Biosensors and Bioelectronics 54 (2014) 323–328
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Layer-by-layer multienzyme assembly for highly sensitive electrochemical immunoassay based on tyramine signal ampliﬁcation strategy Jun Zhou a,b, Juan Tang a, Guonan Chen a, Dianping Tang a,n a Ministry of Education & Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China b Modern Biochemistry Center, Guangdong Ocean University, Zhanjiang 524088, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 11 September 2013 Received in revised form 5 November 2013 Accepted 8 November 2013 Available online 16 November 2013
A new sandwich-type electrochemical immunosensor based on nanosilver-doped bovine serum albumin microspheres (Ag@BSA) with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for quantitative monitoring of biomarkers (carcinoembryonic antigen, CEA, used in this case) by coupling enzymatic biocatalytic precipitation with tyramine signal ampliﬁcation strategy on capture antibody-modiﬁed glassy carbon electrode. Two immunosensing protocols (with and without tyramine signal ampliﬁcation) were also investigated for the detection of CEA and improved analytical features were acquired with tyramine signal ampliﬁcation strategy. With the labeling method, the performance and factors inﬂuencing the electrochemical immunoassay were studied and evaluated in detail. Under the optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005– 80 ng mL 1 toward CEA standards with a low detection limit of 5.0 pg mL 1. Intra- and inter-assay coefﬁcients of variation were below 11%. No signiﬁcant differences at the 0.05 signiﬁcance level were encountered in the analysis of 6 clinical serum specimens and 6 spiked new-born cattle serum samples between the electrochemical immunoassay and the commercialized electrochemiluminescent immunoassay method for the detection of CEA. & 2013 Elsevier B.V. All rights reserved.
Keywords: Electrochemical immunosensor Nanosilver-doped bovine serum albumin microspheres Tyramine signal ampliﬁcation Multienzyme assembly Carcinoembryonic antigen
1. Introduction Tumor markers, usually proteins, are produced by the body in response to cancer growth or by the cancer tissue itself. Sensitive and accurate detection of biomarkers is a fundamental requirement in the ﬁelds of modern biomedicine, clinical diagnostics and therapeutic analysis, which often offers the opportunities for the understanding of disease-related biological processes (Weston and Hood, 2004). Immunoassay based on the antibody–antigen interaction is one of the most important analytical techniques in quantitative monitoring of biomarkers due to the highly speciﬁc molecular recognition event (Lai et al., 2009). Compared with the conventional immunoassays, e.g. by using ﬂuorescence, chemiluminescence, surface-plasmon resonance (SPR), and quartz crystal microbalance (QCM), electrochemical immunosensor has attracted considerable interest because of its intrinsic advantages such as high sensitivity, simple instruments and low power requirements (Li et al., 2008). Various electrochemical immunoassays and
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immunosensors have been developed for the determination of biomarkers (Lin et al., 2012; Das et al., 2006). However, the increasing demand for early and ultrasensitive screening of cancer biomarkers is pushing the enhancement of detection sensitivity by signal ampliﬁcation strategies or coupling various detection technologies (Liu et al., 2012). Nanomaterials provide a promising electrochemical sensing platform, because of the large surface areas for the improvement of mass transport, high loading of receptor molecules for synergistic ampliﬁcation of the target response, and unique biocompatible, electronic, and catalytic properties for the translation of biorecognition events to an electrochemical response (Xu et al., 2012). In recent years, bovine serum albumin (BSA)-mediated synthesis of inorganic nanomaterials has attracted increasing attention due to their advantages of green reaction processing and multifunctionality of the products (Dickerson et al., 2008; Hu et al., 2013). Nanosilver-doped BSA microspheres (Ag@BSA) with the three-dimensional complex structure can not only keep the protein bioactivity and nanoparticle-based conducting property, but also show good stability (e.g., withstanding a wide range of pH changes) and biocompability, thereby enabling them particularly suitable for biological/biomedical applications (Hu et al., 2012).
