Gelation and adhesion behavior of mussel adhesive protein mimetic polymer

Article abstract of DOI:10.1002/pola.26487

An acrylamide-type copolymer containing catechol, amino, and hydroxyl groups was synthesized as a mimetic of the natural mussel adhesive protein (MAP). The obtained copolymer in a phosphate buffer solution (pH = 8.0) formed a hydrogel within 2 h under air, whereas gelation did not proceed under argon atmosphere. We confirmed that the cross-linking reaction of the synthesized MAP mimetic copolymer was triggered by aerobic oxidation of catechol moieties to form an adhesive hydrogel. Two aluminum plates were adhered by the gelation of the MAP mimetic copolymer solution under humid air at room temperature. The interfacial region between the two aluminum plates failed at a lap shear strength of 0.46 MPa due to cohesive failure of the hydrogel. The adhesion strength was dominated by mechanical strength of the hydrogel as well as the interface interaction of catechol groups with substrate surface. 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013. Copyright

Full text of DOI:10.1002/pola.26487

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Gelation and Adhesion Behavior of Mussel Adhesive Protein
Mimetic Polymer
Jin Nishida,1 Motoyasu Kobayashi,1 Atsushi Takahara1,2,3
1JST, ERATO, Takahara Soft Interfaces Project, CE80, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
2Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
3International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, Fukuoka, Japan
Correspondence to: A. Takahara (E-mail: takahara@cstf.kyushu-u.ac.jp)
Received 24 September 2012; accepted 15 November 2012; published online 5 December 2012
DOI: 10.1002/pola.26487
ABSTRACT: An acrylamide-type copolymer containing catechol,
amino, and hydroxyl groups was synthesized as a mimetic of
the natural mussel adhesive protein (MAP). The obtained
copolymer in a phosphate buffer solution (pH ¼ 8.0) formed a
hydrogel within 2 h under air, whereas gelation did not pro-
ceed under argon atmosphere. We confirmed that the cross-
linking reaction of the synthesized MAP mimetic copolymer
was triggered by aerobic oxidation of catechol moieties to
form an adhesive hydrogel. Two aluminum plates were
adhered by the gelation of the MAP mimetic copolymer
solution under humid air at room temperature. The interfacial
region between the two aluminum plates failed at a lap shear
strength of 0.46 MPa due to cohesive failure of the hydrogel.
The adhesion strength was dominated by mechanical strength
of the hydrogel as well as the interface interaction of catechol
C
V
groups with substrate surface.
2012 Wiley Periodicals, Inc.
J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 1058–1065
KEYWORDS: aerobic oxidation; biomimetic; catechol; mussel;
under water adhesion; gels; crosslinking
INTRODUCTION Mussels in nature are able to firmly anchor
to various material surfaces even under wet conditions; how-
ever, it is still difficult to artificially develop a comparable
adhesion technology. Understanding the materials and
mechanisms of the fascinating underwater adhesion system
is useful for developing biological glue that can be applied
for the adhesion of cells or organisms.
investigated.13 For example, polydopamine film was trans-
formed to a surface initiator layer for the surface-initiated
polymerization to form a grafting polymer.14
During last two decades, various types of catechol-contain-
ing polymers have been synthesized to investigate the
nature of mussel adhesive protein (MAP) or the functions
of the catechol group. Yamamoto chemically synthesized a
polypeptide that had a natural fp-1 sequence.15 The adhe-
sion strength of two metal pieces using an aqueous solution
of the polymer was investigated. Deming and coworker
synthesized not only a natural-sequence polypeptide but
also mimetic polypeptides as well.16 They synthesized a
copolymer composed of lysine and DOPA and measured
the adhesion strength of metal adherends. Wilker and
coworkers synthesized catechol-containing polystyrene,
which improved the adhesion strength of aluminum
adherends by the anchoring and cross-linking of catechol
units.17,18 Adhesion in aqueous media has also been investi-
gated using the strong interaction between catechol groups
and metal oxides. Messersmith and coworkers reported that
photocured polyethyleneoxide gel containing catechol
groups successfully adhered the titanium surfaces in
water.19 Geckos pad-inspired microfabricated pillars coated
with catechol-containing polyacrylamide derivatives also
achieved adhesion under wet condition.20
Mussels are able to anchor to underwater surfaces by secre-
tion of byssus adhesive plaque composed of fp-1 to fp-6 pro-
teins.1–4 Each of these proteins has a high content of lysine
and a ‘‘non-standard’’ amino acid, dihydroxyphenylalanine
(DOPA). In particular, the catechol moiety in DOPA is essen-
tial for adhesion.5,6 The catechol unit plays two roles in
adhesion. One is the tethering to surfaces7 through stable
bidentate hydrogen bonding with metal oxides.8–10 The other
is the formation of organs by cross-linking. Oxidation of
catechol units easily undergo by the oxygen in water to give
a catechol quinone. The quinone induces a cross-linking
reaction through radical coupling or nucleophilic attack by
amine.11,12 Therefore, the DOPA is used as a surface modifi-
cation reagent. When the substrate was immersed into the
dopamine aqueous solution, dopamine is readily adsorbed
on the substrate surface to form a unique polydopamine thin
film by the successive oxidation and cross-link reaction.
