Carbon Nanotube Integrated Microdevices for Virus Enrichment And Detection
Open Access
- Author:
- Zhang, Wenlong
- Graduate Program:
- Biomedical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 22, 2019
- Committee Members:
- Siyang Zheng, Dissertation Advisor/Co-Advisor
Siyang Zheng, Committee Chair/Co-Chair
Pak Kin Wong, Committee Member
Jian Yang, Committee Member
Huaguang Lu, Outside Member
Daniel J Hayes, Program Head/Chair - Keywords:
- Infectious disease
Carbon nanotubes
Influenza
microdevice
nano martierials - Abstract:
- Viruses are the most important pathogens causing infectious diseases in both human and animal populations. Existing or newly emerging viral disease outbreaks are unpredictable from mild or less notable to severe or lethal and even devastating pandemic (e.g. 20-50 million victims in 1918 influenza pandemic) and traumatic economic loss (e.g. $490 billion in 1918 influenza pandemic), which impose significant healthcare, economic and social burden. Particularly, newly emerging infectious diseases (EIDs) pose the most catastrophic risk of epidemics due to the low levels of standing immunity in the host populations. Therefore, effective proactive virus detection is the key step for us to prevent viral disease outbreaks, and it is critical to developing target-independent methods for sensitive virus detection in both environmental and clinical samples. However, clinical and environmental samples often have low virus titer, high levels of contaminants, and relatively small volume. Especially in remote or rural areas with limited diagnostic service resources and poor logistic networks, it can pose major challenges in sample preparation to rapidly enrich pure and large amount of genome for virus detection and surveillance. The ability to detect the presence of a viral pathogen in samples is critically essential to monitor potential outbreaks and subsequent quarantine and control decisions. Thus, it will be a great contribution to develop a reliable and efficient virus detection method by using the most advanced nanotechnology techniques for the most effective detection of pathogenic viruses from real-world samples. My present research studies are mainly focused on the application of nano/microfluidics for the detection of viruses. The first work utilized the carbon-nanotube size-tunable-enrichment-microdevice (CNT-STEM) to enrich plant virus particles. In this study, CNT-STEM was used to detect plum pox virus (PPV) in field samples. Compared with conventional antibody-based virus enrichment method, CNT-STEM provides a choice for label-free detection. The effect of inter-tubular distances (ITD) and widths of CNTs forest on the enrichment efficiency for PPV were tested. Four groups of field samples were successfully tested. My research findings indicate that the CNT-STEM device can improve sensitivity by 6-99 times over the standard immunocapture followed with real-time polymerase chain reaction (qPCR) assay. During this first study, it was found that the dead-end design showed the limitation of sample capacity and the drawback in robustness, due to sample clogging and device damage. The dead-end device relied on a single layer structure to isolate the virions. Once it was broken, the whole device failed. The filtration process was also hard to control because it was driven by vacuum force. Moreover, after the filtration process of CNT-STEM, blades were used to retrieve the captured virus particles, which could be a biosafety issue for the operator in case of a high pathogenic virus strain. To overcome the limitations of the first-generation dead-end device, I improved the design of CNT-based microfluidic device and developed a self-regulating continuous flow device (CNT-3D-SPEM). By utilizing the micro spiral structure formed by size-tunable CNTs, this device can isolate the virions in a continuous flow configuration at similar or even higher efficiency than CNT-STEM while having much higher sample capacity for clinical samples. Importantly, the vertically aligned CNTs (VACNTs) in CNT-3D-SPEM has a layer of ZrO2 formed by atomic layer deposition (ALD), which enables the on-chip nucleic acid manipulation by CNT-3D-SPEM. By incorporating active nanoscale zirconia for controlled nucleic acid binding and release, the viral genomes can be purified and amplified on-chip to minimize material loss. Additionally, this characteristics of the CNT-3D-SPEM maintains sample biosafety to avoid personnel exposure to dangerous biohazards. In this study, the inter-tubular distance, the channel width, and the working flow rate of the CNT-3D-SPEM was optimized. Sample capacity was increased to 1-5 mL by CNT-3D-SPEM compared with CNT-STEM of which the sample capacity is ~200 µl. The CNT-STEM showed virus concentration of 8~22 times compared with the conventional direct RNA extraction method. The device can directly enrich virus concentration from the sample with end-to-end efficiency around 60% without any sample preparation off-chip. More importantly, CNT-3D-SPEM can be integrated with the portable MinION nanopore sequencer for virus detection and discovery. After isolation, purification, whole genome amplification and library preparation, the virus genome was sequenced by this portable sequencer with high rate of coverage. This revolutionary technology provides a point-of-care platform to efficiently isolate, purify and identify unknown viruses or emerging new virus strains that could be life-threatening.