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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Aptasensor for electrochemical sensing of angiogenin based on electrode modified by cationic polyelectrolyte-functionalized graphene/gold nanoparticles composites Zhengbo Chen n, Chenmeng Zhang, Xiaoxiao Li, He Ma, Chongqing Wan n, Kai Li, Yuqing Lin Department of Chemistry, Capital Normal University, Beijing 100048, China
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a b s t r a c t
Article history: Received 21 June 2014 Received in revised form 2 October 2014 Accepted 20 October 2014 Available online 22 October 2014
Herein, a label-free and highly sensitive electrochemical aptasensor for the detection of angiogenin was proposed based on a conformational change of aptamer and amplification by poly(diallyldimethyl ammonium chloride) (PDDA)-functionalized graphene/gold nanoparticles (AuNPs) composites-modified electrode. PDDA-functionalized graphene (P-GR) nanosheets as the building block in the self-assembly of GR nanosheets/AuNPs heterostructure enhanced the electrochemical detection performance. The electrochemical aptasensor has an extraordinarily sensitive response to angiogenin in a linear range from 0.1 pM to 5 nM with a detection limit of 0.064 pM. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future. & 2014 Elsevier B.V. All rights reserved.
Keywords: Poly(diallyldimethyl ammonium chloride) Gold nanoparticles Graphene Aptamer Electrochemical Angiogenin
1. Introduction Angiogenin, a 14.4-kDa polypeptide, a member of the pancreatic ribonuclease family, was originally isolated solely based on its ability to induce angiogenesis (Fett et al., 1985). It can strongly stimulate blood vessel formation and its concentration in serum is elevated in patients affected by various types of cancers (Yoshioka et al., 2006). Targeting angiogenin may find a potential therapeutic approach in human malignant melanoma. Thus, sensitive detection of angiogenin has become a very important issue. Serum concentration of angiogenin is commonly detected with antibodybased enzyme-linked immunosorbant assay (Katona et al., 2005). In 1998, AL6, DNA aptamers of angiogenin, was generated by in vitro selection process (Nobile et al., 1998). To date, very few studies have been carried out on detection of angiogenin. At present, enzyme-linked immunosorbent assay (ELISA) is the most widely used immunoassay method in the detection of angiogenin (Zhao et al., 2005; Kishimoto et al., 2005). The fluorescence detection of angiogenin using molecular labels is generally used in most of the cases (Li et al., 2007). However, the poor limit of detection, long assay time, and photo-bleaching effect often limit its potential applications. n
Corresponding authors. E-mail addresses: [email protected] (Z. Chen), [email protected] (C. Wan).
http://dx.doi.org/10.1016/j.bios.2014.10.046 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Recently, electrochemical aptamer-based (E-AB) sensors have emerged as a promising and versatile new biosensor platform (Zhang et al., 2009; Jin et al., 2007). The electrochemical methods play important roles in the development of aptasensors due to their high sensitivity, fast response, simple instrumentation, low production cost, and portability. Among all the voltammetric techniques, SWV has many advantages, such as excellent sensitivity and high speed. This high speed, coupled with computer control and signal averaging, allows for experiments to be performed repetitively and increases the signal-to-noise ratio. Compared to both linear sweep and cyclic voltammetry (CV), SWV has a much broader dynamic range and lower limit of detection because of its efficient discrimination of capacitance current (Li et al., 2010, 2011). As a novel affinity reagent, aptamers can provide the specificity lacking in many extraction matrixes. Aptamers are single-stranded oligonucleotides that bind target molecules with very high affinity in a manner similar to antibodies. However, aptamers possess more advantages over antibodies such as chemical synthesis, selection through the systematic evolution of ligands by exponential enrichment (SELEX) process, easy modification, high stability, target versatility, easy-to-stock, and resistant to denaturation and degradation. All of these unique properties increase the likelihood that aptamers will outperform other affinity reagents (Sefah et al., 2009).
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Graphene, a one-atom thick and two-dimensional closely packed honeycomb lattice, has become a fast rising star among carbon materials, triggering a gold rush to exploit its possible applications in both experimental and theoretical studies, since its discovery by Novoselov and Geim in 2004 (Novoselov et al., 2004). Graphene has remarkably high electron mobility under ambient conditions with reported values in excess of 15,000 cm2/(V s) (Zhang et al., 2005). Furthermore, graphene has a very large specific surface area (theoretical value 2600 m2/g) with low manufacturing cost. These unique properties provide potential applications in synthesizing nanocomposites (Stankovich et al., 2006; Xu et al., 2008; Williarris et al., 2008) and fabricating various microelectrical devices, such as battery (Guo et al., 2009), field-effect transistors (Gilje et al., 2007), electromechanical resonators (Bunch et al., 2007), and ultrasensitive sensors (Schedin et al., 2007), etc. Research on electrical biomolecule detection using graphene and graphene-like materials has gradually increased over the past few years, however, the integration of graphene with noble metal nanoparticles such as gold nanoparticles (AuNPs) (Baby et al., 2010; Hong et al., 2010), is relatively new in biosensor applications. As we know, directly taking advantage of graphene nanosheets' negative charges in the process of self-assembly of these metal nanoparticles has two main difficulties: (1) graphene nanosheets are apt to aggregate, which are caused by strong van der Waals' and π–π interactions between graphene nanosheets; and (2) the negative charge is too weak to assemble AuNPs directly. To overcome the above two obstacles, we employ poly(diallyldimethyl ammonium chloride) (PDDA) as the modifier onto the graphene nanosheets, The positively charged PDDA protected P-GR colloids were obtained via the in situ reduction of GO with abundant oxygen functionalities in the presence of PDDA. The use of PDDA not only alters the electrostatic charges of graphene, but also provides a convenient self-assembly approach for the hybridization of graphene. Owing to the oxygenation of the GR nanosheets in GO, it can weaken the van der Waals interactions between the layers of GO and make them strongly hydrophilic, which thus facilitates their hydration and exfoliation in aqueous media. The reduction process of GO removes oxygen functionalities with partial restoration of
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aromatic grapheme network (sp2) (Gilje et al., 2007), leading to hydrophobic GR sheets. After GR sheets were modified with positively charged PDDA, the obtained PDDA-functionalized graphene (P-GR) nanosheets were hydrophilic and soluble in the presence of a static repulsion force. Herein, as shown in Scheme 1, we present a label-free and highly sensitive electrochemical aptasensor for angiogenin detection based on P-GR nanosheets/AuNPs composites-modified glassy carbon electrode (GCE). The transduction principle is based on electron transfer resistances in the presence of an [Fe(CN)6]3 /4 redox couple. Angiogenin can promote the conversion of the DNA sequence from a loose random coil into the secondary stem-loop structure (Li et al., 2008) (Fig. S1), and leads to an increase in the electron transfer resistance, repelling the [Fe(CN)6]3 /4 redox couple to approach the electrode. Thus, it resulted in a substantial decrease in square-wave voltammetry (SWV) current. The proposed electrochemical aptasensor for angiogenin is very simple, cost-effective, highly sensitive. Of note, it is lable-free, and requires no external modification on the biomolecules. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future.
