Accepted Manuscript Label Electrochemical Immunosensor for Prostate-specific Antigen Based on Functionalized Graphene and Silver Hybridized Mesoporous Silica Yueyun Li, Jian Han, Runhai Chen, Xiang Ren, Qin Wei PII: DOI: Reference:
S0003-2697(14)00428-X http://dx.doi.org/10.1016/j.ab.2014.09.022 YABIO 11877
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
5 July 2014 24 September 2014 30 September 2014
Please cite this article as: Y. Li, J. Han, R. Chen, X. Ren, Q. Wei, Label Electrochemical Immunosensor for Prostatespecific Antigen Based on Functionalized Graphene and Silver Hybridized Mesoporous Silica, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.09.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page Label Electrochemical Immunosensor for Prostate-specific Antigen Based on Functionalized Graphene and Silver Hybridized Mesoporous Silica Yueyun Li1,2*, Jian Han1, Runhai Chen1, Xiang Ren2, Qin Wei2 1. School of Chemical Engineering, Shandong University of Technology, Zibo, 255049, P.R. China 2. Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, P.R. China *: Corresponding author: Email adress: [email protected]; Fax: +86-533-2781664; Tel: +86-533-2781225; The appropriate subject category: Immunologicl Procedures Short title of the paper: Electrochemical Immunosensor for PSA
1
1
Label Electrochemical Immunosensor for Prostate-specific Antigen Based on
2
Graphene and Silver Hybridized Mesoporous Silica
3
Yueyun Li1*, Jian Han1, Runhai Chen1, Xiang Ren2, Qin Wei2
4
1. School of Chemical Engineering, Shandong University of Technology, Zibo,
5
255049, P.R. China
6
2. Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong,
7
School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022,
8
P.R. China
9
*: Corresponding author. Email:[email protected]
10
Abstract
11
Prostate specific antigen (PSA), as the specificity of prostate cancer markers, has been
12
widely used in prostate cancer diagnosis and screening. In this study, we fabricated an
13
electrochemical immunosensor for PSA detection using the amino-functionalized
14
graphene sheet-ferrocenecarboxaldehyde composite materials (NH2-GS@FCA) and
15
silver hybridized mesoporous silica nanoparticles(Ag@NH2-MCM48). Under
16
optimum conditions，the fabricated immunosensor showed a wide linear range with
17
PSA concentration (0.01 to 10.0 ng.mL-1) . Low detection limit (2 pg.mL-1) proved the
18
high sensitivity. In addition, the immunosensor possessed good stability and
19
reproducibility. Moreover, the application to PSA analyse in serum samples yielded
20
satisfactory results.
21
Key words: immunosensor, prostate-specific antigen, silver hybridized mesoporous
22
silica nanoparticles
23
1. Introduction
2
24
Prostate cancer is known as a malignant tumor of male sex glands [1]. It is quite
25
important to diagnose the disease or to monitor the recurrence after treatment. PSA is
26
the most typical tumor marker for clinical diagnosis of prostate cancer, which could
27
provide direct information about the diagnosis of the prostate cancer via its
28
concentration in blood [2]. When the concentration of PSA between 4.0 and 10.0 ng.
29
mL -1, the risk of prostate cancer is 22% to 27%.However, the risk was increased to
30
67% when PSA values was more than 10 ng. mL -1. The internationally accepted
31
threshold value of PSA is 4.0 ng. mL -1[3] Therefore, the quantitative determination of
32
PSA is of great importance in proper diagnosis, prevention, and treatment of prostate
33
cancer.
34
In the past few years, a lot of efficient methods have been developed for the detection
35
of PSA based on immunoreaction, such as enzyme-linked immunosorbent assay [4],
36
time-resolved fluorescence assay [5], chemiluminescence immunoassay [6],
37
bioluminescent immunoassay [7], and electrochemical immunoassay [8]. Compared
38
with the methods mentioned above, electrochemical immunosensors exhibits
39
significant advantages, including high sensitivity, simple instrumentation and easiness
40
of miniaturization. Hence, electrochemical immunosensors, as a kind of effective
41
analytical technique, have been extensively applied in the quantitative detection of
42
biomolecules [9-13].
43
Various nanomaterials such as carbon nanotubes[14-16], gold nanoparticles[17-19],
44
magnetic nanoparticles[21-21] and graphene sheets(GS) [22-23] have been widely
45
employed to fabric sensitive immunosensor. Among these materials, GS have
46
attracted a vast amount of attention because of their excellent properties [24-26], such
47
as large surface area, extraordinary flexibility and electronic transport property.
48
Therefore, as a kind of electrochemical material, some electrochemical sensing and
3
49
biosensing platforms based on graphene were reported [27]. To improve the solubility
50
of graphene and facilitate graphene reaction with other molecules and biological
51
systems, amine can be introduced into GS as ionizable functional groups [28].
