(481g) DEP Isolation of Cancer Related Circulating Cell Free (CCF) DNA Biomarkers Directly From Blood

AIChE 2013

Topical 3: 2013Annual Meeting of the
American Electrophoresis Society

?T3006 ? ?Electrokinetics for Sample
preparation?

DEP
Isolation of Cancer Related Circulating Cell Free (CCF) DNA Biomarkers Directly
from Blood

Michael J. Heller^1,2,*Avery Sonnenberg¹, Jennifer Y. Marciniak¹,
Laura Rassenti³, Emanuela Ghia³, Elaine Skowronski¹,
Sareh Manouchehri¹, George Widhopf³ and Thomas J. Kipps³

University of California San Diego, Department of
Bioengineering¹, Department of Nanoengineering², and UCSD
Moores Cancer Center³, La Jolla, CA, USA 92093-0448

Abstract ? Circulating
cell free (ccf) DNA/RNA biomarkers are now widely used for cancer diagnostics
and management. Unfortunately, the isolation of ccf-DNA/RNA directly from blood
has not been possible, and its isolation from plasma is a long and involved
process. We report on an AC electrokinetic approach for the rapid isolation of
ccf-DNA directly from a small volume of un-processed blood. A dielectrophoretic
(DEP) microarray device (chip) was used to isolate ccf-DNA directly from fresh
blood samples of fifteen Chronic Lymphocytic Leukemia (CLL) patients and three
healthy individuals. Ccf-DNA/RNA from 25uls (a drop) of blood was separated and
concentrated into DEP high-field regions in about three minutes, while blood
cells, proteins and other biomolecules were removed by a fluidic wash.
Concentrated ccf-DNA (SYBR-Green stained) was detected by fluorescence and then
eluted for PCR and DNA sequencing. The complete process, blood sample to
ccf-DNA for PCR, was completed in less than 10 minutes. Eluted ccf-DNA, from
5ul of the original CLL blood sample, was amplified by PCR using IGHV-specific
primers to identify the unique IGHV gene expressed by the leukemic B-cell clone
(ie the major IGHV subgroups expressed by CLL B cells IGHV1, IGHV3, IGHV4). The
PCR results obtained by DEP from CLL blood were comparable to results obtained
using conventional sample preparation of ccf-DNA which starts with one ml of
plasma. The DNA sequencing results from the PCR amplified cff-DNA obtained by
DEP correlated with earlier sequencing results from genomic DNA isolated from
the CLL patient's leukemic B cells. The ability of DEP to provide rapid
isolation and detection of ccf-DNA from the equivalent of a drop of blood
represents a major step forward in the quest for ?liquid biopsy? and point of
care (POC) cancer diagnostics and patient monitoring.   

Introduction - AC
electrokinetic methods like dielectrophoresis (DEP) are well-known techniques
for achieving separations of cells, nanoparticles, and biomolecules. However,
until recently DEP technology had remained impractical for general use with
biological/clinical samples (blood, plasma, serum) and in high conductance buffer
solutions (>1mS/cm). For example, earlier DEP work on separating bacteria
from blood (~7-9 mS/cm) required a 50-fold dilution of the blood sample before
the DEP separation process could be carried out [1]. More recently, we have
developed new DEP devices and methods that allow hmw-DNA, nanoparticles,
bacteriophage and circulating cell free (ccf) DNA to be isolated and detected
from both high ionic strength buffers and blood [2-6]. Using special microarrays
with robust platinum microelectrodes over-coated with novel hydrogel layers
(designed and fabricated by Biological Dynamics, La Jolla, CA) we are now able to
demonstrate the rapid isolation, fluorescent detection and subsequent PCR
amplification and DNA sequencing of cancer related ccf-DNA directly from a 25
ul sample of un-processed blood from Chronic Lymphocytic Leukemia (CLL) patients.

