Enhanced Plasmonic Biosensors of Hybrid Gold Nanoparticle-Graphene Oxide-Based Label-Free Immunoassay
In this study, we propose a modified gold nanoparticle-graphene oxide sheet (AuNP-GO) nanocomposite to detect two different interactions between proteins and hybrid nanocomposites for use in biomedical applications. GO sheets have high bioaffinity, which facilitates the attachment of biomolecules to carboxyl groups and has led to its use in the development of sensing mechanisms. When GO sheets are decorated with AuNPs, they introduce localized surface plasmon resonance (LSPR) in the resonance energy transfer of spectral changes. Our results suggest a promising future for AuNP-GO-based label-free immunoassays to detect disease biomarkers and rapidly diagnose infectious diseases. The results showed the detection of antiBSA in 10 ng/ml of hCG non-specific interfering protein with dynamic responses ranging from 1.45 nM to 145 fM, and a LOD of 145 fM. Considering the wide range of potential applications of GO sheets as a host material for a variety of nanoparticles, the approach developed here may be beneficial for the future integration of nanoparticles with GO nanosheets for blood sensing. The excellent anti-interference characteristics allow for the use of the biosensor in clinical analysis and point-of-care testing (POCT) diagnostics of rapid immunoassay products, and it may also be a potential tool for the measurement of biomarkers in human serum.
KeywordsGold nanoparticle (AuNP) Graphene oxide sheet (GO) Localized surface plasmon resonance (LSPR) Immunoassays
Bovine serum albumin antibody
Bovine serum albumin
Field-emission gun transmission electron microscope
Fourier-transform infrared spectrometer
Graphene oxide sheet
High-resolution transmission electron microscope
Limit of detection
Localized surface plasmon resonance
Reduction graphene oxide
Surface-enhanced Raman scattering
X-ray photoelectron spectroscopy
Carbon molecule-based materials such as carbon nanotubes [1, 2], carbon balls (buckminsterfullerene, C60) , two-dimensional graphene [4, 5, 6], and graphene oxide (GO) [7, 8, 9, 10, 11] have been widely used in biosensors. Among them, the two-dimensional sheet structure of graphene is an ideal material to allow for thin films with high conductivity [12, 13] and excellent optical permeability  characteristics and high biocompatibility [15, 16]. For these reasons, graphene-based material is widely use in biomedical and electrochemical sensing technology [17, 18]. In addition, the photoelectric type of biological sensing technology is mainly based on GO [19, 20, 21]. Because oxide groups can be adjusted to absorb and radiate a light band gap [22, 23], it is commonly used in fluorescence , surface plasmon resonance (SPR) [8, 9, 10, 11, 19, 20, 21], and localized surface plasmon resonance (LSPR) [25, 26] sensing technology. In particular, GO has unique chemical functional groups (epoxy bridges, hydroxyl groups, pairwise carboxyl groups (carboxyl and carbonyl)) which improve the affinity and covalent bonding of biomolecules.
The synthesis of graphene material combined with nanoparticles (such as Pt, Au, Ag, Pd, and ZnO) has been widely studied for the development of new nanocomposite technology. In particular, the use of gold nanoparticles (AuNPs) has been used as a mechanism for the energy transfer of colorimetric and absorption spectroscopy. In addition, during the last decade, research on AuNPs in visible light has highlighted the unique plasmonic resonance characteristics. Because adjusting the size and shape of AuNPs can change the optical absorption wavelength shift, AuNPs can be used for enhanced plasma absorption and signal amplification [27, 28]. Therefore, AuNPs have been extensively used in a wide range of applications such as optoelectronic components because of their special optical and optoelectronic properties to enhance light extraction [29, 30] and light absorption reactions [31, 32, 33]. In addition, AuNPs are biocompatible, and they have been studied for their use in chemical sensing, biomedical imaging, cancer therapy [34, 35], drug carrier [32, 33], photo-thermal therapy [36, 37, 38], contrast agent , radiosensitizer , and biosensing [33, 41, 42, 43] applications.
The functionality of the AuNPs was modified by adding the cross linker to avoid oxidation and act as a carrier of phytochemicals or vectors; thus, this combination can increase biocompatibility and bioactivity [44, 45, 46]. Cross linkers such as cystamine (Cys) or 8-mercaptooctanoic acid (MOA) are activated by carboxylic acid-terminated thiol self-assembled monolayers (SAMs) on a modified Au surface. MOA binds to the Au surface through the thiol linker (-SH end) resulting in monolayers.
