Biomedical subjects

Michael C Breadmore

Publications and source records attributed to Michael C Breadmore.

9 recordsLinked to original sources

PubMed2003

Microchip-based purification of DNA from biological samples.

A microchip solid-phase extraction method for purification of DNA from biological samples, such as blood, is demonstrated. Silica beads were packed into glass microchips and the beads immobilized with sol-gel to provide a stable and reproducible solid phase onto which DNA could be adsorbed. Optimization of the DNA loading conditions established a higher DNA recovery at pH 6.1 than 7.6. This lower pH also allowed for the flow rate to be increased, resulting in a decrease in extraction time from 25 min to less than 15 min. Using this procedure, template genomic DNA from human whole blood was purified on the microchip platform with the only sample preparation being mixing of the blood with load buffer prior to loading on the microchip device. Comparison between the microchip SPE (microchipSPE) procedure and a commercial microcentrifuge method showed comparable amounts of PCR-amplifiable DNA could be isolated from cultures of Salmonella typhimurium. The greatest potential of the microchipSPE device was illustrated by purifying DNA from spores from the vaccine strain of Bacillus anthracis, where eventual integration of SPE, PCR, and separation on a single microdevice could potentially enable complete detection of the infectious agent in less than 30 min.

Adsorption↗

PubMed2003

Hydroxypropyl cellulose as an adsorptive coating sieving matrix for DNA separations: artificial neural network optimization for microchip analysis.

Effective DNA separations in microelectrophoretic systems are complicated by the need to passivate the surface dynamically or covalently. We describe the optimization and utilization of a novel buffer system for fast DNA separations by capillary and microchip electrophoresis without the need for any surface modification or conditioning prior to separation. At concentrations as high as 5%, hydroxypropyl cellulose (HPC) has a relatively low viscosity, allowing for microchip channel filling to be performed with ease. A MES/TRIS buffer system at pH 6.1 eliminates the need for surface preconditioning procedures due to the promotion of hydrogen bonding of HPC with the wall. An additional benefit with this buffer system is the low current observed at high fields when compared to other common DNA separation buffers. An artificial neural network (ANN) was used to model the data and to predict the optimum conditions. Utility of the ANN-optimized system for molecular diagnostic testing was demonstrated by performing microchip separations on DNA samples from patients suspected of having genetic mutations associated with Duchenne muscular dystrophy (DMD). Microchip analysis easily allowed for the patient samples positive for DMD mutations to be distinguished from patient samples negative for the disease.

Adsorption↗

PubMed2003

Capillary electrophoresis evidence for the stereoselective metabolism of itraconazole in man.

Itraconazole (ITC) is a hydrophobic antimycotic drug with three chiral centers that is used clinically as a stereoisomeric mixture. A chiral capillary electrophoretic method for the separation of ITC stereoisomers and those of its main metabolite hydroxyitraconazole (HITC) was developed to determine the stereoselective nature of the ITC to HITC biotransformation. The method is based on the formation of inclusion complexes of the target analytes with the negatively charged sulfated beta-cyclodextrin in the presence of moderate concentrations of methanol in a low-pH phosphate buffer. The addition of polyethylene glycol 4000 was found to be critical in obtaining baseline resolution of eight peaks, two from ITC, four from HITC, and two from R051012 (internal standard), in under 20 min. Application of the developed procedure to serum samples from patients being treated with ITC showed clearly the presence of a stereoselective component in the metabolism of this antimycotic drug. This could be shown from in vitro incubations with single enzyme Supersomes to be in part due to the stereoselective formation of HITC by the human CYP3A4 enzyme. For one patient, monitoring of the ITC and HITC concentrations and peak ratios over a 103 day period of treatment with ITC showed a strong dependency of the chiral ITC ratio to the concentration of ITC, while the dominant enantiomeric ratio of HITC was largely independent of the total HITC concentration.

Cyclodextrins↗

PubMed2003

Towards a microchip-based chromatographic platform. Part 2: sol-gel phases modified with polyelectrolyte multilayers for capillary electrochromatography.

The potential for using polyelectrolyte multilayers (PEMs) to provide chromatographic functionality on continuous silica networks created from sol-gel chemistry has been evaluated by capillary electrochromatography (CEC). Construction of the PEM was achieved by flushing the column with polyelectrolytes of alternative charge, with variation of the properties of the exposed polyelectrolyte providing a unique means to vary the chromatographic surface. Variation of the exposed polyelectrolyte from poly(diallyldimethylammonium chloride) (PDDAC) to dextran sulfate (DS) allowed the direction of the electroosmotic flow (EOF) to be changed and also provided a means to vary the chromatographic capacity. Variation of negative polymer from DS to poly(styrene sulfonate) (PSS) significantly altered the EOF and the migration of peptides, with both the reversed-phase and ion-exchange capacities increasing. An alternative method for changing the column capacity was to change the thickness of the PEM, which was evaluated by anion-exchange CEC. A 70-80% increase in retention was observed for all anions without any increase in EOF suggesting significant penetration of the analytes through the PEM and interaction with buried charges within the PEM.

Dextran Sulfate↗

PubMed2002

On-column ion-exchange preconcentration of inorganic anions in open tubular capillary electrochromatography with elution using transient-isotachophoretic gradients. 3. Implementation and method development.

