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Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay
Analytica Chimica Acta xxx (2015) 1e7
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Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay Hongfang Zhang a, b, *, Danlei Ning a, Lina Ma a, Jianbin Zheng b, ** a Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, PR China b Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi'an 710069, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
An ultrasensitive nonenzymatic electrochemical immunosensor for HIgG detection was developed. AuNRs were used to catalyze the deposition of silver. Detection signal was greatly amplified by self-assembly of AuNRs and the subsequent silver enhancement. This immunosensor exhibited a extremely low detection limit.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 20 October 2015 Accepted 22 October 2015 Available online xxx
A novel electrochemical immunoassay was developed based on the signal amplification strategy of silver deposition directed by gold nanorods (AuNRs), which was in-situ assembled on the sandwich immunocomplex. The superstructure formed by the self-assembly of AuNRs provided abundant active sites for the nucleation of silver nanoparticles. In this pathway, the stripping current of silver was greatly enhanced. Using human immunoglobulin G (HIgG) as a model analyte, the ultrasensitive immunoassay showed a wide linear range of six orders of magnitude from 0.1 fg mL1 to 100 pg mL1, with the low detection limit down to 0.08 fg mL1. The practicality of this electrochemical immunoassay for detection of HIgG in serum was validated with the average recovery of 93.9%. In addition, this enzyme-free immunoassay also has the advantages of acceptable reproducibility and specificity, and thus this immunosensing protocol can be extended to the detection of other low-abundant protein biomarkers. © 2015 Elsevier B.V. All rights reserved.
Keywords: Human IgG Gold nanorod Silver deposition Immunosensor Signal amplification
1. Introduction
* Corresponding author. Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, PR. China. ** Corresponding author. E-mail addresses: [email protected] (H. Zhang), [email protected] (J. Zheng).
In recent years, electrochemical immunoassays for the detection of disease biomarkers have gained increasing attention due to their intrinsic superiority, such as fast speed and low cost, which can favorably meet the clinical requirements [1e3]. What's more, electrochemical immunoassay can offer high sensitivity and low detection limit owing to the convenient incorporation of various signal amplification strategies into the assay protocol [4e9]. Of
http://dx.doi.org/10.1016/j.aca.2015.10.028 0003-2670/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028
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H. Zhang et al. / Analytica Chimica Acta xxx (2015) 1e7
particular interest is the gold nanoparticles (AuNPs)-catalyzed silver deposition because of its inherent signal amplification nature induced by the deposited silver [4,10]. Among various shapes of AuNPs, gold nanorods (AuNRs) attracted substantial attention in recent several years [10e12]. In addition to the unique optical properties [13,14], AuNRs possess general properties similar to AuNPs such as excellent conductivity and biological compatibility, and have several advantages over the spherical AuNPs, including fast electron transfer rate and high surface area [15,16], which inspired researchers to explore the application of AuNRs in electrochemical biosensors [17e19]. Zang et al. [17] established an electrochemical immunosensor for sensitive detection of ofloxacin based on multi-enzyme-antibody functionalized AuNRs. The study of Li's group [18] exhibited that the sensitivity of the proposed immunosensor relied on the amount of label and antibody conjugated on the AuNRs. Du et al. [19] demonstrated that AuNRs could link enzyme and detection antibody at high ratio. These works indicated that AuNRs were excellent nanocarriers for the biomolecular recognition events. Recently, Ju's group [5] developed an immunosensing amplification strategy with ultralow detection limit for a-fetoprotein, by introducing a hosteguest binding reaction into the assembly process of AuNRs superstructure. Thus, the AuNRs based superstructure provided a new avenue for signal amplification in the design of electrochemical immunosensors. Compared with the relatively positive electrochemical oxidization potential of gold, metal silver can be oxidized at a lower potential with a sharp stripping peak [20]. Thus, AuNPs-mediated silver enhancement after specific formation of sandwich immunocomplex has been proposed for sensitive electrochemical immunoassay [3,4]. Herein, AuNRs-directed silver deposition strategy was applied
to design an ultrasensitive electrochemical immunosensor. As shown in Scheme 1, the carbon nanotubes-chitosan composite (CNTs-Chit), owing to excellent conductivity and favorable biocompatibility, was utilized to immobilize the capture antibodies and catalyze the electrochemical oxidation of the deposited silver [5,21,22]. After the formation of sandwich-type immunocomplex, AuNRs was introduced onto the surface of the immunosensor. Then, the multi-layer AuNRs was assembled via the strong covalent AueS linkage. After that, the superstructure-directed silver deposition was performed. Target proteins were quantitatively analyzed using the amplified signal of silver. Using human immunoglobulin G (HIgG) as a model analyte, the proposed method showed a detection limit down to ag mL1 level. 2. Experimental 2.1. Materials and reagents HIgG, mouse anti-human IgG antibody (anti-HIgG) and bovine serum albumin (BSA) were obtained from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijng, China). Chitosan (Chit, MW 56 105, >90% deacetylation) was purchased from Shanghai Yuanju Biotechnology Co. Ltd. (Shanghai, China) and was used as fixative. Carboxylated CNTs (purity >95wt%, Outer diameter 30e50 nm, and length 20 mm) were purchased from Chengdu Organic Chemicals Co. Ltd. (Chengdu, China). Glutaraldehyde (GA), L-ascorbic acid (AA) and hydroquinone (HQ) were purchased from Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). Cetyltrimethylammonium bromide (CTAB), Chloroauric acid, trisodium citrate, and silver nitrate were obtained from Shanghai Reagent Company (Shanghai, China). Sodium borohydride was obtained
Scheme 1. Schematic representation of the preparation of (A) AuNRs-Ab2, (B) the electrochemical immunosensor and sandwich immunoassay procedure.
Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028
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from Sinopharm Chemical Reagent Co. Ltd. (China). Tween-20 was obtained from MP Biomedicals. All other reagents were of analytical grade and used as received. Phosphate buffered saline (PBS, 0.01 M, pH 7.4) was prepared by mixing Na2HPO4 $ 12H2O and KH2PO4. 0.01 M PBS containing 1.0% (w/v) BSA was used as blocking solution. Washing solution (PBST) was prepared by dissolving 0.05% (V/V) Tween-20 in PBS [7]. Deionized and distilled water was used throughout the study. 2.2. Apparatus All electrochemical immunoassays were performed on a CHI660A electrochemical workstation (CH Instrument, China). A three-electrode system was used for all electrochemical immunoassays: glassy carbon electrode (GCE) or modified GCE as the working electrode, a saturated calomel electrode (SCE) and a platinum electrode as reference and counter electrode, respectively. The UVeVis absorption spectrum was observed with a UV2550 UVeVis spectrophotometer (Shimadzu, Japan). Scanning electron microscopy (SEM) images were obtained using a JSM6390A (JEOL, Japan). The transmission electron microscopy (TEM) images were obtained from Tecnai G2 F20 S-TWIN (FEI, USA). The quartz crystal microbalance (QCM) frequency change was monitored by a CHI440c electrochemical quartz crystal microbalance (CH Instrument, China). 2.3. Preparation of gold nanorods labeled anti-human IgG antibody (AuNRs-Ab2) For the adsorption of Ab2 onto the AuNRs (Scheme 1A), the gold nanorods solution (supplementary material) was added into the anti-HIgG solution (0.1 mg mL1) and stirred gently for 3 h, after that, the unattached antibody was separated by centrifugation (4000 rpm for 15 min). The prepared AuNRs-Ab2 was re-dispersed in PBS (0.01 M, pH 7.4) and stored at 4 C before use. 2.4. Fabrication of immunosensor Firstly, 5 mL of the suspension (1.0 mg mL1 in 1.0% Chit solution) of CNTs-Chit nanocomposite was coated on the clean surface of a GCE and dried at room temperature [21]. The modified electrode was then immersed in 5 mL of 5% GA for 2 h, followed by washing with deionized water. After that, 5 mL of 0.1 mg mL1 anti-HIgG (Ab1) was applied to the CNTs-Chit/GCE surface and incubated at 4 C overnight. The loosely adsorbed antibodies were removed sequentially with PBST and PBS. Finally, the modified electrode was blocked by incubating with 5 mL blocking solution for 30 min at 37 C. After washing with PBST and PBS, the immunosensor was obtained and stored at 4 C in a dry environment prior to use. 2.5. Measurement procedure The immunosensor was first incubated a certain concentration of HIgG or serum samples at 37 C for 1 h. After a washing step, 5 mL of AuNRs-Ab2 was dropped onto the immunosensor and incubated for 1 h. Following another washing step, the immunosensor was incubated alternatively with 5 mL of 5.0 mM 1,6-hexanedithiol and AuNRs solution for 10 min for the assemble of second layer AuNRs. By repeating twice the alternative incubation, four layers of AuNRs was assembled. After rinsing with water, silver deposition was performed using 10 mL of silver enhancer solution (supplementary material) for 10 min at room temperature in a dark incubator. Then, the immunosensor was rinsed with water, and the linear sweep voltammogram (LSV) from 0.3 to 0.3 V at 50 mV s1 was recorded in 1.0 M KCl solution in a beaker-type electrochemical cell to record the response.
