Biosensors and Bioelectronics 53 (2014) 465–471
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Competitive-type displacement reaction for direct potentiometric detection of low-abundance protein Bing Zhang, Bingqian Liu, Guonan Chen, Dianping Tang n Key Laboratory of Analysis and Detection for Food Safety, Ministry of Education & Fujian Province, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 August 2013 Received in revised form 10 October 2013 Accepted 11 October 2013 Available online 24 October 2013
Prostate-specific antigen (PSA), one of the indications of possible prostate malignancy, is used as a biomarker for the diagnosis and prognosis of prostate cancer. Herein, we develop a new homogeneous potentiometric immunoassay for sensitive detection of low-concentration PSA without the need of sample separation and washing step. Two nanostructures including positively charged polyethyleneimine-poly(styrene-co-acrylic acid) (PEI-PSAA) nanospheres and negatively charged gold nanoparticles conjugated with anti-PSA antibody (Ab-AuNP) were first synthesized by using mulsifier-free emulsion copolymerization and wet chemistry method, respectively. Thereafter, the as-prepared PEI-PSAA was used as a pseudo hapten for the construction of immunosensing probe based on an electrostatic interaction between PEI-PSAA and Ab-AuNP. Upon target introduction, the added PSA competed with PEI-PASS for Ab-AuNP based on a specific antigen–antibody interaction, and displaced Ab-AuNP from PEI-PASS. The dissociated PEI-PASS was captured through the negatively charged Nafion- modified electrode, thereby resulting in the change of membrane potential. The fabrication process was characterized by using high-resolution transmission electron microscope (HRTEM), scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX), surface plasmon resonance (SPR) and dynamic laser scattering (DLS) technique. Under optimal conditions, the output signal was indirectly proportional to the concentration of target PSA in the sample and exhibited a dynamic range from 0.1 to 50 ng/mL with a detection limit (LOD) of 0.04 ng/mL. Intra- and inter-assay coefficients of variation (CVs) were 6.8 and 7.5%, respectively. In addition, the methodology was evaluated for analysis of 12 clinical serum samples and showed good accordance between the results obtained by the developed immunosensing protocol and a commercialized enzyme-linked immunosorbent assay (ELISA) method. & 2013 Elsevier B.V. All rights reserved.
Keywords: Competitive-type displacement reaction Nanogold-labeled antibody Prostate-specific antigen Potentiometric immunoassay Polyethyleneimine-poly(styrene-co-acrylic acid) microsphere
1. Introduction Immunoassay and immunosensor are usually utilized as a powerful tool for quantitative monitoring of clinically important compounds in the complex biological hierarchy (Lin et al., 2013; Jeong et al., 2013) and substances in the environment (Date et al., 2013; Zhang et al., 2012a; Tang et al., 2013) because of highly sensitive and specific antigen–antibody reaction. Various methods and strategies have been reported for this purpose based on different signal-transducer principles, e.g. fluorescence (Chen et al., 2012), electrochemistry (Wang et al., 2013a, 2013b), electrochemiluminescence (Liang et al., 2012), and colorimetric assay (Wang et al., 2013a, 2013b; Zhou et al., 2012). Recently, ongoing efforts have been made worldwide to develop and improve the clinical immunoassay with the aim of manufacturing portable and affordable diagnostic devices (Zhang et al., 2013). An alternative
n
Corresponding author. Tel.: þ 86 591 2286 6125; fax: þ86 591 2286 6135. E-mail address: [email protected] (D. Tang).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.027
approach that is based on an electrochemical principle and does not require sample separation and washing step would be advantageous because of simple instrumentation and easy signal quantification (Tang et al., 2007). Homogenous immunoassay commonly involves in the immobilization of capture antibody on the nano/microbeads and takes place in the solution, thus allowing the integration of multiple liquid handling processes (Limoges and Degrand, 1993; Kim et al., 2005; Sakashita et al., 1995). Most recently, Akhavan-Tafti et al. (2013) reported a new homogeneous immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase. Without any separation steps, the signal readout could be generated upon addition of a trigger solution. Hu et al. (2012) also designed a separation-free, electrochemical assay format with direct readout that was amenable to highly sensitive and selective quantitation of a wide variety of target proteins. For the successful development of a good homogeneous immunoassay, the construction of immunosensing platform is very crucial. Intrinsically conducting polymers have shown great
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potential for a variety of applications including sensor, anticorrosion coating, battery, capacitor, actuator, and optical device (Bubnova et al., 2012; Talemi et al., 2013). Polystyrene shows promise for commercial applications because of its simple synthesis, high conductivity, environmental stability, and biocompatibility (Uyar et al., 2010; Nakamura et al., 2005). Polystyrene composites have been shown to exhibit easy modification which could lead to biomedical applications such as cell adhesion studies (Palacios et al., 2013) or thermal ablative cancer therapy (Shi et al., 2005). Recent research has looked to develop innovative and powerful novel functionalized nanometer-sized polystyrene microspheres, controlling and tailoring their properties in a very predictable manner to meet the needs of specific applications. Lin et al. (2012) exploited polystyrene microspheres as the labels to build ultrasensitive electrochemical immunosensor for the detection of proteins. Nafion, as a perfluorosulfonate ion-exchange polymer, has been extensively used for preparation of chemically modified electrodes and the construction of ion-exchange membranes (Ensing et al., 2013; Dong et al., 2010). The unique properties of using Nafion are (i) the outstanding chemical and thermal stabilities, (ii) preconcentrating the electroactive and photoactive cations even from dilute solution, and (iii) the multiphase structure consisting of a fluorocarbon hydrophobic phase, a hydrophilic ionic cluster, and interfacial regions (Nieh et al., 2013; Hseih et al., 2014; Siracusano et al., 2013; Matos et al., 2013). The cationic molecules are exchanged into the Nafion film by both electrostatic and hydrophobic interactions due to the sulfonate head groups (–SO3 ) and a fluorocarbon chain (Ladewig et al., 2007; Chen et al., 2008). Herein, we report the proof-of-concept of a new, simple, lowcost, and powerful homogeneous potentiometric immunoassay for sensitive detection of low-abundance proteins based on targetinduced competitive-type displacement reaction between polyethyleneimine- functionalized poly (styrene-co-acrylic acid) microsphere (PEI-PSAA) and nanogold particle- labeled capture antibody (Ab-AuNP). Prostate-specific antigen (PSA), also known as gamma- seminoprotein or kallikrein-3 (KLK3), is a glycoprotein enzyme encoded in humans by the KLK3 gene, which is used as a model analyte for the development of the homogeneous immunoassay. First, the as-synthesized Ab-AuNP with negative charge is attached onto the surface of positively charged PEI-PSAA by the opposite-charged adsorption technique, which is used as an immunosensing probe for the detection of target PSA. Upon addition of target analyte, target PSA competes with PEI-PSAA for Ab-AuNP on the basis of the specific antigen–antibody reaction, and displaces the Ab-AuNP from the PEI-PSAA. The dissociated PEI-PSAA can be captured by the negatively charged Nafionmodified electrode, thus resulting in the change of membrane potential. By monitoring the shift in the potential by using a potentiometry, we can indirectly determine the concentration of target PSA in the sample.
