Conjugation reactions in the preparations of quantum dot-based immunoluminescent probes for analysis of proteins by capillary electrophoresis.

A number of biologically important molecules, such as DNA, proteins, and antibodies, are routinely conjugated with fluorescent tags for high-sensitivity analyses. Here, the application of quantum dots in the place of bright and size-tunable luminophores is studied. Several selected bioconjugation reactions via zero-length cross-linkers, long-chain linkers, and oriented methods for linking of quantum dots with proteins were tested. Anti-ovalbumin, anti-proliferating cell nuclear antigen, anti-hemagglutinin, and anti-CD3 membrane protein as model antibodies and annexin V were used as high-specificity selectors. The reaction yield and efficiency of the prepared immunoluminescent probes were tested by capillary zone electrophoresis with laser-induced fluorescence detection.

Microdevices in mass spectrometry.

Miniaturization of laboratory instrumentation is becoming critical in achieving the speed and throughput required by the current revolutionary progress in biology. This mini review critically summarizes the present status of microfluidic devices designed for use in mass spectrometry.

Two-point fluorescence detection and automated fraction collection applied to constant denaturant capillary electrophoresis.

Constant denaturant capillary electrophoresis (CDCE) has been shown to be a sensitive method to detect point mutations in DNA sequences of 100-bp lengths. Here, we report a significant modifications for the instrumental setup that allows a highly accurate prediction of the elution time of DNA fragments from the capillary and an efficient collection of separated fractions. Fluorescently labeled DNA fragments of TP53 exon 8 wild-type and two mutants (base pair number 14480 and 14525) are detected at two separate points of the same capillary. This permits the precise calculation of the fragment velocity after separation in the heated zone because, at room temperature, all DNA fragments of the same length have the same velocity. Such precision permits the selective collection of separated fragments using an automated fraction collector for additional CDCE analysis or sequencing. Also, the two-point detection allows one to rapidly distinguish between double-stranded and single-stranded DNA fragments of the same length, a process that cannot be achieved with a one-point detection system alone. Both modifications greatly improve the procedure to detect novel mutations by means of CDCE.

DNA sequencing by capillary electrophoresis using short oligonucleotide primer libraries.

Two strategies for DNA sequencing by primer walking using short oligonucleotide primer libraries have been successfully employed along with capillary electrophoresis using replaceable polymer solutions of linear polyacrylamide and fluorescence detection. A 3.5-kb stretch of the single-stranded M13mp18 template was sequenced with T7 PRISM dye-terminator/Sequenase chemistry. An in-house base-calling program offered read lengths of roughly 450 bases with an average of 97.8% accuracy.

Effect of dopamine on gastrointestinal motility during critical illness.

OBJECTIVE: To document the action of dopamine on gastrointestinal motility in mechanically ventilated patients. DESIGN: Crossover, randomized, placebo-controlled study. SETTING: General intensive care unit (ICU) in a university hospital. PATIENTS: Twelve mechanically ventilated patients in a stable hemodynamic condition, with no contraindication to enteral feeding. INTERVENTIONS: Dopamine (4 microg/kg per minute) and placebo were infused over 8 h (4 h fasting, followed immediately by 4 h nasogastric feeding at 100 kcal per hour) on two consecutive days, in a random order. Pressure changes in the gastric antrum (four sites) and in the duodenum (two sites) were recorded by perfused catheter manometry. Each session started with the institution of dopamine or placebo infusion. MEASUREMENTS AND RESULTS: The migrating motor complex and its three successive phases were identified (phase I, period of quiescence; phase II, period of irregular contractile activity; phase III or activity front, period of high-frequency, regular contractions). Contractions and activity fronts at each site were quantified during fasting and feeding. The mean duration of the fasting migrating motor complex was determined in the duodenum, as well as the contribution of each phase (phases I, II, III) to the length of the complete cycle. The propagation characteristics of each activity front were assessed visually. The number of contractions was lower in the antrum (p = 0.024) and phase III motor activity higher in the duodenum [incidence of activity fronts (p = 0.008); number of phase III contractions (p = 0.009)] during dopamine infusion than with placebo. These modifications observed under dopamine were related to decreased antral contractions during fasting (p = 0.050), increased incidence of activity fronts during feeding (p = 0.031), and increased number of phase III contractions during fasting (p = 0.037). In both groups (placebo and dopamine) activity fronts rarely started in the antrum, and abnormally propagated activity fronts were found in the duodenum in some patients. CONCLUSIONS: Low-dose dopamine adversely affects gastroduodenal motility in mechanically ventilated critically ill patients.

