A colorimetric nanosensor based on a selective target-responsive aptamer kissing complex.

Herein, we report a novel approach for the design of a colorimetric aptasensor based on functionalized gold nanoparticle probes. This approach relies on the conjugation of nanoparticles by two functional DNA and RNA hairpins that engage specific kissing (loop-loop) interactions in response to the addition of a small analyte ligand, leading to particle aggregation and then red-to-purple colour change of the colloidal solution.

Toward sensitive immuno-based detection of tau protein by surface plasmon resonance coupled to carbon nanostructures as signal amplifiers.

Interest on Tau protein is fast increasing in Alzheimer's disease (AD) diagnosis. There is the urgent need of highly sensitive and specific diagnostic platforms for its quantification, also in combination with the other AD hallmarks. Up to now, SPR has been poorly exploited for tau detection by immunosensing, due to sensitivity limits at nanomolar level, whereas the clinical requirement is in the picomolar range. Molecular architectures built in a layer-by-layer fashion, biomolecules and nanostructures (metallic or not) may amplify the SPR signal and improve the limit of detection to the desired sensitivity. Mostly gold nanostructures are widely employed to this aim, but great interest is also emerging in Multi Walled Carbon Nanotubes (MWCNTs). Here MWCNTs are modified and then decorated with the secondary antibody for tau protein. Eventually we took advantage from MWCNTs-antibody conjugate to obtain a sandwich-based bioassay with the capability to increase the SPR signal of about 102 folds compared to direct detection and conventional unconjugated sandwich. With respect to these results, we hope to give a strong impulse for further investigation on studying possible roles of carbon nanotubes in optical-based biosensing.

Investigation of low-energy proton effects on aptamer performance for astrobiological applications.

Biochips are promising instruments for the search for organic molecules in planetary environments. Nucleic acid aptamers are powerful affinity receptors known for their high affinity and specificity, and therefore are of great interest for space biochip development. A wide variety of aptamers have already been selected toward targets of astrobiological interest (from amino acids to microorganisms). We present a first study to test the resistance of these receptors to the constraints of the space environment. The emphasis is on the effect of cosmic rays on the molecular recognition properties of DNA aptamers. Experiments on beam-line facilities have been conducted with 2 MeV protons and fluences much higher than expected for a typical mission to Mars. Our results show that this irradiation process did not affect the performances of DNA aptamers as molecular recognition tools.

DNA migration regimes in hydrodynamic chromatography and slalom chromatography: evidence for a transition.

Recently, we hypothesized that DNA separation mechanisms in slalom chromatography (SC) and packed column hydrodynamic chromatography (HDC) could be connected and simultaneously observed 'J. Chromatogr. A 886 (2000) 1'. The present paper describes the migration of various circular and linear double-stranded DNAs in a chromatographic system using a C1 stationary phase and an acetonitrile-phosphate buffer mixture as a mobile phase. The migration dependence on the flow rate for the different species showed the relative contribution of both the SC and HDC separation mechanisms. A transition between the SC and HDC regimes was demonstrated for the first time. In addition, the data were analyzed in terms of polymer migration in a dense structure. Three different behaviors were distinguished in relation to the size and the compactness of the nucleic acid.

Column efficiency and separation of DNA fragments using slalom chromatography: hydrodynamic study and fractal considerations.

Novel equations (Guillaume Y. C.; et al. Anal. Chem. 2000, 72, 853) were developed to describe the large double-stranded DNA molecule retention in slalom chromatography (SC). These equations were applied for the first time to model both the "apparent selectivity" and the resolution between two eluted DNA fragments on a chromatogram. A study of the column efficiency corroborated the fact that slalom chromatography is not based on an adsorption or equilibrium phenomenon, but can be attributed to a hydrodynamic phenomenon. Using a combination of the dynamics of DNA fragment progression in the column and fractal considerations, it was shown that the apparent selectivity depends both on the DNA fragment sizes and mobile-phase flow rate and therefore a balance between two hydrodynamic regimes. A chromatographic response function was also used to obtain the most efficient separation conditions for a mixture of DNA fragments in a minimum analysis time. The chromatographic data confirmed that in SC the flow rate can increase or maintain the separation efficiency with an associated decrease in the analysis time. This constitutes an attractive outcome in relation to the classical chromatographic separation.

