UC Irvine

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.