Talanta 132 (2015) 150–154

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A simple and sensitive impedimetric aptasensor for the detection of tumor markers based on gold nanoparticles signal amplification Xi Liu a, Yun Qin a, Chunyan Deng a,b,n, Juan Xiang a,n, Yuanjian Li b a b

College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, PR China Pharmacology Section, Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha 410083, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 19 June 2014 Received in revised form 27 August 2014 Accepted 31 August 2014 Available online 7 September 2014

A simple and sensitive electrochemical impedimetric aptasensor based on gold nanoparticles (AuNPs) signal amplification was developed for the ultrasensitive detection of tumor markers (mucin 1 protein, MUC1 as a model). The designed cDNA, which is partly complementary with the aptamer of MUC1 was immobilized on the gold electrode. The detection of MUC1 could be carried out by virtue of switching structures of aptamers from DNA/DNA duplex to DNA/target complex. The change of the interfacial feature of the electrode was characterized by electrochemical impedance analysis (EIS) with the redox probe [Fe(CN)6]3  /4  . The quantitative detection of MUC1 protein was obtained from the changes of electron-transfer resistance (ΔRet). Moreover, as the signal enhancer, the aptamer-modified AuNPs (Apt@AuNPs) conjugates was introduced on the electrode by the hybridization of cDNA with aptamer. As expected, the detection sensitivity for MUC1 was greatly improved, which may be due to the specific binding of MUC1 onto the surface of the Apt@AuNPs modified electrode. This proposed simple aptasensor has a low detection limit of 0.1 nM, and also exhibits several advantages of high sensitivity and good selectivity. This present work may provide a general model for the detection of tumor marker based on impedimetric aptasensor. & 2014 Elsevier B.V. All rights reserved.

Keywords: Impedimetric aptasensor Signal amplification Gold nanoparticles Mucin 1

1. Introduction Tumor marker is a substance abnormally expressed in malignancy and benign tumor compared with normal tissues. The content of tumor marker represents the current state of the tumor. The detection of tumor marker is significant for clinical diagnosis and judging those diseases related to tumor. For breast cancer, since human mucin-1 (MUC1) has been found highly overexpressed in breast cancer, it becomes one of the most common tumor markers to diagnose the breast tumor [1]. Therefore, enhancing the detection level of MUC1 plays an important role in the diagnosis of breast cancer. Aptamers are nucleic acid ligands (DNA or RNA) which are artificially synthesized and selected in vitro through SELEX (systematic evolution of ligands by exponential enrichment) [2,3]. They have high specificity and affinity to various target molecules that includes peptides [4], dopamine [5], adenosine and ATP [6], metal ions [7], as well as proteins, such as growth factors [8] and

n Corresponding authors at: College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, PR China. Tel.: þ86 731 88876490; fax: þ86 731 88879616. E-mail addresses: [email protected] (C. Deng), [email protected] (J. Xiang).

http://dx.doi.org/10.1016/j.talanta.2014.08.072 0039-9140/& 2014 Elsevier B.V. All rights reserved.

thrombin [9]. In addition, the immunogenicity and toxicity of aptamers are proved much lower compared with antibodies when they used in biological environment or as a part of therapeutic and diagnostic agents [10], so aptamer has been widely used as a sensing element for developing biosensor. In recent years, aptamers have been screened for targeting and measuring some tumor markers, which have great value in evaluating the progress of cancer, monitoring the therapeutic response and predicting the recurrence [11]. MUC1 aptamers are of great importance in the assessment of breast cancer [12,13]. Up to now, limited researches have been conducted to directly detect the concentration of MUC1. Pang's group designed a fluorescent aptasensor utilizing graphene oxide (GO) to quench the fluorescence of single-stranded dye-labeled MUC1 aptamer, when MUC1 added, the quenched fluorescence was recovered significantly [14]. Yu's group constructed a hairpin DNA aptamers on gold and labeled with methylene blue (MB) for electrochemical measurements [15]. They also reported a method of quantitative detection of MUC1 by labeling a 3-component DNA hybridization system with quantum dot (QD)-based fluorescence readout [16]. Wei et al. performed a method based on electrochemiluminescence (ECL) resonance energy transfer (ERET) to detect MUC1 protein and MCF-7 cancer cells [17]. Among these studies, the electrochemical methods are more remarkable in the

