Abstract
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monomer that provides a head-to-head pyridine building block for the preparation of cationic conjugated polymers. The obtained poly(pyridinium phenylene) polymers display appealing properties such as high electron affinity, charge-transport upon ndoping, and optical response to electron-donating analytes. A simple assay for the optical detection of low micromolar amounts of a variety of analytes in aqueous solution was developed. In particular, caffeine could be measured at a 25 µM detection limit. The reported polymers are also suitable for layer-by-layer (LbL) film formation.
Conjugated polymer semiconductors have advantageous properties (flexibility, low weight, good processability in solution) that make them useful for optoelectronic and electrochemical devices including organic solar cells, [1] [2] [3] [4] [5] [6] [7] [8] light-emitting diodes, 9 field-effect transistors,10-13 chemo-14-15 and biosensors. 16 In contrast to ptype conjugated polymers, which are currently the leading class of organic semiconductors, 17-19 n-type (or electron-transporting) conjugated polymers are still in limited supply [20] [21] [22] [23] [24] [25] [26] and are often hampered by lower performance and stability. This lack of suitable n-type polymeric materials has hindered progress in all polymer organic electronics, and research in polymer photovoltaic devices is largely focused on fullerene-based acceptor molecules, 5, 7 in spite of the poor visible light absorption and bandedge variability of such compounds. 27 In addition to the widespread organic electronics applications, conjugated polymers have been extensively studied for chemical and biological sensing, [28] [29] such as the detection of vapors, [30] [31] [32] anions, 33 or biomolecules.
34-36
Of particular relevance to the present study are the solution-based optical detection schemes for electron-rich bioanalytes.
35-36
The incorporation of electron-deficient N-heterocyclic aromatics into conjugated polymers to produce n-type compounds with high electron affinities was successfully employed in our laboratory 37 and by others. [24] [25] We recently reported a new class of ntype conjugated polymers based on a pyridinium-phenylene scaffold, that possess reversible redox activities, useful electron affinities, and high electrical conductivities. In these compounds, electron-deficient pyridinium rings were introduced by intramolecular cyclization reactions. Building on this success, we now introduce a related class of poly(pyridinium phenylene)s, which are more synthetically accessible, thereby facilitating the development of electrochemical and photophysical applications. Further, we demonstrate that these electron-deficient polymers are sensitive to electron-rich analytes and display fluorescence responses to analytes of biological and physiological interest. In particular, an aqueous solution of one of the reported polymers is able differentiate indole among a series of biologically relevant analytes, and to measure caffeine concentrations down to 25 µM.
Our simplified synthetic scheme conveniently generates monomeric unit 3 with minimial synthetic and purification steps (Scheme 1). The synthesis involves Sonogashira coupling of 5-bromo-2-iodopyridine with propargyl ether to afford precursor 1 in good yield. In the presence of Wilkinson's catalyst (RhCl(PPh 3 ) 3 ) and excess of protected hex-3-yne-1,6-diol (2), diyne 1 undergoes [2+2+2] cycloaddition to afford symmetrical monomer 3. The low yield of this optimized cyclization reaction is mostly attributed to interactions between the metal catalyst and the pyridine rings, and is largely compensated by the simplicity of the synthesis. 38 Furthermore, most of the unreacted alkyne 2 can be recycled.
