Research article Received: 16 February 2015,
Revised: 27 April 2015,
Accepted: 5 May 2015
Published online in Wiley Online Library: 29 May 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3508
Dual-signal amplification strategy for ultrasensitive chemiluminescence detection of PDGF–BB in capillary electrophoresis Jun-Tao Caoa, Hui Wanga, Shu-Wei Renb, Yong-Hong Chenb and Yan-Ming Liua* ABSTRACT: Many efforts have been made toward the achievement of high sensitivity in capillary electrophoresis coupled with chemiluminescence detection (CE-CL). This work describes a novel dual-signal amplification strategy for highly specific and ultrasensitive CL detection of human platelet-derived growth factor–BB (PDGF–BB) using both aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (HRP–AuNPs–aptamer) as nanoprobes in CE. Both AuNPs and HRP in the nanoprobes could amplify the CL signals in the luminol–H2O2 CL system, owing to the excellent catalytic behavior of AuNPs and HRP in the CL system. Meanwhile, the high affinity of aptamer modified on the AuNPs allows detection with high specificity. As proof-of-concept, the proposed method was employed to quantify the concentration of PDGF–BB from 0.50 to 250 fm with a detection limit of 0.21 fm. The applicability of the assay was further demonstrated in the analysis of PDGF–BB in human serum samples with acceptable accuracy and reliability. The result of this study exhibits distinct advantages, such as high sensitivity, good specificity, simplicity, and very small sample consumption. The good performances of the proposed strategy provide a powerful avenue for ultrasensitive detection of rare proteins in biological sample, showing great promise in biochemical analysis. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: dual signal amplification; HRP-AuNPs-aptamer; capillary electrophoresis; chemiluminescence; PDGF-BB
Introduction
1866
Capillary electrophoresis (CE), with the features of high separation efficiency, short analysis time, low sample consumption and flexibility to develop assays using various modes and format, has been applied in the analysis of various analytes primarily including nucleic acids (Choi et al., 2012), proteins (Zhu et al., 2014), carbohydrates (Szabo et al., 2012), pharmaceuticals (Chang et al., 2013a), metal ions (Liu et al., 2001), etc. Among various techniques, CE-based immunoassay has become one of the most important techniques in clinical diagnosis and biochemical analysis since 1991 (Nielsen et al., 1991). However, the antibody adopted in the immunoassay is always temperature-sensitive and irreversibly denatured, and has a limited shelf life. Therefore, introduction of new recognition elements into CE system is an attractive topic. Aptamers have attracted greater interest than antibodies because of their many advantages including high specificity, affinity, nontoxicity, good stability and relatively easy preparation (Ellington and Szostak, 1990). Until now, probe functionalized aptamers have been selected for targeting various analytes such as cancer cells (Zhang et al., 2013b), tumor markers (Cha et al., 2014) and metal ions (Zhang et al., 2013a). Although the aptamer, as an effective recognition element, has been investigated extensively in the construction of various biosensors for electrochemical (Miodek et al., 2013), photoelectrochemical (Zhao et al., 2014), electrochemiluminescent (Li et al., 2013), fluorescent (Müller and König, 2014) and colorimetric analysis (Geng et al., 2014), the CE-based aptamer assay is still in its infancy. On the other hand, the small detection volume in CE often results in poor concentration detection sensitivity; achieving high sensitivity has long been a major goal in
Biomed. Chromatogr. 2015; 29: 1866–1870
CE, especially in the monitoring of rare analytes in biological samples. Therefore, of particular interest here is the possibility of using innovative CE-based aptamer assay formats coupled with simple and ingenious amplification systems to realize ultrasensitive protein detection. Chemiluminescence detection (CL), one of the most sensitive methods of detection, has been proved to be an effective tool in CE analysis (Wang et al., 2010; Zhou et al., 2015; García-Campaña et al., 2009). Nanomaterials, especially noble metal nanoparticles with excellent catalytic activity and conductivity, large surface area, and good biocompatibility, not only could facilitate the immobilization of enzyme and proteins, but also could catalyze some CL reactions (Li et al., 2011; Zhang and Cui, 2014; Huang and Ren, 2012; Qin et al., 2013; Liu et al., 2011). Noble metal nanoparticles serving as signal labels with excellent catalytic behavior have been certified to be applicable in CE-CL based aptamer assays in our previous work (Liu et al., 2014a). With the merits of combining aptamer and Au nanoparticles (AuNPs), the aptamer nanomaterial
* Correspondence to: Y.-M. Liu, College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China. Email: [email protected] a
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China
b
Xinyang Central Hospital, Xinyang 464000, China Abbreviations used: AuNPs, gold nanoparticles; BSA, bovine serum albumin; CE, capillary electrophoresis; CL, chemiluminescence; DN, diabetic nephropathy; HRP, horseradish peroxidase; PBS, phosphate buffer saline; PDGF–BB, human platelet-derived growth factor–BB; PMT, photomultiplier tube.