52
Mesoporous silica materials (MSMs) possessing many advantageous properties, such
53
as large specific surface area, uniform structures and controlled pore size, have been
54
used in immobilization of enzyme [29], improving the electrochemical responses of
55
epinephrine [30]and drug targeting [31]. MCM48 is one of the popular members of
56
MSMs family, which have large surface area and abundant porosity as well as
57
long-range ordered pore structure [32]. Incorporation of different metals into the
58
MCM48 framework can improve the catalytic properties of MCM48 [33-34], which
59
provides more favorable mass transfer kinetics than the pore system of MCM48.Many
60
metal nanomaterial also have been used to amplify the signal in order to improve the
61
sensitivity of electrochemical immunosensor. Among them, silver nanoparticles with
62
biocompatibility, high electrical conductivity have been used in biological markers
63
and amplifying electrochemical signals [35-37].
64
In this work, the amino-functionalized graphene sheet-ferrocenecarboxaldehyde
65
-composite materials (NH2-GS@FCA) and silver hybridized NH2-MCM48
66
nanoparticles (Ag@NH2-MCM48) were synthesized and used for the detection of
67
PSA. The large surface area of NH2-GS increased the fixation capacity and the
68
immobilization effect of antibody. FCA used as mediator promoted the electron
69
transfer. The Ag@NH2-MCM48 showed obvious effect in the H2O2 reduction.
70
Furthermore, NH2-GS@FCA and Ag@NH2-MCM48 improves the electron transfer
71
ability of the immunosensor system. The proposed immunosensor provides a
72
sensitive method with lower detection limit than the other PSA immunosensors
73
[38-39].
4
74
2. Materials and methods
75
2.1 Reagents and apparatus
76
Graphene oxide (GO) was purchased from Shanghai Carbon Co. Ltd. (Shanghai,
77
China).
78
ferrocenecarboxaldehyde (FCA) were purchased from Dingguo Biochemical Reagents
79
(Beijing, China). K3[Fe(CN)6] was purchased from Sinopharm Chemical Reagent
80
Beijing Co., Ltd., China. BSA (96–99%) was purchased from Sigma-Aldrich (USA)
81
and used as received. All other chemicals were of analytical reagents grade and used
82
without further purification. Phosphate buffered saline (PBS, pH 7.4) was used as
83
electrolyte for all electrochemical test. Ultrapure water (obtain from the Milli-Q Element
84
system, Millipore, Billerica, USA) were used throughout the experiments.
85
Transmission electron microscope (TEM) images were obtained from a JEOL
86
JEM-2010 microscope (Japan). Scanning electron microscope (SEM) images were
87
carried on a field emission SEM (ZEISS, Germany). All electrochemical
88
measurements were performed on a CHI760D electrochemical workstation (Shanghai
89
Chenhua Instruments Co., China). Electrochemical impedance spectroscopy (EIS)
90
was obtained from the impedance measurement unit (IM6e, ZAHNER elektrik,
91
Germany). A conventional three-electrode system was used for all electrochemical
92
measurements employing a glassy carbon electrode (GCE, 4 mm in diameter) as the
93
working electrode, a saturated calomel electrode (SCE) as the reference electrode, and
94
platinum wire electrode as the counter electrode, respectively.
95
2.2 Preparation of NH2-GS@FCA
96
The NH2-GS was prepared according to Lai’s method [28]. In a typical experiment,
97
100 mg of GO was added to 40 mL of ethylene glycol under ultrasonication. After
98
further addition of 1 mL of ammonium hydroxide (6mol.L-1), the dark brown mixture
Prostate
specific
antigen
(PSA),
anti-PSA
antibody
(Ab)
and
5
99
was sealed into a teflon-lined stainless steel autoclave followed by hydrothermal
100
treatment at 180 °C for 10 h. After filtered, the dark precipitate (NH2-GS) was washed
101
with distilled water, and dried at 65 ºC for 24 h for further usage. Then, 0.8 mg of
102
NH2-GS and 1.0 mg of FCA were dispersed in 1 mL of 1% carboxymethyl chitosan
103
solution and the mixture was stirred with a magnetic stirrer for 24 h, the resulting
104
fresh product was NH2-GS@FCA. FCA should be combined with NH2-GS through
105
the electrostatic attraction of amino functional groups.
106
2.3. Preparation of Ag@ NH2-MCM48
107
2.3.1 Preparation of NH2-MCM48
108
MCM48 was synthesized following the method reported previously [40-41]. Briefly,
109
under vigorous stirring, 1.0 g tetraethyl orthosilicate (TEOS) was dissolved in 62 ml
110
of water for 10 min. Then 0.5 g KOH was added to the solution, followed by the
111
addition of 0.65 g cetyltrimethyl ammonium bromide (CTAB). The mixture was
112
stirred for 30 min, and then transferred into an autoclave for hydrothermal
113
crystallization for 72 h at 120 ºC. After filtration, the obtained white particles were
114
washed with distilled water and ethanol repeatedly, dried in air, and calcined at 550 ºC
115
for 6 h.
