) and five other veterinary drugs, but showed 52.8% cross-reactivity with clenbuterol. Swine meat, liver and feed samples were spiked with different content of brombuterol and detected by ELISA. The recovery rates and the coefficients of variation of the intra-assay and inter-assay were found to be in the range of 87.3–107.5% and 3.5–11.6% (n = 3), and 85.7–116.0% and 4.1–12.7% (n = 3), respectively. Spiked samples were analyzed by ELISA and HPLC simultaneously. A good correlation between the two methods was obtained. The results demonstrated that the proposed ELISA was a feasible quantitative/screening method for brombuterol analysis in swine meat, liver and feed samples with the properties of high sensitivity, high sample throughput and low cost.