electrochemiluminescence immunosensor for tumor markers based on biological barcode mode with...
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Author's Accepted Manuscript
Electrochemiluminescence immunosensor fortumor markers based on biological barcodemode with conductive nanospheres
Shuping Du, Zhiyong Guo, Beibei Chen, YuhongSha, Xiaohua Jiang, Xing Li, Ning Gan, Sui Wang
PII: S0956-5663(13)00659-3DOI: http://dx.doi.org/10.1016/j.bios.2013.09.041Reference: BIOS6225
To appear in: Biosensors and Bioelectronics
Received date: 22 July 2013Revised date: 19 September 2013Accepted date: 19 September 2013
Cite this article as: Shuping Du, Zhiyong Guo, Beibei Chen, Yuhong Sha,Xiaohua Jiang, Xing Li, Ning Gan, Sui Wang, Electrochemiluminescenceimmunosensor for tumor markers based on biological barcode mode withconductive nanospheres, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2013.09.041
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1
Electrochemiluminescence immunosensor for tumor markers 1
based on biological barcode mode with conductive nanospheres 2
3
Shuping Dua, Zhiyong Guoa,�, Beibei Chena, Yuhong Shaa, Xiaohua Jiangb, Xing Lia, 4
Ning Gana, Sui Wanga 5
6
a Faculty of Materials Science and Chemical Engineering, The State Key Laboratory 7
Base of Novel Functional Materials and Preparation Science, Ningbo University, 8
Ningbo 315211, PR China 9
b School of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, 10
Shenzhen 518055, PR China 11
12
13
Abstract 14
A novel sandwich-type electrochemiluminescence (ECL) immunosensor was 15
developed for highly sensitive and selective determination of tumor markers based on 16
biological barcode mode. N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and the second 17
antibody (Ab2) were simultaneously immobilized on conductive nanospheres to 18
construct ABEI/Ab2-CNSs probes, which could form sandwich immunocomplex by 19
Ab2 and emit ECL signals by ABEI. The gold layer coated on the surface of the 20
conductive nanospheres could extend the outer Helmholtz plane (OHP) of the ECL 21
* Corresponding author. Tel: +86 574 87600798.
E-mail address: [email protected] (Z. Guo).
2
immunosensor effectively. Benefited from it, all ABEI molecules immobilized on 22
conductive nanospheres would act as biological barcode to give in-situ ECL signals 23
without interfering with the activity of the second antibody. In such a case, the 24
sensitivity of the ECL immunosensor would be greatly improved because an antigen 25
molecule would correspond to ECL signals of thousands of ABEI molecules. Using 26
prostate specific antigen (PSA) as a model tumor marker, the ECL intensity was 27
found to increase with the logarithm of PSA concentration with a wide linear range 28
from 0.04 to 10 fg/mL. In addition, specificity, stability, reproducibility, regeneration 29
and application were satisfactory. Therefore, this developed ECL immunosensor has a 30
potential for practical detection of disease-related proteins besides tumor markers in 31
the clinical diagnostics. 32
Keywords: Electrochemiluminescence immunosensor; Tumor markers; Biological 33
barcode mode; Conductive nanospheres (CNSs) 34
35
36
1. Introduction 37
Tumor markers are biochemical substances which are produced by either the 38
tumor itself or the surrounding normal tissue as a response to tumor cells (Freedland, 39
2011; Wickström et al., 2011). Since the presence of tumor markers in serum or other 40
body fluids in response to precancerous or cancerous conditions induces an array of 41
biochemical processes, they are often used for evaluating disease process, recurrence, 42
metastasis and prognosis (Marta et al., 2013; Liu and Ma, 2013; Qu et al., 2013; 43
3
Xiang and Lu, 2012), and the low concentrations of tumor markers may be related to 44
