Highly efficient capture and enumeration of low abundance prostate cancer cells using prostate‐specific membrane antigen aptamers immobilized to a polymeric microfluidic device

Abstract

Prostate tumor cells over-express a prostate specific membrane antigen (PSMA) that can be used as a marker to select these cells from highly heterogeneous clinical samples, even when found in low abundance. Antibodies and aptamers have been developed that specifically bind to PSMA. In this study, anti-PSMA aptamers were immobilized onto the surface of a capture bed poised within a poly(methyl methacrylate), PMMA, microchip, which was fabricated into a high throughput micro-sampling unit (HTMSU) used for the selective isolation of rare circulating prostate tumor cells resident in a peripheral blood matrix. The HTMSU capture bed consisted of 51 ultra-high aspect ratio parallel curvilinear channels with a width similar to the prostate cancer cell dimensions. The surface density of the PSMA-specific aptamers on a UV-modified PMMA microfluidic capture bed surface was determined to be 8.4 × 10 12 molecules/cm 2 . Using a linear velocity for optimal cell capture in the aptamer-tethered HTMSU (2.5 mm/s), a recovery of 90% of LNCaP cells (prostate cancer cell line; used as a model in this example) was found. Due to the low abundance of these cells, the input volume required was 1 mL and this could be processed in approximately 29 min using an optimized linear flow rate of 2.5 mm/s. Captured cells were subsequently released intact from the affinity surface using 0.25% (w/v) trypsin followed by counting individual cells using a contact conductivity sensor integrated into the HTMSU that provided high detection and sampling efficiency (~100%) and did not require staining of the cells for enumeration.

Efficient elusion of viable adhesive cells from a microfluidic system by air foam

“…[1][2][3][4][5][6][7] Despite allowing for the rapid isolation of these clinically relevant cell types, microfluidic methods are facing the challenge of releasing and recovering the isolated target cells for further biological analysis. 8 Due to the nature of low concentration of CTCs in whole cellular population after separation process, the acceptable efficiency of releasing cells should approach 100% for broader applications such as sensitively monitoring therapy response, diagnosis of early stage cancer, and analysis of genetic makeup.…”

“…14,15 For gradient elution methods, although a proteolytic enzyme combined with surfactant can release the captured cells, the surfactant would potentially disrupt the cell membrane and cause the degradation of surface markers, thus limiting the downstream biological analysis of cells. 1,2,16 Moreover, exposed to chemical microenvironments different from physiological level, the phenotypic and functional information of the target cells may be altered. 17 For flow-induced cell detachment, studies found in open literature show that the recovery efficiency of the captured cells ranges from 60% to 80%.…”

Microfluidic Devices for Blood Fractionation

“…Moreover, there is a risk of losing the most aggressive CTCs subpopulation due to epithelial to mesenchymal transition (EMT) process, which leads to a down-regulation of epithelial surface markers and thus the inability for CTCs to bind efficiently [96,97]. Recently, it has been demonstrated that aptamers (single-stranded nucleic acid oligomers) could be used as probes to isolate CTCs with high affinity and specificity [98,99]. As aptamers are synthesized by an in vitro process known as cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment), the surface molecular information of CTCs are not required and different aptamer probes can be generated for any type or subpopulation of CTCs.…”

Bioinspired Pollen‐Like Hierarchical Surface for Efficient Recognition of Target Cancer Cells

“…Polymethyl methacrylate (PMMA), instead of PDMS, was selected for the secondary replication due to its convenience of surface modification and low nonspecificity of cell adhesion. [19] The viscous PMMA prepolymer containing UV curing agent was poured onto the negative PDMS template, and then exposed to UV light for solidification. [20] The positive PMMA replica agreed well with the negative replica ( Figure 2c; Figure S5 in the Supporting Information).…”