pubs.acs.org/NanoLett
Chemical Functionalization of Graphene Enabled by Phage Displayed Peptides Yue Cui,†,§ Sang N. Kim,‡,§ Sharon E. Jones,‡ Laurie L. Wissler,‡ Rajesh R. Naik,*,‡ and Michael C. McAlpine*,† †
Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey 08544, United States, and ‡ Air Force Research Laboratory, Materials & Manufacturing Directorate, Wright-Patterson Air Force Base, Ohio 45433, United States ABSTRACT The development of a general approach for the nondestructive chemical and biological functionalization of graphene could expand opportunities for graphene in both fundamental studies and a variety of device platforms. Graphene is a delicate singlelayer, two-dimensional network of carbon atoms whose properties can be affected by covalent modification. One method for functionalizing materials without fundamentally changing their inherent structure is using biorecognition moieties. In particular, oligopeptides are molecules containing a broad chemical diversity that can be achieved within a relatively compact size. Phage display is a dominant method for identifying peptides that possess enhanced selectivity toward a particular target. Here, we demonstrate a powerful yet benign approach for chemical functionalization of graphene via comprehensively screened phage displayed peptides. Our results show that graphene can be selectively recognized even in nanometer-defined strips. Further, modification of graphene with bifunctional peptides reveals both the ability to impart selective recognition of gold nanoparticles and the development of an ultrasensitive graphene-based TNT sensor. We anticipate that these results could open exciting opportunities in the use of graphene in fundamental biochemical recognition studies, as well as applications ranging from sensors to energy storage devices. KEYWORDS Graphene nanostrips, phage displayed peptides, biomimetic sensors, hybrid materials
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materials.23-25 Phage display24 has emerged as a powerful method for identifying peptide motifs that possess enhanced binding affinity toward an array of substrates, including metals,26 semiconductors,27 polymers,28 and small molecules.29,30 In phage display, a library of approximately 1-2 billion (109) peptide variants is displayed as a fusion with the surface coat protein of bacteriophage, allowing for rapid, combinatorial screening of sequences displaying high affinities toward specific targets. Interestingly, phage display has been used to identify peptides which bind to small carbon-based molecules, including carbon nanotubes31,32 and C60,33 and we have recently demonstrated that phage display can identify patterned carbon-based molecular inks.30 We have also shown that peptides can be linked to silicon nanowire sensors to enable selective recognition of a variety of chemical analytes,34 as well as the self-assembly of bifunctional peptides onto carbon nanotubes to enable selective sensing of trinitrotoluene (TNT).32 Here, we extend these concepts to graphene, by showing for the first time the biochemical functionalization of graphene surfaces via peptides identified by rigorous screening of phage display peptide libraries. The peptides are shown to recognize graphene, even in defined graphene nanostrips. These advances further allow for the development of designer bifunctional peptides for the directed self-assembly of gold nanoparticles onto graphene transistors, and the generation of graphene chemical sensors which respond to parts-per-billion (ppb) levels of TNT.
