(549j) A Novel Device for Highly Efficient Extraction of Nucleic Acids From 100 Microliter Whole Blood Samples

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 µL 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 µL 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 µm tall separation channel with a
volume of 900 µL. 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 µL 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 10°C 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 µL 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 10°C. We hypothesize we can extract all DNA
from 100 µl 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).