- Email: [email protected]
Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample
Biosensors and Bioelectronics 39 (2013) 324–328
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample Honglan Qi a,n, Li Shangguan a, Congcong Li a, Xiaoxia Li b, Qiang Gao a, Chengxiao Zhang a a
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, PR China b College of Chemistry and Chemical Engineering, Yanan University, Yanan 716000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2012 Received in revised form 2 July 2012 Accepted 21 July 2012 Available online 27 July 2012
A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)36 /4 . The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20 ng/mL and a detection limit of 0.3 ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08 ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics. & 2012 Elsevier B.V. All rights reserved.
Keywords: Impedimetric Aptasensor Thrombin Serum
1. Introduction Development of simple, sensitive and selective method for the determination of specific proteins in complex biological matrices such as blood plasma or serum and urine has received more and more attention in clinical and biological fields (Anderson and Anderson, 2002). Recently, short single-stranded DNA and RNA aptamers taken as molecular recognition elements, which are generated from an in vitro selection process called SELEX (systematic evolution of ligands by exponential enrichment), have received considerable interest in protein analysis due to their advantages, such as easy production, excellent controllability and versatility compared with conventional antibodies (Citartan et al., 2012; Ewles and Goodwin, 2011; Famulok et al., 2007; Pu et al., 2011; Willner and Zayats, 2007). As a result, a lot of aptamer-based methods for the detection of proteins have been proposed and extensively studied in the past few years, such as colorimetry ((Huang et al., 2005), fluorescence (Chi et al., 2011; Csordas et al., 2010; Ge et al., 2011; Pavlov et al., 2005; Wang et al., 2011) electrochemistry (Kwon et al., 2011; Li et al., 2011; Radi et al., 2006; Tong et al., 2011; Willner and Zayats, 2007; Yan et al., 2011), etc. Among them, electrochemical technique, especially electrochemical impedance
n
Corresponding author. Tel.: þ86 29 81530726; fax: þ86 29 81530727. E-mail address: [email protected] (H. Qi).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.040
spectroscopy (EIS) technique, becomes increasingly popular in a variety of protein biosensing because it offers several advantages such as simplicity, high sensitivity and serving as an elegant way to interface biorecognition events and signal transduction (Li et al., 2008a, 2008b). Various EIS aptasensors have been designed for determination of proteins (Bogomolova et al., 2009; Li et al., 2008a, 2008b, 2011). The effort is mainly focused on the improvement of sensitivity by employing various amplification strategies and reducing background strategies (Komatsu, 2012). However, these strategies are largely limited due to the challenge of signal transduction and the interferences of nonspecific binding and a high background (Bogomolova et al., 2009). The biological samples often need to be pre-treated, for example, a 2-fold dilution (Baker et al., 2006), 4-fold dilution (Du et al., 2010) or 10-fold dilution (Lao et al., 2009). The direct determination of protein in biological sample is very limited (Zhou et al., 2007). Therefore, how to specifically and sensitively recognize target protein in the presence of thousand folds to million folds excess of non-target proteins is a key problem in the clinical bioassay (Hucknall et al., 2009). To tackle the specificity issues, several approaches have been developed and reported. Surface modifications with antifouling self-assembled monolayers (Li et al., 2007; Emmenegger et al., 2009), grafted polymer layers (Jiang and Cao, 2010; Vaisocherova et al., 2008), and polymer brushes (Yang et al., 2009) were employed to reduce or suppress the non-specific adsorption. For example, poly(carboxybetaine acrylamide)-grafted surface has
H. Qi et al. / Biosensors and Bioelectronics 39 (2013) 324–328
325
Fig. 1. Schematic of impedimetric aptasensor for determination of thrombin.
been used to detect antigen in undiluted human blood serum (Yang et al., 2009). DNA biosensor based on a ternary selfassembled monolayer was developed for direct measurement of target DNA in undiluted and untreated human serum and urine samples (Campuzano et al., 2011). In this paper, we describe a simple, selective and sensitive impedimetric aptasensor for the determination of protein in undiluted serum sample. Thrombin, the main effector protease of the coagulation cascade, was chosen as model protein in this work (Li et al., 2008a, 2008b). Schematic representation of aptasensor with fabrication steps and performance is showed in Fig. 1. The aptasensor was designed by co-assembling antithrombin binding aptamer (TBA) and dithiothreitol (DTT), followed by assembling of 6-mercapto-1-hexanol (MCH) on the surface of gold electrode to form a ternary monolayer. The introduction of target thrombin increased electron transfer resistance of aptasensor. The effects of the different layers on the analytical performances (non-specific adsorption and response sensitivity) were studied. The fabricated aptasensor was also applied to detect thrombin in both buffer solution and undiluted serum sample.
