Silicon Nitride Microsieves Research Articles

Microengineered filters for efficient delivery of nanomaterials into mammalian cells

Intracellular delivery of nanomaterials into the cells of interest has enabled cell manipulation for numerous applications ranging from cell-based therapies to biomedical research. To date, different carriers or membrane poration-based techniques have been developed to load nanomaterials to the cell interior. These biotools have shown promise to surpass the membrane barrier and provide access to the intracellular space followed by passive diffusion of exogenous cargoes. However, most of them suffer from inconsistent delivery, cytotoxicity, and expensive protocols, somewhat limiting their utility in a variety of delivery applications. Here, by leveraging the benefits of microengineered porous membranes with a suitable porosity, we demonstrated an efficient intracellular loading of diverse nanomaterials to different cell types based on inducing mechanical disruption to the cell membrane. In this work, for the first time, we used ultra-thin silicon nitride (SiN) filter membranes with uniform micropores smaller than the cell diameter to load impermeable nanomaterials into adherent and non-adherent cell types. The delivery performance using SiN microsieves has been validated through the loading of functional nanomaterials from a few nanometers to hundreds of nanometers into mammalian cells with minimal undesired impacts. Besides the high delivery efficiency and improved cell viability, this simple and low-cost approach offers less clogging and higher throughput (107 cell min−1). Therefore, it yields to the efficient introduction of exogenous nanomaterials into the large population of cells, illustrating the potential of these microengineered filters to be widely used in the microfiltroporation (MFP) setup.

Microfiltration of oil in water (O/W) emulsions: Effect of membrane microstructure and surface properties

In this work, oil in water (O/W) emulsions prepared with a natural emulsifier and different oil phase concentrations were processed by using Anodisc, cellulose acetate (CA), polyester/nylon, silicon nitride microsieves and polycarbonate track etched (PCTE) membranes of different pore sizes (0.22–0.8μm). The effect of membrane characteristics on properties of the emulsions was assessed by droplet size distribution, ζ-potential and pH. Results suggest that membrane hydrophilicity is critical to avoid breaking during microfiltration of O/W emulsions. Partial or total retention of oil droplets from concentrated emulsions (10–30% oil) occurred with all 0.22μm membranes, except with the 0.26μm microsieves. PCTE membrane (uniform straight pores) performance was function of the d¯droplet/dpore ratio, i.e. (a) if d¯droplet/dpore<1, emulsions fully filtered, (b) if 1.0<d¯droplet/dpore≤1.5, partial breaking of larger droplets occurs and (c) if d¯droplet/dpore≥2, retention and probably coalescence of larger drops led to fouling and total pore blockage. Performance of CA membranes (interconnected pores) was rather related to the oil concentration and the number of droplets found in the emulsion. Breaking into the pores and coalescence onto the 0.22μm CA membrane surface started when processing a 5% oil emulsion with estimated concentration of ≥2.42×1012 oil droplets/mL.

Chemical modification of silicon nitride microsieves for capture of MCF-7 circulating tumor cells of breast cancer

Chemical modification of silicon nitride (SiN) microsieves with glutaraldehyde and 3-glycidoxypropyldimethylethoxysilane (GOPS) for bio-coupling with an antibody to capture MCF-7 circulating tumor cells of breast cancer is reported. In this research, the antibody monoclonal anti-cytokerantin–FITC with fluorescein isothiocyanate label was used due to its good selectivity to MCF-7 circulating tumor cells of breast cancer. Modification efficiency was determined by the variation of contact angle. The increase in contact angle of the microsieves treated with glutaraldehyde and GOPS indicated that the microsieve surface changed from hydrophilic to hydrophobic. These results confirmed the successful immobilization of glutaraldehyde and GOPS onto SiN microsieves. Antibody binding effect was evaluated by fluorescence microscopy. Fluorescent images exhibited that GOPS was more effective than the glutaraldehyde treatment. The GOPS-treated microsieves were then used for capture of MCF-7 cells in phosphate buffered saline (PBS). The fluorescent images proved that the surface modification of SiN microsieves with GOPS helped to increase the efficiency of MCF-7 capture.

Microfiltration of concentrated milk protein dispersions: The role of pH and minerals on the performance of silicon nitride microsieves

The flux behavior of 0.8 and 0.5 μm silicon nitride microsieves was studied in the pH range from 6 to 9 during microfiltration of milk protein dispersions supplemented with a mineral complex. The physicochemical properties of these dispersions were assessed by particle size distribution (PSD), zeta potential and rheological measurements. Microfiltration was carried out in a pilot cross-flow system where back pulsing was used. Results showed that increasing protein concentration from 30 to 90 g L−1 at pH ≈ 7 has a negligible effect on charge and PSD but increased viscosity and strongly reduced performance of both 0.5 and 0.8 μm microsieves. Adding minerals reduced further the 0.8 μm membrane flux and avoid filtration through the 0.5 μm membrane, despite the fact that PSD was ≈0.3 μm in all solutions. Raising the pH of dispersions at 9.0, which is higher than the isoelectric pH of both micelles and microsieves, drastically increased permeate flux of both membranes. Results from this work point out that even under quite high ionic strength conditions, the flux behavior of microsieves is mainly controlled by repulsive electrostatic protein-membrane interactions, hence, the pH of the dispersion and the membrane charge are critical when processing complex dairy products.