Ultrapure water obtained from a Millipore water purification system (Z18 MΩ, Milli-Q) was used in all runs. Phosphate-buffered saline (PBS, 0.1 M, pH 7.4) solution was prepared by mixing 0.1 M NaH2PO4and 0.1 M Na2HPO4, and 0.1 M KCl was added as the supporting electrolyte. Clinical serum samples were made available by Fujian Provincial Hospital, China. 2.2. Preparation of polyethyleneimine-functionalized poly(styreneco-acrylic acid) microspheres (PEI-PSAA) Before modification, poly(styrene-co-acrylic acid) microspheres (PSAA) were synthesized according to the literatures with a little modification (Lin et al., 2012; Zhang et al., 2012b). Briefly, 150 mL of ultrapure water was initially injected into a cleaned threenecked flask submerged in an oil bath, and then the solution was purged with nitrogen for 1 h. Following that, styrene (5.0 g) and acrylic acid (1.0 g) were simultaneously added into the threenecked flask with stirring at 70 1C under the protection of nitrogen. Upon addition of K2S2O8 (0.1 g, 5 mL) solution into the mixture, the emulsifier-free emulsion copolymerization of the styrene and acrylic acid was taken place. After refluxing for 8 h, the resulting PSAA suspension was centrifuged for three times at 8000 rpm for 20 min and washed with distilled water. Afterward, PSAA microspheres were functionalized with the polyethyleneimine (PEI) by simply mixing together. Finally, the as-prepared PEIPSAA microspheres were dispersed into distilled water with a fixed concentration of 20 mg/mL, and stored at 4 1C for further use. 2.3. Synthesis of nanogold-labeled anti-PSA antibody (Ab-AuNP) First, gold nanoparticles (AuNP) with 16 nm in diameter were synthesized by reduction of chlorauric acid with trisodium citrate according to our previous reports (Zhang et al., 2012c; Gao et al., 2013). Initially, 1 mL of 1 wt% HAuCl4 aqueous solution was added to 99 mL of distilled water, and then the mixture was heated to 100 1C. Following that, 2.5 mL of sodium citrate (1 wt%) was quickly dropped into the boiling solution. During this process, the Au(III) was reduced to zero-valent Au0 (Ojea-Jiménez, Puntes, 2009; Ambrosi et al., 2007). The as-synthesized gold colloids were characterized by using transmission electron microscopy (TEM), and the mean size was 16 nm (Tang et al., 2011). Next, the as-prepared gold colloids were used for the labeling of anti-PSA antibody similar to our previous report (Gao et al., 2013). Initially, 1 mL of 16-nm gold colloids (C[Au] E24 nM) was adjusted to pH 8–9 by using Na2CO3, and then 100 μL of 0.1 mg/mL anti-PSA antibody was added to the colloids. The mixture was gently shaken several times, and stored at 4 1C for incubation overnight. Following that, the mixture was centrifuged (14 000 rpm) for 30 min at 4 1C to remove the excess antibody. The obtained soft sediment (i.e. nanogold-labeled anti-PSA antibody, designated as Ab-AuNP) was resuspended into 1 mL of PBS (pH 7.4) containing 1 wt% BSA and stored at 4 1C for further usage.
2. Experimental
2.4. Electrochemical measurement
2.1. Materials and reagents
A gold electrode (2 mm in diameter) was polished repeatedly with 0.3 and 0.05 mm alumina slurry, followed by successive sonication in distilled water and ethanol for 5 min and dried in air. Before modification, the gold electrode was initially cleaned with hot piranha solution (a 3:1 mixture of H2SO4 and H2O2. Caution!) for 10 min, and then continuously scanned within a potential range from 0.3 to 1.5 V in freshly prepared deoxygenated 0.5 M H2SO4 until a voltammogram characteristic of the clean gold electrode was established. Following that, 5 mL of 1 wt % Nafion ethanol solution was thrown on the gold electrode and then removed and parched under an infrared light for 20 min.
Mouse anti-human monoclonal prostate-specific antibody (anti-PSA, designated as Ab, 0.1 mg/mL) was purchased from Amyjet Scientific Inc (Abcam product, Wuhan, China). PSA standards with various concentrations were obtained from Biocell Biotechnol. Co., Ltd. (Zhengzhou, China). Polyethyleneimine (PEI, branched, MW 10,000, 99 wt%), polyethylene glycol (PEG, MW 6000), Nafion (5 wt%), styrene, acrylic acid, and HAuCl4 4 H2O were achieved from Alfa Aesars. All other reagents were of analytical grade and were used without further purification.
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Scheme 1. Schematic illustration of the electrochemical immunoassay based on target-induced competitive-type displacement mode. .
Subsequently, the Nafion-coated electrode was washed and kept in distilled water for 30 min. Scheme 1 represents the fabrication process of the homogeneous immunosensing platform and measurement principle. All electrochemical measurements were carried out with a CHI 620D Electrochemical Workstation (Shanghai, China) with a conventional three-electrode system using a modified gold working electrode, a platinum auxiliary electrode, and an Ag/AgCl reference electrode. Prior to measurement, the immunosensing probe was simply prepared by adding 200 μL of 20 mg/mL PEI-PSAA into 1 mL of the prepared-above Ab-AuNP suspension. During the process, the negatively charged Ab-AuNP was adsorbed onto the surface of positively charged PEI-PSAA. The excess Ab-AuNP was removed by centrifugation at 6000 rpm. Afterward, the asprepared PEI-PSAA/Ab-AuNP was dispersed into 1-mL pH 7.4 PBS for the detection of target PSA. 100 μL of PEI-PSAA/Ab-AuNP suspension was initially added into a homemade micro detection cell (Note: The gold electrode was installed at the bottom of the cell), and then 100 μL of PSA standard/or sample with various concentrations was injected into the detection cell and incubated for 30 min at room temperature. During this process, target PSA competed with PEI-PSAA for Ab-AuNP, thus resulting in the dissociation of PEI-PSAA from Ab-AuNP. The released PEI-PSAA was captured by the negatively charged Nafion membrane immobilized on the electrode. At the same time, the potentiometric measurement was carried out in pH 7.4 PBS by using open-circuit potential. The obtained potential was collected and registered as the signal of the sensor relative to the concentration of target PSA. All electrochemical measurements were done at room temperature (25 71.0 1C). Analyses are always made in triplicate.
