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Investigation of Controllable Nanoscale HeatDenatured Bovine Serum Albumin Films on Graphene Lin Zhou, Kun Wang, Zhenhua Wu, Haidao Dong, Hao Sun, Xuanhong Cheng, Honglian Zhang, Hongbo Zhou, Chunping Jia, Qinghui Jin, Hongju Mao, Jean-Luc Coll, and Jianlong Zhao Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03296 • Publication Date (Web): 15 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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Investigation of Controllable Nanoscale HeatDenatured Bovine Serum Albumin Films on Graphene Lin Zhou,†,‡Kun Wang,†,‡Zhenhua Wu,†,‡Haidao Dong,† Hao Sun,† Xuanhong Cheng, §Hong lian Zhang,† Hongbo Zhou,†Chunping Jia,†Qinghui Jin,† Hongju Mao,†,* Jean-Luc Coll,┴,* Jianlong Zhao†,* †

State Key Laboratory of Transducer Technology; Key Laboratory of Terahertz Solid-State

Technology ,Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China. ‡

University of Chinese Academy of Sciences, Beijing 100039, China.

§

Department of Materials Science and Engineering, Bethlehem, Lehigh University, PA 18015

USA. ┴

INSERM-UJF U823, Institut Albert Bonniot, 38706 Grenoble Cedex, France.

Keywords: Heat Denatured, Bovine Serum Albumin, Graphene, Nanoscale

ACS Paragon Plus Environment

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Figure 1. (a) Denatured BSA adsorbed on the graphene substrate; (b) Contact angle values of several SiO2 substrates before and after transferring graphene; (c) Contact angle image on the SiO2 substrate; (d) Contact angle image on the SiO2 substrate with graphene


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(a) Bare graphene on SiO2 substrate;(b) 1.5 mM native BSA in deionized water heatdenatured for 5 min on graphene and SiO2 substrate; (c) 1.5 mM native BSA in deionized water heat-denatured for 1 min on graphene substrate; (d) 1.5 mM native BSA in deionized water heat-denatured for 3 min on graphene substrate; (e) 1.5 mM native BSA in 10mM PBS heatdenatured for 3 min on graphene substrate; (f) 1.5 mM BSA in 0.1X PBS heat-denatured for 3min on graphene substrate; (g) 0.15 M BSA in deionized water heat-denatured for 3 min on graphene substrate;(h) 15 M BSA in deionized water heat-denatured for 3 min on graphene substrate; Figure 2.


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Figure 3. (a) AFM image of the denatured BSA films on graphene surface; (b) The thickness of BSA layer is approximately 15 nm when measured along the horizontal red line indicated in AFM image (a); (c) Raman spectra of graphene before and after modification of denatured BSA films in deionized water at 80°C for 3 min; (d) Raman spectra of graphene before and after modification of denatured BSA films in deionized water at 80°C for 1 min


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Figure 4. (a) The recorded nitrogen (N1s) spectra of XPS for bare graphene, denatured BSA films in 10 mM PBS, and deionized water; (b) The transfer characteristics of BSA-pH 3.5-doped GFET; (c) The transfer characteristics of BSA-pH 7.4-doped GFET; (d) The transfer characteristics of BSA-pH 9-doped GFET


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Figure 5. (a) The C(1s) peaks of XPS for denatured BSA films in deionized water; (b) The C(1s) peaks of XPS for denatured BSA films in 10 mM PBS; (c) The N(1s) peaks of XPS for denatured BSA films in deionized water; (d) The N(1s) peaks of XPS for denatured BSA films in 10 mM PBS


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Figure 6. (a) ATR-FTIR spectra of denatured BSA films in deionized water; (b) ATR-FTIR spectra of denatured BSA films in 10 mM PBS; (c) Fluorescent image of denatured BSA films with the modification of amino-QDs by EDC and NHS; (d) Control group of amino-QDs without EDC and NHS; (e) Fluorescent image of denatured BSA films by modifying with Cy3-NHS ester; (f) Control group of Cy3-NHS ester-modified graphene


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Investigation of Controllable Nanoscale HeatDenatured Bovine Serum Albumin Films on Graphene Lin Zhou,†,‡Kun Wang,†,‡Zhenhua Wu,†,‡Haidao Dong,† Hao Sun,† Xuanhong Cheng, §Hong lian Zhang,† Hongbo Zhou,†Chunping Jia,†Qinghui Jin,† Hongju Mao,†,* Jean-Luc Coll,┴,* Jianlong Zhao†,* †

State Key Laboratory of Transducer Technology; Key Laboratory of Terahertz Solid-State

Technology ,Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China. ‡

University of Chinese Academy of Sciences, Beijing 100039, China.

