Colloids and Surfaces B: Biointerfaces 116 (2014) 714–719

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Ultrasensitive IL-6 electrochemical immunosensor based on Au nanoparticles-graphene-silica biointerface Guangfeng Wang a,b,∗ , Xiuping He a , Ling Chen a , Yanhong Zhu a , Xiaojun Zhang a,∗ a

College of Chemistry and Materials Science, Key Laboratory for Functional Molecular Solids of the Education Ministry of China, Anhui Key Laboratory of Chem-Biosensing, Center for Nano Science and Technology, Anhui Normal University, Wuhu 241000, PR China Anhui Key Laboratory of Controllable Chemistry Reaction & Material Chemical Engineering, HeFei University of Technology, Hefei 230009, PR China

b

a r t i c l e

i n f o

Article history: Received 30 July 2013 Received in revised form 5 November 2013 Accepted 9 November 2013 Available online 20 November 2013 Keywords: Interleukin-6 Electrochemical immunosensor Graphene Polydopamine

a b s t r a c t An Interleukin-6 (IL-6) electrochemical immunosensor was fabricated based on the Au nanoparticles (AuNP)-graphene-silica sol–gel as immobilization biointerface and AuNP-polydopamine (PDA)@carbon nanotubes (CNT) as the label of HRP-bound antibodies. The AuNP-graphene-silica sol–gel film was prepared in situ and modified on the ITO electrode, providing a stable network for the immobilization of antibody and exhibiting a dynamic working range of 1–40 pg/mL with a low detection limit of 0.3 pg/mL IL-6 (at 3 s). The results of serum samples with the sensor received an acceptable agreement with the ELISA method. Importantly, this method provided a promising ultrasensitive assay strategy for clinical applications. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Interleukin-6 (IL-6), a multifunctional cytokine, has a critical role in the inflammatory response, such as psoriasis, rheumatoid arthritis, cardiovascular disease, and inflammatory bowel disease [1–6]. It is also a suitable biomarker overexpressed by several types of cancer, including head and neck squamous cell carcinoma (HNSCC), which affects nearly 44,000 patients and results in ∼11,000 deaths per year in the US [7,8]. Normally, IL-6 levels are nearly 1000-fold lower than other secreted cancer biomarkers, such as prostate specific antigen (PSA) with normal patient serum levels in the nanograms per milliliter range [9]. Thus, the sensitive determination of IL-6 level with low detection limit is very useful to clinical diagnosis. Due to the advantages of simple preparation, low cost, short response time, small size, and yet accurate and sensitive platform, electrochemical techniques have been well recognized [10]. In the development of electrochemical immunosensing strategies, an efficient biointerface and signal amplification of the immunoconjugates are two key factors [11].

∗ Corresponding author at: Anhui Normal University, Key Laboratory for Functional Molecular Solids of the Education Ministry of China, College of Chemistry and Materials Science, Anhui Key Laboratory of Chem-Biosensing, Center for Nano Science and Technology, Beijing East Road 1#, Wuhu 241000, China. Tel.: +86 553 3869303; fax: +86 553 3869302. E-mail addresses: [email protected], [email protected] (X. Zhang). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.015

As for an efficient biointerface, it first needs to retain the biologic activity of the immobilized biomolecules on the probe surface in its native status [12]. Meanwhile, it should have high immobilized amount and the immobilized firmness of biomolecules [13]. Graphene is an ideal candidate in fabricating the biointerface with extraordinary electronic transport properties and high electrocatalytic activities [14–16]. However, there are three common problems existing in the conventional graphene-based biointerface. First, it is easy for graphene sheet to break off from the surface of the electrode with graphene sheet alone modified [17,18]. Second, in the electrochemical immunosensor with the graphene sheet as the biointerface, antibody is often adsorbed only on the surface of the graphene, and the conjugation is not very stable. Third, graphene will decrease the surface coverage of the electrode due to its two-dimensional network [19–21], which will decrease the loading amount of the protein. However, sol–gel technology provides a unique method to prepare a three-dimensional network suiting for the encapsulation of a variety of bimolecular [22–24]. Proteins entrapped on the growing covalent sol–gel network can retain their functionality and activity, and exhibit improved resistance to thermal and chemical denaturation as well as good storage and operational stability, compared with those specifically adsorbed on a substrate [25,26]. So the film combining graphene and sol–gel will be an effective platform for antibody immobilizaiton. Signal amplification is also very crucial for obtaining high sensitivity of the immunosensor. Au nanoparticles (AuNP) labeled with HRP-bound antibodies are commonly exploited as signal

