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Analyzing Subcellular Liquid Biopsy Markers Using Microfluidic and Nanofluidic Devices
Thippala Gamage, Sachindra Shehani
Thippala Gamage, Sachindra Shehani
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Abstract
Liquid biopsies are gaining significant attention as an attractive alternative to invasive tissue biopsy, specifically when tumor tissue is insufficient or when the patient’s tumor is in the vicinity of a sensitive organ that limits frequent tumor sampling. Microfluidics and nanofluidics are contributing substantially to the biomedical field with miniaturized systems that integrate different operational modules into a single motherboard to deliver effective diagnostic and screening tools for precision medicine. They are an attractive alternative to the standard benchtop assay kits because of unique operational characteristics such as closed architecture to reduce contamination, applicability for automation, and the ability to allow downstream process integration. To fulfill these efforts, we are designing and developing robust and innovative technology that is conducive to point-of-care testing (POCT) and high-scale production at a low-cost using thermoplastics. Among liquid biopsy markers such as circulating tumor cells and extracellular vesicles, cell-free DNA (cfDNA) are blood-based liquid biopsy markers that are fragments of DNA ~ 160 bp long or multiples of this size, circulating in the peripheral blood as a result of events such as secretion, apoptosis, and necrosis. They are attractive biomarkers to investigate since they contain a plethora of genetic information directly associated with the parent cells; these biomarkers can be gathered quickly from minimally invasive techniques such as blood draws. Accordingly, circulating tumor DNA (ctDNA), which is a subclass of cfDNA released by tumor cells, carries clinically actionable mutations that are characteristic of both the primary and metastatic tumor cells from which they were released. Hence, they provide comprehensive mutational profiling that would otherwise be undetectable due to tumor tissue heterogeneity. These mutations such as single nucleotide polymorphisms (SNPs) which can be the origin of certain cancers, can be identified by mutation detection methods such as next-generation sequencing for effective in vitro diagnostics. Therefore, the development of efficient technologies in isolating these biomarkers from body fluids such as blood is of great importance for the progression of targeted therapy tailored to an individual’s genetic makeup, and for screening and disease monitoring in clinical settings. However, these mutational profiling methods of rare biomarkers demand high-quality inputs with minimum contamination to reduce signal background and increase the sensitivity of the analysis. Yet, the availability of effective methods for the isolation of these rare biomarkers indicative of various diseases is limited. The existing commercial methods typically involve extensive sample handling that depends on skilled operators. Hence, due to these analytical and preanalytical factors, there is high interlaboratory variability seen which lowers the reproducibility of such assays targeting liquid biopsy biomarkers. Moreover, due to the low sample input, reported microfluidic assays suffer from the lack of enrichment of rare biomarkers that exist in the presence of high background. Thus, in this dissertation, we first present an efficient microfluidic assay (> 90% recovery in 100-700 bp and > 70 % recovery in 50 bp with 90% recovery in 100-700 bp and > 70 % recovery in 50 bp with 70 % recovery in 50 bp with < 0.01% gDNA contamination) for the extraction of cfDNA that can be coupled to downstream molecular analysis methods such as quantification by real-time PCR and mutation detection by Ligase detection reaction (LDR) and capillary electrophoresis to search for rare ctDNA fragments. We further developed this effort to negate the need for offline analysis by introducing nanofluidic devices that can perform solid phase LDR (spLDR) reactions and identify the different LDR products that are indicative of SNPs by their time-of-flight (TOF) measurements via resistive pulse sensing (RPS). Hence, in chapter three we introduce the nanosensor that is capable of performing the spLDR in its pixel arrays by providing solid support and subsequently separating unlabeled oligonucleotides of different lengths resulting from spLDR by TOF nanopore-based single molecule electrochemical sensing through dual in-plane nanopores. In the same chapter, we also introduce a novel method to tune the nanopore size to increase the S/N ratio for single-molecule sensing and demonstrate an increase in current amplitude by 3.5-fold via high throughput ? DNA translocation. In future directions, we also present a custom-built thermocycler that is capable of effectively transferring thermal energy into the microfluidic reactors to perform on-chip PCR and spLDR reactions. With the emergence of the SARS-CoV-2 pandemic, we addressed the drawbacks of the existing tests that hinder their large-scale global availability by expanding our technology to develop a surface functionalized microfluidic affinity isolation assay with DNA aptamers that effectively identify active SARS-CoV-2 particles in non-invasive and self-collectible saliva samples (100% clinical specificity and 95% clinical sensitivity). In chapter 4, we present the abovementioned reagent-less enrichment technology that can identify infectious COVID-19 patients and can also be rapidly adapted to test for other viruses, their variants, and extracellular vesicles by using alternative affinity agents. Furthermore, we introduce a high-sensitivity extended nano Coulter counter (XnCC) device in chapter 5 that is based on the principle of RPS for rapid label-less enumeration of the affinity-isolated virus particles (VPs) that are hydrodynamically and electrokinetically shuttled to the XnCC. The device consists of five parallel in-plane nanopores with a negative bias voltage applied across it to monitor the change in current using a miniaturized trans-impedance amplifier (TIA) circuit. To improve the throughput and the concentration limit of detection of the device, we also discuss modifying the fluidic network to have 25 nanopores in the future directions section. Moreover, we discuss the integration of the microfluidic affinity device and the XnCC presented in chapter 4 and chapter 5 into a portable instrument with miniaturized electronics, and a fluidic cartridge for rapid (<20 min) at-home or POC testing strategies.
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Date
2022-12-31
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University of Kansas
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Keywords
Analytical chemistry, Biomedical engineering, Nanotechnology, cell-free DNA, COVID-19, Extracellular vesicles, Microfluidics, Nanofluidics, SARS-CoV-2