Single protein nanocapsules (SPNs) Keywords single haemoglobin nanocapsules · artificial blood · biocompatibility · allergic reaction IntroductionProteins usually catalyze biological reactions and have enzymatic function. Enzymes are bioreactors with high specificity and selectivity. But enzymes have a great sensitivity and a relatively brief life-time [1]. The relatively short lifetime of enzymes and their sensibility to change in the environment (pH, temperature, mechanical stress, etc.) tends to limit their biotechnological application. The increase of the lifetime and stability of enzymes are crucial to widen usage of enzymes. Improvement in enzyme stability can reduce the amount of enzyme required, can extend the lifetime of enzymes and can increase the reuse of enzymes [2][3][4][5][6][7]. Enzyme immobilization to the surface or onto the inner cavities of greater structures with adsorption or covalent linkage is an effective strategy to increase its lifetime [2,3]. Covalent linkage between the enzyme molecule and carrier material can reduce the unfolding mechanism of ternary structure of enzyme molecule and increase the stability of enzyme [4]. During the last decade there is a growing interest in minimized-size enzyme carriers [8,9].Single enzyme nanocapsules (SEN), or single protein nanocapsules (SPN) mean that a single protein molecule is covered with a few nanometer thick polymer layer [10][11][12][13][14][15] (Figure 1: Structure). This spatial polymer layer is thin and porous and allows the diffusion of substrate molecules to the active centre of the enzyme. Single enzyme nanoparticles involve the advantages of the multi-point covalent attachment [4] and closing of molecules onto inner cavities [16]. This technique needs a special polymerization step: in situ polymerization starting from the previously modified surface of the protein molecules ("Grafting from" method The quaternary protein structures (e.g. tetrameric haemoglobin molecules), as a functional unit does not change during the preparation of the single protein nanocapsules, i.e. covering enzymes by a thin polymeric layer (Figure 1: Structure). It has been proved that single enzyme nanocapsules, using organic/inorganic hybrid layer [10,12,13,14] organic acrylic copolymer [11,15], hyperhranched polymer layer [18] or superparamagnetic layer [19,20], can essentially stabilize enzymes. 58(Sup), pp. 11-16, 2014 DOI:10.3311 The nano-capsule around the enzymes is multifunctional (Figure 1: Functions). Single protein nanocapsules have 1) stabilization function a) mechanical stability, when under mechanical stress (circular stirring) the single enzyme nanocapsules are more stable than natural enzymes (enzymes without nano-layer) [12,13,14]; b) heat stability: when at extremely high temperature (80°C) single enzyme nanocapsules have greater stability than natural enzymes [12,13,14]. c) pH-stability: at extremely acidic (pH = 1.5) or alkaline (pH = 12.0) pH-values the activity of single enzyme nanocapsules are not reduced essentially, whi...