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3. Results and discussion 3.1. Characterization of AuNRs and AuNRs-Ab2 We presented the TEM image of AuNRs, where rod-shaped nanoparticles with homogeneous size distribution could be observed (Fig. 1A). This holds great promise for the immobilization of antibodies [19]. From Fig. 1B, the UVeVis extinction spectrum of AuNRs (curve a) exhibited two distinct and narrow plasmon resonance bands centered at 520 and 719 nm, which were assigned to the typical transverse and longitudinal plasmon resonances of AuNRs, respectively [23,24]. After interaction with anti-HIgG antibodies, the UVeVis spectrum of AuNRs (curve b) showed a red shift of 17 nm of the transverse band, and an obvious red shift of the longitudinal band, indicating that the antibodies were covalently attached to AuNRs [25]. 3.2. Characterization of the immunosensor Fabrication of the immunosensor was characterized by SEM. From Fig. 1C, well-dispersed nanotubes entangled with each other could be observed. This uniform CNTs-Chit nanostructure greatly increased the reactive surface area and therefore the Ab1 loading amount [5]. Through the specific antibodyeantigen reaction, HIgG and AuNRs-Ab2 were captured on the electrode sequentially. The presence of small clumps of nanoparticles was confirmed by SEM image of AuNRs-Ab2/HIgG/Ab1/CNTs/Chit/GCE (Fig. 1D), which showed the successful attachment of the first layer AuNRs. Then, additional three layers of AuNRs were repeatedly incubated, a remarkable conglomeration of AuNRs was found on the surface of immunosensor from Fig. 1E (Fig. S1 gives the enlarged view to show the AuNRs clearly). After incubated in the silver enhancer solution, the particle size on the nano-granular surface increased obviously as depicted in Fig. 1F, indicating that silver nanoparticles (AgNPs) was catalytically deposited on the surface with AuNRs as nucleation sites [10,26]. The presence of Au or Ag elements on the surface of the immunosensor was directly proved by the intense signals ascribed to Au and Ag on the energy dispersive spectrum of the immunosensor before and after silver deposition (Fig. S2). Previous research has investigated the influence of silver source and silver amplification time on the performance of immunogold silver deposition [27,28]. This work found that the performance was also appreciably affected by the amount of gold catalysts. 3.3. Signal amplification by the self-assembly of AuNRs To study the signal amplification based on the self-assembly of AuNRs and silver enhancement, the stripping peak currents of silver deposited on different layers of AuNRs were recorded. As shown in Fig. 2A, for blank buffer incubation (curve a), neglect peak was observed on the voltammogram. For 1.0 ng mL1 HIgG (curve b), a distinct stripping peak ascribed to the electrochemical reduction of nanosilver was obtained [29], indicating that the deposition of AgNPs induced by the first layer of AuNRs on the immunosensor was connected with the presence of the target protein. After the assembly of one additional layer of AuNRs, the obvious increase of the stripping current of silver for 1.0 ng mL1 HIgG was acquired (curve c), demonstrating the signal amplified effect of the self-assembly of AuNRs on the performance of the immunosensor. Furthermore, the layering provided by additional incubation steps resulted in an enhancement of the peak current of AgNPs, which depended on the number of assembled layer of AuNRs (curve d to f). When the fourth layer of AuNRs was assembled, the stripping current (curve e) of the deposited AgNPs is 5.8 times of it produced by the AuNRs attached only by the immunocomplex (curve b). The results clearly illustrated
Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028
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Fig. 1. UVeVis absorption spectra of AuNRs (curve a) and AuNRs-Ab2 (curve b) (A). TEM image of AuNRs (B). SEM images of CNTs/Chit/GCE (C), AuNRs-Ab2/HIgG/Ab1/CNTs/Chit/GCE (D), (AuNRs)4-Ab2/HIgG/Ab1/CNTs/Chit/GCE (E) and silver deposition on (AuNRs)4-Ab2/HIgG/Ab1/CNTs/Chit/GCE (F). EDS of (AuNRs)4-Ab2/HIgG/Ab1/CNTs/Chit/GCE.
that the assembly of AuNRs enabled an amplified electronic detection of the target proteins. This amplification could be contributed to the abundant catalytic and nucleation sites provided by AuNRs for the deposition of silver [3,4]. Furthermore, there was a roughly linear relationship (shown in Fig. S3) between the peak current and the layer number with a correlation coefficient of 0.98, which was similar with the other layer-by-layer assembly [30,31]. The same trend was also witnessed by QCM monitoring of the assembly of AuNRs on the gold-coated resonator electrode (Fig. S3). The change in QCM frequency with each assembly cycle was linear, as shown in Fig. 2B. According to the Sauerbrey equation, such frequency changes are proportional to the change in mass on the crystal
surface. The results of QCM suggested that the assembly of AuNRs was reproducible, and almost same amount of AuNRs was loaded in each assembly step [32]. This further explained the above linear relationship between the peak current of silver and the layer number. It was undeniable that this immunoassay required a longer analysis time expensed for the assembly of the additional three layers of AuNRs when was compared with the typical sandwichtype immunosensor. However, the total analysis time of this immunoassay was comparable with that of the traditional ELISA [33] and some other electrochemical immunoassays [5,7].
Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028
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Fig. 2. (A) LSV curves of immunosensor after sandwich incubation with 1.0 ng mL1 HIgG (b to f) and AuNRs-Ab2, a is the blank response. (B) Shift of QCM frequency with layer number of assembly.
