Quantification of Ions and Organic Molecules, in Nanoliter Samples, in the Absence of Reference Materials.

A new analytical methodology, using capillary electrophoresis in indirect UV absorbance mode, is developed for the quantification of analytes in the absence of reference materials. The methodology allows the quantification of organic molecules and/or small ions, anionic or cationic, absorbing or not in the UV range, carrying either one or two electric charges. Two methods of data processing were compared. The first is based on the use of a dynamic simulator of electromigration, and the second uses the Kohlrausch regulating function combined with the electroneutrality equation. The experimental conditions presented in this work allow a precise quantification of anions having electrophoretic mobilities (µep) between -22.71 and -36.92 × 10-9 m2 V-1 s-1 and cations with µep between +30.59 and +63.60 × 10-9 m2 V-1 s-1 with percent relative errors lower than -5.52%. The effect of the integration errors on the reliability of the results is discussed in detail.

Aptamer Switches Regulated by Post-Transition/Transition Metal Ions.

We introduced an aptamer switch design that relies on the ability of post-transition/transition metal ions to trigger, through their coordination to nucleobases, substantial DNA destabilization. In the absence of molecular target, the addition of one such metal ion to usual aptamer working solutions promotes the formation of an alternative, inert DNA state. Upon exposure to the cognate compound, the equilibrium is shifted towards the competent DNA form. The switching process was preferentially activated by metal ions of intermediate base over phosphate complexation preference (i.e. Pb2+ , Cd2+ ) and operated with diversely structured DNA molecules. This very simple aptamer switch scheme was applied to the detection of small organics using the fluorescence anisotropy readout mode. We envision that the approach could be adapted to a variety of signalling methods that report on changes in the surface charge density of DNA receptors.

Capillary electrophoretic apparatus for the endpoint detection in microtitration methods.

Titration methods are routinely used in the laboratories for the quantification of acids and bases, for the complexometric determination of metal ions and for the ion-pair titrations of drugs in pharmaceutical control. They also find application in a wide variety of chemical and biochemical studies. However, conventional titration methods (CTM) require large amounts of samples that are not always available. In absence of micro-titrator devices, the application of these methods for expensive samples and for small batch sizes is not possible. In this work, it was demonstrated that the commercial capillary electrophoretic apparatus (CEa) can be used, in a quick and easy way, for the end-point detection in a microtitration process. The proposed methodology exploits the change of the solutions conductivity during the titrations. The equivalent points can be easily located by plotting the change in electrical current as a function of the titrant volume added. More interestingly, only 1.1-1.5 mL of analyte solutions are required to establish the titration curves. The advantages and the limitations of the procedure are discussed in detail.

Inline Coupling of Electrokinetic Preconcentration Method to Taylor Dispersion Analysis for Size-Based Characterization of Low-UV-Absorbing Nanoparticles.

The inline coupling of the field-amplified sample injection (FASI) to Taylor dispersion analysis (TDA) was used to characterize low-UV absorbing carboxylated silica nanoparticles (cNPs). The hydrodynamic diameters (Dh) were measured by using a commercial capillary electrophoresis instrument. The proposed methodology did not require any complicated instruments or chromophoric dye to increase the detection sensitivity. A practical method based on a half-Gaussian fitting was proposed for the data processing. The results obtained by this method were compared with those derived from dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses. From these results, it appeared that the size derived by TDA is in excellent agreement with those measured by DLS and TEM, as demonstrated by stable nanoparticles with narrow size distributions. Intermediate precision relative standard deviations less than 5% were obtained by FASI-TDA. The effect of the FASI-induced cNP peak dispersion on the reliability of the results was discussed in detail.

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low-affinity DNA binders.

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers.

Multiple capillary isotachophoresis with repetitive hydrodynamic injections for performance improvement of the electromigration preconcentration.

