Graphene Oxide Thin Film with Dual Function Integrated into a

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Graphene Oxide Thin Film with Dual Function Integrated Into a Nano-Sandwich Device for In Vivo Monitoring of Interleukin-6 Meng Qi, Jiawei Huang, Hui Wei, Chaomin Cao, Shilun Feng, Qing Guo, Ewa M. Goldys, Rui Li, and Guozhen Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10753 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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ACS Applied Materials & Interfaces

Graphene Oxide Thin Film with Dual Function Integrated Into a Nano-Sandwich Device for In Vivo Monitoring of Interleukin-6

Meng Qi,a Jiawei Huang,b Hui Wei, a Chaomin Cao, a Shilun Feng, c Qing Guo,d Ewa M. Goldys, c Rui Li,b Guozhen Liu a,c,e* 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

Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, 152 Luoyu Road, Wuhan 430079, P. R. China. c

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

d

School of Public Health, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, P. R. China.

e

Department of Molecular Sciences, Macquarie University, North Ryde 2109, Australia

*To whom correspondence should be addressed. Email: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT：：Graphene oxide (GO), with its exceptional physical and chemical properties and biocompatibility, holds a tremendous potential for sensing applications. In this study, GO, acting both as the electron transfer bridge and the signal reporter, was attached on the interface to develop a label-free electrochemical nanosandwich device for detection of interleukin 6 (IL-6). First, a single layer of GO was covalently modified on gold electrodes followed by attachment of anti-IL-6 capture antibody to form the sensing interface. The 4-aminophenyl phosphorylcholine was further attached to the surface of GO to minimise non-specific protein adsorption. For reporting the presence of analyte, the anti-IL-6 detection antibody was covalently modified to the GO which has been integrated with the redox probe Nile blue (NB). Finally, a nano-sandwich assay was fabricated on gold surfaces for detection of IL-6 based on the electrochemical signal of NB. The prepared nanosandwiches demonstrated high selectivity and stability for detection of IL-6 over the range of 1-300 pg mL−1 with the lowest detectable concentration of 1 pg mL−1. The device was successfully used for monitoring of IL-6 secretion in RAW cells and live mice. By tailoring the GO surface with functional components, such devices were able to detect the analyte in vivo without causing inflammatory response. KEYWORDS: graphene oxides, nano-sandwich device, cytokines, in vivo detection, aryldiazonium salt chemistry

INTRODUCTION The choice of interface chemistry is critical for a successful sensor device and the sensing interface together with the recognition elements must be rationally designed to optimise the sensitivity, specificity, selectivity, reproducibility, and response time of the sensor.1-2 Forming such an efficient sensing interface is particularly essential for detecting analytes in a complex matrices such as blood, urine, saliva, and cell culture. 2


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It is desirable for the sensing interface to be integrated with a high surface area film which is thin enough to keep the biomolecules close to the sensing element to allow rapid signal response. Ideally, such film should be universally applicable to multiple analytes, and it should have a suitable chemistry for facile bioconjugation. Moreover, the interface together with the film must be sufficiently robust so that it remains intact during washing and sensing, and the film must not interfere with the transduction mechanism, and must be simple to fabricate. Nanotechnology has a profound impact on biosensor research, and new nanomaterials provide significant opportunities to develop quality biosensing interfaces.3 Graphene oxide (GO) is a two-dimensional nanomaterial which has a layer structure containing sp3 hybridized carbon, high surface area (theoretical value of 2630 m2 g−1), and good conductivity.4 In addition, GO is biocompatible and does not cause inflammatory response in tissues.5 Additionally, GO offers a versatile surface chemistry due to the presence of different types of oxygen-bearing carbonyl, carboxyl, epoxy, and hydroxyl groups. It also combines other exceptional physical, chemical, electrical, and optical characteristics properties which make it one of the best material for the transduction of signals associated with the recognition of analytes and other biosensing applications.6 In particular, GO, enriched with oxygenated functional groups, can serve as a scaffold for surface immobilization of biomolecules. A number of strategies have been used for fabricating GO-based sensing interfaces,7 such as physical adsorption,8 π-π stacking interaction,9 direct π stacking interaction,10 and aryldiazonium salt chemistry.11 However, only a few studies paid attention to the thickness of the GO based nanostructure on sensing interface until recently a thin (~4 nm thick) and conformal GO film was reported on five diverse substrates by spin coating for immunoassays and nucleic acid hybridization assays.12 Such thin layer of GO film on the sensing interface is essential for electrochemical biosensors, since it allows the biomolecules to be close to the sensing interface resulting in the fast electrical communication between the analyte and the electrodes underneath. 3



