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Reduced graphene oxide-based optical sensor for detecting specific protein
Accepted Manuscript Title: Reduced graphene oxide-based optical sensor for detecting specific protein Author: Wen-Shuai Jiang Wei Xin Shuang Xun Shao-Nan Chen Xiao-Guang Gao Zhi-Bo Liu Jian-Guo Tian PII: DOI: Reference:
S0925-4005(17)30579-8 http://dx.doi.org/doi:10.1016/j.snb.2017.03.175 SNB 22157
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
3-11-2016 19-3-2017 29-3-2017
Please cite this article as: W.-S. Jiang, W. Xin, S. Xun, S.-N. Chen, X.G. Gao, Z.-B. Liu, J.-G. Tian, Reduced graphene oxide-based optical sensor for detecting specific protein, Sensors and Actuators B: Chemical (2017), http://dx.doi.org/10.1016/j.snb.2017.03.175 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Reduced graphene oxide-based optical sensor for detecting specific protein Wen-Shuai Jianga, Wei Xina, Shuang Xuna, Shao-Nan Chena, Xiao-Guang Gaoa, Zhi-Bo Liua,b,*, Jian-Guo Tiana,b a
The Key Laboratory of Weak Light Nonlinear Photonics, Ministry of Education, Teda Applied Physics Institute and
The 2011 Project Collaborative Innovation Center for Biological Therapy, Nankai University, Tianjin 300071,
China E-mail addresses: [email protected] (Z. Liu)
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* Correspondences.
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School of Physics, Nankai University, Tianjin 300071, China
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Abstract A sensitive and selective optical biosensor based on reduced graphene oxide (RGO), which uses the polarization-dependent absorption effect of graphene under total internal reflection, is reported for
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determination of rabbit IgG. RGO sheets with the thickness of about 8.1 nm are fabricated by high temperature reduction and used as a sensing film, because of its strong polarization dependent
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absorption. This RGO-based optical sensor shows a satisfactory response to rabbit IgG with a
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minimum concentration of 0.0625 μg/ml. As a contrast, commercial SPR apparatus is used to investigate rabbit IgG with a minimum concentration of 0.3125 μg/ml. Moreover, with
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antigen-antibody binding, this sensor can also achieve label-free and real-time detection. Taking into
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account these factors, the RGO-based optical sensor may be a potential candidate for biosensing.
Keywords: reduced graphene oxide, polarization dependent absorption, optical biosensor, total
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internal reflection, antigen-antibody.
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Highlights A sensitive and selective optical biosensor based on reduced graphene oxide.
This optical sensor shows a high sensitivity for determining the binding of specific protein.
The chip cost of this RGO-based sensor is low.
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1. Introduction Surface plasmon resonance (SPR) biosensors are optical sensors which exploit surface plasmon polariton to probe interactions between an analyte in solution and a biomolecular recognition
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element immobilized on the SPR sensor surface [1,2]. Relying on the measurement of
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binding-induced refractive index change of the solvent near the surface, SPR biosensors can achieve a real-time, label-free and high sensitive detection for target analyte. At present, SPR is a powerful
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analytical technique for characterizing and quantifying biomolecular interactions, and SPR-based
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sensors have been successfully commercialized and are often used to detect analytes related to medical diagnostics [3,4], environmental monitoring [5], and food safety and security [6,7].
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Although SPR sensors, which are modulated by angle, phase and spectrum, own very good
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performance (high sensitive and resolution) with the aid of precision and expensive manipulative equipment [8-11], the expensive devices mean the expensive detection cost in practical application.
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The amplitude modulated SPR sensor can also use to detect the biomolecule, but the performance is typically worse than SPR sensor mentioned above [2, 10-12]. Due to the unique structure, chemical and physics properties, graphene can be easily
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functionalized and modified for the targeted immobilization of bioreceptor units [13-16], and graphene and its derivatives have been applied into some fields related to biological detection [17-20]. Besides, the SPR biosensors combined with graphene have been reported for enhancing the sensitivity [21-25]. Recently, graphene-based refractive index optical sensors with high sensitivity and resolution, which uses the polarization-dependent absorption of graphene under total internal reflection [26, 27], have been reported on for distinguishing cancer cells from normal cells [28], detecting the NO2 gas [29] and investigating the dynamical gas parameters [30]. The detection of 4
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biomolecules has important significance and application in medical diagnosis, environmental monitoring and food safety and security [2-7]. Biomolecular interaction (for example, the binding, dissociation and elution of antigen-antibody) often induce the change of refractive index. However,
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the detection of biomolecules using sensor of polarization-dependent absorption of graphene has not been studied until now. Because RGO can be easily modified and functionalized [28, 31, 32], it is
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possible for detecting biomolecule through the polarization-dependent absorption of graphene under
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total internal reflection.
