Andrew J Gross

Xiaohong Chen

Fabien Giroud

Caroline Abreu

Alan Le Goff

Michael Holzinger

Serge Cosnier

A High Power Buckypaper Biofuel Cell: Exploiting 1,10-Phenanthroline-5,6-dione with FAD-Dependent Dehydrogenase for Catalytically-Powerful Glucose Oxidation

Keywords: bioelectrocatalysis, biocatalyst, paper electrode, enzymatic, FAD-GDH, multicopper oxidases 50

Enzymatic biofuel cells generate electrical energy from renewable sources with high selectivity and environmental benefits compared to lithium batteries and traditional fuel cells. For enzymatic fuel cells to become competitive, major improvements in electrode design are required to enhance power density, voltage output, and stability. Here we have developed a freestanding paper biofuel cell comprising redox molecule embedded multiwalled carbon nanotube papers for electrical wiring of enzymes. The drop-coat and one-pot fabrication methods provide flexibility and permit easy scalability of functionalized bioelectrodes via commercially available materials.

Buckypaper functionalized with 1,10-phenanthroline-5,6-dione (PLQ) as an efficient electron mediator for fungal-derived FADdependent glucose dehydrogenase (FADGDH) shows very high steady-state current densities for glucose oxidation of I max = 5.38 ± 0.54 mA cm -2 at 0.15 V vs SCE at neutral pH. When coupled with a bioinspired protoporphyrin IX buckypaper cathode, the resulting glucose/O 2 fuel cell delivered a power density of 0.65 ± 0.1 mW cm -2 or 24.1 ± 4.7 mW cm -3 at a cell voltage of 0.5 V, limited by the cathode. Galvanostatic and current discharge experiments confirm robust short-term operational performance.

■ INTRODUCTION

Enzymatic biofuels (EBFCs) convert chemical energy into electrical energy under mild conditions from the oxidation of organic fuels such as sugars and alcohols, coupled with the reduction of oxygen using redox enzymes as bioelectrocatalysts. [START_REF] Cosnier | [END_REF][2][3] Biofuel cells offer several advantages over lithium batteries and traditional fuel cells, including the use of renewable, low-cost, and safe fuels from ecological sources and operation at neutral pH and ambient temperature. Furthermore, EBFCs can operate as membraneless fuel cells owing to the high selectivity of enzymes. Particular attention of biofuel cells over the past decade has been directed toward the use of EBFCs as micropower sources which harvest energy from biological fluids for implantable medical devices. [4][5][6][7] In recent years, EBFCs have been successfully implanted in rats, 4 lobsters, 5 and snails. 6 Application of EBFCs for portable and wearable devices is more feasible and has enormous potential. For example, epidermal and contact-lens biofuel cells which harvest energy from sweat or tears using carbon nanotube based electrodes have started to emerge. [8][9][10] Lightweight biofuel cells with small form factors offer the prospect of selfpowered chemical sensors with in situ detection and remediation of toxic chemicals. 11,12 Specific challenges in EBFC electrode design include the development of electrodes which offer (i) stable immobilization of enzymes and mediators with high loadings, (ii) efficient were obtained from Messer. Glucose solutions were left to mutarotate overnight to β-D-glucose prior to use.

Preparation of Unmodified MWCNT Buckypaper and

Redox-Embedded MWCNT Buckypaper Electrodes by

Drop-Coat Method. First, a 1 mg mL -1 MWCNT suspension was prepared by the addition of 150 mg of nonfunctionalized MWCNTs into 150 mL of DMF. The dispersion was then sonicated for 30 min prior to use. After vigorous shaking for 1 min, 66 mL of the suspension was filtered through a Millipore PTFE filter (JHWP, 0.45 μm pore size, Ø = 46 mm) using a vacuum pump, washed with distilled water, and left for 1 h.

After filtration, the resulting unmodified buckypaper with Ø = Average catalytic current densities were obtained by subtracting the background current: the signal obtained without oxygen, for the cathode, and without glucose, for the anode. Fuel cell experiments were performed by recording a linear sweep polarization from the open circuit voltage (OCV) to 0.02 V with the anode connected to the counter and reference leads, and the cathode connected to the working lead. Power densities were obtained by dividing the power delivery by the surface area or volume of one electrode.

Scanning Electron Microscopy. Buckypaper electrodes with Ø = 3 mm were imaged using a FEI/Quanta FEG 250 scanning electron microscope (Hillsboro, OR, USA) operating at an accelerating voltage of 5 kV without metal coating.

Static Water Contact Angles. Water contact angles were obtained at room temperature by delivering a 2 μL droplet of distilled water onto the sample surface on a horizontal stage using a Dataphysics OCA 35 system. Multiple droplet measurements (8 or 9) were recorded per sample type.

■ RESULTS AND DISCUSSION

Characterization of Unmodified MWCNT and Redox-Embedded MWCNT Buckypaper Electrodes. Pristine freestanding buckypaper prepared from filtration of CNT dispersions without additives tends to be fragile and difficult to manipulate. Our initial experiments established that the ability to handle pristine unmodified BP was improved when the amount of MWCNTs in the final product was increased by at least a factor of 2. 30 Redox-embedded BP was subsequently prepared in several steps (see Experimental Section) either by the addition of redox molecules to the MWCNTs dispersion before filtration (one-pot method) or after filtration by dropcoating onto the unmodified BP (drop-coat method). The BP functionalization is based on π-π stacking interactions between aromatic groups and CNT sidewalls. For all buckypapers, flat f1 and reproducible disks were obtained after filtration (Figure f1 1A), drying, and cutting (Figure 1B and C, respectively). BP thicknesses ranged between 230 and 320 μm (see Table S1). The variability in thickness results from differences in vacuum pressure and the MWCNTs dispersion.

Scanning electron microscopy was performed to evaluate the morphology of unmodified BP (BP, Figure 1D) and BP prepared from the one-pot method with redox molecules: protoporphyrin IX (BP PP , Figure 1E), ferriprotoporphyrin IX (BP FePP , Figure 1F), and 1,10-phenanthroline-5,6-dione (BP PLQ , Figure 1G). The SEM images reveal that the BPs comprise a random and entangled network of MWCNTs with nanoscale porosity. The images show subtle evidence that functionalized BPs have a flatter and more compact topography compared to unmodified BP, suggestive of more intimate interactions between nanotubes in the presence of redox molecules via π-π stacking.

Cyclic voltammograms (CVs) of the Fe(CN) 6

3-redox probe recorded at unmodified and BP PP paper electrodes reveal a significant increase in peak current with a decrease in peak-topeak separation (ΔE P ) following modification with protoporphyin molecules (see Figure S1). The dramatic enhancement with chemical functionalization is partially attributed to a physical improvement in the electronic connectivity between carbon nanotubes. The increase in peak current observed is also partially attributed to improved diffusion of Fe(CN) 6

3-/4-in the 3D-structured electrode, facilitated by the increased surface hydrophilicity (Figure S2). Table 1 also reveals that BP FePP prepared via one-pot and 312 drop-coat methods with 0.6 mmol L -1 modifier solution give 313 similar peak-to-peak separation values of ≤65 mV and therefore 314 similar apparent electron transfer kinetics. Significantly larger 315 ΔE P values and therefore sluggish electron transfer kinetics 316 were observed for highly functionalized BP FePP electrodes 317 prepared with 10 mmol L -1 of the modifier. This is consistent 318 with an extensive network of redox molecules being deeply embedded in the CNT structure, which would increase electron tunneling distances and film resistivity.

On the basis of the BP FePP optimization experiments, we prepared PLQ buckypaper via the one-pot method using a modestly high concentration of 2 mmol L -1 . No further optimization was performed. The surface concentration obtained for one-pot BP PLQ was 1.21 ± 0.30 × 10 -7 mol cm -2 , which reveals the possibility to obtain high redox molecule loadings with a near 1 × 10 -7 mol cm -2 concentration. The ΔE P value of 118 mV observed for BP PLQ is consistent with a slow rate of electron transfer, increased BP resistivity, and a large degree of potential inversion for the twoelectron two-proton quinone process. 36 Bioelectrocatalytic O 2 Reduction at BP pp Electrodes with Immobilized BOD Enzyme from Myrothecium verrucaria. For biocathode construction, the multicopper oxidase (MCO) enzyme BOD from Myrothecium verrucaria (Mv) was employed as the catalyst for the four-electron reduction of O 2 to H 2 O. BOD is a promising enzyme for biofuel cells due to its high bioelectrocatalytic activity under mild conditions and formal potential close to that of the O 2 / H 2 O couple (0.816 V vs RHE at pH 0). Bilirubin, as a natural substrate for BOD, and its analogues, have been attached to MWCNT electrodes to facilitate DET with MvBOD for O 2 reduction. 34,37 The interaction is supported by favorable immobilization and orientation. However, a commonly accepted problem with BOD is that significant catalytic activity loss is observed in the presence of H 2 O 2 , a common byproduct of oxidase enzymes found in glucose/O 2 biofuel cells. The use of O 2 -insensitive FADGDH in biofuel cell design can circumvent this issue.

In this study, the porphyrin-BOD system was exploited for the development of buckypaper cathodes. One-pot BP pp electrodes were first prepared then incubated with BOD, washed with phosphate buffer at pH 7.0, then tested for f3 enzyme presence and activity. Figure 3A andB The long-term stability of BP PP -BOD was also assessed by 387 periodically recording chronoamperograms at E app = 0.2 V vs 388 SCE for 30 min over 24 days of storage in phosphate buffer at 389 pH 7.0 (Figure S5). The corresponding plot in Figure 3C of the 390 maximum catalytic current obtained on different days reveals 391 remarkable stability for BP PP -BOD, with the current density decreasing by 10% over the first 7 days and 27% over 24 days.

Stability experiments performed in a similar manner at other MWCNT-MCO enzyme cathodes report 40-45% current loss after 1 week and 45-60% after 20-24 days. 38,39 To clarify, such stability experiments evaluate both operational stability (for 30 min per day) and storage stability. The enhanced stability observed here compared to that with CNT-modified GC electrodes is consistent with improved physical enzyme entrapment into the nanopore-containing CNT matrix via fixation and/or dynamic reorganization effects. 40 A brief assessment of the bioelectrocatalytic performance of The bioelectrocatalytic performance of BP PP for oxygen reduction was also performed at electrodes prepared by the drop-coat method. No significant difference in E onset was observed between drop-coat and one-pot BP PP electrodes.

Likewise, equivalent sigmoidal steady-state current responses (with different current magnitudes) were observed for all BP PP electrodes. For drop-coat BP PP , the maximum average catalytic current only slightly increased from 1.10 ± 0.14 to 1.26 ± 0.11 mA cm -2 with an increase in protoporphyrin modifier from 0.6 mmol L -1 to 10 mmol L -1 .

