Sharp melting transitions in DNA hybrids without aggregate dissolution: proof of neighboring-duplex cooperativity

Similar to DNA-modified gold nanoparticles, comb polymer-DNA hybrids exhibit very sharp melting transitions that can be utilized in highly selective DNA detection systems. Current theories suggest that such sharp melting results from either a phase transition caused by the macroscopic dissolution of the aggregate or neighboring-duplex interactions in the close-packed environment between adjacent DNA duplexes. To delineate the contributions of each of these effects, an aggregate system based on polymer-DNA hybrids was designed to include both polymer-linked and partially untethered duplexes. When this hybridized system was subjected to thermal analysis, both types of duplexes exhibited sharp melting transitions. The very sharp melting transition displayed by the partially untethered DNA duplexes offers proof that neighboring-duplex interactions can indeed induce cooperativity. Contributions of this neighboring-duplex effect, as well as the enhanced stabilization observed in polymer-DNA:polymer-DNA aggregates, can be quantitatively assessed using a simple thermodynamic model. While neighboring-duplex interactions alone can lead to cooperative melting, the enhanced stabilization observed in polymer-DNA aggregates is a function of both neighboring-duplex interactions and multivalent or aggregate properties.     

Ab initio melting curve of copper by the phase coexistence approach

Ab initio calculations of the melting properties of copper in the pressure range 0-100 GPa are reported. The ab initio total energies and ionic forces of systems representing solid and liquid copper are calculated using the projector augmented wave implementation of density functional theory with the generalized gradient approximation for exchange-correlation energy. An initial approximation to the melting curve is obtained using an empirical reference system based on the embedded-atom model, points on the curve being determined by simulations in which solid and liquid coexist. The approximate melting curve so obtained is corrected using calculated free energy differences between the reference and ab initio system. It is shown that for system-size errors to be rendered negligible in this scheme, careful tuning of the reference system to reproduce ab initio energies is essential. The final melting curve is in satisfactory agreement with extrapolated experimental data available up to 20 GPa, and supports the validity of previous calculations of the melting curve up to 100 GPa.     

Sharp melting in DNA-linked nanostructure systems: thermodynamic models of DNA-linked polymers

Sharp melting that has been found for DNA-linked nanostructure systems such as DNA-linked gold nanoparticles enhances the resolution of DNA sequence detection enough to distinguish between a perfect match and single base pair mismatches. One intriguing explanation of the sharp melting involves the cooperative dehybridization of DNA strands between the nanostructures. However, in the DNA-linked gold nanoparticle system, strong optical absorption by the gold nanoparticles hinders the direct observation of cooperativity. Here, with a combination of theory and experiment, we investigate a DNA-linked polymer system in which we can show that the optical profile of the system at 260 nm is directly related to the individual DNA dehybridization profile, providing a clear distinction from other possible mechanisms. We find that cooperativity plays a crucial role in determining both the value of the melting temperature and the shape of the melting profile well away from the melting temperature. Our analysis suggests that the dehybridization properties of DNA strands in confined or dense structures differ from DNA in solution.     

Enhancing the melting properties of small molecule-DNA hybrids through designed hydrophobic interactions: an experimental-computational study

Detailed experimental and computational studies revealed the important role that hydrophobic interactions play in the aqueous assembly of rigid small molecule-DNA hybrid (rSMDH) building blocks into nanoscale cage and face-to-face (ff) dimeric structures. In aqueous environments, the hydrophobic surfaces of the organic cores in these nanostructures are minimized by interactions with the core in another rSMDHs, with the bases in the attached DNA strands, and/or with the base pairs in the final assembled structures. In the case that the hydrophobic surfaces of the cores could not be properly isolated in the assembly process, an ill-defined network results instead of dimers, even at low concentration of DNA. In contrast, if ff dimers can be formed with good minimization of the exposed hydrophobic surfaces of the cores, they are highly stable structures with enhanced melting temperatures and cooperative melting behavior.     

Cooperative melting in caged dimers of rigid small molecule-DNA hybrids

Rigid small-molecule DNA hybrids (rSMDHs) have been synthesized with three DNA strands attached to a rigid tris(phenylacetylene) core. When combined under dilute conditions, complementary rSMDHs form cage dimers that melt at >10 degrees C higher and much sharper than either unmodified DNA duplexes or rSMDH aggregates formed at higher concentrations. With a 2.97 average number of cooperative duplexes, these caged dimers constitute the first example of cooperative melting in well-defined DNA-small-molecule structures, demonstrating the important roles that local geometry and ion concentration play in the hybridization/dehybridization of DNA-based materials.     

