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.

High-throughput microfabricated CE/ESI-MS: automated sampling from a microwell plate.

A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is presented. The approach combines a sample-loading port, a separation channel, and a liquid junction, the latter for coupling the device to the MS with a miniaturized subatmospheric electrospray interface. The microdevice was attached to a polycarbonate manifold with external electrode reservoirs equipped for electrokinetic and pressure-fluid control. A computer-activated electropneumatic distributor was used for both sample loading from the microwell plate and washing of channels after each run. Removal of the electrodes and sample reservoirs from the microdevice structure significantly simplified the chip design and eliminated the need both for drilling access holes and for sample/buffer reservoirs. The external manifold also allowed the use of relatively large reservoirs that are necessary for extended time operation of the system. Initial results using this microfabricated system for the automated CE/ESI-MS analysis of peptides and protein digests are presented.

Automated high-throughput infusion ESI-MS with direct coupling to a microtiter plate.

This paper describes the design and application of instrumentation for automated high-throughput infusion ESI-mass spectrometry. The approach, based on a subatmospheric ESI interface, allows sample introduction from a commercially available microtiter plate without the need for a separate fluid delivery system. The microtiter plate was placed vertically on a three-dimensional translation stage in front of the sampling ESI interface. A single, 7-cm, 20-microm-i.d. fused-silica capillary (total volume, 70 nL), with a tapered tip, served as a combination of sample delivery and spraying capillary. The tapered tip of the capillary was enclosed in a subatmospheric chamber attached in front of the orifice of the mass spectrometer. The sample aspiration rate (flow rate) was regulated by computer-controlled pneumatic valves, which allowed fast switching of the pressure in the subatmospheric ESI chamber. A flow-through wash device was positioned between the microtiter plate and the ESI interface. This design allowed alternate filling of the capillary with (a) sample from the wells and (b) wash solution from the wash device. Sample turnaround times of 10 s/sample, with a 120-nL sample consumption/analysis, and a duty cycle (percentage of total analysis time spent acquiring data) of 40% were achieved. The infusion system was demonstrated in the analysis of preparative HPLC fractions from a small molecule combinatorial library.

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.

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.

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.

Development of multichannel devices with an array of electrospray tips for high-throughput mass spectrometry.

The basic principles of multichannel devices with an array of electrospray tips for high-throughput infusion electrospray ionization mass spectrometry (ESI-MS) have been developed. The prototype plastic devices were fabricated by casting from a solvent-resistant resin. The sample wells on the device were arranged in the format of the standard 96-microtiter well plate, with each sample well connected to an independent electrospray exit port via a microchannel with imbedded electrode. A second plastic plate with distribution microchannels was employed as a cover plate and pressure distributor. Nitrogen gas was used to pressurize individual wells for transport of sample into the electrospray exit port. The design of independent microchannels and electrospray exit ports allowed very high throughput and duty cycle, as well as elimination of any potential sample carryover. The device was placed on a computer-controlled translation stage for precise positioning of the electrospray exit ports in front of the mass spectrometer sampling orifice. High-throughput ESI-MS was demonstrated by analyzing 96 peptide samples in 480 s, corresponding to a potential throughput of 720 samples/h. As a model application, the device was used for the MS determination of inhibition constants of several inhibitors of HIV-1 protease.

Subatmospheric electrospray interface for coupling of microcolumn separations with mass spectrometry.

A modular subatmospheric electrospray interface with fiber optic UV detection close to the electrospray tip was developed for coupling of microcolumn separation techniques with mass spectrometry. The interface was based on a liquid junction with a removable microelectrospray tip. The electrospray tip was enclosed in a subatmospheric chamber attached in front of the sampling orifice of the mass spectrometer. The inlet of the liquid junction was maintained at atmospheric pressure, and thus no pressure drop developed across the separation column. The flow rate of the electrosprayed liquid from the liquid junction reservoir was adjusted by the pressure in the electrospray chamber. In this approach, a continuous and stable electrospray could be achieved without the use of an external pump. Since the electrospray did not depend on fluid delivery from the separation column, coated capillaries without electroosmotic flow as well as capillaries with electroosmotic flow could be used for capillary electrophoresis. In addition, the interface was found to be effective with capillary liquid chromatography. The use of a fiber optic UV detector placed close to the exit of the separation column provided additional detection information and a simple means of troubleshooting. The interface did not significantly influence the quality of the separation, even with columns generating several hundred thousand theoretical plates. Peptide samples in the submicromolar concentration range were detected, corresponding to a limit of detection in the attomole range.

A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry.

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.

Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing.

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Microfabricated devices for capillary electrophoresis-electrospray mass spectrometry.

