Decoration of Reduced Graphene Oxide Nanosheets with

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Decoration of RGO nanosheets with aryldiazonium salt and gold nanoparticles towards a label-free amperometric immunosensor for detecting cytokine TNF-# in live cells Meng Qi, Yin Zhang, Chaomin Cao, Mingxing Zhang, Shenghua Liu, and Guozhen Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02353 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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Analytical Chemistry

Decoration of RGO nanosheets with aryldiazonium salt and gold nanoparticles towards a label-free amperometric immunosensor for detecting cytokine TNF-α in live cells Meng Qi, a Yin Zhang,a Chaomin Cao,a Mingxing Zhang, a Shenghua Liu,a Guozhen Liua, b*

a

Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P. R. China

b

ARC Centre of Excellence in Nanoscale Biophotonics (CNBP), Macquarie University, North Ryde 2109, Australia

*To whom correspondence should be addressed. Email: [email protected]. Tel: +86-27-6786 7535

ABSTRACT In this study, a label-free electrochemical immunosensor was developed for detection of cytokine TNFα. Firstly AuNPs loaded reduced graphene oxides nanocomposites (RGO-ph-AuNP) were prepared, and then a mixed layer of 4-carbxyphenyl and 4-aminophenyl phosphorylcholine (PPC) were modified to the surface of AuNPs for the subsequent modification of anti-TNF-α capture antibody (Ab1) to form the capture surface (Au-RGO-ph-AuNP-ph-PPC(-ph-COOH)) for the analyte TNF-α with the anti-fouling property. For reporting the presence of analyte, the anti-TNF-α detection antibody (Ab2) was modified to the graphene oxides which have been modified with the 4-ferrocenylaniline through diazonium chemistry to form Ab2-GO-ph-Fc. Then a sandwich assay was formed on gold surfaces for the quantitative detection

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of TNF-α based on the electrochemical signal of ferrocene. XPS, TEM, FT-IR, UV-vis and electrochemistry were used for characterization of the stepwise fabrications on the interface. The prepared electrochemical immunosensor were successfully used for the detection of TNF-α over the range of 0.1150 pg mL-1. The lowest detection limit of this immunosensor is 0.1 pg mL-1 TNF-α in 50 mM phosphate buffer at pH 7.0. The fabricated immunosensor provided high selectivity and stability, and can be used to detect TNF-a secreted by live BV-2 cells with comparable accuracy to ELISA but with lower limit of detection.

KEYWORDS Immunosensors, cytokines, AuNPs loaded reduced graphene oxides (RGO), aryldiazonium salts, ferrocene, TNF-α

1. INTRODUCTION Cytokines, low molecular weight (~ 6-70 kDa) soluble proteins secreted from the immune and nonimmune cells are core indicators of the performance of the human immune system.1 They are essential in controlling cell survival, growth, migration, development, differentiation, and function by binding with specific receptors and initiating immune regulation pathways.2 Consequently, monitoring cell functions and cell-to-cell communication by using their cytokine secretions has enormous value in biology and medicine.3 Tumor necrosis factor-alpha (TNF-α) is a kind of cytokine which involves a wide range of pathological and physiological process, such as fever, apoptotic cell death, and cachexia.4 The elevated TNF-α concentrations, especially for several-fold increases, have been found to be associated with a wide range of diseases, such as HIV infection, rheumatoid arthritis, crohn’s disease, severe meningococcemia, and osteosarcoma.5,6 However, such cytokine related immune reactions are dynamic and occur quickly due to cytokine network function.7 The produced cytokine concentration is normally low (in the pM range),8 and possibly there are dominant interference from heterophilic antibodies, rheumatoid factor, and specific or non-specific cytokine binding proteins.9 These measuring challenges have become the driving force of


