This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Article Cite This: ACS Omega 2019, 4, 3287−3297 http://pubs.acs.org/journal/acsodf Versatile Graphene-Based Platform for Robust Nanobiohybrid Interfaces Rebeca Bueno,† Marzia Marciello,†,‡ Miguel Moreno,§ Carlos Sań chez-Sań chez,† Jose ́ I. Martinez,† Lidia Martinez,† Elisabet Prats-Alfonso,∥,⊥ Anton Guimera-̀ Brunet,∥,⊥ Jose A. Garrido,# Rosa Villa,∥,⊥ Federico Mompean,† Mar García-Hernandez,† Yves Huttel,† María del Puerto Morales,† Carlos Briones,§ María F. Loṕ ez,† Gary J. Ellis,¶ Luis Vaź quez,† and Jose ́ A. Martín-Gago*,† † Materials Science Factory, Institute of Materials Science of Madrid (ICMM-CSIC), Sor Juana Inés de la Cruz 3, 28049 Madrid, Spain ‡ Nanobiotechnology for Life Sciences Group, Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy, Complutense University (UCM), Plaza Ramón y Cajal s/n, 28040 Madrid, Spain § Laboratory of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, 28850 Madrid, Spain ∥ Instituto de Microelectrónica de Barcelona IMB-CNM (CSIC) Esfera UAB, Bellaterra, 08193 Barcelona, Spain ⊥ Centro de Investigación Biomédica en Red en Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain # Catalan Institute of Nanoscience and Nanotechnology (ICN2) CSIC and The Barcelona Institute of Science and Technology Campus UAB, Bellaterra, 08193 Barcelona, Spain ¶ Polymer Physics Group, Institute of Polymer Science and Technology (ICTP-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain ABSTRACT: Technologically useful and robust graphene- based interfaces for devices require the introduction of highly selective, stable, and covalently bonded functionalities on the graphene surface, whilst essentially retaining the electronic properties of the pristine layer. This work demonstrates that highly controlled, ultrahigh vacuum covalent chemical functionalization of graphene sheets with a thiol-terminated molecule provides a robust and tunable platform for the development of hybrid nanostructures in different environ- ments. We employ this facile strategy to covalently couple two representative systems of broad interest: metal nanoparticles, via S−metal bonds, and thiol-modified DNA aptamers, via disulfide bridges. Both systems, which have been characterized by a multitechnique approach, remain firmly anchored to the graphene surface even after several washing cycles. Atomic force microscopy images demonstrate that the conjugated aptamer retains the functionality required to recognize a target protein. This methodology opens a new route to the integration of high- quality graphene layers into diverse technological platforms, including plasmonics, optoelectronics, or biosensing. With respect to the latter, the viability of a thiol-functionalized chemical vapor deposition graphene-based solution-gated field-effect transistor array was assessed. 1. INTRODUCTION molecular links are required, preferably via covalent function- Whilst pristine graphene is one of the most relevant materials alization. The most common covalent functionalization of the decade, several important shortcomings must be methodologies for graphene are chemical routes,6−8 mainly overcome before it may step from fundamental physics to based on the reaction between free radicals or dienophiles and applied technology.1 In particular, the absence of an electronic the CC bonds of pristine graphene.9 However, although band gap and its extreme chemical inertness undoubtedly well-developed wet chemistry routes may succeed to link many compromise its use as an active element in electronic devices interesting functional groups to the graphene surface, they or hybrid structures. Molecular functionalization of graphene usually fall short in their usefulness either due to a low degree can provide singular and advantageous properties, and there of functionalization or to extreme disruption of the surface due have been many attempts via nondestructive methodologies to to the harsh nature of the reaction conditions,9,10 resulting in furnish graphene with surface modifications whilst attempting to preserve its extraordinary properties.2−5 Evidently for Received: November 12, 2018 technological applications in environmental operating con- Accepted: January 31, 2019 ditions, such as those related to biosensing, strong and stable Published: February 14, 2019 © 2019 American Chemical Society 3287 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article graphene platforms where excessive defect concentrations degrade the outstanding properties of graphene, thus limiting its applicability. Here, we use a new, recently reported strategy11 for the selective functionalization of graphene based on the controlled formation of atomic vacancies in order to obtain a uniformly covered surface with a covalently bound spacer molecule that is formed from the spontaneous bonding of p-aminothiophenol (pATP) molecules at the vacancies. This results in the controlled decoration of the graphene surface with active thiol moieties that can be directly used to bond diverse nanoarchitectures to graphene. We show that although the functionalization protocol is undertaken in ultrahigh vacuum (UHV), the thiol functional moiety is robust and stable in different environments. As a consequence, it can be used, for example, for the immobilization of metal nanoparticles (NPs), particularly gold NPs (Au NPs) which are known to show a high affinity toward the thiol group.12 The deposition of NPs Figure 1. Schematic illustration of the two-step functionalization on graphene sheets has become a valuable strategy for coupling process: Left panel: UHV linking of the aminothiophenol molecules. graphene with plasmonic nanostructures13 and shows promise (1) Irradiation of the graphene surface with Ar+ at 140 eV; (2) exposure of the graphene surface with ion-beam-induced vacancies to for optoelectronic materials14,15 or in (bio)sensing16 