Scheme 1. Two-step synthesis of monomer 3
Siloxyethyl-substituted homopolymer P1 and copolymer P2 were obtained reacting monomer 3 under Yamamoto and Sonogashira coupling conditions, respectively (Scheme 2). In the case of P1, we observed that microwave irradiation combined with high temperature heating in THF was required in order to enhance the molecular weight of the product. This observation is attributed to an improved solubility of P1 in hot THF. Similarly, we observed no formation of Sonogashira-coupling product when monomer 3 and pentiptycene diacetylene 4 were reacted under standard (e.g., overnight heating to reflux) conditions. It appears that the lower reactivity of the aryl bromide 3 under Sonogashira conditions, although advantageous for the selective synthesis of precursor 1, is significantly enhanced by the use of superheated solvent in a sealed vessel microwave reactor. Polymers P1 and P2 were found to be soluble in common organic solvents, allowing their convenient purification and characterization. Finally, polyelectrolytes P3 and P4 were obtained by subjecting precursors to intramolecular nucleophilic substitution reactions, and purified by reprecipitation from H 2 O/THF (P3) or DMSO/H 2 O followed by washing with H 2 O, MeOH and CH 2 Cl 2 (P4). Homopolymer P3 was found to be soluble in water and trifluoroethanol, whereas copolymer P4 was found to only display significant solubility in DMSO or in formic acid. Meta-stable aqueous solutions of P4 could be prepared by dissolving the polymer in formic acid followed by dilution with water, and these formulations were stable for several hours before precipitation became visible. 
P4

Cl Cl
The polycationic polymers P3 and P4 were characterized by 1 H NMR spectroscopy, absorbance and fluorescence measurements, and by cyclic voltammetry. The optical and electrochemical properties are summarized in Table 1 . The absorbance and emission bands of P3 and P4 are red shifted relative to the precursor polymers P1 and P2, as a result of the enhanced planarity induced by cyclization, and of the incorporation of electron accepting pyridiniums (Figures 1a and b, S7 and S8 ). The pentiptycene fragment of P4 induces broader absorbance and emission bands, a larger Stokes shift, and red-shifted bands compared to P3. The electron affinities (EA) of P3 and P4 are evaluated based on the onset reduction potential in thin film cyclic voltammetry measurements, 39 and are jessica81@example.net and 3.91 eV, respectively. These values are comparable to those of well-known electron transporters PCBM (4.2 eV) 7 and polybenzimidazobenzophenanthroline (BBL, 4.0 eV). 40 The conductivity of a film of polymer P3 was estimated with in situ measurement carried out on interdigitated microelectrodes (Figure 1c) . [41] [42] Inherent nonuniformity in the film limits the accuracy of this method, however these measurements clearly reveal that conductivity appears with reduction of the polymer. Significant conductivity was recorded at moderately reducing potentials (1.7 S cm -1 at -1.36 V vs. SCE), and the fully reduced (neutral) polymer was found to be insulating. 43 Spectroelectrochemical measurements of polymer thin films deposited on ITO-coated electrodes (Figures 1d and S11 ) revealed that upon reduction of the polymers, the bandgap absorbance significantly decreases, and new mid-gap energy states appear, which is in agreement with our conductivity measurements and the polaron-bipolaron theory for conjugated polymer doping. 
(eV).
d IP: Ionization potential calculated from IP = EA + E g (eV). 39 Taking advantage of the polycationic nature of polymers P3 and P4, we investigated the possibility of forming well defined films using the layer-by-layer deposition (LbL) technique 46 in association with anionic poly(styrene sulfonate) (PSS). The absorbance spectra shown in Figure 2a and S12-S13 demonstrate that LbL films are efficiently formed upon alternate exposure of an ITO-coated glass substrate to aqueous solutions of P3 (or P4) and PSS. The absorbance was found to increase linearly with the number of deposited layers, indicating a well-behaved deposition process. Cyclic voltammetry performed on LbL-deposited films (25 bilayers) indicates that the multilayer films remain stable during redox changes. As expected, the peak currents were found to increase linearly with the scan rates (Figure 2b , S14-S15). Spectroelectrochemical experiments reveal electrochromism similar to that observed with polymer thin films, with a significantly improved reversibility in the case of P3 (Figure 2d ). Following application of a sufficiently reducing potential, the initially orange film turns blue due to the reduction of P3. The initial color is recovered upon reoxidization, and the color changes occur within about 5 s. 24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57 58 59 To evaluate