Surface treated CdS QDs and gold nanoparticles

Before screening our library on bare inorganic surfaces directly, we tried to screen it against a priori easier target: nanoparticles coated with a known ligand. This ligand is a small organic molecule and would be in principle antigenic, whereas the antigenicity of inorganics is much less characterised.

Samples were provided by our collaborator, B. Dubetret (ESPCI, Paris). CdS QDs were capped with a novel a DHLA-SB (dihydrolipoic acid-sulfobetaine) ligand. DHLA provides a stable and compact surface to the QDs. This layer is further coated with a zwitterionic group (sulfobetaine) to limit nonspecific electrostatic interactions in biological environment. QDs capped with DHLA-SB are small, exhibit stability with time, pH and salt concentration (Muro, Pons, Lequeux, Fragola, & Sanson, 2010). The above modification of surface chemistry of QDs enabled them for use as probes in many biological imaging applications (long term single molecule tracking, simultaneous multicolor imaging etc). Similarly, gold nanoparticles were also capped with DHLA, and this layer was further coated with PEG. For functionalisation of gold nanoparticles and CdS QDs, surface of these particles can be treated with biotin or streptavidine. We received biotinylated DHLA-SB capped CdS QDs and biotinylated DHLA-PEG capped gold nano particles.

We used classical biotin-streptavidine binding affinity to select antibodies against these materials. First we incubated the biotinylated targets with streptavidine coated paramagnetic beads and later used these magnetic beads (having immobilised targets on their surfaces) for interaction with an antibody phage library. We took precaution to avoid nonspecific selection, by incubation of the antibody phage library with streptavidine coated paramagnetic beads (no immobilised target this time). Further we separated the antibody library and incubated with magnetic beads (having immobilised targets). We used magnetic bar for separation of selected phages. Please refer appendix B for detailed protocol.

The screening results for (DHLA-SB) capped and biotinylated CdS QDs are presented in figure 2.1.1a. The screening results for (DHLA-PEG) capped and biotinylated gold nanoparticles are presented in figure 2.1.1b. Both screenings showed significant increase of target specific binders. Later we performed ELISA experiment to check specificity of selected binders for the targets. The guidelines for analysing the screening results are explained in box 2.1a.

Box 2.1a

1) Graphical bar presents yield during each round of selection - This graph represents the yield at each round of selection. The yield is more precisely defined as the ratio of the number of selected phages over the number of input phages. This ratio is obtained by indirect method of counting number of bacterial colonies obtained after infection of the selected phages from each round of selection and number of bacterial colonies obtained after infection of input phages. We have performed three rounds of selection for all targets. In these graphs, the Y axis represents the yield (ratio of number of selected phages over the number of input phages), whereas the X axis represents the rounds of selection performed.

2) ELISA result - ELISA is a sensitive test that is routinely used in molecular and cell biology laboratories to check antigen – antibody interaction. This assay provides specific color signals in the presence of specific antigen-antibody interaction. If an antibody is not specific to antigen/target, it gives no color response.

Assay results are shown in the 96 well plates. The yellow color intensity increases with increasing binding affinity (i.e. dark yellow indicates a strong binder, light yellow indicates a weak binder, no color indicates no binder). In most of the results, wells A1 and H12 are control group (i.e. no scFv clone, only culture medium).

2.1.1a Screening result for (DHLA-SB) capped and biotinylated CdS QDs

The screening results against DHLA-SB capped and biotinylated CdS QDs as target showed significant enrichment of the target specific antibodies (fig. 2.1.1a). Therefore, we selected 94 random bacterial clones appeared after infection of selected phages from the third round of screening for the target. Further, we performed ELISA experiment to find specificity of these clones for the target (see ELISA result in fig. 2.1.1a). We immobilised the biotinylated targets on streptavidine coated 96 well ELISA plate. We used streptavidine coated ELISA plate for control experiment as well (absence of immobilised targets) to check non-specific selection against streptavidine surface.

Figure 2.1.1a: (A) yield during three rounds of selection for (DHLA-SB) capped and biotinylated CdS QDs. (B) ELISA result (performed in streptavidine coated 96 well plate) a) Experimental plate, streptavidine surface was coated with biotinylated target, b) control plate, uncoated streptavidine surface (absence of target). Wells A1 and H12 represent the control group i.e. no bacterial clone is used, only culture medium.

2.1.1b Screening result for (DHLA-PEG) capped and biotinylated gold nano particles

The screening results against DHLA- PEG capped and biotinylated gold nanoparticles as target showed significant enrichment of the target specific antibodies (fig. 2.1.1b). Therefore, we selected 94 random bacterial clones appeared after infection of selected phages from the third round of screening for the target. Further, we performed ELISA experiment to find specificity of these clones for the target (see ELISA result in fig. 2.1.1b). We immobilised the biotinylated targets on streptavidine coated 96 well ELISA plate. We used streptavidine coated ELISA plate for control experiment as well (absence of immobilised targets) to check non-specific selection against streptavidine surface.

Figure 2.1.1b: (A) yield during three rounds of selection for (DHLA+ PEG) capped and biotinylated gold nanoparticles. (B) ELISA result (performed in streptavidine coated 96 well plate), a) Experimental plate, streptavidine surface was coated with biotinylated target, b) control plate, uncoated streptavidine surface (absence of target). Wells A1 and H12 represent control groups i.e. no bacterial clone is used, only culture medium.

Interpretation of ELISA results of surface treated CdS QDs and gold nanoparticles

ELISA results for both targets showed binding of scFvs in both, the experimental as well as the control plates. The results indicate non-specific selection, thus failure of screens against these targets. The potential explanation for these failures is as follows-

We used streptavidine coated magnetic beads in these screens for immobilisation of biotinylated targets (inorganic nanoparticles). Magnetic beads are micron size beads and they are covalently coated with streptavidine all over their surface. The biotinylated targets are expected to bind streptavidine coated magnetic beads. If biotinylated target fail to bind streptavidine coated magnetic beads, we may raise antibodies against streptavidine coated surface of magnetic beads and we actually ended up in this way. The selection of antibodies against streptavidine surface can be confirmed by their binding in control ELISA plate (control plate used in ELISA experiment is streptavidine coated). THE ELISA result showed that all clones were selected against streptavidine target instead of biotinylated CdS QDs and biotinylated gold nanoparticles.

A possible reason for the failure of immobilisation of biotinylated targets on streptavidine coated paramagnetic beads may be inefficient biotinylation of target surfaces. Unfortunately, we did not check the biotin-coating of the inorganic nanoparticle targets used in this study.

However, on the other extreme if targets are over biotinylated, we might have ended up with selection of anti-biotin antibodies.

Results from these screen pondered more emphasis on sufficient biotinylation of targets.

This might not be efficient in the case of inorganic nanoparticles. So overall this approach is not safe due to increased chances of selection of non-specific antibodies. It motivated us to use the bare surface of semiconductor materials and metal surfaces, which are not stabilised by any surfactants.