Further modification of the polydopamine films was also
C
V
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SCHEME 1 Synthesis of MAP mimetic polymer.
MAPs in nature have not only catechol group but also
hydroxyl and amino groups in the amino acids. Each
functional group must have a unique role in achieving stable
adhesion in an aqueous environment. As mentioned above,
catechol groups worked as anchoring sites on metal oxide
surface. Hydroxyl groups improve the hydrophilicity of the
proteins. Amino groups are expected to promote the cross-
linking reaction through the aerobic oxidation. In this study,
we designed MAP mimetic acrylamide copolymers containing
these three functional groups to investigate the effect of each
groups on the adhesion properties (Scheme 1). Acrylamides
are the useful water soluble monomers for synthesis of the
functional copolymers with desired compositions by a con-
ventional radical polymerization. In addition, water soluble
‘‘vinyl polymers’’ containing catechol moiety has not been
reported so much until now. We synthesized water soluble
acrylamide copolymers containing hydroxyl, amino, and cate-
chol units with various compositions. The relationship
between adhesion properties and the compositions of the
each functional group in the copolymers were investigated.
The effect of oxidant reagent on the gelation process and ad-
hesion ability of the obtained copolymer under wet condition
were also investigated.
calcium hydride before use. Triethylamine (Wako) was dried
over potassium hydroxide. Methanol (MeOH, Wako) was dis-
tilled before use. 2,20-Azobis(isobutyronitrile) (AIBN, Wako)
was recrystallized from methanol before use. N-(2-Hydroxye-
thyl)acrylamide (Tokyo Chemical, Tokyo, Japan) was purified
by flash column chromatography using aluminum oxide
(Merck, Tokyo, Japan) before use. 3-Hydroxytyramine hydro-
chloride, acryloyl chloride, chlorotriethylsilane, N-(tert-butox-
ycarbonyl)-1,6-diaminohexane, and 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) were
purchased from Tokyo Chemical and used as received. Alu-
minum plates (20 ꢀ 50 mm2) for the adhesion tests were
washed with hexane, ethanol, and water and dried under
nitrogen gas. Glass plates (10 ꢀ 40 mm2) were washed with
a mixture of sulfuric acid and hydrogen peroxide (7/3, v/v)
at 100 ꢁC for 1 h, rinsed with water, and dried under nitro-
gen gas. Polytetrafluoroethylene (PTFE) plates (10 ꢀ 40
mm2) were washed with ethanol and dried under nitrogen.
Synthesis of N-(2-[3,4-Dihydroxyphenyl]ethyl)
acrylamide (1)
3-Hydroxytyramine hydrochloride (10.0 g, 52.8 mmol) and
triethylamine (7.31 mL, 52.7 mmol) were dissolved in MeOH
(100 mL), and cooled on an ice bath. THF solution (5 mL) of
acryloyl chloride (5.11 mL, 63.2 mmol) and MeOH solution
(11 mL) of triethylamine (11.0 mL, 79.1 mmol) were
alternately added dropwise to the hydroxyramine solution
maintaining the pH at 9. After adding the reagent, the
reaction mixture was stirred at room temperature for 1 h.
Solvent was removed under vacuo. The residue was dis-
solved in ethyl acetate and washed with 1 M hydrochloric
EXPERIMENTAL
Materials
Dry tetrahydrofuran (THF, Kishida Chem., Osaka, Japan) puri-
fied using an organic solvent pure unit (GlassContour Solvent
Dispensing System, Nikko Hansen, Osaka, Japan) was used.
