Facile Fabrication of Flexible Polymeric Membranes with Micro and Nano Apertures over Large Areas.

Freestanding, flexible and open through-hole polymeric micro- and nanostructured membranes were successfully fabricated over large areas (>16 cm2) via solvent removal of sacrificial scaffolds filled with polymer resin by spontaneous capillary flow. Most of the polymeric membranes were obtained through a rapid UV curing processes via cationic or free radical UV polymerisation. Free standing microstructured membranes were fabricated across a range of curable polymer materials, including: EBECRYL3708 (radical UV polymerisation), CUVR1534 (cationic UV polymerisation) UV lacquer, fluorinated perfluoropolyether urethane methacrylate UV resin (MD700), optical adhesive UV resin with high refractive index (NOA84) and medical adhesive UV resin (1161-M). The present method was also extended to make a thermal set polydimethylsiloxane (PDMS) membranes. The pore sizes for the as-fabricated membranes ranged from 100 µm down to 200 nm and membrane thickness could be varied from 100 µm down to 10 µm. Aspect ratios as high as 16.7 were achieved for the 100 µm thick membranes for pore diameters of approximately 6 µm. Wide-area and uniform, open through-hole 30 µm thick membranes with 15 µm pore size were fabricated over 44 × 44 mm2 areas. As an application example, arrays of Au nanodots and Pd nanodots, as small as 130 nm, were deposited on Si substrates using a nanoaperture polymer through-hole membrane as a stencil.

On-the-Fly Phase Transition and Density Changes of Aqueous Two-Phase Systems on a Centrifugal Microfluidic Platform.

This paper describes on-the-fly physical property changes of aqueous two-phase systems (ATPS) in microfluidic devices. The properties and phases of the ATPS are modulated on-demand by using a centrifugal microfluidic device filled with poly(ethylene glycol) (PEG) and dextran (DEX) solutions. By use of the centrifugal force and active pneumatic controls provided by a centrifugal microfluidic platform (CMP), PEG-DEX mixtures are manipulated and processed inside simple thermoplastic microfluidic devices. First, we experimentally demonstrate an on-chip ATPS transition from two phases to a single phase and vice versa by dynamically changing the concentration of the solution to bring ATPS across the binodal curve. We also demonstrate a density modulation scheme by introducing an ATPS solution mixed with sodium diatrizoate hydrate, which allows to increase the liquid density. By adding precisely metered volumes of water, we spontaneously change the density of the solution on the CMP and show that density marker microbeads fall into the solution according to their corresponding densities. The measured densities of ATPS show a good agreement with densities of microbeads and analytical plots. The results presented in this paper highlight the tremendous potential of CMPs for performing complex on-chip processing of ATPS. We anticipate that this method will be useful in applications such as microparticle-based plasma protein analysis and blood cell fractionation.

Two-level submicron high porosity membranes (2LHPM) for the capture and release of white blood cells (WBCs).

A method modifying a vacuum-assisted UV micro-molding (VAUM) process is proposed for the fabrication of polymer two-level submicron high porosity membranes (2LHPM). The modified process allows for the fabrication of robust, large-area membranes over 5 × 5 cm2 with a hierarchical architecture made from a 200 nm-thick layer having submicron level pores (as small as 500 nm) supported by a 20 µm-thick layer forming a microporous structure with 10-15 µm diameter pores. The fabricated freestanding membranes are flexible and mechanically robust enough for post manipulation and filtration of cell samples. Very high white blood cell (WBC) capture efficiencies (≈97%) from healthy blood samples after red blood cell (RBC) lysis are demonstrated using a 3D-printed filter cartridge incorporated within these 2LHPM. A high release efficiency of ≈95% is also proved using the same setup. Finally, on-filter multistep immunostaining of captured cells is also shown.

Determination of the Relationship between Expression and Functional Activity of Multidrug Resistance-Associated Protein 1 using Scanning Electrochemical Microscopy.

