A Novel Microfluidic Platform for Continuous DNA Extraction and Purification using Laminar Flow Magnetophoresis

Abstract

We present a novel microfluidic platform using laminar-flow magnetophoresis for combined continuous extraction and purification of DNA. All essential unit operations (DNA binding, sample washing and DNA elution) are integrated on one single chip. The key function is the motion of magnetic beads given by the interplay of laminar flow and time-varying magnetic field. The time for extraction was 1 minute. The device is a central part of a complete biochemical system for continuous monitoring of cell-growth in bioreactors. The novel platform allows continuous purification of DNA, but is also applicable to purification of RNA, proteins or cells, including their subsequent real-time analysis in general.

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A NOVEL MICROFLUIDIC PLATFORM FOR CONTINUOUS
DNA EXTRACTION AND PURIFICATION USING
LAMINAR FLOW MAGNETOPHORESIS
Marc Karle1, Junichi Miwa2, Günter Roth2, Roland Zengerle1,2, Felix von Stetten1,2
1HSG-IMIT, Villingen-Schwenningen, Germany
2IMTEK - University of Freiburg, Freiburg, Germany

ABSTRACT
We present a novel microfluidic platform using
laminar-flow magnetophoresis for combined continuous
extraction and purification of DNA. All essential unit
operations (DNA binding, sample washing and DNA
elution) are integrated on one single chip. The key
function is the motion of magnetic beads given by the
interplay of laminar flow and time-varying magnetic field.
The time for extraction was 1 minute. The device is a
central part of a complete biochemical system for
continuous monitoring of cell-growth in bioreactors. The
novel platform allows continuous purification of DNA,
but is also applicable to purification of RNA, proteins or
cells, including their subsequent real-time analysis in
general.

INTRODUCTION
Monitoring of biological agents, including the
pathogenic microorganisms, protein and free nucleic
acids, is highly relevant in the field of security (B-
detection), blood monitoring and process control in
pharmaceutical fermentations. Key requirement to
perform these monitoring tasks is the continuous
processing of biochemical assays. Such continuously
working and automated monitoring systems are currently
not available.
Continuous on-chip PCR systems for amplification
and detection of DNA have already been developed [1]
including a transcription into cDNA for RNA detection
[2]. A concept for continuous molecular enrichment using
segmented flow has also been proposed [3]. However, a
method for continuous DNA or RNA extraction from any
sample such as whole blood or cell cultures was not
achieved. Therefore we develop a novel microfluidic
platform in order to realize a fully integrated microfluidic
continuous extraction and analysis system for on-line
monitoring of cell growth in bioreactors.
Among various techniques for DNA purification, we
use DNA adsorption onto superparamagnetic beads.
Different microfluidic approaches using this method in a
batch-wise manner have already been reported [4-6]. In
contrast our device allows the continuous operation in a
flow through manner. Until now the only existing
continuous magnetophoretic devices are for single-step
cell separation [7;8], while we now report well-controlled
manipulation of magnetic beads through at least three
subsequent assay steps necessary for nucleic acid
purification.

WORKING PRINCIPLE
The working principle is depicted in Fig. 1. The first
step of DNA extraction and purification after sampling
and lysis of the cells is binding of the DNA onto the
superparamagnetic beads in order to separate the DNA
from impurities such as enzymes and/or cell debris. After


Figure 1: Schematic view of the microfluidic chip for
continuous DNA extraction. The rotation of the permanent
magnet is opposite to the direction of the buffer flow.


the separation a washing step is included to remove the
high salt buffers required for DNA binding as well as any
residual impurities. As a last step the beads are
transferred into the elution buffer where the DNA
dissociates from the beads to allow real-time analysis.

Chip design
Our microfluidic chip applies magnetophoresis to
control superparamagnetic beads in different buffer
solutions under continuous laminar flow (Fig. 1).
Bead-bound DNA samples flow through three
chambers for impurity separation, sample washing, and
DNA elution from the beads. The circular arrangement of
the microfluidic structure (Fig. 2), together with a single
rotating permanent magnet positioned below the chip
center, allows the transfer of magnetic beads from one
reagent to the other in each separation chamber.
Subsequently, the magnetic beads transport the DNA
through three separation chambers for the separation of
impurities, sample washing and the final elution of DNA
elution.
The separation chambers consist each of one or more
inlets and two outlets which are different in diameter.
This is a key design feature for the division of the buffer
flow into two fractions of different flow rates; a fraction
of small volume containing a high concentration of
magnetic beads and thus high concentration of sample,
and a large fraction without any magnetic beads but most
of the buffer solution.
978-1-4244-2978-3/09/$25.00 ©2009 IEEE276

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Figure 2: Polycarbonate chip with micro-milled
microchannels. This chip design was used for the DNA
extraction experiment. To illustrate the extraction process
dyed buffer solutions were injected into the chip. Yellow:
Lysis and binding buffer, blue: washing solution, green:
elution buffer, red: dilution buffer.


The magnetic beads exit the separation chambers
through the small outlet and are transferred to the next
section whereas the large fraction of the buffer solution
leaves through the wider outlet that directly leads to the
waste.

Time-varying magnetic field
The circular arranged microchannels guide different
buffer flows from different inlets of the chip around a
single external permanent magnet located beneath the
center of the chip. The permanent magnet is rotated
slowly using a stepping motor to provide the required
periodically changing magnetic field to attract the
magnetic beads in the radially inward direction.
According to the oscillating strength of the magnetic field,
the beads are attracted and stick to the side-wall during
the strong field phase but are released during the weak
field phase. With the circular microfluidic design shown
in Fig. 2, the DNA-bound beads are attracted by
sufficiently strong magnetic force during the strong field
phase at each of the separation chambers. The average
magnetophoretic velocity of beads is determined by the
balance between the flow rate and the strength of
magnetic field, the latter of which can be controlled by
altering the magnet rotation speed. We have also
observed that by rotating the magnet rotation in the
direction opposite to the flow, the streamwise bead
velocity is larger. Thus, we realize efficient separation of
the magnetic beads without immobilizing them on the
walls of the microchannels.