X. Liu et al. / Talanta 132 (2015) 150–154

development of aptasensors due to their fast response, high sensitivity, low cost and the result can be obtained at real-time. As an effective electrochemical method, electrochemical impedance spectroscopy (EIS) can measure the response (current and its phase) of an electrochemical system to an applied oscillating potential as a function of the frequency [18–21]. The immobilization of biomaterials, including enzymes, antigens/antibodies or DNA on electrodes alters the capacitance and interfacial electron transfer resistance of the conductive electrodes. On account of its sensitivity with the changes of interfacial properties, this method has been extensively used to study the electrochemical changes induced by protein binding reactions resulting in interfacial electron transfer kinetics between the redox probe and the electrode changes notably when the binding procedure taking place [22,23]. Therefore, EIS can be used to monitor the different stages of each fabrication step for the biosensor, and the detection of identification process when an immobilized molecule interacts with analytes. Besides, in consequence of the advantages of EIS, for instance, high sensitivity, simplicity and requiring no external modification of the biomolecules, it is an ideal technology for label-free detection. In the present work, a sensitive and label-free impedimetric aptamer-based electrochemical biosensor for the tumor marker of breast cancer (MUC1 as the model) was simply fabricated. A cDNA was used as the linker DNA, which was designed to be complementary with the MUC1 aptamer. Moreover, aptamer functionalized gold nanoparticles (Apt@AuNPs) were used as the signal amplifier and to capture the target molecule MUC1. The introduction of gold nanoparticles is favorable for amplifying electrochemical signals and the resulted electrochemical aptasensor shows high sensitivity, MUC1 can be specifically detected at a concentration down to 0.1 nM. This proposed aptasensor for tumor maker has some advantages of simplicity, high sensitivity, acceptable selectivity, good reproducibility and stability. Therefore, this work would lay a potential foundation for clinical application of the early breast cancer diagnosis, and it is also significant for the development of the electrochemical aptasensor for sensing other tumor markers.

2. Experimental 2.1. Materials and chemicals HAuCl4·3H2O was from Sigma (St. Louis, MO). 6-Mercaptohexanol (MCH), Tris-(hydroxymethyl) aminomethane (Tris), Trisodium citrate was purchased from Bio Basic Inc. NaCl, MgCl2·6H2O were obtained from Amresco. All other reagents are of analytical purity and used as received. All samples and buffer solutions were prepared using ultrapure water obtained from a Milli-Q water purification system (Simplicity 185, Millipore Corp., Billerica, MA). DNA1 (thiolated MUC1 aptamer, 50 –HS-(CH2)6-GCA GTT GAT CCT TTG GAT ACC CTG G-30 ) and cDNA (50 -HS-(CH2)6-TTT TTT ATC CAA AGA-30 ) were manufactured by Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). DNA1 is a 25-base sequence that is MUC1 aptamer, as well as complementary sequence (bold part) to cDNA. Thiolated MUC1 aptamer was diluted to 10 μM in 34 mM Tris–HCl buffer (pH 7.4, 233 mM NaCl, 8.5 mM KCl, 1.7 mM MgCl2, 1.7 mM CaCl2) for use. The obtained solution was stored at 4 1C before use. MUC1 (from the N terminus to the C terminus: APDTRPAPG) was purchased from Shanghai Apeptide Co., Ltd. The peptides were suspended in 10 mM phosphate buffer solution (PBS, pH 7.4) to obtain different concentrations for the subsequent experiment.