the early stage of cancerous conditions. Therefore, it is crucial to develop diagnostic 45
tools for the detection of very low concentrations of these markers in healthy humans, 46
sub-healthy humans or patients to identify the early stage. 47
Lots of methods and strategies have been developed for the detection of tumor 48
markers, including radioimmunoassay (RIA) (Tyan et al., 2013; Su et al., 2011), 49
chemiluminescence immunoassay (CLIA) (Tian et al., 2010; Yang et al., 2010b), 50
enzyme-linked immunosorbent assay (ELISA) (Darwish et al., 2013; Chalupova et al., 51
2013; Saadi et al., 2013), chemiluminescent enzyme immunoassay (CLEIA) (Dong et 52
al., 2012; Xiao et al., 2009) and time-resolved fluorescence immunoassay (TRFIA) 53
(Hou et al., 2012; Lu et al., 2012; Niu et al., 2011), etc. However, there are still some 54
problems challenging their application: (1) the sensitivity is not high enough to 55
determine tumor markers at low level, (2) radioactive or toxic markers are needed, (3) 56
experimental procedures are complex, (4) detection time is long, and (5) instruments 57
used are expensive, and so on. 58
Electrochemiluminescence (ECL) is a valuable detection method, which has 59
been applied extensively due to its acknowledged advantages such as versatility, 60
simplified optical setup, very low background signal, and good temporal and spatial 61
control (Richter, 2004). Ru(bpy)32+ and luminol are the most widely used ECL 62
systems. Besides, N-(4-aminobutyl)-N-ethylisoluminol (ABEI), a derivative of 63
isoluminol with similar ECL mechanism as luminol, is brought to our attention 64
because of its relatively high ECL efficiency as bioassay label compared with luminol 65
4
(Tian et al., 2009; Yang et al., 2002). Based on ECL, electrochemiluminescence 66
immunoassay (ECLIA) has gained much attention in recent years due to its wide 67
dynamic range, high sensitivity, low background, environmentally friendly labels, 68
simple instrumentation and easy methodology. In many cases, a sandwich-type is the 69
commonly used mode of ECLIA. In most of the previous related research work 70
focused on labeling materials and labeling methods such as: using 71
Ru(bpy)32+-encapsulated silica nanosphere (Yang et al., 2010a; Qian et al., 2010), 72
quantum dots functionalized graphene sheet (Liu et al., 2013), dendrimer multiply 73
labeled luminol on Fe3O4 nanoparticles (Li et al., 2013a), 74
N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold nanoparticles (Shen et al., 75
2011; Tian et al., 2009), Au-MSN-HRP-Ab2 composites (Wei et al., 2010) and 76
Ru-AuNPs/graphene (Li et al., 2013b) as labels, basing on energy transfer between 77
quantum dots and quantum dots (Guo et al., 2012), between Ru(bpy)32+ and quantum 78
dots (Hao et al., 2012) and between quantum dots and gold nanoparticles (Qian et al., 79
2013), using quantum dots as labels and graphene as conducting bridge (Guo et al., 80
2013), etc. However, using conductive nanospheres multi-functionalized by the 81
second antibody and luminophore as labels was seldom reported. 82
Biological barcode technology was first reported by Mirkin and his colleagues 83
(Thaxton et al., 2009; Nam et al., 2002; Oh et al., 2006), in which gold nanoparticles 84
multi-functionalized with specific probes can identify target analyte specifically and a 85
large number of oligonucleotide strands. Those oligonucleotide strands with identical 86
sequences playing a role of surrogate target to amplify the detection sensitivity 87
5
effectively are termed as barcode. Due to the efficient amplification, the detection of 88
proteins and DNA by bio-barcode assay can reach the attomolar level (Goluch et al., 89
2006; Nam et al., 2004). However, this ultrasensitive method suffers from 90
