Article pubs.acs.org/ac
An Electrochemically Reduced Graphene Oxide-Based Electrochemical Immunosensing Platform for Ultrasensitive Antigen Detection Al-Monsur Jiaul Haque,† Hyejin Park,† Daekyung Sung,‡ Sangyong Jon,‡ Sung-Yool Choi,§,∥ and Kyuwon Kim†,* †
Department of Chemistry, University of Incheon, Incheon 402-776, Korea. Department of Life Science, Gwangju Institute of Science and Technology, Korea § Electronics and Telecommunications Research Institute, Daejeon, 305-700, Korea ‡
S Supporting Information *
ABSTRACT: We present an electrochemically reduced graphene oxide (ERGO)-based electrochemical immunosensing platform for the ultrasensitive detection of an antigen by the sandwich enzymelinked immunosorbent assay (ELISA) protocol. Graphene oxide (GO) sheets were initially deposited on the amine-terminated benzenediazonium-modified indiun tin oxide (ITO) surfaces through both electrostatic and π−π interactions between the modified surfaces and GO. This deposition was followed by the electrochemical reduction of graphene oxide (GO) for preparing ERGO-modified ITO surfaces. These surfaces were then coated with an Nacryloxysuccinimide-activated amphiphilic polymer, poly(BMA-rPEGMA-r-NAS), through π−π stacking interactions between the benzene ring tethered to the polymer and ERGO. After covalent immobilization of a primary antibody on the polymer-modified surfaces, sandwich ELISA was carried out for the detection of an antigen by use of a horseradish peroxidase (HRP)-labeled secondary antibody. Under the optimized experimental conditions, the developed electrochemical immunosensor exhibited a linear response over a wide range of antigen concentrations with a very low limit of detection (ca. 100 fg/mL, which corresponds to ca. 700 aM). The high sensitivity of the electrochemical immunosensor may be attributed not only to the enhanced electrocatalytic activity owing to ERGO but also to the minimized background current owing to the reduced nonspecific binding of proteins.
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exploited in the design of versatile devices such as field-effect transistors,19 ultrasensitive sensors,20 and electrochemical resonators.21 Graphene oxide (GO) and reduced graphene oxide (RGO) have found potential applications in biosensor fabrication, cellular imaging, and drug delivery owing to their simple synthesis, good water dispersibility, large accessible surface area, and good biocompatibility.22−25 Recent studies reported that the electrochemical biosensing performance of electrodes covered with RGO during detection of electroactive biomolecules in the presence of interfering agents is superior to that of bare electrodes or even electrodes modified with carbon nanotubes.26,27 The superior performance of RGO-modified surfaces in discriminating mixed species was attributed to the excellent electrocatalytic activity because of the large surface area and high density of edge-plane-like defects present in RGO sheets, which allow rapid heterogeneous electron transfer. The commonly reported approach for preparation of RGOmodified electrode surfaces is the predeposition of GO sheets
ince the introduction of the first Clark-type glucose sensor,1 electrochemical biosensors have gained increasing attention and have become one of the predominant analytical techniques in various fields such as clinical diagnosis, food analysis, and environment monitoring because of their excellent sensitivity, selectivity, simplicity, and low cost.2−7 In addition, electrochemical biosensors have good compatibility with advanced micromachining technologies. Moreover, compared to other types of biosensors, they are easy to miniaturize without degrading the analytical performance.8−10 Therefore, the development of highly sensitive and selective electrochemical biosensors using new strategies and sensor platforms generates continual interest. Additionally, a vast majority of recent research on electrochemical biosensors has focused on the incorporation of nanomaterials such as carbon nanotubes and metal nanoparticles in the design of sensor platforms. This incorporation leads to a significant improvement in the sensitivity and selectivity of the sensors.11−15 Graphene, a single-atom-thick and two-dimensional sp2 carbon networking material, has stimulated research interest owing to its remarkable electrical, mechanical, and thermal properties.16−18 The unique characteristics of graphene are © 2012 American Chemical Society
Received: September 29, 2011 Accepted: January 13, 2012 Published: January 13, 2012 1871
dx.doi.org/10.1021/ac202562v | Anal. Chem. 2012, 84, 1871−1878
Analytical Chemistry
Article
protocol for an efficient immunosensor platform based on RGO and ERGO is still highly beneficial. Here, we report a method for fabricating an electrochemical immunosensor platform using RGO as an electrode material. Moreover, we demonstrate that the method can provide a general and an efficient protocol for the ultrasensitive detection of a target protein on RGO surfaces. In this work, we used electrochemically reduced graphene oxide (ERGO), two types of graphenefriendly linkage molecules, ATBD and poly(BPN), and a common sandwich enzyme-linked immunosorbent assay (ELISA) based on electrochemical amplification. ERGOmodified ITO surfaces were produced by the electrochemical reduction of GO sheets deposited on amine-terminated BDelectrografted ITO surfaces. The ERGO-modified ITO surfaces were then coated with the amphiphilic polymer, poly(BPN). This coating process was followed by the covalent immobilization of a primary antibody on the polymer-modified surfaces. Sandwich ELISA was carried out on the modified ITO surfaces for detecting an antigen by use of a horseradish peroxidase (HRP)-labeled secondary antibody. Then an enzyme-catalyzed reaction product, benzoquinone (BQ), was electrochemically reduced at the sensor surfaces, producing a readout signal current proportional to the amount of antigen. ELISA is a sensitive technique for the detection of antigens and has potential applications in medicine and plant pathology as well as a quality-control check in various industries.47 In ELISAbased detection systems, the signal is generated by enzymes that are linked to the detection reagents in fixed proportions to allow accurate quantitation, and the sensitivity of detection depends on amplification of the signal by enzymatic reactions. For the current reading, we investigated and utilized the reaction potential providing minimized background currents by optimizing the experimental conditions. We demonstrated that our immunosensor platform developed for the electrochemical ELISA protocol had excellent sensitivity, a very low detection limit, and a high specificity for mouse IgG.
