Biosensors and Bioelectronics 37 (2012) 94–98
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Aptamer-conjugated silver nanoparticles for electrochemical detection of adenosine triphosphate Leila Kashefi-Kheyrabadi, Masoud A. Mehrgardi n Department of chemistry, University of Isfahan, Isfahan 81746-73441, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 March 2012 Received in revised form 16 April 2012 Accepted 29 April 2012 Available online 8 May 2012
The capability of silver nanoparticles (SNP) as redox tag in the construction of an electrochemical aptasensor for the detection of adenosine triphosphate (ATP) is investigated in the present manuscript. To construct the aptasensor, a well-known ATP binding aptamer (ABA) splits into two segments. The first amino-labeled segment of the aptamer was covalently immobilized on 3-mercaptopropionic acid modified gold electrode surface by the formation of carbodiimide bond. The second segment was modified by SNPs and associated with the first segment in the presence of ATP. The direct oxidation signal of SNPs is followed as the analytical signal to detect ATP. The sandwich assay shows a suitable signal gain and importantly, a good response time. The sensor can detect the concentrations of ATP as low as micromolar scales with a desirable stability under optimum conditions. Furthermore, analog nucleotides including GTP, UTP and CTP, do not show serious interferences and this sensor readily detects its target in a complex media such as human blood plasma. & 2012 Elsevier B.V. All rights reserved.
Keywords: Aptasensor Aptamer Adenosine triphosphate Silver nanoparticle Sandwich assay
1. Introduction The emergence of nanomaterials with tailored composition, structure, and properties such as their unique sizes, unusual physical and chemical properties, variety and relative ease of preparations make them very attractive candidates in various fields of sciences. The attachment of biological recognition elements to nanomaterials, has opened new horizons for the exploitation of modern biosensors (Dillenback et al., 2005; El-Sayed et al., 2005; Graham et al., 2008; Liu et al., 2007; Medintz et al., 2007; Nativo et al., 2008; Wu et al., 2008). The comparable dimensions of nanomaterials and biomacromolecules have adjoined biological systems with nanomaterials, leading to new hybrid nanobiomaterials with synergetic properties and functions. Besides, the unique properties of nanomaterials have attracted much attention in electrochemistry, promising to construct a wide variety of electrochemical biosensors with desirable analytical features. Aptamers are artificial oligonucleic acids selected in vitro using a process termed systematic evolution of ligands by exponential enrichment (SELEX) for binding diverse targets (Ellington and Szostak, 1990; Roberston and Joyce, 1990; Tuerk and Gold, 1990). Their inherent selectivity, affinity, and other
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various advantages over traditional recognition elements make them very good choices to design novel biosensors. Moreover, target versatility, simplicity of in vitro selection, less vulnerability to denaturation, the chemical stability along with flexibility to modification make aptamers promising candidates in the fields of bioassay, biotechnology, biomedical research and nanotechnology (Famulok et al., 2007; Fang and Tan, 2009; Lu and Liu, 2007; Nutiu and Li, 2003; Willner and Zayats, 2007). In recent years, aptamers have specially witnessed a substantial progress in bioanalytical fields. Many different methods of chemical modification for aptamers have been developed to overcome their drawback of inherent instability or to functionalize them with various functional groups. Since, aptamers can be adopted for any target molecule and because of their endless opportunities for chemical modifications, they could be considered as very attractive detection and diagnostic tools in various bioassays. Up to now, many different aptamer-based sensors (aptasensors) for various kinds of targets including small molecules (Ho et al., 