cumbersome steps greatly. 91
Herein, we present a novel sandwich-type electrochemiluminescence (ECL) 92
immunosensor for the ultrasensitive detection of tumor markers based on biological 93
barcode mode, using conductive nanospheres multi-functionalized with the second 94
antibody and luminophore ABEI. Thousands of ABEI molecules were labeled as 95
barcode, and nearly all of them could emit ECL signals efficiently with the help of 96
conductive nanospheres. Therefore, the detection sensitivity was improved greatly, 97
with a detection range of 0.04 to 10 fg/mL using prostate specific antigen (PSA) as 98
model analyte. 99
100
101
2. Experimental 102
2.1. Apparatus 103
A laboratory-built ECL detection system, as described previously (Guo and Gai, 104
2011) was used in this study. A three-electrode system, including bare or modified 105
gold electrode (� = 3 mm), platinum wire electrode and Ag/AgCl electrode as 106
working electrode, counter electrode and reference electrode, respectively was used. 107
Electrochemical impedance spectroscopy (EIS) experiment was performed with a CHI 108
660B electrochemistry workstation (Chenhua Instrument Company, Shanghai, China). 109
6
The morphology of the nanospheres used was characterized using a SU70 scanning 110
electron microscope (SEM, Hitachi, Toyko, Japan). 111
2.2. Reagents and materials 112
N-(4-aminobutyl)-N-ethylisoluminol (ABEI), bovine serum albumin (BSA), 113
tetraethoxysilane (TEOS), glutaraldehyde (GLD) and 2-aminoethanethiol were 114
purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroauric acid 115
(HAuCl4·4H2O), (3-aminopropyl)-triethoxysilane (APS), sodium citrate, 116
hydroxylammonium chloride were obtained from Shanghai Chemical Reagent Co., 117
Ltd. (Shanghai, China). Prostate specific antigen (PSA), the first antibody anti-PSA 118
(Ab1) and the second antibody anti-PSA (Ab2) were purchased from Zhengzhou 119
Biocell Biotechnology Company (Zhengzhou, China), and stored at –20 °C before use. 120
Carbonate buffer solution (CBS, pH 9.6) containing 0.015 mol/L sodium carbonate 121
and 0.035 mol/L sodium bicarbonate, and 1 mmol/L H2O2, was used as the working 122
solution for the ECL measurement. All other reagents were of analytical grade. 123
Ultra-pure water (18 M� cm), obtained from a Heal Force PW ultrapure water system 124
(Heal Force Bio-Meditech Holdings Limited, Hong Kong, China), was used in the 125
experiment throughout. 126
2.3. Synthesis of conductive nanospheres (CNSs) 127
Amino-functionalized SiO2 nanospheres were prepared as described previously 128
(Stober and Fink, 1968; Pan et al., 2012; Jiao et al., 2012) with some modifications. 129
Briefly, ethanol, water and concentrated ammonia-water were mixed to about 100 mL 130
with a volume ratio of 88:8:1, and kept stirred. Then 4.5 mL of TEOS was added and 131
7
allowed to react for 10 h to obtain SiO2 nanospheres. After centrifugal washing with 132
water, the precipitate was dispersed in anhydrous ethanol to form a 50 mg/mL 133
suspension. Finally, amino-functionalized SiO2 nanospheres were obtained by adding 134
2 mL of APS into 20 mL of the above suspension and refluxing for 4 h at 80 °C in 80 135
mL of anhydrous ethanol with stirring, the morphology of which was monitored by 136
SEM, as shown in Fig. 1A. 137
Conductive nanospheres (CNSs) were synthesized according to the literatures 138
(Hu et al., 2005; Wei et al., 2010). A 400 mL solution containing 0.0125 mg/mL gold 139
nanoparticles (AuNPs) with a diameter of 13 nm, prepared as described previously 140
(Polte et al., 2010), and 0.8 mg/mL of amino-functionalized SiO2 nanospheres was 141
stirred for 12 h, washed until no AuNPs could be found in the supernatant, and then 142
aged for 5 d at 4 °C. Subsequently, 20 mL of the obtained solution was mixed with 143
