Analytica Chimica Acta 807 (2014) 120–125
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A non-aggregation colorimetric assay for thrombin based on catalytic properties of silver nanoparticles Jie Li, Wei Li, Weibing Qiang, Xi Wang, Hui Li, Danke Xu ∗ State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, 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 AgNP-based non-aggregation colorimetric aptasensor was first developed. • The colorimetric principle was based on AgNP-catalyzed reductive degradation of RhB. • This assay combined magnetic separation with nanocatalytic amplification. • The detection limit of thrombin was as low as 0.2 nM with excellent specificity.
a r t i c l e
i n f o
Article history: Received 3 August 2013 Received in revised form 1 November 2013 Accepted 7 November 2013 Available online 16 November 2013 Keywords: Silver nanoparticles Catalytic reduction Colorimetric Rhodamine B Aptasensor
a b s t r a c t In this paper, we developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4 ). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well. © 2013 Elsevier B.V. All rights reserved.
1. Introduction It is of great importance to develop new methodologies for specific and sensitive protein detection in many biological and medical diagnostic fields. The advances of nanomaterials have
∗ Corresponding author at: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing 210093, China. Tel.: +86 25 83595835; fax: +86 25 83595835. E-mail address: [email protected] (D. Xu). 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.11.011
provided exciting technologies and novel materials for protein detection based on the unique properties associated with nanoscale phenomena such as plasmon resonance, catalysis and energy transfer [1]. Among various methods, simple colorimetric methods have attracted significant consideration due to its rapidness and simplicity without complicated analytical instruments [2]. In addition, they allow direct and on-site visualization analysis for biological samples by the naked eye. The nanomaterial-based colorimetric mechanism is generally dependent on their inherent optical or catalytic properties. For example, gold nanoparticles (AuNPs) serve as exciting colorimetric reporters, relying on the easily visualized
J. Li et al. / Analytica Chimica Acta 807 (2014) 120–125
colour change in response to the surface plasmon resonance (SPR) absorption between well-spaced individual AuNPs (red) and aggregated AuNPs (blue) [3–9]. In contrary to AuNPs, silver nanoparticles (AgNPs) exhibit higher extinction coefficients than AuNPs at the same size. A collection of AgNP-based aggregation colorimetric assays have also been constructed [10–13]. However, the aggregation-based methods usually suffer from limitations of relatively low sensitivity, which is mainly caused by the lack of amplification of the detection signal [14]. Recently, the intrinsic enzyme-like activity of nanoparticles has become a growing area of interest in colorimetric assays [15,16]. These nanomaterial-based artificial enzymes (nanozymes), such as Fe3 O4 magnetic nanoparticles have been established as highly stable and low-cost alternatives to natural enzymes, which could directly catalyze the oxidation of the corresponding substrates to achieve visual detections [17–19]. Nevertheless, nanozymes have inevitable disadvantages like low efficiency, low selection, and limited types of catalytic reactions, which might restrict their widespread applications [20]. In our previous work, a series of bioassays making full use of metal-enhanced fluorescence (MEF) effect of silver nanostructure have been developed [21–23]. We have gained rich experience in preparation and biofunctionalization of nanosilver. As one of the most fascinating nanomaterials, AgNPs are proved to be effective catalytic materials for various applications because of their large surface-to-volume ratio and electronic properties [24–30]. AgNPs as nanocatalysts have more active sites on their surface than enzymes, which could effectively catalyze specific substrates to produce colorimetric signals [31,32]. Furthermore, they facilitate electron transfer more efficiently and cost much lower than other noble metals (Au, Pd and Pt). To the best of our knowledge, catalytic properties of AgNPs have never been reported in the colorimetric sensing detection of proteins. Thrombin is a key protein that catalyzes many coagulationrelated reactions responsible for blood clotting [33,34]. In this report, thrombin was used as a model analyte and proof-of-concept experiments could be performed. The well-known DNA aptamers that could recognize two different epitopes of the protein were employed as bridge linkers in a sandwich-type complex. The magnetic particles were modified with the aptamers to collect target protein from the sample matrix via a magnetic field; such rapid separation process could be applied for protein detection in biological samples. Inspired by the facts mentioned above, a sandwich-like aptasensor was developed incorporating magnetic particles for protein separation and enrichment, as well as catalytically active AgNPs for colorimetric detection. The typical NaBH4 -mediated catalytic reduction of rhodamine B (RhB) was performed for the visual observation and optical measurement by UV–vis spectroscopy. The results indicated that the target protein could be detected with high sensitivity and excellent specificity using the suggested method.
