University of Bath · and the thicker layer formed after 4 (‘seeding layer’, thickness the surface of a gold electrode by electrochemically generating a surface localised pH drop

Citation for published version:Johnson, EK, Chen, L, Kubiak, PS, McDonald, SF, Adams, DJ & Cameron, PJ 2013, 'Surface nucleated growthof dipeptide fibres', Chemical Communications, vol. 49, no. 77, pp. 8698-8700.https://doi.org/10.1039/c3cc44738c

DOI:10.1039/c3cc44738c

Publication date:2013

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Chemical Communications

Cite this: DOI: 10.1039/c0xx00000x

www.rsc.org/xxxxxx

This journal is

Surface

Eleanor K. Johnson, Cameron

*a


DOI: 10.1039/b

We report the surface

dipeptide films

with a globular structure.

with dipeptide led to growth of

enzymes were

growth solution

Low molecular weight hydrogelators (LMWGs) are small

molecules that can self

structures, trapping water inside the matrix to give hydrogels

LMW hydrogels are currently being investigated as energy

transfer materials

wound healing

gelator. A range of aromatic functionalised dipeptides have been

shown to self-assemble into

ordinate into fibres. Fibr

based LMWG systems enzymatically

changing solvent composition

gelators that assemble at surfaces due to electrostatic interactions

or ligand-receptor interactions have also been reported.

2010, we showed that ultra

be grown by inducing a localised pH drop at the surface of an

electrode.17 The pH was decreased by the release of protons that

accompanied the electrochemical oxidation of 1,4

to 1,4-benzoquinone.

Fmoc-LG (Figure

drops below the pK16). In this communication, we report the nucleated growth of

fibrous N

(Fmoc-LG) gel

grown seeding layer

the seeding layer was placed in contact with a solution of Fmoc

LG at pH 7 (Fig 1S in the ESI shows photos of a seeding layer

and the thicker layer formed after 4

(‘seeding layer’, thickness

the surface of a gold electrode by electrochemically generating a

surface localised pH drop following protocols described

previously (see

thick layer was invisible to the naked eye, but its presence was

confirmed by surface plasmon resonance spectroscopy (SPR,

Figure 1S) and transmission electron microscopy (TEM

2). TEM showed a continuous film that containe

spherical aggregates which were only a few nanometres in

diameter (Figure 1(a)). The seeding layer was gently rinsed with



www.rsc.org/xxxxxx

is © The Royal Society of Chemistry

Surface nucleated

Eleanor K. Johnson, a Lin Chen,


DOI: 10.1039/b000000x

We report the surface nucleated

dipeptide films. The seeding

with a globular structure. Placing the

dipeptide led to growth of

enzymes were incorporated into the gel by

growth solution.


molecules that can self-assemble into complex hierarchical

rapping water inside the matrix to give hydrogels


transfer materials5, 6 and as 3

wound healing7-9. One subset of the LMWG is the dipeptide


assemble into β-

ordinate into fibres. Fibril formation can be induced in dipeptide

based LMWG systems enzymatically

changing solvent composition


receptor interactions have also been reported.

2010, we showed that ultra-thin gel fi


The pH was decreased by the release of protons that


benzoquinone.

Figure 1) is a LMW

drops below the pKa of the terminal carboxylic acid (pK

In this communication, we report the nucleated growth of

N-(9-Fluorenylmethoxycarbonyl)

gels on top of an

grown seeding layers. The thicker gel layer grew over 48 hours if


(Fig 1S in the ESI shows photos of a seeding layer

and the thicker layer formed after 4

(‘seeding layer’, thickness 80



previously (see ESI for full experiment




TEM showed a continuous film that containe





Royal Society of Chemistry

nucleated growth of dipeptide fibres

Lin Chen, b

XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

nucleated growth of

. The seeding-layer was a thin dipeptide film

Placing the seeding

dipeptide led to growth of fibres

incorporated into the gel by


assemble into complex hierarchical



and as 3-D scaffolds for cell growth and

. One subset of the LMWG is the dipeptide


-sheet like structures that further co

il formation can be induced in dipeptide

based LMWG systems enzymatically10-

changing solvent composition14, or via pH drop.


receptor interactions have also been reported.

thin gel films and membranes could




is a LMWG that forms gels when the pH

of the terminal carboxylic acid (pK


Fluorenylmethoxycarbonyl)

80-100 nm thick electrochemically

The thicker gel layer grew over 48 hours if



and the thicker layer formed after 48 hours). T

80-100 nm; Figure



experimental detail).




