3D Nanostructured Palladium-Functionalized Graphene-Aerogel

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3D Nanostructured Palladium Functionalized Graphene Aerogel Supported Fe3O4 for Enhanced Ru(bpy)32+-based Electrochemiluminescent Immunosensing of Prostate Specific Antigen Lei Yang, Yueyuan Li, Yong Zhang, Dawei Fan, Xuehui Pang, Qin Wei, and Bin Du ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11458 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017

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ACS Applied Materials & Interfaces

3D Nanostructured Palladium Functionalized Graphene Aerogel Supported Fe3O4 for Enhanced Ru(bpy)32+-based Electrochemiluminescent Immunosensing of Prostate Specific Antigen

Lei Yang, Yueyuan Li, Yong Zhang, Dawei Fan, Xuehui Pang, Qin Wei, Bin Du.

Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China

* Corresponding author Tel.: +86 531 82765730; Fax: +86 531 82765969; E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: We developed a novel Ru(bpy)32+-based electrochemiluminescence (ECL) immunosensor utilizing palladium nanoparticles (Pd NPs) functionalized graphene aerogel supported Fe3O4 (FGA-Pd) for real sample analysis of prostate specific antigen (PSA). 3D nanostructured FGA-Pd, as a novel ECL carrier, was prepared by in situ reduction. Large amounts of Ru(bpy)32+ could combine with FGA-Pd via electrostatic interaction to establish a brand-new ECL emitter (Ru@FGA-Pd) for improving ECL efficiency. The obtained Ru@FGAPd composite was utilized to label the secondary antibody, which generated strong ECL signals with tripropylamine (TPrA) as coreactant. Furthermore, we demonstrated that the participation of Pd NPs endowed FGA favorable electrocatalytic ability in the luminescent process to produce more excited state [Ru(bpy)32+]* for realizing desirable signal amplification. In addition, the primary antibody was captured by gold nanoparticles (Au NPs) functionalized Fe2O3 nanodendrites (Au-FONDs) which possessed good electrical conductivity and favorable biocompatibility. Under optimum conditions, the fabricated sandwich-type ECL immunosensor showed a sensitive response to PSA with a low detection limit of 0.056 pg/mL (S/N=3) and a calibration range of 0.0001 - 50 ng/mL. Featuring favorable selectivity, stability and repeatability, the proposed immunosensor is expected to blaze a novel trail for the real sample detection of PSA and other biomarkers.

KEYWORDS: Electrochemiluminescence immunosensor, Graphene aerogel supported Fe3O4, Prostate specific antigen, Fe2O3 nanodendrites, Palladium nanoparticles.


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1. INTRODUCTION It is worth noting that prostatic cancer has become one of the most lethal diseases in the world, causing thousands of people lost their lives every year.1 As well known, cancer biomarkers are now playing an increasingly important role in the early detection and monitoring of cancers.2,3 For instance, prostate specific antigen (PSA) is a world-recognized biomarker for clinic evaluation of prostatic cancer. It has been demonstrated that when the concentration of PSA rises to 2 ng/mL, there is more likely to have a prostatic cancer attack in human immune system, even though the normal cutoff value of PSA is 4 ng/mL.1,4 Nowadays, lots of methods have been applied to analyze PSA in human serum, including photoelectrochemical immunoassay,5,6

electrochemical

sensor,7,8

fluoroimmunoassay

and

enzyme-linked

immunosorbent assay and so forth.9-11 Nonetheless, methods with higher sensitivity and better selectivity for the quick and dynamic concentration response of PSA in human serum are still urgently demanded. Till now, electrochemiluminescence (ECL) has aroused widespread concerns in the fields of biochemical analysis,12 environmental pollutant monitoring,13 clinical diagnostics and prognostics of cancers.14-16 Due to the unique superiorities of strong specificity,17 high sensitivity, quick dynamic concentration response and low background noise,18 various ECL systems have been extensively developed, even several commercial ECL systems have already been applied in clinical diagnosis.18 In terms of different luminophores applied in the ECL processes, the ECL systems can be divided into: Ru(bpy)32+-based system, luminol-based system, acridine esterbased system, lucigenin-based system etc.14 With high ECL efficiency, favorable water solubility and excellent stability of chemical property, Ru(bpy)32+ has been a good choice for constructing ECL immunosensors to realize sensitive analysis of tumor markers in various human samples.19