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transduction tools in the electrochemical immunoassays [27–31]. The precondition of the signal amplificaiton by AuNP composites is the attachment of HRP-bound antibodies with AuNP composites. This protocol is simple and well-established; however, the loading label is rather limited and the preparation conditions are often rigorous. Novel and effective materials still need to be studied for the labeling method [32]. In this paper, we tried to utilize polydopamine (PDA), a novel simple, inexpensive, “green” and stable film with AuNP labels, and obtained a satisfying result. 2. Materials and methods 2.1. Materials Gold (III) chloride trihydrate (99.9%), tetraethyl orthosilicate (TEOS) (99.9%), dopamine (DA), and 2-amino2-hydroxymethylpropane-1,3-diol (Tris) were purchased from Shanghai Chemical Reagent Co., Ltd. Carbon nanotubes (CNT) were obtained from the Chengdu Organic Chemistry Institute. Bovine serum albumin (BSA, 96–99%), IL-6, Monoclonal antihuman IL-6 antibody (Ab), HRP, 1-(3(dimethylamino)-propyl)-3-ethylcarbodiimidehydrochloride (EDC), and N-hydroxysulfosuccinimide (NHSS) were purchased from Bomei Co., Ltd. (Hefei, China). All the other reagents were of analytical reagent grade and used without further purification. Clinical serum samples were provided by Cancer Center of the hospital. Phosphate-buffered saline (PBS), 50 mM, with various pH values was prepared by mixing the stock solutions of NaH2 PO4 and Na2 HPO4 , and then adjusting the pH with 0.1 M NaOH and H3 PO4 . Doubly distilled water was purified with a Milli-Q plus 185 equipment of Millipore (Bedford, MA, USA) and used throughout all assays. 2.2. Preparation of AuNP-graphene-silica on ITO chip AuNP-graphene-silica sol–gel composites were prepared according to the literature with a little modification. 0.50 mL colloidal suspension of GO (5 mg/mL, Hummers method), 0.1 mL TEOS, 1 mL ethanol, and 1 mL 1% HAuCl4 were mixed in a vial. After that, the vials were capped and the resulting sols were left at room temperature for a day. The ITO chips were cleaned with acetone, ethanol, and water in sequence. After being immersed in a solution of 1:1 (V/V) ethanol/NaOH (1 M) for 15 min, they were rinsed with water. Thin films were then deposited on the cleaned clips by dipping 10 ␮L sol–gels. Then the composites films were exposed to an atmosphere saturated with vapor of hydrazine monohydrate, which induced the GO into graphene in chemical method. Finally, the films were dried at 100 ◦ C for 3 h in an air. The graphene-silica sol–gel was prepared under the same conditions but without the addition of HAuCl4 . After the modified ITO chips dried under a stream of nitrogen, antibody-IL-6 (Ab1 ) immunoreaction area was prepared on the ITO surface. 50 ␮L of 100 ␮g/mL Ab1 solutions (50 mM PBS, pH 7.4) were dipped onto the immunoreaction area of ITO chip, and then incubated for 12 h at 4 ◦ C to attach Ab1 molecules to the film. After that, they were rinsed with PBST to remove physically absorbed Ab1 . The chips were then blocked with 20 ␮L of 2% BSA in PBS for 1 h at room temperature and washed with PBST. All washing steps were optimized to minimize nonspecific binding (NSB) to achieve the necessary sensitivity. Then the Ab1 -AuNP-graphene-silica platform was formed and was stored at 4 ◦ C when not in use.

