Biosensors and Bioelectronics 70 (2015) 372–375
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Up-conversion fluorescence “off-on” switch based on heterogeneous core-satellite assembly for thrombin detection Xueli Zhao, Si Li, Liguang Xu n, Wei Ma, Xiaoling Wu, Hua Kuang, Libing Wang, Chuanlai Xu State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 11 February 2015 Received in revised form 21 March 2015 Accepted 26 March 2015 Available online 27 March 2015
NaGdF4: Yb, Er nanoparticles, with up-conversion (UC) fluorescence, were used for the first time to build an “off-on” switch based on Au core-UC satellites for thrombin detection. We fabricated the fluorescence sensor using thrombin aptamer modified Au core and complementary sequence modified UC satellites in liquid phase. With optimized assembled conditions, the yield of Au core-UC satellites achieved 80%. The fluorescence of UC nanoparticles quenched when satellite NP attached to Au core NP. Thrombin aptamer on the surface of Au core would bind to targets when thrombin existed in the system, then UC satellites were released and the quenched fluorescence recovered. The sensor showed high specificity for thrombin compared with other biomolecules and the limit of detection reached 3.5 fg/mL. Application of this sensor to detect targets in human serum also achieved satisfactory results. The purpose of this work was to build an ultrasensitive sensor based on Au core-UC satellites for thrombin detection in human serum to achieve diagnosis of diseases. & 2015 Elsevier B.V. All rights reserved.
Keywords: Up-conversion fluorescence Core-satellites Thrombin Aptamer sensor Detection
1. Introduction Thrombin, generated in plasma by the conversion of its precursor, prothrombin, is involved in thrombosis and platelet activation, and plays a significant role in a number of cardiovascular diseases such as cerebral ischemia and infarction (Centi et al., 2007). Thrombin usually leads to vasospasm following subarachnoid hemorrhage. Blood flowing out from ruptured cerebral aneurysm clots around a cerebral artery, releasing thrombin. This can induce an acute and prolonged narrowing of the blood vessel, which might cause cerebral ischemia and infarction (stroke). Thrombin is not present in the blood of healthy individuals, but can be detected in the blood of patients suffering from diseases associated with coagulation abnormalities (Centi et al., 2007; Tennico et al., 2010). Therefore, the detection of thrombin is vital for the diagnosis of related diseases. An aptamer is a single oligonucleotide which is completely engineered in an in vitro selection process, and has high affinity for targets and good stability and storage properties compared to antibodies (Cho et al., 2008). Since the thrombin-binding aptamer (15-mer, 5ʹ-GGTTGGTGTGGTTGG-3ʹ) was selected (Daniel et al., 2013; Li et al., 2008), numerous aptasensors (Chen et al., 2010b; Pavlov et al., 2004; Wang and Liu, 2009; Xiao et al., 2005) have n
Corresponding author. E-mail address: [email protected] (L. Xu).
http://dx.doi.org/10.1016/j.bios.2015.03.068 0956-5663/& 2015 Elsevier B.V. All rights reserved.
been fabricated based on the conformational changes induced by target binding, signals used for quantification ranged from color (Chen et al., 2010a; Chen et al., 2014b; Li et al., 2008; Pavlov et al., 2004), fluorescence (Chang et al., 2010; Chi et al., 2011; Jie and Yuan, 2012; Kim and Lee, 2014; Kong et al., 2013; Wang et al., 2011b; Yan et al., 2011) and electrochemiluminescence (Chen et al., 2014a; Deng et al., 2014; Shan et al., 2011; Wang et al., 2011a) to surface enhanced Raman scattering (SERS) (Z. Wu et al., 2013) and surface plasmon resonance (SPR) (Bai et al., 2013; He et al., 2014; Mani et al., 2011). These methods have the disadvantages of either a high limit of detection or complex operation procedures and limit their application in blood samples (Cho et al., 2008). The aim of this study was to design a fluorescence aptasensor with high sensitivity and the ability to resist interference in a matrix such as human serum. The sensor was based on the signal produced by the assembly and disassembly of gold core and upconversion nanoparticle (UC NP) satellites. Compared with normal fluorescent materials, the fluorescence and chemical properties of UC NPs were more stable, the excitation of 980 nm can avoid the interference of auto fluorescence of biomolecules, such as protein (Wen et al., 2013; H. Wu et al., 2013; Zhang et al., 2014). The low biological toxicity of this sensor also demonstrated significant potential in biological applications such as cancer diagnosis and treatment (Cheng et al., 2013; Li et al., 2013,, 2014; Maji et al., 2014).
