Graphene Oxide Functionalized Biosensor for Detection of Stress-Related Biomarkers.

A graphene oxide (GO)-based cortisol biosensor was developed to accurately detect cortisol concentrations from sweat samples at point-of-care (POC) sites. A reference electrode, counter electrode, and working electrode make up the biosensor, and the working electrode was functionalized using multiple layers consisting of GO and antibodies, including Protein A, IgG, and anti-Cab. Sweat samples contact the anti-Cab antibodies to transport electrons to the electrode, resulting in an electrochemical current response. The sensor was tested at each additional functionalization layer and at cortisol concentrations between 0.1 and 150 ng/mL to determine how the current response differed. A potentiostat galvanostat device was used to measure and quantify the electrochemical response in the GO-based biosensor. In both tests, the electrochemical responses were reduced in magnitude with the addition of antibody layers and with increased cortisol concentrations. The proposed cortisol biosensor has increased accuracy with each additional functionalization layer, and the proposed device has the capability to accurately measure cortisol concentrations for diagnostic purposes.

Multifunctional Cellular Targeting, Molecular Delivery, and Imaging by Integrated Mesoporous-Silica with Optical Nanocrescent Antenna: MONA.

Multifunctional nanoprobes have attracted significant attention in a wide range of disciplines such as nanomedicine, precision medicine, and cancer diagnosis and treatment. However, integrating multifunctional ability in a nanoscale structure to precisely target, image, and deliver with cellular spatial/temporal resolution is still challenging in cellulo applications. This is because the development of such high-precision resolution needs to be carried out without labeling, photobleaching, and structurally segregating live cells. In this study, we present an integrated nanostructure of a mesoporous-silica nanosphere with an optical nanocrescent antenna (MONA) for multifunctional cellular targeting, drug delivery, and molecular imaging with spatiotemporal resolution. MONA comprises a systematically constructed Au nanocrescent (AuNC) antenna as a nanosensor and optical switch on a mesoporous-silica nanosphere as a cargo to molecular delivery. MONA made of antiepithelial cell adhesion molecules (anti-EpCAM)-conjugated AuNC facilitates the specific targeting of breast cancer cells, resulting in a highly focused photothermal gradient that functions as a molecular emitter. This light-driven molecular, doxorubicin (DOX) delivery function allows rapid apoptosis of breast cancer cells. Since MONA permits the tracking of quantum biological electron-transfer processes, in addition to its role as an on-demand optical switch, it enables the monitoring of the dynamic behavior of cellular cytochrome c pivoting cell apoptosis in response to the DOX delivery. Owing to the integrated functions of molecular actuation and direct sensing at the precisely targeted spot afforded by MONA, we anticipate that this multifunctional optical nanoantenna structure will have an impact in the fields of nanomedicine, cancer theranostics, and basic life sciences.

Characterizing Circulating Tumor Cells Isolated from Metastatic Breast Cancer Patients Using Graphene Oxide Based Microfluidic Assay.

The enumeration of circulating tumor cells (CTCs) has shown prognostic importance in patients with breast cancer. However, CTCs are highly heterogeneous with diverse functional properties, which may also be clinically relevant. To provide a comprehensive landscape of the patient's disease, further CTC analysis is required. Here, a highly sensitive and reproducible graphene oxide based CTC assay is utilized to isolate and characterize CTCs from 47 metastatic breast cancer patients. The CTCs are captured with high purity, requiring only a few milliliters of blood and enabling efficient enumeration and subsequent analysis at both the protein and the transcription level. The results show that patient clinical outcomes correlate with the associated CTC profile and clearly demonstrate the potential use of the assay in the clinical setting. Collectively, these findings suggest that beyond simple enumeration, CTC characterization may provide further information that improves the diagnosis of the patients' disease status for proper treatment decisions. Moreover, this thorough validation study will facilitate the translation of the CTC assay into future clinical applications to broaden the utility of liquid biopsy.

High-Throughput Microfluidic Labyrinth for the Label-free Isolation of Circulating Tumor Cells.

We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations.

The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer.