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On this basis, progressive ECL immunosensors relied on Ru(bpy)32+-based nanocomposite are forthcoming in the future. Graphene aerogel (GA), emerged as novel porous carbon materials, has attracted particular interest in many domains, such as sensors and energy storage mediums like fuel cells,20,21 supercapacitors and so on.22,23 Recently, lots of attention has been focused on the great potential of GA and GA-based nanomaterials for being electrocatalytic support in ECL analysis because of their high surface area, favorable electrical conductivity and high porosity.24-27 Compared with traditional graphene sheets (GS), GA with 3D porous structure could properly deal with the severe agglomeration caused by irreversible planar stacking of GS.26 More importantly, the accessibility of electro-active sites for catalysis was improved and the mass transfer rate could be accelerated to a higher level.25 Given that the ECL immunosensors were constructed based on modified electrodes, GA and GA-based nanomaterials are expected to be utilized as ECL substrates to support luminophores and biomolecules. When the modified electrodes were immersed into the electrolyte, the mass transfer between the surface and the coreactant would be facilitated, which was rather conducive to the redox between luminophores and coreactants for stronger ECL emission. In short, GA and GA-based nanomaterials have great potential of being excellent ECL substrate in the following studies. In view of the above, a novel sandwich-typed ECL immunosensor based on Pd NPs functionalized GA supported Fe3O4 (FGA-Pd) was developed. With advantage of the high surface area, the luminophore of Ru(bpy)32+ could combine with FGA-Pd (Ru@FGA-Pd) through electrostatic interaction and the loading could be improved. Pd NPs have always been one of the hottest noble metal catalysts because of the excellent electrocatalytic property. It was found that the introduction of Pd NPs further enhanced the electron-transport ability of FGA and


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the secondary antibody (Ab2) could bind strongly to the surface of Pd NPs.28,29 Then, Au-FONDs were utilized as the ECL sensing platform to label the primary antibody (Ab1). It has been found that the -NH2 groups could bind strongly to Au NPs,20 thus Ab1 could be captured by Au-FONDs. By virtue of the superiorities of Ru@FGA-Pd composite and Au-FONDs, the electron-transfer rate and mass-transfer rate were greatly accelerated during the ECL emission, which gave rise to a higher ECL response in the detection of PSA. More importantly, the proposed ECL immunosensor had satisfying performance in detecting PSA in real samples, which revealed its potential of being a powerful tool for PSA detection in real samples analysis.

2. MATERIALS AND METHODS 2.1. Materials and Reagents Tris (2, 2-bipyridyl) dichlororuthenium (Ⅱ) hexahydrate (Ru(bpy)3Cl2·6H2O), sodium tetrachloropalladate(II) (Na2PdCl4), gold chloride tetrahydrate (HAuCl4·4H2O), sodium hydroxide (NaOH) and sodium citrateand were bought from Sigma-Aldrich (Beijing, China). Potassium ferricyanide (K3[Fe(CN)6]), ferric trichloride (FeCl3) and ferrous chloride tetrahydrate (FeCl2·4H2O) were bought from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). Human serum was provided by a healthy volunteer from hospital. PSA, PSA primary antibody and its secondary antibody, carcinoembryonic antigen (CEA), alpha-fetal protein (AFP) and bovine serum albumin (BSA, 96-99%) were bought from Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). Phosphate buffer solution (PBS) was prepared by adjusting the proportion of 0.1 mol/L of Na2HPO3 and KH2PO3 solution. All the involved chemicals were of analytical grade. In addition, ultrapure water (18.25 MΩ/cm) was employed to finish all the researches. 2.2. Apparatus