§

Department of Materials Science and Engineering, Bethlehem, Lehigh University, PA 18015

USA. ┴

INSERM-UJF U823, Institut Albert Bonniot, 38706 Grenoble Cedex, France.

Keywords: Heat Denatured, Bovine Serum Albumin, Graphene, Nanoscale


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Abstract: Two-dimensional graphene devices are widely used for biomolecule detection. Nevertheless, the surface modification of graphene is critical to achieve the high sensitivity and specificity required for biological detection. Herein, native bovine serum albumin (BSA) in inorganic solution is denatured on the graphene surface by heating, leading to the formation of nanoscale BSA protein films adsorbed on the graphene substrate via π-stacking interactions. This technique yields a controllable, scalable, uniform, and high coverage method for graphene biosensors. Further, the application of such nanoscale heat-denatured BSA films on graphene as a universal graphene biosensor platform is explored. The thickness of heat-denatured BSA films increased with heating time and BSA concentration, but decreased with solvent concentration as confirmed by AFM. The non-covalent interaction between denatured BSA films and graphene was investigated by Raman spectroscopy. BSA can act as a p-type and n-type dopant by modulating pH-dependent net charges on the layered BSA-graphene surface, as assessed by current-voltage measurements. Chemical groups of denatured BSA films, including amino and carboxyl groups, were verified by XPS, attenuated total reflectance-FTIR spectra, and fluorescent labelling. The tailoring of the BSA-graphene surfaces through chemical modification, controlled thickness, and doping type via non-covalent interactions provides a controllable, multifunctional biosensor platform for molecular diagnosis without the possibility of nonspecific adsorption on graphene.

Introduction Graphene, the one-atom-thick layer of sp2-hybridized honeycomb lattice carbon allotrope, has excellent electrical, optical, mechanical, and chemical properties1-3 that make it a promising


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carbon-based material for bioscience applications. Graphene devices have therefore been employed in the detection of proteins,4-5 DNA,6 neural imaging7 and bacteria.8 Nevertheless, for their efficient application as bio-devices, the controlled chemical and spatial assembly of biomolecules on the surface of graphene is a critical factor in their optimized design. Both covalent and non-covalent modification has been used to couple bio-probes on graphene.9 Generally, covalent modification imposes adverse effects on the electrical properties of graphene,9 while non-covalent modification offers the possibility to minimize these effects and, further, to induce desired properties when coupling bio-probes. Non-covalent modification of bare graphene mainly exploits π-stacking interactions and hydrophobic forces,9 and are likely to occur, in principle, directly on the graphene surface due to the availability of aromatic rings in these bio-probes. Some bifunctional non-covalent linkers of graphene, such as 1-pyrenebutanoic acid succinimidyl ester,10 N-hydroxysuccinimide (NHS) ester tripod,11 peptides,12 and bovine serum albumin (BSA),13 can attach on the graphene surface and conjugate bio-probes to detect target molecules. Nevertheless, the precise control of the adsorption coverage, carrier concentration, uniformity, and stability of these organic non-covalent linkers remains a challenge. Moreover, the numerous existing methods present complications and side effects that limit their application. For example, 1-pyrenebutanoic acid succinimidyl ester and NHS ester tripod are sensitive to light and humidity; peptides with certain amino acid sequences, which would adsorb on the graphene, couldn’t cover the graphene uniformly; 12the adsorbing capacity of native BSA on graphene is minimal.13 Therefore, a controllable, stable, uniform, and multifunctional noncovalent modification method is required for the construction of graphene biosensors. Considering the biocompatibility and availability of non-covalent interaction, unspecific adsorption of proteins on graphene is valuable to explore. The adsorption of proteins on solid