In situ 3D characterization of bidisperse cakes using confocal laser scanning microscopy

In situ 3D characterization of a filtration cake obtained with a model bidisperse suspension containing two sizes of fluorescent particles (1µm and 4.8µm) has been performed using Confocal Laser Scanning Microscopy (CLSM). The suspensions were dead-end filtered through 0.8µm pore diameters silicon nitride microsieves, under a constant flow rate. The membrane was put in a specific filtration chamber previously designed [1], allowing direct on-line microscopic observations of particles deposition. The final position of the first particles arriving on the membrane was investigated. Then, the online deposition of the following particles was recorded and, thus, the cake build-up could be analyzed layer by layer. In addition, to analyze the protective effect of a layer of large particles, 1µm particles were filtrated over a previously deposited cake formed with the 4.8µm microspheres.

Reinforced SIL-1 micromembranes integrated on chip: Application to CO2 separation

A novel 4-step microfabrication process is proposed in this work to prepare arrays of c-oriented silicalite (SIL-1) micromembranes on customized silicon nitride (Si3Nx) microsieves. The arrays are integrated on chip and their overall porosity values can be tuned from 1.6% to 19.9%. A low stress Si3Nx microfabricated sieve has been used as support to reinforce via mechanical interlocking and to reduce the effects of the residual stress during membrane processing. The secondary hydrothermal growth over the Si3Nx microsieves also changes the SIL-1 chemistry, improving its affinity towards CO2 adsorption. As a result, the SIL-1/Si3Nx micromembranes integrated on chip facilitate the preferential permeation of CO2 in CO2/H2 mixtures, showing a maximum CO2/H2 separation factor of 16.9 and a CO2 permeance of 8.2×10−7molm−2s−1Pa−1 at ambient conditions.

Study of the separation of yeast by microsieves: In situ 3D characterization of the cake using confocal laser scanning microscopy

In situ 3D characterization of Aquamarijn microsieves fouling was achieved using Confocal Laser Scanning Microscopy (CLSM). A filtration chamber allowing direct microscopic observation of microbial cell deposition and cake characterization, specially designed for in situ observations, was used. Fluorescent dyed Saccharomyces cerevisiae yeast suspensions were filtered through 0.8μm and 2μm pore diameters silicon nitride microsieves under constant flow rate. The on-line yeasts deposition was recorded and the cake construction was followed layer by layer. Based on the 3D image processing, cake properties (particle arrangement, homogeneity, thickness and porosity). The compressibility of the yeast cake was analyzed. Finally, cake removal efficiency was also studied during microsieve cleaning operation.

In situ 3D characterization of monodispersed spherical particle deposition on microsieve using confocal laser scanning microscopy

In situ 3D characterization of Aquamarijn microsieves fouling was achieved using Confocal Laser Scanning Microscopy (CLSM). A filtration chamber allowing direct microscopic observation of particle deposition and cake characterization, specially designed for in situ observations, was used. Suspensions of fluorescent monodisperse spherical particle (1µm and 4.8µm) were filtered under constant flow rate through 0.8µm and 2µm pore diameters silicon nitride microsieves. On-line images of particle deposition were recorded and the cake build up was followed layer by layer. Based on 3D image processing, cake morphology (particle arrangement, homogeneity, thickness and porosity) was characterized and correlated to permeability reduction recorded during the filtration experiments.

Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.

Structural ordering of casein micelles on silicon nitride micro-sieves during filtration

The paper reports on the structure and formation of casein micelle deposits on silicon nitride micro-sieves during the frontal filtration. The most frequent radius of the fractionated casein micelles we use is R=60 nm as detected by static light scattering (SLS) and atomic force microscopy (AFM). We estimate the size and size distribution of the casein micelles which pass through the micro-sieve during the filtration process. A sharpening of the size distribution at the beginning of the filtration process (t=40s) is followed by a broadening and a shift of the most frequent radii towards smaller sizes at later times (t=840 s). The size distribution of the micelles deposited on the micro-sieve during filtration is bimodal and consists of the largest and smallest micelles. At larger filtration times, we observe a shift of both deposited size classes towards smaller sizes. The atomic force micrographs of the reference sample reveal a tendency of the casein micelles to order in a hexagonal lattice when deposited on the micro-sieves by solution casting. The deposition of two size classes can be explained by a formation of a mixed hexagonal lattice with large micelles building up the basis lattice and smaller sizes filling octahedral and tetrahedral holes of the lattice. The accompanied compression with increasing thickness of the casein layer could result from preferential deposition of smaller sizes in the course of the filtration.