151

2.2. Preparation of AuNPs and DNA1-modified AuNPs (apt@AuNPs) AuNPs with the diameter of about 13 nm were prepared by the reduction of HAuCl4 in citrate solution according to reports in the literatures [24,25]. Briefly, 100 mL 0.01% (w/v) HAuCl4 aqueous solution was heated and stirred vigorously with reflux, 2 mL 2% (w/v) trisodium citrate was added rapidly when HAuCl4 aqueous solution became boiled. Color variation was obviously observed and then it was refluxed for an additional 15 min. The solution color turned to wine red, indicating the formation of AuNPs. The solution was cooled to room temperature with continuous stirring. The diameter of AuNPs was determined by UV–vis spectra with an characteristic absorption peak at 520 nm [24,26]. The DNA1-modified AuNPs (Apt@AuNPs) were prepared according to the procedures reported in literatures [27,28]. Briefly, a solution of AuNPs (1 mL) was mixed with the thiolated DNA1 (1 OD) overnight and then slowly brought to 0.05 M NaCl by adding concentrated NaCl solution (3 M), 10 mM phosphate buffer (pH 7.0). Then, after standing for 6–8 h, the concentration of NaCl was brought to 0.1 M and the solution was allowed to age for another 6–8 h. To remove the excess free aptamers, the conjugates were purified by centrifuging for 30 min at 14,000 rpm. The red precipitate was washed, centrifuged, and dispersed in 1.5 mL of 34 mM Tris–HCl buffer solution containing 233 mM NaCl, 8.5 mM KCl, 1.7 mM MgCl2, 1.7 mM CaCl2 (pH 7.4). A characteristic absorption of the Apt@AuNPs conjugates was at 523 nm, which indicates the MUC1 aptamers have been successfully attached onto Au nanoparticles, according to reports in the literature [24]. 2.3. Preparation of the modified electrode Before modification, the bare gold electrode (2 mm in diameter) was polished sequentially with 0.3 mm and 0.05 mm alumina slurry followed by ultrasonic cleaning in ethanol and ultrapure water. Subsequently, the gold electrode was cleaned in piranha solution (v/v 3:1 H2SO4/H2O2) (Caution: piranha solution reacts violently with many organic materials and should be handled with great care), followed by treatment with nitric acid. Afterward, the gold electrode was washed thoroughly with ultrapure water and dried under nitrogen gas. For modification of the electrode, 10 mL of cDNA (10 mM) was dropped onto the cleaned electrode and incubating for 16 h to form the self-assembled monolayer. After modification, the gold electrodes were incubated in 1 mM 6-mercaptohexanol (MCH) solution for 1 h to block the unmodified region of the gold surface. Then a 10 mL droplet of Apt@AuNPs conjugates solution was covered onto the Au/cDNA/MCH electrode and kept for 2 h to obtain the Au/cDNA/MCH/Apt@AuNPs electrode. For comparison, the Au/cDNA/MCH/Apt electrode was also fabricated by dipping 10 mL of thiolated MUC1 aptamer (Apt) solution onto the Au/cDNA/MCH electrode and incubating for 2 h. After each step, the electrode was rinsed thoroughly with ultrapure water and dried under highly pure nitrogen stream prior to electrochemical characterization. For each step of immobilization, the electrochemical impedance measurements were performed in the presence of equimolar [Fe(CN)6]3 /4 as redox probe. 2.4. Apparatus and electrochemical measurements Electrochemical impedance spectroscopy (EIS) measurements were performed on a CHI650D electrochemical workstation. A conventional three-electrode system was used with bare gold electrode as the working electrode, a platinum wire as counter electrode and a saturated calomel electrode (SCE) as reference electrode. All the potentials in this paper were in respect to SCE. All experiments were carried out at laboratory temperature.

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For the detection of MUC1, the modified gold electrodes were immersed in the MUC1 solution (10 mM phosphate buffer solution, pH 7.4) with various concentrations and kept for 2 h, followed by a 5 min washing to remove Apt@AuNPs@MUC1 and nonspecific bound of MUC1. The impedance spectra were obtained in a frequency range from 100 mHz to 100 kHz and the amplitude of 5 mV in 5 mM [Fe(CN)6]3  /4  solution containing 0.1 M KCl. The impedance spectra were plotted in the form of Nyquist plots.

3. Results and discussion 3.1. Design strategy of the aptasensor

Scheme 1. Schematic representation of the electrochemical aptasensor for the detection of MUC1.

C

R R

Z''/k

An electrochemical sensing platform for highly sensitive detection of tumor marker (MUC1 as a model) was proposed. The assay protocol of the electrochemical aptasensor was depicted schematically in Scheme 1. The surface of the gold electrode was first modified with the thiolated cDNA and MCH via the self-assembly. The bold part of the MUC1 aptamer hybridized to the bold part of cDNA. The sensing interface was ready for the detection of MUC1. For Scheme 1A, when MUC1 introduced, the MUC1 aptamercontained DNA1 preferred to form the aptamer-MUC1 complex, which resulted in the dehybridization and the MUC1-aptamer released into the solution (Scheme 1A-II). In this case, it was expected that the EIS of the DNA-modified electrode would decrease. On the other hand, in order to improve the sensitivity of the sensing interface, Apt@AuNPs was employed to hybridize with the MUC1 aptamer, as shown in Scheme 1B. Since Apt@AuNPs can bind more MUC1 molecules, and folding into a well-defined three-dimensional structure, which would lead to the Apt@AuNPs/MUC1 complex of huge weight falling off from the electrode (Scheme 1B-II). This may result in an obvious EIS signal change of the modified electrode, and offering a significant amplification for the detection of MUC1. The design is to fabricate an aptamer based biosensor by relying on the structure-switching properties of aptamers binding to their target molecules, which is applicable for the fabrication of other aptasensor.

