Hydrophilic diazirine polymer for one-step photo-fabrication of proteins on polypropylene surface

Article abstract of DOI:10.3987/COM-08-S(D)60

A facile photochemical fabrication of chemical and biological functionalities on polypropylene surface has been performed by using photoactivatable polymers containing diazirine photophore. The water-soluble polymers have been synthesized from polyamine or polycarboxylic acid by coupling with aryldiazirine derivatives. Upon irradiation, the photoactivatable polymer successfully immobilized proteins such as horseradish peroxidase, lysozyme, and pepsin on the inert surface of polypropylene film in one step. This soft polymer cushion supplied a hydrophilic environment around the enzyme and enhanced the enzyme activity. Specifically, the polymer (HDA-PEI) introduced a hydrophilic spacer between diazirine and polymer main chain significantly increased the activity.

Full text of DOI:10.3987/COM-08-S(D)60

HETEROCYCLES, Vol. 79, 2009
897
HETEROCYCLES, Vol. 79, 2009, pp. 897 - 908. © The Japan Institute of Heterocyclic Chemistry
Received, 27th September, 2008, Accepted, 19th November, 2008, Published online, 27th November, 2008.
DOI: 10.3987/COM-08-S(D)60
HYDROPHILIC DIAZIRINE POLYMER FOR ONE-STEP
PHOTO-FABRICATION OF PROTEINS ON POLYPROPYLENE
SURFACE
Takenori Tomohiro, Norie Tachi, Yusuke Azuma, and Yasumaru
Hatanaka*
Graduate School of Medicine and Pharmaceutical Sciences, University of
Toyama, Sugitani 2630, Toyama 930-0194, Japan
E-mail: yasu@pha.u-toyama.ac.jp
Abstract – A facile photochemical fabrication of chemical and biological
functionalities on polypropylene surface has been performed by using
photoactivatable polymers containing diazirine photophore. The water-soluble
polymers have been synthesized from polyamine or polycarboxylic acid by
coupling with aryldiazirine derivatives. Upon irradiation, the photoactivatable
polymer successfully immobilized proteins such as horseradish peroxidase,
lysozyme, and pepsin on the inert surface of polypropylene film in one step. This
soft polymer cushion supplied a hydrophilic environment around the enzyme and
enhanced the enzyme activity. Specifically, the polymer (HDA-PEI) introduced a
hydrophilic spacer between diazirine and polymer main chain significantly
increased the activity.
INTRODUCTION
The creation of chemical and biological functionalities on polymer surface is a subject of great interest
because most polymers have intrinsically low surface tension. It requires to altering chemical natures of
the surface such as wettability, biocompatibility, and chemical reactivity corresponding to the purpose of
interest. A number of techniques have been developed for patterning of organic substances on polymer
surface such as plasma treatment, graft polymerization,1 photolithography,2 and microcontact printing.3
These methods can directly deposit biomolecules such as proteins onto the surface covalently or
noncovalently, but usually need prior chemical activation of the inert polymer surface. In addition, most
patterning techniques require the masking and the desorption processes of non-substance-addressed area

