An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification.

G Model ACA 233632 No. of Pages 6

Analytica Chimica Acta xxx (2014) xxx–xxx

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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification Jing Zhao a , Meiling Xin a , Ya Cao a , Yongmei Yin b, * , Yongqian Shu b , Wenli Ma c a b c

Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China Institute of Genetic Engineering of Southern Medical University, Guangzhou 510515, PR China

H I G H L I G H T S

G R A P H I C A L A B S T R A C T

An improved electrochemical aptasensor has been proposed for thrombin detection. Both Exo III and dsDNA-templated CuNPs are used for signal amplification. The aptasensor can detect protein in a broad linear range with low detection limit. The aptasensor can easily distinguish thrombin in both buffer and complex sample.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 September 2014 Received in revised form 4 December 2014 Accepted 13 December 2014 Available online xxx

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and doublestranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of o-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 30 terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100 fM to 1 nM with a low detection limit of 20.3 fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Electrochemical aptasensor Thrombin Exonuclease III Double-stranded DNA-templated copper nanoparticles Signal amplification

* Corresponding author. Tel.: +86 2583710040; fax: +86 25 83710040. E-mail address: [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.aca.2014.12.026 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: J. Zhao, et al., An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j. aca.2014.12.026


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J. Zhao et al. / Analytica Chimica Acta xxx (2014) xxx–xxx

1. Introduction Aptamer, which is usually artificial DNA or RNA isolated by an in vitro systematic evolution of ligands by exponential enrichment (SELEX) process, can especially recognize and bind to the target molecules with high affinity, including ions, small molecules, proteins and cells [1,2]. Compared with the traditional recognition element antibody, aptamer is much easier for synthesis and preservation due to the simple structure and high stability [3]. Therefore, aptamer has been increasingly used as an appealing recognition element for the development of new biosensor as an alternative to the antibody-used immunosensor, which is named as aptasensor [4–6]. Among them, benefiting from the unique advantages of electrochemical techniques, the electrochemical aptasensor is the most attractive one and has aroused great attention for the advantages of low background, simple operation, rapid response, high sensitivity and specificity, etc. [7–11]. In order to continuously improve the sensitivity of biological detection, signal amplification strategies have been extensively involved in the design and construction of aptasensors. For example, nucleases that are vital tool in molecular biology have become compelling to assist DNA-based signal amplification for the improvement of the aptasensors [12–15]. So, exonuclease III (Exo III) that can selectively catalyze the stepwise removal of mononucleotides from 30 -terminal of duplex DNA has been the most frequently-used nuclease as it has no requirement for DNA sequences, which has been known to allow one target to interact with multiple DNA probes and thus lead to ultra-high sensitivity in the detection of biomolecules [16–18]. Meanwhile, nanoparticle with particular nano-scale size-dependent properties has been considered as another popular choice for the introduction of signal amplification into the fabrication of aptasensors [19–21]. Besides signal amplification from the large surface-to-volume ratio and well catalytic properties, the nanoparticles with metal components can also be signal tags to induce amplified responses in the electrochemical detection, such as gold nanoparticles, silver nanoparticles and quantum dots. In this case, large amounts of metal ions can be released from the dissolution of these nanoparticles, which can be accurately traced by electrochemical techniques with high sensitivity [22–25]. Recently, a selective formation of copper nanoparticles (CuNPs) by using double-stranded DNA (dsDNA) as templates has been proposed, whereas single-stranded DNA (ssDNA) cannot serve as an effective template to support the formation [26]. Since it was reported by Mokhir et al. in 2010, dsDNA-templated CuNPs have attracted great attention in fluorescence analysis of biomolecules for its facile synthesis and excellent optical properties [26–29]. In this paper, we have employed electrochemical technique as a substitution of fluorescent technique to characterize the formation of dsDNA-templated CuNPs for the development of an improved new aptasensor. Thrombin-binding aptamer (TBA) subunits after ligation reaction can hybridize with probe DNA to be the template for the formation of CuNPs on the electrode surface, while the presence of thrombin can induce Exo III-catalyzed cyclic degradation to inhibit the ligation of TBA subunits. Therefore, highly sensitive and selective detection of target protein can be realized by tracing the amplified electrochemical responses from dsDNAtemplated CuNPs on the electrode surface when combined with copper ions-catalyzed oxidation of o-phenylenediamine (OPD). 2. Experimental 2.1. Materials and reagents OPD, thrombin, ascorbic acid were purchased from Sigma. Exo III and T4 DNA ligase were purchased from New England Biolabs.

