The widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface.Here,we present a simple method(MRBLEs 2.0...The widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface.Here,we present a simple method(MRBLEs 2.0)for the efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding(MRBLEs)that bear functional groups for downstream surface bioconjugation.Bead production in MRBLEs 2.0 relies on the manual mixing of lanthanide/polymer mixtures(each of which comprises a unique spectral code)followed by droplet generation using single-layer,parallel flow-focusing devices and the off-chip batch polymerization of droplets into beads.To streamline downstream analyte coupling,MRBLEs 2.0 crosslinks copolymers bearing functional groups on the bead surface during bead generation.Using the MRBLEs 2.0 pipeline,we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes with high throughput(>150,000/min and can be boosted to 450,000/min).We further demonstrate the efficient conjugation of oligonucleotides and entire proteins to carboxyl MRBLEs and of biotin to amino MRBLEs.Finally,we show that MRBLEs can also be magnetized via the simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space.With the advantages of dramatically simplified device fabrication,elimination of the need for custom-made equipment,and the ability to produce spectrally and magnetically encoded beads with direct surface functionalization with high throughput,MRBLEs 2.0 can be directly applied by many labs towards a wide variety of downstream assays,from basic biology to diagnostics and other translational research.展开更多
基金This work was supported by NIH grants 1DP2GM123641 and R01GM107132.P.M.F.is a Chan Zuckerberg Biohub Investigator and acknowledges the support of a Sloan Research Foundation Fellowship.Y.F.is a Cancer Research Institute Postdoctoral Fellow supported by the Cancer Research Institute Postdoc FellowshipA.K.W was funded by the Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship+1 种基金J.B.H.was funded by grant NNF17OC0025404 from the Novo Nordisk Foundation and the Stanford Bio-X ProgramPart of this work was performed at the Stanford Nano Shared Facilities(SNSF),supported by the National Science Foundation under award ECCS-1542152。
文摘The widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface.Here,we present a simple method(MRBLEs 2.0)for the efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding(MRBLEs)that bear functional groups for downstream surface bioconjugation.Bead production in MRBLEs 2.0 relies on the manual mixing of lanthanide/polymer mixtures(each of which comprises a unique spectral code)followed by droplet generation using single-layer,parallel flow-focusing devices and the off-chip batch polymerization of droplets into beads.To streamline downstream analyte coupling,MRBLEs 2.0 crosslinks copolymers bearing functional groups on the bead surface during bead generation.Using the MRBLEs 2.0 pipeline,we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes with high throughput(>150,000/min and can be boosted to 450,000/min).We further demonstrate the efficient conjugation of oligonucleotides and entire proteins to carboxyl MRBLEs and of biotin to amino MRBLEs.Finally,we show that MRBLEs can also be magnetized via the simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space.With the advantages of dramatically simplified device fabrication,elimination of the need for custom-made equipment,and the ability to produce spectrally and magnetically encoded beads with direct surface functionalization with high throughput,MRBLEs 2.0 can be directly applied by many labs towards a wide variety of downstream assays,from basic biology to diagnostics and other translational research.