Dissemination of cancer stem cells (CSCs) serves as the basis of metastasis. Recently, we demonstrated that circulating prostate cancer targets the hematopoietic stem cell (HSCs) 'niche' in marrow during dissemination. Once in the niche, disseminated tumor cells (DTCs) may remain dormant for extended periods. As the major function of the HSC niche is to maintain stem cell functions, we hypothesized that the niche regulates CSC activities of DTCs. Here we show that DTCs recovered from marrow were significantly enriched for a CSC phenotype. Critically, the conversion of DTCs to CSCs is regulated by niche-derived GAS6 through the Mer/mTOR; molecules previously shown to regulate dormancy. The data demonstrate that the niche plays a significant role in maintaining tumor-initiating prostate cancer in marrow and suggests a functional relationship between CSCs and dormancy. Understanding how the marrow niche regulates the conversion of DTCs to CSCs is critical for the development of therapeutics specifically targeting skeletal bone metastasis and dormancy.

Tunable Thermal-Sensitive Polymer-Graphene Oxide Composite for Efficient Capture and Release of Viable Circulating Tumor Cells.

A highly sensitive microfluidic system to capture circulating tumor cells from whole blood of cancer patients is presented. The device incorporates graphene oxide into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. By decreasing the temperature, captured cells may be released for subsequent analysis.

Microfabrication and in Vivo Performance of a Microdialysis Probe with Embedded Membrane.

Microdialysis sampling is an essential tool for in vivo neurochemical monitoring. Conventional dialysis probes are over 220 µm in diameter and have limited flexibility in design because they are made by assembly using preformed membranes. The probe size constrains spatial resolution and governs the amount of tissue damaged caused by probe insertion. To overcome these limitations, we have developed a method to microfabricate probes in Si that are 45 µm thick × 180 µm wide. The probes contain a buried, U-shaped channel that is 30 µm deep × 60 µm wide and terminates in ports for external connection. A 4 mm length of the probe is covered with a 5 µm thick nanoporous membrane. The membrane was microfabricated by deep reactive ion etching through a porous aluminum oxide layer. The microfabricated probe has cross-sectional area that is 79% less than that of the smallest conventional microdialysis probes. The probes yield 2-20% relative recovery at 100 nL/min perfusion rate for a variety of small molecules. The probe was successfully tested in vivo by sampling from the striatum of live rats. Fractions were collected at 20 min intervals (2 µL) before and after an intraperitoneal injection of 5 mg/kg amphetamine. Analysis of fractions by liquid chromatography-mass spectrometry revealed reliable detection of 14 neurochemicals, including dopamine and acetylcholine, at basal conditions. Amphetamine evoked a 43-fold rise in dopamine, a result nearly identical to a conventional dialysis probe in the same animal. The microfabricated probes have potential for sampling with higher spatial resolution and less tissue disruption than conventional probes. It may also be possible to add functionality to the probes by integrating other components, such as electrodes, optics, and additional channels.

Emerging role of nanomaterials in circulating tumor cell isolation and analysis.

Circulating tumor cells (CTCs) are low frequency cells found in the bloodstream after having been shed from a primary tumor. These cells are research targets because of the information they may potentially provide about both an individual cancer as well as the mechanisms through which cancer spreads in the process of metastasis. Established technologies exist for CTC isolation, but the recent progress and future of this field lie in nanomaterials. In this review, we provide perspective into historical CTC capture as well as current research being conducted, emphasizing the significance of the materials being used to fabricate these devices. The modern investigation into CTCs initially featured techniques that have since been commercialized. A major innovation in the field was the development of a microfluidic capture device, first fabricated in silicon and followed up with glass and thermopolymer devices. We then specifically highlight the technologies incorporating magnetic nanoparticles, carbon nanotubes, nanowires, nanopillars, nanofibers, and nanoroughened surfaces, graphene oxide and their fabrication methods. The nanoscale provides a new set of tools that has the potential to overcome current limitations associated with CTC capture and analysis. We believe the current trajectory of the field is in the direction of nanomaterials, allowing the improvements necessary to further CTC research.

Cascaded spiral microfluidic device for deterministic and high purity continuous separation of circulating tumor cells.