or energy pATP molecules; and (3) covalent bonding of N of pATP to the storage17 applications. The initial graphene substrate generally carbon network at the induced vacancy, leaving the thiol group employed is graphene oxide (GO), which although it is exposed to the medium. Right panel: in-solution process of the thiol significantly more reactive than pristine graphene, allowing the group. Top: scheme of the thiol linkage to the Au NPs; bottom: chemical binding of the NPs to the surface via reduction of the conjugation of the thiol-modified ssDNA aptamer to the thiol group GO and the metal salts, this is at the expense of significantly through a disulfide bond formation. degraded electrical and electronic properties. Although recently a nonchemical Au NP decoration method using 2. RESULTS AND DISCUSSION laser ablation in liquids was reported,18 a GO substrate was still required to efficiently bind the NPs to the surface. The mechanism mediating the functionalization of graphene On the other hand, thiol chemistry is an ideal tool to couple has been understood in terms of the extra charge accumulated a wide range of molecular architectures, in particular in the broken dangling bonds around the single atomic biomolecules through the formation of strong sulfide bridges.19 vacancies that selectively oxidize any molecule containing an One example is nucleic acid aptamers,20 which comprise RNA amino moiety (Figure 1, step1). When pATP molecules are or single-stranded DNA (ssDNA) oligonucleotides selected in injected into the chamber in the gas phase (Figure 1, step 2), vitro from a vast library of synthetic random oligonucleotides21 the N atom from the primary amino group is incorporated into the graphene network (Figure 1, step 3). The nature of the that can bind with high affinity and specificity to a given target molecular bonding to the surface is covalent, as confirmed by molecule. Aptamer-based biosensors have recently emerged as the S 2p and N 1s X-ray photoelectron spectroscopy (XPS) improved biorecognition elements and are increasingly used in core-level peaks (Figure 2b) and density functional theory biotechnology, biomedicine, and environmental control.22,23 (DFT) calculations. The N 1s core level peak can be fitted Moreover, several are the advantages of using graphene-based with a single component at 400.09 eV, in good agreement with platforms for biointerfaces,24,25 among them low noise, the values reported for N within the graphene network and flexibility, and more interestingly, that because of the electronic with a radical.11,27 The calculations show that the molecule nature of graphene, sensing and actuator materials are on the strongly binds within the surface lattice with an anchoring same device. This work opens the door toward the integration energy (energy gain due to the saturation of the C dangling of graphene as a sensing platform. bonds) of 12.21 eV after the reaction is produced, yielding a Finally, it is important to remark that whilst the UHV highly stable final structural configuration (depicted in step 3 environment provides highly controlled preparation of func- of Figure 1). The result of this process is that the free thiol tional material surfaces, these generally fail when exposed to group of the pATP is exposed to the medium. This is opposite the atmosphere. The 13 orders of magnitude difference in to the process that occurs in thiol−ene click chemistry routes, pressure, commonly known as the pressure-gap that separates where the thiol group binds to the pristine graphene the real world from the artificially defined world of surface surface.28−30 In our case, the wide and rich chemistry of the science, is a very difficult barrier to overcome. In this work, thiol groups can be used to further attach other relevant both worlds are successfully combined. UHV protocols and structures and molecules to the graphene sheet, either via S− characterization techniques for covalent attachment and metal bonds or by the formation of disulfide bridges.20 characterization of the process are employed, and solution- The existence of a stable thiol group after steps 1−3 has based and atmospheric protocols are used to incorporate been verified by XPS spectra recorded on the same technologically important nanoarchitectures (see Figure 1). experimental system where the functionalization was per- These conjugated NPs and biomolecules are representative formed that means without exposure to air. The S 2p core level examples of nanobiohybrid nanostructures that may provide peak, shown in Figure 2a, appears at 163.9 eV, confirming the excellent platforms with wide applicability in diverse scientific presence of the thiol moiety, which is easily differentiated from and technological fields.26 S dimers, sulfur oxides, or other S-containing species. The XPS 3288 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article Figure 2. (a) S 2p XPS core-level peak of the G-pATP surface corresponding to free thiol groups (photon energy 400 eV). The solid line corresponds to a fit with a single component (doublet); (b) N 1s XPS core-level peak of the G-pATP surface. The solid line corresponds to a fit with a single component at 400.09 eV, (c) 50 nm × 50 nm STM image of a SiC/H/G-functionalized surface after incorporating pATP. Imaging conditions: V = −339 mV, I = 0.181 nA. Inset: 5 nm × 5 nm. Zoom of one molecule taken at V = 600 mV, I = 0.102 nA; (d) representative Raman spectra of epitaxially-grown graphene on the 4H−SiC surface before (upper trace, red curve) and after (lower trace) incorporation of pATP at the vacancies, showing graphene band assignments. (e) Top: schematic representation of 2,2′ dithiodipyridine (DTP) reaction with thiol groups. Bottom: UV−vis spectrum of the product reaction obtained between pATP-functionalized graphene and DTP. (f) Magnetic field dependence of the Hall resistance channel for the pristine (black dots) and the freshly functionalized G-pATP (red dots), corresponding to our setup to transport by holes and electrons. (g) Top (inset) and perspective views of a computed 3D density-charge redistribution function isosurface (with a positive value of |ρredist(r)| = +10−4 e− a.u.