2. Experimental
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Sigma–Aldrich Co. LLC (USA). Streptavidin coated paramagnetic particles (PMPs ca. 1.0 m) were provided by Promega Co. (Madison, WI, USA). Buffers used in this work: 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 ·12H2 O, 2 mM KH2 PO4 , pH 7.4), 0.5× SSC (75 mM NaCl, 7.5 mM trisodium citrate, pH 7.0), buffer A (50 mM tris–HCl, 2 M NaCl, 0.1% tween 20, pH 7.4), buffer B (20 mM tris–HCl, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.4), protein-free blocking solution (1% content in tris-buffered salines, pH 7.4), stock solution (buffer B containing 0.1% BSA). All the chemicals were of analytical grade. Ultrapure water (≥18.20 M) used throughout the experiments were generated by a millipore water purification system. 2.2. Apparatus The UV–vis absorption spectra were recorded with a Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). Measurement of the absorbance and absorption spectra of rest dye after reduction was conducted on a Synergy NEO HTS multi-mode microplate reader (Biotek, USA). 2.3. Preparation of AgNPs and Apt 29-AgNPs AgNPs with an average diameter of 20 nm were synthesized based on a previously described protocol with some modifications [22]. Briefly, ice cold AgNO3 (2 mM) was added dropwise to twice the volume of NaBH4 (3 mM) with vigorous stir in an ice-water bath. Then the solution was transferred into a hot bath. After being cooled down to room temperature with continuous stir, the prepared AgNPs were stored at 4 ◦ C. The Apt 29-AgNPs were synthesized as follows. 1 mL solution of as-prepared AgNPs was mixed and incubated with Apt 29 (1 nmol) for 16 h at room temperature. Then the mixture was aged in salt and brought to a final concentration of 0.1 M NaCl through a stepwise process. Followed by incubation for 40 h, the solution was centrifuged at 15,000 rpm for 20 min to remove unbound aptamers. The precipitate was washed three times with tris–HCl (10 mM, pH 7.4) and finally dispersed in stock solution at 4 ◦ C for further use. 2.4. Preparation of Apt 15-PMPs Before modification, paramagnetic particles (PMPs) coated with streptavidin were pretreated by washing twice with 0.5× SSC and buffer A, respectively. Then magnetically collected PMPs were redispersed in 300 L of buffer A and mixed with certain amount of Apt 15 (at a ratio of 2 nmol aptamer to 1 mg PMPs). The mixture was incubated for 1 h at 37 ◦ C with gentle shaking. Subsequently, the Apt 15-PMPs were rinsed with buffer B containing 0.1% Tween twice and dispersed in 600 L of blocking solution for 2 h in order to block the nonspecific binding sites. After the washing steps, the Apt 15-PMPs was kept in 600 L of stock solution at 4 ◦ C until use, with a final concentration of about 1 mg mL−1 .
2.1. Materials Oligonucleotides used in this study were synthesized and purified by Sangon Biotech Co. LLC (Shanghai, China) with following sequences. The biotinylated 15-mer anti-thrombin aptamer (denoted as Apt 15): 5 -biotin-A15 -GGT TGG TGT GGT TGG-3 . The thiolated 29-mer anti-thrombin aptamer (denoted as Apt 29): 5 SH-A15 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 . Human serum albumin (HSA), immunoglobulin A (IgA), immunoglobulin G (IgG), lysozyme and bull serum albumin (BSA) were purchased from Biosharp (Japan). Human ␣-thrombin, rhodamine B (RhB), silver nitrate, and sodium borohydride (NaBH4 ) were ordered from
2.5. Fabrication of Apt 15-PMPs/thrombin/Apt 29-AgNPs sandwich complex 20 L of Apt 15-PMPs and 200 L of protein solution with a series of concentration were mixed and incubated for 2 h at 37 ◦ C with gentle shaking. Followed by adding 100 L of Apt 29-AgNPs into the above solution, the mixture was reacted for another 1.5 h to form a sandwich complex. After repetitive washing and separation procedures, unbound AgNPs were removed, and the final sandwich complex was suspended in 40 L of buffer B for the following colorimetric detection.
