Andrew Kamholz

A rapid diffusion immunoassay in a T-sensor

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 μl of reagents and sample, and is applicable to a wide range of analytes.

A ferrofluidic magnetic micropump

A microfluidic pump is described that uses magnetic actuation to push fluid through a microchannel. Operation relies on the use of magnetically-actuated plugs of ferrofluid, a suspension of nanosize ferromagnetic particles. The ferrofluid contacts but is immiscible with the pumped fluid. The prototype circular design demonstrates continuous pumping by regenerating a translating ferrofluidic plug at the conclusion of each pumping cycle. The flow rate can be controlled by adjusting device dimensions or the velocity of an external permanent magnet that directs the motion of the ferrofluid. The ferrofluidic plugs also serve as valves; if the magnetic actuator is stopped, pressure can be maintained with no power consumption. Flow can also be reversed by switching the direction of actuation. The maximum flow rate achieved with minimal backpressure was 45.8 /spl mu/l/min. The maximum pressure head achieved was 135 mm water (1.2 kPa).

Theoretical Analysis of Molecular Diffusion in Pressure-Driven Laminar Flow in Microfluidic Channels

The T-sensor is a microfluidic analytical device that operates at low Reynolds numbers to ensure entirely laminar flow. Diffusion of molecules between streams flowing side by side may be observed directly. The pressure-driven velocity profile in the duct-shaped device influences diffusive transport in ways that affect the use of the T-sensor to measure molecular properties. The primary effect is a position-dependent variation in the extent of diffusion that occurs due to the distribution of residence time among different fluid laminae. A more detailed characterization reveals that resultant secondary concentration gradients yield variations in the scaling behavior between diffusive displacement and elapsed time in different regions of the channel. In this study, the time-dependent evolution of analyte distribution has been quantified using a combination of one- and two-dimensional models. The results include an accurate portrayal of the shape of the interdiffusion region in a representative T-sensor assay, calculation of the diffusive scaling law across the width of the channel, and quantification of artifacts that occur when making diffusion coefficient measurements in the T-sensor.

Optical Measurement of Transverse Molecular Diffusion in a Microchannel

Quantitative analysis of molecular diffusion is a necessity for the efficient design of most microfluidic devices as well as an important biophysical method in its own right. This study demonstrates the rapid measurement of diffusion coefficients of large and small molecules in a microfluidic device, the T-sensor, by means of conventional epifluorescence microscopy. Data were collected by monitoring the transverse flux of analyte from a sample stream into a second stream flowing alongside it. As indicated by the low Reynolds numbers of the system (< 1), flow is laminar, and molecular transport between streams occurs only by diffusion. Quantitative determinations were made by fitting data with predictions of a one-dimensional model. Analysis was made of the flow development and its effect on the distribution of diffusing analyte using a three-dimensional modeling software package. Diffusion coefficients were measured for four fluorescently labeled molecules: fluorescein-biotin, insulin, ovalbumin, and streptavidin. The resulting values differed from accepted results by an average of 2.4%. Microfluidic system parameters can be selected to achieve accurate diffusion coefficient measurements and to optimize other microfluidic devices that rely on precise transverse transport of molecules.

Molecular diffusive scaling laws in pressure-driven microfluidic channels: deviation from one-dimensional Einstein approximations

This study presents a theoretical analysis of the scaling laws for analyte diffusion in a microfluidic chemical analysis device, the T-sensor. Because the flow is pressure-driven, the velocity profile is non-uniform, inducing a distribution in residence time among analyte molecules. Solutions for concentration distribution are given from the device inlet to a downstream distance where variations in the scaling law become negligible. All data were generated using a custom two-dimensional model that describes convection and diffusion in a system of two fluids running side-by-side in a duct. These results deviate substantially from those expected by simpler calculations, including a one-dimensional model. This study lends a better understanding of the transport of analytes diffusing in microchannels and provides a means for determining whether the complicated effects need to be considered in device design and operation.

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.

Analytical Devices Based on Transverse Transport in Microchannels

The manipulation of transport transverse to the flow direction has permitted development of microfluidic devices that allow continuous processing of samples. One of these is a rapid competition immunoassay that relies on apparent changes in the diffusivity of antigen due to binding of the antigen to relatively slowly diffusing antibody. It has also been possible to utilize isoelectric focusing to concentrate and separate different types of analytes into separate flowing streams.