Facile synthesis of novel reduced graphene oxide@polystyrene nanospheres for sensitive label-free electrochemical immunoassay

Qingchun Lan a, Huifang Shen a, Juan Li *a, Chuanli Ren b, Xiaoya Hu *a and Zhanjun Yang *a
aCollege of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou University, Yangzhou 225002, P. R. China. E-mail: zjyang@yzu.edu.cn; xyhu@yzu.edu.cn; lijuan@yzu.edu.cn
bDepartment of Laboratory Medicine and Clinical Medical College of Yangzhou University, Subei Peoples’ Hospital of Jiangsu Province, Yangzhou, 225001, P. R. China

Received 10th October 2019 , Accepted 29th November 2019

First published on 12th December 2019

Herein, we report the synthesis of novel nanosized reduced graphene oxide@polystyrene nanospheres (rGO@PS NSs) through an electrostatic interaction method, and further their exploitation for highly sensitive label-free electrochemical immunoassay applications. These rGO@PS NSs provide a universal and promising carrier platform to develop excellent biosensors for clinical screening and diagnostic applications.

Graphene, a single-atom-thick planar sheet of sp2-bonded carbon atoms, has attracted enormous attention and extensively been used in nanoelectronics,1 solar cells,2 batteries,3 supercapacitors,4 and chemical and biological sensors5 owing to its remarkable physical and chemical properties. Unfortunately, graphene as a promising material with large surface area and excellent electrical conductivity exhibits poor dispersion and can easily agglomerate through π–π stacking and van der Waals interactions.6 Recently, various strategies such as chemical modification and noncovalent functionalization have been used to facilitate the dispersibility of graphene.7 Noncovalent functionalization by blending graphene suspension with polymer solution or emulsion has become a very popular method to obtain a homogeneous distribution of graphene due to the prevention of the destruction of the π-conjugated skeleton and the loss of the electronic properties of graphene. However, some particular treatment steps are also necessary and crucial to avoid reaggregation or restacking of dispersed graphene sheets during the removal of the organic or aqueous media. In fact, the problem of non-uniform dispersion of graphene can be effectively resolved by preparing colloidal graphene composite particles.8 PS spheres, one of the most common colloidal particles, are widely used to synthesize composite materials due to their perfect geometrical shape, uniform size, acid–base resistance properties and various surface chemical performances.9 Until now, few papers have been reported on the fabrication of graphene-based composite particles. Moreover, the resulting composite particles were micro-sized spheres. Nevertheless, design of nanosized graphene composite particles may be more necessary and promising and has not been reported so far.

The sensitive detection of tumor markers plays a considerable role in clinical diagnosis applications. Various immunoassay methods have been developed for detection of tumor markers.10 Among these methods, electrochemical immunoassay has attracted extensive attention because of its special advantages.11 In particular, label-free electrochemical immunoassays have been considered to be one of the most promising methods due to their easy handling, low cost, and rapid assay speed.12 For the construction of an excellent label-free electrochemical immunosensor, the design of an advanced sensing surface is one key factor in efficient immobilization of antibody biomolecules. Over the past few decades, nanomaterials have been widely exploited to modify electrodes for efficient immobilization of biomolecules so as to improve the assay performance of electrochemical immunosensors. To the best of our knowledge, nanosized graphene composite particles have not been used for the immobilization of proteins and biosensing applications.

Herein, we report a facile and effective method to prepare reduced graphene oxide coated polystyrene nanospheres (rGO@PS NSs) by self-assembly of cationic polystyrene nanospheres (PS+) and negatively charged graphene oxide sheets (GO) through electrostatic interaction, followed by hydrazine-reduction of GO. The rGO@PS NSs were for the first time exploited as an excellent electrochemical material for the development of label-free electrochemical immunosensors (illustrated in Scheme 1). The obtained rGO@PS NSs were characterized with various means and exhibited excellent electrochemical properties, good hydrophilicity, larger specific surface area and a high loading capacity of antibodies. The proposed label-free electrochemical immunosensor exhibited a high sensitivity, a wide linear range, and excellent selectivity. The rGO@PS NSs provided an alternative material for biosensing applications.

image file: c9cc07934c-s1.tif
Scheme 1 Preparation procedures of rGO@PS NSs (A) and schematic illustration of the fabrication process of the label-free immunosensor and immunoassay procedure for AFP (B).