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2.6. Colorimetric detection of thrombin The colorimetric detection of thrombin was performed in the RhB/NaBH4 reaction system. In a typical experiment, the catalytic reduction was initiated by adding aqueous solution of RhB (50 L, 0.4 mM) and freshly prepared aqueous solution of NaBH4 (100 L, 80 mM) sequentially to the prepared sandwich complex. After 6 min, 20 L of HCl (0.8 M) was added to terminate the reaction. Upon the magnetic separation procedure, the collected supernatant was observed by the naked eye and measured with absorbance at 558 nm via a microplate reader. 3. Results and discussion 3.1. Mechanism of the AgNP-based colorimetric aptasensor The principle of the AgNP-based colorimetric aptasensor was illustrated in Scheme 1. 15-mer DNA aptamer against thrombin (Apt 15) was first conjugated on magnetic particles (PMPs) through the strong biotin–avidin interaction. As the capture probe, the Apt 15-PMPs could effectively capture target protein from complex sample matrix and achieve fast enrichment by an external magnetic field. Subsequently, thiolated 29-mer aptamer (Apt 29) functionalized AgNPs were introduced to form a typical sandwichtype complex. The colorimetric detection was achieved through the reduction of dye catalyzed by AgNPs. AgNPs play the role of catalyst for the reduction of dyes. This catalytic process could be explained by the electro-chemical mechanism, where AgNPs act as an electron relay and the electron transfers from BH4 − ions to dye via nanoparticles [35]. Rhodamine B (RhB), a type of xanthene and cationic dye possessing a higher catalytic reaction rate than a negatively charged dye due to electrostatic interaction, was chosen for the reaction system. In our detection platform, the sandwich complex as a whole acted as the catalyst to promote the reduction of dyes based on the presence of
AgNPs, thereby producing a colour change. The amount of AgNPs contained in the sandwich complex would increase with the concentration of thrombin, which led to vast degradation of RhB. The degradation degree of the rosy colour was in proportion to the concentration of thrombin. 3.2. Viability of the colorimetric assay for determination of thrombin To demonstrate the utility of the colorimetric sensing strategy, the catalytic performance of particles involved in the detection system was investigated (Fig. 1). The catalytic reduction progress can be easily monitored by the characteristic absorption of RhB at the wavelength of maximum absorbance (max = 558 nm). The control experiment without the involvement of any catalyst was carried out (Fig. 1A). The reaction system contained 0.4 mM RhB and 80 mM NaBH4 . The absorbance of RhB dropped by about 20% in 15 min compared to the initial solution. This phenomenon showed that RhB could be slightly degraded by reductant in the absence of catalyst under the given conditions, which was in accordance with those in the literature [36]. As previously reported, the magnetic nanoparticles might cause background interference because of their intrinsic peroxidaselike catalytic activity [37]. However, under our experimental conditions, the absorbance variation of RhB observed from the time-dependent absorption spectra was consistent with the control test (Fig. 1B and D). Since blocking solution was utilized after the preparation of Apt 15-PMPs, it could effectively decrease the active binding sites on their surface to a great extent. The PMPs proved to be catalytically inactive with no impact on the reductive degradation of RhB. Furthermore, as shown in Fig. 1C, the absorption peak of RhB centred at 558 nm decreased quickly once AgNPs were added and the reduction process was accomplished within 8 min, which indicated the remarkable catalytic performance of AgNPs. The drop
Scheme 1. Schematic representation of the AgNP-based colorimetric aptasensor for thrombin detection.
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Fig. 1. Time-dependent UV–vis absorption spectra of RhB during the reduction process in the absence of catalyst (A), in the presence of (B) PMPs and (C) AgNPs. (D) Plot of absorbance of RhB at 558 nm versus time with different reagents. The reaction system contained RhB (0.4 mM) and NaBH4 (80 mM). Inset in (C) showed chemical structure conversion of RhB during the reduction.
margin on absorbance value of RhB (75%) was approximately 8 times lower than that of the reaction in the absence of catalyst and in the presence of PMPs (Fig. 1D). The catalytic reaction rate of RhB was apparently much faster than the normal reduction rate. As shown in the inset of Fig. 1C, the initially rosy RhB was converted to a colourless quinoid structure. We could conclude that AgNPs performed the function of the sole catalyst in the catalytic reaction system.
3.3. Optimization of experimental conditions In order to achieve the best detection performance, two influencing factors, the reaction time and molar ratio of NaBH4 /RhB were investigated. We utilized the decrease in absorbance of RhB at 558 nm (A0 − A: A0 and A represented the absorbance of initial RhB and rest RhB after reduction) to evaluate the results. The control group contained all the components in the experimental group except thrombin. The signal-to-noise ratio was used to assess the detection performance. Fig. 2 displayed the relationship between different reaction time and A0 − A. The value of A0 − A in the experimental group increased with the reaction time and gradually reached the maximum at 6 min. Meanwhile it was observed that the control group had a slight continuous increase as time went on, which could be caused by the few nonspecific absorption of AgNPs towards PMPs. Then 6 min was taken as the optimal time because of its best signal-tonoise level. The effect of molar ratio of NaBH4 /RhB was studied over the range from 100 to 500 under the optimum reaction time of 6 min (Fig. 3). The absorbance value in the control group revealed a change trend similar to the control in Fig. 2. In the experimental group, A0 − A increased with higher amount of reductant and reached equilibrium when the mole of NaBH4 was 400 times of the mole
of RhB. The highest signal-to-noise value was observed when the molar ratio of NaBH4 /RhB was 400. The more RhB in the system, the deeper colour it revealed, which made it difficult to be discriminated from various solutions. On the contrary, an excess of reductive agent promoted the catalytic reaction so rapidly that would raise the difficulty of detection. Taking both effectiveness and operability into consideration, the molar ratio of 400 was chosen as the appropriate requirement for the colorimetric detection. 3.4. Colorimetric detection of thrombin On the basis of such a colorimetric mechanism mentioned above, it was concluded that the amount of the rest RhB is inversely
Fig. 2. Effect of time course in the absence (grey column) and presence of 1.2 nM thrombin (blue column). Error bars were standard derivations across three repetitive experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effect of molar ratio of NaBH4 /RhB on the absorbance in the absence (grey column) and presence of 1.2 nM thrombin (blue column). Error bars were standard derivations across three repetitive experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Specificity test of the colorimetric assay for thrombin (0.8 nM) by comparing it to the interfering proteins at the 0.8 M level: immunoglobulin A (IgA), immunoglobulin G (IgG), human serum albumin (HSA), lysozyme (Lyso) and the mixed sample containing thrombin (0.8 nM) and IgG (0.8 M) or HSA (0.8 M) was also tested. Error bars were standard derivations across three repetitive experiments.
