Graphene Plasmon-Enhanced IR Biosensing for in Situ Detection of

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Graphene Plasmon Enhanced IR Biosensing for In Situ Detection of Aqueous-Phase Molecules with an Attenuated Total Reflection Mode Bo Zheng, Xin Yang, Jian Li, Cai-Feng Shi, Zhen-Lin Wang, and Xing-Hua Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01715 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 20, 2018

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Analytical Chemistry

Graphene Plasmon Enhanced IR Biosensing for In Situ Detection of Aqueous-Phase Molecules with an Attenuated Total Reflection Mode Bo Zheng,a Xin Yang,b Jian Li,a Cai-Feng Shi,a Zhen-Lin Wang,b and Xing-Hua Xia a, * a

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical

Engineering, Nanjing University, Nanjing, 210023, China, b

School of Physics, Nanjing University, Nanjing, 210093, China.

* Corresponding author, E-mail: [email protected] KEYWORDS: graphene plasmon, nanodisk array, infrared absorption spectroscopy, ATR-SEIRA, aqueous biosensing, label-free detection ABSTRACT: Graphene plasmon has attracted extensive interest due to the unprecedented electromagnetic confinement, long propagation distance and tunable plasmonic frequency. Successful applications of graphene plasmon as infrared sensors have been recently demonstrated, yet mainly focused on solid/solid and solid/gas interfaces analysis. Herein, we for the first time propose a graphene plasmon enhanced infrared sensor based on attenuated total reflection configuration for in situ analysis of aqueous-phase molecules. This IR sensor includes a boron-doped graphene (BG) nanodisk array fabricated on top of a ZnSe prism surface that supports attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRA). Our ATR-SEIRA platform is efficient and straightforward for in situ and label-free monitoring the interaction of biomolecules without interference from the environments, allowing for extracting instant spectroscopic information in a complex biological event. Utilizing the near-field enhancement of graphene plasmon, the binding interaction of L-selectin with its aptamer as demonstration has been investigated to evaluate the specific protein recognition process. The detection limit of target protein reaches 0.5 nM. Our work demonstrates that chemical doped-graphene plasmon combined with ATR-SEIRA is a promising signal enhancement platform for in situ aqueous-phase biosensing.

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Infrared absorption spectroscopy is a label-free and nondestructive analytical technology to quantitatively and qualitatively elucidate molecular information on structure-function relationship. In addition, it can provide a huge amount of molecular information in one single experiment. Thus, in the last few decades, IR absorption spectroscopy has been widely applied in medicine, food safety and life sciences. However, the limited sensitivity of conventional IR spectroscopy restricts its applications in detecting ultrathin films and trace level samples since the huge scale mismatch between infrared light and molecules leads to weak light-matter interaction. A promising way to address this issue is to introduce well-designed metallic substrates with strong electromagnetic near-field confinement. Early substrate design is inspired by surfaced enhanced Raman spectroscopy that utilizes rough noble metal surfaces or nanostructures.1 However, the sizes of such metal structures do not match with IR wavelength and no plasmon can be excited in the mid-IR region, resulting in very weak field confinement and relatively poor enhancement. Recently, well-engineered plasmonic antennas have been applied to enhance IR signals, such as Au nanorods, 2,3 triangles, 4 split-rings5 and holes.6 Remarkably, localized surface plasmonic resonance (LSPR) frequencies of such structures can be tuned to mid-IR region and overlap with the molecular vibrations of objective. With the highly confined and enhanced electromagnetic field of the structures, IR signal enhancement factor up to 104–105 can be achieved.2 Thus, specifically designed antenna with proper plasmonic resonance position becomes the key point for surface enhanced IR spectroscopy. Although the interaction between plasmons in noble metal nanostructures and vibrational modes of molecules has long been used as ultrasensitive probe for trace level sensing, noble metal plasmons cannot fully fulfill the enhancement required in the molecular fingerprint region due to its weak field confinement, narrow spectral resonance and very limited tunability.7 Graphene shows excellent optoelectronic properties and graphene antenna could be a better substitution for metal antennas. With the extremely short plasmon wavelength in mid-infrared, graphene antenna has unprecedented field confinement and provides considerable field enhancement. Compared with metal, graphene is easier to be doped, making it convenient to tune plasmonic resonance frequency of graphene antenna. The plasmon and associated optical fields in graphene can be tuned by varying the carrier density (Fermi level) and/or patterning periodic structures (shape and size).8-10 Common 2