Scheme 1. Schematic view of sandwich-type electrochemical detection of IL-6.

added into 100 mL water and dispersed by sonication 1 min in ice water bath. This mixture was magnetically stirred at room temperature (20 ◦ C) for 36 h. After the coated CNT (PDA@CNT) were filtered and washed by water, 25 mg of it was dispersed into HAuCl4 aqueous solution (25 mL, 1.4 mM). At last, this mixture was mildly stirred at room temperature for 2 h, and then formed AuNPPDA@CNT. The preparation procedure of the HRP-secondary antibodyAuNP-PDA@CNT (HRP-Ab2 -AuNP-PDA@CNT) bioconjugate was as follows. 1 mg AuNP-PDA@CNT composites in 2 mL pH 7.4 PBS were sonicated with 1 mL of freshly prepared 200 mM EDC and 100 mM NHSS for 5 min to obtain a homogeneous dispersion and then centrifuged at 15,000 rpm for 5 min, and the supernatant was discarded. The mixture was gently mixed with 150 ␮L of Ab2 (2.0 ␮g/mL) and 150 ␮L of HRP at 1.0 mg/mL for 2 h at room temperature, after centrifuged at 15,000 rpm for 5 min and the supernatant was discarded. Following that, the precipitation was blocked by 100 ␮L of 2% BSA solution for 30 min to block possible remaining active sites of the AuNP-PDA@CNT and avoid the nonspecific adsorption. Washing was crucial to remove the free Ab2 and HRP and was repeated four times with PBST. Then the bio-precipitate was transferred into 1 mL of 0.05% Tween-20 in PBS (pH 7.4) and vortexed to form a homogeneous dispersion and then stored in the refrigerator at 4 ◦ C. 2.4. Fabrication of the electrochemical immunosensor After aspiration, Ab1 modified electrodes were incubated with 10 ␮L of detecting IL-6 samples for 1 h at 37 ◦ C. After the binding reaction between Ab1 and IL-6, the electrodes were immersed into the 10 ␮L HRP-secondary antibody (HRP-Ab2 ), HRP-Ab2 -AuNPPDA@CNT, or other labeled HRP-Ab2 bioconjugate solution for an incubation time of 1 h. Finally, the chips were washed thoroughly with water to remove nonspecifically bound conjugations. After several washing steps with PBST, the sensor was placed into an electrochemical cell containing 1 mM hydroquinone as mediator in PBS buffer, and 0.4 mM hydrogen peroxide was injected while measuring the current to develop an amperometric signal, for the analysis of IL-6. The way for the immobilization of Ab1 and the immunoassay procedure were shown in Scheme 1. 2.5. Measurement procedure

2.3. Preparation of HRP-Ab2 -AuNP-PDA@CNT bioconjugate First, AuNP-PDA@CNT was synthesized according to the literature [30]. Briefly, 100 mg CNT, 200 mg DA, and 120 mg Tris were

All the electrochemical experiments were carried out at room temperature. Electrochemical impedance spectra (EIS) was performed in 1/15 M PBS (pH 7.4) containing 5 mM

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Fig. 1. SEM images of graphene-silica (A), AuNP-graphene-silica (B), pure CNT (C), PDA@CNT (D), AuNP-PDA@CNT (E) and HRP-Ab2 -AuNP-PDA@CNT (F).

Fe(CN)6 3− /Fe(CN)6 4− and 0.1 M KCl in the frequency range from 0.1 Hz to 100 kHz with 5 mV as the amplitude at a polarization potential of −0.18 V. Amperometric current was obtained at −0.3 V with 3000 rpm after placing electrodes in PBS buffer containing 1 mM hydroquinone and injecting 0.04 mM H2 O2 . Scanning electron microscopy (SEM) images of the electrode surface were obtained using Hitachi S-4800 SEM (operated at 10 kV). FTIR spectra were measured on a FTIR-8400S spectrometer in the 480–4000 cm−1 region using a powered sample on a KBr plate (Japan, Shimadzu). Centrifugation was performed using a HERMLEZ 36 HK apparatus (Wehingen, Germany). 3. Results and discussion 3.1. Characterization Scheme 1 showed the illustration of the immobilization procedures. The sandwich immunoassay protocol based on AuNPgraphene-silica sol–gel biointerface and HRP-Ab2 -AuNP-PDA@CNT label was allowed to improve the performance of the immunoassay in this study. The surface morphology of the sol–gel matrix was an important factor affecting the immobilization of antiIL-6. Fig. 1B showed the morphologies of AuNP-graphene-silica sol–gel films. As shown in the images, AuNP distributed onto the sol–gel evenly. The uniformly open structure provided a significant increase of effective electrode surface for the immobilization of Ab1 which may further resulted in a free binding of the antigen conjugating with Ab2 . However, the sensitivity of the electrochemical immunosensors could be enhanced by using the AuNP-PDA@CNT for labeling HRP-Ab2 bioconjugate. Comparing with the unreacted pure CNT (Fig. 1C), CNT in PDA@CNT became thicker, which showed the CNT may be enwrapped by PDA (Fig. 1D, further proved by IR). In Fig. 1D, we could found that the PDA@CNT was covered with well-distributed AuNP, which provided a significant increment of the binding capacity. After the Ab1 was modified onto the AuNP-PDA@CNT, the surface of these composites became rougher (Fig. 1E). As shown in Fig. 2A, dopamine showed many narrow peaks, a feature of small molecule; while PDA (synthesized at the same condition for CNT@PDA) presented only a few intense absorption features: around 1615 cm−1 from aromatic rings and around

3420 cm−1 from catechol OH groups. The pure CNT material gave weak absorption at the same positions: 1615and 3420 cm−1 , indicating that there was a little acid group on graphene surface and that the graphene structure had an affinity to PDA aromatic rings. These structural features supported the wrapping of CNT by PDA. If the CNT had many acid groups on its surface, the increasing hydrophilicity and the decreasing conjugate structure of CNT would prohibit the smooth coating by PDA. The obtained CNT@PDA showed mainly the intense absorption features of PDA. EIS is always used to probe the features of surface modified electrodes. The semicircle diameter at higher frequencies corresponds to the electron-transfer resistance (Ret). The stepwise construction process of the immunosensor was characterized by EIS. As shown in Fig. 2B, the Ret of bare ITO electrode (curve a) was about 300 . This value remarkably increased to 361  after AuNP-graphenesilica formed onto the ITO surface (curve b). However, then with the Ab1 adsorbed onto the electrode, the Ret increased to 400  (curve c), which suggested the protein formed an additional barrier and further prevented the redox probe. Then the specific interaction of IL-6 and Ab1 made the resistance increase again (curve d). At last, after the HRP-labeled Ab2 interacting with IL-6, interestingly the resistance decreased to 525  (curve e). We suggested that although adsorption of the protein may impede the electron transfer, in our experiment, Ab2 labeled with AuNP-PDA@CNT may improve the electron transfer because of the good electrochemical conductivity of AuNP and CNT. The above results clearly confirmed the success of the assembly on the electrode as expected. 3.2. The optimization of experimental conditions The performance of the amperometric immunosensor is usually influenced by the pH of the buffer solution, the incubation temperature, and the time. Moreover, the signal of this immunosensor is related with the concentration of H2 O2 . In order to achieve high sensitivity, experimental conditions were optimized. The optimization of the variables of the system was shown in Fig. 3. Fig. 3A depicted the effect of varying pH value on the electrocatalytic currents of the resulting immunosensor, and other conditions were the same as above. When the pH value was over 7.4, the current responses declined, resulting that the highest sensitivity was obtained in pH 7.4. Furthermore, Fig. 3B depicted the

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Fig. 2. (A) FTIR spectra (a) of dopamine, CNT, PDA and CNT@PDA; (B) The EIS results of ITO (a), AuNP-graphene-silica/ITO (b), Ab1 /AuNP-graphene-silica/ITO (c), labeledAb2 /IL-6/Ab1 /AuNP-graphene-silica/ITO (d) and IL-6/Ab1 /AuNP-graphene-silica/ITO (e) in 5 mM Fe(CN)6 3−/4− containing 0.1 M KCl solution, respectively.

time-dependent voltammetric waves to form the composition of the proposed sandwich structure. As the interaction time of Ab1 and Ab2 labeled with AuNP-PDA@CNT was prolonged, the currents increased (Only the incubation time changed, other conditions remained). Upon incubation time was more than 60 min, the signal of current showed a level indicating that the reaction may complete in 60 min. Therefore, 60 min was chosen as an optimized incubation time used in the experiments. In Fig. 3C, the response increased and then reached a maximum value at an incubation temperature of 37 ◦ C, which was chosen as the incubation temperature. The proposed immunosensor was incubated in a standard antigen solution for different time at this temperature. Fig. 3D showed that the amperometric current from the sensor reached a steady-state

response rapidly with the addition of H2 O2 from 10 to 40 ␮M, indicating that the response increased with the concentration of H2 O2 . Ultimately, we chose pH 7.4, hybridization time 60 min, incubation temperature 37 ◦ C, and the H2 O2 concentration 40 ␮M, as the optional experimental conditions. 3.3. The immunoassay Fig. 4A showed that the amperometric current from the sensor reached a steady state response rapidly, which increased linearly with IL-6 concentration between 1 and 40 pg/mL. Excellent sensorto-sensor reproducibility was illustrated by the small error bars in Fig. 4B, and the sensitivity as the slope of the calibration plot of

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Fig. 3. Effects of (A) pH of detection solution, (B) incubation time, (C) incubation temperature, and (D) the concentration of H2 O2 at the immunosensor in PBS containing 1 mM hydroquinone.

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Fig. 4. Amperometric results for the immunosensors: (A) Steady state amperometric current at −0.3 V and 3000 rpm after placing electrodes in PBS buffer containing 1 mM hydroquinone and injecting 0.04 mM H2 O2 . (B) Immunosensor calibration plot for IL-6 (n = 3).

Table 1 Performance with different immunosensors array for detection of IL-6. Linear range (pg/mL)

Detection limit (pg/mL)

Compared with ELISA

Real sample analysis

References

20–4 × 103 2–2 × 104 5–5 × 104 5–100 50–500 – 20–40 1–40

10 1 2 1 30 0.5 20 0.3

No No No Yes Yes Yes No Yes

No Yes No Yes Yes Yes Yes Yes

[33] [4] [34] [35] [36] [37] [38] This paper

20.4 nA/pg mL−1 was achieved. This approach provided a detection limit 0.3 pg/mL as 3 times the average noise plus the zero IL-6 control. We compared our results with the literatures’ and it showed that graphene-AuNP sol–gel as the sensor platform and nanolabel using multienzyme-antibody functionalized AuNP-PDA@CNT could greatly improve detection range (as shown in Table 1). The proposed approach was also able to receive an acceptable agreement with the ELISA method. At the same time, this immunosensor assay can provide a promising ultrasensitive assay strategy for clinical diagnosis.

3.5. The reproducibility, stability, and selectivity The proposed immunosensor had a good precision resulting from the low relative standard deviations of 5.1, 4.9, and 5.6% (n = 3) of the immunosensor response to 2, 20, and 40 pg/mL IL-6, respectively. Additionally, the reproducibility of the immunosensor was evaluated by measuring the responses of 3 replicate biosensors. A relative standard deviation (R.S.D.) of around 7.2% was obtained. Thus, the sensor showed a good, reproducible behavior and could be used for reproducible measurements. To monitor the differences among response of the immunosensor to interference degree

3.4. Comparison of different labels

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0.8 HRP-Ab2 AuNP-HRP-Ab2 CNT-HRP-Ab2 HRP-Ab2-AuNP -PDA@CNT

