A Novel Analytical Technique of Skin Photo-stress Biomarker
Using Surface Plasmon Resonance
Masaki Yamaguchi Senior Member, IEEE
Tomoki Shimakura, Akira Date, Makoto Sasaki, Non-member
Graduate School of Engineering, Iwate University
Abstract—It is possible that protein carbonylation in the
stratum corneum might be used as an index of skin photo-stress.
In order to evaluate the relationship between the protein
carbonyl / total protein ratio (carbonylation ratio) and skin
photo-stress, the authors established a methodology by which
protein carbonyl can be quantitatively analyzed using an
optical technique based on surface plasmon resonance (SPR).
Firstly,
the
protein
carbonyl
was
reacted
with
2,4-Dinitrophenylhydrazine (DNP), and the quantity of
DNP-protein carbonyl was determined using an anti-DNP
antibody. The mass of DNP-protein carbonyl was measured
using SPR. A significant difference was observed in the
carbonylation ratio between a sun-protected area (mid-ventral
arm) and a sun-exposed area (upper cheek, p < 0.05). Moreover,
the carbonylation ratio of the sun-exposed area showed a
higher value than that of the sun-protected area. It was
suggested that the carbonylation ratio might be a useful index
of skin photo-stress.
I. INTRODUCTION
W
hen skin is exposed to ultraviolet (UV) light
consisting of UVA (320 – 400 nm) and UVB (290 –
320 nm), reactive oxygen species (ROS) are formed from O2
in the cells. It is well known that ROS causes oxidative
cellular stress, cell injury and DNA damage to the skin. This
photo-stress eventually induces inflammation, skin
photo-aging or malignant tumors. ROS catalyzes oxidative
modification of the proteins. The most common product of
protein oxidation is the protein carbonyl group (protein
carbonyl) [1] – [4]. Thus, the protein carbonyl has a
possibility to be used as a marker for skin photo-stress [5].
One of the most useful methods used to analyze
biomarkers in human samples is immunoassay. However,
this method has some problems in that (i) it is necessary to
synthesize a labeled organic compound for each biomarker,
and (ii) it is often difficult to synthesis the labeling material
and the biomarker. To combat these problems, SPR has been
developed as a label-free immunoassay [6].
The purpose of our research is to clarify the relationship
between the protein carbonyl / total protein ratio
(carbonylation ratio) and skin photo-stress. To achieve this
goal, it is needed to establish a quantitatively analytical
method of protein carbonyl in the stratum corneum.
However, protein carbonyl includes many kinds of protein
(protein carbonyl family), and thus an antibody cannot be
specified. In this study, protein carbonyl was reacted with
2,4-Dinitrophenylhydrazine (DNP) because protein carbonyl
family react easily with DNP. Then, the DNP labeled protein
carbonyl was determined using an anti-DNP antibody. This
method requires the removal of the un-reacted DNP, for
which a gel filtration column was used.
In order to collect the stratum corneum, tape stripping
was employed. The carbonylation ratio per unit area in a
sun-protected area (mid-ventral arm) and a sun-exposed area
(upper cheek) were compared. Finally, the relationship
between the carbonylation ratio and the skin photo-stress
was examined.
II. METHODS
A. Subjects
Ten healthy young male Japanese adults (21 – 23 yr,
22.3 ± 0.7 yr) were enrolled for this study. The study
protocol, which was approved by the Ethical Committee of
Iwate University, was fully explained to all of the subjects,
both orally and in writing, focusing specifically on the
purpose of the study, the precise procedures that would be
used and any possible adverse effects. Signed informed
consent was obtained from each subject enrolled in the
study.
B. Analytical method of protein carbonyl
The principle of the analytical method of protein
carbonyl was shown in Fig.1. A biophysical interaction
analyzer with SPR (Biacore X100, GE Healthcare Japan Co.,
Japan) was used in this study.
In order to specifically detect protein carbonyl, a
stratum corneum sample was reacted with DNP (Activated
DNP, LSL Co. Ltd., Japan) as a pretreatment [7]. The DNP
labeled protein carbonyl (DNP-protein carbonyl) was
synthesized as follows:
O2 N
R
O + H2NNH
Protein C
O2 N
R
 Protein C
NO2
NNH
NO2 + H2O
(1)
In the first step, an anti-DNP antibody (Rabbit
Anti-DNP (IgG), LSL Co. Ltd., Japan) was immobilized
using an amine coupling method on the surface of a sensor
chip (Fig.1. (a)). Carboxymethyl dextran which had been
previously immobilized on the surface was activated by
applying 400 mM N-ethyl-N’- (3-dimethylaminopropyl)
: Carboxymethyl dextran,
: DNP-protein carbonyl
: Anti-DNP antibody,
I
70
Response Units (kRU)
carbodiimide hydrochroride (EDC) and 100 mM
N-hydroxysuccinimide (NHS, activation time: 4 min.). The
anti-DNP antibody, diluted to 50 μg/ml using an acetate
buffer (10 mM, pH5.5), was coupled to the carboxymethyl
dextran for 4 min. Following that, the un-coupled activated
carboxymethyl dextran was blocked by ethanolamine. A
profile of SPR response plotted against time (sensorgram)
was shown in Fig.2. The sensorgram provides real-time
information about the entire interaction, with binding
responses measured in resonance units (RU). The
immobilized quantity of the anti-DNP antibody was 14,148
RU.