TEM showed a continuous film that containe





Royal Society of Chemistry [year]

growth of dipeptide fibres

Peter S. Kubiak,


growth of self-assembled

was a thin dipeptide film

seeding-layer in contact

fibres overnight. Active

incorporated into the gel by adding them to the





D scaffolds for cell growth and



sheet like structures that further co

il formation can be induced in dipeptide12, thermally,13

, or via pH drop. 15-21


receptor interactions have also been reported.22,

lms and membranes could



accompanied the electrochemical oxidation of 1,4-hydroquinone

G that forms gels when the pH

of the terminal carboxylic acid (pKa


Fluorenylmethoxycarbonyl)-L-leucine-glycine

nm thick electrochemically




. The initial gel lay

nm; Figure 2S) was grown on



al detail). The nanometre



Figure 1S) and transmission electron microscopy (TEM, Figure

TEM showed a continuous film that contained numerous





Peter S. Kubiak, a Shane F. McDonald,


assembled

was a thin dipeptide film

in contact

Active

adding them to the



rapping water inside the matrix to give hydrogels1-4.


D scaffolds for cell growth and



sheet like structures that further co-

il formation can be induced in dipeptide-

, thermally,13 by

LMW

gelators that assemble at surfaces due to electrostatic interactions , 23 In

lms and membranes could



hydroquinone

G that forms gels when the pH

a = 5.8

In this communication, we report the nucleated growth of a

glycine

nm thick electrochemically


the seeding layer was placed in contact with a solution of Fmoc-


he initial gel layer

S) was grown on



The nanometre



, Figure

d numerous



NaCl (0.098 mol dm

cm70

buffered saline both at pH7) for 48

of any further current.

60

65

Fig 1

film85

75

80

110

electrochemically onto the TEM grid (the grid was used as the electrode);

(b) shows the development of fibrous structures after the seeding layer

has been left in contact with Fmoc

scale bar on the left hand image is 20 nm and the scale bar on the right

115

The seeding layer was not removed by

that was checked by both SPR and TEM.

gel layer formed on top

showed that fibres were now present in the gel. It is important to

note that the scale bar in Figure 115


Shane F. McDonald,


NaCl (0.098 mol dm

cm-3 of Fmoc-LG (2.4 mmol dm



Fig 1. (a) Schematic showing the surface initiated growth of an Fmoc

film, (b) the structure of Fmoc

layer after 48 hours in solution (

delineated by the circular o

Fig. 2 (a) shows a TEM image of the seeding layer grown



has been left in contact with Fmoc




gel layer formed on top


note that the scale bar in Figure

(a)

Dynamic Article Links

Communication

[journal]

Shane F. McDonald, a Dave J. Adams,

NaCl (0.098 mol dm-3, at pH 7) and then left in contact with ~ 0.5

LG (2.4 mmol dm



Schematic showing the surface initiated growth of an Fmoc

the structure of Fmoc-LG, (c) a photo of a surface initiated gel

layer after 48 hours in solution (The gel was

delineated by the circular o-ring of an electrochemical SPR cell

(a) shows a TEM image of the seeding layer grown



has been left in contact with Fmoc-LG


hand image is 200 nm.



gel layer formed on top of the seeding layer, TEM (Figure


note that the scale bar in Figure


Communication

[journal], [year],

Dave J. Adams,b

and then left in contact with ~ 0.5

LG (2.4 mmol dm-3, either in NaCl or phosph

buffered saline both at pH7) for 48 hours without the application

Schematic showing the surface initiated growth of an Fmoc

, (c) a photo of a surface initiated gel

The gel was formed within an area

ring of an electrochemical SPR cell




LG-OH in solution for 48


hand image is 200 nm.

The seeding layer was not removed by very gentle

that was checked by both SPR and TEM. After 48 hours a thick

of the seeding layer, TEM (Figure


note that the scale bar in Figure 2(a) is 20 nm and in Figure

(b)


Communication

, [vol], 00–00

and Petra J.


, either in NaCl or phosph

hours without the application

Schematic showing the surface initiated growth of an Fmoc-


formed within an area

ring of an electrochemical SPR cell).




OH in solution for 48 hours. The


very gentle rinsing, a fact

After 48 hours a thick

of the seeding layer, TEM (Figure 2(b))


(a) is 20 nm and in Figure 2

►

Communication

00 | 1

and Petra J.