300 mL of HAuCl4/K2CO3 solution containing 0.18 mg/mL HAuCl4 and 0.25 mg/mL 144
K2CO3, followed by adding 2 mmol/L hydroxylammonium chloride aqueous solution 145
till the color of the mixture turned from colorless to reddish brown to obtain the CNSs. 146
The morphology of the obtained CNSs with a diameter of 60 – 90 nm was monitored 147
by SEM and is presented Fig. 1B. 148
149
150
2.4. Preparation of ABEI/Ab2-CNSs probes 151
The schematic diagram of the preparation process of ABEI/Ab2-CNSs probes is 152
illustrated in Fig. 2A. Firstly, a 1 mL mixture of 3 mg/mL CNSs and 7.7 mg/mL of 153
Preferred position for Fig. 1
8
2-aminoethanethiol were incubated for 10 h at room temperature and then centrifuged. 154
Then, the precipitate was dispersed and incubated in 1 mL of 0.12 mg/mL GLD for 1 155
h at room temperature and centrifuged, and the precipitate was then dispersed in 1 mL 156
mixture solution containing 0.2 mg/mL ABEI and 8 �g/mL Ab2. After incubating for 157
another 1 h at room temperature and centrifugation, ABEI/Ab2-CNSs probes were 158
finally obtained. 159
Non-conductive probes were prepared using the same procedures as described in 160
Section 2.4 using amino-functionalized SiO2 nanospheres instead of conductive 161
nanospheres (Fig. 2B). 162
2.5. Fabrication of the ECL immunosensor 163
Fig. 2C presents the fabrication protocol of the ECL immunosensor. Firstly, the 164
bare gold electrode was polished with 1.0, 0.3 and 0.05 �m Al2O3 slurries in sequence, 165
ultrasonicated, and scanned from 0 to 1.6 V in 0.5 mol/L H2SO4 until stable cyclic 166
voltammograms were obtained. Subsequently, the cleaned gold electrode was rinsed 167
and incubated in 0.1 mol/L 2-aminoethanethiol for 10 h at 4 °C. After rinsing, the 168
electrode was incubated in 2.5% GLD for 1 h at 4 °C, rinsed, and incubated in 50 169
�g/mL Ab1 for 12 h at 4 °C. Finally, the electrode obtained was rinsed and incubated 170
in 2% BSA for 1.5 h at 4 °C to block non-specific binding sites. The ECL 171
immunosensor thus obtained was ready for the immunoassay.172
2.6. ECL Detection 173
As shown in Fig. 2C, the ECL immunosensor was incubated in 50 μL of a 174
sample solution containing PSA for 40 min at 37 °C, thoroughly washed with 0.05 175
9
mol/L CBS to remove unbound PSA, and then incubated in 50 �L of ABEI/Ab2-CNSs 176
probes or non-conductive probes at 37 °C for 40 min to form the final sandwich 177
immunocomplex. Then, the electrodes were scanned using cyclic voltammetry in 0.05 178
mol/L CBS containing 1 mmol/L H2O2 from 0 to 1 V with a scan rate of 100 mV/s, 179
and the ECL signals were recorded for measurement. 180
181
182
2.6. Determination of PSA in real samples 183
Samples of human serum and saliva were collected and carefully determined 184
using this method. Samples, in which PSA could not be detected, were selected as 185
blank samples. Real samples were obtained by spiking standard PSA solution in blank 186
human serum and saliva samples, and then used for the assay as mentioned above. 187
188
189
3. Results and discussion 190
3.1. Construction and characteristics of the ECL immunosensor based on biological 191
barcode mode 192
The schematic diagram of the assay process for the proposed ECL 193
immunosensor based on biological barcode mode is depicted in Fig. 2. In case of 194
non-conductive probe (Fig. 2B), thousands of ABEI molecules and the second 195
antibody were immobilized simultaneously on the surface of amino-functionalized 196
SiO2 nanospheres. However, the ECL signal was low (curve b in Fig. 2), because the 197
Preferred position for Fig. 2
10
diameters of amino-functionalized SiO2 nanospheres were tens of nanometers at least, 198
which were much larger than the thickness of outer Helmholtz plane (OHP). OHP is 199