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Cytokines are small soluble proteins secreted from cells which are involved in inflammation and cell proliferation processes through a complex cytokine network.13 Inflammation is generally a disease response, so abnormal level of secreted cytokines may be used as biomarker in diseases such as cancer and as a treatment monitoring tool.14 For example, Interleukin-6 (IL-6) at the centre of this work is a 20 kDa protein, one of many types of cytokines involved in the regulation of the immune and inflammatory responses15. Cytokine detection remains intensively investigated with significant efforts invested into exploring different approaches for detection of cytokines.7 Enzyme-linked immunosorbent assays (ELISA) is the most frequently used method for cytokine quantification. ELISA requires lengthy analysis times, not insignificant sample size, has limited sensitivity in some cases, and it lacks the ability to monitor the cytokines in real time or in a dynamic manner. Electrochemical immunosensors with nanostructured surfaces provide an attractive approach for detecting cytokines owing to their simplicity, low cost, miniaturization and portability. They can also be integrated with nanomaterials with high surface area to yield enhanced specificity and sensitivity when grafted with antibodies.16-18 The nanomaterials (for example, quantum dots19, gold nanoparticles20, graphene based nanomaterials21-22) can also assist in the reporting of the analyte signal in electrochemical sensors. These nanomaterials can be used as the nanocarriers to load the maximum amount of signaling molecules such as horseradish peroxidase23, redox probe ferrocene24 or hemin/G-quadruplex25 to report the electrochemical signal. Recently the preparation and applications of ferrocene/graphene and hemin/graphene nanocomposites for reporting the electrochemical signal in biosensors has been reviewed.26 Loading detection molecules with signaling molecules is required for immunosensors as there is no redox reaction between the antigen and complementary antibody. Zhang et al have proposed an ultrasensitive immunosensor for the detection of IL-6 with detection limit of 0.5 pg mL-1 using quantum dot-based nanoprobes19 Another competitive electrochemical immunosensor for the detection of human IL-6 4


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was based on a heated electrode. This sensor combined highly biocompatible reduced GO and gold-palladium bimetallic nanoparticles as a platform, and high sensitivity silver nanoparticles as nanoprobes.27 The as-prepared immunosensor showed good reproducibility, acceptable stability, and a wide linear response range from 0.1 to 100000 pg mL-1 with a detection limit of 0.059 pg mL-1. For comparison, an alternative electrochemical sensor prepared by Li and coworkers used ferrocene which was physically adsorbed onto porous polyelectrolyte nanoparticles using CaCO3 as template,24 achieving the low detection limit for IL-6 of 1 pg mL-1. Recently, we covalently attached GO to ferrocene molecules through C-C bonds and used the conjugate as a nanoprobe in an immunosensor for the detection of a cardiac marker, troponin-I20, and cytokine TNF-α.28 However, facile oxidation of ferrocene makes it unsuitable for routine analysis under harsh conditions.29 None of these methods demonstrated the capability to detect analyte in vivo. In this work we formed a thin film of GO on gold surface followed by the attachment of biomolecules for developing an immunosensor for detection of IL-6 (Scheme 1). In this nanosandwich design, GO acts as a biocompatible sensing platform for bridging the capture antibody and it also provides enhanced electrical communication between the analyte and the electrodes underneath. Additionally, after being loaded with lots of Nile blue (NB) which is known for its excellent redox properties, GO acts as a nanoprobe for further signal amplification.30 In order to increase the stability of the sensing device, the aryldiazonium salt chemistry was used to fabricate the interface. This immunosensor was successfully used for the detection IL-6 in live RAW cells and in moving animals, where it provided better performance than the traditional ELISA assay in terms of simplicity of preparation, faster response time and higher sensitivity. The results indicated that GO could simultaneously function as a nanoprobe and sensing interface in sandwich electrochemical sensor devices. To our knowledge, it is the first time to report the in vivo cytokine sensing based on GO modified sensing interface. The GO, as a component of the in vivo 5