In this paper, combining the graphene-based optical sensor and easy functionalization of RGO, we
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developed a RGO-based optical biosensor for determining the binding of specific protein. It is an
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important development for this sensor to realize the detection of biomolecules. Under total internal reflection, graphene exhibits a greater absorption for transverse electric (TE) wave than transverse
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magnetic (TM) wave, which is very sensitive to the change of refractive index of the media in contact with the graphene. The change of refractive index induced by the interaction of
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antigen-antibody results in the change of polarization-dependent absorption, which can be measured and recorded by a balanced photo-detector. By adjusting the experimental condition, the RGO-based
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optical biosensor shows a satisfactory response to rabbit IgG with a minimum concentration of 0.0625 μg/ml, comparing with the result of commercial SPR apparatus (angle modulation) with a minimum concentration of 0.3125 μg/ml. This sensor also has advantages of label-free and real-time detection. However, the surface treatment of RGO is more easily and simply than the gold film used by SPR sensor for enhancing surface functionality. Moreover, the RGO-based sensor can be easily integrated and it can be fabricated at low cost, in consideration of the low cost and mass production of GO. 5
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2. Materials and methods 2.1 Materials Goat anti-Rabbit immunoglobulin G (IgG), Rabbit immunoglobulin G (IgG) and Bovine
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immunoglobulin M (IgM) were purchased from Beijing Biosynthesis Biotechnology Co., Ltd.. And bovine serum albumin (BSA), glycine were purchased from Tianjin Unite Stars Biotech Co., Ltd..
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Phosphate Buffer solution (PBS) was purchased from Beijing Zhongshan Golden Bridge
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Biotechnology Co., Ltd.. All solutions were prepared with distilled water. PBS (0.01mol/L, pH=7.4) was diluted by distilled water. Goat anti-Rabbit IgG, Rabbit IgG and Bovine IgM were diluted by
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PBS. 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC, 0.4mol/L) were
purchased from aladdin (Shanghai, China).
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purchased from aladdin (Shanghai, China), and N-Hydroxysuccinimide (NHS, 0.1mol/L) were
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2.2 Fabrication and characterizations of RGO
GO was prepared using a modified Hummers method [33], then 4 mg/ml aqueous solutions of GO
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was fabricated through ultrasonic (2h) and centrifugation (3500 rpm, 1 h). After this, the pre-cleaned quartz substrates were etched by oxygen plasma (1min), and then GO were spin-coated onto the
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quartz substrates. Finally, the coated samples were thermally annealed at 800°C for 1h under the atmosphere of argon (95%) and hydrogen (5%) gases (volume ratio). The thickness of RGO were investigated using atomic force microscope (AFM, Nanoscope Dimension™ 3100). The transmittance was measured by using an ultraviolet-visible-infrared spectrometer (Hitachi 1 U-4100 spectrophotometer). 2.3 The preparation of RGO-based sensor
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Poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow-corning) was used to fabricate the flow cell, due to the good biocompatibility and chemical stability. The pre-polymer of PDMS (10:1, mass ratio) covered the pre-fabrication template, and was placed a 75℃ oven for 2h. Subsequently, the flow cell
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was peeled off from the template. Oxygen plasma was used to clean the redundant RGO through the method of masking. Finally, the binding between flow cell and RGO/quartz was achieved by oxygen
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plasma (20s). In this process, oxygen plasma etching can enhance the oxygen-containing functional
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groups on the top surface of RGO and enhance surface functionality for better biomolecular
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immobilization and sensing performance [31, 32].
2.4 The pretreatment and modification of RGO sensing surface
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After PBS was injected into flow cell, the mixed solution of EDC/NHS was injected into the surface of RGO through flow cell for activating the oxygen-containing functional groups. After 30
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min, PBS was injected into the surface of RGO. Subsequently, the solution of antibody (goat
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anti-rabbit IgG, 250 μg/ml) was injected into flow cell, and antibody was immobilized onto the surface of RGO by covalent and noncovalent attachment. Then, PBS was used to wash the flow cell. After that, BSA (10mg/ml) was injected into flow cell for blocking the nonspecific binding. Finally,
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PBS was injected as the baseline solution. 2.5 The detection of rabbit IgG
After the pretreatment and modification of RGO sensing surface, PBS was injected as the baseline solution. Subsequently, rabbit IgG solution was injected into flow cell, and the binding between rabbit IgG and goat anti-rabbit IgG induced the change of polarization-dependent absorption of graphene. After the reaction reached equilibrium, PBS was injected again. After that, glycine was
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used to elute the rabbit IgG. Finally, PBS was used to wash flow cell and the signal returned the baseline. 3. Results and Discussion
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3.1 Detection methodology The measurements were performed using our homemade equipment. It is working with prism
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configuration to achieve polarization-dependent absorption under total internal reflection, which is
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shown in Fig. 1. Then flow cell/RGO/quartz was put on the base of a K9 prism through suitable
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refractive index matching oil. Light from a He-Ne laser (λ=632.8nm) was firstly converted into linear polarized light using a polarizer, and then was focused onto the RGO by a lens (about 1 mm
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spot size). The reflected light was separated into TE polarized light and TM polarized light using a polarization beam splitter. Because graphene has a greater absorption for TE wave than TM wave,
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the power difference of the separated light could be recorded and compared using a balanced
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photodetector. The interaction between antigen and antibody on the surface of RGO will result in the change of the polarization-dependent absorption of graphene, and a real-time and label-free detection can be achieved. More details can be obtained in our previously reported literature [26, 28]. The inset
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of Fig. 1 gives the schematic of the binding of antigen-antibody on the surface of RGO. 3.2 Characterization of RGO According to the results of ref [28], the sensor owns the better sensitivity when the thickness is 8 nm nearby. Through adjusting the speed of spin-coating and the concentration of GO solution, the thickness of RGO (about 8.1nm) was fabricated, and the result of AFM was shown in Fig. 2(a), which confirmed the thickness of RGO. Moreover, we measured the polarization dependent absorption of RGO with the thickness of about 8.1nm under total internal reflection, and the result 8
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was shown in Fig. 2(b), which showed that RGO fabricated by us exhibited the good polarization dependent absorption. The transmittance was measured using ultraviolet-visible-infrared spectrometer, as is shown in
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Fig. 2(c). The transmittance of RGO fabricated by high temperature reduction is much lower than that of GO. The inset of Fig. 2(c) shows the absorption is about 45% at 550 nm, which is slightly
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lower than that of exfoliated graphene.
