Electroactive composite comprising graphene, a metalloprotein and a conjugate polymer

Abstract

The present invention provides a composite comprising graphene, a conjugated porous organic polymer and a metalloprotein and to methods of making the composite. The invention also relates to articles (e.g. to an electrode) comprising the composite and to uses of the composite, e.g. in heterogeneous catalysis of oxygen reduction reactions, and in oxygen sensing.

Claims

1. A composite comprising graphene, a metalloprotein and a conjugated porous organic polymer, wherein said conjugated porous organic polymer comprises a repeat unit of monomer (IVa) or monomer (IVc): ##STR00014## wherein: X.sub.1 is selected from halide, OH or OTf; X.sub.2 is selected from halide, OH, OTf and alkynyl; each W is independently selected from alkynyl, halide, OH, OTf, CHO, COR, COOR, COOH, NH.sub.2, NHR, NR.sub.2, CONH, CONHR wherein R is C.sub.1-8 alkyl, OH, phenol, halide, aryl or heteroaryl; and m is 0 or an integer between 1 and 4.

2. A composite as claimed in claim 1, wherein said conjugated porous organic polymer is distributed on the surface of said graphene.

3. A composite as claimed in claim 1, wherein said metalloprotein is encapsulated in said conjugated porous organic polymer in said composite.

4. A composite as claimed in claim 1, wherein said conjugated porous organic polymer further comprises a repeat unit derived from a monomer of formula (II): ##STR00015## wherein A is an aromatic ring or ring system; Z.sub.1 is alkynyl; Z.sub.2 is selected from alkynyl, halide, OH and OTf; is a bond which may be present or absent; each Y is independently selected from alkynyl, halide, OH, OTf, CHO, COR, COOR, COOH, NH.sub.2, NHR, NR.sub.2, CONH, CONHR wherein R is C.sub.1-8 alkyl, OH, phenol, halide, aryl or heteroaryl, and n is 0 or an integer between 1 and 4.

5. A composite as claimed in claim 4, wherein Z.sub.1, Z.sub.2 and Y are all alkynyl.

6. A composite as claimed in claim 1, wherein each W is selected from halide, OH and OTf; and m is 0 or 1.

7. A composite as claimed in claim 1, wherein X.sub.1 and X.sub.2 are the same.

8. A composite as claimed in claim 1, wherein X.sub.1 and X.sub.2 are selected from halide, OH and OTf.

9. A composite as claimed in claim 6, wherein said monomer of formula (IVa) is: ##STR00016##

10. A composite as claimed in claim 1, further comprising a unit derived from a compound of formula (VI): ##STR00017## wherein C is an aromatic ring or ring system; U is selected from halide, OH or OTf; is a bond which may be present or absent; V is selected from CHO, COR, COOR, COOH, NH.sub.2, NHR, NR.sub.2, CONH, CONHR wherein R is C.sub.1-8 alkyl, OH, phenol, halide, aryl or heteroaryl; and is 0 or an integer between 1 and 4.

11. A composite as claimed in claim 10, wherein said unit of formula (VI) is selected from monomers (VIa), (VIb), (VIc) and (VId): ##STR00018## wherein U, V and o are as defined in claim 10.

12. A composite as claimed in claim 1, wherein said graphene has an average particle size of 50 nm to 50 micron.

13. A composite as claimed in claim 1, wherein said metalloprotein is a haemoprotein.

14. A method of making a composite as claimed in claim 1 comprising mixing graphene, a metalloprotein and a conjugated porous organic polymer.

15. A method as claimed in claim 14, comprising: mixing graphene, a metalloprotein and monomers for the preparation of a conjugated porous organic polymer in the presence of a catalyst to form a composite; and obtaining said composite.

16. An article comprising a composite as claimed in claim 1.

17. A medical device comprising an electrode which comprises claim 1.

18. A method of catalysing an oxygen reduction reaction comprising: bringing a material to be oxidised into contact with an electrode comprising a composite as claimed in claim 1.

