Substrate-mediated reactors for bioassays
A technology of substrates and biomolecules, applied in the field of substrate-mediated reactors for bioassays, can solve problems such as non-specific amplification, and achieve the effect of improving multiple levels
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Embodiment 1
[0129] Example 1: Multiplex droplet assisted immunoassay
[0130] Protein biomarkers are currently the workhorse of molecular diagnostic laboratories. Multiplexing of many protein markers reduces the labor and costs associated with these tests and increases their predictive power. While existing techniques for multiplex immunoassays suffer from limitations in antibody compatibility between targets, the use of controlled reagent release, utilizing substrate-mediated droplet formation, has been shown to overcome these limitations.
[0131] Validated droplet-assisted immunoassays using multifunctional hydrogel microparticles containing fluorescent recognition regions, reagent storage regions, and target capture regions, as image 3 shown. The target capture region is covalently functionalized using an epitope-specific capture antibody, while the reagent storage region is functionalized via DNA duplex formation using an oligonucleotide-modified detection antibody tethered to the...
Embodiment 2
[0172] Example 2: Physical Separation of Reaction Droplets
[0173] Droplet coalescence or diffusion through immiscible continuous oil relative to biological or chemical agents will result in ineffective separation of individual reactions. The system is designed to assess the extent of droplet merging / coalescence. To assess coalescence, hydrogel particles with covalently bound oligonucleotide probes were encapsulated in an immiscible oil phase by vigorous vortexing for 30 s. A second emulsion was prepared in the aqueous phase consisting of 500 nM oligonucleotides labeled with the Cy5 fluorophore that were reverse complementary to the particle-bound probes. Two emulsions were mixed 1:1, one containing only native particles in buffer and the other containing fluorescent oligonucleotide targets. The resulting mixed emulsion was incubated at 37°C for 60 minutes. The emulsion was then inverted and the fluorescent signal on each particle was analyzed using a Guava 8HT flow cytome...
Embodiment 3
[0175] Example 3: An Exemplary Continuous Phase Allowing Two-Phase Reactive Droplet Systems
[0176] An exemplary embodiment of such a system is described below. In this system, an aqueous phase containing hydrogel particles is mixed with a continuous immiscible oil phase, resulting in the reversible stable encapsulation of individual reactions in the oil phase.
[0177] Hydrogel particles composed of polyethylene glycol and having dimensions of 100-200 micrometers in length and 20-100 micrometers in width and height directions were used. Particles containing at least one capture antibody in a defined particle region and at least one detection antibody tethered in another region using complementary oligonucleotides are added to the biological sample of interest. Each particle type has a specific barcode or other identifying feature associated with the target molecule it detects. Multiple particle types can be used to selectively analyze up to 1000 biological targets simultan...
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