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A RGO-based sensor was fabricated to detect different concentration of NaCl solution. The change of refractive index between H2O and 0.011% NaCl solution can be easily distinguished, as
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3.3 The dynamic process of biomolecular interaction
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shown in Fig. 2(d), which indicates this sensor is very sensitive for change in refractive index.
The sensing film (RGO film) was functionalized by goat anti-rabbit IgG (antibody), and the
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process of goat anti-rabbit IgG (antibody) immobilized on the surface of RGO was monitored by our
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setup. After PBS was injected into the flow cell, the antibody solution (250 μg/ml) was slowly injected by syringe pump. With the injection of antibody, the real-time change of absorption was recorded, as shown in Fig. 3(a). The result indicted antibody was immobilized upon the sensing film.
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Subsequently, PBS was injected again to confirm the functionalization. Fig. 3(b) shows a real-time measuring result of the biosensor after fabrication and then biochemical treatment. The binding, dissociation and elution of specific protein (rabbit IgG), which is important for investigating biomolecular interaction, were shown. First, PBS was injected into the flow cell as a baseline solution. And then with introduction of antigen (25 μg/ml rabbit IgG), binding from antigen-antibody brought about larger absorption of TE wave, and the signal began to increase. Subsequently, PBS was injected again to demonstrate the binding reaction occurred between antigen 9
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and antibody. After this, plenty of antigen molecules which were bound on the surface of RGO were eluted from the sensor surface with the injection of glycine, and the real-time signal began to decrease sharply. Because the glycine solution has a lower refractive index than PBS, the signal
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decreased to under the baseline. Finally, the PBS was injected again, and the signal returned to the baseline, which demonstrated the anti-IgG molecules thoroughly dissociated from the biosensor
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surface in elution process, and the sensor could be reused. The whole process of biomolecular
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interaction between antigen and antibody can be observed clearly, as shown in Fig. 3(b), which is similar to the dynamic process of biomolecular interaction of SPR-based sensor. Hence, it may be a
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candidate except SPR sensor for detecting the biomolecular.
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3.4 The sensitivity and specificity of RGO-based sensor
The sensitivity is an important parameter for a biosensor. For investigating the sensitivity of
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the RGO-based biosensor, different concentration of rabbit IgG solution were separately introduced
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into flow cell at room temperature. The sensitivity of sensor (ΔV) as a function of the concentration of rabbit IgG (μg/ml) was shown in Fig. 4(a), and the minimum concentration of 0.0625 μg/ml rabbit IgG solution can be distinguished with a voltage change of about 0.17 V, which can be observed
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clearly as shown in Fig. 6(d). The sensor has a good linearity within the range of 0.625 μg/ml ~ 25 μg/ml, which was shown in the inset of Fig. 4(a). The calibration curve is y = 0.091x + 0.525 within linear range, and the correlation coefficient is 0.991. Hence, the sensitivity (S) of sensor (slope of the calibration curve) is 0.091 V·ml·μg-1. The repeatability is very important. Further experiments were achieved for confirming the result of the minimum detection concentration. A real-time, continuous measurement result was recorded, which was shown in Fig. 4(c). The results of 0.0625 μg/ml and 0.625 μg/ml were shown in Fig. 4(c), 10
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the change between 0.0625 μg/ml and 0.625 μg/ml can be easily distinguished. The inset of Fig. 4(c) further showed the good repeatability for results of 0.0625 μg/ml. Different sensor chips were used to detect rabbit IgG solution, the result of the minimum concentration was shown in Fig. 5, which
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indicates the results can be repeated well. Using the same principle, methods and devices, the results can be reproduced by different samples at different times. The standard deviation is used as
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reproducibility, which is 7.98%.
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In order to obtain the detection limit and quantification limit of this sensor, we measured 21 samples with blank PBS. The standard deviation of the blank signal (σ) was 0.0012 V, and the
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sensitivity (S) is 0.091 V·ml·μg -1. Hence, the detection limit (DL, 3σ/S) and quantification limit (QL,
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10σ/S) are 0.039 μg/ml and 0.132 μg/ml, respectively.