Mobile-phase-viscosity dependence on DNA separation in slalom chromatography.

Slalom chromatography (SC) is an alternative chromatographic procedure for the separation of relatively large double-stranded DNA molecules and is based on a new principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase with various concentrations of viscosity modifier (i.e. glycerol) and a C1 column as a stationary phase. The DNA molecule retention was accurately described over the glycerol concentration range using a model previously established. It was shown that the eluent viscosity increase enhanced the slalom chromatographic capacity to separate the DNA fragments. A connection between SC and 'hydrodynamic chromatography' processes was predicted to link the two processes in a global separation mechanism based on a non-equilibrium principle.

Mechanism of DNA hydrodynamic separation in chromatography.

An alternative chromatographic procedure for the separation of large double-stranded DNA molecules was discovered recently and called "slalom chromatography". This fractionation is based on a new hydrodynamic process that is determined by the progression of the mobile-phase flow through the interstitial spaces created between the highly packed particles inside the column. Here, the separation is treated as the result of a slowing down of the large double-stranded DNA fragments in relation to their size with the flow direction changing around the particles. A model, based on the concept derived from the reorientation time of macromolecules, was adequate to describe the hydrodynamic phenomenon. This model constitutes an attractive tool to enhance the expansion of this chromatographic procedure and provide valuable information on the dynamic behavior of biological polymers.

Degree of solute inclusion in native beta-cyclodextrin: chromatographic approach

The reversed-phase liquid chromatography retention and separation of a series of D,L dansyl amino acids were investigated over a wide range of salting-out agent (sucrose) concentrations using native beta-cyclodextrin as a chiral stationary phase. An original treatment was developed to determine the number of sucrose molecules (n) excluded from the solute-beta-cyclodextrin cavity interface when the analyte transfer occurred. Using the n values, the relative degrees of compound inclusion were calculated and correlated to the steric bulkiness of the solute. Thermodynamic parameter variations are discussed in relation to the inclusion degree of the dansyl amino acids. This numerical approach is a valuable tool to explore the steric effects implied in the host-guest complex formation.

C60 and C70 HPLC retention reversal study using organic modifiers

A novel equation (Guillaume Y. C. et al. Anal. Chem. 1998, 70, 608) modeling the weak polar solute retention in reversed-phase liquid chromatography (RPLC) was applied to fullerene molecules C60 and C70. In RPLC, with an organic modifier (OM)/water mobile phase, the fullerene cluster solvation energies were calculated for OM = methanol, ethanol, propanol, butanol, and pentanol. An enthalpy-entropy compensation revealed that the type of interactions between fullerenes and the stationary phase was independent of both the fullerene and organic modifier structures. The energetics of OM and OM-water cluster exchange processes in the mobile phase were investigated in relation to the carbon atom number of the hydrophobic chain of the OM. Two linear correlations were found between the Gibbs free energy changes in the solvent exchange processes which confirmed that (i) a reversal elution order existed for C60 and C70 when methanol was changed into ethanol, propanol, butanol, pentanol and that (ii) the mobile phase was dominant in governing selectivity changes in nonpolar solutes.

Optimising mobile phase composition, its flow-rate and column temperature in HPLC using taboo search.

A chemometric methodology is proposed to study the separation of seven p-hydroxybenzoic esters in reversed phase liquid chromatography (RPLC). Fifteen experiments were found to be necessary to find a mathematical model which linked a novel chromatographic response function (CRF) with the column temperature, the water fraction in the mobile phase and its flow rate. The CRF optimum was determined using a new algorithm based on Glover's taboo search (TS). A flow-rate of 0.9 ml min(-1) with a water fraction of 0.64 in the ACN-water mixture and a column temperature of 10 degrees C gave the most efficient separation conditions. The usefulness of TS was compared with the pure random search (PRS) and simplex search (SS). As demonstrated by calculations, the algorithm avoids entrapment in local minima and continues the search to give a near-optimal final solution. Unlike other methods of global optimisation, this procedure is generally applicable, easy to implement, derivative free, conceptually simple and could be used in the future for much more complex optimisation problems.

