Accepted Manuscript Simultaneous electrochemical detection of multiple biomarkers using gold nanoparticles decorated MWCNTs as signal enhancers Dexiang Feng, Lihua Li, Junqing Zhao, Yuzhong Zhang PII: DOI: Reference:
S0003-2697(15)00181-5 http://dx.doi.org/10.1016/j.ab.2015.04.018 YABIO 12047
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
17 January 2015 13 April 2015 16 April 2015
Please cite this article as: D. Feng, L. Li, J. Zhao, Y. Zhang, Simultaneous electrochemical detection of multiple biomarkers using gold nanoparticles decorated MWCNTs as signal enhancers, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.04.018
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1
Simultaneous electrochemical detection of multiple biomarkers using
2
gold nanoparticles decorated MWCNTs as signal enhancers
3 4
Dexiang Fenga,b, Lihua Lia,b, Junqing Zhao a, Yuzhong Zhanga*
5 6 7 8 9
a
College of Chemistry and Materials Science, Anhui Normal University,
Wuhu 241000, People’s Republic of China b
Department of Chemistry, Wannan Medical College, Wuhu 241002,
People’s Republic of China
10 11 12 13 14
Subject category: Imunologicl Pocedures
15
Short title: Electrochemical detection of two tumor markers
16 17 18 19 20 21
* Corresponding author. Tel: +86 553 3869303; Fax: +86 553 3869303
22
E-mail address: [email protected] (Y. Zhang).
23 1
24
Abstract
25
In this work, a novel sandwich-type electrochemical immunosensor has been
26
developed for simultaneous detection of carcinoembryonic antigen (CEA) and
27
α-fetoprotein (AFP) based on metal ions labels. Au nanoparticles decorated multiwall
28
carbon nanotubes (AuNPs@MWCNTs) was used as carriers to immobilize secondary
29
antibody and distinguishable electrochemical tags of Pb2+ and Cd2+ to amplify the
30
signals. Due to the intrinsic property of high surface-to-volume ratio, the
31
AuNPs@MWCNTs could load numerous secondary antibody and lables. Therefore,
32
the multiplexed immunoassay exhibited good sensitivity and selectivity. Experimental
33
results revealed that this sandwich-type immunoassay displayed an excellent linear
34
response with linear range 0.01-60 ng mL−1 for both analytes with the detection limit
35
of 3.0 pg mL−1 for CEA and 4.5 pg mL−1 for AFP (at a signal to noise ratio of 3) ,
36
respectively. The method was successfully applied for the determination of AFP and
37
CEA levels in clinical serum samples.
38 39 40 41 42 43 44
Keywords: Gold nanoparticles decorated multiwall carbon nanotubes; Pb2+ and Cd 2+;
45
Multiplex assay; Carcinoembryonic antigen; α-fetoprotein 2
46
Introduction
47
Precise and early determination of multiplex tumor markers could greatly
48
improve the treatment efficiency of many cancers in clinical diagnosis. Therefore,
49
simultaneous detection of multiplex tumor markers related to a certain cancer in
50
human serum has attracted great attention in biomedical field. In recent years, various
51
immunoassay methods for simultaneous detection of multiplex tumor markers have
52
been developed [1-5]. Among these methods, electrochemical immunoassay has
53
become one of the predominant analytical methods due to high sensitivity, inherent
54
simplicity and low-cost [6-8].
55
In order to realize successfully electrochemical simultaneous multiplexed
56
immunoassays, an important issue is to search distinguishable signal probe as trace
57
labels [9, 10] in multiple labels mode. As ideal multiple-tags for proteins label, it
58
should meet two demands: one is based on the independent response achieved by each
59
target on the identical sensing interface, and the other is based on distinguishable
60
voltammetric signals to avoid interaction with analytes and the sample matrix [11].
61
Nowadays, a large number of signal tags such as enzymes, quantum dots (QDs),
62
oligonucleotide and dyes by loading on carriers for the preparation of labels have been
63
reported in electrochemical immunoassay [12]. Among them, QDs are proved to be
64
promising due to their comparable size and feasibility for surface modification [13].
65
However, QDs-based labels often not only required a tedious preparation process but
66
also involved a complicated detection step of acid dissolving process to obtain the
67
electrochemical signals [14, 15]. As a result, there is still a great challenge in 3
68
developing novel probes with simple preparation process and easy detection steps.
69
Recently, Feng et al. firstly developed a novel multianalyte electrochemical
70
immunosensor for ultrasensitive detection of cardiac troponin (cTnl) and
71
fatty-acid-binding protein (FABP) using metal ions Zn2+ and Cd2+ functionalized
72
titanium phosphate nanospheres as labes [16]. Due to metal ions highly sensitive
73
electrochemical response and easy signal obtaining process, the metal ions as labels
74
are beneficial as reported [17], but this method still involved time-consuming
75
fabrication of titanium phosphate nanospheres for metal ions exchange. Thus, the
76
development of a metal ion label with simple carriers in electrochemical
77
immunoassay is urgent.
78
Herein, we developed a simple electrochemical immunosensor for simultaneous
79
detection of AFP and CEA using the Cd 2+ and Pb2+ as multiple labels with a simple
80
carrier. Using poly (diallyldimethylammonium chloride) (PDDA) as linkage reagents,
81
the Au nanoparticles decorated MWCNTs nanocomposites (AuNPs@MWCNTs) with
82
good electric conductivity and biocompatibility [18-20] were easily fabricated not
83
only as an idea simple carrier but also as an excellent signal enhancer. As expected,
84
the immunosensor can achieve simultaneous detection of CEA and AFP in one
85
detection platform with different voltammetric peaks through square wave
86
voltammetry. The peak currents and the peak positions were dependent on the
87
concentration and type of the corresponding analytes, respectively. In addition, the
88
peak potentials were not too far, which may save the detection time. The obtained
89
immunosensor exhibited sensitive and stable response for detection of multiple tumor 4
90
markers and showed great potential in clinical applications.
91
Experimental
92 93
Materials
94 95
Carcinoembryonic antigen (CEA), CEA antibody (anti-CEA) , α-fetoprotein
96
(AFP) and AFP antibody (anti-AFP) were purchased from Biocell Biotech. Co., Ltd
97
(Zhengzhou, China) and stored in refrigerator at 4 °C. Bovine serum albumin (BSA)
98
and poly (diallyldimethylammonium chloride) (PDDA, 20%, w/w in water, MW 200
99
000-350 000) were purchased from the Sinopharm Chemical Reagent Co., Ltd
100
(Shanghai). Multiwalled carbon nanotubes with carboxylic acid groups (MWCNTs,
101
purity >95%, diameter 20-30 nm, length 10-30 µm) were obtained from Chengdu
102
Institute of Organic Chemistry (Chengdu, China). Acetic acid (HAc), sodium acetate
103
anhydrous (NaAc), Cd(NO3)2, Pb(NO3)2 and chloroauric acid (HAuCl4.4H2O) were
104
obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Phosphate
105
buffer saline (PBS) with various pH values were prepared by mixing the stock
106
solutions of 0.10 mol L-1 Na2HPO4, 0.10 mol L-1 NaH2PO4 and 0.10 mol L-1 KCl, then
107
adjusting the pH with 0.10 mol L-1 NaOH and H3PO4. The washing buffer was pH 7.0
108
PBS containing 0.05% (W/V) Tween (PBST). Blocking solution was 1% BSA. The
109
clinical serum samples were from the clinical laboratory of the Yiji Shan Hospital
110
(Wuhu, China). Twice-quartz-distilled water was used through the study.
