This protocol describes the core methodology for the fabrication of bar-coded hydrogel microparticles, the capture and labeling of protein targets and the rapid microfluidic scanning of particles for multiplexed detection. Multifunctional hydrogel particles made from poly(ethylene glycol) serve as a sensitive, nonfouling and bio-inert suspension array for the multiplexed measurement of proteins. Each particle type bears a distinctive graphical code consisting of unpolymerized holes in the wafer structure of the microparticle; this code serves to identify the antibody probe covalently incorporated throughout a separate probe region of the particle. The protocol for protein detection can be separated into three steps: (i) synthesis of particles via microfluidic flow lithography at a rate of 16,000 particles per hour; (ii) a 3–4-h assay in which protein targets are captured and labeled within particles using an antibody sandwich technique; and (iii) a flow scanning procedure to detect bar codes and quantify corresponding targets at rates of 25 particles per s. By using the techniques described, single- or multiple-probe particles can be reproducibly synthesized and used in customizable multiplexed panels to measure protein targets over a three-log range and at concentrations as low as 1 pg ml−1.
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We acknowledge financial support from the Ragon Institute of Massachusetts General Hospital, the Massachusetts Institute of Technology and Harvard University; grant R21EB008814 from the National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health; and National Science Foundation grant DMR-1006147. R.L.S. is supported by a US National Institutes of Health/National Institute of General Medical Sciences Biotechnology Training Grant.
The authors declare no competing financial interests.
The zip file contains designs for the scanning channel, synthesis channel, and particle mask. For the scanning channel design, see provided AutoCAD file (Flow Focusing Device.dwg in AutoCAD Files.zip) for design. For the synthesis channel design. see the provided AutoCAD file (Synthesis Wafer.dwg in AutoCAD Files.zip) for a wafer containing various types of synthesis channels. For the particle masks, see provided AutoCAD file (Particle Masks.dwg in AutoCAD Files.zip) or Adobe Acrobate file (Particle Mask Sheet.pdf) for a variety of particle masks. (ZIP 153 kb)
See provided AVI file (Supplementary Movie 1 (Doyle).avi). Synthesis of a 4-chemistry particle with monoclonal capture antibodies against Interferon-γ (R&D systems, MAB2852). From top to bottom, the stream compositions are: fluorescent code monomer, blank monomer, IFN-γ antibody probe monomer, blank monomer. Blank monomer is colored with blue food coloring for visible contrast of streams. The antibody concentration in the probe monomer is 2 mg/mL. The code on the mask is "02223". Throughput is approximately 5 particles every second. Recording and playback is set so that the synthesis occurs in real time. (AVI 3288 kb)
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Appleyard, D., Chapin, S., Srinivas, R. et al. Bar-coded hydrogel microparticles for protein detection: synthesis, assay and scanning. Nat Protoc 6, 1761–1774 (2011). https://doi.org/10.1038/nprot.2011.400
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