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experimental schemes for tuning the carrier density include the electrostatic gating and chemical doping. Electrostatic-doping by gate control without further changing of the antenna shape is adopted dominantly to obtain dynamic regulation of graphene plasmon. However, for the separated nano-units (e.g. nanodisks), electrostatic gating needs more sophisticated device. Alternatively, chemical doping is regarded as a feasible and efficient tactics to turn the electronic properties of graphene. Experimentally, up to date, studies on the plasmonic properties of chemical doped and nanostructured graphene are still lacking. For the preparation of periodic structures, electron beam lithography (EBL) is dominantly employed, but it is tedious and high-cost, and thus is not suitable for large scale fabrication. As a feasible experimental strategy, nanosphere lithography (NSL) used in our work is relatively convenient and lowcost. Carefully controlling plasma etching time can prevent the deteriorating of graphene quality. Zhu et al. has successfully fabricated large-area graphene nanodot and antidot arrays by NSL.11 In this work, we tried to tune the plasmonic resonance by controlling the disk size and doping level of graphene nanodisk. It has been reported that graphene is biocompatible to biomolecules. Their interactions originate from weak interactions between biomolecules and the substrates. In the case of graphene, interactions between biomolecules and graphene occur mainly via π-stacking effect. Array of graphene dots provides larger surface area which allows adsorption of large amount of biomolecules and thus higher sensing sensitivity is expected.12 In addition, graphene can be easily functionalized which is promising for immobilization of bioreceptor units.13 Therefore, graphene antenna shows an exciting prospect for biosensing. In the recent five years, graphene plasmon has been successfully applied in surface-enhanced infrared absorption spectroscopy (SEIRAS) for gas sensing, polymer detection and biosensing, exhibiting the flexibility to meet various experimental situations. Despite the exciting and attractive features of graphene as an active plasmonic material for mid-IR photonics, utilizing graphene plasmonics for real device applications is still a major hurdle.14 More researches should be performed to develop new technology to realize its practical application. From another point of view, IR sensors based on graphene plasmon are mainly focused on solid/solid and solid/gas interfaces, e.g. PMMA,15 PEO polymer7 and protein film.16 This is because IR 3



measurements are usually taken on a transmission mode, which is difficult to avoid the large background water absorption from aqueous solution. Nevertheless, for bioanalysis, real-time and in situ detection in aqueous solution is extremely important since it is closer to the internal environment in vivo. Adato and Altug17 demonstrated a plasmonic chip-based technology enabling the in situ monitoring of protein and nanoparticle interactions at high sensitivity in real time by SEIRA-internal reflection technique. Measurement with an attenuated total reflection (ATR) configuration is a possible approach to simultaneously use antenna near-field enhancement and suppress signal from bulk water molecules. Especially, graphene antenna could shrink the original evanescent wavelength, minimizing the optical path to extreme sub-wavelength level and decreasing background water absorption. Herein, we for the first time propose a chemical doped-graphene plasmon enhanced IR biosensor which can be performed in aqueous solution via the ATR-SEIRA. A large area of boron-doped graphene nanodisk array on top of a ZnSe prism surface using the NSL method has been fabricated. Tunable graphene plasmon can be achieved by varying the nanosphere mask size and chemically manipulating the percentage of doped boron in graphene nanodisks. As a demonstration for biosensing, the binding interaction of L-selectin with its aptamer in aqueous environment has been in situ monitored to evaluate the specific protein recognition process. Our work shows that chemical doped-graphene plasmon combined with ATR-SEIRA is a promising signal enhancement platform for in situ aqueous-phase biosensing. ■EXPERIMENTAL SECTION Reagents and Materials. The hemispherical ZnSe ATR prism (the diameter of 20 mm) was bought from Qing Xuan Technology Co. Ltd. (China). Phenylboronic acid and anisole were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Polymethyl methacrylate (PMMA) and bovine serum albumin (BSA) were bought from Sigma-Aldrich. L-selectin was purchased from Sino Biological Inc. (Beijing, China) and used without further purification. The L-selectin aptamer, 5’-SH C6 GCC AAG GTA ACC AGT ACA AGG TGC TAA ACG TAA TGG CTT-3’, was custom synthesized from Sangon Biotechnology Co., Ltd. (Shanghai, China). Hemoglobin was obtained from Boster Biological Technology., Ltd. (Wuhan, China). Polystyrene spheres (PS) were bought from Duke Scientific, Inc. 4