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Tyramine signal ampliﬁcation (TSA) is an enzyme-mediated detection method (Toda et al., 1999). The ampliﬁcation reagent, tyramine, is a phenolic derivative which can serve as a substrate of horseradish peroxidase (HRP) (Kim et al., 2002). HRP catalyzes reporter-conjugated tyramine and active tyramine deposition at the site of the enzyme reaction, accumulating large numbers of reporter molecules to enhance the signals (Bobrow et al., 1989). As a valid approach, tyramine signal ampliﬁcation system has been widely applied to immunohistochemistry, and in situ hybridization, and more recently to cDNA, pathogen detection, and genotyping arrays (Bobrow et al., 1991; Wang et al., 2011; Anderson and Taitt, 2008; Yuan et al., 2012). To the best of our knowledge, however, there is no report focusing on electrochemical detection of tumor markers through tyramine signal ampliﬁcation strategy. Carcinoembryonic antigen (CEA) is a preferred tumor marker to help predict outlook in patients with colorectal cancer (Zhou et al., 2012a). The normal range of blood levels varies between individuals, but levels higher than 3 ng mL 1 are not normal. In the present work, we combine the merits of nanolabels and tyramine signal ampliﬁcation strategy, and devise a new sandwich-type electrochemical immunoassay for the detection of CEA (as a model) by using Ag@BSA microspheres as the labels. Detection antibody and HRP are initially conjugated covalently onto the surface of Ag@BSA microspheres by using glutaraldehyde as the cross-linkage agent, and then the labeling microspheres are used as the trace tags for the detection of CEA on the capture antibody-functionalized probe with a sandwich-type immunoassay format. In the presence of target CEA, the carried HRP can catalyze the deposition of HRP-conjugated tyramine to form a multienzyme system on the probe, thus resulting in the ampliﬁcation of electrochemical signal relative to hydrogen peroxide system. Enhanced sensitivity for the detection of CEA can be achieved by the increment of HRP loading. The aim of this work is to exploit a new signal ampliﬁcation strategy for improving the analytical properties of the conventional sandwich-type electrochemical immunoassays.
2. Experimental 2.1. Materials and reagent Monoclonal rabbit anti-human CEA antibody (anti-CEA, designated as Ab1) and CEA standards were purchased from Biocell Biotechnol. Co., Ltd. (Zhengzhou, China). Polyclonal rabbit antihuman CEA antibody (MW: 150–200 kDa, lyophilized, designated as Ab2) was obtained from Beijing Biosynth. Biotechnol. Co., Ltd. (Bioss, China). Silver nitrate (AgNO3), hydrazine monohydrate (N2H4 H2O), bovine serum albumin (BSA), glutaraldehyde (25 wt%), horseradish peroxidase (HRP), tyramine, β-cyclodextrin (CD) and poly-(ethylene glycol) (PEG) were purchased from Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). N-(3-dimethyla-minopropyl)N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and N-2-hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) were obtained from Sigma-Aldrich (USA). All other reagents were of analytical grade and were used without further puriﬁcation. Ultrapure water obtained from a Millipore water puriﬁcation system (Z18 MΩ, Milli-Q, Millipore) was used in all runs. Phosphatebuffered saline (PBS) solutions with various pH values were prepared by mixing 0.1 M K2HPO4 and 0.1 M KH2PO4, and 0.1 M KCl was used as the supporting electrolyte. Clinical serum samples were made available by Fujian Provincial Hospital, China. 2.2. Synthesis of nanosilver-doped BSA microspheres (Ag@BSA) Ag@BSA microspheres with 150 nm were prepared according to the literature (Hu et al., 2012). Brieﬂy, silver nitrate aqueous
