Rajaram Krishnan

Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions

In biomedical research and diagnostics, it is a significant challenge to directly isolate and identify rare cells and potential biomarkers in blood, plasma and other clinical samples. Additionally, the advent of bionanotechnology is leading to numerous drug delivery approaches that involve encapsulation of drugs and imaging agents within nanoparticles, which now will also have to be identified and separated from blood and plasma. Alternating current (AC) electrokinetic techniques such as dielectrophoresis (DEP) offer a particularly attractive mechanism for the separation of cells and nanoparticles. Unfortunately, present DEP techniques require the dilution of blood/plasma, thus making the technology less suitable for clinical sample preparation. Using array devices with microelectrodes over‐coated with porous hydrogel layers, AC electric field conditions have been found which allow the separation of DNA nanoparticles to be achieved under high‐conductance (ionic strength) conditions. At AC frequencies in the 3000 Hz to 10 000 Hz range and 10 volts peak‐to‐peak, the separation of 10‐µm polystyrene particles into low field regions, and 60‐nm DNA‐derivatized nanoparticles and 200‐nm nanoparticles into high‐field regions was carried out in 149 mM 1×PBS buffer (1.68 S/m). These results may allow AC electrokinetic systems to be developed that can be used with clinically relevant samples under physiological conditions.

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.

An AC electrokinetic method for enhanced detection of DNA nanoparticles.

In biomedical research and diagnostics it is a challenge to isolate and detect low levels of nanoparticles and nanoscale biomarkers in blood and other biological samples. While highly sensitive epifluorescent microscope systems are available for ultra low level detection, the isolation of the specific entities from large sample volumes is often the bigger limitation. AC electrokinetic techniques like dielectrophoresis (DEP) offer an attractive mechanism for specifically concentrating nanoparticles into microscopic locations. Unfortunately, DEP requires significant sample dilution thus making the technology unsuitable for biological applications. Using a microelectrode array device, special conditions have been found for the separation of hmw-DNA and nanoparticles under high conductance (ionic strength) conditions. At AC frequencies in the 3000-10 000 Hz range, 10 mum microspheres and human T lymphocytes can be isolated into the DEP low field regions, while hmw-DNA and nanoparticles can be concentrated into microscopic high field regions for subsequent detection using an epifluorescent system.

Rapid Isolation and Detection of Cell Free Circulating DNA and Other Disease Biomarkers Directly from Whole Blood

The ability to rapidly detect cell free circulating (cfc) DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for early disease detection and nanomedicine. We now show that a microarray dielectrophoretic (DEP) device can be used to rapidly isolate and detect high molecular weight (hmw) DNA nanoparticulates and nanoparticles directly from whole blood. At DEP frequencies of 5–10 kHz both fluorescent-stained hmw-DNA and 40 nm fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP high field 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 hmw-DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260 ng/ml, and the nanoparticles at <9.5 × 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Some initial work now indicates the presence of possible cfc-DNA in CLL patient blood samples.

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

Abstract 1704: AC electrokinetic isolation of cell free high molecular weight DNA (CF-HMW DNA) from serum

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Recent reports of CF-HMW DNA in a variety of diseases has led to interest in its use as a prognostic biomarker, a diagnostic or screening tool for cancer. Apoptotic DNA ( 500bp) that is then trafficked into circulation. Dielectrophoresis, or DEP (movement of particles in a non-uniform AC electric field), based electrokinetic techniques have been used for protein biomarker screening, cell separation, and DNA manipulation. Using specific DEP frequencies, analytes are moved to known locations (high or low field region) based on their inherent dielectric properties. The process allows specific nano-sized particles to be highly concentrated into high field regions, while micron-size cells are held weakly in low field regions. With fluidic washing, cells can be removed while nano-sized particles are held in high field regions. This method can effectively act as a CF-HMW DNA enrichment process for biological fluids. Unfortunately, DEP does not work well in (high conductance) physiological buffers, requiring significant dilution of the sample and, as a result, the target analyte concentration. Here, we describe a novel AC electrokinetic (ACE) technique using a custom microelectrode array and AC parameters to isolate and analyze CF-HMW DNA from high conductance serum. DNA from normal serum samples and the A549 cell line were isolated using Qiagen kits to determine initial DNA quantities. Normal sera were spiked with A549 cell line DNA at concentrations from 0 to 500 ng/ml and subjected to a 20 minute ACE protocol. DNA isolation was visualized in situ using SYBR Green I fluorescence and then eluted for use in secondary analyses. Results showed minimal fluorescence from samples without spike whereas A549 DNA spiked samples showed concentration-dependent fluorescence on the microelectrodes. After elution, levels of DNA were quantitated using ALU-sequence based qPCR and K-ras mutational status was confirmed by ABI CastPCR. Future experiments will compare CF-HMW DNA levels from normal and cancer patients across a variety of solid tumors. The ACE technique enables rapid isolation and detection of CF-HMW DNA directly from biological fluids without prior sample prep and has broad utility for diagnosis and monitoring of cancer and other diseases in which CF-HMW DNA may be used as a marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1704. doi:1538-7445.AM2012-1704