Biomedical subjects

Kevin D Dorfman

Publications and source records attributed to Kevin D Dorfman.

12 recordsLinked to original sources

PubMed2006

Automated microdroplet platform for sample manipulation and polymerase chain reaction.

We present a fully automated system performing continuous sampling, reagent mixing, and polymerase chain reaction (PCR) in microdroplets transported in immiscible oil. Sample preparation and analysis are totally automated, using an original injection method from a modified 96-well plate layered with three superimposed liquid layers and in-capillary laser-induced fluorescence endpoint detection. The process is continuous, allowing sample droplets to be carried uninterruptedly into the reaction zone while new drops are aspirated from the sample plate. Reproducible amplification, negligible cross-contamination, and detection of low sample concentrations were demonstrated on numerous consecutive sample drops. The system, which opens the route to strong reagents and labor savings in high-throughput applications, was validated on the clinically relevant quantification of progesterone receptor gene expression in human breast cancer cell lines.

Automation↗

PubMed2006

DNA electrophoresis in microfluidic post arrays under moderate electric fields.

The dynamics of long DNA moving through microfluidic arrays of micron-sized posts under a moderate electric field are modeled by a Scher-Lax continuous-time random walk. The microscale model consists of a repetitive sequence of three steps: (i) collision with the post and extension into two arms, (ii) electric-field-driven unhooking from the post, and (iii) uniform translation until the next collision. The model features two random variables: the initial offset between the two arms of the polymer during a given collision and the distance traveled between collisions. For experimentally realistic values of the electric field strength and DNA molecular weight, scaling laws indicate that the chain will generally be in a stem-flower conformation when unhooking from the post. Compared to a taut-chain model at the same field strength, the stem-flower conformation reduces the time engaged with the post and increases the collision frequency. Analytical expressions for the mean velocity and dispersivity are derived as a function of the post density, post spacing, free-solution mobility, Kuhn length, and sequence length. The incomplete extension of the chain does not strongly affect the mean velocity, but tends to increase the dispersivity relative to a taut chain. As a result, the separation resolution decreases as the field decreases for a moderate field, in agreement with experiments. The quantitative agreement between the model and experimental data is satisfactory, especially considering that the model contains no adjustable parameters.

DNA↗

PubMed2005

Contamination-free continuous flow microfluidic polymerase chain reaction for quantitative and clinical applications.

We present a method for performing polymerase chain reaction (PCR) using isolated droplets flowing in an immiscible fluorinated solvent system. Thanks to an optimized control of interfacial properties, we could achieve in this capillary-based system reproducible amplification factors, without any detectable contamination between neighboring droplets. The system is readily amenable to further miniaturization and automation and serves as the first step toward a clinically viable, high-throughput, quantitative continuous flow PCR apparatus.

DNA↗

PubMed2005

Non-markovian transport of DNA in microfluidic post arrays.

We present an analytically solvable model for the transport of long DNA through microfluidic arrays of posts. The motion is a repetitive three-part cycle: (i) collision with the post and extension of the arms; (ii) rope-over-pulley post disengagement; and (iii) a random period of uniform translation before the next collision. This cycle, inspired by geometration, is a nonseparable (Scher-Lax) continuous-time random walk on a lattice defined by the posts. Upon adopting a simple model for the transition probability density on the lattice, we analytically compute the mean DNA velocity and dispersivity in the long-time limit without any adjustable parameters. The results compare favorably with the limited amount of experimental data on separations in self-assembled arrays of magnetic beads. The Scher-Lax formalism provides a template for incorporating more sophisticated microscale models.

Bacteriophage T4↗

PubMed2005

Measurement of the surface concentration for bioassay kinetics in microchannels.

We present a simple and versatile method, based on fluorescence microscopy, to reliably measure the concentration of advected molecules in the vicinity of surfaces in microchannels. This tool is relevant to many microfluidic applications such as immunoassays and single-molecule experiments, where one probes the kinetics of a reaction between an immobilized target and a reactant carried by the bulk flow. The characterization of the surface concentration highlights the dominant role of transverse diffusion, which generates an apparent diffusivity at the surface 3-4 orders of magnitude greater than molecular diffusion alone, even close to the point of injection. We directly measure the effects of the longitudinal position along the channel and of the flow rate on the concentration front and develop a simple analytical model that compares well with the data. Finally, we propose a method to properly account for concentration fronts in single-molecule measurements and use it to directly access the kinetics parameters of protamine-induced condensation of DNA.

Biological Assay↗

PubMed2005

Motion of single long DNA molecules through arrays of magnetic columns.

We present a videomicroscopy study of T4 DNA (169 kbp) in microfluidic arrays of posts formed by the self-assembly of magnetic beads. We observe DNA moving through an area of 10 000 microm(2), typically containing 100-600 posts. We determine the distribution of the contact times with the posts and the distribution of passage times across the field of view for hundreds of DNA per experiment. The contact time is well approximated by a Poisson process, scaling like the inverse of the field strength, independent of the density of the array. The distribution of passage times allows us to estimate the mean velocity and dispersivity of the DNA during its motion over distances long compared to our field of view. We compare these values with those computed from a lattice Monte Carlo model and geometration theory. We find reasonable quantitative agreement between the lattice Monte Carlo model and experiment, with the error increasing with increasing post density. The deviation between theory and experiment is attributed to the high mobility of DNA after disengaging from the posts, which leads to a difference between the contact time and the total time lost by colliding. Classical geometration theory furnishes surprisingly good agreement for the dispersivity, while geometration theory with a mean free path significantly overestimates the dispersivity.

Bacteriophage T4↗

PubMed2005

Droplet fusion by alternating current (AC) field electrocoalescence in microchannels.

