Integrated Modular Microfluidic Systems for the Affinity Selection and Comprehensive Analysis of Rare Liquid Biopsy Biomarkers

Abstract

Liquid biopsy is a less invasive sampling technique and is especially beneficial for diseased organs that are anatomically inaccessible for solid tissue biopsy such as the brain, lungs and pancreas. This sampling technique can be analytically challenging due to the multiple processing steps involved to complete the assay and also the mass limits imposed by liquid biopsy markers, such as circulating tumor cells (CTCs), extracellular vesicles (EVs), and cell-free DNA (cfDNA). New microfluidic-based technologies are rapidly evolving and have significantly improved the clinical sensitivity of liquid biopsy assays compared to their benchtop counterparts. Unfortunately many of these assays are only semi-automated and require extensive sample handling, skilled operators, number of cold-storage reagents and centralized laboratories to perform the experiment which generated difficulties when translating the assay into the point of care testing (POCT).Here, we are introducing an integrated modular microfluidic system for the comprehensive analysis of CTCs. The microsystem consists of a CTC selection module, impedance module and an imaging module to isolate, enumerate and immunophenotypic identification of CTCs, respectively. The three individual modules are integrated into a fluidic motherboard that is populated with membrane valves for fluidic directional control. The valving operation is automated to minimize the operator involvement. The individual modules and the motherboard are fabricated in plastics using low-cost replication techniques enabling the use of the system as inexpensive disposables in clinical testing. The individual modules can be easily replaced to reconfigure the system to analyze a different biomarker. We have introduced a 7-aminocoumarin photocleavable (PC) linker strategy to covalently attach the affinity agent (mAb) into the CTC selection module. The PC linker contains a central coumarin moiety and a primary amine group in one end and a carboxylic acid group in the other end. Primary amine group facilitating the surface conjugation and carboxylic acid group enables the covalent attachment of the antibody following EDC/NHS reactions. PC linker cleaves at visible blue light (400-450 nm) obviating the oxidative DNA damage and mRNA expression changes in the desired biomarker. The affinity selected biomarkers were released rapidly (2 min) and efficiently (>90%) using the PC linker strategy. The photo-released CTCs were directed towards the impedance module and finally to the imaging module for the cell enumeration, cell viability assessment and for the immunophenotypic identification. The clinical utility of the system was demonstrated by processing blood samples secured from colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC) patients.PC linker strategy is employed to covalently attach an aptamer specific to SARS-CoV-2 spike (S) protein. The viral particle selection chip was used to affinity select SARS-CoV-2 viral particles using saliva as the specimen. The selected viral particles were photo-released and directed towards the nano coulter counter (nCC) for enumeration. The PC linker-aptamer system could affinity select SARS-CoV-2 viral particles with a high recovery (94 %) and low non-specific attachments (90%) using the PC linker strategy. The photo-released CTCs were directed towards the impedance module and finally to the imaging module for the cell enumeration, cell viability assessment and for the immunophenotypic identification. The clinical utility of the system was demonstrated by processing blood samples secured from colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC) patients.PC linker strategy is employed to covalently attach an aptamer specific to SARS-CoV-2 spike (S) protein. The viral particle selection chip was used to affinity select SARS-CoV-2 viral particles using saliva as the specimen. The selected viral particles were photo-released and directed towards the nano coulter counter (nCC) for enumeration. The PC linker-aptamer system could affinity select SARS-CoV-2 viral particles with a high recovery (94 %) and low non-specific attachments (<4 %). The nCC device is able to detect viral particles in 103-108/mL range. The system was validated by processing 20 clinical samples. We are currently in a process of integrating these two devices into a single chip and develop a hand-held instrument for the early detection of SARS-CoV-2.