Z

d e

b c a

Z'/k

3.2. EIS characterization of the modified electrode

Fig. 1. Electrochemical impedance spectra of (a) the bare Au electrode, (b) the Au/ cDNA electrode, (c) the Au/cDNA/MCH electrode, (d) the Au/cDNA/MCH/ Apt@AuNPs electrode, (e) the Au/cDNA/MCH/Apt electrode in aqueous solution containing 0.1 M KCl and 5 mM [Fe(CN)6]3  /4  (1:1) as the redox probe. The impedance spectra were obtained at the frequency range from 100 mHz to 100 kHz with a DC potential of þ0.224 V. The amplitude was 5 mV.

On the basis of the charge transfer kinetics of the [Fe(CN)6]3  /4 redox probe, faradaic impedance spectra with an [Fe(CN)6]3 /4 redox probe were modeled using the equivalent circuit approach of Radi et al. [22], as shown in the inset of Fig. 1. The circuit includes the commonly existing electrolyte resistance (Rs) and Warburg impedance (Zw) resulting from the diffusion of ions from the bulk of the electrolyte to the interface, double-layer capacitance (Cd), and the electron-transfer resistance (Ret). Among them, Ret can directly and sensitively respond to changes of the electrode interface [29,30]. Whereas a linear section characteristic at the lower frequency is attributable to a diffusion-limited process, a squeezed semicircle portion observed at higher frequencies corresponds to the electron-transfer limited process. Electrochemical impedance spectroscopy (EIS) has been employed to characterize the modified electrode. Fig. 1 displays the EIS results of the (a) bare Au, (b) Au/cDNA, (c) Au/cDNA/MCH, (d) Au/ DNA/MCH/Apt@AuNPs and (e) Au/cDNA/MCH/Apt electrodes in the presence of the equimolar [Fe(CN)6]3  /4  redox probe, respectively. In the Nyquist plots of impedance spectra, a linear section at the lower frequencies is attributable to a diffusion-limited process, and a squeezed semicircle portion observed at higher frequencies would correspond to the electron-transfer limited process. The increase of the diameter of the semicircle reflects the increase of the interfacial charge-transfer resistance (Ret) [31]. It is noted that the bare gold electrode shows a very small semicircle domain

(Ret ¼ 200 Ω, curve a in Fig. 1), indicating a very fast electron transfer process of [Fe(CN)6]3  /4  [29]. The self-assembly of a negatively charged cDNA on the Au electrode surface effectively repels the [Fe(CN)6]3  /4  anions and thus leads to an enhanced electron-transfer resistance (Ret ¼ 3540 Ω, curve b in Fig. 1), which demonstrates that thiolated cDNA has been self-assembled successfully on the electrode. It is also found that the assembly of MCH on the cDNA-modified electrode leads to a significant increase of Ret to 6890 Ω (curve c in Fig. 1). After hybridization with Apt@AuNPs, the value of Ret increases significantly to 12,690 Ω (curve d in Fig. 1). Compared to the Au/DNA/MCH/ Apt@AuNPs electrode, the changes of electron-transfer resistance of the Au/DNA/MCH/DNA1 electrode modified without AuNPs is only varied from 6890 Ω to 8280 Ω (curve e in Fig. 1). This may be attributed to the bulky effect of Apt@AuNPs that hinders the electron-transfer on the surface of the electrode. Besides, the Apt@AuNPs bioconjugates will introduce more aptamers on the electrode, considering the property of the aptamer, it is not only nonconductive, but also the negatively charged phosphate backbone of aptamer repelling [Fe(CN)6]3  /4  anions from the electrode surface [32], which would lead to an obvious change of Ret. Therefore, it is expected that the application of DNAfunctionalized AuNPs can further enhance the sensitivity of the present sensing strategy.

