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.

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 electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood.

BACKGROUND: Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS: We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 µL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS: PCR and DNA sequencing results obtained by DEP from 25 µL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. CONCLUSIONS: Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

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).

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 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 × 10(9) 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.

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.