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A super intramolecular self-enhanced electrochemiluminescence immunosensor based on polymer chains grafted on palladium nanocages Haijun Wang, Ying He, Yaqin Chai* and Ruo Yuan* An intramolecular self-enhanced electrochemiluminescent derivative is prepared by grafting polystyrene (PS)-based polymer chains with pendant Ru(II) luminophore from poly(ethylenimine) (PEI) on the surface of palladium nanocages (PdNCs). In this way, the Ru(II) luminophore and its co-reactive group (amine groups in PEI) exist in the same polymer molecule, which shortens the electronic transmission distance between them and enhances the luminous stability. Meanwhile, through atom transfer radical polymerization (ATRP), the loading amount of Ru(II) luminophore is greatly increased. Therefore, the obtained electrochemiluminescent derivative (PdNC–PEI–PSRu) has high luminous efficiency and stability. Furthermore, due to their special nanostructures of porous walls and hollow interiors, PdNCs have great advantages in high specific surface areas and good electrocatalytic ability, which make them
Received 22nd May 2014 Accepted 27th June 2014 DOI: 10.1039/c4nr02808b www.rsc.org/nanoscale
act as an excellent immobilized platform for PEI and detection antibody. Based on the sandwiched immunoreactions, a sensitive “signal on” electrochemiluminescence immunosensor is constructed for the detection of carbohydrate antigen 15-3 (CA 15-3). As a result, a wide linear range from 0.01 U mL1 to 120 U mL1 is acquired with a relatively low detection limit of 0.003 U mL1.
Introduction With the rising mortality in cancer patients, early diagnosis of disease combined with an effective treatment offers the best chance of survival.1,2 A sensitive and specic method for detecting and quantifying cancer-related biomarkers is essential for early detection of cancer.3 However, recent clinical studies have indicated that some biomarker concentrations are lower than the detection limit of some diagnostic immunoassay technologies. Therefore, more sensitive detection is required for earlier detection of certain diseases. Due to its high sensitivity and wide dynamic concentration response range, electrochemiluminescence (ECL) has received much attention and become a valuable detection method in analytical chemistry.4 Ruthenium(II) tris(2,20 -bipyridyl) (Ru(bpy)32+) and its derivatives, as the most commonly used ECL luminophores, have special advantages in chemical stability, reversible electrochemical behavior, and good compatibility to different pH levels.5,6 Previous research studies indicate that introducing a coreactant into the ECL system can signicantly enhance the ECL intensity and effectively improve the detection sensitivity.7 The common co-reactants of Ru(bpy)32+ and its derivatives are
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China. E-mail: [email protected]; [email protected]; Fax: +86-23-68253172; Tel: +86-023-68252277
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tripropylamine (TPA),8 2-(dibutylamino)-ethanol (DBAE),9 2,5dimethyl-thiophene (DMT)10 and so on. Most of these co-reactants are water soluble small molecules, which are usually added into the detection solution to amplify the ECL signal. However, the biological toxicity and volatile nature of those coreactants sophisticate the operation and increase the measurement error. In order to solve this problem, great efforts have been made by scientists. Sun and the co-workers immobilized the co-reactant on the surface of the electrode by the assistance of aptamers to enhance the ECL signal.11 Kurita and the co-workers used acetylcholinesterase to catalyze acetylcholine to generate thiocholine in situ for ECL signal amplication.12 Although the two methods above could enhance the detection sensitivity compared to the traditional methods, there are still some shortcomings. The intermolecular interaction between the luminescent reagents and the corresponding coreactants presents defects in poor stability, low efficiency of electron transfer which is usually accompanied by the loss of energy. Meanwhile, the complicated operation, high cost and easy inactivation of immobilization of co-reactants and labeling of enzyme limit their application. Therefore, the intramolecular self-enhanced ECL is proposed in this work, which makes luminophores and their co-reactive groups exist in the same molecular structure for shortening the electronic transmission distance, improving the luminous stability and enhancing the luminous efficiency. In order to increase the loading amount and further enhance the ECL signal, some nanomaterials and auxiliary approaches
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have been used in the construction of ECL biosensors. Currently, in order to improve their properties, various types of metal nanostructures have been synthesized, such as nanospheres,13 nanorods,14 nanowheels,15 etc. Due to the relatively lower densities and higher specic surface areas, their performance could be further improved by processing those nanostructures into hollow ones.16 Here, cage-like nanostructures, with porous walls and hollow interiors, are synthesized through galvanic replacement reactions which use silver nanocubes as a template. The higher specic surface areas and good electrocatalytic activity make them become excellent candidates in the fabrication of biosensors. In addition, atom transfer radical polymerization (ATRP) has been proposed as a potential method to achieve both high stability of polymer layers and high gra density for monomer modication.17 In this work, ATRP is employed for local accumulation of the polystyrene (PS)-based monomer. Growth of polymer chains provides numerous binding sites for the coupling of bis(2,20 bipyridine)(5-amino-1,10-phenanthroline)ruthenium(II)dichloride [Ru(bpy)2(5-NH2-1,10-phen)Cl2]. In this way, the loading amount of the Ru(II) complex is greatly increased. The obtained new polymer with a pendant Ru(II) complex has enhanced luminous efficiency. Herein, an intramolecular self-enhanced ECL immunosensor based on polymer chains graed on the palladium nanocage was constructed for the detection of carbohydrate antigen 15-3 (CA 15-3). Palladium nanocages (PdNCs), with porous walls and hollow interiors, were synthesized through a galvanic replacement reaction, which were used to load abundant poly(ethylenimine) (PEI), an effective co-reactant to the ECL of the Ru(II) complex. The resulted PdNC–PEI composite was applied to immobilize 2-bromoisobutyl bromide which was an initiator to the ATRP reaction. Aer the ATRP reaction of 2,5dioxopyrrolidin-1-yl-4-vinylbenzoate (NHVB), polystyrene (PS)based polymer chains graed on the PdNC–PEI composite were obtained. Then, Ru(bpy)2(5-NH2-1,10-phen)Cl2 was decorated onto the as-prepared composite to acquire a new intramolecular self-enhanced electrochemiluminescent derivative (PdNC–PEI–PSRu). Owing to the high specic surface areas, PdNC–PEI–PSRu was applied to immobilize the detection antibody with bovine serum albumin (BSA) blocking the nonspecic adsorption sites. In addition, owing to the enhanced electron transport rate, immobilization ability and structural stability, gold nanoparticle (AuNP) functionalized graphene (Gra) was used as an ECL substrate to immobilize the capture antibody.18 Then, based on the sandwiched immunoreactions, a “signal on” ECL immunosensor was constructed for CA 15-3 by using AuNP functionalized Gra as an ECL substrate and PdNC–PEI– PSRu as a signal tracer. With the intramolecular self-enhanced effect of the prepared electrochemiluminescent derivative, the immunosensor had a good response to CA 15-3.