A novel electrokinetic preconcentration technique based on multiple isotachophoresis (M-ITP) realised in a micro-bored capillary to improve sensitivity for capillary electrophoresis with hydrodynamic injection was developed. The M-ITP operation relies on pressure-assisted pushing of a preconcentrated sample plug after the first ITP process back to the injection end of the capillary, followed by a large volume hydrodynamic injection prior to application of the second ITP step. This operational cycle was repeated as many times as desired with very good repeatability of the peak areas and peak heights at each ITP round (RSD less than 8%). Using imidazole and benzoate as models for cationic and anionic analytes, important insights into the mechanism of this electrokinetic preconcentration process with and without the presence of the electro-osmotic flow (EOF) at acidic and basic conditions were provided. Stacking of the benzoate ion, selected as one model analyte, in the presence of EOF and from a sample plug representing up to 300% of the total capillary length was successfully demonstrated. M-ITP was then demonstrated through the enrichment of the Aß 1-40 amyloid peptide, considered as one of the biomarkers for biochemical diagnosis of Alzheimer's disease. Quantification of Aß 1-40 down to 50nM with UV detection was made possible with 6 M-ITP cycles.

Capillary electrophoretic focusing of covalently derivatized protein induced by surfactant.

In this communication, we present a very simple strategy to focus covalently derivatized proteins for high sensitivity CE analysis by LIF detection. We demonstrated that the covalently tagged protein can be focused just by adding SDS at a concentration above the CMC in the derivatized sample. Under specific injection conditions, SDS concentration below the CMC is also sufficient to induce the focusing of the tagged protein. This method allows the quantification and detection of the covalently tagged protein in a narrow zone with an efficiency approaching 220 000 plates/m. Very good linearity was obtained for the ubiquitin in a concentration range of 2-25 µM.

Size-based characterization of nanoparticle mixtures by the inline coupling of capillary electrophoresis to Taylor dispersion analysis.

Separation of closely related nanoparticles is still a challenging issue for the characterization of complex mixtures for industrial/research applications or regulatory purposes. In this work, the remarkable separating performances of CE were complemented with the absolute size-based determination provided by Taylor dispersion analysis (TDA) for the characterization of nanoparticle mixtures. The inline hyphenation of CE to TDA was successfully implemented for the baseline separation followed by a size-based characterization of a bimodal mixture containing two closely size-related nanolatexes (70nm and 56nm radii). A pixel sensor UV area imager providing three detection points along the capillary was used for a differential measurement of the peak broadening during the Taylor dispersion step. Comparison of this new technique with dynamic light scattering and hydrodynamic chromatography is also discussed.

Chiral resolution capabilities of DNA oligonucleotides.

Herein, we studied the chiral resolution properties of a repertoire of arbitrarily chosen DNA oligonucleotides (ON). Ten oligonucleotidic sequences characterized by diverse base compositions, sizes, and structural features, ranging from secondary structure-free homo-oligonucleotides to duplex, hairpin, and three-way junction architectures, were investigated as potential chiral selectors. Their enantioselective features were assessed by using ONs as running buffer additives in partial-filling capillary electrophoresis. It was shown that all the screened sequences displayed enantiodiscrimination capabilities toward small aromatic compounds. Under (sub)millimolar DNA concentration conditions, the combination of only three oligonucleotidic sequences provided the chiral resolution of around 20 racemates, including drugs, illegal drugs, amino-acids, and nucleosides. This work represents the first demonstration of such analyte selectivity spectrum for nucleic acid-based chiral separation tools.

Physico-chemical characterization of polymeric micelles loaded with platinum derivatives by capillary electrophoresis and related methods.