For a biosensor, specificity is another important parameter. The specificity is demonstrated by
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detecting bovine IgM (100 μg/ml) under the same condition. After the goat anti-rabbit IgG were binding on the surface of RGO-based biosensor, bovine IgG (100 μg/ml) was introduced into the
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flow cell until the signal stabilized. The result was summarized and shown in Fig. 4(b). Compared these results, the voltage change from mismatched bovine IgM is significantly smaller than that from
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rabbit IgG which indicated the selective binding of rabbit IgG. Therefore, the RGO decorated with antibody exhibits specific detection of antigen. And through immobilizing different antibody on the surface of RGO, relevant matched antigen can be detected by this biosensor. 3.5 The comparison with commercial SPR apparatus For further demonstrating the performance of RGO-based biosensor, traditional commercial SPR apparatus (Biacore X100, GE) was used to detect different concentration of antigen solution. The results of three different concentrations of rabbit IgG solution (0.3125, 1.25, 5 μg/ml) were measured 11
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and investigated, in which the minimum detectable concentration is 0.3125 μg/ml, and the results were shown in Fig. 6(a)-(c). The results of RGO-based sensor were shown in Fig. 6(d)-(f), which included the minimum detectable concentration (0.0625 μg/ml) and another two same concentration
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used in commercial SPR apparatus. Compared with these results, the RGO-based sensor provided lower concentration detection.
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4. Conclusion
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In summary, based on the polarization-dependent absorption of graphene, we demonstrate a RGO-based optical sensor for determination of rabbit IgG. Through adjusting the incident angle
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(near to critical angle) and the thickness of RGO (about 8 nm), the sensor shows a satisfactory
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response to rabbit IgG with a minimum concentration of 0.0625 μg/ml, which is more sensitive than commercial SPR apparatus (the minimum concentration of 0.3125 μg/ml). Using the sensor, we can
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achieve a real-time, label-free, high sensitive and selective detection for specific protein. According to the result, we deduce that different specific antibody or antigen should can be detected using the
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sensor through decorating RGO with selective antigen or antibody conjugates. Moreover, the sensor can be fabricated at low cost, considering the use of GO. Further improvement in surface
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modification of graphene or biological structure with nanomaterials, should lead to even higher sensitivity. Taking into consideration of these factors, it should be a good potential candidate for biosensing.
Acknowledgement The authors thank the Natural Science Foundation of China (Grant 11374164), and the National Key 12
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Research and Development Program of China (Grant 2016YFA0200200, 2016YFA0301102)
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Biographies Wenshuai Jiang is a Ph.D. student focusing on graphene and graphene microstructures and their
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optical properties and applications. He received his B.Sc. degree in 2011 from Luoyang Normal University and M.Sc. degrees in optics in School of Physics in 2014 from Nankai University.
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Zhibo Liu received his B.Sc. degree in 2001 and Ph.D. in 2006 in optics from Nankai University. Dr.
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Liu is a Professor in Nankai University. His current research interests are optical properties of graphene and its applications, integration of graphene devices and two dimensional thin films.
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Wei Xin is a Ph.D. student focusing on two dimensional materials (graphene, black phosphorus). He
degrees in optics from Nankai University.
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was awarded a B.Sc. degree from Changchun University of Science and Technology and M.Sc.
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Shuang Xun is a postgraduate student focusing on graphene devices. He was awarded a B.Sc. from Nankai University.
Nankai University.
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Shaonan Chen is a Ph.D. student focusing on graphene biosensor. He was awarded a B.Sc. from
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Xiaoguang Gao is a Ph.D. student focusing on graphene optical sensor. He was awarded a B.Sc. from Shanxi Normal University. Jian-guo Tian received his B.Sc. degree in 1986 and Ph.D. in 1991 in optics from Nankai University. Dr.Tian is a Professor in Nankai University. His current research interests are nanophotonics, metasurfaces, and biophotonics.
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Figures
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Fig. 1. The schematic of RGO-based optical sensor.
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Fig. 2. Characterizations of RGO films. (a) AFM image confirms the thickness of RGO. (b) The polarization dependent absorption of RGO used in our experiment. (c) The transmittance of GO and RGO, the inset of Fig. 2c shows the transmittance in 550 nm. (d) The detection of different concentration of NaCl solution
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Fig. 3. (a) Response curves obtained by immobilization of goat anti-rabbit IgG on the surface of RGO. (b) Response curves obtained from the interaction between antigen and antibody.
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Fig. 4. (a) The sensitivity of RGO-based biosensor, the inset of Fig. 4(a) shows the linear range of the detection of rabbit IgG. (b) The specific detection of RGO-based biosensor. (c) A real-time, repeated measurement of the minimum detection concentration.
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Fig. 5. The repeatability of RGO-based sensor for 0.0625 μg/ml rabbit IgG solution.
