Kang Zhao

Multiple signal amplified electrochemiluminescent immunoassay for Hg2+ using graphene-coupled quantum dots and gold nanoparticles-labeled horseradish peroxidase.

A multiple signal amplification strategy was designed for an ultrasensitive competitive immunoassay for Hg(2+). This strategy was achieved using graphene conjugated with a large number of CdSe quantum dots to enhance the basal signal and enormous horseradish peroxidase (HRP) labeled with gold nanoparticles (AuNPs) to consume the coreactant H2O2 generated in situ. The immunosensor was constructed by immobilization of coating antigen on poly(diallyldimethylammonium chloride)-graphene-CdSe composites (PDDA-GN-CdSe), and a strong electrochemiluminescence (ECL) signal was obtained. When the immunosensor was immersed in antibody-AuNPs-HRP composites, the ECL signal greatly decreased, which was ascribed to the bound enzyme on the electrode surface. The self-produced coreactant H2O2 was consumed by o-phenylenediamine in the presence of enzyme, effectively decreasing the ECL intensity from the quantum dots. The Hg(2+) in solution and the corresponding coating antigen competed for the limited antibody, and thus, the ECL intensity was linearly dependent on the logarithm of the mercury(II) concentration from 0.2 to 1000 ng mL(-1) with a detection limit of 0.06 ng mL(-1). The immunoassay exhibited good stability and accuracy and acceptable reproducibility, indicating that it provides a promising approach for the detection of trace mercury and other small molecular compounds in environmental samples.

Ultrasensitive and Quantitative Detection of a New β-Agonist Phenylethanolamine A by a Novel Immunochromatographic Assay Based on Surface-Enhanced Raman Scattering (SERS)

Phenylethanolamine A (PA) is a new kind of β-agonist, which was illegally used as a feed additive for growth promotion in China. In this study, a novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for the ultrasensitive and quantitative detection of phenylethanolamine A is presented. The principle of this new ICA is similar to that based on colloidal gold particles, but using Au(MBA)@Ag-Ab [e.g., polyclonal antibody of PA labeled Au@Ag core-shell nanoparticles (NPs) sandwiched with a Raman reporter (4-mercaptobenzoic acid, MBA)] as a probe. After ICA procedures, the specific Raman scattering intensity of MBA on the test line was measured for quantitative detection of PA. This assay was completed within 15 min. The IC50 and limit of detection (LOD) values of the ICA for PA detection were 0.06 ng mL(-1) and 0.32 pg mL(-1), respectively, which were 1-3 orders of magnitude lower than those obtained by other immunoassays, indicating the ultrasensitivity of this ICA. There was no cross-reactivity (CR) of the assay with another three β-agonists (ractopamine, clenbuterol, and salbutamol), suggesting high specificity of the SERS-based ICA. A spiking experiment revealed that the recoveries of PA from pig urine samples were in range of 99.9- 101.2% with relative standard deviations (RSDs) of 3.6-5.8%. The results demonstrated that this SERS-based ICA was able to quantitatively detect PA in urine samples with high sensitivity, specificity, precision, and accuracy and might be a powerful method for the analysis of other target analytes in the food area.

Fluorescent nitrogen and sulfur co-doped carbon dots from casein and their applications for sensitive detection of Hg2+ and biothiols and cellular imaging

Fluorescent nitrogen and sulfur co-doped carbon dots (NSCDs) were synthesized by a one-step pyrolysis strategy using casein as carbon, nitrogen and sulfur sources, and characterized by UV-vis spectrum, fluorescent spectrum, X-ray photoelectron spectroscopy (XPS) and FT-IR, etc. The synthesized NSCDs displayed a blue emission under ultraviolet illumination with a quantum yield of 31.8%, and a good aqueous solubility, photostability and biocompatibility. It was found that the fluorescence intensity of NSCDs could be selectively quenched by Hg2+, so NSCDs was used as an effective probe for the detection of Hg2+. The linear range and the limit of detection (LOD) of the fluorescent sensor based on NSCDs for the detection of Hg2+ were 0.01-0.25xa0μM and 6.5xa0nM, respectively. Spiked water samples were detected by the sensor with the recovery of 95.4-106.3% and relative standard deviation (RSD) of 3.6-8.6%. It was also observed that the quenched NSCDs-Hg2+ system could be restored by the addition of biothiols such as l-cysteine (Lcy), homocysteine (Hcy) and glutathione (GSH), thus NSCDs-Hg2+ system was employed as a fluorescent sensor for the detection of biothiols. The linear range and LOD of the NSCDs-Hg2+ system were 1-10xa0μM and 23.6xa0nM for Lcy, 0.2-2.5xa0μM and 12.3xa0nM for Hcy, and 0.1-2.0xa0μM and 16.8xa0nM for GSH, respectively. The NSCDs-Hg2+ system was applied for the detection of biothiols in serum samples with satisfied results. In addition, the study inxa0vitro imaging HeLa cells revealed that the synthesized NSCDs could be used as effective fluorescent probes in cellular imaging without noticeable cytotoxicity.

