VIPs synthesis and characterization

As the carrier material to design the VIPs, we used highly monodisperse SNPs produced following the method developed by Stöber31. The statistical analysis of micrographs acquired using high-resolution field-emission scanning electron microscopy (FESEM) revealed a mean diameter of 410 nm. The high propensity of those nanoparticles to self-assemble into three-dimensional colloidal arrays represents an additional evidence of their high monodispersity (Fig. 2a).

Figure 2: Representative FESEM micrographs and layer growth kinetics. (a) Colloidal self-assembled three-dimensional arrays formed by the starting SNPs, (b) growth kinetics of the recognition layer as a function of the poly-condensation reaction time (mean±s.e.m.). Nanoparticles as they appear (c) before and (d,e) after growth of the recognition layer; (d) VIPs AT and (e) VIPs OM , both with 8 nm-thick recognition layers. (f,g) Micrographs of VIPs OM and VIPs AT once the virions were removed; (h) Close-up view of a VIPs OM . Scale bars represent: a, 5 μm; c–g, 100 nm; and h, 10 nm. Full size image

The first step in VIPs synthesis consisted of grafting the template virions on the surface of the SNPs. In order to provide anchoring amine moieties for this cross-linking, the SNPs were partially modified with aminopropyltriethoxysilane (APTES). A low density of amine groups at the SNP surface is essential to leave enough silanol groups for the further surface-initiated poly-condensation of the recognition layer.

Further coupling of the virions was carried out in water using glutaraldehyde as a homo-bifunctional crosslinker. We used two small RNA plant viruses as model viruses. Tomato bushy stunt virus (TBSV) and turnip yellow mosaic virus (TYMV) are non-enveloped, icosahedral, single-stranded RNA viruses (Fig. 1b). Their capsids are made of 180 copies of protein subunits, with a mass of 40 kDa for TBSV and 20 kDa for TYMV32. The virions have diameters of 33 and 28 nm, molecular weights of 9.0 × 103 and 5.5 × 103 kDa, and isoelectric points of 4.1 and 3.8, respectively.

Once the virions were bound, a silsesquioxane layer was grown from the surface of the SNPs. To produce this recognition layer by following a protein mimetic approach, we selected organosilanes that mimic the lateral chains of amino acids known to be of importance in protein–protein interactions (Fig. 1c)33,34. We hypothesized that these building blocks will self-assemble at the surface of the virions before their covalent incorporation within the recognition layer. This was expected to improve the recognition properties of the VIPs by creating not only a shape but also a chemical imprint that is complementary to the surface of the virion. Hereinafter, the term VIPs OM stands for particles imprinted with TYMV virions and having a recognition layer composed of an organosilanes mixture (OM), and the term non-imprinted particles (NIPs) OM for NIPs produced in the absence of template using the same OM. As controls, we selected two additional formulations, one with tetraethyl orthosilicate (TEOS) alone and one with a mixture of APTES and TEOS (AT). The corresponding VIPs are abbreviated VIPs AT for TYMV-imprinted particles having a recognition layer made of AT, and NIPs AT for those produced under the same conditions in the absence of template.

The kinetics of surface-initiated growth of the silsesquioxane recognition layer in water at 10 °C followed by FESEM revealed that the thickness of the external layer reached only 2 nm after 75 h when the poly-condensation reaction was performed with TEOS alone (Fig. 2b). In the presence of APTES, the layer was 15 nm thick after 10 h. This faster growth can be safely attributed to the catalytic effect of the primary amine function of APTES on the hydrolysis of the organosilanes35. The size of the particles prepared with the mixture of organosilanes increased according to a sigmoidal function; the layer thickness reached 14 nm after 75 h (Fig. 2b). The slower kinetics as compared with that of the APTES/TEOS mixture may be explained by the lower amount of APTES present in a constant total amount of organosilanes. These results demonstrate the possibility of growing an organosilanes layer at the surface of the nanoparticles under aqueous conditions. They also confirmed that neither the crosslinking chemistry of the SNPs (using APTES and glutaraldehyde) nor the presence of the virions at the surface of the SNPs hampered this growth.

