Pushing the limits of detection for proteins secreted from single cells using quantum dots

Abstract

Single cell secretion studies have provided valuable insight into population heterogeneity, but low detection sensitivity requires cells to secrete thousands of molecules. We employed semiconductor quantum dots and a new single particle imaging approach to improve detection sensitivity to only a few secreted molecules per cell.

A Nanoparticle-Based Affinity Sensor that Identifies and Selects Highly Cytokine-Secreting Cells

“…This is an advance over standard single-cell analysis method such as microengraving where conventional sandwich immunofluorescence detection offers sensitivity of ∼1 ng/mL, or 10 pM, for most soluble proteins (Herrera et al., 2019) (for example, Love et al., 2006, reports the lowest detected cytokine concentration to be 4 ng/mL). Recently, microengraving with quantum dot (QD) nanomaterials as assay reporters, chemical amplification (more than one QD per antibody), and single-particle counting achieved the limit of detection of 60 aM for tumor necrosis factor-α (Herrera et al., 2019). This is about two orders of magnitude lower than reported in this work, pointing to possible improvements in the OnCELISA assay where chemical amplification and single-particle counting can also be applied.…”

Measuring single‐cell protein secretion in immunology: Technologies, advances, and applications

“…As already mentioned, one of the main advantages of using micro‐ or nanowells is their high sensitivity due to the small assay volumes, achieving detectable concentrations of secreted proteins within a reasonable timeframe (a few minutes to hours), even for low‐secreting cells. Further advances have been made to obtain increased sensitivity [97]. In microfluidic assays, the achieved sensitivity is generally determined by the used detection antibodies and their concentrations.…”

Chemical Analysis of Single Cells and Organelles

“…Herrera et al discuss the use of a similar on-chip sandwich immunoassay for the detection of TNF-α secretion by single cells. 204 To facilitate the assay, luminescent quantum dots were first modified with tetrazine after which they were conjugated with detection antibodies, modified with trans-cyclooctene. The cycloaddition product has a small footprint, allowing multiple nanoparticles to attach to a single protein target, which amplifies the signal.…”