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device did not cause the inflammatory response. By tailoring the GO surface with functional components, such devices were able to detect analyte in vivo, which provided potentials to monitor cell-secreted products in bioreactors and in living animals.

Scheme 1. (a) The schematics of the preparation of Ab2-GO-NB conjugates. (b) The schematics of fabricated nanosandwiches for the detection of IL-6. The size of molecular species in this diagram is not to scale. MATERIALS AND METHODS Materials. Graphene oxide, hydrochloric acid, sulfuric acid, absolute ethanol, potassium

chloride,

potassium

1-ethyl-3-(3-(dimethylamino)propyl)

ferricyanide, carbodiimide

sodium

nitrite,

hydrochloride

(EDC),

N-hydroxysuccinimide (NHS), 2-(Nmorpholino) ethanesulfonic acid (MES), nile blue, 4-nitroaniline and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. 4-Aminophenyl phosphorylcholine (C11H19N2O4P, PPC) was purchased from Toronto Research Chemicals Inc. Mouse IL-6 (antigen, Ag) and anti-mouse IL-6 monoclonal antibody (capture antibody, Ab1) were purchased from Abm (Canada). Anti-mouse IL-6 polyclonal antibody (detection antibody, Ab2) were purchased from abcam (England). Aqueous solutions were prepared using Milli-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. 6


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Instruments. 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. Atomic Force Microscope (AFM) was conducted by Shimadzu SPM9700. Scanning Electronic Microscopy (SEM) was conducted by FEI Quanta 200. UV-vis absorption data were collected on a Shimadzu UV-vis spectrophotometer model 2450. Fourier transform infrared spectroscopic (FT-IR) measurements were performed with a TENSOR27 FTIR spectrophotometer, and KBr pellet was used to prepare the samples. All Raman spectroscopy was performed on an in Via Renishaw Raman spectroscope, using dry samples on a substrate. A 50x objective was used for all measurements and the system was used unpolarised with a 514.5 nm argon ion laser. Preparation of Ab2-GO-NB. The preparation of Ab2-GO-NB is detailed in Scheme 1 a. GO (0.5 mg) was dissolved in 1.0 mL ice-cold MES (pH 5.5) buffer, to which 3 mM NB dissolved in 300 µL HCl solution (0.5 M) were added. The mixture was stirred in ice bath for 1.5 h to attach NB molecules to GO surfaces by π-π conjugation for the formation of GO-NB conjugates, and then 500 µL MES buffer (pH 5.5) containing 0.4 M EDC and 0.2 M NHS was added to the mixture to activate the carboxylic acid groups on GO-NB. The mixture was stirred in ice bath for another 0.5 h. Then the solution was washed three times by centrifugation, and the collected solid (activated GO-NB) was finally dispersed in 1.0 mL PBS buffer (pH7.4). After that 50 µL PBS buffer (pH 7.4) containing IL-6 polyclonal antibody (50 µg mL-1) was added in 950 µL PBS buffer (pH 7.4) solution containing GO-NB (0.5 mg mL-1). The resulting mixture was stirred at room temperature for 2 h. The obtained solution (300 7