19. A composite as claimed in claim 4, wherein said monomer of formula (II) is selected from monomers (IIa), (IIb) and (IIc): ##STR00019##

20. A composite as claimed in claim 4, wherein said monomer of formula (II) is monomer (IIa): ##STR00020##

Description

DETAILED DESCRIPTION OF THE FIGURES

(1) The invention will now be described with reference to the following non-limiting examples and Figures, wherein:

(2) FIG. 1 is a schematic of the synthesis of a composite of the invention showing immobilisation of Hb on the PyPOP-graphene structure;

(3) FIG. 2 shows the Infrared spectra of Hb as well as PyPOP@G and PyPOP-Hb@G composites synthesised in Example 1;

(4) FIG. 3 shows the .sup.13C-CPMAS NMR spectra of PyPOP@G and PyPOP-Hb@G composites synthesised in Example 1;

(5) FIG. 4 shows the results of X-ray photoelectron spectroscopy (XPS) analysis (Peak Intensity (a.u.) versus Binding Energy (eV)) of PyPOP-Hb@G synthesised in Example 1;

(6) FIG. 5 is a Scanning electron microscopy (SEM) image of the surface of PyPOP-Hb@G synthesised in Example 1;

(7) FIG. 6a is N.sub.2 gas sorption isotherm for PyPOP, PyPOP@G and PyPOP-Hb@G composites synthesised in Example 1;

(8) FIG. 6b is a pore size distribution histogram for PyPOP, PyPOP@G and PyPOP-Hb@G composites synthesised in Example 1, with arrows highlighting the most significant differences;

(9) FIG. 7 shows the cyclic voltammetry (CV) cycles for Hb, PyPOP@G and PyPOP-Hb@G composite of the invention, with the inserts showing a magnified portion of the CV indicating the different onset potentials;

(10) FIG. 8a is an overlay of the disk and ring current Linear Sweep Voltammetry (LSVs) for CPE, PyPOP@G and PyPOP-Hb@G composite;

(11) FIGS. 8b-8d are the LSVs and % 4 electron for (b) Hb, (c) PyPOP@G and (d) PyPOP-Hb@G

EXAMPLES

(12) The examples were performed using the following materials and equipment, unless otherwise stated:

(13) Chemicals: solvents, catalysts and chemicals were purchased from Sigma-Aldrich or Fisher Scientific UK. Brominated aromatics were purchased from Combi-blocks. Graphene was purchased from Alfa-Aesar and used without further purification. Nitrogen gas (99.999%) and carbon dioxide (99.995%) was purchased from Airliquide.

(14) Biological reagents: Human haemoglobin was purchased as a lyophilized powder from Sigma-Aldrich and used without further purification.

(15) Infrared absorption spectra were recorded using a Thermoscientific Nicoletis-10.

(16) .sup.13C NMR spectra were recorded on a 400 MHz SS NMR ADVANCE III spectrometer. .sup.13C CP-MAS were recorded at a resonance frequency of 100 MHz under 13 kHz pining rate using a triple-resonance 4 mm Bruker MAS probe (BrukerBioSpin), at a temperature of 298 K. Cross-polarisation contact time was 2 ms employing ramp 100 for variable amplitude CP. To achieve a sufficient signal-to-noise ratio in a reasonable amount of time, 12 k transients and 24 k were collected with 7 s recycle delay. Exponential line broadening of 10 Hz applied before Fourier Transformation. Bruker Topspin 3.0 software was used for data collection and for spectral analysis.

(17) Elemental analysis for Carbon, Hydrogen and Nitrogen content of samples was conducted using a ThermoScientific Flash 2000.

(18) X-ray photoelectron spectroscopy (XPS) was conducted using a Kratos AXIS Ultra DLD XPS system with a hemispherical energy analyser, and a monochromatic Al Ka source operated at 15 keV and 150 W. The X-rays were incident at an angle of 45° with respect to the surface normal. Samples were placed in small powder sockets on the holder and analysis was performed at a pressure below 1×10.sup.−9 mbar. High resolution core level spectra were measured with pass energy of 40 eV. The XPS experiments were performed using an electron beam directed onto the sample for charge neutralisation. Sample sputtering was performed under Ultra High Vacuum conditions using an ion gun mounted on the XPS analysis chamber. The Ar.sup.+ ions were accelerated to beam energy of 4 keV and the raster size was selected at 6 mm×6 mm.