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surfaces and their subsequent interactions are a major concern in various fields, including medicine, biology, food processing, biotechnology, and nanotechnology. Proteins with a low internal stability, the so-called “soft” proteins such as BSA, tend to adsorb on all surfaces, especially hydrophobic surfaces.14 However, the amount of proteins adsorbed is minimal and hard to control at room temperature. The high temperature treatment of proteins increases the amount of protein adsorbed. Indeed, the adsorption capacity of heat-denatured BSA on hydrophobic polymer lattices and SiO2/Si surfaces,15-16 is greater than that of native BSA. This method could therefore also be suitable for hydrophobic graphene surfaces, as has been verified in the case of forced adsorption of BSA onto a hydrophobic graphite surface by molecular dynamics simulation.17 BSA in tris(hydroxymethyl)aminomethane (Tris) buffer solution denatured on reduced graphene oxide and graphene using chemical vapor deposition (CVD) has been investigated to detect anti-BSA and to dope graphene.18-19 However, the use of heatdenatured BSA to conjugate other bio-probes has not yet been investigated. Further, as an organic molecule, the effect of Tris in denatured BSA could be unclear. Therefore, while denatured BSA has been used to conjugate bio-probes, the interference of chemical groups from Tris could not be excluded. Thus, the adsorption of denatured BSA on a graphene surface in an inorganic solvent under various conditions is of interest. Herein, heat-denatured BSA films adsorbed on graphene are investigated with the aim to construct a universal graphene biosensor platform for various applications. The adsorption of BSA protein dissolved in an inorganic solvent onto a hydrophobic graphene surface via induction of π-stacking interactions through heat denaturation is investigated. Atomic force microscopy (AFM) is utilized to measure the thickness of the denatured BSA films on the graphene surface. The graphene surface was characterized by Raman spectra and X-ray


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photoelectron spectroscopy (XPS) prior to and following modification with denatured BSA. Further, effective charge modulation of the graphene surface by BSA doping at various pH conditions was assessed by current–voltage measurement of the carrier transport properties of BSA-doped graphene transistors. Finally, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra and fluorescent labelling of denatured BSA on graphene surface were used to verify the amino and carboxyl groups on the surface of denatured BSA. Experimental Section Materials. Monolayer graphene films were prepared by CVD (2D Carbon, China) on Cu films prepared on a SiO2/Si substrate (Crystal orientation: , Doping type: P type, Thickness:525 ± 20µm, Resistivity: 1~5×10-3Ω ∙ CM). BSA (Sangon Biotech, China, Purity:>96%) was dissolved in deionized water, 1 mM phosphate buffered solution (PBS) at pH 7, 1 mM phthalate buffer solution at pH 3.5, and 1 mM borate buffer solution at pH 9. Water-soluble sulfo-cyanine 3 N-hydroxysuccinimide (NHS) ester (Lumiprobe, USA) was reactive to amino group on the denatured BSA films.

The denatured BSA layer was activated by 1-ethyl-3-(3-

dimethylaminopropyl)-carbodiimide (EDC, Sigma-Aldrich, USA) and NHS (Sigma-Aldrich, USA) to conjugate with the amino-terminated quantum dots (QD, Jiayuan, China). Deionized water, obtained from Millipore-Q purification system (Millipore, USA), was used for the preparation of all solutions. Graphene Substrate. The CVD graphene-coated Cu foil was spin coated with a poly(methylmethacrylate) (PMMA) film and etched with an ammonium persulfate aqueous solution. Then graphene/PMMA film was transferred to the Si substrate with a 300-nm thick


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thermally grown SiO2 layer. The sample was then immersed in cold acetone to remove the PMMA. Protein Denaturation. Thermal denaturation of BSA and surface immobilization were concurrently conducted by incubating a native BSA solution on the graphene film at 80°C. The denaturation time, solvent concentration, and BSA concentration were modulated to produce denatured BSA films of varying thickness. Depending on the pH of the denaturation solution, BSA could lead to positive or negative charge modulation of the graphene surface. Characterization. The surface topology and thickness of the graphene and denatured BSAmodified graphene films were investigated by AFM (Bruker, USA) in tapping mode. The used cantilever of AFM was RTESP probe from Bruker (Length: 115~135um, Width: 30~40um, Spring constant: 20~80N/m). Raman spectra (Thermo Fisher, USA) were taken to explore the πstacking interactions between denatured BSA and the graphene surface. XPS (Thermo Fisher, USA) and ATR-FTIR (Bruker, USA) spectra and fluorescent modification were used to verify the chemical composition of the denatured BSA surface. The hydrophobicity of the SiO2 substrate with and without the graphene film was measured by a contact angle meter (Kruss, Germany). The carrier transport properties of BSA-doped graphene transistors were measured by a semiconductor parametric analyzer (Keithley 4200, Keithley Instruments Inc., USA).

