RF Remote Blood Glucose Sensor and a Microfluidic Vascular Phantom for Sensor Validation.

Diabetes has become a major health problem in society. Invasive glucometers, although precise, only provide discrete measurements at specific times and are unsuitable for long-term monitoring due to the injuries caused on skin and the prohibitive cost of disposables. Remote, continuous, self-monitoring of blood sugar levels allows for active and better management of diabetics. In this work, we present a radio frequency (RF) sensor based on a stepped impedance resonator for remote blood glucose monitoring. When placed on top of a human hand, this RF interdigital sensor allows detection of variation in blood sugar levels by monitoring the changes in the dielectric constant of the material underneath. The designed stepped impedance resonator operates at 3.528 GHz with a Q factor of 1455. A microfluidic device structure that imitates the blood veins in the human hand was fabricated in PDMS to validate that the sensor can measure changes in glucose concentrations. To test the RF sensor, glucose solutions with concentrations ranging from 0 to 240 mg/dL were injected into the fluidic channels and placed underneath the RF sensor. The shifts in the resonance frequencies of the RF sensor were measured using a network analyzer via its S11 parameters. Based on the change in resonance frequencies, the sensitivity of the biosensor was found to be 264.2 kHz/mg·dL-1 and its LOD was calculated to be 29.89 mg/dL.

Microfluidic Lab-on-a-Chip Based on UHF-Dielectrophoresis for Stemness Phenotype Characterization and Discrimination among Glioblastoma Cells.

Glioblastoma (GBM) is one of the most aggressive solid tumors, particularly due to the presence of cancer stem cells (CSCs). Nowadays, the characterization of this cell type with an efficient, fast and low-cost method remains an issue. Hence, we have developed a microfluidic lab-on-a-chip based on dielectrophoresis (DEP) single cell electro-manipulation to measure the two crossover frequencies: fx01 in the low-frequency range (below 500 kHz) and fx02 in the ultra-high-frequency range (UHF, above 50 MHz). First, in vitro conditions were investigated. An U87-MG cell line was cultured in different conditions in order to induce an undifferentiated phenotype. Then, ex vivo GBM cells from patients' primary cell culture were passed through the developed microfluidic system and characterized in order to reflect clinical conditions. This article demonstrates that the usual exploitation of low-frequency range DEP does not allow the discrimination of the undifferentiated GBM cells from the differentiated one. However, the presented study highlights the use of UHF-DEP as a relevant discriminant parameter. The proposed microfluidic lab-on-a-chip is able to follow the kinetics of U87-MG phenotype transformation in a CSC enrichment medium and the cancer stem cells phenotype acquirement.

Characterization of Glioblastoma Cancer Stem Cells Sorted by Sedimentation Field-Flow Fractionation Using an Ultrahigh-Frequency Range Dielectrophoresis Biosensor.

Cancer stem cells (CSCs) appear to be an essential target for cancer therapies, in particular, in brain tumors such as Glioblastoma. Nevertheless, their isolation is made difficult by their low content in culture or tumors (<5% of the tumor mass) and is essentially based on the use of fluorescent or magnetic labeling techniques, increasing the risk of differentiation induction. The use of label-free separation methods such as sedimentation field-flow fractionation (SdFFF) is promising, but it becomes necessary to consider a coupling with a detection and characterization method for future identification and purification of CSCs from patient-derived tumors. In this study, we demonstrate for the first time the capability of using an ultrahigh-frequency range dielectrophoresis fluidic biosensor as a detector. This implies an important methodological adaptation of SdFFF cell sorting by the use of a new compatible carrier liquid DEP buffer (DEP-B). After SdFFF sorting, subpopulations derived from U87-MG and LN18 cell lines undergo biological characterization, demonstrating that using DEP-B as a carrier liquid, we sorted by SdFFF subpopulations with specific differentiation characteristics: F1 = differentiated cells/F2 = CSCs. These subpopulations presented high-frequency crossover (HFC) values similar to those measured for standard differentiated (around 110 MHz) and CSC (around 80 MHz) populations. This coupling appeared as a promising solution for the development of an online integration of these two complementary label-free separation/detection technologies.

