Multiplexed Protein Quantification with Barcoded Hydrogel Microparticles

摘要

We demonstrate the use of graphically encoded hydrogelnmicroparticles for the sensitive and high-throughputnmultiplexed detection of clinically relevant protein panelsnin complex media. Combining established antibody cap-nture techniques with advances in both microfluidic syn-nthesis and analysis, we detected 1 8 pg/mL amounts ofnthree cytokines (interleuken-2, interleuken-4, and tumornnecrosis factor alpha) in single and multiplexed assaysnwithout the need for filtration or blocking agents. A rangenof hydrogel porosities was investigated to ensure rapidndiffusion of targets and reagents into the particle as wellnas to maintain the structural integrity of particles duringnrinsing procedures and high-velocity microfluidic scan-nning. Covalent incorporation of capture antibodies usingna heterobifunctional poly(ethylene glycol) linker enablednone-step synthesis and functionalization of particles usingnonly small amounts of valuable reagents. In addition tonthe use of three separate types of single-probe particles,nthe flexibility of the stop-flow lithography (SFL) methodnwas leveraged to spatially segregate the three probes fornthe aforementioned target set on an individual encodednparticle, thereby demonstrating the feasibility of single-nparticle diagnostic panels. This study establishes thengel particle platform as a versatile tool for the efficientnquantification of protein targets and significantly advancesnefforts to extend the advantages of both hydrogel sub-nstrates and particle-based arrays to the field of clinicalnproteomics..