Avery Sonnenberg

Dielectrophoretic isolation of DNA and nanoparticles from blood

The ability to effectively detect disease‐related DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for viable diagnostics and therapy monitoring. A DEP method has been developed which allows the rapid isolation, concentration and detection of DNA and nanoparticles directly from human and rat whole blood. Using a microarray device operating at 20 V peak‐to‐peak and 10 kHz, a wide range of high molecular weight (HMW)‐DNA and nanoparticles were concentrated into high‐field regions by positive DEP, while the blood cells were concentrated into the low‐field regions by negative DEP. A simple fluidic wash removes the blood cells while the DNA and nanoparticles remain concentrated in the DEP high‐field regions where they can be detected by fluorescence. HMW‐DNA could be detected at 260 ng/mL, which is a detection level suitable for analysis of disease‐related cell‐free circulating DNA biomarkers. Fluorescent 40 nm nanoparticles could be detected at 9.5 × 109 particles/mL, which is a level suitable for monitoring drug delivery nanoparticles. The ability to rapidly isolate and detect DNA biomarkers and nanoparticles from undiluted whole blood will benefit many diagnostic applications by significantly reducing sample preparation time and complexity.

Dielectrophoretic Isolation and Detection of cfc-DNA Nanoparticulate Biomarkers and Virus from Blood

Dielectrophoretic (DEP) microarray devices allow important cellular nanoparticulate biomarkers and virus to be rapidly isolated, concentrated, and detected directly from clinical and biological samples. A variety of submicron nanoparticulate entities including cell free circulating (cfc) DNA, mitochondria, and virus can be isolated into DEP high‐field areas on microelectrodes, while blood cells and other micron‐size entities become isolated into DEP low‐field areas between the microelectrodes. The nanoparticulate entities are held in the DEP high‐field areas while cells are washed away along with proteins and other small molecules that are not affected by the DEP electric fields. DEP carried out on 20 μL of whole blood obtained from chronic lymphocytic leukemia patients showed a considerable amount of SYBR Green stained DNA fluorescent material concentrated in the DEP high‐field regions. Whole blood obtained from healthy individuals showed little or no fluorescent DNA materials in the DEP high‐field regions. Fluorescent T7 bacteriophage virus could be isolated directly from blood samples, and fluorescently stained mitochondria could be isolated from biological buffer samples. Using newer DEP microarray devices, high‐molecular‐weight DNA could be isolated from serum and detected at levels as low as 8–16 ng/mL.

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma

Conventional methods for the isolation of cancer‐related circulating cell‐free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf‐DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf‐DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf‐DNA from both blood and plasma samples was separated into DEP high‐field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf‐DNA was detected on‐chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf‐DNA from the equivalent of 5 μL of CLL blood and 5 μL of plasma was amplified by PCR using Ig heavy‐chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B‐cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard).

Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology

The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications.

Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays

Common epifluorescent microscopy lacks the sensitivity to detect low levels of analytes directly in clinical samples, such as drug delivery nanoparticles or disease related DNA biomarkers. Advanced systems such as confocal microscopes may improve detection, but several factors limit their applications. This study now demonstrates that combining an epifluorescent microscope with a dielectrophoretic (DEP) microelectrode array device enables the detection of nanoparticles and DNA biomarkers at clinically relevant levels. Using DEP microarray devices, nanoparticles and DNA biomarkers are rapidly isolated and concentrated onto specific microscopic locations where they are easily detected by epifluorescent microscopy. In this study, 40nm nanoparticles were detected down to 2-3 × 10(3) /ul levels and DNA was detected down to the 200 pg/ml level. The synergy of epifluorescent microscopy and DEP microarray devices provides a new paradigm for DNA biomarker diagnostics and the monitoring of drug delivery nanoparticle concentrations.

Device for dielectrophoretic separation and collection of nanoparticles and DNA under high conductance conditions

Most dielectrophoretic (DEP) separations of cells, nanoparticles, and other entities are carried out on microelectrode arrays or in microfluidic device formats. Less work has been directed at designing pipette‐type formats that would allow dipping into and recovering specific analytes from samples in microtiter plate formats. In order to address this important area, we have fabricated micropipette tip devices containing a 2% agarose gel plug, a buffer chamber, and platinum electrode as the DEP collection device, to be used in combination with separate sample wells that contain a circular gold electrode. We demonstrated that 200 nm fluorescent nanoparticles could be isolated into DEP high‐field regions and separated from 10 μm fluorescent microbeads in high conductance buffer (1× PBS) by applying an alternating current at 10 kHz with a peak‐to‐peak voltage (Vpp) of 160 Vpp. The collected nanoparticles were then transferred to a new buffer solution. We also demonstrated the DEP isolation and separation of genomic DNA (>50 kbps) from the 10 μm microbeads in high conductance buffer (1× PBS) with transfer of collected DNA to another solution.

