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Electrochemiluminescence bioassay for thrombin based on dynamic assembly of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles
Electrochimica Acta 125 (2014) 156–162
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Electrochemiluminescence bioassay for thrombin based on dynamic assembly of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles Xiuxia Yu, Hua Cui ∗ CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China
a r t i c l e
i n f o
Article history: Received 12 December 2013 Received in revised form 15 January 2014 Accepted 16 January 2014 Available online 31 January 2014 Keywords: Electrochemiluminescence -(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles Thrombin Aptamer
a b s t r a c t An electrochemiluminescence (ECL) bioassay was developed for the sensitive and selective detection of thrombin, based on dynamic interaction of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized gold nanoparticles (ABEI-AuNPs). Gold nanoparticles by citrate reduction (AuNPs) were firstly assembled onto a gold electrode through 1, 3-propanedithiol, which was further connected with thiolated DNA capture probe. Then biotinylated DNA aptamer probe was assembled onto the modified electrode through the hybridization between capture probe and aptamer probe. After adding target thrombin, aptamer could bind tightly to target molecules to form a tertiary target–aptamer complex with a binding constant greater than DNA duplex, leading to partial extrication of biotinylated DNA aptamer probe from the surface of electrode. Finally, the ABEI-AuNPs coated with streptavidin (SA) were connected with the biotinylated DNA aptamer probe left after specific binding with thrombin to form the aptamerABEI-AuNPs complex on the electrode. When a double-step potential was applied to the electrode, an ECL signal was generated and recorded. The decrease of ECL signal was in proportion to the concentration of thrombin over the range of 1.0 × 10−12 –1.0 × 10−9 M with a detection limit of 3.8 × 10−13 M. The proposed bioassay for the determination of thrombin is sensitive, specific, simple and fast. Finally, being challenged in real blood sample, the proposed bioassay was confirmed to be a good prospect for the detection of thrombin. This work provides a new way to design aptamer-based protocols for the determination of biologically important substances. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Thrombin is a multifunctional serine protease that plays an important role in procoagulant and anticoagulant functions [1,2]. It converts fibrinogen to insoluble strands of fibrin that subsequently forms the fibrin gel for clots [3]. Thrombin also can act as a hormone to regulate platelet aggregation, endothelial cell activation, and other important responses in vascular biology [4]. Normally, thrombin is not present in the blood stream. However, in certain diseases, the concentrations of the protein can reach low picomolar in a patient’s blood [5]. Therefore, sensitive and selective determination of thrombin is very important in clinical research and diagnosis. Aptamers have attracted a great deal of attention as recognition elements in bioassays due to their great advantages in bioassays
∗ Corresponding author. Tel.: +86 551 63606645; fax: +86 551 63600730. E-mail address: [email protected] (H. Cui). 0013-4686/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2014.01.098
such as low cost, good stability, and easy modification [6–9]. Since the labeling procedure would make the experiments relatively complex, time-consuming and might affect the bioaffinity of recognition element for its cognate target, there has been great interest in developing low cost label-free aptamer-based bioassays in recent years [10]. To date, some label-free aptamer-based bioassays for thrombin have been proposed using nanoparticles as sensing platform with various detection methods, such as differential pulse voltammetry (DPV) [11–13], cyclicvoltammetry (CV) [14–16], Electrochemical impedance spectra (EIS) [17], fluorescence [18–20], fluorescence resonance energy transfer (FRET) [21,22] and electrochemiluminescence (ECL) [23]. Although these bioassays show relatively simple operation compared with the conventional labeling methods, they also involve multi-step synthetic reactions and purifications, complicated assembly processes and low sensitivity. The determination of thrombin still remains a challenge in practical applications. Therefore, additional efforts should be made to develop applicable methods that allow sensitive, selective, simple, rapid, and low-cost detection.
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In this work, we developed a novel ECL bioassay for the determination of thrombin based on dynamic interaction of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs). The dynamic interaction can effectively avoid the interference of the matrix because various substances after every step interaction would be stripped off the electrode by simply washing the electrode. The process of the dynamic interaction was characterized by EIS and ECL. The conditions for the detection of thrombin were optimized and the analytical performance of the proposed bioassay was studied. Finally, the applicability of the proposed bioassay for determining thrombin in real blood samples was explored.
ethanol and ultrapure water to remove physically adsorbed 1, 3propanedithiol. 1, 3-Propandithiol was chosen for the assembly of the thiol self-assembled monolayer due to its relatively shorter carbon chain and the thiol self-assembled monolayer formed on the electrode would have smaller contact resistance [26]. Then 50 L 0.01 M MCH solution was added and incubated at 37 ◦ C for 0.5 h to block the gold electrode for the purpose of preventing the nonspecific adsorption of 5 -biotinylated aptamer and thrombin on the gold electrode surface. After that, 50 L aliquots of AuNPs solution were dropped on the thiol functionalized gold electrode for 4 h at 4 ◦ C. After rinsing with ultrapure water, the AuNPs modified electrode was ready for further experiments.
2. Experimental
2.4. Preparation of SA coated ABEI-AuNPs
2.1. Chemicals and materials
The ABEI-AuNPs with good ECL properties were prepared by seed-growth method as described previously and stored at 4 ◦ C [27]. Spherical nanoparticles with a size of approximate 15 nm were obtained. ABEI-AuNPs coated with SA was prepared as follows: 25 L SA (1.0 × 10−3 g/mL) was added to 1 mL re-prepared ABEI-AuNPs, after the mixture was incubated at room temperature for half an hour, 250 L of 5% BSA solution was added to the final concentration of 1% BSA and stirred for 5 min. After that, the as-prepared mixture was centrifuged at 12500 rpm for 15 min (Universal 320, Hettich, Germany), and the red precipitates were dispersed with 200 L of 0.03 M Tris-HCl buffer by shortly ultrasonic operation.