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designing a sensitive assay for measuring cytokines in vivo. The most frequently used method for quantification of cytokines is the conventional enzyme-linked immunosorbent assay (ELISA) because it is cost-effective, high-throughput and relatively sensitive and specific. However, the long sample preparation time, high complexity in sample labeling, and a large amount of sample consumption have compromised clinical applications of ELISA. Thus broad interest exists in developing simple, sensitive and rapid cytokine analysis platforms. We have summarized approaches for the detection of cytokine by immunosensors.10 Recently, a microfluidic microsphere based biosensor for near real-time detection of TNF-α was reported by Konry and coworkers.11 A label-free localized surface plasmon resonance biosensing technique to detect cell-secreted TNF-α cytokines in clinical blood samples was also reported.12 This cellular functional immunoanalysis can detect the cytokines with a minimal blood sample volume (3 µL) and a total assay time 3 times shorter than that of ELISA (5-6 h). Revzin and coworkers has reported a microdevice for detecting local interferon gamma (IFN-γ) release from primary human leukocytes in real time.13 However, most of these methods were expensive with sophisticated instruments and relatively low sensitivity. Electrochemical immunoassays had attracted a variety of attention because of their simple instrumentation, low cost, and good portability, and the introduction of the monoclonal antibody to the nanomaterials modified sensing interface will greatly enhance the detection specificity and sensitivity.14 The nanomaterials, such as graphene oxides (GO)15,16 and gold nanoparticles (AuNPs),17,18 which have excellent electrical signal amplification and versatile functionalization chemistry are very popular in the field of electrochemical biosensors. Hou and co-workers have developed an immunosensor based on AuNP for the detection of TNF-α.19 The authors claim that TNF-α can be determined in a competitive assay from 1 pg mL-1 to 10 ng mL-1 with a detection limit of 0.5 pg mL-1, which is lower than some other studies. Reductive GO (RGO) have similar properties with GO in terms of two-dimensional planar structure and large specific surface area, and the electrical conductivity of RGO is significantly increased comparing to that of GO.20,21 However, RGO is prone to aggregate during the process of GO reduction



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with the loss of a lot of oxygen and thus less dispersible in water. Functionalization of RGO with pphenyl-SO3H molecules (prepared via diazotization) gave a better dispersion of the resultant RGO suspension in polar aprotic solvents without compromising the high electrical conductivity (1250 S m-1) compared to that before functionalization.22 The well dispersion in aqueous solution makes it possible to do further functionization of RGO. Functionalization of RGO including reactions of RGO with organic molecules and decorating RGO with inorganic nanoparticles further strengths their competitive abilities in various applications. Synthetic nanocomposite RGO/AuNPs has already been used to produce electronic storage devices such as nonvolatile memory,23 and have faster energy transfer rate than any one of the two.24,25 In addition, decoration of RGO with AuNPs to achieve the sensing interface have been successfully used for detection of protein immunoglobulin G26 and even small molecules, such as hydrogen peroxide and glucose.27,28 However, the decoration of RGO with AuNPs in these studies was based on Au-NH bonds29, Au-SH bonds30 or just physical adsorption26. No study has investigated the stability of the fabricated device based on RGO and AuNPs nanocomposites. In this study we target to investigate the decoration of RGO with AuNPs through Au-C bonds, which have been proved to be more stable than Au-NH and Au-SH bonds,31 and its subsequent application for sensing with respect to enhance the sensitivity and selectivity. For an immunosensor the most difficult part is to report the analyte signal because there is no redox reaction between antigen and antibody. The most frequently used method is to label enzymes to immenosensors for detection of cytokines.32,33 The drawback of this method is to make the enzyme and the protein active at the same time. In addition, the capacity of the enzyme is limited, as a result, the amount of fixed labels is very low resulting in the compromised sensitivity. Ferrocene derivatives and ferrocenebased polymers show excellent redox properties.34 Thus conjugation of detection antibody and ferrocene species to the nanomaterials will help to load large amount of detection antibody as well as the signal reporter, two important components for sensitive detection.16 Herein, we developed a label-free immunosensor based on a RGO-ph-AuNP-aryldiazonium salt