In the second step, a sample solution including the
DNP-protein carbonyl was added to the sensor chip (Fig.1.
(b)). The change in mass which is proportional to the
specific binding between the antigen (DNP) and the antibody,
was measured with SPR. The concentration of DNP-protein
carbonyl was measured 120 s after the addition of the
sample.
Finally, the DNP-protein carbonyl was dissociated from
the anti-DNP antibody by adding sodium hydroxide solution
(Fig.1. (c)). The sensor chip can then be used again, up to
about 100 times, for this operation.
In order to measure the calibration curve of the DNP
labeled protein, standard DNP labeled protein (DNP-BSA,
LSL Co. Ltd., Japan, 1, 5, 10, 25, 50, 75, 100 and 250 ng/ml)
diluted by a running buffer (HBS-EP+) was used.
II
III
60
50
40
Immobilized anti-DNP
antibody (14,148 RU)
30
20
0
500
1500
1000
Time (s)
2000
Fig.2 Sensorgram showing the response of the
immobilization of the anti-DNP antibody to the sensor chip
surface.
I : Activation of the matrix with EDC/NHS,
II : Immobilization of the anti-DNP antibody,
III : Blocking with ethanolamine.
C. Analysis of total protein
In order to cancel the influence of the volume collected
from each site, total protein (TP) of the stratum corneum
sample was analyzed by Lowry method using a colorimetric
total protein assay kit (DC protein assay, Bio-Rad
Laboratories, Inc., CA) [8]. The Lowry method is little
influenced by the surfactant and has high sensitivity, so it is
suitable for analyzing samples with low concentration. Then,
protein carbonyl / total protein ratio (carbonylation ratio)
was calculated per unit area (cm–2).
(a) Immobilization of anti-DNP antibody
D. Sample collection and human evaluation
Prism
Light source
Detector
(b) Antigen-antibody reaction
(c) Dissociation of antigen
Fig.1 Principle of the analytical method of the protein
carbonyl using SPR.
To clarify the relationship between the carbonylation
ratio and skin photo-stress, stratum corneum samples were
collected from two sites on the body. The mid-ventral arm
(sun-protected area) and the upper cheek (sun-exposed area)
were the areas of skin from which stratum corneum samples
were collected (Fig.3.). The data from the mid-ventral arm
was used as the control, because this area is usually hidden
beneath clothes, so is not exposed to sunlight.
The stratum corneum was collected using a D-Squame
disk (D100, Cuderm Co. TX) with a diameter of 22 mm
(tape stripping method) [9]. The disk was affixed on the skin
for 30 s, and then pulled off. Immediately after, a new disk
was affixed on the same place. This operation was repeated
twice in order to collect samples on 2 disks from the same
area because only a small volume of protein carbonyl can be
collected at once. The 2 disks were inserted into a 750 μl
acetate buffer (4 mM, pH5) containing 0.1% triton X-100.
Ultrasonic wave was applied to the sample using an
ultrasonic cleaner (2210DTH, Branson Ultrasonic Co., CT)
for 30 min. The dissolved solutions were centrifuged using a
centrifugal evaporator (CE1, HitachiKoki Co., Ltd, Japan)
for 4 h at 37 C to obtain a fivefold increase in concentration.
The sample was injected in a microtube in order to get 150
multiple determination obtained at R2 = 0.952. This result
suggested that the validity of this method for quantitatively
analyzing DNP labeled standard proteins.
20
Response Units, RU (RU)
μl of stratum corneum sample solution. With the SPR, there
is a possibility that the buffer solution may have an influence
on the results, so the running buffer of the stratum corneum
sample solution was exchanged to HBS-EP+ using a gel
filtration column (PD SpinTrap G-25, GE Healthcare Japan
Co., Japan).