2009), pharmaceutical drugs (Kashefi-Kheyrabadi and Mehrgardi, 2012), biological macromolecules (Famulok et al., 2007), and even whole cells (Shangguan et al., 2006) have been successfully fabricated. Among them the aptamer-based electrochemical biosensors have been extensively studied. Because of their significant advantages such as simplicity, high sensitivity, low cost, and high stability, these methods play an important role in this field (Baker et al., 2006; Lai et al., 2006; Radi et al., 2005). As mentioned above, multifarious electrochemical aptasensors for detection of small molecules such as ATP and cocaine have been
L. Kashefi-Kheyrabadi, M.A. Mehrgardi / Biosensors and Bioelectronics 37 (2012) 94–98
developed recently (Tang et al., 2008; Zuo et al., 2007; Zuo et al., 2009). ATP is an important substrate in living organisms and is an indicator for the cell viability and the cell injury (Agteresch et al., 1999; Yu et al., 2008). It is well known that ATP is the mediator of energy exchanges that occur in all living cells (Pe´rez-Ruiz et al., 2003). Its presence is necessary for regulating cellular metabolism and biochemical pathways (Deng et al., 2001; Huizenga and Szostak, 1995). From this perspective, detection of ATP with low-affinity aptamer tends to be more challenging than that with high-affinity aptamers. Therefore, detection of ATP is very valuable. Up to now, a variety of aptasensors have been developed for transducing aptamer–ATP interactions into electrochemical (Zuo et al., 2007,, 2009), colorimetric (Wang et al., 2007) and fluorescence (Nutiu and Li, 2003, 2004) signals. As mentioned above metal nanoparticles offer excellent prospects for chemical and biological sensing (Templeton et al., 1999). The application of gold nanoparticles as oligonucleotide labels in DNA bioassays is very common (Mirkin et al., 1996; Storhoff et al., 1998). Also, silver deposition on the gold nanoparticles is used to visualize protein-, antibody- and DNA-conjugated gold nanoparticles in histochemical electron microscopy studies (Taton et al., 2000). Further signal amplification with a silver-enhanced colloidal gold stripping detection strategy has also been investigated for gene analysis (Wang et al., 2001). Recently, using SNPs as electroactive tag for the amplified electrochemical detection of the single base mismatches has been developed in our group (Mehrgardi and Ahangar, 2011). In this manuscript, silver nanoparticle (SNP) has been directly employed as an electrochemical redox tag to fabricate a targetresponsive aptasensor. The voltammetric responses of oxidation of SNPs have been followed as an analytical signal for the detection and quantification of ATP.
2. Experimental 2.1. Chemicals Silver nitrate, sodium borohydride, sodium citrate trihydrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium chloride, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS), 3-mercaptopropionic acid (MPA), 6-aminohexanoic acid (AHA), potassium ferrocyanide, potassium ferricyanide, and sulfuric acid were purchased from commercial sources (Merck or Sigma) in analytical grade. ATP, guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) were obtained from Vivantis Co. (Malaysia). Amino labeled oligonucleotides and thiolated oligonucleotides were obtained from Eurofins MWG/ Operon Co. with the following sequences: F1: 50 –TGCGGAGGAAGGT–(CH2)2–NH-30 ; F2: 50 –HS–(CH2)6–ACCTGGGGGAGTAT-30 All chemicals were used as received. The stock solutions of the aptamers (3 mM) were prepared using PBS buffer solution (pH¼7.0) and kept at 20 1C. Serum samples of healthy human volunteer blood donations were obtained from Esfahan Blood Transfusion Organization (EBTO). All experiments were performed at room temperature. Distilled deionized (DI) water was used in all solution preparations (R ¼18 MO). Electrochemical experiments were carried out using an Autolab PGSTAT30 (ECOchemie, The Netherlands, driven by GPES 4.9 software). A conventional three-electrode system, consisting of gold disk electrode as the working electrode, a platinum wire as an auxiliary electrode and an Ag/AgCl/3.0 M KCl reference electrode