solution (5.0 mL, 50 mM) was initially added into BSA aqueous solution (10 mL, 3.0 mg mL 1) under vigorous stirring at room temperature (RT), and then the mixture was vacuumized and kept static under nitrogen protection for 2 h. Following that, 0.2 mL of hydrazine monohydrate was injected into the vacuumed solution under magnetic stirring. After reaction, the resulting mixture was aged under ambient conditions for 24 h. Subsequently, the suspension was separated by centrifugation at 5000 rpm for 20 min. Finally, the collected solid state products were washed by using distilled water for three times, and dried for further use. 2.3. Conjugation of Ag@BSA microspheres with HRP and Ab2 detection antibody For synthesis of HRP and Ab2-conjugated Ag@BSA microspheres, 100 μL of original glutaraldehyde solution (excess) was initially dropped into 1.0 mL of Ag@BSA suspension under vigorous stirring (1.0 mg mL 1, 0.1 M sodium phosphate buffer, 0.15 M NaCl, pH 6.8), and then the mixture was incubated overnight at RT. Following that, the mixture was centrifuged to remove the excess glutaraldehyde. The obtained sample was redispersed in 1.0 mL of 0.5 M sodium carbonate, pH 9.5. Afterwards, 300 μL of HRP (1.0 mg mL 1) and 10 μL of Ab2 antibody (1.0 mg mL 1) were injected into the mixture. After gently shaking for 10 min, the mixture was transferred to the refrigerator at 4 1C for further reaction (overnight). During this process, HRP and anti-CEA were covalently conjugated onto the surface of Ag@BSA microspheres (Hermanson, 2008; Xin et al., 2002). Finally, the suspension was centrifuged (5000 rpm) for 10 min at 4 1C. The obtained microspheres (designated as HRP-Ag@BSA-Ab2) was re-dispersed in 1.0 mL of 0.1 M PBS (pH 7.4) and stored at 4 1C until use. 2.4. Preparation of HRP-tyramine conjugates HRP-tyramine conjugates were prepared through the typical carbodiimide coupling similar to the literature (Kandimalla et al., 2006). Brieﬂy, 600 μL of 1.0 mg mL 1 HRP was dissolved in N-2hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) (1.5 mL, 50 mM, pH 9.3) buffer, and the pH of the resulting mixture was adjusted to 7.3 with 3.0 M HCl. 15 mg of NHS and 20 mg of EDC were dissolved in the solution followed by continuous stirring for 45 min. Following that, 600 μL of 5.0 mg mL 1 tyramine were added drop by drop into the mixture under continuous stirring at 150 rpm, and left at RT for 12 h. After completion of the incubation, the conjugates were centrifuged for 10 min at 5000 rpm to remove the precipitates. Finally, the obtained conjugates were dialyzed in a dialysis bag against 0.1 M pH 7.4 PBS at RT for 24 h by changing the buffer every 6 h to remove non-conjugated tyramine. The obtained HRP-tyramine conjugates were dispersed into 500μL PBS (0. 1 M, pH 7.4) (conc. 0.5 mg mL 1) and stored at 4 1C for further use. 2.5. Preparation of the electrochemical immunosensor A glassy carbon electrode (GCE) with 3 mm in diameter was polished with 0.3 μm and 0.05 μm alumina, followed by successive sonication in distilled water and ethanol for 5 min, and dried in air. The well-polished electrode was cycled in a 0.1 M H2SO4 solution 5 times in the potential range from 0 to 2 V. During this process, the anodization of the GCE surface resulted in a multilayer oxide ﬁlm having –OH groups or –COOH groups (Collier and Tougas, 1987). Following that, 5 μL of CD aqueous solution (50 mg mL 1) was cast onto the surface of the pretreated GCE and dried at RT to form a CD-modiﬁed GCE (Zhou et al., 2012b; Tang et al., 2012). After washing with distilled water, 30 μL of Ab1 antibody (dilution ratio: 1:50) was thrown on the modiﬁed
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electrode and incubated for 4 h at RT. During this process, Ab1 antibody was immobilized on the CD-modiﬁed GCE because of the β-cyclodextrin capture. Finally, the as-prepared Ab1-CD-GCE was stored at 4 1C when not in use.