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Fig. 6. (a)-(c) The results of commercial SPR biosensing apparatus (Biacore X100, GE), in which the minimum detectable concentration is 0.3125 μg/ml. (d)-(f) The results of RGO-based sensor, in which the minimum detectable concentration is 0.0625 μg/ml.
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S0925-4005(17)30579-8 http://dx.doi.org/doi:10.1016/j.snb.2017.03.175 SNB 22157
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
3-11-2016 19-3-2017 29-3-2017
Please cite this article as: W.-S. Jiang, W. Xin, S. Xun, S.-N. Chen, X.G. Gao, Z.-B. Liu, J.-G. Tian, Reduced graphene oxide-based optical sensor for detecting specific protein, Sensors and Actuators B: Chemical (2017), http://dx.doi.org/10.1016/j.snb.2017.03.175 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Reduced graphene oxide-based optical sensor for detecting specific protein Wen-Shuai Jianga, Wei Xina, Shuang Xuna, Shao-Nan Chena, Xiao-Guang Gaoa, Zhi-Bo Liua,b,*, Jian-Guo Tiana,b a
The Key Laboratory of Weak Light Nonlinear Photonics, Ministry of Education, Teda Applied Physics Institute and
The 2011 Project Collaborative Innovation Center for Biological Therapy, Nankai University, Tianjin 300071,
China E-mail addresses: [email protected] (Z. Liu)
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* Correspondences.
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School of Physics, Nankai University, Tianjin 300071, China
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Abstract A sensitive and selective optical biosensor based on reduced graphene oxide (RGO), which uses the polarization-dependent absorption effect of graphene under total internal reflection, is reported for
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determination of rabbit IgG. RGO sheets with the thickness of about 8.1 nm are fabricated by high temperature reduction and used as a sensing film, because of its strong polarization dependent
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absorption. This RGO-based optical sensor shows a satisfactory response to rabbit IgG with a
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minimum concentration of 0.0625 μg/ml. As a contrast, commercial SPR apparatus is used to investigate rabbit IgG with a minimum concentration of 0.3125 μg/ml. Moreover, with
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antigen-antibody binding, this sensor can also achieve label-free and real-time detection. Taking into
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account these factors, the RGO-based optical sensor may be a potential candidate for biosensing.
Keywords: reduced graphene oxide, polarization dependent absorption, optical biosensor, total
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internal reflection, antigen-antibody.
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Highlights A sensitive and selective optical biosensor based on reduced graphene oxide.
This optical sensor shows a high sensitivity for determining the binding of specific protein.
The chip cost of this RGO-based sensor is low.
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1. Introduction Surface plasmon resonance (SPR) biosensors are optical sensors which exploit surface plasmon polariton to probe interactions between an analyte in solution and a biomolecular recognition
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element immobilized on the SPR sensor surface [1,2]. Relying on the measurement of
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binding-induced refractive index change of the solvent near the surface, SPR biosensors can achieve a real-time, label-free and high sensitive detection for target analyte. At present, SPR is a powerful
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analytical technique for characterizing and quantifying biomolecular interactions, and SPR-based
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sensors have been successfully commercialized and are often used to detect analytes related to medical diagnostics [3,4], environmental monitoring [5], and food safety and security [6,7].
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Although SPR sensors, which are modulated by angle, phase and spectrum, own very good
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performance (high sensitive and resolution) with the aid of precision and expensive manipulative equipment [8-11], the expensive devices mean the expensive detection cost in practical application.
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The amplitude modulated SPR sensor can also use to detect the biomolecule, but the performance is typically worse than SPR sensor mentioned above [2, 10-12]. Due to the unique structure, chemical and physics properties, graphene can be easily
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functionalized and modified for the targeted immobilization of bioreceptor units [13-16], and graphene and its derivatives have been applied into some fields related to biological detection [17-20]. Besides, the SPR biosensors combined with graphene have been reported for enhancing the sensitivity [21-25]. Recently, graphene-based refractive index optical sensors with high sensitivity and resolution, which uses the polarization-dependent absorption of graphene under total internal reflection [26, 27], have been reported on for distinguishing cancer cells from normal cells [28], detecting the NO2 gas [29] and investigating the dynamical gas parameters [30]. The detection of 4
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biomolecules has important significance and application in medical diagnosis, environmental monitoring and food safety and security [2-7]. Biomolecular interaction (for example, the binding, dissociation and elution of antigen-antibody) often induce the change of refractive index. However,
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the detection of biomolecules using sensor of polarization-dependent absorption of graphene has not been studied until now. Because RGO can be easily modified and functionalized [28, 31, 32], it is
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possible for detecting biomolecule through the polarization-dependent absorption of graphene under
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total internal reflection.