2. Experimental section 2.1. Reagents and chemicals The aptamer of angiogenin used in this study was synthesized by TaKaRa Biotechnology (Dalian, China) Co., Ltd. The sequence of oligonucleotide employed is ferrocene-5′-CGG ACG AAT GCT TTG ATG TTG TGC TGG ATC CAG CGT TCA TTC TCA-(CH2)6-(SH)-3′. Sodium tetrachloroaurate (III) (HAuCl4) and sodium citrate were purchased from Sigma-Aldrich (USA). Tris-base was purchased from Sigma-Aldrich. Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Alfa Aesar. 6-Mercapto-1hexanol (MCH) was purchased from J&K Chemical Ltd. All other reagents are of analytical reagent grade. All solutions were prepared with doubly distilled water. Immobilization buffer
Scheme 1. Schematic representation of the overall detection of angiogenin based on a conformational change of aptamer and amplification by P-GR/AuNPs compositesmodified electrode.
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(20 mM Tris–HCl, pH 7.40) containing 10 mM K3Fe(CN)6/K4Fe(CN)6 was chosen as a supporting electrolyte for angiogenin. 2.2. Instrumentation SWV and electrochemical impedance spectroscopy (EIS) were conducted with an advanced electrochemical system (Princeton Applied Research (PARSTAT 2273)) and controlled by the software ZSimpWin (Copyright@1999–2002 EChem software. Author: Bruno Yeum, PdD). All electrochemical experiments were performed using a conventional three-electrode system with a fabricated GCE or GCE (diameter, 3 mm) as the working electrode, Ag/AgCl (sat. KCl) as the reference electrode, and a platinum as counter electrode. All potentials were referred to the reference electrode. The products were characterized with transmission electron microscope (TEM), high-resolution TEM (HRTEM), X-ray powder diffraction (XRD), and Raman spectra. TEM and HRTEM images were obtained on a Hitachi (H-7650, 80 kV) transmission electron microscope. Samples for TEM and HRTEM were prepared by placing two drops of the dispersed samples solution on a carbonfilm-coated copper grid (400 mesh) and then drying under air. XRD was used as a Rigaku Dmax 2200 X-ray diffractometer with Cu Kα radiation (λ ¼1.5416 Å). The samples for Raman measurements were obtained by dropping suspensions of P-GR–AuNPs onto the quartz cuvettes and then drying the cuvettes in air to form films. Then the Raman spectra were recorded at ambient temperature on a Renishaw Raman system model 1000 spectrometer with a 200 mW argon-ion laser at an excitation wavelength of 514.5 nm. pH was measured using a PB-10 Precision pH Meter (Sartorius, Germany), and sonication was carried out with a KuDos Ultrasonic Cleaner (Shanghai, China). 2.3. Preparation of P-GR nanosheets First, the GO was synthesized from natural graphite powder by the modified Hummers method (Hummers and Offeman, 1958; Kovtyukhova et al., 1999). Then, the as-prepared GO was subjected to dialysis for 7 days to completely remove metal ions and acids (Wang et al., 2010; Dong et al., 2010; Choi et al., 2010). Finally, the product was dried in air at room temperature. P-GR nanosheets were prepared via an exfoliation/in situ reduction of GO in the presence of PDDA according to the literature (Fang et al., 2010). First, 20% PDDA (0.5 mL) solution was added to 0.5% GO solution (100 mL) and stirred for 30 min. Then 80% hydrazine hydrate (0.5 mL) was added and stirring was maintained for 24 h at 90 °C. Finally, the black P-GR nanosheets could be obtained by filtration and washing with distilled water, and then redispersed readily in water upon mild sonication, forming a black suspension (0.1 mg mL 1).
2.5. Fabrication of the aptasensor The fabrication of the aptasensor includes three steps as follows: the pretreatment of a GCE, self-assembly of P-GR–AuNPs, and immobilization of aptamer. A GCE was firstly polished with 1, 0.3, 0.05 μm alumina powders and sonicated in ethanol and deionized water. Then, the electrode was subsequently voltammetrically cycled in 0.5 M H2SO4 with the potential between 0.2 and þ 1.5 V at 0.1 V/s until a representative cyclic voltammogram of a clean GCE was obtained. In the second step, 5 mg mL 1 P-GR– AuNPs composite solution (10 μL) was dropped onto the electrode, and dried in the air, followed by being thoroughly rinsed with ethanol and deionized water. The last step is the immobilization of aptamer onto the P-GR–AuNPs-modified GCE via sulphur–Au affinity. The ss-DNA was firstly prepared as follows: the ss-DNA (1 μM each) including 1 mM Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP was employed to reduce disulfide bond oligos) in I-B was heated to 90 °C for 5 min and then gradually cooled to room temperature. Subsequently, the P-GR–AuNPsmodified GCE was incubated in the resulting ss-DNA solution at room temperature for 24 h and then passivated by 1 mM 6-mercapto-1-hexanol (MCH was used to remove nonspecific aptamer adsorption on the P-GR–AuNPs-modified GCE surface) in I-B for 30 min. Finally, the ss-DNAs/P-GR–AuNPs/GCE was rinsed with I-B and then with deionized water, followed by drying under a N2 stream. For angiogenin detection, the ss-DNAs/P-GR–AuNPs/GCE was incubated in a 10 μL droplet of angiogenin of various concentrations in I-B for 30 min at 37 °C. The interfusion of P-GR with AuNPs provided a large specific surface for a large number of aptamers loading, which resulted in extreme sensitivity for angiogenin detection.
