The layer-by-layer (LbL) polyelectrolyte self-assembly encapsulation method has attracted much interest because of its versatility to use various polymers for capsule formation, ability to encapsulate different templates, and capability to control capsule permeability. Traditionally, the LbL method was performed in water as solvent and limited to poorly or non-water-soluble templates. Using the matrix-assisted LbL method, complex mixtures of water-soluble proteins or DNA could be encapsulated within agarose microbeads templates but leakage of biomolecules into the water phase during the LbL process results in low encapsulation efficiency. Recently, the reverse-phase LbL (RP-LbL) method was introduced to perform LbL and encapsulation of water-soluble templates in organic solvents, thus preventing the templates from dissolving and allowing high encapsulation efficiency. However, encapsulation of complex mixtures of biomolecules or other substances with quantitative encapsulation efficiency remained impossible. Here we present a new approach for encapsulation of biomolecules or complex mixtures thereof with almost 100% encapsulation efficiency. The ability of our method to achieve high encapsulation efficiency arises from the combination of two strategies. (1) Using microparticles as surface stabilizer to create stable biomolecule-loaded hydrogel microbeads, termed matrix-assisted colloidosome (MAC), that are able to disperse in oil and organic solvents. (2) Using the RP-LbL method to fabricate polymeric capsule "membranes", thereby preventing diffusion of the highly water-soluble biomolecules. Using an oil phase during emulsification and an organic solvent phase during encapsulation could completely prevent leakage of water-soluble biomolecules and almost 100% encapsulation efficiency is achieved. Microcapsules fabricated with our method retained nearly 100% of encapsulated proteins during a 7 day incubation period in water. The method was demonstrated on model proteins and may be extended to other biomolecules or mixtures. Our method is a valuable addition to the family of encapsulation techniques and can significantly contribute to the fields of bioreactors and bioanalytical microcapsules.

Encoding by encapsulation: A polymeric shell fabrication approach combines biomolecule encapsulation with encoding. Striated polymeric shells, fabricated through an inwards diffusion of poly(allylamine) into the matrices of agarose microbeads, serves to encapsulate the biomolecules within the microcapsule. Encoding is performed through the color and/or thickness permutation of the striated polymeric shells (see picture).

In the quest for greater control over biomacromolecular loading and higher encapsulation efficiencies for biomacromolecule loaded microcapsules we devised a novel approach employing water soluble sacrificial templates. In traditional layer by layer (LbL) methods, aqueous solutions of polyelectrolyte salts in combination with water insoluble sacrificial template materials are used to prepare polyelectrolyte microcapsules that can be loaded with biomacromolecules. Here, we replaced the aqueous phase with pure aliphatic alcohols (Reversed-Phase) to greatly enhance the retention of biomacromolecular cargo close to 100% during microcapsule preparation in this Reverse-Phase Layer by Layer (RP-LbL) process. Formation of stable multilayered polyelectrolyte membranes onto water soluble template materials by sequential deposition of polystyrenesulfonic acid (PSS) and polyallylamine (PA) from pure 1-butanol is reported for the first time. The challenge to exert control over the biomacromolecule concentration within the template material and the resulting microcapsules was addressed by sacrificial template materials. Sacrificial template materials are water soluble and comprise of biomacromolecules embedded into a matrix of small molecular weight molecules such as glucose. Control over the concentration of biomacromolecules in the template material and microcapsules is conveniently exerted by adjusting weight ratios of bimacromolecules to sacrificial template material. This approach is envisioned to be applied alternatively to traditional polyelectrolyte microcapsule preparation techniques in cases where minute losses of expensive biomacromolecules are unfavorable or when accurate control over biomacromolecule concentration is important.