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before/after fabrication of substances. Once the polymer surface is activated, however, it nonspecifically
adsorbs substantive amount of biomolecules that resulting in decreasing detection sensitivity due to the
background noise. Because the surface cannot be cleaned in strong washing condition, the nonspecific
adsorption is especially a serious disadvantage for the high-sensitive detection methods using noncovalent
attachments of proteins on the surface. It should be better to retain the low surface tension outside of area
for fabrication, which should be resistant to nonspecific protein adsorption. Covalent
photoimmobilization, another fabrication technique, of target molecules on the polymer surface is
generally achieved by following two different methods, 1) the target molecules are photocross-linked
after chemical immobilization by photoactivatable compounds on the activated surface, and 2) direct
irradiation on the inert surface after deposition of the target bearing a photoactivatable moiety.
Photochemical method using photoactivatable compounds has an advantage since these functional groups
can be intrinsically activated by UV irradiation. Therefore, fabrication on the surface is limited to the
irradiated area. Photophores such as benzophenone, arylazide,4 and aryldiazirine5,6 that produce high
reactive species, radical, nitrene, or carbene, respectively, and which were often used for cross-linking of
substances to the polymer surface.
For the analysis of biomolecular interactions, it is also required for controlling the distribution of
substances and the spatial environment (hydrophobicity, hydrophilicity, ionic strength, and pH) around
the biomolecules to represent precisely these interactions in vivo. Hydrogels such as polyacrylamide,7
polysaccharide, gelatin8 covalently bounded to the surface gave 3D-biocompatible interface on the
polymer substrates. By arraying of polyacrylamide gel on the glass surface of microchip, sequencing by
hybridization to synthesized oligonucleotides under a liquid phase environment has become possible.9
However, a prior surface treatment process such attachment of functional groups on the glass surface is
unavoidable and the gel on the surface is sometimes easily damaged by mechanical shock. Here we report
a simple photolithographic approach for the creation of chemical and biochemical functionalities on the
polypropylene substrate by employing photoactivatable hydrophilic polymers. Compared to
benzophenone and arylazide, the photolysis of aryldiazirine group used in this report rapidly proceeds by
UVA irradiation and makes a stable bound between spatially proximate molecules. These polymers have
multi-diazirine units in a single molecule that makes covalent bonds to the target molecules as well as the
polymer surface at the same time by irradiation.
RESULTS AND DISCUSSION
Immobilization using photoactivatable polymer
The synthesis of photoactivatable polymer (DA-polymers) has been performed by a coupling reaction of
polymer and diazirine derivatives. The polymer substrates used in this report basically contain amino or

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899
carboxyl groups as functional groups: polyethyleneimine
(PEI),
poly(allylamine
hydrochloride)
(PAH),
poly(L-Lysine hydrobromide) (PLH), and polyacrylic acid
(PAA). Diazirine compounds 1 and 2 were used for the
reaction of polyamines, and compound 3 was used for the
reaction of polycarboxylic acid, respectively (Figure 1).
Compound 1 was prepared according to the previous
report.10 Epoxide derivative 2 was derived from compound 1 using trimethylsulfonium iodide,11 and
sulfonamide derivative 3 was prepared by reaction of 3-phenyl-3-trifluoromethyl-3H-diazirine and
chlorosulfonic acid followed by the treatment with ammonia gas. Incorporation of diazirine photophore
into polymer was performed by use of equivalent molar of diazirine derivative against the corresponding
reactive groups in polymer such as primary amino group of PEI and carboxyl group of PAA. After
purification by gel filtration, the yields of diazirine-incorporation on polyamine were generally 6-39%
against the number of reactive groups in polymer, which calculated by measuring of the absorptions at
360 nm and the elemental analysis. The highest yield of incorporation was given as 75% of a
photoactivatable polyethyleneimine (DA1-PEI) by the reaction of aldehyde derivative 1 and PEI. Another
derivative of polyethyleneimine (DA2-PEI) was obtained by the reaction of epoxide derivative 2 and PEI
in 39% yield of diazirine modification. Similar method using compound 2 was applied for PAH and PLH
to give corresponding photoactivatable polymers in 6, 20%, respectively. In addition, the polymer
containing a hydrophilic spacer (HDA-PEI) was also synthesized by a reaction of PEI and compound 7
that was derived from 3-(3-hydroxyphenyl)-3-trifluoromethyl-3H-diazirine (4) as described in Scheme 1.
Scheme 1. Synthesis of diazirine derivative bearing a hydrophilic linker (7)