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µL) was centrifuged by centrifugal filter (Millipore, 30 kD) for 10 min at 4000 rpm to wish off the unreacted antibody and the collected solid was finally dispersed in 1.0 mL PBS buffer (pH7.4) to get the purified Ab2-GO-NB bioconjugates (0.5 mg mL-1). Preparation of the GO based Immunosensor Interface for Detection of IL-6. The derivatization of the clean Au electrode with 4-nitrophenyl to get Au-ph-NO2 surface (Scheme 1) was achieved by in-situ electrochemical adsorption of aryldiazonium salts. The nitro groups on the Au-ph-NO2 surface were then converted to amine groups in proton solution (VH2O/VEtOH =1:9) to form the Au-ph-NH2 surface. A single layer of GO were modified to the Au surface through C−C coupling by applying the potential between 0 and −0.6 V for 24 cycles at a scan rate of 100 mV s−1 in 0.5 M HCl solution containing GO (0.5 mg mL-1) and NaNO2 (1 mM) to get the Au-ph-GO surface. Then, PPC were modified to the Au-ph-GO surface by applying a potential to the electrode between 0.6 and −0.6 V for three cycles at a scan rate of 100 mV s−1 in the 0.5 M HCl solution containing 1 mM PPC and 1 mM NaNO2. The achieved Au-ph-GO-PPC surface was subsequently soaked in the 0.4 M EDC and 0.2 M NHS in 0.1 M MES buffer for 1 h to activate the carboxylic acid groups on GO followed by adding IL-6 monoclonal antibody (5 µL, 50 µg mL−1) in PBS (pH 7.4) to react for 2 h at 4 °C to get the Au-ph-GO-PPC/Ab1 sensing interface. For AFM and SEM images, the gold wires were immersed in piranha (VH2SO4:VH2O2=3:1) solution and aqua regia (VHCl:VHNO3=3:1) for one night, respectively, to ensure the substrate was clean. Cell Culture and ELISA Measurement. Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U mL−1 of penicillin, 0.1 mg mL−1 of streptomycin was used to culture the mouse RAW cells in a T75 cm2 flask. Cells were harvested when the cell confluence reaches about 80-90%. Deionized PBS and trypsin were used to wash cells and detach the cells from the flask, respectively. The harvested cells in DMEM medium suspension was transferred into a centrifuge tube and centrifuged at 200 rcf for 5 min. The supernatant was then discarded and the cells were 8


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resuspended in fresh DMEM medium for following treatment. The prepared sensor device and BD OptEIATm Mouse IL-6 ELISA kit (BD Bioscience) were used to measure the concentration of IL-6 secreted by RAW cells after LPS stimulation. The cells with the density of 1 x 105 /mL were suspended in 1 mL of warm medium containing 100 ng mL-1 LPS to secret IL-6 for different period of time. Supernatants from cells were collected in triplicate. The Nunc MaxiSorp 96 well plate and Galaxy plate reader were used for ELISA reading. Results are reported as means ± standard deviation.

In Vivo Animal Tests. Male Balb/c mice with 5-6 weeks of age were purchased from Hubei Province Experimental Animal Center (Wuhan, China) and housed in standard environmental conditions (12 h light-dark cycle, 50%-70% humidity, and 20 °C-25 °C). Food and water were provided ad libitum. Mice were quarantined for a week before any administrations. All experimental procedures were approved by the Office of Scientific Research Management of Central China Normal University (Wuhan, China), with a certificate of application for the Use of Animals dated January 31, 2017 (approval ID: CCNU-IACUC-2016-008) Twelve mice were randomly divided into experimental group and control group. In the experimental group, acute inflammation model was established by an intraperitoneal injection of 2 mg kg-1 LPS. Meanwhile, the control group was treated by an intraperitoneal injection of saline. After the stimulation with LPS for 12 h, mice were anesthetized with 1% pentobarbital sodium. Mouse head underwent a gentle hair subtraction following by disinfection with 75% alcoholic cotton swab. Then a skin incision with a length of 2 cm was cut appropriately above the middle of the brain prior to subcutaneous tissue separation to expose the front halogen, chevron and sagittal suture. Afterwards, mouse brain was fixed with brain stereotaxic device, and a small hole was drilled with a skull drill in the vicinity of the sagittal suture (with reference to the mouse brain stereopsis map, AP = 0.5 mm, ML = 1.0 mm, DV = 2.0 mm). Subsequently, the gold wire modified with GO was inserted into the hole for 30-minute reaction before the electrical sensing detection on IL-6. The best stimulation 9



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concentration of LPS was 2 mg kg-1 and the optimal reaction time is 30 min by pre-experiment. After removal of the gold wire, the mice were sacrificed and the brain tissue was made to prepare a 10% tissue homogenate for a parallel IL-6 detection by ELISA.