116
Subsequently, 1 g of MCM48 and 1 mL of 3-aminopropyl trimethoxysilane were
117
dissolved in 80 mL of anhydrous toluene and refluxed for 1.5 h at 70 ºC, The obtained
118
white product was dried at 110 ºC for 1 h to yield the NH2-MCM48.
119
2.3.2 Preparation of Ag@NH2- MCM48
120
Sliver nanoparticles were prepared as described. [42] Briefly, 1 mL (50 mmol.L-1) of
121
silver nitrate and 1 mL of 5% citrate were added in 48 mL ultrapure water with stirring,
122
and then 5mg sodium borohydride was added into the above solution, followed by
123
stirring until the color of solution no longer changed. Subsequently, 20 mg
6
124
NH2-MCM48 was added into the above solution and stirred for 24 h，followed by
125
centrifugation and vacuum drying in 35 ºC to obtain Ag@NH2-MCM48.
126
2.4 Preparation of Ag@NH2-MCM48/Ab 2
127
The synthesized Ag@NH2-MCM48 (1 mg.mL-1) was added to Ab 2 solution (1 mL, 10
128
µg.mL-1) and the mixture was stirred for 24 h. After centrifuged and washed with PBS,
129
the Ag@NH2-MCM48/Ab2 conjugation was re-dispersed in PBS (pH 7.4) and stored
130
at 4 ºC for use. Ag nanoparticles were combined with Ab2 via silver-amino bond [43],
131
led to further sensitivity enhancing. In addition, NH2-MCM48 bearing a large surface
132
area is used to immobilize Ag nanoparticles. Preferred position of Figure 1
133 134
2.5. Fabrication of the immunosensor
135
The preparation process of the immunosensor is shown in Fig. 1. GCE was polished
136
with 1, 0.3, and 0.05 µm alumina powder sequentially and then washed ultrasonically
137
in ethanol and water for a few minutes, respectively. And then, 6 µL of the
138
NH2-GS@FCA mixture was added onto electrode surface and dried. Subsequently, 3
139
µL of 2.5% glutaric dialdehyde (GD) and 6 µL of Ab 1 were added onto electrode
140
surface successively and dried in 4 ºC. After washing with ultrapure water, 3 µL of
141
BSA solution (100 µg .mL-1) was added on the electrode surface to block nonspecific
142
binding sites. Then, the electrodes were incubated with PSA solution for 1 h. Finally,
143
5µL of the prepared Ag@NH2-MCM48/Ab 2 buffer solution (1 mg.mL-1) was added
144
onto the electrode surface and incubated for another 1 h. After washing, the electrode
145
was ready for measurement. The electrode was stored at 4 ºC in phosphate buffer
146
solution in standby.
147
3. Results and discussion
148
3.1. Characterization of Ag@ NH2-MCM48
7
149
Preferred position of Figure 2
150
Fig. 2 shows the SEM and TEM images of Ag@NH2-MCM48. The SEM image (Fig.
151
2a) and TEM image (Fig. 2b) of the Ag@NH2-MCM48 demonstrated that spherical
152
Ag nanoparticles (diameter: 5-35nm) were modified on the surface of NH2-MCM48
153
successfully. Fig. 2b also shows the TEM image of the NH2-MCM48 with ordered
154
silica channels.
155
3.2. Electrochemical characterization of modified electrode
156
Preferred position of Figure 3
157
Electron-impedance spectroscopy (EIS) is a useful tool for probing the features of
158
surface-modified electrodes [44-45]. EIS are recorded in all immobilization steps and
159
shown in Fig.3. It is well known that the high frequency region of the impedance plot
160
shows a semicircle related to the redox probe Fe(CN)63-/4-, followed by a Warburg line
161
in the low frequency region which corresponds to the diffusion step of the overall
162
process [46]. The semicircle portion at higher frequencies corresponds to the
163
electron–transfer limited process, and the linear portion at lower frequencies
164
represents the diffusion-limited process. Clearly, the GCE presented a very small
165
semicircle domain implying a very fast electron-transfer process with a diffusional
166
limiting step (curve a). After NH2-GS@FCA was modified onto the bare electrode,
167
the semicircle became larger (curve b). Then, the resistance (Ret) of the resultant
168
GD/NH2-GS@FCA/GCE film increased (curve c), indicating that GD was modified
169
successfully on to the electrode. After Ab1 was modified onto the electrode (curve d),
170
there was an obvious increase of the semicircle compared with the electrode without
171
Ab 1. This is because the presence of Ab1 hindered the efficiency of electron transfer,
172
suggesting that Ab1 had been immobilized on the surface of the modified electrode.
173
Ret increased in the same way after 1 wt% BSA was used to block nonspecific sites 8
174
(curve e), which might be attributed to the same reason with loading the protein.
