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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2017)

Nucleic acid amplification has applications in diagnostics, sequencing, genetic fingerprinting, among others. Currently nucleic acid amplification is treated as the “gold standard” method for several diagnostics; however, because of the multi-step protocols and the large equipment, these assays are lengthy and require laboratory settings. Digital nucleic acid amplifications assays developed have greatly improved several aspects of nucleic acid amplification by creating a more robust and sensitive assay. This is due to the reduction in background noise and the ability to effectively concentrate target analytes in nano- or picoliter volumes by compartmentalization of these samples. We were able to demonstrate a 69-fold fluorescence change in an isothermal nucleic acid amplification assay, with a >60% increase in fluorescence stability with elevated temperatures over the time course of the reaction, with the use of a unique dye combination of EvaGreen and hydroxynapthol blue (HNB). Due to the improvements in signal, we were able to demonstrate comparable results using a mobile phone based fluorescence plate reader as with that of a benchtop reader. The unique dye combination was then applied to a digital system, demonstrating signal improvements that are crucial to developing a robust assay, giving a higher efficiency (percentage of “on” wells closer to the theoretical value) and a larger difference in fluorescence intensities for “on” versus “off” wells. Lastly, we examined the mechanism of the dye combination to best determine additional ways of improving the signal generation. By sequestering EvaGreen, HNB allows amplification to proceed without interference. Additionally, a F�rster resonance energy transfer (FRET) interaction between the dye molecules, when DNA is absent from the solution, acts to lower the background fluorescence such that a greater fluorescence fold change occurs with DNA amplification. The EvaGreen and HNB have a highly-tuned binding affinity such that prior to DNA amplification, they have FRET interactions, and afterwards in the presence of large amounts of DNA, EvaGreen binding to DNA becomes more favorable. All of these developed technologies and methods work in conjunction to improve upon currently developed techniques for nucleic acid amplification in point of care settings.

    Cover page: Digital Loop-Mediated Isothermal DNA Amplification
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2015)
    A process to surface pattern polydimethylsiloxane (PDMS) with ferromagnetic structures of varying sizes (micrometer to millimeter) and thicknesses (>70 μm) is developed. Their flexibility and magnetic reach are utilized to confer dynamic, additive properties to a variety of substrates, such as coverslips and Eppendorf tubes. It is found that these substrates can generate additional modes of magnetic droplet manipulation, and can tunably steer magnetic-cell organization.
      Cover page: Flexible and Stretchable Micromagnet Arrays for Tunable Biointerfacing
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2019)
      Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications.
        Cover page: Computational cytometer based on magnetically modulated coherent imaging and deep learning.
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