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The next goal was to demonstrate control over the number of the azide groups on the nanoparticle, with the aim of identifying an approach that would yield monovalent azide nanoparticles, and hence, monovalent maleimide functionalisation. This is essential for certain applications, such as single molecule biophysics. To determine the number of maleimide functional groups present on the nanoparticles after the click reaction, a pull-down of the nanoparticles functionalised with a biotinylated peptide was performed. Previously, it has been shown that the percentage of nanoparticles pulled down by affinity resins is proportional to the amount of a functional peptide or ligand present within the monolayer (Levy et al., 2006; Duchesne et al., 2008). In Lévy et al., assuming a Poisson distribution for the number of ligand per nanoparticles, it was estimated that when 10 % of the nanoparticles are bound to resin the nanoparticle population will possess an average of 0.1 functional ligands per nanoparticle, which corresponds to 10 % of the nanoparticles possessing at least one functional ligand (with no more than 0.48% possessing two or more functional ligands) and 90% no functional ligand. Therefore, a titration was performed with gold nanoparticles by varying the molar percentage of azideligands (1 – 0.1 %) in the ligand mix. The N3NPs then underwent the click chemistry in the

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Figure 4.14 Skeletal structure of the final product of the click-chemistry reaction between the nanoparticle bound N3 ligand and the DIBO-Mal strained cyclooctyne.

hour in the dark with mixing on a rotating wheel. This reaction generated maleimide functionality on any nanoparticles possessing azide ligands, and therefore generates DIBO-Maleimide functionalised nanoparticles nanoparticles (DIBO-Mal NPs; Figure 4.14). After removal of excess DIBO-Mal by Sephadex G-25 size-exclusion chromatography, with 1xTBS Tween-20 0.05 % (v/v) as the mobile phase, the nanoparticles were reacted unmodified with a biotinylated peptide (CVVVTGAAHHHHHH-K(biotin)-RKK) for 3 hours. This time was judged to be sufficient for Michael addition of the thiol on the cysteine of the peptide with the maleimide on the nanoparticle. After removal of excess peptide via another round of size-exclusion chromatography the nanoparticles were incubated with Strep-Tactin Sepharose (IBA solutions) overnight. The Strep-Tactin Sepharose will bind to the biotin present within the peptide with high affinity (Kd ~ 100 fM). Therefore, the

quantity of nanoparticles bound to the resin will directly reflect proportion of biotinylated azide ligands within its monolayer. Thus, the amount of nanoparticles left in the supernatant would indicate the amount of particles pulled down when compared with the initial concentration of nanoparticles. The concentration of the nanoparticle in the supernatant was quantified by UV-vis spectrometry, and the percentage of nanoparticles pulled down onto the resin calculated (Figure 4.15). Nanoparticles at all concentrations of N3 ligand were pulled down, with a decrease in

the pull-down percentage from 0.6 % to 0.1% N3 ligand. The concentration at which

10 % pull-down was achieved was 0.15 % (mol/mol, azide ligand: mix matrix ligand mix, Figure 4.15, dotted line). From this point, in order to generate monovalent maleimide functionalised nanoparticles, the initial N3NPs were synthesized with 0.15

% (mol/mol) azide ligand in their monolayer. It is important to note that this titration only holds for the current batches of ligands and nanoparticles. If either constituent

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were to change then the titration would have to be repeated, as not all batches of nanoparticles and ligands are identical.

 4.2.3  Conjugation  of  biomolecules  to  monovalent  maleimide   nanoparticles  via  Michael  addition  reactions  

Having generated the monovalent DIBO-Mal NPs described in the previous section, the next step was to attempt their conjugation to FGF-2 and maleimide modified oligosaccharides. For Michael addition to occur with the maleimide on the DIBO-Mal NP a free thiol is required. Therefore, the reaction should proceed in the same fashion as the reaction with the D-PEG/D-Mal NPs (Section 4.1.2). Hence, the biomolecules selected for the conjugation reactions are identical to those used for the D-PEG/D-Mal NPs, i.e., FGF-2 and maleimide modified heparin-derived hexa- and dodecasaccharides.