111 5
112
Apparatus
113 114
All electrochemical measurements were performed on a CHI 650C
115
electrochemical analyzer (CH Instruments Inc., China) with a conventional
116
three-electrode system composed of a platinum auxiliary electrode, a saturated
117
calomel electrode (SCE) as reference electrode, and a bare Au or modified Au as
118
working electrode.
119
Electrochemical impedance spectra (EIS) were performed in 0.1 M PBS
120
containing 5.0 mM [Fe(CN)63−/4−] and 0.1 M KCl at a pH of 7.0. The frequency
121
ranged from 0.1 to 100 kHz, and the amplitude of the alternate voltage was 5 mV.
122
Morphologies of AuNPs , PDDA functionalized MWCNTs, AuNPs-MWCNTs
123
and AuNPs@Au were obtained with scanning electron microscopy (SEM)
124
(JEOLJSM-6700F, Hitachi, Japan). The morphologies of the materials were measured
125
on a transmission electron microscope (TEM, Hitachi-800).
126 127
Preparation of AuNPs@MWCNTs nanocomposites
128 129
The colloidal AuNPs of 16 nm diameter were prepared according to the previous
130
protocol (Seen in supplementary Fig.S1) [21]. The acid-treated MWCNTs were
131
further functionalized with PDDA according to the reported method [22]. The
132
obtained PDDA-MWCNTs (0.5 mg) (Seen in supplementary Fig.S2) were dispersed
133
in 5.0 mL of as-prepared colloidal AuNPs and stirred for 20 mins, the AuNPs were 6
134
assembled on the surface of PDDA@MWCNTs nanocomposites via electrostatic
135
interaction. After centrifugation, the AuNPs@MWCNTs composites were obtained.
136
(Seen in supplementary Fig.S3), which were further washed with water and
137
redispersed in 2 mL of 50 mM pH 9.0 Tris-HCl solution for further use.
138 139
Preparation of the immunosensing probes
140 141
Firstly, the signal anti-CEA2,1 (Ab 2,1) (200 µL, 1 mg mL-1) was added to the
142
above AuNPs@MWCNTs dispersion and stirred at room temperature for 2 h.
143
Secondly, 400 µL 1% BSA solution was added to the obtained bioconjugates and
144
allowed to react for 2 h. After centrifugation, the resulting immunocomplex was
145
further washed with PBS (0.01 M, pH=7.0) three times. Finally, the prepared
146
immunocomplex was dispersed in 2 mL 10 mM Pb(NO3)2 aqueous solution and
147
shaked for overnight, thus, the Pb2+-Ab 2,1-AuNPs@MWCNTs bioconjugates were
148
synthesized. The Cd 2+- anti-AFP2,2 (Ab2,2)-AuNPs@MWCNTs was synthesized using
149
the similar method.
150 151
Fabrication of the immunosensor
152 153
Before modification, the gold electrode was treated according to our previous
154
reported procedure [23]. The modified electrode was immersed in 0.1 M KNO3
155
solution containing 5.0 mM HAuCl4 and electrochemical deposited 300 s at - 200 mV. 7
156
Thus, AuNPs were distributed on the surface of the gold electrode. The modified
157
electrode was denoted as AuNPs/Au.
158
Immobilization of anti-CEA (Ab1,1) and anti-AFP (Ab 1,2) was performed by
159
dropping a mixture of Ab1,1 and Ab1,2 (10.0 µL, 200 µg mL−1) solution onto the
160
surface of the AuNPs/Au, and kept it for 12 h in a refrigerator at 4 °C, the resulting
161
electrode was washed PBST an PBS to remove physically absored Ab 1. Following
162
that, the modified electrode was incubated with 1% BSA solution for 50 min at 37 °C
163
to block any possible remaining active sites against non-specific adsorption, and
164
washed several times with PBST and PBS. The obtained immunosensor was stored at
165
4 °C prior to use.
166 167
Electrochemical measurements
168 169
The schematic preparation of the immunosensing probes is illustrated in scheme
170
1. SEM was performed to characterize the shape of the AuNPs/MWCNTs (inset of
171
Scheme 1A). The preparation procedure of the immunosensors for CEA and AFP
172
determination as follows: the immunosensor was incubated with the mixture of CEA
173
and AFP solution or serum samples with various concentrations for 50 min at 37 °C,
174
following by washing with PBST and PBS, and then it was incubated with mixture of
175
1:1
176
solution for another 50 min at 37 °C, following by washing with PBST and PBS.
177
Finally, the electrode was transferred into acetate buffer solution (0.2 M, pH=4.5).
diluted
Pb2+-Ab2,1-AuNPs@MWCNTs
8
and
Cd 2+-Ab2,2-AuNPs@MWCNTs
178
The SWV was used to obtain the response signal of the immunosensor. SWV scan
179
from 0 to -800 mV(vs.Ag/AgCl) with a pulse amplitude of 25 mV, a pulse frequency
180
of 15 Hz, and a quiet time of 2 s was performed to record the electrochemical
181
responses at −0.54 and −0.71 V for simultaneous, quantitative measurement of CEA
182
and AFP.
183 184
Results and discussion
185 186
Investigation of the assembled process of the immunosensor with SEM
187
and cyclic voltammetry (CV) techniques
188 189
Fig. 1 A and 1B showed the surface morphologies of bare Au electrode and
190
AuNPs modified Au electrode, respectively. After the electrode was electrodeposited
191
at -0.2 V for 300 s in 0.1 M KNO3 solution containing 5.0 mM HAuCl4, the spherical
192
AuNPs were assembled on the surface of Au electrode, which was beneficial for
193
improving the immobilized amounts of capture antibody due to the good affinity of
194
AuNPs for biomolecules on the electrode surface. The SEM showed a more uniform
195
spherical structure with a regular distribution of AuNPs. As shown in Fig. 1C, the
196
cyclic voltammograms of bare Au electrode and AuNPs modified Au electrode in 0.5
197
M H2SO4 solution, the peak current at AuNPs modified electrode was much higher
198
than that at bare Au electrode. The results revealed that the AuNPs increased the
199
effective surface area of electrode significantly, at the same time, the effective surface 9
200
areas of electrodes can be obtained from the coulombic integration of the reductive
201
waves of gold oxide.
202 203
Electrochemical characterization of the assemble process of the
204
immunosensor
205 206
The CVs characterization of the immunosensor at different step was exhibited in
207
Fig. 2 (A), a pair of distinct redox peaks was observed due to the oxidation and
208
reduction of the redox couple Fe(CN)6-4/-3 on the bare Au electrode (curve a). After the
209
electrode was modified with AuNPs, the peak current of CVs increased sharply (curve
210
b) attribute to the excellent ability of electron transfer of the AuNPs. When the
211
electrode was incubated in the mixture of capture Ab 1,1 and Ab1,2, the mixture of CEA
212
and AFP, the mixture of 1:1 diluted Pb2+-Ab 2,1-AuNPs @MWCNTs and
213
Cd2+-Ab2,2-AuNPs@MWCNTs bioconjugates,
214
decreased step by step (curve c, d, e), this could be ascribed to form a sandwich-type
215
immunocomplex, and the immunocomplex increases with the increment of the CEA
216
and AFP concentration in the sample. The insulating layer of proteins hinders
217
interfacial electron transfer. On the other hand, the stepwise construction process of
218
the immunosensor was characterized with an electrochemical impedance spectrum
219
(EIS) (Fig. 2B). The EIS include a semicircular portion and a linear portion, the
220
diameter of the semicircle at higher frequencies corresponds to the electron-transfer
221
resistance (Ret), and the linear part at lower frequencies corresponds to the diffusion 10
successively.