Inertial microfluidics is an emerging class of technologies developed to separate circulating tumor cells (CTCs). However, defining design parameters and flow conditions for optimal operation remains nondeterministic due to incomplete understanding of the mechanics, which has led to challenges in designing efficient systems. Here, we perform a parametric study of the inertial focusing effects observed in low aspect ratio curvilinear microchannels and utilize the results to demonstrate the isolation of CTCs with high purity. First, we systematically vary parameters including the channel height, width, and radius of curvature over a wide range of flow velocities to analyze its effect on size dependent differential focusing and migration behaviors of binary (10 µm and 20 µm) particles. Second, we use these results to identify optimal flow regimes to achieve maximum separation in various channel configurations and establish design guidelines to readily provide information for developing spiral channels tailored to potentially arbitrary flow conditions that yield a desired equilibrium position for optimal size based CTC separation. Finally, we describe a fully integrated, sheath-less cascaded spiral microfluidic device to continuously isolate CTCs. Human breast cancer epithelial cells were successfully extracted from leukocytes, achieving 86.76% recovery, 97.91% depletion rate, and sustaining high viability upon collection to demonstrate the versatility of the device. Importantly, this device was designed without the cumbersome trail-and-error optimization process that has hindered the development of designing such inertial microfluidic systems.

Sensitive capture of circulating tumour cells by functionalized graphene oxide nanosheets.

The spread of cancer throughout the body is driven by circulating tumour cells (CTCs). These cells detach from the primary tumour and move from the bloodstream to a new site of subsequent tumour growth. They also carry information about the primary tumour and have the potential to be valuable biomarkers for disease diagnosis and progression, and for the molecular characterization of certain biological properties of the tumour. However, the limited sensitivity and specificity of current methods for measuring and studying these cells in patient blood samples prevents the realization of their full clinical potential. The use of microfluidic devices is a promising method for isolating CTCs. However, the devices are reliant on three-dimensional structures, which limits further characterization and expansion of cells on the chip. Here we demonstrate an effective approach to isolating CTCs from blood samples of pancreatic, breast and lung cancer patients, by using functionalized graphene oxide nanosheets on a patterned gold surface. CTCs were captured with high sensitivity at a low concentration of target cells (73 ± 32.4% at 3-5 cells per ml blood).

Electrical transport properties of graphene nanoribbons produced from sonicating graphite in solution.

A simple one-stage solution-based method was developed to produce graphene nanoribbons by sonicating graphite powder in organic solutions with polymer surfactant. The graphene nanoribbons were deposited on a silicon substrate, and characterized by Raman spectroscopy and atomic force microscopy. Single-layer and few-layer graphene nanoribbons with a width ranging from sub-10 nm to tens of nanometers and lengths ranging from hundreds of nanometers to 1 µm were routinely observed. The electrical transport properties of individual graphene nanoribbons were measured in both the back-gate and polymer-electrolyte top-gate configurations. The mobility of the graphene nanoribbons was found to be over an order of magnitude higher when measured in the latter than in the former configuration (without the polymer-electrolyte), which can be attributed to the screening of the charged impurities by the counter ions in the polymer-electrolyte. This finding suggests that the charge transport in these solution produced graphene nanoribbons is largely limited by charge impurity scattering.

Room-temperature high on/off ratio in suspended graphene nanoribbon field-effect transistors.

We have fabricated suspended few-layer (1-3 layers) graphene nanoribbon field-effect transistors from unzipped multi-wall carbon nanotubes. Electrical transport measurements show that current annealing effectively removes the impurities on the suspended graphene nanoribbons, uncovering the intrinsic ambipolar transfer characteristic of graphene. Further increasing the annealing current creates a narrow constriction in the ribbon, leading to the formation of a large bandgap and subsequent high on/off ratio (which can exceed 10(4)). Such fabricated devices are thermally and mechanically stable: repeated thermal cycling has little effect on their electrical properties. This work shows for the first time that ambipolar field-effect characteristics and high on/off ratios at room temperature can be achieved in relatively wide graphene nanoribbons (15-50 nm) by controlled current annealing.