Dimethylformamide (DMF, Wako Pure Chemical, Osaka, Ja-
pan) was dried on a molecular sieve 4A and distilled from
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FIGURE 1 1H NMR spectra of protected polymer (in CD3OD) and target polymer (in D2O).
acid (HCl) and brine. The organic layer was dried over
sodium sulfate, then filtrated, and concentrated by evapora-
tion. The product was dissolved in ethyl acetate to repeat
the recrystallization, giving the compound (1) (7.06 g, 34.1
mmol) in a 65% yield as a white solid.
dissolved in ethyl acetate, washed with 10% citric acid solu-
tion, sat. NaHCO3aq, and brine. The organic layer was dried
on sodium sulfate, filtrated, and dried in vacuo. The resulting
solid was recrystallized from ethyl acetate to give the com-
pound (3) (3.23 g, 12.0 mmol) in 72% yield as a white solid.
1H NMR (400 MHz, CD3OD, d ppm); 6.67 (1H, d, J1 ¼ 8.0),
6.64 (1H, d, J1 ¼ 2.0), 6.52 (1H, dd, J1 ¼ 8.0, J2 ¼ 2.1), 6.19
1H NMR(400 MHz, CDCl3, d ppm); 6.28 (1H, dd, J1 ¼ 17.0, J2 ¼
1.6), 6.10 (1H, dd, J1 ¼ 17.0, J2 ¼ 10.3), 5.80(1H, s), 5.63 (1H,
dd, J1 ¼ 10.3, J2 ¼ 1.6), 4.54 (1H, s), 3.27 (2H, m), 3.12 (2H,
m), 1.55 (2H, m), 1.48 (2H, m), 1.44 (9H, s), 1.35 (4H, m).
(2H, m), 5.62 (1H, dd, J1 ¼ 6.8, J2 ¼ 5.3), 3.40 (2H, t, J1
7.4), 2.66 (2H, t, J1 ¼ 7.4).
¼
Synthesis of N-(2-[3,4-Di-
MAP Mimetic Copolymer Synthesis
triethylsilyloxyphenyl]ethyl)acrylamide (2)
A methanol solution (5 mL) of monomer 2 (218 mg, 0.500
mmol), 3 (135 mg, 0.499 mmol), hydroxyethylacrylamide
(575 mg, 0.499 mmol), and AIBN (19.4 mg, 0.116 mmol)
was degassed by the freeze–thaw method three times. The
reaction vessel was sealed in vacuo, and stirred for 14 h at
60 ꢁC. The reaction mixture was diluted with methanol and
poured into ether to precipitate the resulting polymer, which
was collected by centrifugation (3000 rpm, 10 min), washed
with ether three times, and dried in vacuo. The precopoly-
mer with Mn ¼ 91,600 and Mw/Mn ¼ 3.6 was obtained in a
90% yield.
Triethylchlorosilane (1.75 g, 11.6 mmol) was added drop-
wise to a mixture of compound 1 (1.00 g, 4.83 mmol) and
triethylamine (2.41 mL, 17.4 mmol) dissolved in DMF (10
mL) on an ice bath. The reaction mixture was stirred for 1 h
ꢁ
at 0 C and 2 h at room temperature. Chloroform was added
to the reaction mixture, which was washed with 1 N HClaq,
sat. NaHCO3aq, and brine. The organic layer was dried over
sodium sulfate, filtrated, and dried in vacuo. The crude prod-
uct was purified by silica gel column chromatography using
hexane/ethyl acetate (3/1, v/v) as an eluent. The compound
(2) was obtained as a viscous colorless liquid (2.07 g, 4.75
mmol) in 98% yield.
Aqueous hydrochloric acid solution (2 M, 7 mL) was added to
the methanol solution (7 mL) of precopolymer (730 mg) at 0
ꢁC and was stirred at room temperature for 14 h to proceed
the acidic hydrolysis of silyloxy groups on catechol units and
the removal of Boc groups from amino groups. The reaction
mixture was poured into ether to precipitate the deprotected
polymer, which was collected by centrifugation, washed with
ether three times, and dried in vacuo. The residue was dis-
solved in water and dialyzed to remove the hydrochloric acid
and then lyophilized. The MAP mimetic polymer (608 mg)
was obtained as a white powder. The polymer was stored at
ꢂ20 ꢁC in a freezer and was stable under these this conditions.
1H NMR (400 MHz, CDCl3, d ppm); 6.75 (1H, d, J1 ¼ 8.0),
6.61–6.65 (2H, m), 6.24 (1H, dd, J1 ¼ 17.0, J2 ¼ 1.4), 6.00
(1H, dd, J1 ¼ 16.9, J2 ¼ 10.3), 5.62 (1H, dd, J1 ¼ 10.3, J2
¼
1.4), 5.51 (1H, s), 3.54 (2H, m), 2.72 (2H, t, J1 ¼ 6.8), 0.98
(18H, m,), 0.74 (12H, m).
