Biosensors and Bioelectronics 71 (2015) 164–170
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An electrochemiluminescence immunosensor for thyroid stimulating hormone based on polyamidoamine-norfloxacin functionalized Pd–Au core–shell hexoctahedrons as signal enhancers Yuting Liu, Qiqi Zhang, Haijun Wang, Yali Yuan, Yaqin Chai n, Ruo Yuan n Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2014 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online 11 April 2015
In this work, a novel polyamidoamine-norfloxacin (PAMAM-NFLX) complex and core–shell Pd–Au hexoctahedrons (Pd@Au HOHs) as enhancers are employed for development of a sensitive sandwich-type electrochemiluminescence (ECL) immunosensor to detect thyroid stimulating hormone (TSH). Here, norfloxacin (NFLX) is decorated abundantly on the surface of polyamidoamine (PAMAM) dendrimer via amide linkage to form PAMAM-NFLX complex. Thus, the resultant PAMAM-NFLX can serve as a novel coreactant to efficiently amplify the ECL signal of peroxydisulfate-oxygen (S2O82 -O2) system. Pd@Au HOHs were used as nano-carriers to assemble detection antibody (Ab2) and the PAMAM-NFLX complex. Besides, it can further enhance the ECL signal by promoting the generation of intermediate free radical HO during the ECL reaction of S2O82 -O2 system. The proposed immunosensor shows high sensitivity and specificity, and responds linearly to the concentration of TSH from 0.05 to 20 μIU mL 1 with a low detection limit of 0.02 μIU mL 1 (S/N¼3). Moreover, the immunosensor successfully achieves the detection of TSH in practical human blood serum with desirable results. & 2015 Published by Elsevier B.V.
Keywords: Electrochemiluminescence Peroxydisulfate-oxygen system Hexoctahedrons Co-reactant Thyroid stimulating hormone
1. Introduction Thyroid stimulating hormone (TSH) is secreted by thyrotroph cells in the anterior pituitary gland. In turn, it influences the biosynthesis and secretion of thyroid hormones (triiodothyronine and thyroxine) in human serum (You et al., 2013). A serum TSH concentration below the lower limit of the reference range may indicate subclinical hyperthyroidism (David and Bernadette, 2012). The possible consequences of untreated subclinical hyperthyroidism mainly include adverse cardiac end points, atrial fibrillation, cardiacdysfunction, neuropsychiatric symptoms, reduced bone mineral density, and development toward overt hyperthyroidism (Surks et al., 2004). Therefore, it is imperative to develop some rapid, sensitive and reliable techniques to detect TSH for early intervention and prevention thyroid disease. Nowadays, the “third” generation detection technology with a sensitivity of at least 0.02 μIU mL 1 is used in most of TSH immunoassays (Baloch et al., 2003). Assays with high sensitivity for TSH could be used not only to diagnose hyperthyroidism but also to assess the severity of hyperthyroidism (Lin et al., 2008). n
Corresponding authors. Fax: þ 86 23 68253172. E-mail addresses: [email protected] (Y. Chai), [email protected] (R. Yuan).
http://dx.doi.org/10.1016/j.bios.2015.04.022 0956-5663/& 2015 Published by Elsevier B.V.
To date, majority of immunoassays have been proposed for TSH analysis, such as bioluminescent immunoassay (Sgoutas et al., 1995), chemiluminescence immunoassay (Lin et al., 2008) and fluorescent immunoassay (Papanastasion-Diamandi et al., 1992). Electrochemiluminescence (ECL) immunoassay, which integrates the high specificity of immunoreaction and the excellent sensitivity of ECL technique, has been widely used as a powerful analytical technique for a range of applications with significant advantages (Chen et al., 2014a; Hu and Xu, 2010; Shao et al., 2014). Therefore, the ECL immunosensor is especially attractive for the detection of TSH. According to the previous report, S2O82 solution could produce an interesting ECL emission which was closely related to the dissolved oxygen in solution (Yao et al., 2008). Because of its advantages in simplicity, sensitivity and cheapness, S2O82 -O2 system was increasingly applied for the detection of various analytes (Chen et al., 2014b; Niu et al., 2013). In order to improve the detection sensitivity, a series of signal amplification strategies had been proposed for S2O82 -O2 system (Dai et al., 2014; Han et al., 2014; Niu et al., 2011). For instance, our group developed an ECL immunosensor for the detection of Streptococcus suis Serotype 2 (SS2) by using C60-L-cysteine as a co-reactant and mimic bi-enzyme catalysis to in situ generate O2 for enhancement of ECL response (Wang et al., 2014a). Norfloxacin (NFLX) is a second-generation quinolone antibiotic,
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exhibiting high activity against both Gram-negative and Grampositive bacteria through inhibiting the activity of corresponding DNA gyrase (Nelson et al., 2007). Wang's group reported that NFLX had the ability to amplify cathodic ECL signal of S2O82 -O2 system (Yao et al., 2008). Polyamidoamine (PAMAM) dendrimers, as regular tree-like highly branched macromolecules, possess a high density of surface active groups available for further conjugation (Peng et al., 2008; Wang et al., 2014c). It was reasonable to deduce that amino-terminated PAMAM was capable of enormously improving the loading number of NFLX through covalent binding. In this way, the formed PAMAM-NFLX complex as a novel co-reactant could amplify ECL signal of S2O82 -O2 system more efficiently compared with that of the sole NFLX. Noble metallic nanocrystals have drawn wide attention for many decades due to their high specific surface area, superior biocompatibility, excellent optical performances and outstanding electronic properties (Jie et al., 2010; Wang et al., 2014b; Zhao et al., 2013). More interesting, the structures (especially shapes and facets) of these nanocrystals could be controlled by reaction rates, capping agents, concentrations of reagents, reaction temperatures, etc (Zhang et al., 2012). Among various noble metal nanocrystals, the bimetallic core–shell nanostructures have attracted growing interest, and various invaluable applications in catalysis and sensing have been reported (Mazumder et al., 2010; Tsao et al., 2014; Yang et al., 2014). Hexoctahedral nanocrystals, which are bounded by 48 triangular facets, contain high-density atomic steps and kinks on their surfaces (Tian et al., 2008). As a result, they have a higher surface energy than other nanocrystals with stepped surfaces, such as tetrahexahedral, trisoctahedral, and concave polyhedral nanocrystals (Hong et al., 2012). Herein, convex Pd–Au hexoctahedrons (Pd@Au HOHs) with core–shell structure were synthesized using a facile chemical reduction method through fast growth kinetics and a suitable capping agent (Zhang et al., 2014). The Pd@Au HOHs could act as an outstanding nanocarrier with good biocompatibility and high specific surface area. Furthermore, Pd@Au HOHs were able to promote the ECL reactions of S2O82 -O2 system and amplify ECL signal in view of the presence of active sites on atomic vacancies (Tian et al., 2007). Inspired by above perspectives, we presented a sensitive ECL immunosensor based on the signal amplification of Pd@Au HOHs and PAMAM-NFLX to S2O82 -O2 system for the detection of TSH. Initially, convex Pd@Au HOHs with high-energy surfaces and core– shell structure were synthesized. Next, NFLX was immobilized abundantly on the amino-terminated PAMAM dendrimer by coupling reagents. Then, the obtained Pd@Au HOHs were decorated by PAMAM-NFLX complex via Au–N bond. Using the obtained Pd@Au HOHs-PAMAM-NFLX as bionanolabels, a novel sandwichtype ECL immunosensor was fabricated to detect TSH. The immunosensor exhibited following attractive advantages: firstly, the PAMAM-NFLX complex, which had high loading amount of NFLX, could act as a novel co-reactant to efficiently amplify ECL signal of S2O82 -O2 system. Secondly, Pd@Au HOHs offered an excellent platform for assembling detection antibody (Ab2) and the PAMAM-NFLX complex in this sensor. Lastly, Pd@Au HOHs could further amplify ECL signal of S2O82 -O2 system by promoting the generation of intermediate free radical HO during the ECL reaction of S2O82 -O2 system. With the synergistic signal enhancement effect of PAMAM-NFLX and Pd@Au HOHs, the proposed immunosensor achieved acceptable stability, high sensitivity and specificity for TSH detection. Furthermore, the immunosensor successfully achieved the detection of TSH in practical human blood serum with desirable results.