Experimental section Reagents and apparatus Gold chloride (HAuCl4) was purchased from Kangda Amino Acid company (Shanghai, China). Poly(ethylenimine) (PEI, 50%)
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was from Fluka (Switzerland). Graphene (Gra) was obtained from Nanjing xianfeng nano Co. (Nanjing, China). Bis(2,20 bipyridine)(5-amino-1,10-phenanthroline)ruthenium(II)dichloride [Ru(bpy)2(5-NH2-1,10-phen)Cl2] was obtained from Suna Tech Inc. (Suzhou, China). AgNO3, K2PdCl4, poly(vinyl pyrrolidone) (PVP), ethylene glycol (99.8%), NaOH, toluene and bovine serum albumin (BSA, 96–99%) were purchased from Sigma Chemical (St. Louis, MO, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) were acquired from Shanghai Medpep Co. (Shanghai, China). Carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA 15-3) and its antibody were obtained from Biocell Company (Zhengzhou, China). Streptococcus suis serotype 2 (SS2) was bought from Bioxincheer (Beijing, China). The standard CA 15-3 stock solutions were prepared with PBS (pH 7.4) and stored at 4 C. The serum specimens were obtained from Southwest Hospital (Chongqing, China). Phosphate-buffered solution (PBS) (pH 7.4, 0.1 M) was prepared with 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. Double distilled water was used throughout this study. The ECL emission was monitored with a model MPI-A ECL analyzer (Xi'an Remax Electronic science & Technology Co. Ltd., Xi'an, china). The voltage of the photomultiplier tube (PMT) was set at 800 V and the potential scan was from 0.2 to 1.25 V with a scan rate of 100 mV s1 in the process of ECL detection. Electrochemical impedance spectroscopy (EIS) was carried out with a CHI 660A electrochemical workstation (Shanghai Chenhua Instrument, China). A conventional three-electrode system was used, in which an Ag/AgCl (sat. KCl) was the reference electrode, a platinum wire was the counter electrode and the modied glassy carbon electrode (GCE) was the working electrode. The morphologies of different nanocomposites were characterized by scanning electron microscopy (SEM, S-4800, Hitachi, Japan) at an acceleration voltage of 15–20 kV. X-ray photoelectron spectroscopy (XPS) measurements were carried out using a VG Scientic ESCALAB 250 spectrometer (Thermoelectricity Instruments, USA) and using Al Ka X-ray (1486.6 eV) as the light source.
Synthesis of PdNCs Firstly, silver nanocubes were prepared according to the previous reference with some minor modications.19 Anhydrous ethylene glycol (30 mL) was heated at 160 C for 1 h at rst. Then, 10 mL of an ethylene glycol solution of PVP (0.2 M) and 3.5 mL aliquot of an ethylene glycol solution of AgNO3 (0.29 M) were successively added into the hot ethylene glycol. The reaction mixture was then handled with heating, reuxing and magnetic stirring at 160 C for 40 min. Silver nanocubes were obtained by centrifugation and redispersion. Aer that, using silver nanocubes as templates, PdNCs were obtained through galvanic replacement reaction: K2PdCl4 (5 mL, 0.05 M) was added into the as-prepared silver nanocube solution (20 mL). The mixture was reuxed and heated for 40 min with magnetic stirring during the entire process. Then, PdNCs were obtained by centrifugation and washed with water, and then suspended in distilled water for further use.
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Preparation of Ab and BSA adsorbed PdNC–PEI–PSRu (PdNC–PEI–PSRu@Ab–BSA) The preparation process of the PdNC–PEI–PSRu@Ab–BSA bioconjugate is shown in Scheme 1. Firstly, 3 mL of the as-prepared PdNC solution was mixed with 2 mL PEI (1%), followed by stirring for about 12 h. As a result, PEI functionalized PdNCs (PdNC–PEI) were obtained, in which a lot of PEI existed. Then, 2-bromoisobutyl bromide, an initiator for ATRP reaction, was modied on the as-obtained PdNC–PEI by using EDC and NHS as coupling agents. ATRP reaction of 2,5-dioxopyrrolidin-1-yl-4vinylbenzoate (NHVB) was carried out in DMSO at 80 C with CuBr/1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA) as the catalyst and 2-bromoisobutyl bromide functionalized PdNC–PEI as the initiator to obtain a polystyrene (PS)-based polymer chains graed on PdNC–PEI. Aer that, 5 mg Ru(bpy)2(5-NH2-1,10-phen)Cl2 was added into the as-prepared PdNC–PEI–PS solution and stirred at ambient temperature overnight. The complex of PdNC–PEI–PSRu was acquired by centrifugation (the detailed reaction process of the ATRP reaction is shown in Scheme 2). Then, 0.3 mL Ab and 0.2 mL BSA were slowly added into 3 mL PdNC–PEI–PSRu solution sequentially under soly stirring and incubated for 8 h at 4 C, respectively. Followed by centrifugation at 9000 rpm for 15 min at 4 C to discard excess reagents, the bioconjugate of PdNC–PEI–PSRu@Ab–BSA was obtained.
Scheme 2
The detailed reaction process of the ATRP reaction.
Fabrication of the ECL immunosensor The fabrication process for the ECL immunosensor is schematized in Scheme 3. Prior to modication, the GCE (4 ¼ 4 mm) was polished with 0.3, 0.05 mm alumina slurry respectively, followed by rinsing thoroughly with bi-distilled water and sonicating in ethanol and bi-distilled water separately. The cleaned GCE was rstly coated with 5 mL Gra and gold nanoparticles (AuNPs) homogeneous suspensions dispersed with chitosan (Chi, 0.6%) solution. Aer drying in air, 18 mL capture antibody was dropped onto the electrode followed by incubating at 4 C for 12 h. Then, 16 mL of BSA (1%) solution was placed onto the electrode for 40 min at room temperature to block the remaining active sites. Subsequently, the resulted electrode was incubated in CA 15-3 antigen solution for 20 min at room temperature. Finally, as a sandwich format, the
Scheme 3 Schematic diagram of the preparation and reaction mechanism of the ECL immunosensor.
obtained electrode was immersed in the PdNC–PEI–PSRu@ Ab–BSA solution for immune reaction. The modied electrode was thoroughly cleaned with PBS (pH 7.4) to remove the nonchemisorbed species aer every modied step.