proportional to the amount of thrombin in the system. Therefore, colorimetric detection of thrombin could be accomplished by measuring the absorbance of the rest RhB. The difference of absorbance was defined as A (A = Ablank − A, where Ablank and A are the absorbances of the rest RhB in the absence and presence of thrombin, respectively). Fig. 4 showed the colour and absorption spectra of RhB in the presence of various concentrations of thrombin. In the absence of target, dyes could hardly be reduced and a rosy colour was observed (Fig. 4A). As expected, the colour faded and gradually vanished in the presence of thrombin. Meanwhile, in the UV–vis spectra, the absorbance of RhB at 558 nm continued to decrease with the increase of thrombin concentration until a plateau was reached. A was found to be linear to the concentration of thrombin in the range from 0.4 nM to 1.0 nM (Fig. 4C, inset). The linear equation could be fitted as A = −0.667 + 1.856c (nM) (R2 = 0.986). This strategy allowed for the detection of thrombin at concentration as low as 0.6 nM by the naked eye (Fig. 4A). A limit of detection of 0.2 nM was achieved via UV–vis spectroscopy. Such detection sensitivity at the level of picomole was comparable to many existing aggregation-based and catalyzation-based colorimetric techniques for thrombin [38–41].
3.5. Specificity test of the colorimetric detection
Fig. 4. Colorimetric detection. (A) Photograph for visual detection and (B) UV–vis absorption spectra of RhB corresponding to different concentrations of thrombin (a–i: 0, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.6 nM, respectively). (C) Relationship between difference of absorbance (A) and the concentration of thrombin. A = Ablank − A, where Ablank and A are the absorbance of the rest RhB in the absence and presence of thrombin, respectively. Inset showed the amplification of the linear range from 0.4 nM to 1.0 nM of thrombin. Error bars were standard derivations across three repetitive experiments.
In order to validate that the optical signal was merely generated in response to the target protein, we challenged it with unrelated proteins including immunoglobulin A (IgA), immunoglobulin G (IgG), Human serum albumin (HSA) and lysozyme (Lyso). High optical signals were obtained only when thrombin was tested whereas the absorbance changes were negligible in the presence of even 1000-fold unrelated protein in the assay. In addition, a mixed solution containing 0.8 nM thrombin and 800 nM interfere proteins did not exhibit significant absorbance changes compared with that of thrombin alone. The results essentially suggested that the proposed system revealed high selectivity towards thrombin over other control proteins attributed to the specific capture and binding of aptamers, which might support its potentialities in bioanalytic applications (Fig. 5).
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4. Conclusion In summary, we have successfully proposed a novel and versatile colorimetric platform based on catalytic properties of AgNPs themselves for rapid detection of target protein. This is achieved by integrating capture capacity of the magnetic particles and recognition ability of aptamers. Using this method allows direct visualization detection by the naked eye, thus eliminating the requirement of analytical instrument. More importantly, it overcomes the limitations of aggregation-based systems and provides an attractive alternative to the conventional colorimetric assay, which offers new opportunities for promising applications in analytic diagnose. Acknowledgements
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We acknowledge financial support of the National Basic Research Program of China (973 Program, 2011CB911003), National Natural Foundation of China (Grant no. 21175066, and 21227009), Jiangsu Province Science and Technology Support Program (No. BE2011773), Research Foundation of Jiangsu Province Environmental Monitoring (No. 1116) and the National Science Funds for Creative Research Groups (No. 21121091). References [1] Y. Zhang, Y. Guo, Y. Xianyu, W. Chen, Y. Zhao, X. Jiang, Adv. Mater. 25 (2013) 3802–3819. [2] J. Li, H.E. Fu, L.J. Wu, A.X. Zheng, G.N. Chen, H.H. Yang, Anal. Chem. 84 (2012) 5309–5315. [3] K. Ai, Y. Liu, L. Lu, J. Am. Chem. Soc. 131 (2009) 9496–9497. [4] C. Radhakumary, K. Sreenivasan, Anal. Chem. 83 (2011) 2829–2833. [5] C.D. Medley, J.E. Smith, Z. Tang, Y. Wu, S. Bamrungsap, W. Tan, Anal. Chem. 80 (2008) 1067–1072. [6] L.-Y. Bai, Y.-P. Zhang, N. Chen, J. Chen, X.-M. Zhou, L.-F. Hu, Micro Nano Lett. 6 (2011) 337–341. [7] K. Sato, K. Hosokawa, M. Maeda, J. Am. Chem. Soc. 125 (2003) 8102–8103. [8] P. Baptista, E. Pereira, P. Eaton, G. Doria, A. Miranda, I. Gomes, P. Quaresma, R. Franco, Anal. Bioanal. Chem. 391 (2008) 943–950. [9] J.P. Rosa, J.C. Lima, P.V. Baptista, Nanotechnology 22 (2011) 415202.