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2. Experimental section 2.1. Chemicals and materials Amino-terminated polyamidoamine (PAMAM, G 3.0) dendrimers was from Weihai CY Dendrimer Technology Co. Ltd (Weihai, China). Cetylpyridinium chloride monohydrate (CPC) and cetyltrimethylammonium bromide (CTAB) were obtained from Kelong Chemical Company (Chengdu, China). Gold chloride tetrahydrate (HAuCl4 4H2O) and potassium tetrachloropalladate (K2PdCl4) were purchased from Kangda Amino Acid Company (Shanghai, China). L-Ascorbic acid (AA) and K2S2O8 were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Norfloxaci (NFLX), bovine serum albumin (BSA, 96%–99%), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxy succinimide (NHS) were from Sigma-Aldrich Chem. Co. (St. Louis, MO, USA). TSH antibody (anti-TSH), TSH standards, carbohydrate antigen 15-3 (CA 15-3) and carbohydrate antigen 19-9 (CA 19-9) were bought from Biocell Biotechnol. Co. Ltd. (Zhengzhou, China). The serum specimens were obtained from Ninth People's Hospital (Chongqing, China). Phosphate buffered saline (PBS, pH 7.4, 0.2 M) was prepared using 0.1 M Na2HPO4, 0.1 M KH2PO4 containing 0.1 M KCl. Ferricyanide/ferrocyanide mixed solution ([Fe(CN)6]3 /4 , pH 7.0, 5.0 mM) was employed for cyclic voltammetric (CV) investigation. All aqueous solutions were prepared with deionized water (specific resistance Z 18 MΩ cm 1). 2.2. Apparatus The ECL measurements were monitored through a model MPIA multifunctional chemiluminescent analytical system (Xi'an Remax Electronic Science & Technology Co. Ltd., Xi’ an, China) with the voltage of the photomultiplier tube (PMT) being biased at 800 V and the potential ranging from 0 to –2.0 V during measurements. Depositions and CV measurements were carried out with a CHI 660 A electrochemical workstation (Shanghai Chenhua Instruments, China). All electrochemical and ECL curves were performed with a conventional three-electrode configuration, in which the glassy carbon electrode (GCE, Φ ¼4 mm) acted as working electrode, the platinum wire and the Ag/AgCl (saturated KCl) electrode served as counter electrode and reference electrode, respectively. Transmission electron microscopy (TEM, G2F20STWIN, Tecnai, America) and scanning electron microscopy (SEM, S-4800, Hitachi, Japan) were performed to track the surface morphologies of samples. 2.3. Synthesis of convex Pd@Au HOHs Pd nanocubes (Pd NCs) were utilized for the structure-directing core in synthesis of Pd@Au HOHs. Cubic Pd seeds with a particle size of 22 nm were synthesized according to the literature with some minor modifications (Niu et al., 2008). Briefly, 0.5 mL K2PdCl4 solution (10 mM) was added into 9.42 mL CTAB (12.5 mM) aqueous solution, and then heating was kept at 95 °C for 5 min under stirring. After that, 80 mL of freshly prepared AA (100 mM) aqueous solution was dropped, and the reduction reaction was kept for another 20 min. Finally, the obtained Pd NCs were centrifugally washed with CPC aqueous, followed by dispersing into 10 mL CPC (5 mM) aqueous solution. Convex Pd@Au HOHs with high-energy surfaces were prepared by literature procedures as following (Zhang et al., 2014). Initially, 5 mL CPC (5 mM) aqueous solution was kept at 30 °C for 10 min. Then 100 μL of the obtained Pd NCs solution, 144 mL of freshly made AA (100 mM), and 300 mL HAuCl4 (10 mM) were rapidly added to the CPC solution successively. The color of reaction solution became yellow and eventually
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orange pink in several minutes, which suggested the formation of core–shell hexoctahedrons. Magnetic stirring was maintained throughout the whole process. Then the Pd@Au HOHs were colleted by centrifugation and washed with deionized water several times.
0.2 M). Next, 3 mL BSA (10 mg mL 1) was added into above solution under soft stirring for 4 h at 4 °C to block the non-specific binding sites. The formed NFLX-PAMAM-Pd@Au HOHs-Ab2-BSA bioconjugates were finally collected by centrifugation, then redispersed in 1 mL PBS and stored at 4 °C for further use.
2.4. Preparation of the PAMAM-NFLX complex In order to activate the carboxyl groups of NFLX, the coupling reagents (100 mM NHS and 400 mM EDC in deionized water, 20 μL) were added into NFLX saturated aqueous solution (2 mL) in sequence under stirring for 1 h at room temperature. After that, 20 μL (1 mM) amino-terminated PAMAM was injected in the above solution with stirring for 2 h to combine the carboxyl groups of NFLX with the amino groups of PAMAM to form PAMAM-NFLX complex. 2.5. Preparation of NFLX-PAMAM-Pd@Au HOHs-Ab2-BSA bioconjugates The overall preparation process of bioconjugates was shown schematically in Scheme 1(A). Specifically, the synthesized Pd@Au HOHs were added to the PAMAM-NFLX complex solution, and the resulting mixture was shaken vigorously for 12 h. Through the special interaction between amino group and nobel-metal nanoparticles, the PAMAM-NFLX complex was adsorbed around the Pd@Au HOHs. The resultant functionalized material (Pd@Au HOHs-PAMAM-NFLX) was centrifugally washed to remove redundant reagents, and then redispersed in 1 mL deionized water. Subsequently, 300 μL of a detection TSH antibody (Ab2) solution was dropped into above Pd@Au HOHs-PAMAM-NFLX solution, allowing them to react for 10 h under softly shaking at 4 °C. During this process, the Ab2 was loaded onto the surface of Pd@Au HOHs to form NFLX-PAMAM-Pd@Au HOHs-Ab2 bioconjugates. After that, the obtained mixture was centrifuged at 9000 rpm for 15 min at 4 °C, and then the precipitate was dispersed in 1 mL PBS (pH 7.4,
2.6. Fabrication of the sandwich-type ECL immunosensor Firstly, the GCE was polished carefully with 0.3 and 0.05 μm αAl2O3 slurry, then ultrasonically cleaned with deionized water, ethanol and deionized water in turn. Prior to modification, the electrode was dried with nitrogen at room temperature, followed by immersing in HAuCl4 (1 wt %) solution for electrochemical deposition with a potential of 0.2 V for 30 s to obtain a layer of Au nanoparticles (AuNPs) film. AuNPs modified electrode (AuNPs/ GCE) had better adsorption capacity that could attach antibody molecules more favorable. Subsequently, 18 μL of the capture TSH antibody (Ab1) solution was pipetted onto the GCE and incubated at 4 °C for overnight. Unbound antibodies were removed by rinsing with PBS. After that, the electrode was incubated with 18 μL of 10 mg mL 1 BSA to block the non-specific binding sites. Next, the electrodes were incubated with TSH antigen (Ag) solution at different concentrations for 1 h at room temperature. After the biological recognition reaction between Ab1 and Ag, the obtained electrodes were immersed in a beaker with NFLX-PAMAM-Pd@Au HOHs-Ab2-BSA bioconjugates for immunoreaction for 1 h and subsequently washed with PBS carefully to remove physically absorbed bioconjugates. During the experiments, the electrode was capped to prevent evaporation when necessary, and PBS (pH 7.4, 0.2 M) was used as dilution buffers for the antibody and antigen solutions. Scheme 1 B illustrated the fabrication procedure of ECL immunosensor.