A solid-phase extraction method based on an ion-exchange retention mechanism has been used for in-line preconcentration of inorganic anions prior to their separation by capillary electrophoresis (CE). A single capillary containing a preconcentration and a separation zone has been used in a commercial CE instrument without instrumental modification. Analyte anions were retained on a preconcentration zone comprising an adsorbed layer of cationic latex particles, while separation was achieved in a separation zone comprising fused silica modified by adsorption of a cationic polymer. Elution of the adsorbed analytes was achieved using an eluotropic gradient formed by a transient isotachophoretic boundary between a fluoride electrolyte and a naphthalenedisulfonate electrolyte. Optimization of the electrolyte concentrations, sample injection times, and back-flushing times allowed the successful separation of sub-ppb levels of inorganic anions using a 100-min injection at 2 bar pressure, introducing over 40 capillary volumes of sample. A method based on a 10-min injection allowed a 100-fold increase in sensitivity over conventional hydrodynamic injection for Br-, I-, NO3-, CrO4(2-), and MoO4(2-) with a total analysis time of 25 min. Detection limits were dependent on the injection time but were in the range 2.2-11.6 ppb for a 10-min injection time. This approach was used to determine NO3- in Antarctic ice cores where the analysis could be performed using a sample volume 100 times less than that used for ion chromatography.

Anions↗

PubMed2002

Use of ionic polymers as stationary and pseudo-stationary phases in the separation of ions by capillary electrophoresis and capillary electrochromatography.

One of the problems with capillary electrophoresis is a lack of versatility regarding manipulation of the separation selectivity. A new and potentially universal concept is to introduce an ion-exchange component into a separation so that the migration of analyte ions is influenced by both their electrophoretic mobilities and their chromatographic properties. This may be accomplished by use of capillaries filled with or coated with solid ion-exchange polymers, or by addition of a soluble ionic polymer to the background electrolyte to create a pseudo-stationary phase. While each of these methods achieves the same result, they are not competitive, but rather complementary as the problems associated by one approach are overcome by the others. Recent highlights in the field are used to illustrate the flexibility that this approach provides to electrophoretic separation of ions.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2002

Toward a microchip-based solid-phase extraction method for isolation of nucleic acids.

A silica-based solid-phase extraction system suitable for incorporation into a microchip platform (nu-total analytical system; nu-TAS) would find utility in a variety of genetic analysis protocols, including DNA sequencing. The extraction procedure utilized is based on adsorption of the DNA onto bare silica. The procedure involves three steps: (i) DNA adsorption in the presence of a chaotropic salt, (ii) removal of contaminants with an alcohol/water solution, and (iii) elution of the adsorbed DNA in a small volume of buffer suitable for polymerase chain reaction (PCR) amplification. Multiple approaches for incorporation of this protocol into a microchip were examined with regard to extraction efficiency, reproducibility, stability, and the potential to provide PCR-amplifiable DNA. These included packing microchannels with silica beads only, generating a continuous silica network via sol-gel chemistry, and combinations of these. The optimal approach was found to involve immobilizing silica beads packed into the channel using a sol-gel network. This method allowed for successful extraction and elution of nanogram quantities of DNA in less than 25 min, with the DNA obtained in the elution buffer fraction. Evaluation of the eluted DNA indicated that it was of suitable quality for subsequent amplification by PCR.

DNA↗

PubMed2002

Towards a microchip-based chromatographic platform. Part 1: Evaluation of sol-gel phases for capillary electrochromatography.

Silica monolithic columns suitable for implementation on microchips have been evaluated by ion-exchange capillary electrochromatography. Two different silica monoliths were created from the alkyl silane, tetramethyl orthosilicate (TMOS), by introducing a water-soluble organic polymer, poly(ethylene oxide) (PEO), with varying molecular weights into the prehydrolyzed sol. Silica monoliths created using 10 kDa PEO were found to have a much more closed gel structure with a smaller percentage of pores in the microm size range than gels created using 100 kDa PEO. Additionally, the size of the mesopores in the 100 kDa PEO monolith was 5 nm, while those in the 10 kDa PEO gel were only 3 nm. This resulted in a strong dependence of the electroosmotic flow (EOF) on the ionic strength of the background electrolyte, with substantial pore flow through the nm size pores observed in the 10 kDa PEO gel. The chromatographic performance of the monolithic columns was evaluated by ion-exchange electrochromatography, with ion-exchange sites introduced via dynamic coating with the cationic polymer, poly(diallyldimethylammonium chloride) (PDDAC). Separating a mixture of inorganic anions, the 10 kDa PEO monolithic columns showed a higher effective capacity than the 100 kDa PEO column.

Anions↗

PubMed2002

A simple PDMS-based electro-fluidic interface for microchip electrophoretic separations.

High voltage electrodes for electrophoresis have been integrated into a polymer layer that can be reversibly bound to glass microchips for electrophoretic separations. By using the liquid precursor to the polymer polydimethylsiloxane (PDMS), platinum electrodes and reservoirs can be positioned prior to solidification, providing a simple and flexible method for electrode interface construction. Field strengths up to 875 V cm(-1) over an 8 cm separation channel can be applied to the system without any loss in performance of the interface. The interface can function as an electro-fluidic interface between the high voltage power supply and the separation channel and, when reversibly sealed to an etched glass plate, functions as a cover plate establishing a hybrid PDMS-glass microchip in which the electrodes are directly integrated onto the device. The versatility of this approach is not only demonstrated by separating DNA fragments in a novel buffer sieving matrix, but also with the molecular diagnostic analysis of a variety of DNA samples for Duschenne Muscular Dystrophy and cytomegalovirus (CMV) infection, using both microchip interface configurations.

Cytomegalovirus Infections↗