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modified sensing interface for the detection of TNF-α (Scheme 1). Four functional components were introduced to the sensing interface: (a) aryldiazonium salts, (b) AuNPs decorated RGO nanocomposites (RGO-ph-AuNP), (c) 4-aminophenyl phosphorylcholine (PPC) molecules, (d) TNF-α detection antibody (Ab2) and 4-ferrocenylaniline modified GO nanocomposites (Ab2-GO-ph-Fc). The RGO-ph-AuNP nanocomposites not only helped to amplify the signal because of their improved electron transport properties, but also enhanced the loading amount of capture antibody, which was essential to increase the detection sensitivity. The nanocomposites of Ab2-GO-ph-Fc acted as the signal reporters of the immunosensor. Once specific biorecognition event occurred between TNF-α and the detection antibody in Ab2-GO-ph-Fc, there would be a modulation of the ferrocene electrochemistry. The more cytokine reacted with the detection antibody, the larger electrochemical signal from ferrocene would be obtained. PPC, a zwitterionic molecule, has demonstrated the capability to resist non-specific protein adsorption without passivating the sensing interface,35 and helped to increase the specificity and selectivity. The aryldiazonium salt chemistry was used in the whole fabrication process, targeting to increase the stability of the sensing device.36 The designed system has been successfully used for the detection of TNF-α with high sensitivity, selectivity and stability, and also is applicable to detect cytokines secreted by live cells. To our knowledge, it is the first study to decorate RGO with AuNP through aryldaizonium salt chemistry for development of a label-free amperometric immunosensing interface targeting the detection of cytokines.



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Scheme 1. The schematic of the fabricated immunosensor for detection of TNF-α. The size of species in this diagram is not to scale. 2. EXPERIMENTAL SECTION 2.1 Chemicals and Instruments. Graphite, hydrochloric acid, sulfuric acid, absolute ethanol, potassium chloride, gold trichloride, potassium ferricyanide, ferrocene, trifluoroacetic anhydride, tris(hydroxymethyl)aminomethane (Tris), sodium nitrite, sodium cyanoborohydride, acetonitrile (CH3CN, HPLC

grade),

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride

(EDC),

N-

hydroxysuccinimide (NHS), 2-(N-morpholino) ethanesulfonic acid (MES), 4-nitrophenyl diazonium tetrafluoroborate and lipopolysaccharide were purchased from Sigma-Aldrich. 4-Aminophenyl phosphorylcholine (C11H19N2O4P, PPC) was purchased from Toronto Research Chemicals Inc. The aryldiazonium salt N2+BF4--ph-NO2 and N2+BF4--ph-COOH were prepared according to previous methods.36 4-Ferrocenylaniline was synthesized by following the procedures by Hu et al with some modifications (see detailed method in the Supporting Information).37,38 Mouse tumor necrosis factor-alpha (TNF-α) (antigen, Ag), anti-mouse TNF-α monoclonal antibody (capture antibody, Ab1) and anti-mouse TNF-α polyclonal antibody (detection antibody, Ab2) were purchased from R&D Systems. Aqueous


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solutions were prepared using Mill-Q water. Phosphate buffer solution used in this work contained 0.05 M KCl and 0.05 M K2HPO4/KH2PO4 adjusted to pH 7.0 with NaOH or HCl solution. All electrochemical experiments were conducted using an Electrochemical workstation CHI660E (CHI Instrument, Shanghai). All experiments utilized a Pt secondary electrode and an Ag/AgCl (3.0 M KCl) reference electrode. All voltammetric measurements were obtained with a scan rate of 100 mV s−1. X-ray photoelectron spectra (XPS) were collected from Au plates on a VG multilab 2000 spectrometer with a monochromated Al Kα source (1486.6 eV), hemispherical analyzer, and multichannel detector. The spectra were calibrated on the C1s peak (285.0 eV). Spectra were analyzed using XPSPEAK41 software. Transmission Electron Microscope (TEM) studies were performed with a JEM-2100 (HR) instrument at a voltage of 200 kV. UV-vis absorption data were collected on a Shimadzu UV-vis spectrophotometer model 2450. Fourier transform infrared spectroscopic (FT-IR) measurements were performed with TENSOR27 FTIR spectrophotometer. KBr pellet was used to prepare the samples. 2.2 Preparation of the immunosensor interface for detection of TNF-α. The preparation of RGO-phAuNP nanocomposites and Ab2-GO-ph-Fc were detailed at the Supporting Information Scheme S1. The derivatization of the clean Au electrode with 4-aminophenyl to achieve Au-ph-NH2 (Scheme 1) was based on the method reported previously.39 Then Au-ph-NH2 surface was converted to Au-ph-N2+Cl4- (in situ) by incubation Au-Ph-NH2 surface with 1 mL HCl solution (0.5 M) containing 1 mM NaNO2. The RGO-phAuNP nanocomposites were modified to GC-Ph-N2+Cl4- surface through C-C coupling by applying the potential between 0 V and -1.1 V for twenty four cycles at a scan rate of 100 mV s-1 in the RGO-ph-AuNP nanocomposites solution (1 mg mL-1) to get the Au/RGO-ph-AuNP surfaces. Then the mixed layers of PPC and 4-carboxyphenyl was modified to Au/RGO-ph-AuNP surface by applying a potential to the electrode between 0.6 V and -0.6 V for two cycles at a scan rate of 100 mV s-1 in the 0.5 M HCl solution containing 1 mM N2+BF4--ph-COOH, 1 mM PPC and 2 mM NaNO2. The achieved Au/RGO-ph-AuNPPPC(-ph-COOH) surface was subsequently soaked in the 0.4 M EDC and 0.1 M NHS in 0.1 M MES buffer for 1 h followed by adding TNF-α monoclonal antibody (5 µL, 50 µg mL-1) in PBS (pH 7.4) to



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react for 1 h at 4 °C to get the Au/RGO-ph-AuNP-PPC(-ph-COOH)/Ab1 sensing interface. Subsequently, different concentrations of TNF-α solution were added onto the electrode surface and incubated for 10 min at 4 °C. After extensive wash, 10 µL of the prepared Ab2-GO-ph-Fc solution was applied onto the electrode for another 10 min at 4 °C. Finally, the electrode was washed and dried before measurements. 2.3 Cell culture and ELISA measurement. Mouse BV2 cells were cultured in a T75 cm2 flask containing Dulbecco's Modified Eagle's medium (DMEM) supplement with 10% FBS, 100 U.mL−1 of penicillin, 0.1 mg mL−1 of streptomycin. The cells were cultured to about 80-90% confluence before harvest. During harvest, the cells were washed twice with DPBS followed by trypsinization using 2 mL trypsin to detach the cells from the flask. The trypsin was neutralized by adding 4 mL of fresh supplemented medium, and the harvested cells in DMEM medium suspension was transferred into a centrifuge tube and centrifuged at 200 rcf for 6 min. The supernatant was discarded and the cells were resuspended in fresh medium. The prepared immunosensor and BD OptEIATm Mouse TNF-α ELISA kit (BD Bioscience) were used to measure the concentration of TNF-α secreted by BV-2 cells after lipopolysaccharide (LPS) stimulation. For preparation of TNF-α samples, the cells with the density of 1 x 106 /mL were suspended in 1 mL of warm medium containing 0.11 µg mL-1 LPS from Escherichia coli 026:B6 to secret TNF-α for 0 h, 2 h, 4 h, 6 h, 8 h, and 20 h, respectively. Supernatants from cells were collected in triplicate. The Nunc MaxiSorp 96 well plate and Galaxy plate reader were used for ELISA. Results are reported as means ± standard deviation. 3. RESULTS AND DISCUSSION 3.1 Preparation of the RGO-ph-AuNP nanocomposites. The FT-IR spectroscopy has confirmed the successfully reduction of GO to RGO (Supporting Information Figure S1 a). The premade five RGO compostites (RGO-ph-NO2, RGO-ph-COOH, RGO-ph-NH2, RGO-SO3H, RGO-ph-H) were used to prepare the RGO-ph-AuNP nanocompsities (Scheme S1 a), respectively, which were characterised by FTIR (Supporting Information Figure S1 b). Figure 1 a shows the UV-vis spectra of the obtained five RGOph-AuNP nanocompsities. The obvious adsorption peak at 520 nm, characteristic of colloid gold