−3) for the functionalized system. The density-charge redistribution function is defined as ρredist(r) = ρtot(r) − [ρgr(r) + ρmol(−2H)(r)], where ρtot(r) is the total electronic spatial density charge for the whole, once formed, molecule(−2H)@graphene system, and ρgr(r) and ρmol(−2H)(r) are the total electronic spatial density charges for the graphene with the single atomic vacancy and the pATP(−2H) molecule in the geometric configuration they adopt in the whole system, respectively. spectrum can be fitted with a unique component with a spin- graphene.32,33 In the lower trace, which corresponds to a orbit splitting of 1.15 eV, indicating that all of the molecules pATP-functionalized surface, the defect-induced bands D and are at the same chemical state. Furthermore, these molecules D + D′ clearly emerge at around 1362 and 2962 cm−1, were observed by scanning tunneling microscopy (STM) (on respectively, because of the included N atom of the pATP that the same system where functionalization was performed) to be is sp3 coordinated inducing a distortion of the graphene sheet. well dispersed over the surface, Figure 2c, where the molecules However, the 2D/G ratio remains little changed at >3, appear as bright bumps in the images with typical apparent attesting the structural integrity of the graphene sheet, and the heights of 2.1 ± 0.5 Å (statistics over 150 protrusions), a value incorporation of the N atom into the basal plane is an essential similar to that reported for aminophenol.11 part of this process. Figure 2d presents a representative Raman difference In order to assess the effect of functionalization on the spectrum recorded from a flat graphene terrace of graphene electrical properties, the pristine graphene and G-pATP on 4H−SiC, where the second-order modes of SiC31 have surfaces were compared via a magnetotransport method.34−36 been subtracted to reveal the characteristic graphene bands. In The results indicate that the density of carriers does change the upper trace, which represents the graphene surface prior to because of the charge redistribution after bonding and mobility functionalization, very little evidence for defect-induced modes is reduced by a 24% (Figure 2f). At room temperature, there is was found, confirming the high quality of the as-grown a striking change in the slope of Rxy resistance versus magnetic graphene layer, where G, 2D, and 2D′ bands appear at Δν = field when data from the pristine sample are compared with 1585, 2680, and 3232 cm−1, respectively, and the 2D/G ratio data taken immediately after the production of G-pATP of ∼3.2 correlates very well with quasi-free standing monolayer (Figure 2f). This change in the slope plot at low fields (±1 T) 3289 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article corresponds to a change in the sign of the charge carriers from not alter substantially the robustness scenario of the bonding holes (with an estimated density of 5.2 × 1014 cm−2) in the after the functionalization. pristine sample to electrons (with a density of 1.3 × 1014 cm−2) Once confirmed the robustness of the thiol-functionalized in the functionalized sample. These experiments were graphene upon exposure to both air and liquid environments, performed on an epitaxially grown single-layer graphene we focus on its capacity for conjugation to metal NPs or surface; therefore, the sign of doping is as expected. The biomolecules.40 effects of functionalization are also visible in the highest level In order to couple metal NPs, we take advantage of the of noise apparent in the Hall channel for the functionalized strong and well-studied S−metal bond that forms sponta- sample data. There is also a decrease in the estimated mobility neously and allows to strongly and easily link any noble metal (2390 cm2 V−1 s−1 in the pristine sample and 1600 cm2 V−1 s−1 NP to the functionalized graphene surface.41 For the after functionalization). Further, the G-pATP sample was technological use of graphene/NP assemblies, the NPs need measured again after 1 year storage under ambient conditions, to be securely anchored to a particular surface. The versatility and the electrical properties, in terms of charge carriers and of the present methodology is well suited to this purpose, and density, were essentially preserved. many different types of NPs can be robustly coupled to At such low surface coverage, neither Raman nor infrared graphene. To demonstrate this, two types of Au NPs of spectroscopy could detect the thiol functionalities; thus, their different origins were employed, described in Figure 3: NPs presence was assayed in a liquid environment by dipping a produced in a gold-salt solution and capped with citrate and functionalized graphene surface for 1 h into a DTP solution.37 NPs produced using a multiple ion cluster source (MICS) in The free thiol groups react with DTP forming 2,2′-dipyridyl UHV.42,43 disulfide and releasing 2-mercaptopyridine that quickly tautomerizes into 2-thiopyridone (top part of Figure 2e). The formation of these compounds indicates the presence of free thiols on the graphene surface giving a UV absorption maximum at λ = 343 nm (molar extinction coefficient at 343 nm = 8080 M−1 cm−1), as shown in Figure 2e. The peak observed at 269 nm is associated with unreacted DTP.38 Thus, we can confirm that the pATP molecules are covalently anchored through the amine group to the graphene surface exposing free thiol groups, which are stable in air and available for subsequent reactions. Figure 2g presents the computed 3D isosurface for a positive value of +10−4 e a.u.