The best performing biocathode identified here was the BP PP -BOD prepared using the one-pot method with I max = 1.33 ± 0.17 mA cm -2 . The high performance of this biocathode is clear given that typical values obtained by our group and others for CNT paper-based MCO cathodes are 0.17 to 1.1 mA cm -2 in oxygen-saturated solution. 27,29,30,41,42 Bioelectrocatalytic Glucose Oxidation at BP PLQ Electrodes with Immobilized FADGDH from Aspergillus sp.

For bioanode development, the one-pot method was exploited for preparation of a new type of redox-embedded buckypaper with immobilized FADGDH. To prepare the anode, a MWCNT dispersion containing 2 mmol L -1 of 1,10phenanthroline-5,6-dione (PLQ) was used. The bioanode was obtained by drop-casting 150 μL of 5 mg mL -1 enzyme solution onto the BP PLQ and leaving the solution overnight until the droplet had fully adsorbed. The PLQ molecule has previously been reported as a mediator for the cofactors NADH/NADPH coupled with NAD/NADP-dependent enzymes. 35,43 To the best of our knowledge, the phenanthroline quinone as a free ligand has not been demonstrated as a mediator for FAD-dependent enzymes such as FADGDH. In addition to the low redox potentials, a major advantage of phenanthroline quinone mediators is their nonreactivity toward active-site enzyme amine and thiol groups. 43 In this work, we have investigated the use of PLQ, in the form of a redoxembedded functionalized buckypaper, to electrically connect

onset potential (V) b maximum catalytic current (mA cm -2 ) b,c BP PP -BOD (n = 5)
one-pot (0.6 mmol L -1 ) 0.54 ± 0.01 1.33 ± 0.17 electrode, the BP NQ electrode still exceeds recent high performance NQ-anodes. 22,23 The catalytic current obtained of 5.38 ± 0.54 mA cm -2 at 0.15 V vs SCE for BP PLQ in unstirred solution exceeds the performance of that observed at most bioanodes to date without the use of hydrodynamic conditions. We believe that the BP PLQ anode exhibits the highest catalytic density for glucose oxidation of any paper-based bioanode. At phenothiazine-modified commercial buckypaper with immobilized NADdependent GDH and NAD cofactor, current densities up to 2.6 mA cm -2 at 25 °C have been reported. 25 With the addition of 1 mmol L -1 NAD + in solution, the catalytic current increased to 4.5 mA cm -2 at a high potential of 0.35 V. In addition to the high potential required, the use of NAD in solution is less convenient than if the cofactor is surface-bound or enzymebound. High performing bioanode architectures typically produce no more than 2 mA cm -2 22,44,45 with the exception of a hierarchical porous carbon bioelectrode which exibited enormous densities up to 100 mA cm -2 with rapid convection. 18 With CNT-modified Toray papers with immobilized glucose oxidase and dehydrogenases, current densities in the range 1-3.3 mA cm -2 have been reported. 23,[46][47][48] Evaluation of the steady-state current at a fixed potential of 0.15 V as a function of glucose concentration reveals a linear increase in the range 1 mmol L -1 to 50 mmol L -1 (see calibration plot in Figure S9). As a side note, the linearity of r 2 = 0.996 and the ability to detect beyond the upper limit of 30 mmol L -1 means that the BP PLQ -FADGDH meets basic requirements for a commercial glucose sensor. or 1.97 mW cm -3 is observed. The power outputs for these glucose/O 2 biofuel cells are either very good or exceptional depending on whether the power delivery is divided by the surface area (cm 2 ) or volume (cm 3 ). It is noted that we used equal sized anodes and cathodes in this work and that the power densities were obtained by dividing the power delivery by either the surface area or volume of one electrode. Given that buckypaper is a three-dimensional electrode and size is a crucial parameter for portable biofuel cell applications, the power density in mW cm -3 is arguably more appropriate.

However, for this work we have chosen to remain with the standard mW cm -2 convention for figures. Maximum power outputs for a single EBFC in the literature are between 1.45 and 2.3 mW cm -2 at 0.3 to 0.55 V. 22,23,52 One of these previously reported biofuel cells, based on our estimates, gives up to 3.8 mW cm -3 (1.54 mW cm -2 52 ), and thus the delivery of 24.07 mW cm -3 reported here is markedly high.

The difference in power density observed between the saturated and quiescent oxygen conditions reveals that the biofuel cell is strongly limited by oxygen availability at the cathode. Even in the presence of oxygen-saturated conditions, the biocathode is limiting.

Polarization and power curves were also recorded at freshly prepared biofuel cells in the absence of glucose under oxygensaturated conditions as control experiments (Figure S13). In the absence of the enzyme's substrate, the low power output and a small OCV, namely 0.02 mW cm -2 and 0.53 ± 0.02 V, clearly illustrate and confirm the unambiguous role of FADGDH and the need for glucose for effective biofuel cell performance.

In order to assess the operational stability of the biofuel cell, Similar glucose/oxygen biofuel cell stability has been observed using FADGDH, which, notably, is vastly superior to that observed with the GOx enzyme. 14 The initial induction period is attributed to diffusional equilibration and high charging currents. To further assess the stability, a current of 500 μA was continuously drawn and the evolution of voltage monitored (Figure 6B). Following the short initial induction period, a stable voltage of 0.57 ± 0.01 V is obtained.

  35 mm was left to dry at room temperature. The buckypaper was obtained after its careful removal from the filter paper, then cut into individual electrodes with Ø = 10 mm (geometric surface area of 0.785 cm -2 ) using a metal cutter. To obtain functionalized MWCNT buckypaper electrodes, 150 μL of 0.6 mmol L -1 or 10 mmol L -1 modifier (PP and FePP) in DMF was drop-coated onto unmodified buckypaper surfaces with Ø = 10 mm (each electrode contains 5.39 mg of MWCNTs) and the resulting electrode left to dry overnight at room temperature. Electrical contact was obtained via a metal wire with carbon paste. The back and sides of the electrode were sealed with silicone paste. Preparation of Redox-Embedded MWCNT Buckypaper Electrodes by One-Pot Method. A 1 mg mL -1 MWCNT suspension was first prepared by the addition of 150 mg of nonfunctionalized MWCNTs into 150 mL of DMF. The dispersion was then sonicated for 30 min prior to addition of the modifier (PLQ, NQ, PP, and FePP). The modifier, dissolved in a minimum volume of DMF, was slowly added into the MWCNT suspension to give a final 0.6 mmol L -1 or 2 mmol L -1 modifier concentration. Prior to filtration, the resulting suspension was sonicated for a further 30 min. After rigorous shaking for 1 min, 66 mL of the suspension was filtered, washed, and dried (as above for the unmodified buckypaper) and the resulting buckypaper cut into electrodes with Ø = 10 mm. Electrical contact was obtained via a metal wire with carbon paste. The back and sides of the electrode were sealed with silicone paste. Preparation of Buckypaper Bioelectrodes. First, 5 mg mL -1 stock solutions of BOD and FADGDH were prepared in 0.1 mol L -1 phosphate buffer at pH 7.0 and McIlvaine buffer at pH 7.0 (0.2 mol L -1 Na 2 HPO 4 , 0.1 mol L -1 citric acid), respectively. A total of 150 μL of 5 mg mL -1 enzyme solution (0.75 mg enzyme) was then added to the surface of a buckypaper with Ø = 10 mm, and the enzyme solution was allowed to fully absorb at 4 °C overnight. The electrode was subsequently rinsed with the corresponding buffer before use. Electrochemistry. Electrochemical measurements were performed at room temperature using an Eco Chemie Autolab PGSTAT 100 potentiostat running GPES 4.9 software or a Biologic VMP3Multi Potentiostat with EC-lab software. For half-cell testing, a conventional three-electrode cell setup was used comprising a buckypaper working electrode (Ø = 10 mm), a saturated calomel reference electrode (SCE with sat. KCl), and a Pt wire counter-electrode. The complete fuel cell setup comprised a one-pot protoporphyrin buckypaper with BOD biocathode (BP PP -BOD) and a one-pot phenanthroline quinone buckypaper with FADGDH (BP PLQ -FADGDH) bioanode immersed in 20 mL of McIlvaine buffer at pH 7.0 with an interelectrode distance of 3 mm. Gas flows were set qualitatively and moderately to obtain reproducible conditions.

257

  To investigate the presence and accessibility of porphyrin 258 and PLQ molecules in BP prepared via the one-pot and drop-259 coat methods, cyclic voltammetry was performed on one-sided 260 BP samples with a geometric surface area of 0.785 cm 2 . CVs 261 were first recorded at functionalized electrodes prepared by the 262 one-pot method in argon-saturated 0.1 mol L -1 phosphate 263 f2 buffer at pH 7.0. Figure 2 shows CVs recorded at BP FePP and 264 BP PLQ prepared via the one-pot method. The CVs reveal the 265 presence of well-defined chemically reversible processes. At 266 BP FePP, the one-electron Fe III /Fe II redox couple is observed at 267 E 1/2 = -0.38 V vs SCE (10 mV s -1 ), consistent with reported

Figure 1 .

 1 Figure 1. (A, B) Photographs of freshly prepared BP PP buckypaper (A) after filtration and (B) after filtration, drying, and cutting into the electrode. (C-G) SEM micrographs of (C, E) BP PP , (D) BP, (F) BP FePP , and (G) BP PLQ buckypaper electrodes recorded at (C) 60×, (D-G) 5000× and 40 000× (inset) magnification. All modified electrodes were prepared by the one-pot method.

Figure 2 .