2.6. Electrochemical measurement The analytical procedure for electrochemical immunoassay of CEA is schematically depicted in Scheme 1. The analytical procedure of the electrochemical immunoassay toward CEA standards or samples was carried out with a conventional three-electrode system with a modiﬁed GCE as working electrode, a platinum foil as auxiliary electrode, and a saturated calomel electrode (SCE) as reference electrode. The assay was carried out as follows: (i) 10 μL of standards or samples with various CEA concentrations was dropped onto the Ab1-CD-GCE, and incubated for 40 min at RT to form the antigen–antibody immunocomplex; (ii) 10 μL of the HRPAg@BSA-Ab2 suspension prepared above was dropped onto the immunosensor, and incubated for another 40 min at RT to form a sandwiched immunocomplex; (iii) 5 μL of 2 mM H2O2 and 5 μL of the prepared-above HRP-tyramine conjugates were dropped on the resulting immunosensor in sequence (Note: The aim of using H2O2 was to promote the oxidization of tyramine to form reactive free radicals by HRP), and incubated for 10 min at RT; and (iv) the resulting immunosensor was determined by differential pulse voltammetry (DPV) from 0 to 600 mV (vs. SCE) with a pulse amplitude of 50 mV and a pulse width of 50 ms in pH 7.0 PBS containing 2.5 mM H2O2. After each step, the resulting immunosensor was washed with pH 7.0 PBS. All incubations and measurements were conducted at RT. All data were calculated in triplicate.
Scheme 1. Schematic illustration of (a) HRP-Ag@BSA-Ab2 and (b) HRP-tyramine conjugate, and (c) measurement principle of the electrochemical immunoassay by coupling with tyramine signal ampliﬁcation strategy.
3. Results and discussion 3.1. Characterization of the as-prepared Ag@BSA microspheres The morphology of the as-prepared Ag@BSA microspheres was characterized by using transmission electron microscope (TEM). As shown in Fig. 1(a), the as-synthesized Ag@BSA microspheres exhibited uniform dispersity with a mean size of 150 nm in diameter. Expectedly, we could also observe that a layer of thin BSA membrane was coated on the surface of silver nanocores (inset of Fig. 1(a)). To further conﬁrm the formation of BSA on the silver nanostructres, fourier transform infrared (FT-IR) spectra was also utilized (Fig. 1(b)). As is well known, the IR peaks of pure BSA at 3320, 1660 and 1540 cm 1 are assigned to the stretching vibration of –OH, amide A (mainly –NH stretching vibration), amide I (mainly CQO stretching vibrations) and amide II (the coupling of bending vibration of N–H and stretching vibration of C–N) bands, respectively (Huang et al., 2011). Those special peaks could be found in the spectrum of Ag@BSA microspheres (Fig. 1 (b)), indicating the existence of BSA in the microspheres. The result was consistent with that obtained from TEM observation. Such a nanostructure could provide an inconvenience for conjugation of biomolecules with good biocompatibility, reduce the non-speciﬁc interactions and optimize the growth niches of biomolecules. Meanwhile, the introduction of silver nanostructures could promote the electron transfer during the electrochemical measurement.
3.2. Characteristics of the electrochemical immunosensor To investigate the bioactivity of the labeled HRP/Ab2 on the Ag@BSA microspheres, we also prepared another type of signal tags (i.e., Ab2-labeled Ag@BSA, designated as Ag@BSA-Ab2) by using the mentioned-above method. Thereafter, two signal tags including Ag@BSA-Ab2 and HRP-Ag@BSA-Ab2 were directly used for the detection of 5 ng mL 1 (as an example) on the same-batch Ab1-CD-GCE. As seen from curve ‘a’ in Fig. 2(A), a couple of redox peaks was observed in pH 7.0 PBS when the as-prepared immunosensor was incubated with target CEA and Ag@BSA-Ab2 in turn. The redox peaks mainly derived from the silver/silver dioxide nanocomposites, since colloidal silver is easily oxidized to silver dioxide on the surface under the light (Yuan et al., 2008). Moreover, the peak currents were not almost changed upon addition of 2.5 mM H2O2 in pH 7.0 PBS (curve ‘b’ in Fig. 2(A)). The results indicated that the labeled Ab2 antibody on the Ag@BSA could maintain the native bioactivity for the antigen–antibody reaction. Furthermore, the redox peaks could be also achieved when using
Fig. 1. (a) TEM image (Inset: magniﬁcation image) and (b) FT-IR spectra of the as-prepared Ag@BSA microspheres.
