Frontal analysis capillary electrophoresis: recent advances and future perspectives.

The developments on frontal analysis capillary electrophoresis (FACE) from 2012 to 2017 to study interactions of simple and complex systems are reviewed. Most research papers focused on therapeutic drug-related studies; however, other studies include chemical sensing, drug delivery, inhibitor screening and capillary coating. New ligand-substrate systems such as template-molecularly imprinted polymer systems were reported. Comparison of FACE with other analytical techniques used to investigate binding interaction, and the determination of binding parameters using different isotherm models are also covered. In 2017, eight research papers were reported including new detection by ESI-MS. Future research direction of FACE may include high sensitivity detection and throughput screening of drugs, natural products and biomarkers for clinical diagnosis.

Molecular interactions between warfarin and human (HSA) or bovine (BSA) serum albumin evaluated by isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE).

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: -5.4 and -5.6 Kcal/mol for HSA and -4.3 and -3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively. Further high order interaction events for both albumins are detected only by FA/CE.

Room temperature synthesis and binding studies of solution-processable histamine-imprinted microspheres.

Accurate quantification of histamine levels in food and in biological samples is important for monitoring the quality of food products and for the detection of pathophysiological conditions. In this study, solution processable histamine-imprinted microspheres were synthesized at 30°C via dilute free radical phototochemical polymerization technique using ethylene glycol dimethacrylate (EGDMA) as the crosslinker and methacrylic acid (MAA) as the monomer. The processability of the resulting polymer is dictated by the monomer feed concentration (eg, 4 wt% 80:20 EGDMA:MAA formulation) and solvent (acetonitrile). Whereas, the particle size is influenced by the monomer feed concentration, the presence of template molecule, and independent of the crosslinker content. Evaluation of the binding performance of the photochemically imprinted polymers (PCP) with different crosslinker content (80 and 90 wt%) indicated that the selective binding capacity was notably higher in PCP-80 (N= 16.0 µmol/g) compared to PCP-90 (N= 10.1 µmol/g) when analyzed via frontal analysis capillary electrophoresis (FACE) using Freundlich isotherm. In addition, PCP-80 microspheres are more selective toward histamine than conventional thermal polymers (CTP-80) prepared at 60°C in the presence of structural analogs such as histidine, imidazole, and tryptamine under cross-rebinding and competitive conditions. These results demonstrated that histamine-selective imprinted polymers can be obtained readily using room temperature photochemical polymerization where these materials can be subsequently used as recognition element for optical-based histamine sensing.

A star-shaped poly(2-methyl-2-oxazoline)-based antifouling coating: Application in investigation of the interaction between acetaminophen and bovine serum albumin by frontal analysis capillary electrophoresis.

In this work, an antifouling capillary modified with star-shaped poly(2-methyl-2-oxazoline)-based copolymer was used to study the interaction between acetaminophen (APAP) and bovine serum albumin (BSA) by frontal analysis capillary electrophoresis (FACE). The star-shaped copolymer, poly(ethylene imine)-graft-poly(2-methyl-2-oxazoline) (PEI-g-PMOXA), was immobilized onto the fused-silica capillary inner wall via dopamine-assisted co-deposition strategy, yielding a PEI-g-PMOXA/polydopamine (PDA)-coated antifouling capillary, i.e., an antifouling capillary coated with the PEI-g-PMOXA/PDA co-deposited film. Electroosmotic flow (EOF) mobility of the PEI-g-PMOXA/PDA-coated capillary was almost zero in a wide pH range (3.0-10.0), while the EOF mobility of bare capillary was much larger and increased significantly with pH increasing. When the PEI-g-PMOXA/PDA-coated capillary was exploited to separate a protein mixture including cytochrome c, lysozyme, ribonuclease A and α-chymotrypsinogen A, the theoretical plate numbers were of five orders of magnitude which were about ten-fold higher over those obtained with bare capillary; in addition, the RSD values of migration time were mostly less than 0.7% (30 consecutive runs) which were much smaller than those of bare capillary (c.a. 5.7%). The protein-resistant PEI-g-PMOXA/PDA-coated capillary was then used to investigate the interaction between APAP and BSA by FACE, the binding constant and number of binding sites at 25°C and pH 7.4 (Tris/HCl buffer of 25mM) were 1.39×104M-1 and 1.08, respectively, which were comparable to the results determined by fluorescence spectroscopic measurement (3.18×104M-1 and 1.19, respectively).