Results and discussion Characterization of the different nanomaterials
1 The preparation of the PdNC–PEI–PSRu@Ab–BSA bioconjugate.
Scheme
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The morphologies of different nanomaterials were characterized by SEM. The lamellate mono-layer sheets could be observed clearly in Fig. 1A. Comparing with the Gra, the homogeneous coverage of spherical AuNPs with a diameter of 18 2 nm on the Gra surface could be found in Fig. 1B. According to Fig. 1C, silver nanostructures had cube-like shapes with a diameter of 160 20 nm. As shown in Fig. 1D, a large number of particles stacked cages with a diameter of 250 30 nm were clearly observed aer the galvanic replacement reaction. Using silver
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SEM images of (A) Gra, (B) Gra–AuNPs, (C) Ag nanocubes, and (D) PdNCs.
Fig. 1
Fig. 3 A) EIS profiles of (a) Bare GCE, (b) GCE/Chi–Gra–AuNPs, (c) GCE/Chi–Gra–AuNPs/Ab, (d) GCE/Chi–Gra–AuNPs/Ab/BSA, (e) GCE/ Chi–Gra–AuNPs/Ab/BSA/Ag, and (f) GCE/Chi–Gra–AuNPs/Ab/BSA/ Ag/PdNC–PEI–PSRu@Ab–BSA in 5 mM [Fe(CN)6]3/4 containing 0.1 M KCl. (B) ECL profiles in the fabricated process of the immunosensor before (black curve) and after (red curve) the incubation of PdNC–PEI–PSRu@Ab–BSA bioconjugate (the immunosensor was incubated with 15 U mL1 CA 15-3 and measured in PBS (0.1 M, pH 7.4); the PMT was set at 800 V and the potential scan was from 0.2 to 1.25 V). Fig. 2
XPS analysis for the full region of XPS for PdNC–PEI–PSRu.
nanocubes as templates, the obtained PdNCs retained partial cubic prototype. In order to prove the successfully preparation of PdNC–PEI–PSRu, X-ray photoelectron spectroscopy (XPS) was performed for the elemental analysis. As shown in Fig. 2, the peaks at 284.6 eV, 400.5 eV, 485.9 eV and 530.9 eV could be respectively assigned to C1s, N1s, Ru3p, and O1s XPS spectra, which suggested the presence of the polymer chains functionalized with Ru(II) luminophore and co-reactive group (amine groups). Moreover, the XPS doublet of Pd3d (342.3 eV and 337.2 eV) indicated the existence of the PdNCs. According to the elemental analysis results, we could conrm that the PdNC–PEI–PSRu was successfully prepared.
Characterization of the ECL immunosensor The stepwise fabrication process of the ECL immunosensor was characterized by electrochemical impedance spectroscopy (EIS) in 5 mM [Fe(CN)6]3/4 containing 0.1 M KCl (Fig. 3A). Owing to low resistance to the redox probe dissolved in solution, the bare GCE had a small semicircle (curve a). When Gra and AuNP homogeneous suspensions dispersed with chitosan
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(Chi–Gra–AuNPs) were added dropwise on the GCE, a decreased impedance value was obtained (curve b). Aer the successive immobilization of capture antibody, BSA, and CA 15-3 antigen (15 U mL1), the impedance values increased in order (curves c– e), which is because the formation of protein molecule layers hindered the electron transfer. However, aer the incubation of the PdNC–PEI–PSRu@Ab–BSA bioconjugate, the impedance value was signicantly reduced (curve f), the reason of which was that the materials in the bioconjugate promoted the electron transfer. Furthermore, the proposed immunosensor was characterized by ECL in PBS (0.1 M, pH 7.4). As shown in Fig. 3B, there was almost no ECL response before the incubation of the PdNC–PEI–PSRu@Ab–BSA bioconjugate (black curve) because there was no ECL luminophore. However, due to the existence of the intramolecular self-enhanced electrochemiluminescent derivative, the ECL signal was greatly improved when the PdNC–PEI–PSRu@Ab–BSA bioconjugate was immobilized on the electrode (red curve). Comparison of the immunosensor with different Ab bioconjugates In order to prove the superiority of the immunosensor with the proposed PdNC–PEI–PSRu@Ab–BSA bioconjugate. Contrast
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experiments were conducted by comparing the ECL responses of the immunosensor with different probes under the same conditions. Five kinds of Ab-functionalized probes were prepared: (a) PdNC–PEI–PSRu@Ab–BSA (target probe), (b) PdNC–PEI–Ru@Ab–BSA, (c) PdNC–PSRu@Ab–BSA, (d) PdNP–PEI–PSRu@Ab–BSA (using palladium nanoparticles (PdNPs) instead of PdNCs) and (e) PEI–PSRu@Ab. The same batch of immunosensors were prepared and incubated with 15 U mL1 CA 15-3, then incubated with different Ab-functionalized probe solutions, respectively. As shown in Fig. 4, about 3002.7 a.u. ECL emission was obtained by the immunosensor with the target probe (PdNC–PEI–PSRu@Ab–BSA) compared with background values (Fig. 4A). However, the ECL responses were decreased to 2071.3 a.u., 1059.5 a.u., 932.3 a.u and 488.1 a.u. when the immunosensor was incubated with PdNC–PEI– Ru@Ab–BSA, PEI–PSRu@Ab and PdNC–PSRu@Ab–BSA, respectively (Fig. 4D, B, E and C). The reason could be as follows: rstly, PdNCs, with porous walls and hollow interiors, possessed excellent electrocatalytic ability and high specic surface area, which was better than solid naonostructures such as PdNPs; secondly, the loading amount of Ru(II) luminophore in the target probe was greatly increased through ATRP reaction; thirdly, comparing directly conjugating PEI with antibody, using PdNCs as an immobilized platform had advantages in more binding sites and better electric conductivity; nally, the novel self-enhanced ECL mechanisms of Ru(II) luminophore functionalized PEI greatly enhanced the luminous efficiency.
Fig. 4 The ECL comparison between the immunosensor with different Ab bioconjugates: (A) PdNC–PEI–PSRu@Ab–BSA, (B) PdNC–PEI– Ru@Ab–BSA, (C) PdNC–PSRu@Ab–BSA, (D) PdNP–PEI–PSRu@ Ab–BSA and (E) PEI–PSRu@Ab (the immunosensor was incubated with 15 U mL1 CA 15-3 and measured in PBS (0.1 M, pH 7.4); the PMT was set at 800 V and the potential scan was from 0.2 to 1.25 V).