Cancer cells can develop multidrug resistance (MDR) after prolonged exposure to chemotherapeutic drugs, which is a severe impediment to successful treatment. MDR is typically associated with transmembrane proteins mediating efflux of administered drugs, thereby keeping their intracellular concentration below the threshold required to kill cells. Although expression assays based on flow cytometry and immunostaining have shown that multidrug resistance-associated protein 1 (MRP1) is prevalent in many cancer types, the functional activity of this efflux pump is more difficult to elucidate, especially at the single-cell level. Herein, we report the measurement of MRP1 functional activity in individual cancer cells using scanning electrochemical microscopy (SECM). Cells were cultured onto plastic substrates containing selective adhesion sites. Optical microscopy and SECM revealed that cells adapt to the underlying surface, while MRP1 functional activity increases once the dimensions of the adhesive islands become smaller than those of the cell itself. Time-lapse SECM imaging revealed a suitable window of 30 min to complete each measurement before the cell undergoes blebbing, which is associated with a considerable increase in functional activity. Distinct cell populations were produced by performing a doxorubicin drug challenge on two parental cell lines (e.g., wild-type HeLa cells and MRP1-overexpressing HeLa-R cells). Expression and functional activity of MRP1 were determined using flow cytometry and SECM, and our findings show that these parameters do not directly correlate. This suggests that functional activity may represent a powerful indicator of a cancer cell's response to chemotherapeutic treatment and should improve our understanding of efflux mechanisms based on MRP1.

Fabrication of large-area polymer microfilter membranes and their application for particle and cell enrichment.

A vacuum assisted UV micro-molding (VAUM) process is proposed for the fabrication of freestanding and defect-free polymer membranes based on a UV-curable methacrylate polymer (MD 700). VAUM is a highly flexible and powerful method for fabricating low cost, robust, large-area membranes over 9 × 9 cm2 with pore sizes from 8 to 20 µm in diameter, 20 to 100 µm in thickness, high aspect ratio (the thickness of the polymer over the diameter of the hole is up to 15 : 1), high porosity, and a wide variety of geometrical characteristics. The fabricated freestanding membranes are flexible while mechanically robust enough for post manipulation and handling, which allows them to be cut and integrated as a plastic cartridge onto thermoplastic 3D microfluidic devices with single or double filtration stages. Very high particle capture efficiencies (≈98%) have been demonstrated in the microfluidic devices integrated with polymer membranes, even when the size of the beads is very close to the size of the pores of the microfilter. About 85% of the capture efficiency has been achieved in cancer cell trapping experiments, in which a breast cancer cell line (MDA-MB-231) spiked with phosphate-buffered saline buffer when the pore size of the filter is 8 µm and the device is operated at a flow rate of 0.1 mL min-1.

Combination of Mechanical and Molecular Filtration for Enhanced Enrichment of Circulating Tumor Cells.

Circulating tumor cells (CTCs) have been linked to cancer progression but are difficult to isolate, as they are very rare and heterogeneous, covering a range of sizes and expressing different molecular receptors. Filtration has emerged as a simple and powerful method to enrich CTCs but only captures cells above a certain size regardless of molecular characteristics. Here, we introduce antibody-functionalized microfilters to isolate CTCs based on both size and surface receptor expression. We present a 3D printed filtration cartridge with microfabricated polymer filters with 8, 10, 12, 15, or 20 µm-diameter pores. Pristine filters were used to optimize sample dilution, rinsing protocol, flow rate, and pore size, leading to >80% for the recovery of spiked cancer cells with very low white blood cell contamination (<1000). Then, filters were functionalized with antibodies against either epithelial cell adhesion molecule (EpCAM) or epidermal growth factor receptor (EGFR) and the cartridges were used to enrich breast (MDA-MB-231, MCF-7) and renal (786-O, A-498) cancer cells expressing various levels of EpCAM and EGFR. Cancer cells were spiked into human blood, and when using filters with antibodies specific to a molecular receptor expressed on a cell, efficiency was increased to >96%. These results suggest that filtration can be optimized to target specific CTC characteristics such as size and receptor expression and that a diverse range of CTCs may be captured using particular combinations of pore size, filtration parameters, and antibody functionalization.