Ultradilute Solution Research Articles

Ultrasensitive chemiluminescence detection of sub-f M level Co(II) in capillary electrophoresis

A method of on-line ultrasensitive chemiluminescence detection with capillary electrophoresis for Co(II) is reported. Using our newly developed capillary electrophoresis with chemiluminescence detection system and novel mixing mode of the reagents, the effects of field-amplified injection on detection limits of metal ions were studied in detail. The sub-f M level (1.3·10 −16 M, 1.6×10 −24 mol, 1 molecule) detection of cobalt ions in ultradilute solution was performed. The catalytic behavior of the chemiluminescence reaction of luminol and hydrogen peroxide by cobalt ions and the reaction conditions, such as the concentration of luminol, H 2O 2, and pH of chemiluminescence reagent were investigated. The separation of f M level Co(II) and trace amounts of Ni(II) was performed successfully.

Fluorescence investigations of the thermally induced conformational transition of poly( N-isopropylacrylamide)

Poly( N-isopropylacrylamide), PNIPAM, undergoes phase separation, upon heating under semi-dilute conditions, at 32°C, the lower critical solution temperature (LCST). Upon subsequent cooling, the dispersion rapidly clears again, at the LCST, to form a single, homogeneous phase. In this paper we report that fluorescence techniques, including quenching and time-resolved anisotropy measurements (TRAMS) on ultra-dilute (10 −3 wt%) aqueous solutions of an acenaphthylene labelled sample, provide conclusive evidence that a coil collapse mechanism is implicated in the thermoreversible behaviour: the polymer undergoes a conformational switch from an open coil below 32°C to a compact, globular structure above the LCST. TRAMS reveal that a marked reduction in the segmental mobility of the polymer occurs at the onset of the LCST. In addition, a dramatic change in the accessibility of the label to aqueous-borne quenchers is also apparent at temperatures in excess of 32°C. PNIPAM is capable of solubilising low molar mass species in its compact form: changes in the vibrational fine structure of the emission spectrum of pyrene have been used to monitor uptake and release of the probe. Excited state lifetime measurements have also proven to be sensitive monitors of the conformational switch of PNIPAM: at temperatures greater than 32°C, τ increases to ca. 160 ns which is indicative of pyrene sequestered in a hydrophobic, protective environment. Release of the probe into the aqueous phase results in a dramatic reduction of τ to ca. 130 ns which is characteristic of pyrene dispersed in water. These data highlight the potential of NIPAM based polymers to act as carriers in controlled release applications. Fluorescence techniques have proved capable of monitoring changes in chain mobility, the degree of coil compaction and solubilization capacity (for organic guests) of PNIPAM as it is raised through its LCST. Information of this type is not readily obtained through other techniques and fluorescence approaches should prove invaluable in future investigations of the effects of chemical modification in attempts to manipulate the LCST of NIPAM-based systems.

A Novel Approach to Functionalized Nanoparticles: Self-Crosslinking of Macromolecules in Ultradilute Solution

A Novel Approach to Functionalized Nanoparticles: Self-Crosslinking of Macromolecules in Ultradilute Solution

Time-resolved fluorescence studies of the interactions between the thermoresponsive polymer host, poly( N-isopropylacrylamide), and a hydrophobic guest, pyrene

The time-resolved fluorescence anisotropy behaviour of pyrene solubilized (10 −6 M) in ultra-dilute (10 −3 wt%) aqueous solutions of the thermoresponsive polymer poly( N-isopropylacrylamide), PNIPAM, shows unusual characteristics. Rather than decay to zero (as expected of a freely rotating species in a homogeneous fluid environment) the anisotropy of the emission from the probe attains a minimum but finite value, some 40 ns after excitation of the pyrene. Thereafter, over a timescale of hundreds of nanoseconds, the anisotropy increases: the fluorescence from the solubized pyrene guest becomes more polarized with time! These deviations from “expectation” are significant both in terms of the aqueous solution behaviour of the polymer and basic photophysics. The anisotropy data confirm that, above the lower critical solution temperature (LCST) of PNIPAM, the polymer host (in the form of single, “collapsed” macromolecules) is capable of solubilizing hydrophobic guests. The complexity of the time dependence of anisotropy of fluorescence from pyrene, above the LCST of the system, reflects the heterogeneous nature of the medium in which the probe is dispersed. At one extreme, we have pyrene molecules located within the bulk aqueous phase. At the other extreme, pyrene species are occluded within the hydrophobic interiors of the collapsed PNIPAM globules. The initial rapid loss in emission anisotropy reflects the contribution from shorter lived and rapidly tumbling solutes dispersed largely in the aqueous phase. The subsequent enhancement in the anisotropy at longer times, results from the ever-increasing importance of fluorescence from the longer lived, slowly rotating probes solubilized within the highly viscous cores of the globular conformation of the macromolecule. Below the polymer's LCST, most of the pyrene guest is released to the bulk aqueous phase but a proportion of the organic guests remains associated with the (otherwise) open coils of the polymer. In photophysical terms, the data presented constitute the first example of a study in which the motion of a fluorescent guest, mediated by the influence of its interactions with a synthetic polymer, appears to become more restricted as the time of sampling of its emission, following excitation, increases.

