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Frontal affinity chromatography is a powerful, underappreciated technique for the qualitative (screening) and quantitative ( K d determination) evaluation of biological interactions. Its development has been previously hampered by its sample consumption, limited throughput, and lack of dedicated instrumentation especially at a miniaturized scale. This work describes two original experimental devices allowing nano-frontal affinity chromatography titrations (nano-FAC) to be automatically implemented in the time-saving staircase mode. The first nano-FAC system utilizes a capillary electrophoresis device (7100 CE Agilent system) in the pressurization mode with in situ UV detection. The second nano-FAC experimental setup implements a nano-LC device (Ultimate 3000 Thermo) modified with a 10-port valve equipped with two superloops (loop volume, 5 μL) operating alternatively and automatically in a single run. The benefits and drawbacks of each approach are exemplified using two model protein-ligand interactions (concanavalin A-mannose and concanavalin A-glucose). The two methods result in concordant dissociation constants ( K d ) and number of active site ( B act ) values, obtained in a fully automated manner, with low sample consumption and good throughput.

An integrated, miniaturized and fully automated system was developed for the analysis (preconcentration/purification, separation and detection) of cis-diol containing molecules in complex matrices. This innovative in-line coupling system was achieved via the in-situ and localized synthesis of a short segment of silica-based monolith at the inlet of a 75-μm inner diameter fused silica capillary. The monolithic segment was locally functionalized with an acrylamide derivative of phenylboronic acid by free radical photopolymerization within 10min of irradiation time. Efficiency of the photopolymerization reaction was followed by frontal affinity chromatography of 1,2-dihydroxybenzene (catechol) as cis-diol model solute. An active-site amount of 0.43nmolcm -1 (9.8nmolμL -1 ) of phenylboronic acid moieties was obtained, with a K d value of about 290μM close to reported value for the phenyl boronate-catechol complex. The optimal conditions of use of the miniaturized boronate affinity monolithic column (μBAMC) were determined and adapted to the in-line coupling with capillary electrophoresis. Catechol was specifically preconcentrated in a pH 8.5 phosphate buffer/MeOH (80/20, v/v) mixture. A volume up to 20 times the monolith volume can be percolated with a quantitative recovery yield. Three catecholamines were purified, preconcentrated and in-line separated. Elution from the μBAMC was performed with a small plug of acidic solution, allowing field amplified sample stacking of solutes within the plug before their in-line electrophoretic separation at pH 8.75. This unique in-line coupling was successfully used for the fully automated analysis of catecholamines neurotransmitters in urine samples, highlighting the purification efficiency of the μBAMC and the potential of such a fully integrated approach. In addition to the low sample volume required (less than 2μL), the limits of detection (LOD) accomplished with this coupling were estimated at 9.0, 9.5 and 4.8ngmL -1 for dopamine, adrenaline and noradrenaline respectively, which improves the LOD of theses solutes compared to other CE methods., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A micro-immunoaffinity monolithic column (μIAC) was developed and in-line coupled with capillary zone electrophoresis in a fully automated way with Ochratoxin A as test solute. The in-line micro-immunoaffinity columns based on monolithic methacrylate polymers (EDMA-GMA) were prepared in situ at the inlet end of a PTFE coated fused silica capillary by UV initiated polymerization and subsequently grafted with antibodies. These μIACs were thoroughly characterized. The synthesis of the polymeric support was first demonstrated to be reproducible in terms of permeability, surface properties and efficiency. The antibodies immobilization was then studied by a new original hydrodynamic method (ADECA) allowing the in situ quantitative determination (at a miniaturized scale) of the total amount of immobilized antibodies. The combination of this measurement with the binding capacity of the μIAC allowed, for the first time, the in situ determination of immobilized antibody activity. A total of 260 ± 15 ng (1.6 ± 0.1 pmol) of IgG antibodies/cm in 75 μm i.d. monolithic column (i.e. 18 μgmg(-1)) was obtained with (anti-Ochratoxin A/Ochratoxin A) as antibody/antigen model. 40% of the immobilized antibodies remain active corresponding to a binding capacity of 1.2 ± 0.2 pmol antigen/cm (i.e. 600 pg/cm of our test solute OTA), a very high capacity when dealing with trace analysis and with regard to the detection limits (30 pg and 0.5 pg with UV and LIF detection, respectively). The recovery yields were quantitative with negligible non-specific adsorption and allow analysis of diluted samples (1 ngmL(-1)) for a percolated volume of 10 μL. It was also demonstrated that despite the progressive denaturation of antibodies consecutive to the elution step, the binding capacity of the μIAC remained high enough to implement at least 15 consecutive analyses with the same column and in a fully automated way., (Copyright © 2011 Elsevier B.V. All rights reserved.)