(1,2-diamino-cyclohexane)Platinum(II) ((DACH)Pt) loaded polymeric micelles of poly(ethylene glycol-b-sodium glutamate) (PEG-b-PGlu) are currently studied as a potential candidate to replace oxaliplatin in the treatment of cancers with the aim to reduce side effects like cumulative peripheral distal neurotoxicity and acute dysesthesias. As for all synthetic polymeric drug delivery systems, the characterization of the (co)polymer precursors and of the final drug delivery system (polymeric micelles) is crucial to control the repeatability of the different batches and to get correlation between physico-chemical structure and biological activity. In this work, the use of capillary electrophoresis (CE) and related methods for the characterization of (DACH)Pt-loaded polymeric micelles and their precursor (PEG-b-PGlu copolymer) has been investigated in detail. The separation and quantification of residual PGlu homopolymer in the PEG-b-PGlu sample were performed by free solution capillary zone electrophoresis mode. This mode brought also information on the PEG-b-PGlu copolymer composition and polydispersity. It also permitted to monitor the decomposition of polymeric micelles in the presence of NaCl at room temperature. Interactions between PEG-b-PGlu unimers, on one hand, and polymeric micelles or surfactants, on the other hand, were studied by using the Micellar Electrokinetic Chromatography and Frontal Analysis Capillary Electrophoresis modes. Finally, weight-average hydrodynamic radii of the loaded polymeric micelles and of the PEG-b-PGlu unimers were determined by Taylor Dispersion Analysis (an absolute size determination method that can be easily implemented on CE apparatus).

An improved capillary electrophoresis method for in vitro monitoring of the challenging early steps of Aß1-42 peptide oligomerization: application to anti-Alzheimer's drug discovery.

We report an improved CE method to monitor in vitro the self-assembly of monomeric amyloid ß-peptide (42 amino acids amyloid ß-peptide, Aß1-42 ) and in particular the crucial early steps involved in the formation of the neurotoxic oligomers. In order to start the kinetics from the beginning, sample preparation was optimized to provide samples containing exclusively the monomeric form. The CE method was also improved using a dynamic coating and by reducing the separation distance. Using this method, the disappearance of the monomer as well as the progressive formation of four species during the self-assembly process can now be monitored and quantified over time. The hydrodynamic radius of the species present at the initial kinetics step was estimated around 1.8 nm by Taylor dispersion analysis while SDS-PAGE analyses showed the predominance of the monomer. These results confirmed that the Aß1-42 species present at this initial time was the monomer. Methylene blue, an anti-Alzheimer disease candidate, was then evaluated. In spite of an oligomerization inhibition, the enhanced disappearance of the Aß1-42 monomer provoked by methylene blue was demonstrated for the first time. This method, allowing the monomeric and smallest oligomeric species to be monitored, represents a new accurate and precise way to evaluate compounds for drug discovery.

Suppression of apparent fluid flow in capillary isotachophoresis without recourse to capillary coating.

This manuscript describes a new analytical methodology that allows one to stop the apparent fluid flow in uncoated fused silica capillaries. This method, which requires neither capillary coating nor buffer additive, is based on the use of a 22% polyethylene oxide gel which is placed at the anodic end of the capillary. Its high viscosity precludes its penetration into the capillary and the apparent electro-osmotic pumping effect is stopped. To avoid sample depletion in the gel, the concentration of the background electrolyte in the gel has to be at least 10-fold higher than that of the background electrolyte. This method was applied to isotachophoretic experiments performed in unmodified silica capillaries and at high alkaline pHs. The sharpness of the zone boundaries was perfectly conserved. The separation voltages recorded during the isotachophoretic processes demonstrate a very good stability of the system preventing fluctuations of the analyte migration times between runs. This approach can be virtually applied at any pH value of the background electrolyte, in the presence of organic solvents or surfactants. Using this methodology, it has been possible to detect underivatized Amyloid-ß peptide 1-40, one of the potential Alzheimer's disease biomarkers, at lower concentrations than ever previously achieved using UV detection.

Poly(glycidyl methacrylate)/silver nanocomposite microspheres as a radioiodine scavenger: electrophoretic characterisation of carboxyl- and amine-modified particles.