175
When the PSA antigen captured by its antibody, the Ret increased further (curve f),
176
All the Ab1, BSA and PSA antigen retarded the transfer of electrons toward the
177
electrode surface. After the capture of Ag@NH2-MCM48/Ab2, the Ret decreased
178
obviously (curve g), the formation of hydrophobic immunocomplex layer promoted
179
the
180
Ag@NH2-MCM48/Ab 2. As a result, the fabrication process of the immunosensor was
181
completed successfully.
182
3.3. Optimization of experimental conditions
electron
transfer,
which
indicated
the
successful
capture
of
183
Preferred position of Figure 4
184
Preferred position of Figure 5
185
The experimental conditions, including the working pH (5.85-9.26), concentration of
186
NH2-GS (0.2-1.4 mg.mL-1) and FCA (0.2-1.8 mg.mL-1), were optimized by
187
amperometric i-t test (Fig.4). As shown in Fig. 5a, the maximum current was achieved
188
at pH=7.4 PBS, indicating that the fabricated immunosensor works best in this buffer
189
solution. For the immunosensor, the concentration of NH2-GS plays an important role
190
on its sensitivity. Varying concentrations of NH2-GS between 0.2 and 1.4 mg.mL-1
191
were used to fabricate the immunosensors in pH=7.4 PBS. As seen from Fig.5b, the
192
current change (∆I) reached maximum when the concentration of NH2-GS at 0.8
193
mg.mL-1, so this concentration was used in the following experiments. FCA is used as
194
electron mediator in this study, the concentration of electron mediator plays an
195
important role to the immunosensors sensitivity, different concentrations of FCA in
196
the range of 0.2 to 1.8 mg.mL-1 were used to fabricate the immunosensors. The effect
197
of different concentrations of FCA used for immunosensors on the current response
198
(∆I) was shown in Fig.5C. The response increased maximally to 1.0 mg.mL-1.
9
199
Therefore, 1.0 mg.mL-1 was used as the optimal concentration of FCA.
200
Under the optimum conditions, immunosensors based on Ag@NH2-MCM48/Ab 2 and
201
NH2-GS@FCA were used to detect different concentrations of PSA. The relationship
202
between the current responses toward PSA concentration was shown in Fig.5d. And
203
the equation of the calibration curve was: Y=3.6807X-0.0017, r2=0.9963. The
204
catalytic current increased linearly with the PSA concentration from 0.01 to 10.0 ng.
205
mL-1, and a low detection limit (2pg.mL-1) was obtained, the detection limit is lower
206
than some previous detection for PSA, such as H. Wang et al reported the detection
207
limit is 15pg.mL-1 [1]; K.X. Mao et al reported the detection limit is 13pg.mL-1 [47];
208
and the typical detection limits of commercial ELISA-type assays is 0.08g.L-1 [48].
209
The serum PSA concentration of a normal person and of a cancer patient fell in the
210
linear range of our immunosensor. So, this method can be used for the determination
211
of PSA concentration in human serum, indicating that the immunosensor could be
212
used clinically.
213
The low detection limit maybe attributed to three factors. Firstly, a relatively large
214
amount of Ab 2 had been conjugated onto the Ag@NH2-MCM48-based labels.
215
Generally, when the concentration of PSA was low, the amount of the Ab1 captured
216
by PSA was also low; however, the relatively large amount of Ab2 immobilized onto
217
the labels could greatly increase the probability of Ab2-antigen interactions leading to
218
higher sensitivity. Secondly, as discussed earlier, the Ag@NH2-MCM48 could keep
219
the activity of PSA and catalyze the reduction of H2O2 to produce stronger signal.
220
Thirdly, NH2-GS possesses high conductivity, good electron transfer property and
221
biocompatibility. The mixture of FCA and NH2-GS on the electrode surface which has
222
high space area, amplifies the signal.
223
3.4. Selectivity, reproducibility and stability of the immunosensor
10
224
Selective determination of the immunosensor is important in analyzing biological
225
samples [49]. In this study, we investigated the selectivity of the immunosensor as
226
following. The immunosensor was carried out by incubating the immunosensor in 2
227
ng.mL-1 of PSA solution containing 200 ng. mL-1 of interfering substance (human IgG,
228
lysozyme, α-fetoprotein (AFP) Carcinoembryonic antigen (CEA), Ascorbic acid (AA),
229
BSA and glucose respectively). The results (Fig.6) show that the current variation due
230
to the interfering substances was less than 5.2% of those without interferences. These
231
results demonstrated that the selectivity of the immunosensor was good.
232
Preferred position of Figure 6
233
Preferred position of Figure 7
234
The reproducibility of the immunosensor was also investigated. Five electrodes were
235
prepared for the detection of 2 ng .mL-1 PSA. The relative standard deviation (RSD)
236
of the measurements for the five immunosensors was 6.4%, indicating that the
237
precision and reproducibility of the proposed immunosensor was acceptable.
238
To study the stability of the immunosensor, the current responses were checked
239
periodically (Fig.7). The immunosensor was stored at 4◦C when it was not in use.