Surface phase separation in complex mixed adsorbing systems: an interface-bulk coupling effect

The interfacial thermodynamics and structure of ternary mixtures of the type A+B+solvent are investigated. According to the Gibbs phase rule, the coupling between the bulk phase and the interfacial region-which is related to the reversibility of the adsorption of the corresponding species-is a determinant as to whether phase separation can be observed at the interface. For an n-component adsorbing solution, at least one of the species has to adsorb irreversibly over the experimental time scales in order not to fix more intensive variables than those required to observe surface phase separation. We present results for a lattice model planar interface consisting of the ternary mixture A+B+solvent. The solvent molecules and the type A molecules have fixed chemical potentials at the interface since they are equilibrated with a bulk solution. In contrast, the type B molecules are irreversibly adsorbed at the interface and do not equilibrate with the bulk. Mean-field theory is compared with Monte Carlo simulation. Interestingly, the spinodal line in the interaction-composition plane shows a reentrant on the B-rich phase side. We discuss the implications of these results for surface phase separation of adsorbing mixtures of proteins and low-molecular-weight surfactants.     

Three-stage melting of an annelide-type copper complex. A new type of organized phase: Tegma crystals

The solid—solid phase transition of an annlide type copper complex are described. As the temperature is raised, three different constituents of the complex successively melt. In the temperature range 77–95°C, the polyethylene oxide chains and the paraffinic tail are both in a very mobile state while the copper complex sub-units still form a two-dimensional crystalline array.     

Pervaporation: an integrated evaporation/gas-diffusion approach to analytical continuous separation techniques

A flexible pervaporation module was developed and characterized. The spacers it uses allows the volume of the donor and/or acceptor to be altered as required. The performance of the module was tested as regards both dynamic behaviour and continuous pervaporation in terms of flow-rates, temperature, type of membrane, flow mode, etc. A method for the determination of ethanol in different types of wine was developed and applied to various samples in order to validate the proposed continuous separation approach.     

Fluid phase separation inside a static periodic field: an effectively two-dimensional critical phenomenon

When a fluid with a bulk liquid-vapor critical point is placed inside a static external field with spatial periodic oscillations in one direction, a new phase arises. This new phase-the so-called "zebra" phase-is characterized by an average density roughly between that of the liquid and vapor phases. The presence of the zebra phase gives rise to two new phase transitions: one from the vapor to the zebra phase, and one from the zebra to the liquid phase. At appropriate values of the temperature and chemical potential, the latter two transitions become critical. This phenomenon is called laser-induced condensation [I. O. Go?tze, J. M. Brader, M. Schmidt, and H. Lo?wen, Mol. Phys. 101, 1651 (2003)]. The purpose of this paper is to elucidate the nature of the critical points, using density functional theory and computer simulation of a colloid-polymer mixture. The main finding is that critical correlations develop in two-dimensional sheets perpendicular to the field direction, but not in the direction along the field: the critical correlations are thus effectively two-dimensional. Hence, static periodic fields provide a means to confine a fluid to effectively two dimensions. Away from criticality, the vapor-zebra and liquid-zebra transitions become first-order, but the associated surface tensions are extremely small. The consequences of the extremely small surface tensions on the nature of the two-phase coexistence regions are analyzed in detail.     

Characterization of superlighting polymer-DNA aggregates: a fluorescence and light scattering study

The massive amplification of fluorescence signal observed upon hybridization of as few as five DNA molecules into self-assembled structures formed between a cationic polymer and DNA oligonucleotides is investigated. These superlighting polymer-DNA aggregates were studied by fluorescence spectroscopy, static and dynamic light scattering, and zeta potential measurements in order to characterize the aggregation behavior and to understand the processes involved during DNA detection. Multi-angle laser light scattering was also used to obtain the weight-average aggregate mass (AM), the aggregation number (Nagg), the radius of gyration (Rg), and the dissymmetry ratio (z). These results have been used, together with TEM imaging, to propose a suitable physical model for the aggregates.     