Copyright © 2015 John Wiley & Sons, Ltd.
Dual-signal amplification strategy in CE-CL for PDGF-BB detection conjugation used in CE-CL should be a brilliant prospect for the detection of disease related markers. It is well known that horseradish peroxidase (HRP) could obviously enhance the CL intensity of a luminol–H2O2 system in alkaline solution. Using HRP-labeled antibody as labels, the sensitive detection of various proteins, such as carcinoembryonic antigen ( Jiang et al., 2013), CA-125 (Wang and Ren, 2005), α-fetoprotein (Liu et al., 2007) and bone morhogenic protein-2 (Wang et al., 2004), has been accomplished in CE-CL. These reported works demonstrate that HRP is an effective signal amplifier in the CE-CL system. Accordingly, the appropriate combination of HRP with AuNPs might present a synergetic effect of signal amplification in CE-CL. In this work, we developed an ultrasensitive CL method for human platelet-derived growth factor–BB (PDGF–BB) detection in CE based on a dual signal amplification strategy. AuNPs were modified with HRP and SH-aptamer to form an HRP–AuNPs–aptamer nanoprobe, which is used to specifically recognize the target protein and enhance the detection sensitivity. PDGF–BB, one of potent growth factors, which plays important role in blood vessel formation, embryonic development and cell proliferation, was selected as a model protein. The detection of femtomolar-level PDGF–BB was achieved in CE-CL with our newly developed strategy. The proposed method was applied to the determination of PDGF–BB in human serum samples.
Experimental Materials and reagents PDGF–BB was purchased from Peprotech (USA). The aptamer [5′-SH(CH2)6-TTT TTT TTT TCA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG-3′] for PDGF–BB was synthesized by Shanghai Sangon Biotechnology Co. Ltd (Shanghai, China). Luminol, HRP and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St Louis, MO, USA). The stock solutions of the aptamer and the proteins were prepared with 10 mM pH 7.4 phosphate buffer saline (PBS, containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 8.7 mM Na2HPO4 and 1.4 mM KH2PO4), from which lower concentrations of samples were obtained by serial dilution. AuNPs with an average size of 35 nm were prepared by citrate reduction of HAuCl4 in aqueous solution (Enustun and Turkevich, 1963). All aqueous solutions were prepared using ultrapure water (Kangning Water Treatment Solution Provider, China).
Apparatus and characterization The CE-CL analysis was performed with a homemade CE-CL system as described previously (Liu et al., 2007). Briefly, a DC power supply (0–30 kV, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) was used to drive the electrophoresis. A 50 cm × 75 μm i.d. uncoated fused capillary (Hebei Yongnian Ruifeng Chromatographic Apparatus, China) was used as the separation capillary. After burning off and removing the polyimide on a 5 cm end section of the separation capillary, this end section was etched with 40% HF for 2.5 h and then inserted into a 20 cm × 530 μm i.d. reaction capillary. This transparent section of the separation capillary was located at the detection window by burning 1 cm of the polyimide of the reaction capillary and placed in front of the photomultiplier tube (PMT, CR-135, Binsong Photonics, Beijing, China). The PMT was equipped with a BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China). The post-column CL reagents were siphoned into a tee reservoir with the pair of 40 cm × 320 μm i.d. capillaries. The ground electrode was also put into one joint of the tee. Then the CL reagents mixed and flowed down coaxially along the reaction capillary. When the analytes eluted out from the separation capillary, the CL reaction occurred. The CL signal was collected by the PMT and processed with a computer using BPCL software. The UV–vis spectra were recorded by an UVmini-1240UV-vis spectrophotometer ( Japan). The CL spectra were obtained by a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Walnut Creek).