3. Results and discussion 3.1. Sensing mechanism of the aptasensor A schematic representation of aptasensor with fabrication steps and performance is displayed in Scheme 1. On exposure of the anti-angiogenin aptamer modified electrode to immobilization buffer (I-B: 20 mM Tris–HCl, pH 7.40 with 140 mM NaCl, 20 mM MgCl2 and 20 mM KCl) containing [Fe(CN)6]3 /4 , which serves as a signaling transducer, the negatively-charged aptamer probe acts as an electrostatic barrier that repells the [Fe(CN)6]3 /4 marker and hinders its interfacial electron transfer reaction. After angiogenin was added to the electrochemical cell, the aptamer bound to angiogenin and folded to a secondary stem-loop structure (Fig. S1), resulting in less availability for a redox reaction, which led to even smaller SWV current. 3.2. Characterization of P-GR–AuNPs
2.4. Preparation of P-GR/AuNPs composites P-GR/AuNPs composites were synthesized by depositing AuNPs onto the surface of the P-GR nanosheets through spontaneous chemical reduction of chloroauric acid by sodium citrate. First, 5 mL of 30 mM chloroauric acid solution was added to 85 mL suspension (0.1 mg mL 1) under stirring. 10 mL of 20 g/L sodium citrate was then introduced to the well-stirred mixture when the mixture was heated to the boiling point of water. After refluxing for 10 min, driven by the electrostatic interaction, the citratecapped AuNPs quickly adhered to the surface of the P-GR nanosheets. The black precipitate was collected by centrifugation, which was then rinsed with absolute ethanol for 6 times. Finally, the products were then dried in air at room temperature for further characterization.
The typical TEM images of as-prepared P-GR–AuNPs hybrids at different magnifications were displayed in Fig. 1A and B. The TEM images show that the significant high-loading and uniform distribution of AuNPs on the P-GR nanosheets, making more aptamers adsorb on the P-GR nanosheets. The size of AuNPs ranges from 15 to 30 nm. A lattice resolved HRTEM image of the P-GR– AuNPs composites is shown in Fig. 1C, from which the crystalline features of AuNPs can be clearly observed. The lattice fringe spacing between two adjacent crystal planes of the particle was determined to be 0.204 nm in the HRTEM image, corresponding to the (200) lattice plane of the Au. Fig. 1D illustrates the structural characteristics of the as-prepared P-GR–AuNPs investigated by XRD analysis. The four peaks with d values of 2.36, 2.04, 1.44, 1.23, correspond to the (111), (200), (220) and (311), respectively, are in
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Fig. 1. (A, B) Typical TEM images of P-GR–AuNPs at different magnifications. (C) HRTEM image, (D) X-Ray diffraction pattern, and (E) Raman spectrum of P-GR–AuNPs.
good agreement with the standard values of pure metallic Au (JCPDS, 04-0784). To obtain further information on the structure and topology, Raman spectroscopy was carried out. As shown in Fig. 1E, the Raman spectrum of graphene exhibits the presence of D (1334.8 cm 1), and G (1593.91 cm 1) bands. The D band arises from sp3-hybridized carbon, and the G represents the E2g zone center mode of the crystalline graphite (Ferrari and Robertson, 2000). This means D/G intensity ratio is proportional to the number of defect sites in graphite carbon (Ferrari and Robertson, 2000). The D/G intensity ratio of GR is 1.06, indicating that there are significant edge-plane-like defective sites existing on the surface of grapheme (Ferrari and Robertson, 2000). The P-GR–AuNPs are suitable for electrochemical analysis because of the excellent conductivity of graphene nanosheets. 3.3. Characterization of aptasensor In order to clarify the electrochemical properties of the resulting aptasensor, EIS is an effective method for monitoring the changes in the surface features of the modified electrodes in the assembly process (Li et al., 2009), as shown in Fig. 2. The impedance spectra include a semicircle portion and a linear portion. The semicircle portion at higher frequencies corresponds to the electron-transfer-limited process, and the linear portion at lower frequencies represents the diffusion-limited process. The change in semicircle diameter is a result of the change in the interfacial resistance Rct, due to electrons transferring from the modified electrode to [Fe(CN)6]3 /4 in solution. Curve a represents the electrochemical impedance of the bare GCE, which shows a nearly
Fig. 2. Nyquist plots of impedance spectra at (a) the bare GCE, (b) P-GR/GCE, (c) PGR–AuNPs/GCE, (d) the aptamer/P-GR–AuNPs/GCE, and (e) 10 nM angiogenin/the aptamer/P-GR–AuNPs/GCE. Inset is the equivalent circuit.
