Electrical detection of germination of viable model Bacillus anthracis spores in microfluidic biochips.

In this paper, we present a new impedance-based method to detect viable spores by electrically detecting their germination in real time within microfluidic biochips. We used Bacillus anthracis Sterne spores as the model organism. During germination, the spores release polar and ionic chemicals, such as dipicolinic acid (DPA), calcium ions, phosphate ions, and amino acids, which correspondingly increase the electrical conductivity of the medium in which the spores are suspended. We first present macro-scale measurements demonstrating that the germination of spores can be electrically detected at a concentration of 10(9) spores ml(-1) in sample volumes of 5 ml, by monitoring changes in the solution conductivity. Germination was induced by introducing an optimized germinant solution consisting of 10 mM L-alanine and 2 mM inosine. We then translated these results to a micro-fluidic biochip, which was a three-layer device: one layer of polydimethylsiloxane (PDMS) with valves, a second layer of PDMS with micro-fluidic channels and chambers, and the third layer with metal electrodes deposited on a pyrex substrate. Dielectrophoresis (DEP) was used to trap and concentrate the spores at the electrodes with greater than 90% efficiency, at a solution flow rate of 0.2 microl min(-1) with concentration factors between 107-109 spores ml(-1), from sample volumes of 1-5 microl. The spores were captured by DEP in deionized water within 1 min (total volume used ranged from 0.02 microl to 0.2 microl), and then germinant solution was introduced to the flow stream. The detection sensitivity was demonstrated to be as low as about a hundred spores in 0.1 nl, which is equivalent to a macroscale detection limit of approximately 10(9) spores ml(-1). We believe that this is the first demonstration of this application in microfluidic and BioMEMS devices.

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase.

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.

Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis.

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.

Poly(dimethylsiloxane) microchip for precolumn reaction and micellar electrokinetic chromatography of biogenic amines.

We have demonstrated that precolumn derivatization and capillary electrophoresis separation on a poly(dimethylsiloxane) (PDMS) microchip can be realized as efficient as those on glass microchips. In an optimized condition of micellar electrokinetic chromatography (MEKC), using 25 mM sodium borate buffer (pH 10.0) with 25 mM sodium dodecyl sulfate (SDS) and 5% v/v methanol, the electroosmotic flow in an oxidized PDMS microchip is stabilized within 3% for days. By employing a fluorometric derivatization with o-phthaldialdehyde (OPA) in an optimally designed reaction chamber, four most important biogenic amines occurring in foods, histamine, tyramine, putrescine, and tryptamine, are quantitatively determined in less than 1 min at the levels applicable to real samples. The migration behaviors of anionic OPA-derivatized biogenic amines under the MEKC conditions are analyzed, and it has been found that under our separation conditions, the electrophoretic mobility of the SDS micelles is significantly greater than those of the anions in the aqueous phase. The channel manifold in a PDMS substrate is fabricated using replica molding against a thick photoresist, SU-8, pattern generated by photolithography. The plate with the microchannel pattern is strongly, irreversibly bonded to another PDMS plate by using a new bonding technique, which employs surface oxidation by corona discharge generated from a cheap, handy source, Tesla coil.

Surface modification of poly(dimethylsiloxane) microchannels.

This review looks at the efforts that are being made to modify the surface of poly(dimethylsiloxane) (PDMS) microchannels, in order to enhance applicability in the field of microfluidics. Many surface modifications of PDMS have been performed for electrophoretic separations, but new modifications are being done for emerging applications such as heterogeneous immunoassays and cell-based bioassays. These new modification techniques are powerful because they impart biospecificity to the microchannel surfaces and reduce protein adsorption. Most of these applications require the use of aqueous or polar solvents, which makes surface modification a very important topic.