Self-Assembled Peptide-Carbon Nitride Hydrogel as a Light

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Self-Assembled Peptide-Carbon Nitride Hydrogel as a Light-Responsive Scaffold Material Jong Wan Ko, Woo Seok Choi, Jinhyun Kim, Su Keun Kuk, Sahng Ha Lee, and Chan Beum Park Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00889 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules Revised Manuscript

1

Self-Assembled Peptide-Carbon Nitride Hydrogel as

2

a Light-Responsive Scaffold Material

3

Jong Wan Ko, Woo Seok Choi, Jinhyun Kim, Su Keun Kuk, Sahng Ha Lee, and Chan Beum

4

Park*

5

Department of Materials Science and Engineering, Korea Advanced Institute of Science and

6

Technology (KAIST), 291 Daehak-ro, Daejeon 34141, Republic of Korea.

7

KEYWORDS: peptide hydrogel, self-assembly, diphenylalanine, carbon nitride, biomimetic

8

photosynthesis

9

ACS Paragon Plus Environment

1

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 21 Revised Manuscript

1

ABSTRACT

2

Peptide self-assembly is a facile route to the development of bioorganic hybrid materials that

3

have sophisticated nanostructures towards diverse applications. Here, we report the synthesis of

4

self-assembled peptide (Fmoc-diphenylalanine, Fmoc-FF)/graphitic carbon nitride (g-C3N4)

5

hydrogels for light harvesting and biomimetic photosynthesis through non-covalent interactions

6

between aromatic rings in Fmoc-FF nanofibers and tris-s-triazine in g-C3N4 nanosheets.

7

According to our analysis, the photocurrent density of the Fmoc-FF/g-C3N4 hydrogel was 1.8

8

times higher (0.82 µA cm-1) than that of the pristine g-C3N4. This is attributed to effective

9

exfoliation of g-C3N4 nanosheets in the Fmoc-FF/g-C3N4 network, facilitating photo-induced

10

electron transfers. The Fmoc-FF/g-C3N4 hydrogel reduced NAD+ to enzymatically active NADH

11

under light illumination at a high rate of 0.130 mole g-1 h-1 and drove light-responsive redox

12

biocatalysis. Moreover, the Fmoc-FF/g-C3N4 scaffold could well-encapsulate key photosynthetic

13

components, such as electron mediators, cofactors, and enzymes, without noticeable leakage,

14

while retaining their functions within the hydrogel. The prominent activity of the Fmoc-FF/g-

15

C3N4 hydrogel for biomimetic photosynthesis resulted from the easy transfer of photo-excited

16

electrons from electron donors to NAD+ via g-C3N4 and electron mediators as well as the

17

hybridization of key photosynthetic components in a confined space of the nanofiber network.

18

1. INTRODUCTION

19

Green plants harness sunlight through a cascaded transfer of photo-induced electrons for the

20

biocatalytic synthesis of carbohydrates.1 The natural photosynthetic process takes place in the

21

hierarchically well-organized organelles of chloroplasts,2 where solar energy is absorbed by

22

photosensitizing molecules, such as chlorophylls, and stored in a reduced form of nicotinamide


2

Page 3 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

cofactors (NADPH).3 During the Calvin cycle, a series of NADPH-coupled, redox enzymatic

2

reactions occur to reduce carbon dioxide to carbohydrates.4 In past decades, many efforts have

3

been made to mimic natural photosynthesis for the conversion of solar energy into chemical

4

energy such as fuels and fine chemicals.5 For the implementation of sustainable artificial

5

photosynthesis, key photosynthetic components such as light absorbers, electron mediators, and

6

redox catalysts need to be encapsulated into a well-assembled, hierarchical architecture without

7

the loss of their activities.

8

Here, we report the synthesis of self-assembled, light-harvesting peptide hydrogels for the

9

photochemical regeneration of NAD(P)H to be coupled with redox biocatalysis, as illustrated in

10

Figure 1. Peptide-based building blocks can self-assemble spontaneously into supramolecular

11

nanostructures by intrinsic interactions such as hydrogen bonding and π–π stacking between

12

individual assembling molecules.6,7 Among various peptide-based building blocks, we have

13

chosen diphenylalanine (FF) as a peptide building block for synthesizing light-responsive

14

hydrogel because FF is the most simple peptide motif that can self-assemble into hierarchical

15

nanostructures. Moreover, FF and its derivatives can form diverse nanomaterials (e.g.,

16

nanospheres, nanorods, nanowires, nanotubes, and hydrogels) that exhibit advantageous optical,

17

electrical, thermal properties and high biocompatibility.8 On the other hand, graphitic carbon

18

nitride (g-C3N4) is the most stable phase among carbon nitride allotropes under ambient

19

conditions,9 and have received considerable attention for photocatalytic applications because of

20

their high chemical and thermal stability, adjustability of electronic properties, and

21

photosensitivity.10 However, bulk g-C3N4 suffers from a low charge transfer rate and high

22

electron-hole pair recombination rate, reducing quantum efficiency of photocatalytic reactions.