Figure 3: Instantaneous image of (a) the first separation
chamber and (b) the third separation chamber. The beads
are strongly attracted by the magnetic field and flow in
the vicinity of the inner side wall throughout the device.
The inlet flow velocity of each stream is 12.5 mm·s-1 and
the average velocity of beads estimated from consecutive
images is approximately 1.6 mm·s-1.


Buffer flow protocol
A mixture of cell lysate, binding buffer and
superparamagnetic beads, which are already bound to the
DNA, enters the chip through the 1st inlet (Fig. 2, top
left). In the first separation chamber the beads are
concentrated by the central permanent magnet and
separated from most of the buffer solution containing
impurities (Fig. 3a).
During the transfer to the next section, washing
buffer is added forming a laminar flow with the remaining
binding buffer. The permanent magnet attracts the beads
to the inner part of the channel system and ensures the
transfer of the magnetic beads across the phase interface.
In the second separation chamber the magnetic beads
are again concentrated and most of the buffer is
transferred to the waste, while the magnetic beads leave
through the small outlet towards the third separation
chamber (Fig. 3b).
Here the beads are transferred to the elution buffer, in
which the DNA is released from the bead surface. The
eluate is collected for further analysis. A key feature in
the third separation chamber is the introduction of a
dilution buffer.
The washing buffer contains a high concentration of
ethanol, which is a PCR inhibitor. However, the
hygroscopic nature of ethanol leads to rapid dispersion
throughout the sample flow, and it is thus not possible to
establish a clear laminar interface between the washing
and elution buffers. Therefore we introduce a dilution
buffer that is injected from the opposite side. The purpose
of the dilution buffer is to assimilate the ethanol from the
washing buffer and therefore reduce the ethanol
concentration in the eluate or even exclude ethanol from
the eluate.
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Figure 4: Real-time PCR of extracted genomic E. coli
DNA. Shown in the graph is the result of a DNA
extraction experiment performed both in a test tube and
on the microfluidic chip. The genomic E. coli DNA was
diluted and spiked with herring sperm DNA (therefore the
gap between the standard curve and the tube extraction) .
At E. coli DNA concentrations of 3.15 ng·µl-1 the on-chip
purification achieves 25% of the reference in the test tube.


On the other hand, DNA molecules and magnetic
beads are large enough that they do not diffuse in the
sample solutions so rapidly. Thus, after the mixing of
elution and washing buffer on one side of the third
separation chamber and the mixing of washing and
dilution buffer on the opposite side, the purified DNA
remains in the elution buffer stream to be collected at the
outlet without any loss.

CHIP PRODUCTION
The 2D-layout of the microfluidic channels was
designed with CAD software and converted to G-code
compatible to a precision milling machine. For chip
production polycarbonate was used. The mircochannels
were milled directly into the chip using a 400 µm mill,
with a standard width and depth of 400 µm. After the
milling process the channels were sealed with adhesive
foil.

RESULTS
The flow of sample and buffers of a commercially
available DNA purification kit was controlled by syringe
pumps (neMESYS, cetoni GmbH). The permanent
magnet was mounted below the chip system close to the
surface and rotated by a stepping motor with a rotational
frequency of 1 Hz.
We extracted and purified a sample of genomic DNA
from E. coli DH5αZ1 [9] bacteria on the chip in a flow-
through manner. An inlet flow of 12.5 mm·s-1 lead to an
average bead velocity of 1.6 mm·s-1 and a sample
transition time of approximately 1 minute.
The extracted DNA was successfully amplified off-
chip via real-time PCR to demonstrate that PCR inhibitors
included in the buffers are sufficiently diluted and/or
excluded during the on-chip purification. At a starting
concentration of 3.15 ng·µl-1 the fluorescence signal of the
chip-extracted total DNA crossed the threshold value only
2 cycles later than the reference extraction performed in a
test tube (Fig. 4).

CONCLUSION
We established a microfluidic
continuous DNA purification by magnetophoretic
manipulation of magnetic beads in a time-varying
magnetic field. The platform closes the gap between
continuous cell lysis and continuous DNA amplification
to set up powerful microsystems for the monitoring of
biological agents, pathogenic microorganisms, protein,
free nucleic acids and cell growth in bioreactors.
The magnetic beads enable the transport of the DNA
across the interfaces between co-flowing laminar streams
in a circular channel arrangement around a central rotating
permanent magnet inducing time-varying magnetic field,
which prevents the beads from sticking to the channel
walls and enables controlled transfer of beads between
different extraction reagents. An inlet flow velocity of
12.5 mm·s-1 lead to an average bead velocity of 1.6
mm·s-1. The sample transition time is approximately 1
minute. Compared to a reference extraction in a test tube,
25% of DNA could be detected by Real-Time PCR.
However, we expect significant improvements in the
recovery rate and purity by optimizing the magnet motion
and fluid resistance at each section of the microfluidic
structure.
Applications of this concept for the extraction and
purification of biomolecules are the monitoring of cell
growth in fermenters or continuous safety monitoring of
drinking water against biological contaminations. Other
potential applications are in the field of continuous protein
purification, immuno- and cell based assays or other
biological assays that are compatible with target-binding
to magnetic beads.

ACKNOWLEDGEMENT
The present study is funded by the German Federal
Ministry of Education and Research (16SV3528).

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