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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2019)

Single cell analysis methods are becoming increasingly important since understanding how individual cells process information and respond to stimuli could lead to greater insight into cell heterogeneity. Soluble proteins play a critical role in controlling cell population behavior, but directly monitoring cell secretion is technically challenging. The goal of this work is to develop an extremely sensitive detection platform with multiplexing capabilities to quantify secreted proteins from single cells. Single cell microarrays have been developed that assess protein secretion from isolated single cells, but this platform is currently limited by low detection sensitivity in the ng/ml range (~60 pM) for most proteins. Thus, increasing detection sensitivity would significantly improve this analysis technique by enabling interrogation at earlier time points or in response to more subtle activation factors. Nanomaterial probes have been shown to provide remarkable detection capabilities in cell-based detection applications; especially luminescent quantum dots (QD), with their bright and photostable signals. In this work, we used QD in a sandwich immunoassay to detect secreted soluble cytokines at the single cell level. Using the QD-based detection method, the detection sensitivity was improved to 60 aM; 10E6-fold more sensitive compared to immunofluorescence detection. In single cell experiments, the QD-based platform increased the number of single cells that could be interrogated for TNF-α protein by 3-fold relative to a traditional organic fluorophore, improving detection threshold from 30 pM or 10,000 TNF-α molecules down to only 5 fM or 2 molecules. Furthermore, multiplexing capabilities were adapted to the QD-based platform by using multi-color QD to evaluate simultaneous protein secretion of up to 4 proteins (TNF-α, MCP-1, IL-10, and TGF-β). In single cell experiments, we were able to determine and quantify the type of proteins secreted from each single cell, and consequently, classify the cells as expressing pro-inflammatory or pro-healing characteristics. Additionally, the phasor approach to fluorescence lifetime imaging microscopy (FLIM) was used to assess and correct QD homoquenching.

  • 3 supplemental images
Cover page: Measuring protein secretion from single cells at single molecule resolution using quantum dots and imaging
  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2019)
Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of ∼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.
    Cover page: Pushing the limits of detection for proteins secreted from single cells using quantum dots
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2014)
    Oncolytic viruses (OVs) constitute a promising class of cancer therapeutics which exploit validated genetic pathways known to be deregulated in many cancers. To overcome an immune response and to enhance its potential use to treat primary and metastatic tumors, a method for liposomal encapsulation of adenovirus has been developed. The encapsulation of adenovirus in non-toxic anionic lecithin-cholesterol-PEG liposomes ranging from 140 to 180 nm in diameter have been prepared by self-assembly around the viral capsid. The encapsulated viruses retain their ability to infect cancer cells. Furthermore, an immunoprecipitation (IP) technique has shown to be a fast and effective method to extract non-encapsulated viruses and homogenize the liposomes remaining in solution. 78% of adenovirus plaque forming units were encapsulated and retained infectivity after IP processing. Additionally, encapsulated viruses have shown enhanced transfection efficiency up to 4 × higher compared to non-encapsulated Ads. Extracting non-encapsulated viruses from solution may prevent an adverse in vivo immune response and may enhance treatment for multiple administrations.
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