Sequencing Single RNA Molecules using a Nanofluidic Device with Dual In-Plane Nanopore Sensors and Immobilized Exoribonuclease Enzymes

Wednesday, 29 November 2023 at 09:00

Add to Calendar ▼2023-11-28 17:00:002023-11-28 18:00:00Europe/LondonIdentification of Different Subpopulations of Circulating Tumor Cells and Extracellular Vesicles in the Blood of Cancer Patients using MicrofluidicsLab-on-a-Chip and Microfluidics World Congress 2023 in Laguna Hills, CaliforniaLaguna Hills, CaliforniaSELECTBIOenquiries@selectbiosciences.com

With the development of next generation sequencing (NGS), the field of transcriptomics has seen tremendous advancements opening up opportunities for improved diagnostics of diseases such as cancers and infectious diseases. RNA sequencing enables measurement of single nucleotide variants (SNVs), insertions and deletions, detection of different transcript isoforms, splice variants, and chimeric gene fusions. Although NGS has been useful for unraveling RNA structure and function, several technical difficulties remain including the need for reverse transcription and PCR amplification, which can mask epitranscriptomic modifications. To address NGS issues, we are developing an exciting new sequencing technology called Exonuclease Time-of-Flight (X-ToF) for the label-free detection and identification of single molecules. The hypothesis behind X-ToF is, “individual molecules moving electrokinetically through a 2D nanotube will experience a time-of-flight (ToF) that is dependent upon its molecular identity.”  X-ToF is a nanofluidic device comprised of input/output channels, a nanoscale solid-phase enzymatic bioreactor, and a flight tube equipped with a pair of in-plane nanopores to measure the ToF of a single ribonucleotide monophosphate (rNMP). Each rNMP is produced from an unamplified RNA molecule being clipped with a processive exoribonuclease, XRN1. In this presentation we will discuss the high rate manufacturing of the X-ToF chip with sub-5 nm in-plane nanopores using nano-injection molding from a cyclic olefin polymer (COP) plastic. The in-plane pores are situated at either end of a nanochannel (50 x 50 nm; 10 µm long) that generates current transient signals to detect and deduce the identity of rNMPs. The ToF is dependent on the apparent electrophoretic mobility of the molecule. The identity is determined from the ToF, the current transient amplitudes, and dwell times using multi-parameter Principle Component Analysis (PCA) or machine learning. We will show the ability to detect (detection efficiency ~100%) single rNMPs with identification accuracies >99% in a single read. Finally, we will discuss the generation of solid-phase nano-bioreactors using XRN1, which can cleave ssRNA in the 5' to 3’ direction releasing single rNMPs that are detected and identified using the in-plane nanopore sensor. Unique properties of the immobilized enzyme will be presented in terms of its processivity and kinetic rate of cleaving single RNA molecules.

Identification of Different Subpopulations of Circulating Tumor Cells and Extracellular Vesicles in the Blood of Cancer Patients using Microfluidics

Tuesday, 28 November 2023 at 17:00

Add to Calendar ▼2023-11-28 17:00:002023-11-28 18:00:00Europe/LondonIdentification of Different Subpopulations of Circulating Tumor Cells and Extracellular Vesicles in the Blood of Cancer Patients using MicrofluidicsLab-on-a-Chip and Microfluidics World Congress 2023 in Laguna Hills, CaliforniaLaguna Hills, CaliforniaSELECTBIOenquiries@selectbiosciences.com

Liquid biopsies are an attractive source of biomarkers that can be used to manage a variety of cancer-related diseases. Two liquid biopsy markers found in blood are circulating tumor cells (CTCs) and extracellular vesicles (EVs). The challenge associated with using CTCs or EVs as biomarkers has been that affinity selection using anti-EpCAM antibodies only does not recapitulate the tumor microenvironment entirely providing limited predictive power of the assay. For example, CTCs expressing invasive phenotypes down-regulate epithelial antigens, such as the epithelial cell adhesion molecule – EpCAM. We developed a liquid biopsy selection strategy that employs plastic microfluidic chips modified with antibodies to select two different liquid biopsy marker subpopulations. In addition to the common marker used for positive selection (EpCAM), Fibroblast Activation Protein alpha (FAPa) expressing liquid biopsy markers were also selected. Using the dual selection strategy, both subpopulations are detected from patients with results showing better predictive value compared to selection in which only EpCAM is used. In this presentation, we will show two examples of EpCAM/FAPa selection to monitor response to therapy using CTCs in pancreatic cancer, and determining the best treatment option for breast cancer patients using EVs. The CTCs and EVs were selected using specially designed microfluidic chips that could be operated by a fluid handling robot to support large-scale clinical trials. In the case of CTCs, response to a PARPi therapy was serially monitored to determine treatment efficacy. Results indicated that CTCs were better in monitoring response to the PARPi compared to the serum marker, CA19-9, but only when using both CTC subpopulations. In addition, the CTCs could be sequenced using NGS without the need for single-cell picking. Both EpCAM and FAPa expressing EVs and their mRNA cargo were used to perform molecular subtyping of breast cancer patients. We found the four molecular subtypes (luminal A/B, HER2 enriched, and basal-like) could be successfully identified in patients only when using both EV subpopulations. The EV mRNA copy numbers were analyzed using the nCounter assay without requiring the need for an amplification step.