straight line. After the modification of P-GR nanosheets, owing to nonconductive properties of PDDA, the diameter of the semicircle increased dramatically with a Rct of 2000 Ω (Fig. 2, curve b). After modification with AuNPs, the resulting electrode showed a much lower resistance (Fig. 2, curve c), It can be attributed to the fact that AuNPs can further enhance the electron transfer rate. After the aptamer was immobilized onto the surface of the P-GR– AuNPs/GCE, the Rct value significantly increased to 7000 Ω
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(Fig. 2, curve d). This can be attributed to the fact that [Fe(CN)6]3 /4 received electrostatic repulsive forces from the negatively charged phosphate backbones of DNA. After 10 nM angiogenin was captured onto the surface of the aptamer, the Rct value markedly increased to 11,000 Ω (Fig. 2, curve e), which implies that angiogenin can promote the conversion of the DNA sequence from a loose random coil into the secondary stem-loop structure, and leads to an increase in the electron transfer resistance. By fitting the data (inset of Fig. 2), the sequence of the values of Rct for different electrodes is P-GR/GCE 4P-GR–AuNPs/ GCE. This demonstrates the P-GR–AuNPs/GCE has higher electrochemical activity than P-GR/GCE. The surface structure and morphology of GCE before and after modification of P-GR–AuNPs are characterized by SEM, as shown in Fig. S2(A, B). Compared with Fig. S2A, it can be clearly seen that the P-GR–AuNPs have been successfully absorbed on the surface of the GCE. 3.4. Optimization of the experimental parameters The performance of the developed angiogenin sensor is strongly influenced by the experimental conditions such as incubation temperature, incubation time and pH in the solution. Therefore, each detection parameter was optimized in our study, while keeping the other parameters constant. The incubation temperature of angiogenin has great effects on sensing effect (Fig. S3). The SWV current quickly decreased, reached a minimum at 37 °C, and rose up dramatically when the reaction temperature was above 37 °C. Thus, 37 °C was chosen as an optimal incubation temperature in the experiments. As expected, incubation time and pH in the solution also influence the signal responses of the sensor in the presence of angiogenin. As shown in Fig. S4, it was clearly observed that the SWV peak current decreased with the increase of incubation time. When the incubation time is 30 min, the SWV signal reached saturation. Thus, 30 min was chosen as the optimal in the following experiments. pH value can affect the formation of stem-loop structure, and then control its protein binding. Therefore, pH value can affect the sensitivity of the method, and the effect of pH value was studied over the range from 4.0 to 10.0 (Fig. S5). The peak current decreased with the pH over the range from 4.0 to 7.40, whereas it increased with further increase of pH value. Thereby, pH 7.40 was selected as the optimum pH environment.
Table 1 Comparison between the proposed assay and other reported techniques for the determination of angiogenin. Method
Linear range
LOD
References
Electrochemical FRETa Electrochemical ELISAb Electrochemical Fluorescence
0.1 pM–5 nM 0.5–40 nM 0.01–30 nM N/A 0.2 pM–10 nM N/A
0.064 pM 0.2 nM 1 pM 0.694 nM 0.07 pM 6.9 pM
This work Li et al. (2008) Li et al. (2011) Chon et al. (2013) Shi et al. (2014) Li et al. (2007)
a b
Fluorescence resonance energy transfer. Enzyme-linked immunosorbent assay.
3.5. Performance of the aptasensor To quantitatively assess the detection limit and response range of the angiogenin aptasensor, Fig. 3A shows the SWV plots of P-GR–AuNPs/GCE for angiogenin with different concentrations ranging from 0.1 pM to 5 nM. The introduction of angiogenin at different concentrations to the sensing system induced different decreases in the SWV peak currents since the angiogenin bound to the aptamer and the aptamer folded to the secondary stem-loop structure, repelling the redox marker ions to approach the electrode. Fig. 3B depicts signal suppression is linear with logarithm of angiogenin concentration over a range from 0.1 pM to 5 nM with a correlation coefficient of 0.99, and the limit of detection is 0.064 pM as calculated in terms of the 3s rule (Kaiser, 1973). The values of relative standard deviation from the aptamer/P-GR– AuNPs/GCE for each angiogenin concentrations are listed in Table S1. The detection limit of our proposed angiogenin sensor (0.064 pM) is lower than that in the literature and the linear range of our work, as shown in Table 1. 3.6. The specificity of aptasensor To test the specificity of the electrochemical aptasensor for angiogenin (1 pM) analysis, other proteins including thrombin, and bovine serum albumin (BSA) (each at 1 pM) as the potential interference proteins were tested under the same conditions (Fig. 4). As expected, after the addition of 1 pM angiogenin, a significant decrease of the SWV signal was obtained. However, the SWV signal for the other proteins had little changes after treatment with 1 pM thrombin, and BSA, respectively compared with that obtained containing 1 pM angiogenin, indicating that the
Fig. 3. (A) Square-wave voltammograms of P-GR–AuNPs/GCE to different concentrations of angiogenin. (B) The peak current in the SWV as a function of angiogenin concentration. Inset: The peak current is linear with logarithm of angiogenin concentration over the range from 0.1 pM to 5 nM.
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Scientific Research Project of Beijing Educational Committee (KM201410028006).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.10.046.
References
Fig. 4. The specificity of the electrochemical aptasensor for angiogenin (1 pM) (1 pM thrombin, and 1 pM BSA as the potential interference proteins).
response of the aptasensor to angiogenin was unaffected by presence of other proteins. The result clearly indicated that our method could be used to detect angiogenin with high specificity. 3.7. The reproducibility of the aptasensor Besides sensitivity and selectivity, the reproducibility of the angiogenin biosensor was evaluated. Four different P-GR–AuNPs/ aptamer/GCEs were conducted independently for 10 pM angiogenin SWV response, as shown in Fig. S6. We can see that four repetitive measurements of the aptasensor yielded a reproducible SWV signal with a relative standard deviation (R.S.D.) of these measurements of 2.71%, showing the good reproducibility of the sensor.
4. Conclusion To sum up, we have presented a facile self-assembly method to synthesize 2D P-GR–AuNPs hybrids via an exfoliation/in situ reduction of GO in the presence of PDDA and spontaneous chemical reduction of chloroauric acid by sodium citrate. The AuNPs distributed uniformly on the surface of P-GR nanosheets. The P-GR– AuNPs hybrids were employed as the electrochemical enhanced material for angiogenin sensing, which showed wide linear ranges (0.1 pM to 5 nM) and low detection limits (0.064 pM). The approach provided a convenient, low-cost, sensitive, and specific method for protein detection, which might provide an effective candidate in clinical research.
