Rapid serial analysis of multiple oligonucleotide samples on a microchip using optically-gated injection

Optically-gated injection of fluorescently-labeled DNA has been accomplished for the first time. Rapid, serial analysis of oligonucleotide ladders has been shown on a microchip using this injection technique. Separations of five- and six-component samples have been completed in 60 s or less with a capability to carry out serial injections of these samples every 15 s. The technique has been shown to have better than five base resolution for small oligonucleotides and excellent reproducibility in migration times (< or = 0.75% RSD). Currently, the limit of detection for the system is 0.23 microM. Additionally, multiple unique samples of DNA have been consecutively analyzed in a single separation lane using optical gating. Six consecutive injections of three different samples have been achieved with no sample carryover and a total analysis time of approximately 10 min. These results show the potential of optical gating as an alternative injection technique for high-throughput DNA applications, such as genotyping and monitoring dynamic processes.     

Characterization of etched electrochemical detection for electrophoresis in micron inner diameter capillaries

An enhanced etched electrochemical (EC) detection technique has been developed for CE in micron inner diameter capillaries. The design improvements allow for better alignment between the capillary bore and the electrode. This new method involves utilizing a carbon fiber microelectrode and etching both the carbon fiber and the detection end of a micrometer-sized inner diameter capillary to limit dead volume and analyte diffusion at the amperometric EC detector. To understand the factors affecting enhanced detector efficiency, a detailed examination of the relationship between detector design and performance has been completed by exploring the effects of varying electrode diameter, tip shape, and size, in addition to the etch length of the capillary outlet. The enhanced detection provides peak efficiencies as high as 75000 theoretical plates and estimated detection limits as low as 40 nM for dopamine. This etched detection method should further facilitate volume-limited sample analysis by CE.     

Differential mobility spectrometry of chlorocarbons with a micro-fabricated drift tube

Chlorocarbons were ionized through gas phase chemistry at ambient pressure in air and resultant ions were characterized using a micro-fabricated drift tube with differential mobility spectrometry (DMS). Positive and negative product ions were characterized simultaneously in a single drift tube equipped with a 3 mCi (63)Ni ion source at 50 degrees C and drift gas of air with 1 ppm moisture. Scans of compensation voltage for most chlorocarbons produced differential mobility spectra with Cl(-) as the sole product ion and a few chlorocarbons produced adduct ions, M (.-) Cl(-). Detection limits were approximately 20-80 pg for gas chromatography-DMS measurements. Chlorocarbons also yielded positive ions through chemical ionization in air and differential mobility spectra showed peaks with characteristic compensation voltages for each substance. Field dependence of mobility was determined for positive and negative ions of each substance and confirmed characteristic behavior for each ion. A DMS analyzer with a membrane inlet was used to continuously monitor effluent from columns of bentonite or synthetic silica beads to determine breakthrough volumes of individual chlorocarbons. These findings suggest a potential of DMS for monitoring subsurface environments either on site or perhaps in situ.     

Synthesis of bicyclic pyrimidine derivatives as ATP analogues

A highly efficient and general solid-phase synthesis of bicyclic pyrimidine derivatives that target purine dependent proteins is reported. The synthesis of the key intermediate, 4,6-disubstituted-5-amino-pyrimidine, involved reduction of the corresponding nitro derivatives using 1,1'-dioctyl-viologen in a triphasic milieu. The mild reduction conditions enable the use of any acid labile solid support as well as a wide range of combinatorial substituents, thus enabling the synthesis of large libraries of highly diverse bicyclic pyrimidines. Alternative reduction conditions with tin(II) chloride and structure-reactivity studies are discussed as well.     

CombiDOCK: Structure-based combinatorial docking and library design

We have developed a strategy for efficiently docking a large combinatorial library into a target receptor. For each scaffold orientation, all potential fragments are attached to the scaffold, their interactions with the receptor are individually scored and factorial combinations of fragments are constructed. To test its effectiveness, this approach is compared to two simple control algorithms. Our method is more efficient than the controls at selecting best scoring molecules and at selecting fragments for the construction of an exhaustive combinatorial library. We also carried out a retrospective analysis of the experimental results of a 10×10×10 exhaustive combinatorial library. An enrichment factor of approximately 4 was found for identifying the compounds in the library that are active at 330 nM.     

Analysis of mixed finite element methods on locally refined grids

Summary We consider the mixed finite element method for locally refined triangulations. A local projection operator is defined to satisfy the commutation property that is required in the general theory of mixed methods. Our results can be applied to every known space of arbitrary order over rectangles or triangles. Optimal-order error estimates and superconvergence for the flux along the Gauss lines are established.     

Capillary electrophoresis of single mammalian cells

Capillary electrophoresis (CE) has been established as powerful tool for single cell analysis. Newly developed sampling, separation and detection methods have allowed the investigation of single mammalian cells with CE despite their small size and complex composition. Advances in sample injection techniques include several novel methods for the injection of whole cells and sampling techniques for the study of cellular secretion. CE of single mammalian cells has been applied in a wide range of fields including protein analysis, neuroscience, and oncology. The development of new detection schemes in the analysis of single mammalian cells with CE has included studies of protein expression and the utilization of mass spectrometric and electrochemical detection. Subcellular mammalian cell analysis with CE also has been investigated.     

Parallel separations of oligonucleotides with optically gated sample introduction on multichannel microchips

With the release of the human genome sequence, there has been increasing attention given to other genetic analyses, including the detection of genetic variations and fast sequencing of multiple samples for pharmacogenomics studies. Rapid injections of samples in multiplexed separation channels by optically gated sample introduction are shown here for DNA separation. Serial separations of four amino acids are shown in less than four seconds on a microchip with four multiplexed channels. Five short oligonucleotides have also been rapidly separated in 2% LPA with four channels using this technique. In addition, multiple unique samples have been simultaneously separated and five-base resolution has been demonstrated.