Alexander P. Sassi

Rapid, parallel separations of d1S80 alleles in a plastic microchannel chip.

We have performed fast, parallel separations of alleles of the D1S80 locus in a plastic, multi-channel chip, replicated from a microfabricated master and laminated with a plastic film. The array of 16 channels was filled with a replaceable sieving polymer, and a size-dependent, electrophoretic separation of the DNA fragments was performed in all channels in less than 10 min, representing a 30-fold increase in throughput compared to that on a single-capillary instrument. To detect the fragments in all 16 channels in parallel during the run, we designed and built a scanning, confocal, laser-induced fluorescence system. The electropherograms were then used to determine the sample genotype. To demonstrate the use of multiplexed, microchannel arrays for real-life samples, we amplified D1S80 alleles from genomic DNA extracted from whole blood and separated these alleles by electrophoresis in the plastic chip. Evaluation of the electrophoretic data showed that, using a 300- and a 1,000-base pair fragment as internal mobility markers, 83% of the alleles were assigned correctly, using the allele identification from a single capillary instrument as a reference. This work demonstrates that, with improvements in the microchannel electrophoresis system, it is feasible to perform rapid, parallel genotyping in mass-produced, inexpensive, disposable plastic devices for large-scale applications in medicine and the life sciences.

Partitioning of proteins and small biomolecules in temperature- and pH-sensitive hydrogels

Abstract Experimental partition coefficients were measured for the distribution of selected proteins and small biomolecules between aqueous, buffered solutions and hydrogels. Temperature-sensitive hydrogels were prepared by polymerizing N-isopropylacrylamide (NIPA) alone and by copolymerizing NIPA and a charged monomer. Temperature- and pH-sensitive hydrogels were prepared by copolymerizing NIPA with weakly acidic and/or weakly basic monomers. The effects of temperature, pH and ionic strength on partitioning were investigated. In general, protein partition coefficients ranged from 0 to 10 in buffers on 0.1 M ionic strength. pH had a significant effect on the partition coefficient for a protein into a weakly ionizable polyelectrolyte gel even at this ionic strength.

Monte Carlo simulations of hydrophobic weak polyelectrolytes: Titration properties and pH‐induced structural transitions for polymers containing weak electrolytes

Monte Carlo simulation has been used to study titration and configurational properties of an isolated hydrophobic polymer containing weakly‐ionizable groups. Using a cubic lattice, simulations were performed in the grand canonical ensemble to include the effect of the local charge environment on the ionization of weak electrolyte segments. Properties were studied as a function of polymer hydrophobicity, fraction of ionizable segments, solution ionic strength, and pH. The polymer segments experienced three types of competing interaction: excluded volume, attractive nearest‐neighbor forces which account for the net balance of segment–segment, segment–solvent, and solvent–solvent interactions, and long‐range electrostatic forces between ionized segments, calculated with a screened Debye–Huckel potential. Simulations show that the model chain expands with chain ionization, which depends on solution pH. As the chain becomes increasingly charged, the ionization process becomes more difficult because of rising l...

Monte Carlo simulations of hydrophobic polyelectrolytes. Evidence for a structural transition in response to increasing chain ionization

Monte Carlo simulation has been used to study the configurational properties of a lattice‐model isolated polyelectrolyte with attractive segment–segment interaction potentials. This model provides a simple representation of a hydrophobic polyelectrolyte. Configurational properties were investigated as a function of chain ionization, Debye screening length, and segment–segment potential. For chains with highly attractive segment–segment potentials (i.e., hydrophobic chains), large, global changes in polymer dimensions were observed with increasing ionization. The transformation from a collapsed chain at low ionization to an expanded chain at high ionization becomes increasingly sharp (i.e., occurs over a smaller range of ionization) with increasing chain hydrophobicity. The ionization‐induced structural transitions for this model hydrophobic polyelectrolyte are analogous to pH‐induced transitions seen in real polyelectrolytes and gels. These studies suggest a simple explanation for such transitions based o...

Characterization of size-exclusion effects in highly swollen hydrogels : correlation and prediction

The literature provides several size-exclusion theories to predict solute exclusion by highly swollen hydrogels. Theoretical calculations are compared to the experimental data of Walther et al. (1993) for partitioning of poly(ethylene glycol)s and poly(ethylene oxide)s of various molecular weight into hydrogels made of poly-N-isopropylacrylamide or poly-2-hydroxyethyl methacrylate/dimethylaminoethyl methacrylate. Experimental size-exclusion curves can be correlated almost equally well by theories which characterize the gel as a collection of pores or of fibers; differences between these two theories are important only for partition coefficients near zero or unity. The experimental data of Walther et al. can be predicted best by Schnitzers uniform pore model.