2. Experimental 2.1. Fabrication of aptasensor Materials and apparatus were presented in the supporting information. 10 mL of 10 mM anti-thrombin binding aptamer (TBA, 50 –SH-(CH2)6–GGT TGG TGT GGT TGG-30 ) and 10 mL of 10 mM DTT was mixed in 80 mL immobilization buffer and allowed to stand for 10 min. A cleaned gold electrode was immersed into 100 mL of this mixture and incubated 10 h at 4 1C in a humidified chamber. After washing with washing buffer, the mixed monolayer-modified gold electrode was subsequently treated with 100 mL of 1.0 mM MCH for 1 h to obtain the ternary monolayer
interface (TBA/DTT þMCH). Finally, the resulted modified electrode was thoroughly rinsed with washing buffer. Different binary layers, named as TBA þMCH, TBA þHDT, TBA þHT and TBA þDTT, was prepared by firstly assembling 1.0 mM TBA onto the surface of gold electrode and then backfilling with 1.0 mM MCH, 1.0 mM 1,6-hexanedithiol (HDT), 1.0 mM hexanethiol (HT) and 1.0 mM DTT, respectively. 2.2. Serum sample preparation Thrombin serum samples were prepared by adding small volume of extrinsic thrombin solution (2 mL, 20 mg/mL) in excess volume of undiluted serum samples (healthy adults human serum and fetal calf serum, 2 mL), and further diluted with undiluted serum. In the case of the serum samples analysis, the blank assays corresponded to the serum sample without added extrinsic thrombin. 2.3. Electrochemical measurement The aptasensor was immersed in 100 mL binding buffer or serum sample contained different concentrations of thrombin for 40 min under room temperature and then rinsed with washing buffer. EIS measurements were performed in 5.0 mL of 100 mM phosphate buffer solution (PBS, pH 7.4) containing 5 mM K4[Fe(CN)6] and 5 mM K3[Fe(CN)6]. The concentration of thrombin was quantified by the change of electron transfer resistance of aptasensor.
3. Results and discussion 3.1. Design and comparison of different layer interfaces In the fabrication of apatsensor, the interfacial properties of the mixed monolayer play an important role in the analytical
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reduced (Fig. S-2). The immobilization procedure of multicomponent ternary layer was also monitored by CV and EIS (Fig. S-3). In conclusion, the inclusion of DTT during TBA immobilization can highly suppress background and effectively prevent nonspecific adsorption of BSA. DTT is a four carbon a-o dithiol with hydroxyl groups on the second and third carbons. The two hydroxyl groups on DTT exposed at molecule outer surface provide a hydrophilic microenvironment which is favorable for binding reaction and which also enhances the non-fouling properties of the new monolayer (MacDairmid et al., 2003). Therefore, a novel TBA/DTTþMCH ternary monolayer was chosen as recognition interface for assay of thrombin in the following experiment.
Fig. 2. Comparative reacted efficiency, background noise and S/N characteristics for 2.0 ng/mL target thrombin and 1% of BSA, using the different layer interfaces, in 100 mM PBS buffer solution (pH 7.4) containing 5 mM K4[Fe(CN)6] and 5 mM K3[Fe(CN)6]. The biased potential was 0. 29 V vs Ag/AgCl; the frequency was from 100 kHz to 500 mHz and the amplitude was 5 mV.