on the surfaces to be modified and the subsequent reduction of GO to RGO. Among the various methods reported for reduction of GO, the electrochemical reduction of GO sheets predeposited on electrode surfaces seems to be a promising route for preparing RGO-modified electrode surfaces because it is simple, fast, inexpensive, and more efficient than other methods such as chemical and thermal reduction.28−30 One simple method for preparing GO-deposited surfaces that has recently gained popularity is the deposition of negatively charged GO sheets on positively charged surfaces from aqueous dispersion through electrostatic interactions. In this method, the formation of an almost single-layer assembly of GO sheets on the surfaces is particularly important because the unique properties of graphene are mainly associated with individual sheets.30−32 Therefore, the positively charged surfaces are usually prepared by the formation of self-assembled monolayers (SAMs) of amine-terminated alkoxysilane or alkanethiol molecules.30−32 The electrochemical reduction of benzenediazonium cation (BD) introduced by Delamar et al.33 has been intensively studied over the past few decades and has become one of the best methods for surface functionalization because it permits the rapid formation of stable organic layers on various surfaces such as carbon,34−36 silicon,37 metals,38 and indium tin oxide (ITO).39 The use of amine-terminated benzenediazonium cation (ATBD) could be a potential alternative route for immobilizing GO on solid surfaces because the electrochemical reduction of ATBD produces positively charged surfaces and benzene ring-rich surfaces owing to the partial polymerization reaction during the grafting of ATBD on the electrode. Previously, GO was reported to interact strongly with ssDNA through π−π stacking interactions between the ring structures in the nucleobases of single-stranded DNA and the basal plane of GO.40 Also, such π−π interactions between GO or RGO and aromatic organic molecules have been confirmed by UV− visible spectroscopy study.41,42 Thus the use of ATBD could facilitate the deposition of GO sheets on various surfaces through both electrostatic and π−π stacking interactions. However, this type of approach for GO deposition on the surfaces has not been reported yet. Functional molecules containing aromatic moieties such as pyrene or aniline have been noncovalently attached to RGO surfaces through π−π stacking interactions.41,43−45 One particular advantage of this approach is that the π-electronrich conjugated structure of graphene, which is responsible for its electrical conductivity, is retained after modification; this could be beneficial for electrochemical applications of the modified surface. Recently, we have shown a simple approach for the immobilization of biomolecules on cyclic olefin copolymer (COC) substrates using an amphiphilic polymer, poly(BMA-r-PEGMA-r-NAS), which is referred to in this paper as poly(BPN). It consists of three parts: a hydrophobic residue with a benzene ring (BMA), which anchors the polymer to the surface; a poly(ethylene glycol) methacrylate (PEGMA) component that acts as a protein repellent; and an activated ester, N-acryloxysuccinimide (NAS) as a bioconjugation part.46 On the basis of this previous report, we assume that poly(BPN) may also coat the RGO surfaces in a similar manner through hydrophobic and π−π stacking interactions and that it can be used for the efficient immobilization of receptor biomolecules on RGO-modified electrode surfaces. Although several efforts have been made to increase the performance of biosensors by exploiting the advantages of RGO and ERGO, a systematic study to develop a general
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EXPERIMENTAL SECTION Reagents and Apparatus. Graphite powder (