Sorption of lysozyme by HEMA copolymer hydrogels

Sorption of lysozyme by 2-hydroxyethyl methacrylate (HEMA) copolymer hydrogels was studied as a function of pH and gel composition. Three types of HEMA gels were synthesized : neutral (HEMA), acidic (HEMA + acrylic acid), and basic (HEMA + dimethylaminoethyl methacrylate). Each gel was synthesized at four initial volume fractions to obtain different equilibrium swelling ratios and microstructures. Sorption as a function of time was measured for each gel at pH 7, 7.5, and 8. The rate of uptake by the acidic gels was more rapid than that by the neutral gels : To sorb 90% of the protein required only 1 h for the acidic gels but 15 days for neutral gels. Lysozyme did not adsorb or partition into the basic gels. The fractional approach to equilibrium was most rapid for the more swollen gels, and the effect of pH was small. The results reported here may be useful for rational design of new biomaterials where it is desirable to know the relative magnitude of the effects of composition, synthesis, and pH on protein sorption.

Integrated capillary electrophoresis using glass and plastic chips for multiplexed DNA analysis

Micromachined devices made of plastic have been used for fast electrophoretic separations using short separation distances and high electric field strengths. Unlike their glass counterparts, plastic chips can be manufactured economically and in high volume. Analysis can be performed in single channels, as shown for DNA sequencing mixtures, or in channel arrays as demonstrated for the analysis of ds DNA fragments. Compared to slab gel electrophoresis and capillary electrophoresis, separations are extremely fast with a time-scale under 20 minutes for a sequence analysis and under 2 minutes for fragment analysis. Confocal laser- induced fluorescence provides a sensitive means of detection.

Plastic microfluidic devices: Electrokinetic manipulations, life science applications, and production technologies

Publisher Summary This chapter describes the development and current technological status of a particular subset of microfluidic systems, namely those chips made from organic polymers and utilizing electrokinetic techniques for fluidic motivation and analyte separation. Microfluidic systems are interconnected networks of channels and reservoirs containing nano and microliter volumes. They are particularly well suited to biological assay applications that demand highly parallel, rapid, accurate, low-volume experiments requiring minimal crosscontamination. The DC electrokinetic technique of capillary electrophoresis (CE) is the application of a homogeneous electric field between the ends of a small diameter column, providing a means both to transport and to separate dissolved species in ionically conductive solutions. The surface properties of the plastic are critical to device performance, with changes in morphology or chemistry contributing to variations in adsorption. Typical plastics used for microfluidic devices are thermoplastics. Microfluidic devices demand extreme uniformity of channels that are long compared to their lateral dimensions, as well as accurate replication of micrometer-scale features, challenging production methods. A variety of different plastic devices can be successfully realized using a combination of (MEMS) microfabrication technologies and traditional plastics processing. Thermoplastic microfluidic devices made by any of the molding or embossing methods sealed with a cover to create closed capillaries, minimizing evaporation during analytical applications of the device. Plastic microfluidic devices offer several advantages over glass or silicon structures, including lower processing temperatures, high-volume manufacture at low cost, and simpler extension to multilayer device fabrication.

Partitioning of hexavalent chromium in temperature-sensitive, polyelectrolyte hydrogels

Abstract Partitioning data for hexavalent chromium (Cr(VI)) are presented for systems where thermally sensitive poly-N-isopropylacrylamide (NIPA)-based hydrogels are in contact with aqueous solutions of potassium dichromate (K 2 Cr 2 O 7 ). The poly-NIPA hydrogels contain 0–3% quatemized amine comonomer. Experimental results are given for the effect of gel charge, ionic strength and temperature on the partitioning of Cr(VI) into these NIPA-copolymer gels. At low ionic strength, the partition coefficient increases with the content of quatemized amine. The effect of rising temperature is to increase the partition coefficient. Swelling equilibria in aqueous K 2 Cr 2 O 7 solutions decrease with ionic strength and, at ionic strengths

Disposable Plastic Microfluidic Arrays for Applications in Biotechnology

Plastic microfluidic bioanalytical device arrays offer biochemical compatibility, low-cost mass production, and single-use disposability. We have designed, modeled, prototyped, and manufactured a range of plastic microfluidic devices for bioanalytical applications including DNA sequencing, nucleic acid fragment analysis, and high-throughput screening of pharmaceutical candidate compounds.