Single Molecule Particle Analysis using Nanotechnology

Abstract

Nanotechnology is the area of science that involves creation of devices/materials or systems in the nanometer scale. The last few decades have seen an increasing demand for rapid, sensitive, and cheaper diagnostic tools in healthcare. Advances in fabrication technologies have led to more miniaturized systems that are satisfying the promise of “micro total analysis” or “lab-on-chip” systems by facilitating the integration of multiple processing steps into a single device or multiple task-specific devices into a fluidic motherboard (i.e., modular microfluidics). The field of nanotechnology has the ability to revolutionize medical diagnostics by facilitating point-of-care testing with greater sensitivity even at the single molecule level. This allows for the screening of diseases at an early stage by identifying biomarkers of the diseases that are in extremely low concentrations in the blood (i.e., liquid biopsy). To this realization, we have used thermoplastics as our choice of material to fabricate microfluidic/nanofluidic hybrid systems that can evaluate how well a patient responds to chemotherapy, identify single nucleotide polymorphisms that cause major life threatening diseases such as stroke and caner, and development of nanofluidic devices to enumerate SARS CoV-2 viral particles that causes the novel coronavirus of 2019. We developed a high-throughput nanofluidic circuit on which single DNA molecules can be stretched to near their full contour length in nanochannels (<100 nm). Patients with cancer undergoing chemotherapy have more oxidative damage in their DNA compared to a healthy individual, which is an indicator of their response to therapy. We tested the device using calf thymus DNA standards labelled with a bis-intercalating dye and the abasic sites were labelled with another dye. Thus, the DNA molecules that were stretched in the nanochannels were parked and visualized using a fluorescent microscope. The abasic sites that were labelled were identified with their position in the DNA and the number of abasic sites per 105 nucleotides identified. This technique can be effectively used on samples having mass limits (picograms range) and where PCR cannot be utilized. Higher the number of abasic sites, better the response of the patient to chemotherapy, such as doxorubicin for breast cancer patients. While this nanofluidic circuit was used only to visualize the abnormalities in DNA, the next device we developed, called the nanosensor, facilitates the integration of multiple processes into a single device. The nanosensor was used to identify point mutations in DNA or mRNA responsible for diseases such as cancer and stroke, respectively. The device featured 8 pixel array populated with 1 µm pillars, which act as a solid support for Ligase Detection Reactions (spLDR) that can identify a single nucleotide mutation in a DNA from a large majority of wild type DNA. The spLDR can also identify mRNA transcripts from the design of spLDR primers that specifically recognize a unique transcript. The reaction is performed on the pixel arrays and the products are subsequently shuttled into nanometer flight tubes featuring two in-plane nanopores that act as resistive pulse sensors (RPS) to generate a current drop as the products pass through these pores. The time-of-flight (TOF) between the pores in series are used to distinguish between normal and mutated DNA, thus acting as a diagnostic appropriate for the precision medicine initiative. We were able to successfully fabricate the device, run COMSOL simulations to test operation using both hydrodynamic and electrokinetic flows, which were verified via experimentation to establish the functionality of the device to perform the above mentioned processes. The hydrodynamic flow operations used for spLDR was tested using Rhodamine B and the electrokinetic flow to inject the products of the spLDR into the flight tube was tested using oligonucleotides (25mer). Further, plastic-based nanofluidic devices were extended to detect the presence of SARS-CoV-2 viral particles using a nanopore of 350 nm in effective diameter, which has called a nano-coulter Counter (nCC). Briefly, saliva samples containing the viral particles were run through a microfluidic affinity chip containing pillars with surface-immobilized aptamers specific to the SARS-CoV-2 particles. The captured viral particles were released from the microfluidic chip using a blue light and the elute containing only the SARS viral particles were sent to the nCC, which used the RPS technique to count the number of particles. We designed multiple iterations of the nCC and used COMSOL simulations to guide device development. Using the combined principle of hydrodynamic and electrokinetic flow to introduce the viral particles into the nCC, we were able to detect patients with COVID-19 as well as estimate the viral load in SARS CoV-2 standards based on the frequency of the signals generated by correlating the results to a calibration curve. Thus, this combined multi-chip process can diagnose COVID-19 in <20 min thus venturing as an in-home diagnostic kit in the future by automating the operations into a hand-held device

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