The morphology of the resultant rGO@PS NSs was characterized by scanning electron microscopy and transmission electron microscopy. As shown in the SEM and TEM images in Fig. 1A and C, the PS nanospheres show uniform size with an average diameter of 200 nm, and the surfaces of the spheres are smooth. After being anchored with rGO, the surfaces of the rGO@PS NSs became wrinkled (Fig. 1B and D). Moreover, the rGO@PS NSs still show a uniform morphology and no obvious changes in sphere size. As shown in Fig. S1 (ESI), a yellow-brown GO dispersion was mixed with a milk-white PS suspension, followed by a hydrazine reduction process; the resultant rGO@PS NSs became black suspensions. These results indicate that rGO nanosheets have been successfully attached onto the surfaces of the PS nanospheres via electrostatic interaction.

image file: c9cc07934c-f1.tif
Fig. 1 SEM and TEM images of PS (A and C) and rGO@PS NSs (B and D), and Raman (E) and FT-IR (F) spectra of PS, GO and rGO@PS.

The Raman spectrum and FT-IR spectrum were used to characterize the resultant rGO@PS NSs. As shown in Fig. 1E, the Raman spectrum of the PS nanospheres shows a very strong peak at about 1002 cm−1, which can be assigned to the v1 ring-breathing mode of PS. In contrast, two typical D-bands at 1350 cm−1 and a G band at 1580 cm−1 were observed in the Raman spectrum of GO. After rGO was assembled onto the PS nanospheres, two typical D-bands at 1350 cm−1 and a G band at 1580 cm−1 were also observed in the Raman spectrum of the rGO@PS NSs. The intensity ratio of the D to G bands (ID/IG) in the rGO@PS NSs (1.16) obviously increased compared with that of GO (0.93) under similar conditions.13 Moreover, the characteristic signal of PS almost disappeared from the Raman spectrum of the rGO@PS NSs. These results demonstrated that the reduced graphene oxide sheets were completely coated onto the PS colloidal particles. The reduction of the oxygen-containing groups in GO was also confirmed by FT-IR analysis. As shown in Fig. 1F, four peaks at 697, 757, 1368, and 1448 cm−1 in the FT-IR spectrum of PS are related to the absorption of the benzene ring of PS segments.14 Other peaks at 2921 and 3027 cm−1 correspond to the additional methylene groups of the PS.15 The FT-IR spectrum of GO shows a characteristic absorption peak at 1747 cm−1 (the C[double bond, length as m-dash]O vibration). After reduction of GO assembled onto PS by hydrazine, this characteristic absorption peak at 1747 cm−1 disappeared, indicating the successful synthesis of rGO@PS NS particles.

Scanning electron microscopy was used to characterize the surface morphology of the resultant label-free immunosensor. As shown in Fig. S2 (ESI), the rGO@PS NSs were dispersed into a chitosan film and showed a uniform morphology (Fig. S2A, ESI). When streptavidin was trapped on an rGO@PS NS film (Fig. S2B, ESI), a much rougher surface can be clearly observed due to the attachment of streptavidin. After biotinylated anti-AFP was immobilized on the composite film, the SEM image of biotinylated anti-AFP/streptavidin/rGO@PS NSs (Fig. S2C, ESI) shows bigger aggregates of the loaded proteins than that of the streptavidin-functionalized substrate, suggesting the successful immobilization of biotinylated anti-AFP on the streptavidin-functionalized rGO@PS NS composite platform.

