An aptamer-based assay for thrombin via structure switch based on gold nanoparticles and magnetic nanoparticles

Talanta 80 (2010) 1868–1872

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An aptamer-based assay for thrombin via structure switch based on gold nanoparticles and magnetic nanoparticles Jing Zheng a,b,∗ , Gui-Fang Cheng a , Pin-Gang He a,∗∗ , Yu-Zhi Fang a a b

Department of Chemistry, East China Normal University, Shanghai 200062, China Department of Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China

a r t i c l e

i n f o

Article history: Received 2 February 2009 Received in revised form 8 October 2009 Accepted 18 October 2009 Available online 28 October 2009 Keywords: Aptamer Gold nanoparticle Magnetic nanoparticle Thrombin

a b s t r a c t An aptamer-based assay for thrombin with high specificity and sensitivity was presented. In the protocol, the aptamer for thrombin was immobilized on magnetic nanoparticle, and its complementary oligonucleotide was labeled with gold nanoparticles, then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe, this probe could be used for the specific recognition for thrombin. In the presence of thrombin, the aptamer prefer to form the G-quarter structure with thrombin, resulting in the dissociation of the duplex of the probe and the release of the gold labeled oligonucleotide. Upon this, we were able to detect thrombin through the detection of the electrochemical signal of gold nanoparticles. The strategy combines with the high specificity of aptamer and the excellent characteristics of nanoparticles. This assay is simple, rapid, sensitive and highly specific, it does not require labeling of thrombin, and it could be applied to detect thrombin in complex real sample. The method shows great potential in other protein analysis and in disease diagnosis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The detection of protein is of central importance for diagnosis and treatment of diseases and attracts increasing attention in the biomedical fields [1,2]. In most diagnosis application, specific protein recognition is often accomplished by antibodies [3–4]. Aptamers, the synthetic DNA/RNA selected from combinatorial libraries by SELEX, promise to replace antibodies for their small size, chemical simplicity and flexibility [5–7] and they are considered promising recognition elements for biosensor application. Aptamer-based protein detections have been illustrated such as colorimetric [8], fluorescence [9], quartz crystal microbalance [10] and electrochemical detection [11–13]. In the paper, human thrombin was chosen as the target analyte of interest. Thrombin (activated Factor II) is a specific serine protease involved in the coagulation cascade, which converts soluble fibrinogen into insoluble strands of fibrin and catalyzes many other coagulation-related reactions [14]. The concentration of thrombin in blood during the coagulation progress varies from nM to low ␮M levels [15], while the detection of thrombin is important

∗ Corresponding author at: Department of Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China. Tel.: +86 21 67791214. ∗∗ Corresponding author. Tel.: +86 21 62233798. E-mail addresses: [email protected] (J. Zheng), [email protected] (P.-G. He). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.10.036

for related diagnoses [16]. Therefore, it is necessary to develop an assay for thrombin with high sensitivity, selectivity, and simplicity. Recently, electrochemical [17,18] and optical techniques [19–21] were widely applied in the field of thrombin detection. However, the reliability, selectivity and the sensitivity of the assay should be improved. We show here an analytical strategy based on gold nanoparticles and magnetic nanoparticles that achieves both high specificity and the enhanced sensitivity. Nanoparticles can be used as scaffolds for assembling nanoparticle-based biosensors [22]. Gold nanoparticles are excellent candidate for bioconjugation, owing to the fact that they are compatible, and bind readily to a range of biomolecules such as amino acids, protein, enzymes and DNA [23–25]. Gold nanoparticle-based DNA electrochemical detections have been reported due to the sensitivity and excellent electrochemical signal [26,27]. In addition, magnetic nanoparticles have been widely used in bioanalysis and pharmaceutical detection for the particular properties and magnetism [28]. An electrochemical protocol for detection of thrombin based on the gold nanoparticles and magnetic nanoparticles was proposed. As shown in Fig. 1, the aptamer for thrombin was immobilized on magnetic nanoparticles, and its complementary oligonucleotides was labeled with gold nanoparticles, and then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe (A). In the presence of thrombin, the aptamer prefers to bind with thrombin and cause the dissociation of the probe, liberating the gold nanoparticle-labeled complemen-