RESULTS AND DISCUSSION Characterization

of

GO-NB

Nanocomposites.

The

prepared

GO-NB

nanocomposites were characterised by FT-IR, UV-Vis, Raman spectra, and electrochemistry. The FT-IR spectra (Figure 1 a) of GO shows the characteristic stretching frequencies for oxygen containing functional groups such as O-H (3400 cm−1), C=O (1720 cm−1), and C-O (1105 cm−1). Another band emerges at 1590 cm−1 in the FT-TR spectra of GO-NB, assigned to the N-H bending in NB indicating a successful preparation of GO-NB.31 The UV-Vis absorption spectroscopy also verified the interaction between NB and GO nanosheets. Aqueous GO suspensions displayed a strong peak at 300 nm (Figure 1 b) due to n–π* transition of C=O bonds.32 The spectrum of diluted NB aqueous solution (Figure 1 b) showed two characteristic absorption peaks, around 280 nm in the far-ultraviolet region and around 610 nm in the visible region, indicating the presence of π-π* transitions of aromatic rings and the n-π* transitions of C=N bond, respectively. The characteristic of GO-NB spectrum was similar to that of NB with enhanced absorbance at 636 nm but with red shift, indicating the adsorption of NB on the GO surface as a result of strong interaction between GO and NB. The Raman spectra of GO and GO-NB are compared at Figure 1 c. The Raman peaks corresponding to D band (~1354 cm-1), G band (~1596 cm-1) and 2D band (~2700 cm-1) characteristic of the graphene structure were observed in both GO and GO-NB.33 The relative Raman D/G intensity ratio (ID/IG) is known to be inversely proportional to the average size of the sp2 domains in GO, and it decreases with the removal of the defects.34 The slight decrease of ID/IG intensity between pure GO (ID/IG =0.98) and GO-NB nanocomposite (ID/IG =0.90) suggests a decreased concentration of defects in GO after incorporating NB. Meanwhile, a shift of 3 cm-1 in 10


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the 2D band of GO due to molecular doping with NB was observed for NB-GO. The fingerprint mode of NB around 595 cm−1 region appeared in the Raman spectrum of GO-NB confirming a successful incorporation of NB into GO. In addition, the electrochemistry of GO-NB as redox probes was studied by placing 10 µL of the prepared GO-NB (1 mg mL−1) on a glassy carbon electrode. A well-defined characteristic peak of NB was observed at -0.4 V in Figure 1 d. However, no peaks were observed after adding 10 µL of the pristine GO (1 mg mL−1) verifying the successful attachment of NB on GO.35

Figure 1. (a) FT-IR absorption spectra of GO and GO-NB. (b) UV-Vis absorption spectra of GO, NB and GO-NB suspensions. (c) Raman spectra of GO and GO-NB. (d) The electrochemistry of the glassy carbon surface after dropping down 10 µL of GO-NB and GO, respectively, in 0.05 M PBS at the scan rate of 100 mV s−1. Surface Characterisation of GO Modified Sensing Interface. The prepared sensing interface Au-ph-GO-Ab1 was characterized by AFM, TEM, XPS and electrochemistry, respectively. To prepare the GO based sensing interface, gold surfaces (gold electrode or gold wire) were firstly modified with 4-nitrophenyl using 11



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aryldiazonium salt chemistry (Scheme 1). The electrochemistry for the surface fabrication is shown in Figure S1. Then the terminating nitro groups were electrochemically reduced to amine groups in the protonated solution (EtOH/HCl) to form Au-ph-NH2 (Figure S2). The amine groups on the surface were further reduced to diazonium ions with the presence of NaNO2 in acidic solution. Thus the sheet of GO was anchored to the gold surface by forming C-C bonds through aryldiazonium salt chemistry. AFM was used to study the thickness of GO layer on the gold surface (Figure 2 a). GO flakes were clearly apparent on the substrate, and the average thickness was 1.06 nm ± 0.17 nm (n=4) which agrees with the thickness of a GO single layer between 0.8 and 1.5 nm.36 This result demonstrated that a single layer GO was successfully modified on the gold surface by aryldiazonium salt chemistry. SEM was also used to characterize the GO-modified gold surfaces (Figure 2 b-d). The GO sheets were observed on the GO-modified gold wire (Figure 2 c) suggesting GO was successfully attached to gold surface forming Au-ph-GO as illustrated in Scheme 1. In the SEM image of Au-ph-GO-Ab1, white spots evenly distributed on the GO sheet were observed (Figure 2 d), which might be due to the crystallized IL-6 antibodies Ab2 attached on the surface under the dry conditions. The surface characterization by AFM confirmed that a single layer of GO was fabricated on gold surface, which was available for the attachment of capture antibodies.

12


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Figure 2. (a) AFM image of the single layer of GO on the substrate. (b) SEM image of the Au-ph-NH2 surface. (c) SEM of the Au-ph-GO surface. (d) SEM of the Au-ph-GO-Ab1 surface. XPS measurements were carried out to characterize the stepwise deposited species on the gold surface. As shown in Figure 3 a, the peak at around 405.45 eV in the N1s core level spectra of Au-ph-NO2 is the characteristic peak of the nitrogen in the nitro group, and the peak at around 399.65 eV may be caused by contaminants introduced in the modification procedure37 or atmospheric nitrogen atoms38 and nitrogen in azo groups. The N1s peak at 405.45 eV decreased in the case of the Au-ph-NH2 surface, and the characteristic peak of the nitrogen in amino group appeared at 399.69 eV, suggesting a successful transformation of nitro groups to amine groups (Figure 3 b). It was calculated that about 90% of NO2 groups have been converted to NH2 groups 13



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based on the atomic ratio of N1s species at 399.65 eV and 405.45 eV (9.3:1.4). The C1s scan of Au-ph-GO (Figure 3 c) is dominated by the graphitic carbon peak at 284.63 eV together with peaks at the high binding energy 286.5 eV and 288.5 eV due to carbon atoms of the C-O-C and carboxylic acid group respectively, indicating successful immobilization of GO on the Au surfaces. Figure S3 shows the electrochemistry of gold surface in the case of modification of PPC molecules on GO. In the case of Au-ph-GO-PPC spectra (Figure 3 d), a new peak appeared at 401.9 eV (Figure 3 d) which is assigned to nitrogen of -N(CH3)3 in PPC.39 In addition, a P2p peak at 133.50 eV (Figure 3 e) was also observed. Both these peaks confirm a successful modification of PPC molecules. After modification of Ab1, three N1s species were observed (Figure 3 f). The highest binding energy component at 402.65 eV is tentatively assigned to the unreacted portion of the NHS ester nitrogen after EDC/NHS activation. The peaks at 399.7 eV and 401.4 eV are assigned to the amide nitrogens of the peptide bonds and the nitrogen of -N(CH3)3 in PPC layers, respectively. Additionally, the atomic ratio of N1s species at 399.7 eV and 402.65 eV were calculated to be 6.1: 3.3 for Au-ph-GO-PPC/Ab1 interfaces (Figure 3 f), suggesting that 65% of COOH groups had been activated to form the amide bonds with the attachment of Ab1. This coupling efficiency was slightly higher than the maximum theoretical coupling efficiency (45-57%),40 which might be due to the presence of N1s specie corresponding –N=N- at 399.7 eV.

14


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Figure 3. N1s core level spectra for (a) Au-ph-NO2, (b) Au-ph-NH2, (d) Au-ph-GO-PPC, and (f) Au -ph-GO-PPC/Ab1. (c) C1s core level spectra for Au-ph-GO. (e) P2p core level spectra for Au -ph-GO-PPC. 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. As shown in Figure 4 a, the well-defined peaks characteristic of Fe(CN)63−/Fe(CN)64− were observed on the bare gold electrode. However, the Au-ph-NO2 surface prevents access of the Fe(CN)63−/Fe(CN)64 − redox species to the electrode and hence no faradaic peaks of ferricyanide were observed between -0.2 V 15



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to +0.6 V. A small redox peak current of the redox couple was observed for 4-aminophenyl-modified gold electrode because NO2 is electron-donating but NH2 is electron withdrawing resulting in the partial blocking of the Fe(CN)63−/Fe(CN)64− redox species. An increase in the peak current of the redox couple was observed after the decoration of GO, suggesting that GO-based nanostructure on gold surface increased the electronic coupling to the underlying electrode. After the modification of PPC diazonium salts, the electrode surface was slightly passivated, with a decreased redox peak in Fe(CN)63−/Fe(CN)64− solution, indicating a successful attachment of zwitterion molecules on the sensing interface. The electrochemistry of GO modified sensing interface (Au -ph-GO-PPC/Ab1) after capturing the analyte IL-6 in the PBS solution was recorded in Figure 4 b. No redox peaks were observed before the attachment of the electrochemical signal probe Ab2-GO-NB. However, the redox peaks centered at -0.4 V appeared after incubation with the IL-6 detection antibodyAb2-GO-NB, suggesting that Ab2-GO-NB as the probe was capable to report the electrochemical signal of the sensing interface Au -ph-GO-PPC/Ab1 for detection of IL-6.

Figure 4. (a) The cyclic voltammetry for bare gold electrode and the interfaces of Au-ph-NO2, Au-ph-NH2, Au-ph-GO, Au-Ph-GO-PPC in 0.1 M phosphate buffer saline solution pH 4 containing 0.05 M KCl and 1 mM Fe(CN)63-/Fe(CN)64- with the scan rate of 100 mV s-1. (b) The cyclic voltammetry for GO based sensing interface captured with analyte IL-6 before and after attachment of GO-NB in 0.1 M phosphate 16


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buffer saline solution (pH7.4 containing, 0.05 M KCl). Performance of GO Modified Sensing Interface for Detection of IL-6 In Vitro. In order to further investigate the response of the modified sensing interface Au-ph-GO-PPC/Ab1 to the analyte IL-6, the square wave voltammetry of the sensing interface was recorded after incubation with IL-6 at variable concentrations (Figure 5 a). The redox current of NB from the signal reporter Ab2-GO-NB changed with the concentration of IL-6 correspondingly. As shown in Figure 6 b, the increase of the peak current was proportional to the concentration of IL-6 in the range of 1-300 pg mL−1 with the lowest detectable concentration of 1 pg mL−1, which is within the physiological range of IL-6 (10-75 pg mL-1) and comparable to the IL-6 biosensors reported in the literature (Table S1). As determined from the midpoint of the calibration curve in Figure 6 b, the affinity constant between IL-6 and anti-IL-6 IgG is calculated to be around 1012 M-1. The typical affinity constants Ka for antigen-antibody reactions is in the range of 108 to 1012 M-1,41 so it suggests the detection antibody in this prepared nanosandwich assay has very high affinity with the analyte IL-6. Meanwhile, the diffusion of the signal probe Ab2-GO-NB to the sensing interface was essential to the assay kinetics of the sandwich assay.42 The incubation time of the detection antibody (Ab2-GO-NB) was optimised by recording the current after incubation of the IL-6 captured interface to Ab2-GO-NB (0.5 mg mL-1) for different periods of time. It was observed that the current increased with the incubation time of Ab2-GO-NB and reached a plateau for 30 mins incubation time (Figure S4). The immunoreaction reached equilibrium after 30-60 minutes of incubation time.43 One control was carried out to study the possible non-specific adsorption of Ab2-GO-NB by incubation of Au -ph-GO-PPC/Ab1 surfaces to PB solution without the presence of IL-6 followed by incubation of Ab2-GO-NB for 30 min (Figure S4 b). No obvious redox peak from NB was observed after introducing PPC to the GO based sensing interface, suggesting the nonspecific adsorption of Ab2-GO-NB reduced significantly. The performance the thin film GO-based 17



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immunosensor and general GO-based immunosensor was compared (Figure S5). It shows that the electrochemical response from the thin film GO-based immunosensor was about 4 times higher than that from that general GO-based immunosensor. Thus the performance of the thin film GO-based immunosernsor was superior to that by general GO-based immunosensor.