3

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

According to the literature,11 the degree of exfoliation of g-C3N4 nanosheets significantly affects

2

their electronic band structure and physicochemical properties, including electric conductivity,

3 4 5 6 7 8 9 10 11

Figure 1. A schematic illustration of photosynthesis in green plants (top) and light-driven enzymatic light-energy conversion by self-assembled, light-harvesting hydrogel (bottom). In chloroplast, light-driven regeneration of cofactors (i.e., NADPH) takes place, followed by NADPH-dependent enzymatic reactions in the Calvin cycle. The self-assembled Fmoc-FF hydrogel was hybridized with graphitic carbon nitride (g-C3N4), a photosensitizer, for light energy conversion by NADH-dependent enzymatic reaction with triethanolamine (TEOA) as an electron donor. The photo-excited electrons were transferred from g-C3N4 to NAD+ through an electron mediator {M=[Cp*Rh(bpy)H2O]2+, Cp*=C5Me5, bpy=2,2’-bipyridine}.

12


4

Page 5 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

thermal stability, and optical bandgap. For example, effectively exfoliated g- C3N4 nanosheets

2

demonstrated significantly enhanced photocatalytic performance due to the increment of the

3

electron transfer rate with a larger bandgap.12 In this study, we have synthesized g-C3N4-

4

hybridized Fmoc-FF nanofibers (Fmoc-FF/g- C3N4) that can readily form a hydrogel through π-π

5

interaction between aromatic rings in Fmoc-FF nanofibers and g- C3N4 nanosheets, overcoming

6

van der Waals interaction of C-N groups in the g-C3N4 nanosheets. According to the present

7

work, the high content of water in the Fmoc-FF/g-C3N4 hydrogel ensures easy encapsulation of

8

key photosynthetic components while retaining their activities. Moreover, the supramolecular

9

structure of the Fmoc-FF/g-C3N4 hydrogel allows for the efficient transfer of photo-excited

10

electrons from g-C3N4 to NAD+ via an electron mediator M {[Cp*Rh(bpy)H2O]2+, Cp* = C5Me5,

11

bpy = 2,2’-bipyridine} in a confined space of the nanofiber network. We demonstrate that the

12

light-harvesting peptide hydrogel can regenerate NADH photochemically for driving redox

13

enzymatic reactions using L-glutamate dehydrogenase (GDH), a model NADH-dependent

14

enzyme.

15 16

2. EXPERIMENTAL SECTION

17

2.1. Materials. Fmoc-diphenylalanine (Fmoc-FF) was purchased from Bachem AG

18

(Bubendorf, Switzerland). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), β-nicotinamide adenine

19

dinucleotide hydrate (NAD+), ammonium sulfate [(NH4)2SO4], α-ketoglutarate, L-glutamate

20

dehydrogenase (GDH), urea, sodium phosphate monobasic dihydrate, sodium phosphate dibasic

21

dihydrate, and triethanolamine (TEOA) were purchased from Sigma-Aldrich (St. Louis, USA).

22

All chemicals were used without further purification.


5

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

2.2. Characterization. The Fmoc-FF/g-C3N4 hydrogels were freeze-dried to remove water

2

prior to characterization. The morphologies of each sample were observed using an S-4800 field

3

emission scanning electron microscopy (SEM) (Hitachi Co., Japan) at an electron acceleration

4

voltage of 2.0 kV and a JEM-3011 field emission transmission electron microscope (FE-TEM)

5

(JEOL, Japan) with 300 kV of operating voltage. The X-ray diffraction patterns were recorded

6

using a D/MAX-RB X-ray diffractometer (Rigaku Co., Japan) with a scan rate of 4 ° min-1, a

7

range of 10–70 °, and a Cu Kα radiation wavelength of 1.5418 Å. Absorbance spectra were

8

obtained using a V-650 UV/visible spectrophotometer (Jasco Inc., Japan) with a diffuse-

9

reflectance mode. The FT-IR spectra of samples were obtained using an FT-IR 200

10

spectrophotometer (Jasco Inc., Japan). X-ray photoelectron spectroscopic analysis was carried

11

out using a K-alpha (Thermo Scientific, USA) in the scan range of 0 – 1200 eV.