FGF-2 was incubated at a 3.5 times molar excess over DIBO-Mal NPs with mixing for 3 hours on a rotating wheel. The mixture was then purified via heparin agarose affinity chromatography. Images of the heparin agarose affinity columns can be seen in Figure 4.16. Nanoparticles bearing no maleimide functionality, i.e., a mix matrix monolayer, when incubated with FGF-2 do not bind to the column (Figure 4.16A). This is also the case when DIBO-Mal NPs that have not been incubated with FGF-2 are passed through the column (Figure 4.16B). When the DIBO-Mal NPs are incubated with FGF-2 protein the pink colour of the gold nanoprobe is seen on the heparin agarose column (Figure 4.16C). The nanoparticles can be eluted with 2 M NaCl, which is sufficient to elute FGF-2 from heparin affinity columns.

Figure 4.16 Purification of DIBO-FGF-2 NPs via affinity chromatography with heparin agarose. (A) Mix-matrix nanoparticles incubated with an excess of FGF-2 protein, (B) DIBO-Mal nanoparticles and (C) DIBO-Mal nanoparticles incubated with an excess of FGF-2 protein.

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dithiols, e.g., DTT, then the resulting nanoparticle will have thiol functionality (Section.4.1.2; Figure 4.7). Hence, DIBO-Mal NPs were incubated with an excess of 1 mM DTT for 1 hour to generate thiol functionality, after which excess DTT was removed via Sephadex G-25 size-exclusion chromatography with 1xPBS Tween-20 0.05 % (v/v) as the mobile phase. To see if indeed thiol nanoparticles (DIBO-SH NPs) were generated, they were reacted with small oligosaccharides derived from heparin modified with a maleimide: hexasaccharide (Section 4.1.3), and a dodecasaccharide, a longer oligosaccharide (Gift from Nina Azmi; Azmi, PhD thesis, University of Liverpool, 2012). Nanoparticles with a non-functionalised mix monolayer incubated with the hexa- or dodecasaccharide did not bind to the DEAE Sepharose (Figure 4.17 A and D) and neither did DIBO-SH NPs in the absence of the hexa- and dodecasaccharide (Figure 4.17 B and E). When DIBO-SH nanoparticles had been incubated with the hexa- or dodecasaccharides for 3 hours with mixing, and subsequently used for anion exchange chromatography, nanoparticles bound to the DEAE-Sepharose, indicated by a pink colour at the top of the column (Figure 4.17 C and F). Thus, nanoparticles only bind to the resin when the Michael addition reaction has occurred between DIBO-SH NPs and the maleimide modified oligosaccharides. The hexa- or dodecasaccharides NPs can be eluted from DEAE-Sepharose with 2 M NaCl. The nanoparticles were subsequently buffer exchanged into 1xPBS Tween-20 0.05 % (v/v) via multiple centrifugation steps (35 mins at 13000 rpm), allowing removal of excess hexa- or dodecasaccharides.

Figure 4.17 Purification of DIBO-hexa- and dodecasaccharide NPs via anion exchange chromatography on DEAE-Sepharose. Mix-matrix nanoparticles incubated with an excess MBPH functionalised (A) hexa- and (D) dodecasaccharide. SH NPs on DEAE-Sepharose control for both (B) hexa- and (E) dodecasaccharide experiments. SH NPs incubated with an excess of MBPH functionalised (C) hexa- and (F) dodecasaccharide purified on DEAE-Sepharose.

A

B

C

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nanoparticle to one biological molecule. This will enable single nanoparticle biophysics experiments, using photothermal microscopy, to probe the biological interactions of these molecules. Some proof of principle experiments using the conjugates described above will be examined in the next section.

4.3  Covalent  biomolecule-­‐nanoparticle  conjugates  for  single