We present a system for the electrocoalescence of microfluidic droplets immersed in an immiscible solvent, where the undeformed droplet diameters are comparable to the channel diameter. The electrodes are not in direct contact with the carrier liquid or the droplets, thereby minimizing the risk of cross-contamination between different coalescence events. Results are presented for the coalescence of buffered aqueous droplets in both quiescent and flowing fluorocarbon streams, and on-flight coalescence is demonstrated. The capillary-based system presented here is readily amenable to further miniaturization to any lab-on-a-chip application where the conductivity of the droplets is much greater than the conductivity of the stream containing them, and should aid in the further application of droplet microreactors to biological analyses.

Electricity↗

PubMed2004

Model of RecA-mediated homologous recognition.

We consider theoretically the homology search between a long double-stranded DNA and a RecA-single-stranded DNA nucleofilament, emphasizing the polymeric nature of the search and the ability of double-stranded DNA to overcome the difference in pitch between itself and the nucleofilament by thermally activated stretching from the canonical B state to the metastable, stretched S state. Our analytical first-passage-time analysis agrees well with experimental data, predicts new dependencies on the intracellular fluid viscosity and ionic strength, and strongly suggests that initial homologous recognition involves a three base-pair seed.

Adenosine Triphosphatases↗

PubMed2004

Quantitative microfluidic separation of DNA in self-assembled magnetic matrixes.

We present an experimental study of the microfluidic electrophoresis of long DNA in self-assembling matrixes of magnetic bead columns. Results are presented for the rapid separation of lambda-phage, 2lambda-DNA, and bacteriophage T4 DNA, where separation resolutions greater than 2 between lambda and T4 are achieved in times as short as 150 s. The use of a computer-piloted flow control system and injection results in high reproducibility between separations. We compare the experimentally measured mobility and dispersion with an exactly solvable lattice Monte Carlo model. The theory predicts that the mean velocity scales linearly with the field, the band broadening scales with the inverse of the field, and the resolution is independent of the field for intermediate fields-all of which are in accord with the experimental results. Moreover, reasonable quantitative agreement is achieved for band broadening for longer DNA (2lambda and T4) when the average postengagement time is measured experimentally. This work demonstrates the possibility of achieving fast microfluidic separation of large DNA on a routine basis.

DNA↗

PubMed2004

Semiphenomenological model for the dispersion of DNA during electrophoresis in a microfluidic array of posts.

A lattice Monte Carlo model is proposed for quantifying the dispersion of DNA during microfluidic electrophoresis in a quasiperiodic array of posts similar to that encountered in the microfluidic self-assembly of magnetic bead columns. The transport model is semiphenomenological in the sense that all parameters, such as the post geometry, average collision time, and collision probability, are assumed to be accessible either directly from experiment or from a model of the microscale physics. Asymptotically exact formulas are obtained for the mean velocity and dispersivity using Taylor-Aris dispersion theory, which permits a straightforward analysis of the separation efficiency. The model is applicable to a variety of situations involving collision-retardation processes.

Complex Mixtures↗

PubMed2002

Separation mechanisms underlying vector chromatography in microlithographic arrays.

Micropatterned chips possessing an asymmetric, spatially periodic array of obstacles enable the vector (directional) chromatographic separation of charged particles animated by an external electric field. We apply a network theory to analyze the chip-scale (L-scale) transport of finite-size Brownian particles in such devices and identify those factors that break the symmetry of the chip-scale particle mobility tensor, most importantly the hydrodynamic wall effects between the particles and the obstacle surfaces. Our analysis contrasts with prevailing separation theories, which are limited to effectively point-size particles, for which wall effects are negligible. These theories require a biasing of obstacle-scale (l-scale; l<<L) bifurcation branches within the network. Such bifurcations are shown to constitute but one factor in modeling the vector chromatography of finite-size particles, and not necessarily the dominant factor.

Journal Article↗

PubMed2002

Generalized Taylor-Aris dispersion in discrete spatially periodic networks: microfluidic applications.

A theory is presented for the lumped parameter, convective-diffusive transport of individual, noninteracting Brownian solute particles ("macromolecules") moving within spatially periodic, solvent-filled networks--the latter representing models of chip-based microfluidic chromatographic separation devices, as well as porous media. Using graph-theoretical techniques, the composite medium is conceptually decomposed into a network of channels (the edges) through which the solute is transported by a combination of molecular diffusion and either "piggyback" entrainment within a flowing solvent or an externally applied force field acting upon the solute molecules. A probabilistic choice of egress channel for a solute particle exiting the intersection (vertex) of the channels is furnished by an imperfect mixing model. A spatially periodic, Taylor-Aris-like "method-of-moments" scheme is applied to this transport model, leading to discrete matrix equations for computing the network-scale particle velocity vector U(*) and dispersivity dyadic D(*) in terms of the prescribed microscale transport parameters and network geometry characterizing the basic unit cell of which the spatially periodic device is comprised. The ensuing algebraic equations governing the vertex-based, discrete unit-cell "fields" P(0)(infinity)(i) and B(i) (i=1,2,...,n), whose paradigmatic summations yield U(*) and D(*), constitute discrete analogs of classical continuous macrotransport phenomenological parameters, P(0)(infinity)(r) and B(r), with r a continuous position vector defined within the unit cell. The ease with which these discrete calculations can be performed for complex networks renders feasible parametric studies of potential microfluidic chip designs, particularly those pertinent to biomolecular separation schemes. Application of this discrete theory to the dispersion analysis of pressure-driven flow in spatially periodic serpentine microchannels is shown to accord with existing results previously derived using classical continuous macrotransport theory.

Journal Article↗