Novel thermoplastic microvalves based on an elastomeric cyclic olefin copolymer.

Microfluidic systems combine multiple processing steps and components to perform complex assays in an autonomous fashion. To enable the integration of several bio-analytical processing steps into a single system, valving is used as a component that directs fluids and controls introduction of sample and reagents. While elastomer polydimethylsiloxane has been the material of choice for valving, it does not scale well to accommodate disposable integrated systems where inexpensive and fast production is needed. As an alternative to polydimethylsiloxane, we introduce a membrane made of thermoplastic elastomeric cyclic olefin copolymer (eCOC), that displays unique attributes for the fabrication of reliable valving. The eCOC membrane can be extruded or injection molded to allow for high scale production of inexpensive valves. Normally hydrophobic, eCOC can be activated with UV/ozone to produce a stable hydrophilic monolayer. Valves are assembled following in situ UV/ozone activation of eCOC membrane and thermoplastic valve seat and bonded by lamination at room temperature. eCOC formed strong bonding with polycarbonate (PC) and polyethylene terephthalate glycol (PETG) able to hold high fluidic pressures of 75 kPa and 350 kPa, respectively. We characterized the eCOC valves with mechanical and pneumatic actuation and found the valves could be reproducibly actuated >50 times without failure. Finally, an integrated system with eCOC valves was employed to detect minimal residual disease (MRD) from a blood sample of a pediatric acute lymphoblastic leukemia (ALL) patient. The two module integrated system evaluated MRD by affinity-selecting CD19(+) cells and enumerating leukemia cells via immunophenotyping with ALL-specific markers.

Circulating Tumor Cell Subpopulations Predict Treatment Outcome in Pancreatic Ductal Adenocarcinoma (PDAC) Patients.

There is a high clinical unmet need to improve outcomes for pancreatic ductal adenocarcinoma (PDAC) patients, either with the discovery of new therapies or biomarkers that can track response to treatment more efficiently than imaging. We report an innovative approach that will generate renewed interest in using circulating tumor cells (CTCs) to monitor treatment efficacy, which, in this case, used PDAC patients receiving an exploratory new therapy, poly ADP-ribose polymerase inhibitor (PARPi)-niraparib-as a case study. CTCs were enumerated from whole blood using a microfluidic approach that affinity captures epithelial and mesenchymal CTCs using anti-EpCAM and anti-FAPα monoclonal antibodies, respectively. These antibodies were poised on the surface of two separate microfluidic devices to discretely capture each subpopulation for interrogation. The isolated CTCs were enumerated using immunophenotyping to produce a numerical ratio consisting of the number of mesenchymal to epithelial CTCs (denoted "Φ"), which was used as an indicator of response to therapy, as determined using computed tomography (CT). A decreasing value of Φ during treatment was indicative of tumor response to the PARPi and was observed in 88% of the enrolled patients (n = 31). Changes in Φ during longitudinal testing were a better predictor of treatment response than the current standard CA19-9. We were able to differentiate between responders and non-responders using ΔΦ (p = 0.0093) with higher confidence than CA19-9 (p = 0.033). For CA19-9 non-producers, ΔΦ correctly predicted the outcome in 72% of the PDAC patients. Sequencing of the gDNA extracted from affinity-selected CTC subpopulations provided information that could be used for patient enrollment into the clinical trial based on their tumor mutational status in DNA repair genes.

System Modularity Chip for Analysis of Rare Targets (SMART-Chip): Liquid Biopsy Samples.

Liquid biopsies are becoming popular for managing a variety of diseases due to the minimally invasive nature of their acquisition, thus potentially providing better outcomes for patients. Circulating tumor cells (CTCs) are among the many different biomarkers secured from a liquid biopsy, and a number of efficient platforms for their isolation and enrichment from blood have been reported. However, many of these platforms require manual sample handling, which can generate difficulties when translating CTC assays into the clinic due to potential sample loss, contamination, and the need for highly specialized operators. We report a system modularity chip for the analysis of rare targets (SMART-Chip) composed of three task-specific modules that can fully automate processing of CTCs. The modules were used for affinity selection of the CTCs from peripheral blood with subsequent photorelease, simultaneous counting, and viability determinations of the CTCs and staining/imaging of the CTCs for immunophenotyping. The modules were interconnected to a fluidic motherboard populated with valves, interconnects, pneumatic control channels, and a fluidic network. The SMART-Chip components were made from thermoplastics via microreplication, which lowers the cost of production making it amenable to clinical implementation. The utility of the SMART-Chip was demonstrated by processing blood samples secured from colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) patients. We were able to affinity-select EpCAM expressing CTCs with high purity (0-3 white blood cells/mL of blood), enumerate the selected cells, determine their viability, and immunophenotype the cells. The assay could be completed in <4 h, while manual processing required >8 h.