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We have developed microfluidic devices with pressure-driven injection for electrophoretic analysis of amino acids, peptides, and proteins. The novelty of our approach lies in the use of an externally actuated on-chip peristaltic pump and closely spaced pneumatic valves that allow well-defined, small-volume sample plugs to be injected and separated by microchip electrophoresis. We fabricated three-layer poly(dimethylsiloxane) (PDMS) microfluidic devices. The fluidic layer had injection and separation channels, and the control layer had an externally actuated on-chip peristaltic pump and four pneumatic valves around the T-intersection to carry out sample injection. An unpatterned PDMS membrane layer was sandwiched between the fluidic and control layers as the actuated component in pumps and valves. Devices with the same peristaltic pump design but different valve spacings (100, 200, 300, and 400 μm) from the injection intersection were fabricated using soft lithographic techniques. Devices were characterized through fluorescent imaging of captured plugs of a fluorescein-labeled amino acid mixture and through microchip electrophoresis separations. A suitable combination of peak height, separation efficiency, and analysis time was obtained with a peristaltic pump actuation rate of 50 ms, an injection time of 30 s, and a 200-μm valve spacing. We demonstrated the injection of samples in different solutions and were able to achieve a 2.4-fold improvement in peak height and a 2.8-fold increase in separation efficiency though sample stacking. A comparison of pressure-driven injection and electrokinetic injection with the same injection time and separation voltage showed a 3.9-fold increase in peak height in pressure-based injection with comparable separation efficiency. Finally, the microchip systems were used to separate biomarkers implicated in pre-term birth. Although these devices have initially been demonstrated as a stand-alone microfluidic separation tool, they have strong potential to be integrated within more complex systems. Graphical Abstract Pressure-actuated sample loading and injection for improved microchip electrophoresis analysis

Carbon nanotubes combine a range of properties that make them well suited for use as probe tips in applications such as atomic force microscopy (AFM) 1 , 2 , 3 . Their high aspect ratio, for example, opens up the possibility of probing the deep crevices 4 that occur in microelectronic circuits, and the small effective radius of nanotube tips significantly improves the lateral resolution beyond what can be achieved using commercial silicon tips 5 . Another characteristic feature of nanotubes is their ability to buckle elastically 4 , 6 , which makes them very robust while limiting the maximum force that is applied to delicate organic and biological samples. Earlier investigations into the performance of nanotubes as scanning probe microscopy tips have focused on topographical imaging, but a potentially more significant issue is the question of whether nanotubes can be modified to create probes that can sense and manipulate matter at the molecular level 7 . Here we demonstrate that nanotube tips with the capability of chemical and biological discrimination can be created with acidic functionality and by coupling basic or hydrophobic functionalities or biomolecular probes to the carboxyl groups that are present at the open tip ends. We have used these modified nanotubes as AFM tips to titrate the acid and base groups, to image patterned samples based on molecular interactions, and to measure the binding force between single protein–ligand pairs. As carboxyl groups are readily derivatized by a variety of reactions 8 , the preparation of a wide range of functionalized nanotube tips should be possible, thus creating molecular probes with potential applications in many areas of chemistry and biology.

Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of φX174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 μm) have been explored. By use of channels with an effective length of only 3.5 cm, separations of φX174 Hae III DNA fragments from ≈70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQα alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips.

We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

DNA is expected to play a critical role in biomolecular nanotechnology because nucleic acids offer a range of desirable properties for nanometer scale manipulations. We report here a thorough characterization and optimization of conditions for alignment of well extended single- and double-stranded DNA molecules on mica surfaces. The optimum poly-L-lysine concentration range for surface treatment in DNA alignment experiments is 0.5-2 ppm. DNA is best aligned, and the greatest surface density of DNA molecules is observed in the center of the path traversed by the DNA solution droplet during deposition. The alignment procedure induces only a small amount of fragmentation of deposited DNA molecules. Knowledge of the optimum conditions for nucleic acid alignment on surfaces should facilitate the use of DNA based nanotechnology.

DNA-based nanotechnology is a vibrant and expanding field. The specific molecular recognition properties and large aspect ratio of DNA make the molecule a promising template for bottom-up fabrication of nanowires and nanodevices. Fabricating well-defined DNA-templated nanowires requires aligned surface deposition and specific metallization of DNA molecules. DNA localization on surfaces has been achieved by bulk fluid flow or a moving air-water interface, and localization efficiency has been improved by surface modifications that favor DNA-substrate interaction. DNA-templated nanowires have been constructed from gold, silver, copper, palladium, and platinum, and template modifications have allowed the bottom-up construction of a simple electronic nanodevice. These achievements demonstrate the promising feasibility of using bottom-up nanofabrication to create increasingly sophisticated nanodevices.

Sensory structures of ticks have been studied with the scanning electron microscope. Postulations of certain functions of the sensilla have been made with inferences from the literature. The species examined are: Amblyomma americanum, Argas persicus, Boophilus annulatus, Dermacentor andersoni, D. variabilis, Haemaphysalis leporispalustris, Ixodes kingi, and I. ricinus.

Decomposition of grass leaf litter was studied on a shortgrass prairie using chemicals (HgCl\"2 and CuSO\"4) to prevent microbial activity (abiotic treatment), 53-@mm nylon mesh to exclude mesofauna (microbial treatment), and l-mm nylon mesh to allow the access of mesofauna. After 9 months, 15.2% of the blue grama grass litter was decomposed in the microbial treatment, and 29.4% was decomposed in the microbial plus mesofaunal treatment. After 7 months, 6.2% of the litter had disappeared from the abiotic treatment. There was a general decrease in C:N ratios with the microbial treatment lowest at the end of the experiment. Total available carbohydrates generally decreased with time. Certain mite families fluctuated with seasons. The tydeids were most active in winter and tetranychids were most active in summer. A correlation between abiotic factors and mite families was also observed.