J. Zhou et al. / Biosensors and Bioelectronics 54 (2014) 323–328
Fig. 2. Cyclic voltammograms of the immunosensor toward 5 ng mL 1 CEA by using various signal tags: (A) Ag@BSA-Ab2, (B) HRP-Ag@BSA-Ab2 and (C) HRP-Ag@BSAAb2 þ HRP-tyramine conjugates, respectively, in pH 7.0 PBS in the (a) absence and (b) presence of 2.5 mM H2O2 at 50 mV s 1.
HRP-Ag@BSA-Ab2 as signal tags in pH 7.0 PBS (curve ‘a’ in Fig. 2 (B)). Signiﬁcantly, an obvious catalytic characteristic appeared with an increase of the reduction current and a decrease of the oxidation current in pH 7.0 PBS containing 2.5 mM H2O2 (curve ‘b’ in Fig. 2(B)), which was in accordance with our previous report (Tang et al., 2011). This result suggested that the immobilized HRP on the Ag@BSA could retain high enzymatic catalytic activity and effectively shuttle electrons from the base electrode to the redox center of HRP. The electron transfer pathway occurring at the immunosensor could be assumed to be as the following reaction sequence: HRP(Fe3 þ )þH þ þ e 2HRP(Fe2 þ )
H2O2 þ 2H þ þ 2HRP(Fe2 þ )-2HRP(Fe3 þ )þ2H2O
HRP(Fe3 þ )þH þ þ e -HRP(Fe2 þ )
In the presence of H2O2, HRP(Fe2 þ ) was initially efﬁciently converted to its oxidized form [HRP(Fe3 þ )], and then HRP(Fe3 þ ) was reduced to HRP(Fe2 þ ) at the electrode surface by direct electron transfer (Tang et al., 2011). Since the conjugated amount of HRP-Ag@BSA-Ab2 on the electrode surface increased with the increasing target CEA in the sample, the increased HRP molecules could enhance the electrocatalytic response of the immunosensor relative to H2O2 system. So, the catalytic current was indirectly dependent on the concentration of target CEA. Logically, another question arises as to whether the developed TSA method could really cause the ampliﬁcation of electrochemical signal. The HRP-Ag@BSA-Ab2 was used for the determination of 5 ng mL 1 CEA accompanying with tyramine signal ampliﬁcation strategy. As shown from Fig. 2(C), a stronger shift in the cathodic current (curve ‘b’ vs. curve ‘a’) was acquired in comparison with Fig. 2(B) (curve ‘b’ vs. curve ‘a’). The reason might be most likely as the consequence of the fact that the labeled HRP on the Ag@BSA could convert tyramine derivatives to highly reactive intermediates, resulting in covalent attachment of the HRP-tyramine conjugate to the enzyme, thus increasing the binding amount of HRP. When one antibody on the Ag@BSA reacted with target CEA, all the HRP molecules on the same Ag@BSA were carried over accompanying the microspheres, and participated in the catalytic reaction. The results revealed that the designed tyramine signal ampliﬁcation strategy could be preliminarily used for the detection of CEA targets with signal ampliﬁcation. It has been reported that tyramine could be converted by the oxidation of HRP into a highly reactive short-lived free radical that bond rapidly with proteins in the vicinity of the location of HRP site (Yuan et al., 2012). BSA, as a most common blocking agent in the immunoassay, can also produce stable deposition of tyramine and bring signal interference. In contrast, hydrophilic poly(ethylene glycol) (PEG) was a material known to have a low interfacial free
Fig. 3. DPV responses of the as-prepared Ab1-CD-GCE toward (b) zero analyte and (c) 5 pg mL 1 CEA accompanying the tyramine signal ampliﬁcation strategy in pH 7.0 PBS containing 2.5 mM H2O2 (Note: Curve ‘a’ represents DPV response of the newly prepared Ab1-CD-GCE in pH 7.0 PBS containing 2.5 mM H2O2), respectively.
