(471h) Three Stage Sample Preparation for Biotoxin Detection in Blood

The use of spectroscopic based techniques such as raman spectroscopy or quantum tunneling spectroscopy (Gupta et. al) for analyzing low abundance proteins in blood is difficult due to the high level of background arising from the presence of blood cells and also highly abundant proteins (~1-1000μM) like albumin, IgG, IgA, transferrin, haptoglobin and α-1-antitrypsin making up 90% of the protein mass. The dynamic range of proteins in blood plasma alone spans nine orders of magnitude resulting in difficulties for quantification.  Also, depletion of cells from blood requires centrifugation which is bulky and unsuitable for point of care diagnostics. Finally, the orientation at which the proteins are immobilized on the surface of the sensor also will affect the consistency of the sensor reading and also the signal to noise ratio. We present a three stage on-chip platform for depletion of cells and highly abundant serum proteins in blood, and finally controlling the orientation at which the target proteins become immobilized to the sensor surface.  Our platform consists of three components, the first of which is a microfluidic mixer which mixes beads containing  antibodies against the highly abundant proteins with the whole blood. For microfluidic mixing we use an adapted herringbone structure (Whitesides et. al) to induce chaotic mixing of beads with proteins in the channel. Using the adapted herringbone serpentine channel we achieve depletion of Immunoglobin G all the way down to 35 nanoMolar in concentration.

This complex mixture (consiting of beads, cells, and serum proteins) is then injected into the second component of our microfluidic platform which consists of a filter trench to capture all of the cells and the beads. In order to significantly enhance the trapping of the cells and beads into the filter trench we have integrated a pair of interdigitated electrodes at the top of the channel above the trench in order to use negative dielectrophoresis in order to push all of the micron sized particles (cells and beads which have captured the highly abundant proteins) down into the trench allowing the serum proteins of lower abundance to flow through.  In general dielectrophoresis is incapable of producing forces beyond the low piconewton range. However using atomic layer deposition to deposit a 10 nm pin-hole free layer of SiO2 on top of the electrodes protecting them from corrosion, we were able to demonstrate DEP forces in the nanonewton range.  Using the enhanced negative DEP electrodes in combination with the filter trench, we were able to demonstrate 100% depletion of all micron sized particles in the mixture. 

The third component involves a novel method for tunably controlling the immoblization of proteins on a solid-state surface using electric field. We exploit the dipole property of IgG and its ability to be oriented with field. As a proof of concept, we use fluorescence detection to indirectly verify the modulation of the orientation of proteins bound to the surface. We studied the interaction of fluorescently tagged anti-IgG with surface immobilized IgG controlled by electric field.  Our study demonstrates that the use of electric field can result in up to 40X enhancement in signal to noise ratio compared to normal physical adsorption.