Diane Zebert

Differential blood cell counts obtained using a microchannel based flow cytometer

This paper reports results demonstrating the ability to use single microfabricated silicon flow channels for the differential counting of granulocytes, lymphocytes, monocytes, red blood cells (RBCs), and platelets, in a sample of blood by means of laser light scattering. The microfabrication-based flow cytometer described does not rely on sheath flow in order to align the blood cells.

Results Obtained using A Prototype Microfluidics-Based Hematology Analyzer

Microfluidic laminate-based structures incorporating hydrodynamic focusing and flow channels with dimensions much less than 1 mm were fabricated and used to transport and analyze blood samples. Optically transparent windows integral to the flow channels were used to intercept the sample streams with a tightly focused diode laser probe beam. The size and structure of the blood cells passing through the laser beam determined the intensity and directional distribution of the scattered light generated. Forward and small angle light scattering channels were used to count and differentiate platelets, red blood cells, and various populations of white blood cells. All the blood samples used were characterized using a commercial hematology analyzer for comparison and validation purposes.

Simultaneous Self-Referencing Analyte Determination in Complex Sample Solutions Using Microfabricated Flow Structures (T-Sensors™)

In microfluidic channels, fluids with viscosities similar to or higher than water and flowing at low velocities show laminar behavior. This allows the movement of different layers of fluid and particles next to each other in a channel without mixing other than by diffusion. A sample solution (e.g., whole blood), and a receptor solution (e. g., an indicator solution), and a reference solution (a known analyte standard) are introduced in a common channel (T-Sensor™), and flow next to each other until they exit the structure. Smaller particles such as ions or small proteins diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles (e. g., blood cells) show no significant diffusion within the time the two flow streams are in contact. Two interface zones are formed between the fluid layers. The ratio of a property (e. g., fluorescence intensity) of the two interface zones is a function of the concentration of the analyte, and is largely free of cross-sensitivities to other sample components and instrument parameters. This device allows, for example, one-time or continuous monitoring of the concentration of analytes in microliters of whole blood without the use of membranes or prior removal of blood cells.

Microfluidic approaches to immunoassays

An immunoassay format is presented that takes advantage of the microfluidic properties of the H-FilterTM for measuring sample analyte concentration. The method relies on the diffusion of analyte particles into a region containing beads coated with specific antibody. Competitive binding of labeled analyte and sample analyte with a limited number of binding sites allows measurement of the concentration of sample analyte based on the fraction of labeled analyte bound. The fraction of labeled analyte bound can be determined with a microcytometer by measuring the bead fluorescence intensity on the microcytometer portion of an integrated microfluidic chip. It is not necessary to separate the beads from the mixture because the bead intensity can be determined above the background of unbound labeled antigens. Other advantages include the ability to eliminate large interfering particles from samples, continuous sample monitoring, and the ability to concentrate the beads. Microfluidic immunoassay formats also consume smaller volumes of costly reagents and sample.

Fluorescence and absorbance analyte sensing in whole blood and plasma based on diffusion separation in silicon-microfabricated flow structures

Based on the recently introduced T-Sensor method, we demonstrate the fluorescence-determination of various analytes directly in whole blood and in serum. The method relies on microfluidic flow in silicon structures, diffusion-based separation, and analyte determination using fluorescent and absorption indicator dyes. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without mixing other than by diffusion. A sample solution (e.g., blood), and a receptor solution containing the indicator dye are introduced in a common channel, and flow laminarly next to each other until they exit the structure. Small ions such as H+, and Na+ diffuse rapidly across the channel, whereas larger molecules diffuse more slowly. Larger particles such as blood cells and polymer beads show no significant diffusion within the time the two flow streams are in contact. The fluorescence emission of indicator dyes is a function of the concentration of the analyte molecules and the dye concentration in the interaction zone between the two streams. This device allows continuous monitoring of the concentration of analytes in whole blood without the use of membranes or prior removal of blood cells. This principle is illustrated by the determination of human albumin, total calcium, and pH in whole blood and serum.