On the sampling variance of ultra-dilute solutions

Contemporary analytical methodology allows the determination of substances in ultra-dilute solutions, i.e. picomol per liter (pM) and lower, by employing a wide spectrum of techniques. The reduction of both sample volume and quantification limits made possible the determination of zeptomol amounts of analytes. Among the many problems associated with the analysis of this kind of sample is the relatively high intrinsic sampling variance which restricts the attainable overall analytical precision. This sampling variance is due to the quantized nature of matter. The expected sampling variance can be calculated considering that the limited number of analyte molecules per test-portion is subjected to Poisson distribution.

High performance capillary gel electrophoresis as a method to separate plasmid-DNA cloning vectors with very high resolution (below 100 bp) and its application in molecular biology

A novel high resolution method for the separation of, both, linear and supercoiled circular plasmid DNA in the range of 3000–5600 bp is described. Employing ultradilute solutions of hydroxyethylcellulose (0.070–0.100% w/w) containing no intercalating agent, we were able to resolve plasmids as well as linear fragments with a size difference of about 100 to 55 bp (R=0.5), depending on their size, at a limit-of-detection of about 3 ng (UV/VIS 260 nm, absolute amount). The effect on the separation efficiency of varying polymer and buffer concentration, capillary coating, salt additives and applied voltage has been investigated. The main issue of this study is to explore the applicability of high performance capillary gel electrophoresis (HPCGE) to molecular cloning and related purposes. Our results demonstrate that this method allows convenient monitoring of cloning procedures. It is superior to conventional agarose gel electrophoresis with respect to, both, sensitivity and resolution. Hence, it is especially useful for manipulations which result in only a small difference in fragment length or provide only limited quantities of sample DNA. To summarize, the method described in this paper allows to determine the size, concentration, and purity of plasmid DNA simultaneously in one single step. ©2000 John Wiley & Sons, Inc. J Micro Sep 12: 75–86, 2000

High performance capillary gel electrophoresis as a method to separate plasmid-DNA cloning vectors with very high resolution (below 100 bp) and its application in molecular biology

A novel high resolution method for the separation of, both, linear and supercoiled circular plasmid DNA in the range of 3000–5600 bp is described. Employing ultradilute solutions of hydroxyethylcellulose (0.070–0.100% w/w) containing no intercalating agent, we were able to resolve plasmids as well as linear fragments with a size difference of about 100 to 55 bp (R=0.5), depending on their size, at a limit-of-detection of about 3 ng (UV/VIS 260 nm, absolute amount). The effect on the separation efficiency of varying polymer and buffer concentration, capillary coating, salt additives and applied voltage has been investigated. The main issue of this study is to explore the applicability of high performance capillary gel electrophoresis (HPCGE) to molecular cloning and related purposes. Our results demonstrate that this method allows convenient monitoring of cloning procedures. It is superior to conventional agarose gel electrophoresis with respect to, both, sensitivity and resolution. Hence, it is especially useful for manipulations which result in only a small difference in fragment length or provide only limited quantities of sample DNA. To summarize, the method described in this paper allows to determine the size, concentration, and purity of plasmid DNA simultaneously in one single step. ©2000 John Wiley & Sons, Inc. J Micro Sep 12: 75–86, 2000

Single-molecule analysis of ultradilute solutions with guided streams of 1-µm water droplets

We describe instrumentation for real-time detection of single-molecule fluorescence in guided streams of 1-microm (nominal) water droplets. In this technique, target molecules were confined to droplets whose volumes were comparable with illumination volumes in diffraction-limited fluorescence microscopy and guided to the waist of a cw probe laser with an electrostatic potential. Concentration detection limits for Rhodamine 6G in water were determined to be approximately 1 fM, roughly 3 orders of magnitude lower than corresponding limits determined recently with diffraction-limited microscopy techniques for a chemical separation of similar dyes. In addition to its utility as a vehicle for probing single molecules, instrumentation for producing and focusing stable streams of 1-2-microm-diameter droplets may have other important analytical applications as well.