3. Results and discussion 3.1. Construction and characteristics of the homogeneous immunosensing platform The assay procedure of the homogeneous immunoassay toward target PSA is schematically illustrated in Scheme 1. Branched polyethylenimine (PEI) contains primary, and secondary and
tertiary amino groups. PEI finds many applications in products including detergents, adhesives, water treatment agents and cosmetics. In this work, PEI-functionalized PSAA microsphere was used as a pseudo-hapten for the reaction with the labeled anti-PSA on the AuNP. Similar work on the electrostatic interaction-based immunoassay protocol has been also reported by the Yan's group (Wu et al., 2011). The association of anti-PSA antibody to the surface of AuNP was possibly due to the interaction between cysteine or NH3 þ -lysine residues of protein and gold nanoparticles (Hermanson, 2008). In the absence of target, the formed PEI-PSAA/Ab-AuNP composites were homogenously suspended in pH 7.4 PBS, thus resulting in a steady-state potentiometric response of Nafion-modified electrode. With target introduction, a competitive-type displacement react was executed between PEI-PSAA and target PSA for anti-PSA antibody binding on the AuNP due to the specific antigen–antibody reaction. The dissociated PEI-PSAA could be captured by the negatively charged Nafion membrane immobilized on the electrode, thereby causing the change of membrane potential. The shift in the potential indirectly relied on the concentration of target analyte in the sample. To realize our design, one precondition for the development of the assay protocol was whether the nanostructures with various charges between PEI-PSAA and Ab-AuNP could be formed during the synthesis. Initially, we used the microelectrophoresis (Mk II, Rank Brothers Ltd, England) to monitor these nanocomposites (Fig. 1A). The electrostatic reaction between the positively charged –NH3 þ groups of the PEI-PSAA (ζ potential¼ þ 14.7 mV) and the negatively charged antibodies on the AuNP (ζ potential ¼ 4.63 mV) in pH 7.4 PBS should be responsible for the formation of the inhibition immunosensing probe PEI-PSAA/AbAuNP (ζ potential¼ 2.5 mV). Since the labeled anti-PSA antibody on the AuNP is a kind of proteins, the isoelectric point is about 5.9 obtained by isoelectric focusing (IEF) electrophoresis. In pH 7.4 PBS, the as-prepared Ab-AuNP is negatively charged, which could adsorb the positively charged PEI-PSAA. By the same token, we also investigated the characteristics of the as-synthesized Au-AuNP and PEI-PSAA using UV–vis absorption spectroscopy (UV 1102, Techcomp, China). As shown in Fig. 1B, no absorption peak was observed at PEI-PSAA (curve ‘a’), while there were two
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Fig. 1. (A) Zeta potential profiles of various components; (B) UV–vis absorption spectra of (a) PEI-PSAA, (b) Ab-AuNP, and (c) PEG-neutralized PEI-PSAA after incubation with Ab-AuNP; (C) steady-state potentiometric responses of Nafion-modified electrode (EMF vs. time) after the as-prepared PEI-PSAA/Ab-AuNP immunosensing probes reacted with (a) 0 ng/mL PSA and (b) 50 ng/mL PSA, respectively; and (D) SPR curves of (a) Nafion-coated gold substrate, and (b) Nafion-coated gold substrate after the as-prepared PEI-PSAA/Ab-AuNP reacted with 50 ng/mL PSA in pH 7.4 PBS.
absorption peaks at 281 nm and 552 nm for the prepared Ab-AuNP (curve ‘b’). Compared with the characteristics of pure antibody (278 nm) (Tang et al., 2010) and gold colloids (518 nm) (Zhang et al., 2012c), the shift in the peak also revealed the interaction between antibody and colloidal gold (Liu et al., 2008; Hermanson, 2008). To further clarify the construction of the electrostatic interaction-based immunosensing probe, neutralization of positively charged PEI-PSAA was performed by adding negatively charged PEG into the PEI-PSAA solution consulting to the literature (Wu et al., 2011). The neutralized PEI-PSAA was incubated with negatively charged Ab-AuNP, which was investigated by using UV–vis absorption spectroscopy with the centrifuged products at 8000 rpm. As shown from curve ‘c’ in Fig. 1B, the characteristic peaks for gold colloids and antibody were not observed. The results revealed that the electrostatic interaction-based immunosensing probe could be successfully prepared by using the designed route. To further clarify the feasibility of our developed immunoassay protocol for the detection of target analyte, the newly prepared PEI-PSAA/Ab-AuNP immunosensing probes were used for qualitative monitoring of 0 and 50 ng/mL PSA (as an example), respectively. As seen from Fig. 1C, addition of target PSA (50 ng/mL) in pH 7.4 PBS containing PEI-PSAA/Ab-AuNP led to an obvious shift in the electric motive force (EMF, mV, curve ‘b’) relative to zero analyte (curve ‘a’). The reason might be most likely a consequence of the fact that the dissociated PEI-PSAA from the Ab-AuNP was adsorbed to the Nafion membrane as a result of the competition reaction of target PSA with PEI-PSAA for Ab-AuNP due to the strong and specific affinity of PSA with anti-PSA antibody
(KD ¼9.46 10 10 M) (Uludag and Tothill, 2012). Moreover, the immobilized Nafion membrane on the electrode, a polyanionic ionomer (ζ potential¼ 26.3 mV), documented well ability for trace cations (John and Ramaraj, 1996). After the immunological reaction, the Nafion film-coated electrode could be used as a sensor for the concentration of positively charged PEI-PSAA, thereby triggering the change in the EMF. Furthermore, the capture of Nafion membrane toward PEI-PSAA could also cause the angel change in the surface plasmon resonance (SPR) before (curve ‘a’) and after (curve ‘b’) the PEI-PSAA/Ab-AuNP reacted with 50 ng/mL PSA (Fig. 1D). Based on these results, we might make a conclusion that the designed immunosensing platform could be preliminarily utilized for the analysis of target PSA based on target-induced displacement mode. 3.2. Characterization of target-induced displacement reaction To verify the development of target-induced displacement reaction with the PEI-PSAA/ Ab-AuNP, we used high-resolution transmission electron microscopy (HRTEM) to investigate the immunoassay process. Fig. 2a shows the HRTEM image of the asprepared PEI-PSAA with an average size of 210 nm. When mixing with PEI-PSAA and Ab-AuNP together, many Ab-AuNP nanoparticles were attached on the surface of PEI-PSAA (Fig. 2b), indicating that Ab-AuNP could be immobilized onto the PEI-PSAA through the electrostatic interaction. Moreover, the electrostatic reaction between PEI-PSAA and Ab-AuNP could be also explained by using scanning electron microscope equipped with energy-dispersive X-ray spectroscopy (SEM-EDX) (Fig. 2d and e) and dynamic laser
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Fig. 2. (a)–(c) HRTEM images of (a) PEI-PSAA, (b) PEI-PSAA/Ab-AuNP and (c) probe ‘b’ after incubation with 50 ng/mL PSA; (d) SEM image of PEI-PSAA/Ab-AuNP; (e) EDX plots of PEI-PSAA/Ab-AuNP; (f) SEM image of Nafion-coated substrate after capturing with PEI-PSAA; and DLS data of (g) PEI-PSAA and (h) PEI-PSAA/Ab-AuNP (Insets: The corresponding real samples and original size/phase plots from DLS measurements).