Results and Discussion Native BSA dissolved in various inorganic solutions was incubated on the graphene substrate surface and heat-denatured at 80°C. The heat-denatured BSA films were adsorbed and aggregated on the graphene surface (Figure 1(a)). With the fabrication of graphene devices in mind, monolayer graphene films were grown by CVD and subsequently transferred onto a SiO2


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substrate. The single layer graphene surface containing only carbon atoms was extremely hydrophobic due to the abundance of hydrophobic amino acid residues on the exterior of BSA after denaturation. This hydrophobicity allowed for π-stacking interactions between the graphene and protein layers. The hydrophobicity of the SiO2 substrate under the monolayer graphene film may have also affected the amount of BSA adsorption. Contact angle measurements of several SiO2 substrates before and after graphene transfer were used to confirm the hydrophobicity of the substrate (Figure 1(b)), and showed that the graphene film increased the hydrophobicity of the substrate. The contact angle of the SiO2 substrate alone was approximately 56.6°(Figure 1(c)) and that of SiO2 with graphene was approximately 82.3°(Figure 1(d)). These results indicate that graphene substrates may be more conducive to immobilize denatured BSA films than SiO 2 substrates despite their similar hydrophobic properties.


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Figure 1. (a) Denatured BSA adsorbed on the graphene substrate; (b) Contact angle values of several SiO2 substrates before and after transferring graphene; (c) Contact angle image on the SiO2 substrate; (d) Contact angle image on the SiO2 substrate with graphene Following substrate preparation, native BSA in inorganic solutions was denatured on the substrate at 80°C. Nanoscale denatured BSA films were adsorbed on graphene via π-stacking interactions. A topographic view of the various denatured BSA films on graphene is shown in


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Figure 2 as observed under a light microscope. Figure 2(b)–(d) shows the films produced by 1.5 mM native BSA dissolved in deionized water and denatured for 5, 1, and 3 min on graphene films, respectively. The thickness of the heat-denatured BSA films increased with denaturation time. Conversely, the addition of PBS (10 or 1 mM) to the 1.5 mM BSA denatured on the graphene substrate at 80°C for 3 min (Figure 2(d)–(f)) prevented the formation of heat-denatured BSA films due to an increased ion concentration in the solvent. As expected, the decreased concentration of BSA in deionized water (1.5 mM, 15 M, and 0.15 M BSA; Figure 2(d),(h),(g), respectively) lead to thicker films.

(a) Bare graphene on SiO2 substrate;(b) 1.5 mM native BSA in deionized water heatdenatured for 5 min on graphene and SiO2 substrate; (c) 1.5 mM native BSA in deionized water heat-denatured for 1 min on graphene substrate; (d) 1.5 mM native BSA in deionized water heat-denatured for 3 min on graphene substrate; (e) 1.5 mM native BSA in 10mM PBS heatdenatured for 3 min on graphene substrate; (f) 1.5 mM BSA in 0.1X PBS heat-denatured for 3min on graphene substrate; (g) 0.15 M BSA in deionized water heat-denatured for 3 min on graphene substrate;(h) 15 M BSA in deionized water heat-denatured for 3 min on graphene substrate; Figure 2.