240
After one week, the current response of the immunosensor decreased by 3.7%. After
241
three weeks, there was no obvious signal change (relativestandard deviation
S0003-2697(14)00428-X http://dx.doi.org/10.1016/j.ab.2014.09.022 YABIO 11877
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
5 July 2014 24 September 2014 30 September 2014
Please cite this article as: Y. Li, J. Han, R. Chen, X. Ren, Q. Wei, Label Electrochemical Immunosensor for Prostatespecific Antigen Based on Functionalized Graphene and Silver Hybridized Mesoporous Silica, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.09.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page Label Electrochemical Immunosensor for Prostate-specific Antigen Based on Functionalized Graphene and Silver Hybridized Mesoporous Silica Yueyun Li1,2*, Jian Han1, Runhai Chen1, Xiang Ren2, Qin Wei2 1. School of Chemical Engineering, Shandong University of Technology, Zibo, 255049, P.R. China 2. Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, P.R. China *: Corresponding author: Email adress: [email protected]; Fax: +86-533-2781664; Tel: +86-533-2781225; The appropriate subject category: Immunologicl Procedures Short title of the paper: Electrochemical Immunosensor for PSA
1
1
Label Electrochemical Immunosensor for Prostate-specific Antigen Based on
2
Graphene and Silver Hybridized Mesoporous Silica
3
Yueyun Li1*, Jian Han1, Runhai Chen1, Xiang Ren2, Qin Wei2
4
1. School of Chemical Engineering, Shandong University of Technology, Zibo,
5
255049, P.R. China
6
2. Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong,
7
School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022,
8
P.R. China
9
*: Corresponding author. Email:[email protected]
10
Abstract
11
Prostate specific antigen (PSA), as the specificity of prostate cancer markers, has been
12
widely used in prostate cancer diagnosis and screening. In this study, we fabricated an
13
electrochemical immunosensor for PSA detection using the amino-functionalized
14
graphene sheet-ferrocenecarboxaldehyde composite materials (NH2-GS@FCA) and
15
silver hybridized mesoporous silica nanoparticles(Ag@NH2-MCM48). Under
16
optimum conditions，the fabricated immunosensor showed a wide linear range with
17
PSA concentration (0.01 to 10.0 ng.mL-1) . Low detection limit (2 pg.mL-1) proved the
18
high sensitivity. In addition, the immunosensor possessed good stability and
19
reproducibility. Moreover, the application to PSA analyse in serum samples yielded
20
satisfactory results.
21
Key words: immunosensor, prostate-specific antigen, silver hybridized mesoporous
22
silica nanoparticles
23
1. Introduction
2
24
Prostate cancer is known as a malignant tumor of male sex glands [1]. It is quite
25
important to diagnose the disease or to monitor the recurrence after treatment. PSA is
26
the most typical tumor marker for clinical diagnosis of prostate cancer, which could
27
provide direct information about the diagnosis of the prostate cancer via its
28
concentration in blood [2]. When the concentration of PSA between 4.0 and 10.0 ng.
29
mL -1, the risk of prostate cancer is 22% to 27%.However, the risk was increased to
30
67% when PSA values was more than 10 ng. mL -1. The internationally accepted
31
threshold value of PSA is 4.0 ng. mL -1[3] Therefore, the quantitative determination of
32
PSA is of great importance in proper diagnosis, prevention, and treatment of prostate
33
cancer.
34
In the past few years, a lot of efficient methods have been developed for the detection
35
of PSA based on immunoreaction, such as enzyme-linked immunosorbent assay [4],
36
time-resolved fluorescence assay [5], chemiluminescence immunoassay [6],
37
bioluminescent immunoassay [7], and electrochemical immunoassay [8]. Compared
38
with the methods mentioned above, electrochemical immunosensors exhibits
39
significant advantages, including high sensitivity, simple instrumentation and easiness
40
of miniaturization. Hence, electrochemical immunosensors, as a kind of effective
41
analytical technique, have been extensively applied in the quantitative detection of
42
biomolecules [9-13].
43
Various nanomaterials such as carbon nanotubes[14-16], gold nanoparticles[17-19],
44
magnetic nanoparticles[21-21] and graphene sheets(GS) [22-23] have been widely
45
employed to fabric sensitive immunosensor. Among these materials, GS have
46
attracted a vast amount of attention because of their excellent properties [24-26], such
47
as large surface area, extraordinary flexibility and electronic transport property.
48
Therefore, as a kind of electrochemical material, some electrochemical sensing and
3
49
biosensing platforms based on graphene were reported [27]. To improve the solubility
50
of graphene and facilitate graphene reaction with other molecules and biological
51
systems, amine can be introduced into GS as ionizable functional groups [28].