X. Zhao et al. / Biosensors and Bioelectronics 70 (2015) 372–375
2. Material and methods 2.1. Materials and regents The thiol-modified oligonucleotides (thrombin aptamer (TBApt) and its complementary sequence (TB-Apt C)) (purity 4 95%) used in this study were manufactured by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., China. These oligonucleotides were dissolved in TE buffer (10 mM Tris–hydrochloride buffer, pH 8.0, containing 1.0 mM EDTA, Shanghai Sangon) to give a final concentration of 100 μM. Human α-thrombin (TB), bovine serum albumin (BSA), human prostate-specific antigen (PSA), serum albumin (HSA), immunoglobulin G (IgG), and L-cysteine (L-cys) were purchased from Sigma-Aldrich. Up-conversion nanoparticles (UC NPs) were purchased from Beijing Oneder-Hightech Co. Ltd., China. Deionized (DI) water, obtained using a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France), was used throughout this work. The detailed sequences of the oligonucleotides are as follows: TB-Apt: 5ʹ-HS-TTTTTGGTTGGTGTGGTTGG-3ʹ TB- Apt C: 5ʹ-HS-TTTTTTTAATTATATTAACC-3ʹ
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0.5 nM. A NaNO3 solution of 5 M in water was added to the former solution to produce a NaNO3 concentration of 150 mM. The solution was left for 2 h to complete the modification of TB-Apt C to UC NPs. The modified UC NPs underwent ultrafiltration twice to remove excess DNA. 2.5. Assembly of the Au CORE-UC satellite sensor To assemble the Au core-UC satellite nanostructure, purified TB-Apt-modified AuNPs were mixed with TB-Apt C-modified UC NPs at a 10:1 M excess relative to Au cores in hybridization buffer (10 mM pH 7.2 Tris–HCl). Particles were incubated at room temperature for 8 h. 2.6. Selectivity of the sensor The selectivity of the as-fabricated sensor was evaluated by analyzing its selectivity for bovine serum albumin (BSA), human prostate-specific antigen (PSA), serum albumin (HSA), immunoglobulin G (IgG), and L-cysteine (L-cys). These proteins and amino acid were added to the sensor at a concentration of 1 ng/mL, and the operation procedures were the same as those for thrombin detection.
2.2. Instrumentation
2.7. Recovery test of thrombin in Human serum
Fluorescence spectra were acquired using a F-7000 fluorescence spectrophotometer, scan speed was 240 nm/min. Transmission electron microscopy images were acquired using a JEOL JEM-2100 operating at an acceleration voltage of 200 kV.
As serum is a complex biological matrix containing a wide variety of biomolecules, but no coagulation proteins such as thrombin, human serum was spiked with thrombin in the recovery test for further application. Human serum (obtained from Wuxi No. 2 People's Hospital, All the experiments were performed in compliance with the relevant laws and institutional guidelines, and the experiments were approved by the Ethics Committee of No. 2 People's Hospital) was diluted ten times with 10 mM Tris– HCl buffer (pH 7.2) as the analysis medium. Three concentrations of thrombin in the linear range (10, 20, 100 fg/mL) were spiked for monitoring.