In order to investigate the thickness and surface topography of the denatured BSA films, photoresist was used as a mask to etch the denatured BSA film on the substrate by plasma. Figure 2(d) shows the heat-denatured BSA film formed on graphene with 1.5 mM native BSA in


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deionized water denatured at 80°C for 3 min. The average thickness of this film was approximately 15 nm (Figure 3(a), (b)). However, the thickness of bare graphene is less than 1nm, which was shown in Figure S1(a) and (b). On the other hand, when the denaturation time was 1 min under the same conditions, the thickness of the denatured BSA films was about 9 nm (Figure S1(c) and (e)). However, when the denaturation time was extended to 5 min, the thickness of the denatured BSA films quickly increased to several hundred nanometers with a root-mean-square roughness (RMS) of approximately 16 nm (Figure S2(a)), leading to the roughest surface obtained from the various denaturation conditions in this study. Thus, the longer the denaturation time, the thicker and rougher the denatured BSA films become. The thickness and roughness for other denatured BSA films (1.5 mM native BSA in 1 mM PBS denatured on graphene at 80°C for 3 min in Figures S(3), and 15M native BSA in deionized water denatured on graphene at 80°C for 3 min in Figures S(4)) were observed by AFM. These results of these denatured BSA films indicated a thickness of about 10 nm and a roughness of approximately 3 nm. Although the shape of the globular BSA protein in solution is approximately that of a prolate spheroid 4 nm × 4 nm × 14 nm in dimension,16 due to its unstable structure, conformational changes occur during the denaturation process. Therefore, the thickness of these denatured BSA films may approximate that of monolayer protein molecule on the graphene surface, guaranteeing a thickness in the same order of magnitude with that of biomolecules. From the above results, the thickness of the denatured BSA could be controlled by denaturation time, the solvent used, and the BSA concentration, which could be useful in the design of graphene biosensors.


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The modification of graphene by the denatured BSA films (1.5 mM native BSA in deionized water denatured at 80°C for 3 and 1 min) was investigated by Raman spectra (Figure 3(c),(d)). The intensity ratio between the 2D and G bands showed no obvious difference before and after modification, thus indicating that the substrate was single-layer graphene. The 2D band shifts shown in Figure 3(c),(d) indicate a slight doping of graphene, in accordance with the peptidemodified graphene by π-π interactions.8,

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These results verify a similar interaction between

proteins and peptides with graphene for surface modification.

Figure 3. (a) AFM image of the denatured BSA films on graphene surface; (b) The thickness of BSA layer is approximately 15 nm when measured along the horizontal red line indicated in AFM image (a); (c) Raman spectra of graphene before and after modification of denatured BSA films in deionized water at 80°C for 3 min; (d) Raman spectra of graphene before and after modification of denatured BSA films in deionized water at 80°C for 1 min


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Denatured BSA films that were not easily characterized under a light microscope (Figure 2(e)) were confirmed by XPS. The recorded nitrogen (N1s) spectra of XPS for bare graphene, denatured BSA films in Figure 2(e) (1.5 mM native BSA in 10 mM PBS denatured at 80°C for 3 min), and denatured BSA films in Figure 2(d) (1.5 mM native BSA in deionized water denatured at 80°C for 3 min) are shown in Figure 4(a). The N1s peak of the denatured BSA films in Figure 2(d) was obviously stronger than that in Figure 2(e). Nevertheless, the strong N1s peak of films in Figure 2(e) indicated that the native BSA had also adsorbed on the graphene surface during denaturation, despite this not being easily observed under the microscope. Except for the controllable thickness of the denatured BSA, the type of dopant that the denatured BSA became in graphene could be controlled by pH changes. All proteins, including BSA, exhibit either positive or negative net charges at pH values departing from their isoelectric points (pI). BSA is almost neutrally charged at pH 5.19, 21 Considering the pH value is decreased from 5 to 3, BSA would become positively charged via protonation of the carboxyl groups of amino acids. On the other hand, the rise of pH from 5 to 7 leads to a net negative charge because of the loss of protons from the imidazole groups of amino acids in BSA. Increasing the pH value of the solvent from 7 to 9, results in more negatively charged BSA due to further loss of protons from the ammonium groups of amino acids.19 Taken together, BSA could be used as a charge modulating dopant in a pH-dependent manner and the charge of the BSA layer could be controlled by changing the pH value of the solution. For this purpose, 1.5 mM native BSA dissolved in 1 mM PBS (pH 7.4), 1 mM phthalate buffer solution (pH 3.5), and 1 mM borate buffer solution (pH 9.0) were denatured on the graphene surface at 80°C for 3 min. An electrolyte-gated graphene field effect transistor (GFET) was constructed to investigate the doping effects of graphene by BSA at different pH values, while 1M PBS was chosen as the