scattering (DLS) technique (Fig. 2g and h). As shown from Fig. 2g and h, mixture of the negatively charged Ab-AuNP with the positively charged PEI-PSAA could result in the formation of the larger-sized nanocomposite ( 229.7 nm) in comparison with PEI-PSAA alone ( 210.9 nm). Theoretically, one PEI-PSAA microsphere could adsorb many Ab-AuNP nanoparticles, and the size of the formed PEI-PSAA/Ab-AuNP composite should be 242 nm (210þ 16 2 ¼242 nm, because the average sizes of gold nanoparticles and PEI-PSAA were 16 nm and 210 nm, respectively). However, the size of the formed PEI-PSAA/Ab-AuNP (229.7 nm) was obviously smaller than the theoretical value (242 nm), suggesting that PEI-PSAA was not completely coated by Ab-AuNP. We suspected that the reason might be attributed to the limitation of the electrostatic adsorption force (not so strong). Furthermore, we also observed that the formed PEI-PSAA/Ab-AuNP could be homogeneously dispersed in pH 7.4 PBS, as shown from the insets in Fig. 2g and h. More inspiringly, after 50 ng/mL target PSA was added into the PEI-PSAA/Ab-AuNP system, partial Ab-AuNP nanoparticles were dissociated from the PEI-PSAA, and dispersed in the solution (Fig. 2c), and the dissolved PEI-PSAA from the
nanocomposites could be captured by Nafion-modified electrode (Fig. 2f), thus suggesting that target-responsive displacement format could be utilized for the detection of PSA. 3.3. Potentiometric responses of the immunosensing probe toward PSA standards To simplify the assay process of the developed immunoassay in future, all experiments were carried out at room temperature (25 71.0 1C). At this condition, the sensitivity and working range of the potentiometric immunoassay was evaluated in pH 7.4 PBS by using Nafion-modified gold electrode after the as-prepared PEIPSAA/Ab-AuNP reacted with PSA standards with various concentrations. As seen from Fig. 3a, the electrochemical signal increased with the increasing target PSA in the sample. A linear dependence between the potential and the concentration of PSA was obtained in the range from 0.1 to 50 ng/mL with a detection limit of 0.04 ng/mL estimated at a signal-to-noise ration of 3 (Fig. 3b). The linear regression equation was E/mV¼ 167.9 LnC[PSA] (ng/mL)þ55.11 (R2 ¼ 0.9887, n¼27). Since the threshold value of PSA was 4 ng/mL
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Fig. 3. (a) Time-dependent EMF responses of the developed immunosensing platform toward target PSA with various concentrations, and (b) calibration curve.
Fig. 4. (a) The specificity of the developed immunosensing probe toward PSA, CEA, AFP, LH, TSH and IgG, and (b) comparison of the assayed results for clinical serum samples by using the developed immunoassay and commercialized PSA ELISA kit.
in normal human serum (Wu et al., 2007), the developed immunoassay method could completely meet the requirement of clinical diagnosis toward PSA.
clearly demonstrated the high specificity of the developed immunoassay. 3.5. Analysis of real sample and evaluation of method accuracy
3.4. Reproducibility, stability and selectivity The precision of the developed immunoassay was examined by repeatedly assaying 10 ng/mL PSA (as an example), using identical batched of PEI-PSAA and Ab-AuNP throughout. Experimental results indicated that the coefficient of variation (CV) of the intra-assay between 6 runs was 6.8%, whereas the CV of the inter-assay with various batches was 7.5% (n ¼6), indicating good precision and acceptable fabrication reproducibility. When the asprepared PEI-PSAA/Ab-AuNP was not in use, it was stored in pH 7.4 PBS at 4 1C. No obvious change in the electrochemical signal was observed after storage for two months but a 7.3% decrease of the signal was noticed after 4 months. Further, the specificity of the developed immunoassay was also evaluated by challenging the system with other components, e.g. carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and human IgG. As indicated from Fig. 4a, higher electrochemical signals could be obtained with target PSA than those of other materials. More significantly, the presence of interfering materials in the target PSA solution did not obviously change the electrochemical signal of the developed immunoassay. These results
To evaluate the accuracy of the electrochemical immunoassay, 12 clinical serum specimens, collected from Fujian Provincial Hospital of China according to the rules of the local ethical committee, were detected by using our developed immunoassay. The assayed results were compared with those of using commercialized PSA ELISA kit. The judgment was performed via the use of a regression method between the obtained results of using two methods (Fig. 4b). The regression line was fitted to y¼0.672xþ 0.875 (R2 ¼0.9903, n¼30) where x stands for the PSA concentrations estimated with the developed immunoassay and y stands for those of the reference procedure. The correlation between the two methods was also investigated using t-tests. As shown in Table 1, the texp values in all samples were less than tcrit (tcrit[4, 0.05] ¼ 2.77), thereby revealing a good agreement between two analytical methods.
4. Conclusion In summary, this work describes a new electrochemical immunoassay for the detection of target PSA by coupling target-induced competitive-type displacement reaction with one-step potentiometric measurement. Compared with other electrochemical immunoassays,
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Table 1 Comparison of the assay results for human serum specimens by using the developed immunoassay and the referenced ELISA method. Sample no.
Found by the developed immunoassay (mean7 SD, ng/mL, n¼ 3)
Found by the ELISA (mean7 SD, ng/mL, n ¼3)
texp
1 2 3 4 5 6 7 8 9 10 11 12
57 0.4 15.8 7 0.9 0.7 7 0.3 0.08 7 0.5 12.7 7 0.2 21.6 7 2.1 6.9 7 0.5 1.6 7 0.2 20.3 7 2 0.2 7 2.8 0.8 7 3.2 23.4 7 4.3
5.7 7 0.3 16.4 7 1.3 1.2 7 0.2 0.17 0.6 12.4 7 0.2 197 1.3 7.3 7 0.4 1.4 7 0.2 19.7 7 1.6 0.5 7 1.6 1.17 2.7 18.7 7 2.9
2.42 0.65 2.40 0.04 1.83 1.82 1.08 1.22 0.41 0.16 0.12 1.57
highlight of this work is to avoid complicated reaction schemes and special construction. Meanwhile, the developed electrochemical immunoassay is simple and user-friendly without the need of sample separation and washing steps. Importantly, the method does not need sophisticated instruments, and can be utilized by the public for quantitative monitoring of other biomolecules by controlling the used target antibody, thus representing a versatile detection scheme. Acknowledgements This work was financially supported by the National “973” Basic Research Program of China (2010CB732403), the National Natural Science Foundation of China (41176079, 21075019), the Doctoral Program of Higher Education of China (20103514120003), the National Science Foundation of Fujian Province (2011J06003), the China-Russia Bilateral Scientific Cooperation Research Program (NSFC/RFBR) (21211120157), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116). The authors also thank Prof. Jing Tang for HRTEM assistance. References Akhavan-Tafti, H., Binger, D., Blackwood, J., Chen, Y., Creager, R., Silva, R., Eickholt, R., Gaibor, J., Handley, R., Kapsner, K., Lopac, S., Mazelis, M., McLernon, T., Mendoza, J., Odegaard, B., Reddy, S., Salvati, M., Schoenfelner, B., Shapir, N., Shelly, K., Todtleben, J., Wang, G., Xie, W., 2013. J. Am. Chem. Soc. 135 (11), 4191–4194. Ambrosi, A., Castaňeda, M., Killard, A., Smyth, M., Alegret, S., Merkoci, A., 2007. Anal. Chem. 79 (14), 5232–5240. Bubnova, O., Berggren, M., Crispin, X., 2012. J. Am. Chem. Soc. 134 (40), 16456–16459.