−3 of the density-charge redistribution function (defined in the figure caption) for the G-pATP system and shows how the incorporation of the pATP molecule, via the doubly dehydrogenated N atom within a single atom vacancy (SAV) of the graphene lattice, induces a strong electronic charge spatial localization in the surroundings of the incorporated N atom in a third-order symmetrical way (see the circular inset in Figure 2g), which has been quantified by a Bader population analysis to comprise 0.96 e−. This electronic Figure 3. (a) TEM micrograph showing Au NPs prepared by wet charge accumulation comes from both the rest of the anchored chemistry; (b) size distributions, with the corresponding log-normal molecule (around 0.5 e−) and from charge depletion in fits, of Au NPs prepared under wet chemistry and UHV conditions graphene regions outside the functionalization centers (around obtained from the heights of the corresponding dynamic-mode AFM 0.56 e−). Interestingly, the electronic charge depletion and images; (500 nm × 500 nm) of the Au NPs deposited on graphene via redistribution within the rest of the molecule breaks down the (c) wet chemistry and (d) under UHV conditions. Insets correspond aromatic resonance of the ring, and some electronic charge to characteristic surface profiles of Au NPs. Note that the lateral sizes of the NPs are increased because of tip convolution effects. seems to accumulate in the S atom of the thiol group. This charge accumulation potentially enhances the reactivity of this group, thus positively favoring reaction at the SH site of the Both transmission electron microscopy (TEM) (Figure 3a) anchored molecule. The effect of the solvation has also been and atomic force microscopy (AFM) images (Figure 3c) of studied by DFT calculations. We have repeated some particles prepared by wet chemistry methods show that these calculations having into account the water solvation influence have a tendency to aggregate, whereas those prepared in UHV by the polarizable continuum model (PCM) within the default (Figure 3d) remain completely isolated, with a low diffusion on self-consistent reaction field method as implemented in this substrate. In the latter case, no organic shell or surfactants Gaussian09.39 For this purpose, we have computed again the are required to avoid NP aggregation, which may prove total energy of the full molecule (−2H)@graphene system and advantageous for some plasmonic or optoelectronic applica- of the graphene with the single atomic vacancy and the tions. pATP(−2H) molecule subsystems by separate within the The particle size and size distribution also vary for both PCM model under water solvent conditions. The result of the types of NPs, as obtained from the AFM height measurements, calculations yields an anchoring energy of 11.82 eV, to be undertaken in order to avoid tip convolution effects that compared with the value of 12.21 eV without accounting the coarsen the lateral image size. After measuring more than 50 water solvation environment. This small energy difference does Au NPs from each sample type, the size distribution histogram 3290 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article in Figure 3b was obtained. An average NP size of 5.7 ± 0.6 nm of approximately 8 × 1012 cm−2, which would correspond to was found for Au NPs prepared under UHV conditions and 14 approximately 0.1−1% thiol-functionalized surface.11 Thus, it ± 2 nm for those prepared by wet chemistry. is easy to control the typical average separation between immobilized NPs. Finally, it is important to point out that we have successfully linked Au NPs to graphene surfaces that were functionalized 5 months earlier and stored under ambient conditions, showing a high resistance of the G-pATP surface to ageing. With regard to the development of biohybrid interfaces, we have also explored the suitability of the G-pATP surfaces to immobilize an in vitro selected thiol-derived ssDNA aptamer with high specificity for the recognition of a target protein, where we explored the possibility of forming a stable disulfide bridge bond between the S atom of the free thiol group of the pATP molecule linked to the graphene surface and a terminal S atom present in the thiol-modified aptamer molecule. In this respect, we employed a previously obtained 76 nucleotides- long ssDNA aptamer with selectivity for the protein PCBP-2, a pleiotropic protein that participates in a number of cellular processes including transcriptional and translational regulation. We use the protocol described in the experimental part to first produce an aptamer-modified graphene surface and, second, to expose it to a solution containing target protein molecules. The final dried surfaces are studied in air using AFM, and a typical AFM image of the G-pATP/aptamer/PCBP-2 system is shown in Figure 5. Here, two different types of rounded and compact structures were observed: some small ones with sizes in the Figure 4. AFM images of Au NPs deposited on (a) as-received SiC/ 16−20 nm range and a more reduced number of large H/G substrate, (b) SiC/H/G-pATP substrate following the wet protuberances with lateral sizes in the 28−32 nm range, albeit chemical route, and (c) SiC/H/G-pATP substrate following the UHV the lateral sizes of all of these features may be coarsened by tip physical route (see text). Top row: as-deposited; Bottom row: after 1 convolution effects as well as by the tip load. The different washing cycle. Note that the image sizes in (c) are smaller because of the lower Au NP size. heights observed in the surface profiles corresponding to the path 1-2-3 in the AFM image are shown in Figure 5b. Clearly, the smaller structures show heights in the 0.5−1.5 nm range With regard to the density of NPs deposited, the top row of and likely correspond to free aptamers bound to the G-pATP Figure 4 presents large-scale AFM images for three different surface, whereas the larger structures reach heights