Analysis of protein fractions by micropreparative capillary isoelectric focusing and matrix-assisted laser desorption time-of-flight mass spectrometry.

In this study, the use of capillary isoelectric focusing (cIEF) as a micropreparative tool for protein analysis by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated. A newly designed, automated, collection interface equipped with a fiber-optic UV detector and a sheath flow connection was employed for collection of protein fractions. Multiple fractions were collected during a single cIEF run and further analyzed by MALDI-TOF-MS for mass assignment. The feasibility of the method was tested with a mixture of model proteins with different isoelectric points and molecular masses, and with variants of human hemoglobins differing in pI, but with negligible difference in M(r). Some practical considerations of the collection procedure and subsequent TOF analysis are presented.

Characterization and performance of a neutral hydrophilic coating for the capillary electrophoretic separation of biopolymers.

Polyvinylmethylsiloxanediol (50% vinyl) was synthesized and combined with a cross-linker for static coating onto fused-silica columns. After cross-linking and binding to the surface, linear polyacrylamide was grafted to the double bonds of the siloxanediol; subsequently, this linear polymer matrix was cross-linked with formaldehyde. The grafted neutral polymeric layer provided suppression of electroosmotic flow and minimized adsorption. This combination yielded successful open tube and polymer network separations of proteins, peptides and DNA molecules. Very high efficiencies (ca. 1 x 10(6) plates/m) were achieved for open tube protein separations, and hundreds of consecutive runs were performed with minimal change in migration times.

Automated microanalysis using magnetic beads with commercial capillary electrophoretic instrumentation.

The potential of a new microanalytical method using magnetic beads (MBs) and commercial capillary electrophoresis (CE) instrumentation for performing enzymatic and inhibition assays, as well as for analysis of biological molecules such as antigens, substrates, etc., has been explored. A small quantity of magnetic beads containing immobilized biomolecules was injected into a neutral hydrophilic-coated fused-silica capillary. The short plug (2-3 mm) of beads was held fixed by a magnet placed in the cartridge of the CE system, without the use of frits. The beads could be replaced after each run, eliminating the need to regenerate the solid support. Two protocols were used for analysis: sequential injection (SI) and SI followed by isotachophoretic (ITP) focusing. Alkaline phosphatase (AP) and HIV-protease were used to demonstrate the SI procedure for enzymatic and inhibition assays. The second protocol, SI/ITP, was employed to quantitate an antigen (mouse mAB) using antibodies (sheep IgG towards mouse AB) immobilized on the beads. The MB-CE method, requiring only femtomole (fmol) quantities of material, can potentially be employed in diagnostic and forensic assays, kinetic studies and searching for inhibitors, ligands, receptors, etc.

Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved. Typically, 4 x 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6% T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electro-osmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.

Central and extrapontine myelinolysis in a patient in spite of a careful correction of hyponatremia.

We report the case of a 54-year-old alcoholic female patient who was hospitalized for neurologic alterations along with a severe hyponatremia (plasma Na+: 97 mEq/l). She suffered from potomania and was given, a few days before admission, a thiazide diuretic for hypertension. A careful correction of plasma Na+ levels was initiated over a 48-hour period (rate of correction < 10 mEq/l/24h) in order to avoid brain demyelination. After a 2-day period of clinical improvement, her neurologic condition started to deteriorate. By the 5th day of admission, she became tetraplegic, presented pseudobulbar palsy, ataxia, strabism, extrapyramidal stiffness and clouding of consciousness. Scintigraphic and MRI investigations demonstrated pontine and extrapontine lesions associated with Gayet-Wernicke encephalopathy. After correction of ionic disorders (hyponatremia, hypokaliemia) and vitamin B (thiamine) deficiency, the patient almost completely recovered without notable disabilities. This case illustrates that profound hyponatremia, in a paradigm of slow onset, can be compatible with life. It also demonstrates that demyelinating lesions, usually considered as a consequence of a too fast correction of hyponatremia, may occur despite the strict observance of recent guidelines. There is increasing evidence to suggest that pontine swelling and dysfunction may sometimes occur in alcoholic patients even in absence of disturbance in plasma Na+ levels. It is therefore of importance, while managing a hyponatremic alcoholic patient, to identify additional risk factors (hypokaliemia, hypophosphoremia, seizure-induced hypoxemia, malnutrition with vitamin B deficiency) for brain demyelination and to correct them appropriately.