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Current / µA

To further investigate the effect of the synthesized AuNPPDA@CNT on the sensitivity of the electrochemical immunosensors, a comparative study of the amperometric responses of immunoreaction was carried out by using HRP-Ab2 , AuNP-HRPAb2 , CNT-HRP-Ab2 , and HRP-Ab2 -AuNP-PDA@CNT as a secondary conjugating antibody in the sandwich immunoassay, respectively. As shown in Fig. 5, we could find that the use of AuNP-PDA@CNT showed much greater amperometric changes than those obtained at the other label methods. Some possible explanations may contribute to these observations. First, CNT and AuNP with high capability of electron transfer doped into the bionanocomposites effectively shuttled the electrons from the base electrode surface to the redox center of HRP. Second, the immobilization density of HRPAb2 bound could enhance due to the high surface-to-volume ratio of bionanocomposites. Third, PDA may make more CNT and AuNP attach, with more HRP loading effectively and amplified output of the amperometric signal. Apparently, the above results showed that the use of AuNP-PDA@CNT could generate better performance in the detection of IL-6.

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Fig. 5. Amperometric responses of the immunosensor with the variously secondary labeled antibody toward different IL-6 concentrations: (a) HRP-Ab2 , (b) AuNP-HRPAb2 , (c) CNT-HRP-Ab2 , and (d) HRP-Ab2 -AuNP-PDA@CNT in pH 7.4 PBS containing 1 mM hydroquinone and, then injecting 0.04 mM H2 O2 .

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Table 2 Comparison with ELISA in real samples. IL-6 standard (pg/mL)

2

20

40

Detection (pg/mL)

Standard deviation

ELISA

Proposed immunosensor

ELISA

Proposed immunosensor

2.2 2.4 2.1 23.4 21.2 25.6 42.6 42.3 41.3

2.3 1.9 1.8 27.5 24.2 23.8 42.5 38.2 42.3

0.125

0.216

1.797

1.658

0.557

1.982

or crossing recognition level, alpha-fetoprotein, human chorionic gonadotropin, and prostate-specific antigen with various concentrations were injected into the detection system, respectively. The interference degree among lineage-different tumor markers was 2.2–8.8% implying that the selectivity of the developed immunosensor was acceptable.

[2] [3] [4] [5] [6] [7] [8]

3.6. Application in real samples

[9] [10]

To investigate the possibility of the newly developed technique applied for clinical analysis, real samples were examined by the developed immunoassay. The results and the relative deviations between the two methods were shown in Table 2. Compared with standard ELISA assays, our method gave excellent correlations indicating that the fabricated immunosensors were hopeful for future fabrication of bioelectronic arrays.

[11] [12] [13] [14] [15] [16] [17]

4. Conclusion

[18]

This article described a new biointerface based on grapheneAuNP sol–gel and nanolabel using AuNP-PDA@CNT. The grapheneAuNP sol–gel biointerface was stable and effective for Ab1 immobilization, and the AuNP-PDA@CNT could be conveniently used for the label of secondary antibodies in the sandwich-type immunoassay format. Compared with conventional immobilizaiton and labeled methods, the immunoassay exhibited high sensitivity and low detection limit. Highlight of this work is to improve the stability and loading amount. The AuNP-PDA@CNT label facilitates the electron transfer between the analyte and the base electrode with the signal amplification. The “green” operation and ultrasensitivity of the developed methodology provide a promising potential in clinical diagnosis.

[19] [20] [21]

Acknowledgments

[32]

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[33]

This work was financially supported by the projects (21005001 and 21073001) from National Natural Science Foundation of China, Anhui Provincial Natural Science Foundation (1208085QB28), Natural Science Foundation of Anhui (KJ2012A139) and the project of Anhui Key Laboratory of Controllable Chemistry Reaction & Material Chemical Engineering (OFCC0905). References [1] J. Van Snick, Annu. Rev. Immunol. 8 (1990) 253.

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Ultrasensitive IL-6 electrochemical immunosensor based on Au nanoparticles-graphene-silica biointerface.

An Interleukin-6 (IL-6) electrochemical immunosensor was fabricated based on the Au nanoparticles (AuNP)-graphene-silica sol-gel as immobilization bio...
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