The 5 -thiolated DNA capture probe (5’-TTTTTCCAACCACACCA3’), and 5 -biotinylated 15-mer aptamer for thrombin (5’-biotinGGTTGGTGTGGTTGG-3 ) were purchased from Shanghai Sangon Biotechnology Co. Let.(Shanghai, China) and purified by HPLC. Bovine serum albumin (BSA), hIgG and streptavidin (SA) were purchased from Solarbio (Beijing, China). ABEI, thrombin, lysozyme, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 6-Mercapto1-hexanol (MCH) and 1, 3-propanedithiol were obtained from Sigma-Aldrich (USA). ABEI stock solution was prepared by dissolving ABEI in 0.1 M NaOH and kept at 4 ◦ C. HAuCl4 stock solution (2% HAuCl4 , w/w) was prepared by dissolving 1.0 g of HAuCl4 ·4H2 O (Shanghai Reagent, China) in 412 mL of purified water and stored at 4 ◦ C. Different concentrations of thrombin were prepared in the assemble buffer (2.0 × 10−2 M pH 7.4 Tris–HCl containing 0.1 M NaCl and 5.0 × 10−3 M MgCl2 ). All other reagents were of analytical grade. Ultrapure water was prepared by a Millipore Milli-Q system and used throughout. 2.2. Apparatus ECL were performed with a homemade ECL/Electrochemical cell system, including a model CHI 760D electrochemistry workstation (Chenhua, China), an H-type electrochemical cell (homemade), a model CR-105 photomultiplier tube (PMT) (Bingsong, China) and a model RFL-1 luminometer (Ruimai, China). EIS experiment was performed with a CHI 760D electrochemistry workstation (Chenhua, China). A PST-60 HL plus Thermo Shaker (Biosan, Latvia) was used to control the temperature of the reactions. 2.3. Preparation of AuNPs modified electrode AuNPs with a diameter of 16 nm [24] were prepared by the reduction of 0.01% (w/v) HAuCl4 with trisodium citrate (1%) [25] and stored at 4 ◦ C. 1 mL AuNPs colloid was added with 5% (w/w) BSA solution to the final concentration of 1% BSA, and then stirred for 5 min. The AuNPs coated with BSA was centrifuged at 12500 rpm for 15 min (Universal 320, Hettich, Germany), and the red precipitates were dispersed with 0.03 M Tris-HCl buffer by shortly ultrasonic operation. The bare gold electrode was polished to mirror-like surface with 1.0, 0.3, 0.05 m Al2 O3 in turn, rinsed with anhydrous ethanol and ultrapure water in turn for three times by using ultrasound. Then the gold electrode was electrochemically cleaned using cyclic voltammetry in 0.5 M H2 SO4 between 0 and 1.6 V until reproducible cyclic voltammograms were obtained. Subsequently, the cleaned electrode was thoroughly rinsed with water and immersed in a 5 × 10−3 M 1, 3-propanedithiol ethanol solution and incubated at room temperature for 20 h. After that the thiol self-assembled monolayer on the surface of the gold electrode was rinsed with
2.5. Dynamic assembly of aptamer, thrombin and ABEI-AuNPs on AuNPs modified electrode First, 75 L 1.0 × 10−5 M 5 -thiolated DNA capture probe and 75 L 1.0 × 10−2 M TCEP were mixed in advance and incubated at room temperature for 0.5 h. Then the mixture was combined with buffer (5.0 × 10−2 M pH 7.4 Tris–HCl containing 0.3 M NaCl) to the final concentration of 2.5 × 10−6 M thiolated DNA capture probe. Then, 50 L aliquots of 5 -thiolated DNA capture probe were dropped with a pipette on the AuNPs modified electrode at 4 ◦ C for 4 h and rinsed by washing buffer (1.0 × 10−2 M pH 7.4 Tris–HCl containing 0.1 M NaCl) to remove the absorbed capture probe. Finally, 50 L aliquots of the DNA aptamer probe were dropped with a pipette on the electrode at 37 ◦ C for 1 h and rinsed with washing buffer. The gold electrode modified with aptamer probe was ready for the further experiments. 50 L aliquots of thrombin solution or the assemble buffer instead of thrombin were dropped on the modified electrode at 37 ◦ C for 40 min, followed by a thorough washing with the same buffer in order to remove any unbound thrombin. 50 L aliquots of the ABEI-AuNPs coated with SA were then dropped with a pipette on the modified electrode at 37 ◦ C for 1 h and rinsed with washing buffer. The ABEI-AuNPs coated with SA were connected with the biotinylated DNA aptamer probe left after specific binding with thrombin to form the aptamer-ABEI-AuNPs complex. The “aptamer/ABEI-AuNPs conjugate” was obtained on the electrode. 2.6. ECL measurement ECL measurements were carried out with the homemade ECL system. A three-electrode system was used, being composed of the modified gold electrode described above as working electrode, a platinum wire as counter electrode, and a silver wire as the quasi-reference electrode (AgQRE). A 1.75 × 10−3 M H2 O2 solution containing 0.02 M carbonate buffer solution (CBS, pH 10.0) was used as working solution for the detection of thrombin. During measurements, a 3.0 mL portion of the working solution and blank solution without H2 O2 were added to the working compartment
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Fig. 1. A schematic for proposed ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs.
and the auxiliary compartment of the ECL cell, respectively. When a double-step potential (30 s pulse period, 0.1 s pulse time, 0.9 V pulse potential and 0 V initial pulse potential) was applied to the working electrode, an ECL signal was generated and recorded. The generation of ECL response resulted from electrochemical reactions of ABEI on the surface of the ABEI-AuNPs in the presence of H2 O2 . 3. Results and discussion 3.1. Strategy for the ECL bioassay Fig. 1 depicted schematically the proposed ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs. First, 1, 3-propanedithiol and AuNPs were self-assembled on the surface of the gold electrode successively. Then the thiolated DNA capture probe was subsequently assembled on the modified electrode via Au–S bond between the AuNPs and thiolated DNA capture probe, followed by the hybridization of biotinylated DNA aptamer probe with the DNA capture probe. The AuNPs provided amplification function for the detection of thrombin due to an increase in amount of DNA capture probes immobilized on the electrode. After adding target molecule thrombin, aptamer could bind tightly to target molecules to form a tertiary target–aptamer complex with a binding constant greater than DNA duplex, leading to partial extrication of biotinylated DNA aptamer probe from the surface of electrode. Finally, ABEI-AuNPs coated with SA were combined with the biotinylated DNA aptamer probe left on the surface of the electrode via SA-biotin interaction, and the aptamer/ABEI-AuNPs conjugates immobilized on the electrode were eventually formed. In the absence of thrombin (blank), a strong ECL signal would be observed. The ECL intensity of the blank was recorded as I0 . In the presence of thrombin, a remarkable decrease in ECL signals was observed due to the formation of aptamer–thrombin complex and ECL signal I1 was detected. In this way, the I (I= I0 - I1 ) of two events was obtained and can be used to quantify thrombin. 3.2. Characterization of the modified electrode EIS is an important tool for elucidating complicated electrochemical processes due to its advantage of separating different rate
processes in the frequency domain, and therefore provides a better insight on the interfacial reaction and mass transport processes in electrochemical systems [28]. EIS was used to monitor the fabrication procedures of ECL bioassay. Fig. 2 shows the results of EIS at different fabrication stages in 1.0 × 10−3 M Fe(CN)6 4-/3− solution (0.1 M PBS). Nyquist plot of the EIS measurement showed a semicircle in the high-frequency region while showed a straight line in the low-frequency region, demonstrating that the electrode process was controlled by electron transfer at high frequency and by diffusion at low frequency. The smaller the semicircle, the smaller the electron-transfer resistance (Ret ). Significant differences in the EIS spectra were observed during the step-by-step modification. It can be seen that when 1, 3-propanedithiol was assembled on a bare gold electrode, the electron transfer resistance increased remarkably (curve b) compared with a bare gold electrode (curve a), which is due to that 1, 3-propanedithiol could perturb the interfacial electron-transfer [29]. When AuNPs were further modified on the 1, 3-propanedithiol/gold electrode, the EIS value had a
Fig. 2. EIS a) on a bare Au electrode, b) on a 1, 3-propanedithiol/Au electrode, c) on an AuNPs/1, 3-propanedithiol/Au electrode, (d) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode, (e) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode after reacting with thrombin to form thrombin-aptamer complex and partial extrication of DNA aptamer probe from the surface of electrode, (f) on an ABEI-AuNPs/DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode in PBS (0.1 M, pH 7.0) containing [Fe(CN)6 ]3- + [Fe(CN)6 ]4− (both 1.0 × 10−3 M).