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nanoparticles, was observed for the RGO-ph-COOH, RGO-ph-NO2, and RGO-ph-NH2 although the peak for RGO-ph-NH2 is smaller. No obvious peak at 520 nm was observed for RGO-ph-SO3H and RGO-ph-H. In order to further confim the successful loading of AuNPs on RGO, the prepared RGO-ph-AuNP nanocomposites (1 mg mL-1, 10 µL) were droped down to a glassy carbon electrode surface followed by electrochmical measurment in H2SO4 (Figure 1 b). As expected that RGO did not show any Faradaic peaks between -0.3 and 1.5 V versus Ag/AgCl. For the RGO-ph-AuNP nanocompostites prepared using three ayrldiazonium salts (RGO-ph-COOH, RGO-ph-NO2, and RGO-ph-NH2) an oxidation peak (centred at 1.2 V) and a reduction peak (centred at 0.8 V) were observed, which corresponded to the oxidation and reduction peaks of gold on the surface, respectively, hence showing that AuNPs were successfully attached to the surface. Ignorable peaks were observed for RGO-ph-AuNP prepared by using RGO-phSO3H and RGO-ph-H, which was consistent with the results of UV-vis (Figure 1 a). Then the stability of the formed RGO-ph-AuNP nanocomposites was investigated by applying multiple cycles (Figure 1 c). Interestingly the AuNP reductive peak for RGO-ph-NO2 remained almost the same but that for RGO-phCOOH and RGO-ph-NH2 disappeared after 10 cycles. We suggested that RGO-ph-NO2 were reduced to RGO-ph-NH2 which was then reduced to RGO-ph-N2+Cl- by NaBH4 followed by the attachment of AuNP by forming Au-C bonds. The AuNP decorated on RGO-ph-COOH was due to the physical adsorption, which came off after multiple cycles. However RGO-ph-NH2 was reduced to RGO-ph-H by NaBH4,16 and the limited amount of AuNPs which disappeared after multiple cycles were attached. Thus controlling the amount of NaBH4 was essential for obtaining RGO-ph-AuNP nanocomposites (Figure 1 d), and it was observed that reaction with 10 mM of NaBH4 helped to decorate the RGO with maximum of AuNPs based on RGO-ph-NO2. The RGO-ph-AuNP nanocomposites using RGO-ph-NO2 as the template to complex AuNPs resulted in the largest amount of AuNPs on RGO than that using other RGO-aryldiazonium salt conjugates (Figure 1 c). On the basis of the size of the reduction peak at about 0.8 V, the total surface area of AuNPs on the RGO-ph-AuNP surface was estimated to be 0.27 cm2. Thus these nanocomposites (using RGO-ph-NO2) were used for the following further studies.



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Figure 1. (a) UV-vis spectra for the corresponding RGO-ph-AuNP nanocomposites (using five different aryldiazonium salt modified RGO). (b) The electrochemical behavior of RGO-ph-AuNPs nanocomposites (using five different aryldiazonium salt modified RGO) on glassy carbon electrode in 0.05 M H2SO4 for the first scanning lap with the scan rate of 100 mV s-1. (c) The peak area at 0.8 V for RGO-ph-AuNP nanocomposites (using five different aryldiazonium salt modified RGO) adsorbed on the glassy carbon electrode for the first scanning lap and the tenth scanning lap. (d) The relationship between the peak area at 0.8 V in 0.05 M H2SO4 and the amount of NaBH4 added for making RGO-ph-AuNP nanocomposites (using RGO-ph-NO2). 3.2 Characterization of RGO-ph-AuNPs. The decoration of AuNPs on RGO nanosheets was further confirmed by high-resolution transmission electron microscopy (HRTEM). The RGO nanosheets were decorated with the spherical AuNPs in high density (Figure 2, and the particle size of the AuNPs is within the range of 1-10 nm (average size of 4.8 ± 0.3 nm), which was smaller than that of the reported AuNPs on RGO nanosheets using ascorbic acid as a reducing agent under ultrasonication.40 The measured fringe


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lattices of AuNPs corresponding to the (111) plane and the (200) plane were found to be 0.24 nm, and 0.20 nm, respectively (Figure 2 d), which corresponded well to the spacing between (111) plane of face centered cubic (fcc) gold (0.235 nm) (JCPDS card No. 04-0784), and in good agreement with the d spacing of Au (200) (0.20 nm).41