12

Spectrofluorometric analysis was conducted using an RF-5301PC (Shimadzu Co., Japan) with an

13

excitation wavelength of 320 nm. The rheology experiments were conducted using a HAAKE

14

Rheostress 600 rheometer (Thermo Scientific, USA). A parallel-plate (diameter 20 mm, gap 2

15

mm) was used for the measurement. The rheological tests were performed at a frequency of 0.2

16

Hz with a strain of 0.5%. All the rheological measurements were conducted at 25 °C.

17

2.3. Synthesis of graphitic carbon nitride (g-C3N4). We calcinated 10 g of urea dissolved in

18

5 ml of deionized water (resistivity: 18.2 MΩ) in an alumina crucible at 550 °C (heating rate of

19

0.5 °C min-1) under air for 3 hours. The obtained powder was washed with DI water and dried

20

under vacuum conditions at room temperature.

21

2.4. Synthesis of Fmoc-FF/g-C3N4 hydrogels. For the preparation of the Fmoc-FF stock

22

solution, 100 mg of Fmoc-FF was dissolved in 1 mL of HFIP. Calculated amounts of g-C3N4

23

(20, 50, and 100 mg) were dispersed into the Fmoc-FF stock solution (100 mg mL-1). The


6

Page 7 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

mixtures were diluted with DI water to reach the final concentration of 10 mg mL-1. The

2

transparent (Fmoc-FF) and bright yellow colored (Fmoc-FF/g-C3N4) hydrogels were instantly

3

formed. The hydrogels were freeze-dried to remove water and HFIP prior to further experiments.

4

2.5. Photochemical nicotinamide adenine dinucleotide (NADH) regeneration. The

5

photochemical regeneration of NADH was conducted under xenon lamp irradiation (450 W) at

6

room temperature. During the experiments, the ratio of irradiation area to reactor volume was

7

maintained at 1 cm2 per cm3. The samples (placed in a 4 ml UV-cuvette) were positioned parallel

8

to the light beam at a distance of 5 cm from a 450 W xenon lamp (3.25 mW cm-2). The reaction

9

solution was prepared by dissolving 1 mM of NAD+ and 250 µM of [Cp*Rh(bpy)H2O]2+ (M) in

10

a phosphate buffer (100 mM, pH 7.0) containing 15 w% triethanolamine. The calculated amount

11

of Fmoc-FF/g-C3N4 samples and g-C3N4 powder were dispersed in the reaction solution. The

12

Fmoc-FF/g-C3N4 hydrogel instantly formed with NAD+, and M. The concentration of NADH in

13

the reaction solution was obtained by measuring the absorbance of the reaction solution at 340

14

nm using a V-650 UV/visible spectrophotometer (Jasco Inc., Japan).

15

2.6. Photoenzymatic conversion of α-ketoglutarate to L-glutamate. The photoenzymatic

16

synthesis of L-glutamate was coupled with photoregeneration of NADH using Fmoc-FF/g-C3N4

17

hydrogels. The reaction solution for the photoenzymatic reaction consisted of freeze-dried Fmoc-

18

FF/g-C3N4 (3.34–10 mg, 1.67 mg of g-C3N4), NAD+ (1 mM), [Cp*Rh(bpy)H2O]2+ (250 µM), α-

19

ketoglutarate (5 mM), (NH4)2SO4 (100 mM), and GDH (40 U) in 3 mL of phosphate buffer (100

20

mM, pH 7.0) containing 15 w% triethanolamine. The L-glutamate production was analyzed by

21

using a 1260 Infinity liquid chromatography system (Agilent Technologies, USA).

22

2.7. Leakage test for photosynthetic components from Fmoc-FF/g-C3N4 hydrogel. The

23

Fmoc-FF/g-C3N4 hydrogels, which accommodated M, NAD+, and GDH, was immersed in a


7

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

phosphate buffer (10 mM, pH 7.0) and incubated under ambient conditions. The absorbance

2

spectra of supernatants were obtained using a V-650 UV/visible spectrophotometer (Jasco Inc.,

3

Japan) to measure the concentrations of M, and NAD+. We plotted the time profiles of relative

4

amounts of M and NAD+ in the Fmoc-FF/g-C3N4 hydrogel by estimating their concentrations in

5

the supernatants from the normalized absorbance (A/A0) measured at the absorption peaks

6

corresponding to M (λ=313 nm) and NAD+ (λ=214 nm). We determined the amount of proteins

7

in the supernatant using bicinchoninic acid (BCA) assay kit (Pierce, USA).

