Development of Analytical Methods for Intracellular Ions and Nucleic Acid Detection

細胞內離子分析和核酸檢測方法的開發

Student thesis: Doctoral Thesis

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Award date13 Nov 2023

Abstract

The smallest unit of life, or cell, comprises numerous complex components required to maintain its normal function, from various ions, macromolecules, and nucleic acids. A healthy cell needs to maintain the balance of these components, and deviation from the homeostasis condition will lead to the development of diseases. Conversely, numerous diseases cause dysregulation in the cellular process, disrupting the cellular machinery and leading to more damage to the cells, tissues and eventually to the whole biological system. The anomalies caused by diseases are reflected in the changes in cellular components, which may be exploited to offer a promising new perspective on studying or diagnosing diseases. The distinct metabolic change in cancers, for example, has been reported to cause the accumulation of sodium ions or elevated chloride channel expression in cancer cells. Thus, cells with aberrant ion concentrations might be an indicator of disease. Meanwhile, other diseases might be caused by infectious agents, which could damage the cells by hijacking cells to proliferate, secrete harmful toxins and interfere with the normal cellular mechanisms.

Despite the potential, intracellular ion measurement requires demanding preparations or bulky instrumentation, making adapting for disease diagnosis difficult. Moreover, standard cell preservation methods could impact the intracellular ion composition or require prolonged storage in low temperatures. Thus, we developed a method to measure intracellular ions in a single cell with a cell preservation method to accommodate cell measurement up to the single-cell level. Intracellular ions were quantified with laser-induced breakdown spectroscopy (LIBS) and X-ray fluorescence (XRF) for bulk sample measurement of heavier ions. The samples were prepared by a novel cell preservation based on freeze-drying, which preserved the cell content and fixed their position, as confirmed by the cell staining result before and after freeze-drying. Additionally, the dried samples can be stored for a prolonged time in room temperature conditions, providing an easy way to store numerous samples at a low cost. Single-cell ion measurement was successfully done on the LIBS platform to detect Li+, Na+, K+ and Mg2+, with Li+ quantification realized, capable of measuring 0.5 mM intracellular ion in one cell. Further tests on XRF and scanning electron microscope - energy dispersive X-ray (SEM-EDS) could quantify intracellular Cl- ion as low as 0.9 mM.

Aside from intracellular ions, nucleic acid detection is commonly used to diagnose various diseases, especially infection-related diseases. PCR is commonly used to detect specific nucleic acids in the sample. However, PCR requires a controlled environment and complex instruments, making it impractical for high test loads or field use. The isothermal amplification method, for example, loop-mediated isothermal amplification (LAMP), is simpler, more robust and can be performed at a constant temperature. The simplicity of the system enables the adaptation of nucleic acid detection along with other new technology, such as liquid marble (LM), which has shown its potential for biomedical purposes. The integration of LAMP with iron-oxide nanoparticles (IONPs)-coated magnetic LMs demonstrated that the LMs are manipulable, capable of detecting nucleic acid using magnetic-based remote heating up to 10 minutes faster than standard LAMP reaction in a thermocycler, and the results can be observed by color change, forgoing the need for expensive instruments and bringing it closer to point-of-care test (POCT) adaptation.

Although LAMP has proven advantageous for simple nucleic acid detection and is compatible with multiple device platforms, its color-based result interpretation is still prone to false positive cases, especially in a pandemic where many tests must be performed. Integration of lateral flow assay (LFA) with LAMP system allows rapid, low-cost SARS-CoV-2 detection with minimal instrument and adaptable for field use, with higher sensitivity than rapid antigen tests (RATs) but simpler operation than qPCR. However, most reports on LAMP-LFA do not consider the effect of the modified primers combination on LAMP-LFA reaction efficiency. Therefore, we designed a novel optimization method for the LAMP-LFA tag screening and successfully developed multiple LFA-adaptable primer sets with efficient amplification.

Further improvement on the outstanding issues of LAMP-LFA assay was achieved by multiplexing the individual LAMP-LFA primer sets in one reaction to simultaneously detect two SARS-CoV-2 target genes (E and N) or one target gene with a human gene target POP7 in a single tube reaction. The multiplexes permit sample quality checks through internal control tests or dual-target amplification to minimize the effect of new mutations in one of the targets. Herein, we reported the utilization of the amplicon melting temperature (Tm) curve for optimizing the multiplex reaction to achieve optimal amplification on both multiplex components. All multiplexes (E-POP7, N-POP7 and E-N) successfully detected both targets, and the melting curve optimization demonstrated a great potential for maximizing each multiplex component to achieve the most favorable condition for the multiplex reaction.