In addition, research on plasmon metal material has also been widely reported. For example, plasmonic metal core shell nanoparticles , nanostars , and fluorine-doped tin oxide nanoparticles  have been shown to enhance the energy band gap, which makes them desirable in solar cell and sensing applications.
Moreover, the use of AuNP-GO hybrids based on chemical synthesis [50, 51, 52] and electrostatic self-assembly  has been reported in sensor, energy, and catalytic applications. In recent years, an increasing amount of research has focused on the use of AuNP-GO hybrids in biosensors. These hybrids have been shown to be useful in the development of electrochemical [54, 55, 56, 57, 58, 59] and surface-enhanced Raman scattering (SERS) [56, 59] platform technology to improve the application in biological assays. However, there are currently no relevant reports on the use of naked eye or colorimetric rapid immunoassay biosensor technology. For example, electrochemical DNA biosensors based on AuNP-GO hybrids have been used to detect breast cancer biomarkers to allow for an early diagnosis. With this biosensor, the detection limit (LOD) of 0.16 nM was obtained with a sensitivity of 378 nA/nM for the ERBB2 biomarker . In addition, an AuNP dotted reduction graphene oxide (rGO-AuNP) nanocomposite-based electrochemical aptasensor has been used to selectively detect a concentration of 3,3′4,4′-polychlorinated biphenyls (PCB77) between 1 pg L− 1 and 10 μg L− 1, with a LOD of 0.1 pg L− 1 . Moreover, AuNP-GO hybrids have been used as an electrochemical-based biosensor to detect hydrogen peroxide (H2O2), with food dynamic responses ranging from 0.1 to 2.3 mM, and an LOD of 0.01 mM . Another good example is the utilization of AuNP-GO [56, 59] and AuNP-graphene  hybrids for SERS-based biosensors in diverse applications, as well as SERS-measured bioimaging.
In this study, we propose an alternative method of chemical synthesis and electrostatic self-assembly of AuNP-GO hybrids using layer-by-layer self-assembly. We also analyze the biological detection sensitivity of the modified combined AuNPs and GO sheets and their protein immune response. We designed two kinds of AuNP-GO-based protein label-free immunoassays and evaluated their response time and sensitivity in antigen-antibody interactions. The excellent sensing features of graphene-AuNP composites include ultra-high sensitivity and the affinity of biomolecule interactions that influence the detection of a diverse range of biomolecules with high specificity. These features imply that these composites have a promising role in future applications, and the potential to be the preferred route of disease detection in clinical diagnosis applications.
Graphite was purchased from Graphene Supermarket (Graphene Laboratories Inc., Reading, MA, USA). GO sheets were obtained from graphite flakes by using a modified Hummer’s method  followed by ultrasonic shattering for 5 h for a flake size of 0.1–1 μm and thickness of 1.1 nm. Cystamine dihydrochloride (Cys, 96%), hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O), ACS, 99.99% (metals basis), and Au 49.5% min were purchased from Alfa Aesar Co. (USA). Sodium citrate (HOC(COONa)(CH2COONa)2·2H2O) was purchased from J.T. Baker Chemical Co. (USA). Bovine serum albumin (BSA, SI-B4287, Sigma-Aldrich, USA), Anti-Bovine Albumin antibody produced in rabbit (antiBSA, SI-B1520, Sigma-Aldrich, USA), N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Sigma-Aldrich Inc. (USA). The immunoglobulins (Ig) of antiBSA antibody structure were produced by B lymphocytes and secreted into the plasma. The monomeric forms of Ig molecules were glycoproteins with a molecular weight of about 150 kDa. Each Ig monomer was capable of binding two antigen molecules. All reagents and solvents were used without further purification.
We used three different temperature conditions at 550, 400, and 100 °C with boiling times of 5, 5, and 120 minutes, respectively, to control reduction of the nanoparticles. These different temperatures reduced the nanoparticles to obtain the same absorption spectrum at 520 nm as clearly seen in Additional file 1: Figure S1.