Bovine Serum Albumin (BSA), ovalbumin (OVA) and hemoglobin (Hb) were purchased from Dingguo biotech. All other chemicals used were analytical grade. Milli-Q water (>18.0 MV) was used in all experiments, which was purified by a Milli-Q ultrapure water system (Millipore purification pack). DNA oligonucleotides were synthesized by Sangon (Shanghai, China) and the sequences are as follows. TBA subunit 1: 50 -P-AGTCCGTGGTAGGGC-30 ; TBA subunit 2: 50 -TAGGTTGGGGTGACT-30 ; Probe DNA: 50 -SH-TTTTTTCACGGACTAGTCACCCC-30 .

2.2. The preparation of probe DNA modified electrode The substrate gold electrode (3 mm diameter) was first polished on sand paper and then silk with alumina oxide powder (particle sizes are about 1.0, 0.3 mm and 0.05 mm in sequence). Then, the residual organics were removed by ultrasonicating the electrode in both ethanol and double-distilled water for 5 min, respectively. Finally, the electrode was electrochemically cleaned in 0.5 M H2SO4 by scanning from 0 V to 1.6 V. Afterward, the gold electrode was immediately incubated with a solution containing 1 mM probe DNA for 16 h at room temperature and then treated with 1 mM 6-mercapto-1-hexanol (MCH) for 1 h. After being rinsed by doubledistilled water thoroughly, probe DNA modified electrode was prepared for use.

2.3. Exo III-catalyzed degradation and dsDNA-templated formation of CuNPs 100 nM TBA subunit 1 and TBA subunit 2 were firstly incubated with desired concentration of thrombin at 37 C for 1 h to help TBA correctly bind to thrombin. Then, TBA subunits was incubated with 0.025 U mL1 Exo III at 37 C for 20 min in NEB 1 buffer solution (10 mM Bis Tris Propane–HCl, 10 mM MgCl2, pH 7.0), which was terminated by heating at 70 C for 20 min. Afterward, probe DNA modified electrode was immersed into the resulting solution and incubated at 16 C for 30 min after the addition of 1 mM ATP and 3.2 U mL1 ligase for the ligation of TBA subunits on the electrode surface. After being thoroughly rinsed by double-distilled water, the electrode was immersed into a solution containing 200 mM Cu2+ and 100 mM ascorbic acid at the room temperature for 15 min to form CuNPs, which was followed by being thoroughly rinsed with ethylene diamine tetraacetic acid (EDTA) buffer to remove nonspecific adsorbed Cu2+.

2.4. Electrochemical analysis For the electrochemical measurement, CuNPs-coated electrode was firstly dipped in a 0.5 mM HNO3 solution at room temperature for 1 h to dissolve CuNPs, and the resulting solution was then incubated with 1 mg mL1 OPD at 80 C water bath for 15 min to oxidize OPD. Differential pulse voltammetry (DPV) and chronocoulometry (CC) were performed on a model 660 C electrochemical analyzer with a three-electrode electrochemical system at room temperature. The three-electrode system consists of a working electrode, a saturated calomel reference electrode (SCE), and a platinum wire as the counter electrode. In order to maintain the solution anaerobic, all the electrolytes were thoroughly deoxygenated by bubbling high-purity nitrogen for at least 10 min, and a stream of nitrogen was blown gently across the surface of the solution throughout all the measurements.