Scheme 1. (A) The preparation procedure of PAMAM-NFLX complex and NFLX-PAMAM- Pd@Au HOHs-Ab2-BSA bioconjugates. (B) Schematic illustration of the ECL immunosensor and the possible luminescence mechanism.
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3. Results and discussion 3.1. Characterization of the Pd@Au HOHs-PAMAM-NFLX composite The morphological changes in the synthetic process of Pd@Au HOHs-PAMAM -NFLX composite were monitored using SEM and TEM. As shown in Fig. 1A and B, the synthesized Pd@Au HOHs, in a homogeneous distribution, had a polyhedral shape with a diameter of 907 10 nm. While in the presence of PAMAM-NFLX complex, it could be clearly observed that the Pd@Au HOHs gathered into big clusters (Fig. 1C and D). The increased aggregation degree of Pd@Au HOHs indicated that the existence of PAMAM-NFLX complex on the surface of Pd@Au HOHs. In order to further prove the successful synthesis of Pd@Au HOHs-PAMAM-NFLX composite, X-ray photoelectron spectroscopy (XPS) was used for elemental analysis (Fig. S1). The peaks at about 343 eV and 90 eV could be attributed to the binding energy of Pd3d and Au4f (Fig. S1E and F, respectively), confirming the presence of Pd@Au HOHs. The peaks at 694.0 eV, 535.7 eV and 404.1 eV could be respectively assigned to F1s, O1s and N1s (Fig. S1B, C and D), which clearly showed the presence of PAMAM-NFLX complex. This result strongly verified that the Pd@Au HOHs-PAMAM-NFLX composite were successfully prepared.
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(curve c, curve d and curve e). The reason might be that the protein biomacromolecules on the electrode hindered the diffusion of S2O82 toward the electrode surface. Finally, after NFLX-PAMAMPd@Au HOHs-Ab2-BSA bioconjugates were incubated on the electrode surface, an anticipated great enhancement of ECL signal was appeared (curve f), because the Ab2 bioconjugates were captured successfully on the electrode surface and exhibited excellent performance for ECL analysis. Moreover, the procedure of the electrode modification was monitored by CV experiment in [Fe(CN)6]3 /4 solution. Fig. 2B displayed electrochemical behaviors of the immunosensor at different modification stages. As shown in curve a, the bare GCE exhibited a couple of reversible redox peaks. After AuNPs were electrodeposited on the electrode surface, the peak current values (curve b) increased obviously since AuNPs could facilitate the electron transfer. Whereas the introductions of Ab1 (curve c), BSA (curve d) and TSH (curve e) to AuNPs/GCE induced continuous decreases of peak currents. The reason might be that these biomacromolecules with poor conductivity could hinder the electron transfer between modified electrode and electrolyte. The entire CV data sufficiently demonstrated that the electrode was modified as expect. 3.3. The possible mechanisms for signal amplification
3.2. ECL and electrochemical characterization of the immunosensor To characterize the modified process of the sandwich-type immunosensor step by step, the ECL responses of electrode after each fabricated procedure were recorded in 3 mL PBS containing 0.1 M S2O82 (Fig. 2A). After the AuNPs were electrodeposited on the pretreated bare GCE, the peak intensity on AuNPs/GCE (curve b) exhibited much higher signal compared to the bare GCE (curve a), the reason was that AuNPs could promote the electrochemical reaction efficiency of S2O82 -O2 system in the ECL process. However, after Ab1, BSA and TSH being immobilized onto the AuNPs/ GCE in succession, the decreased ECL responses were observed
The possible mechanisms for the ECL of S2O82 -O2 system were outlined using the following equations (Yao et al., 2008): S2O82 þe -SO4 þSO42
(1)
SO4 þ H2O-HO þHSO4
(2)
HO-HOO þ H2O
(3)
O2 þH2O þe -HOO þ HO SO4
þHOO-HSO4
þ 1(O2)2n
(4) (5)
Fig. 1. SEM (A) and TEM (B) images showing the formation of convex Pd@Au HOHs; SEM (C) and TEM (D) images showing the formation of Pd@Au HOHs-PAMAM-NFLX composite.
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Fig. 2. (A) The ECL responses of the modified electrodes step by step: (a) bare GCE, (b) AuNPs/GCE, (c) Ab1/AuNPs/GCE, (d) BSA/Ab1/AuNPs/GCE, (e) TSH/BSA/Ab1/AuNPs/GCE, and (f) NFLX-PAMAM-Pd@Au HOHs-Ab2/TSH/BSA /Ab1/AuNPs/GCE. Scanning from 2.0 to 0 V in 0.1 M K2S2O8 (pH 7.4 PBS). Scan rate: 0.1 V s 1. (B) Typical cyclic voltammograms of the electrode at different stages in 5.0 mM [Fe(CN)6]3 /4 solution for (a) bare GCE, (b) AuNPs/GCE, (c) Ab1/AuNPs/GCE, (d) BSA/Ab1/AuNPs/GCE, and (e) TSH/BSA/Ab1/AuNPs/GCE. Scanning from 0.2 to 0.6 V. Scan rate: 0.05 V s 1. 1
(O2)2n-23O2 þhν
(6)
Convex Pd@Au HOHs played a great role in amplifying ECL signals of S2O82 -O2 system. Attributing to the abundance of atoms at kink and step sites on high-energy surfaces, the atomic vacancies from the surface could provide active sites for the generation of HO (Zhang et al., 2014). As a result, the local concentration of HO around the electrode surface increased rapidly, which was in favor of the change in Eq. (3), thus enhancing the ECL activity remarkably. In addition, the existence of novel co-reactant PAMAM-NFLX complex further enhanced the ECL intensity of S2O82 -O2 system. Concretely, PAMAM-NFLX was chemically oxidized by the strong oxidant SO4 generated in Eq. (1) to produce the radical cation of PAMAM-NFLX (PAMAM-NFLX þ ). Then PAMAM-NFLX þ underwent deprotonation to form a radical (PAMAM-NFLX). Due to its strong reducing ability, PAMAM-NFLX could reduce dissolved
oxygen in the solution to generate HO2 and oxidized product of PAMAM-NFLX (PAMAM-NFLXox) (Eq 9). Subsequently, part of the HO2 could further combine by virtue of collision to be 1(O2)2n (Eq 10). In this way, the ECL emission was increased as more 1(O2)2n decomposed into 3O2. PAMAM-NFLX þ SO4
-PAMAM-NFLX þ þ SO42
(7)
PAMAM-NFLX þ - PAMAM-NFLX þH þ
(8)
PAMAM-NFLX þO2 þH2O-PAMAM-NFLXox þHO2
(9)
2HO2 þ2HO2-1(O2)2n þH2O2 (O2)2n-23O2 þhν
1
(10) (11)
Fig. 3. ECL-time profiles after the sandwich immunoreaction of the resultant immunosensor with various Ab2 bioconjugates: (A) Pd NCs-PAMAM-NFLX labled Ab2, (B) AuNPs-PAMAM-NFLX labled Ab2, (C) Pd@Au HOHs-PAMAM labled Ab2, and (D) Pd@Au HOHs-PAMAM-NFLX labled Ab2 (target sensor) toward 2.0 μIU mL 1 TSH (red line) in PBS 7.4 containing 0.1 M K2S2O8. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.4. Comparisons of immonosensors with different Ab2 bioconjugates To confirm the amplifying effect of PAMAM-NFLX and Pd@Au HOHs in the S2O82--O2 ECL system, several different bioconjugates were synthesized using the same modified methods. The corresponding bioconjugates were Pd NCs-PAMAM- NFLX labled Ab2, AuNPs-PAMAM-NFLX labled Ab2, Pd@Au HOHs-PAMAM labled Ab2 and Pd@Au HOHs-PAMAM-NFLX labled Ab2 (target sensor). They were employed for the detection of 2.0 μIU mL 1 TSH antigen at the same experiment conditions. The change of the ECL signal (ΔI) before and after the sandwich immunoreaction between Ab2 bioconjugates and TSH antigen varied with different sensors, and the results were shown in Fig. 3. Specifically, the ΔI values of the four sensors were 5637 a.u. (A), 4928 a.u. (B), 3328 a.u. (C), and 7522 a.u. (D). Through comparison, it can be estimated that the NFLX-PAMAM-Pd@Au HOHs composite synthesized in this work had a remarkable advantage in constructing ECL immunosensors. Accordingly, both PAMAM-NFLX and Pd@Au HOHs designed in this target sensor played essential roles in signal amplification, which much favored for the high sensitivity of detection. 