Abstract
Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales and determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample.
Highlights
The correlation between the concentration of protein captured on the glass slide Γ ' (τ ) and the protein concentration on the surface of the single cell C0, which are calculated at a certain incubation time of the microengraving immunoassay, gives protein adsorption isotherms
Controlling mechanisms for protein adsorption dynamics change as functions of secretion rates, binding affinity K D and bulk concentration C 0* from diffusion to mixed kinetic-diffusion control
Protein bulk concentration is low, and the influence of molecular diffusion is more important in protein adsorption dynamics
Summary
The microengraving immunoassay is based on intaglio printing, in which a glass slide is temporarily sealed to the array of nanowells to capture proteins secreted by confined cells in both a multiplexed and quantitative manner. This immunoassay enabled identification and recovery of antigen-specific cells with highest secretion rates for clonal expansion [14,15]. When proteins are secreted from a single cell in microengraving immunoassay (Figure 1A), they diffuse through microwells to the sublayer of the sealing glass slide and bind onto it. Where τK is defined as characteristic kinetic time necessary for proteins in the sublayer to bind onto the surface of the glass slide in absence of molecular diffusion.
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