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Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A non-aggregation colorimetric assay for thrombin based on catalytic properties of silver nanoparticles Jie Li, Wei Li, Weibing Qiang, Xi Wang, Hui Li, Danke Xu ∗ State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, 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 AgNP-based non-aggregation colorimetric aptasensor was first developed. • The colorimetric principle was based on AgNP-catalyzed reductive degradation of RhB. • This assay combined magnetic separation with nanocatalytic amplification. • The detection limit of thrombin was as low as 0.2 nM with excellent specificity.
a r t i c l e
i n f o
Article history: Received 3 August 2013 Received in revised form 1 November 2013 Accepted 7 November 2013 Available online 16 November 2013 Keywords: Silver nanoparticles Catalytic reduction Colorimetric Rhodamine B Aptasensor
a b s t r a c t In this paper, we developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4 ). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well. © 2013 Elsevier B.V. All rights reserved.
1. Introduction It is of great importance to develop new methodologies for specific and sensitive protein detection in many biological and medical diagnostic fields. The advances of nanomaterials have
∗ Corresponding author at: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing 210093, China. Tel.: +86 25 83595835; fax: +86 25 83595835. E-mail address: [email protected] (D. Xu). 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.11.011
provided exciting technologies and novel materials for protein detection based on the unique properties associated with nanoscale phenomena such as plasmon resonance, catalysis and energy transfer [1]. Among various methods, simple colorimetric methods have attracted significant consideration due to its rapidness and simplicity without complicated analytical instruments [2]. In addition, they allow direct and on-site visualization analysis for biological samples by the naked eye. The nanomaterial-based colorimetric mechanism is generally dependent on their inherent optical or catalytic properties. For example, gold nanoparticles (AuNPs) serve as exciting colorimetric reporters, relying on the easily visualized
J. Li et al. / Analytica Chimica Acta 807 (2014) 120–125
colour change in response to the surface plasmon resonance (SPR) absorption between well-spaced individual AuNPs (red) and aggregated AuNPs (blue) [3–9]. In contrary to AuNPs, silver nanoparticles (AgNPs) exhibit higher extinction coefficients than AuNPs at the same size. A collection of AgNP-based aggregation colorimetric assays have also been constructed [10–13]. However, the aggregation-based methods usually suffer from limitations of relatively low sensitivity, which is mainly caused by the lack of amplification of the detection signal [14]. Recently, the intrinsic enzyme-like activity of nanoparticles has become a growing area of interest in colorimetric assays [15,16]. These nanomaterial-based artificial enzymes (nanozymes), such as Fe3 O4 magnetic nanoparticles have been established as highly stable and low-cost alternatives to natural enzymes, which could directly catalyze the oxidation of the corresponding substrates to achieve visual detections [17–19]. Nevertheless, nanozymes have inevitable disadvantages like low efficiency, low selection, and limited types of catalytic reactions, which might restrict their widespread applications [20]. In our previous work, a series of bioassays making full use of metal-enhanced fluorescence (MEF) effect of silver nanostructure have been developed [21–23]. We have gained rich experience in preparation and biofunctionalization of nanosilver. As one of the most fascinating nanomaterials, AgNPs are proved to be effective catalytic materials for various applications because of their large surface-to-volume ratio and electronic properties [24–30]. AgNPs as nanocatalysts have more active sites on their surface than enzymes, which could effectively catalyze specific substrates to produce colorimetric signals [31,32]. Furthermore, they facilitate electron transfer more efficiently and cost much lower than other noble metals (Au, Pd and Pt). To the best of our knowledge, catalytic properties of AgNPs have never been reported in the colorimetric sensing detection of proteins. Thrombin is a key protein that catalyzes many coagulationrelated reactions responsible for blood clotting [33,34]. In this report, thrombin was used as a model analyte and proof-of-concept experiments could be performed. The well-known DNA aptamers that could recognize two different epitopes of the protein were employed as bridge linkers in a sandwich-type complex. The magnetic particles were modified with the aptamers to collect target protein from the sample matrix via a magnetic field; such rapid separation process could be applied for protein detection in biological samples. Inspired by the facts mentioned above, a sandwich-like aptasensor was developed incorporating magnetic particles for protein separation and enrichment, as well as catalytically active AgNPs for colorimetric detection. The typical NaBH4 -mediated catalytic reduction of rhodamine B (RhB) was performed for the visual observation and optical measurement by UV–vis spectroscopy. The results indicated that the target protein could be detected with high sensitivity and excellent specificity using the suggested method.