Acknowledgments All authors gratefully acknowledge the financial support of the Natural Natural Science Foundation of China (No. 21371123) and
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Aptasensor for electrochemical sensing of angiogenin based on electrode modified by cationic polyelectrolyte-functionalized graphene/gold nanoparticles composites Zhengbo Chen n, Chenmeng Zhang, Xiaoxiao Li, He Ma, Chongqing Wan n, Kai Li, Yuqing Lin Department of Chemistry, Capital Normal University, Beijing 100048, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 June 2014 Received in revised form 2 October 2014 Accepted 20 October 2014 Available online 22 October 2014
Herein, a label-free and highly sensitive electrochemical aptasensor for the detection of angiogenin was proposed based on a conformational change of aptamer and amplification by poly(diallyldimethyl ammonium chloride) (PDDA)-functionalized graphene/gold nanoparticles (AuNPs) composites-modified electrode. PDDA-functionalized graphene (P-GR) nanosheets as the building block in the self-assembly of GR nanosheets/AuNPs heterostructure enhanced the electrochemical detection performance. The electrochemical aptasensor has an extraordinarily sensitive response to angiogenin in a linear range from 0.1 pM to 5 nM with a detection limit of 0.064 pM. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future. & 2014 Elsevier B.V. All rights reserved.
Keywords: Poly(diallyldimethyl ammonium chloride) Gold nanoparticles Graphene Aptamer Electrochemical Angiogenin
1. Introduction Angiogenin, a 14.4-kDa polypeptide, a member of the pancreatic ribonuclease family, was originally isolated solely based on its ability to induce angiogenesis (Fett et al., 1985). It can strongly stimulate blood vessel formation and its concentration in serum is elevated in patients affected by various types of cancers (Yoshioka et al., 2006). Targeting angiogenin may find a potential therapeutic approach in human malignant melanoma. Thus, sensitive detection of angiogenin has become a very important issue. Serum concentration of angiogenin is commonly detected with antibodybased enzyme-linked immunosorbant assay (Katona et al., 2005). In 1998, AL6, DNA aptamers of angiogenin, was generated by in vitro selection process (Nobile et al., 1998). To date, very few studies have been carried out on detection of angiogenin. At present, enzyme-linked immunosorbent assay (ELISA) is the most widely used immunoassay method in the detection of angiogenin (Zhao et al., 2005; Kishimoto et al., 2005). The fluorescence detection of angiogenin using molecular labels is generally used in most of the cases (Li et al., 2007). However, the poor limit of detection, long assay time, and photo-bleaching effect often limit its potential applications. n
Corresponding authors. E-mail addresses: [email protected] (Z. Chen), [email protected] (C. Wan).
http://dx.doi.org/10.1016/j.bios.2014.10.046 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Recently, electrochemical aptamer-based (E-AB) sensors have emerged as a promising and versatile new biosensor platform (Zhang et al., 2009; Jin et al., 2007). The electrochemical methods play important roles in the development of aptasensors due to their high sensitivity, fast response, simple instrumentation, low production cost, and portability. Among all the voltammetric techniques, SWV has many advantages, such as excellent sensitivity and high speed. This high speed, coupled with computer control and signal averaging, allows for experiments to be performed repetitively and increases the signal-to-noise ratio. Compared to both linear sweep and cyclic voltammetry (CV), SWV has a much broader dynamic range and lower limit of detection because of its efficient discrimination of capacitance current (Li et al., 2010, 2011). As a novel affinity reagent, aptamers can provide the specificity lacking in many extraction matrixes. Aptamers are single-stranded oligonucleotides that bind target molecules with very high affinity in a manner similar to antibodies. However, aptamers possess more advantages over antibodies such as chemical synthesis, selection through the systematic evolution of ligands by exponential enrichment (SELEX) process, easy modification, high stability, target versatility, easy-to-stock, and resistant to denaturation and degradation. All of these unique properties increase the likelihood that aptamers will outperform other affinity reagents (Sefah et al., 2009).
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Graphene, a one-atom thick and two-dimensional closely packed honeycomb lattice, has become a fast rising star among carbon materials, triggering a gold rush to exploit its possible applications in both experimental and theoretical studies, since its discovery by Novoselov and Geim in 2004 (Novoselov et al., 2004). Graphene has remarkably high electron mobility under ambient conditions with reported values in excess of 15,000 cm2/(V s) (Zhang et al., 2005). Furthermore, graphene has a very large specific surface area (theoretical value 2600 m2/g) with low manufacturing cost. These unique properties provide potential applications in synthesizing nanocomposites (Stankovich et al., 2006; Xu et al., 2008; Williarris et al., 2008) and fabricating various microelectrical devices, such as battery (Guo et al., 2009), field-effect transistors (Gilje et al., 2007), electromechanical resonators (Bunch et al., 2007), and ultrasensitive sensors (Schedin et al., 2007), etc. Research on electrical biomolecule detection using graphene and graphene-like materials has gradually increased over the past few years, however, the integration of graphene with noble metal nanoparticles such as gold nanoparticles (AuNPs) (Baby et al., 2010; Hong et al., 2010), is relatively new in biosensor applications. As we know, directly taking advantage of graphene nanosheets' negative charges in the process of self-assembly of these metal nanoparticles has two main difficulties: (1) graphene nanosheets are apt to aggregate, which are caused by strong van der Waals' and π–π interactions between graphene nanosheets; and (2) the negative charge is too weak to assemble AuNPs directly. To overcome the above two obstacles, we employ poly(diallyldimethyl ammonium chloride) (PDDA) as the modifier onto the graphene nanosheets, The positively charged PDDA protected P-GR colloids were obtained via the in situ reduction of GO with abundant oxygen functionalities in the presence of PDDA. The use of PDDA not only alters the electrostatic charges of graphene, but also provides a convenient self-assembly approach for the hybridization of graphene. Owing to the oxygenation of the GR nanosheets in GO, it can weaken the van der Waals interactions between the layers of GO and make them strongly hydrophilic, which thus facilitates their hydration and exfoliation in aqueous media. The reduction process of GO removes oxygen functionalities with partial restoration of
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aromatic grapheme network (sp2) (Gilje et al., 2007), leading to hydrophobic GR sheets. After GR sheets were modified with positively charged PDDA, the obtained PDDA-functionalized graphene (P-GR) nanosheets were hydrophilic and soluble in the presence of a static repulsion force. Herein, as shown in Scheme 1, we present a label-free and highly sensitive electrochemical aptasensor for angiogenin detection based on P-GR nanosheets/AuNPs composites-modified glassy carbon electrode (GCE). The transduction principle is based on electron transfer resistances in the presence of an [Fe(CN)6]3 /4 redox couple. Angiogenin can promote the conversion of the DNA sequence from a loose random coil into the secondary stem-loop structure (Li et al., 2008) (Fig. S1), and leads to an increase in the electron transfer resistance, repelling the [Fe(CN)6]3 /4 redox couple to approach the electrode. Thus, it resulted in a substantial decrease in square-wave voltammetry (SWV) current. The proposed electrochemical aptasensor for angiogenin is very simple, cost-effective, highly sensitive. Of note, it is lable-free, and requires no external modification on the biomolecules. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future.
