(314b) Lab-on-a-Chip Sample Preparation for Subcellular Analysis: a Technique to Rapidly Rupture Erythrocytes in a Dielectrophoretic Microdevice

Authors: 
Minerick, A. R., Mississippi State University
Reeves, S., Mississippi State University


Laboratories-on-a-chip and micro Total Analytical Systems (microTAS) are seen as one of the key growth industries for the 21st century. These systems are attractive due to the promise of raw unprocessed samples entering the device with quantitative analysis results as an output from the device. Because of their small size, these microdevices require small sample volumes and conceivable can return the results rapidly (within a matter of minutes). Such devices also have the potential to decrease the cost of analysis for applications such as medical testing, do not require technician operation, and can be adapted as point of care devices for use at home to monitor vital indicators for diseases.

Electrokinetic tools have been identified as the most promising forces to manipulate and quantify analytes within the microdevices. This work examines the use of special electric fields to reliably and rapidly rupture erythrocytes for subsequent subcellular analysis. Dielectrophoresis (DEP) is the movement of particles in a non-uniform alternating current field. Under the influence of a non-uniform AC field, charged particles become polarized. By tuning the AC frequency, membrane instabilities can be created thus causing the cell to lyse.

The fresh human blood was obtained via venipuncture by a certified phlebotomist; samples were stored in K2 EDTA anticoagulant at 5?C. The blood was diluted with a 0.143M sodium phosphate saline buffer (PBS) just prior to experimentation in a microdevice consisting of a glass slide constructed out of perpendicularly positioned 100 micron platinum wire. Dielectrophoretic fields were applied via the microdevice electrodes. A Zeiss Axiovert 200M inverted light microscope with a high resolution Axiocam camera was used to record images of the experiment every twenty seconds. The frequencies were varied from 1 kHz ? 5 kHz and the field intensity for a series of dependence experiments was varied from 1Vpp/200?m to 6Vpp/200?m. The total number of RBC in each image was analyzed manually in 1 minute intervals over the 8 minute experiment. These counts were tabulated in a spreadsheet and graphed using a number fraction (number of RBC at a specific time/number of RBC at time=0) as a function of time.

Results from this process show that there exists a relationship between the age of the blood sample and the applied field. The optimal field density for rupturing human blood was 5Vpp/200?m. At this field density, 50% of the cells were ruptured within 220 seconds and complete sample rupture was accomplished after only 360 seconds. Varying the frequency of the process showed that it indeed had an effect on rupturing. The age dependency of the rupturing for a single sample was conducted over a five day period to reveal any statistical dependencies on age.