2.3. Synthesis of gold nanoparticles Gold nanoparticles (327 1 nm) were synthesized by a seedmediated growth method. Seeds (Au NPs of 13 71 nm) were synthesized by a routine method: 2 mL of 38.8 mM trisodium citrate was quickly added to a boiling solution of HAuCl4 (40 mL, 0.5 mM) with vigorous stirring and refluxed until there were no more color changes in the solution. Then, 7.5 mL of 5.3 mM ascorbic acid was injected into the mixed solution (2 mL–10 mM HAuCl4, 0.1 mL–10 mM AgNO3, 42.5 mL H2O, 4 mL seeds) with a constant-flow pump at a speed of 0.6 mL/min under vigorous stirring. A BPS (bis(p-sulfonatophenyl) phenylphosphine dihydrate, dipotassium salt) solution was added to the as-synthesized Au NPs to a final concentration of 2.5 mg/mL and stirred in darkness for 12 h. TEM image of Au NPs was shown in Fig. S1a. 2.4. Oligonucleotide-functionalization of AuNPs and UC NPs Au NPs were modified with thiolated TB-Apt using a previously reported method (Rosi et al., 2006). AuNPs (327 1 nm) were suspended in 10 mM PB (pH 7.2) and 0.01% Tween20. TB-Apt (dissolved in TE buffer) was added to the AuNPs at a final ratio of 500 oligonucleotides per particle. The mixed solution was left for 2 h, a NaCl solution of 5 M in water was added (1 μL NaCl solution to 100 μL solution) with an interval of 30 min to bring the final concentration of NaCl to 400 mM at room temperature. The TBApt-modified AuNPs were washed three times with 10 mM PB buffer. The ligand of UC NPs purchased from Beijing Oneder-Hightech Co. Ltd. was PEG2000 bearing a maleimide group at one end and two phosphate groups at the other end ( Fig. S1b, Liu et al., 2013). The maleimide groups on the surface of UC NPs allowed further modification. The UC NPs were diluted using 10 mMpH 7.2 Tris– HCl buffer to a final concentration of 50 pM. TB-Apt C (100 μM) was added to the UC NPs solution to a final concentration of
3. Results and discussion 3.1. Establishment of fluorescence “off-on” switch for thrombin detection As shown in Scheme 1, thrombin-aptamer (TB-Apt)-modified gold nanoparticles (AuNPs) were mixed with thrombin-aptamer complementary (TB-Apt C)-modified UC NPs, and the UC satellites assembly with Au cores led to core-satellites architecture, and the fluorescence of UC NPs was quenched. However, when thrombin was added to the system, the aptamer on the surface of AuNPs binding to the target then released the UCNPs thus the quenched fluorescence recovered, and a fluorescence “off-on” switch for thrombin determination was obtained. During the assembly process, the fluorescence intensity was monitored and the corresponding assembly product was characterized by TEM. TEM images at different hybridization times (1, 2, 4 and 8 h) are shown in Fig. S2. The number of UC satellites around the Au core increased with prolonged hybridization time. Two layers of UC NPs can be observed around some Au NPs (Fig. S2b, d), which may be formed from the collapse of three dimensional structure of core-satellite during the drying in the sample preparation process for TEM characterization. The assembly of Au core-UC satellites took 8 h and a yield of up to 80% was achieved. The UC NPs showed three fluorescence peaks. The peak at 543 nm was chosen as the signal peak, because it was nearest the plasmonic band of AuNPs which exhibited excellent sensitivity.
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X. Zhao et al. / Biosensors and Bioelectronics 70 (2015) 372–375
Scheme 1. Scheme of TB detection with Au core-UC satellites assemblies.
