Suqing Zhao

Cd-doped ZnO quantum dots-based immunoassay for the quantitative determination of bisphenol A.

Bisphenol A (BPA) is a ubiquitous environmental contaminant in food products and aquatic ecosystems. Its endocrine and developmental toxicity presents a serious concern to human health and an effective high-throughput method for its detection is desirable. In this paper, water-soluble quantum dots (QDs) have been conjugated covalently with BPA antibodies and the conjugate has been utilized in a competitive fluorescence-linked immunoassay (FLISA). Cd-doped ZnO QDs were functionalized with poly(amidoamine) (PAMAM) dendrimers, as evidenced by ultraviolet absorption spectrum and fluorescence emission spectra analyses, and this led to their successful transfer into aqueous solution. Biological mass spectrometry demonstrated that the bisphenol A antibodies were successfully coupled to the water-soluble QDs, and the structures of these conjugates kept intact. The FLISA method allowed for BPA determination in a linear working range of 20.8-330.3 ng mL(-1) with the limit of detection (LOD) of 13.1 ng mL(-1). The recoveries of BPA from water samples were from 85.92% to 109.62%. In conclusion, a rapid and sensitive FLISA was developed by utilizing novel QD coupling method and validated for use in aqueous samples.

An indirect competitive enzyme-linked immunosorbent assay for bisphenol-A based on the synthesis of a poly-L-lysine–hapten conjugate as a coating antigen

Bisphenol-A (BPA) is commonly suspected to act as an endocrine disruptor, thus it is important to detect BPA residues in water and food samples. An indirect competitive ELISA (ic-ELISA) method was developed in this study based on synthesis of a poly-L-lysine (PLL)–hapten conjugate as a coating antigen. The performance of the PLL–4,4-bis(4-hydroxyphenol) valeric acid (BVA) conjugate in the immunoassay was studied. The established method showed that the PLL–BVA coated format could improve the sensitivity of the ic-ELISA. The limit of detection was 0.5 ng mL−1. IC50 value was 14.5 ng mL−1, which was 9-fold lower than that ovalbumin (OVA)–BVA-coated methods. A good detection range from 1.3 ng mL−1 to 292.2 ng mL−1 was obtained. The recoveries from spiked samples were from 86.7% to 109.2%. The reliability of ELISA was validated by a high-performance liquid chromatography. These data demonstrated that the established ic-ELISA is a potential technique for detection of BPA.

Development of a Highly Specific Fluorescence Immunoassay for Detection of Diisobutyl Phthalate in Edible Oil Samples.

The diisobutyl phthalate (DiBP) hapten containing an amino group was synthesized successfully, and the polyclonal antibody against 4-amino phthalate-bovine serum albumin (BSA) was developed. On the basis of the polyclonal antibody, a rapid and sensitive indirect competitive fluorescence immunoassay (icFIA) has been established to detect DiBP in edible oil samples for the first time. Under the optimized conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R(2) = 0.991), exhibiting a detection limit of 5.82 ng/mL. In this assay, the specific results showed that other similar phthalates did not significantly interfere with the analysis, with the cross-reactivity less than 1.5%, except for that of DiBAP. Thereafter, DiBP contamination in edible oil samples was detected by icFIA, with the recovery being from 79 to 103%. Furthermore, the reliability of icFIA was validated by gas chromatography-mass spectrometry (GC-MS). Therefore, the developed icFIA is suitable for monitoring DiBP in some edible oil samples.

Synthesis and characterization of new Cd-doped ZnO/ZnS core–shell quantum dots with tunable and highly visible photoluminescence

Novel Cd-doped ZnO/ZnS core–shell quantum dots (QDs) were successfully synthesized via a simple two-step chemical solution method. The morphologies and structures of the product and the precursor were verified using X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray analysis (EDX) and X-ray photoelectron spectroscopy (XPS). The exciton dynamics and fluorescence lifetime were studied by time-resolved photoluminescence spectroscopy (TRPL). The thickness of ZnS was controlled by modifying the concentration of thioacetamide (TAA). As the TAA content was increased, the UV-Vis absorption spectra of these novel QDs red shifted toward longer wavelengths; this shift is attributed to the leakage of excitons from the ZnO core to the ZnS shell. The PL emission spectra of the novel QDs blue shifted toward shorter wavelengths as the TAA content was increased, and when the molar ratio of the Cd-doped ZnO to TAA reached 5:1.5. For these novel QDs, the luminous intensity was observed to significantly increase with increasing Cd content at a Cd doping of 20% or less, and a small red shift in the peak was also observed with increasing Cd content. At appropriate Cd doping and ZnS coating, the absolute quantum yield can reach 21% for Cd-doped ZnO/ZnS QDs from 3% and 8% for undoped ZnO/ZnS and Cd-doped ZnO QDs, respectively. All results indicate that the described synthesis method is appropriate for the preparation of Cd-doped ZnO/ZnS core–shell quantum dots with tunable and highly visible photoluminescence.