Synthesis of AuNPs
The method used to obtain AuNPs was based on the use of sodium citrate as a reducing agent to reduce tetrachloroaurate taurine ions in water. A volume of 15 mL of HAuCl4·3H2O solution containing 1 mM of Au was refluxed, and 1.8 mL of 38.8 mM sodium citrate (Na3C6H5O7) solution was added to the boiling (550 °C, 1100 rpm) solution. The reduction of the gold ions by the citrate ions was complete after 5 min, and the solution was further boiled for 30 min (400 °C, 900 rpm) and then left to cool to room temperature [36, 61, 62]. This method yields spherical AuNPs with an average diameter of about 15 nm, and a reduced concentration of 0.8 mL of 38.8 mM sodium citrate can be used to produce AuNPs with an average diameter of about 60 nm [63, 64]. The chemical reaction is as follows: HAuCl4(aq) + C6H5O7Na3(aq) → Au(s) + CO2 + HCOOH.
Preparation of GO Based on an Antigen Target
Preparation of AuNPs and AuNP-GO Based on an Antibody Probe
We used AuNPs prepared using the GO sheet method to modify the AuNP-GO nanocomposite. The AuNPs were prepared using the sodium citrate reduction method, and the size of the 60-nm AuNPs was modified using Cys (5 mM). We then used EDC/NHS to activate the –COOH groups on the surface of the GO sheets. The Cys attached to the AuNPs included –NH2 groups covalently coupled with –COOH groups on the surface of the GO sheets. The AuNPs on the surface of the GO sheets were immobilizing using a Cys linker to promote covalent bonding reactions between the AuNPs and GO. Covalent coupling offers a stable and easy method for bonding surface functionalization on GO sheets. The GO sheets were thoroughly rinsed with deionized water to remove unbonded GO at the AuNP-linker surface. The AuNP-Cys formed a new composite of AuNP-GO, followed by immobilization with different dilution concentrations from 100 μg/ml to 1 pg/ml of antibody (antiBSA) probe protein to form AuNP-GO-antiBSA, as shown in Fig. 2b.
Characterization of AuNPs and GO Sheets
The dispersion and morphology of AuNP-GO sheets were characterized using a 300-kV field-emission gun transmission electron microscope (FEG-TEM; Tecnai G2F30S-Twin, Philips-FEI, Amsterdam, Netherlands) and a high-resolution transmission electron microscope (HR-TEM) on a FEI Tecnai G20 system (Hillsboro, OR, USA). The dispersion and morphology of the AuNP-GO and GO sheets were characterized using a JEOL JSM-7800F Prime Extreme-resolution Analytical Field Emission Scanning Electron Microscope (JEOL Inc., USA). The ultraviolet-visible (UV-vis) transmittance spectrum of a double beam spectrophotometer was observed using a UV-vis spectrophotometer (U-2900, Hitachi High-Technologies Corporation, Tokyo, Japan) with a wavelength from 200 to 1100 nm at room temperature. Raman measurements were performed using a microscopic Raman system (MRI, Protrustech Co., Ltd., Taiwan). An air-cooled spectrometer (AvaSpec-ULS2048L) with 1800 lines/mm grating and 50-μm slit was used as a detector. Fourier-transform infrared spectrometer (FTIR) measurements were made using a Bruker Vertex 80v spectrometer in attenuated total reflection (ATR) mode, and a DTGS detector (64 scans) with a resolution of 2 cm− 1 on a KBr pellet in a vacuum at a pressure of around 6 Pa. The Instrumentation Center at National Tsing Hua University provided support for this work. X-ray photoelectron spectroscopy (XPS) was performed using the facilities at the National Synchrotron Radiation Research Center, Hsinchu, Taiwan. The photoelectron spectroscopy experiments were performed using a 09A2 U5-spectroscopy beamline for XPS. Photons with fixed energies of 380 and 900 eV were used for C (1s) and Co (2p) throughout the core-level photoelectron spectroscopy experiments. The experiments were carried out in total-electron-yield mode at a 6-m high-energy spherical grating monochromator. The photons were incident to the normal surface, and photoelectrons were collected at an angle of 58° from the normal surface. The binding energies in all spectra referred to the Au 4f7/2 core level at 84.0 eV. After subtracting the linear background, the spectra were fitted with mixed Gaussian–Lorentzian functions based on a nonlinear least-square algorithm .
Results and Discussion
Structure and Morphology Analysis
Characterizations of GO by XPS, Raman, and FTIR Spectroscopy
Analysis of GO and AuNP with Protein Interaction Properties
Analysis of AuNP-antiBSA and AuNP-GO-antiBSA Based on Immunoassay Interactions
During the quantitation experiment, we added a fixed concentration of 10 ng/ml of human chorionic gonadotropin (hCG) protein to act as an interferer. The results showed that the fixed interferer hCG protein on the immunoassay calibration curves were fitted by f(x) = 0.843 + 0.113× (correlation coefficient, R2 = 0.89) for the AuNP-GO probe with antiBSA interactions (Fig. 7a), and f(x) = 0.722 + 0.051× (correlation coefficient, R2 = 0.73) for the GO and AuNP-GO based on the immunoassay (Fig. 7b).