The redox peaks
222
process. It could be observed that the bare electrode displayed a small semicircle with
223
a Ret of about 250 Ω (curve a). After the bare electrode modified with AuNPs, the Ret
224
decreased to 120Ω(curve b), indicating that the AuNPs was assembled on the
225
electrode surface. However, as the electrode was incubated in the mixture of capture
226
Ab 1,1 and Ab1,2, the mixture of CEA and AFP, the mixture of 1:1 diluted
227
Pb2+-Ab 2,1-AuNPs@MWCNTs and Cd 2+-Ab 2,2-AuNPs@MWCNTs bioconjugates,
228
successively, the Ret increased step by step (curve c, d, e). The results were in good
229
agreement with the results obtained from CVs. Based on above results, the
230
simultaneous detection of CEA and AFP was possible.
231 232
Optimization of experimental conditions
233 234
The electrochemical performance of the immunosensor would be influenced by
235
many factors. Therefore, some experiment parameters were investigated (such as pH,
236
incubation time). Fig. 3A and B show the pH value could affect electrochemical
237
behavior of the Cd2+ and Pb2+. The pH value of detection solution not only has a great
238
influence on the activity of the antigens and antibodies, but also on the
239
electrochemical behavior of Cd 2+ and Pb2+. It could be observed clearly that the
240
reduction peak current of Cd 2+ and Pb 2+ was increased with the pH value increased
241
from 3.0 to 4.5, and then decreased. When the pH was 4.5, the reduction peak current
242
of Cd2+ and Pb 2+ reached the maximum value. This might be because metal ions
243
combined with hydroxide ions and became inactive at pH above 4.5. Herein, pH of 11
244
4.5 was chosen in this study.
245
Fig. 3C and D showed the response of the immunosensor changed with the
246
incubation times range from 10-60 min. It could be clearly observed that the reduction
247
current of Cd 2+ and Pb2+ increased with increasing incubation time and trended to
248
reach a plateau after 50 min, exhibiting a saturated binding between the antigen and
249
the primary antibody on electrode surface. Therefore, subsequent experiments
250
employed 50 min as the optimum time for all the incubation steps of the assay.
251 252
Analytical performance
253 254
Under optimized assay conditions, the reduction peaks of Pb2+ and Cd2+ were
255
found to be proportional to the concentration of CEA and AFP in the incubation
256
solution. Under the optimum conditions, the SWV peaks for simultaneous detection of
257
CEA and AFP increased with the increment of CEA and AFP concentrations. The
258
calibration plots displayed a good linear relationship between the reduction peaks and
259
the concentration of analytes in the range of 0.01-60 ng mL-1 for both CEA and AFP
260
(seen Fig. 4B and C). The regression coefficients were 0.9911 and 0.9962,
261
respectively. The detection limit of CEA and AFP were 3.0 pg mL−1 and 4.5 pg mL−1
262
(at 3σ), respectively. Which were lower than those of metal ions tagged
263
immunocolloidal gold (4.6 pg mL-1 for CEA and 3.1 pg mL-1 for AFP) [8], HRP
264
functionalized Pt hollow nanospheres (50 pg mL-1 for CEA and 80 pg mL-1 for AFP)
265
[24], CdS/DNA and PbS/DNA nanochains as labels (3.3 pg mL-1 for CEA and 7.8 pg 12
266
mL-1 for AFP) [25], SiO2@C-dots label-electrochemiluminescence (6.0 pg mL-1 for
267
CEA and 5.0 pg mL-1 for AFP) [26], immunochromatographic test strip (2.0 ng mL-1
268
for CEA and 3.0 ng mL-1 for AFP) [27], and time-resolved immunofluorometric assay
269
(240 pg mL-1) quantum dot barcode-based electrochemical immunoassay (3.3 pg mL-1)
270
for CEA [28, 29] reported in previous studies. The results indicated that the
271
multiplexed electrochemical immunoassay enabled wide linear ranges and low LODs.
272
Furthermore, the proposed immunosensor exhibited a satisfactory electrochemical
273
performance, some possible explanations may contribute to these observations. Firstly,
274
the AuNPs@MWCNTs nanocomposites as good carriers have a large area to provide
275
a biocompatible microenvironment for the immobilization of antibody and further
276
loading a large amount of lables [30]. Secondly, the outstanding electric conductivity
277
of AuNPs@MWCNTs nanocomposites can also accelerate the electron transfer.
278
Finally, the amplification of the amperometric signal output was mainly ascribed to
279
the excess Pb2+ and Cd2+ with favorable electron conductivity and chemical stability
280
were loaded on the surface of AuNPs@MWCNTs nanocomposites.
281 282
Cross-reactivity,
283
immunosensor
specificity,
reproducibility
and
stability of
the
284 285
The cross-reactivity of the immunosensor was examined by comparing the SWV
286
responses of two analytes to those containing only one analyte. Firstly, antibodies of
287
CEA and AFP were immobilized on the electrodes. Then, two control tests were 13
288
carried out as follows: (i) immunosensors was incubated with CEA only or with AFP
289
only. (ii) AFP and CEA were simultaneously monitored. Finally, they were
290
bioconjugated
291
-AuNPs@MWCNTs probes to perform the sandwich-type immunoreaction. The
292
responses of SWV were listed in Table 1. The results indicated that the detection of
293
CEA and AFP exhibited low interference and the cross-reactivity between two
294
analytes was negligible.
Pb2+-antiCEA-AuNPs@MWCNTs
with
or
Cd 2+-anti-AFP
295
The specificity of the immunosensor played an important role in analyzing
296 297
biological
samples
without
separation,
298
immunoglobulin G (IgG), BSA, glucose (Glu) and ascorbic acid (AA) were used as
299
the interferes to evaluate the specificity. To test the specificity of the immunosensor,
300
1.0 ng mL-1 CEA and AFP were mixed with 50 ng mL-1 of IgG, BSA, Glu, and AA,
301
respectively. Fig. 5 showed SWV response of pure CEA and AFP, and that obtained
302
from CEA and AFP containing an interferential substance. When the immunosensor
303
detected the mixture of 1 ng mL-1 of CEA or AFP and 50 ng mL-1 of another interferes,
304
the response signal of the mixture changed a little in contrast with CEA or AFP alone,
305
which showed that the proposed immunosensor had good selectivity for CEA and
306
AFP.
