Author ORCID Identifier
Semester
Fall
Date of Graduation
2024
Document Type
Dissertation
Degree Type
PhD
College
Eberly College of Arts and Sciences
Department
Chemistry
Committee Chair
Peng Li
Committee Member
Stephen J. Valentine
Committee Member
Glen P. Jackson
Committee Member
Oluwatobi Odeleye
Committee Member
Lori Hazlehurst
Abstract
Nucleic acid tests are key tools for the detection and diagnosis of many diseases. In many cases, the amplification of nucleic acids is required to reach a detectable level. Although nucleic acid tests have great success, there is a need to make nucleic acid amplification tests more accessible to a point-of-care (POC) setting for diagnosis, prognosis, and monitoring of infectious diseases. Traditionally, these tests are performed through bulk Polymerase Chain Reaction (PCR) and are monitored in real time with a change in fluorescence intensity. With these bulk testing methods, only a qualitative or semi-quantitative result is possible. For more exact quantification of results, digital nucleic acid amplification tests can be utilized. Although exact quantification is a benefit for monitoring and prognosis for infectious diseases, these tests are performed in large laboratory settings that can be controlled. As a result, there are limitations for applying the technology to POC settings due to three main aspects: compartmentalization, amplification, and detection.
There are two main categories for compartmentalization of a bulk reaction solution for digital tests, microwells and droplets. With microwell arrays, the individual reaction portions are the same in size leading to simple quantification of results. However, the analysis is limited to only the number of microwells used in the assay. In contrast, any number of droplets can be used for more accurate results, but it is difficult to ensure all droplets are monodisperse. To provide a simple and portable system for droplet generation, we have developed the vibrating sharp-tip capillary. With the vibrating sharp-tip capillary device simple, tunable, on-demand, and monodisperse droplet generation is possible with a large range of droplet sizes from a single device. In addition, the large range of droplet sizes possible allows for a high dynamic range assay. Because of the development of the vibrating sharp-tip capillary, the compartmentalization of nucleic acids for digital assays is possible at POC.
The next aspect to address is the amplification of nucleic acids. Traditionally, nucleic acid amplification tests have been performed with PCR, however there are several isothermal amplification techniques available for POC applications. Loop-mediated isothermal amplification (LAMP) is considered the standard for isothermal amplification of nucleic acids. With LAMP, loop structures are formed with the use of 4-6 primer sequences. When the loop structures form, amplification of the nucleic acid target occurs. We have incorporated LAMP with the vibrating sharp-tip capillary device for a high dynamic range (~2 to 6000 copies/µL) digital droplet loop-mediated isothermal amplification (ddLAMP) system. As LAMP is isothermal, only a simple heat source is needed for implementation at POC. However, the method still utilizes fluorescence detection, indicating further work was needed to adapt the detection method to POC.
To further improve the ddLAMP assay, cellphone-based detection was investigated. The LAMP reaction is unique among isothermal amplification techniques, due to the formation of a precipitate byproduct, Magnesium Pyrophosphate. We found that this precipitate is visible under a bright-field microscope, with a direct match to the fluorescence images. Furthermore, when a simple microscope lens was attached to a cellphone, the precipitate was visible in the center of the droplets. The presence of the precipitate was more noticeable with large droplet diameters larger than approximately 250 μm. The precipitate became less distinguishable with smaller droplet diameters. As a result, the minimum droplet diameter usable with current method is at approximately 100 μm, although droplet diameters at approximately 70 μm were detected in some instances. When the same droplet samples were analyzed using fluorescence and bright field cellphone detection, a similar dynamic range was determined. For fluorescence detection, the dynamic range was determined to be approximately 3 to 650 copies of DNA target per microliter. In comparison, the dynamic range for cellphone detection was determined to be 2 to 700 copies of DNA target per microliter. With further improvements, the dynamic range could be pushed even further allowing for a high dynamic range ddLAMP system for use at POC.
Although LAMP is isothermal and requires only a simple heat source, the temperature required is relatively high (approximately 65 °C). As a result, some softer isothermal amplification techniques have been developed such as Recombinase Polymerase Amplification (RPA). In this method, the sample is held at a constant low temperature (approximately 40 °C) for 20 to 25 minutes, compared to 2.5 hours for PCR or 1 hour for LAMP. As a result, RPA could greatly reduce the assay time in POC settings, allowing for more immediate treatment. However, there are some issues to overcome for implementation of digital RPA at POC. RPA is a chemically initiated process and can begin at room temperature with the addition of the Magnesium Acetate coenzyme. This limits the capabilities of sample loading and compartmentalization for digital analysis. To overcome the compartmentalization issue, a vibrating sharp-tip theta capillary device was developed. With the theta capillary device, two separate solutions are introduced through the individual channels of the device. As the droplets form at the tip of the device, the two solutions mix to form the overall reaction solution.
In addition to RPA, the theta capillary device has the potential to implement many other methods of digital analysis, such as digital Enzyme-Linked Immunosorbent Assay (ELISA). With a sandwich-based digital ELISA, magnetic beads would be treated with a capture antibody, the target, and a detection antibody prior to loading into one channel of the theta capillary device. The substrate solution would then be loaded into the other channel of theta capillary device, allowing for digital detection of the target. In addition, the reaction of Catalase and Hydrogen Peroxide to form oxygen gas could be used in conjunction with the theta capillary. The Catalase enzyme could be used to tag the target nucleic acid sequence or protein and distributed in solution loaded into one channel. A Hydrogen Peroxide solution could then be added to the second channel. As the two solutions mix, oxygen gas is formed and trapped within the droplets. It would also be a benefit to enclose the droplet reservoirs, as under current conditions, the droplet reservoirs are open which can lead to spillage of mineral oil.
Recommended Citation
Fike, Bethany J., "A Portable, Digital Nucleic Acid Amplification Test for Use at the Point of Care" (2024). Graduate Theses, Dissertations, and Problem Reports. 12703.
https://researchrepository.wvu.edu/etd/12703