performance of aptasensor (Dharuman and Hahn, 2008). In this work, different binary and multi-component ternary layers prepared with the different backfillers using one-step or two-step sequential assembly processes were examined in connection to impedimetric assay. The results were shown in Fig. 2 and Table S-1. As can be observed from Fig. 2 and Table S-1, only TBA was immobilized onto the surface of gold electrode, the Ret was 26680 O. The increase is attributed to electrostatic repulsion between negatively charged TBA modified on electrode surface and [Fe(CN)6]3 /4 in solution (Li et al., 2008a, 2008b). The Ret decreased to 26160 O, 11810 O and 3478 O for TBA þMCH, TBA þHT, TBAþDTT binary layer, respectively. This is because that MCH, HT and DTT as backfillers can be used to displace nonspecific interactions between TBA and gold electrode surface and to cause the TBA to stand up from the surface (Herne and Tarlov, 1997; Li et al., 2008a, 2008b; Campuzano et al., 2011). This is confirmed by the density of TBA onto gold electrode (1.2 1013, 6.5 1012, 4.4 1012 and 4.5 1012 molecules.cm 2, TBA, TBAþDTT, TBAþMCH and TBAþHT, respectively, Table S-1), which is calculated by using adsorption of cationic [Ru(NH3)6]3 þ species onto the negative phosphate backbone of TBA (Steel et al.,1998). For TBAþHDT, the Ret surprisingly increases to 104700 O and density of TBA decreases to 6.6 1012 molecules.cm 2. Compared with TBAþ DTT, common binary layer (TBAþHDT, TBAþMCH and TBAþ HT) displayed large background current, as a consequence, a low S/N ratio for target thrombin is obtained. This is consistent with the capacitance data (Table S-1). The capacitance data in Table S-1 can be analyzed according to the parallel plate capacitor model in which the modified electrodes are modeled to be a capacitor with gold electrode surface and electrolyte solution forming two conducting plates of the capacitor (Miller et al., 1991; Bard and Faulkner, 1980). As to estimate the degree of non-specific binding to different layers, BSA was chosen as non-specific protein. As shown in Fig. 2, TBAþDTT binary interface was more resistant to BSA, and other binary interfaces (TBAþMCH, TBAþHDT and TBAþHT) have high susceptibility to BSA. The contact angle data further confirms this result (Fig. S-1). Furthermore, the multicomponent ternary layer obtained by further backfilling TBA/DTT binary layer (obtained by co-assembly processes) with MCH shows low background (214 O) and higher S/N signal (3.1). The distinctive difference in the topography of TBA (a), TBAþMCH (b) and TBA/DTTþMCH (c) surface can be observed. Pinhole was blackfilled by MCH and the roughness was
3.2. Analytical performance of the aptasensor Fig. 3A shows Nyquist plots of faradic impedance spectra of the aptasensor (TBA/DTTþ MCH) for different concentrations of thrombin in buffer solution. From the inset of Fig. 3A, it is
Fig. 3. Nyquist plots of aptasensor to different concentrations of thrombin in buffer solution (A) and undiluted serum (B). Inset: calibration curve of thrombin. (A) (a) 0 g/mL; (b) 1.0 ng/mL; (c) 2.0 ng/mL; (d) 5.0 ng/mL; (e) 8.0 ng/mL; (f) 10 ng/mL; (g) 20.0 ng/mL. (B) (a) 0 g/mL; (b) 0.14 ng/mL; (c) 0.28 ng/mL; (d) 0.56 ng/mL; (e) 1.12 ng/mL; (f) 2.25 ng/mL; (g) 7.5 ng/mL; (h) 12 ng/mL; (i)20 ng/mL. The detected conditions were same as Fig. 2.