Preparation of HRP–AuNPs–aptamer nanoprobes AuNPs were coated with aptamer and HRP according to the following method. Before conjugation, the pH of the AuNPs solution (1 mL, 10 3.2 × 10 M) was adjusted to 8.2 with K2CO3. Subsequently, 3.0 μL of HRP (10 μM) and 1.5 μL of aptamer (10 μM) were added into the above solution. The mixture was then kept for 4 h at room temperature under gentle shaking, blocked by 0.1% BSA for 30 min, and thereafter centrifuged at 10,000 rpm for 15 min. After the supernatant was discarded, the oiled drop was washed with 10 mM PBS (pH 7.4) containing 0.05% (v/v) Tween 20 and 0.1% BSA, recentrifuged and the resulting HRP–AuNPs–aptamer conjugates were resuspended in the PBS containing 0.05% (v/v) Tween 20 and 0.1% BSA and stored at 4 °C until use. The preparation process of the HRP– AuNPs–aptamer nanoprobes is shown in Scheme 1(A).
Conjugation of HRP–AuNPs–aptamer nanoprobes with PDGF–BB Aliquots of 10 μL HRP–AuNPs–aptamer nanoprobes were added into 10 μL PDGF–BB at a certain concentration. After gentle shaking for 30 min at 37 °C, the mixture was diluted with 20 μL 10 mM Tris–HCl (pH 7.4, containing
Biomed. Chromatogr. 2015; 29: 1866–1870
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1867
Scheme 1. (A) Fabrication procedure of the horseradish peroxidase (HRP) modified gold nanoparticles (HRP–AuNPs–aptamer) nanoprobe and (B) scheme illustration of the CE-CL method for platelet-derived growth factor–BB (PDGF–BB) detection based on the dual signal amplification strategy.
J.-T. Cao et al. 140 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1 mM CaCl2) and subsequently analyzed by CE-CL. The procedure is illustrated in Scheme 1 (B).
Capillary treatment and electrophoresis procedure The new separation capillary was treated sequentially with 2.0 M CH3OH– NaOH, 1.0 M NaOH, 1.0 M HCl, and H2O for 20 min, and finally with electrophoretic buffer for 20 min. At the beginning of each day, the capillary was reconditioned with 0.1 M NaOH and water and equilibrated with the electrophoretic buffer for 3 min successively to maintain an active and reproducible inner surface. After three consecutive injections, the capillary was rinsed sequentially with 0.1 M NaOH, H2O, and electrophoretic buffer for 2 min. A 25 mM Tris–HCl solution at pH 10.0 containing 0.40 mM luminol and 0.09% Tween 20 was used as electrophoretic buffer. Solutions of 50 mM H2O2 and 30 mM pH 11.0 NaHCO3 introduced from two vials were used as the post-column CL reagents. The samples were introduced by electrokinetic injections at 12 kV for 6 s and separated at 22 kV.
HRP is widely used as antibody label reagent to catalyze the luminol–H2O2 system. It can be estimated that the co-catalysis of HRP and AuNPs would produce dual signal amplification in the luminol–H2O2 CL system. To explore this synergetic effect, series experiments were designed and the results are depicted in Fig. 2. It can be seen that the mixture of luminol and H2O2 produced weak CL (curve a), whereas the CL intensity was enhanced in the presence of AuNPs (curve b) or HRP (curve c), indicating the effective catalytic effect of AuNPs and HRP. The maximum CL emission was observed in the presence of luminol, H2O2, AuNPs and HRP (curve d), indicative of the cosensitized effect of the two catalysts on the system. Although the CL emission decreased in the presence of HRP–AuNPs–aptamer nanoprobes (curve e) compared with curve d, the dual signal amplification was still achieved. Optimization of CL detection
Preparation of human serum samples Human blood samples from four healthy volunteers and three patient volunteers with diabetic nephropathy (DN) were kindly provided by Xinyang Central Hospital (Xinyang, China). The serum samples were separated from the fresh blood samples by centrifugation at 1000 rpm for 15 min. These serum samples were stored at 20 °C before use. The peak areas were used for quantification.