Synthesis of N-[N0-(Tert-butoxycarbonyl)-6-aminohexyl]
acrylamide (3)
To a solution of N-(tert-butoxycarbonyl)-1,6-diaminohexane
(3.01 g, 13.9 mmol) and triethylamine (2.88 mL, 20.8 mmol)
diluted with THF (30 mL), THF solution (2 mL) of acryloyl
chloride (1.34 mL, 16.6 mmol) was added dropwise at 0 ꢁC,
and the mixture was stirred at room temperature for 1 h.
After the reaction mixture was evaporated, the residue was
Characterization
1H NMR spectra were recorded in chloroform-d (CDCl3),
CD3OD, or D2O on a Bruker AV400N (1H 400.13 MHz, 13C
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TABLE 1 Code Name of the MAP Mimetic Copolymers and
on the aluminum plate and was bound with the other plate.
The adhesion area was 100 mm2. The substrates were
allowed to stand under humid air (>90% relative humidity)
at room temperature for 24 h. The sample was held at both
ends with two mechanical chucks connected to a load cell
and a tensile tester base anchor. The crosshead speed was
set to 1 mm/min in tensile mode. The lap shear strength
was defined as the force corresponding to the failure force
divided by an adhesion area of 100 mm2.
Contents of Catechol and Amine Units
Contents (mol %)
Polymer Code
Namea
x/y/z
(Molar Ratio)b
Catechol
Ammonium
P(10H-1C-1A)
P(5H-1C-1A)
P(6H-1C)
P(11H-1C)
P(20H-1C-1A)
P(21H-1C)
P(10H)
10/1/1
5/1/1
0.08
0.14
0.14
0.08
0.045
0.045
0.00
0.00
0.08
0.14
0.00
0.00
0.045
0.00
0.00
0.08
6/1/0
11/1/0
20/1/1
21/1/0
10/0/0
10/0/1
RESULTS AND DISCUSSION
Synthesis of MAP Mimetic Polymer
MAPs in nature are mainly composed of certain amino acids
containing hydroxyl, amino, and catechol groups. Each func-
tional group plays a unique role in achieving stable adhesion
in an aqueous environment. Catechol groups form bidentate
type hydrogen bonds with metal oxide surfaces to anchor
the proteins. Catechol and amino groups are used for cross-
linking reaction of proteins followed by aerobic oxidation.
Hydroxyl groups are useful for improving water solubility of
the proteins. Based on this knowledge, the target material
was designed as an acrylamide terpolymer containing
catechol, amino, and hydroxyl groups. To obtain this target
polymer, triethylsilyl-protected catechol monomer 2 and
Boc-protected amino monomer 3 were copolymerized with
hydroxyl monomer (H) by AIBN-initiated radical polymeriza-
tion. 1H NMR spectrum of the polymer [Fig. 1(a)] showed
characteristic peaks attributed to each monomer unit, such
as triethylsilyl groups (1.5 and 1.0 ppm) and aromatic pro-
ton (6.6 ppm) in catechol monomer 2, tert-butyl group (1.4
ppm) in amino monomer 3, and oxymethylene group (3.6
ppm) in hydroxyl monomer H. All protection groups were
successively removed by acid treatment simultaneously.
P(10H-1A)
a
Remarks: (H) ¼ N-(2-hydroxyethyl) acrylamide, (C) ¼ N-(2-[3,4-dihy-
droxyphenyl]ethyl) acrylamide, (A)
¼
6-(acrylamidehexyl)ammonium
chloride.
b
Molar ratio of x/y/z corresponds to H/C/A unit ratio in the copolymers,
respectively.
100.61 MHz) spectrometer, with tetramethylsilane or solvent
as an internal standard. Number- and weight-average molec-
ular weights (Mw and Mn) were estimated by size-exclusion
column chromatography on
a Shimazu HPLC system
(Shimadzu Corporation, Kyoto, Japan), equipped with two
consecutive polymethylmethacrylate gel columns (TSKgel
SuperAW4000 and TSKgel SuperAW5000, Tosoh Corporation,
Tokyo, Japan) and refractive index and ultraviolet (254 nm)
detectors, using DMF solution of 0.01 M LiBr at an eluent
flow rate of 1.0 mL/min, calibrated with polystyrene stand-
ards. Water content of adhesive layers was determined from
weight loss during heating, recorded by a Pyris TGA (Perki-
nElmer Japan, Kanagawa, Japan).
