Label-free Capture Research Articles

The Shape Effect of Acoustic Micropillar Array Chips in Flexible Label-Free Separation of Cancer Cells.

The precise isolation of circulating tumor cells (CTCs) from blood samples is a potent tool for cancer diagnosis and clinical prognosis. However, CTCs are present in extremely low quantities in the bloodstream, posing a significant challenge to their isolation. In this study, we propose a non-contact acoustic micropillar array (AMPA) chip based on acoustic streaming for the flexible, label-free capture of cancer cells. Three shapes of micropillar array chips (circular, rhombus, and square) were fabricated. The acoustic streaming characteristics generated by the vibration of microstructures of different shapes are studied in depth by combining simulation and experiment. The critical parameters (voltage and flow rate) of the device were systematically investigated using microparticle experiments to optimize capture performance. Subsequently, the capture efficiencies of the three micropillar structures were experimentally evaluated using mouse whole blood samples containing cancer cells. The experimental results revealed that the rhombus microstructure was selected as the optimal shape, demonstrating high capture efficiency (93%) and cell activity (96%). Moreover, the reversibility of the acoustic streaming was harnessed for the flexible release and capture of cancer cells, facilitating optical detection and analysis. This work holds promise for applications in monitoring cancer metastasis, bio-detection, and beyond.

A dual-blocker aided and dual-label-free electrochemical biosensor based on mbHCR/rGO nanocomplexes for ultrasensitive DNA detection

Heterogeneous electrochemical DNA biosensors have attracted huge attention due to their enhanced signal sensitivity, compared to homogeneous biosensors. However, the high cost of probe labeling and the reduced recognition efficiency associated with current heterogeneous electrochemical biosensors confine their potential applications. In the present work, a dual-blocker assisted and dual-label-free heterogeneous electrochemical strategy based on multi-branched hybridization chain reaction (mbHCR) and reduced graphene oxide (rGO) was fabricated for ultrasensitive detection of DNA. The target DNA could trigger the mbHCR of two DNA hairpin probes, resulting in the generation of multi-branched long chain of DNA duplexes with bidirectional arms. One direction of the multi-branched arms in the mbHCR products were then bound to the label-free capture probe on the gold electrode through multivalent hybridization with enhanced recognition efficiency. The other direction of multi-branched arms in mbHCR product could adsorb rGO via π-π stacking interactions. Two DNA blockers were ingeniously designed to block the binding of excessive H1-pAT on electrode and to prevent the adsorption of rGO by residual unbound capture probes. As a result, with the electrochemical reporter methylene blue selectively intercalated into the long chain of DNA duplex and absorbed on rGO, a remarkable electrochemical signal rise was observed. Thus, a dual-blocker aided and dual-label-free electrochemical strategy for ultrasensitive DNA detection is readily realized with the merit of cost-effective. The as-developed dual-label-free electrochemical biosensor has great potential to be employed in nucleic acid related medical diagnostics.

Engineered multifunctional metal-phenolic nanocoatings for label-free capture and "self-release" of heterogeneous circulating tumor cells.

Immunomagnetic beads have been widely explored as an important analytical tool for the rapid and sensitive detection of circulating tumor cells (CTCs). However, their clinical application is seriously hindered by the tedious preparation procedures and heterogeneous nature of CTCs. To this end, a designed multifunctional platform named Fe3O4@TA/CuII superparamagnetic nanoparticles (SPMNPs) is expected to have the following features: (i) the formation of a tannic acid-copper (II) ion (TA/CuII) coating which could be accomplished by a one-step method is very simple; (ii) the TA/CuII coating shows high affinity for heterogeneous CTCs and good resistance to nonspecific adhesion of blood cells; (iii) "self-release" of the captured cells could be achieved as the TA/CuII coating gradually degrades in the cell culture environment without any additional interventions. Therefore, the resulting Fe3O4@TA/CuII SPMNPs could capture various CTCs (MCF-7, HepG2 and HeLa cells) with different expression levels of the epithelial cell adhesion molecule (EpCAM). And the capture efficiency and cell purity can reach 88% and 87%, respectively. In addition, 68% of the captured cells are self-released after 6 h of incubation and most of the released cells show high cell proliferation activity. In particular, Fe3O4@TA/CuII SPMNPs can successfully detect 1-13 CTCs from 1 mL of blood of 14 patients with 6 types of cancers. Hence, we expect that the as-prepared Fe3O4@TA/CuII SPMNPs with simple, efficient, and universal yet cost-efficient characteristics could act as a promising analytical tool for clinical CTC detection.