Silver nanoparticles possess potent antibacterial properties and have extremely high affinities to radioiodine. For several applications, it is essential to anchor the nanoparticles to microparticles or solid surfaces to make them insoluble while retaining their unique properties. This current work is related to the design of anionic and cationic macroporous polymer microspheres based on poly(glycidyl methacrylate) (PGMA) obtained using a multistep swelling polymerisation. According to scanning electron microscopy, the microspheres were monodisperse in size and 4.2 µm in diameter. The presence of the carboxyl and amino groups in the PGMA-COOH and PGMA-NH2 microspheres was confirmed by FT-IR spectroscopy. Capillary electrophoresis (CE) and pressure-assisted capillary electrophoresis (PACE) were used to study the electrophoretic behaviour of both types of microparticles. The electrophoretic mobility of the microparticles was changed into ζ potential using Smoluchowski modelling. Finally, silver-containing microspheres were prepared by reducing silver nitrate in the presence of the microspheres, and they proved effective for scavenging radioiodide ions from a model medium.

On-line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid.

Electrophoretically mediated microanalysis was implemented for on-line fluorescence derivatization of ubiquitin (UBI), a potential biomarker of Alzheimer disease. Ubiquitin was labeled on its amino groups by the Fluoprobes® 488 N-hydroxysuccinimide. The pH of the BGE, the applied electric field, and the washing process of the capillary were optimized. The best derivatization yield was obtained at pH 9.5 under an electric field of ∼210 V/cm. The concentration and volume of the fluorescent dye as well as the sample medium and volume of injected UBI were also optimized. In order to improve further the LOD of UBI, electrophoretically mediated microanalysis was performed in a wider (100 µm id) and longer (110 cm) capillary. As expected, these capillary dimensions improved the analytical sensitivity of UBI when compared to the 50 µm id capillary. Under the optimized conditions, this analytical methodology allowed the analysis of free UBI in standard solution with a LOD of ∼15 nM. Finally, the on-line fluorescent derivatization of UBI was applied to the detection and the quantification of UBI in cerebrospinal fluid (CSF) samples. Due to the high salt concentration of biological samples, desalting of CSF was required prior to the CE analysis. Quantification of UBI in CSF either by a direct evaluation of peak areas or using the standard addition method was achieved.

Study of interactions between oppositely charged dendrigraft poly-L-lysine and human serum albumin by continuous frontal analysis capillary electrophoresis and fluorescence spectroscopy.

Dendrigraft poly-L-lysine (DGL) are biomacromolecules of great interest for many applications including antibacterial activity, drug delivery systems, gene therapy and production of antibodies. As human serum albumin (HSA) is the most abundant serum protein, the study of interactions between these two compounds is crucial for the use of DGL in drug or gene delivery systems. The present work aims at determining the number of binding sites and the corresponding successive equilibrium constants between DGL of generation 3 (G3) and HSA in physiological conditions. To meet this end, continuous frontal analysis capillary electrophoresis (FACCE) and fluorescence spectroscopic methods were implemented and compared. FACCE was performed on a polycationic modified capillary in combination with a co-pressure that allowed for selectively introducing the free G3 from the G3/HSA mixtures. FACCE studies demonstrated that HSA has 2 binding sites with DGL G3 with the following successive constants K1=31.2×10(3) M(-1) and K2=30.6×10(3) M(-1). For a 1 g/L concentration in G3 and assuming a plasmatic HSA concentration of 40g/L, these binding constants lead to only 5% free DGL in the medium. It was also shown that the interactions between G3 and HSA corresponded to a model of cooperative sites. These results are in good agreement with the presence of two negatively charged domains in the HSA. Good fitting of the fluorescence spectroscopy data was obtained using the equilibrium constants derived from FACCE. Nevertheless, due to the high number of fitting parameters, it was difficult to fit the fluorescence spectroscopic data independently of the results obtained by FACCE.

Monitoring surface functionalization of dendrigraft poly-L-lysines via click chemistry by capillary electrophoresis and Taylor dispersion analysis.