2. Experimental
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Sigma–Aldrich Co. LLC (USA). Streptavidin coated paramagnetic particles (PMPs ca. 1.0 m) were provided by Promega Co. (Madison, WI, USA). Buffers used in this work: 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 ·12H2 O, 2 mM KH2 PO4 , pH 7.4), 0.5× SSC (75 mM NaCl, 7.5 mM trisodium citrate, pH 7.0), buffer A (50 mM tris–HCl, 2 M NaCl, 0.1% tween 20, pH 7.4), buffer B (20 mM tris–HCl, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.4), protein-free blocking solution (1% content in tris-buffered salines, pH 7.4), stock solution (buffer B containing 0.1% BSA). All the chemicals were of analytical grade. Ultrapure water (≥18.20 M) used throughout the experiments were generated by a millipore water purification system. 2.2. Apparatus The UV–vis absorption spectra were recorded with a Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). Measurement of the absorbance and absorption spectra of rest dye after reduction was conducted on a Synergy NEO HTS multi-mode microplate reader (Biotek, USA). 2.3. Preparation of AgNPs and Apt 29-AgNPs AgNPs with an average diameter of 20 nm were synthesized based on a previously described protocol with some modifications [22]. Briefly, ice cold AgNO3 (2 mM) was added dropwise to twice the volume of NaBH4 (3 mM) with vigorous stir in an ice-water bath. Then the solution was transferred into a hot bath. After being cooled down to room temperature with continuous stir, the prepared AgNPs were stored at 4 ◦ C. The Apt 29-AgNPs were synthesized as follows. 1 mL solution of as-prepared AgNPs was mixed and incubated with Apt 29 (1 nmol) for 16 h at room temperature. Then the mixture was aged in salt and brought to a final concentration of 0.1 M NaCl through a stepwise process. Followed by incubation for 40 h, the solution was centrifuged at 15,000 rpm for 20 min to remove unbound aptamers. The precipitate was washed three times with tris–HCl (10 mM, pH 7.4) and finally dispersed in stock solution at 4 ◦ C for further use. 2.4. Preparation of Apt 15-PMPs Before modification, paramagnetic particles (PMPs) coated with streptavidin were pretreated by washing twice with 0.5× SSC and buffer A, respectively. Then magnetically collected PMPs were redispersed in 300 L of buffer A and mixed with certain amount of Apt 15 (at a ratio of 2 nmol aptamer to 1 mg PMPs). The mixture was incubated for 1 h at 37 ◦ C with gentle shaking. Subsequently, the Apt 15-PMPs were rinsed with buffer B containing 0.1% Tween twice and dispersed in 600 L of blocking solution for 2 h in order to block the nonspecific binding sites. After the washing steps, the Apt 15-PMPs was kept in 600 L of stock solution at 4 ◦ C until use, with a final concentration of about 1 mg mL−1 .
2.1. Materials Oligonucleotides used in this study were synthesized and purified by Sangon Biotech Co. LLC (Shanghai, China) with following sequences. The biotinylated 15-mer anti-thrombin aptamer (denoted as Apt 15): 5 -biotin-A15 -GGT TGG TGT GGT TGG-3 . The thiolated 29-mer anti-thrombin aptamer (denoted as Apt 29): 5 SH-A15 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 . Human serum albumin (HSA), immunoglobulin A (IgA), immunoglobulin G (IgG), lysozyme and bull serum albumin (BSA) were purchased from Biosharp (Japan). Human ␣-thrombin, rhodamine B (RhB), silver nitrate, and sodium borohydride (NaBH4 ) were ordered from
2.5. Fabrication of Apt 15-PMPs/thrombin/Apt 29-AgNPs sandwich complex 20 L of Apt 15-PMPs and 200 L of protein solution with a series of concentration were mixed and incubated for 2 h at 37 ◦ C with gentle shaking. Followed by adding 100 L of Apt 29-AgNPs into the above solution, the mixture was reacted for another 1.5 h to form a sandwich complex. After repetitive washing and separation procedures, unbound AgNPs were removed, and the final sandwich complex was suspended in 40 L of buffer B for the following colorimetric detection.