Biomimetic microcavity interfaces for a label-free capture of pathogens in the fluid bloodstream by vortical crossflow filtration.

Bacterial sepsis is a lethal disease triggered by microbial pathogens. The blood pathogen load is a major contributor to both disease severity and mortality in patients with sepsis blood. Therefore, it is crucial to reduce the load of pathogens, in particular the drug-resistant pathogens. In this work, inspired by the crossflow filtration mechanism in suspension-feeding fish, we developed a biomimetic microcavity interface to mimic a porous gill-raker surface as a blood-cleansing dialyzer for sepsis therapy, which can rapidly, safely and efficiently clear bacteria from the fluidic blood. The microcavity interface consists of microcavity arrays, the innerface of which contains nanowire forests. By precisely controlling the pore size of the microcavity and directing the axial travel of the fluid, the bacteria can be isolated from the whole blood without disturbing any blood components or blocking the blood cell transportation. In addition, the three-dimensional nanowire forests assist in the formation of vortices with reduced blood flow velocity and increased resistance to bacterial deposition in situ. Functional modification is not required to recognize the bacteria specifically in our designed dialyzer. Moreover, the microcavity interface clears over 95% bacteria from a fluid blood sample without inducing protein adsorption or complement and platelet activation when contacting the fluid blood. The study supports this biomimetic microcavity interface to be a promising extracorporeal blood-cleansing device in clinical settings.

Natural Fish Trap-Like Nanocage for Label-Free Capture of Circulating Tumor Cells.

Nanomaterials have achieved several breakthroughs in the capture of circulating tumor cells (CTCs) over the past decades. However, artificial fabrication of label‐free nanomaterials used for high‐efficiency CTC capture is still a challenge. Through billions of years of evolution and natural selection, various complicated and precise hierarchical structures are developed. Here, a novel fish trap‐like “nanocage” structure derived from the natural Chrysanthemum pollen is reported and a nanocage‐featured film for the label‐free capture of CTCs and CTC clusters is constructed. The nanocage‐featured film effectively captures 92% rare cancer cells with a broad spectrum of cancer types, due to the synergistic effect of nanocage‐CTC filopodia matching, high contact area, and strong adhesion force between the cancer cells and the nanocage. Furthermore, the nanocage‐featured film successfully detects CTCs and CTC clusters in 2 or 4 mL blood taken from 21 cancer patients (stages I–IV) suffering from various types of cancers. This novel, abundant, and economical fish trap‐like “nanocage” may provide new perspectives for the application of natural nanomaterials in clinical CTC capture and analysis.

Rapid Size-Based Isolation of Extracellular Vesicles by Three-Dimensional Carbon Nanotube Arrays.

Recent discoveries reveal that extracellular vesicles (EVs) play an important role in transmitting signals. Although this emerging transcellular pathway enables a better understanding of neural communication, the lack of techniques for effectively isolating EVs impedes their studies. Herein, we report an emergent high-throughput platform consisting of three-dimensional carbon nanotube arrays that rapidly capture different EVs based on their sizes, without any labels. More importantly, this label-free capture maintains the integrity of the EVs when they are excreted from a host cell, thus allowing comprehensive downstream analyses using conventional approaches. To study neural communication, we developed a stamping technique to construct a gradient of nanotube herringbone arrays and integrated them into a microdevice that allowed us processing of a wide range of sample volumes, microliters to milliliters, in several minutes through a syringe via manual hand pushing and without any sample preparation. This microdevice successfully captured and separated EVs excreted from glial cells into subgroups according to their sizes. During capture, this technology preserved the structural integrity and originality of the EVs that enabled us to monitor and follow internalization of EVs of different sizes by neurons and cells. As a proof of concept, our results showed that smaller EVs (∼80 nm in diameter) have a higher uptake efficiency compared to larger EVs (∼300 nm in diameter). In addition, after being internalized, small EVs could enter endoplasmic reticulum and Golgi but not the largest ones. Our platform significantly shortens sample preparation, allows the profiling of the different EVs based on their size, and facilitates the understanding of extracellular communication. Thus, it leads to early diagnostics and the development of novel therapeutics for neurological diseases.