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radicals electro-oxidized by ABEI with H2 O2 by the catalysis of AuNPs [31]. 3.3. Optimization of experimental conditions
Fig. 3. ECL signals under pulse potential. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. ECL signals were obtained a) on a bare Au electrode, b) on a 1, 3-propanedithiol/Au electrode, c) on an AuNPs/1, 3-propanedithiol/Au electrode, (d) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode, (e) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode after reacting with thrombin to form thrombin-aptamer complex and partial extrication of DNA aptamer probe from the surface of electrode, (f) on an ABEI-AuNPs/DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
significant decline (curve c), which might be due to that the AuNPs could facilitate electron-transfer [30]. The fast electrode charge transfer kinetics on an AuNPs modified gold electrode originated from the enlarged surface area of the AuNPs monolayer. Next, when the DNA capture probe and the DNA aptamer probe were assembled successively on the modified electrode above, as expected, the electron transfer resistance increased (curve d). Generally, negatively charged oligonucleotides repel the negatively charged redox probe Fe(CN)6 4-/3− and thus the interfacial electron transfer resistance will increase. Then, in the presence of thrombin (1.0×10−10 M), the aptamer–thrombin complex formed and extricated from the electrode so that the electron transfer resistance decreased (curve e). Finally, when the ABEI-AuNPs was assembled on the electrode, the EIS value decreased (curve f), which might be also due to that the AuNPs could facilitate electron-transfer. The results above indicated the electrodes modified with sensing interface were fabricated as expected. The ECL behavior of the bioassay was studied with a doublestep pulse potential in the ECL working solution. We examined the ECL signal of the modified electrodes during different stages to gain a clear idea of the ECL signal generation as shown in Fig. 3. No ECL responses were observed on a bare gold electrode (curve a), 1, 3-propanedithiol/gold electrode (curve b), AuNPs/1, 3-propanedithiol/gold electrode (curve c), DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode (curve d), DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode after reacting with thrombin to form thrombin-aptamer complex and partial extrication of DNA aptamer probe from the surface of electrode (curve e), respectively. In contrast, a strong ECL signal was observed as shown in curve f of Fig. 3 on the modified electrode (ABEI-AuNPs/DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode), indicating that the ABEI-AuNPs was successfully assembled on the electrode and the ECL signals were from ABEIAuNPs. The ECL signal was generated by the reaction of ABEI
When a double-step potential was applied to the electrode, a pulse ECL signal was obtained. The pulse ECL intensity reached a stable value in the second to tenth periods in every experiment. The average intensity of nine pulse ECL signal in the stable area was used as an analytical signal for the detection of target thrombin. To obtain the maximal ECL intensity, the effects of pH value, H2 O2 concentration, pulse period, pulse time, pulse potential and initial potential on the ECL intensity were investigated. As we know, the ECL performance of luminol and its derivatives greatly depends on pH of the solution. The effect of pH in the range of 6.2–8.6 (PBS, 0.02 M) and 8.6–11.3 (CBS, 0.02 M) was examined. As shown in Fig. 4A, the optimal pH 10.0 was obtained, at which point the ECL intensity reached its maximal value. Therefore, pH 10.0 was selected using in the following experiments. Fig. 4B shows the change of ECL intensity with the concentration of H2 O2 . The ECL intensity increased with the increase of the concentration of H2 O2 and reached maximum at 1.75 × 10−3 M. This trend might be caused by of the co-oxidation function of H2 O2 [32]. When the H2 O2 concentration was higher than 1.75 × 10−3 M, the ECL intensity decreased. Therefore, the optimized concentration of H2 O2 was 1.75 × 10−3 M for the following experiments. The electrochemical parameters play very important roles due to that the ECL reaction is initiated by an electrochemical reaction at the electrode surface. Pulse wave was used as the work waveform in the present work, because pulse wave probably produce more photons than other waveforms at the same time [33]. The effect of the pulse period on the ECL intensity was investigated in the range of 10–40 s as shown in Fig. 5A. The ECL signal showed a positive correlation with the pulse period. This phenomenon was attributed to the more effective diffusion of H2 O2 in the longer pulse period. Considering shorter determining time and higher ECL intensity, a pulse period of 30 s was chosen in the following experiments. The effect of the pulse time on the ECL intensity was examined in the range of 0.01–0.16 s as shown in Fig. 5B. The ECL intensity showed a positive correlation with the pulse time at a lower pulse time. It was clear that when the pulse time increased over 0.1 s, the ECL intensity decreased rapidly at the second period, and could not reach a stable ECL signal in the following period. An explanation for this phenomenon was that when pulse time lasted a relatively long time, the diffusion layer on the surface of electrode became thicker and was hard to recover in the next pulse. Thus, 0.1 s was chosen as the best pulse time. The effect of the pulse potential is shown in Fig. 5C over the range of 0.5–1.0 V (versus SCE). Because the electro-oxidation of ABEI was much faster at higher electrode potential, a positive correlation between pulse potential and ECL intensity was also observed. However, if a high potential above 0.9 V was used, the ECL intensity went down oppositely. Wang and coworkers [34] proposed that Au-S bond between the gold electrode and the 1, 3-propanedithiol molecules might be broken in high potential. Thus, a pulse potential of 0.9 V was adopted. The effect of the initial potential in the range of -0.3 - 0.3 V was also studied. As shown in Fig. 5D, when the initial potential was 0 V, the ECL intensity reached its maximum value. This is probably attributed to a better diffusion controlled reaction on the surface of the modified electrode in initial potential of 0 V. Therefore, an initial potential of 0 V was adopted.
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Fig. 4. Effect of pH (A) and H2 O2 concentration (B) on ECL intensity. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V.
In summary, the optimized conditions for the ECL determination are as follows: 30 s pulse period, 0.9 V pulse potential, 0.1 s pulse time and 0 V initial potential. 3.4. Analytical performance Under the optimized conditions, the quantitative behavior of the fabricated ECL bioassay for thrombin was assessed by measuring the dependence of I upon the concentration of thrombin. The calibration curve for the determination of thrombin is shown in Fig. 6. It is obvious that the ECL intensity decreased with an increase of the concentration of thrombin. I was linear with the logarithm of concentration of thrombin over the range of 1.0 × 10−12 –1.0 × 10−9 M. The regression equation was I = 15282 + 1214 × log C (unit of C is
M) with a correlation coefficient of 0.996. The I was the relative ECL intensity calculated by I0 - I1 , where I0 and I1 are the ECL intensity without and with thrombin, respectively. The detection limit for thrombin at a signal-to-noise ratio of 3 (S/N = 3) was estimated to be 3.8 × 10−13 M. The precisions of the bioassay are investigated. The relative standard deviations of seven replicate determinations of 1 × 10−10 M thrombin with different electrodes was 2.98% (n = 7), showing good reproducibility. A comparison between the proposed ECL bioassay and other previously reported label-free bioassays based on aptamer for thrombin is listed in Table 1. It can be seen that the sensitivity of the presented ECL bioassay is superior to most previously reported label-free bioassays for thrombin using nanoparticles as sensing platform. Although the present ECL bioassay exhibits higher detection limit than the bioassay based
Fig. 5. Effect of (A) pulse period, (B) pulse time, (C) pulse potential, and (D) initial potential on ECL intensity. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
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Table 1 A comparison of the proposed ECL bioassay with other label-free aptamer-based bioassays for thrombin using nanoparticles as sensing platform. Analytical method DPV DPV DPV CV CV CV EIS Fluorescence Fluorescence Fluorescence FRET FRET ECL ECL a b
Sensing platform Hemin and AuNPs Fc-AuNPsa Graphene Silver nanoparticles Tb–Grab and AuNPs Thionine and AuNPs AuNPs CdTe quantum dots CdTe quantum dots AuNPs quantum dots fluorescein amidite luminol-AuNPs ABEI-AuNPs
Detection range (nM) 0-200 0.012-1200 0.000001-0.0001 1-100 0.001-80 0.12–46 0.1–30 200-1600 1.4-21 1-10 0.0625-0.1875 0.005-50 0.001-1
Detection limit (pM) 100 100 0.00045 1000 0.33 40 13 50000 700 140 1000 31.3 1.7 0.38
Reference 11 12 13 14 15 16 17 18 19 20 21 22 23 This work
Fc-AuNPs: ferrocene-coated gold nanoparticles. Tb–Gra: toluidine blue–grapheme.