307
308 14
some
interferes
such
as
human
309
To estimate the reproducibility of the simultaneous multianalyte immunoassay,
310
the intra-assay precision was investigated by detecting five times every 5 h at four
311
different concentrations of CEA and AFP (0.05, 2, 20, and 60 ng mL−1) using identical
312
immunosensor. The coefficient of variations was 5.5%, 6.8%, 7.9%, and 9.2% at 0.05,
313
2, 20, and 60 ng mL−1 of CEA and AFP, respectively. Similarly, the inter-assay
314
precision was investigated by measuring four different concentrations of CEA and
315
AFP (0.05, 2, 20 and 60 ng mL−1) using five immunosensors. The coefficient of
316
variations was 9.6%, 8.8%, 6.1% and 8.6%, respectively, suggesting the
317
immunosensor possessed acceptable precision and reproducibility. In addition, when
318
the immunosensor was stored at 4◦ C, the stability of the immunosensor was examined
319
by testing the response after three weeks, over 90.1% of the initial responses remained
320
after three weeks for both CEA and AFP. The slow decrease in response seemed to be
321
related to the gradual deactivation of the immobilized antibody on the sensor platform.
322
The immunosensor showed acceptable stability and was favored as dual labels for
323
simultaneous detection of dual biomarkers based on SWV measurments.
324 325
Application of the immunosensor in human serum
326 327
The capability to detect practical samples is a major concern during the
328
development of a clinical diagnostic platform. Detailed process of practical samples
329
prepared as follows: the blood samples were from the venous blood and without
330
added anticoagulant, then, it was placed in dry test tube to stand for one hour. Next, 15
331
blood samples were centrifuged (2000 rpm×5 min) and precipitated, and we took the
332
upper solution to detect. In order to examine the applicability of the immunosensor for
333
practical analyses, the recovery experiments were performed by standard addition
334
methods. The standard samples of CEA and AFP were dissolved in the healthy human
335
serum (the concentrations range of the CEA and AFP are within 0.01 to 60 ng mL-1)
336
and detected the CEA and AFP in serum. The recovery is obtained within
337
94.65%-108% and 94.2%-105.7%, respectively, indicating the method is suitable for
338
serum sample analysis. (Seen in Table S1)
339
Importantly, to investigate the possibility of the newly developed method to be
340
applied for clinical analysis, several real samples were examined by the developed
341
immunoassay and the ELISA methods for determination of CEA and AFP. The serum
342
samples came from normal persons and cancer patients. These results were shown in
343
Table 2. The value obtained was in agreement with that of the ELISA methods,
344
indicating the immunosensor could be applied to serum analysis.
345 346
Conclusions
347 348
In summary, we have developed a simple and reliable electrochemical
349
immunosensor for simultaneous detection of CEA and AFP based on metal ion as
350
lables and AuNPs@MWCNTs as simple carrier and signal enhancers. Highlights of
351
the
352
nanocomposites with its exceptionally high surface area, good conductivity and
developed
immunoassay
were
as
16
follows:
firstly,
AuNPs@MWCNTs
353
biocompatibility was used as an excellent carrier for immobilizing antibodies and
354
further loading a large amount of lables. Secondly, high-content metal ions labels
355
could be detected without acid dissolution using SWV analysis technique, thus,
356
amplifying the current response effectively. Moreover, the immunosensor showed
357
good precision, high sensitivity, acceptable stability and reproducibility.
358 359
Acknowledgments This work was supported by the Special Foundation for
360
excellent doctoral fostering of Anhui Normal University-Organic Chemistry (No.
361
003061425), the National Natural Science Foundation of China (No. 20675002) and
362
the National Natural Science Foundation for Young Scholars of Anhui Province of
363
China (No. 1408085QB40).
364 365 366 367 368 369 370 371 372 373 374 17
375
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457
Multiplexed sandwich immunoassays using flow-injection electrochemiluminescence
458
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459
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464
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467
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468
electronic coding of a cancer marker, Anal. Chem. 82, (2010) 1138-1141.
469
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470
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471
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472
Sens. Actuators, B 193, (2014) 451-460.
473 474 475 476 477 478 479 480 481 482 483 484 22
485
Captions to figures:
486 487
Scheme 1 (A) The preparation procedure of immunosensing probes. (B) The
488
fabrication process of the immunosensors.
489 490
Fig. 1 SEM images of bare Au electrode (A), AuNPs/Au (B), cyclic voltammograms
491
of bare Au electrode and AuNPs modified electrode in 0.5 M H2SO4 solution (C)
492 493
Fig. 2 (A) CV responses and (B) EIS of the different modified electrodes in 0.1 M
494
PBS containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] containing 0.1 M KCl, respectively.
495
bare Au (a), electrode modified successively with AuNPs (b), mixture of capture
496
anti-CEA and anti-AFP (c), mixture of 10 ng mL-1 CEA and AFP (d), mixture of
497
Pb2+-Ab 2,1-AuNPs @MWCNTs and Cd2+-Ab2,2-AuNPs@MWCNTs (e)
498 499
Fig. 3 Effect of the pH (A) and (B), the incubation time of (C) and (D) on the
500
response of the immunosensor to CCAE=CAFP = 20 ng mL−1
501 502
Fig. 4 (A) SWV of the immunsensors for different concentrations of CEA and AFP
503
(0.01, 0.05, 2.0, 8, 20, 30, 45, 60 ng mL-1), calibration curves of the multiplexed
504
immunoassay toward (B) CEA and (C) AFP in 0.2 M HAc-NaAc (pH 4.5).
505 506
Fig. 5 Specificity of the immunosensor conditions: CCEA =1 ng mL-1, Cinterferents =50 ng 23
507
mL-1 (BSA, Glu, IgG and AA)
508 509
Table 1 Interference degree or cross-talk level
510 511
Table 2 Comparison of CEA and AFP using the proposed immunosensor and
512
reference methods
513 514 515 516
24
517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545
Scheme 1
25
546 547
548 549 550 551
Fig. 1
26
552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570
Fig. 2
27
571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592
Fig. 3
28
593 594 595
596 597 598 599 600
Fig. 4
29
601 602 603
604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628
Fig. 5
30
629 630 631
Scheme:
632 633 634
We have developed a simple and reliable electrochemical immunosensor for
635
simultaneous detection of CEA and AFP based on metal ion as lables and Au
636
nanoparticles decorated MWCNTs as signal enhancers. Due to the intrinsic property
637
of high surface-to-volume ratio, the AuNPs@MWCNTs could load numerous
638
secondary antibody and lables. So, the multiplexed immunoassay exhibited good
639
sensitivity and selectivity.
640 641
31
642 Sample type
Concentration (ng mL-1 )
Current shift at CEA position (µA)a,b
CEA
AFP
CE+AFP
Current shift at AFP position (µA)a,c
2
8.72
0.07
60
58.98
0.02
2
0.03
6.32
60
0.06
50.31
2+2
8.43
6.01
60+60
59.76
49.97
643
a
The average value of three measurements in n 0.2 M HAc-NaAc (pH 4.5).
644
b
The SWV peak current was 23.12 µA for zero CEA analyte.
645
c
The SWV peak current was 14.02 µA for zero AFP analyt.