1
Removal of each protection group was confirmed by H NMR
A gelation process was evaluated by a rheometer (Anton
Paar Physica MCR 101) with parallel geometry (diameter 50
mm). MAP mimetic polymer solution (5 wt %) in phosphate
buffer (0.1 M, pH ¼ 8.0) was prepared and quickly put on a
sample stage (thinkness: 0.2 mm). Then storage modulus
(G0) and loss modulus (G00) were measured at 5 min intervals
spectrum [Fig. 1(b)]. Signals from the tert-butyl group and
triethylsilyl group disappeared completely. Monomer ratio in
the obtained polymer was estimated from this spectrum. The
peaks at 3.5 ppm (ACH2AOH) for hydroxyl group, at 2.5
ppm (ACH2AAr) for catechol group, and at 2.8 ppm
(ACH2ANH2) for amino group were used for the estimation.
The ratio of each peak area was determined as 10/0.97/
0.97, which agreed well with feed ratio (10/1/1). These
results clearly indicate that the target polymer was quantita-
tively obtained. The monomer units of 2 and 3 in the copoly-
mer were successfully converted to catechol unit (C) and
ammonium chloride unit (A), respectively.
ꢁ
(strain 1%, frequency 1 Hz, 25 C).
The adhesion strength was determined by measuring the
lap shear adhesion force with a tensile tester (Shimadzu
EZ-Graph) at 298 K in an ambient atmosphere. An amount
of 10 mg of 10 wt % solution of MAP mimetic polymer in
phosphate buffer solution (0.1 M, pH ¼ 8.0) was mounted
FIGURE 2 Result of the gelation test of MAP polymer solution in phosphate buffer (pH ¼ 8.0). (a) Gel formed in air with 2 h. (b)
Gel formed tyrosynase containing solution with 20 min. (c) Polymer solution under argon atmosphere after 24 h.
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FIGURE 3 Time course of storage modulus (G0) and loss modu-
lus (G00) (strain 1%, frequency 1 Hz, 25 ꢁC) during gelation of MAP
mimetic P(10H-1C-1A) phosphate buffer solution (pH ¼ 8.0).
FIGURE 4 Lap shear adhesive strength of aluminum plates
using MAP mimetic copolymers with various molar ratio of
hydroxyl, catechol, and ammonium functional groups. Adhe-
sive was aqueous solution of copolymer containing tyrosynase
and buffers (pH ¼ 8). Lap shear adhesive strength was meas-
ured under air atmosphere at 25 ꢁC.
We prepared several types of copolymers having various
comonomer ratio of x/y/z corresponding to the unit ratio of
H/C/A, as listed in Table 1. The copolymer samples were
named after the comonomer ratio, such as P(10H-1C-1A),
which is the copolymer consisting of H, C, and A monomer
units at the molar ratio of 10/1/1.
orange and viscous to lose fluidity. It took 2 h to form a
hydrogel eventually [Fig. 2(a)]. Conversely, when tyrosinase
was added to the P(10H-1C-1A), the viscosity quickly
increased. A gel was formed within 20 min [Fig. 2(b)]. This
result clearly indicates that the tyrosinase accelerated oxida-
tion rate to induce rapid gel formation.
Main component of mussel adhesion protein (fp-1) is com-
posed from decapeptide repeating units and contain one
Dopa and two lysine residues in the repeating unit.1 We set
the catechol group composite almost same with fp-1, and
same ratio of amino group was adopted. In this report, gela-
tion and adhesion properties of P(10H-1C-1A) were mainly
discussed.
However, gelation did not occur under oxygen-free condition
even if the tyrosinase was contained. Figure 2(c) represents
gelation test under argon atmosphere. After oxygen gas in
the buffer solution was removed by argon bubbling, MAP
mimetic P(10H-1C-1A) was dissolved in the deoxygenized
buffer solution under argon atmosphere. In this case, the
polymer solution kept fluidity even after 24 h, and no gela-
tion occurred. These results clearly indicate that gelation
was triggered by aerobic oxidation of catechol units. Synthe-
sized mimetic polymer showed aerobic gelation properties
like a native adhesive protein.
Gelation of MAP Mimetic Polymer Solution by
Aerobic Oxidation
To achieve adhesion, fluid adhesives have to be solidified for
formation of adhesive layer and anchoring adhesives to the
surfaces.21 Therefore, gelation of polymer solution is essen-
tial in this system. Gelation of polymer solution was investi-
gated first.