, either in NaCl or phosphate

hours without the application

-LG


formed within an area



hours. The


rinsing, a fact

After 48 hours a thick

(b))


2(b)

2

is 200nm

spherical

(tens of nanometres

of diameters, which were up to several microns in length.

5

10

15

20

nucleated and grew on the pre

represents a steady increase in

The pH of the grow

remained at pH 7. The pH of the 25

with both pH paper and by inserting a pH probe designed for gels

into the film) was 6.5 after 48 hours. Importantly, control

experiments showed that

seeding layer. In a second set of control experiments, all of the

experimental steps (electrochemistry, rinsing and 48 hour 30

monitoring) were carried out in the absence of hydroquinone.

Hydroquinone is necessary to g

drop that induces formation of the seeding layer. Without

hydroquinone, no seeding layer formed and no thicker gel layer

grew on the surface.35

spontaneous assembly of

using SPR, a technique which can be used to measures changes in

refractive index on top of a metal surface.

wave of the surface plasmon only extends ~ 200 nm into the 40

material above the metal,

thickness of gel layers above 200 nm. SPR c

to prove the existence of the seeding layer

used to measure changes in the gel density as hierarchical

structures developed during the 48 hour growth period. The real 45

time change in

shown in Figure

related to an increase in structure density, e.g. the formation of

aggregates or fibres at the surface.

50

gel layer at the metal surface remained unchanged, followed by

44 hours where the

steadily with time.

1.348

44 hours55

film remained s

no changes were occurring.

| Journal Name

is 200nm. The

spherical aggregates,

tens of nanometres


Fig. 3 The real time change in

nucleated and grew on the pre

represents a steady increase in

corresponding shift in minimum angle as the thick gel layer formed.

The pH of the grow

remained at pH 7. The pH of the



experiments showed that


experimental steps (electrochemistry, rinsing and 48 hour





grew on the surface.

Both the growth of the seeding layer and the subsequent

spontaneous assembly of



wave of the surface plasmon only extends ~ 200 nm into the

material above the metal,




structures developed during the 48 hour growth period. The real

time change in gel refractive index measured over

shown in Figure



Interestingly there was a four


44 hours where the

steadily with time.

1.348 which correspond

44 hours (ESI). The

film remained static during this time, just that close to the surface


Journal Name, [year], [vol]

he thicker film (Fig. 2(b))

aggregates, although the diameters were

tens of nanometres). The film also contained fibres with a range


The real time change in refractive index

nucleated and grew on the pre-seeded surface. The

represents a steady increase in gel


The pH of the growth solution was measured after 48 hours and

remained at pH 7. The pH of the



experiments showed that no gel layer grew in the absence of the







grew on the surface.


spontaneous assembly of a thicker layer were followed in




material above the metal, SPR cannot be used to measure the





gel refractive index measured over

shown in Figure 3. The increase in



Interestingly there was a four


44 hours where the amount of structure in the gel increased

steadily with time. The refractive index increased from 1.338 to

which corresponds to a fivefold increase in gel density over

The time lag does not necessarily indicate that the

tatic during this time, just that close to the surface


[vol], 00–00

thicker film (Fig. 2(b)) still contained some

although the diameters were

. The film also contained fibres with a range


refractive index as the thick gel layer

seeded surface. The refractive index

density with time. Insert shows the


th solution was measured after 48 hours and

remained at pH 7. The pH of the 1-2mm thick gel film (measured



no gel layer grew in the absence of the




Hydroquinone is necessary to generate the surface localised pH




a thicker layer were followed in


refractive index on top of a metal surface.24-26


SPR cannot be used to measure the

thickness of gel layers above 200 nm. SPR can

to prove the existence of the seeding layer (Fig.



gel refractive index measured over

he increase in refractive index



Interestingly there was a four-hour time lag during which the


amount of structure in the gel increased

The refractive index increased from 1.338 to

fivefold increase in gel density over

time lag does not necessarily indicate that the


still contained some

although the diameters were much larger



as the thick gel layer

refractive index shift




2mm thick gel film (measured







enerate the surface localised pH




a thicker layer were followed in-


As the evanescent



an, however, be used

(Fig. 2S). It was also



gel refractive index measured over 48 hours is

refractive index is directly


hour time lag during which the







This journal is © The Royal Society of Chemistry [year]

still contained some

much larger


as the thick gel layer

shift




2mm thick gel film (measured







enerate the surface localised pH




-situ


As the evanescent



, however, be used

. It was also



48 hours is

rectly


hour time lag during which the







A nucleation and growth mechanism

previously been reported

a pH trigger, fibre growth was imaged, although the nucleation 140

step was below the temporal and spatial resolution of confocal

microscopy.

fibres that

sites. In amyloid plaque formation, a nucleation and growth type

mechanism is frequently suggested.145

formation show

phase in which the nuclei are extended into fibrillar structures.