646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668
Table 1
32
669 670 Sample no. Multiplexed immunoassaya (ng mL-1) CEA
671 672 673
AFP
ELISA (ng mL-1)
Relative deviation (%)
CEA
AFP
CEA
AFP
1
0.63±0.23
0.58±0.45
0.60
0.60
+5.00
-1.67
2
0.98±0.36
1.10±0.25
1.00
1.00
+2.00
-1.00
3
18.45±0.42
21.21±0.32
20.00
20.00
-7.75
+6.05
4
41.12±0.72
38.58±0.24
40.00
40.00
+2.80
+3.55
5
58.36±0.64
61.08±0.56
60.00
60.00
-2.77
+1.80
Table 2
674 675
33
S0003-2697(15)00181-5 http://dx.doi.org/10.1016/j.ab.2015.04.018 YABIO 12047
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
17 January 2015 13 April 2015 16 April 2015
Please cite this article as: D. Feng, L. Li, J. Zhao, Y. Zhang, Simultaneous electrochemical detection of multiple biomarkers using gold nanoparticles decorated MWCNTs as signal enhancers, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.04.018
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.
1
Simultaneous electrochemical detection of multiple biomarkers using
2
gold nanoparticles decorated MWCNTs as signal enhancers
3 4
Dexiang Fenga,b, Lihua Lia,b, Junqing Zhao a, Yuzhong Zhanga*
5 6 7 8 9
a
College of Chemistry and Materials Science, Anhui Normal University,
Wuhu 241000, People’s Republic of China b
Department of Chemistry, Wannan Medical College, Wuhu 241002,
People’s Republic of China
10 11 12 13 14
Subject category: Imunologicl Pocedures
15
Short title: Electrochemical detection of two tumor markers
16 17 18 19 20 21
* Corresponding author. Tel: +86 553 3869303; Fax: +86 553 3869303
22
E-mail address: [email protected] (Y. Zhang).
23 1
24
Abstract
25
In this work, a novel sandwich-type electrochemical immunosensor has been
26
developed for simultaneous detection of carcinoembryonic antigen (CEA) and
27
α-fetoprotein (AFP) based on metal ions labels. Au nanoparticles decorated multiwall
28
carbon nanotubes (AuNPs@MWCNTs) was used as carriers to immobilize secondary
29
antibody and distinguishable electrochemical tags of Pb2+ and Cd2+ to amplify the
30
signals. Due to the intrinsic property of high surface-to-volume ratio, the
31
AuNPs@MWCNTs could load numerous secondary antibody and lables. Therefore,
32
the multiplexed immunoassay exhibited good sensitivity and selectivity. Experimental
33
results revealed that this sandwich-type immunoassay displayed an excellent linear
34
response with linear range 0.01-60 ng mL−1 for both analytes with the detection limit
35
of 3.0 pg mL−1 for CEA and 4.5 pg mL−1 for AFP (at a signal to noise ratio of 3) ,
36
respectively. The method was successfully applied for the determination of AFP and
37
CEA levels in clinical serum samples.
38 39 40 41 42 43 44
Keywords: Gold nanoparticles decorated multiwall carbon nanotubes; Pb2+ and Cd 2+;
45
Multiplex assay; Carcinoembryonic antigen; α-fetoprotein 2
46
Introduction
47
Precise and early determination of multiplex tumor markers could greatly
48
improve the treatment efficiency of many cancers in clinical diagnosis. Therefore,
49
simultaneous detection of multiplex tumor markers related to a certain cancer in
50
human serum has attracted great attention in biomedical field. In recent years, various
51
immunoassay methods for simultaneous detection of multiplex tumor markers have
52
been developed [1-5]. Among these methods, electrochemical immunoassay has
53
become one of the predominant analytical methods due to high sensitivity, inherent
54
simplicity and low-cost [6-8].
55
In order to realize successfully electrochemical simultaneous multiplexed
56
immunoassays, an important issue is to search distinguishable signal probe as trace
57
labels [9, 10] in multiple labels mode. As ideal multiple-tags for proteins label, it
58
should meet two demands: one is based on the independent response achieved by each
59
target on the identical sensing interface, and the other is based on distinguishable
60
voltammetric signals to avoid interaction with analytes and the sample matrix [11].
61
Nowadays, a large number of signal tags such as enzymes, quantum dots (QDs),
62
oligonucleotide and dyes by loading on carriers for the preparation of labels have been
63
reported in electrochemical immunoassay [12]. Among them, QDs are proved to be
64
promising due to their comparable size and feasibility for surface modification [13].
65
However, QDs-based labels often not only required a tedious preparation process but
66
also involved a complicated detection step of acid dissolving process to obtain the
67
electrochemical signals [14, 15]. As a result, there is still a great challenge in 3
68
developing novel probes with simple preparation process and easy detection steps.
69
Recently, Feng et al. firstly developed a novel multianalyte electrochemical
70
immunosensor for ultrasensitive detection of cardiac troponin (cTnl) and
71
fatty-acid-binding protein (FABP) using metal ions Zn2+ and Cd2+ functionalized
72
titanium phosphate nanospheres as labes [16]. Due to metal ions highly sensitive
73
electrochemical response and easy signal obtaining process, the metal ions as labels
74
are beneficial as reported [17], but this method still involved time-consuming
75
fabrication of titanium phosphate nanospheres for metal ions exchange. Thus, the
76
development of a metal ion label with simple carriers in electrochemical
77
immunoassay is urgent.
78
Herein, we developed a simple electrochemical immunosensor for simultaneous
79
detection of AFP and CEA using the Cd 2+ and Pb2+ as multiple labels with a simple
80
carrier. Using poly (diallyldimethylammonium chloride) (PDDA) as linkage reagents,
81
the Au nanoparticles decorated MWCNTs nanocomposites (AuNPs@MWCNTs) with
82
good electric conductivity and biocompatibility [18-20] were easily fabricated not
83
only as an idea simple carrier but also as an excellent signal enhancer. As expected,
84
the immunosensor can achieve simultaneous detection of CEA and AFP in one
85
detection platform with different voltammetric peaks through square wave
86
voltammetry. The peak currents and the peak positions were dependent on the
87
concentration and type of the corresponding analytes, respectively. In addition, the
88
peak potentials were not too far, which may save the detection time. The obtained
89
immunosensor exhibited sensitive and stable response for detection of multiple tumor 4
90
markers and showed great potential in clinical applications.
91
Experimental
92 93
Materials
94 95
Carcinoembryonic antigen (CEA), CEA antibody (anti-CEA) , α-fetoprotein
96
(AFP) and AFP antibody (anti-AFP) were purchased from Biocell Biotech. Co., Ltd
97
(Zhengzhou, China) and stored in refrigerator at 4 °C. Bovine serum albumin (BSA)
98
and poly (diallyldimethylammonium chloride) (PDDA, 20%, w/w in water, MW 200
99
000-350 000) were purchased from the Sinopharm Chemical Reagent Co., Ltd
100
(Shanghai). Multiwalled carbon nanotubes with carboxylic acid groups (MWCNTs,
101
purity >95%, diameter 20-30 nm, length 10-30 µm) were obtained from Chengdu
102
Institute of Organic Chemistry (Chengdu, China). Acetic acid (HAc), sodium acetate
103
anhydrous (NaAc), Cd(NO3)2, Pb(NO3)2 and chloroauric acid (HAuCl4.4H2O) were
104
obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Phosphate
105
buffer saline (PBS) with various pH values were prepared by mixing the stock
106
solutions of 0.10 mol L-1 Na2HPO4, 0.10 mol L-1 NaH2PO4 and 0.10 mol L-1 KCl, then
107
adjusting the pH with 0.10 mol L-1 NaOH and H3PO4. The washing buffer was pH 7.0
108
PBS containing 0.05% (W/V) Tween (PBST). Blocking solution was 1% BSA. The
109
clinical serum samples were from the clinical laboratory of the Yiji Shan Hospital
110
(Wuhu, China). Twice-quartz-distilled water was used through the study.