Two fundamental approaches for the coupling of microfabricated devices to electrospray mass spectrometry (ESI-MS) have been developed and evaluated. The microdevices, designed for electrophoretic separation, were constructed from glass by standard photolithographic/wet chemical etching techniques. Both approaches integrated sample inlet ports, preconcentration sample loops, the separation channel, and a port for ESI coupling. In one design, a modular, reusable microdevice was coupled to an external subatmospheric electrospray interface using a liquid junction and a fused silica transfer capillary. The transfer capillary allowed the use of an independent electrospray interface as well as fiber optic UV detection. In the second design, a miniaturized pneumatic nebulizer was fabricated as an integral part of the chip, resulting in a very simple device. The on-chip pneumatic nebulizer provided control of the flow of the electrosprayed liquid and minimized the dead volume associated with droplet formation at the electrospray exit port. Thus, the microdevice substituted for a capillary electrophoresis instrument and an electrospray interface--traditionally two independent components. This type of microdevice is simple to fabricate and may thus be developed either as a part of a reusable system or as a disposable cartridge. Both devices were tested on CE separations of angiotensin peptides and a cytochrome c tryptic digest. Several electrolyte systems including a transient isotachophoretic preconcentration step were tested for separation and analysis by an ion trap mass spectrometer.

On-line MALDI-TOF MS using a continuous vacuum deposition interface.

In this work, a new interface for continuous on-line MALDI-TOF MS is presented. The sample, mixed with a suitable matrix, was transported into the evacuated source chamber of the mass spectrometer at liquid flow rates of 100-400 nL/min. The liquid sample matrix was deposited on a rotating quartz wheel and transported to the repeller, where laser desorption took place. Rapid evaporation of the solvent (water or methanol) on the surface of the wheel resulted in formation of a thin, approximately 50-micron-wide, sample trace. Scanning electron microscopic photographs of the vacuum-dried trace revealed the deposited material to consist of an amorphous film. Furthermore, sample uniformity along the trace, in conjunction with its narrow width, resulted in excellent signal reproducibility, with detection limits in the attomole range. The interface permitted the on-line coupling of microcolumn separation techniques with MALDI MS, as demonstrated in the capillary electrophoresis MALDI-TOF MS analysis of a 12-peptide mixture. The approach offers the potential for rapid separation and trace analysis of complex mixtures.

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.

DNA cycle sequencing of a common restriction fragment of Staphylococcus aureus bacteriophages by capillary electrophoresis using replaceable linear polyacrylamide.

The nucleotide sequence of a part of a 4.9 kbp common restriction fragment isolated from Staphylococcus aureus bacteriophage (bacterial virus) 3A has been determined by capillary electrophoresis (CE). The fast separation of sequencing fragments in linear polyacrylamide solution at a temperature of 55 degrees C allowed the reading of more than 650 bases of sequence in 60 min. The single strand (ss)DNA fragments were prepared by cycle sequencing with fluorescently labeled dideoxy-terminators on the cloned bacteriophage DNA template. With respect to analysis speed, sequence read-length, low sample consumption and automation, CE offers a simple, labor-saving and inexpensive procedure for DNA sequencing. Operating the CE columns at elevated temperature proved to be a rapid procedure capable of extending sequence read-length. The resulting sequence of the common restriction fragment can be used for the preparation of specific primers and oligonucleotide hybridization probes for identification of Staphylococcus aureus bacteriophages and/or prophages belonging to the bacteriophage species 3A.

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.

Multichannel microchip electrospray mass spectrometry.

Microfabricated multiple-channel glass chips were successfully interfaced to an electrospray ionization mass spectrometer (ESI-MS). The microchip device was fabricated by standard photolithographic, wet chemical etching, and thermal bonding procedures. A high voltage was applied individually from each buffer reservoir for spraying sample sequentially from each channel. With the sampling orifice of the MS grounded, it was found that a liquid flow of 100-200 nL/min was necessary to maintain a stable electrospray. The detection limit of the microchip MS experiment for myoglobin was found to be lower than 6 x 10(-8) M. Samples in 75% methanol were successfully analyzed with good sensitivity, as were aqueous samples. The parallel mutliple-channel microchip system allowed ESI-MS analysis of different samples of standard peptides and proteins in one chip.

Mutational spectrometry without phenotypic selection: human mitochondrial DNA.

By first separating mutant from nonmutant DNA sequences on the basis of their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that allows observation of point mutations in biological samples at fractions at or above 10-6. Using this method, we have observed the hotspot point mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues. To date, 19 mutants have been isolated, their fractions ranging from 4x10-4 down to the limit of detection. We performed specific tests to determine if the observed signals were artefacts arising from contamination, polymerase errors during PCR or DNA adducts created during the procedure. We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, were included with true mutants in our separation steps and converted to mutants during PCR. We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at least in part from mismatch intermediates or cellular DNA adducts. This technology is therefore sufficient for the observation of the spectrum of point mutations in human mitochondrial DNA and is a tool for discovering the primary causes of these mutations.