 2 Figure 2. (A,B) CVs recorded at (A) BP FePP and (B) BP PLQ buckypaper electrodes (one-pot method) in Ar saturated 0.1 mol L -1 phosphate buffer at pH 7 at different scan rates. (C, D) Corresponding plots of anodic and cathodic peak current versus scan rate.

  show CVs recorded in argon and oxygen at one-pot BP PP -BOD and BP-BOD, respectively. Under oxygen, both types of electrode exhibited similar onset potential of E onset = 0.54 ± 0.01 V vs SCE and 0.52 ± 0.01 V vs SCE, respectively. The onset potentials are close to that of the predicted T1 copper site of MvBOD responsible for substrate oxidation and successive electron transfers (E 1/2(T1) = 0.48 V vs SCE at pH 7.0). 34 The onset potentials are close to the ideal thermodynamic reduction potential for O 2 of 0.572 V vs SCE at pH 7.0 (assuming a potential difference of 244 mV between RHE and SCE reference electrodes). The onset potential values are therefore very attractive for biofuel cell applications and consistent with efficient single-proton single-electron DET between the BP and the enzyme. The catalytic wave observed in oxygen at BP PP confirms DET accompanied by the electrocatalytic reduction of oxygen via

  BP PP -BOD was also performed to demonstrate enzyme inhibition at the biocathode. CVs recorded in the presence and absence of 40 mmol L -1 H 2 O 2 in phosphate buffer at pH 7.0 in oxygen are shown in Figure S6 and clearly reveal the loss of oxygen reduction current due to inhibition of immobilized bilirubin oxidase by H 2 O 2 . The catalytic current was suppressed in the presence of H 2 O 2 and remained suppressed after transfer to a fresh phosphate buffer solution, confirming that the enzyme's activity was inhibited and not restored. The absence of oxygen reduction in these experiments validates the importance of the active "wired" enzyme as the biocatalyst. Chronoamperometry performed at BP PP -BOD in phosphate buffer at pH 7.0 in oxygen (Figure S6) revealed the rapid nature of enzyme inhibition by H 2 O 2 and the good stability of the biocathode in the presence of a physiologically relevant concentration of NaCl (100 mM).

Figure 3 .

 3 Figure 3. (A,B) CVs of bioelectrocatalytic O 2 reduction recorded at (A) BP PP (one-pot method) and (B) BP buckypaper electrodes after BOD enzyme immobilization in (--) Ar saturated and (-) O 2 saturated 0.1 mol L -1 phosphate buffer at pH 7.0 at scan rate = 0.2 mV s -1 . (C) Evolution of maximum catalytic current density as a function of time for BP PP -BOD. Chronoamperograms recorded at E app = 0.2 V vs SCE and current densities obtained after 30 min. Error bars correspond to one standard deviation from two electrodes.

BPa

  Number of independent buckypaper electrode samples analyzed. b Parameters obtained from the forward sweep of CVs recorded at 0.2 mV s -1 . c Current obtained at 0.2 and 0.15 V at BOD and FADGDH electrodes, respectively.

Figure

  Figure 4C shows that the steady-state currents increased with increasing concentration to a plateau at 170 mmol L -1 glucose; hence 170 mmol L -1 was adopted in experiments as the concentration to maximize catalytic current output, limited by the enzymatic reaction and catalyst surface coverage. Limiting current values were reached within 30 s (Figure S10), consistent with fast mass transport of glucose at the electrode. Estimated values for the apparent Michaelis-Menten and velocity constants of K m = 40.4 mmol L -1 and V max = 6.1 mA cm -2 , respectively, are obtained. The estimated K m is similar to that observed for fungal FADGDHs (K m = 35 mmol L -1 49 ), and hence the BP PLQ electrode maximizes electrocatalysis without significantly affecting the enzymes' binding constant for glucose. The stability of the BP PLQ -FADGDH bioanode was assessed over 10 days by periodically recording chronoamperograms at E app = 0.15 V vs SCE for 30 min in McIlvaine buffer at pH 7.0 (Figure S11). The plot in Figure 4D of the maximum catalytic

Figure 4 .

 4 Figure 4. (A) Scheme illustrating bioelectrocatalytic oxidation of glucose on BP PLQ buckypaper via MET with the active site of FADGDH. (B) CVs recorded at a BP PLQ buckypaper electrode after FADGDH immobilization in (--) 0 mmol L -1 and (-) 170 mmol L -1 glucose in Ar saturated McIlvaine buffer at pH 7.0 at scan rate = 0.2 mV s -1 . (C) Plot of average current density obtained for different glucose concentrations recorded at three BP PLQ -FADGDH electrodes. (D) Evolution of maximum catalytic current density as a function of time for BP PLQ -FADGDH with 170 mmol L -1 glucose. (C, D) Chronoamperograms recorded at E app = 0.15 V vs SCE and current densities obtained after 30 min. Error bars correspond to one standard deviation from at least two electrodes.

  potentiostatic and galvanostatic tests were performed for 30 min. This duration may, for example, be considered as an adequate operational time for a single use self-powered biosensor. The stability of the biofuel cell was first examined by applying a mild fixed voltage of 0.2 V and monitoring the current in oxygen-saturated solution. The power obtained from the recorded current revealed a stable power output of 295 ± f6 25 μW cm -2 after an initial 30 s induction period (Figure6A).

Figure 5 .

 5 Figure 5. (A) Scheme illustrating the single compartment buckypaper biofuel cell comprising an O 2 reducing BP PP -BOD cathode and glucose oxidizing BP PLQ -FADGDH anode. (B, C) Biofuel cell polarization (-•-) and power curves (-) recorded under (B) oxygen-saturated and (C) quiescent oxygen conditions in McIlvaine buffer at pH 7.0 with 170 mmol L -1 glucose. Polarization voltammograms were recorded at 0.2 mV s -1 . Error bars correspond to one standard deviation from three biofuel cells.

Figure 6 .

 6 Figure 6. (A) Power generated from current discharge at 0.2 V and (B) voltage generated at 600 μA cm -2 (500 μA) for 30 min operation of the BP PP -BOD/BP PLQ -FADGDH biofuel cell in O 2 saturated McIlvaine buffer at pH 7.0 with 170 mmol L -1 glucose. Error bars correspond to one standard deviation from three biofuel cells.

Table 1 .

 1 Electrochemical Parameters for BP FePP and BP PLQ Electrodes Prepared by One-Pot and Drop-Coating Functionalization Methods buckypaper electrode (number a ) fabrication method modifier concentration (mmol L -1 ) surface concentration b (10 -7 mol cm -2 ) ΔE p (mV) E 1/2 (V) Number of independent buckypaper electrode samples analyzed. b Surface concentrations obtained from the anodic peak of CVs recorded at 10 mVs -1 in Ar.371 BOD. On the basis of repeat measurements, an average 372 maximum current density of 0.34 ± 0.15 mA cm -2 is observed 373 at BP compared to 1.33 ± 0.17 mA cm -2 for BP PP (one-pot 374 method), confirming substantially enhanced performance for 375 the protoporphyrin buckypaper. At BP, an ill-defined "residual 376 slope" current is observed in oxygen, attributed to unfavorable 377 orientation of adsorbed enzymes. In contrast, at the BP PP 378 electrode, well-defined steady-state voltammograms are ob-379 served consistent with fast mass transport, enzyme catalysis, 380 and heterogeneous electron transfer at the buckypaper 381 electrode.37 It is noted that a scan rate of 0.2 mV s -1 was 382 employed due to the superior steady-state signals with low 383 capacitance (see FigureS4for catalytic currents recorded at 384 different scan rates). A summary of the catalytic parameters

	BP FePP (n = 3)	one-pot	0.6	1.38 ± 0.58	65	-0.37
	BP FePP (n = 3)	drop-coat	0.6	0.48 ± 0.25	55	-0.35
	BP FePP (n = 7)	drop-coat	10	6.43 ± 3.58	215	-0.36
	BP PLQ (n = 3)	one-pot	2	1.21 ± 0.30	118	-0.13

a t2 385 obtained at different bioelectrodes is shown in Table

2

.

386

Table 2 .

 2 Catalytic Parameters for BP PP -BOD Cathode and BP PLQ -FADGDH Anode Prepared by One-Pot and Drop-Coating Functionalization Methods

	buckypaper	fabrication method
	electrode (number a )	(modifier concentration)

■ ACKNOWLEDGMENTS This work was supported by LabEx ARCANE (ANR-11-LABX-0003-01) and ANR-15-JTIC-0002-01. The region Auvergne-Rhone-Alpes is acknowledged for the Ph.D. funding of C.A. The authors would also like to thank the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG) and PFT "Surfaces Functionalization and Transduction" for providing facilities and PMIEL "Microscopy" for SEM imaging. We are very grateful to Christine Lancelon Pin for assistance with SEM imaging.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.


Dr Fabien Giroud
email: fabien.giroud@univ-grenoble-alpes.fr

A J Gross

D Faggion Junior

M Holzinger

C E Maduro De Campos

S Acuña

J B Domingos
email: josiel.domingos@ufsc.br

S Cosnier

Junior D Faggion

J J S Acuña

Pr Josiel

B Domingos

Hydrazine Electrooxidation with PdNPs and Its Application for a Hybrid Self-Powered Sensor and N 2 H 4 Decontamination

published or not. The documents may come

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Introduction

Hydrazine, N2H4, is extensively used commercially for the production of agricultural herbicides and pesticides, as a rocket propellant in the aerospace industry, as the refining agent for precious ores recovery, and as a corrosion inhibitor (oxygen scavenger) in water boiler rooms. 1,2 The use of N2H4 as a fuel has attracted huge interest since the 1970s largely due its high solubility, rapid kinetics and low oxidation potential which enable large current densities not possible with other fuels such as hydrogen and ethanol. 3 The theoretical electromotive force for a N2H4/O2 fuel cell is relatively large with a value of 1.56 V compared to other fuel/oxidant couples (1.23 V for H2/O2, 1.20 V for glucose/O2 and 1.15 V for ethanol/O2 systems) and therefore desirable for increasing overall performances. [4][5][6] N2H4 is also a "carbonfree" reducing agent due to its efficient conversion into N2 gas and water unlike more traditional fuels which release CO2 during their oxidation process. Hydrazine can also be stored in liquid form and will not participate in increasing greenhouse gas levels as is the case for other fuels such as hydrogen gas.

As a result of widespread industrial use, hydrazine and its derivatives are frequently found in our environment and this poses health risks. Hydrazine is a documented toxic environmental contaminant and is known for its potential human carcinogenic and mutagenic effects. 7,8 Even acute poisoning can result in adverse health conditions ranging from dizziness to damage of the central nervous system. 8 Since large volumes of hydrazine are consumed on a daily basis and this consumption will likely increase with time, there is a great need to develop methods of detecting and remediating hydrazine in the environment.

Current methods of dealing with high levels of hydrazine in the environment include chemical treatment with oxidizers such as sodium/calcium hypochlorite (bleach). Thermal or chemical methods typically give incomplete oxidation and may create more hazardous conditions than before the treatment. [START_REF] Bowman | NASA, Handbook on Hypergolic Propellant Discharges and Disposal[END_REF] The development of disposal techniques which completely convert hydrazine fuel or environmental solutions to non-toxic compounds is therefore highly desirable and only a few (bio)chemical approaches exist. [START_REF] Ou | [END_REF]11 However, these techniques only intend to phase out hydrazine from fuel or contaminated solutions without taking advantage of its high energy density.

Several electrochemical systems have now been developed for detection and/or electrocatalytic oxidation of hydrazine in aqueous solutions. However, the electrochemical response at unmodified electrodes is hindered by slow electron transfer kinetics and a very large oxidation overpotential requirement, leading to poor sensitivity and reproducibility. A range of electrodes have been developed to address these limitations including chemically-modified electrodes with metal complexes as efficient electrocatalysts.