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For further experiments were used DA1-PEI and DA2-PEI from the aspect of the cost of polymer and the
incorporation yield of diazirine. We also prepared other photoactivatable polyamine derivatives by using
3-(4-bromomethylphenyl)-3-trifluoromethyl-3H-diazirine, the incorporation of diazirine was relatively
low for three kinds of polyamines (3-14%). Other type of polymer, photoactivatable polycarboxylic acid
(DA-PAA), was synthesized by a coupling reaction of compound 3 and PAA, containing
N-acylsulfonamide groups. The pKa value of general N-acylsulfonamide is close to that of general
carboxylic acid, which results in the formation of an anion under the physiological conditions. Whereas,
another photoactivatable polycarboxylic acid synthesized by using compound 6 easily aggregated in an
aqueous solution because of increasing the hydrophobicity due to diazirine moiety although it has a
hydrophilic spacer resulting the low yield of diazirine incorporation (7%).
Upon irradiation of 360 nm light (15 W x 2), diazirine group produces a carbene, a high-reactive
electrophilic species, and immediately reacts with spatially proximate chemical bond. Therefore, diazirine
derivatives has been recognized to satisfy most of the chemical criteria required for photoaffinity
labeling12 and polymer surface fabrication.5,13 The photolysis was carried out after DA-PEI or original
PEI were spotted on the film following the dryness in vacuo. There was nothing left on the surface after
washing in both cases without irradiation and with irradiation of original PEI. The yield of
immobilization of DA-PEI was determined by
measuring the amount of primary amino group after
the treatment of ninhydrin reagents. The yield was
depended on the concentration of deposited polymer
as well as the irradiation time as shown in Figure 2,
and the photolysis was completed within 40 min. By
the photolysis using 1% w/v of polymer solution, the
quantity was reached to ca. 9 x 104 / nm2 of amino
groups immobilized on the surface. Although the
cross-linking yield of diazirine is usually low since
carbene species can react with water, the value is
about 104-fold larger than that of commercial
amine-modified chips.
Protein fabrication and the activity
This methodology was then applied for immobilization of proteins including horseradish peroxidase
(HRP),14 lysozyme, and pepsin by using DA1-PEI for the former two proteins and DA-PAA for the latter
one, respectively. These polymers were selected according to the stable pH region of proteins. After

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901
incubation of DA1-PEI and HRP following
dryness of the film, immobilization of proteins
was performed by UV irradiation on ice. Figure 3
shows the quantity of HRP immobilized on the
film, which is estimated by the amount of the
heme moiety extracted in organic solution
according to the previous method.15 While there
are small amount of products detected by the
treatment from a non-irradiated spot, the amount
of immobilized HRP was obviously increased
with the irradiation time and then became
constant after 60 minutes. It also depended on the
amount of DA1-PEI and was maximized in the
solution at the molar ratio of protein/polymer as
1:4 (ca. 70% of the deposited amount, Figure 3).
The following photolysis in this report has been
performed in a solution containing 4 equivalent
moles of the photoactivatable polymer by
60-minute
irradiation.
The
activity
of
immobilized HRP on a polypropylene film was
determined by the method of Trinder.16 The
optimum activity of immobilized HRP was
obtained at pH 9 while native HRP showed it at
pH 7 (data not shown). The immobilized HRP
remained its activity in 40% of initial value after
keeping in a buffer (0.1 M Tris-HCl, pH 9) at
4°C for a week. In addition, the stability of HRP
against organic solvents was tested in 20% (v/v)
of aqueous organic solvents including MeCN, acetone, and ethanol (Figure 4). The immobilized HRP
exhibited rather strong resistance against such organic solvents than non-immobilized HRP, and kept
more than 80% of activity in aqueous solution. This is a similar phenomenon to that of a previous
report.17 However, the activity was decreased by the enzymatic reaction during repeated measurements.
The immobilized HRP should be damaged by hydrogen peroxide used in the reaction. Furthermore, the
relative enzyme activity of immobilized HRP against native HRP (non-immobilized HRP) was

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determined from the pseudo-first-order reaction
rate constants in these initial reactions. It was
calculated as ca. 0.1% compared to that of native
HRP. It is because the reaction surface is quite
small on the polymer while native HRP was
homogeneously dissolved in a solution. In
addition, 4-aminoantipyrine, a substrate for the
enzyme reaction, may slowly diffuse into the
polymer. In case using DA2-PEI instead of
DA1-PEI, the immobilized HRP showed higher
relative activity as ca. 2%. It might be due to the
increasing of hydrophilic space produced by
hydroxyl group in DA2-PEI polymer. To
increase the swelling of the polymer on the film
and to gain the space for the interaction, the
hydrophilic spacer was added between the
photophore and polymer main chain. The yield of
HDA-PEI immobilization on a polypropylene
film was smaller than that of DA1-PEI (Figure 5),
but the relative activity of HRP immobilized with
HDA-PEI increased 6-fold in comparison with
that of DA-PEI (black columns in Figure 6). We
also evaluated the activity by luminol emission
catalyzed by HRP. This method requests a small
quantity of reagents because of the high sensitive
detection as well as the simple treatment. It gave
similar level of the enzyme activity to that of
native HRP as 75 and 98% for immobilized HRP
using DA-PEI and HDA-PEI, respectively. However, the enzyme activity of HRP for oxidation is
basically depended on the activity of the heme moiety. It may not be an appropriate example for
evaluation of the protein fabrication. Therefore, we then applied this method for immobilization of
lysozyme that catalyzes hydrolysis of beta (1-4) glycosidic bond of N-acetylmuramic acid and
N-acetylglucosamine. The activity of immobilized lysozyme was evaluated by the previous method using
Micrococcus lysodeikticus.18 The relative activities of immobilized lysozyme were 0.1 and 2.8% for