An examination of the morphology of the particles produced with AT reveals the presence of open cavities with an average diameter of ~20 nm at the surface of the VIPs for a layer thickness of 8 nm (Fig. 2d). This suggests that the growth of the layer had started at the surface of the SNPs but was hindered by the virions, resulting in the formation of crater-like imprints (Fig. 2d). The virions were destroyed by the physical treatment of the particles before and/or during the FESEM imaging, thus leaving empty imprints. Particles synthesized in the presence of the silane mixture were distinguishable from predecessors by the presence of protuberances measuring about 32 nm in diameter (Fig. 2e) and by a thin shell around every virion that was not removed under FESEM sample preparation and imaging conditions. Thus, poly-condensation started not only at the surface of the SNPs but also at the surface of the virions that act as a template for this reaction. This observation supported our hypothesis that the selected silanes interacted with the entire surface of the virions before they were incorporated in the recognition layer. The ability of silicatein proteins to act as templates/catalysts for the biomineralization of silica has been demonstrated36. Numerous biomimetic synthetic silica systems have been reported and are mainly based on the modification of the protein to introduce catalytic/template sequences36,37,38. Here, we demonstrated that the template effect could be obtained by using a mixture of organosilanes that self-assemble around the native virus. By submitting these VIPs to an ultrasonic treatment under acidic conditions, this shell was broken without altering the recognition layer. FESEM micrographs showed that the protuberances observed previously were eliminated (Fig. 2f). The cleaned particles exhibited empty cavities at their surface, meaning that the virus imprinting method was successful. Also, the particles produced using the APTES/TEOS mixture resulted in being completely cleaned after the ultrasonic treatment (Fig. 2g). Images of the cavities taken at a higher magnification revealed their hexagonal shape (with an edge length of 11 nm), which originated from that of the template virus (Fig. 2h). Altogether, these results confirmed that the three-dimensional icosahedral architecture of the template virions was preserved under the mild conditions used throughout the full chemical synthesis.

Virus-binding assays

The binding performances of the synthesized VIPs were assessed in aqueous batch rebinding assays. VIPs and NIPs were incubated with TBSV or TYMV virions under well-defined conditions and, following centrifugation, the proportion of unbound virions was quantified using an enzyme-linked immunosorbent assay (ELISA) (Fig. 3).

Figure 3: Binding of virions of the templated TYMV and non-templated TBSV to VIPs and NIPs. Symbols are for TYMV (open squares) and for TBSV (solid squares). Binding time, selectivity, composition and thickness of the recognition layer were compared. (a–d) Four types of particles were assayed: (a) VIPs OM , (b) NIPs OM , (c) VIPs AT and (d) NIPs AT ; OM and AT particles with 8-mm-thick recognition layers. (e–h) Nanoparticles with recognition layers of increasing thicknesses (mean±s.e.m.) were assayed: (e) VIPs OM , (f) NIPs OM , (g) VIPs AT and (h) NIPs AT . All values are presented normalized in percentage of initial virus concentration (mean±s.e.m.). Full size image

The results of the binding experiments revealed that, starting from an initial virus concentration of 65 pM, after 30 min, as much as 95% of TYMV were bound to VIPs OM possessing 8-nm-thick recognition layers (834 μg ml−1 or 100 μg per 120 μl; Fig. 3a). Under the same conditions, these nanoparticles after 30 min bound no more than 12% of TBSV (Fig. 3a). Thus, VIPs OM specifically bind the template virions and almost none of the virions of another icosahedral virus possessing a comparable particle diameter and isoelectric point. Quantification of the intrinsic binding of NIPs possessing the same chemical composition as VIPs but having no imprints resulted in 21% of TYMV virions and 6% of TBSV virions bound after 45 min, respectively (Fig. 3b). Thus, TYMV adsorbs on NIPs OM more than TBSV does, but the difference between both viruses is too small to explain the strong effect observed with the VIPs OM . The selectivity of the VIPs, at a target concentration in the pM range, is essentially owing to the presence of the virion imprints at the surface of the nanoparticles.