energy and was capable of resisting nonspeciﬁc adsorption of biomolecules including proteins (Li et al., 2005). In this work, PEG was used as surface inactivation agent for the effectiveness in passivating the surface that met the requirement for TSA. To clarify this point, the asprepared immunosensor was used for detection of 0 and 5 pg mL 1 CEA (as an example), respectively (Fig. 3). Curve ‘a’ represents the DPV response ( 0.05 μA) of the newly prepared Ab1-CD-GCE in pH 7.0 PBS containing 2.5 mM H2O2. When the immunosensor was incubated with 0 pg mL 1 CEA and excess HRP-Ag@BSA-Ab2 with the TSA in sequence, the DPV peak current was 0.47 μA (curve ‘b’). Relative to curve ‘a’, the peak current was slightly increased. The reason might be ascribed to the fact that very few HRP-Ag@BSA-Ab2 or HRP-tyramine conjugates were nonspeciﬁcally adsorbed onto the surface of the Ab1CD-GCE. Compared with that of using 5 pg mL 1 CEA as target analyte (curve ‘c’), however, the DPV peak current was only 15.6% increase [(ib–ia)/(ic–ia)100%E15.6%]. Such a high increase in the peak current might be attributed to the use of low-concentration target CEA (because the detection limit of the electrochemical immunosensor was 5 pg mL 1 CEA as seen from Section 3.4). Thus, we might expect that the value (15.6%) could be decreased when using the highconcentration CEA as target analyte. Considering this point, the nonspeciﬁc absorption of the electrochemical immunosensor should be ignorable. 3.3. Optimization of experimental conditions To acquire an optimal electrochemical response, some experimental parameters including incubation time and incubation temperature for the antigen–antibody reaction, pH of assay solution and the deposition time of HRP-tyramine conjugates were
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studied. Fig. 4(a) represents the current responses of the electrochemical immunoassay in the PBS with various pH values containing 2.5 mM H2O2 toward 5.0 ng mL 1 CEA (as an example). The currents increased with the increasing pH values from 4.5 to 7.0, and then decreased. Highly acidic or alkaline surroundings would damage the immobilized protein, especially in alkalinity. So, the optimal current was obtained at pH 7.0 PBS. Considering the practical application, all experiments were performed at RT (2571.0 1C). At this condition, the cathodic currents of the immunosensor toward 5.0 ng mL 1 CEA increased with the increment of incubation time and leveled off after 40 min (Fig. 4(b)) (Note: To avoid confusion, the incubation times of the immunosensor with CEA were paralleled with those of the immunosensor-CEA with HRP-Ag@BSA-Ab2). Longer incubation time did not signiﬁcantly increase the response. Thus, 40 min was selected as the incubation time for the antigen–antibody reaction. As mentioned above, the tyramine could be converted into a highly reactive short-lived free radical by the oxidation of HRP. When exposing the HRP-Ag@BSA-Ab2 in HRP-labeled tyramine solution, the covalent attachment of tyramine to HRP resulted in the deposition of HRP-tyramine conjugates on the electrode surface. Usually, it takes some time for the accumulation of HRPtyramine conjugates. As shown in Fig. 4(c), an acceptable signal was obtained at 10 min. Longer incubation time did not cause the large change in the current. Therefore, 10 min was employed for the deposition time of HRP-tyramine conjugates. 3.4. Analytical performance Under optimal conditions, the sensitivity and dynamic range of the electrochemical immunosensor were evaluated toward CEA
standards in pH 7.0 PBS containing 2.5 mM H2O2 by coupling with tyramine signal ampliﬁcation strategy. The DPV peak currents increased with the increasing CEA concentration (Fig. 5(a)). A calibration plot displayed a good linear relationship between the DPV peak currents and the logarithm of CEA concentration in the range from 0.005 to 80 ng mL 1, and the detection limit (LOD) calculated from the linear dependence of the signal on the concentration of the analyte was 5.0 pg mL 1. For comparison, we also investigated the analytical properties of the immunosensor by using HRP-Ag@BSA-Ab2 without tyramine signal ampliﬁcation. The linear range and detection limit were 0.01–80 ng mL 1 and 10 pg mL 1 CEA, respectively (Fig. 5(b)). Although the linear range and LOD of using tyramine signal ampliﬁcation strategy were not obviously improved, the sensitivity (i.e., the slope of the regression equation) ( 1.6171, Fig. 5(a)) was largely higher than that without tyramine signal ampliﬁcation ( 0.9562, Fig. 5(b)). The results also revealed that the developed immunoassay based on tyramine signal ampliﬁcation strategy could display stronger electrochemical signal in comparison with conventional enzyme immunoassay toward the same-concentration target analyte, thus resulting in the ampliﬁcation of detectable electrochemical signal. More favorably, the advantage of such a highly sensitive immunosensor could exactly differentiate from two very close CEA levels. The precision of the electrochemical immunosensor was evaluated by calculating the intra- and inter-batch variation coefﬁcients (CVs, n¼3). Experimental results indicated that the CVs of the assays using the same-batch immunosensor and HRP-Ag@BSA-Ab2 were 8.5%, 10.2%, and 5.7% at 0.1, 5, and 60 ng mL 1 CEA, respectively. The sensor-to-sensor reproducibility was also studied by using HRPAg@BSA-Ab2 and immunosensors with various batches. The CVs were 9.3%, 10.6%, and 8.9% at the above-mentioned analyte levels.