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Competitive-type displacement reaction for direct potentiometric detection of low-abundance protein Bing Zhang, Bingqian Liu, Guonan Chen, Dianping Tang n Key Laboratory of Analysis and Detection for Food Safety, Ministry of Education & Fujian Province, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 August 2013 Received in revised form 10 October 2013 Accepted 11 October 2013 Available online 24 October 2013
Prostate-specific antigen (PSA), one of the indications of possible prostate malignancy, is used as a biomarker for the diagnosis and prognosis of prostate cancer. Herein, we develop a new homogeneous potentiometric immunoassay for sensitive detection of low-concentration PSA without the need of sample separation and washing step. Two nanostructures including positively charged polyethyleneimine-poly(styrene-co-acrylic acid) (PEI-PSAA) nanospheres and negatively charged gold nanoparticles conjugated with anti-PSA antibody (Ab-AuNP) were first synthesized by using mulsifier-free emulsion copolymerization and wet chemistry method, respectively. Thereafter, the as-prepared PEI-PSAA was used as a pseudo hapten for the construction of immunosensing probe based on an electrostatic interaction between PEI-PSAA and Ab-AuNP. Upon target introduction, the added PSA competed with PEI-PASS for Ab-AuNP based on a specific antigen–antibody interaction, and displaced Ab-AuNP from PEI-PASS. The dissociated PEI-PASS was captured through the negatively charged Nafion- modified electrode, thereby resulting in the change of membrane potential. The fabrication process was characterized by using high-resolution transmission electron microscope (HRTEM), scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX), surface plasmon resonance (SPR) and dynamic laser scattering (DLS) technique. Under optimal conditions, the output signal was indirectly proportional to the concentration of target PSA in the sample and exhibited a dynamic range from 0.1 to 50 ng/mL with a detection limit (LOD) of 0.04 ng/mL. Intra- and inter-assay coefficients of variation (CVs) were 6.8 and 7.5%, respectively. In addition, the methodology was evaluated for analysis of 12 clinical serum samples and showed good accordance between the results obtained by the developed immunosensing protocol and a commercialized enzyme-linked immunosorbent assay (ELISA) method. & 2013 Elsevier B.V. All rights reserved.
Keywords: Competitive-type displacement reaction Nanogold-labeled antibody Prostate-specific antigen Potentiometric immunoassay Polyethyleneimine-poly(styrene-co-acrylic acid) microsphere
1. Introduction Immunoassay and immunosensor are usually utilized as a powerful tool for quantitative monitoring of clinically important compounds in the complex biological hierarchy (Lin et al., 2013; Jeong et al., 2013) and substances in the environment (Date et al., 2013; Zhang et al., 2012a; Tang et al., 2013) because of highly sensitive and specific antigen–antibody reaction. Various methods and strategies have been reported for this purpose based on different signal-transducer principles, e.g. fluorescence (Chen et al., 2012), electrochemistry (Wang et al., 2013a, 2013b), electrochemiluminescence (Liang et al., 2012), and colorimetric assay (Wang et al., 2013a, 2013b; Zhou et al., 2012). Recently, ongoing efforts have been made worldwide to develop and improve the clinical immunoassay with the aim of manufacturing portable and affordable diagnostic devices (Zhang et al., 2013). An alternative
n
Corresponding author. Tel.: þ 86 591 2286 6125; fax: þ86 591 2286 6135. E-mail address: [email protected] (D. Tang).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.027
approach that is based on an electrochemical principle and does not require sample separation and washing step would be advantageous because of simple instrumentation and easy signal quantification (Tang et al., 2007). Homogenous immunoassay commonly involves in the immobilization of capture antibody on the nano/microbeads and takes place in the solution, thus allowing the integration of multiple liquid handling processes (Limoges and Degrand, 1993; Kim et al., 2005; Sakashita et al., 1995). Most recently, Akhavan-Tafti et al. (2013) reported a new homogeneous immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase. Without any separation steps, the signal readout could be generated upon addition of a trigger solution. Hu et al. (2012) also designed a separation-free, electrochemical assay format with direct readout that was amenable to highly sensitive and selective quantitation of a wide variety of target proteins. For the successful development of a good homogeneous immunoassay, the construction of immunosensing platform is very crucial. Intrinsically conducting polymers have shown great
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potential for a variety of applications including sensor, anticorrosion coating, battery, capacitor, actuator, and optical device (Bubnova et al., 2012; Talemi et al., 2013). Polystyrene shows promise for commercial applications because of its simple synthesis, high conductivity, environmental stability, and biocompatibility (Uyar et al., 2010; Nakamura et al., 2005). Polystyrene composites have been shown to exhibit easy modification which could lead to biomedical applications such as cell adhesion studies (Palacios et al., 2013) or thermal ablative cancer therapy (Shi et al., 2005). Recent research has looked to develop innovative and powerful novel functionalized nanometer-sized polystyrene microspheres, controlling and tailoring their properties in a very predictable manner to meet the needs of specific applications. Lin et al. (2012) exploited polystyrene microspheres as the labels to build ultrasensitive electrochemical immunosensor for the detection of proteins. Nafion, as a perfluorosulfonate ion-exchange polymer, has been extensively used for preparation of chemically modified electrodes and the construction of ion-exchange membranes (Ensing et al., 2013; Dong et al., 2010). The unique properties of using Nafion are (i) the outstanding chemical and thermal stabilities, (ii) preconcentrating the electroactive and photoactive cations even from dilute solution, and (iii) the multiphase structure consisting of a fluorocarbon hydrophobic phase, a hydrophilic ionic cluster, and interfacial regions (Nieh et al., 2013; Hseih et al., 2014; Siracusano et al., 2013; Matos et al., 2013). The cationic molecules are exchanged into the Nafion film by both electrostatic and hydrophobic interactions due to the sulfonate head groups (–SO3 ) and a fluorocarbon chain (Ladewig et al., 2007; Chen et al., 2008). Herein, we report the proof-of-concept of a new, simple, lowcost, and powerful homogeneous potentiometric immunoassay for sensitive detection of low-abundance proteins based on targetinduced competitive-type displacement reaction between polyethyleneimine- functionalized poly (styrene-co-acrylic acid) microsphere (PEI-PSAA) and nanogold particle- labeled capture antibody (Ab-AuNP). Prostate-specific antigen (PSA), also known as gamma- seminoprotein or kallikrein-3 (KLK3), is a glycoprotein enzyme encoded in humans by the KLK3 gene, which is used as a model analyte for the development of the homogeneous immunoassay. First, the as-synthesized Ab-AuNP with negative charge is attached onto the surface of positively charged PEI-PSAA by the opposite-charged adsorption technique, which is used as an immunosensing probe for the detection of target PSA. Upon addition of target analyte, target PSA competes with PEI-PSAA for Ab-AuNP on the basis of the specific antigen–antibody reaction, and displaces the Ab-AuNP from the PEI-PSAA. The dissociated PEI-PSAA can be captured by the negatively charged Nafionmodified electrode, thus resulting in the change of membrane potential. By monitoring the shift in the potential by using a potentiometry, we can indirectly determine the concentration of target PSA in the sample.