on differential pulse voltammetry (DPV) [13], the assembly procedure of the proposed method is simpler and faster and does not need complex reaction steps during the detection, making the method low-cost, easy to operate and time-saving. Additionally, it has been reported that the physiological concentrations of thrombin in resting and activated blood range from low picomolar to low micromolar [5,35,36]. Therefore, the sensitivity of the proposed bioassay for thrombin is good enough for the detection of thrombin in real blood samples. 3.5. Selectivity of the ECL bioassay The ECL biosensor was assumed to be highly selective towards thrombin as the sensing was relied on thrombin–aptamer recognition [37]. If other proteins instead of thrombin were employed on the bioassay, DNA aptamer probe would not form a tertiary complex. To affirm the selectivity of the ECL bioassay, other proteins such as hIgG, BSA, and lysozyme with same concentration (1.0 × 10−9 M) instead of thrombin were used for the bioassay to evaluate the selectivity of the ECL bioassay. As can be seen in Fig. 7, there was no distinct difference between blank signal and I1 . The results indicated that no target-induced-aptamer happened in our ECL bioassay with other proteins. On the other hand, a much weaker ECL response was observed when 1.0 × 10−10 M thrombin was used for the ECL bioassay. The cross sensitivity of
Fig. 7. A comparison of ECL responses of thrombin with those of different interfering species. a: blank; b: 1.0 × 10−9 M BSA c: 1.0 × 10−9 M lysozyme; d: 1.0 × 10−9 M IgG; e: 1.0 × 10−10 M thrombin; f: 1.0 × 10−10 M thrombin with 1.0 × 10−9 M BSA, lysozyme and IgG. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
the ECL bioassay in a mixture of four different proteins including thrombin, BSA, lysozyme and hIgG was also examined. The intensity of ECL signal in the mixture was comparable with that in pure thrombin solution. These results demonstrate that the developed ECL bioassay is selective for the detection of thrombin. 3.6. Application of the ECL bioassay for the detection of thrombin in human plasma sample
Fig. 6. Linear relationship between ECL response and logarithm of thrombin concentration. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
To test the validation of the ECL bioassay for the real sample matrix, analysis of thrombin were implemented by determining spiked thrombin in human blood serum [38]. The human plasma samples collected from the Hospital of University of Science and Technology of China were measured using the proposed ECL bioassay. Considering the high sensitivity of the method, the pretreated human blood plasma sample was diluted 100 times before analysis. The samples in Table 2 were diluted and the standard solutions of thrombin with appropriate concentration were added. The results as shown in Table 2 demonstrated good recoveries (97.0–107.5%) and relative standard deviation (1.1–3.9%, n = 3), indicating that the
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Table 2 Determination of thrombin in blood serum samples using the ECL bioassay. Serum samples
Thrombin found (pM)
Thrombin added (pM)
Total thrombin detected (pM)a
Recovery/%
1 2 3
3.5 ± 0.7 1.6 ± 0.3 1.9 ± 0.3
20 20 20
22.9 ± 2.4 23.1 ± 3.9 22.4 ± 1.1
97.0 107.5 102.5
a The data given are the detection results from 100-fold diluted human serum samples.
present ECL bioassay is applicable for the determination of thrombin in real sample matrix. 4. Conclusions In conclusion, an ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs for the detection of thrombin has been developed. The presented ECL bioassay is sensitive, specific, simple and fast. It exhibits a wide dynamic range from 1.0 × 10−12 M to 1.0 × 10−9 M with a low detection limit of 3.8 × 10−13 M, which is superior to most previously reported label-free aptamer-based bioassays for thrombin. Moreover, the interfering species in the matrix can be well separated from the detection system by simply washing the electrode during the dynamic interaction. Thus the method is of high sensitivity and small matrix effects since many substances in the matrix could enhance and inhibit the CL of luminol and analogues leading to the interference. It has been successfully applied to the detection of thrombin in real human serums. This work provides a new way to design aptamer-based protocols for the determination of biologically important substances. In principle, the proposed strategy might also be applicable for the determination of other biologically important substances by using corresponding aptamer. Acknowledgements The support of this research by the National Natural Science Foundation of PR China (Grant nos. 21173201, 21075115, 20625517 and 20573101), the Opening Fund of State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, CAS (Grant no. SKLEAC201110) and the Fundamental Research Funds for the Central Universities (Grant nos. WK2060190007) are gratefully acknowledged. References [1] C.A. Holland, A.T. Henry, H.C. Whinna, F.C. Church, Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin, FEBS Lett. 484 (2000) 87. [2] L.C. Bock, L.C. Griffin, J.A. Latham, E.H. Vermaas, J.J. Toole, Selection of singlestranded DNA molecules that bind and inhibit human thrombin, Nature 355 (1992) 564. [3] D.M. Tasset, M.F. Kubik, W. Steiner, Oligonucleotide inhibitors of human thrombin that bind distinct epitopes, J. Mol. Biol. 272 (1997) 688. [4] S.R. Coughlin, Thrombin signalling and protease-activated receptors, Nature 407 (2000) 258. [5] J. Bichler, J.A. Heit, W.G. Owen, Detection of thrombin in human blood by exvivo hirudin, Thromb. Res. 84 (1996) 289. [6] M.A. Syed, S. Pervaiz, Advances in aptamers, Oligonucleotides 20 (2010) 215. [7] W. Cheng, L. Ping, Y.L. Chen, F. Yan, H.X. Ju, Y.B. Yin, A facile scanometric strategy for ultrasensitive detection of protein using aptamer-initiated rolling circle amplification, Chem. Commun. 46 (2010) 6720. [8] L.H. Tang, Y. Liu, M.M. Ali, D.K. Kang, W.A. Zhao, J.H. Li, Colorimetric and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme, Anal. Chem. 84 (2012) 4711. [9] Y. Wang, Z.H. Li, T.J. Weber, D.H. Hu, C.T. Lin, J.H. Li, Y.H. Lin, In situ live cell sensing of multiple nucleotides exploiting DNA/RNA aptamers and graphene oxide nanosheets, Anal. Chem. 85 (2013) 6775. [10] A. Sassolas, L.J. Blum, B.D. Leca-Bouvier, Electrochemical aptasensors, Electroanalysis 21 (2009) 1237.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Electrochemiluminescence bioassay for thrombin based on dynamic assembly of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles Xiuxia Yu, Hua Cui ∗ CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China