The attachment of UC satellites to the Au core turned off the UC fluorescence, and the addition of thrombin then turn on the fluorescence. The TB-Apt C modified on the UC NPs was not the full complementary oligonucleotide of TB-Apt, which provided the chance for thrombin to compete with the UC NPs. For quantification of thrombin, a standard solution was added to the assembly system at five final concentrations, 10, 20, 50, 100 and 1000 fg/mL. The solution was incubated at 37 °C for 30 min. From Fig. 1a to f, it can be seen that an increasing number of attached satellites escaped from the Au core. The higher the concentration of target, the lower the degree Au core-UC satellites assembly. The spectra of the recovered UC fluorescence were obtained using a F-7000 fluorescence spectrophotometer with a 980 nm laser (Fig. 2a). Calibration curves were acquired by plotting FL543 (the mean value of five parallel tests) as a logarithmic function of thrombin concentration, which exhibited a good linear range from 10 to 1000 fg/mL with a linear relationship of R2 ¼0.997 (Fig. 2b). The limit of detection (LOD ¼3.3 SD/S, where SD is the standard deviation of the result and S is the slope of the calibration curve, each point was repeated for five times to calculate the mean value as the final data point for calibration curve.) of thrombin was calculated to be 3.5 fg/mL, lower than a previously reported fluorescence sensor (Chen et al., 2013; Kim and Lee, 2014; Yuan et al., 2014; Zheng et al., 2012). Compared with other sensors, complex operation procedures were avoided and a lower limit of detection was obtained (Table S1). The high affinity and specificity of aptamer, good quenching efficiency of the Au core for the up-conversion nanoparticle and wonderful fluorescence recovery when targets exist contributed to the ultrasensitivity of the sensor. Also, the partially complementary sequence on UC particle made the structure more sensitive for thrombin. 3.2. Selectivity of the sensor
Fig. 1. TEM images of Au core-UC satellites assemblies in the existence of different concentrations of TB: (a) 0 fg/mL, (b) 10 fg/mL, (c) 20 fg/mL, (d) 50 fg/mL, (e) 100 fg/mL, (f) 1000 fg/mL. Scale bar was 50 nm.
Control experiments where the AuNPs and UC NPs without DNA functionalization were mixed at the same molar ratio as the hybridization system were performed. The changes in fluorescence intensity after the UC NPs and AuNPs were mixed are shown in Fig. S3. The quenching efficiency of AuNPs in the fluorescence of UC NPs was weak and the fluorescence was almost the same at 12 h. However, the fluorescence intensity at 543 nm (FL543) during fabrication, decreased from 1290 to 320 in 8 h (Fig. S4) with a quenching efficiency of 75.2%. The distance between Au and UC was 8.5 nm (25 bp), and the quenching effect was notable.
As shown in Fig. S5, only the addition of thrombin or thrombin and the mixture of five other substances enhanced the fluorescence with a thrombin concentration as low as 1 pg/mL, while the others showed almost no difference from the original sensor denoted as blank. These results indicated that the sensor showed good specificity for thrombin determination. 3.3. Recovery of thrombin spiked in Human serum The fluorescence spectra of thrombin in the recovery experiment are shown in Fig. S6. Five parallel tests for each spiked concentration were carried out to achieve an average as the final result (Table S2). The recovery was between 87% and 109% which suggested high capability of the Au core-UC satellite based fluorescence sensor in a complex matrix.
Fig. 2. TB detection based on fluorescence recovery with the disassembly of Au core-UC satellites. Fluorescence spectra (a) for different concentrations of TB and the calibration curve (b) for TB detection.
X. Zhao et al. / Biosensors and Bioelectronics 70 (2015) 372–375
4. Conclusions In summary, we constructed an up-conversion fluorescent sensor based on Au core-UC satellites for the detection of thrombin. The selectivity of the sensor for thrombin was demonstrated and a limit of detection of 3.5 fg/mL was achieved. The application of this sensor in the spiked human serum recovery test indicated good potential for disease diagnosis.