Pressure refilled polyacrylamide columns for the separation of oligonucleotides by capillary electrophoresis.

The separation of oligonucleotides by capillary zone electrophoresis (CZE) was studied in fused silica separation capillaries filled by linear (noncrosslinked) polyacrylamide (PAA) solutions, introduced into the capillary from the stock by pressure after each analysis. The time-consuming in-capillary polymerization step could thus be avoided, and fast and reproducible repetition of the analyses was assured. The PAA concentrations varied within the range of 3-10% and both the reproducibility of the analyses and the stability of the solution in the capillary, with and without a chemically treated inner wall, were tested. Ferguson plots were used to assess the size selectivity of the separation.

Applicability of dynamic change of pH in the capillary zone electrophoresis of proteins.

A method to extend the separation power of CZE is described. The method is based on the separation of sample components at two different pH values during one separation run, and involves dynamic buffering of the pH inside a separation capillary by controlling the flow of H+ ions from the anodic electrode chamber. By changing the anolyte in the chamber, a dynamic pH step is generated, which proceeds rapidly along the capillary and establishes the required new pH value. The use of the method has been demonstrated by the cationic separation of a model mixture of proteins.

Separation of some triazine herbicides and their solvolytic products by capillary zone electrophoresis.

The separation of triazine herbicides and their solvolytic products by capillary zone electrophoresis in mixed water-ethanol background electrolytes is described, allowing the analysis of picomole amounts of a triazine mixture in less than 15 min.

Capillary zone electrophoretic separation of cyclodextrins with indirect UV photometric detection.

A method for the separation of cyclodextrins by capillary zone electrophoresis with indirect detection is described. It is based on the formation of inclusion complexes with benzoate which simultaneously forms a UV absorbing constituent of the background electrolyte used for the separation.

Determination of halofuginone in feedstuffs by the combination of capillary isotachophoresis and capillary zone electrophoresis in a column-switching system.

A method has been developed for the determination of the coccidiocidic drug halofuginone in feedstuff concentrates which is based on the combination of capillary isotachophoresis and capillary zone electrophoresis in the column-switching mode. The high load capacity of the isotachophoretic step and high sensitivity of the zone electrophoretic step enabled analysis of up to 25 microliters of sample solution containing as little as 10(-8) M halofuginone with excellent reproducibility (R.S.D. about 1%). Attention was paid to the possibility of the existence of transient local isotachophoresis in the zone electrophoretic step, and experimental and theoretical methods of revealing zones migrating isotachophoretically in the background electrolyte were shown.

Ultrafast DNA analysis by capillary electrophoresis/laser-induced fluorescence detection.

The limits of ultrafast DNA analysis by CE were determined by investigating the influence of the effective capillary length and the electric field strength on the analysis time for a given peak resolution (10 bp). In accordance with theory, the use of a fast ramp power supply for narrow plug electrokinetic injection was found to be essential to minimize the extra column effects on peak dispersion. Two major column dispersion factors, longitudinal diffusion and thermal dispersion, were determined experimentally, as well as the influence of the electric field strength on the electrophoretic mobilities and diffusion coefficients of DNA. It was found that higher field strengths can be applied with lower thermal dispersion than predicted by classical CE models. This was attributed to the faster mass transport in the radial direction due to field-induced DNA orientation. Short capillaries (approximately 3-7 cm effective length) and moderate to high electric field strengths (approximately 600-800 V/cm) were used to perform a series of fast DNA separations. The dsDNA fragment standards phiX174/HaeIII and pBR322/HaeIII were separated within 30 s. The possibility for fast mutation detection was demonstrated using constant denaturant capillary electrophoresis (CDCE) for the analysis of a single base mutation in mitochondrial DNA in 72 s. The potential for fast DNA sequencing was illustrated by separating 300 ssDNA fragments within 180 s.

Recent developments in isotachophoresis.

This paper is summarizing the contributions to the analytical capillary isotachophoresis published during the period 1981-1984. It characterizes the present state of the method and covers theory, fundamental analytical aspects, instrumentation and applications. Special attention was payed to the fundamental analytical aspects, and a detailed discussion is given of the selection of electrolyte systems, stability of zones and separability of substances. The present commercial instrumentation is also briefly described.