XPS spectra and FT-IR spectroscopy were used to further analyze the fabricated immunosensor.16 As shown in Fig. S2D (ESI), the rGO@PS NSs/chitosan composite (curve a) shows a distinct N1s peak at 399.85 eV. Compared with the rGO@PS NSs/chitosan composite, the N1s peak intensity of the streptavidin-functionalized rGO@PS NSs/chitosan composite obviously increased (curve b), verifying the successful modification of streptavidin. After immobilizing biotinylated AFP antibodies on the biofunctionalized substrate, the N1s peak intensity of the antibody loaded substrate further greatly increased, indicating the presence of AFP antibodies in the streptavidin/rGO@PS NSs/chitosan film. The FT-IR spectral results also confirmed the fabrication of the immunosensor. As shown in Fig. S2E (ESI), the FT-IR spectrum of the streptavidin-functionalized rGO@PS NSs/chitosan composite (curve b) shows two obvious absorption peaks assigned to the amide I and II bands at 1660 and 1545 cm−1 in comparison with the rGO@PS NSs/chitosan composite (curve a). After the modification of biotinylated AFP antibodies (curve c), two absorption peaks at 1659 and 1540 cm−1 were also observed, and the peak intensity greatly increased. This further confirms the successful immobilization of biotinylated anti-AFP on the rGO@PS NSs/chitosan film.

The biocompatibility of a sensing interface is positively related to its hydrophilicity, and can be evaluated by measuring the static water contact angle of the substrate.17 Fig. S3 (ESI) shows the contact angles of the bare GCE (a), rGO@PS NSs/chitosan (b), streptavidin/rGO@PS NSs/chitosan (c) and biotinylated antibodies/streptavidin/rGO@PS NSs/chitosan (d). Their contact angle values were 68.8°, 49.3°, 40.6° and 33.8°, respectively. Compared with the bare GCE, the rGO@PS NSs/chitosan modified GCE shows an obviously decreased contact angle due to its increasing hydrophilicity. When streptavidin/rGO@PS NSs/chitosan was modified on the electrode, a further decrease of the contact angle could be observed. The excellent hydrophilicity of the streptavidin/rGO@PS NSs/chitosan film provides a favorable environment to maintain the bioactivity of the loaded antibody molecules. After the biotinylated AFP antibody was immobilized onto the biofunctionalized substrate, it displayed the lowest contact angle, suggesting the successful immobilization of the biotinylated antibodies onto the biosensing surface.

The electrochemical behavior of the label-free immunosensor was investigated in a pH 6.5 PBS solution containing 0.1 M KCl and 5.0 mM [Fe(CN)6]3−/4− by cyclic voltammetry (Fig. S5, ESI). As shown in the cyclic voltammogram of rGO@PS NSs/chitosan/GCE (curve a), the greatest redox peak was observed in the potential range from −0.2 to 0.6 V. After streptavidin was trapped on the composite film, the peak current of streptavidin/rGO@PS NSs/chitosan/GCE was greatly decreased owing to the poor conductivity of streptavidin (curve b). When the biotinylated anti-AFP was immobilized onto the electrode through the biotin–avidin system, biotin-anti-AFP/streptavidin/rGO@PS NSs/chitosan/GCE (curve c) shows an obvious decrease of peak current, indicating that the biotinylated antibody was successfully immobilized onto the surface of the streptavidin/rGO@PS NSs/chitosan modified electrode. After the residual reactive sites of the label-free immunosensor were blocked with BSA solution, the peak current was further declined (curve d). Finally, the fabricated label-free immunosensor was incubated with the AFP sample, the oxidation peak current of the immunosensor was decreased obviously (curve e). This phenomenon is ascribed to the formed immunocomplex which blocked the electron transfer of the electroactive probe. An electrochemical immunosensor is therefore proposed for label-free detection of tumor markers based on the decrease in oxidation peak current.

Under the optimized conditions, the quantitative detection of standard AFP samples was investigated using the proposed label-free immunosensor. As shown in Fig. 2A, the DPV current response decreased with increasing AFP concentration. The calibration plot in Fig. 2B shows a good linear relationship between the reduction peak current and the AFP concentration over the range of 0.1 to 100 ng mL−1 with a detection limit of 0.03 ng mL−1 (S/N = 3). The fitted linear regression equation was Y (μA) = 119.26–0.635X (ng mL−1), and the linear regression coefficient was R2 = 0.9952. The performance of the proposed label-free AFP immunosensor was compared to those of previous methods, see Table S1 (ESI). It can be seen that the value of the detection limit of the biosensor is much lower than those of the previous methods. To evaluate the practical application ability of the label-free immunosensor, clinical human serum samples were measured by the proposed method and the reference method. The reference method is an ECL immunoassay, which was conducted by the Jiangsu Institute of Cancer Research. Prior to the measurement, the serum samples were appropriately diluted to the calibration range of the immunosensor with 0.01 M pH 6.5 PBS. Each sample was detected five times, and the results are listed in Table S2 (ESI). The obtained results demonstrate an acceptable relative error of less than 8.65% between these two methods, which confirms that the proposed immunosensor can be applied in the determination of real-life clinical samples.