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was used for all experiments. Differential pulse voltammetry (DPV) was applied to electrochemical detection and quantification of ATP over a range from 0.2 to þ0.5 V and recorded at the scan rate of 20 mV s 1 and the pulse amplitude of 25 mV. Electrochemical impedance spectroscopy (EIS) experiments were performed at open circuit potential (OCP) as constant DC potential. The applied AC potential was 5 mV. 2.2. Synthesis of silver nanoparticles (SNPs) SNPs were prepared according to a procedure previously reported in the literature (Mulfinger et al., 2007; Wang et al., 1998). Briefly, an excess amount of sodium borohydride is needed both for chemical reduction of the ionic silver and to stabilize the silver nanoparticles. A 5.00 mL aliquot of silver nitrate (1.0 mM) was added drop by drop ( 1 drop/s) to 15.0 mL of 2.0 mM sodium borohydride solution at 0 1C. The reaction mixture was stirred vigorously on a magnetic stirrer. The solution turned to light yellow after the addition of 2.0 mL of silver nitrate. It turned to brighter yellow when the rest of the silver nitrate solution was added. The colloid was continuously stirred while it was allowed to warm to room temperature. In order to minimize the aggregation of Ag NPs, 60 mg sodium citrate tri-hydrate was then added to the resulting solution. SNPs could be stored in a dark bottle (4 1C) for several days. The sizes of the SNPs were verified through transmission electron microscopy (TEM) analysis (Zeiss, EM 900). It was equal to 10 73 nm as is shown in Fig. S1 (Supporting Information). 2.3. Fabrication of ATP sensor Prior to sensor fabrication, gold disk electrodes with a geometric area of 0.03 cm2 (Azar electrode Co., Iran) were polished with 0.05 mm alumina slurry, rinsed with deionized water and sonicated in water for a couple of minutes, to remove bound particulates. They were then electrochemically cleaned by a series of oxidation and reduction cycles in 0.5 M H2SO4 until reproducible voltammograms were observed (Hoare, 1984). The real surface area of each electrode was obtained based on the amount of charge consumed during the reduction of the gold surface oxide monolayer in 0.05 M H2SO4, assuming 482 mC cm 2 charge for reduction of one monolayer of AuO to Au(111) (Oesch and Janata, 1983). The surface area of the gold electrode obtained was equal to 0.06270.005 cm2. Scheme 1 shows the different steps of fabrication of the silver nanoparticle based sensor. In the first step, a layer of MPA was self-assembled on the surface of Au electrode by incubating it in 2 mL of 50 mM aqueous solution of MPA for 18 h. After washing the electrode, the carboxylic groups on the surface were activated using 20 mM EDC and 30 mM NHS solutions in phosphate buffer (pH 5.5) for 2 h (Ahangar and Mehrgardi 2011). Thereafter, the electrode was washed and 15 ml of F1 was dropped onto the electrode surface for 2 h. In this step the covalent bond forms between activated carboxylic groups of MPA and amino labeled oligonucleotides. The electrode was rinsed with DI water and subsequently passived with 6-AHA (2 mM) for 2 h to displace nonspecifically bound oligonucleotides. Finally the functionalized electrode was rinsed with DI water and incubated in 10 mM phosphate buffer (pH 7.0) prior to use. On the other hand, to reduce the disulfide bond of F2, a 20 mL aliquot of 3 mM F2 with 5 mL of 0.5 mM TCEP was incubated in the dark for 1 h. Then 20 mL of SNPs solution was added to the deprotected F2 and kept for 8 h for the assembling of SNPs to thiol group of F2 segment. Finally a mixture of 10 mL of ATP and 10 mL of SNPs-F2 was dropped for 5 min onto the surface of
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Scheme 1. Schematic presentation of different steps for fabrication of the silver nanoparticle based aptasensor.
modified electrode which have been prepared in the previous step. Modification steps were monitored by the EIS technique. For EIS analysis, a solution of 0.5 mM potassium ferricyanide and potassium ferrocyanide (1:1) in 0.1 M KCl was used.