H. Qi et al. / Biosensors and Bioelectronics 39 (2013) 324–328
apparent that the Ret has a fine linear relationship with the concentration of thrombin from 1.0 ng/mL to 20 ng/mL. The regression equation was WRet (O)¼76.74 C (ng/mL)þ487.43, and regression coefficient was 0.9865. The relative standard deviation (RSD) is 4.3% for five measurements of 2.0 ng/mL thrombin with one electrode and RSD is 5.3% for 2.0 ng/mL thrombin with 10 different electrodes. The detection limit for thrombin is 0.3 ng/mL (Kaiser, 1973), which is much lower than previously reported in our previous work (Li et al., 2008b) by using gold nanoparticle amplification (as shown in Table S-2). Since the clinically meaningful concentration of thrombin in resting and activated blood ranges from nanomolar to micromolar per milliliter, respectively (Mann et al., 2003; Hemker et al., 2006), the sensitivity of the proposed method is satisfied for practical application. Therefore, a high sensitivity and a good reproducibility of the developed aptasensor were obtained. 3.3. Electrochemical detection of thrombin in serum samples We found that the novel ternary recognition interfaces allow the direct detection of trace target thrombin in undiluted serum sample. A measurable blank signal was observed when incubated in serum samples and different serum samples give different signals (Fig. S-4). Similar results were also obtained in Refs. (Xiao et al., 2005; Wang et al., 2010). Although some blank signal was observed, the impendence was also increased with increase of concentration of extrinsic thrombin (Fig. 3B, Fig. S-5-6). Fig. 3B shows the Nyquist plots of faradic impedance spectra of the aptasensor for different concentrations of extrinsic thrombin in one undiluted serum sample. From the inset of Fig. 3B, it is apparent that Ret has a fine linear relationship with the logarithm of the concentration of thrombin from 0.14 ng/mL to 20.0 ng/mL. The regression equation was WRet (O)¼987.8 l gC (pg/mL) 1225, and regression coefficient was 0.9932. Wider linear range can be obtained by using different immobilization aptamer density (Zhang et al., 2008). Good recovery results (Table S-3) suggest the feasibility of aptasensor for the determination of thrombin in undiluted serum sample. The detection limit for thrombin was 0.08 ng/mL, which is lower than buffer solution. This is may be due to the unspecific affinity of substances in the biological samples (Song et al., 2009). The association constant (Ka) of the immobilized aptamer with thrombin in undiluted serum is 7.8 107 M 1 according to the method in Ref. (Li et al., 2008b), similar with that in buffer solution (Li et al., 2008b).
4. Conclusions A simple, sensitive and anti-fouling impedimetric aptasensor for determination of thrombin was developed by employing a ternary monolayer, incorporating TBA, MCH, and DTT as recognition monolayer. The novel interface offers high sensitivity and direct measurement of attomole (ng/mL) levels of target thrombin in serum with good antifouling characteristics. This work provides a promising strategy for real-world clinical application with impressive sensitivity and antifouling characteristics.
Acknowledgements Financial supports from National Nature Science Foundation of China (no. 20805028), the Fundamental Research Funds for the Central Universities (no.GK201002027), Natural Science Basic Research Plan in Shaanxi Province of China (Program no.2010JM2020) and Program for Changjiang Scholars and Innovative Research Team in University (IRT 1070) are gratefully
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acknowledged. We also thank Dr. Bingling Li for helpful discussion.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.07.040.
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Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample Honglan Qi a,n, Li Shangguan a, Congcong Li a, Xiaoxia Li b, Qiang Gao a, Chengxiao Zhang a a
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, PR China b College of Chemistry and Chemical Engineering, Yanan University, Yanan 716000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2012 Received in revised form 2 July 2012 Accepted 21 July 2012 Available online 27 July 2012
A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)36 /4 . The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20 ng/mL and a detection limit of 0.3 ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08 ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics. & 2012 Elsevier B.V. All rights reserved.
Keywords: Impedimetric Aptasensor Thrombin Serum
1. Introduction Development of simple, sensitive and selective method for the determination of specific proteins in complex biological matrices such as blood plasma or serum and urine has received more and more attention in clinical and biological fields (Anderson and Anderson, 2002). Recently, short single-stranded DNA and RNA aptamers taken as molecular recognition elements, which are generated from an in vitro selection process called SELEX (systematic evolution of ligands by exponential enrichment), have received considerable interest in protein analysis due to their advantages, such as easy production, excellent controllability and versatility compared with conventional antibodies (Citartan et al., 2012; Ewles and Goodwin, 2011; Famulok et al., 2007; Pu et al., 2011; Willner and Zayats, 2007). As a result, a lot of aptamer-based methods for the detection of proteins have been proposed and extensively studied in the past few years, such as colorimetry ((Huang et al., 2005), fluorescence (Chi et al., 2011; Csordas et al., 2010; Ge et al., 2011; Pavlov et al., 2005; Wang et al., 2011) electrochemistry (Kwon et al., 2011; Li et al., 2011; Radi et al., 2006; Tong et al., 2011; Willner and Zayats, 2007; Yan et al., 2011), etc. Among them, electrochemical technique, especially electrochemical impedance