3.5. Optimizing the incubation time of immunosensor External experimental parameters are notable factors in affecting the performance of ECL immunosensor. Using 2 μIU mL 1 TSH as an example, we investigated the effect of incubation time between Ab1 and TSH on ECL response of immunosensor. Seen from Fig. S2, the ECL intensity increased with the incubation time, and then reached a plateau at the time of 1 h. Consequently, 1 h was chosen as the optimal incubation time in the process for further experiments. In addition, the effect of the incubation time between TSH and the NFLX-PAMAM-Pd@Au HOHs-Ab2 bioconjugates on ECL response was shown in Fig. S3. It was found that the ECL intensity increased with an increasing incubation time from 20 to 50 min and reached a maximum at about 1 h. Accordingly, the optimal incubation time was 1 h. 3.6. Analytical performance of TSH immunosensor Under optimal conditions, the typical ECL responsive curves to different TSH concentrations in the proposed sensing platform were shown in Fig. 4. ECL intensity (from curve a to h) gradually increased with increasing concentration of TSH. As shown in the inset of Fig. 4, the calibration plot exhibited a good linear relationship between ECL intensity and logarithmic value of TSH concentration in the range of 0.05–20 μIU mL 1. The related regression equation of ECL signals could be expressed as
Fig. 4. ECL-time profiles of the immunosensor in the presence of different concentrations of TSH (a–h). The concentrations of TSH: (a) 0.05 μIU mL 1, (b) 0.1 μIU mL 1, (c) 0.5 μIU mL 1, (d) 1 μIU mL 1, (e) 2 μIU mL 1, (f) 5 μIU mL 1, (g) 10 μIU mL 1, and (h) 20 μIU mL 1. All ECL signals were measured in PBS 7.4 containing 0.1 M S2O82 . Inset: calibration curve for TSH determination.
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I¼ 7362.3 þ2730.7 lg c (I represents the ECL intensity, c stands for the concentration of TSH) with a correlation coefficient of 0.996. A linear range from 0.05 μIU mL 1 to 20 μIU mL 1 with a detection limit of 0.02 μIU mL 1 (calculated by LOD¼ 3SB/m, where SB is the standard deviation of the blank measures, and m is the analytical sensitivity, which can be estimated as the slope of calibration plot at lower concentration ranges) for TSH was achieved (IUPAC, 1976). The results demonstrated that the ECL immunosensor was applicable to the quantitative detection of TSH. 3.7. Stability, reproducibility and selectivity of the ECL immunosensor To investigate the stability of the proposed immunosensor for TSH determination, ECL responses to several different concentrations of TSH antigen were recorded and presented in Fig. S4. The ECL intensity enhanced with increasing concentration and a stable signal under consecutive potential scans was obtained for each concentration used, which demonstrated that the immunosensor possessed good stability. The relative standard deviations (R.S.D.) of intra- and inter-assays were used to evaluate the reproducibility. The ECL responses were obtained by measuring 2 μIU mL 1 TSH. Experimental results revealed that the RSD of intra-assay (n ¼5) were 3.0% and inter-assay were 3.9%. Both of them were not more than 5%, which showed acceptable reproducibility of the fabricated immunosensor. The selectivity and specificity of the immunosensor for TSH determination was tested via comparing the ECL signal changes brought by adding two other interfering proteins (CA 15-3, CA 199). The assay was carried out under the same experimental procedures. As shown in Fig. 5, when CA 15-3 (10 U mL 1) and CA 199 (10 U mL 1) solutions were incubated alone, the ECL responses were almost the same with the blank sample. Furthermore, when the immunosensor was incubated with 2 μΙU mL 1 TSH containing different interfering substances, no significant changes were observed compared to the TSH (2 μΙU mL 1) existed alone. All these results indicated a high selectivity and specificity of the proposed immunosensor toward TSH. 3.8. Preliminary analysis of real samples To test the applicability of the proposed immunosensor in real samples, recovery experiments were investigated by means of standard addition methods. The practical human blood serum was spiked with various concentrations of the target TSH. Then these
Fig. 5. Selectivity of the proposed immunosensor to TSH by comparing it to the interfering proteins. (a) blank solution (0 μIU mL 1 target), (b) CA 15-3 (10 U mL 1), (c) CA 19-9 (10 U mL 1), (d) TSH (2 μIU mL 1), (e) a mixture containing TSH (2 μIU mL 1) and CA 15-3 (10 U mL 1), (f) a mixture containing TSH (2 μIU mL 1) and CA 19-9 (10 U mL 1), (g) a mixture containing TSH (2 μIU mL 1), CA 15-3 (10 U mL 1) and CA 19-9 (10 U mL 1).
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Table 1 Assay results of clinical serum samples using the proposed method and reference method for the detection of TSH. Sample
1
2
3
4
Reference method (μIU mL 1) Proposed method (μIU mL 1) Relative error (%)
0.200 0.210 5.0
2.03 2.10 3.4
3.40 3.24 4.7
17.07 16.01 6.2
target antigens were analyzed in the same manner as described before. The recovery was obtained by calculating the found amount/added amount ratio. As shown in Table S1, the acceptable recoveries (between 91.4% and 107.0%) firmly demonstrated that the immunosensor met the requirement in testing real biological samples. In order to further validate the designed immunosensor, four human serum samples from different people were analyzed by the proposed immunosensor. Prior to the analysis, all of the samples were gently shaken for 5 min at room temperature and then were measured without dilution. The assay results were compared with the referenced values obtained from the commercialized ECL immunoassay (ECLIA, provided by the Third Military Medical University). As shown in Table 1, the assay results between the two methods showed an acceptable agreement, and the relative deviations were in the range from 6.2% to 5.0%, further indicating the feasibility for clinical application.
4. Conclusions This work described the construction of a sensitive ECL immunosensor which was achieved by using the novel coreactant of PAMAM-NFLX complex and convex Pd@Au HOHs for signal amplification to S2O82--O2 system. The PAMAM-NFLX complex possessed high loading amount of NFLX so that it could amplify the ECL signal efficiently. Convex Pd@Au HOHs, which had superior biocompatibility and high specific surface area, were acted as an ideal nano-carrier to immobilize Ab2 and PAMAM-NFLX complex. Additionally, they could further amplify the ECL signals by promoting the generation of intermediate free radical. Moreover, the immunosensor successfully achieved the detection of TSH in practical human blood serum with desirable results, showing potential applications in clinical analysis.
Acknowledgments This work was financially supported by the NNSF of China (21275119), Chongqing Postdoctoral Research Project (Xm2014022), and the Fundamental Research Funds for the
Central Universities (XDJK2014A012, XDJK2015A002), China.