raphene is a single-atom-thick, sp2 carbon-based material that has attracted significant recent interest due to its remarkable electrical,1,2 optical,3,4 mechanical,5 sensing,6-9 and thermal10 properties. Due to its single-atomic-layer structure, isolation of graphene has only recently been achieved, via epitaxial growth,11 chemical vapor deposition,12 chemical exfoliation,13 and mechanical exfoliation.1,7,8,14 Despite these advances in the isolation of graphene, the ability to generically tailor its chemical properties has been limited by its delicate structure. For example, covalent functionalization15,16 can trigger symmetry breakage of the graphene lattice, thereby altering its properties. Further, noncovalent chemical modification strategies17 may be limited in scope of applicability. A general method for chemical and biological modification of graphene with specific binding motifsswhile retaining the excellent properties of graphenesis thus highly desired. Oligopeptides are robust biorecognition molecules that display broad chemical diversity (acidity, hydrophobicity, etc.), which allows them to be chemically engineered to bind specific targets, including inorganic materials,18 biomaterials,19 nanotubes,20 and small molecules.21 Further, with their relatively small molecular weight and ability to be linked into multifunctional networks,22,23 peptides can form complex, self-assembled hybrid conjugates with a variety of * Corresponding authors,
[email protected] and
[email protected]. § These authors contributed equally to this work. Received for review: 07/21/2010 Published on Web: 10/13/2010
© 2010 American Chemical Society
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quences emerged for both types of graphene/graphite flakes. Interestingly, analysis of the resulting sequences found them to be enriched in glutamine, histidine, and methionine when compared to the observed library frequency (see Figure S1 in Supporting Information). Conversely, carbon nanotube binding peptides (CNTBPs) are enriched in aromatic residues such as tyrosine, phenylalanine, histidine, and tryptophan, when compared to the observed library frequency.22 Aromatic residues in the GBP and CNTBP sequences interact with nanotubes and graphene via π-π interactions, but the higher abundance of these residues in CNTBPs can be attributed to the nonplanar CNT surface.35 The CNTBPs also appear to have hydrophobic and hydrophilic block domains, whereas GBPs appear to alternate hydrophilic and hydrophobic amino acid residues (see Figure S2 in the Supporting Information). We attribute these differences to the planar structure of graphene/graphite flake surfaces relative to the tightly curved nanostructures of CNTs, suggesting that the GBPs must fold to maximize their interaction with planar graphene surfaces. Detailed binding and theoretical modeling experiments are currently underway to investigate these effects further and to better understand the contributions of aromatic versus aliphatic acids in interactions with nonplanar and planar carbon surfaces.36 The identification of peptides which bind to graphene/ graphite flakes provided a reduced set of peptides which could then be tested for specific interactions with substrateimmobilized graphene sheets. Here, mechanical exfoliation of graphite (Kish graphite, Covalent Materials, San Jose, CA) onto a SiO2/Si substrate was used to produce few-layer graphene sheets.1,7,8,14 As shown in Figure 2a, the sheets were colorimetrically identified by optical microscopy, and topologically characterized via atomic force microscopy (AFM), which indicated a total thickness of approximately 1 nm. Given the interlayer separation in graphite of 0.335 nm, this suggests that the thickness is e4 graphene layers. Raman spectroscopy further confirmed the few-layer thickness of the graphene (see Figure S3 in the Supporting Information).37 Next, GBPs were allowed to interact with the graphene layers to determine their relative affinities. Figure 2b shows resulting AFM scans of the graphene sheets after exposure to the phage. M13 phages are 6.5 nm wide and 900 nm long and thus appear as thin lines in the AFM images after binding to the graphene surfaces. As shown in Figure 2b, 7-mer (left image) and 12-mer (right image) phages consisting of the sequences EPLQLKM (GBP1) and TMGFTAPRFPHY (GBP2), respectively, display affinities to the graphene surfaces. Significantly, this is in contrast to the background SiO2/Si substrates, for which no phage binding was observed (see Figure S4 in the Supporting Information). Other phage expressing peptide sequences identified by graphene screening, including QQQLSTH (GBP3) and YHRMPQALSAME (GBP4), showed similar results (see Figure S5 in the Supporting Information).
FIGURE 1. Identification of phage displayed peptides binding to graphene/graphite flakes. (a) Schematic illustration of the generalized screening protocol for identifying phage displayed peptides which recognize graphene/graphite flakes. (b) Summary of consensus graphene binding peptides (GBPs) to various graphene/graphite flakes.