3. Results and discussion 3.1. The principle of the aptasensor The principle of the aptasensor can be used to fabricate many sensors, and we have chosen thrombin, which is not only a vital serine protease in blood coagulation and hemostasis, but also a tumor maker for the indication of tumor growth and metastasis [30,31], as the target protein to reveal the principle of our detection. Fig. 1 may illustrate the principle of our aptasensor. In the absence of target protein, both TBA subunits (TBA subunit 1 and 2) can maintain the single-stranded configuration, so Exo III that is not active on single-stranded DNA cannot catalyze the digestion of the aptamer strands. Because these two TBA subunits contain eight and nine complementary bases with probe DNA respectively, a ligatable nick can be produced from the transient hybridization of TBA subunits with the probe DNA. In the presence of T4 DNA ligase that can repair the single strand break in duplex DNA by catalyzing the formation of phosphodiester bond, the two TBA subunits can be joined together by the catalysis of T4 DNA ligase and thus produce a long duplex DNA through the hybridization with probe DNA. Compared to the short and unstable duplex DNA from separate hybridization of TBA subunit with probe DNA, the newly-formed long duplex DNA from ligation reaction is much more stable at the room temperature because of the high melting temperature, so CuNPs can be formed on the electrode surface by using the long duplex DNA as template. Afterward, Cu2+ released from acid-dissolution of CuNPs can catalyze the oxidation of OPD to an electro-active 2,3-diaminophenazine (DAP), resulting in an amplified electrochemical signal [32]. In the presence of thrombin, two TBA subunits can selfassemble into an integrated aptamer for the recognition and binding with the target protein, so a short duplex domain consisting of four complementary base pairs can be stabilized as a part of the thrombin-TBA subunits complex. Subsequently, Exo III can recognize and selectively catalyze the degradation of TBA subunit 2 within the duplex region in 30 to 50 direction, thereby

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separating the thrombin-TBA subunits complex to release thrombin and TBA subunits [33]. With the recycle of thrombin, increased TBA subunit 2 can collaborate with TBA subunit 1 and then be partially digested by Exo III. Because four bases of TBA subunit 2 at the ligation site have been removed by Exo III, the ligation of TBA subunit 1 and 2 by the catalysis of T4 DNA ligase will be inhibited due to the lack of the ligatable nick. In this case, both TBA subunit 1 and digested TBA subunit 2 cannot hybridize with probe DNA to produce stable duplex DNA. Since the single-stranded probe DNA cannot serve as the efficient template for the formation of CuNPs on the electrode surface, the formation of dsDNA-templated CuNPs and the subsequent generation of electrochemical responses can be inhibited. Therefore, sensitive and specific detection of thrombin can be successfully achieved by electrochemical characterization of the formation of dsDNA-templated CuNPs on the electrode surface. 3.2. Electrochemical studies of our aptasensor by CC [Ru(NH3)6]3+ that can bind to the phosphodiester groups of DNA molecules through electrostatic adsorption has been widely used to quantify the surface-confined DNA, so we have firstly employed an electrochemical technique CC to characterize the status of DNA strands on the electrode surface by using [Ru(NH3)6]3+ as a signaling transducer [34,35]. As shown in Fig. 2, a low surface charge density can be observed at the probe DNA modified electrode, indicating the adsorption of [Ru(NH3)6]3+ onto probe DNA on the electrode surface. After the ligation reaction, the ligation of two TBA subunits can form a stable long duplex DNA through the hybridization with probe DNA, so more [Ru(NH3)6]3+ can be adsorbed to newly-formed duplex DNA and thus largely increase the surface charge density. Similarly, in the absence of thrombin, two TBA subunits without Exo III-catalyzed degradation can be connected together to form dsDNA through the hybridization with probe DNA, so a high surface charge density similar to the above-mentioned DNA strands after ligation reaction can be obtained. However, in the presence of thrombin, TBA subunit 2 will

Fig. 1. Schematic illustration of the electrochemical aptasensor based on Exo III and dsDNA-templated CuNPs assisted signal amplification.



