Uniformly sized silica-coated magnetic nanoparticles (magnetite@silica) are synthesized in a simple one-pot process using reverse micelles as nanoreactors. The core diameter of the magnetic nanoparticles is easily controlled by adjusting the w value ([polar solvent]/[surfactant]) in the reverse-micelle solution, and the thickness of the silica shell is easily controlled by varying the amount of tetraethyl orthosilicate added after the synthesis of the magnetite cores. Several grams of monodisperse magnetite@silica nanoparticles can be synthesized without going through any size-selection process. When crosslinked enzyme molecules form clusters on the surfaces of the magnetite@silica nanoparticles, the resulting hybrid composites are magnetically separable, highly active, and stable under harsh shaking conditions for more than 15 days. Conversely, covalently attached enzymes on the surface of the magnetite@silica nanoparticles are deactivated under the same conditions.
A one-column chromatograph with recycle analogous to a four-zone simulated moving bed (SMB),
called “the analogue”, was developed for binary separations. The analogue has one chromatography column connected to a number of tanks equal to the number of steps in the SMB cycle.
For example, the analogue to a four-zone SMB with two columns per zone would have eight
tanks. The analogue's simple design gives it great flexibility. The analogues and corresponding
SMBs were simulated using Aspen Chromatography (v 11.1) for the separation of dextran T6−fructose and dextran T6−raffinose mixtures, which are linear systems, and for the separation
of binaphthol enantiomers, which is a nonlinear system. Because of mixing in the tanks, lower
purities were obtained with the analogue than with the SMB at equal productivities and
desorbent to feed, D/F, values. Therefore, dividing the tanks into several smaller tanks increased
the product purities. By increasing D/F from 1.0 to 2.6 (for one column per zone) or to 2.3 (for
two columns per zone), the analogue achieved the same purities as the SMB. These increases in
D/F are significantly less than the increased amounts of desorbent and adsorbent required for
a chromatograph without recycle to obtain the same purity as an SMB.
A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.
We have developed a reliable fabrication method of forming micron scale metal patterns on poly(dimethylsiloxane) (PDMS) using a pattern transfer process. A metal stack layer consisting of Au-Ti-Au layers, providing a weak but reliable adhesion, was deposited on a silicon wafer. The metal stack layer was then transferred to a PDMS substrate using serial and selective etching. We demonstrate that features as small as 2 microm were reliably transferred on to the PDMS substrate for use as interconnects and electrodes for biosensors and flexible electronics application.
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