3.4. Analytical performance The as-prepared immunosensor was challenged with different concentrations of HIgG. The peak current on the LSV of the sensor increases gradually with the increase of the amount of HIgG in the concentration range of 0.1 fg mL1 to 1.0 ng mL1 (Fig. 3A). The calibration plot shows a good linear relationship between the peak currents and the root values of the analyte concentrations in the wide range from 0.1 fg mL1 to 100 pg mL1 (Fig. S4). The linear regression equation was expressed as I ¼ 24.324 þ 0.896 C1/2 (I: mA, C: pg mL1) with a correlation coefficient of 0.996. What's more, LSV curves of the immunosensor obtained in blank buffer and 0.1 fg mL1 HIgG were compared (Fig. 3B). The average peak current of five individual blank experiments was 4.562 mA with the relative standard deviation (RSD) less than 10%. Compared to the low background, the response of the immunosensor toward 0.1 fg mL1 HIgG gave a bigger current of 14.61 mA, which supplied a convincing evidence for the minimum detection level of the immunosensor. Therefore, the limit of detection at a signal-to-noise ratio of 3 was estimated to be 0.08 fg mL1. The comparison of the performance of the proposed HIgG immunosensor with other electrochemical sensors (Table 1) was listed, demonstrating that our sensor exhibited a low detection limit. Previous work has proved extensively the catalytic function of nanogold to reduce silver ions to metallic silver in the presence of a reducing agent [27,28]. Lin and
co-workers demonstrated that high loading of nanogold greatly amplified the detection signal and improved the detection sensitivity [20]. The ultralow detection limit of the immunosensor was definitely contributed to the super nanostructure assembled by AuNRs which supplied efficient active sites for the deposition of silver. To examine the specificity of the immunosensor, BSA, Hb and HSA were incubated and detected individually with the amplification strategy. The current measured with the HIgG immunosensor toward the other proteins at the same concentration level exhibited variations negligibly small when was compared with the current response toward HIgG (Fig. S5). These results demonstrated that high sensitivity of the immunosensor was obtained without sacrificing the specificity. To investigate the repeatability of the immunoassay, a series of measurements were conducted at five immunosensors prepared with the same GCE. The current responses of the sensor toward HIgG at two different concentration levels were recorded. The RSD were 3.9% and 2.5% for the measurements of 10 fg mL1 and 1.0 pg mL1 HIgG, respectively. Furthermore, about 92% of their initial signal response was remained for 1.0 pg mL1 HIgG after the immunosensors were stored for 2 weeks at 4 C. The reproducibility of the immunosensor prepared with newly prepared materials, a different GCE and operated by a different researcher was investigated. The relative deviation of the current response for
Fig. 3. LSV curves toward HIgG with different concentrations using the proposed immunosensor. (A) Curves a to k correspond to HIgG at concentrations from 0.1 fg mL1 to 100 pg mL1. The inset shows the magnification curves of a to f. (B) Comparison of LSV responses of the immunosensor toward (a) blank buffer of five individual experiments and (b) 0.1 fg mL1 HIgG.
Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028
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Table 1 Comparison of different immunosensors for detection of HIgG. Sensing platforma
Linear range (pg mL1)
Detection limit (pg mL1)
Ref.
SPCE/Chit-Ab1/HIgG/Ab2-AuNPs-ALP/AgNPs GCE/Chit/AuNPs/Ab1/HIgG/Ab2-CNT-Gox GCE/AuNPs-GN-Ab1/HigG/Ab2-PNW-Fc GCE/AgNW-Chit/Ab1/HIgG/HRP-Ab2-ZnO-TH SPCE/CNT-Ab1/HIgG/Ab2-AuNPs-PDA/silica GCE/CNFs-PAMAM-Ab1/HIgG/Ab2-Ag@AuNRs GCE/CuSNWs-Chit/Ab1/HIgG (AA in solution) GCE/CNTs-Chit-Ab1/HIgG/Ab2-(AuNRs)4/AgNPs
10e250000 0.1e100 0.01e100 10e200000 10e0000 0.001e1000 1e320000 0.0001e100
4.8 0.05 0.005 4 6.9 0.0005 0.1 0.00008
[4] [6] [8] [9] [12] [29] [34] This work
a SPCE: screen-printed carbon electrode; ALP: alkaline phosphatase; Gox: glucose oxidase; GN: graphene; PNW-Fc: ferrocene-peptide nanowire; AgNW: silver nanowire; HRP: horseradish peroxidase; TH: thionine; PDA: polydopamine; CNFs: carbon nanofibers; PAMAM: polyamidoamine; CuSNWs: CuS nanowires; AA: ascorbic acid.
Table 2 Results of real sample assay and recoveries. Initial HIgG in sample 1
C (pg mL
)
20.7
Measured after addition RSD (%, n ¼ 3)
Added (fg mL1)
Found (fg mL1)
Recovery (%)
RSD (%, n ¼ 3)
3.3
10 100 10000
10.7 117.0 9660.7
107 117 96.6
4.9 4.2 4.7
1.0 pg mL1 HIgG was 4.1%. These results suggested the potential application of the immunosensing strategy for the clinical detection of protein markers.
3.5. Detection of HIgG in serum samples To evaluate the practicality of the immunosensing method, HIgG concentration in human serum was determined, and different amounts of HIgG were added into the serum samples for recovery tests. The peak current on the voltammogram increases by different degrees when compared to the signal obtained in non-spiked serum (Fig. S6). The test results from three repeated experiments were listed in Table 2. The measured serum HIgG level was in consistence with the result in the literature [12]. The recovery of the standard addition experiments was between 89.0% and 96.4% with RSD less than 5%, which validated the feasibility of the proposed immunosensor for low-abundance biomarkers detection in real human serum samples.
4. Conclusions In summary, by introducing the cascade of AuNRs onto the sensor surface via self-assembly, we developed an excellent electrochemical immunoassay platform based on the signal amplification strategy of silver enhancement. The abundant silver deposition sites supplied by AuNRs allowed a highly sensitive immunoassay method. The immunoassay exhibited excellent analytical performance for the measurement of HIgG with an ultralow detection limit. The acceptable recoveries in human serum showed the potential application of the immunosensor in clinical diagnostics. The amplification strategy described here indicates unlimited potential to detect the other protein biomarkers with further development.
Acknowledgment The authors gratefully acknowledge the financial support of this project by the National Natural Science Foundation of China (No. 21275116).