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Fig. 1. Schematic representation of the assay. (A) The duplex structure of magnetic nanoparticle-aptamer/gold nanoparticle-oligonucleotide. (B) In the presence of thrombin, the dissociation of the duplex structure and the liberating of the gold nanoparticles labeled complementary oligonucleotides. (C) After the magnetic separation, the released gold nanoparticle-labeled complementary oligonucleotide to produce an electrochemical signal.

tary oligonucleotide (B). The basic principle for the strategy is that aptamers could form defined tertiary structure when binding to target protein, and also able to bind to complementary DNA sequence to form a duplex structure. Therefore, when the target protein and the complementary oligonucleotide are introduced, the aptamer is in a conformational equilibrium between the duplex structure and the tertiary structure [29,30]. It prefers to bind to the target protein and thus resulting in the specific recognition of the target [31,32]. Then after magnetic separation, high specific and sensitive detection of thrombin was achieved in connection to measurement of the electrochemical signal of the released gold nanoparticlelabeled complementary oligonucleotide (C). The electrochemical signal of gold is proportional to the amount of thrombin and the quantitative assay of thrombin was accomplished by using this proposed electrochemical sensor. In this strategy, gold nanoparticle offered excellent electrochemical signal and magnetic nanoparticle was used for separation. The assay responds rapidly and specifically to thrombin, which shows extensive application in protein monitoring, in disease diagnosis as well as other research fields. 2. Experimental

of PBS buffer solution and 4.6 mg of EDAC was added. The resulting mixture was stirred for 8 h for the amidation between amino aptamer and carboxyl functionalized magnetic nanoparticles. After magnetic separation, the magnetic nanoparticles with immobilized aptamer was washed three times with 500 ␮L of PBS, unbounded aptamer was removed by using a magnet, the resulting solution was resuspended in 500 ␮L of PBS buffer and stored at 4 ◦ C. 2.3. Preparation of the gold nanoparticles Gold nanoparticles were prepared according to the literature [33]. All glassware used in the following procedure was cleaned in a bath of freshly prepared 3:1 HNO3 –HCl, rinsed thoroughly in twicedistilled water and dried in air. HAuCl4 and sodium citrate solution needs to be filtered through a 0.22 ␮m microporous membrane filter prior to use. Gold nanoparticles were prepared by adding 2.5 mL of sodium citrate solution to 100 mL of boiling aqueous solution containing 1 mL of 1% (w/w) HAuCl4 , and stirred for 30 min; within the time, the color of the solution changed from grey, blue, purple, to wine red. The mixture continued to stir for 10 min after removal from the heater. The preparation was stored in dark glass bottles at 4 ◦ C for further use.

2.1. Apparatus and reagents 2.4. Preparation of gold nanoparticle-labeled oligonucleotide Differential pulse voltammetry (DPV) was performed using a CHI Instruments model 832 Electrochemical Analyzer (CHI Instrument Inc., USA). The electrochemical system comprised of a working electrode of glassy carbon electrode, an Ag/AgCl reference electrode, and a platinum wire counter electrode. SEM data were recorded using a JEOL JSM-6700F Field Emission Scanning Electron Microscope (JEOL, Japan). The oligonucleotides used in this study were purchased from Sangon Biotechnology Inc. (Shanghai, China) with the following sequences: the oligonucleotide with amino-group at 5 end (aptamer) : 5 H2 N-(CH2 )6 –ATAGGTTGGTGTGGTTGG; complementary oligonucleotide with mercapto-group at 5 end: (5 SH(CH2 )6 CCAACCACACCAACC); thrombin, bovine plasma albumin (BSA) and lysozyme were purchased from Sigma–Aldrich. Magnetic nanoparticle (50 mg/mL, carboxyl functionalized, average diameter 100 nm) was purchased from Chemicell Inc. (Berlin, Germany). Sodium citrate, AuCl3 HCl·4H2 O, ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDAC), imidazole, PBS (0.1 M, pH 7.3), Tris–HCl buffer and other reagents were commercially available and were all of analytical reagents grade, and ultrapure water was used from Aquapro system (specific resistance is 13 M cm). 2.2. Immobilization of the aptamer onto magnetic nanoparticles The immobilization of the aptamer onto magnetic nanoparticles was carried out on, briefly, magnetic nanoparticles were washed three times with PBS buffer (pH 7.3) and resuspended in 500 ␮L of PBS buffer, then ultrasonicated for 30 min after 4.3 mg imidazole was added. After 2.0 OD of aptamer was dissolved in 100 ␮L