Acknowledgments This work is financially supported by the National Natural Science Foundation of China (21471068, 31400848), the Key Programs from MOST (2012YQ09019410, 2012BAD29B05), and grants from the Natural Science Foundation of Jiangsu Province, MOF and MOE (BE2013613, BE2013611).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.03.068.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Up-conversion fluorescence “off-on” switch based on heterogeneous core-satellite assembly for thrombin detection Xueli Zhao, Si Li, Liguang Xu n, Wei Ma, Xiaoling Wu, Hua Kuang, Libing Wang, Chuanlai Xu State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 11 February 2015 Received in revised form 21 March 2015 Accepted 26 March 2015 Available online 27 March 2015
NaGdF4: Yb, Er nanoparticles, with up-conversion (UC) fluorescence, were used for the first time to build an “off-on” switch based on Au core-UC satellites for thrombin detection. We fabricated the fluorescence sensor using thrombin aptamer modified Au core and complementary sequence modified UC satellites in liquid phase. With optimized assembled conditions, the yield of Au core-UC satellites achieved 80%. The fluorescence of UC nanoparticles quenched when satellite NP attached to Au core NP. Thrombin aptamer on the surface of Au core would bind to targets when thrombin existed in the system, then UC satellites were released and the quenched fluorescence recovered. The sensor showed high specificity for thrombin compared with other biomolecules and the limit of detection reached 3.5 fg/mL. Application of this sensor to detect targets in human serum also achieved satisfactory results. The purpose of this work was to build an ultrasensitive sensor based on Au core-UC satellites for thrombin detection in human serum to achieve diagnosis of diseases. & 2015 Elsevier B.V. All rights reserved.
Keywords: Up-conversion fluorescence Core-satellites Thrombin Aptamer sensor Detection
1. Introduction Thrombin, generated in plasma by the conversion of its precursor, prothrombin, is involved in thrombosis and platelet activation, and plays a significant role in a number of cardiovascular diseases such as cerebral ischemia and infarction (Centi et al., 2007). Thrombin usually leads to vasospasm following subarachnoid hemorrhage. Blood flowing out from ruptured cerebral aneurysm clots around a cerebral artery, releasing thrombin. This can induce an acute and prolonged narrowing of the blood vessel, which might cause cerebral ischemia and infarction (stroke). Thrombin is not present in the blood of healthy individuals, but can be detected in the blood of patients suffering from diseases associated with coagulation abnormalities (Centi et al., 2007; Tennico et al., 2010). Therefore, the detection of thrombin is vital for the diagnosis of related diseases. An aptamer is a single oligonucleotide which is completely engineered in an in vitro selection process, and has high affinity for targets and good stability and storage properties compared to antibodies (Cho et al., 2008). Since the thrombin-binding aptamer (15-mer, 5ʹ-GGTTGGTGTGGTTGG-3ʹ) was selected (Daniel et al., 2013; Li et al., 2008), numerous aptasensors (Chen et al., 2010b; Pavlov et al., 2004; Wang and Liu, 2009; Xiao et al., 2005) have n
Corresponding author. E-mail address: [email protected] (L. Xu).
http://dx.doi.org/10.1016/j.bios.2015.03.068 0956-5663/& 2015 Elsevier B.V. All rights reserved.
been fabricated based on the conformational changes induced by target binding, signals used for quantification ranged from color (Chen et al., 2010a; Chen et al., 2014b; Li et al., 2008; Pavlov et al., 2004), fluorescence (Chang et al., 2010; Chi et al., 2011; Jie and Yuan, 2012; Kim and Lee, 2014; Kong et al., 2013; Wang et al., 2011b; Yan et al., 2011) and electrochemiluminescence (Chen et al., 2014a; Deng et al., 2014; Shan et al., 2011; Wang et al., 2011a) to surface enhanced Raman scattering (SERS) (Z. Wu et al., 2013) and surface plasmon resonance (SPR) (Bai et al., 2013; He et al., 2014; Mani et al., 2011). These methods have the disadvantages of either a high limit of detection or complex operation procedures and limit their application in blood samples (Cho et al., 2008). The aim of this study was to design a fluorescence aptasensor with high sensitivity and the ability to resist interference in a matrix such as human serum. The sensor was based on the signal produced by the assembly and disassembly of gold core and upconversion nanoparticle (UC NP) satellites. Compared with normal fluorescent materials, the fluorescence and chemical properties of UC NPs were more stable, the excitation of 980 nm can avoid the interference of auto fluorescence of biomolecules, such as protein (Wen et al., 2013; H. Wu et al., 2013; Zhang et al., 2014). The low biological toxicity of this sensor also demonstrated significant potential in biological applications such as cancer diagnosis and treatment (Cheng et al., 2013; Li et al., 2013,, 2014; Maji et al., 2014).