Graphene is an atomically thin material possessing an extremely large surface energy, which has limited the preparation of pure graphene in air or aqueous media without the use of surfactant molecules. As a result, the identification of graphene-specific peptides was narrowed from a collection of peptides which displayed high affinities toward various graphite flakes. A schematic of the phage display protocol for identifying sp2 carbon-specific peptides is shown in Figure 1a. We used three different forms of commercial graphene/graphite samples as targets for panning against peptide libraries: TIMREX SLP30 Primary Synthetic Potato graphite (TIMCAL, Westlake, OH, surface area ) 8.0 m2/g); N006 nano graphene platelets (Angstron Materials, Dayton, OH, surface area up to ∼2675 m2/g); and AFM standard highly ordered pyrolytic graphite (HOPG, SPI, West Chester, PA, surface area not available). Briefly, 7-mer or 12-mer peptide phage libraries were incubated with SLP30, N006, or HOPG graphene/graphite flakes in Trisbuffered saline containing 0.1-0.8% Tween-20 (TBST) for 1 h at room temperature. The graphite particles were then washed several times with TBST buffer. The phages were eluted from the particles by addition of glycine-HCl (pH 2.2) for 5 min, neutralized with Tris-HCl, pH 9.1, amplified, and subjected to additional pannings. Eluted phages were then amplified in E. coli, and the process repeated for up to five rounds of biopanning, under increasingly stringent conditions, to obtain phage clones expressing peptides having the highest binding affinities to the graphene/graphite samples. After the final round of panning, DNA sequence analysis of the isolated phage clones yielded heptameric or dodecameric graphene-binding peptides (GBPs). As shown in the table in Figure 1b, dominant sequences developed after multiple rounds of panning against SLP30 graphene/graphite flakes. In the case of N006 and HOPG graphene/graphite flakes, no heptameric dominant sequences were found; however, identical dodecameric se© 2010 American Chemical Society
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FIGURE 2. Immobilization of graphene and recognition characterization. (a) AFM image of a few-layer graphene sheet (scale bar 2 µm). The inset shows an optical image containing the characteristic purple color of graphene (scale bar 500 nm). (b) AFM images of phages displaying GBP1 7-mer (left) and GBP2 12-mer (right) binding to graphene (scale bars 1 µm). (c) AFM (left) and SEM (right) images of AuNPs on GBP1-A3 self-assembled onto graphene. (scale bars 400 nm).
Next, to further demonstrate the ability of these selected peptides (without the phage body) to assemble on graphene surfaces, bifunctional designer peptides were synthesized containing the graphene-binding motif along with a gold nanoparticle (AuNP) binding domain. The 12-mer goldbinding/mineralizing A3 peptide, AYSSGAPPMPPF,26 was linked to GBP1 to produce a bifunctional graphene-AuNP peptide, EPLQLKM-GGGG-AYSSGAPPMPPF (GBP1-A3) (Peptide 2.0 Inc., Chantilly, VA). Here, the tetraglycine linker (GGGG) is a spacer included to retain the independent accessibility of the flanking peptide domains for binding to different surfaces. By designing a bifunctional peptide, the decoration of graphene surfaces by GBP1 can be visualized © 2010 American Chemical Society
through the binding of Au nanoparticles via the A3 peptide domain. Peptides were dissolved in deionized water at a concentration of 0.5 mg/mL. Twenty microliters of the peptide solutions were dropped onto the graphene and incubated for 20 min in an enclosed chamber (RH ∼ 100%), followed by extensive washing with deionized water and drying. Figure 2c shows the result after exposure of the peptides first to a graphene sheet, followed by incubation with a concentrated solution of 5 nm diameter AuNPs. Significantly, AFM (left) and scanning electron microscopy (SEM, right) images show selective binding of the AuNPs to the bifunctional peptides immobilized on the graphene surface. By contrast, a comparison with bare (peptide-free) 4561
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FIGURE 3. Recognition of nanopatterned graphene. (a) Schematic illustration of the fabrication process for generating nanostrips of graphene. (1) Graphene is exfoliated onto a Si/SiO2 substrate and coated with a thin film of Ni and photoresist. (2) The photoresist is micropatterned, and (3) the Ni film is electrochemically undercut etched to form NiNWs. (4) An oxygen plasma treatment translates the NiNWs into nanographene, and (5) the nickel is finally removed. (b) AFM images of NiNW patterns (left) and graphene NW patterns (right) following incubation with the graphene-binding phage GBP1 (scale bars 2 µm).