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Fig. 2. Chronocoulometric curves obtained at probe DNA modified electrode, the modified electrode after ligation reaction in the presence of two TBA subunits and the cases that two TBA subunits has been treated by Exo III in the presence or absence of 1 nM thrombin. The test solution: 10 mM Tris–HCl buffer (pH 7.4) containing 50 mM [Ru(NH3)6]3+.

be partially digested by Exo III with the cycle of thrombin and TBA subunit 1, so the formation of stable duplex DNA from the ligation reaction can be inhibited on the electrode surface, and a relatively low surface charge density similar to that at probe DNA modified electrode can be obtained. In this context, CC studies have preliminarily confirmed the formation of duplex DNA from the ligation reaction and the inhibition by the presence of target protein.

in the absence of thrombin ascribing to the produce of DAP from Cu2+-catalyzed oxidation of OPD, which may decrease along with the addition of thrombin. The results are consistence with our expectation, and very reasonable. In the absence of thrombin, TBA subunits without Exo III-catalyzed degradation can be ligated together to form stable duplex DNA with probe DNA, so large amounts of dsDNA-templated CuNPs formed on the electrode surface can induce amplified electrochemical responses via combination with Cu2+-catalyzed oxidation of OPD. In the presence of thrombin, the binding of thrombin and TBA subunits can initiate the Exo III-catalyzed cyclic digestion of TBA subunit 2, so the ligation of TBA subunits and the further formation of CuNPs can be inhibited, leading to the decreased electrochemical responses. Moreover, the increase of thrombin concentration can accelerate the partial digestion of 30 terminal of TBA subunit 2, so the electrochemical response decreases monotonically with the addition of thrombin. Fig. 4 has shown the peak currents obtained in the presence of different concentrations of thrombin, and the inset has shown a linear relationship between peak current and the logarithm of the concentration of thrombin in the range from 100 fM to 1 nM. The regression equation is y = 0.9018–0.1584x, where y is the peak current (mA); x is the logarithm of the concentration of thrombin (pM), r = 0.995. The detection limit is 20.3 fM defined as 3s (where s is the standard deviation of the zero standards), which is much lower than the previous methods by using time-resolved fluorescence aptamer-based sandwich assay (7.7 nM), SERS-based magnetic aptasensor (0.27 pM) or hemin/G-quadruplex-based pseudobienzyme electrochemical detection (0.15 pM) [36–38]. The relative standard deviation for three times of repetitive analysis of different concentrations of thrombin can all within 5%, and the average of the relative standard deviations is 2.21%, demonstrating that well reproducibility of the proposed method.

3.3. The detection of thrombin by our aptasensor with DPV 3.4. The selectivity of our aptasensor Since duplex DNA from the hybridization of the ligated TBA subunits and probe DNA can be the efficient template for the formation of CuNPs, and Cu2+ released from acid-dissolution of CuNPs can catalyze the oxidation of OPD to produce amplified electrochemical responses, we have then employed another electrochemical technique DPV to characterize the generation of oxidation product DAP as well as detect the presence of thrombin. As shown in Fig. 3, a high DPV response can be obtained at 0.108 V

In order to evaluate the specificity of our aptasensor, we have used BSA, OVA and Hb as control proteins. As shown in Fig. 5, the peak current with 1 nM aspecific proteins (BSA, OVA or Hb) are much higher than that with 1 nM thrombin, which are quite close to that obtained in the absence of thrombin. So the comparisons have clearly shown that the control proteins have nearly no effect on the detection by using our aptasensor,

Fig. 3. DPV responses obtained with the addition of different concentrations of thrombin. Potential scan range: 0 to 0.3 V, potential step: 4 mV, amplitude: 50 mV. The reported DPV curves have been baseline correction.

Fig. 4. The peak currents obtained in the presence of different concentrations of thrombin. Inset shows a linear relationship between peak current and the logarithm of the concentration of thrombin from 100 fM to 1 nM. Error bars represent the standard deviations of three repetitive measurements.




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Acknowledgements This work is supported by the National Natural Science Foundation of China (Grant No. 31200745), the Innovation Foundation of Shanghai University (Grant No. sdcx2012036) and the Innovation Program of Shanghai Municipal Education Commission (Grant No. 14YZ026).

References

Fig. 5. The peak currents obtained in the absence or presence of 1 nM thrombin, BSA, OVA and Hb. Error bars represent the standard deviations of three repetitive measurements.

Table 1 The comparisons of thrombin concentrations detected by our method with given concentrations in serum samples. Samples

Detected concentration Standard concentration Relative error in 10% blood serum (pM) (pM) (%)

1 2 3 4

1038 104.9 10.48 1.021

1000 100 10 1

3.8 4.9 4.8 2.1

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