More recently, the use of composite carbon electrodes with metal particles and nanoparticles have been developed as effective composite catalytic electrodes. [12][13][14][15][16][17] Palladium nanoparticles are currently of high interest as electrocatalysts for direct hydrazine oxidation owing to their rate-enhancing electronic properties. 18 However, despite the attraction of such electrocatalysts for hydrazine oxidation for sensing or energy production purposes, to the authors' best knowledge, no reports exist on their integration into hybrid biofuel cell systems. Furthermore, such hybrid systems could be developed into self-powered systems, a concept first reported by Katz and coworkers, where the fuel cell output is used as the sensor signal. 19 In this work, we aim to demonstrate that CNT-supported PdNPs catalysts can be used for the consumption of aqueous hydrazine as a possible method of environmental remediation whilst, at the same time, generating clean electrical power and/or sensing the concentration of hydrazine present. Initial investigations were performed to optimize PdNPs loadings on MWCNT surfaces. Since hydrazine is known to scavenge dissolved oxygen, bilirubin oxidase (BOx) biocathodes made from gas diffusion layer (GDL) electrodes were utilized for construction of a hybrid hydrazine/O2 fuel cell and self-powered sensor. This fuel cell configuration was adapted to overcome low O2 concentration in aqueous electrolyte solution to improve overall catalytic performances.

Keywords: Pd nanoparticles, hydrazine oxidation, oxygen reduction, electrocatalysis, biofuel cell

Materials and Methods

Chemicals and materials.

Polyvinylpyrrolidone (PVP, MW ~ 55,000 g mol -1 ), L-ascorbic acid, potassium bromide, potassium tetrachloropalladate(II), 1-pyrenemethylamine hydrochloride (PMA), 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), N-hydroxysuccinimide (NHS), hydrazine solution (N2H4; 35 wt % in H2O) and 1-methyl-2pyrrolidone (NMP), disodium citrate, and sodium phosphate were obtained from Sigma Aldrich and used as received. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was purchased from Fluka. Bilirubin oxidase (BOx) from Myrothecium verrucaria (E.C. 1.3.3.5) was purchased from Amano Enzyme (Japan). Reagent grade acetonitrile (CH3CN) was received from VWR Chemicals Commercial grade thin multi-walled carbon nanotubes (MWCNTs) (NC3100, 9.5 nm diameter, purity > 95 %,) were obtained from Nanocyl (Belgium). Gas diffusion layers were non-treated carbon cloth electrodes and were purchased from PAXITECH (France). Distilled deionized water (15 MΩ) was obtained from a PURELAB ELGA purification system. Oxygen and argon were purchased from Air Liquide (France). Non-treated carbon gas diffusion layer (GDL) electrode were purchased from Paxitech (France).

Oxidizing electrode fabrication.

The anodes used for N2H4 oxidation were made by layer-by-layer deposition of MWCNTs with cubic PdNPs. A 2.5 mg mL -1 MWCNT dispersion in NMP was prepared by 30 min sonication to achieve a homogeneous black suspension. The black suspension was deposited onto the glassy carbon electrode (GCE) to give an electrode disc with a geometric surface area of 0.126 or 0.196 cm 2 for 10 or 20 µL droplet deposits, respectively, followed by deposition of various loadings of PdNPs (4 mg mL -1 in NMP). The load of PdNPs was adjusted by depositing different solution volumes onto the electrode surface. The distribution of the deposited cubic PdNPs on the MWCNT electrode surface was verified by scanning electron microscopy (SEM) with a QUANTA FEG 250 (FEI) without coating the substrates with metal. Secondary electron and backscattered electron detection modes were used to assess topography and chemical composition of the samples, respectively. The morphologies of the cubic PdNPs were previously characterized by transmission electron microscopy (TEM) using a JEM-1011 TEM transmission electron microscope operated at 100 kV. For the analysis, one aliquot of the dispersed samples in ethanol was deposited onto a 400-square mesh copper grid with carbon film and dried under vacuum.

Bioreducing electrode fabrication.

MWCNT enzyme-modified electrodes were prepared from a 2.5 mg mL -1 suspension in NMP after 20 min sonication and vigorous shaking immediately before use. The black suspension was deposited onto the GCE to give an electrode disc with a geometric surface area of 0.126 cm 2 for a 20 µL droplet. The solvent was then removed over several hours under vacuum. Next, the electrodes were incubated in a 10 mmol L -1 PMA solution in CH3CN for 1 h and thoroughly rinsed with CH3CN then deionized water. Subsequently, electrodes were left to incubate in a 4 mmol L -1 DTNB, 4 mmol L -1 NHS and 2 mmol L -1 EDC solution (0.2 mol L -1 phosphate buffer (PB), pH 7.0) for 5 h. The electrodes were rinsed with deionized water to remove any loosely bound species then soaked in McIlvaine buffer (pH 7.0). Modified-(DTNB-Pyr)/MWCNT electrodes were then incubated overnight at 4 ⁰C in 40 μL of BOx solution (2.5 mg mL -1 ) prepared in McIlvaine buffer (pH 7.0). Before testing, electrodes were thoroughly rinsed with deionized water and soaked in fresh McIlvaine buffer solution. For preparation of GDL-based air-breathing biocathodes for sensing, the same preparation methodology was applied as that used for GC electrodes. To ensure the biocathode was not the limiting electrode, three droplets of 20 µL each were deposited onto the GDL surface. The geometric surface area of the GDL-based biocathodes was SGDL = 0.378 cm 2 .

Electrochemistry.

A Biologic VMP3 Multi Potentiostat controlled with EC-lab software was used to perform electrochemical experiments. A three-electrode setup was used for half-cell characterization comprising a glassy carbon electrode (Ø = 3 mm, ALS Co., Japan) as the working electrode, platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. Prior to modification, the GCE working electrodes were polished with a 2 μm diamond paste purchased from Presi (France), rinsed successively with deionized water, acetone, and ethanol, then left to sonicate for 5 min in ethanol. McIlvaine buffer solutions (pH range: 3 ≤ pH ≤ 9) were used as supporting electrolytes. All solutions for N2H4 oxidation experiments at the half-cell anode were purged with nitrogen gas and a nitrogen flow was maintained on top of the solution during analysis. All experiments were conducted at room temperature unless stated otherwise.

Hybrid hydrazine sensor, removal and power system.

The fuel cell/sensor set-up comprised of a rounded poly(methyl methacrylate) tube and a flat poly(methyl methacrylate) piece assembled over a rubber joint to form the fuel cell reservoir (see Figure S1). For air-the breathing cell, a 5 mm diameter hole was made into the flat piece to expose the BOx-modified air-breathing biocathode to air. The complete hybrid biofuel cell and sensor system consisted of a PdNPs/ MWCNT anode and a BOx/(DTNB-Pyr)/MWCNT cathode or BOx/(DTNB-Pyr)/MWCNT/GDL biocathode. The distance between the electrodes was approximatively 1 cm. Oxygen flow was qualitatively maintained but not monitored to obtain reproducible steady state currents between different experiments. When the airbreathing GDL biocathode was used, no additional O2 was provided to the electrolyte. Testing was performed with a relatively slow scan rate of 2 mV s -1 using linear sweep voltammetry. All experiments were performed in a 5 mL volume of McIlvaine buffer (pH 7.0) solution.

Results and Discussion

Scanning electron microscopy characterization of PdNPs and PdNPs/MWCNT-based electrodes.

The cubic PdNPs used here with average size of 22.5 ± 2.6 nm were synthesized and characterized as described in our recent work. 20 Figure 1A shows a TEM micrograph and the corresponding correlated size distribution for the PdNPs, which confirms good dimension homogeneity and a high yield of cubic shapes.

SEM imaging was subsequently performed to assess the presence and distribution of the PdNPs on the MWCNT-based electrodes prepared by deposition from the nanoparticle solution as described in the Experimental Section. Figure 1B and 1C shows SEM micrographs recorded at an unmodified MWCNT electrode and a PdNP-modified MWCNT electrode, respectively. A well dispersed and homogeneous three-dimensional CNT network is observed for the unmodified MWCNT electrodes. For the PdNP modified MWCNT electrodes, the CNT network was maintained and the appearance of bright spots associated with individual and aggregated nanoparticles were observed at high magnification. The relatively uniform distribution of bright spots was observed at multiple regions of the surface indicating an even coverage of PdNPs across the MWCNT electrode surface. The increased brightness observed across parts of the carbon nanotube network for the PdNP-modified MWCNT electrodes is attributed to polymeric material associated with the as-prepared nanoparticle solution. Overall, the electron microscopy analysis confirms the presence and good homogeneous distribution of PdNPs on the surface of the modified electrodes. The CV for bare MWCNT electrodes did not present any faradaic signal scanning in the range from -0.85 V to 0.3 V vs. SCE at 10 mv s -1 . For the PdNP-modified MWCNT electrodes, the voltammograms in the same range reveal the appearance of electroactivity in the range of -0.7 to -0.4 V. The redox peaks observed at negative potentials are ascribed to the characteristic peaks of hydrogen adsorption and desorption on palladium, as reported in different literature reports. 21,22 Upon scanning of the PdNPs/MWCNT electrode at more positive potentials, a broad oxidation wave was observed with an onset potential of around 0.5 V and current maximum at 0.6 V, corresponding to hydroxide (PdOH) and oxide (PdO) formation on the Pd surface, as reported previously. 21 After formation of the oxide, a new reduction peak appeared on the reverse sweep near 0.0 V, corresponding to palladium oxide reduction. The reduction peak currents were found to be linearly dependent on the scan rate, sustaining the presence of an adsorbed species on the MWCNT electrodes and, furthermore, confirming the good adhesion of the nanoparticles on the MWCNT surface.

INSERT FIGURE 1

Electrochemical characterization of PdNPs/MWCNT-based electrodes.

INSERT FIGURE 2

The hydrogen evolution region for MWCNT electrodes modified with cubic PdNPs was investigated in McIlvaine buffer over a pH range from 3.0 to 9.0. Figure 3 shows second scan CVs recorded in N2 saturated solution at different pHs. The oxidation peak potentials shifted in a linear manner towards more negative values with increasing pH, giving an average slope of -55 ± 2 mV per pH unit.

INSERT FIGURE 3

In order to maximize the surface coverage of PdNPs we investigated the effect of PdNP loading on the MWCNT electrode by monitoring the increase in peak current corresponding to palladium oxidation.