a r t i c l e
i n f o
Article history: Received 12 December 2013 Received in revised form 15 January 2014 Accepted 16 January 2014 Available online 31 January 2014 Keywords: Electrochemiluminescence -(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles Thrombin Aptamer
a b s t r a c t An electrochemiluminescence (ECL) bioassay was developed for the sensitive and selective detection of thrombin, based on dynamic interaction of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized gold nanoparticles (ABEI-AuNPs). Gold nanoparticles by citrate reduction (AuNPs) were firstly assembled onto a gold electrode through 1, 3-propanedithiol, which was further connected with thiolated DNA capture probe. Then biotinylated DNA aptamer probe was assembled onto the modified electrode through the hybridization between capture probe and aptamer probe. After adding target thrombin, aptamer could bind tightly to target molecules to form a tertiary target–aptamer complex with a binding constant greater than DNA duplex, leading to partial extrication of biotinylated DNA aptamer probe from the surface of electrode. Finally, the ABEI-AuNPs coated with streptavidin (SA) were connected with the biotinylated DNA aptamer probe left after specific binding with thrombin to form the aptamerABEI-AuNPs complex on the electrode. When a double-step potential was applied to the electrode, an ECL signal was generated and recorded. The decrease of ECL signal was in proportion to the concentration of thrombin over the range of 1.0 × 10−12 –1.0 × 10−9 M with a detection limit of 3.8 × 10−13 M. The proposed bioassay for the determination of thrombin is sensitive, specific, simple and fast. Finally, being challenged in real blood sample, the proposed bioassay was confirmed to be a good prospect for the detection of thrombin. This work provides a new way to design aptamer-based protocols for the determination of biologically important substances. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Thrombin is a multifunctional serine protease that plays an important role in procoagulant and anticoagulant functions [1,2]. It converts fibrinogen to insoluble strands of fibrin that subsequently forms the fibrin gel for clots [3]. Thrombin also can act as a hormone to regulate platelet aggregation, endothelial cell activation, and other important responses in vascular biology [4]. Normally, thrombin is not present in the blood stream. However, in certain diseases, the concentrations of the protein can reach low picomolar in a patient’s blood [5]. Therefore, sensitive and selective determination of thrombin is very important in clinical research and diagnosis. Aptamers have attracted a great deal of attention as recognition elements in bioassays due to their great advantages in bioassays
∗ Corresponding author. Tel.: +86 551 63606645; fax: +86 551 63600730. E-mail address: [email protected] (H. Cui). 0013-4686/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2014.01.098
such as low cost, good stability, and easy modification [6–9]. Since the labeling procedure would make the experiments relatively complex, time-consuming and might affect the bioaffinity of recognition element for its cognate target, there has been great interest in developing low cost label-free aptamer-based bioassays in recent years [10]. To date, some label-free aptamer-based bioassays for thrombin have been proposed using nanoparticles as sensing platform with various detection methods, such as differential pulse voltammetry (DPV) [11–13], cyclicvoltammetry (CV) [14–16], Electrochemical impedance spectra (EIS) [17], fluorescence [18–20], fluorescence resonance energy transfer (FRET) [21,22] and electrochemiluminescence (ECL) [23]. Although these bioassays show relatively simple operation compared with the conventional labeling methods, they also involve multi-step synthetic reactions and purifications, complicated assembly processes and low sensitivity. The determination of thrombin still remains a challenge in practical applications. Therefore, additional efforts should be made to develop applicable methods that allow sensitive, selective, simple, rapid, and low-cost detection.
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In this work, we developed a novel ECL bioassay for the determination of thrombin based on dynamic interaction of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs). The dynamic interaction can effectively avoid the interference of the matrix because various substances after every step interaction would be stripped off the electrode by simply washing the electrode. The process of the dynamic interaction was characterized by EIS and ECL. The conditions for the detection of thrombin were optimized and the analytical performance of the proposed bioassay was studied. Finally, the applicability of the proposed bioassay for determining thrombin in real blood samples was explored.
ethanol and ultrapure water to remove physically adsorbed 1, 3propanedithiol. 1, 3-Propandithiol was chosen for the assembly of the thiol self-assembled monolayer due to its relatively shorter carbon chain and the thiol self-assembled monolayer formed on the electrode would have smaller contact resistance [26]. Then 50 L 0.01 M MCH solution was added and incubated at 37 ◦ C for 0.5 h to block the gold electrode for the purpose of preventing the nonspecific adsorption of 5 -biotinylated aptamer and thrombin on the gold electrode surface. After that, 50 L aliquots of AuNPs solution were dropped on the thiol functionalized gold electrode for 4 h at 4 ◦ C. After rinsing with ultrapure water, the AuNPs modified electrode was ready for further experiments.
2. Experimental
2.4. Preparation of SA coated ABEI-AuNPs
2.1. Chemicals and materials
The ABEI-AuNPs with good ECL properties were prepared by seed-growth method as described previously and stored at 4 ◦ C [27]. Spherical nanoparticles with a size of approximate 15 nm were obtained. ABEI-AuNPs coated with SA was prepared as follows: 25 L SA (1.0 × 10−3 g/mL) was added to 1 mL re-prepared ABEI-AuNPs, after the mixture was incubated at room temperature for half an hour, 250 L of 5% BSA solution was added to the final concentration of 1% BSA and stirred for 5 min. After that, the as-prepared mixture was centrifuged at 12500 rpm for 15 min (Universal 320, Hettich, Germany), and the red precipitates were dispersed with 200 L of 0.03 M Tris-HCl buffer by shortly ultrasonic operation.