Appendix A. Suplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.022.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
An electrochemiluminescence immunosensor for thyroid stimulating hormone based on polyamidoamine-norfloxacin functionalized Pd–Au core–shell hexoctahedrons as signal enhancers Yuting Liu, Qiqi Zhang, Haijun Wang, Yali Yuan, Yaqin Chai n, Ruo Yuan n Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2014 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online 11 April 2015
In this work, a novel polyamidoamine-norfloxacin (PAMAM-NFLX) complex and core–shell Pd–Au hexoctahedrons (Pd@Au HOHs) as enhancers are employed for development of a sensitive sandwich-type electrochemiluminescence (ECL) immunosensor to detect thyroid stimulating hormone (TSH). Here, norfloxacin (NFLX) is decorated abundantly on the surface of polyamidoamine (PAMAM) dendrimer via amide linkage to form PAMAM-NFLX complex. Thus, the resultant PAMAM-NFLX can serve as a novel coreactant to efficiently amplify the ECL signal of peroxydisulfate-oxygen (S2O82 -O2) system. Pd@Au HOHs were used as nano-carriers to assemble detection antibody (Ab2) and the PAMAM-NFLX complex. Besides, it can further enhance the ECL signal by promoting the generation of intermediate free radical HO during the ECL reaction of S2O82 -O2 system. The proposed immunosensor shows high sensitivity and specificity, and responds linearly to the concentration of TSH from 0.05 to 20 μIU mL 1 with a low detection limit of 0.02 μIU mL 1 (S/N¼3). Moreover, the immunosensor successfully achieves the detection of TSH in practical human blood serum with desirable results. & 2015 Published by Elsevier B.V.
Keywords: Electrochemiluminescence Peroxydisulfate-oxygen system Hexoctahedrons Co-reactant Thyroid stimulating hormone
1. Introduction Thyroid stimulating hormone (TSH) is secreted by thyrotroph cells in the anterior pituitary gland. In turn, it influences the biosynthesis and secretion of thyroid hormones (triiodothyronine and thyroxine) in human serum (You et al., 2013). A serum TSH concentration below the lower limit of the reference range may indicate subclinical hyperthyroidism (David and Bernadette, 2012). The possible consequences of untreated subclinical hyperthyroidism mainly include adverse cardiac end points, atrial fibrillation, cardiacdysfunction, neuropsychiatric symptoms, reduced bone mineral density, and development toward overt hyperthyroidism (Surks et al., 2004). Therefore, it is imperative to develop some rapid, sensitive and reliable techniques to detect TSH for early intervention and prevention thyroid disease. Nowadays, the “third” generation detection technology with a sensitivity of at least 0.02 μIU mL 1 is used in most of TSH immunoassays (Baloch et al., 2003). Assays with high sensitivity for TSH could be used not only to diagnose hyperthyroidism but also to assess the severity of hyperthyroidism (Lin et al., 2008). n
Corresponding authors. Fax: þ 86 23 68253172. E-mail addresses: [email protected] (Y. Chai), [email protected] (R. Yuan).
http://dx.doi.org/10.1016/j.bios.2015.04.022 0956-5663/& 2015 Published by Elsevier B.V.
To date, majority of immunoassays have been proposed for TSH analysis, such as bioluminescent immunoassay (Sgoutas et al., 1995), chemiluminescence immunoassay (Lin et al., 2008) and fluorescent immunoassay (Papanastasion-Diamandi et al., 1992). Electrochemiluminescence (ECL) immunoassay, which integrates the high specificity of immunoreaction and the excellent sensitivity of ECL technique, has been widely used as a powerful analytical technique for a range of applications with significant advantages (Chen et al., 2014a; Hu and Xu, 2010; Shao et al., 2014). Therefore, the ECL immunosensor is especially attractive for the detection of TSH. According to the previous report, S2O82 solution could produce an interesting ECL emission which was closely related to the dissolved oxygen in solution (Yao et al., 2008). Because of its advantages in simplicity, sensitivity and cheapness, S2O82 -O2 system was increasingly applied for the detection of various analytes (Chen et al., 2014b; Niu et al., 2013). In order to improve the detection sensitivity, a series of signal amplification strategies had been proposed for S2O82 -O2 system (Dai et al., 2014; Han et al., 2014; Niu et al., 2011). For instance, our group developed an ECL immunosensor for the detection of Streptococcus suis Serotype 2 (SS2) by using C60-L-cysteine as a co-reactant and mimic bi-enzyme catalysis to in situ generate O2 for enhancement of ECL response (Wang et al., 2014a). Norfloxacin (NFLX) is a second-generation quinolone antibiotic,
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exhibiting high activity against both Gram-negative and Grampositive bacteria through inhibiting the activity of corresponding DNA gyrase (Nelson et al., 2007). Wang's group reported that NFLX had the ability to amplify cathodic ECL signal of S2O82 -O2 system (Yao et al., 2008). Polyamidoamine (PAMAM) dendrimers, as regular tree-like highly branched macromolecules, possess a high density of surface active groups available for further conjugation (Peng et al., 2008; Wang et al., 2014c). It was reasonable to deduce that amino-terminated PAMAM was capable of enormously improving the loading number of NFLX through covalent binding. In this way, the formed PAMAM-NFLX complex as a novel co-reactant could amplify ECL signal of S2O82 -O2 system more efficiently compared with that of the sole NFLX. Noble metallic nanocrystals have drawn wide attention for many decades due to their high specific surface area, superior biocompatibility, excellent optical performances and outstanding electronic properties (Jie et al., 2010; Wang et al., 2014b; Zhao et al., 2013). More interesting, the structures (especially shapes and facets) of these nanocrystals could be controlled by reaction rates, capping agents, concentrations of reagents, reaction temperatures, etc (Zhang et al., 2012). Among various noble metal nanocrystals, the bimetallic core–shell nanostructures have attracted growing interest, and various invaluable applications in catalysis and sensing have been reported (Mazumder et al., 2010; Tsao et al., 2014; Yang et al., 2014). Hexoctahedral nanocrystals, which are bounded by 48 triangular facets, contain high-density atomic steps and kinks on their surfaces (Tian et al., 2008). As a result, they have a higher surface energy than other nanocrystals with stepped surfaces, such as tetrahexahedral, trisoctahedral, and concave polyhedral nanocrystals (Hong et al., 2012). Herein, convex Pd–Au hexoctahedrons (Pd@Au HOHs) with core–shell structure were synthesized using a facile chemical reduction method through fast growth kinetics and a suitable capping agent (Zhang et al., 2014). The Pd@Au HOHs could act as an outstanding nanocarrier with good biocompatibility and high specific surface area. Furthermore, Pd@Au HOHs were able to promote the ECL reactions of S2O82 -O2 system and amplify ECL signal in view of the presence of active sites on atomic vacancies (Tian et al., 2007). Inspired by above perspectives, we presented a sensitive ECL immunosensor based on the signal amplification of Pd@Au HOHs and PAMAM-NFLX to S2O82 -O2 system for the detection of TSH. Initially, convex Pd@Au HOHs with high-energy surfaces and core– shell structure were synthesized. Next, NFLX was immobilized abundantly on the amino-terminated PAMAM dendrimer by coupling reagents. Then, the obtained Pd@Au HOHs were decorated by PAMAM-NFLX complex via Au–N bond. Using the obtained Pd@Au HOHs-PAMAM-NFLX as bionanolabels, a novel sandwichtype ECL immunosensor was fabricated to detect TSH. The immunosensor exhibited following attractive advantages: firstly, the PAMAM-NFLX complex, which had high loading amount of NFLX, could act as a novel co-reactant to efficiently amplify ECL signal of S2O82 -O2 system. Secondly, Pd@Au HOHs offered an excellent platform for assembling detection antibody (Ab2) and the PAMAM-NFLX complex in this sensor. Lastly, Pd@Au HOHs could further amplify ECL signal of S2O82 -O2 system by promoting the generation of intermediate free radical HO during the ECL reaction of S2O82 -O2 system. With the synergistic signal enhancement effect of PAMAM-NFLX and Pd@Au HOHs, the proposed immunosensor achieved acceptable stability, high sensitivity and specificity for TSH detection. Furthermore, the immunosensor successfully achieved the detection of TSH in practical human blood serum with desirable results.