Assessment of the binding performance of histamine-imprinted microspheres by frontal analysis capillary electrophoresis.

Frontal analysis capillary electrophoresis was used to evaluate the binding performance of molecularly imprinted microspheres (MIM) toward its template histamine and analogs at pH 7, and compared to the high performance liquid chromatographic method. In both methods, batch binding was employed and the binding parameters were calculated from the measured concentration of unbound amine analytes and afforded comparable histamine equilibrium dissociation constants (Kd ∼ 0.4 mM). FACE was easily carried out at shorter binding equilibration time (i.e. 30 min) and without the need to separate the microspheres, circumventing laborious and, in the case of the system under study, inefficient sample filtration. It also allowed for competitive binding studies by virtue of its ability to distinctly separate intact microspheres and all tested amines which could not be resolved in HPLC. Kd 's for nonimprinted (control) microspheres (NIM) from FACE and HPLC were also comparable (∼ 0.6 mM) but at higher histamine concentrations, HPLC gave lower histamine binding. This discrepancy was attributed to inefficient filtration of the batch binding samples prior to HPLC analysis resulting in an over-estimation of the concentration of free histamine brought about by the presence of unfiltered histamine-bound microspheres.

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis.

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×109 ) and binding constant (Kb = 1×104 L/mol) of the interaction between RAW264.7 and naringin.

Molecular interactions between some non-steroidal anti-inflammatory drugs (NSAID's) and bovine (BSA) or human (HSA) serum albumin estimated by means of isothermal titration calorimetry (ITC) and frontal analysis capillary electrophoresis (FA/CE).

The interactions between some non-steroidal anti-inflammatory drugs, NSAIDs, (naproxen, ibuprofen and flurbiprofen) and bovine (BSA) or human (HSA) serum albumin have been examined by means of two complementary techniques, isothermal titration calorimetry (ITC) and frontal analysis/capillary electrophoresis (FA/CE). It can be concluded that ITC is able to measure with high precision the strongest drug-albumin interactions but the higher order interactions can be better determined by means of FA/CE. Then, the combination of both techniques leads to a complete evaluation of the binding profiles between the selected NSAIDs and both kind of albumin proteins. When BSA is the binding protein, the NSAIDs show a strong primary interaction (binding constants: 1.5 × 10(7), 8 × 10(5) and 2 × 10(6) M(-1) for naproxen, ibuprofen and flurbiprofen, respectively), and also lower affinity interactions of the same order for the three anti-inflammatories (about 1.7 × 10(4) M(-1)). By contrast, when HSA is the binding protein two consecutive interactions can be observed by ITC for naproxen (9 × 10(5) and 7 × 10(4) M(-1)) and flurbiprofen (5 × 10(6) and 6 × 10(4) M(-1)) whereas only one is shown for ibuprofen (9 × 10(5) M(-1)). Measurements by FA/CE show a single interaction for each drug being the ones of naproxen and flurbiprofen the same that those evaluated by ITC as the second interaction events. Then, the ability of both techniques as suitable complementary tools to establish the whole interaction NSAIDs-albumin profile is experimentally demonstrated and allows foreseeing suitable strategies to establish the complete drug-protein binding profile. In addition, for the interactions analyzed by means of ITC, the thermodynamic signature is established and the relative contributions of the enthalpic and entropic terms discussed.