15
250 ng/ml
10
100 ng/ml
5
10 ng/ml
0
1 ng/ml
-5
-10
-10 0
(a) Mid-ventral arm (sun-protected)
60
Time (s)
Fig.4 Response of the biophysical interaction analyzer with
SPR using DNP labeled standard protein.
n=3
Response Units, RU (RU)
15
RU = 0.8105 × DNP 0.4544
R2 = 0.952
10
5
0
(b) Upper cheek (sun-exposed)
Fig.3 Skin areas for collection of the stratum corneum
samples.
120
1
10
100
1000
DNA labeled standard protein, DNP (ng/ml)
Fig.5 Calibration curve of the biophysical interaction
analyzer with SPR using DNP labeled standard protein.
E. Statistical analysis
Within-group comparisons were performed using the
paired Student’s t-test. A P-value < 0.05 was taken to
represent statistical significance. Unless otherwise stated, all
data are expressed as the mean  standard deviation (SD).
III. RESULTS AND DISCUSSION
A. Analytical method for DNP labeled proteins
Figure 4 showed the direct response of the biophysical
interaction analyzer with SPR using DNP labeled standard
protein. The sample was added at 0 s. The response unit
(RU) at each concentration was analyzed at 120 s. The RU
for 1, 10, 100 and 250 ng/ml showed 1.4, 2.2, 7.8 and 14.4
RU, respectively.
The results of a regression analysis gave a calibration
curve shown in Fig.5: RU = 0.8105 × DNP 0.4544. Within
the range of DNP between 1 – 250 ng/ml, the coefficient of
B. Carbonylation ratio in human skin
The protein carbonyls ranged between 0.05 – 0.64
ng/cm2 in all of the human samples. The mean protein
carbonyls of the mid-ventral arm and the upper cheek were
0.20 ± 0.12 and 0.31 ± 0.16 ng/cm2, respectively (Fig.6. (a)).
A significant difference was observed between the two areas
(p < 0.05), and the protein carbonyls of the upper cheek was
higher than that of the mid-ventral arm.
When data of TP less than two SDs from the mean were
removed (n = 9), the TP ranged between 1.61 – 7.26 μg/cm2
in all of the human samples. The mean TP of the mid-ventral
arm and the upper cheek were 4.14 ± 1.41 and 4.26 ± 1.88
μg/cm2, respectively (Fig.6. (b)). A significant difference
was not observed between them. It was considered that the
volume of stratum corneum collected from each site was
different.
In order to cancel out the influence of collected volume
of protein, the carbonylation ratio  100 (%) was calculated
(Fig.6. (c)). The carbonylation ratio ranged between 0.002 –
0.015 % in all of the human samples. The mean
carbonylation ratio of the mid-ventral arm and the upper
cheek were 0.004 ± 0.002 and 0.008 ± 0.004 %, respectively.
A significant difference was observed between the two sites
(p < 0.05), and the carbonylation ratio of the upper cheek
was higher than that of the mid-ventral arm. It was suggested
that the proteins in the stratum corneum might be
carbonylated by sun-exposure. Moreover, calculating the
protein carbonyl / TP ratio can be useful to cancel out the
influence of the collecting technique.
Protein carbonyls (ng/cm2)
p < 0.05
0.5
0.4
0.3
0.2
0.1
IV. CONCLUSION
0
A method for quantitatively analyzing protein carbonyl
in stratum corneum ranging between 0.05 – 0.64 ng/cm2 was
established using SPR. Our results indicated that the
carbonylation ratio might be a useful index of skin
photo-stress.
In the future, more studies are needed to evaluate the
effects of several other important factors such as gender, race
and the environment.
[4]
[5]
[6]
[7]
[8]
[9]
Total protein, TP (μg/cm2)
5
4
3
2
1
Sun-protected area
(Mid-ventral arm)
Sun-exposed area
(Upper cheek)
(b) Total protein, TP
n=9
0.016
Carbonylation ratio  100 (%)
[3]
n=9
6
0
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[2]
Sun-exposed area
(Upper cheek)
7
Part of this research was supported by the FY 2009 –
2010 Subsidies for projects to promote the enhancement of
manufacturing technology for small and medium enterprises
(SMEs) (project leader: Masaki Yamaguchi) from Small and
Medium Enterprise Agency, Minister of Economy, Trade and
Industry, Japan.
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Sun-protected area
(Mid-ventral arm)
(a) Protein carbonyl
ACKNOWLEDGMENT
[1]
n = 10
0.6
p < 0.05
0.012
0.008
0.004
0
Sun-protected area
(Mid-ventral arm)
Sun-exposed area
(Upper cheek)
(c) Carbonylation ratio
Fig.6 Comparison of carbonylation ratio between
sun-protected and sun-exposed areas in normal subjects.