Figure 4 depicts the anodic current peak height recorded at -0.45 V as a function of the volume of PdNPs drop-casted for MWCNT electrodes prepared from two different volumes of 2.5 mg mL -1 . The palladium loading on the PdNPs/MWCNT-GC modified electrodes investigated therefore varied. The plots in Figure 4 reveal that the palladium oxidation current obtained for electrodes prepared by addition of between 1-5 µL of PdNPs solution (4 mg mL -1 ) reaches a limiting current after addition of 1 μL of nanoparticle solution for the MWCNT electrodes prepared with a smaller CNT solution volume (10 µL) of CNTs (ie 10 % vPdNPs/vCNT). However, for the MWCNT electrodes prepared with a larger volume (20 µL) of CNTs (ie 12.5 % vPdNPs/vCNT), larger oxidation currents were obtained after addition of between 2.5 to 5 μL of nanoparticle solution (4 mg mL -1 ), with a limiting current after addition of 2.5 μL of solution. These experiments clearly show that that the amount of palladium oxide current and therefore PdNP loading can be controlled by finely adjusting the volumes of PdNP and CNT solutions using during electrode fabrication. Based on these results, nanocomposite PdNP electrodes with high PdNPs loadings were prepared for electrocatalytic oxidation of hydrazine. The electrodes used to study hydrazine oxidation were prepared using 10 % vPdNPs/vCNT and 12.5 % vPdNPs/vCNT ratios.

INSERT FIGURE 4

PdNPs-based MWCNT electrodes for electrocatalytic N2H4 oxidation.

Since we previously showed that oxygen can be effectively reduced by PdNPs (22.5 nm diameter) at potentials below 0.25 V vs. SCE in aqueous solution 20 , we investigated these PdNPs for the oxidation of hydrazine on MWNT-modified electrodes at neutral pH and in deaerated solution. Figure 5A shows typical CVs recorded at unmodified MWCNTs and PdNPs/MWCNT electrodes with cubic PdNPs in deaerated McIlvaine buffer (pH 7.0) solution in the absence and presence of 8 mmol L -1 N2H4. At unmodified MWCNT electrodes, the onset potential for N2H4 oxidation was observed at 0 V. In contrast, the PdNP-modified MWCNT electrodes exhibited a drastic change in the electrocatalytic properties. The presence of the metallic nanoparticles caused a shift in the oxidation onset potential towards a more negative potential at around -0.4 V and with the hydrazine oxidation peak at Ep = -0.2 V. Comparison of the two different electrodes prepared using "small" (10 % vPdNPs/vCNT) or "large" (12.5 % vPdNPs/vCNT) amounts of PdNPs revealed, surprisingly, that the electrodes with the smaller amount of PdNPs gave the highest electrocatalytic current, which reached 2.80 ± 0.13 mA cm -2 compared to 2.04 ± 0.34 mA cm -2 .

During electrocatalytic oxidation, a larger amount of gas bubble formation was observed for the electrodes prepared with the larger amount of PdNPs. The lower catalytic current observed is attributed to impeded hydrazine diffusion and reduced electroactive surface area due to the gas bubbles at the surface. Next the catalytic performance of the PdNPs was assessed over a wide concentration range for PdNPs/MWCNTmodified electrodes prepared with the smaller amount of PdNPs. A linear calibration plot was obtained from CVs for the catalytic oxidation current at 0.1 V as a function of increasing hydrazine concentration.

This experiment shows that in the μmol L -1 to mmol L -1 concentration range there are no significant limitations due to substrate diffusion, catalytic surface area or catalyst degradation with repeat electrochemical cycling over a wide concentration range and at very high mmol L -1 concentrations of hydrazine. It is noted that for these experiments the catalytic current was measured from the reverse sweep at 0.1 V as this provides a more stable and reliable picture of the current achieved under continuous operation of electrode. High electrooxidation currents were recorded which reached a current maximum of 7.13 ± 0.31 mA cm -2 at 100 mmol L -1 N2H4 solution.

INSERT FIGURE 5

BOx-based bioelectrode for O2 reduction.

In order to develop the self-powered sensor, an enzymatic biocathode was employed as the cathode to pair with the optimized PdNPs/MWCNT-modified electrode as the anode. The standard biocathode used here for the enzymatic four-electron oxygen reduction reaction was described in our recent work. 23 The biocathode exploits the use of negatively charged (5,5′-dithiobis(2-nitrobenzoic acid)) groups to provoke preferential orientation of the BOx enzyme towards the surface and thus facilitate efficient direct electron transfer for the oxygen reduction reaction. Figure 6 shows the CVs recorded at a GC-based BOx/(DTNB-Pyr)/MWCNT in quiescent solution and under oxygen purging. Firstly, for both oxygen conditions, the onset potential for the oxygen reduction reaction is Eonset = 0.52 ± 0.01 V which is close to the redox potential of the laccase T1 copper center responsible for the intramolecular electron transfer to the T2/T3 site at which O2 is reduced to H2O. Comparison of the CVs recorded in quiescent solution and under oxygen purging reveal the drastic difference in catalytic current possible as a function of oxygen content.

In buffer solution the dissolved oxygen is typically [O2] ≈ 0.2 mmol L -1 whereas under oxygen bubbling saturated oxygen levels of [O2]sat ≈ 1.2 mmol L -1 are possible. Under continuous oxygen saturation conditions, the maximum achievable current densities were 1.34 ± 0.15 mA cm -2 at 0.1 V, which compares to 0.06 ± 0.01 mA cm -2 at 0.1 V with only dissolved oxygen. After the addition of 8 mmol L -1 of hydrazine under oxygen saturation conditions with bubbling, a decrease of the observed resting potential from 0.52 V to 0.34 V was observed. Moreover, the voltammograms show that the O2 reduction onset potential was lowered to more negative potentials (Eonset = 0.35 ± 0.01 V) and with a noticeable decrease in cathodic current. This observation reveals that there is a competition between oxygen reduction from the BOx enzyme immobilized on the electrode surface and the electrooxidation of N2H4 in solution at the electrode surface (see Figure 5). Nevertheless, at high overpotential with Eapp = 0 V to 0.15 V, the maximum steady-state current densities obtained were identical to that recorded in the absence of hydrazine.

INSERT FIGURE 6

Hydrazine self-powering sensor and remediation system.

The complete hybrid N2H4 self-powered sensor was built by combining a PdNPs/MWCNT modified electrode as the anode and a BOx/(DTNB-Pyr)/MWCNT biocathode supported either on GC or GDL. In initial experiments, the GC-based biocathode was employed and oxygen was continuously purged to keep the oxygen solution concentration constant and to maximize power output (see cell configuration in Scheme 1). The bioanode was prepared using the optimized anode prepared using the 10 % vPdNPs/vCNT ratio which gave the highest catalytic activity for hydrazine oxidation. In the absence of hydrazine, the open circuit voltage (OCV) of the fuel cell / sensor was 147 ± 7 mV. Upon polarization of the electrodes between OCV and 0 V a maximum power density of Pmax = 0.2 µW cm -2 was obtained (Figure S2). Next, polarization curves were recorded for 10 different concentrations in the range of 0.2 to 15 mmol L -1 of hydrazine and the three parameters of OCV, Pmax and current density (Jmax) were monitored, and importantly, all three parameters increased with increasing hydrazine concentration. For hydrazine sensing, the maximum power density was measured from linear sweep voltammograms after each individual hydrazine injection into the solution, then and a calibration curve was recorded presenting Pmax = f ([N2H4]) (Figure 7A). It can be clearly observed that the Pmax varies linearly with N2H4 concentration in the range of 0 to 4 mmol L -1 before saturation is reached at a hydrazine concentration ≥ 6 mmol L -1 where Jmax = 937 ± 23 µA cm -2 and Pmax = 211 ± 30 µW cm -2 , respectively. Finally, with the aim of alleviating the need for an oxygen source to operate the sensor device, for a more realistic sensor which could be applied at contaminated hydrazine sites, a second configuration of biocathode was employed. The biocathode was prepared as before but on a GDL substrate rather than GC (see cell in Figure S1) and in complete quiescent solution rather than under continuous oxygen bubbling. The advantage of the GDL is that oxygen can be brought to the cell system naturally via diffusion from the air giving increased currents compared to simple quiescent solution. Furthermore, the GDL electrodes further benefit from being lighter and more portable than typical GCE working electrodes. The device with the GDL-based anode was operated and the data analyzed as described for the first self-powered sensor, and the results obtained were in agreement with those obtained previously (see Figure 7A). The only significant difference was regarding the limiting current obtained for this type of device which was slightly higher for the cell setup using the GDL-based anode. Additionally, the shapes of the power curves clearly indicate a diffusion limitation of the system at low concentration of N2H4 due to the absence of mechanic convection from the O2 purging. When GCE-based biocathodes this type of diffusion limitation was not observed due to very high rates of mass transport (see Figure S2).

INSERT FIGURE 7

Conclusions

A self-powered sensor has been developed which allows the electrochemical detection of hydrazine in solution without any additional external power supply and using a known environmental contaminant and natural oxygen as the fuels. Due to their remarkable catalytic properties, 22.5 nm diameter PdNPs immobilized onto a MWCNT matrix were used for the electrooxidation of the contaminant, hydrazine, in aqueous solution. This electrode was employed as an anode and combined with a BOx-based electrode as the cathode to form the self-powered sensor as a novel hybrid biofuel cell. This sensor exhibited a linear power response with increasing concentration in the range 0 to 4 mmol L -1 in continuously saturated O2 solution with a regular immersed biocathode with a glassy carbon support. To provide better insight into the practical use of the sensor as a future transportable sensing device, the biocathode was replaced with an air-breathing biocathode that alleviated the need of O2 supply from the system. The linear sensing range was equivalent to the one obtained using glassy carbon. In addition to providing the ability to detect hydrazine in solution, this hybrid self-powering device could be used "in the field" for the remediation of contaminated areas while generating green electrical current with the production of nitrogen gas and water as by products. As a hybrid fuel cell, the open circuit voltage, maximum current density and the maximum power density were in the range of 0.5 V, 1.2 mA cm -2 and 0.25 mW cm -2 , respectively, in quiescent solution. Ongoing research must be assessed to decrease the cross reaction of hydrazine oxidation with the biocatalytic oxidation reaction at the cathode which reduces the open circuilt voltage potential possible. Furthermore, a more efficient cell design for efficient gas removal from the oxidizing electrode is desired. Future work will address application of the sensor in contaminated waste waters.

Figure 2

 2 Figure 2 shows typical cyclic voltammograms (CVs) recorded at unmodified MWCNTs and PdNPs/MWCNT electrodes with cubic PdNPs (22.5 nm) in deaerated McIlvaine buffer (pH 7.0) solution.