2. Experimental section 2.1. Reagents and chemicals The aptamer of angiogenin used in this study was synthesized by TaKaRa Biotechnology (Dalian, China) Co., Ltd. The sequence of oligonucleotide employed is ferrocene-5′-CGG ACG AAT GCT TTG ATG TTG TGC TGG ATC CAG CGT TCA TTC TCA-(CH2)6-(SH)-3′. Sodium tetrachloroaurate (III) (HAuCl4) and sodium citrate were purchased from Sigma-Aldrich (USA). Tris-base was purchased from Sigma-Aldrich. Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Alfa Aesar. 6-Mercapto-1hexanol (MCH) was purchased from J&K Chemical Ltd. All other reagents are of analytical reagent grade. All solutions were prepared with doubly distilled water. Immobilization buffer
Scheme 1. Schematic representation of the overall detection of angiogenin based on a conformational change of aptamer and amplification by P-GR/AuNPs compositesmodified electrode.
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(20 mM Tris–HCl, pH 7.40) containing 10 mM K3Fe(CN)6/K4Fe(CN)6 was chosen as a supporting electrolyte for angiogenin. 2.2. Instrumentation SWV and electrochemical impedance spectroscopy (EIS) were conducted with an advanced electrochemical system (Princeton Applied Research (PARSTAT 2273)) and controlled by the software ZSimpWin (Copyright@1999–2002 EChem software. Author: Bruno Yeum, PdD). All electrochemical experiments were performed using a conventional three-electrode system with a fabricated GCE or GCE (diameter, 3 mm) as the working electrode, Ag/AgCl (sat. KCl) as the reference electrode, and a platinum as counter electrode. All potentials were referred to the reference electrode. The products were characterized with transmission electron microscope (TEM), high-resolution TEM (HRTEM), X-ray powder diffraction (XRD), and Raman spectra. TEM and HRTEM images were obtained on a Hitachi (H-7650, 80 kV) transmission electron microscope. Samples for TEM and HRTEM were prepared by placing two drops of the dispersed samples solution on a carbonfilm-coated copper grid (400 mesh) and then drying under air. XRD was used as a Rigaku Dmax 2200 X-ray diffractometer with Cu Kα radiation (λ ¼1.5416 Å). The samples for Raman measurements were obtained by dropping suspensions of P-GR–AuNPs onto the quartz cuvettes and then drying the cuvettes in air to form films. Then the Raman spectra were recorded at ambient temperature on a Renishaw Raman system model 1000 spectrometer with a 200 mW argon-ion laser at an excitation wavelength of 514.5 nm. pH was measured using a PB-10 Precision pH Meter (Sartorius, Germany), and sonication was carried out with a KuDos Ultrasonic Cleaner (Shanghai, China). 2.3. Preparation of P-GR nanosheets First, the GO was synthesized from natural graphite powder by the modified Hummers method (Hummers and Offeman, 1958; Kovtyukhova et al., 1999). Then, the as-prepared GO was subjected to dialysis for 7 days to completely remove metal ions and acids (Wang et al., 2010; Dong et al., 2010; Choi et al., 2010). Finally, the product was dried in air at room temperature. P-GR nanosheets were prepared via an exfoliation/in situ reduction of GO in the presence of PDDA according to the literature (Fang et al., 2010). First, 20% PDDA (0.5 mL) solution was added to 0.5% GO solution (100 mL) and stirred for 30 min. Then 80% hydrazine hydrate (0.5 mL) was added and stirring was maintained for 24 h at 90 °C. Finally, the black P-GR nanosheets could be obtained by filtration and washing with distilled water, and then redispersed readily in water upon mild sonication, forming a black suspension (0.1 mg mL 1).
2.5. Fabrication of the aptasensor The fabrication of the aptasensor includes three steps as follows: the pretreatment of a GCE, self-assembly of P-GR–AuNPs, and immobilization of aptamer. A GCE was firstly polished with 1, 0.3, 0.05 μm alumina powders and sonicated in ethanol and deionized water. Then, the electrode was subsequently voltammetrically cycled in 0.5 M H2SO4 with the potential between 0.2 and þ 1.5 V at 0.1 V/s until a representative cyclic voltammogram of a clean GCE was obtained. In the second step, 5 mg mL 1 P-GR– AuNPs composite solution (10 μL) was dropped onto the electrode, and dried in the air, followed by being thoroughly rinsed with ethanol and deionized water. The last step is the immobilization of aptamer onto the P-GR–AuNPs-modified GCE via sulphur–Au affinity. The ss-DNA was firstly prepared as follows: the ss-DNA (1 μM each) including 1 mM Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP was employed to reduce disulfide bond oligos) in I-B was heated to 90 °C for 5 min and then gradually cooled to room temperature. Subsequently, the P-GR–AuNPsmodified GCE was incubated in the resulting ss-DNA solution at room temperature for 24 h and then passivated by 1 mM 6-mercapto-1-hexanol (MCH was used to remove nonspecific aptamer adsorption on the P-GR–AuNPs-modified GCE surface) in I-B for 30 min. Finally, the ss-DNAs/P-GR–AuNPs/GCE was rinsed with I-B and then with deionized water, followed by drying under a N2 stream. For angiogenin detection, the ss-DNAs/P-GR–AuNPs/GCE was incubated in a 10 μL droplet of angiogenin of various concentrations in I-B for 30 min at 37 °C. The interfusion of P-GR with AuNPs provided a large specific surface for a large number of aptamers loading, which resulted in extreme sensitivity for angiogenin detection.