Gold nanoparticle-labeled oligonucleotide was prepared as literature [23], briefly, gold nanoparticle solution was mixed with 1 OD of mercapto-oligonucleotide for 16 h, and the solution was centrifugated for at least 25 min at 16,128 × g to remove the excess oligonucleotide. The wine red precipitate was washed with 0.1 M PBS and redispersed in 0.1 M PBS. Then, the resulting solution was stored in the refrigerator for further use. 2.5. Preparation of the probe of magnetic nanoparticle-aptamer/gold nanoparticle-oligonucleotide The probe of magnetic nanoparticle-aptamer/gold nanoparticle-oligonucleotide was prepared as follows, after 600 ␮L of the gold nanoparticle-labeled oligonucleotides were added in the solution containing 40 ␮L of magnetic nanoparticleaptamer in PBS buffer, then 0.585 g of NaCl was added to retain a certain salinity for hybridization, and the volume was adjusted to 1000 ␮L by adding PBS buffer. The hybridization was carried out for 1 h at 37 ◦ C. The final magnetic nanoparticle-aptamer/gold nanoparticle-oligonucleotide were washed five times with 200 ␮L of PBS buffer and resuspended in 300 ␮L of PBS buffer for further use. 2.6. Experimental method for the recognition of thrombin The specific recognition for thrombin was carried out in an electrochemical cell containing 15 ␮L of the probe, adding 10 ␮L of definite concentration of thrombin and the volume was adjusted

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3.2. The SEM identification for the displacement assay Two samples were prepared according to the protocol, sample a contains thrombin, sample b contains no thrombin as a control experiment. After the incubation, wash, the samples were diluted by 600 ␮L distilled water respectively. 30 ␮L of the resultant solution was used for SEM. Fig. 3(b) shows apparently that gold nanoparticle adhered on the magnetic nanoparticles, owing to the fact that the magnetic nanoparticle-aptamer bind to the gold nanoparticle-oligonucleotide through the hybridization. While no gold nanoparticles was observed in sample a (Fig. 3 (a)), which has identified that in the present of the target protein, the aptamer prefers to bind to thrombin, and therefore the gold nanoparticle-labeled oligonucleotide released from the duplex structure.

3.3. Optimization for the assay

Fig. 2. DPV response of (a) in the absence of thrombin; (b) in the presence of 3.36 × 10−11 M thrombin.

to 100 ␮L by adding PBS buffer (pH 7.3) containing Mg2+ (1 mM Mg2+ ). The incubation was carried out for 20 min at 37 ◦ C, after the magnetic separation, the supernatant fluid was dissolved in 0.1 M HCl solution for detection. 2.7. Electrochemical measurements All electrochemical experiments were directly performed in an electrochemical cell, with a glassy carbon working electrode (diameter = 5.0 mm), an Ag/AgCl (saturated KCl) reference electrode and a platinum auxiliary electrode. The supernatant fluid was dissolved in 1 mL of 0.1 MHCl solution and the electrochemical oxidation of gold was performed at +1.25 V for 120 s in the solution. Immediately after the electrochemical oxidation, differential pulse voltammetry was performed with the scan range from +0.65 V to 0.25 V (scan rate 20 mV/s, pulse width 0.05), resulting in an analytical signal due to the reduction of AuCl4 − , which relates to the amount of the gold nanoparticle-oligonucleotide released upon the target protein. The DPV peak height at a potential of +0.43 V of the reduction of AuCl4 − was used in all of the measurements. 3. Results and discussion 3.1. The recognition for thrombin Upon the presence of the target protein, the probe of magnetic nanoparticle-aptamer/gold nanoparticle-oligonucleotide dissociated and cause the release of the gold nanoparticle-labeled oligonucleotide, which can be investigated by the DPV responses of the gold nanoparticles. As shown in Fig. 2(a), without thrombin for incubation, the peak current of DPV at +0.43 V (vs Ag/AgCl) could be neglected. For that without thrombin, gold nanoparticle-oligonucleotide would not dissociate from the duplex structure, therefore no DPV response of gold was observed. When 3.36 × 10−11 M of thrombin was added, 4.2340 ␮A of peak current of DPV was observed (Fig. 2(b)). Because the aptamer is in a conformational equilibrium between duplex structure and the G-quarter structure, it prefers to bind with thrombin to form the G-quarter structure, and liberate the gold nanoparticle-labeled oligonucleotide.