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The CHI760D electrochemical workstation (Chenhua, China) was used to accomplish all the electrochemical measurements, and the standard three-electrode system was employed: the modified GCE used as the working electrode, the Ag/AgCl electrode worked as the reference electrode while the platinum electrode worked as the counter electrode. The IM6e electrochemical Interface (Zahner, Germany) and the model MPI-F flow injection chemiluminescence detector (Remax, China) were used to conduct all the electrochemical impedance spectroscopy (EIS) measurements and all the ECL measurements, respectively. The field emission scanning electron microscope (SEM) (Zesis, Germany) was applied for characterization of SEM images and EDX spectrums. The JEOL JEM-2100F transmission electron microscope (TEM) (Japan) was used for the characterization of TEM and HRTEM images. The Zeta potential was tested on Zetasizer Nano ZS 90 (Britain). The Lambda 35 UV/Vis Spectrometer (Perkin-Elmer, United States) was used to complete the characterization of the UV-vis absorption spectrum. And the D8 focus diffractometer (Bruker AXS, Germany) was used to complete the X-ray diffraction (XRD) patterns. 2.3. Preparation of Au-FONDs Composite The FONDs composite was synthesized referring to a previously reported method with a slight modification.30 In the beginning, 40 mL of ultrapure was continuously stirred with the dissolving of 0.1974 g of K3[Fe(CN)6] to form a mixed solution. Then, NaOH solution (0.1 mol/L) was used to adjust the pH value to 12. After 10 min of the reaction, the obtained mixture was moved into a clean autoclave and kept the reaction at 140 °C for 24 h. After washing the formed precipitate with alcohol, ultrapure water for 3 times and dried under vacuum at 50 °C for 4 h, the FONDs product was synthesized successfully.


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Au NPs sol was synthesized successfully according to Frens’s method.31 Au-FONDs (2 mg/mL) were prepared by oscillating the mixed solution (2 mL) that containing 1 mL of FONDs (2 mg/mL) and 1 mL of Au NPs sol for 24 h. After that, the excess Au NPs were removed out of the solution by centrifugation. The synthesized Au-FONDs product was redispersed in 1 mL of ultrapure water. 2.4. The Preparation of Ru@FGA-Pd Composite The 3D porous FGA nanocomposite was followed by a reported method with a slight modification.32 (The detailed preparation procedures of FGA and Pd NPs sol are shown in Supporting Information). Ru@FGA-Pd (2 mg/mL) was synthesized as follows: Firstly, oscillating the mixed solution (2 mL) containing 1 mL of FGA (2 mg/mL) and 1 mL of Pd NPs sol for 24 h. After that, the redundant Pd NPs were removed out of the solution by centrifugation. The obtained FGA-Pd composite was redispersed in 1 mL of ultrapure water. Next, 0.4 mL of Ru(bpy)32+ solution (2.5 mmol/L) was mixed with the obtained FGA-Pd solution. After oscillating for 24 h and redundant Ru(bpy)32+were removed out of solution by centrifugation, the final product of Ru@FGA-Pd were obtained and redispersed in 1 mL of PBS( pH 7.4). 2.5. Preparation Process of Ab2 Bioconjugate (Ru@FGA-Pd-Ab2) The preparation process of Ru@FGA-Pd-Ab2 bioconjugate was showed as follows: Firstly, 100 μL of Ab2 (10 μg/mL) was mixed with 1 mL of Ru@FGA-Pd solution (2 mg/mL). After oscillating the mixture at 4 °C for 24 h and centrifugation, the finally obtained Ru@FGA-Pd-Ab2 bioconjugatewas redispersed in 1 mL of PBS (pH 7.4) and stored at 4 °C. 2.6. Fabrication Process of the ECL immunosensor


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Scheme 1. Fabrication process of the proposed ECL immunosensor