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2. Experimental section 2.1. Chemicals and materials Amino-terminated polyamidoamine (PAMAM, G 3.0) dendrimers was from Weihai CY Dendrimer Technology Co. Ltd (Weihai, China). Cetylpyridinium chloride monohydrate (CPC) and cetyltrimethylammonium bromide (CTAB) were obtained from Kelong Chemical Company (Chengdu, China). Gold chloride tetrahydrate (HAuCl4 4H2O) and potassium tetrachloropalladate (K2PdCl4) were purchased from Kangda Amino Acid Company (Shanghai, China). L-Ascorbic acid (AA) and K2S2O8 were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Norfloxaci (NFLX), bovine serum albumin (BSA, 96%–99%), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxy succinimide (NHS) were from Sigma-Aldrich Chem. Co. (St. Louis, MO, USA). TSH antibody (anti-TSH), TSH standards, carbohydrate antigen 15-3 (CA 15-3) and carbohydrate antigen 19-9 (CA 19-9) were bought from Biocell Biotechnol. Co. Ltd. (Zhengzhou, China). The serum specimens were obtained from Ninth People's Hospital (Chongqing, China). Phosphate buffered saline (PBS, pH 7.4, 0.2 M) was prepared using 0.1 M Na2HPO4, 0.1 M KH2PO4 containing 0.1 M KCl. Ferricyanide/ferrocyanide mixed solution ([Fe(CN)6]3 /4 , pH 7.0, 5.0 mM) was employed for cyclic voltammetric (CV) investigation. All aqueous solutions were prepared with deionized water (specific resistance Z 18 MΩ cm 1). 2.2. Apparatus The ECL measurements were monitored through a model MPIA multifunctional chemiluminescent analytical system (Xi'an Remax Electronic Science & Technology Co. Ltd., Xi’ an, China) with the voltage of the photomultiplier tube (PMT) being biased at 800 V and the potential ranging from 0 to –2.0 V during measurements. Depositions and CV measurements were carried out with a CHI 660 A electrochemical workstation (Shanghai Chenhua Instruments, China). All electrochemical and ECL curves were performed with a conventional three-electrode configuration, in which the glassy carbon electrode (GCE, Φ ¼4 mm) acted as working electrode, the platinum wire and the Ag/AgCl (saturated KCl) electrode served as counter electrode and reference electrode, respectively. Transmission electron microscopy (TEM, G2F20STWIN, Tecnai, America) and scanning electron microscopy (SEM, S-4800, Hitachi, Japan) were performed to track the surface morphologies of samples. 2.3. Synthesis of convex Pd@Au HOHs Pd nanocubes (Pd NCs) were utilized for the structure-directing core in synthesis of Pd@Au HOHs. Cubic Pd seeds with a particle size of 22 nm were synthesized according to the literature with some minor modifications (Niu et al., 2008). Briefly, 0.5 mL K2PdCl4 solution (10 mM) was added into 9.42 mL CTAB (12.5 mM) aqueous solution, and then heating was kept at 95 °C for 5 min under stirring. After that, 80 mL of freshly prepared AA (100 mM) aqueous solution was dropped, and the reduction reaction was kept for another 20 min. Finally, the obtained Pd NCs were centrifugally washed with CPC aqueous, followed by dispersing into 10 mL CPC (5 mM) aqueous solution. Convex Pd@Au HOHs with high-energy surfaces were prepared by literature procedures as following (Zhang et al., 2014). Initially, 5 mL CPC (5 mM) aqueous solution was kept at 30 °C for 10 min. Then 100 μL of the obtained Pd NCs solution, 144 mL of freshly made AA (100 mM), and 300 mL HAuCl4 (10 mM) were rapidly added to the CPC solution successively. The color of reaction solution became yellow and eventually
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orange pink in several minutes, which suggested the formation of core–shell hexoctahedrons. Magnetic stirring was maintained throughout the whole process. Then the Pd@Au HOHs were colleted by centrifugation and washed with deionized water several times.
0.2 M). Next, 3 mL BSA (10 mg mL 1) was added into above solution under soft stirring for 4 h at 4 °C to block the non-specific binding sites. The formed NFLX-PAMAM-Pd@Au HOHs-Ab2-BSA bioconjugates were finally collected by centrifugation, then redispersed in 1 mL PBS and stored at 4 °C for further use.
2.4. Preparation of the PAMAM-NFLX complex In order to activate the carboxyl groups of NFLX, the coupling reagents (100 mM NHS and 400 mM EDC in deionized water, 20 μL) were added into NFLX saturated aqueous solution (2 mL) in sequence under stirring for 1 h at room temperature. After that, 20 μL (1 mM) amino-terminated PAMAM was injected in the above solution with stirring for 2 h to combine the carboxyl groups of NFLX with the amino groups of PAMAM to form PAMAM-NFLX complex. 2.5. Preparation of NFLX-PAMAM-Pd@Au HOHs-Ab2-BSA bioconjugates The overall preparation process of bioconjugates was shown schematically in Scheme 1(A). Specifically, the synthesized Pd@Au HOHs were added to the PAMAM-NFLX complex solution, and the resulting mixture was shaken vigorously for 12 h. Through the special interaction between amino group and nobel-metal nanoparticles, the PAMAM-NFLX complex was adsorbed around the Pd@Au HOHs. The resultant functionalized material (Pd@Au HOHs-PAMAM-NFLX) was centrifugally washed to remove redundant reagents, and then redispersed in 1 mL deionized water. Subsequently, 300 μL of a detection TSH antibody (Ab2) solution was dropped into above Pd@Au HOHs-PAMAM-NFLX solution, allowing them to react for 10 h under softly shaking at 4 °C. During this process, the Ab2 was loaded onto the surface of Pd@Au HOHs to form NFLX-PAMAM-Pd@Au HOHs-Ab2 bioconjugates. After that, the obtained mixture was centrifuged at 9000 rpm for 15 min at 4 °C, and then the precipitate was dispersed in 1 mL PBS (pH 7.4,
2.6. Fabrication of the sandwich-type ECL immunosensor Firstly, the GCE was polished carefully with 0.3 and 0.05 μm αAl2O3 slurry, then ultrasonically cleaned with deionized water, ethanol and deionized water in turn. Prior to modification, the electrode was dried with nitrogen at room temperature, followed by immersing in HAuCl4 (1 wt %) solution for electrochemical deposition with a potential of 0.2 V for 30 s to obtain a layer of Au nanoparticles (AuNPs) film. AuNPs modified electrode (AuNPs/ GCE) had better adsorption capacity that could attach antibody molecules more favorable. Subsequently, 18 μL of the capture TSH antibody (Ab1) solution was pipetted onto the GCE and incubated at 4 °C for overnight. Unbound antibodies were removed by rinsing with PBS. After that, the electrode was incubated with 18 μL of 10 mg mL 1 BSA to block the non-specific binding sites. Next, the electrodes were incubated with TSH antigen (Ag) solution at different concentrations for 1 h at room temperature. After the biological recognition reaction between Ab1 and Ag, the obtained electrodes were immersed in a beaker with NFLX-PAMAM-Pd@Au HOHs-Ab2-BSA bioconjugates for immunoreaction for 1 h and subsequently washed with PBS carefully to remove physically absorbed bioconjugates. During the experiments, the electrode was capped to prevent evaporation when necessary, and PBS (pH 7.4, 0.2 M) was used as dilution buffers for the antibody and antigen solutions. Scheme 1 B illustrated the fabrication procedure of ECL immunosensor.