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3.3. Signal amplification of AuNPs for sensing MUC1 In order to prove the signal amplification of AuNPs in the electrochemical aptasensor for sensing tumor biomarker, we investigated the changes of the electron-transfer resistance (ΔRet) of the two modified electrode, (a) Au/cDNA/MCH/DNA1 electrode and (b) Au/cDNA/MCH/ Apt@AuNPs electrode in the equimolar [Fe(CN)6]3 /4 solution after incubation with 10 nM MUC1 for 2 h. As shown in Fig. 2, the ΔRet of the Au/cDNA/MCH/Apt@AuNPs electrode (ΔRet ¼4960 Ω in Fig. 2b) is about 7 times higher than that of the Au/cDNA/MCH/Apt electrode (ΔRet ¼690 Ω in Fig. 2a). This demonstrates that for sensing the MUC1, the Au/cDNA/MCH/Apt@AuNPs electrode exhibits much stronger impedance signal than the Au/cDNA/MCH/Apt. This can be ascribed to that more aptamers introduced by Apt@AuNPs can bind more MUC1, forming huge molecular weight of Apt@AuNPs/MUC1. Once the Apt@AuNPs/MUC1 falls down from the electrode can lead to much more obvious decrease of ΔRet. Therefore, it is believed that the introduction of Apt@AuNPs is effective to improve the sensitivity of the aptasensor.

3.4. The EIS detection of MUC1 In this work, different concentrations of the target protein, MUC1, was detected by the EIS method using the Au/cDNA/MCH/ Apt@AuNPs electrode. The electrochemical measurements were conducted after the Au/cDNA/MCH/Apt@AuNPs electrode was treated with (a) 0 nM, (b) 0.5 nM, (c) 1 nM and (d) 5 nM MUC1 for 2 h respectively, the corresponding results are shown in Fig. 3. It can be observed from Fig. 3 that the Ret values of the modified electrode decrease with the increase of the concentration of MUC1.

Ret/

b

153

This is due to that more MUC1 protein was introduced on the modified electrode, and more Apt@AuNPs/MUC1 bioconjugates with high molecular weight would be formed. When the modified electrode was rinsed thoroughly with ultrapure water, the formed bioconjugates would fall down from the surface of the modified electrode because of the gravity, leading to a marked decrease of Ret. Therefore, it can be elucidated that the designed electrochemical aptasensor is feasible and effective for the sensitive detection of tumor biomarkers (MUC1 as a model). Herein, the detection sensitivity of the present aptasensor was further investigated according to the above procedure. Fig. 4 represents the impedance response of the sensing platform to different concentrations of MUC1. As illustrated in Fig. 4, a series of MUC1 solutions from 0.1 nM to 50 nM were investigated, it can be seen that the ΔRet values of the Au/cDNA/MCH/Apt@AuNPs electrode increase obviously with the increase of MUC1 concentration from 0.1 nM to 10 nM, and the ΔRet values no longer increase significantly as the concentration increases from 10 nM to higher concentrations. Meanwhile, as the insert of Fig. 4 displayed, there is a linear relationship between ΔRet and the concentration of MUC1 ranging from 0.5 nM to 10 nM, and the regression equation was ΔRet (Ω)¼0.8128 þ0.3884 cMUC1 (nM) (r ¼0.9658). This demonstrates that the designed aptasensor possesses the high detection sensitivity with a linear range, and MUC1 can be detectable at a concentration as low as 0.1 nM, which is much lower than that of some existing electrochemical aptasensor for MUC1. For example, Zhao et al. reported a “signal-on” electrochemical aptasensor for determination of two tumor markers with the detection of MUC1 is 20 nM [33]. Hu et al. developed a electrochemical aptamer biosensor based on HO-functionalized AuNPs amplification coupled with enzyme-linkage reactions with a detection limit of 2.2 nM [34]. Meanwhile, the detection limit of this label-free impedimetric aptasensor is also much simpler and more sensitive than the sensor based on other methods,for example, a detection limit of 250 nM was obtained by using quantum dot-labeling fluorescence method [16] or by using aptamer–antibody sandwich enzyme immunoassay [35]. 3.5. Selectivity, reproducibility and accuracy

a c

Ret/K

Z''/k

Fig. 2. The changes of the electron-transfer resistance (ΔRet) of (a) the Au/cDNA/ MCH/Apt electrode, (b) the Au/cDNA/MCH/Apt@AuNPs electrode after incubation with 10 nM MUC1 for 2 h.

From the practical point, a biosensor should be not only sensitive but also specific. In order to evaluate the selectivity of the aptasensor, control experiments were performed using different proteins, including carcinoembryonic antigen (CEA) and tumor necrosis factor-α (TNF-α) to replace MUC1. The Au/cDNA/MCH/ Apt@AuNPs modified electrode was incubated with 1 μM CEA, 1 μM TNF-α and 0.01 μM MUC1 for 2 h, respectively. As shown in

Ret/k

a

b

d c /nM MUC1

c Z'/k Fig. 3. Electrochemical impedance spectra of the Au/cDNA/MCH/Apt-AuNPs electrode after incubation with (a) 0 nM, (b) 0.5 nM, (c)1 nM, (d) 5 nM MUC1 for 2 h.

