James P. Brody

Biotechnology at low Reynolds numbers.

The shrinking of liquid handling systems to the micron and submicron size range entails moving into the area of small Reynolds numbers. The fluid dynamics in this regime are very different from the macroscale. We present an intuitive explanation of how the different physics of small Reynolds numbers flow, along with microscopic sizes, can influence device design, and give examples from our own work using fluid flow in microfabricated devices designed for biological processing.

Diffusion-based extraction in a microfabricated device

Abstract Microfabricated fluid systems allow complex chemical analyses to be performed on sub-nanoliter volumes. However, many common laboratory procedures, including filtration, have yet to be robustly implemented in micro-fluid systems. A device has been developed to separate particles and molecules based on their diffusion coefficients; the process is demonstrated using a micromachined device with fluid channels as small as 20 μm. A simple model predicts exponential dependence of the output concentration on diffusion coefficient in certain regimes. Experiments confirm the model.

p62 overexpression in breast tumors and regulation by prostate-derived Ets factor in breast cancer cells

p62 is a multifunctional cytoplasmic protein able to noncovalently bind ubiquitin and several signaling proteins, suggesting a regulatory role connected to the ubiquitin–proteasome pathway. No studies to date have linked p62 protein expression with pathological states. Here we demonstrate the overabundance of p62 protein in malignant breast tissue relative to normal breast tissue. The proteasome inhibitor PSI increased p62 mRNA and protein; however, PSI treatment of breast epithelial cells transfected with the p62 promoter did not affect promoter activity. High levels of prostate-derived Ets factor (PDEF) mRNA have been identified in breast cancer compared to normal breast. Only the PSA and maspin promoters have been identified as targets of this transcription factor. Here we show that PDEF stimulates the p62 promoter through at least two sites, and likely acts as a coactivator. PSI treatment abrogates the PDEF-stimulated increase of p62 promoter activity by 50%. Thus, multiple mechanisms for the induction of p62 exist. We conclude that (1) p62 protein is overexpressed in breast cancer; (2) p62 mRNA and protein increase in response to PSI, with no change of basal promoter activity; (3) PDEF upregulates p62 promoter activity through at least two sites; and (4) PSI downregulates PDEF-induced p62 promoter activation through one of these sites.

A planar microfabricated fluid filter

Abstract Many blood tests must be performed on plasma without cellular matter present. In the standard laboratory protocol, pure plasma is obtained through centrifugation. In order to produce a miniaturized blood sensor, a method to separate plasma other than centrifugation is needed. We describe the design, fabrication, and testing of a fluid filter that fulfils this need, and also has some features of general interest. Results from a particular device show that we can easily remove 16 μm diameter spheres from the fluid. This filter can be reusable, can potentially remove particles as small as 0.1 μm, and is easily fabricated.

A self-assembled microlensing rotational probe

A technique to measure microscopic rotational motion is presented. When a small fluorescent polystyrene microsphere is attached to a larger polystyrene microsphere, the larger sphere acts as a lens for the smaller microsphere and provides an optical signal that is a strong function of the azimuthal angle. We demonstrate the technique by measuring the rotational diffusion constant of the microsphere in solutions of varying viscosity and discuss the feasibility of using this probe to measure rotational motion of biological systems.

Applying Microfluidic Chemical Analytical Systems to Imperfect Samples

Over the last 4 years our group has been involved in developing a series of devices for chemical separation and analysis. These devices share a theme of utilizing the low Reynolds number properties of liquids flowing at slow speeds in small channels. These devices allow some types of function that are not possible in larger devices because of the possibility of bringing flows together without convective mixing. We have taken two somewhat different approaches to coping with samples that contain particles that are incompatible with one or more analytical methods to be used.

Prominent microscopic effects in microfabricated fluidic analysis systems

Microfabricated fluidic systems allow complex chemical analyses to be performed on sub-nanoliter volumes of sample. Compared to macroscopic systems, these devices offer many advantages, including the promise of performing some analytical functions more rapidly and on smaller samples. However, miniaturization of analytic instruments is not simply a matter of reducing their size. At small scales, different effects become more prominent, rendering some processes inefficient and others useless. The small scales also permit the creation of novel devices, such as the H- filter, which we are using to extract analytes from whole blood. Fluid flow in microfluidic systems is entirely dominated by viscous forces, making diffusion the sole mechanism of mixing. In addition, a larger fraction of molecules are lost to surface adsorption as devices shrink. This paper examines some of the issues involved in device miniaturization, specifically those phenomena that become increasingly dominant.

Using poly(ethylene glycol) silane to prevent protein adsorption in microfabricated silicon channels

Microfluidic devices fabricated in silicon are quickly finding use in many areas of technology. Exploration of new applications of this technology has shown both advantages and disadvantages to extreme miniaturization of chemical assays. While accuracy, efficiency and smaller sample volumes are among the advantages, interactions between the walls of the micro-channels and the fluid or particles it contains are among the disadvantages. Our group is applying this technology to chemical and biological warfare (CBW) agent purification and detection. We present preliminary result towards achieving a long-term antifouling surface in our detection system. A microfluidic device was anisotropically etched in a (100) silicon wafer and attached to a Pyrex glass slip to create an enclosed channel. Poly(ethylene glycol) (PEG) silane was covalently bonded to the hydroxyls of an oxide layer on the silicon device and the Pyrex cover slip. Fluorescently labeled ovalbumin, a CBW simulant, was in contact with an unmodified and PEG-modified channel. The extent of adsorption was determined using fluorescence microscopy.

A Planar Microfabricated Fluid Filter

Many blood tests must be performed on plasma without cellular matter present. In the standard laboratory protocol, pure plasma is obtained through centrifugation. In order to produce a miniaturized blood sensor, a method to separate plasma other than centrifugation is needed. We describe the design, fabrication, and testing of a fluid filter that fulfills this need, and also has some features of general interest. Results from a particular device show that we can easily remove 16/spl mu/m diameter spheres from the fluid. This filter can be reusable, can potentially remove particles as small as 0.1 /spl mu/m, and is easily fabricated.

Hydrodynamic activation and sorting of white blood cells in a microfabricated lattice

We demonstrate a novel hydrodynamic shear activation of leucocyte adhesion, using physiological flow conditions and a microfabricated array of channels with length scales similar to those of human capillaries. Vital chromosome stains and cell specific fluorochrome labeled antibodies reveal that the eventual adhesion of the leukocytes to the silicon array displays a strong dependence on cell type and nuclear morphology, with granulocytes activating more rapidly with distance and penetrating a smaller distance than lymphocytes. Further, the granulocytes interact with the lymphocytes in a self-exclusionary manner under shearing flow with the eventual separation of the two cell types in the array. Such arrays of microfabricated obstacles thus have an interesting potential for sorting white blood cells by type from a 10 microliter drop of whole blood.