8 9

3. RESULTS AND DISCUSSION

10

3.1. Self-assembly of Supramolecular Fmoc-FF and Fmoc-FF/g-C3N4 Hydrogels. We

11

confirmed the formation of sp2-bonded graphitic carbon nitride using SEM, TEM, X-ray

12

photoelectron spectroscopy (XPS), and photoluminescence (PL) (Figure S1). To prepare

13

exfoliated g-C3N4, we dispersed bulk g-C3N4 using a homogenizer for 6 hours at 300 W

14

intensity. We injected a mixture of Fmoc-FF monomers and dispersed g-C3N4 (dissolved in HFIP

15

solvent) into deionized water to induce spontaneous assembly of a free-standing Fmoc-FF/g-

16

C3N4 hydrogel. The Fmoc-FF/g-C3N4 hydrogel was opaque and bright yellow-colored, as shown

17

in Figure 2A. We expected the g-C3N4 nanosheets to be hybridized onto Fmoc-FF nanofibers

18

during the hydrogel formation through π-π interaction between tris-s-triazine in g-C3N4 and

19

phenyl rings in Fmoc-FF. According to our electron microscopic analysis of freeze-dried Fmoc-

20

FF and Fmoc-FF/g-C3N4 hydrogels (Figures 2B, 2C), the fibrous Fmoc-FF strands were

21

interwoven and well-hybridized with g-C3N4 nanosheets. For a more detailed analysis of the

22

hybrid structure, we prepared Fmoc-FF/g-C3N4 hydrogels in the different weight ratios of 1:1

23

(Fmoc-FF/g-C3N4 #1), 1:0.5 (Fmoc-FF/g-C3N4 #2), and 1:0.2 (Fmoc-FF/g-C3N4 #3) and


8

Page 9 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

analyzed them using Fourier transform infrared (FT-IR) spectroscope and X-ray diffractometer

2

(XRD). The FT-IR spectra of Fmoc-FF/g-C3N4 hydrogels in Figure 3A show prominent peaks at

3

1660 cm-1 irrespective of the ratios of Fmoc-FF to g-C3N4, which correspond to the anti-parallel

4

configuration of peptides and β-sheet structure of Fmoc-FF nanofibers.13 The characteristic peaks

5

from 1100 to 1600 cm-1 also matched with the typical stretching vibration modes for C=N and C-

6

N in g-C3N4.12 The broad peak centered at around 3300 cm-1 is induced by the N-H stretches,

7 8 9 10 11 12

Figure 2. Photograph, SEM, and TEM images of Fmoc-FF/g-C3N4 hydrogels. (A) Photographic image of the Fmoc-FF/g-C3N4 hydrogel showing its bright yellowish color. (B) SEM image of the Fmoc-FF/g-C3N4 hydrogel exhibiting macro/mesoporous networks. (C) The TEM images of the Fmoc-FF/g-C3N4 hydrogel show that individual Fmoc-FF nanofibers are well-hybridized with g-C3N4 sheets.


9

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

indicating hydrogen bonds in the C-N layers of g-C3N4. We examined crystal structures of the

3

pristine Fmoc-FF, g-C3N4, and Fmoc-FF/g-C3N4 using powder XRD. In the XRD spectra of g-

4

C3N4 and Fmoc-FF/g-C3N4 (Figure 3B), the coherence of the main peaks for g-C3N4 (at 13.1°

5

and 27.6°) indicates that crystal structures of g- C3N4 remain the same in the hydrogel. The XRD

6

spectra for the pristine Fmoc-FF and the Fmoc-FF/g-C3N4 hybrids exhibited characteristic peaks

7

at 4.8-4.9 Å corresponding to the inter-strand distance of Fmoc-FF nanofibers and at 3.4 Å for

8 9 10

Figure 3. (A) FT-IR spectra of Fmoc-FF/g-C3N4 hydrogels in the different weight ratios of 1:1 (Fmoc-FF/g-C3N4 #1), 1:0.5 (Fmoc-FF/g-C3N4 #2), and 1:0.2 (Fmoc-FF/g-C3N4 #3). (B) X-ray


10

Page 11 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

diffraction patterns of Fmoc-FF/g-C3N4 #2 (red), bulk g-C3N4 (blue), and Fmoc-FF hydrogel (dark grey).

3 4

the π-π stacking between the aromatic rings.14 This result suggests that the incorporation of g-

5

C3N4 sheets into the Fmoc-FF network does not affect the intermolecular structure of Fmoc-FF.

6

In addition, we conducted rheology measurements to examine the mechanical strength of Fmoc-

7

FF and Fmoc-FF/g-C3N4 hydrogels. Whereas pristine Fmoc-FF hydrogel exhibited an average

8

storage modulus (G’) of 243.4 Pa, Fmoc-FF/g-C3N4 hydrogels showed much enhanced G’ values

9

in a range of 4000-13000 Pa (Figure S2A). The hydrogels containing more g-C3N4 exhibited

10

higher mechanical strength. The increments in the hydrogel strength were attributed to the

11

increased crosslinking between Fmoc-FF fibers and g-C3N4, resulting in the larger diameter of

12

fibrils with the increasing content of g-C3N4 in the hydrogel (Figures S2B-S2E).