graphene surfaces shows substantially lower concentrations of bound AuNPs (see Figure S6 in the Supporting Information). Similar results were obtained using 50 nm AuNPs (see Figure S7 in the Supporting Information). Nanopatterned graphene38,39 has recently garnered significant interest due to the ability of geometrically confined one-dimensional graphene strips to display interesting properties, such as enhanced electrical gating.2 We generated nanoscale patterns of graphene as a means of definitively demonstrating the relative selectivity of the identified peptide sequences. Our patterning approach, which we term photolithography and etching for nanoscale lithography (PENCiL), will be described in detail elsewhere.40 Briefly, nanographene strips were defined using low-resolution photolithography, followed by electrochemical etching. A schematic illustration of the process is shown in Figure 3a. First, graphene nanosheets were exfoliated onto oxidized Si as described above. Next, the wafer containing the graphene was plated with a thin film of Ni (100 nm), a redox-active metal. The Ni layer is finally coated with a layer of photoresist. The PENCiL process then proceeds stepwise as follows: First, resist micropatterns are generated by photolithography to reveal 2.5 µm center-to-center spaced, 1 µm wide windows. Second, these windows behaved as electrochemical etch masks for undercutting the Ni sandwich to generate Ni nanowires (NiNWs). The chip and a Pt counter electrode © 2010 American Chemical Society
were attached to electrical leads, connected to opposite poles of a potentiostat and placed in a bath of 85% phosphoric acid (H3PO4). Applying 5 V on the potentiostat for 3 min, followed by removal of the photoresist with acetone, defined an undercut Ni metal layer which was shaped into arrays of metal nanowires. The widths of the NiNWs varied with etching conditions (see Figure S8 in the Supporting Information), with achievable diameters of sub-50 nm.40 Finally, the Ni wires served as protective nanomasks for translation via plasma oxidation (100 mTorr, 150 W, 2 min) into the underlying graphene, with a final Ni-removal step (HNO3, 60 °C, 30 min) to reveal the defined graphene nanostrips (GNSs). Figure 3b shows AFM images of NiNW (left) and GNS (right) patterns following incubation with the graphenebinding phages displaying GBP1. Before removal of the Ni, no phage was observed on the NiNW patterns in the AFM image, demonstrating that the identified GBP1 phage shows no binding affinity toward NiNWs. In contrast, revealed GNSs (diameter ∼300 nm) incubated with GBP1 phages show clear binding of the phage along the length of the GNSs. Importantly, no phage particles are seen between the graphene NWs, showing that the selective binding to the graphene nanostrips is significantly enhanced relative to both the NiNWs, as well as the background Si/SiO2 substrate. This result clearly shows the recognition capabilities of the 4562
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phy was employed for the fabrication of source/drain addressing electrodes to the GFET, followed by evaporation of Ti/Au and lift-off. A four-probe station equipped with a Keithley semiconductor parameter analyzer was used to characterize the GFET device performance. First, we investigated the performance of a GFET with self-assembled AuNPs via the GBP1-A3 bifunctional peptide. In the case of GFETs, the electrodes serve as source and drain, while the heavily doped Si substrate serves as gate. Figure 4b shows the device output characteristics (gate voltage Vgs versus source-drain current Ids) of the bare graphene, of graphene after immobilization of GBP1-A3 bifunctional peptides, and of hybrid graphene-GBP1-A3 after incubation with AuNPs on the same graphene device. The results show that the bare graphene sheets behave as p-type FETs, which has been seen previously.38,41 After immobilization of GBP1-A3 peptide on the GFET, the current increased slightly. More significantly, subsequent assembly of AuNPs showed a much larger current increase. We attribute this result to the field effect induced by negatively charged AuNPs,42 which are insulated from the GFETs by the assembled peptides, such that the overall device behaves as a leakage