of Fe3+
5.1 Chapter Summary
Inspired by nature’s exploitation of the 1,2-dihydroxybenzene (or catechol) unit in mammalian and bacterial siderophores, this chapter reports the first example of a nanoparticle sensing system which utilises the strong catechol-Fe3+ binding motif to trigger nanoparticle aggregation, promoting a powerful optical response. Gold nanoparticles were functionalised with RAFT polymerisation-prepared, water- soluble poly(N-hydroxyethylacrylamide) containing a catechol moiety at the α-
chain-end. A strong red-to-purple colorimetric response occurred in the presence of Fe3+ at serum concentrations (8 – 25 µM) in saline solution. The addition of sodium chloride was critical in generating a strong optical output, as was the length of the polymer used to coat the nanoparticles. This behaviour was also demonstrated to be selective for Fe3+ over a host of other biologically relevant ions. Therefore, these highly selective and sensitive nanocomposites provide a route towards a simple, cheap and highly applicable Fe3+ biosensor.
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5.2 Introduction
Iron is the most abundant metal in the body with the average adult requiring a total of around 5 g, using it for a range of functions including oxygen transport, electron transport and metabolic processes.1 The in vivo concentration is, however, carefully
regulated to avoid serious health implications. For example, anaemia is caused by insufficient dietary intake and absorption of iron, whilst elevated iron concentrations, such as those present in genetic disorders such as hemochromatosis, can produce excess levels of reactive oxygen species via Fenton chemistry.2, 3 Undesirable iron
levels have been linked to a variety of diseases including Alzheimer's and Parkinson's,4 making the effective detection of iron species a critical area of study.
The ability to sequester iron in the human body is complicated by the poor bioavailability of the ferric ion (Fe3+), which predominates in aerobic conditions forming complexes exhibiting a solubility of approximately 10-18 M.5 To overcome this, it is sequestered within the porphyrin ring of heme and as a co-factor in haemoglobin and myoglobin, whilst its passage into cells depends heavily on transport proteins such as transferrin.6, 7 Bacteria also require sufficient levels of the metal to survive and grow, abstracting it from their mammalian hosts with siderophores - low molecular weight complexes that bind iron with an association constant in excess of 1050 M allowing effective competition with the host for iron sequestration.7, 8
A large number of bacterial siderophores are known in comparison to the single mammalian siderophore, siderocalin,6 which counteracts bacterial uptake by sequestering the bacterial siderophore-iron complex.9-11 The majority of these species are divided into three groups: α-hydroxycarboxylates, hydroxamates and
168 catecholates, examples of which are deoxymugineic acid, rhodotorulic acid and enterobactin respectively (Figure 5.1). These functionalities have inspired the development of a number of compounds such as deferoxamine, deferiprone and deferasirox for use in iron chelation therapies,12, 13 and have provided a focus for investigations into the use of siderophore (mimics) as pharmaceutical and antibiotic drug targeting devices.14-17
Figure 5.1 Chemical structures (and classification) of example siderophores: (A) Deoxymugineic acid (α-hydroxycarboxylates); (B) rhodotorulic acid (hydroxamate) and (C) enterobactin (catecholate).
The development of sensors capable of detecting a variety of metal ions is a popular field of research.18, 19 Such detection is important for a variety of applications such as environmental monitoring, clinical toxicology and as a diagnostic tool.20-22 Several sensing platforms/responses have been described to date, including proteins and DNA,23, 24 metal electrodes25, 26 and graphene oxide,27 whilst the ability to trigger a fluorescent response upon metal ion binding has received significant study.28, 29 A further, particularly attractive platform is that of gold nanoparticles (AuNPs) due to their facile preparation, control over size, ease of surface functionalisation through
169 simple coating strategies and, most importantly for sensing applications, the resulting impact on optical properties.30-32 Specifically, the extent of nanoparticle dispersion/aggregation has a significant effect on the nanoparticle colour due to shifts in surface plasmon resonance (SPR). This signature has been applied for the detection of a variety of metal ions including lithium,33 mercury,34, 35 potassium,36 aluminium,37 lead38 and chromium.39 However, there are few instances of the application of AuNPs capable of detecting iron which have been reported. Bai et al.
functionalised gold nanoparticles with a hydrophilic ligand, to ensure water solubility, and a 4-piperazinyl-1,8-napthalimide-based ligand which was able to complex with Fe3+ generating a colorimetric and fluorescent output.40 Tripathy et al.