Partial thermal imidization of polyelectrolyte multilayer cell tethering surfaces (TetherChip) enables efficient cell capture and microtentacle fixation for circulating tumor cell analysis.

The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.

Wide subwavelength grating waveguide photonic integrated circuit sensor system

We are studying the viability of a chip-scale integrated photonics based sensor assay array for wearable soldier health monitoring and environmental hazard warning applications. We propose using label-free analyte capture material clad wide subwavelength grating (SWG) waveguides as the sensor elements in a multiplexed array to probe the myriad variety and concentration ranges of chemical hazards and biomarker health molecules expected in these applications. We present here the simulated behavior of both rectangular and tapered wide SWG geometries. We discuss their transmission spectra, effective index, bulk sensitivity, and cladding dependence on waveguide width alongside strip and slot waveguide geometries. Experimental transmission and effective index measurements validating our simulation techniques are also given.

Abstract 1598: EGFR mutational detection in vortex-enriched CTCs, ctDNA, and comparison to tumor tissue in non-small cell lung cancer (NSCLC) patients

Abstract Background Lung cancer is the leading cause of cancer-related mortality worldwide and 85% cases are NSCLC. Epidermal growth factor receptor (EGFR) mutations occur in 10-30% of NSCLC patients1. EGFR tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutation, have shown dramatic clinical benefits. EGFR assays are mainly performed on tumor biopsies, which carry risks and expense and are not always successful1. In order to identify the development of secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKI's and an indication for therapy with a 3rd generation drug, effective and non-invasive monitoring is needed. Liquid biopsies containing circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), allow such monitoring over the course of the therapy2. The Vortex Biosciences' VTX-1 Liquid Biopsy System enables the label-free capture of CTCs from blood samples, with high CTC recovery, purity and compatibility with downstream genomic assays on CTCs and plasma3,4. Our combined CTC and ctDNA EGFR assay was applied to blood samples from NSCLC patients. Method ctEGFR kit (EntroGen) was selected and validated for the detection of EGFR major mutations in a single PCR reaction, for both ctDNA and CTCs. Several blood collection tubes (BCT: EDTA, CellSave, LBGard and Streck) were tested using spiked cells, considering CTC recovery, DNA yield, and EGFR profiling. 20 blood samples were studied from 15 NSCLC patients. Plasma was extracted for ctDNA assay. CTCs were isolated from the plasma-depleted-blood using the VTX-1, immunostained and enumerated5. EGFR mutations were then detected in CTC+ctDNA, and compared to the tissue results. Results The VTX-1 provided a similar CTC recovery from plasma-depleted-blood and whole blood, enabling CTC+ctDNA EGFR profiling from the same tube of blood. Among the BCTs tested, LBGard obtained the best CTC capture and EGFR mutation detection performance. 10/15 patients showed the same mutations between tissue and CTC+ctDNA. For one patient with two blood draws without mutation in tissue, no mutation was detected in the first draw, while a T790M mutation was identified 6 months later. For another patient, an Exon19 deletion was detected in the ctDNA+CTC but not in the tissue, and a repeat draw confirmed the result. 4/15 patients had mutations in tissue that were missed in CTC+ctDNA. Conclusion Performing EGFR mutation analysis on the combination of ctDNA and VTX-1-collected CTCs may offer an improved sensitivity of detection over analysis of only the ctDNA, and will be potentially a useful tool for monitoring treatment and medication guidance of NSCLC patients. [1] Calabuig-Fariñas et al. Transl Lung Cancer Res. 2016. [2] Sundaresan TK et al. Clin Cancer Res. 2016. [3] Kidess-Sigal E et al. Oncotarget 2016. [4] Haiyan E L et al. npj Genomic Medicine (2017) 2:34. [5] Che J. et al., Oncotarget (2016) Citation Format: Elodie Sollier-Christen, Haiyan E. Liu, Meghah Vuppalapaty, Michael Chiu, James Che, Charles Wilkerson, Nasim Barzanian, Steve Crouse, James Carroll, Melissa Matsumoto, Edward B. Garon, Jonathan W. Goldman. EGFR mutational detection in vortex-enriched CTCs, ctDNA, and comparison to tumor tissue in non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1598.

