Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.

Chitosan as an Alternative to Oil-Based Materials for the Fabrication of Lab-on-a-Chip.

Given the growing importance of lab-on-a-chip in a number of fields, such as medical diagnosis or environmental analysis, the fact that the current fabrication process relies mainly on oil-based polymers raises an ecological concern. As an eco-responsible alternative, we presented, in this article, a manufacturing process for microfluidic devices from chitosan, a bio-sourced, biodegradable, and biocompatible polysaccharide. From chitosan powder, we produced thick and rigid films. To prevent their dissolution and reduce their swelling when in contact with aqueous solutions, we investigated a film neutralization step and characterized the mechanical and physical properties of the resulting films. On these neutralized chitosan films, we compared two micropatterning methods, i.e., hot embossing and mechanical micro-drilling, based on the resolution of microchannels from 100 µm to 1000 µm wide. Then, chitosan films with micro-drilled channels were bonded using a biocompatible dry photoresist on a glass slide or another neutralized chitosan film. Thanks to this protocol, the first functional chitosan microfluidic devices were prepared. While some steps of the fabrication process remain to be improved, these preliminary results pave the way toward a sustainable fabrication of lab-on-a-chip.

MagPure chip: an immunomagnetic-based microfluidic device for high purification of circulating tumor cells from liquid biopsies.

The isolation of circulating tumor cells (CTCs) directly from blood, as a liquid biopsy, could lead to a paradigm shift in cancer clinical care by providing an earlier diagnosis, a more accurate prognosis, and personalized treatment. Nevertheless, CTC-specific challenges, including their rarity and heterogeneity, have hampered the wider use of CTCs in clinical studies. Microfluidic-based isolation technologies have emerged as promising tools to circumvent these limitations but still fail to meet the constraints of high purity and short processing time required to ensure compatibility with clinical follow-up. In this study, we developed an immunomagnetic-based microfluidic device, the MagPure chip, to achieve the negative selection of CTCs through the depletion of white blood cells (WBCs) and provide highly purified samples for subsequent analysis. We demonstrate that the MagPure chip depletes all magnetically labeled WBCs (85% of WBCs were successfully labeled) and ensures a CTC recovery rate of 81%. In addition, we show its compatibility with conventional biological studies, including 2D and 3D cell culture, as well as phenotypic and genotypic analyses. Finally, we successfully implemented a two-step separation workflow for whole blood processing by combining a size-based pre-enrichment system (ClearCell FX1®) with the MagPure chip as a subsequent purification step. The total workflow led to high throughput (7.5 mL blood in less than 4 h) and high purity (947 WBCs per mL remaining, 99.99% depletion rate), thus enabling us to quantify CTC heterogeneity in size and tumor marker expression level. This tumor-marker-free liquid biopsy workflow could be used in a clinical context to assess phenotype aggressiveness and the prognosis rate.

Microfluidic-Based Technologies for CTC Isolation: A Review of 10 Years of Intense Efforts towards Liquid Biopsy.

The selection of circulating tumor cells (CTCs) directly from blood as a real-time liquid biopsy has received increasing attention over the past ten years, and further analysis of these cells may greatly aid in both research and clinical applications. CTC analysis could advance understandings of metastatic cascade, tumor evolution, and patient heterogeneity, as well as drug resistance. Until now, the rarity and heterogeneity of CTCs have been technical challenges to their wider use in clinical studies, but microfluidic-based isolation technologies have emerged as promising tools to address these limitations. This review provides a detailed overview of latest and leading microfluidic devices implemented for CTC isolation. In particular, this study details must-have device performances and highlights the tradeoff between recovery and purity. Finally, the review gives a report of CTC potential clinical applications that can be conducted after CTC isolation. Widespread microfluidic devices, which aim to support liquid-biopsy-based applications, will represent a paradigm shift for cancer clinical care in the near future.

Biosensors for circulating tumor cells (CTCs)-biomarker detection in lung and prostate cancer: Trends and prospects.

