Do Circulating Tumor Cells, Exosomes, and Circulating Tumor Nucleic Acids Have Clinical Utility?

ABSTRACT: Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of CTCs and circulating tumor DNA (ctDNA) are likely to improve cancer diagnosis, treatment, and minimal residual disease monitoring. However, more trials are required to validate the clinical utility of precise molecular markers for a variety of tumor types. This review focuses on the clinical utility of CTCs and ctDNA testing in patients with solid tumors, including somatic and epigenetic alterations that can be detected. A comparison of methods used to isolate and detect CTCs and some of the intricacies of the characterization of the ctDNA are also provided.

Do Circulating Tumor Cells, Exosomes, and Circulating Tumor Nucleic Acids Have Clinical Utility? Gold, Bert et al. The Journal of Molecular Diagnostics , Volume 17 , Issue 3 , 209 - 224

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

This study demonstrates the potential clinical relevance of this Biological Dynamics technologyor many future clinical diagnostic applications, including to virus and other pathogens detection. The authors showed rapid isolation and detection of SYBR Green stained cell-free DNA from 20 µL whole blood samples from Chronic Lymphocytic Leukemia (CLL) patients.

Overall the results of this study support the enormous potential of DEP as a “seamless sample-to-answer” technique for the rapid detection of cfc-DNA and nanoparticulate biomarkers directly from blood and other complex biological samples.

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.

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Publication Info: Electrophoresis. 2013 Apr; 34(7): 1076–1084. doi:  10.1002/elps.201200444

Dielectrophoretic Isolation of DNA and Nanoparticles From Blood

In this article, the authors used Biological Dynamics technology to isolate, concentrate and detect DNA and nanoparticles directly from human and rat whole blood. This ability to work directly with high conductivity solutions dramatically shortens and simplifies sample preparation for many diagnostic applications.

Sonnenberg, A., Marciniak, J. Y., Krishnan, R. and Heller, M. J. (2012), Dielectrophoretic Isolation of DNA and Nanoparticles From Blood. ELECTROPHORESIS, 33: 2482–2490. doi: 10.1002/elps.201100700

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ABSTRACT

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.

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

BIological Dynamic founders contribute a chapter to the 6th CNAPS book that summarizes proceedings of this international conference held on 9-11 November 2009 in Hong Kong.

Circulating Nucleic Acids in Plasma and Serum by Gahan, Peter B. R 10.1007/978-90-481-9382-0_3 Rapid Isolation and Detection of Cell Free Circulating DNA and Other Disease Biomarkers Directly from Whole Blood Springer Netherlands 8 2011-01-01 Krishnan, Rajaram, Heller, Michael J. 247-257 English

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ABSTRACT

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 × 10^9 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.

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Rapid Detection of Cancer-DNA Biomarkers and Nanoparticles

This is one of the early articles by founders of Biological Dynamics, where they demonstrated the feasibility of isolation  hmw-DNA and fluorescent nanoparticles directly from blood using AC electric fields to manipulate cells and nanoparticles on DEP device. 

Rapid detection of Cancer-DNA Biomarkers and Nanoparticles, Michael Heller, Raj Krishnan and Avery Sonnenberg. Biomedical Optics & Medical Imaging 19 August 2010, SPIE Newsroom. DOI: 10.1117/2.1201007.003153

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Interaction of nanoparticles at the DEP microelectrode interface under high conductance conditions

This was a first work, where Biological Dynamic's founders were able to demonstrate possibility of isolation of nanoparticles from a high conductivity solution using hydrogel over-coated microelectrodes.

Krishnan, R., Dehlinger, D.A., Gemmen, G.J., Mifflin, R.L., Esener, S.C., and Heller, M.J. (2009), Interaction of nanoparticles at the DEP microelectrode interface under high conductance conditions. Electrochem commun., 11: 1661–1666. doi: 10.1016/j.elecom.2009.06.033

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ABSTRACT

The separation of nanoparticles from micron size particles in high conductance buffers was achieved using an AC dielectrophoretic (DEP) microarray device with hydrogel over-coated microelectrodes. While nanoparticles could be selectively concentrated into high field regions directly over the platinum microelectrodes, micro-bubbling and electrode darkening was also observed. For similar experiments using un-coated microelectrodes, SEM analysis showed severe erosion of the platinum microelectrodes and fusion of nanoparticles due to the aggressive electrochemistry.

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An AC Electrokinetic Method for Enhanced Detection of DNA Nanoparticles

This was a first work, where Biological Dynamic's founder were able to demonstrate possibility of no-dilution isolation of nanoparticles from a high conductivity solution using special conditions applied to DEP field.

Krishnan, R. and Heller, M. J. (2009), An AC Electrokinetic Method for Enhanced Detection of DNA Nanoparticles. J. Biophoton., 2: 253–261. doi: 10.1002/jbio.200910007

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ABSTRACT

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.

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

This was the first article that proved that AC electrokinetics could be used to isolate nanoparticles in high conductivity solutions. 

Krishnan, R., Sullivan, B. D., Mifflin, R. L., Esener, S. C. and Heller, M. J. (2008), Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions. ELECTROPHORESIS, 29: 1765–1774. doi: 10.1002/elps.200800037

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ABSTRACT

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-microm 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 1xPBS 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.

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