Fig. 4. Dependence of cathodic currents of the immunosensor on (a) pH of PBS, (b) incubation time for the antigen–antibody reaction, and (c) precipitation time for HRPtyramine conjugates (5 ng mL 1 CEA used in this case).
Fig. 5. Calibration plots of the electrochemical immunosensor toward CEA standards by using HRP-Ag@BSA-Ab2 as signal tags (a) with and (b) without the tyramine signal ampliﬁcation (Insets: the corresponding DPV curves in pH 7.0 PBS containing 2.5 mM H2O2), and (c) the speciﬁcity of the electrochemical immunosensor.
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Hence, the repeatability and intermediate precision of the developed immunoassay was acceptable. To evaluate the speciﬁcity of the electrochemical immunosensor, we challenged the system with other low-abundance proteins, such as alpha-fetoprotein (AFP), human IgG, and prostate-speciﬁc antigen (PSA). As seen from Fig. 5(c), low signals were acquired toward these components only at much higher concentrations compared to the target analyte. Therefore, the speciﬁcity of the electrochemical immunoassay was acceptable. In addition, the stability of the electrochemical immunosensor was satisfactory, and as much as 96.3% and 88.6% of the initial electrochemical signal was maintained after storage of the immunosensor and HRP-Ag@BSA-Ab2 at 4 1C for 14th day and 28th day, respectively. 3.5. Analysis of real samples and evaluation of method accuracy The as-prepared electrochemical immunosensor was employed for the analysis of real samples including 6 clinical serum specimens and 6 spiked blank new-born cattle serum samples with different CEA concentrations. The assay results were compared with those obtained by using the commercialized Electrochemiluminescent (ECL) immunoassay (provided by the Hospital). The results are listed in Table S1 of the Supporting Information. Statistical comparison of the experimental results between two methods was performed using a t-test for comparison of means preceded by the application of an F-test. As seen from Table S1, the texp values in all cases at the 0.05 signiﬁcance level were below than tcrit (tcrit[4, 0.05] ¼2.77) between two methods, thereby indicating that the developed electrochemical immunosensor could be regarded as an optional protocol for quantitative monitoring of CEA in biological ﬂuids.
4. Conclusions In this work, we devise a new sandwich-type immunoassay protocol for sensitive electrochemical determination of CEA, as a model protein, by coupling enzymatic biocatalytic precipitation of HRP-conjugated tyramine with the Ag@BSA labeling strategy. Experimental results revealed that the electrochemical immunoassay coupling with tyramine signal ampliﬁcation could exhibit higher sensitivity in comparison with conventional Ag@BSA labeling method. Highlights of this work are to (i) utilize highly conductive Ag@BSA microspheres with good biocompatibility for the labeling of biomolecules and (ii) employ the enzymatic biocatalytic in-situ precipitation of HRP-tyramine conjugates for signal ampliﬁcation. Further work should be focused on the detection of other low-abundance proteins by controlling the target antibody, thus presenting the versatility of the assay scheme.