13

3.2. Photoresponsivity of Fmoc-FF/g-C3N4 Hydrogels. To investigate the photoresponsive

14

property of Fmoc-FF/g-C3N4 hydrogels, we obtained UV-visible spectra of Fmoc-FF/g-C3N4

15

hydrogels. Figure 4A shows that Fmoc-FF/g-C3N4 hydrogels exhibit a strong absorption peak at

16

around 400 nm that stems from the band edge position of g-C3N4. Shown in the inset figure are

17

Kubelka-Munk remission function plots [i.e., (αhν)2 vs. photon energy] corresponding to each

18

UV-visible spectrum, which indicates that optical bandgaps of Fmoc-FF/g-C3N4 and g-C3N4 are

19

2.86–2.88 eV and 2.70 eV, respectively. We attribute the increased bandgap of Fmoc-FF/g-C3N4

20

to a quantum confinement effect. This should occur with the exfoliation of g-C3N4 nanosheets

21

during the formation of Fmoc-FF/g-C3N4 hydrogel through the π-π interaction between the

22

graphitic structure in g-C3N4 and the aromatic rings in Fmoc-FF nanofibers.15 Exfoliated g-C3N4

23

is known to exhibit enhanced photoresponse compared to its bulk counterpart due to an increased

24

number and extended lifetime of charge carriers.16 To verify photo-driven electron transfer in the


11

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Fmoc-FF/g-C3N4, we measured photocurrent profiles under light on/off modes in a phosphate

2

buffer (100 mM, pH 7.0) that included triethanolamine (TEOA) as an electron donor. As shown

3

in Figure 4B, Fmoc-FF/g-C3N4 exhibited a much higher photocurrent density than that of

4

dispersed g-C3N4 during each light-on period. We attribute the enhanced photo-activity of Fmoc-

5

FF/g-C3N4 to the increment of charge carriers along with the facile electron transfer of exfoliated

6

g-C3N4 in the hydrogel. The cathodic photocurrent density of Fmoc-FF/g-C3N4 #2 hydrogel was

7

higher than those of Fmoc-FF/g-C3N4 #1 and #3, indicating enhanced charge separation in the

8

Fmoc-FF/g-C3N4 #2.

9


12

Page 13 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3

Figure 4. (A) Absorbance spectra of g-C3N4 and Fmoc-FF/g-C3N4 hydrogels in different weight ratios of 1:1 (Fmoc-FF/g-C3N4 #1), 1:0.5 (Fmoc-FF/g-C3N4 #2), and 1:0.2 (Fmoc-FF/g-C3N4 #3). (B) Photoresponse profiles of Fmoc-FF/g-C3N4 hydrogels, dispersed g-C3N4, and bare ITO glass.

4 5

3.3. Light-driven NADH Regeneration by Fmoc-FF/g-C3N4 Hydrogels. We explored the

6

capacity of self-assembled Fmoc-FF/g-C3N4 hydrogels for light-driven regeneration of the

7

enzymatically active form of NADH from NAD+, an essential progress for performing

8

biomimetic photosynthesis. According to the J-V characteristic in Figure 5A, the cathodic

9

current increased upon light irradiation on the Fmoc-FF/g-C3N4 hydrogel. We further verified

10 11 12

Figure 5. (A) Linear-sweep voltammograms of the Fmoc-FF/g-C3N4 #2 under dark and light illumination with or without the presence of M and NAD+ in electrolyte. (B) Photochemical


13

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

NADH regeneration by Fmoc-FF/g-C3N4 hydrogels, dispersed g-C3N4, and bulk g-C3N4 under light irradiation.

3 4

photo-excited electron transfers from g-C3N4 to NAD+ via the electron mediator M by

5

introducing M and NAD+ to the electrolyte solution. Note that M is required here because of its

6

high stereoselectivity in the reduction of NAD+ to the enzymatically active 1,4-NAD(P)H by

7

hydride transfer with high efficiency.4 The cathodic current increased with the addition of M,

8

indicating that the photo-excited electrons from Fmoc-FF/g-C3N4 reduced M. A further increase

9

in the current density was observed in the presence of both M and NAD+ at a reduction potential

10

of ~0.6 V (vs. Ag/AgCl). These results suggest that the Fmoc-FF/g-C3N4 hydrogel can

11

effectively transfer photo-excited electrons from the electron donor (i.e., TEOA) to the electron