n
Corresponding author. Tel.: þ86 29 81530726; fax: þ86 29 81530727. E-mail address: [email protected] (H. Qi).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.040
spectroscopy (EIS) technique, becomes increasingly popular in a variety of protein biosensing because it offers several advantages such as simplicity, high sensitivity and serving as an elegant way to interface biorecognition events and signal transduction (Li et al., 2008a, 2008b). Various EIS aptasensors have been designed for determination of proteins (Bogomolova et al., 2009; Li et al., 2008a, 2008b, 2011). The effort is mainly focused on the improvement of sensitivity by employing various amplification strategies and reducing background strategies (Komatsu, 2012). However, these strategies are largely limited due to the challenge of signal transduction and the interferences of nonspecific binding and a high background (Bogomolova et al., 2009). The biological samples often need to be pre-treated, for example, a 2-fold dilution (Baker et al., 2006), 4-fold dilution (Du et al., 2010) or 10-fold dilution (Lao et al., 2009). The direct determination of protein in biological sample is very limited (Zhou et al., 2007). Therefore, how to specifically and sensitively recognize target protein in the presence of thousand folds to million folds excess of non-target proteins is a key problem in the clinical bioassay (Hucknall et al., 2009). To tackle the specificity issues, several approaches have been developed and reported. Surface modifications with antifouling self-assembled monolayers (Li et al., 2007; Emmenegger et al., 2009), grafted polymer layers (Jiang and Cao, 2010; Vaisocherova et al., 2008), and polymer brushes (Yang et al., 2009) were employed to reduce or suppress the non-specific adsorption. For example, poly(carboxybetaine acrylamide)-grafted surface has
H. Qi et al. / Biosensors and Bioelectronics 39 (2013) 324–328
325
Fig. 1. Schematic of impedimetric aptasensor for determination of thrombin.
been used to detect antigen in undiluted human blood serum (Yang et al., 2009). DNA biosensor based on a ternary selfassembled monolayer was developed for direct measurement of target DNA in undiluted and untreated human serum and urine samples (Campuzano et al., 2011). In this paper, we describe a simple, selective and sensitive impedimetric aptasensor for the determination of protein in undiluted serum sample. Thrombin, the main effector protease of the coagulation cascade, was chosen as model protein in this work (Li et al., 2008a, 2008b). Schematic representation of aptasensor with fabrication steps and performance is showed in Fig. 1. The aptasensor was designed by co-assembling antithrombin binding aptamer (TBA) and dithiothreitol (DTT), followed by assembling of 6-mercapto-1-hexanol (MCH) on the surface of gold electrode to form a ternary monolayer. The introduction of target thrombin increased electron transfer resistance of aptasensor. The effects of the different layers on the analytical performances (non-specific adsorption and response sensitivity) were studied. The fabricated aptasensor was also applied to detect thrombin in both buffer solution and undiluted serum sample.
2. Experimental 2.1. Fabrication of aptasensor Materials and apparatus were presented in the supporting information. 10 mL of 10 mM anti-thrombin binding aptamer (TBA, 50 –SH-(CH2)6–GGT TGG TGT GGT TGG-30 ) and 10 mL of 10 mM DTT was mixed in 80 mL immobilization buffer and allowed to stand for 10 min. A cleaned gold electrode was immersed into 100 mL of this mixture and incubated 10 h at 4 1C in a humidified chamber. After washing with washing buffer, the mixed monolayer-modified gold electrode was subsequently treated with 100 mL of 1.0 mM MCH for 1 h to obtain the ternary monolayer
interface (TBA/DTT þMCH). Finally, the resulted modified electrode was thoroughly rinsed with washing buffer. Different binary layers, named as TBA þMCH, TBA þHDT, TBA þHT and TBA þDTT, was prepared by firstly assembling 1.0 mM TBA onto the surface of gold electrode and then backfilling with 1.0 mM MCH, 1.0 mM 1,6-hexanedithiol (HDT), 1.0 mM hexanethiol (HT) and 1.0 mM DTT, respectively. 2.2. Serum sample preparation Thrombin serum samples were prepared by adding small volume of extrinsic thrombin solution (2 mL, 20 mg/mL) in excess volume of undiluted serum samples (healthy adults human serum and fetal calf serum, 2 mL), and further diluted with undiluted serum. In the case of the serum samples analysis, the blank assays corresponded to the serum sample without added extrinsic thrombin. 2.3. Electrochemical measurement The aptasensor was immersed in 100 mL binding buffer or serum sample contained different concentrations of thrombin for 40 min under room temperature and then rinsed with washing buffer. EIS measurements were performed in 5.0 mL of 100 mM phosphate buffer solution (PBS, pH 7.4) containing 5 mM K4[Fe(CN)6] and 5 mM K3[Fe(CN)6]. The concentration of thrombin was quantified by the change of electron transfer resistance of aptasensor.