X. Zhao et al. / Biosensors and Bioelectronics 70 (2015) 372–375
2. Material and methods 2.1. Materials and regents The thiol-modified oligonucleotides (thrombin aptamer (TBApt) and its complementary sequence (TB-Apt C)) (purity 4 95%) used in this study were manufactured by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., China. These oligonucleotides were dissolved in TE buffer (10 mM Tris–hydrochloride buffer, pH 8.0, containing 1.0 mM EDTA, Shanghai Sangon) to give a final concentration of 100 μM. Human α-thrombin (TB), bovine serum albumin (BSA), human prostate-specific antigen (PSA), serum albumin (HSA), immunoglobulin G (IgG), and L-cysteine (L-cys) were purchased from Sigma-Aldrich. Up-conversion nanoparticles (UC NPs) were purchased from Beijing Oneder-Hightech Co. Ltd., China. Deionized (DI) water, obtained using a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France), was used throughout this work. The detailed sequences of the oligonucleotides are as follows: TB-Apt: 5ʹ-HS-TTTTTGGTTGGTGTGGTTGG-3ʹ TB- Apt C: 5ʹ-HS-TTTTTTTAATTATATTAACC-3ʹ
373
0.5 nM. A NaNO3 solution of 5 M in water was added to the former solution to produce a NaNO3 concentration of 150 mM. The solution was left for 2 h to complete the modification of TB-Apt C to UC NPs. The modified UC NPs underwent ultrafiltration twice to remove excess DNA. 2.5. Assembly of the Au CORE-UC satellite sensor To assemble the Au core-UC satellite nanostructure, purified TB-Apt-modified AuNPs were mixed with TB-Apt C-modified UC NPs at a 10:1 M excess relative to Au cores in hybridization buffer (10 mM pH 7.2 Tris–HCl). Particles were incubated at room temperature for 8 h. 2.6. Selectivity of the sensor The selectivity of the as-fabricated sensor was evaluated by analyzing its selectivity for bovine serum albumin (BSA), human prostate-specific antigen (PSA), serum albumin (HSA), immunoglobulin G (IgG), and L-cysteine (L-cys). These proteins and amino acid were added to the sensor at a concentration of 1 ng/mL, and the operation procedures were the same as those for thrombin detection.
2.2. Instrumentation
2.7. Recovery test of thrombin in Human serum
Fluorescence spectra were acquired using a F-7000 fluorescence spectrophotometer, scan speed was 240 nm/min. Transmission electron microscopy images were acquired using a JEOL JEM-2100 operating at an acceleration voltage of 200 kV.
As serum is a complex biological matrix containing a wide variety of biomolecules, but no coagulation proteins such as thrombin, human serum was spiked with thrombin in the recovery test for further application. Human serum (obtained from Wuxi No. 2 People's Hospital, All the experiments were performed in compliance with the relevant laws and institutional guidelines, and the experiments were approved by the Ethics Committee of No. 2 People's Hospital) was diluted ten times with 10 mM Tris– HCl buffer (pH 7.2) as the analysis medium. Three concentrations of thrombin in the linear range (10, 20, 100 fg/mL) were spiked for monitoring.