In this paper, combining the graphene-based optical sensor and easy functionalization of RGO, we
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developed a RGO-based optical biosensor for determining the binding of specific protein. It is an
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important development for this sensor to realize the detection of biomolecules. Under total internal reflection, graphene exhibits a greater absorption for transverse electric (TE) wave than transverse
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magnetic (TM) wave, which is very sensitive to the change of refractive index of the media in contact with the graphene. The change of refractive index induced by the interaction of
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antigen-antibody results in the change of polarization-dependent absorption, which can be measured and recorded by a balanced photo-detector. By adjusting the experimental condition, the RGO-based
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optical biosensor shows a satisfactory response to rabbit IgG with a minimum concentration of 0.0625 μg/ml, comparing with the result of commercial SPR apparatus (angle modulation) with a minimum concentration of 0.3125 μg/ml. This sensor also has advantages of label-free and real-time detection. However, the surface treatment of RGO is more easily and simply than the gold film used by SPR sensor for enhancing surface functionality. Moreover, the RGO-based sensor can be easily integrated and it can be fabricated at low cost, in consideration of the low cost and mass production of GO. 5
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2. Materials and methods 2.1 Materials Goat anti-Rabbit immunoglobulin G (IgG), Rabbit immunoglobulin G (IgG) and Bovine
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immunoglobulin M (IgM) were purchased from Beijing Biosynthesis Biotechnology Co., Ltd.. And bovine serum albumin (BSA), glycine were purchased from Tianjin Unite Stars Biotech Co., Ltd..
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Phosphate Buffer solution (PBS) was purchased from Beijing Zhongshan Golden Bridge
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Biotechnology Co., Ltd.. All solutions were prepared with distilled water. PBS (0.01mol/L, pH=7.4) was diluted by distilled water. Goat anti-Rabbit IgG, Rabbit IgG and Bovine IgM were diluted by
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PBS. 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC, 0.4mol/L) were
purchased from aladdin (Shanghai, China).
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purchased from aladdin (Shanghai, China), and N-Hydroxysuccinimide (NHS, 0.1mol/L) were
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2.2 Fabrication and characterizations of RGO
GO was prepared using a modified Hummers method [33], then 4 mg/ml aqueous solutions of GO
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was fabricated through ultrasonic (2h) and centrifugation (3500 rpm, 1 h). After this, the pre-cleaned quartz substrates were etched by oxygen plasma (1min), and then GO were spin-coated onto the
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quartz substrates. Finally, the coated samples were thermally annealed at 800°C for 1h under the atmosphere of argon (95%) and hydrogen (5%) gases (volume ratio). The thickness of RGO were investigated using atomic force microscope (AFM, Nanoscope Dimension™ 3100). The transmittance was measured by using an ultraviolet-visible-infrared spectrometer (Hitachi 1 U-4100 spectrophotometer). 2.3 The preparation of RGO-based sensor
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Poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow-corning) was used to fabricate the flow cell, due to the good biocompatibility and chemical stability. The pre-polymer of PDMS (10:1, mass ratio) covered the pre-fabrication template, and was placed a 75℃ oven for 2h. Subsequently, the flow cell
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was peeled off from the template. Oxygen plasma was used to clean the redundant RGO through the method of masking. Finally, the binding between flow cell and RGO/quartz was achieved by oxygen
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plasma (20s). In this process, oxygen plasma etching can enhance the oxygen-containing functional
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groups on the top surface of RGO and enhance surface functionality for better biomolecular
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immobilization and sensing performance [31, 32].
2.4 The pretreatment and modification of RGO sensing surface
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After PBS was injected into flow cell, the mixed solution of EDC/NHS was injected into the surface of RGO through flow cell for activating the oxygen-containing functional groups. After 30
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min, PBS was injected into the surface of RGO. Subsequently, the solution of antibody (goat
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anti-rabbit IgG, 250 μg/ml) was injected into flow cell, and antibody was immobilized onto the surface of RGO by covalent and noncovalent attachment. Then, PBS was used to wash the flow cell. After that, BSA (10mg/ml) was injected into flow cell for blocking the nonspecific binding. Finally,
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PBS was injected as the baseline solution. 2.5 The detection of rabbit IgG
After the pretreatment and modification of RGO sensing surface, PBS was injected as the baseline solution. Subsequently, rabbit IgG solution was injected into flow cell, and the binding between rabbit IgG and goat anti-rabbit IgG induced the change of polarization-dependent absorption of graphene. After the reaction reached equilibrium, PBS was injected again. After that, glycine was
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used to elute the rabbit IgG. Finally, PBS was used to wash flow cell and the signal returned the baseline. 3. Results and Discussion
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3.1 Detection methodology The measurements were performed using our homemade equipment. It is working with prism
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configuration to achieve polarization-dependent absorption under total internal reflection, which is
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shown in Fig. 1. Then flow cell/RGO/quartz was put on the base of a K9 prism through suitable
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refractive index matching oil. Light from a He-Ne laser (λ=632.8nm) was firstly converted into linear polarized light using a polarizer, and then was focused onto the RGO by a lens (about 1 mm
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spot size). The reflected light was separated into TE polarized light and TM polarized light using a polarization beam splitter. Because graphene has a greater absorption for TE wave than TM wave,
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the power difference of the separated light could be recorded and compared using a balanced
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photodetector. The interaction between antigen and antibody on the surface of RGO will result in the change of the polarization-dependent absorption of graphene, and a real-time and label-free detection can be achieved. More details can be obtained in our previously reported literature [26, 28]. The inset
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of Fig. 1 gives the schematic of the binding of antigen-antibody on the surface of RGO. 3.2 Characterization of RGO According to the results of ref [28], the sensor owns the better sensitivity when the thickness is 8 nm nearby. Through adjusting the speed of spin-coating and the concentration of GO solution, the thickness of RGO (about 8.1nm) was fabricated, and the result of AFM was shown in Fig. 2(a), which confirmed the thickness of RGO. Moreover, we measured the polarization dependent absorption of RGO with the thickness of about 8.1nm under total internal reflection, and the result 8
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was shown in Fig. 2(b), which showed that RGO fabricated by us exhibited the good polarization dependent absorption. The transmittance was measured using ultraviolet-visible-infrared spectrometer, as is shown in
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Fig. 2(c). The transmittance of RGO fabricated by high temperature reduction is much lower than that of GO. The inset of Fig. 2(c) shows the absorption is about 45% at 550 nm, which is slightly
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lower than that of exfoliated graphene.