3. Results and discussion 3.1. Sensing mechanism of the aptasensor A schematic representation of aptasensor with fabrication steps and performance is displayed in Scheme 1. On exposure of the anti-angiogenin aptamer modified electrode to immobilization buffer (I-B: 20 mM Tris–HCl, pH 7.40 with 140 mM NaCl, 20 mM MgCl2 and 20 mM KCl) containing [Fe(CN)6]3 /4 , which serves as a signaling transducer, the negatively-charged aptamer probe acts as an electrostatic barrier that repells the [Fe(CN)6]3 /4 marker and hinders its interfacial electron transfer reaction. After angiogenin was added to the electrochemical cell, the aptamer bound to angiogenin and folded to a secondary stem-loop structure (Fig. S1), resulting in less availability for a redox reaction, which led to even smaller SWV current. 3.2. Characterization of P-GR–AuNPs
2.4. Preparation of P-GR/AuNPs composites P-GR/AuNPs composites were synthesized by depositing AuNPs onto the surface of the P-GR nanosheets through spontaneous chemical reduction of chloroauric acid by sodium citrate. First, 5 mL of 30 mM chloroauric acid solution was added to 85 mL suspension (0.1 mg mL 1) under stirring. 10 mL of 20 g/L sodium citrate was then introduced to the well-stirred mixture when the mixture was heated to the boiling point of water. After refluxing for 10 min, driven by the electrostatic interaction, the citratecapped AuNPs quickly adhered to the surface of the P-GR nanosheets. The black precipitate was collected by centrifugation, which was then rinsed with absolute ethanol for 6 times. Finally, the products were then dried in air at room temperature for further characterization.
The typical TEM images of as-prepared P-GR–AuNPs hybrids at different magnifications were displayed in Fig. 1A and B. The TEM images show that the significant high-loading and uniform distribution of AuNPs on the P-GR nanosheets, making more aptamers adsorb on the P-GR nanosheets. The size of AuNPs ranges from 15 to 30 nm. A lattice resolved HRTEM image of the P-GR– AuNPs composites is shown in Fig. 1C, from which the crystalline features of AuNPs can be clearly observed. The lattice fringe spacing between two adjacent crystal planes of the particle was determined to be 0.204 nm in the HRTEM image, corresponding to the (200) lattice plane of the Au. Fig. 1D illustrates the structural characteristics of the as-prepared P-GR–AuNPs investigated by XRD analysis. The four peaks with d values of 2.36, 2.04, 1.44, 1.23, correspond to the (111), (200), (220) and (311), respectively, are in
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Fig. 1. (A, B) Typical TEM images of P-GR–AuNPs at different magnifications. (C) HRTEM image, (D) X-Ray diffraction pattern, and (E) Raman spectrum of P-GR–AuNPs.
good agreement with the standard values of pure metallic Au (JCPDS, 04-0784). To obtain further information on the structure and topology, Raman spectroscopy was carried out. As shown in Fig. 1E, the Raman spectrum of graphene exhibits the presence of D (1334.8 cm 1), and G (1593.91 cm 1) bands. The D band arises from sp3-hybridized carbon, and the G represents the E2g zone center mode of the crystalline graphite (Ferrari and Robertson, 2000). This means D/G intensity ratio is proportional to the number of defect sites in graphite carbon (Ferrari and Robertson, 2000). The D/G intensity ratio of GR is 1.06, indicating that there are significant edge-plane-like defective sites existing on the surface of grapheme (Ferrari and Robertson, 2000). The P-GR–AuNPs are suitable for electrochemical analysis because of the excellent conductivity of graphene nanosheets. 3.3. Characterization of aptasensor In order to clarify the electrochemical properties of the resulting aptasensor, EIS is an effective method for monitoring the changes in the surface features of the modified electrodes in the assembly process (Li et al., 2009), as shown in Fig. 2. The impedance spectra include a semicircle portion and a linear portion. The semicircle portion at higher frequencies corresponds to the electron-transfer-limited process, and the linear portion at lower frequencies represents the diffusion-limited process. The change in semicircle diameter is a result of the change in the interfacial resistance Rct, due to electrons transferring from the modified electrode to [Fe(CN)6]3 /4 in solution. Curve a represents the electrochemical impedance of the bare GCE, which shows a nearly
Fig. 2. Nyquist plots of impedance spectra at (a) the bare GCE, (b) P-GR/GCE, (c) PGR–AuNPs/GCE, (d) the aptamer/P-GR–AuNPs/GCE, and (e) 10 nM angiogenin/the aptamer/P-GR–AuNPs/GCE. Inset is the equivalent circuit.
