Dielectrophoretic isolation and detection of cfc‐<scp>DNA</scp> nanoparticulate biomarkers and virus from blood

Sonnenberg, Avery; Marciniak, Jennifer Y.; McCanna, James P.; Krishnan, Rajaram; Rassenti, Laura Z.; Kipps, Thomas J.; Heller, Michael

doi:10.1002/elps.201200444

Cited by 70 publications

(63 citation statements)

References 63 publications

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“…The adaptation of new AC electrokinetic dielectrophoretic devices for isolating cancer-related cf-nucleic acid biomarkers represents a promising approach that overcomes many of the limitations of conventional sample preparation methods [31, 101,119]. DEP is the movement of particles suspended in a nonuniform electric field.…”

Section: Dep Isolation Of Cancer Biomarkersmentioning

confidence: 99%

Detecting cancer biomarkers in blood: challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids

Lewis

Heineck

Heller

2015

Expert Review of Molecular Diagnostics

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As we move into the era of individualized cancer treatment, the need for more sophisticated cancer diagnostics has emerged. Cell-free (cf) nucleic acids (cf-DNA, cf-RNA) and other cellular nanoparticulates are now considered important and selective biomarkers. There is great hope that blood-borne cf-nucleic acids can be used for 'liquid biopsies', replacing more invasive tissue biopsies to analyze cancer mutations and monitor therapy. Conventional techniques for cf-nucleic acid biomarker isolation from blood are generally time-consuming, complicated and expensive. They require relatively large blood samples, which must be processed to serum or plasma before isolation of biomarkers can proceed. Such cumbersome sample preparation also limits the widespread use of powerful, downstream genomic analyses, including PCR and DNA sequencing. These limitations also preclude rapid, point-of-care diagnostic applications. Thus, new technologies that allow rapid isolation of biomarkers directly from blood will permit seamless sample-to-answer solutions that enable next-generation point-of-care molecular diagnostics.

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Section: Dep Isolation Of Cancer Biomarkersmentioning

confidence: 99%

Detecting cancer biomarkers in blood: challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids

Lewis

Heineck

Heller

2015

Expert Review of Molecular Diagnostics

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“…The use of DEP under high conductance conditions has now been clearly demonstrated for separations of nanoparticles and high molecular weight (HMW) DNA [35] and cancer-related cell free circulating (cfc) DNA and virus [36] directly from un-diluted whole blood. The design of new DEP systems that complement the common epifluorescent microscope will result in improved assays and diagnostics for detecting very low levels of DNA biomarkers, nanoparticles and virus in both clinical and environmental samples.…”

Section: Biophotonicsmentioning

confidence: 99%

“…The separation of polystyrene nanoparticles in high conductance buffers and blood has been demonstrated through prior work [33][34][35][36], but cannot be reliably repeated in blood for lower levels. The nanoparticles used in this research are coated with carboxylate groups, which increase the surface potential and provide a net negative charge.…”

Section: Separation Of Dna From Whole Bloodmentioning

confidence: 99%

Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays

McCanna¹,

Sonnenberg²,

Heller³

2013

Journal of Biophotonics

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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 × 103/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. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…However, the isolation of ccf-DNA in these studies required long and involved processes. We have previously demonstrated rapid semiquantitative fluorescence detection results for the DEP isolation of DNA spiked into serum that spanned 8 to 500 ng/mL, which would be a useful dynamic range for measuring ccf-DNA in clinical samples (37). …”

Section: Discussionmentioning

confidence: 99%

“…The devices still could not be used with whole blood samples and, more importantly, did not provide isolation of DNA from the sample. We have developed an electrokinetic technique that allows nanoscale entities, including high molecular weight DNA and nanoparticles, to be isolated from high-conductance (>10 mS/cm) solutions (33–35) and whole blood samples (36), and ccf-DNA from blood samples (37). In this study, we show fluorescent analysis, PCR, and Sanger sequencing results for ccf-DNA isolated by DEP from 25-μL samples of unprocessed CLL patient blood.…”

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confidence: 99%

Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood

Sonnenberg¹,

Marciniak²,

Rassenti³

et al. 2014

Clinical Chemistry

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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.

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Dielectrophoretic isolation and detection of cfc‐DNA nanoparticulate biomarkers and virus from blood

Cited by 70 publications

References 63 publications

Detecting cancer biomarkers in blood: challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids

Detecting cancer biomarkers in blood: challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids

Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays

Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood

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