111 5
112
Apparatus
113 114
All electrochemical measurements were performed on a CHI 650C
115
electrochemical analyzer (CH Instruments Inc., China) with a conventional
116
three-electrode system composed of a platinum auxiliary electrode, a saturated
117
calomel electrode (SCE) as reference electrode, and a bare Au or modified Au as
118
working electrode.
119
Electrochemical impedance spectra (EIS) were performed in 0.1 M PBS
120
containing 5.0 mM [Fe(CN)63−/4−] and 0.1 M KCl at a pH of 7.0. The frequency
121
ranged from 0.1 to 100 kHz, and the amplitude of the alternate voltage was 5 mV.
122
Morphologies of AuNPs , PDDA functionalized MWCNTs, AuNPs-MWCNTs
123
and AuNPs@Au were obtained with scanning electron microscopy (SEM)
124
(JEOLJSM-6700F, Hitachi, Japan). The morphologies of the materials were measured
125
on a transmission electron microscope (TEM, Hitachi-800).
126 127
Preparation of AuNPs@MWCNTs nanocomposites
128 129
The colloidal AuNPs of 16 nm diameter were prepared according to the previous
130
protocol (Seen in supplementary Fig.S1) [21]. The acid-treated MWCNTs were
131
further functionalized with PDDA according to the reported method [22]. The
132
obtained PDDA-MWCNTs (0.5 mg) (Seen in supplementary Fig.S2) were dispersed
133
in 5.0 mL of as-prepared colloidal AuNPs and stirred for 20 mins, the AuNPs were 6
134
assembled on the surface of PDDA@MWCNTs nanocomposites via electrostatic
135
interaction. After centrifugation, the AuNPs@MWCNTs composites were obtained.
136
(Seen in supplementary Fig.S3), which were further washed with water and
137
redispersed in 2 mL of 50 mM pH 9.0 Tris-HCl solution for further use.
138 139
Preparation of the immunosensing probes
140 141
Firstly, the signal anti-CEA2,1 (Ab 2,1) (200 µL, 1 mg mL-1) was added to the
142
above AuNPs@MWCNTs dispersion and stirred at room temperature for 2 h.
143
Secondly, 400 µL 1% BSA solution was added to the obtained bioconjugates and
144
allowed to react for 2 h. After centrifugation, the resulting immunocomplex was
145
further washed with PBS (0.01 M, pH=7.0) three times. Finally, the prepared
146
immunocomplex was dispersed in 2 mL 10 mM Pb(NO3)2 aqueous solution and
147
shaked for overnight, thus, the Pb2+-Ab 2,1-AuNPs@MWCNTs bioconjugates were
148
synthesized. The Cd 2+- anti-AFP2,2 (Ab2,2)-AuNPs@MWCNTs was synthesized using
149
the similar method.
150 151
Fabrication of the immunosensor
152 153
Before modification, the gold electrode was treated according to our previous
154
reported procedure [23]. The modified electrode was immersed in 0.1 M KNO3
155
solution containing 5.0 mM HAuCl4 and electrochemical deposited 300 s at - 200 mV. 7
156
Thus, AuNPs were distributed on the surface of the gold electrode. The modified
157
electrode was denoted as AuNPs/Au.
158
Immobilization of anti-CEA (Ab1,1) and anti-AFP (Ab 1,2) was performed by
159
dropping a mixture of Ab1,1 and Ab1,2 (10.0 µL, 200 µg mL−1) solution onto the
160
surface of the AuNPs/Au, and kept it for 12 h in a refrigerator at 4 °C, the resulting
161
electrode was washed PBST an PBS to remove physically absored Ab 1. Following
162
that, the modified electrode was incubated with 1% BSA solution for 50 min at 37 °C
163
to block any possible remaining active sites against non-specific adsorption, and
164
washed several times with PBST and PBS. The obtained immunosensor was stored at
165
4 °C prior to use.
166 167
Electrochemical measurements
168 169
The schematic preparation of the immunosensing probes is illustrated in scheme
170
1. SEM was performed to characterize the shape of the AuNPs/MWCNTs (inset of
171
Scheme 1A). The preparation procedure of the immunosensors for CEA and AFP
172
determination as follows: the immunosensor was incubated with the mixture of CEA
173
and AFP solution or serum samples with various concentrations for 50 min at 37 °C,
174
following by washing with PBST and PBS, and then it was incubated with mixture of
175
1:1
176
solution for another 50 min at 37 °C, following by washing with PBST and PBS.
177
Finally, the electrode was transferred into acetate buffer solution (0.2 M, pH=4.5).
diluted
Pb2+-Ab2,1-AuNPs@MWCNTs
8
and
Cd 2+-Ab2,2-AuNPs@MWCNTs
178
The SWV was used to obtain the response signal of the immunosensor. SWV scan
179
from 0 to -800 mV(vs.Ag/AgCl) with a pulse amplitude of 25 mV, a pulse frequency
180
of 15 Hz, and a quiet time of 2 s was performed to record the electrochemical
181
responses at −0.54 and −0.71 V for simultaneous, quantitative measurement of CEA
182
and AFP.
183 184
Results and discussion
185 186
Investigation of the assembled process of the immunosensor with SEM
187
and cyclic voltammetry (CV) techniques
188 189
Fig. 1 A and 1B showed the surface morphologies of bare Au electrode and
190
AuNPs modified Au electrode, respectively. After the electrode was electrodeposited
191
at -0.2 V for 300 s in 0.1 M KNO3 solution containing 5.0 mM HAuCl4, the spherical
192
AuNPs were assembled on the surface of Au electrode, which was beneficial for
193
improving the immobilized amounts of capture antibody due to the good affinity of
194
AuNPs for biomolecules on the electrode surface. The SEM showed a more uniform
195
spherical structure with a regular distribution of AuNPs. As shown in Fig. 1C, the
196
cyclic voltammograms of bare Au electrode and AuNPs modified Au electrode in 0.5
197
M H2SO4 solution, the peak current at AuNPs modified electrode was much higher
198
than that at bare Au electrode. The results revealed that the AuNPs increased the
199
effective surface area of electrode significantly, at the same time, the effective surface 9
200
areas of electrodes can be obtained from the coulombic integration of the reductive
201
waves of gold oxide.
202 203
Electrochemical characterization of the assemble process of the
204
immunosensor
205 206
The CVs characterization of the immunosensor at different step was exhibited in
207
Fig. 2 (A), a pair of distinct redox peaks was observed due to the oxidation and
208
reduction of the redox couple Fe(CN)6-4/-3 on the bare Au electrode (curve a). After the
209
electrode was modified with AuNPs, the peak current of CVs increased sharply (curve
210
b) attribute to the excellent ability of electron transfer of the AuNPs. When the
211
electrode was incubated in the mixture of capture Ab 1,1 and Ab1,2, the mixture of CEA
212
and AFP, the mixture of 1:1 diluted Pb2+-Ab 2,1-AuNPs @MWCNTs and
213
Cd2+-Ab2,2-AuNPs@MWCNTs bioconjugates,
214
decreased step by step (curve c, d, e), this could be ascribed to form a sandwich-type
215
immunocomplex, and the immunocomplex increases with the increment of the CEA
216
and AFP concentration in the sample. The insulating layer of proteins hinders
217
interfacial electron transfer. On the other hand, the stepwise construction process of
218
the immunosensor was characterized with an electrochemical impedance spectrum
219
(EIS) (Fig. 2B). The EIS include a semicircular portion and a linear portion, the
220
diameter of the semicircle at higher frequencies corresponds to the electron-transfer
221
resistance (Ret), and the linear part at lower frequencies corresponds to the diffusion 10
successively.
