The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of ∼3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymer's damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the device's cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.

A circulating tumor cell (CTC) selection microfluidic device was integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for the detection of mutations contained within the genomic DNA of the CTCs. Molecular profiling of CTCs can provide important clinical information that cannot be garnered simply by enumerating the selected CTCs. We evaluated our approach using SW620 and HT29 cells (colorectal cancer cell lines) seeded into whole blood as a model system. Because SW620 and HT29 cells overexpress the integral membrane protein EpCAM, they could be immunospecifically selected using a microfluidic device containing anti-EpCAM antibodies immobilized to the walls of a selection bed. The microfluidic device was operated at an optimized flow rate of 2 mm s−1, which allowed for the ability to process 1 mL of whole blood in <40 min. The selected CTCs were then enzymatically released from the antibody selection surface and hydrodynamically transported through a pair of Pt electrodes for conductivity-based enumeration. The efficiency of CTC selection was found to be 96% ± 4%. Following enumeration, the CTCs were hydrodynamically transported at a flow rate of 1 μL min−1 to an on-chip electromanipulation unit, where they were electrophoretically withdrawn from the bulk hydrodynamic flow and directed into a receiving reservoir. Using an electric field of 100 V cm−1, the negatively charged CTCs were enriched into an anodic receiving reservoir to a final volume of 2 μL, providing an enrichment factor of 500. The collected CTCs could then be searched for point mutations using a PCR/LDR/capillary electrophoresis assay. The DNA extracted from the CTCs was subjected to a primary polymerase chain reaction (PCR) with the amplicons used for a ligase detection reaction (LDR) to probe for KRAS oncogenic point mutations. Point mutations in codon 12 of the KRAS gene were successfully detected in the SW620 CTCs for samples containing <10 CTCs in 1 mL of whole blood. However, the HT29 cells did not contain these mutations, consistent with their known genotype.

We present a critical review of microfluidic technologies and material effects on the analyses of circulating tumour cells (CTCs) selected from the peripheral blood of cancer patients. CTCs are a minimally invasive source of clinical information that can be used to prognose patient outcome, monitor minimal residual disease, assess tumour resistance to therapeutic agents, and potentially screen individuals for the early diagnosis of cancer. The performance of CTC isolation technologies depends on microfluidic architectures, the underyling principles of isolation, and the choice of materials. In this review, we present a critical review of the fundamental principles used in these technologies and discuss their performance. We also give context to how CTC isolation technologies enable downstream analysis of selected CTCs in terms of detecting genetic mutations and gene expression that could be used to gain information that may affect patient outcome.

In this manuscript, we discuss the development and clinical use of a thermoplastic modular microsystem for the high-throughput analysis of CTCs directly from whole blood. The modular system offers some innovative features that address challenges currently associated with many CTC platforms; it can exhaustively process 7.5 ml of blood in less than 45 min with recoveries >90%. In addition, the system automates the post-selection CTC processing steps and thus, significantly reduces assay turnaround time (from selection to enumeration <1.5 h as compared to >8 h for many reported CTC platforms). The system is comprised of 3 functional modules including; (i) a thermoplastic CTC selection module composed of high aspect ratio (30 μm × 150 μm) channels containing anti-EpCAM antibodies that is scalable in terms of throughput by employing channel numbers ranging from 50 to 320 – the channel number is user selected to accommodate the volume of blood that must be processed; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for the placement of released cells into a 2D array within a common imaging plane for phenotypic identification. To demonstrate the utility of this system, blood samples from patients with local resectable and metastatic pancreatic ductal adenocarcinoma (PDAC) were analyzed. We demonstrate the ability to select EpCAM positive CTCs from PDAC patients in high purity (>86%) and with excellent yields (mean = 53 CTCs per ml for metastatic PDAC patients) using our modular system. In addition, we demonstrate the ability to detect CTCs in PDAC patients with local resectable disease (mean = 11 CTCs per ml).

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges.