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Graphene oxides signal reporters based multifunctional immunosensing platform for amperometric profiling of multiple cytokines in serum Hui Wei, Shengnan Ni, Chaomin Cao, Guang-Fu Yang, and Guozhen Liu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00365 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018
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Graphene oxides signal reporters based multifunctional immunosensing platform for amperometric profiling of multiple cytokines in serum Hui Wei,a,b Shengnan Ni,a,b Chaomin Cao,a,b Guangfu Yang,a,b Guozhen Liu a,b,c,d* 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
International Joint Research Center for Intelligent Biosensor Technology and Health, Central China Normal University, Wuhan 430079, P. R. China
c
Graduate School of Biomedical Engineering, ARC Centre of Excellence in Nanoscale Biophotonics, Faculty of Engineering, University of New South Wales, Sydney 2052, Australia d
Australian Centre for NanoMedicine, University of New South Wales, Sydney 2052, Australia
*Corresponding author:
[email protected]; Tel: +61 2 9385 0714
ABSTRACT Cytokines are small proteins and form complicated cytokine network to report the status of our health. Thus accurate profiling and sensitive quantification of multiple cytokines is essential to have the comprehensive and accurate understanding of the complex physiological and pathological conditions in body. In this study we demonstrated a robust electrochemical immunosensor for the simultaneous detection of three cytokines IL-6, IL-1β, and TNF-α. Firstly, graphene oxides (GO) were loaded with redox probes nile blue (NB), methyl blue (MB), and ferrocene (Fc), followed by covalent attachment of anti-cytokine antibodies for IL-6, IL-1β, and TNF-α, respectively to obtain Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc, acting as the signal reporters. The sensing interface was fabricated by attachment of mixed layers of 4-carboxylic phenyl and 4-aminophenyl phosphorylcholine (PPC) to glassy carbon surfaces. After that, capture monoclonal antibody for IL-6, IL-1β, and TNF-α was modified to the carboxylic acid terminated sensing interface. And finally a sandwich assay was developed. The quantitative detection of three cytokines was achieved by
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observing the change in electrochemical signal from signal reporters Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc, respectively. The designed system has been successfully used for detection of three cytokines (IL-6, IL-1β, and TNF-α) simultaneously with desirable performance in sensitivity, selectivity and stability, and the recovery of 93.6%-105.5% was achieved for determining cytokines spiked in the whole mouse serum. Keywords： ： Cytokines, multiplex detection, graphene oxides, electrochemistry, immunosensors, redox probes
Cytokines are small proteins or glyco-protein messenger molecules that immune system cells use to send signals to each other.1 Detection of cytokines secretions is essential to monitor cell-to-cell communication for understanding biology and medicine because they provide insight into physiological processes and disease pathways, and can serve as biomarkers for various diseases.2 Until now more than 200 kinds of cytokine families, such as interleukins, growth factors, chemokines, interferons, and a host of others, have been identified. According to their functions in our immune system, cytokines can be divided into pro-inflammatory or anti-inflammatory cytokines.3 In order to response a specific cellular stimuli, cells can affect the neighbored cells by secreting varieties of different cytokines to convey a unique signal. However, most cytokines have multiple and diverse biological functions.4 These properties make the cytokine network even complicated. One biological phenomenon is normally the interactions between different cytokines. For example, TNF-α produced by activated macrophages with stimulation of lipopolysaccharide (LPS) is a significant mediator of acute inflammation to produce fever and promotes proteins production. Additionally, there is abundant evidence showing that TNF-α is one of the important pro-inflammatory cytokines with IL-1β, IL-6 involving in the process of pathological pain.5 Recent study suggests that TNF-α, IFN-ϒ and interleukins are predictive markers of anti-programmed cell-death protein-1 treatment in advanced non-small cell lung cancer.6 There is limited value for measurement of a single cytokine because cytokines form complicated cytokine networks to act a biological symptom in human body. So development of the reliable, low cost, easy to operating, and reproducible technology for the measurement of multiple cytokines is urgently needed. Cytokine sensing is challenging due to complex cytokine network and low concentration (~pM range) under physiological conditions.7 The most common approach for cytokine quantification is the ACS Paragon Plus Environment
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traditional ELISA assay,8 which is reliable, but time-consuming (6 h) and unable to do simultaneous multiple cytokine detection. Quantitative analysis of multiple analytes by immunoassays can provide advantages
of
high
specificity
and
cost-effectiveness.
A
significant
advancement
in
cytokine/chemokine profiling was achieved by using fluorescent bead-based technology.9 The routinely practiced methods for detection of multiplex cytokines are based on Luminex multiplex technologies,10 which provide a vastly enhanced method of cytokine discovery and identification, allowing for the testing of dozens of cytokines at once, from a single sample. Simultaneous detection of VEGF, IL-8, and TIMP-1 has been achieved by a fluorescently encoded microbeads-based platform.11 These high-quality multiplex technologies have proven to be at least as accurate as traditional methods while being much more time- and cost-efficient, and they are accelerating medical research in numerous ways. However, these beads-based assays are suffering the limitation of sensitivity, and cross reaction between different cytokines. Their wide application is compromised by the limited number of distinguishable codes in the same array. Then a novel encoding method based on quantum dots and magnetic nanoparticles (NPs) with nanosphere structure was developed to increase encoding capacity by manipulating the magnetic field.12 This method has been applied for detection of IgG with the sensitivity of 1 fM and demonstrated the feasibility of NPs as encoded carriers in multiplex immunoassays.13 Recently, a high throughput, label-free, multiarrayed localized surface plasmon resonance (LSPS)-based microfluidic optical biosensor has been developed for parallel multiplex detection of IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α, in a complex serum matrix on a single device chip.14 Additionally, a multiplexed cytokine immunoassay utilizing electron beam lithography to directly write antibodies on silicon substrates has been used to detect IL-6 and TNF-α by LSPS with dark-field microscopy.15 Surface-enhanced Raman spectroscopy (SERS) based Raman tags provide new opportunities of the optical encoding systems for high levels of multiplexing,16 which shows particular promise for sensitive cytokine detection due to the high sensitivity. However, besides the limited reproducibility, challenges with SERS technology for multiplex biosensing have to be met by developing new nanoprobes and instrumentation. Silicon photonic micro-ring resonators has demonstrated great potential for detection of multiple cytokines because of their tunable waveguides.17-18 Comparing to other different signaling strategies such as fluorescence immunoassay, SERS, silicon photonic MR and other methods, electrochemical techniques have their advantages because ACS Paragon Plus Environment
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of their simplicity, low cost, speed, possibility for high throughput detection, and high sensitivity particularly in amperometric based measurements.19-21 Revzin’s group has developed the two cytokine sensing platforms based on electrochemical methods.22-23 One example is to integrate the microfluidic chips to the aptamer based sensing surface for simultaneous detection of IFN-γ and TNF-α.23 In this study, redox probers anthraquinonoid and methylene blue were labeled to IFN-γ aptamers and TNF-α aptamers, respectively. Conjugation of IFN-γ or TNF-α with the corresponding aptamer triggered the conformation change of aptamers leading to changes in electrochemistry of redox probes. However, the sensitivity of aptasensors were compromised due to the single redox probe labeled on one recognition molecule (aptamer) comparing to that of immunosensors.24 In this study, we target to develop a robust electrochemical immunosensing platform based on sandwich assays for simultaneous detection of cytokines IL-6, IL-1β, and TNF-α (Scheme 1). Firstly, three graphene oxides (GO) based signal reporters were prepared by loading the redox probe nile blue (NB),25 MB, and ferrocene (Fc) to GO, respectively, to achieve GO-NB, GO-MB, and GO-Fc nanocomposites. Then anti-cytokine antibodies for IL-6, IL-1β, and TNF-α were covalently attached to GO-NB, GO-MB, and GO-Fc, respectively, to obtain Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc, acting as the signal reporters. The sensing interface was fabricated by attachment of mixed layers of 4-carboxylic phenyl and 4-aminophenyl phosphorylcholine (PPC) to the glassy carbon (GC) surface by C-C covalent bond through aryldiaznium salt chemistry.26 After that, capture monoclonal antibody for IL-6, IL-1β, and TNF-α were immobilized to the carboxylic acid terminated sensing interface by forming amide bonds. And finally a sandwich assay was developed. Once specific biorecognition event occurred between cytokines and the detection antibodies in signal reporters, there would be a modulation of electrochemistry from specific redox probes. The quantitative detection of three cytokines was achieved by observing the change in electrochemical signal from signal reporters Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc, respectively. The more cytokine reacted with the detection antibody, the larger electrochemical signal from signal reporter would be obtained. PPC, a zwitterionic molecule was used here to resist non-specific protein adsorption and subsequently increase the specificity and selectivity. It orders to achieve the stable sensing device, aryldiazonium salt chemistry was used for surface coupling of reagents.26 After The designed system has been successfully used for simultaneous detection of three cytokines (IL-6, IL-1β, and TNF-α) with desirable performance, and also is applicable to detect cytokines in serum. To our knowledge, it is ACS Paragon Plus Environment
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the first study on an amperometric immunosensor for simultaneous detection of at least three cytokines.
Scheme 1. The schematics of fabrication of the immunosensor for multiplex detection of three cytokines IL-6, IL-1β and TNF-α. EXPERIMENTAL SECTION Materials. Single layer of graphene oxides, sodium nitrite, nile blue(NB), methylene blue(MB), ferrocene(Fc), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC), potassium ferricyanide, absolute ethanol, lipopolysaccharide (LPS)
hydrochloric acid, potassium chloride,
N-hydroxysuccinimide (NHS), 2-(Nmorpholino) ethanesulfonic acid (MES), and 4-aminobenzoic acid were purchased from Sigma-Aldrich. Anti-mouse IL-6 monoclonal antibody and mouse IL-6 were purchased from Abcam (Canada). Anti-mouse IL-6 polyclonal antibody were purchased from Abcam (England). Anti-mouse TNF-α polyclonal antibody, anti-mouse IL-1β/IL-1F2 polyclonal antibody, anti-mouse TNF-α monoclonal antibody, anti-mouse IL-1β/IL-1F2 monoclonal antibody, mouse tumor necrosis factor-alpha (TNF-α) and mouse IL-1β/IL-1F2 were purchased from R&D Systems. Phosphate buffer saline solution (pH 7.4) containing 0.05 M KCl and 0.05 M K2HPO4/KH2PO4 was used in this work. The 4-aminophenyl phosphorylcholine (C11H19N2O4P, PPC) was purchased from Toronto Research Chemicals Inc. Aqueous solutions were prepared using Milli-Q water.
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Instruments. A CHI660E system (CHI Instrument, Shanghai) was used for all electrochemical measurements. Glassy carbon (GC) disk electrodes (3 mm) were purchased from Gaoss Union (China). A SCE (3.0 M KCl) reference electrode and a Pt secondary electrode were used for all electrochemical measurements. UV-Vis absorption data were collected on a Shimadzu UV-Vis spectrophotometer model 2450. X-ray photoelectron spectra (XPS) were collected from carbon plates on a VG multilab 2000 spectrometer with a monochromated Al Kα source (1486.6 eV), hemispherical analyzer, and multichannel detector. A TENSOR27 FTIR spectrophotometer was used for Fourier Transform Infrared Spectroscopic (FT-IR) measurements. Preparation of Ab2-GO-NB and Ab2-GO-MB. According to the method reported by Gao et al,27 in which nile blue was loaded to reduced graphene oxide, we successfully prepared the graphene oxide loaded with nile blue.25 The detailed procedures for making Ab2-GO-NB here were exactly the same as those we reported previously.25 The protocol to prepare for Ab2-GO-MB (Scheme S1 b) is the same as that for Ab2-GO-NB except that NB and IL-6 polyclonal antibody were replaced by MB and IL-1β polyclonal antibody, respectively.28-29 Preparation of Ab2-GO-Fc. The protocol for making Ab2-GO-Fc is based on the method reported in the literature30, which is detailed at Scheme S1 c. Specifically, the cyclopentadienyl rings of Fc was attached to the aromatic sheets of GO via the π-π interactions between GO and Fc. GO (4 mg) was suspended in a 500 µL:500 µL mixture of ethanol and deionized water to form a homogeneous suspension (4 mg mL-1) by sonication for 1 h. And then ferrocene (5 mg) was dissolved in ethanol solution (5 mg mL-1) followed by addition to the homogeneous suspension quickly and shaken violently. The mixture was stirred at room temperature for 1.5 h, to which 0.4 M EDC and 0.1 M NHS dissolved in 500 µL MES buffer (pH5.5) was added with stirring for another 0.5 h to activate carboxylic acid groups on GO. After washing three times by centrifugation, the collected solid (GO-Fc) was dispersed in 1.0 mL PBS buffer (pH7.4). Finally, 50 µL PBS buffer (pH7.4) containing TNF-α polyantibody (50 µg mL-1) was added to 1 mL GO-Fc (pH7.4) buffer solution with stirring for 2 h at room temperature. In order to wash off the unreacted antibody the resulting solution (300 µL) was centrifuged (10 min, 4000 rpm) by centrifugal filter (Millipore, 30 kD) and finally resuspended in 1.0 mL PBS buffer (pH7.4) to achieve Ab2-GO-Fc for storage before usage. Preparation of Immunosensing Interface for Detection of Three Cytokines. Mixed layers of 4-aminobenzoic acid and PPC were modified to the clean GC surfaces by scanning the potential ACS Paragon Plus Environment
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range from 0.6 V to -1.0 V for 2 cycles in corresponding diazonium salts (1 mM NH2-ph-COOH, 1 mM PPC and 2 mM NaNO2) in 0.5 M HCl solution to achieve the mixed layer modified interface GC-ph-COOH/PPC. In order to activate carboxylic acid groups, the modified GC surface was incubated in 0.1 M MES buffer containing 0.4 M EDC and 0.1 M NHS for 1 h. After rinsing with PBS (pH 7.4) buffer solution, the activated GC surfaces were dried under N2 followed by adding mixture of IL-6, IL-1β and TNF-α monoclonal antibody (5 µL, 50 µg mL−1 for each antibody) in PBS (pH 7.4) to react for 2 h at 4 °C to create GC-ph-COOH(PPC)/Ab1 as shown in Scheme 1. Serum Sample Measurement. The freezed mouse serum was purchased from Bioscience, China. The received serum samples were apportioned into 0.5 mL aliquots, and stored at –20°C to avoid freeze-thaw cycles. The aliquots waiting for analysis were maintained at 2–8°C while handling. Three cytokines with certain concentration were spiked into serum samples followed by electrochemical measurement. RESULTS AND DISCUSSION Characterization of GO Based Nanocomposites. The GO was used as received and characterized by the AFM in Figure S1. The characterization of GO-NB has been reported in our previous study,25 and Figure 1 illustrated the characterization of GO-MB and GO-Fc nanocomposites by FT-IR, UV-Vis and electrochemistry. After binding MB to GO to form GO-MB, the stretching frequency of C=C bonds shifted from 1650 nm (GO) to 1643 nm (GO-MB) in FT-IR spectra (Figure 1 a), which is consistent with that in the literature.28 The interaction between MB and GO was further confirmed by UV-Vis absorption spectroscopy. The emerged adsorption peaks at 233 nm of GO suspensions in Figure 1 b was attributed to n-π* transition of C=O bonds. The two main adsorption peaks at 294 nm (ultraviolet region) and 670 nm (visible region) of diluted MB solution corresponded to π-π* transitions of aromatic rings. The enhanced UV-Vis spectrum absorbance at 630 nm of GO-MB suggested the strong coupling of GO with MB.28 Furthermore, a well-assigned MB characteristic peak was captured at -0.236 V (Figure 1 c), for GO-MB nanoconjugates illustrating the success in preparation of GO-MB.29 Comparing to the FT-IR spectra (Figure 1 d) of GO, another three new band emerged at 1157, 810, 956 cm−1 in the FT-IR spectra of GO-Fc nanocomposites, which confirmed that GO-Fc was achieved by π-π stacking interactions between GO and Fc.31 As shown in UV-Vis absorption spectroscopy of GO-Fc (Figure 1 e). The broad peak at 233 nm was assigned to π system of GO. And Fc exhibited a ACS Paragon Plus Environment
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striking peak at around 205 nm. After GO hybridization with Fc, a new peak appeared at 207 nm, which was at the similar position to that of Fc. In addition, after attachment of Fc, the broad band of GO shifted to 252 nm suggesting the effective interaction between GO and Fc.31 A well-assigned Fc characteristic redox peak in Figure 1 f was observed at +0.24 V, indicating that Fc was attached to GO.31
Figure 1. (a) FT-IR spectra of GO and GO-MB. (b) UV-Vis spectra of GO, MB, and GO-MB. (c) The electrochemistry of GO-MB and GO. (d) FT-IR spectra of GO and GO-Fc. (e) UV-Vis spectra of GO, Fc, and GO-Fc. (f) The electrochemistry of GO-Fc and GO. Surface Characterisation of the Modified Sensing Interfaces. Electrochemical characterization of GC electrode before and after modification of mixed layers of COOH-ph and PPC has been detailed in Figure S2. It confirmed that mixed layers of 4-carboxylphenyl and PPC had been modified on GC surfaces. The surface coverage of 4-carboxylphenyl and PPC was calculated to be 7.62 ×10-10 mol cm-2 corresponding to a single layer of aryldiazonium salt on electrode surfaces as reported previously.32 The XPS measurement (Figure 2) further characterized the stepwise modified deposition species of modified GC surfaces. The measured C1s core level spectrum for GO-ph-COOH(PPC) included two peaks at around 284.63 eV for graphitic carbon and 288.5 eV corresponding to carbon species of the carboxyl groups on the surface (Figure 2 a).33 The N1s peak of –N(CH3)3 in PPC at around 401.9 eV (Figure 2 d) was observed on the surface of GO-ph-COOH(PPC).34-35 Moreover, the peak at 133.50 eV (Figure 2 f) was assigned to the P2p of
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PPC on the surface of GO-ph-COOH(PPC). These peaks suggested a mixed layer of –COOH-ph and PPC was achieved on GC surfaces. According to the peak area of at 288.5 eV (-COOH) and at 133.50 eV (phosphorus species), the ratio of -COOH-ph and PPC molecules modified on the surface of GC-ph-COOH(PPC) was 9:1. After activation of -COOH groups on GC-ph-COOH(PPC) surface with EDC/NHS, three additional N1s species were observed (Figure 2 e). The three energy peaks at 402.4 eV, 400.7 eV, and 399.84 eV was assigned to the nitrogen of the NHS ester, the protonated tertiary amine of EDC, and the imine and secondary amine of EDC.36 After modification of capture antibodies (Ab1), the C1s peak at 288.5 eV shifted to 287.89 eV suggesting the formation of -CO-NH- bonds (Figure 2c) due to the attachment of antibodies. And the N1s and P2p species increased
significantly
for
the
capture
antibodies
attached
sensing
interface
(GC-ph-COOH(PPC)/Ab1) (Figure 2 g, h). The N12 peak at 399.85 eV (Figure 2 f) was assigned to the amide nitrogens of peptide bonds.36 The p2p at 133.50 eV was assigned to the phosphorus species in antibodies. These results suggested that we have successfully achieved the sensing interface GC-ph-COOH(PPC)/Ab1.
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Figure 2. C1s core level spectra for interfaces of (a) GC-ph-COOH(PPC), (b) GC-ph-COOH(PPC) after activation with EDC/NHS, (c) GC-ph-COOH(PPC)/Ab1. N1s core level spectra for interfaces of (d)
GC-ph-COOH(PPC),
(e)
GC-ph-COOH(PPC)
after
activation
with
EDC/NHS,
(f)
GC-ph-COOH(PPC)/Ab1. P2p core level spectra for interfaces of (g) GC-ph-COOH(PPC) and GC-ph-COOH(PPC)/Ab1. (h) The wide XPS scanning spectra for interfaces of GC-ph-COOH(PPC), GC-ph-COOH(PPC) after activation with EDC/NHS, and GC-ph-COOH(PPC)/Ab1. Sensitivity of the Prepared Immunosensors for Detection of Three Cytokines. To study the performance of the sandwich assay for detection of cytokines, GC-ph-COOH(PPC)/Ab1 sensing interface was incubated with three cytokines IL-6, TNF-α and IL-1β（250 pg mL-1) followed by incubation of Ab2-GO-NB (50 µg mL−1), Ab2-GO-MB (50 µg mL−1), and Ab2-GO-Fc (50 µg mL−1) for 30 min, and then washing with PBS. The electrochemical response of GC-ph-COOH(PPC)/Ab1 sensing interface before and after incubation with three cytokines IL-6, TNF-α and IL-1β was shown in Figure 3. Without the presence of three cytokines IL-6, TNF-α and IL-1β, no faradic peak was observed (Figure 3 a). However, three pairs redox peaks which are characteristic of NB (-0.4 V), MB (-0.2 V) ACS Paragon Plus Environment
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and Fc (0.2 V) appeared after exposure to the analyte IL-6, IL-1β and TNF-α. Square wave voltammetry (SWV), a more sensitive electrochemical method, was applied to quantitatively detect three cytokines IL-6, IL-1β and TNF-α (Figure 3 b).
Figure 3. a) Cyclic voltammetry and b) Square wave voltammetry of GC-ph-COOH(PPC)/Ab1 sensing interface in phosphate buffer pH7.4 before and after incubation with three cytokines IL-6, IL-1β and TNF-α（250 pg mL-1) followed by incubation of Ab2-GO-NB (50 µg mL−1), Ab2-GO-MB (50 µg mL−1), and Ab2-GO-Fc (50 µg mL−1) for 30 min, and then wash with PBS. The response of GC-ph-COOH (PPC)/Ab1 to three cytokines (IL-6, IL-1β and TNF-α) was recorded by SWV (Figure 4). It was observed that the NB peak current increased with the IL-6 concentration and the sensitivity was calculated to be 27 pA per pg mL-1 (or 382 pA per pg mL-1 per cm-2 by considering the surface area of GC electrode of 0.071 cm2) (Figure 4 a), The achieved linear range of IL-6 was 5-150 pg mL−1 with the detection limit of 5 pg mL-1 and correlation coefficients of 0.9315 (Figure 4 b). The MB current from signal reporter Ab2-GO-MB increased with the concentration of IL-1β with the sensitivity of 0.8 pA per pg mL-1 (or 11 pA per pg mL-1 per cm-2 by considering the surface area of GC electrode of 0.071 cm2) (Figure 4 c), and the increase in the peak current consisted with the increase of IL-1β concentration in the range of 5-200 pg mL−1 with the detection limit of 5 pg mL-1 and correlation coefficients of 0.9712 in phosphate buffer solution (Figure 4 d). The ferrocene current from signal reporter Ab2-GO-Fc corresponded to concentration of TNF-α with the sensitivity of 1 pA per pg mL-1 (or 14 pA per pg mL-1 per cm-2 by considering the surface area of GC electrode of 0.071 cm2) (Figure 4 e), As illustrated in Figure 4 f, the increase in current with a linear concentration range of 5-200 pg mL-1 and correlation coefficients of 0.929. The detection limit was TNF-α is 5 pg mL−1. The performance of herein immunosensor for detection of ACS Paragon Plus Environment
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IL-6, IL-1β and TNF-α was compared with that of sensors reported previously (Table S1). It suggested that herein developed sensor provided high sensitivity while having the multiplex sensing capability.
Figure 4. SWV for detection of (a) IL-6, (c) IL-1β and (e) TNF-α with concentrations of 0, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 400 and 500 pg mL-1, respectively. Calibration curves for detection of (b) IL-6, (d) IL-1β and (f) TNF-α in PBS (pH 7.4). Arrow shows the increase direction of cytokine concentration. Error bar in the calibration curve indicates the standard deviation for three individual measurements. Selectivity, Specificity and Stability of Prepared Immunosensors for Detection of Three Cytokines. To demonstrate the specificity of the fabricated sensor, electrochemistry of GC-ph-COOH(PPC)/Ab1 sensing interface was monitored with the presence of four interfering proteins such as bovine serum albumin (BSA) (2 mg mL-1), mouse IgG (1 mg mL-1), prostate specific antigen (PSA) (1 mg mL-1), and cancer antigen 125 (CA-125) (1 mg mL-1), respectively.
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Then, the response of this prepared immunosensor to these proteins was compared with the response to 50 pg mL-1 IL-6, IL-1β and TNF-α, respectively. Figure 5 a, b, c show that the signal from target cytokines (IL-6, IL-1β and TNF-α) was more than 80% of the signal from interfering proteins, suggesting that these proteins (BSA, IgG, PSA, CA-125) did not show significant interference and the sensor demonstrated high specificity for detection of target cytokines. In order to further explore the selectivity of prepared immunosensor, four potentially interfering proteins, including PSA (1 mg mL-1), BSA (2 mg mL-1), mouse IgG (1 mg mL-1), and CA-125 (1 mg mL-1), were added to the detection buffer solution with the presence of 50 pg mL-1 cytokines (IL-6, IL-1β, and TNF-a). Then, the response of this prepared immunosensor to each cytokine was recorded (Figure 5 d, e, f), and the current for interference proteins was above 86% of the original signal, indicating almost no interference from these species was observed (