The redox peaks
222
process. It could be observed that the bare electrode displayed a small semicircle with
223
a Ret of about 250 Ω (curve a). After the bare electrode modified with AuNPs, the Ret
224
decreased to 120Ω(curve b), indicating that the AuNPs was assembled on the
225
electrode surface. However, as the electrode was incubated in the mixture of capture
226
Ab 1,1 and Ab1,2, the mixture of CEA and AFP, the mixture of 1:1 diluted
227
Pb2+-Ab 2,1-AuNPs@MWCNTs and Cd 2+-Ab 2,2-AuNPs@MWCNTs bioconjugates,
228
successively, the Ret increased step by step (curve c, d, e). The results were in good
229
agreement with the results obtained from CVs. Based on above results, the
230
simultaneous detection of CEA and AFP was possible.
231 232
Optimization of experimental conditions
233 234
The electrochemical performance of the immunosensor would be influenced by
235
many factors. Therefore, some experiment parameters were investigated (such as pH,
236
incubation time). Fig. 3A and B show the pH value could affect electrochemical
237
behavior of the Cd2+ and Pb2+. The pH value of detection solution not only has a great
238
influence on the activity of the antigens and antibodies, but also on the
239
electrochemical behavior of Cd 2+ and Pb2+. It could be observed clearly that the
240
reduction peak current of Cd 2+ and Pb 2+ was increased with the pH value increased
241
from 3.0 to 4.5, and then decreased. When the pH was 4.5, the reduction peak current
242
of Cd2+ and Pb 2+ reached the maximum value. This might be because metal ions
243
combined with hydroxide ions and became inactive at pH above 4.5. Herein, pH of 11
244
4.5 was chosen in this study.
245
Fig. 3C and D showed the response of the immunosensor changed with the
246
incubation times range from 10-60 min. It could be clearly observed that the reduction
247
current of Cd 2+ and Pb2+ increased with increasing incubation time and trended to
248
reach a plateau after 50 min, exhibiting a saturated binding between the antigen and
249
the primary antibody on electrode surface. Therefore, subsequent experiments
250
employed 50 min as the optimum time for all the incubation steps of the assay.
251 252
Analytical performance
253 254
Under optimized assay conditions, the reduction peaks of Pb2+ and Cd2+ were
255
found to be proportional to the concentration of CEA and AFP in the incubation
256
solution. Under the optimum conditions, the SWV peaks for simultaneous detection of
257
CEA and AFP increased with the increment of CEA and AFP concentrations. The
258
calibration plots displayed a good linear relationship between the reduction peaks and
259
the concentration of analytes in the range of 0.01-60 ng mL-1 for both CEA and AFP
260
(seen Fig. 4B and C). The regression coefficients were 0.9911 and 0.9962,
261
respectively. The detection limit of CEA and AFP were 3.0 pg mL−1 and 4.5 pg mL−1
262
(at 3σ), respectively. Which were lower than those of metal ions tagged
263
immunocolloidal gold (4.6 pg mL-1 for CEA and 3.1 pg mL-1 for AFP) [8], HRP
264
functionalized Pt hollow nanospheres (50 pg mL-1 for CEA and 80 pg mL-1 for AFP)
265
[24], CdS/DNA and PbS/DNA nanochains as labels (3.3 pg mL-1 for CEA and 7.8 pg 12
266
mL-1 for AFP) [25], SiO2@C-dots label-electrochemiluminescence (6.0 pg mL-1 for
267
CEA and 5.0 pg mL-1 for AFP) [26], immunochromatographic test strip (2.0 ng mL-1
268
for CEA and 3.0 ng mL-1 for AFP) [27], and time-resolved immunofluorometric assay
269
(240 pg mL-1) quantum dot barcode-based electrochemical immunoassay (3.3 pg mL-1)
270
for CEA [28, 29] reported in previous studies. The results indicated that the
271
multiplexed electrochemical immunoassay enabled wide linear ranges and low LODs.
272
Furthermore, the proposed immunosensor exhibited a satisfactory electrochemical
273
performance, some possible explanations may contribute to these observations. Firstly,
274
the AuNPs@MWCNTs nanocomposites as good carriers have a large area to provide
275
a biocompatible microenvironment for the immobilization of antibody and further
276
loading a large amount of lables [30]. Secondly, the outstanding electric conductivity
277
of AuNPs@MWCNTs nanocomposites can also accelerate the electron transfer.
278
Finally, the amplification of the amperometric signal output was mainly ascribed to
279
the excess Pb2+ and Cd2+ with favorable electron conductivity and chemical stability
280
were loaded on the surface of AuNPs@MWCNTs nanocomposites.
281 282
Cross-reactivity,
283
immunosensor
specificity,
reproducibility
and
stability of
the
284 285
The cross-reactivity of the immunosensor was examined by comparing the SWV
286
responses of two analytes to those containing only one analyte. Firstly, antibodies of
287
CEA and AFP were immobilized on the electrodes. Then, two control tests were 13
288
carried out as follows: (i) immunosensors was incubated with CEA only or with AFP
289
only. (ii) AFP and CEA were simultaneously monitored. Finally, they were
290
bioconjugated
291
-AuNPs@MWCNTs probes to perform the sandwich-type immunoreaction. The
292
responses of SWV were listed in Table 1. The results indicated that the detection of
293
CEA and AFP exhibited low interference and the cross-reactivity between two
294
analytes was negligible.
Pb2+-antiCEA-AuNPs@MWCNTs
with
or
Cd 2+-anti-AFP
295
The specificity of the immunosensor played an important role in analyzing
296 297
biological
samples
without
separation,
298
immunoglobulin G (IgG), BSA, glucose (Glu) and ascorbic acid (AA) were used as
299
the interferes to evaluate the specificity. To test the specificity of the immunosensor,
300
1.0 ng mL-1 CEA and AFP were mixed with 50 ng mL-1 of IgG, BSA, Glu, and AA,
301
respectively. Fig. 5 showed SWV response of pure CEA and AFP, and that obtained
302
from CEA and AFP containing an interferential substance. When the immunosensor
303
detected the mixture of 1 ng mL-1 of CEA or AFP and 50 ng mL-1 of another interferes,
304
the response signal of the mixture changed a little in contrast with CEA or AFP alone,
305
which showed that the proposed immunosensor had good selectivity for CEA and
306
AFP.