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903
DA-PEI and HDA-PEI, respectively (white columns in Figure 6). HDA-PEI was significantly enhanced
the enzymatic reaction of lysozyme at 28-fold higher reaction rate than DA-PEI as a similar result with
the case of HRP. It might be due to the larger hydrophilic space around lysozyme or increasing of the
stability. Moreover, pepsin was also treated in a similar method by using DA-PAA. While pepsin was not
efficiently immobilized on the film, the immobilized pepsin exhibited its activity.
CONCLUSION
The soft polymer cushion on the organic and inorganic substrate has been an important process for
fabrication of DNA chip,19 biological system on membrane.20 The present photolithographic method
using photoactivatable hydrophilic polymer illustrated a very simple fabrication of polymer cushion as a
basis matrix as well as one-step fabrication of biological functionalities on the surface of inert polymer. It
also gained higher S/N value for the analysis because the variety of functions was fabricated only within
deposited area on the surface. The specific activity of fabricated enzymes increased by the introduction of
a hydrophilic spacer between a photophore and polymer main chain. The flexibility and hydrophilicity of
spacers could provide more spaces within a polymer to enhance the diffusion rate of the substances. This
technique will contribute to control immobilization of proteins on polymer surface that is important
implications in many areas of science and technology.
EXPERIMENTAL
1
Melting points were measured on Yanaco MP-S3 micro melting point apparatus and are uncorrected. H
NMR spectra were recorded at 500 MHz on Varian UNITY plus 500 spectrometer. IR spectra were
recorded on JASCO FT/IR-460plus. UV-Vis absorption spectra were measured on JASCO V-550. MS
spectra were obtained by using a JEOL JMS-GCMATE. Chemiluminescence detection was performed by
ChemiDoc system (Bio-Rad Lab. Inc.). Polyethyleneimine (PEI, molecular weight: 10,000),
poly(L-Lysine hydrobromide) (PLH, molecular weight: 5,000 - 15,000), and polyacrylic acid (PAA,
molecular weight: 25,000), pepsin were purchased from Wako Pure Chemical Industries, Ltd.
Poly(allylamine hydrochloride) (PAH, molecular weight: 15,000) and horseradish peroxidase (HRP) were
purchased from Sigma-Aldrich Corp. Polypropylene (PP) film was obtained from Shin-kobe Electric
Machinery Co., Ltd. Streptavidin-HRP was obtained from GE Healthcare UK Ltd. All chemicals were of
analytical grade and were used without further purification.
3-(4-Oxiranylphenyl)-3-trifluoromethyl-3H-diazirine (2): Trimethylsulfonium iodide (0.95 g, 4.67
mmol), potassium hydroxide (0.52 g, 9.34 mmol) was stirred in MeCN (4 mL) at 60 °C under nitrogen for
10
min.
To
the
suspension
was
added
a
MeCN
solution
(5
mL)
of
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzaldehyde21 (1, 1.0 g, 4.67 mmol) and water (20 mL). The