have prepared a colorimetric detection system based on gold, whereby the addition of iron(III) in the presence of thiourea and hydrochloric acid catalyses leaching of the gold, damping the SPR and hence resulting in a visible colour change.41 Wu et
al. have functionalised gold nanoparticles with pyrophosphate which underwent a
pink-to-violet change in the presence of Fe3+, showing selectivity for Fe3+ over a host of other ions.42
Further inspired by the structure and action of siderophores, we hypothesised that the binding between catechols43 and Fe3+ may be sufficient to affect the aggregation behaviour of a gold nanoparticle suspension in order to generate an optical response. To date, catechol groups have been used in the formulation of nanoparticles as a way of stabilising the metallic cores, but not such that the catechol unit is readily available to undergo further chemistry.44, 45 They have also been grafted onto mesoporous silica nanoparticles46 and have been used as a stabiliser in the synthesis of gold nanoparticles from their parent Au(III) salt due to the redox chemistry involved with the interchange of catechol to quinone groups.47-49 To the best of our
170 knowledge however, there are few examples whereby (gold) nanoparticles have been prepared with a catechol group protruding from the particle surface, allowing active use.50 This is surprising given developments in controlled (living) radical polymerisation (C(L)RP) methodologies over the past two decades have enabled the routine generation of vast arrays of polymeric architectures with high levels of control over functionality, chain-end structure and topology. Moreover, polymers derived from the Reversible Addition-Fragmentation Chain Transfer (RAFT) methodology51 are ideally suited for further reaction with gold nanoparticles given the inherent affinity between gold and the thiol groups derived from the ω-end of the preceding polymer chains.32 It is therefore possible, through judicial selection of the chain transfer agent used, to install the desired catechol functional group at the α-end of a polymer chain which will be subsequently available on the gold nanoparticle surface.
Herein, inspired by the structure and biological importance of siderophores, we detail the first example of a gold nanoparticle-based system capable of sensing subtle changes in physiological Fe3+ concentration through the powerful catechol-Fe3+ binding motif.
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5.3 Results and Discussion
To prepare catechol-functional polymers, the chain-transfer agent described in chapter 4 was employed.52 To effectively furnish catechol-functional gold nanoparticles, a water-soluble polymer was first required, hence N-hydroxyethyl
acrylamide (HEA) was selected for use here. This polymer was also deliberately chosen as it does not possess thermo-responsive behaviour and hence removes any potential complication of particle aggregation behaviour given the transition temperature of thermo-responsive polymers is known to change when tethered to gold nanoparticles.53 The polymerisation conditions used afforded rapid polymerisation rates (at least 75 % conversion after 35 mins) and polymers of a range of molecular weights were prepared and characterised by SEC and 1H NMR spectroscopy (Table 5.1, Figure 5.2). Narrow dispersities (≤ 1.20) were observed, an attractive feature of the RAFT technique.
Table 5.1 Characterisation of pHEA samples prepared in this study.
Polymer [M]:[CTA] Conversion (%)a
Mn(th) (g.mol-1)a Mn(SEC) (g.mol-1)b Mw/Mnb pHEA-1 30 83.1 2900 4800 1.14 pHEA-2 75 78.5 6800 9700 1.17 pHEA-3 200 74.7 17200 17500 1.20 pHEA-4c 75 74.1 6400 10200 1.16 a
Determined by 1H NMR spectroscopy relative to an internal standard (mesitylene);
b
Determined by SEC (DMF inc. 5 mM NH4BF4) relative to PMMA standards; cA
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Figure 5.2 SEC characterisation of pHEA-1/2/3/4.
Nanoparticles can be routinely synthesized from Au3+ salts and are ideally suited for RAFT-derived polymers given the strong affinity between thiols, which are derived from the ω-trithiocarbonate terminus, and gold. This can be used to prepare self- assembled monolayers and has been previously exploited to generate large nanoparticle libraries.54 Commercially available citrate-coated nanoparticles with a diameter of 40 nm (AuNP40) were used as a starting material and coated with the polymers by a simple mixing procedure, washing several times by centrifugation to remove excess polymer yielding pHEA-1/2/3@AuNP40 (Scheme 5.1).
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Scheme 5.1 Synthetic scheme for the preparation of catechol-functional gold nanoparticles: (i) NHS/DIC, THF, 0 °C r.t, 16 hrs; (ii) Dop.HCl/TEA, MeOH, r.t, 48 hrs; (iii) HEA, 3, ACVA, MeOH/Toluene, 70 °C, 35 mins; (iv) Polymer/AuNP, 5 °C, 16 hrs.
Successful functionalisation of the nanoparticles was confirmed by several techniques. UV-visible spectrophotometry revealed a distinctive red shift in maximum wavelength from 525 nm for the uncoated particles to between 531-534 nm following polymer addition, implicit of a change in refractive index of the coating (Figure 5.3A/E/I/M). DLS analysis also indicated an increase in particle size from 41.5 ± 0.2 nm for the uncoated particles to between 80 and 126 nm when coated with polymer, with the size increasing with polymer length (Figure 5.3B/F/J/N). TEM confirmed particle sizes of ~ 40 nm (Table 5.2, Figure 5.3C/G/K/O) with narrow size distributions in all cases. Due to the low degrees of surface loading employed herein, the surface polymer coating was not clearly observed by this technique. Particle sizes are larger for polymer-coated particles due to the presence of the hydrophilic polymer layer contributing to the particles’ hydrodynamic diameter as the functionalised particles undergo Brownian motion. The larger particle sizes observed for pHEA-1/2@AuNP40 by DLS may also suggest