of 3 nm and cases: Au NPs deposited on pristine, nonfunctionalized can be assigned to aptamer/PCBP-2 complexes. Analysis of the graphene (column A), and Au NPs deposited on G-pATP heights of 400 features (not shown) like those shown in Figure surfaces, following either the wet chemical (column B) or the 5a presented two maxima at around 0.9 and 3.1 nm, UHV physical (column C) route. The figure shows that more corresponding to aptamer and aptamer/protein structures, NPs are seen in the functionalized surface, with 1.2 × 1010 respectively. These data are consistent with our experimental cm−2 and 3 × 1010 cm−2 for chemical and physical deposition, conditions in which an aptamer/protein ratio of 10:1 was respectively, than that for the pristine G/Au system, with 4 × employed, and only a limited fraction of the aptamers is 109 cm−2, suggesting that the deposition of Au NPs is relatively expected to be bound to the target PCBP-2. enhanced by the presence of pATP. It should be pointed out that one of the added values of In most studies of decoration of high-quality graphene with using a biosensing system based on aptamers instead of NPs, these are simply deposited on the surface from antibodies is their reversibility with temperature.22 The solutions,44,45 although a recent study shows −COO−- modified Au NPs electrostatically linked to graphene surfaces functionalized via thiol−ene click chemistry, but NP stability or resistance was not addressed.29,46 To test the binding strength of the Au NPs to the G surfaces, the respective samples are subjected to a washing cycle, the corresponding images being displayed in the bottom row of Figure 4. Clearly, in the case of the pristine G surface, almost all of the NPs were removed from the surface. However, for the G-pATP surfaces, the density of NPs does not appear to change, within the Figure 5. (a) 500 nm × 500 nm AFM image taken in dynamic mode statistical error, after washing for both chemical and physical in air of G-pATP/aptamer/PCBP-2, after a washing step with routes. This is due to the high strength of the covalent link selection buffer (SB). The line profile connecting points 1, 2, and 3 in between the thiol group and the Au NPs. the image is shown in panel (b). The image displays 0.5−1 nm-height Furthermore, an important advantage of our methodology is and 16−20 nm-wide features (that can be assigned to free aptamers), that the number of available thiol moieties on the surface can as well as a smaller number of 1.5−3 nm-height and 28−32 nm-wide be relatively easily tuned up to a maximum estimated density features (corresponding to aptamers bound to the target protein). 3291 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article Figure 6. (a) Optical microscopy image of the solution-gated field-effect transistor (gSGFET) device with the graphene transistors displayed (red cross indicates a transistor), (b) Transistor for a 40 × 20 μm channel size (red square on the graphene channel) and (c) cross section of the gSGFET device. The gate−source polarization is applied on the gate, and the drain−source polarization is applied on the drain. The Ids is measured on the drain side. (d−g) Characterization curves of the CVD transistor before and after functionalization with pATP. (d) I−V transfer curves for 40 × 40 μm transistor channel size, (e) CNP values at different channel lengths before and after functionalization and the difference between both, (f) gm curves for 40 × 40 μm channel size, and (g) gm values at different channel lengths before and after functionalization and the difference between both. denaturing temperature used for our aptamer (with a length of functionalized G surface consists of a reductive elimination of 76 nucleotides) without compromising its integrity is 95 °C. hydrogen of the S−H bond by the metal surface. However, in Unlike antibodies, which undergo irreversible denaturation the case of G-pATP functionalization with the thiolated above 37 °C, aptamers can refold into their original, active aptamer, it relies on the oxidation of both sulfhydryl groups conformations when optimal temperature (in our case, 25 °C) reaching a disulfide bridge.47 Gold-thiolate and disulfide bond is restored. Therefore, the aptamer is fully denatured at 95 °C, cleavage occur via reduction. Hence, the solvent would only and the target molecule (in this case, the protein PCBP-2) is affect if it contains reductant agents, which is not our case. completely released, and then the surface immobilized Additionally, because basic pH could also promote gold- aptamers can be reused. The G-pATP system is perfectly thiolate dissociation, we employed pH 7.4 for the functional- capable of resisting this temperature level, which allows ization process.48 Finally, the strong affinity between gold and repeated reutilization of the same G-pATP/aptamer sensing the thiol group and the S−S bond strength guarantee the platform. It is important to note that the developed chemistry robustness of the functionalization and binding processes, in this work is perfectly capable of resisting this denaturation leading to the stability of the different platforms developed in temperature. this work under the different experimental conditions.11 The binding mechanism is different in both cases. In the 2.1. Perspectives, a Platform for gFET. As it has been case of metal NPs, they are based on the strong affinity of the shown, the presented methodology exhibits potential applic- sulfur-containing compounds for noble metal surfaces, ability in two systems of broad interest, namely, metal NPs and specifically gold. The mechanism of binding NPs to the (bio)sensing. The use of gFET as sensing devices has shown 3292 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article certain advantages in biomedical applications, such as coupling capacitance, although further evaluations need to be described for PoC (point of