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HETEROCYCLES, Vol. 79, 2009
reaction mixture was stirred for 1 h and poured into cold Et2O. The organic phase was washed with water,
brine, and dried over MgSO4. After removal of solvent, the residue was purified by chromatography
1
(hexane:CHCl3 = 1:2) on silica gel to afford compound 3 as a pale yellow oil (0.56 g, 52%). H NMR
(CDCl3):  7.31 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 7.9 Hz, 2H), 3.81 (dd, J = 2.6, 3.8 Hz, 1H), 3.17 (dd, J =
5.6, 3.8 Hz, 1H), 2.75 ppm (dd, J = 2.6, 5.6 Hz, 1H); IR (liquid film):  1343, 1233, 1156 cm-1; UV-vis
(hexane) max = 228 ( 15700), 268 ( 490), 342 (sh,  290), 357 ( 390), 371 nm ( 290); MS (m/z, %):
229 (M+, 10), 201 (100).
3-(4-Aminosulfonylphenyl)-3-trifluoromethyl-3H-diazirine (3): Chlorosulfonic acid (23.3 g, 214
mmol, caution:
a
hygroscopic and
a
powerful lachrymator) was slowly added into
3-phenyl-3-trifluoromethyl-3H-diazirine (8.0 g, 43.0 mmol) placed in a 100mL flask followed by stirring
at rt for 30 min using a CaCl2 drying tube. The mixture was carefully poured on ice and the product was
extracted with Et2O. Ammonia gas was passed through the ethereal solution until the solution become
basic, and nitrogen was then bubbled to remove excess ammonia. After neutralization with conc. HCl, the
reaction mixture was washed with water, brine, and dried over MgSO4. After removal of solvent, the
residue was purified by chromatography (hexane:EtOAc = 2:1) on silica gel to afford compound 1 as a
white solid. The product was recrystallized from EtOAc to give a white needle (8.6 g, 76%), mp
1
133-134.5 °C. H NMR (500 MHz, CDCl3):  7.97 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 4.84
ppm (s, 2H); IR (KBr):  3388, 3257, 3102, 1331 cm-1; UV-vis (CHCl3) max = 241 ( 10000), 272 (
850), 280 ( 760), 344 ( 355), 357 nm (sh,  280); MS (m/z, %): 266 (M+, 20), 237 (100).
3-[2-[2-(2-tert-Butoxycarbonylaminoethoxy)ethoxy]ethoxyphenyl]-3-trifluoromethyl-3H-diazirine
(5): To a solution of 3-(3-hydroxyphenyl)-3-trifluoromethyl-3H-diazirine 4 (1.12 g, 4.85 mmol), K2CO3
(0.56 g, 4.00 mmol) in DMF (5 mL) was added 2-[2-(tert-butoxycarbonylaminoethoxy)ethoxy]ethyl
bromide (1.38 g, 4.00 mmol). The mixture was stirred at 60 °C overnight under argon. The solvent was
removed in vacuo. The mixture was extracted with EtOAc three times, and the combined organic phase
was then washed with brine and dried over MgSO4. After removal of the solvent, the residue was purified
by chromatography (hexane:Et2O = 1:4) on silica gel to afford compound 1 as a yellow liquid (0.88 g,
1
48%). H NMR (CDCl3):  7.30 (t, J = 9.0 Hz, 1H), 6.96 (dd, J = 2.6, 9.0 Hz, 1H), 6.77 (d, J = 9.0 Hz,
2H), 6.72 (s, 1H), 4.99 (br s, 1H), 4.13 (t, J = 5.4 Hz, 2H), 3.84 (t, J = 5.4 Hz, 2H), 3.63-3.73 (m, 4H),
3.55 (t, J = 5.7 Hz, 2H), 3.28-3.36 (m, 2H), 1.43 ppm (s, 9H); IR (liquid film):  3359, 1712 cm-1;
UV-vis (CHCl3) max = 240 ( 1490), 279 ( 1910), 355 ( 320), 369 (sh,  270);
MS (m/z, %): 434
(M+, 50), 334 (100).
3-[2-[2-(2-Bromoacethylaminoethoxy)ethoxy]ethoxyphenyl]-3-trifluoromethyl-3-H-diazirine
(7):
The compound 5 (0.43 g, 1.0 mmol) was treated with 50% TFA-CH2Cl2 (1 mL) at rt for 1 h. After
evaporated the solvent, the residue was dissolved in CH2Cl2 (1 mL) and then adjusted pH to ca. 9 by