The fabrication process of the ECL immunosensor was shown in Scheme 1. Before the modification, alumina slurries (1.0 μm, 0.3 μm and 0.05 μm) were sequentially used to polish the bare GCE (4 mm in diameter) to a mirror finish, which was washed with ultrapure water. Then, the cleaned GCE was first modified with 10 μL of Au-FONDs solution and dried at room temperature. Then, 10 μL of Ab1 (10 μg/mL) was incubated on the formed film at 4 °C. After washing the resulting electrode with the PBS to remove the physically absorbed Ab1, 10 μL of BSA (1%) was coated on the formed film to block the remaining nonspecific binding sites. Then, the redundant BSA was removed by washing the obtained electrode with PBS. Finally, the prepared electrode was stored at 4 °C for the further research. 2.7. Measurements of the Fabricated ECL Immunosensor


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At first, the prepared electrode was incubated with PSA at 37 °C for 1 h. Subsequently, 10 μL of Ru@FGA-Pd-Ab2 was incubated onto the formed PSA/BSA/Ab1-Au-FONDs/GCE surface at 37 °C for 40 min to fabricate the immunocomplex. The sandwich-type ECL immunosensor was constructed completely and ready for the following measurements. The ECL measurements were carried out in10 mL of PBS (pH 7.4) containing 55 mmol/L TPrA at room temperature. The ECL signals were recorded on the MPI-F ECL analyzer and the voltage of photomultiplier tube was set at 300 V. Cyclic voltammetry (CV) was applied for the electrochemical measurements and the scan voltage was set from 0 to 1.2 V and the scan rate was set at 100 mV/s.

3. RESULTS AND DISCUSSION 3.1. Characterizations of Different Nanomaterials

Figure 1. SEM images of FONDs (A), Au-FONDs (B) and FGA (C); XRD pattern (D) of FGA; TEM (E) and HRTEM images (F) of FGA-Pd composite.


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The morphologies of FONDs, Au-FONDs, FGA and FGA-Pd composite were characterized by SEM images. In Figure 1A, the pure FONDs with an average size of 500 nm were presented. The successfully prepared FONDs exhibited high surface area which could be loaded with much of Au NPs. As expected, there were large amounts of Au NPs distributed uniformly on the surface of FONDs (Figure 1B), indicating the transmission rate of the electrons on the surface of the FONDs could be greatly accelerated. The EDX spectrum of Au-FONDs was shown in Figure S1 and the corresponding UV-vis absorption spectrum was shown in Figure S2 with a characteristic absorption peak of Au NPs appeared at 524 nm, which further demonstrated the successful functionalization of Au NPs. The SEM image of FGA with a 3D porous structure was presented in Figure 1C, from which lots of Fe3O4 NPs could be seen distributed in pores and cross-linked areas of GA. The inserted picture exhibited the magnetic property of FGA. Depending on this, FGA could be separated very quickly from the solution during the preparation process, which saved a lot of time. Then, XRD pattern was used to find out the crystallization of FGA. From Figure 1D, it can be observed that a hump appeared in the range of 22.8-25.4°, which was caused by the network of GA. Notably, several sharp peaks were found at 2θ= 30.2°, 35.5°, 43.2°, 53.4°, 57.1° and 62.6°, which were corresponded to the (220), (311), (400), (422), (511), and (440) planes of Fe3O4 NPs, respectively. The results above revealed the successful preparation of FGA. After the introduction of Pd NPs, the morphology of FGA-Pd composite was characterized by TEM. From Figure 1E, it could be clearly seen that a lot of Pd NPs were covered uniformly on the surface of porous FGA. And the EDX spectrum of FGA-Pd was shown in Figure S3. Moreover, the HRTEM was used to further demonstrate the successful functionalization of Pd NPs. As seen in


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Figure 1F, the fringes displayed an interplanar spacing of 0.22 nm, which was corresponding to the lattice spacing of face (111) centered cubic (fcc) Pd. Zeta potentials of Fe3O4 sol, FGA (10 mg/mL), Pd NPs, FGA-Pd (10 mg/mL), Ru(bpy)32+ and Ru@FGA-Pd (10 mg/mL) were shown in Figure S4. Due to the fact that the FGA-Pd composite was negatively charged while the Ru(bpy)32+ was positively charged, the declination of the zeta potential of FGA-Pd indicated that the Ru(bpy)32+ had linked to the surface of FGAPd closely via the electrostatic interaction, further proving the successful preparation of Ru@FGA-Pd. 3.2. Discussion of ECL-signal Amplification Strategy The possible ECL mechanism of the proposed ECL-signal amplification strategy was discussed according to several reported works.12,14,16 The ECL properties of Ru@FGA composite and Ru@FGA-Pd composite were investigated. In Figure S5, the ECL signal of Ru@FGA was enhanced remarkably after the Pd NPs were loaded on FGA composite, which could be made clear by: the introduction of negatively charged Pd NPs endowed FGA a stronger ability to connect with more Ru(bpy)32+ via electrostatic interaction to generate stronger ECL signal than FGA; FGA-Pd exhibited good electrocatalytic ability to accelerate the transportation of electrons between the Ru(bpy)33+ (eq 1) and TPrA•(eq 2) to produce more exited state [Ru(bpy)32+]*(eq 3). When more [Ru(bpy)32+]* jumped back to the ground state Ru(bpy)32+ (eq 4), higher ECL responses were generated. Ru(bpy)32+ → e- + Ru(bpy)33+

(1)

TPrA → e- + TPrA+→ TPrA• + H+

(2)

Ru(bpy)33+ + TPrA• → (more) [Ru(bpy)32+]*+ TPrA

(3)

[Ru(bpy)32+]* → Ru(bpy)32+ + hv

(4)


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3.3. Electrochemical Characterization of the ECL Immunosensor

Figure 2. The CV curves (A) and the corresponding EIS responses (B) of: (a) bare GCE, (b) Au-FONDs/GCE, (c) Ab1/Au-FONDs/GCE, (d) BSA/Ab1/Au-FONDs/GCE, (e) PSA/BSA/Ab1/Au-FONDs/GCE, (f) Ru@FGAPd-Ab2/PSA/BSA/Ab1/Au-FONDs/GCE in 0.1 mol/L KCl solution containing 2.5 mmol/L [Fe(CN)6]4-/3- ； ECL-time curves (C) of bare GCE, Au-FONDs, Ru@FGA-Pd, Ru@FGA-Pd-Ab2 and their corresponding ECL-potential curves (D), all the modified electrodes were detected in 10 mL of PBS (pH 7.4) containing 55 mmol/L TPrA.

CV was used to accomplish the electrochemical characterization and the curves were shown in Figure 2A. It could be clearly seen that curve a exhibited well-defined redox peak in [Fe(CN)6]3-/4- solution. After the Au-FONDs composite was modified on GCE, there was no apparent increased peak current (curve b) appeared, which could be explained by the good electrical conductivity of Au-FONDs. However, when the as-formed surface was covered with Ab1, BSA and PSA orderly, the corresponding redox peak currents (curve c, d and e) declined successively. That could be explained by that Ab1, BSA and PSA are nonconductive proteins, so


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their sequent modifications would make the electron transfer process much harder. When the Ab2 bioconjugate was modified onto the formed film (curve f), there was an obvious increase in peak current due to the excellent electrical conductivity of Ru@FGA-Pd composite. Meanwhile, the EIS was used to further investigate the assembly course of the immunosensor. As illustrated in Figure 2B, the EIS of bare GCE (curve a) displayed a straight line with a very small semicircle, indicating a diffusional process. When the bare GCE was coated with Au-FONDs (curve b), the resistance was much smaller than the bare GCE, which benefited from the excellent electrical conductivity of Au-FONDs. As the Ab1, BSA and PSA were coated on the GCE orderly, the corresponding resistances (curve c, d and e) were increased ulteriorly because of the hindrance of the nonconductive proteins. However, the resistance decreased when the Ab2 bioconjugate was incubated onto the GCE surface, which could be explained by the favorable electron-transport ability of Ru@FGA-Pd composite. All the results above were completely consistent with the CV curves, indicating that the immunosnesor was successfully constructed step by step. The ECL responses of different nanocomposite used as the ECL probes were shown in Figure 2C. When the bare electrode was modified with Au-FONDs, weak ECL responses were generated. This may attributed to the very poor ECL property of Au-FONDs. Nonetheless, strong ECL signal was generated when the Ru@FGA-Pd was employed as the ECL probe, indicating the excellent ECL nature of Ru(bpy)32+ and favorable supporting ability and catalytic property of FGA-Pd composite. After the Ab2 combined with Ru@FGA-Pd, the ECL signal decreased due to the nonconductive property of proteins, which also proved the successful construction of Ab2 bioconjugate.


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3.4. Optimization of the Experimental Conditions

Figure 3. Optimization of the pH value of the PBS (A), concentration of TPrA (B), volume of Ru(bpy)32+ (C)and incubation time of Ab2 bioconjugate (D). All the immunosenosrs were incubated with 0.1 ng/mL of PSA.

To fulfill the best performance of the proposed ECL immunosensor, experimental conditions including pH value of the PBS and the concentration of TPrA were optimized in this work. In the beginning, pH value of the PBS was studied. As presented in Figure 3A, the maximum value of the ECL intensity appeared at 7.4 in a range from 5.8 to 8.5. Thus, 7.4 was chosen to be the optimum pH value of the PBS buffer to obtain high ECL intensity. In addition, the optimum concentration of TPrA was studied in the PBS with optimum pH value. As illustrated in Figure 3B, the maximum value appeared at 55 mmol/L, thus the optimum concentration of TPrA was 55 mmol/L.


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More importantly, the optimized volume of Ru(bpy)32+ and the incubation time of the Ab2 bioconjugate were some other vital factors on the performance of immunosensors. Experiment on the research of the optimized volume of Ru(bpy)32+ was conducted in the 10 mL of PBS (pH 7.4) containing 55 mmol/L of TPrA. As presented in Figure 3C, the value of the ECL intensity got to the maximum rapidly and tended to be stable at 400 μL. Thus, 400 μL of Ru(bpy)32+ was used in the following research. As seen in Figure 3D, the ECL intensity achieved the maximum value at 40 min in that case where the Ab2 could bind with PSA to an optimum extent. And the sluggish decline after the maximum could be made clear by the inactivated property of proteins. Therefore, the optimum incubation time was set at 40 min for the subsequent study. 3.5. Analysis of PSA

Figure 4. Calibration curve (A) and the corresponding ECL-potential curves (B) of the proposed ECL immunosensors incubated with: 0.1 pg/mL, 0.5 pg/mL, 1 pg/mL, 5 pg/mL, 50 pg/mL, 0.1 pg/mL, 0.5 ng/mL, 10 ng/mL, 30 ng/mL, 50 ng/mL of PSA. All the immunosensors were detected in 10 mL of PBS (pH 7.4) containing 55 mmol/L TPrA.

The proposed ECL immunosensors were incubated with different concentrations of PSA under best conditions. The relationship between the concentration of PSA (log c) and the ECL response (IECL) was presented in Figure 4A. The linear relation could be displayed as IECL=764.2


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log c + 4164 with a correlation coefficient of 0.9976. The ECL immunosesnor showed a wide linear range of 0.0001 - 50 ng/mL with a low detection limit of 0.056 pg/mL (S/N=3). And corresponding ECL intensity-potential curves were presented in Figure 4B. It was worth noticing the high sensitivity of the proposed immunosensor after comparing with other previous works which were listed in Table 1.3,6,9,11,33-36 This may attribute to the outstanding properties of the Au-FONDs and Ru@FGA-Pd composite, which played an irreplaceable role in exhibiting satisfying detection with wider linear range and lower detection limit than other reported methods. Table 1. The comparison of PSA detection using other methods with the proposed ECL immunosensor.