Viscoelasticity of the copolymer solution during the gelation
process was measured by rotational rheometer. MAP mimetic
P(10H-1C-1A) dissolved in phosphate buffer solution (pH ¼
8.0) was placed between the stage and the parallel plate
under air, then time course of G0 and G00 was measured
(Fig. 3). The G0 and G00 of the solution increased with time
until the 1000 min, indicating that the cross-linking reaction
A solution of P(10H-1C-1A) formed a gel under ambient air
conditions. This polymer had catechol moieties, cross-linking
reaction triggered by aerobic oxidation of catechol groups in
the polymer. The synthesized P(10H-1C-1A) was dissolved in
basic phosphate buffer solution. The pH of the solution was
set at 8.0. It is known that the oxidation reaction of catechol
is accelerated under basic conditions,22 such as sea water of
which pH is almost 8. The solution gradually became deep
of P(10H-1C-1A) proceeded to form
oxidation.
a gel by aerobic
TABLE 2 Lap Shear Adhesive Strength of MAP Mimetic
Adhesion of Aluminum Plates by Aqueous Gel Formation
Triggered by Aerobic Oxidation
Polymers and Reference Polymers
A portion of the aqueous solution of P(10H-1C-1A) contain-
ing tyrosinase and buffers was put on an aluminum plate
and sandwiched with two aluminum plates. The sample was
fixed by clamps and stored under humid atmosphere at
room temperature for 24 h. Two substrates were adhered
through gelation of the polymer solution. Lap shear adhesion
strength is summarized in Table 2. The aluminum plates
adhered by synthesized MAP mimetic P(10H-1C-1A) broke at
Polymers
Lap Shear Adhesive Strength
P(10H-1C-1A)
Synthesized MAPa
0.46 6 0.11 MPa
0.25 MPa
P(10H)
No adhesion
0.024 6 0.01 MPa
P(10H-1A) þ adipic acid þ EDC
a
Ref. 14.
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TABLE 3 Lap Shear Adhesive Strength of Various Substrates
P(11H-1C). We supposed that amino groups promoted the
oxidation of catechol and cross-linking reaction improving
the mechanical strength of the resulting hydrogel.
Using P(10H-1C-1A)
Lap Shear Adhesive
Adhesion Strength of Various Substrates by MAP
Substrates
Strength (MPa)
Mimetic Polymer
Stainless steel
Glass
0.45 6 0.02
0.34 6 0.08
It is known that catechol units strongly interact with metal
oxides surface through stable bidentate hydrogen bonding,
whereas interactions of catechol with polymers are weak.23
We adapted stainless steel, glass, and PTFE plates for
adhesion test using test pieces. MAP mimetic P(10H-1C-1A)
solution containing tyrosinase and buffers was put between
two substrates, clumped, and stored under humid air for 24
h. As shown in Table 3, lap shear adhesion strength for
stainless steel and glass was 0.45 and 0.34 MPa, respectively.
Adhesion layer was broken with cohesive failure manner,
which is the same behavior observed with aluminum plate
adhesion. These results indicate that the interaction between
the polymer gel and these material surfaces is much stronger
than the shear strength of gel. Conversely, the lap shear
strength of PTFE adhesion was 0.011 MPa. When the sub-
strate was separated by lap shear test, the gel layer peeled
off smoothly from one substrate. The failure took place at
the interface between gel and PTFE surface.
PTFE
0.011 6 0.001
a lap shear strength of 0.46 MPa. This value was similar to
the adhesion strength of synthesized MAP proteins reported
previously by Yamamoto.15 Broken hydrogel remained on the
failed face of each aluminum plate, indicating that cohesive
failure took place at the polymer gel. The polymer interacted
so strongly with the aluminum oxide surface that it broke
inside the hydrogel layer.
As a reference experiment, P(10H) and P(10H-1A) bearing
no catechol unit were used for adhesives instead of MAP
mimetic polymer. A solution of the homopolymer P(10H)
was sandwiched between two aluminum plates and stored
under humid air for 24 h. Even after 24 h, the P(10H)
remained as a viscous solution between the aluminum plates
with no adhesion. A mixture of P(10H-1A), adipic acid, and
EDC formed adhesive gel layer of the aluminum plates;
however, the lap shear adhesion strength was lower level as
0.024 MPa. Therefore, adhesion by MAP mimetic polymer
requires two factors; gel formation and strong interaction of
polymers with substrate surface owing to catechol groups.
Mechanical strength of MAP mimetic polymer gel formed
between the substrates would be independent on the substrate
materials. Therefore, low adhesion strength on PTFE substrate
must be caused by the weak interaction of catechol groups in
the gel and PTFE surface. Conversely, catechol units strongly
interacted with metal oxide. The lap shear adhesion strength
was dominated by mechanical strength of the hydrogel because
the gel ruptured before the interface interaction was broken.
