Huisheng Zhuang

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Two novel and high-throughput enzyme-linked immunosorbent assays (ELISA) were developed for determining tonalide in human blood samples. For establishing the proposed methods, the tonalide hapten and immunogen primarily were prepared. After the immunization, the polyclonal antibody and biotinylated polyclonal antibody were obtained. The biotin-streptavidin system and polyamidoamine-gold nanoparticle conjugation were applied respectively to establish thebiotin-streptavidin-ELISA and Au-polyamidoamine-ELISA. For reducing the background interference, some factors and procedures were also discussed and optimized. Under the optimal conditions, the IC₁₀ of biotin-streptavidin-ELISA was 0.046 µg/L and the IC₁₀ of Au-polyamidoamine-ELISA was 0.016 µg/L. The Au-polyamidoamine-ELISA was used to determine tonalide in human blood and the recovery values and coefficients of variation were acceptable. In this study, tonalide was detected in 92.2% of the samples; the median and the maximum values were 0.62 and 1.76 µg/L, respectively. In general, due to the specificity of the antibody and novel nanoprobe design, this Au-polyamidoamine-ELISA simplified the sample preparation and provided a useful and potential way for trace determination of tonalide. The results also suggested that tonalide concentrations were not significantly relative to gender, but the high concentrations of tonalide in human blood should encourage further toxicological studies.

Polycyclic musks (PCMs) in the aquatic environment and organisms have become an emerging environmental issue because of their potential risk. The most used method for polycyclic musk determination is gas chromatography-mass spectrometry (GC-MS) with different sample extractions, which are somewhat expensive to operate, complex and laborious. In this study, a novel and ultrasensitive real-time polymerase chain reaction (PCR) assay with multiple signal amplification of carboxylic-DNA by gold nanoparticle-polyamidoamine conjugation (Au-PAMAM) was developed for determining polycyclic musks in fish. Hapten and immunogen were specially prepared. Polyclonal antibodies were produced based on the optimal immunisation, and the antibodies were characterised. Due to PAMAM’s unique nanostructure of numerous functional amino groups, polyclonal antibody and carboxylic-DNA were immobilised by Au-PAMAM conjugation to develop the antibody-Au-PAMAM-DNA probes, which were used as a signal DNA amplifier in the PCR system. Compared with real-time immuno-PCR, this biological probe-amplified immuno-PCR (BPAI-PCR) assay had higher sensitivity due to the probes’ higher ratio of signal DNA. Finally, the BPAI-PCR assay was applied to analyse AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene,Tonalide) concentrations in fish samples in the range from 1 pg/L to 10 ng/L, giving an of LOD 0.61 pg/L. In general, due to the specificity of the antibody and novel nanoprobe design, this BPAI-PCR assay provided a potential way for trace analysis of AHTN in the aquatic organisms. The high concentrations of AHTN found in cultivated fish should encourage further toxicological studies. GRAPHICAL ABSTRACT

A specific BaP hapten was synthesized and BaP antigens (immunogen, coating antigen) were prepared using diverse methods. Based on these works, a streptavidin-horseradish peroxidase-based enzyme-linked immunological analysis was developed and firstly used to detect BaP. Several physiochemical factors that may influence the assay performance were optimized in the assay. Under optimal conditions, a detection limit of 0.0094 ng mL⁻¹ was obtained and good linearity was achieved within a range of 0.03–35.40 ng mL⁻¹. Satisfactory recovery of spiked samples (91.12–109.23%) was obtained and the coefficient of variation was acceptable. Finally, the detection results of BaP in environmental and food samples were consistent with those obtained using high-performance liquid chromatography. The proposed immunoassay is reliable and has great potential for detecting trace amounts of BaP.

In this paper, a sensitive biotin–streptavidin (BS)-ELISA was developed for determining the polybrominated diphenyl ethers in PM_2.5. For establishing this proposed BS-ELISA, we prepared the biotinylated antibody primarily. And for reducing the background interference, some influencing factors and procedures for this immunoassay were also discussed and optimised. Under the optimal conditions, the IC₅₀ = 0.53 ng/mL; IC₁₀ was 0.002 ng/mL; and the results were almost consistent with those using the gas chromatography and electron capture detection (GC-ECD). Less procedures and simpler sample preparation were required for this method compared with the GC-ECD. The results showed that the highest value of BDE-47 concentration occurred in December, which might reflect the combination of heating and industrial pollution. In our analysis, we studied the Pearson correlations between BDE-47/PM_2.5 and gaseous pollutants (such as NO₂, SO₂, CO, O₃, PM₁₀ and PM_2.5). BDE-47 showed a higher correlation with NO₂ than that with PM₁₀, PM_2.5 and SO₂, which implied that the BDE-47 emission process might be accompanied by the emission of NO₂. Moreover, during the Spring Festival, the concentration of BDE-47 in PM_2.5 decreased significantly, whereas the PM_2.5 changed little. This suggested factories and vehicles might be the major contributors to BDE-47 emissions (but not to PM_2.5).

Extensive polybrominated diphenyl ethers (PBDEs) use has resulted in its increasingly widespread presence in the environment. PBDEs release from existing products can still persist and accumulate in the environment as well as in human and wildlife magnifying through the food web. Due to its ultra-trace amount in the environment, a novel ultrasensitive nano-rt-iPCR assay has been developed to determine polybrominated diphenyl ethers in natural samples. Numerous amino-DNA and polyclonal antibody (anti-PBDE) were immobilized onto the single-walled carbon nanotubes (SWCNTs) to form antibody-SWCNTs-DNA signal amplifier used in the proposed immunoassay system. Compared with rt-iPCR, this nano-rt-iPCR assay had a higher ratio of signal DNA, which meant higher signal measured and lower detection limit. This proposed nano-rt-iPCR assay was used to determine PBDEs in water samples ranging from 0.5 pg/L to 0.5ng/L; giving the LOD 1 pg/L. To the best of our knowledge, this nano-rt-iPCR is the most sensitive method for PBDEs detection. Because of that, this method needs no pre-concentration or extractions, using sample sizes as low as 10 µL. In general, this nano-rt-iPCR method will be a useful and potential way for batch detection of ultra-trace PBDEs in the aquatic environment.