Click chemistry by copper-mediated azide/alkyne cycloaddition reaction is known as a very efficient synthetic pathway for chemical coupling and for surface functionalization. The attractiveness of this reaction is due to the selectivity of the reaction of azides and alkynes. Nevertheless, the control of the kinetics of the reaction and the characterization of the reaction products requires the use of adequate analytical techniques. This is especially the case for the monitoring of the functionalization of polymeric compounds and the control of the homogeneity of the chemical composition of these products. In this work, the third generation of dendrigraft poly-L-lysines derivatized with acetylenic groups was decorated with tryptophan-PEG-azide arms via triazole ring formation. Pressure assisted capillary electrophoresis was used to monitor the progress and the kinetics of the click chemistry reaction. As expected the click product was obtained with excellent yields within 20 min reaction time at room temperature. Diffusion coefficients and hydrodynamic radii of dendrigraft poly-L-lysines and their click reaction products were determined by Taylor dispersion analysis. Finally, capillary electrophoresis was found very useful to check the purity and the homogeneity in the chemical composition of the decorated structures.

Field enhanced bacterial sample stacking in isotachophoresis using wide-bore capillaries.

The isotachophoretic analysis of different bacterial strains was studied using capillaries with different internal diameters from 50 to 250 µm. Several injection modes were investigated and compared in order to improve the limit of detection of bacteria by capillary isotachophoresis. A system suitability test obtained from the separation voltage was developed to ensure reliable results. As expected, the use of wider bore capillaries improved the analytical sensitivity of the isotachophoretic method when compared to the 50 µm capillary. With the optimized conditions, the isotachophoretic method presented in this work allows the quantification of Erwinia carotovora (Gram negative bacteria) with a limit of detection as low as ~3000 cells mL(-1). The proposed methodology does not require any additive in the electrolyte such a fluorescent or chromophoric dye to reach these limits of detection.

Taylor dispersion analysis with two detection points on a commercial capillary electrophoresis apparatus.

This work describes a simple technical modification for doing Taylor dispersion analysis with two UV detection points on a commercial capillary electrophoresis apparatus. So far, double UV detection was only possible using specific detectors that are external to the capillary electrophoresis apparatus. In this work, the detection interface of the capillary electrophoresis apparatus was easily modified to allow the introduction and the superposition of two capillary windows in the same interface (at the same detection point). This modification made possible the double detection of the sample zone in Taylor dispersion analysis using a loop. The peak dispersion using the modified interface was similar to that obtained on a non-modified UV interface. Diffusion coefficients (and the corresponding hydrodynamic radii) of small molecule and proteins were determined in good agreement with values of the literature and with RSD lower than 5%.

Study of antibacterial activity by capillary electrophoresis using multiple UV detection points.

A new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gram-negative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm ×100 µm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-L-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-L-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications.

Simultaneous electrokinetic and hydrodynamic injection for high sensitivity bacteria analysis in capillary electrophoresis.

A repeatable preconcentration electrophoretic methodology for the analysis of bacteria was developed. This method is based on an isotachophoretic mode coupled with a simultaneous hydrodynamic-electrokinetic injection in conditions of field-amplified sample injection. This electrophoretic method allows the quantification of Enterobacter cloacae (studied as a model of Gram negative bacteria) with a limit of detection of 2 × 10(4) cells/mL. With the optimized conditions, a preconcentration factor of about 500-fold was obtained as compared to a standard hydrodynamic injection. The RSD (n = 5) on the migration time and on the peak area were 3% and 5%, respectively. This capillary electrophoretic methodology has been applied for the quantification of microbes in natural water (river and natural spring waters). Filtration of the sample prior to injection was required to remove ions present in the water and to keep the field-amplified sample injection condition at the injection. Filtrated bacteria were then recovered in terminating electrolyte diluted 10 times with water. Good agreements were obtained between cellular ATP measurements and the proposed CE methodology for the quantification of bacteria in waters.