3. Results and discussion 3.1. Design and comparison of different layer interfaces In the fabrication of apatsensor, the interfacial properties of the mixed monolayer play an important role in the analytical
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reduced (Fig. S-2). The immobilization procedure of multicomponent ternary layer was also monitored by CV and EIS (Fig. S-3). In conclusion, the inclusion of DTT during TBA immobilization can highly suppress background and effectively prevent nonspecific adsorption of BSA. DTT is a four carbon a-o dithiol with hydroxyl groups on the second and third carbons. The two hydroxyl groups on DTT exposed at molecule outer surface provide a hydrophilic microenvironment which is favorable for binding reaction and which also enhances the non-fouling properties of the new monolayer (MacDairmid et al., 2003). Therefore, a novel TBA/DTTþMCH ternary monolayer was chosen as recognition interface for assay of thrombin in the following experiment.
Fig. 2. Comparative reacted efficiency, background noise and S/N characteristics for 2.0 ng/mL target thrombin and 1% of BSA, using the different layer interfaces, in 100 mM PBS buffer solution (pH 7.4) containing 5 mM K4[Fe(CN)6] and 5 mM K3[Fe(CN)6]. The biased potential was 0. 29 V vs Ag/AgCl; the frequency was from 100 kHz to 500 mHz and the amplitude was 5 mV.
performance of aptasensor (Dharuman and Hahn, 2008). In this work, different binary and multi-component ternary layers prepared with the different backfillers using one-step or two-step sequential assembly processes were examined in connection to impedimetric assay. The results were shown in Fig. 2 and Table S-1. As can be observed from Fig. 2 and Table S-1, only TBA was immobilized onto the surface of gold electrode, the Ret was 26680 O. The increase is attributed to electrostatic repulsion between negatively charged TBA modified on electrode surface and [Fe(CN)6]3 /4 in solution (Li et al., 2008a, 2008b). The Ret decreased to 26160 O, 11810 O and 3478 O for TBA þMCH, TBA þHT, TBAþDTT binary layer, respectively. This is because that MCH, HT and DTT as backfillers can be used to displace nonspecific interactions between TBA and gold electrode surface and to cause the TBA to stand up from the surface (Herne and Tarlov, 1997; Li et al., 2008a, 2008b; Campuzano et al., 2011). This is confirmed by the density of TBA onto gold electrode (1.2 1013, 6.5 1012, 4.4 1012 and 4.5 1012 molecules.cm 2, TBA, TBAþDTT, TBAþMCH and TBAþHT, respectively, Table S-1), which is calculated by using adsorption of cationic [Ru(NH3)6]3 þ species onto the negative phosphate backbone of TBA (Steel et al.,1998). For TBAþHDT, the Ret surprisingly increases to 104700 O and density of TBA decreases to 6.6 1012 molecules.cm 2. Compared with TBAþ DTT, common binary layer (TBAþHDT, TBAþMCH and TBAþ HT) displayed large background current, as a consequence, a low S/N ratio for target thrombin is obtained. This is consistent with the capacitance data (Table S-1). The capacitance data in Table S-1 can be analyzed according to the parallel plate capacitor model in which the modified electrodes are modeled to be a capacitor with gold electrode surface and electrolyte solution forming two conducting plates of the capacitor (Miller et al., 1991; Bard and Faulkner, 1980). As to estimate the degree of non-specific binding to different layers, BSA was chosen as non-specific protein. As shown in Fig. 2, TBAþDTT binary interface was more resistant to BSA, and other binary interfaces (TBAþMCH, TBAþHDT and TBAþHT) have high susceptibility to BSA. The contact angle data further confirms this result (Fig. S-1). Furthermore, the multicomponent ternary layer obtained by further backfilling TBA/DTT binary layer (obtained by co-assembly processes) with MCH shows low background (214 O) and higher S/N signal (3.1). The distinctive difference in the topography of TBA (a), TBAþMCH (b) and TBA/DTTþMCH (c) surface can be observed. Pinhole was blackfilled by MCH and the roughness was
3.2. Analytical performance of the aptasensor Fig. 3A shows Nyquist plots of faradic impedance spectra of the aptasensor (TBA/DTTþ MCH) for different concentrations of thrombin in buffer solution. From the inset of Fig. 3A, it is
Fig. 3. Nyquist plots of aptasensor to different concentrations of thrombin in buffer solution (A) and undiluted serum (B). Inset: calibration curve of thrombin. (A) (a) 0 g/mL; (b) 1.0 ng/mL; (c) 2.0 ng/mL; (d) 5.0 ng/mL; (e) 8.0 ng/mL; (f) 10 ng/mL; (g) 20.0 ng/mL. (B) (a) 0 g/mL; (b) 0.14 ng/mL; (c) 0.28 ng/mL; (d) 0.56 ng/mL; (e) 1.12 ng/mL; (f) 2.25 ng/mL; (g) 7.5 ng/mL; (h) 12 ng/mL; (i)20 ng/mL. The detected conditions were same as Fig. 2.