Artificial multiple criticality and phase equilibria: an investigation of the PC-SAFT approach

The perturbed-chain statistical associating fluid theory (PC-SAFT) is studied for a wide range of temperature, T, pressure, p, and (effective) chain length, m, to establish the generic phase diagram of polymers according to this theory. In addition to the expected gas-liquid coexistence, two additional phase separations are found, termed "gas-gas" equilibrium (at very low densities) and "liquid-liquid" equilibrium (at densities where the system is expected to be solid already). These phase separations imply that in one-component polymer systems three critical points occur, as well as equilibria of three fluid phases at triple points. However, Monte Carlo simulations of the corresponding system yield no trace of the gas-gas and liquid-liquid equilibria, and we conclude that the latter are just artefacts of the PC-SAFT approach. Using PC-SAFT to correlate data for polybutadiene melts, we suggest that discrepancies in modelling the polymer density at ambient temperature and high pressure can be related to the presumably artificial liquid-liquid phase separation at lower temperatures. Thus, particular care is needed in engineering applications of the PC-SAFT theory that aims at predicting properties of macromolecular materials.     

(Micellar) electrokinetic chromatography: an interesting solution for the liquid phase separation dilemma

High-performance liquid chromatography (HPLC) is a well-established method in modern analysis. The method is simple, very robust and is applicable to the majority of components to be analyzed in contrast to gas chromatography. Low efficiency and small peak capacity are sore points of HPLC when complex mixtures have to be separated. The reason for this dilemma is the small diffusion coefficient of the analytes in the liquid mobile phase compared to a gaseous phase. This review, complemented by exemplary calculated data and some latest results of our own research, illustrates the dilemma of liquid phase chromatography to achieve high efficiencies under reasonable conditions. It is shown that (micellar) electrokinetic chromatography, offering fast and efficient separations, is a very promising solution for this dilemma. Additional features of this method are possibilities of on-line analyte concentration, coupling to mass spectrometry and the easy change of selectivities by applying various separation additives. The pros and cons of electrokinetic chromatography are pointed out and some application examples are given.     

Cobalt-mediated approach for the synthesis of terpene-based hybrids

[structure: see text] Dicobalt-beta-pinene hybrids of types I and II have been prepared using a Nicholas reaction between propargyl derivatives, obtained from commercial (1R)-(-)-myrtenal, and different aromatic nucleophiles. The method is suitable for the preparation of densely functionalized bio-organometallic natural product-based hybrids, as demonstrated by the preparation of a beta-pinene-neoclerodane hybrid.     

Proteolysis in microfluidic droplets: an approach to interface protein separation and peptide mass spectrometry

A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic micro-reaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, non-specific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry.     

Scaling behavior of nonisothermal phase separation

The phase separation process in a critical mixture of polydimethylsiloxane and polyethylmethylsiloxane (PDMS/PEMS, a system with an upper critical solution temperature) was investigated by time-resolved light scattering during continuous quenches from the one-phase into the two-phase region. Continuous quenches were realized by cooling ramps with different cooling rates kappa. Phase separation kinetics is studied by means of the temporal evolution of the scattering vector qm and the intensity Im at the scattering peak. The curves qm(t) for different cooling rates can be shifted onto a single mastercurve. The curves Im(t) show similar behavior. As shift factors, a characteristic length Lc and a characteristic time tc are introduced. Both characteristic quantities depend on the cooling rate through power laws: Lc approximately kappa(-delta) and tc approximately kappa(-rho). Scaling behavior in isothermal critical demixing is well known. There the temporal evolutions of qm and Im for different quench depths DeltaT can be scaled with the correlation length xi and the interdiffusion coefficient D, both depending on DeltaT through critical power laws. We show in this paper that the cooling rate scaling in nonisothermal demixing is a consequence of the quench depth scaling in the isothermal case. The exponents delta and rho are related to the critical exponents nu and nu* of xi and D, respectively. The structure growth during nonisothermal demixing can be described with a semiempirical model based on the hydrodynamic coarsening mechanism well known in the isothermal case. In very late stages of nonisothermal phase separation a secondary scattering maximum appears. This is due to secondary demixing. We explain the onset of secondary demixing by a competition between interdiffusion and coarsening.     