Human Medulloblastoma Cell Lines: Investigating on Cancer Stem Cell-Like Phenotype.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite the progress of new treatments, the risk of recurrence, morbidity, and death remains significant and the long-term adverse effects in survivors are substantial. The fraction of cancer stem-like cells (CSCs) because of their self-renewal ability and multi-lineage differentiation potential is critical for tumor initiation, growth, and resistance to therapies. For the development of new CSC-targeted therapies, further in-depth studies are needed using enriched and stable MB-CSCs populations. This work, aimed at identifying the amount of CSCs in three available human cell lines (DAOY, D341, and D283), describes different approaches based on the expression of stemness markers. First, we explored potential differences in gene and protein expression patterns of specific stem cell markers. Then, in order to identify and discriminate undifferentiated from differentiated cells, MB cells were characterized using a physical characterization method based on a high-frequency dielectrophoresis approach. Finally, we compared their tumorigenic potential in vivo, through engrafting in nude mice. Concordantly, our findings identified the D283 human cell line as an ideal model of CSCs, providing important evidence on the use of a commercial human MB cell line for the development of new strategic CSC-targeting therapies.

A New Label-Free Approach to Glioblastoma Cancer Stem Cell Sorting and Detection.

Cancer stem cells (CSCs) play critical roles in cancer, making them important targets for new diagnostic and therapeutic approaches. Since CSCs are heterogeneous and not abundant in tumors, and few specific markers for these cells currently exist, new methods to isolate and characterize them are required. To address this issue, we developed a new label-free methodology to isolate, enrich, and identify CSCs from an heterogeneous tumor cell subpopulation using a cell sorting method (sedimentation field flow fractionation, SdFFF) and a biosensor as a detector. Enrichment was optimized using an original protocol and U87-MG glioblastoma cells cultured in a normal (N) or defined (D) medium (± fetal bovine serum, FBS) under normoxic (N, pO2 = 20%) or hypoxic (H, pO2 < 2%) conditions to obtain four cell populations: NN, NH, DN, and DH. After elution of CSCs via SdFFF using the hyperlayer mode (inertial elution mode for micrometer-sized species), we isolated eight subpopulations with distinct CSC contents based on phenotypical and functional properties, ranging from NN F1 with a lower CSC content to DH F3 with a higher CSC content. Reflecting biological differences, the intrinsic intracellular dielectric permittivity increased from NN to DH conditions. The largest difference in electromagnetic signature was observed between NN F1 and DH F3, in which the CSC content was lowest and highest, respectively. The results demonstrate that microwave dielectric spectroscopy can be used to reliably and efficiently distinguish stem cell characteristics. This new instrumental and methodological approach is an important innovation that allows both enrichment and detection of CSCs, opening the door to novel diagnostic and therapeutic approaches.

Improved sedimentation field-flow fractionation separation channel for concentrated cellular elution.

SdFFF is now commonly used for cell sorting. Nevertheless, as with many other separation methods, SdFFF Hyperlayer elution leads (1) to sample dilution resulting in cell loss which could restrict further use; and (2) to a high output flow rate impacting detector sensitivity and selectivity. In order to limit these problems, we proposed modifications of the SdFFF separation channel consisting both in downscaling and the insertion of an outlet stream splitter. This last system corresponded to a strip which divides the flow rate output into two parts, one containing concentrated cells in a reduced volume and flow rate, the other containing the excess mobile phase useless for further cell manipulation, detection and characterization. For the first time we have shown that splitter implementation and downscaling respected channel flowing and resulted in Hyperlayer elution of around 95% of cells in less than 50% of input flow rate. Improved cell sorting was demonstrated by enrichment (∼10 times) of cancer stem cells from WiDr cells with two times less quantity of injected cells.