Production and Characterization of Biotinylated Anti-fenitrothion Nanobodies and Development of Sensitive Fluoroimmunoassay.

A simple and sensitive fluoroimmunoassay (FIA) based on a heavy-chain antibody (VHH) for rapid detection of fenitrothion was developed. A VHH library was constructed from an immunized alpaca, and one clone recognizing fenitrothion (namely, VHHjd8) was achieved after careful biopanning. It was biotinylated by fusing with the Avi tag and biotin ligase to obtain a fusion protein (VHHjd8-BT), showing both binding capacity to fenitrothion and the streptavidin poly-horseradish peroxidase conjugate (SA-polyHRP). Based on a competitive assay format, the absorbance spectrum of oxidized 3,3',5,5'-tetramethylbenzidine generated by SA-polyHRP overlapped the emission spectrum of carbon dots, which resulted in quenching of signals due to the inner-filter effect. The developed FIA showed an IC50 value of 1.4 ng/mL and a limit of detection of 0.03 ng/mL, which exhibited 15-fold improvement compared with conventional enzyme-linked immunosorbent assay. The recovery test of FIA was validated by standard GC-MS/MS, and the results showed good consistency, indicating that the assay is an ideal tool for rapid screening of fenitrothion in bulk food samples.

Ultrasensitive immunosensor for acrylamide based on chitosan/SnO2-SiC hollow sphere nanochains/gold nanomaterial as signal amplification.

An electrochemical immunosensor for ultrasensitive detection of acrylamide (AA) in water and food samples was developed. SnO2-SiC hollow sphere nanochains with high surface area and gold nanoparticles with good electroconductivity were fabricated onto the surface of a glassy carbon electrode pre-coated with chitosan. The coating antigen (AA-4-mercaptophenylacetic acid-ovalbumin conjugate, AA-4-MPA-OVA) was immobilized on the electrode. Polyclonal antibody specific for AA-4-MPA was conjugated to gold nanorod (AuNR) as primary antibody (AuNR-Ab1). Horseradish peroxidase labelled anti-rabbit antibody produced in goat was conjugated to AuNR as secondary antibody (HRP-AuNR-Ab2). For detection, the analyte (AA-4-MPA) in sample competed with coating antigen for binding with AuNR-Ab1. After washing, HRP-AuNR-Ab2 was added to capture the AuNR-Ab1, and the electrical signal was obtained by addition of hydroquinone and H2O2. After investigation of the binding ability on nanomaterials and optimization of competitive immunoassay conditions, the proposed immunosensor exhibited a sensitive response to AA with a detection limit of 45.9 ±â€¯2.7 ng kg-1, and working range of 187 ±â€¯12.3 ng kg-1 to 104 ±â€¯8.2 µg kg-1 for drinking water samples. Recoveries of AA from spiked samples were ranged from 86.0% to 115.0%. The specificity, repeatability and stability of the immunosensor were also proved to be acceptable, indicating its potential application in AA monitoring.

Development of a progesterone immunosensor based on thionine-graphene oxide composites platforms: Improvement by biotin-streptavidin-amplified system.

Progesterone (P4) is a kind of hormone that can cause neuropathic disturbances in humans when the concentration overpasses a certain degree. In this work, an electrochemical immunosensor capable of detecting P4 sensitively and selectively was developed. Thionine-graphene oxide (Thi-GO) composites with excellent biocompatibility were synthesized and coated to a clear glassy carbon electrode. P4 coating antigen (P4-OVA) was immobilized to the electrode, then sample as well as biotinylated antibody (biotin-P4 Ab) were added. The free P4 can compete with P4-OVA for binding to biotin-P4 Ab. After the further addition of streptavidin-HRP, H2O2 was introduced to develop electrical signal for quantitative determination of P4. After careful optimization of assay conditions, the proposed immunosensor showed a linear range from 0.02 to 20ngmL-1 for P4 in milk samples. The averaged recoveries from spiked samples ranged from 84.0% to 102.0%, which correlated well with standard HPLC-MS/MS. The biosensor also showed good specificity, reproducibility and stability, indicating its potential application in monitoring of P4 in a simple and low cost manner.