Fluorescence Correlation Spectroscopy for Rapid Multicomponent Analysis in a Capillary Electrophoresis System

We describe a new technique for performing multicomponent analysis using a combination of capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS), which we refer to as CE/FCS. FCS is a highly sensitive and rapid optical technique that is often used to perform multicomponent analysis in static solutions based on the different diffusion times of the analyte species through the detection region of a tightly focused laser beam. In CE/FCS, transit times are measured for a mixture of analytes continuously flowing through a microcapillary in the presence of an electric field. Application of an electric field between the inlet and outlet of the capillary alters the transit times, depending on the magnitude and polarity of the applied field and the electrophoretic mobilities of the analytes. Multicomponent analysis is accomplished without the need to perform a chemical separation, due to the different electrophoretic mobilities of the analytes. This technique is particularly applicable to ultradilute solutions of analyte. We have used CE/FCS to analyze subnanomolar aqueous solutions containing mixtures of Rhodamine 6G (R6G) and R6G-labeled deoxycytosine triphosphate nucleotides. Under these conditions, fewer than two molecules were typically present in the detection region at a time. The relative concentrations of the analytes were determined with uncertainties of ∼10%. Like diffusional FCS, this technique is highly sensitive and rapid. Concentration detection limits are below 10(-)(11) M, and analysis times are tens of seconds or less. However, CE/FCS does not require the diffusion coefficients of the analytes to be significantly different and can, therefore, be applied to multicomponent analysis of systems that would be difficult or impossible to study by diffusional FCS.

The dynamics of ultradilute polymer solutions in transient flow: Comparison of dumbbell-based theory and experiment

Predictions of the Chilcott–Rallison FENE dumbbell model for polymer configuration in start-up flow of a co-rotating two-roll mill, are compared with birefringence measurements in a 40 ppmw solution of high molecular weight polystyrene. Two versions of the Chilcott–Rallison model are considered, one with a constant bead friction and the other with bead friction that is assumed to increase in proportion to the end-to-end dimension of the dumbbell. Parameters for the model were determined via independent experiments. We show that the flow, at this concentration level, is unchanged from the form for a Newtonian fluid. Measurements and predictions of the birefringence are compared at the stagnation point of the flow, where a high degree of polymer stretch is possible. The Chilcott–Rallison model with constant bead friction, and an approximation to the inverse Langevin spring function, gives excellent agreement with the experimental results.

Capillary electrophoresis of herbicides: IV. Evaluation of octylmaltopyranoside chiral surfactant in the enantiomeric separation of fluorescently labeled phenoxy acid herbicides and their laser-induced fluorescence detection.

A novel chiral nonionic surfactant, namely octyl-b-D-maltopyranoside (OM), was evaluated in chiral capillary electrophoresis of fluorescently labeled phenoxy acid herbicides. The labeling of the analytes with 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) permitted a concentration detection limit of 5 x 10-10 M using laser-induced fluorescence detection. This limit of detection allowed the determination of ultradiluted solutions of the ANDSA-derivatized phenoxy acid herbicides whose concentration was a low as 10-11 M (i.e. 2.2 ppt) by applying the concept of field-amplified sample stacking (FASS). The sample injection by FASS did not adversely affect separation efficiencies, resolution and reproducibility of the electrophoretic system. The tagging of the phenoxy acid herbicides with ANDSA increased the hydrophobicity of the analytes, thus favoring an enhanced solubilization of the derivatized herbicides in the OM micellar phase. The net results of this effect were a much shorter analysis time and an improved enantiomeric resolution of the derivatives when compared to underivatized phenoxy acid herbicides. The optimum surfactant concentration required for maximum resolution decreased with increasing hydrophobicity of the analyte, with the least hydrophobic analyte requiring higher surfactant concentration. Because of the two permanently charged sulfonic acid groups of the ANDSA tag, the pH of the running electrolyte had little effect on the enantiomeric resolution of the derivatized herbicides. Due to its salting-out effect and increasing the micellized surfactant concentration, increasing the ionic strength of the running electrolyte increased the enantiomeric resolution of the least hydrophobic analytes. Conversely, increasing the percent methanol in the running electrolyte decreased the enantiomeric resolution of the least hydrophobic analytes due to a decrease strength of solute-micelle association. For hydrophobic analytes, existed an optimum percent of methanol existed for maximum enantiomeric resolution.

Ultrafast high resolution separation of large DNA fragments by pulsed‐field capillary electrophoresis

Pulsed-field capillary electrophoresis (PFCE) in buffers containing ultradilute polymer solutions is used to separate long chain dsDNA in less than 4 min. Separations are shown to work with chain lengths below 10 kbp and greater than 1.5 Mbp. Several pulse protocols have been examined. If running time is to be minimized, a field inversion with higher peak amplitude in the forward direction than in the reverse, but with equal pulse durations, provides the best resolution. Other protocols can provide higher resolution, but only with longer running times.