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Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay Hongfang Zhang a, b, *, Danlei Ning a, Lina Ma a, Jianbin Zheng b, ** a Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, PR China b Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi'an 710069, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
An ultrasensitive nonenzymatic electrochemical immunosensor for HIgG detection was developed. AuNRs were used to catalyze the deposition of silver. Detection signal was greatly amplified by self-assembly of AuNRs and the subsequent silver enhancement. This immunosensor exhibited a extremely low detection limit.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 20 October 2015 Accepted 22 October 2015 Available online xxx
A novel electrochemical immunoassay was developed based on the signal amplification strategy of silver deposition directed by gold nanorods (AuNRs), which was in-situ assembled on the sandwich immunocomplex. The superstructure formed by the self-assembly of AuNRs provided abundant active sites for the nucleation of silver nanoparticles. In this pathway, the stripping current of silver was greatly enhanced. Using human immunoglobulin G (HIgG) as a model analyte, the ultrasensitive immunoassay showed a wide linear range of six orders of magnitude from 0.1 fg mL1 to 100 pg mL1, with the low detection limit down to 0.08 fg mL1. The practicality of this electrochemical immunoassay for detection of HIgG in serum was validated with the average recovery of 93.9%. In addition, this enzyme-free immunoassay also has the advantages of acceptable reproducibility and specificity, and thus this immunosensing protocol can be extended to the detection of other low-abundant protein biomarkers. © 2015 Elsevier B.V. All rights reserved.
Keywords: Human IgG Gold nanorod Silver deposition Immunosensor Signal amplification
1. Introduction
* Corresponding author. Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, PR. China. ** Corresponding author. E-mail addresses: [email protected] (H. Zhang), [email protected] (J. Zheng).
In recent years, electrochemical immunoassays for the detection of disease biomarkers have gained increasing attention due to their intrinsic superiority, such as fast speed and low cost, which can favorably meet the clinical requirements [1e3]. What's more, electrochemical immunoassay can offer high sensitivity and low detection limit owing to the convenient incorporation of various signal amplification strategies into the assay protocol [4e9]. Of
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Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028
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H. Zhang et al. / Analytica Chimica Acta xxx (2015) 1e7
particular interest is the gold nanoparticles (AuNPs)-catalyzed silver deposition because of its inherent signal amplification nature induced by the deposited silver [4,10]. Among various shapes of AuNPs, gold nanorods (AuNRs) attracted substantial attention in recent several years [10e12]. In addition to the unique optical properties [13,14], AuNRs possess general properties similar to AuNPs such as excellent conductivity and biological compatibility, and have several advantages over the spherical AuNPs, including fast electron transfer rate and high surface area [15,16], which inspired researchers to explore the application of AuNRs in electrochemical biosensors [17e19]. Zang et al. [17] established an electrochemical immunosensor for sensitive detection of ofloxacin based on multi-enzyme-antibody functionalized AuNRs. The study of Li's group [18] exhibited that the sensitivity of the proposed immunosensor relied on the amount of label and antibody conjugated on the AuNRs. Du et al. [19] demonstrated that AuNRs could link enzyme and detection antibody at high ratio. These works indicated that AuNRs were excellent nanocarriers for the biomolecular recognition events. Recently, Ju's group [5] developed an immunosensing amplification strategy with ultralow detection limit for a-fetoprotein, by introducing a hosteguest binding reaction into the assembly process of AuNRs superstructure. Thus, the AuNRs based superstructure provided a new avenue for signal amplification in the design of electrochemical immunosensors. Compared with the relatively positive electrochemical oxidization potential of gold, metal silver can be oxidized at a lower potential with a sharp stripping peak [20]. Thus, AuNPs-mediated silver enhancement after specific formation of sandwich immunocomplex has been proposed for sensitive electrochemical immunoassay [3,4]. Herein, AuNRs-directed silver deposition strategy was applied
to design an ultrasensitive electrochemical immunosensor. As shown in Scheme 1, the carbon nanotubes-chitosan composite (CNTs-Chit), owing to excellent conductivity and favorable biocompatibility, was utilized to immobilize the capture antibodies and catalyze the electrochemical oxidation of the deposited silver [5,21,22]. After the formation of sandwich-type immunocomplex, AuNRs was introduced onto the surface of the immunosensor. Then, the multi-layer AuNRs was assembled via the strong covalent AueS linkage. After that, the superstructure-directed silver deposition was performed. Target proteins were quantitatively analyzed using the amplified signal of silver. Using human immunoglobulin G (HIgG) as a model analyte, the proposed method showed a detection limit down to ag mL1 level. 2. Experimental 2.1. Materials and reagents HIgG, mouse anti-human IgG antibody (anti-HIgG) and bovine serum albumin (BSA) were obtained from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijng, China). Chitosan (Chit, MW 56 105, >90% deacetylation) was purchased from Shanghai Yuanju Biotechnology Co. Ltd. (Shanghai, China) and was used as fixative. Carboxylated CNTs (purity >95wt%, Outer diameter 30e50 nm, and length 20 mm) were purchased from Chengdu Organic Chemicals Co. Ltd. (Chengdu, China). Glutaraldehyde (GA), L-ascorbic acid (AA) and hydroquinone (HQ) were purchased from Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). Cetyltrimethylammonium bromide (CTAB), Chloroauric acid, trisodium citrate, and silver nitrate were obtained from Shanghai Reagent Company (Shanghai, China). Sodium borohydride was obtained
Scheme 1. Schematic representation of the preparation of (A) AuNRs-Ab2, (B) the electrochemical immunosensor and sandwich immunoassay procedure.
Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028
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from Sinopharm Chemical Reagent Co. Ltd. (China). Tween-20 was obtained from MP Biomedicals. All other reagents were of analytical grade and used as received. Phosphate buffered saline (PBS, 0.01 M, pH 7.4) was prepared by mixing Na2HPO4 $ 12H2O and KH2PO4. 0.01 M PBS containing 1.0% (w/v) BSA was used as blocking solution. Washing solution (PBST) was prepared by dissolving 0.05% (V/V) Tween-20 in PBS [7]. Deionized and distilled water was used throughout the study. 2.2. Apparatus All electrochemical immunoassays were performed on a CHI660A electrochemical workstation (CH Instrument, China). A three-electrode system was used for all electrochemical immunoassays: glassy carbon electrode (GCE) or modified GCE as the working electrode, a saturated calomel electrode (SCE) and a platinum electrode as reference and counter electrode, respectively. The UVeVis absorption spectrum was observed with a UV2550 UVeVis spectrophotometer (Shimadzu, Japan). Scanning electron microscopy (SEM) images were obtained using a JSM6390A (JEOL, Japan). The transmission electron microscopy (TEM) images were obtained from Tecnai G2 F20 S-TWIN (FEI, USA). The quartz crystal microbalance (QCM) frequency change was monitored by a CHI440c electrochemical quartz crystal microbalance (CH Instrument, China). 2.3. Preparation of gold nanorods labeled anti-human IgG antibody (AuNRs-Ab2) For the adsorption of Ab2 onto the AuNRs (Scheme 1A), the gold nanorods solution (supplementary material) was added into the anti-HIgG solution (0.1 mg mL1) and stirred gently for 3 h, after that, the unattached antibody was separated by centrifugation (4000 rpm for 15 min). The prepared AuNRs-Ab2 was re-dispersed in PBS (0.01 M, pH 7.4) and stored at 4 C before use. 2.4. Fabrication of immunosensor Firstly, 5 mL of the suspension (1.0 mg mL1 in 1.0% Chit solution) of CNTs-Chit nanocomposite was coated on the clean surface of a GCE and dried at room temperature [21]. The modified electrode was then immersed in 5 mL of 5% GA for 2 h, followed by washing with deionized water. After that, 5 mL of 0.1 mg mL1 anti-HIgG (Ab1) was applied to the CNTs-Chit/GCE surface and incubated at 4 C overnight. The loosely adsorbed antibodies were removed sequentially with PBST and PBS. Finally, the modified electrode was blocked by incubating with 5 mL blocking solution for 30 min at 37 C. After washing with PBST and PBS, the immunosensor was obtained and stored at 4 C in a dry environment prior to use. 2.5. Measurement procedure The immunosensor was first incubated a certain concentration of HIgG or serum samples at 37 C for 1 h. After a washing step, 5 mL of AuNRs-Ab2 was dropped onto the immunosensor and incubated for 1 h. Following another washing step, the immunosensor was incubated alternatively with 5 mL of 5.0 mM 1,6-hexanedithiol and AuNRs solution for 10 min for the assemble of second layer AuNRs. By repeating twice the alternative incubation, four layers of AuNRs was assembled. After rinsing with water, silver deposition was performed using 10 mL of silver enhancer solution (supplementary material) for 10 min at room temperature in a dark incubator. Then, the immunosensor was rinsed with water, and the linear sweep voltammogram (LSV) from 0.3 to 0.3 V at 50 mV s1 was recorded in 1.0 M KCl solution in a beaker-type electrochemical cell to record the response.