Results: Figure 1 (below) shows the
electrokinetic DEP device (Biological Dynamics) used for the separation of
ccf-DNA from CLL patient blood. The DEP microarray device/chip is a 10 x 12 mm
silicon die patterned structure with an interdigitated array of 1000 (60 µm
dia.) platinum microelectrodes on 160 um center-center pitch. The microarray is
over-coated with a 200nm-500nm porous hydrogel layer. A printed circuit board
is attached to the chip using electrically conductive pressure sensitive
adhesive which provides electrical contact to standard pogo pin connectors. The
PCB cutout forms the sides of the flow cell and an acrylic window
with fluidic input and output ports forms the top cover, resulting in
a 20 ul flow cell volume.  Typically, ~25 ul of the sample would be
aspirated to completely fill the flow cell and the fluidic ports. For the
actual process, the chip was first pre-treated with approximately 25 ul of 0.5X
PBS and run at 2Vrms, 5Hz sine waveform for 15 second to improve the hydrogel
porosity. The fluid was aspirated and 25 ul of undiluted sample (blood) was
then applied to the chip flow cell and run at 12 Volts peak to peak (Vp-p)
at 10 kHz (sine wave) for 3 minutes.

Figure 1 ? Electrokinetic DEP
microarray chip device designed and fabricated by Biological Dynamics for
carrying out separations in high conductance buffers and clinical samples
(blood, plasma, serum, etc.)

A
checker board field geometry is formed by the AC bias with the positive DEP high-field
regions occurring on the microelectrodes, and negative DEP low-field regions
occurring between microelectrodes. With the AC voltage ON, the flow cell
was washed with TE buffer for 5 minutes at 100 ul/min flow rate.  After
the wash step, the power was turned OFF allowing the captured DNA to diffuse off
the hydrogel. The sample was then aspirated from the flow cell and stored at 4^o
C in a microcentrifuge tube. In order to visualize real time capture on the
microelectrode array, the CLL and normal blood samples were treated with 5x
SYBR Green I fluorescent dye which stains the double stranded DNA. Bright
field and fluorescent images of the microelectrode pads were then acquired using
a CCD camera with a 10x objective, FITC fluorescent filters, and a 470nm LED
excitation source. Figure 2 shows representative fluorescent images of the SYBR
Green stained ccf-DNA from one of the normal blood samples along with two CLL patient
blood samples. MATLAB was used to create 3D fluorescent intensity images which
provide better quantification of the relative amount of ccf-DNA that has been
concentrated onto the microelectrodes by the DEP process. In general, fluorescent
DNA levels were higher in all 15 CLL patient samples than in the normal blood
samples; and in most cases they were significantly higher. The concentration of
ccf-DNA in the eluted samples (25ul) was also determined by PicoGreen fluorescent
analysis. Again the fluorescent DNA levels were higher in all 15 CLL patient samples
than in the normal blood samples, and in most cases the concentrations were
significantly higher.  Subsequent PCR amplification
for ccf-DNA (equivalent to 5ul blood) isolated by DEP from all fifteen CLL
blood samples compared well with the results obtained from ccf-DNA isolated by the
Qiagen process starting with 1ml of CLL patient plasma. Finally, DNA sequencing
carried out on the PCR amplified ccf-DNA isolated by DEP correlated very well
for all 15 CLL patient samples with sequencing results (Gold Standard) from
genomic DNA obtained from the patients B-lymphocyte cells which was isolated
from 5 ml of blood. 

Figure 2 - Fluorescent images of
the SYBR green stained ccf-DNA from a normal blood sample and two CLL patient
blood samples. Also shown (far left) are 3D fluorescent intensity images
created by MATLAB.

Discussion and Conclusions

The
use of circulating cell-free (ccf) DNA biomarkers for ?liquid biopsy? will unquestionably be important for future point of
care (POC) cancer diagnostics and management. Unfortunately, the time and
process complexity required for the isolation of ccf-DNA from plasma by
conventional methods is a major limitation for developing POC diagnostic
formats. Additionally, these processes add significant cost to the assays which
will also limit their use. We have now shown that an AC electrokinetic DEP
microarray device can be used to isolate ccf-DNA directly from CLL patient
blood in less than 10 minutes. The DEP process is not only significantly faster
than the conventional methods, but requires only three steps. We have also
shown that DEP allows ccf-DNA to be isolated from a relatively small sample
volume of only 25 uls, which is equivalent to a drop of blood.

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