Results and discussion
To achieve high sensitivity, the concentration of luminol, H2O2 and NaHCO3 and the pH of NaHCO3 need to be optimized. To screen the proper concentrations of luminol and H2O2, concentrations of luminol ranging from 0.2 to 0.7 mM, and H2O2 from 20 to 70 mM were tested. The maximum CL responses were obtained with 0.4 mM luminol and 50 mM H2O2. Next, the effects of pH and concentration of NaHCO3 were investigated. The results show that the optimum concentration and pH are 30 mM and pH 11.0, respectively.
Characterization of the HRP–AuNPs–aptamer nanoprobe
Optimization of CE procedure
Figure 1 presents the UV spectra of AuNPs (curve a) and HRP– AuNPs–aptamer (curve b). Compared with the bare AuNPs with a strong absorption at 526 nm, the absorption peak of AuNPs after modification with HRP and aptamer red shifted, owing to the decoration of biomolecules on the AuNPs. The results demonstrate the effect of the HRP–AuNPs–aptamer nanoprobes.
The effective separation of HRP–AuNPs–aptamer and HRP– AuNPs–aptamer–PDGF–BB complex is a critical step in the present work. The influence of the parameters such as the concentration and pH of electrophoretic buffer, concentration of surfactant and separation voltage on the resolution (R) and the CL intensity was examined. A solution of 25 mM Tris–HCl electrophoretic buffer at pH 10.0 produced the maximum CL intensity. To reduce the adsorption of the protein on the inner wall of capillary, Tween 20 was selected as an additive for dynamic coating of capillary wall and the concentration of Tween 20 was evaluated. The maximum resolution and CL intensity
The dual signal amplification strategy In recent years, the AuNPs has been substantiated as a perfect catalyst for the luminol CL system (Li et al., 2011; Zhang and Cui, 2014; Huang and Ren, 2012). In conventional CL immunoassay,
1868
Figure 1. UV–vis absorption spectra of the bare AuNPs (a) and the HRP– AuNPs–aptamer (b).
wileyonlinelibrary.com/journal/bmc
Figure 2. CL spectra of luminol + H2O2 (a), luminol + H2O2 + AuNPs (b), luminol + H2O2 + HRP (c), luminol + H2O2 + AuNPs + HRP (d), and luminol + H2O2 + HRP–AuNPs–aptamer (e).
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1866–1870
Dual-signal amplification strategy in CE-CL for PDGF-BB detection were observed with 0.09% Tween 20. By weighing the CL intensity, resolution and migration time, 22 kV was selected as the optimized separation voltage.
Performance of HRP–AuNPs–aptamer in the CE-CL system To clarify the advantages of HRP–AuNPs–aptamer in the assay, AuNPs–aptamer was synthesized by the similar procedure for HRP–AuNPs–aptamer without addition of HRP. The AuNPs– aptamer and HRP–AuNPs–aptamer were injected into the CE-CL system under the optimum conditions. The typical electropherograms of AuNPs–aptamer and HRP–AuNPs–aptamer are shown in Fig. 3. In comparison with the AuNPs–aptamer, the CL intensity of the HRP–AuNPs–aptamer was enhanced. The dual amplification effect of the HRP–AuNPs–aptamer in CE-CL is in agreement with that observed in bulk solution by CL spectra.
Analytical performance
Figure 3. Electropherograms of AuNPs–aptamer (a) and HRP–AuNPs– aptamer (b). Conditions: electrophoretic buffer, 25 mM Tris and 0.09% Tween 20 at pH 10.0; the postcolumn CL reagents, 50 mM H2O2 and 30 mM pH 11.0 NaHCO3; electrokinetic injection with 12 kV for 6 s; separation voltage, 22 kV; separation capillary, 50 cm × 75 μm i.d.
