Characterization methods

In terms of characterization, a significant number of analytical techniques has been used to characterize folded macromolecules. However, most of them can provide only circumstantial evidence of compacted nano-object characteristics. A combination of different characterization techniques is generally required to fully characterize folded macromolecules. The main analytic tools used to either evidence the compaction process or characterize the resulting morphology are reported in this section.

Size Exclusion Chromatography (SEC) remains one of the crucial analytic tools to prove the formation of compacted macromolecules. First, this characterization method allows to gain insight into the formation of intramolecular vs. intermolecular crosslinking bond formations.

Furthermore, upon compaction, an increase in retention time from the polymer precursor to folded polymer can be observed, indicating a decrease in apparent molecular weight.¹⁸⁰ This shift in elution volume is typical of a hydrodynamic volume reduction caused by intramolecular crosslinking bond formations.¹⁸¹ However, while this technique demonstrates a reduction in hydrodynamic volume, the quantification of this change is impossible with standard SEC measurements alone. Quantitative information can be obtained by coupling SEC chromatography with multiple in-line detectors, such as Multi-Angles Light Scattering (MALS).¹⁸² Often, the analytical data obtained by SEC can be verified by Dynamic Light Scattering (DLS).^183,184 Proton NMR spectroscopy is also an important analytic tool, since this characterization enables to monitor the appearance or disappearance of signals corresponding to the formed crosslinking bonds. However, this technique does not differentiate the formation of intramolecular and intermolecular bond. Nevertheless, others NMR spectroscopies such as Diffusion Ordered Spectroscopy (DOSY) can indicate a decrease in hydrodynamic volume.

DOSY experiments reveal the diffusion coefficient of a macromolecule in solution, which is inversely proportional to the hydrodynamic volume.¹²⁷ The formation of folded nanoparticles

26 should lead to an increase of the diffusion coefficient, as evidence of the formation of single chain compaction. Regarding complex macromolecular folding obtained by formation of several crosslinks, one challenging aspect of characterization is accurately deciphering their compact morphology, which is in some cases dependant on solvent and concentration.¹⁶⁸ Characterization of the morphology can be achieved by using solution-free microscopy techniques, such as Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM), to gain insight into the size and shape of folded macromolecules. These microscopies are powerful methods since both allow the direct visualization of the folded nano-objects.

However, the absence of solvent can potentially modify the initial morphology. Also, interactions with the analytical substrate can occur and even yield in misleading images.¹⁸⁵ Hence, characterization of single chain compaction in solution is also achieved using small-angle neutron scattering (SANS) and small-small-angle X-ray scattering (SAXS) by measuring essentially the radius of gyration.

The synthesis and characterization of folded macromolecules represents an important initial step toward advanced nano-architectures that are capable of mimicking the complexity found in natural systems.¹⁶⁹ Such compacted systems are believed to make major contributions to a wide range of fields, from nanomedicine to sensing, catalysis, and other diverse uses.⁵ Already some proof-of-concept applications of single-chain folded macromolecules are reported in literature, as synthetic materials capable of exhibiting functions.^6,125,186 Compacted nano-objects can be used as enzyme-mimic systems: such synthetic collapsed polymers are exhibiting an internal cavity, which can be used as nanoreactor and/or in which catalysis can take place. For example, an impressive study showed that an amphiphilic grafted triblock copolymer can fold in water via intramolecular hydrogen bonds, leading to the formation of an internal compartment. The resulting hydrophobic cavity proved to efficiently catalyse the transfer hydrogenation reaction of ketones.¹⁸⁷ Similar copolymers were also able to facilitate the oxidation of secondary alcohols into their corresponding ketones.¹⁸⁸ Modelling enzymes with synthetic polymers offers the opportunity to improve the catalysis efficiency by controlling polymer solubility or even increasing catalytic site accessibilities.¹⁶⁸ Moreover, some folded macromolecules have been reported as effective molecular sensors for metal ions.

In a recent study, linear copolymers exhibiting pendant bipyridine-BTA units were synthesized by ring opening metathesis polymerisation and used to detect copper ions.¹⁴³ The polymers fold intramolecularly via π–π interactions into fluorescent, compartmentalised particles in a mixture of organic solvents. The fluorescence intensity was found to depend on the bipyridine content in

27 the polymer precursor.¹⁴³ Quenching of fluorescence was observed in the presence of metal ions, such as copper, since copper exhibits high affinity toward the bipyridine units and thus were encapsulated in the macromolecules. Such characteristic endows these nanoparticles as promising materials in sensor applications to detect the presence of metal ions.

Although remarkable progress has been made for the fabrication of folded polymers with promising functions, current synthetic strategies remains limited in terms of sophisticated design, folding control and unique characterization techniques.¹⁶⁹ Another impediment seen in most syntheses is the requirement of dilute polymer solutions, which complicate the practicality of scalable or industrial production of folded macromolecules. It is certainly clear, despite some obstacles, that folded macromolecules are a firmly established research topic in modern polymer science and the field of single-chain technology will undoubtedly continue to evolve and access synthetic materials with unprecedented properties.

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