269572 A Novel Device for Highly Efficient Extraction of Nucleic Acids From 100 Microliter Whole Blood Samples

Wednesday, October 31, 2012: 5:30 PM
411 (Convention Center )
Lewis A. Marshall, Chemical Engineering, Stanford University, Stanford, CA and Juan G. Santiago, Stanford University, Stanford, CA

A NOVEL DEVICE FOR HIGHLY EFFICIENT EXTRACTION OF

NUCLEIC ACIDS FROM 100 MICROLITER WHOLE BLOOD SAMPLES

Lewis A. Marshall and Juan G. Santiago

Stanford University

Stanford, CA 94305, USA

We report on an effort to bridge the divide between nanoliter-scale microfluidic processes and milliliter-scale tissue sample preparation. We have designed a novel device to electrokinetically extract nucleic acids from 100 L of lysed whole blood with high efficiency in less than 30 minutes. We performed this extraction using isotachophoresis (ITP), a technique that achieves selective, rapid pre-concentration and separation of molecules based on electrophoretic mobility [1]. Previous efforts to extract nucleic acids from complex biological samples using ITP have typically operated in standard etched glass microchannels or capillary systems with separation capacity equivalent to 100 nL of blood or less. As shown in Figures 1 and 2, we designed and fabricated a novel device with a separation capacity of 100 L blood while maintaining a sample processing time of less than 30 min. We achieve this increase in throughput in a high-aspect ratio geometry which rejects Joule heating while providing sufficient buffering capacity for electrokinetic processing. The device was designed for a theoretical nucleic acid extraction efficiency of 100%, and we have so far demonstrated a preliminary efficiency of 25%.

The device consists of stereolithographically-defined reservoirs (Figure 2) attached to 50 x 75 mm glass layers that define the length and width of the channel. These glass layers are spaced to form a 250 m tall separation channel with a volume of 900 L. The device employs photolithographically fabricated phaseguides [2] to enable consistent loading of fluid into the channel without bubble formation. Each reservoir contains porous plastic spacers to hydrodynamically isolate sample and buffer chemistries. To prepare the device for operation, we loaded leading electrolyte (LE) buffer into the separation channel, and high-concentration solution into the buffering reservoirs. We pipette in 100 L of nucleic acid solution into the sample reservoir and apply electric field to perform sample preparation. The nucleic acids are electrophoretically transferred through the channel toward the negative electrode, where they elute into the clean LE buffer. In Figure 3, we visualize operation of the device using alexa fluor 488 and fluorescein. We also demonstrated the device efficiency by processing a sample spiked with synthetic DNA oligonucliotides. We collected the output samples and analyzed them using quantitative PCR. We show recovery of 25% of dispensed synthetic DNA based on qPCR threshold cycle (as shown in Figure 4).

The device throughput is limited by heat dissipation. We deigned the device geometry to minimize temperature rise during extraction, and analyzed the theoretical steady-state temperatures. We measured steady-state temperature using thermocouples inserted into the device as a function of operational current. As shown in Figure 5, the device can operate at up to 10 mA while maintaining a temperature within 10C of room temperature.

Flexibility in samples is a necessary component in nucleic acid sample preparation in microfluidic devices. Samples containing rare sequences are subject to Poisson statistics, so small processed volumes may not contain important targets. To our knowledge, our system is unique in addressing separation capacity, extraction efficiency, and throughput in on-chip nucleic acid extraction.


Figure 1. Image of device showing the wide central channel connecting two plastic reservoirs on each side. Blue strips are photoresist phaseguides to aid device loading. Porous spacers form a sample inlet, sample outlet, and two buffer/electrode reservoirs.

Figure 2. CAD drawing showing details of the fabricated reservoir. The device uses a stereolithographically defined plastic chamber. A porous plastic spacer separtes this into two reservoirs for electrode buffer and sample volumes. Stereolithography allows us to quickly and cheaply fabricate complex geometries for our devices.

Figure 3. Fluorescence visualizaton of device operation. High-mobility Alexa Fluor 488 focuses into the ITP zone, while the lower-mobility fluorescein trails behind as an unfocused zone. The distance between the DNA simulant here (alexa fluor) increases from contaminants (fluorescein). The target sample ions (Alex Fluor) travel toward the anode, where they are collected, and the fluorescein discarded.

Figure 4. Off-chip quantitative PCR curves showing the purity and abundance of extracted DNA from a 100 L sample. Based on the threshold cycle of the extracted DNA sample, we calculate a preliminary yield of 25% from the sample. We hypothesize we can increase this to very near 100% extraction efficiency.

Figure 5. Measured temperature in the extraction device (maximum temperature in channel) as a function of applied current. The device throughput is limited by Joule heating, but it can operate at currents as high as 10 mA with a temperature rise of approximately 10C. We hypothesize we can extract all DNA from 100 l by balancing focusing dynamics, temperature, and buffering capacity.

REFERENCES:

1.     "Purification of nucleic acids from whole blood using isotachophoresis," Persat, A., Marshall, L. A., and Santiago, J. G. Analytical Chemistry 81, 9507 (2009).

2.     "Phaseguides: a paradigm shift in microfluidic priming and emptying," Vulto, P., Podszun, S., Meyer, P., Hermann, C., Manz, A., and Urban, G. A. Lab Chip 11, 1596 (2011).


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