A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection

Abstract

We report a high-sensitivity cell secretome detection method using competitive immuno-aggregation and a micro-Coulter counter. A target cell secretome protein competes with anti-biotin-coated microparticles (MPs) to bind with a biotinylated antibody (Ab), causing decreased aggregation of the functionalized MPs and formation of a mixture of MPs and aggregates. In comparison, without the target cell secretome protein, more microparticles are functionalized, and more aggregates are formed. Thus, a decrease in the average volume of functionalized microparticles/aggregates indicates an increase in cell secretome concentration. This volume change is measured by the micro-Coulter counter, which is used to quantitatively estimate the cell secretome concentration. Vascular endothelial growth factor (VEGF), one of the key cell secretome proteins that regulate angiogenesis and vascular permeabilization, was used as the target protein to demonstrate the sensing principle. A standard calibration curve was generated by testing samples with various VEGF concentrations. A detection range from 0.01 ng/mL to 100.00 ng/mL was achieved. We further demonstrated the quantification of VEGF concentration in exogenous samples collected from the secretome of human mesenchymal stem cells (hMSCs) at different incubation times. The results from the assay agree well with the results of a parallel enzyme-linked immunoabsorbent assay (ELISA) test, indicating the specificity and reliability of the competitive immuno-aggregation assay. With its simple structure and easy sample preparation, this assay not only enables high sensitivity detection of VEGF but also can be readily extended to other types of cell secretome analysis as long as the specific Ab is known.

Microfluidics for Peptidomics, Proteomics, and Cell Analysis

“…Recently, MF has become a powerful technique for single-cell-omics analyses due to the MF technology’s advantages of flow, sensitivity, and precision [ 196 ]. Here, recent advances in MF single-cell oocyte analysis, including various designs of microfluidic platforms, lysis strategies, and oocyte analysis techniques, have become essential biomolecular tools [ 196 , 197 ].…”

“…However, the new research still faces challenges [ 198 ]. First, in single-cell isolation, not all advantages, such as high throughput and effective cell isolation, simple chip design and fabrication, and integrated multistep operation, can be maintained because each isolation method has its advantages and limitations [ 196 , 197 ]. Second, in omics analysis, barcoding technologies used for cell/molecule labeling can increase throughput and significantly reduce cost, but they are often biased (e.g., the 3-terminal cDNA ends) and can lead to loss of important signals.…”

“…Second, in omics analysis, barcoding technologies used for cell/molecule labeling can increase throughput and significantly reduce cost, but they are often biased (e.g., the 3-terminal cDNA ends) and can lead to loss of important signals. To address this challenge, mature third-generation sequencing technology can reverse this trend by increasing read length and single-molecule sequencing [ 196 , 197 ].…”