(19) Scanning electron microscopy images were acquired on a JEOL JEM-2100 at 200 KV.

(20) Gas sorption analysis was conducted on a Micrometrics ASAP2020. The variable temperate CO.sub.2 isotherms were recorded in an insulated dewar connected to an LAUDA A-8 circulating chiller. The surface areas were determined from the nitrogen adsorption isotherms collected at 77 K by applying the Brunauer-Emmett-Teller and Langmuir models. Pore size analysis was conducted using a slit NLDFT pore model system by assuming a carbon finite pores surface.

(21) Cyclic voltammetry CPE electrode were coated with PyPOP-Hb@G

Example 1

(22) Synthesis of a conjugated porous-organic polymer and synthesis of a composite of the invention

(23) Pyrimide based porous organic polymer (PyPOP) was synthesised employing the following procedure: A solution of dimethylformamide (15 mL) and trimethylamine (2 mL) was degassed in a 100 mL pressure vial using the freeze-pump-thaw method for three cycles, and maintained under a nitrogen atmosphere. To the degassed solution was added 4,6-dibromopyrimidine (23 mg, 0.1 mmol), and 1,3,5-triethynylbenzene (15 mg, 0.1 mmol), and the vial sonicated for 30 minutes. Copper (I) iodide (5 mg, 0.026 mmol), triphenylphosphine (5 mg, 0.019 mmol) and PdCl.sub.2(PPh.sub.3).sub.2 (5 mg, 0.014 mmol) were then added, and the vial sealed under a flow of nitrogen. The reaction mixture was stirred at 80° C. for 24 hours. The resulting mixture was filtered under vacuum through a sintered glass funnel, and the solid washed with acetonitrile. The solid was then suspended in acetonitrile and stirred at 60° C. in a sealed vial for 6 hours, and filtered under vacuum as previously described. The resulting solid was dried at 110° C. for 5 minutes to afford PyPOP.

(24) A PyPOP and graphene composite (POP@G) was synthesised using the procedure described above except that graphene powder (8 mg) was added to the reaction mixture at the same time as the brominated aromatics. The reaction yielded a dark olive-black solid (30 mg, 96% yield).

(25) A composite of the present invention (PyPOP-Hb@G) was synthesised using the procedure as described above wherein the reaction mixture additionally comprised haemoglobin (5 mg) to afford PyPOP-Hb@G as a solid (33 mg).

Example 2

(26) Infrared spectroscopy was used to confirm the presence of PyPOP and Hb in the PyPOP@G and PyPOP-Hb@G composites synthesised in Example 1.

(27) Analysis of the IR spectrum of PyPOP@G revealed the presence of stretch frequencies at 2215 cm.sup.−1 and 1564 cm.sup.−1, corresponding to the characteristic stretch frequency of a diaryl substituted alkyne (v.sub.C≡C) and pyrimidine (v.sub.C═N) respectively (FIG. 2). Notably, no peak was observed for an unsubstituted terminal alkyne (v.sub.C-H˜3200 cm.sup.−1) indicating the cross coupling reaction as described in Example 1 was successful, and that PyPOP was successfully synthesised. A weak absorption peak at 1650 cm.sup.−1 was also observed, and assigned to the amide (v.sub.C═O) stretch frequency in Hb. This weak peak at 1650 cm.sup.−1 was found to be absent in the IR spectrum of PyPOP@G, providing confidence in attributing the peak to Hb (FIG. 2).

Example 3

(28) Solid State .sup.13C-CPMAS NMR spectrometry was used to evaluate the presence of immobilised Hb in PyPOP-Hb@G synthesised in Example 1.