straight line. After the modification of P-GR nanosheets, owing to nonconductive properties of PDDA, the diameter of the semicircle increased dramatically with a Rct of 2000 Ω (Fig. 2, curve b). After modification with AuNPs, the resulting electrode showed a much lower resistance (Fig. 2, curve c), It can be attributed to the fact that AuNPs can further enhance the electron transfer rate. After the aptamer was immobilized onto the surface of the P-GR– AuNPs/GCE, the Rct value significantly increased to 7000 Ω
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(Fig. 2, curve d). This can be attributed to the fact that [Fe(CN)6]3 /4 received electrostatic repulsive forces from the negatively charged phosphate backbones of DNA. After 10 nM angiogenin was captured onto the surface of the aptamer, the Rct value markedly increased to 11,000 Ω (Fig. 2, curve e), which implies that angiogenin can promote the conversion of the DNA sequence from a loose random coil into the secondary stem-loop structure, and leads to an increase in the electron transfer resistance. By fitting the data (inset of Fig. 2), the sequence of the values of Rct for different electrodes is P-GR/GCE 4P-GR–AuNPs/ GCE. This demonstrates the P-GR–AuNPs/GCE has higher electrochemical activity than P-GR/GCE. The surface structure and morphology of GCE before and after modification of P-GR–AuNPs are characterized by SEM, as shown in Fig. S2(A, B). Compared with Fig. S2A, it can be clearly seen that the P-GR–AuNPs have been successfully absorbed on the surface of the GCE. 3.4. Optimization of the experimental parameters The performance of the developed angiogenin sensor is strongly influenced by the experimental conditions such as incubation temperature, incubation time and pH in the solution. Therefore, each detection parameter was optimized in our study, while keeping the other parameters constant. The incubation temperature of angiogenin has great effects on sensing effect (Fig. S3). The SWV current quickly decreased, reached a minimum at 37 °C, and rose up dramatically when the reaction temperature was above 37 °C. Thus, 37 °C was chosen as an optimal incubation temperature in the experiments. As expected, incubation time and pH in the solution also influence the signal responses of the sensor in the presence of angiogenin. As shown in Fig. S4, it was clearly observed that the SWV peak current decreased with the increase of incubation time. When the incubation time is 30 min, the SWV signal reached saturation. Thus, 30 min was chosen as the optimal in the following experiments. pH value can affect the formation of stem-loop structure, and then control its protein binding. Therefore, pH value can affect the sensitivity of the method, and the effect of pH value was studied over the range from 4.0 to 10.0 (Fig. S5). The peak current decreased with the pH over the range from 4.0 to 7.40, whereas it increased with further increase of pH value. Thereby, pH 7.40 was selected as the optimum pH environment.
Table 1 Comparison between the proposed assay and other reported techniques for the determination of angiogenin. Method
Linear range
LOD
References
Electrochemical FRETa Electrochemical ELISAb Electrochemical Fluorescence
0.1 pM–5 nM 0.5–40 nM 0.01–30 nM N/A 0.2 pM–10 nM N/A
0.064 pM 0.2 nM 1 pM 0.694 nM 0.07 pM 6.9 pM
This work Li et al. (2008) Li et al. (2011) Chon et al. (2013) Shi et al. (2014) Li et al. (2007)
a b
Fluorescence resonance energy transfer. Enzyme-linked immunosorbent assay.
3.5. Performance of the aptasensor To quantitatively assess the detection limit and response range of the angiogenin aptasensor, Fig. 3A shows the SWV plots of P-GR–AuNPs/GCE for angiogenin with different concentrations ranging from 0.1 pM to 5 nM. The introduction of angiogenin at different concentrations to the sensing system induced different decreases in the SWV peak currents since the angiogenin bound to the aptamer and the aptamer folded to the secondary stem-loop structure, repelling the redox marker ions to approach the electrode. Fig. 3B depicts signal suppression is linear with logarithm of angiogenin concentration over a range from 0.1 pM to 5 nM with a correlation coefficient of 0.99, and the limit of detection is 0.064 pM as calculated in terms of the 3s rule (Kaiser, 1973). The values of relative standard deviation from the aptamer/P-GR– AuNPs/GCE for each angiogenin concentrations are listed in Table S1. The detection limit of our proposed angiogenin sensor (0.064 pM) is lower than that in the literature and the linear range of our work, as shown in Table 1. 3.6. The specificity of aptasensor To test the specificity of the electrochemical aptasensor for angiogenin (1 pM) analysis, other proteins including thrombin, and bovine serum albumin (BSA) (each at 1 pM) as the potential interference proteins were tested under the same conditions (Fig. 4). As expected, after the addition of 1 pM angiogenin, a significant decrease of the SWV signal was obtained. However, the SWV signal for the other proteins had little changes after treatment with 1 pM thrombin, and BSA, respectively compared with that obtained containing 1 pM angiogenin, indicating that the
Fig. 3. (A) Square-wave voltammograms of P-GR–AuNPs/GCE to different concentrations of angiogenin. (B) The peak current in the SWV as a function of angiogenin concentration. Inset: The peak current is linear with logarithm of angiogenin concentration over the range from 0.1 pM to 5 nM.
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Scientific Research Project of Beijing Educational Committee (KM201410028006).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.10.046.
References
Fig. 4. The specificity of the electrochemical aptasensor for angiogenin (1 pM) (1 pM thrombin, and 1 pM BSA as the potential interference proteins).
response of the aptasensor to angiogenin was unaffected by presence of other proteins. The result clearly indicated that our method could be used to detect angiogenin with high specificity. 3.7. The reproducibility of the aptasensor Besides sensitivity and selectivity, the reproducibility of the angiogenin biosensor was evaluated. Four different P-GR–AuNPs/ aptamer/GCEs were conducted independently for 10 pM angiogenin SWV response, as shown in Fig. S6. We can see that four repetitive measurements of the aptasensor yielded a reproducible SWV signal with a relative standard deviation (R.S.D.) of these measurements of 2.71%, showing the good reproducibility of the sensor.
4. Conclusion To sum up, we have presented a facile self-assembly method to synthesize 2D P-GR–AuNPs hybrids via an exfoliation/in situ reduction of GO in the presence of PDDA and spontaneous chemical reduction of chloroauric acid by sodium citrate. The AuNPs distributed uniformly on the surface of P-GR nanosheets. The P-GR– AuNPs hybrids were employed as the electrochemical enhanced material for angiogenin sensing, which showed wide linear ranges (0.1 pM to 5 nM) and low detection limits (0.064 pM). The approach provided a convenient, low-cost, sensitive, and specific method for protein detection, which might provide an effective candidate in clinical research.
Acknowledgments All authors gratefully acknowledge the financial support of the Natural Natural Science Foundation of China (No. 21371123) and
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