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905
addition of Et3N. -Bromoacetyl bromide (200 L, 2.3 mmol) was added to the mixture followed by
stirring at rt for 2 h. The mixture was washed with water and dried over MgSO4. After removal of the
solvent, the residue was purified by chromatography (CH2Cl2:EtOAc = 2:1) on silica gel to afford
compound 1 as a yellow liquid (74 mg, 16%). 1H NMR (CDCl3):  7.30 (t, J = 9.0 Hz, 1H), 6.97 (dd, J =
2.9, 9.0 Hz, 1H), 6.91 (br s, 1H), 6.78 (d, J = 9.0 Hz, 1H), 6.72 (s, 1H), 4.14 (t, J = 4.8 Hz, 2H), 3.87 (t, J
= 4.8 Hz, 2H), 3.86 (s, 2H), 3.71-3.74 (m, 2H), 3.66-3.69 (m, 2H), 3.60 (t, J = 5.2 Hz, 2H), 3.50 ppm (q, J
= 5.4 Hz, 2H); IR (liquid film):  3301, 1666 cm-1; UV-vis (CHCl3) max = 240 ( 1720), 279 ( 1830),
355 ( 360), 369 (sh,  300); MS (m/z, %): 453 (M+, 8), 455 (M+, 8), 346 (50), 157 (100).
Synthesis of photoactivatable polyethyleneimine using compound 1 (DA1-PEI): To a solution of PEI
(6.0
g)
in
acetic
acid
(125
mL)
was
added
a
solution
of
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzaldehyde 1 (6.2 g, 30.0 mmol) in MeOH (50 mL), and the
reaction mixture was stirred at 50 °C for 24 h. Excess amount of sodium cyanoborohydride and MeOH
(175 mL) was added and the mixture was stirred at 50 °C for 24 h under argon. After removal of the
solvent, the residue was dissolved in water (300 mL) followed by washing with CHCl3. The aqueous
solution was reduced to about 80 mL and was then dialyzed (cellulose ester membrane, 1,000 molecular
weight cut off). The solvent was removed in vacuo to give a pale yellow solid (6.7 g, 75% yield of
diazirine modification).
Synthesis of photoactivatable polymers using compound 2 (DA2-PEI): A solution of
3-(4-oxiranylphenyl)-3-trifluoromethyl-3H-diazirine 2 (30 mg), PEI (17 mg) in CHCl3 (2 mL) was stirred
at 50 °C for 24 h under dark. The reaction was monitored by thin-layer chromatography. The solvent was
removed under reduced pressure, and the residue was dissolved in water (1 mL). The solution was
subjected to gel filtration on Sephadex G15 (Column: inner diameter 0.75 cm, height 36 cm, volume 63.6
cm3) eluted with water. Each fraction was measured the absorption at 360 nm and 254 nm. The polyamine
fractions were recovered and freeze-dried to yield 15.8 mg of yellow viscous liquid (DA2-PEI). Yield of
diazirine modification on polymer was calculated against number of primary amines in polymer was 39%
calculated by the absorption at 360 nm corresponding to 3-phenyl-3-trifluoromethyl-3H-diazirine moiety.
Similar method was applied for PAH and PLH to give corresponding photoactivatable polymers in 6,
20%, respectively.
Synthesis of photoactivatable polymers using compound 4 (DA-PAA): A solution of PAA (18.8 mg,
0.26 mmol) and N-methylimidazol (22 mg, 0.26 mmol) in DMF solution (4 mL) was added
3-(4-sulfonamidyl)-3-trifluoromethyl-3H-diazirine 3 (35 mg, 0.13 mmol) and diisopropylcarbodiimide
(66 mg, 0.52 mmol) and was stirred at 50 °C for 24 h under dark. The decrease of the compound 3 was
monitored by thin-layer chromatography. The solvent was removed under reduced pressure, and the
residue was dissolved in water that was washed with CHCl3. After reducing to 1 mL, it was subjected to

