Studies on the Effect of Graphene Oxide Deposited on Gold and Nickel Microsieves on Prostate Cancer Cells DU 145.

This work shows the effect of graphene oxide deposition on microsieves' surfaces of gold and nickel foils, on DU 145 tumor cells of the prostate gland. The sieves were made by a laser ablation process. The graphene oxide (GO) deposition process was characterized by the complete covering of the inner edges of the microholes and the flat surface between the holes with GO. Electron microscanning studies have shown that due to the deposition method applied, graphene oxide flakes line the interior of the microholes, reducing the unevenness of the downstream surfaces during the laser ablation process. The presence of graphene oxide was confirmed by Fourier infrared spectroscopy. During the screening (sieving) process, the microsieves were placed in a sieve column. Gold foil is proven to be a very good material for the screening of cancer cells, but even more so after screening as a substrate for re-culture of the DU 145. This allows a potential recovery of the cells and the development of a targeted therapy. The sieved cells were successfully grown on the microsieves used in the experiment. Graphene oxide remaining on the surface of the nickel sieve has been observed to increase the sieving effect. Although graphene oxide improved separation efficiency by 9.7%, the nickel substrate is not suitable for re-culturing of the Du 145 cells and the development of a targeted therapy compared to the gold one.

Microsieves for the detection of circulating tumor cells in leukapheresis product in non-small cell lung cancer patients.

BACKGROUND: Circulating tumor cells (CTC) in non-small cell lung cancer (NSCLC) patients are a prognostic and possible therapeutic marker, but have a low frequency of appearance. Diagnostic leukapheresis (DLA) concentrates CTC and mononuclear cells from the blood. We evaluated a protocol using two VyCAP microsieves to filter DLA product of NSCLC patients and enumerate CTC, compared with CellSearch as a gold standard. METHODS: DLA was performed in NSCLC patients before starting treatment. DLA product equaling 2×108 leukocytes was diluted to 9 mL with CellSearch dilution buffer in a Transfix CTC tube. Within 72 hours the sample was filtered with a 7 µm pore microsieve and subsequently over a 5µm pore microsieve. CTC were defined as nucleated cells which stained for cytokeratin, but lacked CD45 and CD16. CellSearch detected CTC in the same volume of DLA. RESULTS: Of 29 patients a median of 1.4 mL DLA product (range, 0.5-4.1) was filtered (2% of total product) successfully in 93% and 45% of patients using 7 and 5 µm pores, respectively. Two DLA products were unevaluable for CTC detection. Clogging of the 5 µm but not 7 µm microsieves was positively correlated with fixation time (ρ=0.51, P<0.01). VyCAP detected CTC in 44% (12/27) of DLA products. Median CTC count per mL DLA was 0 [interquartile range (IQR): 0-1]. CellSearch detected CTC in 63% of DLA products (median =0.9 CTC per mL DLA, IQR: 0-2.1). CTC counts detected by CellSearch were significantly higher compared with VyCAP (P=0.05). CONCLUSIONS: VyCAP microsieves can identify CTC in DLA product, but workflows need to be optimized.

The Influence of Laser Ablation Parameters on the Holes Structure of Laser Manufactured Graphene Paper Microsieves.

The graphene paper microsieves can be applied in the filtration of biological fluids or separation of solid particles from exploitation fluids. To produce graphene paper microsieves for specific applications, good control over fabrication should be achieved. In this study, a laser ablation method using a picosecond laser was applied to fabricate graphene paper microsieves. Holes in the microsieves were drilled using pulsed laser radiation with a pulse energy from 5 to 100 µJ, a duration of 60 ps, a wavelength of 355 nm, and a repetition rate of 1 kHz. The impact method was applied using 10 to 100 pulses to drill one hole. To produce holes of a proper diameter which could separate biological particles of a certain size (≥10 µm), optimum parameters of graphene paper laser ablation were defined using the MATLAB software taking into account laser pulse energy, repetition rate, and a desired hole diameter. A series of structural tests were carried out to determine the quality of an edge and a hole shape. Experimental results and Laguerre-Gauss calculations in MATLAB were then compared to perform the analysis of the distribution of diffraction fringes. Optimum experimental parameters were determined for which good susceptibility of the graphene paper to laser processing was observed.

Magnetic Cell Centrifuge Platform Performance Study with Different Microsieve Pore Geometries.