Rapid detection of cancer related DNA nanoparticulate biomarkers and nanoparticles in whole blood

The ability to rapidly detect cell free circulating (cfc) DNA, cfc-RNA, exosomes and other nanoparticulate disease biomarkers as well as drug delivery nanoparticles directly in blood is a major challenge for nanomedicine. We now show that microarray and new high voltage dielectrophoretic (DEP) devices can be used to rapidly isolate and detect cfc-DNA nanoparticulates and nanoparticles directly from whole blood and other high conductance samples (plasma, serum, urine, etc.). At DEP frequencies of 5kHz-10kHz both fluorescent-stained high molecular weight (hmw) DNA, cfc-DNA and fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP highfield regions over the microelectrodes, while blood cells move to the DEP low field-regions. The blood cells can then be removed by a simple fluidic wash while the DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260ng/ml and the nanoparticles at <9.5 x 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Disease specific cfc-DNA materials could also be detected directly in blood from patients with Chronic Lymphocytic Leukemia (CLL) and confirmed by PCR genotyping analysis.

Nanoparticles: Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology (Small 38/2015).

M. J. Heller and co-workers describe the use of electrode arrays to collect select nanoparticles from a complex mixture. On page 5088, they explain how their dielectrophoresis collection chip pulls the desired nanoparticles down to the electrode surface, allowing them to be collected from complex fluid mixtures such as biological blood samples. Recovering nanoparticles is a major challenge in many fields, so this technique is expected to find broad interest across the nanoparticle community.

Abstract 1514: An AC electrokinetic microarray device for isolation of cell circulating free nucleic acid from the blood of cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Circulating cell-free (ccf) DNA is becoming a biomarker of interest for cancer diagnosis and monitoring. The isolation of ccf-DNA from plasma may be used as a “liquid biopsy” and could replace more invasive tissue biopsies for analysis of cancer-related mutations. Conventional methods for isolation of ccf-DNA from plasma are costly, time-consuming, and require a significant amount of sample processing, which could result in loss of important biomarkers. An AC electrokinetic microarray device has been developed to reduce the time and cost needed for sample processing. In an earlier study, this microarray device was used to rapidly (15 minutes) isolate ccf-DNA from 25 μL of unprocessed blood from 15 chronic lymphocytic leukemia (CLL) patients to compare PCR and sequencing results with classical ccf-DNA isolation processes and gold standard isolation of DNA directly from lymphocytes. Our objective now is to determine if there is a correlation between the on-chip fluorescence imaging data of the ccf-DNA with the lymphocyte cell counts. Blood was collected from CLL patients in lithium heparin collection tubes. SYBR Green was added to the CLL blood sample to label the ds-DNA. A 25 μL aliquot of the CLL blood was inserted into the AC electrokinetic microarray device. A 10 kHz sinusoidal waveform was applied at 11 volts peak-to-peak for three minutes without fluid flow. With the field still on, the chip was washed for five minutes with 1x TE at a rate of 100 µL/min to remove other blood components. Once the wash was complete, a fluorescence image was taken for analysis. The white blood cell counts and % lymphocyte data were used to calculate the concentration of lymphocytes in each patient sample, and the fluorescence images were analyzed for average fluorescence in a defined region of interest. The preliminary data shows some correlation between the number of lymphocytes in the patient blood and the fluorescence calculated from the images. It is important to note that while the sample size was small, the data shows promise and calls for a larger study. Citation Format: Jennifer Marciniak, Avery Sonnenberg, Laura Rassenti, Emanuela Ghia, George Widhopf, Elaine Skowronski, Sareh Manouchehri, Thomas J. Kipps, Michael J. Heller, David J. Charlot, Rajaram Krishnan. An AC electrokinetic microarray device for isolation of cell circulating free nucleic acid from the blood of cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2014-1514