Terminal supraparticle assemblies from similarly charged protein molecules and nanoparticles

Abstract

Self-assembly of proteins and inorganic nanoparticles into terminal assemblies makes possible a large family of uniformly sized hybrid colloids. These particles can be compared in terms of utility, versatility and multifunctionality to other known types of terminal assemblies. They are simple to make and offer theoretical tools for designing their structure and function. To demonstrate such assemblies, we combine cadmium telluride nanoparticles with cytochrome C protein and observe spontaneous formation of spherical supraparticles with a narrow size distribution. Such self-limiting behaviour originates from the competition between electrostatic repulsion and non-covalent attractive interactions. Experimental variation of supraparticle diameters for several assembly conditions matches predictions obtained in simulations. Similar to micelles, supraparticles can incorporate other biological components as exemplified by incorporation of nitrate reductase. Tight packing of nanoscale components enables effective charge and exciton transport in supraparticles and bionic combination of properties as demonstrated by enzymatic nitrate reduction initiated by light absorption in the nanoparticle.

Figure 1. Assembly of cadmium telluride nanoparticles(CdTe NPs) and cytochrome C (CytC)

(a–c) Scanning electron miscroscopy (SEM) images of CdTe/CytC assemblies with molar ratio of CdTe:CytC as 1:1(a), 2:1(b), and 6:1(c). 1:6 (d). (e) Transmission electron microscopy (TEM) image of CdTe/CytC supraparticles (SPs). (f) Size distribution of the self-assembled SPs by dynamic light scattering (DLS). Scale bars are 500 nm (a,c), 1 µm (b,d), and 200 nm (e).

Figure 2. Dependences of SP diameters on media parameters

(a–d) TEM images of SPs made in the solutions with (a) 0.1 M, (b) 0.5 M, (c) 1.0 M and (d) 2.0 M of NaCl. Scale bars are 100 nm. (e) Dependence of SP diameters on the NaCl concentration of NaCl and on inverse screening length in simulation (inset). (f) Temperature dependence of the SP diameters in experiment and in simulation (inset). The error bars in the experimental results (e,f) were obtained from the standard deviation (std) values from multiple experiments. Those in the simulation plots (insets in e and f) are the corresponding std of the SP size distribution.

Figure 3. Structural Characterization of CdTe/CytC SPs

(a) High-resolution TEM (HR-TEM) image of the SP. Lighter areas correspond to CytC-rich phase (white-dashed circles) while the darker ones correspond to CdTe-rich phase. Scale bar is 10 nm. (b) TEM tomography images of CdTe/CytC SP: X-Y slices (i-vi) of the SP, shown in every 4.8 nm through the volume. 3D surface reconstruction (vii) and cross-section (viii) of the SP (see Supplementary Movie 1,2). Scale bar is 20 nm. (c) X-ray energy dispersive spectroscopy (XEDS) spectrum for an SP in (a). (d) Schematics of the formation of CdTe-CytC SPs. (e) Circular dichroisum (CD) spectra for CytC (red, 6 µM), CdTe NPs (black, 6 µM), 1:1 mixture of CdTe/CytC after 72 (blue) hrs.

Figure 4. Mechanism of self-assembly process

(a) Pair potential between CdTe NPs and CytC according to Derjaguin, Landau, Verwey, and Overbeek (DLVO) and extended-DLVO (E-DLVO) theories: V_DLVO (red); V_E-_DLVO1 (green); V_E-_DLVO2 (blue)(Supplementary Methods). (b) ζ-potential values for the assembly of CdTe NPs with CytC at different time intervals. Error bars indicate the std values from multiple measurements. (c) Spherical assemblies formed by a mixture of 1000 NPs (yellow) and 1000 CytC units (blue) at ρσ³ = 0.25. (d) Distribution of SP sizes and of the asphericity parameter (A_S) (inset) of a system composed of 8000 NPs and 8000 CytC units (Supplementary Fig. 12a). A_S = 0 corresponds to perfect spheres. Error bars indicate 10 equilibrated, independent configurations separated by intervals of 2000 τ. (e, f) Snapshots of mixtures with NP/CytC molar ratios of 2:1 and 6:1, respectively. (g) A snapshot of a system identical to (c), but without the inter-SP charge-charge repulsion renormalization. The images in (c,e,f and g) were generated using the software VMD. (h) Experimental and simulated data fitted against decaying laws, i.e. f(x) ~ exp(−x), and plotted against dimensionless inverse screening length (κ*) and normalized diameters (D*) (see Supplementary Methods for details)

Figure 5. Functional integration of SPs with photoenzymatic activity

(a) Formation of nitrite for SP-nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NRed) (red) excited at 470 nm and for NADPH-NRed (green) and SP-NADPH-NRed in dark (blue). Inset: Formation of nitrite for NADPH-NRed being excited at 470 nm in presence of only one of SP components: either CdTe NPs (black) or CytC (purple) when no hybrid SP were formed. Errors bars are based on triplicate experimental measurements. (b) Schematics of the reactions upon the photo-excitation of SP-NADPH-NRed. Nitrate reductase is represented as a surface model from PyMol software, PDB entry is 2BIH. TEM images of (c) SP-NADPH-NRed after 20 min and (d) after 40 min of the of photoenzymatic reaction. Scale bars are 100 nm.