3.3.1. Effect of the temperature and time The temperature of the incubation has an influence on the dissociation of the probe of magnetic nanoparticle-aptamer/gold nanoparticle-oligonucleotide and the displacement reaction in the presence of thrombin. According to the experimental scheme, two samples were prepared, one contained 1.12 × 10−11 M of thrombin, another contained no thrombin as control. The effect of the temperature was investigated in the range of 17–57 ◦ C and the interval is 5 ◦ C. The experimental results were shown that in the absence of thrombin (Fig. 4A (curve a)), the peak current could be neglected in the range of 17–42 ◦ C, for the probe would not dissociate during the range of 17–42 ◦ C, and it began to dissociate at 45 ◦ C. While in the presence of thrombin (Fig. 4A (curve b)), during the range of 17–37 ◦ C, the peak current gradually increased with the increase of the temperature, and a higher response was obtained at 37 ◦ C, thus 37 ◦ C was chosen in the experiment for the determination. Afterwards, the peak current increased swiftly with the increase of the temperature, owing to the fact that the probe may dissociate after 45 ◦ C, resulting in the rapid increase of the peak current. These experimental results showed that the specific recognition for thrombin at 37 ◦ C was induced by thrombin, not by the dissociation of the complex. The time dependence of the assay was also studied in the range of 5–30 min at 37 ◦ C (Fig. 4B), the electrochemical signal we obtained increased swiftly with the time increased, reached at a platinum at 20 min, then decreased after 25 min. We deduced from the experiments that 20 min will assure the fully dissociation of the duplex and the formation of the G-quarter structure. The probable reason of the signal decreased after 25 min is that the activity of thrombin may decreased after 25 min, therefore affect the binding affinity toward aptamer. Therefore 20 min was chosen in the experiment.

3.3.2. Effect of the buffer The effect of buffer in the assay was compared by carrying out the assay in Tris–HCl, PBS and PBS containing MgCl. The experimental results revealed that the signal obtained in PBS buffer was significantly higher than in Tris–HCl, which indicates that PBS buffer was more favorable for the incubation. This is consistent with the report that PBS buffer is suitable for the incubation of thrombin [34]. The results also showed that the signal achieved in PBS containing MgCl was higher than in PBS because Mg2+ could stabilize the G-quarter structure of the aptamer-protein. It was reported that Mg2+ , Ca2+ , Na+ , K+ have a great influence on the formation of the G-quarter structure of the aptamer [35], thus PBS containing 0.001 M Mg2+ was chosen as the reaction buffer.

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Fig. 3. SEM of the specific recognition for thrombin. (a) In the presence of thrombin; (b) in the absence of thrombin.

Fig. 4. (A) Effect of the temperature (a) on the dissociation of the probe, (b) on the displacement assay in the presence of thrombin; (B) effect of the time on the displacement assay, the temperature applied in the experiments is 37 ◦ C. The error bar indicates the RSD of five independent experiments, the concentration of thrombin applied in the experiments is 1.12 × 10−11 M.