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A RGO-based sensor was fabricated to detect different concentration of NaCl solution. The change of refractive index between H2O and 0.011% NaCl solution can be easily distinguished, as
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3.3 The dynamic process of biomolecular interaction
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shown in Fig. 2(d), which indicates this sensor is very sensitive for change in refractive index.
The sensing film (RGO film) was functionalized by goat anti-rabbit IgG (antibody), and the
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process of goat anti-rabbit IgG (antibody) immobilized on the surface of RGO was monitored by our
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setup. After PBS was injected into the flow cell, the antibody solution (250 μg/ml) was slowly injected by syringe pump. With the injection of antibody, the real-time change of absorption was recorded, as shown in Fig. 3(a). The result indicted antibody was immobilized upon the sensing film.
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Subsequently, PBS was injected again to confirm the functionalization. Fig. 3(b) shows a real-time measuring result of the biosensor after fabrication and then biochemical treatment. The binding, dissociation and elution of specific protein (rabbit IgG), which is important for investigating biomolecular interaction, were shown. First, PBS was injected into the flow cell as a baseline solution. And then with introduction of antigen (25 μg/ml rabbit IgG), binding from antigen-antibody brought about larger absorption of TE wave, and the signal began to increase. Subsequently, PBS was injected again to demonstrate the binding reaction occurred between antigen 9
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and antibody. After this, plenty of antigen molecules which were bound on the surface of RGO were eluted from the sensor surface with the injection of glycine, and the real-time signal began to decrease sharply. Because the glycine solution has a lower refractive index than PBS, the signal
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decreased to under the baseline. Finally, the PBS was injected again, and the signal returned to the baseline, which demonstrated the anti-IgG molecules thoroughly dissociated from the biosensor
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surface in elution process, and the sensor could be reused. The whole process of biomolecular
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interaction between antigen and antibody can be observed clearly, as shown in Fig. 3(b), which is similar to the dynamic process of biomolecular interaction of SPR-based sensor. Hence, it may be a
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candidate except SPR sensor for detecting the biomolecular.
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3.4 The sensitivity and specificity of RGO-based sensor
The sensitivity is an important parameter for a biosensor. For investigating the sensitivity of
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the RGO-based biosensor, different concentration of rabbit IgG solution were separately introduced
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into flow cell at room temperature. The sensitivity of sensor (ΔV) as a function of the concentration of rabbit IgG (μg/ml) was shown in Fig. 4(a), and the minimum concentration of 0.0625 μg/ml rabbit IgG solution can be distinguished with a voltage change of about 0.17 V, which can be observed
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clearly as shown in Fig. 6(d). The sensor has a good linearity within the range of 0.625 μg/ml ~ 25 μg/ml, which was shown in the inset of Fig. 4(a). The calibration curve is y = 0.091x + 0.525 within linear range, and the correlation coefficient is 0.991. Hence, the sensitivity (S) of sensor (slope of the calibration curve) is 0.091 V·ml·μg-1. The repeatability is very important. Further experiments were achieved for confirming the result of the minimum detection concentration. A real-time, continuous measurement result was recorded, which was shown in Fig. 4(c). The results of 0.0625 μg/ml and 0.625 μg/ml were shown in Fig. 4(c), 10
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the change between 0.0625 μg/ml and 0.625 μg/ml can be easily distinguished. The inset of Fig. 4(c) further showed the good repeatability for results of 0.0625 μg/ml. Different sensor chips were used to detect rabbit IgG solution, the result of the minimum concentration was shown in Fig. 5, which
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indicates the results can be repeated well. Using the same principle, methods and devices, the results can be reproduced by different samples at different times. The standard deviation is used as
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reproducibility, which is 7.98%.
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In order to obtain the detection limit and quantification limit of this sensor, we measured 21 samples with blank PBS. The standard deviation of the blank signal (σ) was 0.0012 V, and the
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sensitivity (S) is 0.091 V·ml·μg -1. Hence, the detection limit (DL, 3σ/S) and quantification limit (QL,
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10σ/S) are 0.039 μg/ml and 0.132 μg/ml, respectively.