FiguresFigure 1 :

 1 Figures

  Figure 1: (A) TEM images of 22.5 nm cubic PdNPs synthesized in the presence of 11 mmol L -1 HCl and the correlated histogram of size distribution (inset). SEM images of (B) an unmodified MWCNT surface and (C) a MWCNT/cubic PdNPs surface. The images were recorded using a secondary electron detector (Everhart-Thornley; ETD) to visualize the in-depth details of the MWCNT deposit.

Figure 2 :

 2 Figure 2: (A) Cyclic voltammograms recorded in the hydrogen evolution region at an unmodified MWCNT GC electrode (dashed line) and PdNPs/MWCNT-modified GC electrode (black solid line) in McIlvaine buffer solution (pH 7.0); (red solid line) cyclic voltammograms recorded at PdNPs/MWCNTmodified GC electrode in the PdO region. Voltammograms recorded (second scans shown) in deaerated solution at scan rate = 10 mVs -1 . (B) Cyclic voltammograms of PdNPs/MWCNT-modified GC electrode recorded at different scan rate from 5 to 200 mV s -1 (the black arrow indicates increasing scan rates). Inset: Plot of cathodic peak current for PdO reduction versus the scan rate.

Figure 3 :

 3 Figure 3: Representative cyclic voltammograms for PdNPs/MWCNT-modified GC electrodes under deaerated solution in McIlvaine buffer at different pH values (dashed line represents pH 7.0). (Inset) Corresponding plot of the anodic peak potential associated with hydrogen adsorption over the full pH range. Error bars represent one standard deviation from triplicate experiments. Voltammograms recorded (second scans shown) in deaerated solution at scan rate = 10 mVs -1 .

Figure 4 :

 4 Figure 4: Anodic current peak height recorded at -0.45 V as a function of the PdNPs volume used for surface modification for (■) 20 µL MWCNT electrodes and (▲) 10 µL MWCNT electrodes. Error bars represent one standard deviation from triplicate experiments.

Figure 5 :

 5 Figure 5: (A) Representative cyclic voltammograms for (dashed line) MWCNT-modified electrodes, (solid black line) 10 % vPdNPs/vCNT PdNPs/MWCNT-modified electrodes and (red solid line) 12.5 % vPdNPs/vCNT PdNPs/MWCNT-modified electrodes under deaerated solution in McIlvaine buffer (pH 7.0) in the presence of 8 mmol L -1 N2H4, at 10 mV s -1 . (B) Calibration curve obtained at 10 % vPdNPs/vCNT PdNPs/MWCNT-modified electrodes from cyclic voltammograms recorded at increasing concentrations of N2H4. Currents were taken at 0.1 V vs. SCE during the backward scan. Error bars represent one standard deviation from triplicate experiments.

Figure 6 :

 6 Figure 6: Representative cyclic voltammograms for BOx/(DTNB-Pyr)/MWCNT-modified electrodes under quiescent solution in the absence of N2H4 (dashed line) and under oxygen purging without (black solid line) or in the presence (blue solid line) of 8 mmol L -1 N2H4 solution in McIlvaine buffer (pH 7.0) at 10 mV s -1 . Error bars represent one standard deviation from triplicate experiments.

Figure 7 :

 7 Figure 7: (A) (■) Calibration curve for N2H4 sensing from 0 to 15 mmol L -1 obtained from maximum power density for each N2H4 concentration using the optimized anode and GC-based biocathode. (Inset: linear range for N 2 H 4 quantification). (•) Calibration curve for N 2 H 4 sensing obtained with the GDL-based biocathode device. Error bars represent one standard deviation from triplicate experiments. (B) Representative power curves for the self-powered N2H4 sensor utilizing GDL-based biocathodes in quiescent solution in the presence of increasing concentration of N2H4 from 0.2 to 15 mmol L -1 as shown with the black dashed arrow. The curves were obtained from linear polarization curves recorded at 2 mV s - 1 from OCV to 0 V as shown with the red dashed line.

Acknowledgments

The authors are grateful to CNPq and CAPES for financial support received under the COFECUB 2014 Program to complete this collaborative project. Additionally, this work was also supported by the Central Laboratory of Electron Microscopy (LCME) (TEM analysis) and the multiuser facility LDRX (XRD patterns) at UFSC. Further support was granted by the projects CAROUCELL (ANR-13-BIME-0003-02) and Labex ARCANE (ANR-11-LABX-0003-01). The authors would also like to thank the platforms "Surfaces Functionalization and Transduction" of the scientific structure "Nanobio" for providing facilities and "Microscopy" of the ICMG structure for SEM imaging.


Andrew J Gross

Raoudha Haddad

Christophe Travelet

Eric Reynaud

Pierre Audebert

Redouane Borsali

Serge Cosnier

Redox-Active Carbohydrate-Coated Nanoparticles: Self-Assembly of a Cyclodextrin-Polystyrene Glycopolymer with Tetrazine-Naphthalimide

The controlled self-assembly of precise and well-defined photochemically and electrochemically active carbohydrate-coated nanoparticles offers the exciting prospect of biocompatible catalysts for energy storage/conversion and biolabeling applications. Here an aqueous nanoparticle system has been developed with a versatile outer layer for host-guest molecule encapsulation via β-cyclodextrin inclusion complexes. A β-cyclodextrin-modified polystyrene polymer was first obtained by copper nanopowder click chemistry. The glycopolymer enables self-assembly and controlled encapsulation of tetrazine-naphthalimide, as a model redox-active agent, into nanoparticles via nanoprecipitation. Cyclodextrin hostguest interactions permit encapsulation and internanoparticle cross-linking for the formation of fluorescent compounds and clustered self-assemblies with chemically reversible electroactivity in aqueous solution. Light scattering experiments revealed stable particles with hydrodynamic diameters of 138 and 654 nm for nanoparticles prepared with tetrazine, of which 95% of the nanoparticles represent the smaller objects by number. Dynamic light scattering revealed differences as a function of preparation method in terms of size, 3-month stability, polydispersity, radius of gyration, and shape factor. Individual self-assemblies were visualized by atomic force microscopy and fluorescence microscopy and monitored in real-time by nanoparticle tracking analysis. UV-vis and fluorescence spectra provided insight into the optical properties and critical evidence for host-guest encapsulation as evidenced by solvachromatism and enhanced tetrazine uptake. Cyclic voltammetry was used to investigate the electrochemical properties and provided further support for encapsulation and an estimate of the tetrazine loading capacity in tandem with light scattering data. 50 ides (maltoheptaose, 10 dextrans, 9,11 and amylose 12 with 51 synthetic polymers such as polystyrenes, 9-13 poly(ε-52 caprolactone)s, 14 poly(ethylene glycol)s 15 and poly(methyl 53 methacrylate)s 16 ). Amphiphilic block copolymer formation is 54 generally achieved using combinations of controlled living 55 radical polymerizations, transition-metal-mediated coupling 56 chemistry, and tedious purification procedures. 7 Many methods 57 report the self-assembly of amphiphilic block copolymers into 58 nanoparticles by direct dissolution into a selective solvent for 59 one of the block moieties. 9,11,17,18 However, with this approach 60 it is difficult to avoid random aggregation and control particle 61 size at the nanoscale. Clever manipulation strategies involving 62 indirect dissolution methods such as nanoprecipitation facilitate 63 the preparation of well-defined micellar nanoparticles with

■ INTRODUCTION

The development of biocompatible nanoassembled particles with controlled chemical and physical properties has attracted significant attention for applications such as imaging, biosensing, drug delivery, and biocatalysis. 1,[START_REF] Nicolas | Functionalization Strategies and Biomedical Applications of Targeted Biodegradable/Biocompatible Polymer-Based Nanocarriers for Drug Delivery[END_REF] Nanoparticles such as micelles capable of carrying and delivering redox molecules in a selective manner have shown great benefits including enhanced bioavailability, [START_REF] Qu | Carbohydrate-Based Micelle Clusters which Enhance Hydrophobic Drug Bioavailability by up to 1 Order of Magnitude[END_REF] stimuli-responsiveness, [START_REF] Li | Polymeric Assemblies and Nanoparticles with Stimuli-Responsive Fluorescence Emission Characteristics[END_REF] and reduced toxicity. [START_REF] Farokhzad | Targeted Nanoparticle-Aptamer Bioconjugates for Cancer Chemotherapy in vivo[END_REF] However, polymer synthesis and particle assembly methods are often complicated and time-consuming. 6 Furthermore, challenges remain for in vivo and in vitro applications including solubility and stability issues.

The preparation of amphiphilic glycopolymers provides a route to biocompatible and bioinspired nanoparticles but remains relatively undeveloped because of their tricky preparation with many synthesis steps. [START_REF] Xu | Unimolecular Micelles of Amphiphilic Cyclodextrin-Core Star-like Block Copolymers for Anticancer Drug Delivery[END_REF][START_REF] Zhang | Silica-Based Nanocapsules: Synthesis, Structure Control and Biomedical Applications[END_REF][START_REF] Kakuchi | Glycoconjugated Polymer. 4. Synthesis and Aggregation Property of Well-Defined End-Functionalized Polystyrene with β-Cyclodextrin[END_REF] Several groups have now reported on the preparation and self-assembly of block copolymers containing polysaccharides and oligosacchar-UV-vis absorption spectra were recorded in quartz cuvettes using a PerkinElmer UV-Lambda 650 spectrophotometer, and fluorescence spectra were recorded using a PerkinElmer LS50.

Synthesis of β-Cyclodextrin-Modified Polystyrene Polymer.

The amphiphilic glycopolymer was obtained by click chemistry. The first step was to functionalize each block: the hydrophobic block, ωhydroxyl-terminated polystyrene, was functionalized to form ω-azidoterminated polystyrene via the ω-tosyl terminated polystyrene intermediate with an overall yield of 75% according to the method of Fallais and co-workers. 31 The hydrophilic block, native βcyclodextrin, was monofunctionalized into mono-6-alkyne-β-cyclodextrin via the mono-6-tosyl-β-cyclodextrin intermediate with an overall yield of 7% according to the procedure developed by Moutard. 32,33 Both blocks with their complementary chemical functions (alkyne and azido) were subsequently linked together via a click reaction in heterogeneous media catalyzed by Cu nanopowder 34,35 to obtain the β-cyclodextrin-modified polystyrene polymer (M n = 4900 g mol -1 , PDI = 1.01) (Scheme S1). To perform the click reaction, a degassed solution containing the azido-functionalized polystyrene (1 equiv, 8.1 g, 1.8 × 10 -3 mol) and the alkynylfunctionalized cyclodextrin (1.2 equiv, 2.7 g, 2.2 × 10 -3 mol) in DMF was first prepared. An excess of Cu nanopowder (1.8 equiv, 260 mg, 3.2 × 10 -3 mol) was added to the solution and the mixture stirred under Ar at 65 °C for several hours until the azide 1 H NMR signal disappeared. At the end of the reaction, the crude heterogeneous mixture was filtered off under diatomaceous earth, and the obtained filtrate was stirred several hours in the presence of the Cuprisorb resin.