considered as the position of electronic exchange between solvated ions and the 200
electrode in electrode kinetics, i.e., OHP is the place where electrochemically active 201
species have to reach. Electrochemical reactions would occur when the distance 202
between electrochemically active species and the electrode is no more than that 203
between OHP and the electrode; otherwise no electrochemical reaction would occur 204
(Bard and Faulkner, 2000). Therefore, most of ABEI molecules immobilized on the 205
surface of amino-functionalized SiO2 nanospheres were beyond OHP, and they could 206
not emit ECL signals, termed as ‘ineffective ABEI’. Therefore, it is understandable 207
that the ECL signal was not so high. As to ABEI/Ab2-CNSs probe (Fig. 2A), 208
thousands of ABEI molecules and the second antibody were immobilized 209
simultaneously on the surface of conductive nanospheres. After the sandwich 210
immunocomplex was constructed, most of ABEI/Ab2-CNSs probes were close to or 211
even contacted the surface of the electrode. Thus, OHP would be extended due to the 212
existence of gold layer on the surface of conductive nanospheres, similar to that the 213
electrode surface was extended. Under such a circumstance, nearly all ABEI 214
molecules immobilized on the surface of conductive nanospheres were in the area 215
between the OHP and the electrode, and they could emit ECL signals, termed as 216
‘effective ABEI’. Therefore, at the same concentration of tumor marker PSA, the ECL 217
intensity was improved about 7 times (curve a in Fig. 2). 218
11
Compared with conventional sandwich-type ECLIA method, in which 219
luminophore is labeled directly on the second antibody, this proposed ECL 220
immunosensor based on biological barcode mode has two significant advantages: (1) 221
The sensitivity could be improved greatly. An antigen molecule corresponds to the 222
ECL signal emitted by thousands of luminophore molecules labeled as barcode on the 223
surface of conductive nanospheres. In contrast, in the conventional method, an antigen 224
molecule corresponds to the ECL signal emitted by no more than tens of luminophore 225
molecules. (2) The immunoactivity of the second antibody would not be affected 226
because luminophore molecules were labeled on the surface of conductive 227
nanospheres. In case of conventional one, at most tens of luminophore molecules 228
could be labeled to maintain the immunoactivity of the second antibody (Fung and 229
Wong, 2001; Pei et al., 2013; Zhang et al., 2008). In addition, this proposed ECL 230
immunosensor based on biological barcode mode was much easier than biological 231
barcode immunoassay, because luminophore as barcode could give real-time ECL 232
signal while the latter must employ cockamamie PCR amplification steps. 233
Electrochemical impedance spectroscopy (EIS) was employed to monitor the 234
interface properties of the electrodes in the assembly process. The impedance 235
spectrum comprises a line at low-ac modulation frequency and a semicircle at high-ac 236
modulation frequency, while the latter indicates the electron transfer resistance Ret. As 237
shown in Fig. 3A, Ret increased a little compared with the bare gold electrode (curve a) 238
when 2-aminoethanethiol and GLD was assembled (curve b). Then, Ret increased 239
remarkably when Ab1, BSA, PSA and non-conductive probe were immobilized 240
12
successively (curves c, d, e and f), due to their gradual increase of hindrance to the 241
interfacial electron transfer (Li et al., 2008). Finally, in contrast to non-conductive 242
probes, Ret decreased obviously when ABEI/Ab2-CNSs probes were assembled 243
successfully on the modified electrode (curve g) owing to the gold layer coated on the 244