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electrolyte. The Dirac points of the BSA-doped GFET shifted toward negative or positive voltages depending on the pH at the time of BSA deposition. BSA-pH 3.5-modified GFET exhibited p-doped transport behavior, which showed that the positive shift of the Dirac point was about 11 mV (Figure 4(b)). BSA-pH 7- and BSA-pH 9-modified GFET exhibited n-doped transport behaviors, which showed that the negative shifts of the Dirac points were 24 mV (Figure 4(c)) and 42 mV (Figure 4(d)), respectively. These transfer characteristics of BSA-doped GFET were all measured twice. Thus, the carrier concentration of graphene could be controlled by concentration- and pH-dependent net charge modulation of protein molecules.

Figure 4. (a) The recorded nitrogen (N1s) spectra of XPS for bare graphene, denatured BSA films in 10 mM PBS, and deionized water; (b) The transfer characteristics of BSA-pH 3.5-doped GFET; (c) The transfer characteristics of BSA-pH 7.4-doped GFET; (d) The transfer characteristics of BSA-pH 9-doped GFET


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Following the achievement of uniform nanoscale denatured BSA films adsorbed on the graphene surface, the surface groups of the denatured BSA films were investigated to explore their potential application as biosensors. XPS was carried out to explore the elemental composition and oxidation states of the species at the denatured BSA film surfaces before and after modification. Two different heat-denatured BSA films were formed on graphene with 1.5 mM native BSA dissolved in deionized water or 10 mM PBS and denatured at 80°C for 3 min. The carbon (1s) peaks (Figure 5(a), (b)) for the two films revealed that this peak could be attributed to three components. One was the bulk C peak attributed to the alkyl chain at 284.8 eV, which was also observed on the bare graphene surface (Figure S(5)). The second peak, at approximately 286.1 eV, was assigned to carbon bonded to sulfur,22 indicating that thiol groups may exist on the denatured BSA film surface. These results verified heat-denatured BSA films were adsorbed on the solid surface and aggregated by S-S bond formation and S-S exchange reaction with other molecules, while other molecules aggregated in the bulk solution in a manner competitive to surface aggregation.14 The third peak, observed at approximately 288.1 eV, indicated amide bonds or carboxyl groups on the denatured BSA film surface.23 Similarly, the nitrogen 1s peak of these two denatured BSA films both had a binding energy of 400.0 eV. Denatured BSA films formed in deionized water and in 10 mM PBS exhibited a small full-width-half-maximum value of 1.37 eV (Figure 5(c)) and 1.4 eV (Figure 5(d)), respectively. The main nitrogen peak at 400.0 eV corresponded to the unprotonated amino group (-NH2). Therefore, native BSA denatured on the graphene surface led to the presence of amino, carboxyl, and thiol groups on the heat-denatured BSA film surface, indicating the potential application of denatured BSA-modified graphene for biomolecule conjugation.


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Figure 5. (a) The C(1s) peaks of XPS for denatured BSA films in deionized water; (b) The C(1s) peaks of XPS for denatured BSA films in 10 mM PBS; (c) The N(1s) peaks of XPS for denatured BSA films in deionized water; (d) The N(1s) peaks of XPS for denatured BSA films in 10 mM PBS ATR-FTIR spectra of denatured BSA films were also measured to confirm the groups for conjugation. A strong amide II band was observed at 1540–1550 cm–1 and a weaker shoulder at 1510–1525 cm–1 (Figure 6(a),(b)). Peptides and proteins with an antiparallel β-sheet structure have strong amide II bands between 1510 and 1530 cm–1, whereas a parallel β-sheet structure is found at somewhat higher frequencies (1530–1550 cm–1).24 Proteins known to have an α-helical conformation have strong amide I bands between 1650 and 1655 cm–1. Thus, the peak at 1652 cm−1 indicates a substantial α-helical fraction in the denatured BSA films. The apparent shoulders around 1630 cm−1 and 1685 cm−1 indicated, however, a larger fraction of β-sheet


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configurations, than those for native BSA, possibly related to some aggregation in solution and subsequently in the film. These results reveal that the denaturation process could change the conformation of BSA and increase the β-sheet fraction of BSA to allow easier adsorption on the graphene surface. Beside XPS and ATR-FTIR spectra, the immobilization of fluorescent groups was also performed to verify the presence of amino and carboxyl groups on denatured BSA films. The amino-terminated QDs reacted with the carboxyl groups on denatured BSA films, which were activated by EDC and NHS. The fluorescent image of QD-modified denatured BSA films is shown in Figure 6(c), while the control group of the denatured BSA film just incubated with QDs without EDC and NHS is shown in Figure 6(d). These results indicate that the surface of denatured BSA films is rich in carboxyl groups. Similarly, amino groups on the denatured BSA films were verified in Figure 6(e), and sulfo-cyanine3 NHS ester was chosen as the amino reactive cross-linking agent. When this cross-linking agent was incubated with pure graphene, no obvious fluorescence was observed (Figure 6(f)). Nevertheless, when incubated with 1.5 mM native BSA in deionized water denatured at 80°C for 3 min in the presence of binding sulfocyanine3 NHS ester, fluorescence was observed (Figure 6(e)). Further, when the denaturation time was increased to 5 min, the fluorescence intensity was even stronger. The same concentration BSA in 10 mM PBS was denatured on graphene to form thinner films, and aminoterminated QDs and sulfo-cyanine3 NHS ester were also immobilized on this film (Figure S6(c) and Figure S6(e)), respectively. Due to the resonant energy transfer from fluorescent groups to single-layer graphene,25 the fluorescence intensity of thinner denatured BSA films in Figure S6(c),(e) was much weaker than that of denatured BSA films in Figure 6(c),(e). The results of


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fluorescent characterization proved that the surface of the denatured BSA films is rich in amino and carboxyl groups.

Figure 6. (a) ATR-FTIR spectra of denatured BSA films in deionized water; (b) ATR-FTIR spectra of denatured BSA films in 10 mM PBS; (c) Fluorescent image of denatured BSA films with the modification of amino-QDs by EDC and NHS; (d) Control group of amino-QDs without EDC and NHS; (e) Fluorescent image of denatured BSA films by modifying with Cy3-NHS ester; (f) Control group of Cy3-NHS ester-modified graphene


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Conclusions Native BSA in inorganic solutions denatured on graphene surfaces provided a simple, scalable, uniform, and high-coverage method to modify graphene surfaces. The heat-denatured BSA films adsorb onto the hydrophobic graphene surface via π-stacking interactions and are therefore possible biosensors. The thickness of the nanoscale denatured BSA films increases with increasing denaturation time and concentration of native BSA, but has an inverse relationship with solvent ion concentration, as confirmed by AFM. The concentration and pH-dependent net charge of BSA at various pH values denatured on the graphene surface could control the doping carrier concentration of graphene. The denatured BSA films were seen to be rich in amino and carboxyl groups, and therefore the possibility to bind biomolecules of a comparable size to denatured BSA films offers a controllable and multi-functional platform for graphene biosensor applications. ASSOCIATED CONTENT Supporting Information. The AFM images and surface topography of bare graphene, denatured BSA films, X-Ray photoelectron spectroscopy (XPS) of bare graphene, and fluorescent characterization of denatured BSA Films were included in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Corresponding author. Tel: +86-21-62511070-8705; Fax: +86-21-62511070-8714; E-mail: [email protected].


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*Corresponding author. Tel: +33 4 76 54 95 53; Fax: +33 4 76 54 94 13; E-mail: [email protected] *Corresponding author. Tel: +86-21-62511070-8702; Fax: +86-21-62511070-8714; E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by Grants from the Science and Technology Commission of Shanghai Municipality (16410711800) and the National Science Foundation of China (No.61571429, 61571077, 61571428, 61271162 and 61401442). ACKNOWLEDGMENT The authors are grateful to the support of grants from the Science and Technology Commission of Shanghai Municipality (16410711800) and the National Science Foundation of China (No.61571429, 61571077, 61571428, 61271162 and 61401442). We thank the suggestions from Professor Xuanhong Cheng of Lehigh University. ABBREVIATIONS BSA, bovine serum albumin; Tris, tris(hydroxymethyl)aminomethane; CVD, chemical vapor deposition; AFM, Atomic force microscopy; ATR-FTIR, Attenuated total reflectance-Fourier transform infrared ;PBS, phosphate buffered solution; NHS, N-hydroxysuccinimide; EDC,1-


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ethyl-3-(3-dimethylaminopropyl)-carbodiimide; RMS, root-mean-square roughness; PMMA, poly(methylmethacrylate) ; GFET, graphene field effect transistor; XPS, X-ray photoelectron spectroscopy. REFERENCES 1. Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A., Electric field effect in atomically thin carbon films. Science 2004, 306 (5696), 666-669. 2. Nair, R. R. B., P.;Grigorenko, A. N.; Novoselov,K. S.; Booth,T. J.;Stauber, T. ; Peres,N. M. R.;A. K. Geim, Fine Structure Constant Defines Visual Transparency of Graphene. science 2008, 320, 1308. 3. Lee, C. G. W., X.D.; Kysar,J. W. ;Hone,J. , Measurement of the Elastic Properties and Intrinsic Strength of Monolayer Graphene. science 2008, 321, 385-388. 4. Wang, Y.; Li, Y.; Tang, L.; Lu, J.; Li, J., Application of graphene-modified electrode for selective detection of dopamine. Electrochemistry Communications 2009, 11 (4), 889-892. 5. Ohno, Y. M., K.; Yamashiro,Y. ; Matsumoto,K. , Electrolyte-Gated Graphene FieldEffect Transistors for Detecting pH and Protein Adsorption. Nano Lett. 2009, 9 (9), 3318-3322. 6. Dong, X.; Shi, Y.; Huang, W.; Chen, P.; Li, L. J., Electrical detection of DNA hybridization with single-base specificity using transistors based on CVD-grown graphene sheets. Adv Mater 2010, 22 (14), 1649-1653. 7. Kuzum, D.; Takano, H.; Shim, E.; Reed, J. C.; Juul, H.; Richardson, A. G.; de Vries, J.; Bink, H.; Dichter, M. A.; Lucas, T. H.; Coulter, D. A.; Cubukcu, E.; Litt, B., Transparent and flexible low noise graphene electrodes for simultaneous electrophysiology and neuroimaging. Nat Commun 2014, 5, 5259. 8. Mannoor, M. S.; Tao, H.; Clayton, J. D.; Sengupta, A.; Kaplan, D. L.; Naik, R. R.; Verma, N.; Omenetto, F. G.; McAlpine, M. C., Graphene-based wireless bacteria detection on tooth enamel. Nat Commun 2012, 3, 763. 9. Georgakilas, V.; Otyepka, M.; Bourlinos, A. B.; Chandra, V.; Kim, N.; Kemp, K. C.; Hobza, P.; Zboril, R.; Kim, K. S., Functionalization of graphene: covalent and non-covalent approaches, derivatives and applications. Chem Rev 2012, 112 (11), 6156-6214. 10. Ohno, Y. M., K.;Matsumoto,K., Label-Free Biosensors Based on Aptamer-Modified Graphene Field-Effect Transistors. J. AM. CHEM. SOC. 2010, 132, 18012–18013. 11. Mann, J. A.; Alava, T.; Craighead, H. G.; Dichtel, W. R., Preservation of antibody selectivity on graphene by conjugation to a tripod monolayer. Angew Chem Int Ed Engl 2013, 52 (11), 3177-80. 12. Cui, Y.; Kim, S. N.; Jones, S. E.; Wissler, L. L.; Naik, R. R.; McAlpine, M. C., Chemical functionalization of graphene enabled by phage displayed peptides. Nano Lett 2010, 10 (11), 4559-65. 13. Xu, G.; Abbott, J.; Qin, L.; Yeung, K. Y.; Song, Y.; Yoon, H.; Kong, J.; Ham, D., Electrophoretic and field-effect graphene for all-electrical DNA array technology. Nat Commun 2014, 5, 4866. 14. Nakanishi, K., Sakiyama,T. , Imamura,K., On the Adsorption of Proteins on Solid Surfaces, a Common but Very Complicated Phenomenon. J. Biosci.Bioeng 2001, 93, 233-244.


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35x15mm (300 x 300 DPI)


Investigation of Controllable Nanoscale Heat ... - ACS Publications

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