coupled capacitor. As a result, binding of negatively charged AuNPs on p-type GFETs increases the conductance. This ability to nondestructively and selectively direct the assembly of nanoparticles on graphene may provide opportunities for applications including magnetic and catalytic graphene devices. Second, we investigated how the assembly of recognition peptides affects the performance of graphene chemical sensors. Electronic noses based on nanoscale materials such as semiconducting nanowires,43-46 nanotubes,32,47 and graphene6-8 have been shown to boast parts per billion sensitivities, a consequence of the high surface areas of these materials. In some cases, these nanosensors have shown selectivity to certain molecules, via the use of chemoselective polymer coatings,48,49 surface chemistry modifications,44 and functionalization with proteins,43,45 peptides,32,34 or nucleic acid aptamers.50 For example, we have recently shown the rational design and test of a CNT sensor based on noncovalent attachment of a bifunctional peptide. The sensor was selective toward a specific target molecule, trinitrotoluene (TNT),32 wherein the TNT tetrapeptide binding domain (WFVI) was derived from the binding pocket of the honeybee odor binding protein ASP1. TNT is a wellknown chemical explosive, and thus the detection of TNT is critical for security-related applications. Here, the TNT binding domain was linked to a representative graphene binding peptide to form EPLQLKM-GGGGWFVI (GBP1-ASP1), in order to determine the effect of the immobilized peptide on the activity of a GFET sensor toward TNT. A representative GFET device was chosen and annealed in 400 °C H2/Ar mixture for 1 h to yield an ultraclean graphene surface in the GFET. TNT binding onto the graphene surface can take place via direct (nonspecific) physisorption
FIGURE 4. Characterization of a hybrid graphene transistor (GFET) and sensor modified with bifunctional peptides. (a) Schematic of the GFET device design, containing a bifunctional graphene-AuNP binding/mineralizing peptide. (b) Device output characteristics (gate voltage Vgs vs source-drain current Ids) of the bare graphene (black curve), of graphene after immobilization of GBP1-A3 bifunctional peptides (red curve), and of the hybrid graphene after incubation with AuNPs (blue curve). The inset shows the graphene device, in which a graphene strip bridges two electrodes (scale bar 5 µm). (c) Electrical responses of bare (black), GBP1-functionalized (blue), and GBP1-ASP1 functionalized (red) graphene sensors to 12 ppb of TNT, injected at time 30 s.
phage displayed peptides, even toward nanoconfined graphene strips. In order to investigate the altered functionality of “hybrid graphene”sthat is, graphene chemically modified with peptidessrelative to native graphene, peptides were selfassembled onto graphene transistors and sensors. As shown in Figure 4a, graphene field-effect transistors (GFETs)2 and nanosensors were fabricated on heavily doped Si wafers containing an insulating SiO2 layer. Electron beam lithogra© 2010 American Chemical Society
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Materials (No. DMR-0819860). S.N.K. acknowledges support via a National Research Council Research Associateship Award. R.R.N. acknowledges support of this work by the Air Force Office of Scientific Research and the Materials and Manufacturing Directorate (AFRL/RX). M.C.M. acknowledges support of this work by the Air Force Office of Scientific Research via a Young Investigator Grant (No. FA9550-09-10096) and as a fellow of the American Asthma Foundation (No. 09-0038). The authors thank Dr. Slocik at AFRL for the preparation and reduction of gold nanoparticles.
or via selective adsorption at peptide binding sites. To separate these two processes, we included in our sensing experiments a bare graphene sensor and a sensor modified with only GBP1 as controls. The source-drain current was monitored at a fixed source-drain voltage (0.5 V) while the same device was exposed to 12 ppb of TNT vapor before and after functionalization with peptide. Figure 4c shows a normalized Ids plot of the result (the devices experienced a constant baseline drift which was eliminated from the plots). The bare GFET shows negligible response (