surface of CNSs that improved the electrical conductivity of the electrode interface 245
and reduced Ret effectively. 246
The ECL behaviors of the immunosensor were recorded step by step in 0.05 247
mol/L pH 9.6 CBS containing 1.0 mmol/L H2O2. As shown in Fig. 3B, no ECL signal 248
was found when 2-aminoethanethiol, GLD, Ab1, BSA and PSA were immobilized on 249
the electrode successively (curves a-e), due to the absence of luminophore. When 250
non-conductive probes were connected to PSA/Ab1/GLD/2-aminoethanethiol/Au 251
electrode, ECL signal was observed owing to the ECL reaction of ABEI molecules 252
(curve f). However, the ECL intensity was not high because the nanospheres were so 253
big that most of ABEI molecules immobilized on them were far away from the 254
electrode surface and beyond the space domain of the OHP. With conductive 255
nanospheres (curve g), the ECL intensity was increased about 7 times, attributing to 256
the gold layer coated on the surface of CNSs that extend the OHP effectively, and 257
thus nearly all the ABEI molecules immobilized on CNSs participated in ECL 258
reactions. 259
260
261
3.2. Optimization of experimental conditions 262
Preferred position for Fig. 3
13
The performance of the immunosensor mainly depends on pH value of working 263
solution, concentration of H2O2, incubation time and time interval between two cyclic 264
voltammetric periods. To obtain an optimal ECL signal, the effects of the above 265
factors were investigated by detecting PSA solution at the concentration of 0.4 fg/mL. 266
The effect of pH on the ECL intensity was examined in the range of 8.94 – 9.90 267
in 0.05 mol/L CBS. As shown in Fig. 4A, the maximum ECL intensity was obtained 268
when the pH was 9.6. The ECL reaction of ABEI in alkaline solution was markedly 269
improved by the addition of H2O2 (Fig. 4B). The reason is that ABEI deprotonates in 270
alkaline solution to form an anion that can undergo electrochemical oxidation. The 271
intermediate species obtained undergoes further electro-oxidation in the presence of 272
H2O2 to produce an excited state, which produces the ECL emission finally (Richter, 273
2004; Arai et al., 1999). In addition, the presence of H2O2 not only enhanced the 274
sensitivity of the ECL reaction greatly, but also made the ECL reaction perform at a 275
relatively low potential (Yang et al., 2002). Therefore, the ECL intensity increased 276
with the increase of the H2O2 concentration and reached the maximum at 1 mmol/L. 277
The effect of incubation time was also investigated (Fig. 4C). When the incubation 278
time was longer than 40 min, the ECL intensity did not increase with the increasing 279
incubation time because the reaction was almost completed. Finally, the time interval 280
between two cyclic voltammetric periods was examined, and the results are shown in 281
Fig. 4D. The ECL intensity decreased when the time interval was 40 s or 50 s; 282
whereas, the ECL signal tended to be stable when the time interval was 60 s or more, 283
due to more effective diffusion of H2O2 in a longer time interval. Therefore, the 284
14
optimal experimental conditions of this assay were as follows: 0.05 mol/L CBS at pH 285
9.6 containing 1.0 mmol/L H2O2, 40 min incubation time and 60 s time intervals 286
between two cyclic voltammetric periods. 287
288
289
3.3. Sensitivity and linear range 290
As presented in Fig. 5A, the response ECL intensity increased with an increase 291
in the concentration of PSA (curve a�j). A good linear relationship between the ECL 292
intensity (y) and the logarithm of the analyte PSA concentrations (CPSA) in the range 293
from 0.04 to 10 fg/mL (n = 5) was observed (Fig. 5B). The regression equation was y 294
= 1480.48 + 370.29*log CPSA (fg/mL) with a correlation coefficient r of 0.9987; 295