Scheme 1. (A) The preparation procedure of PAMAM-NFLX complex and NFLX-PAMAM- Pd@Au HOHs-Ab2-BSA bioconjugates. (B) Schematic illustration of the ECL immunosensor and the possible luminescence mechanism.
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3. Results and discussion 3.1. Characterization of the Pd@Au HOHs-PAMAM-NFLX composite The morphological changes in the synthetic process of Pd@Au HOHs-PAMAM -NFLX composite were monitored using SEM and TEM. As shown in Fig. 1A and B, the synthesized Pd@Au HOHs, in a homogeneous distribution, had a polyhedral shape with a diameter of 907 10 nm. While in the presence of PAMAM-NFLX complex, it could be clearly observed that the Pd@Au HOHs gathered into big clusters (Fig. 1C and D). The increased aggregation degree of Pd@Au HOHs indicated that the existence of PAMAM-NFLX complex on the surface of Pd@Au HOHs. In order to further prove the successful synthesis of Pd@Au HOHs-PAMAM-NFLX composite, X-ray photoelectron spectroscopy (XPS) was used for elemental analysis (Fig. S1). The peaks at about 343 eV and 90 eV could be attributed to the binding energy of Pd3d and Au4f (Fig. S1E and F, respectively), confirming the presence of Pd@Au HOHs. The peaks at 694.0 eV, 535.7 eV and 404.1 eV could be respectively assigned to F1s, O1s and N1s (Fig. S1B, C and D), which clearly showed the presence of PAMAM-NFLX complex. This result strongly verified that the Pd@Au HOHs-PAMAM-NFLX composite were successfully prepared.
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(curve c, curve d and curve e). The reason might be that the protein biomacromolecules on the electrode hindered the diffusion of S2O82 toward the electrode surface. Finally, after NFLX-PAMAMPd@Au HOHs-Ab2-BSA bioconjugates were incubated on the electrode surface, an anticipated great enhancement of ECL signal was appeared (curve f), because the Ab2 bioconjugates were captured successfully on the electrode surface and exhibited excellent performance for ECL analysis. Moreover, the procedure of the electrode modification was monitored by CV experiment in [Fe(CN)6]3 /4 solution. Fig. 2B displayed electrochemical behaviors of the immunosensor at different modification stages. As shown in curve a, the bare GCE exhibited a couple of reversible redox peaks. After AuNPs were electrodeposited on the electrode surface, the peak current values (curve b) increased obviously since AuNPs could facilitate the electron transfer. Whereas the introductions of Ab1 (curve c), BSA (curve d) and TSH (curve e) to AuNPs/GCE induced continuous decreases of peak currents. The reason might be that these biomacromolecules with poor conductivity could hinder the electron transfer between modified electrode and electrolyte. The entire CV data sufficiently demonstrated that the electrode was modified as expect. 3.3. The possible mechanisms for signal amplification
3.2. ECL and electrochemical characterization of the immunosensor To characterize the modified process of the sandwich-type immunosensor step by step, the ECL responses of electrode after each fabricated procedure were recorded in 3 mL PBS containing 0.1 M S2O82 (Fig. 2A). After the AuNPs were electrodeposited on the pretreated bare GCE, the peak intensity on AuNPs/GCE (curve b) exhibited much higher signal compared to the bare GCE (curve a), the reason was that AuNPs could promote the electrochemical reaction efficiency of S2O82 -O2 system in the ECL process. However, after Ab1, BSA and TSH being immobilized onto the AuNPs/ GCE in succession, the decreased ECL responses were observed
The possible mechanisms for the ECL of S2O82 -O2 system were outlined using the following equations (Yao et al., 2008): S2O82 þe -SO4 þSO42
(1)
SO4 þ H2O-HO þHSO4
(2)
HO-HOO þ H2O
(3)
O2 þH2O þe -HOO þ HO SO4
þHOO-HSO4
þ 1(O2)2n
(4) (5)
Fig. 1. SEM (A) and TEM (B) images showing the formation of convex Pd@Au HOHs; SEM (C) and TEM (D) images showing the formation of Pd@Au HOHs-PAMAM-NFLX composite.
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Fig. 2. (A) The ECL responses of the modified electrodes step by step: (a) bare GCE, (b) AuNPs/GCE, (c) Ab1/AuNPs/GCE, (d) BSA/Ab1/AuNPs/GCE, (e) TSH/BSA/Ab1/AuNPs/GCE, and (f) NFLX-PAMAM-Pd@Au HOHs-Ab2/TSH/BSA /Ab1/AuNPs/GCE. Scanning from 2.0 to 0 V in 0.1 M K2S2O8 (pH 7.4 PBS). Scan rate: 0.1 V s 1. (B) Typical cyclic voltammograms of the electrode at different stages in 5.0 mM [Fe(CN)6]3 /4 solution for (a) bare GCE, (b) AuNPs/GCE, (c) Ab1/AuNPs/GCE, (d) BSA/Ab1/AuNPs/GCE, and (e) TSH/BSA/Ab1/AuNPs/GCE. Scanning from 0.2 to 0.6 V. Scan rate: 0.05 V s 1. 1
(O2)2n-23O2 þhν
(6)
Convex Pd@Au HOHs played a great role in amplifying ECL signals of S2O82 -O2 system. Attributing to the abundance of atoms at kink and step sites on high-energy surfaces, the atomic vacancies from the surface could provide active sites for the generation of HO (Zhang et al., 2014). As a result, the local concentration of HO around the electrode surface increased rapidly, which was in favor of the change in Eq. (3), thus enhancing the ECL activity remarkably. In addition, the existence of novel co-reactant PAMAM-NFLX complex further enhanced the ECL intensity of S2O82 -O2 system. Concretely, PAMAM-NFLX was chemically oxidized by the strong oxidant SO4 generated in Eq. (1) to produce the radical cation of PAMAM-NFLX (PAMAM-NFLX þ ). Then PAMAM-NFLX þ underwent deprotonation to form a radical (PAMAM-NFLX). Due to its strong reducing ability, PAMAM-NFLX could reduce dissolved
oxygen in the solution to generate HO2 and oxidized product of PAMAM-NFLX (PAMAM-NFLXox) (Eq 9). Subsequently, part of the HO2 could further combine by virtue of collision to be 1(O2)2n (Eq 10). In this way, the ECL emission was increased as more 1(O2)2n decomposed into 3O2. PAMAM-NFLX þ SO4
-PAMAM-NFLX þ þ SO42
(7)
PAMAM-NFLX þ - PAMAM-NFLX þH þ
(8)
PAMAM-NFLX þO2 þH2O-PAMAM-NFLXox þHO2
(9)
2HO2 þ2HO2-1(O2)2n þH2O2 (O2)2n-23O2 þhν
1
(10) (11)
Fig. 3. ECL-time profiles after the sandwich immunoreaction of the resultant immunosensor with various Ab2 bioconjugates: (A) Pd NCs-PAMAM-NFLX labled Ab2, (B) AuNPs-PAMAM-NFLX labled Ab2, (C) Pd@Au HOHs-PAMAM labled Ab2, and (D) Pd@Au HOHs-PAMAM-NFLX labled Ab2 (target sensor) toward 2.0 μIU mL 1 TSH (red line) in PBS 7.4 containing 0.1 M K2S2O8. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.4. Comparisons of immonosensors with different Ab2 bioconjugates To confirm the amplifying effect of PAMAM-NFLX and Pd@Au HOHs in the S2O82--O2 ECL system, several different bioconjugates were synthesized using the same modified methods. The corresponding bioconjugates were Pd NCs-PAMAM- NFLX labled Ab2, AuNPs-PAMAM-NFLX labled Ab2, Pd@Au HOHs-PAMAM labled Ab2 and Pd@Au HOHs-PAMAM-NFLX labled Ab2 (target sensor). They were employed for the detection of 2.0 μIU mL 1 TSH antigen at the same experiment conditions. The change of the ECL signal (ΔI) before and after the sandwich immunoreaction between Ab2 bioconjugates and TSH antigen varied with different sensors, and the results were shown in Fig. 3. Specifically, the ΔI values of the four sensors were 5637 a.u. (A), 4928 a.u. (B), 3328 a.u. (C), and 7522 a.u. (D). Through comparison, it can be estimated that the NFLX-PAMAM-Pd@Au HOHs composite synthesized in this work had a remarkable advantage in constructing ECL immunosensors. Accordingly, both PAMAM-NFLX and Pd@Au HOHs designed in this target sensor played essential roles in signal amplification, which much favored for the high sensitivity of detection. 