3.6. The Specificity, Stability and Repeatability of the ECL Immunosensor With the purpose of testifying the specificity of the ECL immunosensors, three of the most common interfering proteins in human serum including CEA, AFP and BSA with a specific concentration of 10 ng/mL were used to accomplish this research. As illustrated in Figure 5A, there were no significant differences among the ECL responses detected from PSA sample (0.1 ng/mL) and mixed samples and the relative standard deviation (RSD) of 1.42%, suggesting favorable specificity of the proposed ECL immunosensor. Moreover, stability is another


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important factor for the performance of the immunosensor. Electrodes modified with 0.1 ng/mL PSA were detected. According to the Figure 5B, it was found that no significant changes appeared in ECL signal with a RSD of 3.15%, which illustrated the good stability of the proposed ECL immunosensors. In addition, the repeatability of the ECL immunosensors (Figure 5C) was also inspected by detecting five ECL immunosensors incubated with PSA (0.1 ng/mL). With the result of RSD = 2.31%, the proposed ECL immunosensors exhibited a favorable reproducibility.

Figure 5. (A) Specificity of the proposed ECL immunosenosr: blank, CEA (10 ng/mL), AFP (10 ng/mL), BSA (10 ng/mL), PSA (0.1 ng/mL), mixture (containing 10 ng/mL of CEA, 10 ng/mL of AFP, 10 ng/mL of BSA and 0.1 ng/mL of PSA ). (B) Stability of the ECL immunosensor for 10 cycles. (C) Repeatability of five developed ECL immunosensors incubated with 0.1 ng/mL PSA.

3.7. Analysis of PSA in Human Serum


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In order to investigate the feasibility and accuracy of the immunosensor, the human serum was appropriately diluted with PBS (pH 7.4) before the detection. The diluted samples with an original concentration of 0.92 ng/mL were used for the research. As shown in Table 2, the recovery (between 93% and 102%) and RSD (between 0.91% and 3.58%) were acceptable, proving the potential of being a promising tool in detecting PSA in real samples. Table2. The recoveries of PSA in human serum samples were detected by using the proposed ECL immunosensor in 10 mL of PBS (pH 7.4) containing 55 mmol/L TPrA.

4. CONCLUSION In this work, a brand-new ECL immunosensor was constructed for PSA determination by means of combining the advantages of Ru@FGA-Pd and Au-FONDs nanocomposite. Firstly, FGA-Pd composite with excellent electrical conductivity and biocompatibility could combine with large amounts of Ru(bpy)32+ and captured Ab2 because of its high surface area and high porosity. More importantly, the FGA-Pd nanocomposite exhibited excellent catalytic ability for the redox between Ru(bpy)32+ and TPrA, which was conducive to amplify the ECL signal and improve the sensitivity of the immunosensor. Moreover, Au-FONDs with good electrical conductivity and biocompatibility were utilized as a novel ECL sensing platform to capture Ab1, which accelerated the electron-transfer of the ECL immunosensor remarkably. Featuring


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favorable specificity, stability and repeatability, this developed ECL immunosensor is expected to be a powerful tool for the sensitive analysis of PSA and other biomarkers.

ASSOCIATED CONTENT Supporting Information

The following files are available free of charge. The detailed preparation procedures of FGA and Pd NPs sol; EDX spectrum of Au-FONDs ; UV-vis spectrum of Au-FONDs and pure FONDs; EDX spectrum of FGA-Pd composite; Zeta potentials of Fe3O4 gel, FGA, Pd NPs, FGA-Pd, Ru(bpy)32+, Ru@FGA-Pd composite; ECL-time curves (A) of the Ru@FGA-Pd and Ru@FGA and their corresponding ECL-potential curves (B). (PDF)

AUTHOR IFORMATION Corresponding author *E-mail: [email protected]. *Tel.: +86 531 82765730. *Fax: +86 531 82765969. Notes: The authors declare no competing financial interest.

ACKNOLEDGEMENTS This work was supported by the National Key Scientific Instrument and Equipment Development Project of China (No.21627809), National Natural Science Foundation of China (No. 21575050 and 21375047).

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Quenching

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3D Nanostructured Palladium-Functionalized Graphene-Aerogel

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