A Chemical Method for Specific Capture of Circulating Tumor Cells Using Label-Free Polyphenol-Functionalized Films

The capture of circulating tumor cells (CTCs) mainly depends on the biological affinity and differences in physical properties between tumor cells and peripheral blood mononuclear cells (PBMCs), but the effective capture of CTCs using only a chemical method is still rare. Inspired by the excellent anti-inflammatory effects of polyphenols, we used polyphenol tannic acid-functionalized (TA-functionalized) film to develop a simple and yet effective chemical strategy for label-free capture of CTCs. The film could be easily prepared within minutes by metal–polyphenol coordination interaction while tactfully integrating the effects of repelling PBMC adhesion and enhancing cancer cell adhesion. The galloyl groups of TA were found to play a crucial role in the enhanced adhesion of cancer cells to the film. As a result of the preference of TA film for cancer cells over PBMCs, the film could effectively capture a broad range of cancer cells without the aid of capture antibodies (yield >76%). Importantly, the viabil...

Recent advances and perspectives on capture and concentration of label-free rare cells for biomedical science and engineering research

Abstract Capture and concentration of the rare cells from a heterogeneous population plays an important role in the development of disease diagnostics, therapeutics, and biomedical research/applications. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer therapy and regenerative medicine. Large-scale supply of the rare cells at high purity and viability is essential, especially on stem cells, cancer stem cells (CSCs), and circulating tumor cells (CTCs). Conventional capture and concentration methods providing efficient and high throughput isolations are introduced; however, most of them require well-defined biochemical markers and immunolabeling procedures which are complicated, uneconomical, and often unreliable. In contrast, the emerging label-free cell capture methods based on biophysical and biomechanical properties of the desired cell population are growing. In this review, we aim to describe the status of label-free rare cell capture and concentration techniques and to highlight exciting new approaches in this field. Label-free rare cell isolation methods using intrinsic biophysical properties such as filtration, size, electrical polarizability, dielectrophoresis, optical dielectrophoresis, and hydrodynamic chromatography are introduced. Moreover, the microfluidic systems adopted to precisely handle cells and interface with other tools have been described earlier; thus, new approaches dealing with merging label-free selection methods and microfluidic systems are merely summarized. Furthermore, the capture and concentration methods using biochemical and materials properties such as cell-materials interaction, spheroid formation, and stem cells/CSCs niche mimicking are highlighted particularly. Advancing cell isolation technique provides the opportunity to avoid the shortcoming of using biochemical labels and presents better flexibility for subsequent characterization, such as on site screening, high throughput screening, and personalized medicine.

Abstract 1715: EGFR mutational detection in ctDNA, Vortex-enriched CTCs and comparison to tumor tissue in non-small-cell-lung-cancer (NSCLC) patients