12

mediator (i.e., M) and to the cofactor (i.e., NAD+). Next, we conducted light-driven regeneration

13

of NADH from NAD+ using Fmoc-FF/g-C3N4 hydrogels with M, NAD+, and TEOA. After a

14

three-hour incubation with NAD+ (1 mM) under light illumination, 62.7% conversion of NAD+

15

to NADH was obtained with Fmoc-FF/g-C3N4 #2 with an average conversion rate of 0.130 mole

16

g-1 h-1, while dispersed g-C3N4 and bulk g-C3N4 showed only 35.8% and 0.4% conversions,

17

respectively (Figure 5B). We attribute the significant enhancement in photochemical NADH

18

regeneration performance by Fmoc-FF/g-C3N4 to its higher photoresponse, which facilitates

19

rapid transfer of photo-excited electrons from TEOA to NAD+ via g-C3N4 and M.

20

In natural photosynthesis, photo-induced electron transfers occur within hierarchical

21

architectures that encapsulate key components, such as light harvesters, electron mediators,

22

cofactors, and catalysts. For the development of sustainable and cost-effective photosynthetic

23

platforms, it is critical to assemble such components into a 3D scaffold material while retaining

24

their functions. Many of the previous studies for light-driven enzymatic photosynthesis,


14

Page 15 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

however, have been conducted using homogeneous reaction solutions containing the components

2

in a colloidal state.17 In this study, we demonstrate that all of the components for the photo-

3

induced electron transfer and redox biocatalysis can be well-retained within the Fmoc-FF/g-C3N4

4

hydrogel network with minimal loss. For the leakage tests, we incorporated M, NAD+, and

5

enzyme (i.e., L-glutamate dehydrogenase, GDH) during the formation of the Fmoc-FF/g-C3N4

6

hydrogel and then incubated the resulting hydrogel in a phosphate buffer (10 mM, pH 7.0) at

7

room temperature. According to the changes in the absorbance spectra of the supernatant, we

8

observed that over 90% of M and 99% of NAD+ remained in the Fmoc-FF/g-C3N4 hydrogel after

9

a seven-hour incubation (Figures S3A, S3B). Furthermore, our protein assay using bicinchoninic

10

acid indicated that there was no GDH released to the supernatant solution (Figure S3C). These

11

results indicate that M, NAD+, and GDH were successfully encapsulated in the Fmoc-FF/g-C3N4

12

hydrogel through non-covalent interactions. The comparison of molecular structures of g-C3N4

13

and M suggests a possibility of π-π stacking and hydrogen bonding between tris-s-triazine in g-

14

C3N4 and the bipyridine ring in M. According to the literature,18 graphitic materials such as

15

graphene can readily adsorb M and its derivatives through π-π (or π-cation) interactions.

16

Furthermore, negatively charged Fmoc-FF nanofibers by the deprotonation of carboxylic groups

17

at a neutral pH give rise to retain NAD+ within Fmoc-FF/g-C3N4 hydrogels. Due to the large size

18

of enzymes, supramolecular enzyme-hydrogel hybrids can be easily prepared using peptide-

19

based hydrogelators.19 These multiple effects are synchronized to form the M-NAD+-GDH-

20

Fmoc-FF/g-C3N4 hybrid hydrogel that functions as an all-in-one photosynthetic platform.

21

3.4. Light-driven Redox Biocatalysis using Fmoc-FF/g-C3N4 Hydrogels. We successfully

22

coupled photochemical NADH regeneration with redox enzymatic reaction using Fmoc-FF/g-

23

C3N4 hydrogels and GDH as a model NADH-dependent enzyme that can convert α-ketoglutarate


15

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

into L-glutamate (Figure 6A). The maximum conversion yield (52.04%) with Fmoc-FF/g-C3N4

2

was more than two-fold higher than that of dispersed g-C3N4. L-Glutamate production rate by

3

Fmoc-FF/g-C3N4 #2 hydrogel was estimated to be approximately 0.43 mM h-1. In the case of

4

bulk g-C3N4, we did not observe any biocatalytic conversions by GDH, which is ascribed to the

5

poor NADH regeneration capacity of bulk g-C3N4 as shown in Figure 5B. According to the

6

literature,20 the amphiphilic nature and nanosized internal channels in supramolecular hydrogels

7 8 9 10 11

Figure 6. (A) Photoenzymatic conversion of α-ketoglutarate to L-glutamate by GDH coupled with photocatalytic NADH regeneration using Fmoc-FF/g-C3N4 hydrogels, dispersed g-C3N4, and bulk g-C3N4. (B) Time profile of light-driven GDH reaction by Fmoc-FF/g-C3N4 #2 during light on/off periods.