Consequently, the improvement of physical strength of the
hydrogel would be expected to give large lap shear strength.
MAP contains lysine unit as well as the Dopa. The amino
group in lysine work as pH-controller keeping the adhesion
environment to be basic condition to promote the oxidation
of Dopa. However, the further detail role is still unclear. In
this study, adhesion strength of the copolymer with or
without amino group was investigated. As shown in Figure 4,
copolymer solution containing amino group showed higher
lap shear adhesive strength. The pH of the polymer solution
was adjusted to be 8, ammonium salt in polymer was con-
verted to amino group. P(5H-1C-1A) and P(10H-1C-1A)
revealed slightly higher adhesion strength compared with
copolymer without amino group, such as P(6H-1C) and
Effect of Water on Adhesion by Aerobic Oxidation
The functions and structure of this polymer were learned from
the natural system of MAPs. Mussels adhere to surfaces even
in the aqueous environment using these adhesive proteins
containing catechol groups. MAP mimetic polymer inspired by
MAPs is also expected to work as an adhesive in aqueous
condition. Thus, the effect of water on adhesion of the MAP
mimetic polymer gel was examined by following two
TABLE 4 Lap Shear Adhesive Strength of P(10H-1C-1A)a
Lap Shear Adhesive
Water Content
(%)
Oxidation
Adhesion Processb
Strength (MPa)
Aerobic oxidation
Aerobic oxidation
Aerobic oxidation
In humid air for 24 h
In water for 24 hc
0.46 6 0.11
0.036 6 0.16
0.22 6 0.01
10.5
83.1
13.8
In humid air for 24 h and
in water for 24 hd
H2O2 þ Fe3þ
H2O2 þ Fe3þ
In humid air for 24 h
In water for 24 hc
0.48 6 0.14
0.15 6 0.05
14.0
16.8
a
Under air at 25 ꢁC.
b
At 25 ꢁC.
c
Adhesion Procedure 1.
d
Adhesion Procedure 2.
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FIGURE 5 (a) Gradient gel formed by aerobic oxidation of aqueous solution of P(10H-1C-1A) in the presence of tyrosynase and
buffers (pH ¼ 8) under air for 24 h at 25 ꢁC, and (b) swollen state of the resulting gradient gel in citric acid solution at 25 ꢁC.
procedures. First one is adhesion in water. (Procedure 1) Im-
mediately after the 10 wt % P(10H-1C-1A) solution containing
tyrosinase was placed between the two aluminum plates, the
plates were immersed in water. Twenty-four hours later, lap
shear strength of the resulting plates was measured by tensile
tester under air without any drying. The other method is
adhesion in humid air and successive immersion in water.
(Procedure 2) A solution of P(10H-1C-1A) with tyrosinase was
placed between two aluminum plates and stored under humid
atmosphere for 24 h. The adhered aluminum plates were
immersed in water for 24 h, and then the lap shear strength
was measured under air without any drying. In the case of
Procedure 1, the aluminum plates were adhered but signifi-
FIGURE 6 Homogeneous gel prepared from aqueous solution
cantly low adhesion strength to be 0.036 MPa. This value was
MAP mimetic P(10H-1C-1A) containing tyrosynase and buffers
almost one-tenth the adhesion strength compared with gela-
(pH ¼ 8) by oxidation with 1 mol % of H2O2 and Fe3þ for 5 min
tion under humid air. In the case of Procedure 2, the lap shear
at 25 ꢁC.
adhesion strength was 0.22 MPa, maintaining half the value
compared with that before water immersion. In each case,
immersed in water for 24 h (Sample c, Procedure 2), the
polymer gel remained at the face of the aluminum plates after
water content was 13.8%. The water content was increased
the lap shear test. This means cohesive failure, and rupture
by comparing with Sample a, but still lower than Sample c.
was caused by the collapse of the hydrogel. The difference in
adhesion strength between the two procedures was attributed
to the mechanical strength of the gel. The hydrogel prepared
in water was more fragile than the gel prepared in humid air.
Relationship between lap shear adhesion strength and water
contents in the gel is shown in Table 4. When the substrates
were adhered in water (Sample b), the MAP mimetic
polymer formed a gel with 83% content of water and low
mechanical strength leading to low adhesion strength of
0.036 MPa. When the substrates were adhered under humid
air for 24 h (Sample a), the hydrogel having relatively high
strength was formed to show 0.46 MPa. Dipping the adhered
substrates into water increased the water content in the gel
(Sample c) to reduce the adhesion strength to 0.22 MPa.