On the basis of the optimal conditions, the free HRP–AuNPs– aptamer and the HRP–AuNPs–aptamer–PDGF–BB complex were well separated within 3 min (shown in Fig. 4). For the linearity and the limit of detection, a series of PDGF–BB solutions were tested. The results show that the peak area increased linearly with the increasing PDGF–BB concentrations in the range from 0.50 to 250 fM. The linear regression equation is A = 61,098 + 415C with a correlation coefficient of R = 0.994, where A is the peak area of HRP–AuNPs–aptamer-PDGF–BB complex and C is the concentration of PDGF–BB. The detection limit (signal-to-noise ratio = 3) of PDGF–BB was estimated to be 0.21 fM, which is
Revised: 27 April 2015,
Accepted: 5 May 2015
Published online in Wiley Online Library: 29 May 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3508
Dual-signal amplification strategy for ultrasensitive chemiluminescence detection of PDGF–BB in capillary electrophoresis Jun-Tao Caoa, Hui Wanga, Shu-Wei Renb, Yong-Hong Chenb and Yan-Ming Liua* ABSTRACT: Many efforts have been made toward the achievement of high sensitivity in capillary electrophoresis coupled with chemiluminescence detection (CE-CL). This work describes a novel dual-signal amplification strategy for highly specific and ultrasensitive CL detection of human platelet-derived growth factor–BB (PDGF–BB) using both aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (HRP–AuNPs–aptamer) as nanoprobes in CE. Both AuNPs and HRP in the nanoprobes could amplify the CL signals in the luminol–H2O2 CL system, owing to the excellent catalytic behavior of AuNPs and HRP in the CL system. Meanwhile, the high affinity of aptamer modified on the AuNPs allows detection with high specificity. As proof-of-concept, the proposed method was employed to quantify the concentration of PDGF–BB from 0.50 to 250 fm with a detection limit of 0.21 fm. The applicability of the assay was further demonstrated in the analysis of PDGF–BB in human serum samples with acceptable accuracy and reliability. The result of this study exhibits distinct advantages, such as high sensitivity, good specificity, simplicity, and very small sample consumption. The good performances of the proposed strategy provide a powerful avenue for ultrasensitive detection of rare proteins in biological sample, showing great promise in biochemical analysis. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: dual signal amplification; HRP-AuNPs-aptamer; capillary electrophoresis; chemiluminescence; PDGF-BB
Introduction
1866
Capillary electrophoresis (CE), with the features of high separation efficiency, short analysis time, low sample consumption and flexibility to develop assays using various modes and format, has been applied in the analysis of various analytes primarily including nucleic acids (Choi et al., 2012), proteins (Zhu et al., 2014), carbohydrates (Szabo et al., 2012), pharmaceuticals (Chang et al., 2013a), metal ions (Liu et al., 2001), etc. Among various techniques, CE-based immunoassay has become one of the most important techniques in clinical diagnosis and biochemical analysis since 1991 (Nielsen et al., 1991). However, the antibody adopted in the immunoassay is always temperature-sensitive and irreversibly denatured, and has a limited shelf life. Therefore, introduction of new recognition elements into CE system is an attractive topic. Aptamers have attracted greater interest than antibodies because of their many advantages including high specificity, affinity, nontoxicity, good stability and relatively easy preparation (Ellington and Szostak, 1990). Until now, probe functionalized aptamers have been selected for targeting various analytes such as cancer cells (Zhang et al., 2013b), tumor markers (Cha et al., 2014) and metal ions (Zhang et al., 2013a). Although the aptamer, as an effective recognition element, has been investigated extensively in the construction of various biosensors for electrochemical (Miodek et al., 2013), photoelectrochemical (Zhao et al., 2014), electrochemiluminescent (Li et al., 2013), fluorescent (Müller and König, 2014) and colorimetric analysis (Geng et al., 2014), the CE-based aptamer assay is still in its infancy. On the other hand, the small detection volume in CE often results in poor concentration detection sensitivity; achieving high sensitivity has long been a major goal in
Biomed. Chromatogr. 2015; 29: 1866–1870
CE, especially in the monitoring of rare analytes in biological samples. Therefore, of particular interest here is the possibility of using innovative CE-based aptamer assay formats coupled with simple and ingenious amplification systems to realize ultrasensitive protein detection. Chemiluminescence detection (CL), one of the most sensitive methods of detection, has been proved to be an effective tool in CE analysis (Wang et al., 2010; Zhou et al., 2015; García-Campaña et al., 2009). Nanomaterials, especially noble metal nanoparticles with excellent catalytic activity and conductivity, large surface area, and good biocompatibility, not only could facilitate the immobilization of enzyme and proteins, but also could catalyze some CL reactions (Li et al., 2011; Zhang and Cui, 2014; Huang and Ren, 2012; Qin et al., 2013; Liu et al., 2011). Noble metal nanoparticles serving as signal labels with excellent catalytic behavior have been certified to be applicable in CE-CL based aptamer assays in our previous work (Liu et al., 2014a). With the merits of combining aptamer and Au nanoparticles (AuNPs), the aptamer nanomaterial
* Correspondence to: Y.-M. Liu, College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China. Email: [email protected] a
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China
b
Xinyang Central Hospital, Xinyang 464000, China Abbreviations used: AuNPs, gold nanoparticles; BSA, bovine serum albumin; CE, capillary electrophoresis; CL, chemiluminescence; DN, diabetic nephropathy; HRP, horseradish peroxidase; PBS, phosphate buffer saline; PDGF–BB, human platelet-derived growth factor–BB; PMT, photomultiplier tube.