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(Shanghai, China). Other reagents were of analytical grade and used without further purification. All solutions were prepared with Millipore water (Purelab Classic Corp., USA). Characterizations. Infrared transmittance spectra were measured on a Thermo Fisher Nicolet 6700 FTIR spectrometer equipped with a liquid-nitrogen-cooled MCT detector. All spectra were recorded at 4000 ~ 400 cm−1 with the resolution of 4 cm-1. ATR-SEIRAS spectra were collected on the Thermo Fisher Nicolet 6700 FTIR spectrometer with a homemade ATR accessory (Figure S1). Unpolarized IR radiation was totally reflected at the hemispherical ZnSe prism/solution interface with an incident angle θ=75° and was detected with a liquid-nitrogen-cooled MCT detector. An ATR-SEIRAS spectrum without introducing the target biomolecule was used as the background. Scanning electron microscopy (FE-SEM, S-4800, Hitachi) was used to characterize the morphology of graphene disk array without spraying gold conducting film at an accelerating voltage of 5 kV. Transmission electron microscopy (TEM) and HRTEM using a Hitachi-2100 TEM facility with a 200 kV accelerating voltage were further used to characterize the morphology of graphene sample. The atomic force microscopic (AFM) image was performed on Fastscan AFM (Bruker, Inc.) using ScanAsyst mode in air. Raman spectra were collected on a Renishaw InVia micro-Raman system with an excitation wavelength of 488 nm (Renishaw, UK). The laser spot size was about 1 μm X-ray photoelectron spectroscopy (K-alpha, USA) was analyzed on a Thermo Fisher X-ray photoelectron spectrometer system. UV-vis spectroscopy was conducted by Nanodrop-2000C spectrometer (Thermo Fisher Scientific). O2 plasma etching was performed on a ULVAC CE300I (Japan, ULVAC). Synthesis and transfer of boron-doped graphene. Boron-doped graphene (BG) was grown on copper foil via chemical vapor deposition method.18 Copper foil (99.8 % purity, 25 μm thick) was cleaned by acetone, ethanol, 10% HCl and water in sequence, and then loaded in the center of a furnace. Phenylboronic acid power (30 mg) was placed upstream at the center of mini-heater. The copper foil was annealed at 1000 oC for half an hour under 10 sccm H2 with a pressure of 16 Pa, then the furnace was cooled down to the growth temperature. In order to tune the boron doping level, the growth temperature was manipulated at 800 and 950 oC, respectively. Phenylboronic acid powder was sublimated at 130 oC and the vapor was transported to the copper foil by H2 carrier. The growth was 5



maintained for 20 min. After growth, turn off the heater power, move away the furnace quickly, keep the H2 flowrate and cool to room temperature. Boron-doped graphene films were transferred onto SiO2 (300 nm)/Si or glass substrates using the wet PMMA assisted transfer procedure. The BG/Cu was spin-coated by a PMMA layer (100 μL, 4% in anisole, 3000 rpm for 1 min). Then, the Cu foil was etched by Marble’s reagent (CuSO4: HCl: H2O=10 g: 50 mL: 50 mL) for 2 h. The residue Cu of the resulted film was further removed by 0.05 M (NH4)2S2O8 solution. Next, the PMMA/BG film was rinsed by water for several times to remove the etchant residue. After rinsing, the PMMA/BG film was picked up by the cleaned substrate and dried at room temperature. Finally, the PMMA was removed by acetone-rinsing and annealing at 400 oC under the mixed gas of Ar (180 sccm) and H2 (20 sccm) at 185 Pa. It is noted that in many literature, the PMMA was later removed with acetone after the transfer of PMMA/graphene.19-23 Here, we found that PMMA residues still obviously appeared by only acetone-rinsing (red circle in Figure S2). The PMMA residues will deteriorate the quality of graphene. Thus, some published works adopted 500 °C to remove the protective polymer layer atop the graphene.24,25 However, annealing would reduce the doping level of the graphene. Hence, in our work, after acetone-rinsing, the relatively low temperature of 400 ℃ was applied to remove the protective polymer layer and keep the doping level. Preparation and transfer of the boron-doped graphene nanodisk array. A monolayer of polystyrene spheres (diameter of 200 nm) was deposited on the BG-coated glass substrate. Then, O2 plasma was employed to etch the PS spheres and the exposed graphene using a ULVAC CE300I (The main parameters are APC Press2.60 Pa, Trigger Press 3.50 Pa, PFC Press 300 Pa, MFC22 flow (O2) 10 sccm, Antenna RF Power 100 W, Antenna AMC NO. 1 Pos, Bias RF Power 10 W and Bias AMC NO. 1 Pos.). After sonication in toluene and annealing (400 oC, Ar/H2=180 sccm/20 sccm) processes, the residue PS was removed. Periodically BG nanodisk array was eventually fabricated on the glass substrate surface. Note that less procedures will reduce the defect. But, O2 plasma etching on the metal substrate in the chamber of instrument (ULVAC CE300I, Japan) is prohibited. Thus, we first transfer the BG film from Cu foil to a glass substrate, then etch it into BG nanodisk array. The transfer procedure of BG nanodisk array from the glass substrate to ZnSe substrate is as following. 6


