Biomedical subjects

Thomas N Chiesl

Publications and source records attributed to Thomas N Chiesl.

5 recordsLinked to original sources

PubMed2006

A bio-barcode assay for on-chip attomolar-sensitivity protein detection.

Functionalized nanoparticles hold great promise in realizing highly sensitive and selective biodetection. We report a single disposable chip which is capable of carrying out a multi-step process that employs nanoparticles--a bio-barcode assay (BCA) for single protein marker detection. To illustrate the capability of the system, we tested for the presence of prostate specific antigen (PSA) in buffer solution and goat serum. Detection was accomplished at PSA concentrations as low as 500 aM. This corresponds to only 300 copies of protein analytes using 1 microL total sample volume. We established that the on-chip BCA for PSA detection offers four orders of magnitude higher sensitivity compared to commercially available ELISA-based PSA tests.

Animals↗

PubMed2006

Self-associating block copolymer networks for microchip electrophoresis provide enhanced DNA separation via "inchworm" chain dynamics.

We describe a novel class of DNA separation media for microchip electrophoresis, "physically cross-linked" block copolymer networks, which provide rapid (<4.5 min) and remarkably enhanced resolution of DNA in a size range critical for genotyping. Linear poly(acrylamide-co-dihexylacrylamide) (LPA-co-DHA) comprising as little as 0.13 mol % dihexylacrylamide yields substantially improved electrophoretic DNA separations compared to matched molar mass linear polyacrylamide. Single-molecule videomicroscopic images of DNA electrophoresis reveal novel chain dynamics in LPA-co-DHA matrixes, resembling inchworm movement, to which we attribute the increased DNA resolution. Substantial improvements in DNA peak separation are obtained, in particular, in LPA-co-DHA solutions at polymer/copolymer concentrations near the interchain entanglement threshold. Higher polymer concentrations yield enhanced separations only for small DNA molecules (<120 base pairs). Hydrophobically cross-linked networks offer advantages over conventional linear polymers based on enhanced separation performance (or speed) and over chemically cross-linked gels because hydrophobic cross-links can be reversibly broken, allowing facile microchannel loading.

DNA↗

PubMed2006

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9.

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.

Acrylic Resins↗

PubMed2005

A modular microfluidic architecture for integrated biochemical analysis.

Microfluidic laboratory-on-a-chip (LOC) systems based on a modular architecture are presented. The architecture is conceptualized on two levels: a single-chip level and a multiple-chip module (MCM) system level. At the individual chip level, a multilayer approach segregates components belonging to two fundamental categories: passive fluidic components (channels and reaction chambers) and active electromechanical control structures (sensors and actuators). This distinction is explicitly made to simplify the development process and minimize cost. Components belonging to these two categories are built separately on different physical layers and can communicate fluidically via cross-layer interconnects. The chip that hosts the electromechanical control structures is called the microfluidic breadboard (FBB). A single LOC module is constructed by attaching a chip comprised of a custom arrangement of fluid routing channels and reactors (passive chip) to the FBB. Many different LOC functions can be achieved by using different passive chips on an FBB with a standard resource configuration. Multiple modules can be interconnected to form a larger LOC system (MCM level). We demonstrated the utility of this architecture by developing systems for two separate biochemical applications: one for detection of protein markers of cancer and another for detection of metal ions. In the first case, free prostate-specific antigen was detected at 500 aM concentration by using a nanoparticle-based bio-bar-code protocol on a parallel MCM system. In the second case, we used a DNAzyme-based biosensor to identify the presence of Pb(2+) (lead) at a sensitivity of 500 nM in <1 nl of solution.

Biochemistry↗

PubMed2005

Poly(acrylamide-co-alkylacrylamides) for electrophoretic DNA purification in microchannels.

We have created a family of water-soluble block copolymers of acrylamide and N-alkylacrylamides that are designed to selectively remove proteins from DNA via microchannel electrophoresis. It was hypothesized that the inclusion of hydrophobic subunits in the polymer chain, in sufficient concentration, could lead to protein adsorption due to hydrophobic interactions. A series ofN-alkylacrylamide comonomers with varying alkyl chain lengths (C4, C6, C8) and also an N,N-dialkyl group (C6-C6) were synthesized via reactions between acryloyl chloride and the respective alkylamines. Copolymers were synthesized using an aqueous "micellar" polymerization technique, which involves dissolving acrylamide in the aqueous phase while hydrophobic monomers are solubilized in sodium dodecyl sulfate micelles. Copolymers comprising up to 4 mol % of a hydrophobic subunit (as verified by (1)H NMR) were synthesized. Polymer molecular weights were determined by tandem gel permeation chromatography-multiangle laser light scattering, and ranged from 1.5 x 10(6) to 4.3 x 10(6). Capillary electrophoresis analysis of bovine serum albumin and beta-lactoglobulin migration in these matrixes revealed that the octylacrylamide and dihexylacrylamide copolymers show the most significant extent of protein adsorption while butylacrylamides show no noteworthy adsorption trend. All copolymer matrixes studied allowed the passage of a dsDNA digest, and displayed some DNA sieving ability at 0.5% (w/w) in TTE (50 mM Tris, 50 mM TAPS, 2 mM EDTA, pH 8.4) buffer. These matrixes are demonstrated in on-chip experiments to adsorb protein, in a step toward meeting the front-end processing goals of mu-TAS for genetic analysis applications.

Acrylamides↗