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2.6. Colorimetric detection of thrombin The colorimetric detection of thrombin was performed in the RhB/NaBH4 reaction system. In a typical experiment, the catalytic reduction was initiated by adding aqueous solution of RhB (50 L, 0.4 mM) and freshly prepared aqueous solution of NaBH4 (100 L, 80 mM) sequentially to the prepared sandwich complex. After 6 min, 20 L of HCl (0.8 M) was added to terminate the reaction. Upon the magnetic separation procedure, the collected supernatant was observed by the naked eye and measured with absorbance at 558 nm via a microplate reader. 3. Results and discussion 3.1. Mechanism of the AgNP-based colorimetric aptasensor The principle of the AgNP-based colorimetric aptasensor was illustrated in Scheme 1. 15-mer DNA aptamer against thrombin (Apt 15) was first conjugated on magnetic particles (PMPs) through the strong biotin–avidin interaction. As the capture probe, the Apt 15-PMPs could effectively capture target protein from complex sample matrix and achieve fast enrichment by an external magnetic field. Subsequently, thiolated 29-mer aptamer (Apt 29) functionalized AgNPs were introduced to form a typical sandwichtype complex. The colorimetric detection was achieved through the reduction of dye catalyzed by AgNPs. AgNPs play the role of catalyst for the reduction of dyes. This catalytic process could be explained by the electro-chemical mechanism, where AgNPs act as an electron relay and the electron transfers from BH4 − ions to dye via nanoparticles [35]. Rhodamine B (RhB), a type of xanthene and cationic dye possessing a higher catalytic reaction rate than a negatively charged dye due to electrostatic interaction, was chosen for the reaction system. In our detection platform, the sandwich complex as a whole acted as the catalyst to promote the reduction of dyes based on the presence of
AgNPs, thereby producing a colour change. The amount of AgNPs contained in the sandwich complex would increase with the concentration of thrombin, which led to vast degradation of RhB. The degradation degree of the rosy colour was in proportion to the concentration of thrombin. 3.2. Viability of the colorimetric assay for determination of thrombin To demonstrate the utility of the colorimetric sensing strategy, the catalytic performance of particles involved in the detection system was investigated (Fig. 1). The catalytic reduction progress can be easily monitored by the characteristic absorption of RhB at the wavelength of maximum absorbance (max = 558 nm). The control experiment without the involvement of any catalyst was carried out (Fig. 1A). The reaction system contained 0.4 mM RhB and 80 mM NaBH4 . The absorbance of RhB dropped by about 20% in 15 min compared to the initial solution. This phenomenon showed that RhB could be slightly degraded by reductant in the absence of catalyst under the given conditions, which was in accordance with those in the literature [36]. As previously reported, the magnetic nanoparticles might cause background interference because of their intrinsic peroxidaselike catalytic activity [37]. However, under our experimental conditions, the absorbance variation of RhB observed from the time-dependent absorption spectra was consistent with the control test (Fig. 1B and D). Since blocking solution was utilized after the preparation of Apt 15-PMPs, it could effectively decrease the active binding sites on their surface to a great extent. The PMPs proved to be catalytically inactive with no impact on the reductive degradation of RhB. Furthermore, as shown in Fig. 1C, the absorption peak of RhB centred at 558 nm decreased quickly once AgNPs were added and the reduction process was accomplished within 8 min, which indicated the remarkable catalytic performance of AgNPs. The drop
Scheme 1. Schematic representation of the AgNP-based colorimetric aptasensor for thrombin detection.
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Fig. 1. Time-dependent UV–vis absorption spectra of RhB during the reduction process in the absence of catalyst (A), in the presence of (B) PMPs and (C) AgNPs. (D) Plot of absorbance of RhB at 558 nm versus time with different reagents. The reaction system contained RhB (0.4 mM) and NaBH4 (80 mM). Inset in (C) showed chemical structure conversion of RhB during the reduction.
margin on absorbance value of RhB (75%) was approximately 8 times lower than that of the reaction in the absence of catalyst and in the presence of PMPs (Fig. 1D). The catalytic reaction rate of RhB was apparently much faster than the normal reduction rate. As shown in the inset of Fig. 1C, the initially rosy RhB was converted to a colourless quinoid structure. We could conclude that AgNPs performed the function of the sole catalyst in the catalytic reaction system.
3.3. Optimization of experimental conditions In order to achieve the best detection performance, two influencing factors, the reaction time and molar ratio of NaBH4 /RhB were investigated. We utilized the decrease in absorbance of RhB at 558 nm (A0 − A: A0 and A represented the absorbance of initial RhB and rest RhB after reduction) to evaluate the results. The control group contained all the components in the experimental group except thrombin. The signal-to-noise ratio was used to assess the detection performance. Fig. 2 displayed the relationship between different reaction time and A0 − A. The value of A0 − A in the experimental group increased with the reaction time and gradually reached the maximum at 6 min. Meanwhile it was observed that the control group had a slight continuous increase as time went on, which could be caused by the few nonspecific absorption of AgNPs towards PMPs. Then 6 min was taken as the optimal time because of its best signal-tonoise level. The effect of molar ratio of NaBH4 /RhB was studied over the range from 100 to 500 under the optimum reaction time of 6 min (Fig. 3). The absorbance value in the control group revealed a change trend similar to the control in Fig. 2. In the experimental group, A0 − A increased with higher amount of reductant and reached equilibrium when the mole of NaBH4 was 400 times of the mole
of RhB. The highest signal-to-noise value was observed when the molar ratio of NaBH4 /RhB was 400. The more RhB in the system, the deeper colour it revealed, which made it difficult to be discriminated from various solutions. On the contrary, an excess of reductive agent promoted the catalytic reaction so rapidly that would raise the difficulty of detection. Taking both effectiveness and operability into consideration, the molar ratio of 400 was chosen as the appropriate requirement for the colorimetric detection. 3.4. Colorimetric detection of thrombin On the basis of such a colorimetric mechanism mentioned above, it was concluded that the amount of the rest RhB is inversely
Fig. 2. Effect of time course in the absence (grey column) and presence of 1.2 nM thrombin (blue column). Error bars were standard derivations across three repetitive experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effect of molar ratio of NaBH4 /RhB on the absorbance in the absence (grey column) and presence of 1.2 nM thrombin (blue column). Error bars were standard derivations across three repetitive experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Specificity test of the colorimetric assay for thrombin (0.8 nM) by comparing it to the interfering proteins at the 0.8 M level: immunoglobulin A (IgA), immunoglobulin G (IgG), human serum albumin (HSA), lysozyme (Lyso) and the mixed sample containing thrombin (0.8 nM) and IgG (0.8 M) or HSA (0.8 M) was also tested. Error bars were standard derivations across three repetitive experiments.
