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The concentration of glycocholic acid (GCA) in urine and blood is an important biomarker for liver cancer. Monitoring of GCA depends to a large extent on the availability of appropriate analytical techniques. In this work, based on the immobilization of GCA-OVA onto the sensor chip surface, a label-free competitive inhibition immunoassay for the determination of GCA with the surface plasmon resonance (SPR) technique was developed. The proposed SPR immunosensor is simple to prepare, recyclable and exhibits excellent sensitivity to GCA (a linear range of 13.3–119.4 ng mL−1and a limit of detection (LOD) of 2.5 ng mL−1), which was 14 times lower than that of the traditional immunoassay. Excellent recoveries and correlation between these two methods were observed (R2= 0.995). Hence, it can be proved that the SPR immunosensor could be used to achieve rapid and sensitive quantitative detection of GCA in real urine samples and meet clinical needs.

A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for glycocholic acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli(E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC50) and limit of detection (LOD) for GCA were 216 ng mL−1and 37.0 ng mL−1, respectively, and the linear response range extended from 71.0 to 657 ng mL−1. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. colistrain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.

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 IC50of 140 ng mL−1and a dynamic range of 11.32–904.21 ng mL−1approximately. 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.