52
Mesoporous silica materials (MSMs) possessing many advantageous properties, such
53
as large specific surface area, uniform structures and controlled pore size, have been
54
used in immobilization of enzyme [29], improving the electrochemical responses of
55
epinephrine [30]and drug targeting [31]. MCM48 is one of the popular members of
56
MSMs family, which have large surface area and abundant porosity as well as
57
long-range ordered pore structure [32]. Incorporation of different metals into the
58
MCM48 framework can improve the catalytic properties of MCM48 [33-34], which
59
provides more favorable mass transfer kinetics than the pore system of MCM48.Many
60
metal nanomaterial also have been used to amplify the signal in order to improve the
61
sensitivity of electrochemical immunosensor. Among them, silver nanoparticles with
62
biocompatibility, high electrical conductivity have been used in biological markers
63
and amplifying electrochemical signals [35-37].
64
In this work, the amino-functionalized graphene sheet-ferrocenecarboxaldehyde
65
-composite materials (NH2-GS@FCA) and silver hybridized NH2-MCM48
66
nanoparticles (Ag@NH2-MCM48) were synthesized and used for the detection of
67
PSA. The large surface area of NH2-GS increased the fixation capacity and the
68
immobilization effect of antibody. FCA used as mediator promoted the electron
69
transfer. The Ag@NH2-MCM48 showed obvious effect in the H2O2 reduction.
70
Furthermore, NH2-GS@FCA and Ag@NH2-MCM48 improves the electron transfer
71
ability of the immunosensor system. The proposed immunosensor provides a
72
sensitive method with lower detection limit than the other PSA immunosensors
73
[38-39].
4
74
2. Materials and methods
75
2.1 Reagents and apparatus
76
Graphene oxide (GO) was purchased from Shanghai Carbon Co. Ltd. (Shanghai,
77
China).
78
ferrocenecarboxaldehyde (FCA) were purchased from Dingguo Biochemical Reagents
79
(Beijing, China). K3[Fe(CN)6] was purchased from Sinopharm Chemical Reagent
80
Beijing Co., Ltd., China. BSA (96–99%) was purchased from Sigma-Aldrich (USA)
81
and used as received. All other chemicals were of analytical reagents grade and used
82
without further purification. Phosphate buffered saline (PBS, pH 7.4) was used as
83
electrolyte for all electrochemical test. Ultrapure water (obtain from the Milli-Q Element
84
system, Millipore, Billerica, USA) were used throughout the experiments.
85
Transmission electron microscope (TEM) images were obtained from a JEOL
86
JEM-2010 microscope (Japan). Scanning electron microscope (SEM) images were
87
carried on a field emission SEM (ZEISS, Germany). All electrochemical
88
measurements were performed on a CHI760D electrochemical workstation (Shanghai
89
Chenhua Instruments Co., China). Electrochemical impedance spectroscopy (EIS)
90
was obtained from the impedance measurement unit (IM6e, ZAHNER elektrik,
91
Germany). A conventional three-electrode system was used for all electrochemical
92
measurements employing a glassy carbon electrode (GCE, 4 mm in diameter) as the
93
working electrode, a saturated calomel electrode (SCE) as the reference electrode, and
94
platinum wire electrode as the counter electrode, respectively.
95
2.2 Preparation of NH2-GS@FCA
96
The NH2-GS was prepared according to Lai’s method [28]. In a typical experiment,
97
100 mg of GO was added to 40 mL of ethylene glycol under ultrasonication. After
98
further addition of 1 mL of ammonium hydroxide (6mol.L-1), the dark brown mixture
Prostate
specific
antigen
(PSA),
anti-PSA
antibody
(Ab)
and
5
99
was sealed into a teflon-lined stainless steel autoclave followed by hydrothermal
100
treatment at 180 °C for 10 h. After filtered, the dark precipitate (NH2-GS) was washed
101
with distilled water, and dried at 65 ºC for 24 h for further usage. Then, 0.8 mg of
102
NH2-GS and 1.0 mg of FCA were dispersed in 1 mL of 1% carboxymethyl chitosan
103
solution and the mixture was stirred with a magnetic stirrer for 24 h, the resulting
104
fresh product was NH2-GS@FCA. FCA should be combined with NH2-GS through
105
the electrostatic attraction of amino functional groups.
106
2.3. Preparation of Ag@ NH2-MCM48
107
2.3.1 Preparation of NH2-MCM48
108
MCM48 was synthesized following the method reported previously [40-41]. Briefly,
109
under vigorous stirring, 1.0 g tetraethyl orthosilicate (TEOS) was dissolved in 62 ml
110
of water for 10 min. Then 0.5 g KOH was added to the solution, followed by the
111
addition of 0.65 g cetyltrimethyl ammonium bromide (CTAB). The mixture was
112
stirred for 30 min, and then transferred into an autoclave for hydrothermal
113
crystallization for 72 h at 120 ºC. After filtration, the obtained white particles were
114
washed with distilled water and ethanol repeatedly, dried in air, and calcined at 550 ºC
115
for 6 h.