The nuclei can be small oligomers, spherical aggregates or

micellar species. If pre

solution, there i150

follows.

proteins has also been induced by the presence of a mica

surface.

In the experi

mechanisms are contributing to the nucleated growth of the 230

thicker film. Firstly there may be enough protons remaining

inside the seeding layer to create a local pH close to the pK

the Fmoc

6.5, even though it had been sitting in a solution at pH 7 for 48

hours. This suggests that some protons remained trapped within 235

the film.

of dipeptides and other a

environment.

pKas considerably higher than the

been reported240

reported for a range of

surface bound molecules.

used to measure surface p

SAMs; it was found tha

functionalised monolayers shifted about 2 pH units more alkaline 245

when compared to the p

there was a relationship between surface roughness and apparent

pKa for 3

with shifts of up to 4 pH units relative to the solution p

possible.250

shifts of up to 2 pH units for benzoic acid covalently bound to

different carbon surfaces.

entropically controlled due to changes in the solvent ordering at

the surface upon ionisation. The hydrophobicity/hydrophilicity of

the surface also played a role.255

moldm

already slightly above the pK

take a small increase in the pK

nucleated growth to occur.

In a final experiment, surface160

used to incorporate horse

films, by

for experimental details

into diphenylalanine nanotubes and the authors found that

encapsulation enhanced the st165

and bacteria have also been incorporated in Fmoc

films.

layer was removed from the growth solution and rinsed.

incorporation of HRP

simple colorimetric assay, namely the conversion of o170



previously been reported

a pH trigger, fibre growth was imaged, although the nucleation


microscopy.28 Spheres were initially formed and then replaced by

fibres that appeared to grow from a small number of nucleation


mechanism is frequently suggested.

formation show a time lag while nuclei are created, then a growth



micellar species. If pre

solution, there is no time

follows.30 Fibre formation by the mi


surface.31

In the experiments described above it is likely that two

mechanisms are contributing to the nucleated growth of the



the Fmoc-LG. As mentioned a


hours. This suggests that some protons remained trapped within

the film. Secondly there is evidence in the literature that the pK

of dipeptides and other a

environment. Dipeptides with environment specific or ‘apparent’

s considerably higher than the

been reported. 32-35

reported for a range of

surface bound molecules.

used to measure surface p

SAMs; it was found tha

functionalised monolayers shifted about 2 pH units more alkaline

when compared to the p


for 3-mercaptopropionic acid monolayers on gold electrodes


possible.37 Abiman


different carbon surfaces.



the surface also played a role.

moldm-3 NaCl was measured to

already slightly above the pK

take a small increase in the pK


In a final experiment, surface

used to incorporate horse

films, by simply adding it to the second growth solution (

for experimental details


encapsulation enhanced the st


films.41 After 48 hours, a 1


incorporation of HRP

simple colorimetric assay, namely the conversion of o



previously been reported in mixed DMSO/water systems.



Spheres were initially formed and then replaced by

appeared to grow from a small number of nucleation


mechanism is frequently suggested.

a time lag while nuclei are created, then a growth



micellar species. If pre-formed nuclei are added to a protein

s no time-lag and fibre formation rapidly

Fibre formation by the mi


ments described above it is likely that two




As mentioned above the final pH of the gel was



Secondly there is evidence in the literature that the pK

of dipeptides and other acids may be strongly modified by their

Dipeptides with environment specific or ‘apparent’

s considerably higher than the

Surface induced p

reported for a range of self-assembled monolayers (SAMs) and

surface bound molecules.36-39 Impedance spectroscopy has been

used to measure surface pKa values for a range of functionalised

SAMs; it was found that the p


when compared to the pKa in solution.


mercaptopropionic acid monolayers on gold electrodes


Abiman et al. investigated the reasons behind p


different carbon surfaces.36 They concluded that the p



the surface also played a role. The pK

NaCl was measured to be 6.1 by a pH titration (Fig

already slightly above the pKa in pure water (5.8). It would only

take a small increase in the pKa of the molecules at the surface for


In a final experiment, surface-

used to incorporate horse radish peroxidase (HRP) inside the gel

adding it to the second growth solution (

for experimental details). HRP has previously been incorporated


encapsulation enhanced the stability of the enzyme.