Microfiltration of whole milk with silicon microsieves: Effect of process variables

The impact of high-frequency cross-flow back-pulsing on microsieves permeation performance during the microfiltration of whole milk is described in this work. Silicon nitride microsieves (0.8 μm rectangular) combined with a dynamic permeate cross-flow back-pulsing technique to control fouling were used. Results showed that the transmembrane pressure (TMP pos) and the back-pulsing frequency were the process variables that most influenced microsieves performance. Permeation rates in the range of 5000 up to 27,000 L h −1 m −2 which are one order of magnitude higher than those reported for skim milk microfiltration were obtained depending on the process conditions selected. It was concluded that higher permeation rates are obtained when the back-pulse pressure, i.e., the negative TMP is set equal to the positive TMP, both at 150 mBar and the back-pulsing frequency at 15 Hz.

Microfiltration with silicon nitride microsieves and high frequency backpulsing

Silicon nitride microsieves are microfiltration membranes manufactured with techniques commonly used in the semi-conductor industry for wafer production. These membranes are characterised by the uniform pore size and the high porosity. Furthermore, due to their extremely thin selective layer and the relative open support structure, the clean water fluxes are usually very much higher (an order of magnitude of at least 100 times higher) than the conventional microfiltration. This extremely high fluxes causes the microsieves to be extremely susceptible to fouling and flux decline during filtration. As such, a new operating technique, i.e. high frequency backpulsing is introduced to keep the microsieve surface cleaned and to maintain the high fluxes. This paper discussed the mechanism used to create backpulses at very high frequency and the impact of such high frequency backpulses on particulate and nonparticulate fouling of the microsieves were studied with different model solutions.

Flux stabilization of silicon nitride microsieves by backpulsing and surface modification with PEG moieties

The influence of the surface properties of chemically modified silicon nitride microsieves on the filtration of protein solutions and defatted milk is described in this research. Prior to membrane filtrations, an antifouling polymer based on poly(ethylene glycol), poly(TMSMA-r-PEGMA) was synthesized and applied on silicon-based surfaces like silicon, silicon nitride, and glass. The ability of such coating to repel proteins like bovine serum albumin (BSA) was confirmed by ellipsometry and confocal fluorescence microscopy. In BSA and skimmed milk filtrations no differences could be seen between unmodified and PEG-coated membranes (decreasing permeability in time). On the other hand, reduced fouling was observed with PEG-modified microsieves in combination with backpulsing and air sparging.

Protein aggregate deposition and fouling reduction strategies with high-flux silicon nitride microsieves

Membrane morphology is a key factor in fouling. Characteristics such as a very thin selective layer, well-structured pore size and shape, and smooth surfaces would be ideal for low-fouling membranes. Microsieves, inorganic membranes with high and controlled porosity, offer all the previous features. The goal of this study was the investigation of the mechanism behind flux decline in crossflow filtration of bovine serum albumin (BSA) solutions with microsieve membranes. Subsequently, several strategies to reduce flux decline were also analyzed. The flux decline rate was pH-dependent, with a more severe reduction at pH close to the isoelectric point. In-line pre-filtering of the BSA solution led to stable fluxes, which indicated that pore blocking was responsible for the decrease in performance. Modelling of the filtration data confirmed a complete pore blocking mechanism. At pH where the decline was lower (6.8), water backflushing helped to recover the flux partially. Water forward flushing was only effective at relatively high permeation rates. As an alternative method, permeate backpulsing was investigated. The permeated volume was successfully increased and the flux decrease retarded when backpulsing was applied. If the permeate was backpulsed at higher frequencies, lower fouling rates were achieved, because aggregates could be removed faster from the microsieve surface.

The role of wetting on the water flux performance of microsieve membranes

Microsieve membranes are microfabricated devices that possess a high and controlled porosity. Due to the extremely high product throughput, microsieves are very sensitive systems in terms of flux decline during clean water measurements. The goal of this investigation was to study the influence of the surface properties of silicon nitride microsieves and the hydrodynamic parameters on the clean water flux performance. First, we studied the properties of bare silicon nitride dices. Contact angles and XPS measurements clearly demonstrated that silicon nitride experiences aging and variations in its surface properties, making it necessary to hydrophilize and homogenize the substrates. To understand the role of the surface properties on the water permeation, the water flux through unmodified, hydrophilized and hydrophobized silicon nitride microsieves was studied as a function of the wetting procedure and the presence of air in the feed liquid. For most surfaces, a good wetting protocol and the exclusion of contaminating particles and air from the system led to stable fluxes. Hydrophobized membranes presented low performance when they were poorly wetted. In these cases, air was massively deposited on the surface, since the low working pressures made the filtration sensitive to air bubbles. For highly hydrophobized membranes (contact angle, θ = 112°) no stable fluxes could be measured due to rapid dewetting.

Nanostructured Ion-Selective MCM-48 Membranes

Templated MCM-48 silica was prepared using CTAB as surfactant. The MCM-48 powders and thin films were characterized by different techniques. MCM-48 layers were deposited on macroporous α-alumina supports and silicon nitride microsieves. The water permeability of MCM-48 was compared with the permeability of conventional mesoporous γ-alumina membranes. The applicability of MCM-48 as ion-selective electric field-driven switchable interconnect for microfluidic devices was demonstrated.