Cancer is one of the leading cause of death worldwide. Lung cancer (LCa) and prostate cancer (PCa) are the two most common ones particularly among men with about 20% of aggressive metastatic form leading to shorter overall survival. In recent years, circulating tumor cells (CTCs) have been investigated extensively for their role in metastatic progression and their involvement in reduced overall survival and treatment responses. Analysis of these cells and their associated biomarkers as "liquid biopsy" can provide valuable real-time information regarding the disease state and can be a potential avenue for early-stage detection and possible selection of personalized treatments. This review focuses on the role of CTCs and their associated biomarkers in lung and prostate cancer, as well as the shortcomings of conventional methods for their isolation and analysis. To overcome these drawbacks, biosensors are an elegant alternative because they are capable of providing valuable multiplexed information in real-time and analyzing biomarkers at lower concentrations. A comparative analysis of different transducing elements specific for the analysis of cancer cell and cancer biomarkers have been compiled in this review.

Self-Assembled Permanent Micro-Magnets in a Polymer-Based Microfluidic Device for Magnetic Cell Sorting.

Magnetophoresis-based microfluidic devices offer simple and reliable manipulation of micro-scale objects and provide a large panel of applications, from selective trapping to high-throughput sorting. However, the fabrication and integration of micro-scale magnets in microsystems involve complex and expensive processes. Here we report on an inexpensive and easy-to-handle fabrication process of micrometer-scale permanent magnets, based on the self-organization of NdFeB particles in a polymer matrix (polydimethylsiloxane, PDMS). A study of the inner structure by X-ray tomography revealed a chain-like organization of the particles leading to an array of hard magnetic microstructures with a mean diameter of 4 µm. The magnetic performance of the self-assembled micro-magnets was first estimated by COMSOL simulations. The micro-magnets were then integrated into a microfluidic device where they act as micro-traps. The magnetic forces exerted by the micro-magnets on superparamagnetic beads were measured by colloidal probe atomic force microscopy (AFM) and in operando in the microfluidic system. Forces as high as several nanonewtons were reached. Adding an external millimeter-sized magnet allowed target magnetization and the interaction range to be increased. Then, the integrated micro-magnets were used to study the magnetophoretic trapping efficiency of magnetic beads, providing efficiencies of 100% at 0.5 mL/h and 75% at 1 mL/h. Finally, the micro-magnets were implemented for cell sorting by performing white blood cell depletion.

Nanonewton Magnetophoretic Microtrap Array for Microsystems.

Here we report on the development of a lab-on-chip that integrates a dense array of micrometer-sized magnetic traps, with each individual trap generating a magnetic force as high as a few nN on standard superparamagnetic beads. The composite materials embedding traps are prepared from the microstructural engineering of a mixture between iron microparticles and polydimethylsiloxane. This approach breaks with standard microfabrication technologies: it is inexpensive, relatively easy to implement, and offers the ability to modulate the magnetic properties of the composites on a customized basis. The magnetic forces acting on the superparamagnetic beads have been measured following two approaches: first, on-chip through the hydrodynamic determination of the holding magnetic force, simultaneously on a large population of traps; and second, ex situ, by atomic force microscopy equipped with a colloidal probe, on individual traps. The experimental results have been compared with calculations from finite element modeling. Despite the geometrical simplification of the modeled system, both experiments and calculations give consistent values of force, ranging from 0.5 to 5 nN. These findings show that in operando determination of forces is a robust method that gives a high throughput overview of the forces acting in the device. It further demonstrates that the use of such functional composite materials can be a relevant alternative to standard microfabrication technologies, as it leads to competitive magnetophoretic performances.

Magnetic filaments for anisotropic composite polymers.

The use of contactless magnetic forces meets numerous needs in microelectromechanical systems (MEMS) or microfluidic devices. In this view, heterogeneous materials integrating magnetic nanostructures within a non-magnetic matrix such as polymer can produce local variations of magnetic field, at the sub-micrometer scale. Here we report on the synthesis of magnetic composites using electrospun nanofilaments and a polydimethylsiloxane (PDMS) matrix. Varying the precursor nature and heat treatment conditions, we obtained single phase filaments of Fe, FeNi, and MFe2O4 (M = Co, Fe, Ni). Thanks to a fine investigation of their structure and morphology, it was possible to measure from magnetically-soft (µ0HC ⩽ 5 mT) to relatively hard (µ0HC up to 93 mT, MR/MS up to 0.5) behaviors. The common one-dimensional shape of these filaments leads to an anisotropic magnetic response. This can be exploited to achieve self-organization of the filaments in arrays within the non-magnetic matrix. We show the first step towards the development of magnetically anisotropic membranes of PDMS with 0.23 wt% Fe filaments. These composite materials are promising for implementing magnetic functions in microsystems while circumventing complex micro-fabrication steps.