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The possible luminous mechanisms of the self-enhanced ECL derivative The possible mechanisms to the ECL of PdNC–PEI–PSRu were described with the following equations: PdNC–PEI–PSRu(II) 2e / PdNC–PEI_+–PSRu(III)
(1)
PdNC–PEI_+–PSRu(III) / PdNC–PEI_–PSRu(III) + H+
(2)
PdNC–PEI_–PSRu(III) / PdNC–PEI–PSRu*(II)
(3)
PdNC–PEI–PSRu*(II) / PdNC–PEI–PSRu(II) + hv
(4)
With the potential scan from 0.2 to 1.25 V, both the luminophore and the co-reactive groups in the complex of PdNC–PEI–PSRu(II) are oxidized. Then, aer the intramolecular electron transfer and energy transmission, the strong oxidant intermediate (PdNC–PEI_–PSRu(III)) changes to the excited state (PdNC–PEI–PSRu*(II)). Finally, the enhanced ECL emission was obtained with PdNC–PEI–PSRu*(II) returning back to PdNC–PEI–PSRu(II). ECL detection of CA 15-3 with the obtained immunosensor The quantitative measurement of the proposed ECL immunosensor to CA 15-3 antigen was explored by using a sandwichtype format. Fig. 5 shows the relationship between the ECL intensity and deferent concentrations of CA 15-3, in which the ECL intensity increased with the increasing concentration of CA 15-3 (Fig. 5, curves a–h). The inset in Fig. 5 presented the calibration curve of the immunosensor to CA 15-3, where the ECL intensity had positive correlation to the logarithm of CA 15-3 concentrations. The linear equation was I ¼ 1993.0 + 830.4 log c with a correlation coefficient of 0.9956 (where I is the ECL intensity and c is the concentration of CA 15-3). The linear range for CA 15-3 was from 0.01 U mL1 to 120 U mL1 with a relatively low detection limit of 0.003 U mL1. Table 1 shows the detection results of immunosensors with previous reports.20–22 Compared to other methods, the immunosensor had a lower
ECL profiles of the immunosensor in the presence of different concentrations of CA 15-3 (a–h). Inset: calibration curve for CA 15-3 detection. The concentrations of CA 15-3: (a) 0.01 U mL1, (b) 0.1 U mL1, (c) 1 U mL1, (d) 5 U mL1, (e) 15 U mL1, (f) 30 U mL1, (g) 60 U mL1, and (h) 120 U mL1. All ECL signals were measured in PBS (0.1 M, pH 7.4). Fig. 5
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Table 1 Comparison of our research with other methods for CA 15-3 detection
Method
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Electrochemical immunoassay Chemiluminescence Optical sensing ECL
Linear range Detection limit (U mL1) (U mL1) References 1–100
0.3
20
0–250 1.25–25 0.01–120
0.035 0.4 0.003
21 22 Present work
detection limit, and might hold a new promise for highly sensitive bioassays applied in clinical detection. Selectivity, stability and reproducibility of the ECL immunosensor
Table 2
Recovery results of the proposed immunosensor in human
serum
Sample number
Added (U mL1)
Found (U mL1)
Recovery (%)
1 2 3 5
1 5 15 30
0.9382 5.078 16.27 27.87
93.82 101.6 108.5 92.89
(between 92.89% and 108.5%) was obtained, which suggested that the proposed immunosensor provided a potential tool for determining CA 15-3 in real biological samples.
Conclusions
The selectivity of the immunosensor was monitored by the assistance of some interfering agents such as carcinoembryonic antigen (CEA) and Streptococcus suis serotype 2 (SS2). As shown in Fig. 6A, the ECL signal was similar to that of the blank sample when the immunosensor was incubated in the SS2 solution. And no obvious changes were observed on comparing the ECL with interfering agents to that obtained in the pure CA 15-3, which indicated that the immunosensor possessed good selectivity for the detection of CA 15-3. Simultaneously, the stability of the ECL immunosensor was evaluated under consecutive cyclic potential scans for 34 cycles. As shown in Fig. 6B, the ECL intensity did not show any obvious changes with relative standard deviations (RSD) of 1.86%. The outstanding stability may be attributed to the excellent luminous stability of the intramolecular self-enhanced electrochemiluminescent derivative. The reproducibility of the proposed immunosensor was evaluated by the RSD (ECL response) of intra- and inter-assays. The RSDs of the intra- and inter-assay were not more than 5%, which implied that the reproducibility of the proposed immunoassay was acceptable.
In this work, an intramolecular self-enhanced ECL immunosensor based on polymer chains graed on the PdNCs was constructed for cancer-related biomarkers. The prepared complex of PdNC–PEI–PSRu was a novel electrochemiluminescent derivative which included the Ru(II) luminophore and its co-reactive group (amine groups) in the same polymer molecule. Through this design, the electronic transmission distance between the luminophore and its co-reactive group was greatly shortened, by which the luminous efficiency and stability were enhanced remarkably. Moreover, by the assistance of ATRP reaction and PdNCs with high specic surface areas, the loading amounts of Ru(II) luminophore and co-reactive group (amine groups) were signicantly improved. Based on this self-enhanced electrochemiluminescent derivative, the proposed ECL immunosensor had a sensitive response to CA 15-3 with good selectivity and reproducibility, acceptable precision and accuracy. This work provided a new method for signal amplication of ECL and would extend the application of ECL in bioanalysis.
Preliminary analysis of real samples
Acknowledgements
Recovery experiments were explored in human serum to monitor the feasibility of the ECL immunosensor by the standard addition method. According to Table 2, the recovery
This work was nancially supported by the NNSF of China (21075100, 21275119), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), Ministry of Education of China (Project 708073), Natural Science Foundation of Chongqing City (CSTC-2010BB4121, CSTC-2011BA7003) and the Fundamental Research Funds for the Central Universities (XDJK2012A004, XDJK2013A008, and XDJK2013A027).