307
308 14
some
interferes
such
as
human
309
To estimate the reproducibility of the simultaneous multianalyte immunoassay,
310
the intra-assay precision was investigated by detecting five times every 5 h at four
311
different concentrations of CEA and AFP (0.05, 2, 20, and 60 ng mL−1) using identical
312
immunosensor. The coefficient of variations was 5.5%, 6.8%, 7.9%, and 9.2% at 0.05,
313
2, 20, and 60 ng mL−1 of CEA and AFP, respectively. Similarly, the inter-assay
314
precision was investigated by measuring four different concentrations of CEA and
315
AFP (0.05, 2, 20 and 60 ng mL−1) using five immunosensors. The coefficient of
316
variations was 9.6%, 8.8%, 6.1% and 8.6%, respectively, suggesting the
317
immunosensor possessed acceptable precision and reproducibility. In addition, when
318
the immunosensor was stored at 4◦ C, the stability of the immunosensor was examined
319
by testing the response after three weeks, over 90.1% of the initial responses remained
320
after three weeks for both CEA and AFP. The slow decrease in response seemed to be
321
related to the gradual deactivation of the immobilized antibody on the sensor platform.
322
The immunosensor showed acceptable stability and was favored as dual labels for
323
simultaneous detection of dual biomarkers based on SWV measurments.
324 325
Application of the immunosensor in human serum
326 327
The capability to detect practical samples is a major concern during the
328
development of a clinical diagnostic platform. Detailed process of practical samples
329
prepared as follows: the blood samples were from the venous blood and without
330
added anticoagulant, then, it was placed in dry test tube to stand for one hour. Next, 15
331
blood samples were centrifuged (2000 rpm×5 min) and precipitated, and we took the
332
upper solution to detect. In order to examine the applicability of the immunosensor for
333
practical analyses, the recovery experiments were performed by standard addition
334
methods. The standard samples of CEA and AFP were dissolved in the healthy human
335
serum (the concentrations range of the CEA and AFP are within 0.01 to 60 ng mL-1)
336
and detected the CEA and AFP in serum. The recovery is obtained within
337
94.65%-108% and 94.2%-105.7%, respectively, indicating the method is suitable for
338
serum sample analysis. (Seen in Table S1)
339
Importantly, to investigate the possibility of the newly developed method to be
340
applied for clinical analysis, several real samples were examined by the developed
341
immunoassay and the ELISA methods for determination of CEA and AFP. The serum
342
samples came from normal persons and cancer patients. These results were shown in
343
Table 2. The value obtained was in agreement with that of the ELISA methods,
344
indicating the immunosensor could be applied to serum analysis.
345 346
Conclusions
347 348
In summary, we have developed a simple and reliable electrochemical
349
immunosensor for simultaneous detection of CEA and AFP based on metal ion as
350
lables and AuNPs@MWCNTs as simple carrier and signal enhancers. Highlights of
351
the
352
nanocomposites with its exceptionally high surface area, good conductivity and
developed
immunoassay
were
as
16
follows:
firstly,
AuNPs@MWCNTs
353
biocompatibility was used as an excellent carrier for immobilizing antibodies and
354
further loading a large amount of lables. Secondly, high-content metal ions labels
355
could be detected without acid dissolution using SWV analysis technique, thus,
356
amplifying the current response effectively. Moreover, the immunosensor showed
357
good precision, high sensitivity, acceptable stability and reproducibility.
358 359
Acknowledgments This work was supported by the Special Foundation for
360
excellent doctoral fostering of Anhui Normal University-Organic Chemistry (No.
361
003061425), the National Natural Science Foundation of China (No. 20675002) and
362
the National Natural Science Foundation for Young Scholars of Anhui Province of
363
China (No. 1408085QB40).
364 365 366 367 368 369 370 371 372 373 374 17
375
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electronic coding of a cancer marker, Anal. Chem. 82, (2010) 1138-1141.
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473 474 475 476 477 478 479 480 481 482 483 484 22
485
Captions to figures:
486 487
Scheme 1 (A) The preparation procedure of immunosensing probes. (B) The
488
fabrication process of the immunosensors.
489 490
Fig. 1 SEM images of bare Au electrode (A), AuNPs/Au (B), cyclic voltammograms
491
of bare Au electrode and AuNPs modified electrode in 0.5 M H2SO4 solution (C)
492 493
Fig. 2 (A) CV responses and (B) EIS of the different modified electrodes in 0.1 M
494
PBS containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] containing 0.1 M KCl, respectively.
495
bare Au (a), electrode modified successively with AuNPs (b), mixture of capture
496
anti-CEA and anti-AFP (c), mixture of 10 ng mL-1 CEA and AFP (d), mixture of
497
Pb2+-Ab 2,1-AuNPs @MWCNTs and Cd2+-Ab2,2-AuNPs@MWCNTs (e)
498 499
Fig. 3 Effect of the pH (A) and (B), the incubation time of (C) and (D) on the
500
response of the immunosensor to CCAE=CAFP = 20 ng mL−1
501 502
Fig. 4 (A) SWV of the immunsensors for different concentrations of CEA and AFP
503
(0.01, 0.05, 2.0, 8, 20, 30, 45, 60 ng mL-1), calibration curves of the multiplexed
504
immunoassay toward (B) CEA and (C) AFP in 0.2 M HAc-NaAc (pH 4.5).
505 506
Fig. 5 Specificity of the immunosensor conditions: CCEA =1 ng mL-1, Cinterferents =50 ng 23
507
mL-1 (BSA, Glu, IgG and AA)
508 509
Table 1 Interference degree or cross-talk level
510 511
Table 2 Comparison of CEA and AFP using the proposed immunosensor and
512
reference methods
513 514 515 516
24
517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545
Scheme 1
25
546 547
548 549 550 551
Fig. 1
26
552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570
Fig. 2
27
571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592
Fig. 3
28
593 594 595
596 597 598 599 600
Fig. 4
29
601 602 603
604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628
Fig. 5
30
629 630 631
Scheme:
632 633 634
We have developed a simple and reliable electrochemical immunosensor for
635
simultaneous detection of CEA and AFP based on metal ion as lables and Au
636
nanoparticles decorated MWCNTs as signal enhancers. Due to the intrinsic property
637
of high surface-to-volume ratio, the AuNPs@MWCNTs could load numerous
638
secondary antibody and lables. So, the multiplexed immunoassay exhibited good
639
sensitivity and selectivity.
640 641
31
642 Sample type
Concentration (ng mL-1 )
Current shift at CEA position (µA)a,b
CEA
AFP
CE+AFP
Current shift at AFP position (µA)a,c
2
8.72
0.07
60
58.98
0.02
2
0.03
6.32
60
0.06
50.31
2+2
8.43
6.01
60+60
59.76
49.97
643
a
The average value of three measurements in n 0.2 M HAc-NaAc (pH 4.5).
644
b
The SWV peak current was 23.12 µA for zero CEA analyte.
645
c
The SWV peak current was 14.02 µA for zero AFP analyt.
646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668
Table 1
32
669 670 Sample no. Multiplexed immunoassaya (ng mL-1) CEA
671 672 673
AFP
ELISA (ng mL-1)
Relative deviation (%)
CEA
AFP
CEA
AFP
1
0.63±0.23
0.58±0.45
0.60
0.60
+5.00
-1.67
2
0.98±0.36
1.10±0.25
1.00
1.00
+2.00
-1.00
3
18.45±0.42
21.21±0.32
20.00
20.00
-7.75
+6.05
4
41.12±0.72
38.58±0.24
40.00
40.00
+2.80
+3.55
5
58.36±0.64
61.08±0.56
60.00
60.00
-2.77
+1.80
Table 2
674 675
33