Copyright © 2015 John Wiley & Sons, Ltd.
Dual-signal amplification strategy in CE-CL for PDGF-BB detection conjugation used in CE-CL should be a brilliant prospect for the detection of disease related markers. It is well known that horseradish peroxidase (HRP) could obviously enhance the CL intensity of a luminol–H2O2 system in alkaline solution. Using HRP-labeled antibody as labels, the sensitive detection of various proteins, such as carcinoembryonic antigen ( Jiang et al., 2013), CA-125 (Wang and Ren, 2005), α-fetoprotein (Liu et al., 2007) and bone morhogenic protein-2 (Wang et al., 2004), has been accomplished in CE-CL. These reported works demonstrate that HRP is an effective signal amplifier in the CE-CL system. Accordingly, the appropriate combination of HRP with AuNPs might present a synergetic effect of signal amplification in CE-CL. In this work, we developed an ultrasensitive CL method for human platelet-derived growth factor–BB (PDGF–BB) detection in CE based on a dual signal amplification strategy. AuNPs were modified with HRP and SH-aptamer to form an HRP–AuNPs–aptamer nanoprobe, which is used to specifically recognize the target protein and enhance the detection sensitivity. PDGF–BB, one of potent growth factors, which plays important role in blood vessel formation, embryonic development and cell proliferation, was selected as a model protein. The detection of femtomolar-level PDGF–BB was achieved in CE-CL with our newly developed strategy. The proposed method was applied to the determination of PDGF–BB in human serum samples.
Experimental Materials and reagents PDGF–BB was purchased from Peprotech (USA). The aptamer [5′-SH(CH2)6-TTT TTT TTT TCA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG-3′] for PDGF–BB was synthesized by Shanghai Sangon Biotechnology Co. Ltd (Shanghai, China). Luminol, HRP and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St Louis, MO, USA). The stock solutions of the aptamer and the proteins were prepared with 10 mM pH 7.4 phosphate buffer saline (PBS, containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 8.7 mM Na2HPO4 and 1.4 mM KH2PO4), from which lower concentrations of samples were obtained by serial dilution. AuNPs with an average size of 35 nm were prepared by citrate reduction of HAuCl4 in aqueous solution (Enustun and Turkevich, 1963). All aqueous solutions were prepared using ultrapure water (Kangning Water Treatment Solution Provider, China).
Apparatus and characterization The CE-CL analysis was performed with a homemade CE-CL system as described previously (Liu et al., 2007). Briefly, a DC power supply (0–30 kV, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) was used to drive the electrophoresis. A 50 cm × 75 μm i.d. uncoated fused capillary (Hebei Yongnian Ruifeng Chromatographic Apparatus, China) was used as the separation capillary. After burning off and removing the polyimide on a 5 cm end section of the separation capillary, this end section was etched with 40% HF for 2.5 h and then inserted into a 20 cm × 530 μm i.d. reaction capillary. This transparent section of the separation capillary was located at the detection window by burning 1 cm of the polyimide of the reaction capillary and placed in front of the photomultiplier tube (PMT, CR-135, Binsong Photonics, Beijing, China). The PMT was equipped with a BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China). The post-column CL reagents were siphoned into a tee reservoir with the pair of 40 cm × 320 μm i.d. capillaries. The ground electrode was also put into one joint of the tee. Then the CL reagents mixed and flowed down coaxially along the reaction capillary. When the analytes eluted out from the separation capillary, the CL reaction occurred. The CL signal was collected by the PMT and processed with a computer using BPCL software. The UV–vis spectra were recorded by an UVmini-1240UV-vis spectrophotometer ( Japan). The CL spectra were obtained by a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Walnut Creek).