Notes and references (A) Comparison of ECL responses with different antigens: blank; SS2 (20 ng mL1); CA 15-3 (15 U mL1); a mixture containing CA 15-3 (15 U mL1) and SS2 (20 ng mL1); a mixture containing CA 15-3 (15 U mL1), SS2 (20 ng mL1) and CEA (20 ng mL1). (B) The ECL stability of the proposed immunosensor under consecutive cyclic potential scans. Fig. 6
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A super intramolecular self-enhanced electrochemiluminescence immunosensor based on polymer chains grafted on palladium nanocages Haijun Wang, Ying He, Yaqin Chai* and Ruo Yuan* An intramolecular self-enhanced electrochemiluminescent derivative is prepared by grafting polystyrene (PS)-based polymer chains with pendant Ru(II) luminophore from poly(ethylenimine) (PEI) on the surface of palladium nanocages (PdNCs). In this way, the Ru(II) luminophore and its co-reactive group (amine groups in PEI) exist in the same polymer molecule, which shortens the electronic transmission distance between them and enhances the luminous stability. Meanwhile, through atom transfer radical polymerization (ATRP), the loading amount of Ru(II) luminophore is greatly increased. Therefore, the obtained electrochemiluminescent derivative (PdNC–PEI–PSRu) has high luminous efficiency and stability. Furthermore, due to their special nanostructures of porous walls and hollow interiors, PdNCs have great advantages in high specific surface areas and good electrocatalytic ability, which make them
Received 22nd May 2014 Accepted 27th June 2014 DOI: 10.1039/c4nr02808b www.rsc.org/nanoscale
act as an excellent immobilized platform for PEI and detection antibody. Based on the sandwiched immunoreactions, a sensitive “signal on” electrochemiluminescence immunosensor is constructed for the detection of carbohydrate antigen 15-3 (CA 15-3). As a result, a wide linear range from 0.01 U mL1 to 120 U mL1 is acquired with a relatively low detection limit of 0.003 U mL1.
Introduction With the rising mortality in cancer patients, early diagnosis of disease combined with an effective treatment offers the best chance of survival.1,2 A sensitive and specic method for detecting and quantifying cancer-related biomarkers is essential for early detection of cancer.3 However, recent clinical studies have indicated that some biomarker concentrations are lower than the detection limit of some diagnostic immunoassay technologies. Therefore, more sensitive detection is required for earlier detection of certain diseases. Due to its high sensitivity and wide dynamic concentration response range, electrochemiluminescence (ECL) has received much attention and become a valuable detection method in analytical chemistry.4 Ruthenium(II) tris(2,20 -bipyridyl) (Ru(bpy)32+) and its derivatives, as the most commonly used ECL luminophores, have special advantages in chemical stability, reversible electrochemical behavior, and good compatibility to different pH levels.5,6 Previous research studies indicate that introducing a coreactant into the ECL system can signicantly enhance the ECL intensity and effectively improve the detection sensitivity.7 The common co-reactants of Ru(bpy)32+ and its derivatives are
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China. E-mail: [email protected]; [email protected]; Fax: +86-23-68253172; Tel: +86-023-68252277
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tripropylamine (TPA),8 2-(dibutylamino)-ethanol (DBAE),9 2,5dimethyl-thiophene (DMT)10 and so on. Most of these co-reactants are water soluble small molecules, which are usually added into the detection solution to amplify the ECL signal. However, the biological toxicity and volatile nature of those coreactants sophisticate the operation and increase the measurement error. In order to solve this problem, great efforts have been made by scientists. Sun and the co-workers immobilized the co-reactant on the surface of the electrode by the assistance of aptamers to enhance the ECL signal.11 Kurita and the co-workers used acetylcholinesterase to catalyze acetylcholine to generate thiocholine in situ for ECL signal amplication.12 Although the two methods above could enhance the detection sensitivity compared to the traditional methods, there are still some shortcomings. The intermolecular interaction between the luminescent reagents and the corresponding coreactants presents defects in poor stability, low efficiency of electron transfer which is usually accompanied by the loss of energy. Meanwhile, the complicated operation, high cost and easy inactivation of immobilization of co-reactants and labeling of enzyme limit their application. Therefore, the intramolecular self-enhanced ECL is proposed in this work, which makes luminophores and their co-reactive groups exist in the same molecular structure for shortening the electronic transmission distance, improving the luminous stability and enhancing the luminous efficiency. In order to increase the loading amount and further enhance the ECL signal, some nanomaterials and auxiliary approaches
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have been used in the construction of ECL biosensors. Currently, in order to improve their properties, various types of metal nanostructures have been synthesized, such as nanospheres,13 nanorods,14 nanowheels,15 etc. Due to the relatively lower densities and higher specic surface areas, their performance could be further improved by processing those nanostructures into hollow ones.16 Here, cage-like nanostructures, with porous walls and hollow interiors, are synthesized through galvanic replacement reactions which use silver nanocubes as a template. The higher specic surface areas and good electrocatalytic activity make them become excellent candidates in the fabrication of biosensors. In addition, atom transfer radical polymerization (ATRP) has been proposed as a potential method to achieve both high stability of polymer layers and high gra density for monomer modication.17 In this work, ATRP is employed for local accumulation of the polystyrene (PS)-based monomer. Growth of polymer chains provides numerous binding sites for the coupling of bis(2,20 bipyridine)(5-amino-1,10-phenanthroline)ruthenium(II)dichloride [Ru(bpy)2(5-NH2-1,10-phen)Cl2]. In this way, the loading amount of the Ru(II) complex is greatly increased. The obtained new polymer with a pendant Ru(II) complex has enhanced luminous efficiency. Herein, an intramolecular self-enhanced ECL immunosensor based on polymer chains graed on the palladium nanocage was constructed for the detection of carbohydrate antigen 15-3 (CA 15-3). Palladium nanocages (PdNCs), with porous walls and hollow interiors, were synthesized through a galvanic replacement reaction, which were used to load abundant poly(ethylenimine) (PEI), an effective co-reactant to the ECL of the Ru(II) complex. The resulted PdNC–PEI composite was applied to immobilize 2-bromoisobutyl bromide which was an initiator to the ATRP reaction. Aer the ATRP reaction of 2,5dioxopyrrolidin-1-yl-4-vinylbenzoate (NHVB), polystyrene (PS)based polymer chains graed on the PdNC–PEI composite were obtained. Then, Ru(bpy)2(5-NH2-1,10-phen)Cl2 was decorated onto the as-prepared composite to acquire a new intramolecular self-enhanced electrochemiluminescent derivative (PdNC–PEI–PSRu). Owing to the high specic surface areas, PdNC–PEI–PSRu was applied to immobilize the detection antibody with bovine serum albumin (BSA) blocking the nonspecic adsorption sites. In addition, owing to the enhanced electron transport rate, immobilization ability and structural stability, gold nanoparticle (AuNP) functionalized graphene (Gra) was used as an ECL substrate to immobilize the capture antibody.18 Then, based on the sandwiched immunoreactions, a “signal on” ECL immunosensor was constructed for CA 15-3 by using AuNP functionalized Gra as an ECL substrate and PdNC–PEI– PSRu as a signal tracer. With the intramolecular self-enhanced effect of the prepared electrochemiluminescent derivative, the immunosensor had a good response to CA 15-3.