A phase separation fluoroimmunoassay of estradiol

A competitive indirect fluoroimmunoassay of free estradiol (E₂) was established based on the thermal sensitivity of hydrogel–‐poly‐N‐isopropylacrylamide. Free estradiol was covalently bound to bovine serum albumin (BSA) to form complete antigen (E₂‐BSA), which was in turn labeled by fluorescein isothiocyanate (FTTC) as the fluorescence probe. The anti‐ E₂ monoclonal antibody (McAb) was prepared by an in vivo method, and coupled with N‐isopropylacrylamide (NIPA) to make an immune copolymer, poly‐N‐isopropylacylamidemonoclonal antibody (pNIPA‐McAb), for the determination of free E₂. The immunoassay method was based on the competitive binding of free E₂ and fluoresceinated antigen (E₂‐BSA‐FTTC) with limited amount of pNIPA‐McAb. When the immunological reaction was over, precipitation and centrifugal procedures were carried out to separate pNIPA‐McAb‐E₂‐BSA‐FTTC from other constituents in solution. The precipitate pNIPA‐McAb‐E₂‐BSA‐FTTC was dissolved in solution and then the fluorescence intensity was measured. The calibration curve covered a range of 78–500 ng/mL for free E₂. The recoveries were 91.2–107.2%.     

Effect of packing parameter on phase diagram of amphiphiles: an off-lattice Gibbs ensemble approach

We determine the phase diagram of several amphiphilic molecules as a function of the amphiphilic parameter alpha defined as the ratio of the volume of hydrophilic to hydrophobic segments using the Gibbs ensemble Monte Carlo method supplemented by configurational bias scheme. Specifically, we study amphiphilic molecules h(1)t(7), h(2)t(6), and h(3)t(5), for which alpha=0.14, 0.33, and 0.60 respectively, and demonstrate that the former two exhibit phase separation while h(3)t(5) forms micelles, supporting the contention that alpha=0.5 is the border line for phase separation and micellization, as observed in previous lattice Monte Carlo studies [Panagiotopoulos et al., Langmuir 18, 2940 (2002)]. Further, we study the phase separation in amphiphilic molecules as a function of the packing parameter by varying the size of the hydrophilic head for each molecule. We find that a larger hydrophilic head lowers the critical temperature T(c), and raises the critical density rho(c).     

A novel approach in drug discovery: synthesis of estrone--talaromycin natural product hybrids

Hetero-Diels-Alder reaction of the steroidal exocyclic enol ethers 14 and 15, obtained from the secoestrones 8 and 9 by reduction, iodoetherification, and elimination, with ethyl O-benzoyldiformylacetate (16) leads to the spiroacetals 17 and 18 as a mixture of four diastereomers. Reduction of the major diastereomers 17a and 18a with DIBAH and subsequent hydrogenation yields the novel natural product hybrids 21, 23, 24, and 25, which possess the structural features of the steroid estrone (7) and the mycotoxin talaromycin 6.     

Simultaneous concentration and separation of microorganisms: insulator-based dielectrophoretic approach

Microanalytical methods offer attractive characteristics for rapid microbial detection and concentration. There is a growing interest in the development of microscale separation techniques. Dielectrophoresis (DEP), a nondestructive electrokinetic transport mechanism, is a technique with great potential for microbe manipulation, since it can achieve concentration and separation in a single step. DEP is the movement of particles due to polarization effects in nonuniform electric fields. The majority of the work on dielectrophoretic manipulation of microbes has employed alternating current fields in arrays of microelectrodes, an approach with some disadvantages. An alternative is to employ insulator-based DEP (iDEP), a dielectrophoretic mode where nonuniform fields are produced by employing arrays of insulating structures. This study presents the concentration and fractionation of a mixture of bacteria and yeast cells employing direct current-iDEP in a microchannel containing an array of cylindrical insulating structures. Negative dielectrophoretic trapping of both types of microorganisms was demonstrated, where yeast cells exhibited a stronger response, opening the possibility for dielectrophoretic differentiation. Simultaneous concentration and fractionation of a mixture of both types of cells was carried out analogous to a chromatographic separation, where a dielectropherogram was obtained in less than 2 min by applying an electric field gradient and achieving concentration factors in the order of 50 and 37 times the inlet concentration for Escherichia coli and Saccharomyces cerevisiae cells, respectively. Encouraging results were also obtained employing a sample of water taken from a pond. The findings demonstrated the great potential of iDEP as a rapid and effective technique for intact microorganism concentration and separation.