The 5 -thiolated DNA capture probe (5’-TTTTTCCAACCACACCA3’), and 5 -biotinylated 15-mer aptamer for thrombin (5’-biotinGGTTGGTGTGGTTGG-3 ) were purchased from Shanghai Sangon Biotechnology Co. Let.(Shanghai, China) and purified by HPLC. Bovine serum albumin (BSA), hIgG and streptavidin (SA) were purchased from Solarbio (Beijing, China). ABEI, thrombin, lysozyme, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 6-Mercapto1-hexanol (MCH) and 1, 3-propanedithiol were obtained from Sigma-Aldrich (USA). ABEI stock solution was prepared by dissolving ABEI in 0.1 M NaOH and kept at 4 ◦ C. HAuCl4 stock solution (2% HAuCl4 , w/w) was prepared by dissolving 1.0 g of HAuCl4 ·4H2 O (Shanghai Reagent, China) in 412 mL of purified water and stored at 4 ◦ C. Different concentrations of thrombin were prepared in the assemble buffer (2.0 × 10−2 M pH 7.4 Tris–HCl containing 0.1 M NaCl and 5.0 × 10−3 M MgCl2 ). All other reagents were of analytical grade. Ultrapure water was prepared by a Millipore Milli-Q system and used throughout. 2.2. Apparatus ECL were performed with a homemade ECL/Electrochemical cell system, including a model CHI 760D electrochemistry workstation (Chenhua, China), an H-type electrochemical cell (homemade), a model CR-105 photomultiplier tube (PMT) (Bingsong, China) and a model RFL-1 luminometer (Ruimai, China). EIS experiment was performed with a CHI 760D electrochemistry workstation (Chenhua, China). A PST-60 HL plus Thermo Shaker (Biosan, Latvia) was used to control the temperature of the reactions. 2.3. Preparation of AuNPs modified electrode AuNPs with a diameter of 16 nm [24] were prepared by the reduction of 0.01% (w/v) HAuCl4 with trisodium citrate (1%) [25] and stored at 4 ◦ C. 1 mL AuNPs colloid was added with 5% (w/w) BSA solution to the final concentration of 1% BSA, and then stirred for 5 min. The AuNPs coated with BSA was centrifuged at 12500 rpm for 15 min (Universal 320, Hettich, Germany), and the red precipitates were dispersed with 0.03 M Tris-HCl buffer by shortly ultrasonic operation. The bare gold electrode was polished to mirror-like surface with 1.0, 0.3, 0.05 m Al2 O3 in turn, rinsed with anhydrous ethanol and ultrapure water in turn for three times by using ultrasound. Then the gold electrode was electrochemically cleaned using cyclic voltammetry in 0.5 M H2 SO4 between 0 and 1.6 V until reproducible cyclic voltammograms were obtained. Subsequently, the cleaned electrode was thoroughly rinsed with water and immersed in a 5 × 10−3 M 1, 3-propanedithiol ethanol solution and incubated at room temperature for 20 h. After that the thiol self-assembled monolayer on the surface of the gold electrode was rinsed with
2.5. Dynamic assembly of aptamer, thrombin and ABEI-AuNPs on AuNPs modified electrode First, 75 L 1.0 × 10−5 M 5 -thiolated DNA capture probe and 75 L 1.0 × 10−2 M TCEP were mixed in advance and incubated at room temperature for 0.5 h. Then the mixture was combined with buffer (5.0 × 10−2 M pH 7.4 Tris–HCl containing 0.3 M NaCl) to the final concentration of 2.5 × 10−6 M thiolated DNA capture probe. Then, 50 L aliquots of 5 -thiolated DNA capture probe were dropped with a pipette on the AuNPs modified electrode at 4 ◦ C for 4 h and rinsed by washing buffer (1.0 × 10−2 M pH 7.4 Tris–HCl containing 0.1 M NaCl) to remove the absorbed capture probe. Finally, 50 L aliquots of the DNA aptamer probe were dropped with a pipette on the electrode at 37 ◦ C for 1 h and rinsed with washing buffer. The gold electrode modified with aptamer probe was ready for the further experiments. 50 L aliquots of thrombin solution or the assemble buffer instead of thrombin were dropped on the modified electrode at 37 ◦ C for 40 min, followed by a thorough washing with the same buffer in order to remove any unbound thrombin. 50 L aliquots of the ABEI-AuNPs coated with SA were then dropped with a pipette on the modified electrode at 37 ◦ C for 1 h and rinsed with washing buffer. The ABEI-AuNPs coated with SA were connected with the biotinylated DNA aptamer probe left after specific binding with thrombin to form the aptamer-ABEI-AuNPs complex. The “aptamer/ABEI-AuNPs conjugate” was obtained on the electrode. 2.6. ECL measurement ECL measurements were carried out with the homemade ECL system. A three-electrode system was used, being composed of the modified gold electrode described above as working electrode, a platinum wire as counter electrode, and a silver wire as the quasi-reference electrode (AgQRE). A 1.75 × 10−3 M H2 O2 solution containing 0.02 M carbonate buffer solution (CBS, pH 10.0) was used as working solution for the detection of thrombin. During measurements, a 3.0 mL portion of the working solution and blank solution without H2 O2 were added to the working compartment
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Fig. 1. A schematic for proposed ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs.
and the auxiliary compartment of the ECL cell, respectively. When a double-step potential (30 s pulse period, 0.1 s pulse time, 0.9 V pulse potential and 0 V initial pulse potential) was applied to the working electrode, an ECL signal was generated and recorded. The generation of ECL response resulted from electrochemical reactions of ABEI on the surface of the ABEI-AuNPs in the presence of H2 O2 . 3. Results and discussion 3.1. Strategy for the ECL bioassay Fig. 1 depicted schematically the proposed ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs. First, 1, 3-propanedithiol and AuNPs were self-assembled on the surface of the gold electrode successively. Then the thiolated DNA capture probe was subsequently assembled on the modified electrode via Au–S bond between the AuNPs and thiolated DNA capture probe, followed by the hybridization of biotinylated DNA aptamer probe with the DNA capture probe. The AuNPs provided amplification function for the detection of thrombin due to an increase in amount of DNA capture probes immobilized on the electrode. After adding target molecule thrombin, aptamer could bind tightly to target molecules to form a tertiary target–aptamer complex with a binding constant greater than DNA duplex, leading to partial extrication of biotinylated DNA aptamer probe from the surface of electrode. Finally, ABEI-AuNPs coated with SA were combined with the biotinylated DNA aptamer probe left on the surface of the electrode via SA-biotin interaction, and the aptamer/ABEI-AuNPs conjugates immobilized on the electrode were eventually formed. In the absence of thrombin (blank), a strong ECL signal would be observed. The ECL intensity of the blank was recorded as I0 . In the presence of thrombin, a remarkable decrease in ECL signals was observed due to the formation of aptamer–thrombin complex and ECL signal I1 was detected. In this way, the I (I= I0 - I1 ) of two events was obtained and can be used to quantify thrombin. 3.2. Characterization of the modified electrode EIS is an important tool for elucidating complicated electrochemical processes due to its advantage of separating different rate
processes in the frequency domain, and therefore provides a better insight on the interfacial reaction and mass transport processes in electrochemical systems [28]. EIS was used to monitor the fabrication procedures of ECL bioassay. Fig. 2 shows the results of EIS at different fabrication stages in 1.0 × 10−3 M Fe(CN)6 4-/3− solution (0.1 M PBS). Nyquist plot of the EIS measurement showed a semicircle in the high-frequency region while showed a straight line in the low-frequency region, demonstrating that the electrode process was controlled by electron transfer at high frequency and by diffusion at low frequency. The smaller the semicircle, the smaller the electron-transfer resistance (Ret ). Significant differences in the EIS spectra were observed during the step-by-step modification. It can be seen that when 1, 3-propanedithiol was assembled on a bare gold electrode, the electron transfer resistance increased remarkably (curve b) compared with a bare gold electrode (curve a), which is due to that 1, 3-propanedithiol could perturb the interfacial electron-transfer [29]. When AuNPs were further modified on the 1, 3-propanedithiol/gold electrode, the EIS value had a
Fig. 2. EIS a) on a bare Au electrode, b) on a 1, 3-propanedithiol/Au electrode, c) on an AuNPs/1, 3-propanedithiol/Au electrode, (d) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode, (e) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode after reacting with thrombin to form thrombin-aptamer complex and partial extrication of DNA aptamer probe from the surface of electrode, (f) on an ABEI-AuNPs/DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode in PBS (0.1 M, pH 7.0) containing [Fe(CN)6 ]3- + [Fe(CN)6 ]4− (both 1.0 × 10−3 M).