proportional to the amount of thrombin in the system. Therefore, colorimetric detection of thrombin could be accomplished by measuring the absorbance of the rest RhB. The difference of absorbance was defined as A (A = Ablank − A, where Ablank and A are the absorbances of the rest RhB in the absence and presence of thrombin, respectively). Fig. 4 showed the colour and absorption spectra of RhB in the presence of various concentrations of thrombin. In the absence of target, dyes could hardly be reduced and a rosy colour was observed (Fig. 4A). As expected, the colour faded and gradually vanished in the presence of thrombin. Meanwhile, in the UV–vis spectra, the absorbance of RhB at 558 nm continued to decrease with the increase of thrombin concentration until a plateau was reached. A was found to be linear to the concentration of thrombin in the range from 0.4 nM to 1.0 nM (Fig. 4C, inset). The linear equation could be fitted as A = −0.667 + 1.856c (nM) (R2 = 0.986). This strategy allowed for the detection of thrombin at concentration as low as 0.6 nM by the naked eye (Fig. 4A). A limit of detection of 0.2 nM was achieved via UV–vis spectroscopy. Such detection sensitivity at the level of picomole was comparable to many existing aggregation-based and catalyzation-based colorimetric techniques for thrombin [38–41].
3.5. Specificity test of the colorimetric detection
Fig. 4. Colorimetric detection. (A) Photograph for visual detection and (B) UV–vis absorption spectra of RhB corresponding to different concentrations of thrombin (a–i: 0, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.6 nM, respectively). (C) Relationship between difference of absorbance (A) and the concentration of thrombin. A = Ablank − A, where Ablank and A are the absorbance of the rest RhB in the absence and presence of thrombin, respectively. Inset showed the amplification of the linear range from 0.4 nM to 1.0 nM of thrombin. Error bars were standard derivations across three repetitive experiments.
In order to validate that the optical signal was merely generated in response to the target protein, we challenged it with unrelated proteins including immunoglobulin A (IgA), immunoglobulin G (IgG), Human serum albumin (HSA) and lysozyme (Lyso). High optical signals were obtained only when thrombin was tested whereas the absorbance changes were negligible in the presence of even 1000-fold unrelated protein in the assay. In addition, a mixed solution containing 0.8 nM thrombin and 800 nM interfere proteins did not exhibit significant absorbance changes compared with that of thrombin alone. The results essentially suggested that the proposed system revealed high selectivity towards thrombin over other control proteins attributed to the specific capture and binding of aptamers, which might support its potentialities in bioanalytic applications (Fig. 5).
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4. Conclusion In summary, we have successfully proposed a novel and versatile colorimetric platform based on catalytic properties of AgNPs themselves for rapid detection of target protein. This is achieved by integrating capture capacity of the magnetic particles and recognition ability of aptamers. Using this method allows direct visualization detection by the naked eye, thus eliminating the requirement of analytical instrument. More importantly, it overcomes the limitations of aggregation-based systems and provides an attractive alternative to the conventional colorimetric assay, which offers new opportunities for promising applications in analytic diagnose. Acknowledgements
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We acknowledge financial support of the National Basic Research Program of China (973 Program, 2011CB911003), National Natural Foundation of China (Grant no. 21175066, and 21227009), Jiangsu Province Science and Technology Support Program (No. BE2011773), Research Foundation of Jiangsu Province Environmental Monitoring (No. 1116) and the National Science Funds for Creative Research Groups (No. 21121091). References [1] Y. Zhang, Y. Guo, Y. Xianyu, W. Chen, Y. Zhao, X. Jiang, Adv. Mater. 25 (2013) 3802–3819. [2] J. Li, H.E. Fu, L.J. Wu, A.X. Zheng, G.N. Chen, H.H. Yang, Anal. Chem. 84 (2012) 5309–5315. [3] K. Ai, Y. Liu, L. Lu, J. Am. Chem. Soc. 131 (2009) 9496–9497. [4] C. Radhakumary, K. Sreenivasan, Anal. Chem. 83 (2011) 2829–2833. [5] C.D. Medley, J.E. Smith, Z. Tang, Y. Wu, S. Bamrungsap, W. Tan, Anal. Chem. 80 (2008) 1067–1072. [6] L.-Y. Bai, Y.-P. Zhang, N. Chen, J. Chen, X.-M. Zhou, L.-F. Hu, Micro Nano Lett. 6 (2011) 337–341. [7] K. Sato, K. Hosokawa, M. Maeda, J. Am. Chem. Soc. 125 (2003) 8102–8103. [8] P. Baptista, E. Pereira, P. Eaton, G. Doria, A. Miranda, I. Gomes, P. Quaresma, R. Franco, Anal. Bioanal. Chem. 391 (2008) 943–950. [9] J.P. Rosa, J.C. Lima, P.V. Baptista, Nanotechnology 22 (2011) 415202.
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