indicating that the proposed immunosensor has an excellent analytical performance, 296
including very low detection level and wide linear ranges over two orders. To our best 297
knowledge, the sensitivity of this method for PSA is much higher than the lowest 298
detection limit of 0.6 pg/mL reported in the previous literatures (Choi et al., 2013; 299
Ahmed and Azzazy, 2013; Li et al., 2013c; Dey et al., 2012). 300
301
302
3.4. Specificity, stability, reproducibility and regeneration of the immunosensor 303
The ECL immunosensor was considered of having a high specificity for PSA, 304
which depended on the specific binding between PSA and its corresponding antibody. 305
Various species of interfering proteins including human immunoglobulin (hIgG), 306
Preferred position for Fig. 4
Preferred position for Fig. 5
15
BSA, alfa-fetoprotein (AFP) and carcino-embryonic antigen (CEA) were used to 307
further investigate the specificity of the proposed immunosensor. Results showed that 308
no ECL signal could be found when hIgG, BSA, AFP and CEA at the concentration 309
of 1 �g/mL were detected. Therefore, the specificity of the immunosensor developed 310
was acceptable. 311
The stability of the immunosensor stored in 0.01 M PBS (pH 7.4) containing 312
0.1% NaN3 at 4 °C was investigated by periodical checking of its relative activity. 313
The initial value of ECL signal for 1 fg/mL PSA was obtained when the 314
immunosensors were constructed freshly. After a storage period of a month, the ECL 315
intensity for the detection of 1 fg/mL PSA was 90.5% of the initial value. Thus, the 316
immunosensor has acceptable storage stability. 317
The reproducibility of the immunosensor was evaluated by detecting 1 fg/mL 318
PSA with five equally prepared immunosensors. The relative standard deviation (RSD) 319
of the measurements for the five immunosensors was 6.9%, indicating the excellent 320
precision and reproducibility of the immunosensor. 321
Regeneration of the immunosensor was examined by detecting 1 fg/mL PSA 322
with a same immunosensor. The immunosensor was regenerated by dipping into 0.2 323
mol/L glycine-hydrochloric acid (Gly-HCl) buffer solution (pH 2.8) for 8 min to 324
break the antibody-antigen linkage. The consecutive measurements were repeated ten 325
times, an average recovery of 91.8% and an intra-assay RSD of 8.2% were acquired. 326
The results demonstrated that the proposed immunosensor could be regenerated and 327
used for at least ten times. 328
16
3.5. Application of ECL immunosensor in human serum and saliva samples 329
Determination of the low concentrations of tumor marker in the human serum 330
and saliva is very important in the early diagnosis of cancer. In order to investigate the 331
applicability and reliability of the prepared ECL immunosensor for clinical 332
applications, recovery experiments were performed by detection of PSA spiked in 333
human serum and saliva samples. As shown in Table 1, an acceptable recovery 334
obtained was in the range of 97.3 111.3%, indicating that the developed ECL 335
immunosensor is an efficient tool for ultrasensitive determination of PSA in human 336
serum and saliva. 337
338
339
340
341
4. Conclusions 342
In this work, an ultrasensitive ECL immunosensor based on biological barcode 343
mode for the detection of very low concentrations of the tumor marker PSA was 344
constructed. ABEI/Ab2-CNSs probes was used to (1) recognize tumor markers with 345
Ab2, (2) emit ECL signal with ABEI acting as barcode, and (3) extend OHP with gold 346
layer coated to make all ABEI immobilized effective. In such a case, a tumor marker 347
antigen corresponded to the ECL signal emitted by thousands of ABEI molecules, 348
lowering the detection limit down to fg/mL level. In addition, the specificity, stability, 349