3
3. Results and discussion 3.1. Characterization of AuNRs and AuNRs-Ab2 We presented the TEM image of AuNRs, where rod-shaped nanoparticles with homogeneous size distribution could be observed (Fig. 1A). This holds great promise for the immobilization of antibodies [19]. From Fig. 1B, the UVeVis extinction spectrum of AuNRs (curve a) exhibited two distinct and narrow plasmon resonance bands centered at 520 and 719 nm, which were assigned to the typical transverse and longitudinal plasmon resonances of AuNRs, respectively [23,24]. After interaction with anti-HIgG antibodies, the UVeVis spectrum of AuNRs (curve b) showed a red shift of 17 nm of the transverse band, and an obvious red shift of the longitudinal band, indicating that the antibodies were covalently attached to AuNRs [25]. 3.2. Characterization of the immunosensor Fabrication of the immunosensor was characterized by SEM. From Fig. 1C, well-dispersed nanotubes entangled with each other could be observed. This uniform CNTs-Chit nanostructure greatly increased the reactive surface area and therefore the Ab1 loading amount [5]. Through the specific antibodyeantigen reaction, HIgG and AuNRs-Ab2 were captured on the electrode sequentially. The presence of small clumps of nanoparticles was confirmed by SEM image of AuNRs-Ab2/HIgG/Ab1/CNTs/Chit/GCE (Fig. 1D), which showed the successful attachment of the first layer AuNRs. Then, additional three layers of AuNRs were repeatedly incubated, a remarkable conglomeration of AuNRs was found on the surface of immunosensor from Fig. 1E (Fig. S1 gives the enlarged view to show the AuNRs clearly). After incubated in the silver enhancer solution, the particle size on the nano-granular surface increased obviously as depicted in Fig. 1F, indicating that silver nanoparticles (AgNPs) was catalytically deposited on the surface with AuNRs as nucleation sites [10,26]. The presence of Au or Ag elements on the surface of the immunosensor was directly proved by the intense signals ascribed to Au and Ag on the energy dispersive spectrum of the immunosensor before and after silver deposition (Fig. S2). Previous research has investigated the influence of silver source and silver amplification time on the performance of immunogold silver deposition [27,28]. This work found that the performance was also appreciably affected by the amount of gold catalysts. 3.3. Signal amplification by the self-assembly of AuNRs To study the signal amplification based on the self-assembly of AuNRs and silver enhancement, the stripping peak currents of silver deposited on different layers of AuNRs were recorded. As shown in Fig. 2A, for blank buffer incubation (curve a), neglect peak was observed on the voltammogram. For 1.0 ng mL1 HIgG (curve b), a distinct stripping peak ascribed to the electrochemical reduction of nanosilver was obtained [29], indicating that the deposition of AgNPs induced by the first layer of AuNRs on the immunosensor was connected with the presence of the target protein. After the assembly of one additional layer of AuNRs, the obvious increase of the stripping current of silver for 1.0 ng mL1 HIgG was acquired (curve c), demonstrating the signal amplified effect of the self-assembly of AuNRs on the performance of the immunosensor. Furthermore, the layering provided by additional incubation steps resulted in an enhancement of the peak current of AgNPs, which depended on the number of assembled layer of AuNRs (curve d to f). When the fourth layer of AuNRs was assembled, the stripping current (curve e) of the deposited AgNPs is 5.8 times of it produced by the AuNRs attached only by the immunocomplex (curve b). The results clearly illustrated
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Fig. 1. UVeVis absorption spectra of AuNRs (curve a) and AuNRs-Ab2 (curve b) (A). TEM image of AuNRs (B). SEM images of CNTs/Chit/GCE (C), AuNRs-Ab2/HIgG/Ab1/CNTs/Chit/GCE (D), (AuNRs)4-Ab2/HIgG/Ab1/CNTs/Chit/GCE (E) and silver deposition on (AuNRs)4-Ab2/HIgG/Ab1/CNTs/Chit/GCE (F). EDS of (AuNRs)4-Ab2/HIgG/Ab1/CNTs/Chit/GCE.
that the assembly of AuNRs enabled an amplified electronic detection of the target proteins. This amplification could be contributed to the abundant catalytic and nucleation sites provided by AuNRs for the deposition of silver [3,4]. Furthermore, there was a roughly linear relationship (shown in Fig. S3) between the peak current and the layer number with a correlation coefficient of 0.98, which was similar with the other layer-by-layer assembly [30,31]. The same trend was also witnessed by QCM monitoring of the assembly of AuNRs on the gold-coated resonator electrode (Fig. S3). The change in QCM frequency with each assembly cycle was linear, as shown in Fig. 2B. According to the Sauerbrey equation, such frequency changes are proportional to the change in mass on the crystal
surface. The results of QCM suggested that the assembly of AuNRs was reproducible, and almost same amount of AuNRs was loaded in each assembly step [32]. This further explained the above linear relationship between the peak current of silver and the layer number. It was undeniable that this immunoassay required a longer analysis time expensed for the assembly of the additional three layers of AuNRs when was compared with the typical sandwichtype immunosensor. However, the total analysis time of this immunoassay was comparable with that of the traditional ELISA [33] and some other electrochemical immunoassays [5,7].
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Fig. 2. (A) LSV curves of immunosensor after sandwich incubation with 1.0 ng mL1 HIgG (b to f) and AuNRs-Ab2, a is the blank response. (B) Shift of QCM frequency with layer number of assembly.
