Letter Cite This: ACS Sens. XXXX, XXX, XXX−XXX
pubs.acs.org/acssensors
Sandwich-Type Electrochemiluminescence Sensor for Detection of NT-proBNP by Using High Efficiency Quench Strategy of Fe3O4@PDA toward Ru(bpy)32+ Coordinated with Silver Oxalate Li Shi,† Xiaojian Li,† Wenjuan Zhu,† Yaoguang Wang,† Bin Du,‡ Wei Cao,† Qin Wei,*,† and Xuehui Pang*,† †
Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering and ‡School of Resources and Environment, University of Jinan, Jinan 250022, Shandong, China S Supporting Information *
ABSTRACT: Heart failure (HF) is a burgeoning public health problem trigged by a heart circulation disorder. N-terminal proB-type natriuretic peptide (NT-proBNP) has been acknowledged as a prognostic biomarker for cardiac disease. Herein, a sandwichtype electrochemiluminescence (ECL) immunosensor was introduced for sensitive detection of NT-proBNP. Gold nanoparticle modified graphene oxide-Ru(bpy)32+/Ag2C2O4 was used as a luminophore and a desirable platform for immobilization of the captured antibodies. The more stable immobilization of plentiful Ru(bpy)32+ could be implemented by direct covalent bonding chelation with Ag2C2O4. More importantly, significant quenching can be achieved by introducing polydopamine (PDA) coated Fe3O4 onto the electrode via sandwich immunoreactions. The quenching mechanism mainly showed that the excited states of Ru(bpy)32+ could be annihilated by quinone units in PDA via energy transfer. The ECL quenching efficiency was logarithmically related to the concentration of the NT-proBNP in the range from 0.0005 ng/mL to 100.0 ng/mL with a detection limit of 0.28 pg/mL. Furthermore, this specific immunosensor presented good stability and repeatability as well as selectivity, which offers a guiding significance in both fundamental and clinical diagnosis of NT-proBNP. KEYWORDS: N-terminal pro-B-type natriuretic peptide, electrochemiluminescence, polydopamine, quenching, immunosensor
H
enhancement, and simpler experimental design when compared with liquid-phase Ru(bpy)32+ in ECL system.7 Therefore, numerous Ru(bpy)32+ immobilization approaches onto the solid electrode surface have been studied for ECL biosensor construction.8,9 Due to the intrinsic property of Ru(bpy)32+, Pointillart and his co-workers have discovered that Ru(bpy)32+ could be embedded into three-dimensional (3D) CuNi oxalate through coordination reaction of Ru(bpy)32+ and oxalate.10 Compared to the physical absorption,11 or encapsulation of Ru(bpy)32+,12 the more stable immobilization of plentiful Ru(bpy)32+ could be implemented by direct covalently bonded chelating. Silver oxalate (Ag2C2O4) has topological properties and special layered structures with monoclinic unit cells P21/c.13 Each C2O42− in silver oxalate is coordinated to six Ag+ cations. Specifically, these oxalate anions could form a framework with the (2 0 0) plane layers and extend the channels along [1 0 0] with Ag−Ag dimers which locate inside these channels.14
eart failure (HF) is identified as the ability of the heart being unable to meet the circulatory demands of the body.1 Recently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been characterized as a new biochemical marker of HF disease for diagnostics and prognostics.2 However, there are few reports related to sensitive determination of NT-proBNP. Consequently, designing a specific and sensitive identification of NT-proBNP would be of great significance for major HF guidelines. Electrochemiluminescence (ECL) is an emerging method which combines electrochemistry and chemiluminescence, and is emitted through electron-transfer reaction of generated species occurring from the vicinity of an electrode.3,4 Due to its excellent advantages including low background signal, high sensitivity, and fast sample analysis, ECL has been a promising bioanalytical technique and is widely applied in areas of immunoassay.3,5 It is universally acknowledged that Ru(bpy)32+, as the conventional ECL luminophore, has plenty of unique properties including chemical stability, high ECL efficiency, and superior biocompatibility in the ECL bioanalytical system.6 Immobilization of Ru(bpy)32+ on a solid electrode surface has many advantages, including less reagent used, better ECL signal © XXXX American Chemical Society
Received: November 2, 2017 Accepted: December 11, 2017 Published: December 11, 2017 A
DOI: 10.1021/acssensors.7b00809 ACS Sens. XXXX, XXX, XXX−XXX
Letter
ACS Sensors
coordination reaction, which were supported by GO to form GO-Ru(bpy)32+/Ag2C2O4. Then, Au nanoparticles were also utilized to form Au@GO-Ru(bpy)32+/Ag2C2O4 (Au@GO-Ru/ Ag) composites as the sensing platform, by which could further improve the ECL immunosensor sensitivity. Furthermore, a simple method was selected to synthesize the Fe3O4@PDA and employ them as the Ru(bpy)32+ quencher. Finally, the fabricated sensing platform performed not only with high sensitivity and accuracy, but also with foreseeable prospects for clinical detection of NT-proBNP.