care) biosensing of biomolecules49 undertaken to fully assess this behavior. Thus, it can be or electrical brain recording.50 However, for using the gFET as concluded that this is a valid functionalization strategy to tailor biosensors, the graphene channel has to be chemically the CVD graphene surface in a device with thiol-terminated modified to graft bioreceptors that will detect the specific moieties, and it opens the door to versatile thiol chemistry to a biomarkers. In this specific application,50 keeping the electrical broad range of biosensing experiments using this type of performance after the functionalization procedures is really devices. important to ambition dual sensing applications, recording and sensing biomolecules in parallel.51 3. CONCLUSIONS In order to advance toward the implementation of these functionalized graphene surfaces into devices showing In summary, we present here a facile and controllable electrical read out for (bio)sensing applications, the impact methodology to decorate a graphene surface with covalently of this controlled surface functionalization methodology on the linked organic spacers containing exposed thiol functionalities electrical properties of graphene needs to be evaluated. For this that can be employed in diverse chemical linking strategies. In purpose, we have fabricated and characterized prototypical particular, we illustrate the linking of metal NPs and aptamers, graphene solution-gated field-effect transistors (gSGFET),52 bisensor-relevant biomolecules, to a graphene surface. This which were measured before and after functionalization with methodology opens the door to the growth of controllable and pATP. stable nanobiohybrid structures on graphene platforms, with a Figure 6a shows an optical image of a device consisting of a broad applicability in plasmonic, biotechnology, and bio- 25-transistor array, where a series of single-layer chemical medicine. vapor deposition (CVD)-graphene channels are connected by two metallic contacts, the drain and the source, with a 4. EXPERIMENTAL METHODS reference electrode (Ag/AgCl) immersed in an electrolyte 4.1. Graphene Surface Functionalization. Although we solution, which is used as the gate terminal that modulates the have employed several types of graphene surfaces, all with conductivity of the graphene channel. The image in Figure 6b similar results, most of the experiments reported were shows a close up of one of the graphene channels and the gold performed on high-quality graphene layers epitaxially grown contacts of the transistor corresponding to the area marked by on 4H−SiC(0001) by CVD with or without H under the a red cross in Figure 6a. A schematic representation of the superficial graphene layer. These surfaces behave as quasi free- device is shown in Figure 6c. The standard electrical standing monolayer graphene, with Hall mobility values above characterization is derived from the gSGFET transfer curve 8 >000 cm2 V−1 s−1.54 On the other hand, the immobilization (Figure 6d), where the drain−source current, Ids, is obtained as protocol has also been performed directly on devices, where a function of the gate−source voltage, Vgs (−0.2−0.6 V), for a CVD graphene sheets were obtained by electrochemical fixed drain−source voltage, Vds (0.1 V). From this curve, both delamination and transferred onto SiO2 wafers. the charge neutrality point (CNP), corresponding to the The main steps in the functionalization process are voltage at the minimum Ids current and minimum of the curve, illustrated in Figure 1. The strategy used to covalently link and the transconductance (gm), corresponding to the slope of thiol moieties to the graphene basal plane was adapted from a the I−V curve, can be evaluated. The latter, which is the recently reported protocol used to couple para-aminophenol derivative of the Ids versus Vgs (Figure 6d), is related to the molecules.11 It begins in the UHV chamber by creating a geometrical parameters of the transistor (width to length ratio) network of single atomic vacancies on the surface via gentle and the graphene charge mobility. Further, the CNP can be Ar+ sputtering of the graphene surface (Figure 1, step 1).55,56 related to the doping of the graphene: a p-doped surface shifts The functionalization experiments were performed in situ in the CNP to more positive values of Vgs, whereas an n-doped UHV with a base pressure below 2 × 10−10 mbar. In the first surface shifts to more negative ones.53 This doping arises step, the substrate was degassed, followed by an annealing principally from the nature of the molecules attached to the cycle at 250 °C for 15 min to remove any physiosorbed surface and the charges trapped near the graphene lattice. The contamination from the surface. Vacancies were created in the transfer curves (Ids vs Vgs) obtained before and after graphene lattice by bombarding with argon ions (Ar+) for 90 s functionalization for a 40 μm long gSGFET are shown in using an electron-impact ion gun. The acceleration energy of Figure 6d, where it can be observed that the functionalization the ions inside the ion gun was 140 eV, the sample current was mainly affects the CNP, shifting it to lower potentials, whereas 1 μA, and the gas pressure during this process was maintained the other parameters remain constant. In Figures 6e−g, the at 1 × 10−7 mbar. The sample was then annealed at 500 °C for values of CNP and gm obtained before and after functionaliza- 5 min. Before dosing pATP molecules (Sigma-Aldrich, purity tion are shown, as well as the variation evaluated for each 99%), these were purified by pumping with a turbomolecular transistor before and after functionalization. The average pump (P = 10−8 mbars) for >6 h to eliminate any impurities. variation in CNP observed over the 25 transistors is 45.7 ± 9.8 Subsequently, the ion-treated graphene surface was exposed to mV, suggesting that the molecular coverage