Experimental section Reagents and apparatus Gold chloride (HAuCl4) was purchased from Kangda Amino Acid company (Shanghai, China). Poly(ethylenimine) (PEI, 50%)
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was from Fluka (Switzerland). Graphene (Gra) was obtained from Nanjing xianfeng nano Co. (Nanjing, China). Bis(2,20 bipyridine)(5-amino-1,10-phenanthroline)ruthenium(II)dichloride [Ru(bpy)2(5-NH2-1,10-phen)Cl2] was obtained from Suna Tech Inc. (Suzhou, China). AgNO3, K2PdCl4, poly(vinyl pyrrolidone) (PVP), ethylene glycol (99.8%), NaOH, toluene and bovine serum albumin (BSA, 96–99%) were purchased from Sigma Chemical (St. Louis, MO, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) were acquired from Shanghai Medpep Co. (Shanghai, China). Carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA 15-3) and its antibody were obtained from Biocell Company (Zhengzhou, China). Streptococcus suis serotype 2 (SS2) was bought from Bioxincheer (Beijing, China). The standard CA 15-3 stock solutions were prepared with PBS (pH 7.4) and stored at 4 C. The serum specimens were obtained from Southwest Hospital (Chongqing, China). Phosphate-buffered solution (PBS) (pH 7.4, 0.1 M) was prepared with 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. Double distilled water was used throughout this study. The ECL emission was monitored with a model MPI-A ECL analyzer (Xi'an Remax Electronic science & Technology Co. Ltd., Xi'an, china). The voltage of the photomultiplier tube (PMT) was set at 800 V and the potential scan was from 0.2 to 1.25 V with a scan rate of 100 mV s1 in the process of ECL detection. Electrochemical impedance spectroscopy (EIS) was carried out with a CHI 660A electrochemical workstation (Shanghai Chenhua Instrument, China). A conventional three-electrode system was used, in which an Ag/AgCl (sat. KCl) was the reference electrode, a platinum wire was the counter electrode and the modied glassy carbon electrode (GCE) was the working electrode. The morphologies of different nanocomposites were characterized by scanning electron microscopy (SEM, S-4800, Hitachi, Japan) at an acceleration voltage of 15–20 kV. X-ray photoelectron spectroscopy (XPS) measurements were carried out using a VG Scientic ESCALAB 250 spectrometer (Thermoelectricity Instruments, USA) and using Al Ka X-ray (1486.6 eV) as the light source.
Synthesis of PdNCs Firstly, silver nanocubes were prepared according to the previous reference with some minor modications.19 Anhydrous ethylene glycol (30 mL) was heated at 160 C for 1 h at rst. Then, 10 mL of an ethylene glycol solution of PVP (0.2 M) and 3.5 mL aliquot of an ethylene glycol solution of AgNO3 (0.29 M) were successively added into the hot ethylene glycol. The reaction mixture was then handled with heating, reuxing and magnetic stirring at 160 C for 40 min. Silver nanocubes were obtained by centrifugation and redispersion. Aer that, using silver nanocubes as templates, PdNCs were obtained through galvanic replacement reaction: K2PdCl4 (5 mL, 0.05 M) was added into the as-prepared silver nanocube solution (20 mL). The mixture was reuxed and heated for 40 min with magnetic stirring during the entire process. Then, PdNCs were obtained by centrifugation and washed with water, and then suspended in distilled water for further use.
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Preparation of Ab and BSA adsorbed PdNC–PEI–PSRu (PdNC–PEI–PSRu@Ab–BSA) The preparation process of the PdNC–PEI–PSRu@Ab–BSA bioconjugate is shown in Scheme 1. Firstly, 3 mL of the as-prepared PdNC solution was mixed with 2 mL PEI (1%), followed by stirring for about 12 h. As a result, PEI functionalized PdNCs (PdNC–PEI) were obtained, in which a lot of PEI existed. Then, 2-bromoisobutyl bromide, an initiator for ATRP reaction, was modied on the as-obtained PdNC–PEI by using EDC and NHS as coupling agents. ATRP reaction of 2,5-dioxopyrrolidin-1-yl-4vinylbenzoate (NHVB) was carried out in DMSO at 80 C with CuBr/1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA) as the catalyst and 2-bromoisobutyl bromide functionalized PdNC–PEI as the initiator to obtain a polystyrene (PS)-based polymer chains graed on PdNC–PEI. Aer that, 5 mg Ru(bpy)2(5-NH2-1,10-phen)Cl2 was added into the as-prepared PdNC–PEI–PS solution and stirred at ambient temperature overnight. The complex of PdNC–PEI–PSRu was acquired by centrifugation (the detailed reaction process of the ATRP reaction is shown in Scheme 2). Then, 0.3 mL Ab and 0.2 mL BSA were slowly added into 3 mL PdNC–PEI–PSRu solution sequentially under soly stirring and incubated for 8 h at 4 C, respectively. Followed by centrifugation at 9000 rpm for 15 min at 4 C to discard excess reagents, the bioconjugate of PdNC–PEI–PSRu@Ab–BSA was obtained.
Scheme 2
The detailed reaction process of the ATRP reaction.
Fabrication of the ECL immunosensor The fabrication process for the ECL immunosensor is schematized in Scheme 3. Prior to modication, the GCE (4 ¼ 4 mm) was polished with 0.3, 0.05 mm alumina slurry respectively, followed by rinsing thoroughly with bi-distilled water and sonicating in ethanol and bi-distilled water separately. The cleaned GCE was rstly coated with 5 mL Gra and gold nanoparticles (AuNPs) homogeneous suspensions dispersed with chitosan (Chi, 0.6%) solution. Aer drying in air, 18 mL capture antibody was dropped onto the electrode followed by incubating at 4 C for 12 h. Then, 16 mL of BSA (1%) solution was placed onto the electrode for 40 min at room temperature to block the remaining active sites. Subsequently, the resulted electrode was incubated in CA 15-3 antigen solution for 20 min at room temperature. Finally, as a sandwich format, the
Scheme 3 Schematic diagram of the preparation and reaction mechanism of the ECL immunosensor.
obtained electrode was immersed in the PdNC–PEI–PSRu@ Ab–BSA solution for immune reaction. The modied electrode was thoroughly cleaned with PBS (pH 7.4) to remove the nonchemisorbed species aer every modied step.