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.

A competitive immunoassay for ultrasensitive detection of Hg(2+) in water, human serum and urine samples using immunochromatographic test based on surface-enhanced Raman scattering.

An immunochromatographic test (ICT) strip was developed for ultrasensitive competitive immunoassay of Hg(2+). This strategy was achieved by combining the easy-operation and rapidity of ICT with the high sensitivity of surface-enhanced Raman scattering (SERS). Monoclonal antibody (mAb) against Hg(2+) and Raman active substance 4-mercaptobenzoic acid (MBA) dual labelled gold nanoparticles (GNPs) were prepared as an immunoprobe. The Raman scattering intensity of MBA on the test line of the ICT strip was measured for quantitative determination of Hg(2+). The ICT was able to directly detect Hg(2+) without complexing due to the specific recognition of the mAb with Hg(2+). The IC50 and limit of detection (LOD) of the assay for Hg(2+) detection were 0.12xa0ngxa0mL(-1) and 0.45xa0pgxa0mL(-1), respectively. There was no cross-reactivity (CR) of the assay with other nineteen ions and the ICT strips could be kept for 5 weeks without loss of activity. The recoveries of the assay for water, human serum and urine samples spiked with Hg(2+) were in range of 88.3-107.3% with the relative standard deviations (RSD) of 1.5-9.5% (nxa0=xa03). The proposed ICT was used for the detection of Hg(2+) in urine samples collected from Occupational Disease Hospital and the results were confirmed by cold-vapor atomic fluorescence spectroscopy (CV-AFS). The assay exhibited high sensitivity, selectivity, stability, precision and accuracy, demonstrating a promising method for the detection of trace amount of Hg(2+) in environmental water samples and biological serum and urine samples.

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.

Ultrasensitive detection of the β-adrenergic agonist brombuterol by a SERS-based lateral flow immunochromatographic assay using flower-like gold-silver core-shell nanoparticles

AbstractThe β-adrenergic agonist brombuterol (BB) is illicitly used as an additive in animal feed to enhance the lean meat-to-fat ratio. The authors describe an ultrasensitive lateral flow immunochromatographic assay (LFIA) based on the use of surface enhanced Raman scattering (SERS) for the determination of brombuterol in swine meat and urine samples. Flower-like gold-silver core-shell bimetallic nanoparticles (referred to as AuNF@Ag) displaying strong SERS enhancement were synthesized, characterized and used as the substrate for the preparation of the LFIA. Polyclonal antibody against brombuterol was immobilized on the surface of the AuNF@Ag particles carrying the Raman reporter 4-mercaptobenzoic acid (MBA). After performing an LFIA, the Raman scattering intensity of MBA on the test line was measured and used for quantitation of brombuterol. Figures of merit of this assay procedure include (a) duration of LFIA process of 15xa0min; (b) an IC50 value (e.g. the concentration of brombuterol producing 50% of signal inhibition in standard curve) of 380xa0pg mL-1; and (c) a limit of detection as low as 0.5xa0pg mL-1. The LFIA is selective over the molecules salbutamol, ractopamine, phenylethanolamine A, isoproterenol and phenylephrine, but shows a 8.5% cross-reactivity to clenbuterol, probably due to the high structural similarity. Swine meat and urine samples spiked with different amounts of brombuterol were analyzed by this method and gave recoveries between 95.8 and 108.0%, and relative standard deviations between 2.0 and 6.3% (for nxa0=xa03).n Graphical abstractSchematic presentation of the lateral flow immunochromatographic assay (LFIA) based on surface enhanced Raman scattering (SERS) using flower-like gold-silver core-shell nanoparticles. It is capable of detecting brombuterol in swine meat and urine samples with high sensitivity and specificity.