(29) The .sup.13C spectra of PyPOP-Hb@G revealed the presence of resonance peaks at δ 175 ppm, and broad resonance peaks at δ 50 ppm and δ 25 ppm (FIG. 3). Each of these peaks were found to be absent in the .sup.13C spectrum of PyPOP@G. The additional resonance peaks observed in the PyPOP-Hb@G spectrum were subsequently found to be good agreement with those reported for Hb. This confirmed the presence of immobilised Hb in the PyPOP-Hb@G composite (FIG. 3). The resonance peak at δ 175 ppm was assigned to amide carbonyls, and the peaks at δ 50 ppm and δ 25 ppm to aliphatic groups, of the immobilised Hb proteins. These spectra indicated that Hb had been successfully immobilised within the composite to form PyPOP-Hb@G.

Example 4

(30) Elemental analysis was used to analyse the carbon, nitrogen and oxygen content of each composite synthesised in Example 1, and compared with that of graphene and PyPOP. The results recorded are shown in the table below (Table 1).

(31) TABLE-US-00001 TABLE 1 % C % N % O G 94.30 — — PyPOP 71.55 21.14  2.66 PyPOP@G 76.40 6.64 2.95 PyPOP-Hb@G 75.98 8.03 3.02

(32) These data indicated that the carbon content increased when PyPOP@G composites were synthesised from PyPOP. This can be attributed to the incorporation of carbon rich graphene. In addition, the nitrogen content decreased as expected for the same reasons. Elemental analysis of PyPOP-Hb@G revealed an increase in nitrogen content compared to PyPOP@G, consistent with the presence of nitrogen-rich proteins. These data further supported immobilisation of Hb in the PyPOP-Hb@G composition.

Example 5

(33) X-ray photoelectron spectroscopy (XPS) was used to analyse the surface composition of PyPOP-Hb@G, as synthesised in Example 1.

(34) XPS spectra for the sample were recorded, the sample was then sputtered with Ar ions to probe the composition beneath the surface of the sample, and the spectra recorded again. The binding energies of electrons in the C.sup.1s, N.sup.1s and O.sup.1s orbitals before and after sputtering were then plotted (FIG. 4). The C.sup.1s spectra revealed distinct peaks at 284.6 eV (corresponding to aromatic C—C bonds), 285.5 and 286.6 eV (corresponding to pyrimidine C—N bonds) and 288.3 eV (corresponding to carbonyl C═O bonds). After sputtering the C.sup.1s spectrum revealed the presence of a new peak at 288.7 eV, which was assigned to C—N amide bonds. This binding energy is characteristic of amide bonds in proteins, and indicated that Hb was immobilised within the composite structure, and was not primarily located on the surface. Similar data were recorded in the N.sup.1s XPS spectra, wherein after sputtering a new peak at 402.4 eV (C—N amide bonds) was observed, again indicating the presence of Hb within the composite. The O.sup.1s XPS spectrum revealed the presence of a peak at 532.6 eV (C—O acid/amide bonds) and 533.9 eV (C—OH acid/water) which increased in intensity following sputtering. Collectively, these data indicated that Hb was immobilised within the PyPOP structure, and hence the characteristic C.sup.1s and N.sup.1s protein signals were not observed at the composite surface.

Example 6

(35) Scanning electron microscopy (SEM) was used to analyse the surface of PyPOP-Hb@G synthesised in Example 1.

(36) The SEM image revealed a rough, heterogeneous surface with no discernible segregation between graphene and the POP (FIG. 5).

Example 7

(37) To evaluate the porous structure of the composite synthesised in Example 1, N.sub.2 sorption isotherms for each of PyPOP, PyPOP@G and PyPOP-Hb@G were recorded.