on the surface about 10 Langmuir of pATP (1 L = 10−7 mbar s). The G- does not induce a large doping in the graphene lattice. The pATP surfaces were either maintained in UHV for further in broad dispersion in gm observed in the measurements before situ reaction with Au NPs or removed from the chamber and and after functionalization is due to the different channel stored under ambient conditions for ex situ reaction with Au lengths of the transistors. From the results obtained, the gm NPs or aptamers. after functionalization is slightly reduced to a nonrelevant value 4.2. NP Fabrication. Citrate-stabilized gold NPs were of 0.401 ± 0.340 m S, suggesting that the charge mobility in synthesized according to the Turkevich method.57 The Au the graphene lattice is unaffected by the process. Indeed, the content in the samples was determined by inductively coupled small gm reduction could be attributed to variations in the plasma-optical emission spectrometry (ICP-OES, PerkinElmer 3293 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article Optima 2100 DVICP) after digestion of the Au NP suspension The 5′ thiol group of the aptamer was introduced to with a mixture of HNO3:HCl (1:3 ratio v/v) and dilution with establish a disulfide bond with the free thiol group of the G- ultrapure water. Graphene (1 cm2) functionalized with pATP pATP. The terminal sulfur atoms of thiolated DNA have a was placed into a small plastic vial containing 2.5 mL of the tendency to form dimers in solution, especially in the presence aqueous suspension (0.1 mg Au/mL) of Au NPs and left, of oxygen. Therefore, dithiothreitol (DTT) was used as a without agitation, for 15 h. Next, the graphene sample was reducing or “deprotecting” agent for thiolated DNA. Typically, introduced into a Petri dish in the presence of 5 mL of water a 10 mM DTT solution in 100 mM sodium phosphate buffer and stirred in an orbital shaker for 15 min. Subsequently, in (pH 8.3) was mixed with a 100 μM thiolated aptamer solution order to eliminate any residues of unconjugated Au NPs, the and allowed to react for 1 h at room temperature. Then, DTT graphene surface was thoroughly washed with ultrapure water. was removed by filtration using a 3 K Amicon Ultracentrifugal Finally, the washed sample was dried in an oven at 50 °C for 2 filter. Subsequently, 30 μL of the thiolated ssDNA aptamer (at h. The same procedure was applied to pristine, non- 1 μM concentration in selection buffer, SB, composed of 100 functionalized graphene. The NPs on the graphene surfaces mM NaCl, 6 mM MgCl2 and 100 mM HEPES pH 7.4) was were observed by TEM and AFM. renatured (by incubation at 95 °C for 10 min, and then at 37 Au NPs were fabricated in UHV via a gas-phase synthesis °C for 10 min). The renatured, thiolated ssDNA was deposited route using a MICS,43 equipped with three 1″ magnetrons. In onto the G-pATP surface and left to react via disulfide this case, one of the magnetrons was loaded with an Au target bridge47,64,65 in a humidity chamber for 20 min at controlled (99.99% purity). To ensure minimal contamination, all gas temperature (25 °C). Then, the G-pATP/aptamer surface was pipes used to inject Ar (95.5% purity) are made of stainless gently washed with ultrapure, DEPC-treated milliQ water, and steel and vacuum-sealed. The MICS is connected to the main finally air-dried. The aptamer-functionalized graphene samples UHV chamber, where the substrate is located, through a small were subsequently incubated with 30 μL of a 100 nM solution orifice with a nozzle. The distance of the sample to the MICS (in SB) of recombinant PCPB-2 protein for 20 min, in a exit during deposition is 200 mm. The working parameters humidity chamber at 25 °C. Then, the sample was gently employed in the MICS for Au NP fabrication were a power of washed with SB to discard the protein molecules that could 8 W; an aggregation distance to the exit slit of 70 mm; and have nonspecifically bound to the surface. Finally, the G- total Ar flux of 100 sccm with an Ar flux through the Au pATP/aptamer/protein samples were AFM imaged in air, as magnetron of 30 sccm. Once formed, the Au NPs travel previously described.66 through the vacuum to the main chamber where they soft land 4.4. Characterization Techniques. XPS was undertaken at the PEARL Beamline of the Swiss Light Source. The on the surface of the substrate, ensuring the absence of beamline produces a photon flux of 2 × 1011 ph/s at 1 keV on deformation of the Au NPs due to their low energy.58 The base sample. XPS spectra were fitted with an Igor Pro macro using pressure in the system was below 5 × 10−10 mbar. Voigt (Lorentzian and Gaussian) curve profiles. In all cases, a 4.3. Binding of Aptamers to G-pATP and Target Shirley-type background was subtracted from the raw data. Protein Recognition. Aptamers are RNA or ssDNA Raman spectra were obtained in the Raman laboratory of the oligonucleotides that are selected in vitro from a vast library characterization service of the ICTP−CSIC employing a of synthetic oligonucleotides (in general, from 1013 to 1015) Renishaw inVia Reflex-dispersive Raman spectrometer incor- with random sequence21 using an amplification-selection porating a Leica microscope, using a 100× (N.A. = 0.85) method termed “systematic evolution of ligands by exponential objective with a 514.5 nm (2.41 eV) laser power of approx. 