Abstract Background: Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutation, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks and expense and are not always successful. In order to identify the development of secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKI’s and an indication for therapy with a 3rd generation drug, effective and non-invasive monitoring is needed. Liquid biopsy biomarkers, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), allow such monitoring over the course of the therapy. Interestingly, ctDNA or CTC analysis alone had less sensitivity vs. combining both, with a genotyping of 70% and 80% for CTCs and ctDNA respectively, but 100% when combined2. Vortex technology is a platform enabling label-free capture of CTCs from blood samples and genomic assays downstream3. The aim of this study is to demonstrate the sensitivity of a combined CTC and ctDNA assay through Vortex using blood samples spiked with molecularly-characterized lung cancer cell lines and then to apply this technique to matched blood and tumor samples from NSCLC patients. Method: Lung cancer cell lines with different EGFR mutations (A549: wild type, H1975: L858R+ and T790M+, HCC827: 19del+) were used to validate our CTC workflow. Blood samples and matched tumor tissues were collected from NSCLC patients. Plasma was extracted first for ctDNA assay. CTCs were isolated from the plasma-depleted-blood using Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. DNA from CTCs, plasma and matched tumor tissue was analyzed for EGFR mutations 19del, L858R and T790M using the ctEGFR kit from EntroGen. Results: Mutant DNA could be identified at a quantity as low as 0.5 ng (~83 cells), with a sensitivity ranging from 0.1% to 2% for a total DNA varying from 25ng (~4 CTCs among 4000 WBCs) to 1ng (~4 CTCs among 200 WBCs). We demonstrated the ability of Vortex technology to enrich CTCs from metastatic NSCLC patients. Processing of plasma-depleted-blood showed the same capture efficiency when compared to whole blood. This makes possible the detection of EGFR mutations on CTC samples collected by Vortex technology. > 10 NSCLC patients are being enrolled in this study and results will be presented at AACR. Conclusion: The ctEGFR mutation assay performed well on both Vortex-enriched CTCs and ctDNA, enabling a low cost approach to analyze EGFR mutation from a single blood tube. This non-invasive EGFR mutation analysis will be potentially a useful tool for monitoring treatment and medication guidance of NSCLC patients. 1. Calabuig-Fariñas et al. Transl Lung Cancer Res. 2016. 2. Sundaresan TK et al. Clin Cancer Res. 2016. 3. Kidess-Sigal E et al. Oncotarget 2016. Citation Format: Haiyan E. Liu, Meghah Vuppalapaty, Clementine A. Lemaire, Charles Wilkerson, Steve C. Crouse, Jonathan W. Goldman, Elodie Sollier-Christen. EGFR mutational detection in ctDNA, Vortex-enriched CTCs and comparison to tumor tissue in non-small-cell-lung-cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1715. doi:10.1158/1538-7445.AM2017-1715

Abstract 2822: Establishing the use of Vortex technology for investigating circulating tumor cells in mouse models of breast cancer

Abstract Background: Circulating tumor cells (CTCs) are precursors of metastatic disease and are an important indicator of the disease progression and outcome of many cancers. Though several recent studies have advanced our understanding of CTCs, many critical areas of CTC biology remain largely unexplored. Murine models of cancer offer a unique opportunity to address this issue. However, small sample volumes, low number of CTCs, difficulty to access ports for blood collection are challenges that diminish the utility of mouse models for studying CTCs. In our previous studies, we had validated the use of Vortex technology for the label-free capture of CTCs from blood samples of different cancers. In this current study, we now report our recent, unpublished data on the adaptation of Vortex platform for enriching and characterizing murine and human tumor cells from mouse blood. Methods: By spiking tumor cells into small volumes of mouse blood (200-400 µl) from cardiac puncture, we tested the impact of i) different dilutions of mouse blood (10X, 20X, 40X); ii) varying numbers of tumor cells; iii) different cell types (MDA-MB-231, 4T1 and EMT6); and iv) sample recycling on capture efficiency and purity. Blood was processed through the Vortex plastic chip and, to accurately identify and enumerate the enriched tumor cells, we developed a staining protocol that labels human- or murine-specific cytokeratin, vimentin, and CD45. Results: Our results reveal that over a range of dilutions tested, just after one cycle, 10X dilution of mouse blood was superior, yielding a capture efficiency of 38.83% and a purity of 33% for MDA-MB-231 cells, compared to 20X (34.16% efficiency, 31.1% purity), and 40X (31.83% efficiency, 31% purity) dilutions. We are currently testing whether recycling the blood flow-through would increase the recovery. Even when as many as ~7500 tumor cells were spiked in mouse blood, the Vortex platform was able to successfully enrich tumor cells with high consistency and purity. Importantly, we were now able to successfully isolate human and murine tumor cell lines with varying levels of epithelial cell adhesion molecule (EpCAM) expression (4T1 - high, MDA-MB-231 - moderate to low) spiked into mouse blood. Experiments confirming the viability and growth rate of cancer cells isolated from mouse blood using MTT assay will also be presented. Conclusion: In summary, our results reveal the use of the Vortex technology for studying CTCs in murine models of cancer, with the ability to handle volumes of blood as low as 200uL while having a capacity to capture extremely large number of CTCs. While this study was focused on breast cancer, our workflow is easily adapted to other malignancies. Studies are underway to grow viable CTCs from tumor bearing animals and isolated by this platform for subsequent growth in 3D culture systems for future biologic and drug testing studies. Citation Format: Vishnu Ramani, Rakhi Gupta, Melanie Triboulet, Corinne Renier, Steve C. Crouse, Elodie Sollier-Christen, Stefanie S. Jeffrey. Establishing the use of Vortex technology for investigating circulating tumor cells in mouse models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2822. doi:10.1158/1538-7445.AM2017-2822