The mixture was finally precipitated in cold MeOH to give a white powder with a yield of 90% (mass = 8 g). The modified polymer structure was confirmed using NMR and MALDI analysis (Figure S1).

Preparation of PS-CD NP and PS-CD-Tz NP Nanoparticles via

Nanoprecipitation. Polymer solutions were prepared by dissolving either 5 mg of PS-CD or 5 mg of PS-CD together with 1 mg of tetrazine in 5 g of a THF/H 2 O solution (80:20 v/v %, unless otherwise stated). For nanoprecipitation by method A, 1 g of the PS-CD solution was slowly added dropwise to 40 g of Milli-Q water under stirring at 500 rpm at room temperature. For nanoprecipitation by method B, 40 g of water was slowly added to 1 g of the PS-CD solution under stirring at 500 rpm at room temperature. The solutions were subsequently stirred at 500 rpm at room temperature for 1.5 h and then preconcentrated to 4 g by evaporation under reduced pressure at 50 °C using a rotary evaporator. The nanoparticle suspensions were stored at room temperature in the dark.

Static Light Scattering (SLS)/Dynamic Light Scattering (DLS). Light scattering measurements were performed using an ALV/CGS-8FS/N069 goniometer with an ALV/LSE-5004 multiple-τ digital corrector with a 125 ns initial sampling time and a 35 mW red HeNe linearly polarized laser operating at λ = 632.8 nm. Nanoparticle suspensions were loaded into 10-mm-diameter quartz cells thermostated at 25 ± 0.1 °C. Data were collected using the digital ALV correlator control software at observation angles relative to the transmitted beam of 90/150°(scattering vector moduli 1.87 × 10 -2 / 2.56 × 10 -2 nm -1 ) for a counting time of typically 180 s. In DLS measurements, relaxation time distributions were determined using a Contin analysis of the autocorrelation functions. Polydispersity values were calculated from the cumulant analysis of the DLS autocorrelations (g [START_REF] Nicolas | Functionalization Strategies and Biomedical Applications of Targeted Biodegradable/Biocompatible Polymer-Based Nanocarriers for Drug Delivery[END_REF] -1).

Diffusion coefficients were obtained from eq 1 where D is the diffusion coefficient, τ is the relaxation time, and q is the modulus of the scattering vector:

τ = D q 1 2 (1)
Hydrodynamic diameters, D h , were calculated using the Stokes-Einstein equation (eq 2), where K B is the Boltzmann constant, T is the temperature, η is the medium viscosity, and D is the diffusion coefficient:

πη = D K T D 3 h B (2)
The percentage of small nanoparticles was calculated using eq 3, assuming that the nanoparticles behave like hard spheres in water and have the same density, where N small refers to the number of small nanoparticles (fast relaxation mode), τ small is the relaxation time corresponding to the small nanoparticles, and S small is the surface area under the peak related to the small nanoparticles: 36

τ τ = ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ ⎛ ⎝ ⎜ ⎜ ⎞ ⎠ ⎟ ⎟ N N S S small big big small 3 small big (3)
For SLS measurements, scattering intensities were corrected by the background solvent signal. The diameter of gyration, D g , was determined from eq 4, where n is the number of repetitive units and b is the Kuhn length:

= ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ D nb 2 6 g 2 0.5 (4)
Electrochemistry. Electrochemical measurements were performed at room temperature using an Eco Chemie Autolab PGSTAT 100 potentiostat running GPES 4.9 software. A conventional threeelectrode cell setup was used for all electrochemical experiments comprising a glassy carbon working electrode (GC, 3 mm diameter), a saturated calomel reference electrode (SCE), and a Pt wire counter electrode. The concentration of tetrazine-naphthalimide was estimated according to the Randles-Sevcik equation (eq 5) where I p is the peak current, n is the number of electrons transferred in the redox event, F is the Faraday constant, C is the concentration of electroactive species, D is the diffusion coefficient (here estimated from dynamic light scattering), A is the geometric electrode area, ν is the scan rate, R is the gas constant, and T is room temperature:

ν = ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ I nFAC nF D RT p 0.5 (5)
Nanoparticle Tracking Analysis (NTA). NTA was carried out using a Nanosight LM10HS optical microscope setup equipped with a blue-purple laser (λ ex = 405 nm), a camera, and a chamber mounted on a modified microscope stage (Nanosight, Amesbury, U.K.). The original suspensions of nanoparticles were diluted with Milli-Q water and introduced into the chamber with a syringe. Video clips of the nanoparticles subjected to their natural Brownian motion were captured over 60 s at 25.0 °C and analyzed using analytical software version 2.1, giving access to the number-weighted size distributions. (See the Supporting Information for 10 s video clips.) Atomic Force Microscopy (AFM). Twenty microliters of a nanoparticle suspension was dropped onto a Si(100) substrate, and the sample was left to dry at room temperature overnight in a desiccator. AFM measurements were performed using a Dimension Icon (Bruker, Santa Barbara, CA) with SCANASIST-Air (Bruker, Santa Barbara, CA) probes in peak-force mode with a scan rate of 1 Hz. The data was processed using Gwyddion 2.41 microscope software.

Fluorescence Microscopy. Twenty microliters of a nanoparticle suspension was dropped onto a Si(100) substrate, and the sample was left to dry at room temperature overnight in a desiccator. Fluorescence microscopy images were recorded using an Olympus BX61 microscope coupled with an Olympus DP30BW camera and CellP imaging software. A DAPI (4′,6-diamidino-2-phenylindole) filter set was used: DAPI with λ ex = 340-380 nm and λ em = 450-490 nm. Images were recorded with an exposure time of 500 ms.

■ RESULTS AND DISCUSSION

For the preparation of nanoparticles in aqueous solution via nanoprecipitation, we focused our initial studies on optimizing the solvent system for both blocks of the PS-CD glycopolymer. Tetrahydrofuran was chosen as the organic solvent because of its good solubility toward polystyrenes and Tz, water miscibility, and good evaporation rate. Static light scattering Figure 2A shows the DLS autocorrelation function (g [START_REF] Nicolas | Functionalization Strategies and Biomedical Applications of Targeted Biodegradable/Biocompatible Polymer-Based Nanocarriers for Drug Delivery[END_REF] -1) 305 and relaxation time distribution for the PS-CD NP suspensions. 306 For PS-CD NP prepared by methods A (Figure 2Ai) and B 307 (Figure 2Aii), unimodal distributions of relaxation times were 308 observed corresponding to mean hydrodynamic diameters of 309 D h = 240 and 378 nm with polydispersities of Đ = 1.34 and 310 1.45, respectively. The particles prepared by method A are 311 therefore smaller and less polydisperse than those prepared by 312 method B. The PS-CD NP particles prepared by method A are 313 comparable in size to previously reported compound micelles 314 with D h ≈ 160 nm prepared by the self-assembly of 315 polystyrene-containing diblock polymers. [START_REF] Otsuka | Self-Assembly of Maltoheptaose-block-Polystyrene into Micellar Nanoparticles and Encapsulation of Gold Nanoparticles[END_REF][START_REF] He | Controlled Drug Release System Based on Cyclodextrin-Conjugated Poly(lactic acid)-b-Poly(ethylene glycol) Micelles[END_REF] Figure 2B 316 shows the DLS autocorrelation function (g [START_REF] Nicolas | Functionalization Strategies and Biomedical Applications of Targeted Biodegradable/Biocompatible Polymer-Based Nanocarriers for Drug Delivery[END_REF] -1) and 317 relaxation time distribution for the PS-CD-Tz NP suspensions 318 prepared by methods A (Figure 2Bi) and B (Figure 2Bii). The 319 relaxation time distribution plots, reflecting mass-weighted 320 distributions, show the appearance of two particle sizes for PS-321 CD-Tz NP . The PS-CD-Tz NP suspension obtained by method B 322 exhibits greater polydispersity (Đ = 1.43) and contains 323 microscale particles (D h = 2940 nm). In contrast, smaller 324 nanoparticles with a lower polydispersity (Đ = 1.32) are 325 obtained by method A, and thus this method of nano-326 precipitation was adopted for further analysis.

327

The smaller particle size of D h = 138 nm observed for PS-328 CD-Tz NP prepared by method A is attributed to small self-329 assemblies that have decreased in size compared to the PS-330 CD NP nanoparticles prepared by the same method as a result of 331 the encapsulation of Tz molecules and the shrinking of the 332 more hydrophobic core. A change in self-assembly behavior of 333 the PS-CD polymer in the presence of Tz was expected because 334 the cone-shaped hydrophobic cavities of the β-cyclodextrin 335 groups drive the 1:1 inclusion of guest Tz molecules via 1,8-naphthalimide (equatorial inclusion) and 1,2,4,5-tetrazine groups. 29,37 A decrease in size with encapsulation has previously been reported for self-assembled nanoparticles with encapsulated hydrophobic molecules. [START_REF] He | Controlled Drug Release System Based on Cyclodextrin-Conjugated Poly(lactic acid)-b-Poly(ethylene glycol) Micelles[END_REF] The larger nanoparticles of PS-CD-Tz NP with D h = 654 nm, observed only after self-assembly in the presence of Tz, are attributed to nanoparticle clusters. surprising given that (i) NTA gives access to number-weighted distributions of which only ∼4.9% are the clusters and (ii) only a few nanoparticles are imaged, as can be seen in Figure S3 and the video clips.

Further analysis of the DLS and SLS scattering data was performed to shed light on the shape of the nanoparticles. First, the diameter of gyration, D g , was determined from SLS angular f4 dependence experiments via Guinier plots (Figure 4). Values of D g = 326 and 258 nm were obtained from the slope of the linear fit in the low qD g region for PS-CD NP and PS-CD-Tz NP , respectively. Next, the shape factor, ρ, was obtained from the ratio ρ = D g /D h , as proposed by Burchard. 38 The ρ values of 1.4 for PS-CD NP and 1.9 for PS-CD-Tz NP fall between those of vesicles (ρ = 1) and rigid rods (ρ ≥ 2.0), and thus PS-CD NP and PS-CD-Tz NP particles can be ascribed to particles whose D overall shape is spherical (1.4) and polydisperse spheres (1.9), respectively. 38 An important issue largely overlooked in previous studies on the self-assembly of saccharide-based polymers is their stability. In this study, we tested the long-term stability of PS-CD NP and PS-CD-Tz NP suspensions by monitoring the particle size change after storage in the dark at ambient temperature for 3 months. The DLS data (Figure S4) reveal relatively small increases in hydrodynamic radii for PS-CD-Tz NP compared to markedly larger increases in radii for PS-CD NP particles. The apparent low stability of the nonencapsulated PS-CDNP particles is attributed to nonspecific hydrophobic interactions of β-cyclodextrin groups at the periphery of the nanoparticles, which give larger aggregations over time. The higher stability of PS-CD-Tz NP reflects the saturation of these β-cyclodextrin groups, which in turn suppresses particle aggregation over time. Crucially, the stability of PS-CD-Tz NP suggests little evidence of the presence of Tz molecules in solution that would likely increase particle aggregation.