2 enrichment” or SELEX,59 which can bind with high affinity and mW at the sample surface, and multiple scans in order to specificity to a given target molecule. Aptamers possess a obtain adequate signal-to-noise ratios. specific three-dimensional structure in solution that depends The in-air AFM measurements were performed in the on their sequence and on the physicochemical features of the dynamic mode with a Nanoscope IIIA (Veeco) system, an folding buffer, including temperature, pH, ionic strength, and Agilent 5500 PicoPlus, and a Cervantes system from Nanotec concentration of divalent cations. Electronica S.L. under ambient conditions. We employed We have used a 76 nucleotides-long ssDNA aptamer specific silicon tips with nominal force constants of 40 N/m and softer to the protein PCBP-2 (also known as hnRNP E2 or CP-2), silicon cantilevers of 2−4 N/m for imaging the aptamer/ which is a member of the cellular heterogeneous nuclear protein complexes. ribonucleoprotein family that mediates relevant biological TEM measurements were performed using a JEOL JEM processes including mRNA stabilization, transcriptional 1011 with a Gatan ES1000Ww camera. TEM samples were regulation, translational control, and apoptotic program prepared by placing one drop of a dilute suspension of Au NPs activation.60−62 Among the PCBP-2-specific aptamers pre- in water on a carbon-coated copper grid, and the solvent was viously obtained and analyzed by means of a variant of the slowly evaporated at room temperature. SELEX process,63 we selected the ssDNA aptamer with the Magnetotransport was investigated over macroscopic highest affinity for the target protein, with a dissociation dimensions (10 mm × 10 mm) in the square van der Pauw constant Kd of 8.4 nM. It is termed 05DS10-21, and its configuration using a Quantum Design 9T PPMS (R) and nucleotide sequence (5′ thiol-modified, purchased from IBA press-on contacts from Wimbush Scientific (R). Resistance GmbH, Gö t tingen, Germany) is 5′-GCGGATCCA- measurements were collected in helium atmosphere (5 Torr) GACTGGTGTGGAGGTTAGCCGAAACACGTA- along the sample edges (xx and yy) and diagonal (xy) TACGCGTATTTATCCTCGGGCCCTAAAGA- directions. CAAGCTTC-3′. The recombinant protein PCBP-2 used as 4.5. Theoretical Computational Methods. Physical and the target in this work (purchased from Abnova, Taipei, electronic properties of the pATP molecules incorporated Taiwan) is a 362 amino acids-long globular protein that within a SAV created in the graphene were using a set of includes a GST-tag at its N-terminal end (MW of 65.56 kDa). accurate DFT-based calculations by the plane-wave scheme 3294 DOI: 10.1021/acsomega.8b03152 ACS Omega 2019, 4, 3287−3297 ACS Omega Article implemented in the QUANTUM ESPRESSO simulation Author Contributions package.67 All calculations accounted for dispersion forces Rebeca Bueno and Marzia Marciello contributed equally to within the DFT + D approach via an empirical efficient vdW this work. The manuscript was written through contributions R-6 correction.68,69 The revised version of the generalized of all authors. All authors have given approval to the final gradient-corrected approximation of Perdew, Burke, and version of the manuscript. Ernzerhof (rPBE) has been used to account for the Notes exchange−correlation effects,70 and Kohn−Sham equations The authors declare no competing financial interest. ■ were solved using a periodic supercell geometry. Rabe− Rappe−Kaxiras−Joannopoulos ultrasoft pseudopotentials71 ACKNOWLEDGMENTS have been adopted to model the ion−electron interaction in This work was supported by the European Union’s Horizon the H, C, N, and S atoms. The Brillouin zone was sampled by 2020 research and innovation programme under grant employing an optimal [4 × 4 × 1] Monkhorst−Pack grid.72 agreement No 696656 (Graphene Flagship-core 1) and no One-electron wave functions were expanded on a basis of 785219 (Graphene Flagship −core 2); UE FP7 ideas: ERC planewaves with energy cutoffs of 450 and 550 eV for the (grant ERC-2013-SYG-610256 Nanocosmos) and Spanish kinetic energy and the electronic density, respectively, which MINECO grants MAT2014-54231-C4-1-P, MAT2014- have been adjusted to achieve sufficient accuracy to guarantee 54231-C4-4-P, MAT2017-85089-C2-1-R, MAT2014-59772- a full convergence in total energy and density. C2-2-P, and BIO2016-79618-R (funded by EU under the 4.6. gSFET Device Fabrication and Testing. CVD FEDER programme), as well as the Nanoavansens program graphene transistors were fabricated using graphene layers from the Community of Madrid (S2013/MIT-3029). This grown by a CVD process using a 25 μm thick copper foil of work has made use of the Spanish ICTS Network MICRO- 99.8% metal basis obtained from Alfa Aesar. The copper foils NANOFABS partially supported by MINECO and also the were cleaned with acetic acid, then rinsed with deionized (DI) ICTS NANBIOSIS, more specifically the Micro-Nano water, followed by cleaning cycles with acetone, DI water, and Technology Unit of the CIBER in Bioengineering, Biomate- isopropyl alcohol. The sample dimensions were 6 × 5 cm2, and rials & Nanomedicine (CIBER-BBN) at the IMB-CNM. We the growth conditions were 10 min at 750 °C, 2 sccm H2 and 5 are grateful to Matthias Muntwiler for his assistance with min at 800 °C, 25 sccm CH4/H2. A 700 nm poly(methyl experiments in the PEARL beamline in the SLS facility. Finally, methacrylate) (PMMA) layer was deposited on top of the we acknowledge the TEM and ICP services at the CNB and graphene surface. The graphene layer was separated from ICMM institutes, respectively. CSS acknowledges the MINE- copper using an electrochemical delamination process CO for a Juan de la Cierva Incorporación grant (IJCI-2014- following the procedure described by Rosa et al.73 without 19291). M. Marciello is grateful to the Comunidad de Madrid the use of a frame. After transfer, the layers were baked at 180 (CM) and European Social Fund (ESF) for supporting her °C for 2 min and the PMMA was subsequently removed by research work through the I+D Collaborative Programme in Biomedicine NIETO-CM (B2017-BMD3731). ■ rinsing with acetone and isopropanol. A Si/SiO2 wafer was used as a substrate. The first layer of Ti/Au metal contacts was deposited by electron-beam vapor REFERENCES deposition and subsequently structured by optical lithography. (1) Singh, V.; Joung, D.; Zhai, L.; Das, S.; Khondaker, S. I.; Seal, S. 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