A novel approach to study the inclusion mechanism of imidazole derivatives in micellar chromatography.

A chromatographic approach was proposed to describe the existence of surfactant micelles in a surfactant/hydroorganic phosphate buffer mobile phase. Using this mixture as a mobile phase, a novel mathematical theory is presented to describe the inclusion mechanism of imidazole derivatives in surfactant micelles. Using this model, enthalpy, entropy and the Gibbs free energy were determined for two chromatographic chemical processes: (i) the transfer of the imidazole derivative from the mobile phase to the stationary phase; and (ii) the imidazole derivative inclusion in surfactant micelles. These thermodynamic data indicate that the main parameter determining chromatographic retention is distribution of the imidazole derivatives to micelles of surfactant while the interaction with the stationary phase play a minor role.

Effect of temperature on DNA fractionation in slalom chromatography.

Slalom chromatography is an alternative chromatographic procedure for the analysis of relatively large double-stranded DNA molecules and is based on a hydrodynamic principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase (flow rate equal to 0.3 ml/min) and a C1 column as a stationary phase at various temperatures. It was shown that the temperature constituted an important parameter for the separation of the DNA fragments in slalom chromatography. The DNA hydrodynamic behavior with the temperature was related to the variation in the fluid viscosity and the modification of the elastic properties of the biopolyrner.

Characterization of solute binding at human serum albumin site II and its geometry using a biochromatographic approach.

Chiral recognition mechanism relationships for binding at site II on human serum albumin (HSA) were investigated using D, L dansyl amino acids. Sodium phosphate salt was used as a solute-HSA interaction modifier. A new model was developed using a biochromatographic approach to describe the variation in the transfer equilibrium constant with the salt concentration, i.e., the nature of the interactions. The solute binding was divided into two salt concentration ranges c. For the low c values, below 0.03 M, the nonstereoselective interactions constituted the preponderant contribution to the variation in the solute binding with the salt concentration. For the high c values, above 0.03 M, the solute binding was governed by the hydrophobic effect and the stereoselective interactions. The different contributions implied in the binding process provided an estimation of both the surface charge density (sigma/F) and the surface area of the site II binding cavity accessible to solvent, which were found to be equal to around 10.10(-7) mol/m(2) and 2 nm(2). As well, the excess of sodium ions excluded by the solute transfer from the surface area of the pocket were about(-0.7) for dansyl norvaline and (-0.8) for dansyl tryptophan.

Chemometric approach to the treatment of benzodiazepine separation and peak broadening in capillary electrophoresis.

A chemometric methodology was used to study capillary efficiency and the separation of ten benzodiazepines in capillary electrophoresis. The resolution between two adjacent peaks on the electropherogram was estimated and the overall quality of the separation was assessed by means of a new response function. The nature (methanol or acetonitrile) and proportion of the organic modifier both in the background electrolyte and the sample buffer and the injection time were considered. The results predicted that if the sample had a lower dielectric constant than the background electrolyte buffer then a much larger injection volume could be used. The computer optimization routine was experimentally validated and the result demonstrated that the fastest electrophoretic reparation was obtained with acetonitrile (7 min instead of 9 min with methanol).

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin.

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van't Hoff plots. An enthalpy-entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy-Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.

Symmetry breaking during the formation of beta-cyclodextrin-imidazole inclusion compounds: capillary electrophoresis study.