A fluorescence polarization immunoassay method for detection of the bisphenol A residue in environmental water samples based on a monoclonal antibody and 4′-(aminomethyl)fluorescein

Based on a sensitive monoclonal antibody against bisphenol A (BPA) and a new tracer named BVA–AMF, a homogeneous fluorescence polarization immunoassay (FPIA) was developed and applied in the determination of bisphenol A in environmental water samples. BVA was selected as the hapten to couple with bovine serum albumin and the conjugate was used as the immunogen for the monoclonal antibody production. Three fluorescein-labeled BVA tracers with different structures (BVA–AMF, BVA–EDF, and BVA–lysFITC) were synthesized. Under the same optimal conditions, BVA–AMF showed the highest sensitivity for FPIA and the detection of BPA exhibited a limit of detection of 5.60 ng mL−1, an IC50 of 140 ng mL−1 and a dynamic range of 11.32–904.21 ng mL−1 approximately. In this assay, several similar compounds were shown of little significantly with the cross-reactivity being less than 0.15%. Four different kinds of water samples were analyzed, with recoveries being 87.91–114.28%. The detection standard curve of BPA exhibited a good linearity (R2 = 0.9913, n = 3). Compared with ELISA and HPLC methods, FPIA showed reliability and a high correlation with ELISA of 0.9964 and HPLC of 0.9971. The proposed immunoassay technique is suitable for detection of BPA in authentic environmental water samples.

A direct competitive inhibition time-resolved fluoroimmunoassay for the detection of unconjugated estriol in serum of pregnant women

Unconjugated estriol (uE3) is one of the most important serum markers for prenatal screening. The abnormally low content of uE3 is used as an indicator of fetal DS (Down syndrome) during the second trimester in pregnant women. In the present study, we developed a time-resolved fluoroimmunoassay to detect uE3 by employing microtiter plates with pre-captured primary antibodies. E3-3-CME-BSA (estriol-3-carboxymethyl ether-bovine serum albumin) conjugates served as labels and Eu3+ (europium) as the probe for signal detection. The detection limit of this assay was 0.35 nmol L−1. The within-run and between-run imprecision values for serum control detection were less than 5.0% and 6.0% respectively. The mean recovery was 102.6%. The long-term stability (2–8 °C, 15 months) and thermostability (37 °C, 10 days) were excellent. The uE3 concentrations measured by the present assay in 1168 Chinese maternal serum samples correlated well with those obtained by the chemiluminescence immunoassay assay (r = 0.948). The reference range in normal singleton pregnancies in Southern China was established which provided reference data to adjust the uE3 medians for biochemical screening.

A hydrophilic conjugate approach toward the design and synthesis of ursolic acid derivatives as potential antidiabetic agent

In this study, a series of novel ursolic acid (UA) derivatives were designed and synthesized successfully via conjugation of hydrophilic and polar groups at 3-OH and/or 17-COOH position. Molecular docking studies were carried out with the binding of UA and acabose in the active site of α-glucosidase, in order to prove that the hydrophilic/polar moieties can interact with the hydrophobic group of the catalytic pocket and form hydrogen bonds. The bioactivities of these synthesized compounds against α-glucosidase were determined in vitro. Kinetic studies were performed to determine the mechanism of inhibition by compounds 3, 4, 10 and 11. The results indicated that most of the target compounds have significant inhibitory activity, and the compound 3, 4, 10 and 11 were potent inhibitors of α-glucosidase, with the IC50 values of 0.149 ± 0.007, 0.223 ± 0.023, 0.466 ± 0.016 and 0.298 ± 0.021 μM, respectively. These compounds were more potent than parent compound and acarbose. The kinetic inhibition studies revealed that compound 3 and 4 were mix-type inhibitors while compound 10 and 11 were non-competitive inhibitors. Furthermore, the molecular docking studies for these two kinds of compounds suggested that free carboxylic group at either C-3 or C-28 position could remarkably improve inhibitory activity. It is noteworthy that the exploration of relationship between hydrophilic and polar groups of these structures and the hydrophobic group in catalytic pocket is benefited from our rational design of potent α-glucosidase inhibitor.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies

Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 μg/mL, with an IC50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

Synthesis and Evaluation of Novel Triterpene Analogues of Ursolic Acid as Potential Antidiabetic Agent

Ursolic acid (UA) is a naturally bioactive compound that possesses potential anti-diabetic activity. The relatively safe and effective molecule intrigued us to further explore and to improve its anti-diabetic activity. In the present study, a series of novel UA analogues was synthesized and their structures were characterized. Their bioactivities against the α-glucosidase from bakers yeast were determined in vitro. The results suggested that most of the analogues exhibited significant inhibitory activity, especially analogues 8b and 9b with the IC50 values of 1.27 ± 0.27 μM (8b) and 1.28 ± 0.27 μM (9b), which were lower than the other analogues and the positive control. The molecular docking and 2D-QSAR studies were carried out to prove that the C-3 hydroxyl could interact with the hydrophobic region of the active pocket and form hydrogen bonds to increase the binding affinity of ligand and the homology modelling protein. Thus, these results will be helpful for understanding the relationship between binding mode and bioactivity and for designing better inhibitors from UA analogues.

A highly sensitive and direct competitive enzyme-linked immunosorbent assay for the detection of di-(2-ethylhexyl) phthalate (DEHP) in infant supplies

A sensitive and specific direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was studied in this paper for the detection of di-(2-ethylhexyl) phthalate (DEHP) based on an antigen-coating format. The DEHP-specific polyclonal antibody was raised in rabbits and used to construct the dc-ELISA for the measurement of DEHP. The conjugates of the antibody with horseradish peroxidase (HRP) were used as the detection probe. Under optimal conditions, the assay had a detection limit (LOD) of about 0.0042 ng mL−1, with an apparent linear range of 10−3−103 ng mL−1 (R2 = 0.998). The cross-reactivity with six other structurally related phthalate esters was below 1%. The recoveries of DEHP ranged from 80.8% to 119.2% indicating that the method was successfully applied for the determination of DEHP in infant supplies.