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Spin a PMMA (100 μL, 4% in anisole, 3000 rpm for 1 min) layer onto the BG nanodisk array/glass, then, dry it at 100 oC for 5 min. The film of PMMA/BG nanodisk array will float at the surface of the solution by etching the glass using HF/H2O solution. The resulting film was rinsed by water for several times. After that, the film was pick up with a cleaned ZnSe substrate (plate or hemisphere) and dried at atmosphere. Finally, the BG nanodisk array deposited on ZnSe substrate was obtained by removing the PMMA by acetone-rinsing and annealing (400oC, Ar/H2 =180 sccm/20 sccm). Fabrication of the graphene based IR sensor. BG nanodisk array on glass substrate was transferred onto a hemishperical ZnSe prism by PMMA-assisted method as described above and installed in a homemade accessory seen in Figure S1. This sensing interface was then immersed in 250 nM L-selectin aptamer + 1 M NaCl solution overnight to create a homogeneous DNA layer captured by BG as a result of the strong π-π interaction and van der Waals forces between graphene and DNA. Next day, remove the solution in the container by microsyringe carefully. Then rinse the surface for 5 min by injecting pure PBS buffer solution. After washing, add 300 μL 1% BSA into the detection cell, the uncoated sites were blocked with BSA. Subsequently, the sensing platform was washed by injecting PBS carefully to eliminate weakly bound biomolecules. The background spectrum was recorded in 400 μL 50 mM PBS (pH=7.4). Finally, the target protein injected into the solution was specifically captured to the sensing interface, generating featured absorption bands. Simulation. Numerical simulations were performed by a commercial finite-element method (FEM) software package (COMSOL Multiphysics, Burlington, Mass., USA) to theoretically explore the optical properties of the structure. In the calculations, the refractive index of zinc selenide is assumed to be 2.4. Considering the contribution from both the intraband and the interband transitions, we used the wellknown Kubo formula to describe the conductivity (σ) of graphene with temperature T=300 K and scattering rate Γ=0.11 meV. Thus, the corresponding complex permittivity of graphene can be expressed as: εg=1+iσ/ (ωε0dg), where ε0 is the permittivity of vacuum. The thickness of the monolayer graphene (dg) is set as 0.5 nm. Periodic boundary conditions are employed to mimic periodicity. ■RESULTS AND DISCUSSION Design of boron-doped graphene plasmonic biosensor. Aptamers due to its small size and high 7



stability are often regarded as molecular recognition probes for the protein.26-28 Herein, we select Lselectin and its aptamer as model system to investigate their interaction in aqueous environment using graphene plasmonic enhancement substrate by ATR-SEIRAS. As illustrated in Scheme 1, the entire experimental design includes the following procedures: (1) preparation of the BG nanodisk array by NSL, (2) transfer of the BG nanodisk array to ZnSe prism, and (3) construction of biosensor using a model system of L-selectin protein with its aptamer in water.

Scheme 1. Schematic illustration of preparation of boron-doped graphene plasmonic biosensor.