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gel filtration on Sephadex G15 eluted with water. Each fraction was measured the absorption at 360 nm
and 254 nm. The polyamine fractions were recovered and freeze-dried to give 15.8 mg of pale yellow
viscous liquid. The yield of diazirine modification on polymer was 39%.
Synthesis of photoactivatable polymers using compound 7 (HDA-PEI): The compound 7 (22.7 mg)
and diisopropylethylamine (6.5 mg) were added to a DMF solution (2 mL) of PEI (6.5 mg), and the
mixture was stirred at rt overnight. The solvent was removed under reduced pressure, and the residue was
dissolved in water (1 mL). The solution was subjected to gel filtration on Sephadex G15 eluted with water.
Each fraction was measured the absorption at 360 nm and 254 nm. The polymer fractions were recovered
and freeze-dried to give pale yellow viscous liquid (20.5 mg, 20% yield of diazirine modification).
Immobilization of polymer on a polypropylene surface: Aqueous solution of photoactivatable polymer
(1% w/v, 10 L per dot) was spotted onto a polypropylene film (thickness 0.2 cm) and dried for 20 min
under reduced pressure. After photolysis by UV irradiation at a distance of 4 cm from blacklight lamp
(360 nm, 15 W × 2) for 30 min, the film was thoroughly washed with ion-exchanged water, 1 M
hydrochloric acid, and was rinsed with pure water. The immobilization of polyamines was estimated by
measuring of the amount of primary amine on the film by a ninhydrin reaction. In case of polycarboxylic
acid, the film was treated with N-hydroxysuccimide (0.5 M) and DCC (0.5 M) in DMF for 4 h at rt, and
was then washed with DMF. The film was incubated in a DMF solution of diethylene glycol
bis(3-aminopropyl) Et2O overnight at rt followed by washing with DMF. The amount of amino groups
was estimated by ninhydrin reaction.
Immobilization of HRP and lysozyme on a polypropylene film using DA-PEI: A mixed solution of an
aqueous solution of DA1-PEI (1% w/v, 5 L) and an aqueous solution of HRP (1% w/v, 5 L) was
spotted onto a polypropylene film and dried. After irradiation with UV for 30 min, the film was
ultrasonicated for 1 min in a Tris-HCl buffer (0.1 M, pH 9) in a plastic bag. The same procedure has been
performed for Lysozyme.
Enzyme assay of HRP: Firstly, chemiluminescence of luminol catalyzed by HRP on the film was
detected. The amount of HRP immobilized on the film was determined by measuring the amount of heme
moiety on the film.14 The film was thoroughly shaken in a cold 0.1 M HCl - 2-butanone (1: 1 v/v) for 30
min, the heme moiety was extracted twice in organic layer, and the amount was calculated from the
absorption at 384 nm. The activity of HRP on the film was evaluated by the modified method of the
previous report.16 The film was floated and stirred in a reaction solution (3.0 mL) containing
4-aminoantipyrine with phenol (0.625 mM) and hydrogen peroxide (0.85 mM) in a 0.1 M Tris-HCl buffer
(pH 9) at 25 °C followed by monitoring the absorption at 510 nm.
Enzyme assay of lysozyme: The activity of immobilized lysozyme was evaluated by the previous
method using Micrococcus lysodeikticus.18 The film was floated and stirred in a reaction solution (3.0

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907
mL) containing Micrococcus lysodeikticus (0.87 mg) in a 0.1 M potassium phosphate buffer (pH 7) at
25 °C followed by monitoring the absorption at 410 nm.
Immobilization and enzyme assay of pepsin on a polypropylene film using DA-PAA: A mixed
solution of an aqueous solution of DA-PAA (1% w/v, 40 L) and a 0.01 M HCl solution of pepsin (1%
w/v, 5 L) was spotted onto a polypropylene film and dried. After irradiation with UV for 60 min, the
film was ultrasonicated for 1 min in a sodium acetate buffer (0.1 M, pH 3) in a plastic bag. The activity of
pepsin on the film was evaluated by the enzyme activity using hemoglobin according to the previous
report.22 The film was floated and stirred in a reaction solution at 37 °C followed by monitoring the
absorption of peptide fragments dissolved in trichloroacetic acid at 280 nm after filtration.
ACKNOWLEDGEMENTS
This research was financially supported by the Ministry of Education, Culture, Sports, Science and
Technology through Special Coordination Funds for Promoting Science and Technology, and also
Grants-in-Aid for Scientific Research (18390036 and 20390032), CLUSTER (Cooperative Link of
Unique Science and Technology for Economy Revitalization), and the Fugaku Trust for Medicinal
Research.
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