capable of absolute quantification of secreted proteins due to the
lack of calibration approaches. In droplet cytometry, cytokine-
capture beads are usually confined in the same droplet for the
capture of secreted proteins, which, however, leads to an uneven
distribution of fluorescence among individual droplets (intensity
peaks around individual beads), posing obstacles in calibration.
(2) The cell loading rates in individual droplets need to be further
optimized. In the process of forming droplets for single-cell encap-
sulation, as time goes by, there is a concern of cell sedimentation,
leading to variations in the concentrations of cell suspensions, and
thus the cell loading rates in the individual droplet cannot be finely
tuned. Although cell-suspension solutions with enhanced densities
(e.g., percoll) were used to address this issue, the addition of percoll
may lead to enhanced auto-fluorescence, bringing another concern
in fluorescence-based detections.5 Microengraving
As an enabling technique, microengraving proposed by the Love
group effectively isolates individual cells to realize the absolute
quantification of secreted proteins in a time sequence
[43, 95–107]. As shown in Fig.1d, single cells, suspended in
media, are deposited into a large array of polymeric microwells,
which are then sealed by a glass slides coated with pre-coated with
capture reagents, sequentially. The resulting glass slides are then
interrogated with laser-based fluorescence scanners to quantify the
protein copy numbers.
Based on microengraving, antigen-specific antibodies [95, 96,
105 ], cytokines [42, 96–98, 101, 102], and chemokines [43]
secreted at the single-cell level were reported. Although microen-
graving can enable absolute quantification of multiple proteins
secreted by individual cells, compared to flow cytometry, it requires
a careful manipulation of glass slides without stimulating or even
dislodging single cells within microwells. This process requires
highly trained technicians and cannot be further increased in the
throughput, easily translated to function in an automatic manner,
limiting its widespread applications in single-cell proteomics.6 Single-Cell Barcoding Microchip
Both flow cytometry and mass cytometry cannot realize the abso-
lute quantification of intracellular proteins at the single-cell level
due to the lacks of calibration approaches. To address this issue,
Heath et al. proposed large-array microchambers (single-cell bar-
coding microchips), which enables absolute quantification of both
cytosolic and membrane proteins at the single-cell level300 Beiyuan Fan et al.