Results and discussion Characterization of the different nanomaterials
1 The preparation of the PdNC–PEI–PSRu@Ab–BSA bioconjugate.
Scheme
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The morphologies of different nanomaterials were characterized by SEM. The lamellate mono-layer sheets could be observed clearly in Fig. 1A. Comparing with the Gra, the homogeneous coverage of spherical AuNPs with a diameter of 18 2 nm on the Gra surface could be found in Fig. 1B. According to Fig. 1C, silver nanostructures had cube-like shapes with a diameter of 160 20 nm. As shown in Fig. 1D, a large number of particles stacked cages with a diameter of 250 30 nm were clearly observed aer the galvanic replacement reaction. Using silver
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SEM images of (A) Gra, (B) Gra–AuNPs, (C) Ag nanocubes, and (D) PdNCs.
Fig. 1
Fig. 3 A) EIS profiles of (a) Bare GCE, (b) GCE/Chi–Gra–AuNPs, (c) GCE/Chi–Gra–AuNPs/Ab, (d) GCE/Chi–Gra–AuNPs/Ab/BSA, (e) GCE/ Chi–Gra–AuNPs/Ab/BSA/Ag, and (f) GCE/Chi–Gra–AuNPs/Ab/BSA/ Ag/PdNC–PEI–PSRu@Ab–BSA in 5 mM [Fe(CN)6]3/4 containing 0.1 M KCl. (B) ECL profiles in the fabricated process of the immunosensor before (black curve) and after (red curve) the incubation of PdNC–PEI–PSRu@Ab–BSA bioconjugate (the immunosensor was incubated with 15 U mL1 CA 15-3 and measured in PBS (0.1 M, pH 7.4); the PMT was set at 800 V and the potential scan was from 0.2 to 1.25 V). Fig. 2
XPS analysis for the full region of XPS for PdNC–PEI–PSRu.
nanocubes as templates, the obtained PdNCs retained partial cubic prototype. In order to prove the successfully preparation of PdNC–PEI–PSRu, X-ray photoelectron spectroscopy (XPS) was performed for the elemental analysis. As shown in Fig. 2, the peaks at 284.6 eV, 400.5 eV, 485.9 eV and 530.9 eV could be respectively assigned to C1s, N1s, Ru3p, and O1s XPS spectra, which suggested the presence of the polymer chains functionalized with Ru(II) luminophore and co-reactive group (amine groups). Moreover, the XPS doublet of Pd3d (342.3 eV and 337.2 eV) indicated the existence of the PdNCs. According to the elemental analysis results, we could conrm that the PdNC–PEI–PSRu was successfully prepared.
Characterization of the ECL immunosensor The stepwise fabrication process of the ECL immunosensor was characterized by electrochemical impedance spectroscopy (EIS) in 5 mM [Fe(CN)6]3/4 containing 0.1 M KCl (Fig. 3A). Owing to low resistance to the redox probe dissolved in solution, the bare GCE had a small semicircle (curve a). When Gra and AuNP homogeneous suspensions dispersed with chitosan
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(Chi–Gra–AuNPs) were added dropwise on the GCE, a decreased impedance value was obtained (curve b). Aer the successive immobilization of capture antibody, BSA, and CA 15-3 antigen (15 U mL1), the impedance values increased in order (curves c– e), which is because the formation of protein molecule layers hindered the electron transfer. However, aer the incubation of the PdNC–PEI–PSRu@Ab–BSA bioconjugate, the impedance value was signicantly reduced (curve f), the reason of which was that the materials in the bioconjugate promoted the electron transfer. Furthermore, the proposed immunosensor was characterized by ECL in PBS (0.1 M, pH 7.4). As shown in Fig. 3B, there was almost no ECL response before the incubation of the PdNC–PEI–PSRu@Ab–BSA bioconjugate (black curve) because there was no ECL luminophore. However, due to the existence of the intramolecular self-enhanced electrochemiluminescent derivative, the ECL signal was greatly improved when the PdNC–PEI–PSRu@Ab–BSA bioconjugate was immobilized on the electrode (red curve). Comparison of the immunosensor with different Ab bioconjugates In order to prove the superiority of the immunosensor with the proposed PdNC–PEI–PSRu@Ab–BSA bioconjugate. Contrast
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experiments were conducted by comparing the ECL responses of the immunosensor with different probes under the same conditions. Five kinds of Ab-functionalized probes were prepared: (a) PdNC–PEI–PSRu@Ab–BSA (target probe), (b) PdNC–PEI–Ru@Ab–BSA, (c) PdNC–PSRu@Ab–BSA, (d) PdNP–PEI–PSRu@Ab–BSA (using palladium nanoparticles (PdNPs) instead of PdNCs) and (e) PEI–PSRu@Ab. The same batch of immunosensors were prepared and incubated with 15 U mL1 CA 15-3, then incubated with different Ab-functionalized probe solutions, respectively. As shown in Fig. 4, about 3002.7 a.u. ECL emission was obtained by the immunosensor with the target probe (PdNC–PEI–PSRu@Ab–BSA) compared with background values (Fig. 4A). However, the ECL responses were decreased to 2071.3 a.u., 1059.5 a.u., 932.3 a.u and 488.1 a.u. when the immunosensor was incubated with PdNC–PEI– Ru@Ab–BSA, PEI–PSRu@Ab and PdNC–PSRu@Ab–BSA, respectively (Fig. 4D, B, E and C). The reason could be as follows: rstly, PdNCs, with porous walls and hollow interiors, possessed excellent electrocatalytic ability and high specic surface area, which was better than solid naonostructures such as PdNPs; secondly, the loading amount of Ru(II) luminophore in the target probe was greatly increased through ATRP reaction; thirdly, comparing directly conjugating PEI with antibody, using PdNCs as an immobilized platform had advantages in more binding sites and better electric conductivity; nally, the novel self-enhanced ECL mechanisms of Ru(II) luminophore functionalized PEI greatly enhanced the luminous efficiency.
Fig. 4 The ECL comparison between the immunosensor with different Ab bioconjugates: (A) PdNC–PEI–PSRu@Ab–BSA, (B) PdNC–PEI– Ru@Ab–BSA, (C) PdNC–PSRu@Ab–BSA, (D) PdNP–PEI–PSRu@ Ab–BSA and (E) PEI–PSRu@Ab (the immunosensor was incubated with 15 U mL1 CA 15-3 and measured in PBS (0.1 M, pH 7.4); the PMT was set at 800 V and the potential scan was from 0.2 to 1.25 V).
