Fully integrated glass microfluidic device for performing high-efficiency capillary electrophoresis and electrospray ionization mass spectrometry.

A microfabricated device has been developed in which electrospray ionization is performed directly from the corner of a rectangular glass microchip. The device allows highly efficient electrokinetically driven separations to be coupled directly to a mass spectrometer (MS) without the use of external pressure sources or the insertion of capillary spray tips. An electrokinetic-based hydraulic pump is integrated on the chip that directs eluting materials to the monolithically integrated spray tip. A positively charged surface coating, PolyE-323, is used to prevent surface interactions with peptides and proteins and to reverse the electroosmotic flow in the separation channel. The device has been used to perform microchip CE-MS analysis of peptides and proteins with efficiencies over 200,000 theoretical plates (1,000,000 plates/m). The sensitivity and stability of the microfabricated ESI source were found to be comparable to that of commercial pulled fused-silica capillary nanospray sources.

On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry.

Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using "wrong-way-round" electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/microL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.

A microfabricated fluidic device for performing two-dimensional liquid-phase separations.

A microfabricated fluidic device that combines micellar electrokinetic chromatography and high-speed open-channel electrophoresis on a single structure for the rapid automated two-dimensional analysis of peptides has been devised and demonstrated. The microchip operates by rapidly sampling and analyzing effluent in the second dimension from the first dimension. Second-dimension analyses are performed and completed every few seconds, with total analysis times of less than 10 min for tryptic peptides. The peak capacity of the two-dimensional separations has been estimated to be in the 500-1000 range. The orthogonality of the separation techniques, an important factor for maximizing peak capacity or resolution elements, was verified by examining each technique independently for peptide separations. The two-dimensional separation strategy was found to greatly increase the resolving power over that obtained for either dimension alone.

Novel microfabricated device for electrokinetically induced pressure flow and electrospray ionization mass spectrometry.

A novel microchip device for electrospray ionization has been fabricated and interfaced to a time-of-flight mass spectrometer. Fluid is electrokinetically transported through the chip to a fine fused-silica capillary inserted directly into a channel at the edge of the device. Electrospray is established at the tip of the capillary, which assures a stable, efficient spray. The electric potential necessary for electrospray generation and the voltage drop for electroosmotic pumping are supplied through an electrically permeable glass membrane contacting the fluidic channel holding the capillary. The membrane is fabricated on the microchip using standard photolithographic and wet chemical etching techniques. Performance relative to other microchip electrospray sources has been evaluated and the device tested for potential use as a platform for on-line electrophoretic detection. Sensitivity was found to be approximately three orders of magnitude better than spraying from the flat edge of the chip. The effect of the capillary on electroosmotic flow was examined both experimentally and theoretically.

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling.

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.

Subattomole-Sensitivity Microchip Nanoelectrospray Source with Time-of-Flight Mass Spectrometry Detection.

A microfabricated microfluidic device coupled with a nanospray tip for electrospray ionization of dilute solutions is described. The device has been interfaced with a time-of-flight mass spectrometer and evaluated for sensitive, high-speed detection of peptides and proteins. The electrospray voltage was applied through the microchip to the nanospray capillary that was attached at the end of a microfabricated channel. Fluid delivery rates were 20-30 nL min(-)(1) through the hybridized structure without any pressure assistance. On-line microchip electrophoresis has been demonstrated and the effect of the capillary-chip junction on band broadening examined. Full mass spectra are acquired within 10-20 ms at 50-100 spectra s(-)(1) storage rates. Detection of subattomole levels of sample from a 100 nM solution is demonstrated for infusion experiments.

Durable gold-coated fused silica capillaries for use in electrospray mass spectrometry.

A simple procedure for preparing gold-coated silica capillaries for use in electrospray ionization mass spectrometry is described. The tip of the capillary is mechanically tapered to a fine point, and a thin film of gold is vapor deposited on the outer surface following treatment with an organofunctional silane. The performance characteristics of these durable capillaries as continuous infusion sources are examined, and their utility in on-line capillary electrophoresis mass spectrometry is demonstrated.

Gaseous myoglobin ions stored at greater than 300 K.