Dynamics of T2 DNA during electrophoresis in entangled and ultradilute hydroxyethyl cellulose solutions

Dynamics of T2 DNA during electrophoresis in entangled and ultradilute hydroxyethyl cellulose solutions

Dynamics of T2 DNA during electrophoresis in entangled and ultradilute hydroxyethyl cellulose solutions

Dynamics of T2 DNA during electrophoresis in entangled and ultradilute hydroxyethyl cellulose solutions

DNA conformational dynamics in polymer solutions above and below the entanglement limit

Video microscopy of nucleic acids (DNA) undergoing electrophoresis in hydroxyethyl cellulose (HEC) sieving buffers demonstrates previously unobserved shape-changing interactions between DNA and HEC molecules. We provide the first visual demonstration of entanglement between DNA and one or several discrete HEC molecules, which has been postulated to occur in ultradilute polymer solutions. Typically, nucleic acids appear to become entangled with HEC at a single region only, in both dilute and fully entangled HEC solutions. Fluctuations of the center of mass velocity of a DNA molecule and its correlation with conformation are revealed from analyses of the image data. These observations account for the success of recently reported rapid, high-resolution dc and pulsed-field capillary electrophoretic separations of nucleic acids in ultradilute hydroxyethyl cellulose solutions and hydroxyethyl cellulose/poly(ethylene oxide) solutions.

Rapid pulsed field capillary electrophoretic separation of megabase nucleic acids.

Pulsed field capillary electrophoresis in ultradilute solutions of sieving polymers has been used to separate nucleic acid fragments as long as 1.6 million base pairs. Hydroxyethyl cellulose solutions are used for separations in the size range 8000-50,000 base pairs. Longer chain nucleic acids are separated in mixed hydroxyethyl cellulose/poly(ethylene oxide) solutions. Separations are rapid (12-13 min).

Capillary Electrophoresis Separation Techniques and Mechanisms in Dilute Polymer Matrices

AbstractCapillary electrophoresis in ultradilute derivatized solutions is used to separate nucleic acid fragment mixtures. Pulsed field techniques are used to improve resolution. Protocols in which pulse parameters are varied during the course of the separation are used to increase the range of fragment sizes separated. Fluorescence microscopy is used to study the separation mechanism in ultradilute solution.

The use of coated and uncoated capillaries for the electrophoretic separation of DNA in dilute polymer solutions

We show that both uncoated and polyacrylamide-coated capillaries provide separation of large DNA restriction fragments (2.0-23.1 kbp) by capillary electrophoresis in dilute cellulosic polymer solutions. Uncoated capillaries, however, provide significantly better resolution of DNA fragments, particularly when ultra-dilute polymer solutions are used. This is because electroosmotic flow in uncoated capillaries increases the residence time of DNA in the capillary, without significantly contributing to band-broadening. At a given field strength and polymer concentration in the buffer, the electrophoretic mobilities of DNA restriction fragments in coated capillaries are virtually identical to those previously measured in uncoated capillaries. It is concluded that the fused silica surface of the capillary does not play a significant role in the mechanism of DNA separation by capillary electrophoresis in uncrosslinked polymer solutions. Thus, the separation of large DNA which has been observed to occur in ultra-dilute polymer solutions arises primarily from entanglement interactions between the cellulosic polymers and DNA restriction fragments which occur within the bulk of the polymer solution.

A transient entanglement coupling mechanism for DNA separation by capillary electrophoresis in ultradilute polymer solutions.

Using capillary electrophoresis, large DNA molecules (2.0-23.1 kbp) may be rapidly separated in ultradilute polymer solutions (< 0.002% w/w) under a high-voltage, steady field (265 V/cm). At this polymer concentration, the separation mechanism appears to be significantly different from that postulated to occur in crosslinked gels. Based on experimental results obtained with DNA restriction fragments and with negatively charged latex microspheres, we conclude that the Ogston and reptation models typically used to describe gel electrophoresis are not appropriate for DNA separations in such dilute polymer solutions. Electrophoresis experiments employing solutions of both small and large hydroxyethyl cellulose polymers highlight the importance of polymer length and concentration for the optimum resolution of DNA fragments varying in size from 72 bp to 23.1 kbp. A transient entanglement coupling mechanism for DNA separation in dilute polymer solutions is developed, which suggests that there is no a priori upper size limit to DNA that can be separated by capillary electrophoresis in a constant field.

Microstructure of crystalline-amorphous type block copolymer prepared from ultra-dilute solution

The microstructures of crystalline-amorphous type block copolymers, poly(tetrahydrofuran- b-isoprene), prepared from ultra-dilute solution (0.001 wt%) were studied by transmission electron microscopy. The obtained shape was different from the shape observed in the case of amorphous-amorphous type block copolymers; the shape was deformed by poly(tetrahydrofuran) crystallization.