Figure 2. (a-c) TEM images of the prepared RGO-ph-AuNP nanocomposites in different magnifications. (d) HRTEM image along with fringe spacing of Au (111) and Au (200) planes in the enlarged form. 3.3 Characterization of GO-ph-Fc. The FT-IR spectra (Figure 3 a) of GO shows the characteristic stretching frequencies for oxygen containing functional groups such as O−H (3400 cm−1), C=O（1720 cm, C−O (1105 cm−1). In the spectra of GO-ph-Fc, another three new bands emerged at 3017 cm-1, 1488

1

cm-1, and 859 cm-1 respectively, which were assigned to the fingerprint of ferrocene suggesting the successful preparation of GO-ph-Fc.42 The electrochemistry was also used to characterize GO-ph-Fc (Figure 3 b). A well-defined characteristic peak of ferrocene was observed at +0.2 V on the cyclic



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voltammogram of glassy carbon electrode after dropping down 10 µL 1 mg mL-1 GO-ph-Fc (blue), while no peaks was observed on that after dropping down 10 µL 1 mg mL-1 GO (red), further confirming that ferrocene molecules were modified on GO successfully.

Figure 3. (a) FT-IR adsorption spectra of GO and GO-ph-Fc. (b) The electrochemistry of the glassy carbon surface after dropping down 10 µL GO-ph-Fc and GO, respectively in 0.05 M PBS at the scan rate of 100 mV s-1. 3.4 XPS characterization of the stepwise fabricated sensing interfaces. XPS measurements were carried out to further characterize the deposited species after stepwise modification of the gold surface. The N1s peak at around 405.9 eV is assigned to the nitrogen of the nitro group on Au-ph-NO2 (Figure 4 a). However, a small N1s peak at around 399.8 eV indicates the presence of nitrogen species with lower binding energy arising from nitrogen atoms in azo groups as observed previously.43 After reduction of NO2 groups, the N1s peak for-NO2 groups disappeared while the peak at 400.2 eV was observed suggesting the successful transition of nitro groups to amine groups (Figure 4 b). For the N1s species on the RGO-ph-AuNP interface, only a tiny peak at about 399.8 eV remained arising from the few azo groups left and indicating the successfully reduction of amine groups (Figure 4 c). After attachment of PPC, an additional peak at 401.8 eV was emerged (Figure 4 d) corresponding to the nitrogen of -N(CH3)3 in PPC layers. Meanwhile, a P2p peak at 133.58 eV (Figure 4 e) was emerged indicating the presence of phosphate. These two emerged peaks suggest the successful modification of PPC molecules to the sensing


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interface. After modification of Ab1, two additional N1s species were observed (Figure 4 f). The highest binding energy component at 402.6 eV can be reasonably assigned to the unreacted portion of the NHS ester nitrogen, which is understandable since the EDC/NHS binding efficiency is normally 50-60%. The peak at 400.3 eV was assigned to the amide nitrogens of the peptide bonds.44

Figure 4. N1s core level spectra for (a) Au-ph-NO2, (b) Au-ph-NH2, (c) Au/RGO-ph-AuNP, (d) Au/RGOph-AuNP-PPC/(-ph-COOH), (f) Au/RGO-ph-AuNP-PPC/(-ph-COOH)/Ab1. (e) P2p core level spectra for Au/RGO-ph-AuNP-PPC/(-ph-COOH). (g) XPS survey spectra for stepwise modification of the sensing interfaces. The C1s core level spectra were plotted in the Supporting Information Figure S2. There is no difference