Besides, it has reported that ruthenium cations could lead to optically active 3D compounds through chiral template effect.10 This result emphasizes the ability of Ru(bpy)32+ to template the self-assembling of an anionic network with the desired topology. Hence, these unique properties of silver oxalate and Ru(bpy)32+ could allow them to combine together strongly via coordination reaction, which exhibited high-efficiency and stable ECL emission. Otherwise, considering the lower solubility of pure Ag2C2O4,15 AgCO3 was adopted as a sacrificial template for successful preparation of Ag2C2O4 via an anion exchange process. In order to further increase the ECL intensity, graphene oxide was synthesized in this work. Graphene oxide (GO) has attracted marvelous attention for its extraordinary in-plane electrical conductivity and ultrahigh specific surface area, which enables it to become the admirable support material in ECL sensors.16,17 Gold nanoparticles (Au NPs) with spectacular biocompatibility could directly combine with target biomolecules via covalent coupling between gold and amino group. In addition, due to their outstanding conductivity, Au NPs could accelerate electron transfer that further enhance the ECL signal dramatically. Thus, Au NPs were also selected in the design of this immunosensor. In order to enhance the sensitivity of the ECL immunosensor, a quenching strategy was developed based on Fe3O4@PDA quenching the ECL of Ru(bpy)32+ in this work. Polydopamine (PDA) is a kind of adhesive-inspired biomimetic polymer with excellent coating properties, remarkable biocompatibility, and favorable biodegradability. In recent research, it is certain that PDA consists of dihydroxy-indole, indolequinone, and DA units by which the excited states of Ru(bpy)32+ (Ru(bpy)32+*) could be quenched through energy transfer.18−21 Ferroferric oxide (Fe3O4) is famous for its superior biocompatibility, low toxicity, and environmental benignity.22 Moreover, Fe3O4 nanoparticles, in core−shell structure, have exhibited excellent tunable absorption frequency and the integration of chemical composition.23 Thus, a kind of composite microspheres, with Fe3O4 as cores and PDA as shells, was designed and synthesized. Consequently, PDA coated Fe3O4 (Fe3O4@PDA) microspheres were used as labels to quench the ECL signal. In this work, a sandwich-type ECL immunosensor was developed for the NT-proBNP determination as illustrated in Figure 1. Ag2C2O4 and Ru(bpy)32+ combined together through
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RESULTS AND DISCUSSION Characterizations of Different Nanomaterials. Figure 2A was the SEM image of the as-prepared GO. It was obvious
Figure 2. SEM image of GO (A), GO-Ag2CO3 (B), EDS image of GO-Ru/Ag (C), SEM image of Au@GO-Ru/Ag (D), Fe 3 O 4 nanoshperes (E), and Fe3O4@PDA (F).