Adhesion strength closely related with water content in the
MAP mimetic polymer gel. However, we should consider why
water content of the gel formed in water (Sample b, Proce-
dure 1) was much higher than that obtained under humid
air (Sample c, Procedure 2).
Strength of the gel seems to be related with degree of swel-
ling (water contents) of the gel. Water contents of adhesive
gel layers formed in humid air (Sample a), formed in water
(Sample b, Procedure 1), formed in humid air then immersed
in water (Sample c, Procedure 2) were compared. Water
content of the hydrogels was valuated after the adhesion
test. The P(10H-1C-1A) gel layer was peeled away from the
aluminum surfacꢁes. This sample wasꢁheated from room tem-
perature to 600 C at the rate of 10 C/min, and weight loss
was recorded by thermogravimetric analyzer (TGA). Water
mass was determined from the weight loss during heating
from room temperature to 150 ꢁC, and polymer mass was
determined during heating from 150 to 600 ꢁC. The water
content of the gel formed under humid air (Sample a) was
10.5%, which is much lower value compared with initial
solution of MAP mimetic polymer containing 90% of water.
The water evaporated from the polymer solution during
gelation process even under humid air conditions. Con-
versely, when adhesion was conducted in water by Proce-
dure 1, water content in the gel (Sample b) was 83.1%. In
addition, when adhesion was conducted in humid air then
We carefully observed the gel prepared by aerobic oxidation
again. The color distribution in the gel formed by P(10H-1C-
1A) was not uniform, which can be seen in Figures 1(a),
1(b), and 5(a). In general, the brown color appears by the
oxidation and cross-linking reaction of catechol groups.24
The characteristic deep brown color appeared near air–water
interfaces, but inside of the hydrogel apart from the air
interface were still pale yellow [Fig. 5(a)]. This gradient
distribution of brown color suggested that cross-linking
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densities were high near the air interface and low at inside
of the hydrogel. The gradient of cross-linking density was
also confirmed by the swelling test. When the gel was pulled
out from the bottle and placed in the water, the faint color
region swelled more than the deeper color region, as shown
in Figure 5(b). These results suggest that the oxidation of
catechol groups near the air interface region efficiently took
place to give densely cross-linked gel. Conversely, hydrogel
with low cross-linking density was formed inside the solu-
tion. This heterogeneous gel structure might be formed in
the gap between the aluminum substrates to result in the
low adhesive strength of MAP mimetic polymer. To improve
the gel formation, we use reagent oxidization instead of
aerobic oxidation.
reagents. Aluminum, stainless steel, and glass plates were
adhered through the gelation of the polymer solution. Cohe-
sive failure took place at the gel layer because of higher adhe-
sive interaction between gel and substrate surface than the
mechanical strength of the gel. The copolymer with higher
content of catechol and amino groups showed higher adhesion
strength. These results indicate that adhesion strength was
mainly dominated by the mechanical strength of the gel. Ad-
hesion in water was also achieved by addition of the oxidizing
agent Fe(NO3)3 in the copolymer solution. These results
clearly show that the synthesized polymer acts like the
adhesive proteins. By mimicking nature’s system, unique gel
formation and adhesion properties were successfully intro-
duced in an artificial acrylamide polymer. We expect that this
polymer system will become a useful platform for construct-
ing functional materials that work in water.
Adhesion of Aluminum Plates by Gelation of MAP Mimetic
Polymer Solution Triggered by Reagent Oxidation
For oxidizing the catechol groups in the polymer, a combina-
tion of hydrogen peroxide (oxidant) and Fe3þ (catalyst) was
selected. The mixture of 5 wt % aqueous solution of MAP
mimetic polymer, equimolar hydrogen peroxide, and 1 mol
% Fe(NO3)3 became viscose rapidly to form hydrogel within
few minutes, as shown in Figure 6. As we expected, the
brown color of the gel was uniform. Rapid gel formation
without oxygen was achieved.
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A portion of 10 wt % aqueous solution of MAP mimetic
P(10H-1C-1A) containing hydrogen peroxide and Fe(NO3)3
was sandwiched between two aluminum plates and then
stored under humid atmosphere at room temperature for 24
h. Lap shear strength of the adhered sample was 0.48 MPa,
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adhesion in water was improved as follows. Aqueous P(10H-
1C-1A) solution containing hydrogen peroxide and Fe(NO3)3
as oxidant was placed between two aluminum plates, then
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