16

Page 17 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

can facilitate mass transfer of small hydrophilic molecules. Furthermore, the high water content

3

(0.6 ml mg-1) of the Fmoc-FF/g-C3N4 hydrogel would ensure efficient diffusional transportation

4

of hydrophilic substrates and products during the enzymatic reaction. We attribute the enhanced

5

photobiocatalytic reaction with Fmoc-FF/g-C3N4 to the efficient cofactor regeneration by the

6

relevant configuration of light harvester (i.e., g-C3N4) and photosynthetic components (i.e., M,

7

NAD+, and GDH) embedded in the 3D Fmoc-FF hydrogel network accompanied by the rapid

8

mass transfer of substrates. We further examined light-driven enzymatic reactions in the Fmoc-

9

FF/g-C3N4 hydrogel under light on/off modes with 90 min intervals for 12 hours. As shown in

10

Figure 6B, the Fmoc-FF/g-C3N4 hydrogel containing M, NAD+, and GDH steadily produced L-

11

glutamate under light illumination using TEOA as an electron donor, while the conversion of α-

12

ketoglutarate became sluggish during light-off periods. The light-responsive biocatalytic

13

synthesis of L-glutamate shows that the light-driven enzymatic reaction using Fmoc-FF/g-C3N4

14

hydrogels was sustainable without significant loss of photobiocatalytic activity over an extended

15

time.

16 17

4. CONCLUSION

18

We have assembled light-responsive Fmoc-FF/g-C3N4 hydrogels that encapsulate electron

19

mediators, cofactors, and biocatalysts to make all-in-one machinery for biomimetic

20

photosynthesis. The non-covalent interactions between Fmoc-FF nanofibers and g-C3N4

21

nanosheets resulted in the spontaneous formation of highly photoactive Fmoc-FF/g-C3N4

22

hydrogel embedded with exfoliated g-C3N4 nanosheets. The Fmoc-FF/g-C3N4 hydrogel exhibited

23

a good photoactivity (0.82 µA cm-1) compared to dispersed g-C3N4 (0.45 µA cm-1), which not

24

only allowed for light-driven regeneration of enzymatically active NADH from NAD+ but also


17

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

facilitated

2

synergistically expedited photo-induced electron transfers between electron donors and GDH via

3

g-C3N4, M (electron mediator), and NAD+ (enzyme cofactor). This work presents a facile

4

approach for the self-assembly of bioorganic hybrid materials capable of converting light energy

5

into valuable chemicals through redox biocatalysis.

redox

enzymatic

turnover.

The

photosynthetic

Fmoc-FF/g-C3N4 hydrogel

6 7

ASSOCIATED CONTENT

8

Supporting Information. The Supporting Information is available free of charge via the

9

Internet. Additional SEM and TEM images, absorbance and PL spectra, XPS spectra, rheological

10

data, and leakage test.

11

AUTHOR INFORMATION

12

Corresponding Author

13

*E-mail: [email protected].

14

Notes

15

The authors declare no competing financial interest.

16

ACKNOWLEDGMENT

17

This study was supported by the National Research Foundation via the Creative Research

18

Initiative Center (Grant No. NRF-2015R1A3A2066191), Republic of Korea.

19

REFERENCES


18

Page 19 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

(1)

(a) Zhang, T.; Lin, W. Chem. Soc. Rev. 2014, 43, 5982-5993 (b) Berardi, S.; Drouet, S.;

2

Francas, L.; Gimbert-Surinach, C.; Guttentag, M.; Richmond, C.; Stoll, T.; Llobet, A. Chem.

3

Soc. Rev. 2014, 43, 7501-7519 (c) Wiechen, M.; Najafpour, M. M.; Allakhverdiev, S. I.; Spiccia,

4

L. Energy Environ. Sci. 2014, 7, 2203-2212.

5

(2)

(a) Wang, M.; Zhan, W. Acc. Chem. Res. 2016, 49, 2551-2559 (b) Hasan, K.; Milton, R.

6

D.; Grattieri, M.; Wang, T.; Stephanz, M.; Minteer, S. D. ACS Catalysis 2017, 7, 2257-2265 (c)

7

Bedford, N. M.; Winget, G. D.; Punnamaraju, S.; Steckl, A. J. Biomacromolecules 2011, 12,

8

778-784.

