High Sensitivity Extended Nano-Coulter Counter for Detection of Viral Particles and Extracellular Vesicles.

We present a chip-based extended nano-Coulter counter (XnCC) that can detect nanoparticles affinity-selected from biological samples with low concentration limit-of-detection that surpasses existing resistive pulse sensors by 2-3 orders of magnitude. The XnCC was engineered to contain 5 in-plane pores each with an effective diameter of 350 nm placed in parallel and can provide high detection efficiency for single particles translocating both hydrodynamically and electrokinetically through these pores. The XnCC was fabricated in cyclic olefin polymer (COP) via nanoinjection molding to allow for high-scale production. The concentration limit-of-detection of the XnCC was 5.5 × 103 particles/mL, which was a 1,100-fold improvement compared to a single in-plane pore device. The application examples of the XnCC included counting affinity selected SARS-CoV-2 viral particles from saliva samples using an aptamer and pillared microchip; the selection/XnCC assay could distinguish the COVID-19(+) saliva samples from those that were COVID-19(-). In the second example, ovarian cancer extracellular vesicles (EVs) were affinity selected using a pillared chip modified with a MUC16 monoclonal antibody. The affinity selection chip coupled with the XnCC was successful in discriminating between patients with high grade serous ovarian cancer and healthy donors using blood plasma as the input sample.

Microfluidic Device for On-Chip Immunophenotyping and Cytogenetic Analysis of Rare Biological Cells.

The role of circulating plasma cells (CPCs) and circulating leukemic cells (CLCs) as biomarkers for several blood cancers, such as multiple myeloma and leukemia, respectively, have recently been reported. These markers can be attractive due to the minimally invasive nature of their acquisition through a blood draw (i.e., liquid biopsy), negating the need for painful bone marrow biopsies. CPCs or CLCs can be used for cellular/molecular analyses as well, such as immunophenotyping or fluorescence in situ hybridization (FISH). FISH, which is typically carried out on slides involving complex workflows, becomes problematic when operating on CLCs or CPCs due to their relatively modest numbers. Here, we present a microfluidic device for characterizing CPCs and CLCs using immunofluorescence or FISH that have been enriched from peripheral blood using a different microfluidic device. The microfluidic possessed an array of cross-channels (2-4 µm in depth and width) that interconnected a series of input and output fluidic channels. Placing a cover plate over the device formed microtraps, the size of which was defined by the width and depth of the cross-channels. This microfluidic chip allowed for automation of immunofluorescence and FISH, requiring the use of small volumes of reagents, such as antibodies and probes, as compared to slide-based immunophenotyping and FISH. In addition, the device could secure FISH results in <4 h compared to 2-3 days for conventional FISH.