116
Subsequently, 1 g of MCM48 and 1 mL of 3-aminopropyl trimethoxysilane were
117
dissolved in 80 mL of anhydrous toluene and refluxed for 1.5 h at 70 ºC, The obtained
118
white product was dried at 110 ºC for 1 h to yield the NH2-MCM48.
119
2.3.2 Preparation of Ag@NH2- MCM48
120
Sliver nanoparticles were prepared as described. [42] Briefly, 1 mL (50 mmol.L-1) of
121
silver nitrate and 1 mL of 5% citrate were added in 48 mL ultrapure water with stirring,
122
and then 5mg sodium borohydride was added into the above solution, followed by
123
stirring until the color of solution no longer changed. Subsequently, 20 mg
6
124
NH2-MCM48 was added into the above solution and stirred for 24 h，followed by
125
centrifugation and vacuum drying in 35 ºC to obtain Ag@NH2-MCM48.
126
2.4 Preparation of Ag@NH2-MCM48/Ab 2
127
The synthesized Ag@NH2-MCM48 (1 mg.mL-1) was added to Ab 2 solution (1 mL, 10
128
µg.mL-1) and the mixture was stirred for 24 h. After centrifuged and washed with PBS,
129
the Ag@NH2-MCM48/Ab2 conjugation was re-dispersed in PBS (pH 7.4) and stored
130
at 4 ºC for use. Ag nanoparticles were combined with Ab2 via silver-amino bond [43],
131
led to further sensitivity enhancing. In addition, NH2-MCM48 bearing a large surface
132
area is used to immobilize Ag nanoparticles. Preferred position of Figure 1
133 134
2.5. Fabrication of the immunosensor
135
The preparation process of the immunosensor is shown in Fig. 1. GCE was polished
136
with 1, 0.3, and 0.05 µm alumina powder sequentially and then washed ultrasonically
137
in ethanol and water for a few minutes, respectively. And then, 6 µL of the
138
NH2-GS@FCA mixture was added onto electrode surface and dried. Subsequently, 3
139
µL of 2.5% glutaric dialdehyde (GD) and 6 µL of Ab 1 were added onto electrode
140
surface successively and dried in 4 ºC. After washing with ultrapure water, 3 µL of
141
BSA solution (100 µg .mL-1) was added on the electrode surface to block nonspecific
142
binding sites. Then, the electrodes were incubated with PSA solution for 1 h. Finally,
143
5µL of the prepared Ag@NH2-MCM48/Ab 2 buffer solution (1 mg.mL-1) was added
144
onto the electrode surface and incubated for another 1 h. After washing, the electrode
145
was ready for measurement. The electrode was stored at 4 ºC in phosphate buffer
146
solution in standby.
147
3. Results and discussion
148
3.1. Characterization of Ag@ NH2-MCM48
7
149
Preferred position of Figure 2
150
Fig. 2 shows the SEM and TEM images of Ag@NH2-MCM48. The SEM image (Fig.
151
2a) and TEM image (Fig. 2b) of the Ag@NH2-MCM48 demonstrated that spherical
152
Ag nanoparticles (diameter: 5-35nm) were modified on the surface of NH2-MCM48
153
successfully. Fig. 2b also shows the TEM image of the NH2-MCM48 with ordered
154
silica channels.
155
3.2. Electrochemical characterization of modified electrode
156
Preferred position of Figure 3
157
Electron-impedance spectroscopy (EIS) is a useful tool for probing the features of
158
surface-modified electrodes [44-45]. EIS are recorded in all immobilization steps and
159
shown in Fig.3. It is well known that the high frequency region of the impedance plot
160
shows a semicircle related to the redox probe Fe(CN)63-/4-, followed by a Warburg line
161
in the low frequency region which corresponds to the diffusion step of the overall
162
process [46]. The semicircle portion at higher frequencies corresponds to the
163
electron–transfer limited process, and the linear portion at lower frequencies
164
represents the diffusion-limited process. Clearly, the GCE presented a very small
165
semicircle domain implying a very fast electron-transfer process with a diffusional
166
limiting step (curve a). After NH2-GS@FCA was modified onto the bare electrode,
167
the semicircle became larger (curve b). Then, the resistance (Ret) of the resultant
168
GD/NH2-GS@FCA/GCE film increased (curve c), indicating that GD was modified
169
successfully on to the electrode. After Ab1 was modified onto the electrode (curve d),
170
there was an obvious increase of the semicircle compared with the electrode without
171
Ab 1. This is because the presence of Ab1 hindered the efficiency of electron transfer,
172
suggesting that Ab1 had been immobilized on the surface of the modified electrode.
173
Ret increased in the same way after 1 wt% BSA was used to block nonspecific sites 8
174
(curve e), which might be attributed to the same reason with loading the protein.
175
When the PSA antigen captured by its antibody, the Ret increased further (curve f),
176
All the Ab1, BSA and PSA antigen retarded the transfer of electrons toward the
177
electrode surface. After the capture of Ag@NH2-MCM48/Ab2, the Ret decreased
178
obviously (curve g), the formation of hydrophobic immunocomplex layer promoted
179
the
180
Ag@NH2-MCM48/Ab 2. As a result, the fabrication process of the immunosensor was
181
completed successfully.