Furthermore, our experimental results showed that the detection strategy allowed for surface regeneration with no loss in specificity (four regenerations) and that it could also be used to detect antiBSA protein with dynamic responses ranging from 1.45 nM, 145 pM, 14.5 pM, 1.45 pM, 145 fM, and 0 fM. The results demonstrated that with a decreased concentration of antiBSA (from 1.45 nM to 145 fM) and even without the presence of antiBSA (0 fM), the spectral absorption intensity did not change the minimum level of quantitation. The hCG protein interfered with the antibody recognition in the immunoassay to a limited extent, possibly due to non-specific adsorption. This implies a very low cross-reactivity of the hCG protein and non-specific interactions at a low adsorption. In the practical quantitative analysis with immunoassays, a LOD of 145 fM for antiBSA was achieved in both buffer and interference protein samples.
We successfully demonstrated a GO-bound AuNP biocompatible nanocomposite in a biosensing mechanism in a rapid and label-free immunoassay for biomolecule interactions. The results showed that the AuNP-GO nanocomposite was biocompatible and exhibited LSPR extinction to biomolecules, which could promote the absorption spectra characteristic peaks, accelerate the reaction of molecules, and enhance the stability of chemical covalent bonds during immobilization. For the detection of antiBSA protein, the limit of detection of the GO and AuNP-GO based on the immunoassay was as low as 145 fM. Among the AuNP-GO biosensors, GO immobilized in the AuNP-GO nanocomposite showed the highest bioaffinity, with good sensitivity, low detection limit, and fast response toward the protein immunoassay. The results of our experiments showed that a fixed concentration 10 ng/ml of hCG protein as an interferer did not affect the test response. Given the growing trend of applying biosensors in POCT, LSPR for AuNP-GO nanocomposite technology is a highly promising and versatile tool for use in immunoassays. Combining the properties of AuNPs and GO sheets to develop new nanocomposites for the synthesis of smart materials shows promise for the development of user-friendly diagnosis applications. In the future, AuNP-GO nanocomposites may be used in innovative immunoassays, rapid detection reagents, and miniaturization, which may in turn make LSPR technology an irreplaceable tool for routine clinical analysis and POCT diagnostics.
The authors would like to thank Miss Yi-Chun Li and the team at Protrustech Co., Ltd., Taiwan, for their assistance in Raman measurements (Micro Raman Identify, MRI). We thank Dr. Yao-Jane Hsu’s group for their help in analyzing XPS spectra (National Synchrotron Radiation Research Center, Beamline 09A2) and the Instrumentation Center at National Tsing Hua University (FTIR) Li-Kang Chu’s group provided support for this work.
The authors would like to thank the Ministry of Science and Technology of the Republic of China, Taiwan, for financially supporting this research under Contract No. MOST 103-2221-E-003-008, MOST 104-2314-B-195-015 and MOST 105-2221-E-003-027.
Availability of Data and Materials
All data are fully available without restriction.
All authors contributed to the analysis and discussion of the data and writing the manuscript. N-FC conceived and designed the experiments; C-CC performed the synthesis of AuNP experiments; N-FC, C-CC, and C-DY performed the experiment immunoassay and contributed in data analysis and discussion. C-DY, C-CC, and Y-SK, conducted the XPS, FTIR, TEM, SEM, and Raman of the samples and contributed in data analysis and discussion. W-RW performed the synthesis of the three different temperature (550, 400 and 100 °C) AuNPs samples. N-FC contributed reagents, materials, and analysis tools; N-FC wrote the paper. All authors read and approved the final manuscript.
Nan-Fu Chiu is an associate professor of Institute of Electro-Optical Science and Technology, National Taiwan Normal University. Chi-Chu Chen is a Master’s student of the Institute of Electro-Optical Science and Technology, National Taiwan Normal University. Cheng-Du Yang is a Master’s student of the Institute of Electro-Optical Science and Technology, National Taiwan Normal University. Yu-Sheng Kao is a Master’s student of the Institute of Electro-Optical Science and Technology, National Taiwan Normal University. Wei-Ren Wu is a Master’s student of the Institute of Electro-Optical Science and Technology, National Taiwan Normal University.
The authors declare that they have no competing interests.
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