3.4. Analytical performance The as-prepared immunosensor was challenged with different concentrations of HIgG. The peak current on the LSV of the sensor increases gradually with the increase of the amount of HIgG in the concentration range of 0.1 fg mL1 to 1.0 ng mL1 (Fig. 3A). The calibration plot shows a good linear relationship between the peak currents and the root values of the analyte concentrations in the wide range from 0.1 fg mL1 to 100 pg mL1 (Fig. S4). The linear regression equation was expressed as I ¼ 24.324 þ 0.896 C1/2 (I: mA, C: pg mL1) with a correlation coefficient of 0.996. What's more, LSV curves of the immunosensor obtained in blank buffer and 0.1 fg mL1 HIgG were compared (Fig. 3B). The average peak current of five individual blank experiments was 4.562 mA with the relative standard deviation (RSD) less than 10%. Compared to the low background, the response of the immunosensor toward 0.1 fg mL1 HIgG gave a bigger current of 14.61 mA, which supplied a convincing evidence for the minimum detection level of the immunosensor. Therefore, the limit of detection at a signal-to-noise ratio of 3 was estimated to be 0.08 fg mL1. The comparison of the performance of the proposed HIgG immunosensor with other electrochemical sensors (Table 1) was listed, demonstrating that our sensor exhibited a low detection limit. Previous work has proved extensively the catalytic function of nanogold to reduce silver ions to metallic silver in the presence of a reducing agent [27,28]. Lin and
co-workers demonstrated that high loading of nanogold greatly amplified the detection signal and improved the detection sensitivity [20]. The ultralow detection limit of the immunosensor was definitely contributed to the super nanostructure assembled by AuNRs which supplied efficient active sites for the deposition of silver. To examine the specificity of the immunosensor, BSA, Hb and HSA were incubated and detected individually with the amplification strategy. The current measured with the HIgG immunosensor toward the other proteins at the same concentration level exhibited variations negligibly small when was compared with the current response toward HIgG (Fig. S5). These results demonstrated that high sensitivity of the immunosensor was obtained without sacrificing the specificity. To investigate the repeatability of the immunoassay, a series of measurements were conducted at five immunosensors prepared with the same GCE. The current responses of the sensor toward HIgG at two different concentration levels were recorded. The RSD were 3.9% and 2.5% for the measurements of 10 fg mL1 and 1.0 pg mL1 HIgG, respectively. Furthermore, about 92% of their initial signal response was remained for 1.0 pg mL1 HIgG after the immunosensors were stored for 2 weeks at 4 C. The reproducibility of the immunosensor prepared with newly prepared materials, a different GCE and operated by a different researcher was investigated. The relative deviation of the current response for
Fig. 3. LSV curves toward HIgG with different concentrations using the proposed immunosensor. (A) Curves a to k correspond to HIgG at concentrations from 0.1 fg mL1 to 100 pg mL1. The inset shows the magnification curves of a to f. (B) Comparison of LSV responses of the immunosensor toward (a) blank buffer of five individual experiments and (b) 0.1 fg mL1 HIgG.
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Table 1 Comparison of different immunosensors for detection of HIgG. Sensing platforma
Linear range (pg mL1)
Detection limit (pg mL1)
Ref.
SPCE/Chit-Ab1/HIgG/Ab2-AuNPs-ALP/AgNPs GCE/Chit/AuNPs/Ab1/HIgG/Ab2-CNT-Gox GCE/AuNPs-GN-Ab1/HigG/Ab2-PNW-Fc GCE/AgNW-Chit/Ab1/HIgG/HRP-Ab2-ZnO-TH SPCE/CNT-Ab1/HIgG/Ab2-AuNPs-PDA/silica GCE/CNFs-PAMAM-Ab1/HIgG/Ab2-Ag@AuNRs GCE/CuSNWs-Chit/Ab1/HIgG (AA in solution) GCE/CNTs-Chit-Ab1/HIgG/Ab2-(AuNRs)4/AgNPs
10e250000 0.1e100 0.01e100 10e200000 10e0000 0.001e1000 1e320000 0.0001e100
4.8 0.05 0.005 4 6.9 0.0005 0.1 0.00008
[4] [6] [8] [9] [12] [29] [34] This work
a SPCE: screen-printed carbon electrode; ALP: alkaline phosphatase; Gox: glucose oxidase; GN: graphene; PNW-Fc: ferrocene-peptide nanowire; AgNW: silver nanowire; HRP: horseradish peroxidase; TH: thionine; PDA: polydopamine; CNFs: carbon nanofibers; PAMAM: polyamidoamine; CuSNWs: CuS nanowires; AA: ascorbic acid.
Table 2 Results of real sample assay and recoveries. Initial HIgG in sample 1
C (pg mL
)
20.7
Measured after addition RSD (%, n ¼ 3)
Added (fg mL1)
Found (fg mL1)
Recovery (%)
RSD (%, n ¼ 3)
3.3
10 100 10000
10.7 117.0 9660.7
107 117 96.6
4.9 4.2 4.7
1.0 pg mL1 HIgG was 4.1%. These results suggested the potential application of the immunosensing strategy for the clinical detection of protein markers.
3.5. Detection of HIgG in serum samples To evaluate the practicality of the immunosensing method, HIgG concentration in human serum was determined, and different amounts of HIgG were added into the serum samples for recovery tests. The peak current on the voltammogram increases by different degrees when compared to the signal obtained in non-spiked serum (Fig. S6). The test results from three repeated experiments were listed in Table 2. The measured serum HIgG level was in consistence with the result in the literature [12]. The recovery of the standard addition experiments was between 89.0% and 96.4% with RSD less than 5%, which validated the feasibility of the proposed immunosensor for low-abundance biomarkers detection in real human serum samples.
4. Conclusions In summary, by introducing the cascade of AuNRs onto the sensor surface via self-assembly, we developed an excellent electrochemical immunoassay platform based on the signal amplification strategy of silver enhancement. The abundant silver deposition sites supplied by AuNRs allowed a highly sensitive immunoassay method. The immunoassay exhibited excellent analytical performance for the measurement of HIgG with an ultralow detection limit. The acceptable recoveries in human serum showed the potential application of the immunosensor in clinical diagnostics. The amplification strategy described here indicates unlimited potential to detect the other protein biomarkers with further development.
Acknowledgment The authors gratefully acknowledge the financial support of this project by the National Natural Science Foundation of China (No. 21275116).
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Please cite this article in press as: H. Zhang, et al., Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.028