Ultrapure water obtained from a Millipore water purification system (Z18 MΩ, Milli-Q) was used in all runs. Phosphate-buffered saline (PBS, 0.1 M, pH 7.4) solution was prepared by mixing 0.1 M NaH2PO4and 0.1 M Na2HPO4, and 0.1 M KCl was added as the supporting electrolyte. Clinical serum samples were made available by Fujian Provincial Hospital, China. 2.2. Preparation of polyethyleneimine-functionalized poly(styreneco-acrylic acid) microspheres (PEI-PSAA) Before modification, poly(styrene-co-acrylic acid) microspheres (PSAA) were synthesized according to the literatures with a little modification (Lin et al., 2012; Zhang et al., 2012b). Briefly, 150 mL of ultrapure water was initially injected into a cleaned threenecked flask submerged in an oil bath, and then the solution was purged with nitrogen for 1 h. Following that, styrene (5.0 g) and acrylic acid (1.0 g) were simultaneously added into the threenecked flask with stirring at 70 1C under the protection of nitrogen. Upon addition of K2S2O8 (0.1 g, 5 mL) solution into the mixture, the emulsifier-free emulsion copolymerization of the styrene and acrylic acid was taken place. After refluxing for 8 h, the resulting PSAA suspension was centrifuged for three times at 8000 rpm for 20 min and washed with distilled water. Afterward, PSAA microspheres were functionalized with the polyethyleneimine (PEI) by simply mixing together. Finally, the as-prepared PEIPSAA microspheres were dispersed into distilled water with a fixed concentration of 20 mg/mL, and stored at 4 1C for further use. 2.3. Synthesis of nanogold-labeled anti-PSA antibody (Ab-AuNP) First, gold nanoparticles (AuNP) with 16 nm in diameter were synthesized by reduction of chlorauric acid with trisodium citrate according to our previous reports (Zhang et al., 2012c; Gao et al., 2013). Initially, 1 mL of 1 wt% HAuCl4 aqueous solution was added to 99 mL of distilled water, and then the mixture was heated to 100 1C. Following that, 2.5 mL of sodium citrate (1 wt%) was quickly dropped into the boiling solution. During this process, the Au(III) was reduced to zero-valent Au0 (Ojea-Jiménez, Puntes, 2009; Ambrosi et al., 2007). The as-synthesized gold colloids were characterized by using transmission electron microscopy (TEM), and the mean size was 16 nm (Tang et al., 2011). Next, the as-prepared gold colloids were used for the labeling of anti-PSA antibody similar to our previous report (Gao et al., 2013). Initially, 1 mL of 16-nm gold colloids (C[Au] E24 nM) was adjusted to pH 8–9 by using Na2CO3, and then 100 μL of 0.1 mg/mL anti-PSA antibody was added to the colloids. The mixture was gently shaken several times, and stored at 4 1C for incubation overnight. Following that, the mixture was centrifuged (14 000 rpm) for 30 min at 4 1C to remove the excess antibody. The obtained soft sediment (i.e. nanogold-labeled anti-PSA antibody, designated as Ab-AuNP) was resuspended into 1 mL of PBS (pH 7.4) containing 1 wt% BSA and stored at 4 1C for further usage.
2. Experimental
2.4. Electrochemical measurement
2.1. Materials and reagents
A gold electrode (2 mm in diameter) was polished repeatedly with 0.3 and 0.05 mm alumina slurry, followed by successive sonication in distilled water and ethanol for 5 min and dried in air. Before modification, the gold electrode was initially cleaned with hot piranha solution (a 3:1 mixture of H2SO4 and H2O2. Caution!) for 10 min, and then continuously scanned within a potential range from 0.3 to 1.5 V in freshly prepared deoxygenated 0.5 M H2SO4 until a voltammogram characteristic of the clean gold electrode was established. Following that, 5 mL of 1 wt % Nafion ethanol solution was thrown on the gold electrode and then removed and parched under an infrared light for 20 min.
Mouse anti-human monoclonal prostate-specific antibody (anti-PSA, designated as Ab, 0.1 mg/mL) was purchased from Amyjet Scientific Inc (Abcam product, Wuhan, China). PSA standards with various concentrations were obtained from Biocell Biotechnol. Co., Ltd. (Zhengzhou, China). Polyethyleneimine (PEI, branched, MW 10,000, 99 wt%), polyethylene glycol (PEG, MW 6000), Nafion (5 wt%), styrene, acrylic acid, and HAuCl4 4 H2O were achieved from Alfa Aesars. All other reagents were of analytical grade and were used without further purification.
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Scheme 1. Schematic illustration of the electrochemical immunoassay based on target-induced competitive-type displacement mode. .
Subsequently, the Nafion-coated electrode was washed and kept in distilled water for 30 min. Scheme 1 represents the fabrication process of the homogeneous immunosensing platform and measurement principle. All electrochemical measurements were carried out with a CHI 620D Electrochemical Workstation (Shanghai, China) with a conventional three-electrode system using a modified gold working electrode, a platinum auxiliary electrode, and an Ag/AgCl reference electrode. Prior to measurement, the immunosensing probe was simply prepared by adding 200 μL of 20 mg/mL PEI-PSAA into 1 mL of the prepared-above Ab-AuNP suspension. During the process, the negatively charged Ab-AuNP was adsorbed onto the surface of positively charged PEI-PSAA. The excess Ab-AuNP was removed by centrifugation at 6000 rpm. Afterward, the asprepared PEI-PSAA/Ab-AuNP was dispersed into 1-mL pH 7.4 PBS for the detection of target PSA. 100 μL of PEI-PSAA/Ab-AuNP suspension was initially added into a homemade micro detection cell (Note: The gold electrode was installed at the bottom of the cell), and then 100 μL of PSA standard/or sample with various concentrations was injected into the detection cell and incubated for 30 min at room temperature. During this process, target PSA competed with PEI-PSAA for Ab-AuNP, thus resulting in the dissociation of PEI-PSAA from Ab-AuNP. The released PEI-PSAA was captured by the negatively charged Nafion membrane immobilized on the electrode. At the same time, the potentiometric measurement was carried out in pH 7.4 PBS by using open-circuit potential. The obtained potential was collected and registered as the signal of the sensor relative to the concentration of target PSA. All electrochemical measurements were done at room temperature (25 71.0 1C). Analyses are always made in triplicate.