Figure 5. (a) SWV curves for Au -ph-GO-PPC/Ab1 surfaces for the detection of IL6 with concentrations of 0, 1, 2, 5, 12, 25, 50, 75, 100, 150, 200, 300 pg mL−1, respectively. (b) A calibration curve showing the variation in relative current with the log concentration of IL-6 in 0.05 M phosphate buffer (pH 7.4). In addition, this nano-sandwich assay showed good reproducibility, with a relative standard deviation of 5.3% for the response of 6 independently prepared immunosensors to 50 pg mL-1 IL-6. To investigate the selectivity of fabricated nanosandwich assay, IL-6 was detected in the presence of five potentially interfering proteins, such as bovine serum albumin (BSA), prostate specific antigen (PSA), cancer antigen 125 (CA-125), and mouse IgG (Figure S5 a). The result indicated that these common components in body fluids did not have significant effect to the performance of nanosandwich for detection of IL-6. The ignorable non-specific protein adsorption might be due to the presence of PPC which has the capability to resist non-specific protein adsorption.39 The stability of the immunosensor was investigated by recording the response of the sensing interface Au-ph-GO-PPC/Ab1 to 18


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100 pg mL-1 IL-6 after storing Au-ph-GO-PPC/Ab1 in dry state at 4 oC for 30 days. The nanosandwich retained 92% of the signal of the freshly prepared sensing interface Au-ph-GO-PPC/Ab1, indicating that the sensing interface was stable. In addition, there was no obvious change in electrochemistry after scanning the nanosandwich assay Au-ph-GO-PPC/Ab1/IL-6/Ab2-GO-NB in the phosphate buffer solution for 10 cycles (Figure S5 b). This confirms that the nanosandwich assay was stable under the investigated conditions. Performance of the Gold Wire Based Nano-sandwich Device for Detection of IL-6 in Ex Vivo and In Vivo. To evaluate the performance of the prepared sensing interface, the nanosandwich assay was fabricated on gold wire and the functionalized gold wire device was used for detection of IL-6 secreted by live RAW cells (Figure 6). The supernatant of RAW cells was collected for IL-6 analysis after LPS (100 ng mL-1) stimulation for 2, 4, 6, 8, 12 and 24 h. The supernatant samples were diluted 2-fold with cell culture media DMEM and subsequently analysed by a commercial ELISA kit and herein prepared nanosandwich assay, respectively. After incubation of the gold wire device in the supernatant for 30 min, many white spots were observed on the surface of gold as shown in SEM image (Figure 6 a), suggesting the attachment of proteins. As shown in Figure 6 b, the concentration of IL-6 increased with the duration of the LPS treatment and the maximum concentration of IL-6 (345 pg mL-1 per 105 cells) was obtained after 8 h LPS stimulation. Similar detection patterns based on IL-6 concentration in ELISA to that in our electrochemical assay were obtained. However, the sensitivity and the assay time in these two methods was substantially different. The lowest detectable concentration of IL-6 was 1 pg mL-1 for our nanosandwich assay, which was lower than that for the IL-6 ELISA from BD Biosciences (4 pg mL-1). The nanosandwich device based on gold wire is rigid and sensitive. We envisage it to be used as the dipstick-in probe for monitoring cytokine secretion in bioreactors.

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Figure 6. (a) SEM of the gold wire modified with the capture antibody (GW-ph-GO-Ab1) after incubation with the RAW cell supernatant. (Insert: picture of the original gold wire). (b) The IL-6 secretion profile of RAW cells after LPS stimulation for the commercial ELISA and the herein fabricated gold wire based cytokine assay. Detection of IL-6 content in mouse brain (n=6, *** means p

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