After 48 hours, a 1-2 mm thick gel layer formed,


incorporation of HRP in the gel was proved by carry



A nucleation and growth mechanism for Fmoc

in mixed DMSO/water systems.






mechanism is frequently suggested.29, 30 The kinetics of amyloid




formed nuclei are added to a protein

lag and fibre formation rapidly

Fibre formation by the miss-folding of silk






bove the final pH of the gel was




cids may be strongly modified by their


s considerably higher than the pKa of the free dipeptide have

Surface induced pKa shifts have also been

assembled monolayers (SAMs) and

Impedance spectroscopy has been

values for a range of functionalised

t the pKa of both acid and amine


in solution.38 Burris et al.




investigated the reasons behind p


They concluded that the p



The pKa of Fmoc

be 6.1 by a pH titration (Fig

in pure water (5.8). It would only

of the molecules at the surface for

-templated gel formation was

radish peroxidase (HRP) inside the gel

adding it to the second growth solution (

). HRP has previously been incorporated


ability of the enzyme.


2 mm thick gel layer formed,


was proved by carry



Fmoc-LG gels has

in mixed DMSO/water systems.27 Using






The kinetics of amyloid






folding of silk-like





inside the seeding layer to create a local pH close to the pKa of







of the free dipeptide have

shifts have also been




of both acid and amine


et al. showed that



with shifts of up to 4 pH units relative to the solution pK

investigated the reasons behind pK


They concluded that the pKa shift was



of Fmoc-LG in 0.1

be 6.1 by a pH titration (Fig 3S),



templated gel formation was


adding it to the second growth solution (See ESI



ability of the enzyme.40 Enzymes

and bacteria have also been incorporated in Fmoc-F/gelatin

2 mm thick gel layer formed, the

layer was removed from the growth solution and rinsed. The

was proved by carrying out a



has

Using






The kinetics of amyloid






like





of




Secondly there is evidence in the literature that the pKa



of the free dipeptide have

shifts have also been




of both acid and amine


showed that



Ka

Ka


ift was



0.1

),



templated gel formation was


See ESI



Enzymes

F/gelatin

the

he

ing out a

simple colorimetric assay, namely the conversion of o-

This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 3

phenylenediamine (OPD) to 2,3-diaminophenazine (DAP) in the

presence of HRP and H2O2 (ESI). DAP has a strong orange

colour and its appearance is easily monitored by UV-Vis. The gel

was placed in a solution containing OPD and H2O2 and the colour

change in the gel was monitored by UV-Vis in reflectance mode. 5

10

15

20

Fig. 3 The increase in absorbance at 450 nm due to the formation of DAP

after the addition of OPD and H2O2 at 18 minutes. The purple circles

show the change for a gel that does not contain HRP (DAP forms in

contact with oxygen); the blue squares show the change for a gel

incorporating HRP. 25

The solution containing the OPD and H2O2 was not observed to

change colour, but the gel turned a uniform orange, suggesting

that active HRP was evenly distributed. Interestingly the

conversion of OPD to DAP was ~10 times slower inside the gel

than in solution (Fig 4S). 30

In conclusion, the nucleated growth of a hydrogel layer on top

of a nanometre thick seeding layer has been shown. It is likely

that the nucleated growth occurs due to some protons trapped

within the seeding layer lowering the local pH and a modification

of the apparent pKa of the Fmoc-LG due to the surface 35

environment. The growth of gel layers in contact with a pH 7

solution has been used to allow the enzyme HRP to be

incorporated into the gel.

Notes and references

PJC acknowledges the EPSRC for funding (EP/H026304/1). DJA 40

and LC acknowledge the EPSRC for funding (EP/G012741/1). a Department of Chemistry, University of Bath, BA2 7AY. E-mail:

[email protected] b Department of Chemistry, University of Liverpool, L69 7ZD,

U.K. 45

† Electronic Supplementary Information (ESI) available:

Experimental detail, SPR curves for growth of hydrogel layers,

Fresnel fitting of layers, calibration calculations for enzyme

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