that the GO presented the sheet-like structure with the large thickness, smooth surface, and wrinkled edge. As shown in Figure 2B, Ag2CO3 nanospheres was ideally dispersed on the surface of GO. After introduced C2O42− and Ru(bpy)32+, GORu/Ag composites were synthesized, which could be proven by EDS analysis. As shown in Figure 2C, the C, O, Ag, and Ru elements were observed, indicating that GO-Ru/Ag nanoparticle composites were prepared successfully. The morphology of the Au@GO-Ru/Ag was characterized by SEM (Figure 2D). As can be seen in the SEM image, it was clear that large amounts of Au NPs and Ru/Ag NPs were dispersed uniformly on GO surface attributed to the large specific surface area. Ru/ Ag NPs still preserved the original morphology of Ag2CO3. EDS spectrum was employed to further analyze the decoration of Au NPs (Figure S-1), and characteristic elements of Au could be observed. Pure Fe3O4 nanospheres were observed in Figure 2E. It could be seen that regular spheres were presented with the diameter distribution of about 250 nm. On the surface of these particles, there were small and bumpy nanocrystals. After polymerizing with DA, monodisperse Fe3O4@PDA were obtained with 300 nm average diameter (Figure 2F). Obviously, Fe3O4@PDA was prepared successfully. The UV−vis absorbance spectra of Ru(bpy)32+, Au NPs, Ag2C2O4, and Au@GO-Ru/Ag were explored to testify the formation of Au@GO-Ru/Ag composites. From Figure S-2, the prominent absorption band of Ru(bpy)32+ (curve a) was observed at about 453 nm due to the spin allowed dπ(Ru)−π(ligand)* metal-to-ligand charge transfer (MLCT) transitions. In addition, owing to the π−π* electron transfers of pyridine ring, there was a sharp characteristic absorption peak at about 289 nm.24 The absorption peak of pure Au NPs was at
Figure 1. Schematic illustration of proposed ECL immunosensor for NT-proBNP determination. B
DOI: 10.1021/acssensors.7b00809 ACS Sens. XXXX, XXX, XXX−XXX
Letter
ACS Sensors approximately 514 nm (curve b). The broad absorption peak of Ag2C2O4 was observed at 289 nm (curve c). When Au@GORu/Ag was generated, a major absorption peak around 519 nm was observed again (curve d), and the absorption peak at 289 nm became wider, which was influenced by Ag2C2O4. Generally speaking, these characteristic absorption peaks indicated the successful fabrication of Au@GO-Ru/Ag. Ru(bpy)32+ Emission and Quenching Mechanism. The ECL behavior of Au@GO-Ru/Ag was attributed to Ru(bpy)32+ and Au NPs could enhance the intensity of this ECL immunosensor. The mechanism of Ru(bpy)32+ at cathode has been proposed. Shortly, excited states (Ru(bpy)32+*) was generated by activating the Ru(bpy)32+ in two parallel pathways. The ECL excitation route of Ru(bpy)32+ could occur by reduction-initiated oxidative excitation pathway (eqs 2 and 3) or the oxidation-initiated reductive excitation pathway (eqs 4 and 5) as follows: S2 O82 − + e− → SO4 2 − + SO4•−
(1)
Ru(bpy)32 + + e− → Ru(bpy)3+
(2)
Ru(bpy)3+ + SO4•− → Ru(bpy)32 +* + SO4 2 −
(3)
Ru(bpy)32 + + SO4•− → Ru(bpy)33 + + SO4 2 −
(4)
Ru(bpy)33 + + e− → Ru(bpy)32 +*
(5)
With Fe3O4@PDA immobilized onto the electrode, the ECL signal of Au@GO-Ru/Ag decreased dramatically. Excited states of Ru(bpy)32+* can be annihilated by quinone units in PDA via energy transfer (eq 7).25 In this reason, the PDA took the responsible for the luminescence quenching.
Figure 3. (A) ECL response of the ECL sensor to different concentrations of NT-proBNP: 0.0005, 0.001, 0.005, 0.05, 0.5, 10, 30, and 100 ng/mL. (B) Calibration curve for NT-proBNP determination. error bars = SD (n = 5). (C) Repeatability of the proposed ECL immunosensor with 10 ng/mL NT-proBNP. (D) ECL response of the immunosensor to (1) 0.5 ng/mL NT-proBNP + 50 ng/mL lgG, (2) 0.5 ng/mL NT-proBNP + 50 ng/mL PSA, (3) 0.5 ng/ mL NT-proBNP + 50 ng/mL CEA, (4) 0.5 ng/mL NT-proBNP + 50 ng/mL BSA, (5) 0.5 ng/mL NT-proBNP + 50 ng/mL insulin, (6) 0.5 ng/mL NT-proBNP. (E) ECL intensity of the immunosensor incubated with 0.5 ng/mL NT-proBNP. (F) Long-term storage stability of the immunosensor incubated with 0.5 ng/mL NT-proBNP. (Error bar = SD, n = 5.)
Performance of the Immunosensor. The ECL signals exhibited relativity with the NT-proBNP concentration which was shown in Figure 3A. It was revealed that with the increase of NT-proBNP concentration, ECL intensity decreased. The ECL signal was correlated with the logarithmic value of NTproBNP concentration and a linear response range from 0.0005 ng/mL to 100.0 ng/mL with a correlation coefficient of 0.992 (Figure 3B). The linear regression equation was I = 4470.97 − 1553.98 lg [c (ng/mL)]. The detection limit was calculated at 0.28 pg/mL according to the IUPAC rules. Compared with previous work (Table S-2), this proposed biosensor had a lower detection limit and wider linear range. Repeatability, Selectivity, and Stability of ECL Response. In the sensing field, excellent repeatability was treated as one of the most significant points for extending potential application. In Figure 3C, five successive assays for detection of NT-proBNP (10 ng/mL) were carried out in 10 mL of 0.1 M PBS (pH 7.4) containing 0.1 M KCl and 0.1 M K2S2O8. The RSD was less than 1%, which illustrated an acceptable level of reproducibility for the immunosensor.