Multiply-charged myoglobin ions retaining the prosthetic heme group have been formed by electrospray, injected into a quadrupole ion trap, and stored for up to one second prior to mass analysis. Collisional activation experiments indicate that these ions readily fragment into the charged heme group and the complementary apomyoglobin ion. No fragmentation is observed, however, upon ion storage in the presence of a neutral bath gas at 1 × 10(-3) torr for up to one second. The significance of this observation is that these non-covalently-bound ions, in which both the heme group and the polypeptide carry charge, are kinetically stable for over one second at room temperature and, perhaps, at higher temperatures. This suggests that other biologically relevant ions derived using electrospray and bound by non-covalent interactions can be studied using the various tools available with ion storage mass spectrometers and by other techniques that employ relatively high pressure environments for the study of gaseous ions.

Supercritical fluid extraction of chemical warfare agent simulants from soil.

Chemical warfare agent simulants are efficiently recovered from 2-ppm spikes in 1 g of Rocky Mountain Arsenal Standard Soil using methanol-carbon dioxide (5:95) at 300 atm for 2 min at 60 degrees C. Recoveries (n = 3) were 79 +/- 23% for dimethylmethylphosphonate, 93 +/- 14% for 2-chloroethylethyl sulfide, 92 +/- 13% for diisopropylfluorophosphate and 95 +/- 17% for diisopropylmethylphosphonate. Recoveries are higher than, but less precise than those achieved from a 5-min ultrasonic micro-scale extraction using methanol. Much less laboratory waste is generated than the current standard organic solvent extraction method (33 g of soil shaken with 100 ml of chloroform).

Teacher competency testing: what are special education teachers expected to know?

Teacher competency testing (TCT) is a current manifestation of education reform; 48 of the 50 states are actively planning or implementing teacher testing programs, and 25 (plus the District of Columbia) are developing or administering tests in specific subject areas to include special education. This article presents the results of a nationwide survey that addressed TCT in special education. Responses were collected from 46 states and the District of Columbia. Analyses of results revealed that states vary in their use of teacher competency tests and that specific special education teacher competency tests are used or being actively studied in about half the states. Implications for the practice of special education are provided.

Teacher competency testing: what teachers of students with LD need to know.

In 1964, North Carolina became the first state to enact a testing program for initial certification of teachers. Today, almost all states and the District of Columbia are actively planning or implementing teacher testing programs; six are administering tests in the specific area of learning disabilities. The objectives and content areas of knowledge included in competency testing programs within this specialized area of teacher competence were analyzed, and comparisons were completed across knowledge areas and among states. In general, much similarity exists in what teachers of students with learning disabilities in different states are expected to know. This information represents a strong foundation for planning, implementing, and evaluating training programs for all teachers.

Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection.

Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.

Retention modification of nucleic acid constituents in reversed-phase high-performance liquid chromatography.

Secondary equilibria in reversed-phase liquid chromatography have been investigated as a means of enhancing selectivity and optimizing separations of nucleic acid constituents. The retention behavior of various nucleotides, nucleosides and modified compounds has been examined as a function of five different metal ion additives in the mobile phase: K+, Mg2+, Mn2+, Ni2+ and Zn2+. Complexation of the solute molecules with the metal ions changes the electronic structure and alters solute-solvent interactions. Alkali and alkaline earth metals bind primarily to phosphate groups while transition metals also interact with the N7 of purine bases. All nucleotides were found to be eluted very close to the void volume of the high-performance liquid chromatographic column without any metal additive, but retention increased as the concentration of a given cation increased. The transition metals were found to have the greatest effect, with affinities for nucleotide monophosphates on the order of 100 times greater than potassium, and 10 times that of magnesium. Differences in affinity based upon phosphate structure (i.e., cyclic vs. linear), phosphate position (e.g., 2'- vs. 3'-monophosphates), and base modification were also noted. The retention of most nucleosides, unlike the charged compounds, remained relatively constant as the ionic strength or type of cation was varied. Also, improvements were obtained in the resolution of some oligonucleotides with the addition of divalent ions to a potassium buffer mobile phase.

Taking the practicum beyond the public school door.

A unique teacher preparation component is operated by the Albany State College Graduate Program in Special Education at the nearby Turner Job Corps Center. Student teachers learn a foundation of remedial interventions appropriate to most mildly handicapped young adult learners in a comprehensive, residential teaching environment.