3. Results and discussion 3.1. Construction and characteristics of the homogeneous immunosensing platform The assay procedure of the homogeneous immunoassay toward target PSA is schematically illustrated in Scheme 1. Branched polyethylenimine (PEI) contains primary, and secondary and
tertiary amino groups. PEI finds many applications in products including detergents, adhesives, water treatment agents and cosmetics. In this work, PEI-functionalized PSAA microsphere was used as a pseudo-hapten for the reaction with the labeled anti-PSA on the AuNP. Similar work on the electrostatic interaction-based immunoassay protocol has been also reported by the Yan's group (Wu et al., 2011). The association of anti-PSA antibody to the surface of AuNP was possibly due to the interaction between cysteine or NH3 þ -lysine residues of protein and gold nanoparticles (Hermanson, 2008). In the absence of target, the formed PEI-PSAA/Ab-AuNP composites were homogenously suspended in pH 7.4 PBS, thus resulting in a steady-state potentiometric response of Nafion-modified electrode. With target introduction, a competitive-type displacement react was executed between PEI-PSAA and target PSA for anti-PSA antibody binding on the AuNP due to the specific antigen–antibody reaction. The dissociated PEI-PSAA could be captured by the negatively charged Nafion membrane immobilized on the electrode, thereby causing the change of membrane potential. The shift in the potential indirectly relied on the concentration of target analyte in the sample. To realize our design, one precondition for the development of the assay protocol was whether the nanostructures with various charges between PEI-PSAA and Ab-AuNP could be formed during the synthesis. Initially, we used the microelectrophoresis (Mk II, Rank Brothers Ltd, England) to monitor these nanocomposites (Fig. 1A). The electrostatic reaction between the positively charged –NH3 þ groups of the PEI-PSAA (ζ potential¼ þ 14.7 mV) and the negatively charged antibodies on the AuNP (ζ potential ¼ 4.63 mV) in pH 7.4 PBS should be responsible for the formation of the inhibition immunosensing probe PEI-PSAA/AbAuNP (ζ potential¼ 2.5 mV). Since the labeled anti-PSA antibody on the AuNP is a kind of proteins, the isoelectric point is about 5.9 obtained by isoelectric focusing (IEF) electrophoresis. In pH 7.4 PBS, the as-prepared Ab-AuNP is negatively charged, which could adsorb the positively charged PEI-PSAA. By the same token, we also investigated the characteristics of the as-synthesized Au-AuNP and PEI-PSAA using UV–vis absorption spectroscopy (UV 1102, Techcomp, China). As shown in Fig. 1B, no absorption peak was observed at PEI-PSAA (curve ‘a’), while there were two
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Fig. 1. (A) Zeta potential profiles of various components; (B) UV–vis absorption spectra of (a) PEI-PSAA, (b) Ab-AuNP, and (c) PEG-neutralized PEI-PSAA after incubation with Ab-AuNP; (C) steady-state potentiometric responses of Nafion-modified electrode (EMF vs. time) after the as-prepared PEI-PSAA/Ab-AuNP immunosensing probes reacted with (a) 0 ng/mL PSA and (b) 50 ng/mL PSA, respectively; and (D) SPR curves of (a) Nafion-coated gold substrate, and (b) Nafion-coated gold substrate after the as-prepared PEI-PSAA/Ab-AuNP reacted with 50 ng/mL PSA in pH 7.4 PBS.
absorption peaks at 281 nm and 552 nm for the prepared Ab-AuNP (curve ‘b’). Compared with the characteristics of pure antibody (278 nm) (Tang et al., 2010) and gold colloids (518 nm) (Zhang et al., 2012c), the shift in the peak also revealed the interaction between antibody and colloidal gold (Liu et al., 2008; Hermanson, 2008). To further clarify the construction of the electrostatic interaction-based immunosensing probe, neutralization of positively charged PEI-PSAA was performed by adding negatively charged PEG into the PEI-PSAA solution consulting to the literature (Wu et al., 2011). The neutralized PEI-PSAA was incubated with negatively charged Ab-AuNP, which was investigated by using UV–vis absorption spectroscopy with the centrifuged products at 8000 rpm. As shown from curve ‘c’ in Fig. 1B, the characteristic peaks for gold colloids and antibody were not observed. The results revealed that the electrostatic interaction-based immunosensing probe could be successfully prepared by using the designed route. To further clarify the feasibility of our developed immunoassay protocol for the detection of target analyte, the newly prepared PEI-PSAA/Ab-AuNP immunosensing probes were used for qualitative monitoring of 0 and 50 ng/mL PSA (as an example), respectively. As seen from Fig. 1C, addition of target PSA (50 ng/mL) in pH 7.4 PBS containing PEI-PSAA/Ab-AuNP led to an obvious shift in the electric motive force (EMF, mV, curve ‘b’) relative to zero analyte (curve ‘a’). The reason might be most likely a consequence of the fact that the dissociated PEI-PSAA from the Ab-AuNP was adsorbed to the Nafion membrane as a result of the competition reaction of target PSA with PEI-PSAA for Ab-AuNP due to the strong and specific affinity of PSA with anti-PSA antibody
(KD ¼9.46 10 10 M) (Uludag and Tothill, 2012). Moreover, the immobilized Nafion membrane on the electrode, a polyanionic ionomer (ζ potential¼ 26.3 mV), documented well ability for trace cations (John and Ramaraj, 1996). After the immunological reaction, the Nafion film-coated electrode could be used as a sensor for the concentration of positively charged PEI-PSAA, thereby triggering the change in the EMF. Furthermore, the capture of Nafion membrane toward PEI-PSAA could also cause the angel change in the surface plasmon resonance (SPR) before (curve ‘a’) and after (curve ‘b’) the PEI-PSAA/Ab-AuNP reacted with 50 ng/mL PSA (Fig. 1D). Based on these results, we might make a conclusion that the designed immunosensing platform could be preliminarily utilized for the analysis of target PSA based on target-induced displacement mode. 3.2. Characterization of target-induced displacement reaction To verify the development of target-induced displacement reaction with the PEI-PSAA/ Ab-AuNP, we used high-resolution transmission electron microscopy (HRTEM) to investigate the immunoassay process. Fig. 2a shows the HRTEM image of the asprepared PEI-PSAA with an average size of 210 nm. When mixing with PEI-PSAA and Ab-AuNP together, many Ab-AuNP nanoparticles were attached on the surface of PEI-PSAA (Fig. 2b), indicating that Ab-AuNP could be immobilized onto the PEI-PSAA through the electrostatic interaction. Moreover, the electrostatic reaction between PEI-PSAA and Ab-AuNP could be also explained by using scanning electron microscope equipped with energy-dispersive X-ray spectroscopy (SEM-EDX) (Fig. 2d and e) and dynamic laser
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Fig. 2. (a)–(c) HRTEM images of (a) PEI-PSAA, (b) PEI-PSAA/Ab-AuNP and (c) probe ‘b’ after incubation with 50 ng/mL PSA; (d) SEM image of PEI-PSAA/Ab-AuNP; (e) EDX plots of PEI-PSAA/Ab-AuNP; (f) SEM image of Nafion-coated substrate after capturing with PEI-PSAA; and DLS data of (g) PEI-PSAA and (h) PEI-PSAA/Ab-AuNP (Insets: The corresponding real samples and original size/phase plots from DLS measurements).