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in C1s species between Au-ph-NO2 and Au-ph-NH2. The C1s scan of the Au/RGO-ph-AuNP surface (Figure S2 a) included three peaks (the graphitic carbon peak at 284.6 eV, a broader peak at 285.5 eV, and a small peak at 288.2 eV). The peak at 265.5 eV might be due to the presence of low levels of oxidised carbon species and peak at 288.2 eV was due to the presence of trace amount of -COOH on the edge of RGO. Meanwhile, a C1s peak was fitted to the spectra at 284.2 eV although a good fitting could be obtained without this peak, which was assigned to the -C-Au- carbon species.36 For the Au/RGO-phAuNP-PPC/(-ph-COOH) interface, the signal for the peak at 284.2 eV increased obviously suggesting the -ph-PPC and -ph-COOH components were attached to AuNPs but not to RGO resulting in the increased C-Au- carbon species. 3.5 Performance of the fabricated immunosensors for the detection of TNF-α. Before the detection of TNF-γ, cyclic voltammetry was employed to investigate the electrochemistry of the stepwise fabricated electrode interfaces in Fe(CN)63−/Fe(CN)64− redox couple in pH7.4 PBS buffer (Supporting Information Figure S3). The attachment of the RGO-ph-AuNP nanocomposites could increase the electronic coupling to the underlying electrode, which was favorable for the following analyte detection with increased sensitivity. As shown in Scheme 1, after the immobilization of Ab1 onto the prepared interface Au/RGOph-AuNP-PPC(-ph-COOH)/Ab1 followed by the capture of TNF-α, Ab2-GO-ph-Fc could be easily attached to the electrode surface via the specific Ag-Ab interaction to form a sandwich structure. Based on the geometric area of a gold disk electrode (7.07 × 10-2 cm2), the diameter of the AuNPs determined by TEM (4.8 nm), and the total surface area of AuNPs on Au electrode (0.27 cm-2), the density of AuNPs on the gold electrode could be estimated to be 1.5 × 1012 AuNP cm-2, which was 300 times higher than the density of AuNPs (50 × 108 AuNP cm-2) directly modified on GC disk electrode with the same electrode area (7.07 × 10-2 cm2) reported previously.31 Thus larger number of AuNPs on the surface corresponded to the bigger surface area for loading the Ab1, suggesting the RGO-ph-AuNP was helpful to conjugate large number of Ab1. On the basis of the footprint area of a benzene ring (3.78 × 10-18 cm2), the surface area for a single AuNP (1.8 × 10-13 cm2), and the 1:1 molar ratio of 4-carboxyphenyl and PPC molecules modified


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on AuNPs, one AuNP could accommodate 2.38 × 104 4-carbxyphenyl molecules. Subsequently 1.4 × 104 4-carbxyphenyl molecules could be activated by EDC/NHS to react with Ab1 based on 60% efficiency of EDC/NHS coupling. Thus the calculated concentration of Ab1 modified on the sensing interface was about 3.8 × 10-8 mol cm-2. In order to test the response of the modified sensing interface Au/RGO-ph-AuNP-PPC(-ph-COOH)/Ab1 to the analyte, the sensing interface was firstly incubated with 50 pg mL-1 TNF-α. The ferrocene current of detection antibody (Ab2-GO-Fc) changed with the concentration of TNF-α correspondingly (Figure 5 a). The optimized incubation time was 10 min (Figure S4) and this quick response time might be due to the presence of large number of Ab1 (3.8 × 10-8 mol cm-2) on the sensing interface.45 As shown in Figure 5 b the increase in the peak current was proportional to the concentration of TNF-α in the range of 0.1~150 pg mL-1 with correlation coefficients of 0.9893 and a lowest detectable concentration of 0.1 pg mL-1. Table S1 compared the limit of detection (LOD) of herein sensor with that of reported sensors for detection of TNF-α. The herein sensor provided the lowest LOD than others reported so far although the linear range was not the widest. The use of RGO-ph-AuNP nanocomposites in this study could not only increase the Ab1 loading due to the large surface area but also could facilitate the electron transfer, which resulted in the achieved high sensitivity (0.065 µA/pg mL-1).

Figure 5. (a) SWV curves for Au/RGO-ph-AuNP-PPC(-ph-COOH)/Ab1 surfaces for the detection of TNF-α with concentrations of 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 150, and 200 pg mL-1,



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respectively. (b) A calibration curve showing the variation in relative current with the concentration of TNF-α in 0.05 M phosphate buffer (pH 7.4). To investigating the selectivity of fabricated sensor, five potentially interfering compounds, including bovine serum albumin (BSA), prostate specific antigen (PSA), cancer antigen 125 (CA-125), and mouse IgG, were added (each with the concentration of 150 pg mL-1) to the detection buffer solution, respectively. Then the response of this prepared sensor to 50 pg mL-1 TNF-α was recorded (Figure 6 a). No significant effect from these five non-specific proteins were detected. The retained current percentage for all nonspecific proteins was above 92%), suggesting an ignorable degree of interference for these species tested in relation to the control test (

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