2.3. Synthesis of gold nanoparticles Gold nanoparticles (327 1 nm) were synthesized by a seedmediated growth method. Seeds (Au NPs of 13 71 nm) were synthesized by a routine method: 2 mL of 38.8 mM trisodium citrate was quickly added to a boiling solution of HAuCl4 (40 mL, 0.5 mM) with vigorous stirring and refluxed until there were no more color changes in the solution. Then, 7.5 mL of 5.3 mM ascorbic acid was injected into the mixed solution (2 mL–10 mM HAuCl4, 0.1 mL–10 mM AgNO3, 42.5 mL H2O, 4 mL seeds) with a constant-flow pump at a speed of 0.6 mL/min under vigorous stirring. A BPS (bis(p-sulfonatophenyl) phenylphosphine dihydrate, dipotassium salt) solution was added to the as-synthesized Au NPs to a final concentration of 2.5 mg/mL and stirred in darkness for 12 h. TEM image of Au NPs was shown in Fig. S1a. 2.4. Oligonucleotide-functionalization of AuNPs and UC NPs Au NPs were modified with thiolated TB-Apt using a previously reported method (Rosi et al., 2006). AuNPs (327 1 nm) were suspended in 10 mM PB (pH 7.2) and 0.01% Tween20. TB-Apt (dissolved in TE buffer) was added to the AuNPs at a final ratio of 500 oligonucleotides per particle. The mixed solution was left for 2 h, a NaCl solution of 5 M in water was added (1 μL NaCl solution to 100 μL solution) with an interval of 30 min to bring the final concentration of NaCl to 400 mM at room temperature. The TBApt-modified AuNPs were washed three times with 10 mM PB buffer. The ligand of UC NPs purchased from Beijing Oneder-Hightech Co. Ltd. was PEG2000 bearing a maleimide group at one end and two phosphate groups at the other end ( Fig. S1b, Liu et al., 2013). The maleimide groups on the surface of UC NPs allowed further modification. The UC NPs were diluted using 10 mMpH 7.2 Tris– HCl buffer to a final concentration of 50 pM. TB-Apt C (100 μM) was added to the UC NPs solution to a final concentration of
3. Results and discussion 3.1. Establishment of fluorescence “off-on” switch for thrombin detection As shown in Scheme 1, thrombin-aptamer (TB-Apt)-modified gold nanoparticles (AuNPs) were mixed with thrombin-aptamer complementary (TB-Apt C)-modified UC NPs, and the UC satellites assembly with Au cores led to core-satellites architecture, and the fluorescence of UC NPs was quenched. However, when thrombin was added to the system, the aptamer on the surface of AuNPs binding to the target then released the UCNPs thus the quenched fluorescence recovered, and a fluorescence “off-on” switch for thrombin determination was obtained. During the assembly process, the fluorescence intensity was monitored and the corresponding assembly product was characterized by TEM. TEM images at different hybridization times (1, 2, 4 and 8 h) are shown in Fig. S2. The number of UC satellites around the Au core increased with prolonged hybridization time. Two layers of UC NPs can be observed around some Au NPs (Fig. S2b, d), which may be formed from the collapse of three dimensional structure of core-satellite during the drying in the sample preparation process for TEM characterization. The assembly of Au core-UC satellites took 8 h and a yield of up to 80% was achieved. The UC NPs showed three fluorescence peaks. The peak at 543 nm was chosen as the signal peak, because it was nearest the plasmonic band of AuNPs which exhibited excellent sensitivity.
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Scheme 1. Scheme of TB detection with Au core-UC satellites assemblies.