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radicals electro-oxidized by ABEI with H2 O2 by the catalysis of AuNPs [31]. 3.3. Optimization of experimental conditions
Fig. 3. ECL signals under pulse potential. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. ECL signals were obtained a) on a bare Au electrode, b) on a 1, 3-propanedithiol/Au electrode, c) on an AuNPs/1, 3-propanedithiol/Au electrode, (d) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode, (e) on a DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode after reacting with thrombin to form thrombin-aptamer complex and partial extrication of DNA aptamer probe from the surface of electrode, (f) on an ABEI-AuNPs/DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
significant decline (curve c), which might be due to that the AuNPs could facilitate electron-transfer [30]. The fast electrode charge transfer kinetics on an AuNPs modified gold electrode originated from the enlarged surface area of the AuNPs monolayer. Next, when the DNA capture probe and the DNA aptamer probe were assembled successively on the modified electrode above, as expected, the electron transfer resistance increased (curve d). Generally, negatively charged oligonucleotides repel the negatively charged redox probe Fe(CN)6 4-/3− and thus the interfacial electron transfer resistance will increase. Then, in the presence of thrombin (1.0×10−10 M), the aptamer–thrombin complex formed and extricated from the electrode so that the electron transfer resistance decreased (curve e). Finally, when the ABEI-AuNPs was assembled on the electrode, the EIS value decreased (curve f), which might be also due to that the AuNPs could facilitate electron-transfer. The results above indicated the electrodes modified with sensing interface were fabricated as expected. The ECL behavior of the bioassay was studied with a doublestep pulse potential in the ECL working solution. We examined the ECL signal of the modified electrodes during different stages to gain a clear idea of the ECL signal generation as shown in Fig. 3. No ECL responses were observed on a bare gold electrode (curve a), 1, 3-propanedithiol/gold electrode (curve b), AuNPs/1, 3-propanedithiol/gold electrode (curve c), DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode (curve d), DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode after reacting with thrombin to form thrombin-aptamer complex and partial extrication of DNA aptamer probe from the surface of electrode (curve e), respectively. In contrast, a strong ECL signal was observed as shown in curve f of Fig. 3 on the modified electrode (ABEI-AuNPs/DNA aptamer probe/DNA capture probe/AuNPs/1, 3-propanedithiol/gold electrode), indicating that the ABEI-AuNPs was successfully assembled on the electrode and the ECL signals were from ABEIAuNPs. The ECL signal was generated by the reaction of ABEI
When a double-step potential was applied to the electrode, a pulse ECL signal was obtained. The pulse ECL intensity reached a stable value in the second to tenth periods in every experiment. The average intensity of nine pulse ECL signal in the stable area was used as an analytical signal for the detection of target thrombin. To obtain the maximal ECL intensity, the effects of pH value, H2 O2 concentration, pulse period, pulse time, pulse potential and initial potential on the ECL intensity were investigated. As we know, the ECL performance of luminol and its derivatives greatly depends on pH of the solution. The effect of pH in the range of 6.2–8.6 (PBS, 0.02 M) and 8.6–11.3 (CBS, 0.02 M) was examined. As shown in Fig. 4A, the optimal pH 10.0 was obtained, at which point the ECL intensity reached its maximal value. Therefore, pH 10.0 was selected using in the following experiments. Fig. 4B shows the change of ECL intensity with the concentration of H2 O2 . The ECL intensity increased with the increase of the concentration of H2 O2 and reached maximum at 1.75 × 10−3 M. This trend might be caused by of the co-oxidation function of H2 O2 [32]. When the H2 O2 concentration was higher than 1.75 × 10−3 M, the ECL intensity decreased. Therefore, the optimized concentration of H2 O2 was 1.75 × 10−3 M for the following experiments. The electrochemical parameters play very important roles due to that the ECL reaction is initiated by an electrochemical reaction at the electrode surface. Pulse wave was used as the work waveform in the present work, because pulse wave probably produce more photons than other waveforms at the same time [33]. The effect of the pulse period on the ECL intensity was investigated in the range of 10–40 s as shown in Fig. 5A. The ECL signal showed a positive correlation with the pulse period. This phenomenon was attributed to the more effective diffusion of H2 O2 in the longer pulse period. Considering shorter determining time and higher ECL intensity, a pulse period of 30 s was chosen in the following experiments. The effect of the pulse time on the ECL intensity was examined in the range of 0.01–0.16 s as shown in Fig. 5B. The ECL intensity showed a positive correlation with the pulse time at a lower pulse time. It was clear that when the pulse time increased over 0.1 s, the ECL intensity decreased rapidly at the second period, and could not reach a stable ECL signal in the following period. An explanation for this phenomenon was that when pulse time lasted a relatively long time, the diffusion layer on the surface of electrode became thicker and was hard to recover in the next pulse. Thus, 0.1 s was chosen as the best pulse time. The effect of the pulse potential is shown in Fig. 5C over the range of 0.5–1.0 V (versus SCE). Because the electro-oxidation of ABEI was much faster at higher electrode potential, a positive correlation between pulse potential and ECL intensity was also observed. However, if a high potential above 0.9 V was used, the ECL intensity went down oppositely. Wang and coworkers [34] proposed that Au-S bond between the gold electrode and the 1, 3-propanedithiol molecules might be broken in high potential. Thus, a pulse potential of 0.9 V was adopted. The effect of the initial potential in the range of -0.3 - 0.3 V was also studied. As shown in Fig. 5D, when the initial potential was 0 V, the ECL intensity reached its maximum value. This is probably attributed to a better diffusion controlled reaction on the surface of the modified electrode in initial potential of 0 V. Therefore, an initial potential of 0 V was adopted.
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Fig. 4. Effect of pH (A) and H2 O2 concentration (B) on ECL intensity. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V.
In summary, the optimized conditions for the ECL determination are as follows: 30 s pulse period, 0.9 V pulse potential, 0.1 s pulse time and 0 V initial potential. 3.4. Analytical performance Under the optimized conditions, the quantitative behavior of the fabricated ECL bioassay for thrombin was assessed by measuring the dependence of I upon the concentration of thrombin. The calibration curve for the determination of thrombin is shown in Fig. 6. It is obvious that the ECL intensity decreased with an increase of the concentration of thrombin. I was linear with the logarithm of concentration of thrombin over the range of 1.0 × 10−12 –1.0 × 10−9 M. The regression equation was I = 15282 + 1214 × log C (unit of C is
M) with a correlation coefficient of 0.996. The I was the relative ECL intensity calculated by I0 - I1 , where I0 and I1 are the ECL intensity without and with thrombin, respectively. The detection limit for thrombin at a signal-to-noise ratio of 3 (S/N = 3) was estimated to be 3.8 × 10−13 M. The precisions of the bioassay are investigated. The relative standard deviations of seven replicate determinations of 1 × 10−10 M thrombin with different electrodes was 2.98% (n = 7), showing good reproducibility. A comparison between the proposed ECL bioassay and other previously reported label-free bioassays based on aptamer for thrombin is listed in Table 1. It can be seen that the sensitivity of the presented ECL bioassay is superior to most previously reported label-free bioassays for thrombin using nanoparticles as sensing platform. Although the present ECL bioassay exhibits higher detection limit than the bioassay based
Fig. 5. Effect of (A) pulse period, (B) pulse time, (C) pulse potential, and (D) initial potential on ECL intensity. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
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Table 1 A comparison of the proposed ECL bioassay with other label-free aptamer-based bioassays for thrombin using nanoparticles as sensing platform. Analytical method DPV DPV DPV CV CV CV EIS Fluorescence Fluorescence Fluorescence FRET FRET ECL ECL a b
Sensing platform Hemin and AuNPs Fc-AuNPsa Graphene Silver nanoparticles Tb–Grab and AuNPs Thionine and AuNPs AuNPs CdTe quantum dots CdTe quantum dots AuNPs quantum dots fluorescein amidite luminol-AuNPs ABEI-AuNPs
Detection range (nM) 0-200 0.012-1200 0.000001-0.0001 1-100 0.001-80 0.12–46 0.1–30 200-1600 1.4-21 1-10 0.0625-0.1875 0.005-50 0.001-1
Detection limit (pM) 100 100 0.00045 1000 0.33 40 13 50000 700 140 1000 31.3 1.7 0.38
Reference 11 12 13 14 15 16 17 18 19 20 21 22 23 This work
Fc-AuNPs: ferrocene-coated gold nanoparticles. Tb–Gra: toluidine blue–grapheme.