182
3.3. Optimization of experimental conditions
electron
transfer,
which
indicated
the
successful
capture
of
183
Preferred position of Figure 4
184
Preferred position of Figure 5
185
The experimental conditions, including the working pH (5.85-9.26), concentration of
186
NH2-GS (0.2-1.4 mg.mL-1) and FCA (0.2-1.8 mg.mL-1), were optimized by
187
amperometric i-t test (Fig.4). As shown in Fig. 5a, the maximum current was achieved
188
at pH=7.4 PBS, indicating that the fabricated immunosensor works best in this buffer
189
solution. For the immunosensor, the concentration of NH2-GS plays an important role
190
on its sensitivity. Varying concentrations of NH2-GS between 0.2 and 1.4 mg.mL-1
191
were used to fabricate the immunosensors in pH=7.4 PBS. As seen from Fig.5b, the
192
current change (∆I) reached maximum when the concentration of NH2-GS at 0.8
193
mg.mL-1, so this concentration was used in the following experiments. FCA is used as
194
electron mediator in this study, the concentration of electron mediator plays an
195
important role to the immunosensors sensitivity, different concentrations of FCA in
196
the range of 0.2 to 1.8 mg.mL-1 were used to fabricate the immunosensors. The effect
197
of different concentrations of FCA used for immunosensors on the current response
198
(∆I) was shown in Fig.5C. The response increased maximally to 1.0 mg.mL-1.
9
199
Therefore, 1.0 mg.mL-1 was used as the optimal concentration of FCA.
200
Under the optimum conditions, immunosensors based on Ag@NH2-MCM48/Ab 2 and
201
NH2-GS@FCA were used to detect different concentrations of PSA. The relationship
202
between the current responses toward PSA concentration was shown in Fig.5d. And
203
the equation of the calibration curve was: Y=3.6807X-0.0017, r2=0.9963. The
204
catalytic current increased linearly with the PSA concentration from 0.01 to 10.0 ng.
205
mL-1, and a low detection limit (2pg.mL-1) was obtained, the detection limit is lower
206
than some previous detection for PSA, such as H. Wang et al reported the detection
207
limit is 15pg.mL-1 [1]; K.X. Mao et al reported the detection limit is 13pg.mL-1 [47];
208
and the typical detection limits of commercial ELISA-type assays is 0.08g.L-1 [48].
209
The serum PSA concentration of a normal person and of a cancer patient fell in the
210
linear range of our immunosensor. So, this method can be used for the determination
211
of PSA concentration in human serum, indicating that the immunosensor could be
212
used clinically.
213
The low detection limit maybe attributed to three factors. Firstly, a relatively large
214
amount of Ab 2 had been conjugated onto the Ag@NH2-MCM48-based labels.
215
Generally, when the concentration of PSA was low, the amount of the Ab1 captured
216
by PSA was also low; however, the relatively large amount of Ab2 immobilized onto
217
the labels could greatly increase the probability of Ab2-antigen interactions leading to
218
higher sensitivity. Secondly, as discussed earlier, the Ag@NH2-MCM48 could keep
219
the activity of PSA and catalyze the reduction of H2O2 to produce stronger signal.
220
Thirdly, NH2-GS possesses high conductivity, good electron transfer property and
221
biocompatibility. The mixture of FCA and NH2-GS on the electrode surface which has
222
high space area, amplifies the signal.
223
3.4. Selectivity, reproducibility and stability of the immunosensor
10
224
Selective determination of the immunosensor is important in analyzing biological
225
samples [49]. In this study, we investigated the selectivity of the immunosensor as
226
following. The immunosensor was carried out by incubating the immunosensor in 2
227
ng.mL-1 of PSA solution containing 200 ng. mL-1 of interfering substance (human IgG,
228
lysozyme, α-fetoprotein (AFP) Carcinoembryonic antigen (CEA), Ascorbic acid (AA),
229
BSA and glucose respectively). The results (Fig.6) show that the current variation due
230
to the interfering substances was less than 5.2% of those without interferences. These
231
results demonstrated that the selectivity of the immunosensor was good.
232
Preferred position of Figure 6
233
Preferred position of Figure 7
234
The reproducibility of the immunosensor was also investigated. Five electrodes were
235
prepared for the detection of 2 ng .mL-1 PSA. The relative standard deviation (RSD)
236
of the measurements for the five immunosensors was 6.4%, indicating that the
237
precision and reproducibility of the proposed immunosensor was acceptable.
238
To study the stability of the immunosensor, the current responses were checked
239
periodically (Fig.7). The immunosensor was stored at 4◦C when it was not in use.
240
After one week, the current response of the immunosensor decreased by 3.7%. After
241
three weeks, there was no obvious signal change (relativestandard deviation