scattering (DLS) technique (Fig. 2g and h). As shown from Fig. 2g and h, mixture of the negatively charged Ab-AuNP with the positively charged PEI-PSAA could result in the formation of the larger-sized nanocomposite ( 229.7 nm) in comparison with PEI-PSAA alone ( 210.9 nm). Theoretically, one PEI-PSAA microsphere could adsorb many Ab-AuNP nanoparticles, and the size of the formed PEI-PSAA/Ab-AuNP composite should be 242 nm (210þ 16 2 ¼242 nm, because the average sizes of gold nanoparticles and PEI-PSAA were 16 nm and 210 nm, respectively). However, the size of the formed PEI-PSAA/Ab-AuNP (229.7 nm) was obviously smaller than the theoretical value (242 nm), suggesting that PEI-PSAA was not completely coated by Ab-AuNP. We suspected that the reason might be attributed to the limitation of the electrostatic adsorption force (not so strong). Furthermore, we also observed that the formed PEI-PSAA/Ab-AuNP could be homogeneously dispersed in pH 7.4 PBS, as shown from the insets in Fig. 2g and h. More inspiringly, after 50 ng/mL target PSA was added into the PEI-PSAA/Ab-AuNP system, partial Ab-AuNP nanoparticles were dissociated from the PEI-PSAA, and dispersed in the solution (Fig. 2c), and the dissolved PEI-PSAA from the
nanocomposites could be captured by Nafion-modified electrode (Fig. 2f), thus suggesting that target-responsive displacement format could be utilized for the detection of PSA. 3.3. Potentiometric responses of the immunosensing probe toward PSA standards To simplify the assay process of the developed immunoassay in future, all experiments were carried out at room temperature (25 71.0 1C). At this condition, the sensitivity and working range of the potentiometric immunoassay was evaluated in pH 7.4 PBS by using Nafion-modified gold electrode after the as-prepared PEIPSAA/Ab-AuNP reacted with PSA standards with various concentrations. As seen from Fig. 3a, the electrochemical signal increased with the increasing target PSA in the sample. A linear dependence between the potential and the concentration of PSA was obtained in the range from 0.1 to 50 ng/mL with a detection limit of 0.04 ng/mL estimated at a signal-to-noise ration of 3 (Fig. 3b). The linear regression equation was E/mV¼ 167.9 LnC[PSA] (ng/mL)þ55.11 (R2 ¼ 0.9887, n¼27). Since the threshold value of PSA was 4 ng/mL
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Fig. 3. (a) Time-dependent EMF responses of the developed immunosensing platform toward target PSA with various concentrations, and (b) calibration curve.
Fig. 4. (a) The specificity of the developed immunosensing probe toward PSA, CEA, AFP, LH, TSH and IgG, and (b) comparison of the assayed results for clinical serum samples by using the developed immunoassay and commercialized PSA ELISA kit.
in normal human serum (Wu et al., 2007), the developed immunoassay method could completely meet the requirement of clinical diagnosis toward PSA.
clearly demonstrated the high specificity of the developed immunoassay. 3.5. Analysis of real sample and evaluation of method accuracy
3.4. Reproducibility, stability and selectivity The precision of the developed immunoassay was examined by repeatedly assaying 10 ng/mL PSA (as an example), using identical batched of PEI-PSAA and Ab-AuNP throughout. Experimental results indicated that the coefficient of variation (CV) of the intra-assay between 6 runs was 6.8%, whereas the CV of the inter-assay with various batches was 7.5% (n ¼6), indicating good precision and acceptable fabrication reproducibility. When the asprepared PEI-PSAA/Ab-AuNP was not in use, it was stored in pH 7.4 PBS at 4 1C. No obvious change in the electrochemical signal was observed after storage for two months but a 7.3% decrease of the signal was noticed after 4 months. Further, the specificity of the developed immunoassay was also evaluated by challenging the system with other components, e.g. carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and human IgG. As indicated from Fig. 4a, higher electrochemical signals could be obtained with target PSA than those of other materials. More significantly, the presence of interfering materials in the target PSA solution did not obviously change the electrochemical signal of the developed immunoassay. These results
To evaluate the accuracy of the electrochemical immunoassay, 12 clinical serum specimens, collected from Fujian Provincial Hospital of China according to the rules of the local ethical committee, were detected by using our developed immunoassay. The assayed results were compared with those of using commercialized PSA ELISA kit. The judgment was performed via the use of a regression method between the obtained results of using two methods (Fig. 4b). The regression line was fitted to y¼0.672xþ 0.875 (R2 ¼0.9903, n¼30) where x stands for the PSA concentrations estimated with the developed immunoassay and y stands for those of the reference procedure. The correlation between the two methods was also investigated using t-tests. As shown in Table 1, the texp values in all samples were less than tcrit (tcrit[4, 0.05] ¼ 2.77), thereby revealing a good agreement between two analytical methods.
4. Conclusion In summary, this work describes a new electrochemical immunoassay for the detection of target PSA by coupling target-induced competitive-type displacement reaction with one-step potentiometric measurement. Compared with other electrochemical immunoassays,
B. Zhang et al. / Biosensors and Bioelectronics 53 (2014) 465–471
Table 1 Comparison of the assay results for human serum specimens by using the developed immunoassay and the referenced ELISA method. Sample no.
Found by the developed immunoassay (mean7 SD, ng/mL, n¼ 3)
Found by the ELISA (mean7 SD, ng/mL, n ¼3)
texp
1 2 3 4 5 6 7 8 9 10 11 12
57 0.4 15.8 7 0.9 0.7 7 0.3 0.08 7 0.5 12.7 7 0.2 21.6 7 2.1 6.9 7 0.5 1.6 7 0.2 20.3 7 2 0.2 7 2.8 0.8 7 3.2 23.4 7 4.3
5.7 7 0.3 16.4 7 1.3 1.2 7 0.2 0.17 0.6 12.4 7 0.2 197 1.3 7.3 7 0.4 1.4 7 0.2 19.7 7 1.6 0.5 7 1.6 1.17 2.7 18.7 7 2.9
2.42 0.65 2.40 0.04 1.83 1.82 1.08 1.22 0.41 0.16 0.12 1.57
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