An indirect competitive fluorescence immunoassay using a DNA/dye conjugate as antibody multiple labels was developed on 96-well plates for the identification and quantification of 2,2＇,4,4＇-tetrabromodiphenyl ether （BDE-47） in aqueous samples. A hapten, 2,4,2＇- tribromodiphenyl ether-4＇-aldehyde, was synthesized, and was conjugated to bovine serum albumin to form a coating antigen. Specific recognition of the antigen by anti-PBDE antiserum was confirmed by a surface plasmon resonance measurement. In the immunoassay, the coating antigen was adsorbed on a 96-well plate first, and a sample, antiserum and biotinylated goat anti-rabbit secondary antibody were then added and reacted sequentially. A biotinylated, double-stranded DNA with 219 base pairs was attached to the secondary antibody by using streptavidin as a molecular bridge. In situ multiple labeling of the antibody was accomplished after addition of a DNA-binding fluorescent dye, SYBR Green I. The working range of the immunoassay for the BDE-47 standard was 3.1-390 ~tg/L, with an IC50 value of 15.6 Ixg/L. The calculated LOD of the immunoassay is 0.73 Ixg/L. The immunoassay demonstrated relatively high selectivity for BDE-47, showing very low cross-reactivity （〈 3%） with BDE-15, BDE-153 and BDE-209. With a spiked river water sample containing 50 Izg/L BDE-47, quantification by the immunoassay was 41.9 ~tg/L, which compared well with the standard GC-ECD method （45.7 Ixg/L）. The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.
Zi-Yan FanYoung Soo KeumQing-Xiao LiWeilin L.ShelverLiang-Hong Guo
Bisphenol A （BPA） is a chemical with high production volume and wide applications in many industries. Although BPA is known as an endocrine disruptor, its toxic mechanisms have not been fully characterized. Due to its structural similarity to thyroid hormones thyroxine （T4） and triiodothyronine （T3）, one possible mechanism of BPA toxicity is disruption of hormone transport by competitive binding with the transport proteins. In this study, the binding interactions of BPA, T4, and T3 with three thyroid hormone transport proteins, human serum albumin （HSA）, transthyretin （TTR）, and thyroxine-binding globulin （TBG） were investigated by fluorescence measurement. Using two site-specific fluorescence probes dansylamide and dansyl-L-proline, the binding constants of BPA with HSA at drug site I and site II were determined as 2.90 × 10^4 and 3.14 × 10^4 L/mol, respectively. By monitoring the intrinsic fluorescence of tryptophan, a binding constant of 4.70 ×10^3 L/mol was obtained. Similarly, by employing 8-anilino-l-naphthalenesulfonic acid as fluorescence probe, the binding affinity of BPA with TTR and TBG was measured to be 3.10 × 10^5 and 5.90× 10^5 L/mol, respectively. In general, BPA showed lower binding affinity with the proteins than T3 did, and even lower affinity than T4. Using these binding constants, the amount of BPA which would bind to the transport proteins in human plasma was estimated. These results suggest that the concentrations of BPA commonly found in human plasma are probably not high enough to interfere with T4 transport.
Nanomaterials have been used increasingly in a wide variety of applications, and some of them have shown toxic effects on experimental animals and cells. In this study, a previously established photoelectrochemical DNA sensor was employed to rapidly detect DNA damage induced by polystyrene nanosphere （PSNS） suspensions. In the sensor, a double-stranded DNA film was assembled on a semiconductor electrode, and a DNA intercalator, Ru（bpy）2（dppz）2＋ （bpy = 2,2＇-bipyridine, dppz = dipyrido[3,2-a：2＇,3＇-c]phenazine） was used as the photoelectrochemical signal indicator. After the DNA-modified electrode was exposed to 2.0 mg/mL PSNS suspension, photocurrent of DNA-bound Ru（bpy）2（dppz）2＋ decreased by about 20%. The decrease is attributed to the chemical damage of DNA and consequently less binding of Ru（bpy）2（dppz）2＋ molecules to the electrode. Gel electrophoresis of DNA samples incubated with PSNS suspension confirmed DNA damage after the chemical exposure. However, in both photoelectrochemical and gel electrophoresis experiments, extensively washed PSNS did not induce any DNA damage, and the supernatant of PSNS suspension exhibited comparable DNA damage as the unwashed PSNS suspension. Furthermore, UV-visible absorption spectrum of the supematant displayed a pattern very similar to that of styrene oxide （SO）, a compound which has been shown to induce DNA damage by forming covalent DNA adducts. It is therefore suggested that styrene oxide and other residual chemicals in the PSNS may be responsible for the observed DNA damage. The results highlight the importance of full characterization of nanomaterials before their toxicity study, and demonstrate the utility of photoelectrochemical DNA sensors in the rapid assessment of DNA damage induced by chemicals and nanomaterials.
Silica nanoparticles are most commonly modified with amino-silanes, followed by post-modification activation for protein immobilization. In this work, epoxy-functionalized silica nanoparticles were prepared by modification with glycidyloxypropyl trimethoxysilane (GPTMS) for direct protein immobilization. Silica nanoparticles possessed an average size of 46 nm, but increased to 63 nm after GPTMS modification. Reaction time, reaction temperature and GPTMS content had no significant effect on particle size. Zeta potential of SiO2 changed from ?26mV to +38mV after modification. Fourier-transformed infrared spectroscopy revealed alkyl C-H bending and stretching bands at 2944 cm-1, 1343 cm-1 and 1465 cm-1, respectively, for the modified nanoparticles. Fluorescein cadaverine was found to bind to GPTMS-modified SiO2, but not to bare SiO2, indicating the chemical reactivity of epoxy groups on the modified nanoparticle with amines. Finally, fluorescently labeled bovine serum albumin (BSA) was used as a model protein to investigate the capacity of epoxy-SiO2 nanoparticles for protein immobilization. The results showed that more proteins were immobilized on the particle with longer reaction time, higher NaCl concentration, lower pH, and less GPTMS content. More importantly, proteins bound to epoxy-SiO2 nanoparticle were highly stable. Under optimized reaction conditions, as much as 25 mg BSA/g nanoparticle was covalently attached to the nanoparticle. The epoxy silane modification of silica nanoparticles offers a reactive surface for one-step and high-density protein immobilization.