on differential pulse voltammetry (DPV) [13], the assembly procedure of the proposed method is simpler and faster and does not need complex reaction steps during the detection, making the method low-cost, easy to operate and time-saving. Additionally, it has been reported that the physiological concentrations of thrombin in resting and activated blood range from low picomolar to low micromolar [5,35,36]. Therefore, the sensitivity of the proposed bioassay for thrombin is good enough for the detection of thrombin in real blood samples. 3.5. Selectivity of the ECL bioassay The ECL biosensor was assumed to be highly selective towards thrombin as the sensing was relied on thrombin–aptamer recognition [37]. If other proteins instead of thrombin were employed on the bioassay, DNA aptamer probe would not form a tertiary complex. To affirm the selectivity of the ECL bioassay, other proteins such as hIgG, BSA, and lysozyme with same concentration (1.0 × 10−9 M) instead of thrombin were used for the bioassay to evaluate the selectivity of the ECL bioassay. As can be seen in Fig. 7, there was no distinct difference between blank signal and I1 . The results indicated that no target-induced-aptamer happened in our ECL bioassay with other proteins. On the other hand, a much weaker ECL response was observed when 1.0 × 10−10 M thrombin was used for the ECL bioassay. The cross sensitivity of
Fig. 7. A comparison of ECL responses of thrombin with those of different interfering species. a: blank; b: 1.0 × 10−9 M BSA c: 1.0 × 10−9 M lysozyme; d: 1.0 × 10−9 M IgG; e: 1.0 × 10−10 M thrombin; f: 1.0 × 10−10 M thrombin with 1.0 × 10−9 M BSA, lysozyme and IgG. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
the ECL bioassay in a mixture of four different proteins including thrombin, BSA, lysozyme and hIgG was also examined. The intensity of ECL signal in the mixture was comparable with that in pure thrombin solution. These results demonstrate that the developed ECL bioassay is selective for the detection of thrombin. 3.6. Application of the ECL bioassay for the detection of thrombin in human plasma sample
Fig. 6. Linear relationship between ECL response and logarithm of thrombin concentration. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.75 × 10−3 M H2 O2 .
To test the validation of the ECL bioassay for the real sample matrix, analysis of thrombin were implemented by determining spiked thrombin in human blood serum [38]. The human plasma samples collected from the Hospital of University of Science and Technology of China were measured using the proposed ECL bioassay. Considering the high sensitivity of the method, the pretreated human blood plasma sample was diluted 100 times before analysis. The samples in Table 2 were diluted and the standard solutions of thrombin with appropriate concentration were added. The results as shown in Table 2 demonstrated good recoveries (97.0–107.5%) and relative standard deviation (1.1–3.9%, n = 3), indicating that the
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Table 2 Determination of thrombin in blood serum samples using the ECL bioassay. Serum samples
Thrombin found (pM)
Thrombin added (pM)
Total thrombin detected (pM)a
Recovery/%
1 2 3
3.5 ± 0.7 1.6 ± 0.3 1.9 ± 0.3
20 20 20
22.9 ± 2.4 23.1 ± 3.9 22.4 ± 1.1
97.0 107.5 102.5
a The data given are the detection results from 100-fold diluted human serum samples.
present ECL bioassay is applicable for the determination of thrombin in real sample matrix. 4. Conclusions In conclusion, an ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs for the detection of thrombin has been developed. The presented ECL bioassay is sensitive, specific, simple and fast. It exhibits a wide dynamic range from 1.0 × 10−12 M to 1.0 × 10−9 M with a low detection limit of 3.8 × 10−13 M, which is superior to most previously reported label-free aptamer-based bioassays for thrombin. Moreover, the interfering species in the matrix can be well separated from the detection system by simply washing the electrode during the dynamic interaction. Thus the method is of high sensitivity and small matrix effects since many substances in the matrix could enhance and inhibit the CL of luminol and analogues leading to the interference. It has been successfully applied to the detection of thrombin in real human serums. This work provides a new way to design aptamer-based protocols for the determination of biologically important substances. In principle, the proposed strategy might also be applicable for the determination of other biologically important substances by using corresponding aptamer. Acknowledgements The support of this research by the National Natural Science Foundation of PR China (Grant nos. 21173201, 21075115, 20625517 and 20573101), the Opening Fund of State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, CAS (Grant no. SKLEAC201110) and the Fundamental Research Funds for the Central Universities (Grant nos. WK2060190007) are gratefully acknowledged. References [1] C.A. Holland, A.T. Henry, H.C. Whinna, F.C. Church, Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin, FEBS Lett. 484 (2000) 87. [2] L.C. Bock, L.C. Griffin, J.A. Latham, E.H. Vermaas, J.J. Toole, Selection of singlestranded DNA molecules that bind and inhibit human thrombin, Nature 355 (1992) 564. [3] D.M. Tasset, M.F. Kubik, W. Steiner, Oligonucleotide inhibitors of human thrombin that bind distinct epitopes, J. Mol. Biol. 272 (1997) 688. [4] S.R. Coughlin, Thrombin signalling and protease-activated receptors, Nature 407 (2000) 258. [5] J. Bichler, J.A. Heit, W.G. Owen, Detection of thrombin in human blood by exvivo hirudin, Thromb. Res. 84 (1996) 289. [6] M.A. Syed, S. Pervaiz, Advances in aptamers, Oligonucleotides 20 (2010) 215. [7] W. Cheng, L. Ping, Y.L. Chen, F. Yan, H.X. Ju, Y.B. Yin, A facile scanometric strategy for ultrasensitive detection of protein using aptamer-initiated rolling circle amplification, Chem. Commun. 46 (2010) 6720. [8] L.H. Tang, Y. Liu, M.M. Ali, D.K. Kang, W.A. Zhao, J.H. Li, Colorimetric and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme, Anal. Chem. 84 (2012) 4711. [9] Y. Wang, Z.H. Li, T.J. Weber, D.H. Hu, C.T. Lin, J.H. Li, Y.H. Lin, In situ live cell sensing of multiple nucleotides exploiting DNA/RNA aptamers and graphene oxide nanosheets, Anal. Chem. 85 (2013) 6775. [10] A. Sassolas, L.J. Blum, B.D. Leca-Bouvier, Electrochemical aptasensors, Electroanalysis 21 (2009) 1237.
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