(38) Calculations using the Brauner-Emmet-Teller (BET) model enabled evaluation of the surface area of each sample. BET calculations revealed that each composition had comparable surface areas: PyPOP-Hb@G 445 m.sup.2/g; PyPOP@G 582.7 m.sup.2/g; and PyPOP 664 m.sup.2/g (FIG. 6a). Application of the non-local density function theory (NLDFT) model of carbon finite pores to the early absorption points in the data for each composition allowed the pore size distributions (PSD) to be calculated. Analysis of PyPOP-Hb@G revealed diminished distribution of pores with 32 Å, 42 Å and 50-60 ∪ diameters, as compared to PyPOP and PyPOP@G (FIG. 6b). Cross sections of 49-64 ∪ correspond to that reported for Hb. These data therefore indicate that Hb was occupying correspondingly shaped pores within the PyPOP. The inclusion of Hb within the larger pores may have affected formation of the PyPOP polymer chains, resulting in diminished pores within the 32 ∪ to 42 ∪ range.

Example 8

(39) Cyclic voltammetry (CV) was used to evaluate the utility of PyPOP@G and PyPOP-Hb@G in the oxygen reduction reaction (ORR).

(40) CV was performed using an oxygen-saturated 0.1 M potassium hydroxide solution as the electrolyte, and electrode potentials were recorded using a silver chloride electrode (Ag|AgCl) as a reference, and a carbon paste electrode (CPE) as a control electrode. The oxygen reduction reaction is known to be poor at a CPE, making it an appropriate control.

(41) Evaluation of PyPOP@G revealed an enhanced reduction onset potential (−0.186 V vs Ag|AgCl) compared to the CPE for the ORR. This observation can be attributed to an increased number of active sites within the porous composite structure for the reaction to occur (FIG. 7). Furthermore, an increase in current density was also observed, which was attributed to the high surface area of the porous composite compared to the reference electrode.

(42) Evaluation of Hb immobilised on CPE in a sol-gel film, under the same conditions, revealed two reduction plateaus with onset potentials of −0.217 V and −0.52 V (vs Ag|AgCl) (FIG. 7). The onset potential at −0.52 V was attributed to reduction of oxygen by the Fe.sup.2+ haem centre to form Fe.sup.3+ and superoxide. The second onset potential at −0.217 V was attributed to reduction of Fe.sup.3+ to Fe.sup.2+, which overlapped with a second oxygen reduction reaction. The onset potential of the second reduction falls within the range reported for Hb(Fe.sup.3+/Fe.sup.2+) reduction under basic conditions. The cathodic peak P.sub.c for the second reduction is labelled in FIG. 7 at −0.75V, and can therefore be assigned to reduction of Fe.sup.3+ to Fe.sup.2+ by peroxide intermediates formed in the first reduction of oxygen.

(43) PyPOP-Hb@G was evaluated under the same electrode configuration. Cyclic voltammetry revealed a considerable enhancement in oxygen reduction activity, demonstrated by an increased current density and a lower onset potential (−0.16 V vs Ag|AgCl), compared to PyPOP@G and Hb. The decrease in onset potential (anodic shift) was attributed to the presence of Hb within the PyPOP pores. PyPOP provides a localised concentration of oxygen i.e. reservoirs of oxygen within the porous structure, and therefore a supply of oxygen is provided in the immediate vicinity of the immobilised Hb. The porphyrin centres of Hb can additionally stimulate the active sites within the PyPOP by readily reacting with, and catalysing the ORR. The high concentration of oxygen trapped within the PyPOP therefore overcomes some unfavourable processes including desolvation of oxygen to reach the active sites.

(44) The onset potential (−0.16 V vs Ag|AgCl, −0.828 vs RHE) for PyPOP-Hb@G in the ORR was found to be comparable to that reported for Platinum on Carbon (0.809 vs RHE), and comparable with various other metal, and non-noble metal, based electrocatalysts. The cathodic peak P.sub.c at −0.676 V was more pronounced for PyPOP-Hb@G compared to that observed for the Hg/CPE electrode. Following degassing of the electrolyte solution, to form an oxygen-free solution, the cathodic peak P.sub.c was still observed (FIG. 7, dashed line). The absence of first onset potential (i.e oxygen reduction and oxidation of Fe.sup.2+ to Fe.sup.3+) in the degassed electrolyte can be attributed to rapid reaction of Fe.sup.2+ with traces of oxygen trapped in the composite, as was observed with the Hb immobilised electrode. The cathodic peak P.sub.c was therefore assigned to the reduction of Fe.sup.3+ to Fe.sup.2+. These data further indicated the enhanced catalytic activity of PyPOP-Hb@G in the ORR compared to Hb.