The attachment of UC satellites to the Au core turned off the UC fluorescence, and the addition of thrombin then turn on the fluorescence. The TB-Apt C modified on the UC NPs was not the full complementary oligonucleotide of TB-Apt, which provided the chance for thrombin to compete with the UC NPs. For quantification of thrombin, a standard solution was added to the assembly system at five final concentrations, 10, 20, 50, 100 and 1000 fg/mL. The solution was incubated at 37 °C for 30 min. From Fig. 1a to f, it can be seen that an increasing number of attached satellites escaped from the Au core. The higher the concentration of target, the lower the degree Au core-UC satellites assembly. The spectra of the recovered UC fluorescence were obtained using a F-7000 fluorescence spectrophotometer with a 980 nm laser (Fig. 2a). Calibration curves were acquired by plotting FL543 (the mean value of five parallel tests) as a logarithmic function of thrombin concentration, which exhibited a good linear range from 10 to 1000 fg/mL with a linear relationship of R2 ¼0.997 (Fig. 2b). The limit of detection (LOD ¼3.3 SD/S, where SD is the standard deviation of the result and S is the slope of the calibration curve, each point was repeated for five times to calculate the mean value as the final data point for calibration curve.) of thrombin was calculated to be 3.5 fg/mL, lower than a previously reported fluorescence sensor (Chen et al., 2013; Kim and Lee, 2014; Yuan et al., 2014; Zheng et al., 2012). Compared with other sensors, complex operation procedures were avoided and a lower limit of detection was obtained (Table S1). The high affinity and specificity of aptamer, good quenching efficiency of the Au core for the up-conversion nanoparticle and wonderful fluorescence recovery when targets exist contributed to the ultrasensitivity of the sensor. Also, the partially complementary sequence on UC particle made the structure more sensitive for thrombin. 3.2. Selectivity of the sensor
Fig. 1. TEM images of Au core-UC satellites assemblies in the existence of different concentrations of TB: (a) 0 fg/mL, (b) 10 fg/mL, (c) 20 fg/mL, (d) 50 fg/mL, (e) 100 fg/mL, (f) 1000 fg/mL. Scale bar was 50 nm.
Control experiments where the AuNPs and UC NPs without DNA functionalization were mixed at the same molar ratio as the hybridization system were performed. The changes in fluorescence intensity after the UC NPs and AuNPs were mixed are shown in Fig. S3. The quenching efficiency of AuNPs in the fluorescence of UC NPs was weak and the fluorescence was almost the same at 12 h. However, the fluorescence intensity at 543 nm (FL543) during fabrication, decreased from 1290 to 320 in 8 h (Fig. S4) with a quenching efficiency of 75.2%. The distance between Au and UC was 8.5 nm (25 bp), and the quenching effect was notable.
As shown in Fig. S5, only the addition of thrombin or thrombin and the mixture of five other substances enhanced the fluorescence with a thrombin concentration as low as 1 pg/mL, while the others showed almost no difference from the original sensor denoted as blank. These results indicated that the sensor showed good specificity for thrombin determination. 3.3. Recovery of thrombin spiked in Human serum The fluorescence spectra of thrombin in the recovery experiment are shown in Fig. S6. Five parallel tests for each spiked concentration were carried out to achieve an average as the final result (Table S2). The recovery was between 87% and 109% which suggested high capability of the Au core-UC satellite based fluorescence sensor in a complex matrix.
Fig. 2. TB detection based on fluorescence recovery with the disassembly of Au core-UC satellites. Fluorescence spectra (a) for different concentrations of TB and the calibration curve (b) for TB detection.
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4. Conclusions In summary, we constructed an up-conversion fluorescent sensor based on Au core-UC satellites for the detection of thrombin. The selectivity of the sensor for thrombin was demonstrated and a limit of detection of 3.5 fg/mL was achieved. The application of this sensor in the spiked human serum recovery test indicated good potential for disease diagnosis.
Acknowledgments This work is financially supported by the National Natural Science Foundation of China (21471068, 31400848), the Key Programs from MOST (2012YQ09019410, 2012BAD29B05), and grants from the Natural Science Foundation of Jiangsu Province, MOF and MOE (BE2013613, BE2013611).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.03.068.
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