Characterizations of the boron-doped graphene. In mid-infrared region, doped graphene exhibits distinct optical properties, including long propagation length, low intrinsic damping, extreme field confinement and enhancement.29 In the present work, we study the plasmon properties of chemical doped-graphene nanodisk array. First of all, boron-doped graphene is synthesized by chemical vapor deposition (CVD) using phenylboronic acid (C6H7BO2) as the sole precursor illustrated in Figure 1a as previously reported. 18 We find that the boron doping level can be easily tuned by manipulating the growth temperature. However, it should be noted that lower temperature results in poorer quality of graphene, e.g., a non-uniform film is observed at the growth temperature of 700 oC (Figure S3). This phenomenon has been observed previously.19 Thus, to synthesize high quality graphene, the graphene growth was performed at 800 and 950 oC, respectively. The resulting graphene films are denoted as BG800 and BG-950, respectively. Furthermore, we tried to vary boron doping by the addition of boric acid. But, the results show that the repeatability and uniformity are poor. In order to prepare more homogeneous and high-quality boron-doped graphene, phenylboronic acid is used as the sole precursor. The optical images of the two samples (Figure S4a and b) reveal large-area, continuous and homogenous 8


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films of graphene deposited on SiO2/Si surfaces. A clear boundary line (in red dashed frame of Figure S4a) is seen between graphene and SiO2/Si substrate. The Raman spectra of BG-800 and BG-950 samples (Figure S4c) both display a prominent D band (∼ 1351 cm-1) and G band (~1596 cm-1). The 2D band of BG-800 sample shows a slight red-shift (~ 2704 cm-1) compared with that of BG-950 sample (2709 cm-1). The intensity ratio of the D to G bands of the BG-800 sample (~0.94) is also larger than that of the BG-950 sample (~0.66). We attribute these phenomena to the increase of elastically scattered photoexcited electron from more boron atoms incorporated in the graphene lattice. For the doped graphene, the I 2D /I G ratio will decrease compared to that of pristine graphene (~2).30-33 As the increase of the doping level, the intensity ratio of the 2D to G bands of the BG-800 sample (~0.71) is lower than that of the BG-950 sample (~0.92). The digital photo of the BG-800 film transferred onto a quartz substrate is displayed in Figure 1b (Inset, BG film in blue dashed frame). The ultraviolet-visible spectroscopy (UV-vis) was conducted to reveal the optical transparency of the BG-800 sample on quartz substrate. Taking blank quartz as the reference, the UV-vis spectrum exhibits an optical transmittance of ~97.1 % at 550 nm, which is comparable to the pristine monolayer graphene. Transmission electron microscopy (TEM) was applied to observe the microstructure of the as-synthesized graphene. As displayed in Figure 1c, the imaged graphene edge shows one carbon layer, demonstrating the monolayer nature of our BG film. 18 Selected area electron diffraction (SAED) in Figure 1d presents a single set of hexagonal symmetry, implying the single-crystalline nature of the detected domains. X-ray photoelectron spectroscopy (XPS) measurements were performed to analyze the chemical composition and bonding form of the BG films. The B1s peak of phenylboronic acid precursor is located at 192.3 eV (Figure S5). For the BG-800 sample, the B1s peak appears at ~190.9 eV which can be ascribed to the main bonding form of BC3 (Figure S6a). The B atomic percentages from three random positions of each film are presented in Table S1. It is clear that the boron content increases with the decrease of growth temperature. But remarkably, the boron doping in our graphene films is not very homogeneous, which does not agree with the observations from Liu and the coworkers.18 As for C atoms, the pronounced C1s peak at 284.6 eV can be assigned to the sp2-C bonding and the small peak at 286.2 eV in the C1s spectrum corresponds to C-O species (Figure S6b). 9