For a biosensor, specificity is another important parameter. The specificity is demonstrated by
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detecting bovine IgM (100 μg/ml) under the same condition. After the goat anti-rabbit IgG were binding on the surface of RGO-based biosensor, bovine IgG (100 μg/ml) was introduced into the
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flow cell until the signal stabilized. The result was summarized and shown in Fig. 4(b). Compared these results, the voltage change from mismatched bovine IgM is significantly smaller than that from
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rabbit IgG which indicated the selective binding of rabbit IgG. Therefore, the RGO decorated with antibody exhibits specific detection of antigen. And through immobilizing different antibody on the surface of RGO, relevant matched antigen can be detected by this biosensor. 3.5 The comparison with commercial SPR apparatus For further demonstrating the performance of RGO-based biosensor, traditional commercial SPR apparatus (Biacore X100, GE) was used to detect different concentration of antigen solution. The results of three different concentrations of rabbit IgG solution (0.3125, 1.25, 5 μg/ml) were measured 11
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and investigated, in which the minimum detectable concentration is 0.3125 μg/ml, and the results were shown in Fig. 6(a)-(c). The results of RGO-based sensor were shown in Fig. 6(d)-(f), which included the minimum detectable concentration (0.0625 μg/ml) and another two same concentration
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used in commercial SPR apparatus. Compared with these results, the RGO-based sensor provided lower concentration detection.
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4. Conclusion
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In summary, based on the polarization-dependent absorption of graphene, we demonstrate a RGO-based optical sensor for determination of rabbit IgG. Through adjusting the incident angle
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(near to critical angle) and the thickness of RGO (about 8 nm), the sensor shows a satisfactory
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response to rabbit IgG with a minimum concentration of 0.0625 μg/ml, which is more sensitive than commercial SPR apparatus (the minimum concentration of 0.3125 μg/ml). Using the sensor, we can
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achieve a real-time, label-free, high sensitive and selective detection for specific protein. According to the result, we deduce that different specific antibody or antigen should can be detected using the
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sensor through decorating RGO with selective antigen or antibody conjugates. Moreover, the sensor can be fabricated at low cost, considering the use of GO. Further improvement in surface
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modification of graphene or biological structure with nanomaterials, should lead to even higher sensitivity. Taking into consideration of these factors, it should be a good potential candidate for biosensing.
Acknowledgement The authors thank the Natural Science Foundation of China (Grant 11374164), and the National Key 12
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Research and Development Program of China (Grant 2016YFA0200200, 2016YFA0301102)
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Biographies Wenshuai Jiang is a Ph.D. student focusing on graphene and graphene microstructures and their
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optical properties and applications. He received his B.Sc. degree in 2011 from Luoyang Normal University and M.Sc. degrees in optics in School of Physics in 2014 from Nankai University.
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Zhibo Liu received his B.Sc. degree in 2001 and Ph.D. in 2006 in optics from Nankai University. Dr.
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Liu is a Professor in Nankai University. His current research interests are optical properties of graphene and its applications, integration of graphene devices and two dimensional thin films.
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Wei Xin is a Ph.D. student focusing on two dimensional materials (graphene, black phosphorus). He
degrees in optics from Nankai University.
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was awarded a B.Sc. degree from Changchun University of Science and Technology and M.Sc.
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Shuang Xun is a postgraduate student focusing on graphene devices. He was awarded a B.Sc. from Nankai University.
Nankai University.
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Shaonan Chen is a Ph.D. student focusing on graphene biosensor. He was awarded a B.Sc. from
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Xiaoguang Gao is a Ph.D. student focusing on graphene optical sensor. He was awarded a B.Sc. from Shanxi Normal University. Jian-guo Tian received his B.Sc. degree in 1986 and Ph.D. in 1991 in optics from Nankai University. Dr.Tian is a Professor in Nankai University. His current research interests are nanophotonics, metasurfaces, and biophotonics.
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Figures
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Fig. 1. The schematic of RGO-based optical sensor.
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Fig. 2. Characterizations of RGO films. (a) AFM image confirms the thickness of RGO. (b) The polarization dependent absorption of RGO used in our experiment. (c) The transmittance of GO and RGO, the inset of Fig. 2c shows the transmittance in 550 nm. (d) The detection of different concentration of NaCl solution
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Fig. 3. (a) Response curves obtained by immobilization of goat anti-rabbit IgG on the surface of RGO. (b) Response curves obtained from the interaction between antigen and antibody.
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Fig. 4. (a) The sensitivity of RGO-based biosensor, the inset of Fig. 4(a) shows the linear range of the detection of rabbit IgG. (b) The specific detection of RGO-based biosensor. (c) A real-time, repeated measurement of the minimum detection concentration.
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Fig. 5. The repeatability of RGO-based sensor for 0.0625 μg/ml rabbit IgG solution.
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Fig. 6. (a)-(c) The results of commercial SPR biosensing apparatus (Biacore X100, GE), in which the minimum detectable concentration is 0.3125 μg/ml. (d)-(f) The results of RGO-based sensor, in which the minimum detectable concentration is 0.0625 μg/ml.
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