UV-vis and fluorescence spectra were recorded to probe the ability of the PS-CD polymer to encapsulate tetrazinenaphthalimide during self-assembly. Three solutions were investigated: as-prepared PS-CD NP , PS-CD-Tz NP (Tz = 0.06 mg mL -1 ), and a saturated tetrazine-naphthalimide in water solution (H 2 O-Tz) prepared by addition of 0.3 mg Tz in 3 mL (Tz < 0.1 mg mL -1 ). The absorption spectrum for PS-CD NP in the absence of Tz shows weak background absorption due to f5 the polymer but no well-defined absorption peaks (Figure 5Ai). In contrast, both PS-CD-Tz NP (Figure 5Aii) and H 2 O-Tz (Figure 5Aiii) solutions exhibit a strong absorption band at 340-380 nm, ascribed to both the imide group and the π-π* band transition of the tetrazine group. Significantly larger absorption is observed for PS-CD-Tz NP compared to the H 2 O-Tz, consistent with improved solubilization of the Tz via hostguest encapsulation. The small hypsochromic shift (blue shift) from λ max = 353 nm for H 2 O-Tz to 341 nm for PS-CD-Tz NP is consistent with the Tz molecules shifting from a polar to a nonpolar environment. The shift therefore provides further evidence of the inclusion of Tz in the hydrophobic βcyclodextrin cavities. Such small blue shifts in UV-vis spectra due to host-guest complexation have previously been reported for 4-amino-1,8-naphthalimides with β-cyclodextrin molecules. 37 Another remarkable feature of the fluorescence emission of the encapsulated molecules is that both the imide antenna and the tetrazine acceptor emit together (producing almost white light), proving that the energy transfer is partially 428 blocked as a consequence of the encapsulation process. This is In the absence of Tz, a typical background response of a GC 456 f6 electrode in aqueous solution was observed (Figure 6Ai). For 457 PS-CD-Tz NP nanoparticles, a single well-defined and chemically 458 reversible redox couple at E 1/2 = -0.32 V vs SCE was observed 459 (Figure 6Aii). This system is attributed to a two-electron, two-460 proton reduction of 1,4-tetrazine to its protonated form, 1,4-461 dihydrotetrazine. 29,39 This behavior is analogous to the two-

  252 experiments were recorded for a series of PS-CD solutions 253 prepared in THF with water content of between 0 and 50% 254 (Figure S2). Very low scattering intensities were observed in 255 the presence of 5 to 20% water, consistent with desirable 256 polymer chain dissolution and few aggregates in a mixed 257 solvent. In the presence of ≥20% water, a significant increase in 258 scattering intensity was observed because of the unwanted 259 formation of self-assemblies. β-Cyclodextrin-modified polymer 260 solutions were therefore investigated in 10 and 20% water 261 solutions. However, further observations proved that suspen-262 sions based on 10% water solutions were more unstable, with 263 microscopic particles visible after just a few days of storage, and 264 thus the 80:20 THF/water ratio was adopted for further 265 investigations. 266 We explored the controlled self-assembly of PS-CD (1 mg 267 g -1 ) in the presence and absence of Tz (0.2 mg g -1 ) in THF/ 268 f1 H 2 O via nanoprecipitation in a large excess of water (Figure 269 f1 1A). For the PS-CD with Tz solution, the PS-CD/Tz mole-to-270 mole ratio used was 1:3. Herein, suspensions obtained from PS-271 CD and PS-CD with Tz will be referred to as PS-CD NP and PS-272 CD-Tz NP , respectively. Two methods of nanoprecipitation were 273 explored: method A, where 1 g of PS-CD polymer solution was 274 slowly added to 40 g of water (solvent-to-water method), and 275 method B, where 40 g of water was slowly added to 1 g of PS-276 CD polymer solution (water-to-solvent method). For both 277 methods A and B, the same precipitation and evaporation 278 procedures were performed (Experimental Section) to obtain 279 aqueous PS-CD NP and PS-CD-Tz NP suspensions. 280 The ability of the β-cyclodextrin-modified polystyrene 281 solutions to spontaneously assemble into nanoparticles was 282 initially confirmed by atomic force microscopy and fluorescence 283 microscopy after solution drop-casting and drying on a silicon 284 wafer (Figure 1B,C). AFM imaging of the PS-CD-Tz NP sample 285 prepared by method A revealed the presence of individual 286 nanoparticles with an average particle diameter of 167 ± 30 nm 287 (n = 89 particles). Importantly, the brief AFM analysis proved 288 the successful formation of nanoparticle self-assemblies using 289 our nanoprecipitation protocol. Figure 1C shows a fluorescence

Figure 1 .

 1 Figure 1. (A) Synthesis route for the preparation of encapsulated nanoparticles (PS-CD-Tz NP ). (B, C) Images of PS-CD-Tz NP particles prepared by method A on silicon recorded by (B) AFM in peak-force mode at 1 Hz and (C) fluorescence microscopy with a DAPI filter and a 500 ms exposure time.

  The formation of clusters is attributed to the cross-linking of individual particles via host-guest interactions between Tz molecules and host β-cyclodextrin groups located at the periphery of the particles. (Both tetrazine and naphthalimide functional groups can function as guests.) Particle aggregation due to nonspecific interactions 3 must be considered and likely contributes to the self-assembly of the larger particle distribution. Regardless of the nature of formation of the larger particles, their presence in terms of number is very small compared to the smaller particles. On the basis of 12 DLS measurements at 90°, the estimated number of smaller objects is 95.1% (N small ≈ 19.1N big ), according to eq 3. To complete the nanoparticle sizing experiments, nanoparticle tracking analysis was performed. Nanoparticles were illuminated with a 405 nm blue laser and tracked individually f3 (Figure 3, Figure S3 and corresponding video clips in the Supporting Information). Nanoparticles that undergo Brownian motion were taken into account, and average D h,NTA values of 195 nm for PS-CD NP and 105 nm for PS-CD-Tz NP were acquired. These values are smaller than those obtained by DLS because of the fact that number-weighted distributions are obtained. Nevertheless, the sizes obtained by NTA are complementary, with the corresponding particle sizes obtained by DLS (D h = 240 and 138 nm). The absence of the larger cluster distribution for PS-CD-Tz NP by NTA is not at all

Figure 2 .

 2 Figure2. DLS autocorrelation (g[START_REF] Nicolas | Functionalization Strategies and Biomedical Applications of Targeted Biodegradable/Biocompatible Polymer-Based Nanocarriers for Drug Delivery[END_REF] -1) measured at 90°and relaxation time distribution of aqueous suspensions of (A) PS-CD NP prepared by (i) method A and (ii) method B and (B) PS-CD-Tz NP prepared by (i) method A and (ii) method B.

Figure 3 .

 3 Figure 3. Hydrodynamic number-weighted size distributions by NTA of (A) PS-CD NP and (B) PS-CD-Tz NP suspensions prepared by method A.

  429 in sharp contrast to the situation encountered when the 430 molecules are dissolved in an organic solvent, where the energy 431 transfer occurs with a ca. 90% efficiency and only tetrazine 432 fluorescence is observed. 28 433 Fluorescence spectra of PS-CD-Tz NP nanoparticles were 434 initially recorded at different emission wavelengths. Strong 435 fluorescence was observed only upon excitation in the UV 436 region at λ ex = 353 nm (Figure 5B) and λ ex = 340 nm (data not 437 shown) and not at higher wavelengths (for example, λ ex = 518 438 and 544 nm, data not shown). The classical strong fluorescence 439 of 1,8-naphthalimide was observed between 370 and 420 nm 440 along with a weak fluorescence band at 550-600 nm due to the 441 tetrazine moiety. Further evidence for the improved uptake of 442 fluorescent Tz molecules via molecular encapsulation was 443 obtained by comparison of the fluorescence spectra recorded at 444 λ ex = 340 nm for PS-CD NP , PS-CD-Tz NP , and H 2 O-Tz solutions 445 (Figure 5C). No fluorescence was observed for nanoparticles 446 free of Tz (Figure 5Ci), as expected. A clear enhancement in 447 fluorescence was, however, observed at λ em = 570 nm for PS-448 CD-Tz NP (Figure 5Cii) compared to the Tz-saturated H 2 O-Tz 449 solution, reflecting the improved solubility of tetrazine-450 naphthalamide in the nanoparticle solution compared to water. 451 Cyclic voltammograms (CVs) of the solutions were also 452 performed to test the electroactivity of the nanoparticles. CVs 453 were recorded at glassy carbon electrodes without supporting 454 electrolyte to avoid aggregation due to increased ionic strength.

  455

  462 electron reduction of quinones to hydroquinones in aqueous 463 solution. It should be noted that the CV recorded in H 2 O-Tz 464 solution (Figure S5i) shows an almost identical E 1/2 but with an 465 extremely weak peak intensity, reflecting the trace presence of

Figure 4 .

 4 Figure 4. SLS Guinier plots of the scattering intensity of (i) PS-CD NP and (ii) PS-CD-Tz NP suspensions prepared by method A.

Figure 5 .

 5 Figure 5. (A) UV-vis absorption spectra for (i) PS-CD NP , (ii) PS-CD-Tz NP , and (iii) H 2 O-Tz. (B, C) Fluorescence emission spectra recorded at λ ex = 353 nm for (B) PS-CD-Tz NP at 365 to 650 nm and (C) (i) PS-CD NP , (ii) PS-CD-Tz NP , and (iii) H 2 O-Tz at 525 to 680 nm.

DOI: 10.1021/acs.langmuir.6b03512Langmuir XXXX, XXX, XXX-XXX

■ ACKNOWLEDGMENTS

This work was supported by LABEX ARCANE (ANR-11-LABX-0003-01) and platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG). We gratefully acknowledge the assistance of Hugues Bonnet with AFM imaging.

*E-mail: serge.cosnier@univ-grenoble-alpes.fr.

Notes

The authors declare no competing financial interest.