The detection and analysis of circulating tumor cells (CTCs) plays a crucial role in clinical practice. However, the heterogeneity and rarity of CTCs make their capture and separation from peripheral blood very difficult while maintaining their structural integrity and viability. We previously demonstrated the effectiveness of the Magnetic Cell Centrifuge Platform (MCCP), which combined the magnetic-labeling cell separation mechanism with the size-based method. In this paper, a comparison of the effectiveness of different microsieve pore geometries toward MCCP is demonstrated to improve the yield of the target cell capture. Firstly, models of a trapped cell with rectangular and circular pore geometries are presented to compare the contact force using finite element numerical simulations. The device performance is then evaluated with both constant pressure and constant flow rate experimental conditions. In addition, the efficient isolation of magnetically labeled Hela cells with red fluorescent proteins (target cells) from Hela cells with green fluorescent protein (background cells) is validated. The experimental results show that the circular sieves yield 97% purity of the target cells from the sample with a throughput of up to 2 µL/s and 66-fold sample enrichment. This finding will pave the way for the design of a higher efficient MCCP systems.

Scanning Electron Microscopy of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles.

To explore morphological features of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs), we developed a protocol for scanning electron microscopy (SEM) of CTCs and tdEVs. CTCs and tdEVs were isolated by immunomagnetic enrichment based on their Epithelial Cell Adhesion Molecule (EpCAM) expression or by physical separation through 5 µm microsieves from 7.5 mL of blood from Castration-Resistant Prostate Cancer (CRPC) patients. Protocols were optimized using blood samples of healthy donors spiked with PC3 and LNCaP cell lines. CTCs and tdEVs were identified among the enriched cells by fluorescence microscopy. The positions of DNA+, CK+, CD45- CTCs and DNA-, CK+, CD45- tdEVs on the CellSearch cartridges and microsieves were recorded. After gradual dehydration and chemical drying, the regions of interest were imaged by SEM. CellSearch CTCs retained their morphology revealing various shapes, some of which were clearly associated with CTCs undergoing apoptosis. The ferrofluid was clearly distinguishable, shielding major portions of all isolated objects. CTCs and leukocytes on microsieves were clearly visible, but revealed physical damage attributed to the physical forces that cells exhibit while entering one or multiple pores. tdEVs could not be identified on the microsieves as they passed through the pores. Insights on the underlying mechanism of each isolation technique could be obtained. Complete detailed morphological characteristics of CTCs are, however, masked by both techniques.

Hierarchical Porous Polybenzimidazole Microsieves: An Efficient Architecture for Anhydrous Proton Transport via Polyionic Liquids.

Liquid-induced phase-separation micromolding (LIPSµM) has been successfully used for manufacturing hierarchical porous polybenzimidazole (HPBI) microsieves (42-46% porosity, 30-40 µm thick) with a specific pore architecture (pattern of macropores: ∼9 µm in size, perforated, dispersed in a porous matrix with a 50-100 nm pore size). Using these microsieves, proton-exchange membranes were fabricated by the infiltration of a 1H-3-vinylimidazolium bis(trifluoromethanesulfonyl)imide liquid and divinylbenzene (as a cross-linker), followed by in situ UV polymerization. Our approach relies on the separation of the ion conducting function from the structural support function. Thus, the polymeric ionic liquid (PIL) moiety plays the role of a proton conductor, whereas the HPBI microsieve ensures the mechanical resistance of the system. The influence of the porous support architecture on both proton transport performance and mechanical strength has been specifically investigated by means of comparison with straight macroporous (36% porosity) and randomly nanoporous (68% porosity) PBI counterparts. The most attractive results were obtained with the poly[1-(3H-imidazolium)ethylene]bis(trifluoromethanesulfonyl)imide PIL cross-linked with 1% divinylbenzene supported on HPBI membranes with a 21-µm-thick skin layer, achieving conductivity values up to 85 mS cm-1 at 200 °C under anhydrous conditions and in the absence of mineral acids.

Constructing Straight Polyionic Liquid Microchannels for Fast Anhydrous Proton Transport.

Polymeric ionic liquids (PILs) have triggered great interest as all solid-state flexible electrolytes because of safety and superior thermal, chemical, and electrochemical stability. It is of great importance to fabricate highly conductive electrolyte membranes capable to operate above 120 °C under anhydrous conditions and in the absence of mineral acids, without sacrificing the mechanical behavior. Herein, the diminished dimensional and mechanical stability of poly[1-(3H-imidazolium)ethylene]bis(trifluoromethanesulfonyl)imide has been improved thanks to its infiltration on a polybenzimidale (PBI) support with specific pore architecture. Our innovative solution is based on the synergic combination of an emerging class of materials and sustainable large-scale manufacturing techniques (UV polymerization and replication by microtransfer-molding). Following this approach, the PIL plays the proton conduction role, and the PBI microsieve (SPBI) mainly provides the mechanical reinforcement. Among the resulting electrolyte membranes, conductivity values above 50 mS·cm-1 at 200 °C and 10.0 MPa as tensile stress are shown by straight microchannels of poly[1-(3H-imidazolium)ethylene]bis(trifluoromethanesulfonyl)imide cross-linked with 1% of dyvinylbenzene embedded in a PBI microsieve with well-defined porosity (36%) and pore diameter (17 µm).