The selectivity of the proposed immunosensor for NTproBNP detection was evaluated to illustrate its practicability. Immunoglobulin G (lgG), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), bull serum albumin (BSA), and insulin were selected as potential interferents (50 ng/mL) and mixed with 0.5 ng/mL of NT-proBNP. As shown in Figure 3D, the corresponding ECL response variation was less than 5% which was negligible interference. This result indicated favorable selectivity and superior specificity of the proposed immunosensor to NT-proBNP. To further monitor the stability, the ECL response of the immunosensor with 0.5 ng/mL of NT-proBNP was investigated under 8 cycles of continuous potential scans, shown in Figure 3E. The relative standard deviation (RSD) was 1.46% which meant the excellent stability of the immunosensor. Moreover, the stability of long-term storage was studied and the results were presented in Figure 3F. The ECL intensity of the immunosensor kept around 91.7% of the beginning value at 4 °C after 20 days, which indicated immunosensor was reasonable and acceptable. The above results testified that the immunosensor had attractive stability and important practical value. Clinical Analysis. The feasibility of the immunosensor was one of the major concerns for practical application, which could be evaluated by a recovery test.26,27 To check its practical application, NT-proBNP in human serum sample were diluted
Ru(bpy)32+*
After these excitation steps, relaxed (eq 6) to the ground state inducing the emission peaking. Ru(bpy)32 +* → Ru(bpy)32 + + hν
(6)
C
DOI: 10.1021/acssensors.7b00809 ACS Sens. XXXX, XXX, XXX−XXX
Letter
ACS Sensors Notes
with PBS (pH 7.4) until a level was detected using the designed ECL immunosensor with standard addition methods. As shown in Table S-3, different concentrations of the standard NTproBNP solution (1.00, 2.00, and 3.00 ng/mL) were added into serum samples. The recoveries of the immunosensor were in the range of 97.0−101.7%, and the RSD was 1.92−3.22%. In addition, the accuracy test of designed ECL immunosensor was conducted via ELISA kit. The diluted human serum sample was determined five times in parallel by the ELISA kit. The results are shown in the Table S-4. To further ensure the precision and accuracy of the immunosensor, the F-test and t test were evaluated. F-test can reflect the precision of the two methods and judge whether they have a significant difference.28 F value was calculated as 1.08 via formula 8, which was far less than the theoretical F value (F = 6.39 at 95% confidence limits). The results implied that ELISA kit and this ECL immunosensor do not have any significant difference. F=
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the National Key Scientific Instrument and Equipment Development Project of China (No.21627809), National Natural Science Foundation of China (Nos. 21575050 and 21375047).
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2 s big 2 ssmall
(8)
29
The t-test is carried out to assess whether there is a bigger error between the ECL method and ELISA analysis. The t value is calculated at 0.59 by the eqs 9 and 10 and less than 2.78 (P = 0.95, α = 0.05, f = 4). By the t-test, it could be found that the mean values of two methods are not obviously different, indicating the system error can be ignored. s=
t=
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∑ (x1i − x1̅ )2 + ∑ (x 2i − x 2̅ )2 (n1 − 1) + (n2 − 1) |x1̅ − x 2̅ | s
n1n2 n1 + n2
(9)
(10)
CONCLUSIONS In conclusion, an effective quenching pattern of immunosensor was developed for the detection of NT-proBNP based on PDA to Ru(bpy)32+ via energy transfer. Ru(bpy)32+ could coordinate with Ag2C2O4, which not only provided a prolonged stability on ECL emission of Ru(bpy)32+, but also allows new perspective for enhanced immobilization Ru(bpy)32+ on application of sensor field. The designed system enables repaid responses for NT-proBNP detection, making it an efficient method in real-time sensing. Also, this detection pattern likely provides a promising technique for other biomarker detection in clinical diagnosis.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.7b00809. Experimental Section (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
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Qin Wei: 0000-0002-3034-8046 D
DOI: 10.1021/acssensors.7b00809 ACS Sens. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acssensors.7b00809 ACS Sens. XXXX, XXX, XXX−XXX