To assess the influence of the chemical composition of the recognition layer, VIPs with an 8 nm recognition layer synthesized with AT were assayed under the same conditions. VIPs AT bound 80% of the TYMV virions in 30 min while binding of TBSV virions was <5% (Fig. 3c). As for NIPs, they bound significantly more TYMV virions (that is, 40% in 30 min) than TBSV virions (5% in 30 min), showing that their binding performances were lower than those of the VIPs prepared with an APTES/TEOS mixture (Fig. 3d). This pointed to the importance of the chemical composition of the recognition layer and supported our hypothesis of an organosilanes chemical imprinting.

According to our initial hypothesis, increasing the recognition layer thickness (limited to the radius of the virion) should significantly increase the surface area available for interactions per virion and, hence, the number of potential interaction points between hosts and guests. To verify the influence of the thickness of the recognition layer on the affinity of VIPs for their template, we produced VIPs OM and VIPs AT with layers of various thicknesses. In batch assays, the binding of the template to the VIPs OM increased with the thickness of the recognition layer (625 μg ml−1 or 75 μg per 120 μl). The fraction of bound virus reached 34% for a layer thickness of 3 nm, 74% for one of 9 nm and 100% for a layer of 14 nm (Fig. 3e). The non-template virus (TBSV) was bound only to a limited extent to particles with thin recognition layers: 10% of the non-template virus, while particles with a layer of 14 nm had bound to 30%. In binding assays done with NIPs OM (Fig. 3f), only about 10% of each virus was bound. VIPs AT particles bound specifically to their template (Fig. 3g), and there was no major effect from recognition layer thickness. As much as 40% of the virions of TYMV were bound to particles with a recognition layer of 6 nm and 70% to the particles with recognition layers of 8, 11, 14 or 16 nm. None of the NIPs AT bound either virus (Fig. 3h). Again, these results confirmed that the affinity of the VIPs material for its template virus can be tuned by varying the thickness of the recognition layer.

Competition binding assay in serum and rebinding

To assess the selectivity of the VIPs OM and the effect of a more complex matrix, we performed competition binding assays in buffer and human serum (HS) at different dilutions (Fig. 4a). HS is a complex matrix possessing, in addition to a high total protein concentration (60–85 g l−1, predominantly albumin and immunoglobulins), lipids, metabolites, vitamins, regulatory factors and electrolytes. It has to be added that for non-diluted HS, the ELISA test used did not show a consistent response and therefore prevented us from evaluating the VIPs OM -binding performances in these conditions. The assays performed in buffer revealed that 84% of the template TYMV was bound to VIPs OM while only 10% of the non-template TBSV was bound to the particles. This result confirmed the selectivity of VIPs OM for its template. The competition assays performed in HS showed that VIPs OM are specifically binding 45, 64 and 88% of the template TYMV at 1:10, 1:50 and 1:100 HS dilutions, respectively. The non-template TBSV binding on the particles is of 6, 16 and 18% at 1:10, 1:50 and 1:100 HS dilution, respectively. These results confirmed that the VIPs OM maintained their capabilities of discriminating between the two viruses in complex matrix, such as HS.

Figure 4: Competition binding assay of the templated TYMV and non-templated TBSV to VIPs OM in complex matrices and FESEM of VIPs OM after a rebinding assay. (a) Symbols are for TYMV (white bars) and for TBSV (black bars). The competition assays were performed with VIPs OM particles with a 8-nm-thick recognition layer in buffer containing bovine serum albumin (75 μg ml−1) and in different dilution of HS (1:10, 1:50 and 1:100). All values are presented normalized in percentage of initial virus concentration (mean±s.e.m.). (b) VIPs OM particles after the binding assay with the template TYMV. Scale bar represent 100 nm. Full size image