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3. Results and discussion
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3.1. Electrochemical characterization of sensing interface EIS not only represents a suitable transduction technique to follow the interfacial interactions of biomolecules, but it also provides a very powerful method for the characterization of structural features of sensing interfaces (Katz and Willner, 2003). EIS is a nondestructive technique that makes it extremely attractive for electrochemical biosensing assays (Elbaz et al., 2009; Radi et al., 2005; Rodriguez et al., 2005; Zayats et al., 2006). The immobilization of biomaterial films on the conductive or semi-conductive supports changes the capacitance and resistance properties of the solid support-electrolyte interface (Katz and Willner, 2003). Indeed, the presence of a biomaterial on the electrode surface decreases the double-layer capacitance and retards the interfacial electron-transfer kinetics (Daniels and Pourmand, 2007). These effects depend on the molecular and immobilization characteristics of the recognition layer on the surface. Fig. 1 shows the impedance spectra of the various modification steps of the electrode surface. Upon the formation of MPA selfassembled monolayer (SAM) on the electrode surface, the negative charges on the surface of the electrode are developed in neutral pHs. The electrostatic repulsion between negatively charged redox indicator [Fe(CN)6]3 /4 and the DNA phosphate backbone negative charges on the electrode surface, increases the interfacial electron transfer resistances (Fig. 1b). A similar trend is observed upon the covalent binding of the F1 and subsequent hybridization with the F2 (Fig. 1c and d respectively). Negatively charged phosphate backbone of DNA is responsible for these changes. This repulsion causes a bigger increase in the interfacial electron-transfer resistance of the probes (Radi et al., 2005).
Zim/Ω
In the present manuscript, a target responsive electrochemical aptasensor which uses silver nanoparticle as an electroactive tag for the detection of ATP is introduced. An in-vitro selected 27-mer ATP binding aptamer (ABA), which owns high affinity for ATP while, not for its analogs, GTP, CTP, and UTP was employed. ABA was split into two segments according to the previously described protocol in the literatures (Zuo et al., 2009). One of them (F1) was attached to the MPA modified gold electrode via EDC/NHS chemistry. The second one (F2) was labeled with the SNP as redox tag. These two modified segments fold around ATP molecules and form an associated complex, which creates a sandwich assay aptasensor for the detection of ATP (Scheme 1).
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a
b
e
c
d
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0
0
5000
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Zre/Ω Fig. 1. Characterization of various modification steps of the electrode surface by EIS: Bare Au (a), Au/MPA (b), Au/MPA/NHAPT (c), Au/MPA/NHAPT/AHA/ATP (1 mM)/SHAPT (d), and Au/MPA/NHAPT/AHA/ATP (1 mM)/SHAPT-SNP (e).
Finally, in the last step of modification, SNPs are attached to the thiol group of the F2. By attachment of SNPs to F2, charge transfer kinetics would improve and consequently charge transfer resistance would diminish (Fig. 1e). This is in consistence with previously published reports (Fu et al., 2005; Li et al., 2009; Liang et al., 2009; Mehrgardi and Ahangar, 2011).
3.2. Quantitation of aptamer surface density Aptamer coverage at the electrode surface can be estimated using cationic redox molecules that are electrostatically associated with the negatively charged DNA backbone (Steel et al., 1998). Cations provide charge compensation for the anionic phosphate groups in DNA backbone. Briefly, the modified electrode with aptamer was placed in the hexaamine ruthenium(III) (RuHex) solution with low ionic strength (0.05 M KCl). The amount of cationic redox probe, RuHex, was then measured using the chronocoulometry technique, under equilibrium condition (Bard and Faulkner, 2001). By referring to the Cottrell equation, the chronocoulometric intercept at t¼ 0 is sum of the double layer charge and the charge due to the reaction of species that adsorbed on the electrode surface. The surface excess terms are calculated by the difference between the chronocoulometric intercepts for the identical potential step experiments in the presence and absence of the redox probe. Aptamer surface density can be
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estimated using the following equation:
Gapt ¼ G0 ðz=mÞ where Gapt is the aptamer surface density in mol/cm2, m is the number of bases in aptamer and z is the charge of RuHex. Finally the surface density of aptamer has been obtained equal to 10 70.5 pmol cm 2. 3.3. Voltametric investigations Association of two ABA segments in the presence of ATP accumulates SNPs on the electrode surface and they can be easily monitored by following silver nanoparticle oxidation peaks using voltammetric techniques. Also, in the absence of ATP, a small oxidation peak at 0.15 V is observed, probably, due to the interactions of F1 and F2. The oxidation peak currents of SNPs not only depend on the concentration of ATP, but also depend on the concentration of F2-SNPs. The lower concentrations of F2SNPs destabilize the associated complex, reducing its affinity for ATP; on the other hand, their higher concentrations cause more sensor’s background currents. It was found that optimum concentration of F2-SNPs for the proposed aptasensor is equal to 3 mm (Fig. S2). Under optimum conditions, the associated complex is stabilized by the addition of ATP molecules, leading to a large increase in faradic current. Fig. 2 shows a monotonic increase in redox currents with increasing ATP concentrations until saturation at the concentrations upto 3 mM ATP. This aptasensor easily detects ATP at concentrations as low as 1 mM (Fig. 3). Detection sensivity of the aptasensor is favorably comparable to other reported ATP aptasensors (Zuo et al., 2007,, 2009).