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The possible luminous mechanisms of the self-enhanced ECL derivative The possible mechanisms to the ECL of PdNC–PEI–PSRu were described with the following equations: PdNC–PEI–PSRu(II) 2e / PdNC–PEI_+–PSRu(III)
(1)
PdNC–PEI_+–PSRu(III) / PdNC–PEI_–PSRu(III) + H+
(2)
PdNC–PEI_–PSRu(III) / PdNC–PEI–PSRu*(II)
(3)
PdNC–PEI–PSRu*(II) / PdNC–PEI–PSRu(II) + hv
(4)
With the potential scan from 0.2 to 1.25 V, both the luminophore and the co-reactive groups in the complex of PdNC–PEI–PSRu(II) are oxidized. Then, aer the intramolecular electron transfer and energy transmission, the strong oxidant intermediate (PdNC–PEI_–PSRu(III)) changes to the excited state (PdNC–PEI–PSRu*(II)). Finally, the enhanced ECL emission was obtained with PdNC–PEI–PSRu*(II) returning back to PdNC–PEI–PSRu(II). ECL detection of CA 15-3 with the obtained immunosensor The quantitative measurement of the proposed ECL immunosensor to CA 15-3 antigen was explored by using a sandwichtype format. Fig. 5 shows the relationship between the ECL intensity and deferent concentrations of CA 15-3, in which the ECL intensity increased with the increasing concentration of CA 15-3 (Fig. 5, curves a–h). The inset in Fig. 5 presented the calibration curve of the immunosensor to CA 15-3, where the ECL intensity had positive correlation to the logarithm of CA 15-3 concentrations. The linear equation was I ¼ 1993.0 + 830.4 log c with a correlation coefficient of 0.9956 (where I is the ECL intensity and c is the concentration of CA 15-3). The linear range for CA 15-3 was from 0.01 U mL1 to 120 U mL1 with a relatively low detection limit of 0.003 U mL1. Table 1 shows the detection results of immunosensors with previous reports.20–22 Compared to other methods, the immunosensor had a lower
ECL profiles of the immunosensor in the presence of different concentrations of CA 15-3 (a–h). Inset: calibration curve for CA 15-3 detection. The concentrations of CA 15-3: (a) 0.01 U mL1, (b) 0.1 U mL1, (c) 1 U mL1, (d) 5 U mL1, (e) 15 U mL1, (f) 30 U mL1, (g) 60 U mL1, and (h) 120 U mL1. All ECL signals were measured in PBS (0.1 M, pH 7.4). Fig. 5
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Table 1 Comparison of our research with other methods for CA 15-3 detection
Method
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Electrochemical immunoassay Chemiluminescence Optical sensing ECL
Linear range Detection limit (U mL1) (U mL1) References 1–100
0.3
20
0–250 1.25–25 0.01–120
0.035 0.4 0.003
21 22 Present work
detection limit, and might hold a new promise for highly sensitive bioassays applied in clinical detection. Selectivity, stability and reproducibility of the ECL immunosensor
Table 2
Recovery results of the proposed immunosensor in human
serum
Sample number
Added (U mL1)
Found (U mL1)
Recovery (%)
1 2 3 5
1 5 15 30
0.9382 5.078 16.27 27.87
93.82 101.6 108.5 92.89
(between 92.89% and 108.5%) was obtained, which suggested that the proposed immunosensor provided a potential tool for determining CA 15-3 in real biological samples.
Conclusions
The selectivity of the immunosensor was monitored by the assistance of some interfering agents such as carcinoembryonic antigen (CEA) and Streptococcus suis serotype 2 (SS2). As shown in Fig. 6A, the ECL signal was similar to that of the blank sample when the immunosensor was incubated in the SS2 solution. And no obvious changes were observed on comparing the ECL with interfering agents to that obtained in the pure CA 15-3, which indicated that the immunosensor possessed good selectivity for the detection of CA 15-3. Simultaneously, the stability of the ECL immunosensor was evaluated under consecutive cyclic potential scans for 34 cycles. As shown in Fig. 6B, the ECL intensity did not show any obvious changes with relative standard deviations (RSD) of 1.86%. The outstanding stability may be attributed to the excellent luminous stability of the intramolecular self-enhanced electrochemiluminescent derivative. The reproducibility of the proposed immunosensor was evaluated by the RSD (ECL response) of intra- and inter-assays. The RSDs of the intra- and inter-assay were not more than 5%, which implied that the reproducibility of the proposed immunoassay was acceptable.
In this work, an intramolecular self-enhanced ECL immunosensor based on polymer chains graed on the PdNCs was constructed for cancer-related biomarkers. The prepared complex of PdNC–PEI–PSRu was a novel electrochemiluminescent derivative which included the Ru(II) luminophore and its co-reactive group (amine groups) in the same polymer molecule. Through this design, the electronic transmission distance between the luminophore and its co-reactive group was greatly shortened, by which the luminous efficiency and stability were enhanced remarkably. Moreover, by the assistance of ATRP reaction and PdNCs with high specic surface areas, the loading amounts of Ru(II) luminophore and co-reactive group (amine groups) were signicantly improved. Based on this self-enhanced electrochemiluminescent derivative, the proposed ECL immunosensor had a sensitive response to CA 15-3 with good selectivity and reproducibility, acceptable precision and accuracy. This work provided a new method for signal amplication of ECL and would extend the application of ECL in bioanalysis.
Preliminary analysis of real samples
Acknowledgements
Recovery experiments were explored in human serum to monitor the feasibility of the ECL immunosensor by the standard addition method. According to Table 2, the recovery
This work was nancially supported by the NNSF of China (21075100, 21275119), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), Ministry of Education of China (Project 708073), Natural Science Foundation of Chongqing City (CSTC-2010BB4121, CSTC-2011BA7003) and the Fundamental Research Funds for the Central Universities (XDJK2012A004, XDJK2013A008, and XDJK2013A027).
Notes and references (A) Comparison of ECL responses with different antigens: blank; SS2 (20 ng mL1); CA 15-3 (15 U mL1); a mixture containing CA 15-3 (15 U mL1) and SS2 (20 ng mL1); a mixture containing CA 15-3 (15 U mL1), SS2 (20 ng mL1) and CEA (20 ng mL1). (B) The ECL stability of the proposed immunosensor under consecutive cyclic potential scans. Fig. 6
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