A mathematical model was developed for the estimation of binding constants by capillary electrophoresis. The effective electrophoretic mobility in a solute mixture is dependent on the cyclodextrin concentration in the background electrolyte (BGE) as well as the stoichiometry and the binding constant of the guest-cyclodextrin complex. As well, a determination of the degree of complexation, nc (the percent of complexed guest) could be carried out. The model was applied to a series of imidazole derivatives. Thermodynamic data for the solute complexation mechanism were calculated. Different van't Hoff plot shapes of the degree of complexation were observed with different BGE pH values, indicating a change in the solute complexation mechanism. Enthalpy-entropy compensation revealed that the solute complexation mechanism was independent of the imidazole derivative molecular structure, the same at pH = 4.5, 5.0, 5.5, 6.0, and 6.5 but changed at pH = 7.0 and 7.5. Topological defects formed during a symmetry-breaking transition could be responsible for the modification of the structure of the cyclodextrin cavity and explained the nc variations in relation to pH and temperature.

Reanalysis of solute retention on immobilized human serum albumin using fractal geometry.

Earlier experimental data for sucrose dependence of dansyl amino acid retention on immobilized human serum albumin (Peyrin E.; et al. Anal. Chem. 1998, 70, 2812) are reexamined within a fractal framework. A mathematical development based on the fractal geometry of Mandelbrot is proposed to provide a more realistic picture of the molecular association between the ligand and the site II cavity. The fractal dimension D of the cavity surface is calculated using the theoretical approach from previous data. Results show that the surface morphology of the cavity is strongly influenced by the surface tension effects of sucrose molecules, the salting-out agent leveling the surface irregularities. In addition, dansyl amino acid retention and thermodynamic parameter variations are discussed using this fractal concept of surface fluctuations.

Chromatographic approach to study beta-cyclodextrin as a promoter of the penetration of bifonazole into keratinic tissue.

A high-performance liquid chromatographic method for the determination of bifonazole in dimethyl sulfoxide solvent was developed to study its penetration into sheephoof. The analytical method was linear over the concentration range studied, i.e., from 0.1 mg/ml to 1 mg/ml. The relative standard deviation was less than 2%. The data obtained showed that complex forming with beta-cyclodextrin greatly improved the penetration of bifonazole.

Effect of tetrabutylammonium chloride as eluent modifier on the retention and enantioselectivity of d,l-dansyl amino acids using immobilized human serum albumin.

The influence of tetrabutylammonium (TBA) as a hydrophobic charged additive of the mobile phase was investigated in a chromatographic system involving d,l-dansyl amino acids as the test solute enantiomers and immobilized human serum albumin as the chiral stationary phase. By varying the column temperature, van't Hoff plots for solute retention and enantioselectivity were performed and thermodynamic parameters were calculated. An enthalpy-entropy compensation study revealed that the type of interaction between the analyte and stationary phase was independent of TBA concentration in the eluent. The counterion dependence on retention indicated that the coulombic interactions between dansyl amino acid and the site II binding cavity were of crucial importance in this association process. Also, the increasing variations of chiral discrimination with a TBA addition were attributed, by an analysis of the thermodynamic parameter trends, to a great facilitation of enantioselective H-bonding between solute and polar residues at the cavity rim.

Chemometric method to optimize chiral separation of imidazole derivatives by capillary electrophoresis.

A chemometric methodology was proposed to optimize the migration time, height equivalent to a theoretical plate and separation of a mixture of a series of imidazole compounds by capillary electrophoresis. The optimization process was based on a special polynomial from 9 or 18 preliminary experiments. This method connects a general simplex method to a computer. A simplex two or three optimization-capillary electrophoresis (STO-CE) method has been developed in our laboratory. The most efficient separation was achieved with acetonitrile-phosphate buffer, pH 4.70, (5.30+94.70 (v/v)) with a beta-cyclodextrin concentration in the background electrolyte equal to 5.80 mM and a capillary temperature of 35 degrees C. Similar results were obtained using simple step-wise scanning. The higher relative difference obtained for these values with these two methods (simplex and step-wise scanning) was 5% for the beta-cyclodextrin concentration factor.