image file: c9cc07934c-f2.tif
Fig. 2 (A) DPV responses of the immunosensor toward different concentrations of AFP (from a–j: 0, 0.1, 0.5, 1, 10, 20, 40, 60, 80, and 100 ng mL−1 AFP). (B) Linear relation between the current response and AFP concentration (n = 5 for each point). (C) The specificity study of the rGO@PS NS modified immunosensor toward AFP (a), AFP + AA (b), AFP + CEA (c), AFP + CA125 (d), and AFP + glucose (e). (D) Current responses of the label-free biosensor after storage for 0, 7, 14, 21, and 28 days at a concentration of 10 ng mL−1 AFP (n = 5 for each point).

The selectivity test was performed in order to evaluate the anti-interference ability of the label-free immunosensor. The immunosensor was incubated with AFP (10 ng mL−1) solution that contains 100 ng mL−1 of interfering substances including AA, CEA, CA125, and glucose, respectively. As shown in Fig. 2C, the changes in current response caused by the interfering agents were less than 5% in comparison with the single AFP, which indicates that the label-free immunoassay has high specificity and can be used for the selective determination of AFP. Besides, the reproducibility was investigated by the coefficient of variation of the intra- and inter-assays. The intra-assay used five prepared electrodes to detect the same concentration of AFP (10 ng mL−1). All the electrodes exhibit similar current responses and the relative standard deviation (RSD) is less than 3%. The inter-assay was investigated by using the same electrode to test repetitively five times at a certain concentration of AFP (10 ng mL−1); the RSD is 5.8%. All the results demonstrated acceptable reproducibility of the proposed immunosensor. Finally, the stability of the label-free immunosensor was investigated by analysing its current responses periodically after a period of storage (shown in Fig. 2D). The immunosensor was measured every week, and the current response retained 96% of its initial current after being kept at 4 °C in 0.01 M of PBS (pH 7.4) for 4 weeks, indicating the acceptable stability of the label-free immunosensor.

In summary, a novel label-free electrochemical immunosensing platform was constructed for highly sensitive detection of tumor markers based on streptavidin-functionalized rGO@PS NSs. The rGO@PS NSs were synthesized by self-assembly of positively charged PS microspheres and negatively charged GO sheets through electrostatic interaction, followed by hydrazine reduction. Various means were used to characterize the rGO@PS NSs, which exhibit excellent electrochemical properties, good hydrophilicity, larger specific surface area and a high loading capacity of antibodies. This label-free immunoassay strategy can be easily achieved by immobilization of the capture antibodies on rGO@PS NSs through a biotin–avidin system. The efficiency and capacity of antibody immobilization on the rGO@PS NS interfaces were greatly enhanced. Besides, the proposed label-free immunosensor is simple, fast, and efficient and has high sensitivity, good selectivity, and acceptable reproducibility. The rGO@PS NSs provide a universal and promising avenue for the development of immunoassay applications.

This work was financially supported by the National Natural Science Foundation of China (21575125, 21575124, 21475116, 81573220), the Natural Science Foundation of Jiangsu Province (BK20191434), the 333 Project and Qinglan Project of Jiangsu Province and the High-end Talent Support Program of Yangzhou University (to Zhanjun Yang and Juan Li), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD) and the Six Talent Peaks Project of Jiangsu Province (to Zhanjun Yang and Juan Li), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (no. KYCX17_1876) and the Open Research Fund of State Key Laboratory of Analytical Chemistry for Life Science (no. SKLACLS1915).

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Electronic supplementary information (ESI) available: Experimental details, experimental conditions and some results. See DOI: 10.1039/c9cc07934c

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