9 10 11 12 13 14 15

(3)

(a) Rudolf, M.; Kirner, S. V.; Guldi, D. M. Chem. Soc. Rev. 2016, 45, 612-630 (b) Wang,

Y.; Li, S.; Liu, L.; Lv, F.; Wang, S. Angew. Chem. 2017, 129, 5392-5395. (4)

(a) Kim, J. H.; Nam, D. H.; Park, C. B. Curr. Opin. Biotech. 2014, 28, 1-9 (b) Lee, S. H.;

Kim, J. H.; Park, C. B. Chem. Eur. J. 2013, 19, 4392-4406. (5)

(a) Tachibana, Y.; Vayssieres, L.; Durrant, J. R. Nat. Photon. 2012, 6, 511-518 (b)

Kärkäs, M. D.; Johnston, E. V.; Verho, O.; Åkermark, B. Acc. Chem. Res. 2014, 47, 100-111. (6)

(a) Fleming, S.; Ulijn, R. V. Chem. Soc. Rev. 2014, 43, 8150-8177 (b) Knowles, T. P. J.;

16

Buehler, M. J. Nat. Nanotech. 2011, 6, 469-479 (c) Hamley, I. W. Biomacromolecules 2014, 15,

17

1543-1559.

18 19 20

(7)

(a) Kim, S.; Kim, J. H.; Lee, J. S.; Park, C. B. Small 2015, 11, 3623-3640 (b) Kim, Y.; Li,

W.; Shin, S.; Lee, M. Acc. Chem. Res. 2013, 46, 2888-2897. (8)

Yan, X.; Zhu, P.; Li, J. Chem. Soc. Rev. 2010, 39, 1877-1890.


19

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3 4

(9)

Zhu, J.; Xiao, P.; Li, H.; Carabineiro, S. A. C. ACS Appl. Mater. Interfaces 2014, 6,

16449-16465. (10) (a) Zhao, Z.; Sun, Y.; Dong, F. Nanoscale 2015, 7, 15-37 (b) Zheng, Y.; Liu, J.; Liang, J.; Jaroniec, M.; Qiao, S. Z. Energy Environ. Sci. 2012, 5, 6717-6731.

5

(11) Wang, Y.; Wang, X.; Antonietti, M. Angew. Chem., Int. Ed 2012, 51, 68-89.

6

(12) Yang, S.; Gong, Y.; Zhang, J.; Zhan, L.; Ma, L.; Fang, Z.; Vajtai, R.; Wang, X.; Ajayan,

7 8 9 10 11

P. M. Adv. Mater. 2013, 25, 2452-2456. (13) Zhou, M.; Smith, A. M.; Das, A. K.; Hodson, N. W.; Collins, R. F.; Ulijn, R. V.; Gough, J. E. Biomaterials 2009, 30, 2523-2530. (14) Mu, X.; Eckes, K. M.; Nguyen, M. M.; Suggs, L. J.; Ren, P. Biomacromolecules 2012, 13, 3562-3571.

12

(15) Kim, J. H.; Nam, D. H.; Lee, Y. W.; Nam, Y. S.; Park, C. B. Small 2014, 10, 1272-1277.

13

(16) (a) Bai, X.; Wang, L.; Zong, R.; Zhu, Y. J. Phys. Chem. C 2013, 117, 9952-9961 (b) She,

14

X.; Xu, H.; Xu, Y.; Yan, J.; Xia, J.; Xu, L.; Song, Y.; Jiang, Y.; Zhang, Q.; Li, H. J. Mater.

15

Chem. A 2014, 2, 2563-2570 (c) Zhang, S.; Li, J.; Wang, X.; Huang, Y.; Zeng, M.; Xu, J. ACS

16

Appl. Mater. Interfaces 2014, 6, 22116-22125.

17 18 19

(17) (a) Ko, J. W.; Lee, B. I.; Chung, Y. J.; Park, C. B. Green Chem. 2015, 17, 4167-4172 (b) Shi, Q.; Yang, D.; Jiang, Z.; Li, J. J. Mol. Catal. B: Enzym. 2006, 43, 44-48. (18) Lee, J. S.; Lee, S. H.; Kim, J.; Park, C. B. J. Mater. Chem. A 2013, 1, 1040-1044.


20

Page 21 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

(19) Wan, L.; Chen, Q.; Liu, J.; Yang, X.; Huang, J.; Li, L.; Guo, X.; Zhang, J.; Wang, K. Biomacromolecules 2016, 17, 1543-1550.

3

(20) (a) Wang, Q.; Yang, Z.; Zhang, X.; Xiao, X.; Chang, C. K.; Xu, B. Angew. Chem., Int. Ed

4

2007, 46, 4285-4289 (b) Wang, Q.; Yang, Z.; Gao, Y.; Ge, W.; Wang, L.; Xu, B. Soft Matter

5

2008, 4, 550-553.

6 7

Table of Contents Graphic

8


21

Self-Assembled Peptide-Carbon Nitride Hydrogel as a Light

Recommend Documents