Preparation of HRP–AuNPs–aptamer nanoprobes AuNPs were coated with aptamer and HRP according to the following method. Before conjugation, the pH of the AuNPs solution (1 mL, 10 3.2 × 10 M) was adjusted to 8.2 with K2CO3. Subsequently, 3.0 μL of HRP (10 μM) and 1.5 μL of aptamer (10 μM) were added into the above solution. The mixture was then kept for 4 h at room temperature under gentle shaking, blocked by 0.1% BSA for 30 min, and thereafter centrifuged at 10,000 rpm for 15 min. After the supernatant was discarded, the oiled drop was washed with 10 mM PBS (pH 7.4) containing 0.05% (v/v) Tween 20 and 0.1% BSA, recentrifuged and the resulting HRP–AuNPs–aptamer conjugates were resuspended in the PBS containing 0.05% (v/v) Tween 20 and 0.1% BSA and stored at 4 °C until use. The preparation process of the HRP– AuNPs–aptamer nanoprobes is shown in Scheme 1(A).
Conjugation of HRP–AuNPs–aptamer nanoprobes with PDGF–BB Aliquots of 10 μL HRP–AuNPs–aptamer nanoprobes were added into 10 μL PDGF–BB at a certain concentration. After gentle shaking for 30 min at 37 °C, the mixture was diluted with 20 μL 10 mM Tris–HCl (pH 7.4, containing
Biomed. Chromatogr. 2015; 29: 1866–1870
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1867
Scheme 1. (A) Fabrication procedure of the horseradish peroxidase (HRP) modified gold nanoparticles (HRP–AuNPs–aptamer) nanoprobe and (B) scheme illustration of the CE-CL method for platelet-derived growth factor–BB (PDGF–BB) detection based on the dual signal amplification strategy.
J.-T. Cao et al. 140 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1 mM CaCl2) and subsequently analyzed by CE-CL. The procedure is illustrated in Scheme 1 (B).
Capillary treatment and electrophoresis procedure The new separation capillary was treated sequentially with 2.0 M CH3OH– NaOH, 1.0 M NaOH, 1.0 M HCl, and H2O for 20 min, and finally with electrophoretic buffer for 20 min. At the beginning of each day, the capillary was reconditioned with 0.1 M NaOH and water and equilibrated with the electrophoretic buffer for 3 min successively to maintain an active and reproducible inner surface. After three consecutive injections, the capillary was rinsed sequentially with 0.1 M NaOH, H2O, and electrophoretic buffer for 2 min. A 25 mM Tris–HCl solution at pH 10.0 containing 0.40 mM luminol and 0.09% Tween 20 was used as electrophoretic buffer. Solutions of 50 mM H2O2 and 30 mM pH 11.0 NaHCO3 introduced from two vials were used as the post-column CL reagents. The samples were introduced by electrokinetic injections at 12 kV for 6 s and separated at 22 kV.
HRP is widely used as antibody label reagent to catalyze the luminol–H2O2 system. It can be estimated that the co-catalysis of HRP and AuNPs would produce dual signal amplification in the luminol–H2O2 CL system. To explore this synergetic effect, series experiments were designed and the results are depicted in Fig. 2. It can be seen that the mixture of luminol and H2O2 produced weak CL (curve a), whereas the CL intensity was enhanced in the presence of AuNPs (curve b) or HRP (curve c), indicating the effective catalytic effect of AuNPs and HRP. The maximum CL emission was observed in the presence of luminol, H2O2, AuNPs and HRP (curve d), indicative of the cosensitized effect of the two catalysts on the system. Although the CL emission decreased in the presence of HRP–AuNPs–aptamer nanoprobes (curve e) compared with curve d, the dual signal amplification was still achieved. Optimization of CL detection
Preparation of human serum samples Human blood samples from four healthy volunteers and three patient volunteers with diabetic nephropathy (DN) were kindly provided by Xinyang Central Hospital (Xinyang, China). The serum samples were separated from the fresh blood samples by centrifugation at 1000 rpm for 15 min. These serum samples were stored at 20 °C before use. The peak areas were used for quantification.