reproducibility, regeneration and application of this immunosensor were validated. 350
Preferred position for Table 1
17
Therefore, the proposed immunosensor based on biological barcode mode not only 351
built a potential detection plateau for disease-related proteins in the clinical 352
diagnostics, but also opened a new avenue to label a large number of tags in 353
immunoassay. 354
355
356
Acknowledgments 357
Financial supports from National Natural Science Foundation of China 358
(81273130, 81072336), Science and Technology Department of Zhejiang Province of 359
China (2012R405061, 2012C23101), Ningbo Science and Technology Bureau 360
(2011C50037), Key project of Shenzhen Polytechnic (2210K3070014) and 361
“Qianbaishi Candidate” fund for Higher Education of Guangdong Province are 362
gratefully acknowledged. This work was also sponsored by K.C. Wong Magna Fund 363
in Ningbo University. 364
365
366
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460
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Figure legend 461
462
Fig. 1. The SEM image of (A) SiO2 nanospheres and (B) SiO2/Au conductive 463
nanospheres (CNSs). 464
Fig. 2. The schematic diagram for (A) the preparation of the ABEI/Ab2-CNSs probe 465
with SiO2/Au conductive nanosphere, (B) the preparation of the 466
non-conductive probe with amino-functionalized SiO2 nanosphere and (C) the 467
fabrication of the ECL immunosensor using the ABEI/Ab2-CNSs probe and 468
the non-conductive probe respectively. 469
Fig. 3. EIS (A) and ECL profiles (B) of (a) bare Au electrode, (b) (a) + 470
2-aminoethanethiol + GLD, (c) (b) + Ab1, (d) (c) + BSA, (e) (d) + PSA (1 471
fg/mL), (f) (e) + non-conductive probes, and (g) (e) + ABEI/Ab2-CNSs probes. 472
Experimental conditions of EIS: 5 mmol/L Fe(CN)64-/3- solution (0.05 mol/L 473
PBS, pH 7.0); frequency range: between 0.01 and 100,000 Hz; signal 474
amplitude: 5 mV. Experimental conditions of ECL: 0.05 mol/L CBS at pH 9.6 475
containing 1.0 mmol/L H2O2; 40 min incubation time; and 60 s time intervals 476
between two cyclic voltammetric periods. 477
Fig. 4. Effect of (A) pH value; (B) H2O2 concentration; (C) incubation time; and (D) 478
time interval between two cyclic voltammetric periods on the ECL intensity. 479
Fig. 5. (A) ECL profiles of the immunosensor in the presence (a�j) of different 480
concentrations of PSA (fg/mL): (a) 0.04; (b) 0.1; (c) 0.2; (d) 0.3; (e) 0.4; (f) 1; 481
(g) 2; (h) 3; (i) 4; (j) 10. (B) Calibration curve for PSA determination. The 482
23
experimental conditions were as follows: 0.05 mol/L CBS at pH 9.6 483
containing 1.0 mmol/L H2O2, 40 min incubation time and 60 s time intervals 484
between two cyclic voltammetric periods. 485
Table 1 Recovery tests for PSA in spiked human serum and saliva samples ( sx � , n = 486
3). 487
488
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Table 1 Recovery tests for PSA in spiked human serum and saliva samples ( sx � , n = 489
3). 490
Samples Added (fg/mL) Found (fg/mL) Recovery (%)
Serum 1 0.040 0.0445 ± 0.0046 111.3
Serum 2 0.40 0.423 ± 0.040 105.8
Serum 3 4.0 3.89 ± 0.26 97.3
Saliva 1 0.040 0.0432 ± 0.0041 108.0
Saliva 2 0.40 0.427 ± 0.038 106.8
Saliva 3 4.0 4.11 ± 0.22 102.8
491 492
493
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� An ECL immunosensor based on biological barcode mode was proposed. 494 � Conductive nanospheres (CNSs) could extend the outer Helmholtz plane 495
effectively. 496 � ABEI/Ab2-CNSs probes were constructed by immobilizing ABEI and Ab2497
on CNSs. 498 � ABEI on CNSs give in-situ ECL signal as biological barcode without 499
interfering Ab2.500 � PSA could be detected in the concentration range from 0.04 to 10 fg/mL. 501
502
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