EGFR-mutation detection in VTX-1-enriched CTCs, ctDNA, and comparison to tumor in patients with non-small cell lung cancer (NSCLC).

e20525 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI's) have shown dramatic clinical benefits when EGFR mutations are detected. These assays are mainly performed on tumor biopsies, which carry risks, are expensive and are not always successful. Moreover, secondary mutations, causing resistance to 1st & 2nd generation TKI’s develop during treatment and therefore mutations require ongoing monitoring. Circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) are promising for monitoring the patient over time and show enhanced sensitivity when combined1. The VTX-1 Liquid Biopsy System enables label-free capture of CTCs from blood and genomic assays downstream2. In this study, we demonstrate the sensitivity of a combined EGFR assay on VTX-1-enriched CTCs and ctDNA, using blood spiked with cancer cells & DNA and then with NSCLC patients. Methods: NSCLC cells (A549: wt, H1975: L858R+ & T790M+, HCC827: 19del+) were used to validate the assay. H1975 cells and HCC827 DNA were spiked in blood. Plasma was extracted and the plasma depleted blood was processed through VTX-1. The collected plasma and enriched cells were subjected to the detection of 19del, L858R and T790M EGFR mutations using the EntroGen ctEGFR kit. Later, blood and tumor were collected from NSCLC patients and analyzed for EGFR mutations on biopsies, ctDNA and CTCs. Results: Mutant DNA was detected for an input as low as 0.5 ng (~83 cells), with a sensitivity from 0.1% to 2% for a total DNA of 25ng (~4 cancer cells & 4000 WBCs) to 1ng (~4 cancer cells & 200 WBCs). Processing of plasma-depleted-blood with Vortex showed the same efficiency compared to whole blood. Mutations were detected from enriched cells and plasma respectively for 100 H1975 cells and 0.5ng of HCC827 DNA spiked in 2mL blood. NSCLC patients are being enrolled and results will be presented at the conference. Conclusions: The ctEGFR assay performed well on VTX-1 enriched cells and ctDNA, enabling a low cost approach to analyze EGFR mutations from a single blood tube with high sensitivity, potentially being a useful tool for guiding treatment of NSCLC patients. 1.Sundaresan, et al. Clin Cancer Res 2016. 2. Kidess-Sigal, et al. Oncotarget 2016.

AC dielectrophoretic manipulation and electroporation of vaccinia virus using carbon nanoelectrode arrays.

This paper reports the capture and detection of vaccinia virus particles based on AC dielectrophoresis (DEP) and electrochemical impedance measurements employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) versus a macroscopic indium-tin-oxide (ITO) transparent electrode in a "points-and-lid" configuration. The nano-DEP device was fabricated by bonding two SU-8 covered electrodes patterned using photolithography. The bottom electrode contains a 200 × 200 μm2 active region on a randomly distributed NEA and the top electrode contains a microfluidic channel in SU-8 spin-coated on ITO to guide the flow of the virus solution. The real-time impedance change was measured during DEP capture and validated with fluorescence microscopy measurements. The NEA was able to capture virus particles with a rather low AC voltage (∼8.0 V peak-to-peak) at 1.0 kHz frequency as the particles were passed through the fluidic channel at high flow velocities (up to 8.0 mm/s). A concentration detection limit as low as ∼2.58 × 103 particles/mL was obtained via impedance measurements after only 54 sec of DEP capture. At the low AC frequencies (50.0 Hz or less), the high electric field at the exposed VACNF tips induced electroporation of the DEP-captured virus particles, which was validated by fluorescence emission from the dyes staining lipophilic membrane and internal nucleic acid, respectively. This study suggests the possibility of integration of a fully functional electronic device for rapid, reversible and label-free capture and detection of pathogenic viruses, with a potential of generating electroporation to the captured the virus particles for further biochemical study.

Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube–CTC chip

We demonstrate the rapid and label-free capture of breast cancer cells spiked in blood using nanotube-antibody micro-arrays. 76-element single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (anti-EpCAM), Anti-human epithelial growth factor receptor 2 (anti-Her2) and non-specific IgG antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester. Following device functionalization, blood spiked with SKBR3, MCF7 and MCF10A cells (100/1000 cells per 5 μl per device, 170 elements totaling 0.85 ml of whole blood) were adsorbed on to the nanotube device arrays. Electrical signatures were recorded from each device to screen the samples for differences in interaction (specific or non-specific) between samples and devices. A zone classification scheme enabled the classification of all 170 elements in a single map. A kernel-based statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping series to classify device electrical signals that corresponded to plain blood (control) or SKBR3 spiked blood (case) on anti-Her2 functionalized devices with ∼90% sensitivity, and 90% specificity in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2 functionalized devices. Screened devices that gave positive electrical signatures were confirmed using optical/confocal microscopy to hold spiked cancer cells. Confocal microscopic analysis of devices that were classified to hold spiked blood based on their electrical signatures confirmed the presence of cancer cells through staining for DAPI (nuclei), cytokeratin (cancer cells) and CD45 (hematologic cells) with single cell sensitivity. We report 55%–100% cancer cell capture yield depending on the active device area for blood adsorption with mean of 62% (∼12 500 captured off 20 000 spiked cells in 0.1 ml blood) in this first nanotube–CTC chip study.

Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays

We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1–17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for Ebola and Marburg detection.

Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.

Detection of circulating tumor cells in patients with hepatocellular carcinoma

To explore the detection efficiency of circulating tumor cells (CTCs) in patients with hepatocellular carcinoma (HCC). Immunomagnetic negative enrichment by nanometer magnetic beads and label-free capture with Captor(TM) system were used to isolate and enrich CTCs from peripheral blood of HCC patients, and epithelial and HCC markers were applied to identify CTCs by immunofluorescence staining. CTCs were detected in 50 HCC patients before and after hepatectomy to test the method for isolation, enrichment and identification. The dynamic changes of pre- and post-operative CTCs' numbers were compared. The clinical data were analyzed using SPSS 19.0 software. Negative enrichment methods by nanometer magnetic beads and label-free capture using Captor(TM) system were both suitable for CTCs isolation and enrichment in HCC patients. The positive detection rate of CTCs in HCC patients via negative enrichment was 96.0% (48/50), the preoperative median number of CTCs was 16 per 7.5 ml blood, and the postoperative median number was 17 per 7.5 ml blood. Both negative enrichment and Captor(TM) system are suitable for isolation and enrichment of CTCs in HCC patients. There is a significant difference in the numbers of CTCs before and after operation, and dynamic detection of CTCs will provide helpful prognostic information for HCC patients in clinics.

Effect of interdome spacing on the resonance properties of plasmonic nanodome arrays for label-free optical sensing

In this paper, we report on experimental and theoretical studies that investigate how the structural properties of plasmonic nanodome array devices determine their optical properties and sensing performance. We examined the effect of the interdome gap spacing within the plasmonic array structures on the performance for detection of change in local refractive index environment for label-free capture affinity biosensing applications. Optical sensing properties were characterized for nanodome array devices with interdome spacings of 14 nm, 40 nm, and 79 nm, as well as for a device where adjacent domes are in contact. For each interdome spacing, the extinction spectrum was measured using a broadband reflection instrumentation, and finite-difference-time-domain (FDTD) simulation was used to model the local electric field distribution associated with the resonances. Based on these studies, we predict that nanodome array devices with gap between 14 nm to 20 nm provide optimal label-free capture affinity biosensing performances, where the dipole resonance mode exhibits the highest overall surface sensitivity, as well as the lowest limit of detection.