3.4. Specificity of the assay As signaling is based on a specific, binding-induced conformational change, the sensor should be relatively insensitive to nonspecific binding, such as adsorption. In order to test this, we have shown that 1.49 × 10−6 M of BSA and 3.13 × 10−6 M of lysozyme did not exhibit any measurable signal (Fig. 5(a) and (b)). Similarly, when thrombin coexisted with other protein, 1.49 × 10−6 M of BSA (Fig. 5(d)) or 3.13 × 10−6 M of lysozyme (Fig. 5(e)), did not exhibit any apparent signal change compared with thrombin (Fig. 5(c)). These results imply that the dissociation of the duplex, and the subsequent increase of the peak current require the formation of a specific aptamer–thrombin complex. Therefore the sensor is highly selective, quite affinity toward the target protein. It is quite insensitive to nonspecific binding and thus readily detects physiological thrombin levels, even in complex sample such as blood serum. 3.5. Quantitative detection of thrombin The illustrated error bars represent the RSD of five measurements conducted at each thrombin concentration. To assess the sensitivity and the linear range of the optimized assay, the target thrombin was serially diluted and analyzed. In Fig. 6, the increased electric current generated by gold was observed depending on the increased concentration of thrombin.

Fig. 5. Specificity of the assay (a) with 1.49 × 10−6 M BSA (b) with 3.13 × 10−6 M lysozyme (c) with 1.12 × 10−11 M thrombin (d) with 1.12 × 10−11 M thrombin and 1.49 × 10−6 M of BSA coexisted (e) with 1.12 × 10−11 M thrombin and 3.13 × 10−6 M lysozyme coexisted. The error bars indicates the RSD of five independent experiments.

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Table 1 Assay results of thrombin in plasma. Sample

Thrombin amount found after dilution (M)

Added amount (M)

Found amount (M)

Recovery (%)

1 2 3

7.47 × 10−12 7.48 × 10−12 8.33 × 10−12

5.60 × 10−12 5.60 × 10−12 5.60 × 10−12

13.45 × 10−12 12.58 × 10−12 13.52 × 10−12

106.8 91.07 92.70

Thrombin amount in plasma (M) 7.76 × 10−7

and magnetic nanoparticles exhibit excellent electrochemical characteristics to assure the high sensitivity. The simplicity and the selectivity were achieved by the aptamer-based structure switch system. On the other hand, it is fit for the detection of real sample, as a result of the high specificity to human thrombin, even in a complex blood serum. The assay provides a new platform for the specific recognition of other proteins. It is also believed that the facile method developed will find more potential biological applications due to its simple, fast, specificity and stability. Acknowledgment This research was supported by grants from the NSFC (Grant No. 20675031), and from Shanghai Science and Technology Committee (Grant No. 06PJ14032). References

Fig. 6. Dependence of the peak current on the concentration of target thrombin, the concentration of the thrombin is 1.47 × 10−12 M to 4.48 × 10−11 M. The illustrated error bars represent the RSD of five measurements conducted at each thrombin concentration.

The dynamic range of the assay covers physiologically relevant concentrations, which range from 1.47 × 10−12 M to 4.48 × 10−11 M. The resulting linear regression is Y = 0.12427 + 0.12958X (X stands for the value of the 10−12 M, and Y is the value of the DPV current, unit: ␮A), with a correlation coefficient of 0.997. As low as 6.616 × 10−13 M of the target thrombin can be detected with a signal-to-background ratio of 2.7 with the proposed assay. The reproducibility of the assay assessed by analyzing samples containing 4.48 × 10−11 M of target thrombin was 4.87% (n = 5). The amount of thrombin in real plasma sample was detected. Thrombin is a plasma protein, and all of the thrombin in plasma is present in the form of its precursor, prothrombin. Trypsin was added in 100 ␮L of the plasma to convert the prothrombin to thrombin. Calcium ion was added to retain 1 M. After proper dilution, the electrochemical detection were carried out as the procedure mentioned above. Three different plasma samples were detected, the amount of thrombin found in plasma and the recovery were presented in Table 1. Data show that the concentration of thrombin in plasma is 7.76 × 10−7 M. 4. Conclusion In conclusion, the present study described a novel and efficient detection procedure for thrombin. In the sensing system, the electrochemical signals with the concentration of thrombin over a range from 1.47 × 10−12 M to 4.48 × 10−11 M were detected and the detection limit obtained was 6.616 × 10−13 M. This aptamerbased assay demonstrated several advantages. Gold nanoparticles

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