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apparent that the Ret has a fine linear relationship with the concentration of thrombin from 1.0 ng/mL to 20 ng/mL. The regression equation was WRet (O)¼76.74 C (ng/mL)þ487.43, and regression coefficient was 0.9865. The relative standard deviation (RSD) is 4.3% for five measurements of 2.0 ng/mL thrombin with one electrode and RSD is 5.3% for 2.0 ng/mL thrombin with 10 different electrodes. The detection limit for thrombin is 0.3 ng/mL (Kaiser, 1973), which is much lower than previously reported in our previous work (Li et al., 2008b) by using gold nanoparticle amplification (as shown in Table S-2). Since the clinically meaningful concentration of thrombin in resting and activated blood ranges from nanomolar to micromolar per milliliter, respectively (Mann et al., 2003; Hemker et al., 2006), the sensitivity of the proposed method is satisfied for practical application. Therefore, a high sensitivity and a good reproducibility of the developed aptasensor were obtained. 3.3. Electrochemical detection of thrombin in serum samples We found that the novel ternary recognition interfaces allow the direct detection of trace target thrombin in undiluted serum sample. A measurable blank signal was observed when incubated in serum samples and different serum samples give different signals (Fig. S-4). Similar results were also obtained in Refs. (Xiao et al., 2005; Wang et al., 2010). Although some blank signal was observed, the impendence was also increased with increase of concentration of extrinsic thrombin (Fig. 3B, Fig. S-5-6). Fig. 3B shows the Nyquist plots of faradic impedance spectra of the aptasensor for different concentrations of extrinsic thrombin in one undiluted serum sample. From the inset of Fig. 3B, it is apparent that Ret has a fine linear relationship with the logarithm of the concentration of thrombin from 0.14 ng/mL to 20.0 ng/mL. The regression equation was WRet (O)¼987.8 l gC (pg/mL) 1225, and regression coefficient was 0.9932. Wider linear range can be obtained by using different immobilization aptamer density (Zhang et al., 2008). Good recovery results (Table S-3) suggest the feasibility of aptasensor for the determination of thrombin in undiluted serum sample. The detection limit for thrombin was 0.08 ng/mL, which is lower than buffer solution. This is may be due to the unspecific affinity of substances in the biological samples (Song et al., 2009). The association constant (Ka) of the immobilized aptamer with thrombin in undiluted serum is 7.8 107 M 1 according to the method in Ref. (Li et al., 2008b), similar with that in buffer solution (Li et al., 2008b).
4. Conclusions A simple, sensitive and anti-fouling impedimetric aptasensor for determination of thrombin was developed by employing a ternary monolayer, incorporating TBA, MCH, and DTT as recognition monolayer. The novel interface offers high sensitivity and direct measurement of attomole (ng/mL) levels of target thrombin in serum with good antifouling characteristics. This work provides a promising strategy for real-world clinical application with impressive sensitivity and antifouling characteristics.
Acknowledgements Financial supports from National Nature Science Foundation of China (no. 20805028), the Fundamental Research Funds for the Central Universities (no.GK201002027), Natural Science Basic Research Plan in Shaanxi Province of China (Program no.2010JM2020) and Program for Changjiang Scholars and Innovative Research Team in University (IRT 1070) are gratefully
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acknowledged. We also thank Dr. Bingling Li for helpful discussion.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.07.040.
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