Xurography-based microfluidic algal biosensor and dedicated portable measurement station for online monitoring of urban polluted samples.

A critical need exists to develop rapid, in situ, and real-time tools to monitor the impact of pollution discharge toxicity on aquatic ecosystems. The present paper deals with the development of a novel, simple-to-use, low-cost, portable, and user-friendly algal biosensor. In this study, a complete and autonomous portable fluorimeter was developed to assess the A-chlorophyll fluorescence of microalgae, inserted by capillarity into low-cost and disposable xurography-based microfluidic chips. Three microalgae populations were used to develop the biosensor: Chlorella vulgaris, Pseudokirchneriella subcapitata, and Chlamydomonas reinhardtii. Biosensor feasibility and sensitivity parameters, such as algal concentration and light intensity, were optimized beforehand to calibrate the biosensor sensitivity with Diuron, a pesticide known to be very toxic for microalgae. Finally, the biosensor was employed in 10 aqueous urban polluted samples (7 urban wet-weather discharges and 3 wastewater) in order to prove its reliability, reproducibility, and performance in the detection of toxic discharges in the field.

Dielectrophoretic capture of low abundance cell population using thick electrodes.

Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O2 plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force ([Formula: see text]) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 µl/h and 78.7% at 80 µl/h, for 100 µm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10).

Magnetophoretic manipulation in microsystem using carbonyl iron-polydimethylsiloxane microstructures.

This paper reports the use of a recent composite material, noted hereafter i-PDMS, made of carbonyl iron microparticles mixed in a PolyDiMethylSiloxane (PDMS) matrix, for magnetophoretic functions such as capture and separation of magnetic species. We demonstrated that this composite which combine the advantages of both components, can locally generate high gradients of magnetic field when placed between two permanent magnets. After evaluating the magnetic susceptibility of the material as a function of the doping ratio, we investigated the molding resolution offered by i-PDMS to obtain microstructures of various sizes and shapes. Then, we implemented 500 µm i-PDMS microstructures in a microfluidic channel and studied the influence of flow rate on the deviation and trapping of superparamagnetic beads flowing at the neighborhood of the composite material. We characterized the attraction of the magnetic composite by measuring the distance from the i-PDMS microstructure, at which the beads are either deviated or captured. Finally, we demonstrated the interest of i-PDMS to perform magnetophoretic functions in microsystems for biological applications by performing capture of magnetically labeled cells.

Recurrent tamponade and aortic dissection in syphilis.

Syphilitic cardiovascular disease has been described since the 19th century, mainly on autopsy series. Major clinical manifestations are aortic aneurysm, aortic insufficiency, and coronary ostial stenosis. The diagnosis of syphilitic cardiovascular disease is based mainly on positive serologic tests and overt clinical manifestations. We present here a rare and unusual clinical presentation of a tertiary syphilis with recurrent tamponade and type B aortic dissection, whose positive diagnosis was made by polymerase chain reaction on pericardial fluid analysis.

PCB/polymer based micro-fluidic system for NMR spectroscopy for nanoliters sample volume.

In this work, we report on the realization of an innovating micro system for NMR spectroscopy on small sample volume (30-100 nL). We propose a micro system based on Printed Circuit Board (PCB) technology for the NMR probe associated to a micro fluidic system made with polymer (COC). The comparison of several samples during the same NMR experiments could provide more precise information. In that context, we have realized a micro-fluidic system with two cavities, each cavity presenting a volume of 37 nl. The fabrication process is described, and first results are reported. The tight sealing of the micro-fluidic system has been demonstrated and preliminary NMR experiment results are presented.

Synthesis of a thermally stable hybrid acene-thiophene organic semiconductor via a soluble precursor.

An acene fused-thiophene hybrid p-semiconductor exhibiting high thermal stability has been synthesized via a soluble precursor bearing sterically interacting trimethylsilyl groups. [structure: see text]