Results and discussion
To achieve high sensitivity, the concentration of luminol, H2O2 and NaHCO3 and the pH of NaHCO3 need to be optimized. To screen the proper concentrations of luminol and H2O2, concentrations of luminol ranging from 0.2 to 0.7 mM, and H2O2 from 20 to 70 mM were tested. The maximum CL responses were obtained with 0.4 mM luminol and 50 mM H2O2. Next, the effects of pH and concentration of NaHCO3 were investigated. The results show that the optimum concentration and pH are 30 mM and pH 11.0, respectively.
Characterization of the HRP–AuNPs–aptamer nanoprobe
Optimization of CE procedure
Figure 1 presents the UV spectra of AuNPs (curve a) and HRP– AuNPs–aptamer (curve b). Compared with the bare AuNPs with a strong absorption at 526 nm, the absorption peak of AuNPs after modification with HRP and aptamer red shifted, owing to the decoration of biomolecules on the AuNPs. The results demonstrate the effect of the HRP–AuNPs–aptamer nanoprobes.
The effective separation of HRP–AuNPs–aptamer and HRP– AuNPs–aptamer–PDGF–BB complex is a critical step in the present work. The influence of the parameters such as the concentration and pH of electrophoretic buffer, concentration of surfactant and separation voltage on the resolution (R) and the CL intensity was examined. A solution of 25 mM Tris–HCl electrophoretic buffer at pH 10.0 produced the maximum CL intensity. To reduce the adsorption of the protein on the inner wall of capillary, Tween 20 was selected as an additive for dynamic coating of capillary wall and the concentration of Tween 20 was evaluated. The maximum resolution and CL intensity
The dual signal amplification strategy In recent years, the AuNPs has been substantiated as a perfect catalyst for the luminol CL system (Li et al., 2011; Zhang and Cui, 2014; Huang and Ren, 2012). In conventional CL immunoassay,
1868
Figure 1. UV–vis absorption spectra of the bare AuNPs (a) and the HRP– AuNPs–aptamer (b).
wileyonlinelibrary.com/journal/bmc
Figure 2. CL spectra of luminol + H2O2 (a), luminol + H2O2 + AuNPs (b), luminol + H2O2 + HRP (c), luminol + H2O2 + AuNPs + HRP (d), and luminol + H2O2 + HRP–AuNPs–aptamer (e).
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1866–1870
Dual-signal amplification strategy in CE-CL for PDGF-BB detection were observed with 0.09% Tween 20. By weighing the CL intensity, resolution and migration time, 22 kV was selected as the optimized separation voltage.
Performance of HRP–AuNPs–aptamer in the CE-CL system To clarify the advantages of HRP–AuNPs–aptamer in the assay, AuNPs–aptamer was synthesized by the similar procedure for HRP–AuNPs–aptamer without addition of HRP. The AuNPs– aptamer and HRP–AuNPs–aptamer were injected into the CE-CL system under the optimum conditions. The typical electropherograms of AuNPs–aptamer and HRP–AuNPs–aptamer are shown in Fig. 3. In comparison with the AuNPs–aptamer, the CL intensity of the HRP–AuNPs–aptamer was enhanced. The dual amplification effect of the HRP–AuNPs–aptamer in CE-CL is in agreement with that observed in bulk solution by CL spectra.
Analytical performance
Figure 3. Electropherograms of AuNPs–aptamer (a) and HRP–AuNPs– aptamer (b). Conditions: electrophoretic buffer, 25 mM Tris and 0.09% Tween 20 at pH 10.0; the postcolumn CL reagents, 50 mM H2O2 and 30 mM pH 11.0 NaHCO3; electrokinetic injection with 12 kV for 6 s; separation voltage, 22 kV; separation capillary, 50 cm × 75 μm i.d.
On the basis of the optimal conditions, the free HRP–AuNPs– aptamer and the HRP–AuNPs–aptamer–PDGF–BB complex were well separated within 3 min (shown in Fig. 4). For the linearity and the limit of detection, a series of PDGF–BB solutions were tested. The results show that the peak area increased linearly with the increasing PDGF–BB concentrations in the range from 0.50 to 250 fM. The linear regression equation is A = 61,098 + 415C with a correlation coefficient of R = 0.994, where A is the peak area of HRP–AuNPs–aptamer-PDGF–BB complex and C is the concentration of PDGF–BB. The detection limit (signal-to-noise ratio = 3) of PDGF–BB was estimated to be 0.21 fM, which is