3.5. Optimizing the incubation time of immunosensor External experimental parameters are notable factors in affecting the performance of ECL immunosensor. Using 2 μIU mL 1 TSH as an example, we investigated the effect of incubation time between Ab1 and TSH on ECL response of immunosensor. Seen from Fig. S2, the ECL intensity increased with the incubation time, and then reached a plateau at the time of 1 h. Consequently, 1 h was chosen as the optimal incubation time in the process for further experiments. In addition, the effect of the incubation time between TSH and the NFLX-PAMAM-Pd@Au HOHs-Ab2 bioconjugates on ECL response was shown in Fig. S3. It was found that the ECL intensity increased with an increasing incubation time from 20 to 50 min and reached a maximum at about 1 h. Accordingly, the optimal incubation time was 1 h. 3.6. Analytical performance of TSH immunosensor Under optimal conditions, the typical ECL responsive curves to different TSH concentrations in the proposed sensing platform were shown in Fig. 4. ECL intensity (from curve a to h) gradually increased with increasing concentration of TSH. As shown in the inset of Fig. 4, the calibration plot exhibited a good linear relationship between ECL intensity and logarithmic value of TSH concentration in the range of 0.05–20 μIU mL 1. The related regression equation of ECL signals could be expressed as
Fig. 4. ECL-time profiles of the immunosensor in the presence of different concentrations of TSH (a–h). The concentrations of TSH: (a) 0.05 μIU mL 1, (b) 0.1 μIU mL 1, (c) 0.5 μIU mL 1, (d) 1 μIU mL 1, (e) 2 μIU mL 1, (f) 5 μIU mL 1, (g) 10 μIU mL 1, and (h) 20 μIU mL 1. All ECL signals were measured in PBS 7.4 containing 0.1 M S2O82 . Inset: calibration curve for TSH determination.
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I¼ 7362.3 þ2730.7 lg c (I represents the ECL intensity, c stands for the concentration of TSH) with a correlation coefficient of 0.996. A linear range from 0.05 μIU mL 1 to 20 μIU mL 1 with a detection limit of 0.02 μIU mL 1 (calculated by LOD¼ 3SB/m, where SB is the standard deviation of the blank measures, and m is the analytical sensitivity, which can be estimated as the slope of calibration plot at lower concentration ranges) for TSH was achieved (IUPAC, 1976). The results demonstrated that the ECL immunosensor was applicable to the quantitative detection of TSH. 3.7. Stability, reproducibility and selectivity of the ECL immunosensor To investigate the stability of the proposed immunosensor for TSH determination, ECL responses to several different concentrations of TSH antigen were recorded and presented in Fig. S4. The ECL intensity enhanced with increasing concentration and a stable signal under consecutive potential scans was obtained for each concentration used, which demonstrated that the immunosensor possessed good stability. The relative standard deviations (R.S.D.) of intra- and inter-assays were used to evaluate the reproducibility. The ECL responses were obtained by measuring 2 μIU mL 1 TSH. Experimental results revealed that the RSD of intra-assay (n ¼5) were 3.0% and inter-assay were 3.9%. Both of them were not more than 5%, which showed acceptable reproducibility of the fabricated immunosensor. The selectivity and specificity of the immunosensor for TSH determination was tested via comparing the ECL signal changes brought by adding two other interfering proteins (CA 15-3, CA 199). The assay was carried out under the same experimental procedures. As shown in Fig. 5, when CA 15-3 (10 U mL 1) and CA 199 (10 U mL 1) solutions were incubated alone, the ECL responses were almost the same with the blank sample. Furthermore, when the immunosensor was incubated with 2 μΙU mL 1 TSH containing different interfering substances, no significant changes were observed compared to the TSH (2 μΙU mL 1) existed alone. All these results indicated a high selectivity and specificity of the proposed immunosensor toward TSH. 3.8. Preliminary analysis of real samples To test the applicability of the proposed immunosensor in real samples, recovery experiments were investigated by means of standard addition methods. The practical human blood serum was spiked with various concentrations of the target TSH. Then these
Fig. 5. Selectivity of the proposed immunosensor to TSH by comparing it to the interfering proteins. (a) blank solution (0 μIU mL 1 target), (b) CA 15-3 (10 U mL 1), (c) CA 19-9 (10 U mL 1), (d) TSH (2 μIU mL 1), (e) a mixture containing TSH (2 μIU mL 1) and CA 15-3 (10 U mL 1), (f) a mixture containing TSH (2 μIU mL 1) and CA 19-9 (10 U mL 1), (g) a mixture containing TSH (2 μIU mL 1), CA 15-3 (10 U mL 1) and CA 19-9 (10 U mL 1).
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Table 1 Assay results of clinical serum samples using the proposed method and reference method for the detection of TSH. Sample
1
2
3
4
Reference method (μIU mL 1) Proposed method (μIU mL 1) Relative error (%)
0.200 0.210 5.0
2.03 2.10 3.4
3.40 3.24 4.7
17.07 16.01 6.2
target antigens were analyzed in the same manner as described before. The recovery was obtained by calculating the found amount/added amount ratio. As shown in Table S1, the acceptable recoveries (between 91.4% and 107.0%) firmly demonstrated that the immunosensor met the requirement in testing real biological samples. In order to further validate the designed immunosensor, four human serum samples from different people were analyzed by the proposed immunosensor. Prior to the analysis, all of the samples were gently shaken for 5 min at room temperature and then were measured without dilution. The assay results were compared with the referenced values obtained from the commercialized ECL immunoassay (ECLIA, provided by the Third Military Medical University). As shown in Table 1, the assay results between the two methods showed an acceptable agreement, and the relative deviations were in the range from 6.2% to 5.0%, further indicating the feasibility for clinical application.
4. Conclusions This work described the construction of a sensitive ECL immunosensor which was achieved by using the novel coreactant of PAMAM-NFLX complex and convex Pd@Au HOHs for signal amplification to S2O82--O2 system. The PAMAM-NFLX complex possessed high loading amount of NFLX so that it could amplify the ECL signal efficiently. Convex Pd@Au HOHs, which had superior biocompatibility and high specific surface area, were acted as an ideal nano-carrier to immobilize Ab2 and PAMAM-NFLX complex. Additionally, they could further amplify the ECL signals by promoting the generation of intermediate free radical. Moreover, the immunosensor successfully achieved the detection of TSH in practical human blood serum with desirable results, showing potential applications in clinical analysis.
Acknowledgments This work was financially supported by the NNSF of China (21275119), Chongqing Postdoctoral Research Project (Xm2014022), and the Fundamental Research Funds for the
Central Universities (XDJK2014A012, XDJK2015A002), China.
Appendix A. Suplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.022.
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