[108–126]. As shown in Fig.1e, the single-cell barcoding micro-
chips include thousands of individually addressed microchambers
to realize single-cell trapping, lysis, and protein capture by
pre-printed antibodies on the surface of the chambers. Then the
fluorescent immunosandwich approach is used for absolute quanti-
fication. This powerful approach has been used to quantify multiple
key cytokines at the single-cell level, reporting cellular heterogene-
ities in both immunology [111] and tumor biology [114, 115].
As an approach capable of absolute quantification of both
surface and cytosolic proteins of single cells in large arrays, there
are two practical concerns. When running experiments with this
approach, it is possible that large portions of single cells may stick to
the bottom surfaces of cellular inlets, and thus this technique may
not be directly used to characterize rare cells (e.g., circulating
tumor cells). In addition, the potential of trapping multiple cells
within individual chambers is a key concern. If a manual check of
the number of cells within each chamber is conducted, it is defi-
nitely time-consuming. If a very low concentration of cell suspen-
sion is used in the cell loading step, a large number of empty
chambers can be generated. Thus this approach suffers from limited
throughput, although instrumentation can address this issue to an
extent.7 Discussion and Future Development
In this study, we summarized key technical approaches enabling the
quantification of single-cell proteins. Although significant improve-
ments have been made within the last decade, there are still several
important issues that should be addressed in the near future.
The absolute quantification of intracellular proteins in a high-
throughput manner is a key issue in the field of single-cell proteo-
mics. Currently, both flow cytometry and mass cytometry cannot
produce absolute copy numbers of interested intracellular proteins
due to the lack of calibration beads. In addition, the step of intra-
cellular staining in flow or mass cytometry also brings concerns that
not all of the targeted intracellular proteins are bound with
corresponding antibodies. As an enabling technique, single-cell
barcoding microchip can quantify intracellular proteins, which,
however, suffers from limited throughput. Thus, more efforts are
suggested to be devoted in this field to realize the absolute quanti-
fication of single-cell intracellular proteins to construct single-cell
data, functioning as a reference for future analysis.
Other issues in the field of single-cell proteomics may include
detection multiplexity, resolution, and throughput. With the help
of mass cytometry, 100 of proteins can be simultaneously charac-
terized, which provide key insights in understanding cellular signal
transductions. Further increases in the type of proteins that can beSingle-Cell Protein Assays: A Review 301