its utility as a sensory material, we investigated the response of copolymer P4 to a series of analytes of biological interest. Although P4 is relatively weakly emissive in solution, its high ionization potential (and associated high excited-state electron affinity) is particularly well-suited to interact with electronrich analytes. Indeed solutions of P4 in water (15 µg mL -1 , formate buffer, pH 3.6) give responses to 100 µM of electrondonating aromatic analytes (Figure 3a) . The majority of the heterocycles give an enhancement in the emission, however indole induces a 48% decrease in the fluorescence intensity. This quenching is consistent with our previous work on high ionization potential conjugated polymers and was rationalized by electrontransfer quenching from the electron-donating analyte to the polymer. [35] [36] In order to gain more insight into the quenching mechanism, we performed Stern-Volmer quenching experiments, observing both the emission intensity and lifetime as a function of the indole concentration. The linear increase in quenching with the concentration of indole illustrated in Figure 3b reveals a SternVolmer constant K SV = 9.5(0.6)⋅10 3 M -1 and an estimated detection limit of 25 µM. The fluorescence lifetime (τ = 0.4 ns) was found to be invariant as indole was added to the solution, pointing towards a static quenching mechanism. The fact that the other investigated analytes produced an increase in the emission intensity admittedly was unexpected and suggests a more complex interaction mechanism between the analytes and the polymer. Copolymer P4 has large hydrophobic surfaces and we assume that intraand intermolecular hydrophobic interactions have a strong impact on the polymer structure in solution, that can be influenced by the presence of interacting aromatic molecules. For example, although phenylalanine does not possess an easily oxidizable aromatic ring, it still induces a strong fluorescence increase. Further, the importance of the hydrophobic iptycenes in the sensing process is confirmed by the fact that homopolymer P3 shows no significant optical response in the presence of the tested analytes. Nevertheless, the fact that indole responds differently than any other studied analyte (including closely-related tryptophan, that induces an 11% increase in fluorescence intensity) potentially provides a powerful level of selectivity, where a single specific analyte is detected against several others.
The second analyte of particular interest was caffeine. In the presence of 100 µM of caffeine, the fluorescence intensity of the polymer solution increased by 38%, the largest measured increase over our panel of analytes. A titration study performed with various concentrations of caffeine allowed to estimate a detection limit of 25 µM (Figures 3c and S17) , which makes our sensing scheme competitive with formerly reported schemes for the solution-based optical sensing of caffeine. [47] [48] [49] In particular, a detection limit of 50 µM was recently reported using polysulfonated pyrenes as molecular probes. 47 We were able to use P4 to estimate the caffeine content of a series of everyday products. In this procedure, the selected samples were extracted (coffee and decaffeinated coffee, soda, energy drink, and a painkiller) into dichloromethane and evaporated, in order to minimize the influence of the matrix. The extracts were then redissolved in formate buffer and added into aqueous solutions of polymer P4. The change in fluorescence intensity was measured, and the caffeine content in the samples was then calculated using the calibration curve displayed in Figure 3c . This procedure yielded caffeine concentrations within a reasonable margin of error for most samples, but coffee and decaffeinated coffee displayed erroneously high values (Figure 4) . These discrepancies may be the result of additional analytes present in the beverages that interfere in a non-selective fashion with P4 and contribute to the fluorescence increase. In summary, we have described a simple synthetic pathway and characterization of cationic conjugated polymers that possess interesting optical and electrochemical properties. These materials have high electron affinities, useful ionization potentials and large Stokes shift (in particular in the case of the iptycene containing copolymer P4). They can be conveniently processed using simple layer-by-layer methodologies, and we also demonstrated that such polymers can act as optical probes for various analytes of biological interest in aqueous solutions, such as indole and caffeine. Further studies to elucidate the interaction mechanisms behind the fluorescence responses are required in order to fine-tune the sensing abilities of these compounds. 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 

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