Fig. 3. Signal increase percentages of ATP aptasensor vs. logarithm of ATP concentrations. The data points and error bars correspond to the average and standard deviations from three independent measurements.
3.4. Selectivity,stability and reproducibility The present aptasensor shows an excellent selectivity for ATP molecule. This sensor was challenged with 1 mM ATP and other ATP analogs, UTP, GTP, and CTP (all are of 1 mM concentrations). The oxidation peak currents of SNPs for all other nucleotides were as low as the background signal, 10-fold lower than that for 1 mM ATP (Fig. 4). Furthermore, in order to investigate the ability
Fig. 4. Signal gain for UTP, GTP and CTP (all are of 1 mM concentrations) and also for pretreated plasma (P-Plasma) containing 1 mM ATP.
3 mM 2 mM 1 mM 500 µM 100 µM 10 µM 1 µM 0
Fig. 2. Differential pulse voltammograms of ATP aptasensor in the presence of various concentrations of ATP.
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of the aptasensor to detect ATP under realistic conditions, it was measured in the human blood plasma sample. The plasma was deproteinized by trichloroacetic acid by a previously reported procedure (Berg et al., 2002) and diluted 10-folds using 10 mM phosphate buffer (pH 7.0). Then ATP was spiked into diluted deproteinized plasma and employed in the electrochemical experiments. As Fig. 4 shows ATP aptasensor strongly supports the detection of its target even in the complicated matrix under realistic conditions. The stability of the aptasensor was examined by incubation of the F1-modified electrode in PBS 1X (pH 7.0) at 4 1C. After 5 days, a decrease of 4.2% was observed for the detection of the same ATP concentration, implying good electrode stability. In order to evaluate the inter-day variations of the aptasensor, a series of three electrodes was examined for the detection of 500 mM ATP. The relative standard deviation (RSD) of 2.3% for these experiments has been obtained. This shows that the reproducibility of the aptasensor is quite reliable.
4. Conclusion A new strategy for signaling of an electrochemical aptasensor for detection of ATP, based on oxidation of SNPs, has been introduced. High sensitivity, signal on strategy, and short response time, are the most important advantages of the present aptasensor. In this assay, the recognition layer is formed by immobilization of the first segment of aptamer on the electrode surface. In the presence of target, the second segment that has been modified by SNPs, associates with the first one and accumulates the nanoparticles on the electrode surface. The oxidation signal of SNPs is followed as the analytical signal to detect ATP. This aptasensor can detect the concentrations of ATP at micromolar scales with an acceptable response time. Furthermore, analog nucleotides do not show serious interferences and this sensor readily detects its target in complex media such as human blood plasma. Furthermore, ATP aptasensor shows a desirable stability.
Acknowledgments The authors gratefully appreciate the financial support for this project by the research council of the University of Isfahan. Also we would like to express our sincere acknowledgments to Esfahan Blood Transfusion Organization (EBTO) for providing serum samples of healthy human volunteer blood donations.
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.2012.04.045.
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