Ultrasensitive and Specific Detection of Salbutamol in Swine Feed, Meat, and Urine Samples by a Competitive Immunochromatographic Test Integrated with Surface-Enhanced Raman Scattering

Salbutamol (SAL), a kind of β-agonist which can enhance the lean meat-to-fat ratio, has been inhibited as an additive used in animal feeds for livestock production in many countries due to its harmful effect to the consumers. In this study, an ultrasensitive and specific competitive immunochromatographic test (ICT) integrated with surface-enhanced Raman scattering (SERS) for the detection of SAL was described. The immunoprobe was prepared by immobilizing polyclonal antibody against SAL on the surface of Au@Ag nanoparticles in which the Raman reporter (4-mercaptobenzoic acid, MBA) had been sandwiched. After ICT procedures, the specific SERS signals generated from MBA on the test line of the ICT strip were measured for the quantitative determination of SAL. The assay was completed in 15xa0min. The IC50 and the limit of detection (LOD) values of the assay for SAL were 0.028xa0ngxa0mL−1 and 3.0xa0pgxa0mL−1, respectively. There was no cross-reactivity (CR) of the assay with other three β-agonists (clenbuterol, phenylethanolamine A, and ractopamine), showing high specificity of the assay. Spiking experiments indicated that the average recoveries (nu2009=u20093) of SAL from swine feed, meat, and urine samples were in ranges of 98.4–105.2xa0% with the relative standard deviations (RSDs) of 1.7–7.8xa0%. The results demonstrated that the proposed ICT was a feasible method for ultrasensitive and specific detection SAL in swine feed, meat and urine samples, and could be extended for the detection of other target analytes.

Multiple signal amplified electrochemiluminescent immunoassay for brombuterol detection using gold nanoparticles and polyamidoamine dendrimers-silver nanoribbon

Electrochemiluminescent (ECL) immunosensor with multiple signal amplification was designed based on gold nanoparticles (AuNPs), polyamidoamine dendrimers (PAMAM) and silver-cysteine hybrid nanoribbon (SNR). Low toxic l-cysteine capped CdSe QDs was chosen as the ECL signal probe. To verify the proposed ultrasensitive ECL immunosensor for β-adrenergic agonists (β-AA), we detected Brombuterol (Brom) as a proof-of-principle analyte. Therein, AuNPs as the substrate can simplify the experiment process, accelerate the electron transfer rate, and carry more coating antigen (Ag-OVA) to enlarge ECL signal. On one hand, SNR on the surface of electrode can avoid the aggregation of AuNPs, and SNR-PAMAM-AuNPs also can be acted as a good accelerator for electron transfer. On the other hand, PAMAM (16 -NH2) functionalized SNR (SNR-PAMAM) with numerous amino groups could be employed to bond abundant actived QDs to further amplify ECL signal. The new immunosensor can offer a simple, reliable, rapid, and selective detection for Brom, which have a dynamic range of 0.005-700xa0ngxa0mL-1 with a low detection limit at 1.5xa0pgxa0mL-1. The proposed biosensor will extend the application of nanomaterials in ECL immunoassays and open a new road for the detection of Brom and other β-AA in the future.

Ultrasensitive electrochemiluminescent brombuterol immunoassay by applying a multiple signal amplification strategy based on a PAMAM-gold nanoparticle conjugate as the bioprobe and Ag@Au core shell nanoparticles as a substrate

AbstractA multiple amplified electrochemiluminescence immunosensor is described for the determination of the illicit β-adrenergic agonist brombuterol. Firstly, cystein-coated silver nanowires (SCNW) were modified with polyamidoamine dendrimers (PAMAM) and then immobilized on a glassy carbon electrode (GCE). Then, Ag@Au core/shell nanoparticles (NPs) were deposited on the surface of the modified GCE via gold-nitrogen bonds. The use of Cys-coated silver nanowires accelerates the electron transfer process and also prevents the aggregation of the Ag@Au NPs. The nanocomposites on the GCE have a large surface and outstanding electrical conductivity. It can carry a large amount of coating antigen to amplify the ECL signal. Moreover, PAMAM-Au-CdSe quantum dot bioprobes were prepared, and the unique dendrimer-encapsulated gold nanoparticles with numerous functional amino groups were employed to load abundant activated CdSe quantum dots for further enhancement of the ECL signal. On the basis of signal amplification of the SCNW-PAMAM-Ag@Au-based immunosensor and the PAMAM-Au-CdSe quantum dot bioprobes. The assay has a wide linear range that extends over the 0.001–1000xa0ng·mL−1 brombuterol concentration range and a lower detection limit of 37xa0fg·mL−1. The method is well reproducible, stable and specific. It was applied to the determination of brombuterol in spiked samples of pork and feed where it gave satisfactory recoveries.n Graphical abstractSchematic of a multiple signal amplification electrochemiluminescent immunoassay for ultrasensitive detection of the β-adrenergic agonist brombuterol. It is based on PAMAM-Au-CdSe QDs as bioprobes and SCNWs-PAMAM-Ag@Au as substrates.