Example 9

(45) Linear sweep voltammetry (LSV) employing the rotating ring disk electrode (RRDE) technique was used to evaluate the mechanism of oxygen reduction on PyPOP-Hb@G.

(46) The oxygen reduction mechanism on porphyrin (N.sub.4—Fe) species is reported to proceed via a 4-electron pathway. It was hypothesised that the increased ORR activity of PyPOP-Hb@G, as described in Example 8, was a result of inner sphere electron transfer (ISET) mechanisms facilitated by the porphyrin core of Hb. CPE and PyPOP@G were used as controls. LSVs were recorded at a fixed rotation speed of 1600 rpm, and the electrolyte was an oxygen-saturated 0.1 M potassium hydroxide solution (FIG. 8a).

(47) The disk current observed during the oxygen reduction reaction for CPE and PyPOP@G were both similar, wherein PyPOP@G demonstrated enhanced disk current attributed to the larger surface area of the porous structure. The enhanced ring current observed for PyPOP@G compared to CPE was attributed to enhanced production and oxidation of hydrogen peroxide. In comparison PyPOP-Hb@G demonstrated a reduced onset potential for the disk current, associated with the presence of immobilised Hb in the porous structure, and an increased ring current, associated with greater ORR activity. These data are in support of the data described in Example 8.

(48) Utilising both the ring current and disk current recorded, the proportion of the current consumed in a 4-electron oxygen reduction pathway was calculated (FIGS. 8b-d). Interestingly, for each of CPE, PyPOP@G and PyPOP-Hb@G, the ring current and disk current were shown to have an inverse relationship as the voltage was increased. The area between the onset potential of the ring current and the onset potential of the disk current indicated the onset of the 4-electron pathway through the ISET mechanism (FIG. 8b-d). The difference in voltage (ΔE) between the onset potential of the ring current, and the onset potential of the disk current therefore provided a voltage range in which onset of the 4-electron pathway became the dominant reduction pathway. PyPOP-Hb@G (ΔE=80 mV) demonstrated an earlier onset of the 4-electron ISET mechanism compared to PyPOP (ΔE=50 mV) and Hb (ΔE=58 mV) (FIG. 8b-d). These data suggest that a synergy between PyPOP and Hb resulted in enhancement of 4-electron ISET activity on PyPOP-Hb@G, compared to PyPOP or Hb alone.

Example 10

(49) To further evaluate the ORR mechanism on PyPOP-Hb@G, LSVs utilising the rotating disk electrode (RDE) technique were performed, under variable disk rotation speeds. The CPE and PyPOP@G demonstrated no disk current dependence on the rate of rotation (FIG. 8a, area A), and can therefore be attributed to a kinetically controlled ORR. However, at increased voltages (FIG. 8a, area B), the charge transfer was enhanced, and the ORR became an oxygen-diffusion controlled process. As porous PyPOP@G has a larger surface area compared to CPE, and therefore greater capacity to store oxygen therein, PyPOP@G demonstrated enhanced disk current at higher voltages.

(50) The disk current recorded for the ORR over the Hb and PyPOP-Hb@G electrode surfaces were shown to depend on the disk rotation speed for potentials in area A (FIG. 8b-c). The relationship between disk current and disk rotation speed was more pronounced for the PyPOP-Hb@G electrode compared to the Hb electrode alone. These data indicated synergism between the Hb and PyPOP, wherein PyPOP acts as an oxygen reservoir to supply Hb, thereby enhancing the ORR. The Koutecky-Levich equation (B=0.62nFC.sub.oD.sub.o.sup.2/3v.sup.−1/6 wherein n=number of electrons) was then applied to the PyPOP-Hb@G LSV RDE curves to plot a linear graph. The value of n, the number of electrons involved in the ORR, was then calculated using the Koutecky-Levich plot to be 4, consistent with the more-efficient 4-electron ORR pathway.