In-line coupling of an aptamer based miniaturized monolithic affinity preconcentration unit with capillary electrophoresis and Laser Induced Fluorescence detection.

A composite 30-cm capillary was prepared. The head of the capillary was a 1.5-cm original and miniaturized aptamer-based monolithic affinity support that was in-line coupled to the end of the capillary used for capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The device was used for the preconcentration, separation and quantification of ochratoxin A (OTA) as a test solute. The 1.5-cm preconcentration unit consists of a fritless affinity monolithic bonded with 5'-SH-modified oligonucleotide aptamers. A vinyl spacer was used for thiol-ene photoclick chemistry with a 5min irradiation at 365nm. Photografting allowed to confine the binding reaction to the desired silica monolithic segment, upstream the empty section of the CE capillary using an UV mask. The photografting procedure was optimized preparing 10-cm capillary monoliths for nano-LC. The retention factors of cationic solutes in ion-exchange nano-LC allowed to follow the aptamer binding on the monolith. The reproducibility of the photografting process was satisfactory with inter-capillary variation lower than 10%. The aptamer bonding density can be increased by successive graftings of 100μM aptamer concentration solution (5pmol/cm/grafting). The optimal conditions to successfully perform the in-line coupling (preconcentration, elution and separation of OTA) with the composite capillary were adjusted depending on individual requirements of each step but also insuring compatibility. Under optimized conditions, OTA was successfully preconcentrated and quantified down to 0.1pg (percolation of 2.65μL of a 40ng/L OTA solution). A quantitative recovery of OTA (93±2%) was achieved in a single elution of 30pg percolated OTA amount. The reproducibility of the overall process was satisfactory with a relative standard deviation lower than 10% with negligible non-specific adsorption. This device was applied for the preconcentration and analysis of OTA in beer and wine at the ppb level within a total analysis time of 30min.