Acknowledgments Support by the “973” National Basic Research Program of China (2010CB732403), the Research Fund for the National Science Foundation of Fujian Province (2011J06003), the Doctoral Program of Higher Education of China (20103514120003), the National Natural Science Foundation of China (21075019 & 41176079), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116) is gratefully acknowledged. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.032. References Anderson, G., Taitt, C., 2008. Biosens. Bioelectron. 24, 324–328. Bobrow, M., Harris, T., Shaughnessy, K., Litt, G., 1989. J. Immunol. Methods. 125, 279–285. Bobrow, M., Shaughnessy, K., Litt, G., 1991. J. Immunol. Methods. 137, 103–112. Collier, W., Tougas, T., 1987. Anal. Chem. 59, 396–399. Das, J., Aziz, M., Yang, H., 2006. J. Am. Chem. Soc. 128, 16022–16023. Dickerson, M., Sandhage, K., Naik, R., 2008. Chem. Rev. 108, 4935–4978. Hermanson, G.T., 2008. Bioconjugate Techniques, 2nd ed. Academic Press, San Diego, pp. 797–800. Hu, C., Yang, D., Wang, Z., Huang, P., Wang, X., Chen, D., Cui, D.X., Yang, M., Jia, N., 2013. Biosens. Bioelectron. 41, 656–662. Hu, C., Yang, D., Xu, K., Cao, H., Wu, B., Cui, D., Jia, N., 2012. Anal. Chem. 84, 10324–10331. Huang, P., Yang, D., Zhang, C., Lin, J., He, M., Bao, L., Cui, D., 2011. Nanoscale 3, 3623–3626. Kandimalla, V., Tripathi, V., Ju, H., 2006. Biomaterials 27, 1167–1174. Kim, S., Jung, K., Shin, Y., Lee, K., Yoon, Y., Choi, Y., Oh, K., Kim, M., Chung, D., Song, H., Park, S., 2002. Histochem. J. 34, 97–103. Lai, G., Yan, F., Ju, H., 2009. Anal. Chem. 81, 9730–9736. Li, L., Chen, S., Zheng, J., Ratner, B., Jiang, S., 2005. J. Phys. Chem. B 109, 2934–2941. Li, X., Yang, X., Zhang, S., 2008. TRAC Trends Anal. Chem. 27, 543–553. Lin, D., Wu, J., Wang, M., Yan, F., Ju, H., 2012. Anal. Chem. 84, 3662–3668. Liu, Y., Liu, Y., Feng, H., Wu, Y., Joshi, L., Zeng, X., Li, J., 2012. Biosens. Bioelectron. 35, 63–68. Tang, J., Tang, D., Su, B., Li, Q., Qiu, B., Chen, G., 2011. Electrochim. Acta 56, 3773–3780. Tang, J., Hou, L., Tang, D., Zhang, B., Zhou, J., Chen, G., 2012. Chem. Commun. 48, 8180–8182. Toda, Y., Kono, K., Abiru, H., Kokuryo, K., Endo, M., Yaegashi, H., Fukumoto, M., 1999. Pathol. Int. 49, 479–483. Wang, N., Gibbons, C., Freeman, R., 2011. J. Histochem. Cytochem. 59, 382–390. Weston, A., Hood, L., 2004. J. Proteome Res. 3, 179–196. Xin, W., Juan, C., Waldemar, G., 2002. Anal. Chem. 74, 5039–5046. Xu, L., Wang, S., Dong, H., Liu, G., Wen, Y., Wang, S., Zhang, X., 2012. Nanoscale 4, 3786–3790. Yuan, L., Xu, L., Liu, S., 2012. Anal. Chem. 84, 10737–10744. Yuan, P., Zhuo, Y., Chai, Y., Ju, H., 2008. Electroanalysis 20, 1839–1842. Zhou, J., Zhuang, J., Miro, M., Gao, Z., Chen, G., Tang, D., 2012a. Biosens. Bioelectron. 35, 394–400. Zhou, J., Xu, M., Tang, D., Gao, Z., Tang, J., Chen, G., 2012b. Chem. Commun. 48, 12207–12209.