MUC1

/nM

Fig. 4. The calibration curve corresponding to the detection of MUC1 based on the changes of electron-transfer resistance (ΔRet) at the Au/cDNA/MCH/Apt@AuNPs electrode. The inset shows the linear curve of the aptasensor for MUC1.

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Fig. 5, it is noted that incubation of the Au/cDNA/MCH/Apt@AuNPs electrode with TNF-α (Fig. 5b) or CEA (Fig. 5c) does not produce significant changes of ΔRet response as compared to the case of MUC1 (Fig. 5a). These results demonstrate that the developed aptasensor has a sufficient specificity to its target molecule, MUC1. The reproducibility of the aptasensor was also investigated at the MUC1 concentration of 10 nM. Every five freshly prepared modified electrodes have been used for the detection of 10 nM MUC1. All electrodes exhibited similar electrochemical response and the relative standard deviation is 4.1% for MUC1 detection. In addition, the reproducibility of measurements for the same electrode was investigated at the MUC1 concentration of 10 nM, and the relative standard deviation for six times is 4.6%. This demonstrates the aptasensor for MUC1 detection is highly reproducible. The long-term stability of the sensing interface is further studied since it is an important issue for the practical implementation of MUC1 detection. The modified electrode, which was stored in Tris–HCl at 4 1C for 10 days, could still be used for the measurement of MUC1 without significant change of Ret. This implies that the proposed aptasensor has a sufficient stability for detection of MUC1. Additionally, the accuracy of the impedimetric aptasensor has been evaluated by the standard addition method. A series of samples were prepared by adding MUC1 of different concentrations to human blood serum (obtained from Cancer Hospital of Hunan Province, China). The analytical results for MUC 1 are shown in Table 1. From Table 1, it can be seen that the recovery and relative standard deviation values are acceptable. This implies that the impedimetric aptasensor has a good accuracy, which also indicates a promising feature for the analytical application in complex biological samples.

a

4. Conclusion In this paper, a simple electrochemical impedimetric aptasensor for the detection of MUC1 protein has been developed based on AuNPs signal amplification. EIS method has been demonstrated to be a sensitive way to monitor the changes on the surface of the electrode during the process of building the aptasensor. With this aptasensor, MUC1 can be detected at a concentration down to 0.1 nM. The electrochemical aptasensor possesses good selectivity, reproducibility and stability. Besides, taking advantage of EIS technology, this label-free aptasensor is not only convenient for building and detection, it is also satisfying the fast speed and good detection limit. By changing aptamers of different target molecules, this method can be used to detect many kinds of tumor markers, so it may have potential prospect for other studies. Acknowledgment Partial support of this work by the National Nature Science Foundation of China (21273288, 20773165, 21005090, 13JJ3004), the Fundamental Research Funds for the Central Universities (2011JQ1004) and the China Postdoctoral Science Foundation (2012M510136, 2013T60774) is acknowledged. References [1] [2] [3] [4] [5] [6] [7] [8]

R et /

[9] [10] [11] [12] [13] [14] [15] [16] [17] [18]

MUC1

b

c

TNF-

CEA

[19] [20] [21]

Fig. 5. The changes of the electron-transfer resistance (ΔRet) of the Au/cDNA/MCH/ Apt@AuNPs electrode after incubation with (a) 0.01 μM MUC1, (b) 1 μM TNF-α, and (c) 1 μM CEA for 2 h. The Ret of the Au/cDNA/MCH/Apt@AuNPs electrode was considered as the background.

[22] [23] [24] [25]

Table 1 Determination of MUC1 added in human blood serum (n ¼5) with the proposed aptasensora. Serum Concentration of samples MUC1 added (nM)

Concentration obtained with aptasensor (nM)

RSD (%)b

Recovery (%)

1 2 3 4

0.59 2.58 6.86 9.56

3.7 4.1 4.5 5.2

90.8 92.1 91.5 90.2

0.65 2.80 7.50 10.5 a

Impedance measurements were carried out within the range of 100 kHz– 100 mHz at the formal potential of the [Fe(CN)6]3  /4  (1:1). The amplitude of the alternate voltage was 5 mV. b RSD is relative standard deviation.

[26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

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A simple and sensitive impedimetric aptasensor for the detection of tumor markers based on gold nanoparticles signal amplification.

A simple and sensitive electrochemical impedimetric aptasensor based on gold nanoparticles (AuNPs) signal amplification was developed for the ultrasen...
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