Figure 1. (a) Schematic diagram of preparation for boron-doped graphene by CVD method. The red, grey, yellow, and greyish white spheres represent boron, carbon, oxygen and hydrogen atoms, respectively. (b) Digital photo of BG-800 on a quartz (Inset, in blue rectangle frame) and corresponding UV-vis spectrum. (c) HRTEM image of BG-800 edges that shows one carbon layer on a TEM grid. (d) Hexagonal SAED pattern of the monolayer BG-800. All the above characterization results suggest that large-area boron-doped graphene films have been successfully synthesized. Subsequently, NSL technique was employed as template to obtain large-scale nanostructured BG arrays illustrated in Figure S7. After the BG film is transferred to SiO2 (300 nm thickness)/Si or glass substrates, a self-assembly monolayer of PS nanosphere (200 nm diameter) is packed on top of the BG film. Then, they are exposed in O2 plasma for etching, resulting in periodically arranged BG nanodisk array after removing the residue of PS spheres with sonication and annealing processes. Figure 2a and b show the SEM images of BG nanodisk array on the SiO2 /Si substrates prepared using different plasma etching time. The BG nanodisk arrays with the diameter of about 100 nm and 60 nm correspond to etching time of 90 s and 110 s, respectively. It is clear that the BG nanodisks are highly arranged. Raman characterizations reveal that the D band (1355 cm-1), G band (1598 cm-1) 10


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and 2D band (2709 cm-1) of the structured BG-800 sample (Figure 2c) show slight upshifts of 4 cm-1, 2 cm-1 and 5 cm-1, respectively, as compared to the pristine unstructured BG-800 sample. In addition, the ID/IG ratio (~1.02) for the nanodisk array increases, which may be resulted from the rich variation on the boundaries of graphene nanodisks and defects introduced by etching process. The atomic force microscopic (AFM) image of the nanodisk array with the corresponding AFM height profiles is shown in Figure 2d and e. The BG nanodisks have a uniform thickness of 1.5 nm, which is similar to the monolayer graphene nanoribbon array.7

Figure 2. SEM images of the BG nanodisk array with the diameters of 60 and 100 nm obtained under plasma etching for a duration of 110s (a) and 90s (b), respectively. (c) Raman spectrum of the BG nanodisk array shown in (a). (d) AFM image of the BG nanodisk array. Disk diameter is about 60 nm. (e) Line-scan profile of the image shown in (d), corresponding to the white line. The BG nanodisks have a thickness of 1.5 nm. (f) SEM image of BG nanodisk array transferred from glass onto ZnSe plate.

Optical properties of the boron-doped graphene nanodisk array. As demonstrated by theory and experiments,34-37 graphene plasmon is sensitive to the surround dielectric environment. Moreover, phonon of the substrate at mid-IR region, e.g., SiO2 at 806 and 1068 cm-1, will severely couple with the 11



graphene plasmon, leading to strongly impair the electromagnetic energy on the top surface of graphene as reported by Hu. 7 Compared with SiO2/Si, ZnSe has lower cut-off wavenumber and absence of surface phonons in mid-IR range, making it helpful for probing molecular vibrations in the fingerprint range. Hence, ZnSe is chosen as a mid-IR spectral window to carry out subsequent experiments. Since it is inconvenient to directly fabricate nanodisk array on top of a prism, the as-synthesized BG nanodisk array on glass substrate is artificially transferred to ZnSe surface (details refer to Methods). As the SEM image shown in Figure 2f, well-ordered BG nanodisk array is successfully transferred onto the ZnSe substrate. Hence, our transfer method is beneficial to the fundamental research and application of optical and electric fields. The mid-infrared transmittance response of the BG nanodisk array on ZnSe substrate was measured on a Fourier-transform infrared spectrometer (FTIR). A bare ZnSe substrate was used to collect the background spectrum. As shown in Figure 3a, the FTIR spectra of the BG nanodisk arrays with plasma etched for 90 s and 110 s exhibit prominent plasmonic resonances around 510 cm-1 and 613 cm-1 denoted in the pink shadows, respectively, which is obviously distincted from the spectrum of bare ZnSe region (Figure S8). Although the exact Fermi level (Ef) of the structured graphene is hard to be measured, it can still be inferred to be approximate -0.2 eV from the numerical simulation as shown in the pink area of Figure 3b, which match well with the experimental results. The simulated results also suggest that the plasmonic resonance frequency of BG nanodisk shifts to higher frequency with the increase of the Fermi level when setting the same size. In addition, with the same Fermi energy of the nanodisk arrays, the position and intensity of the resonances are sensitive to the nanodisk diameter as displayed in Figure 3a, showing that the resonance peak blue-shifts with the decrease of nanodisk diameter, which is well consistent with the numerical simulations in Figure 3c. This phenomenon is also in good agreement with the previous report.25,38 The broaden peak observed experimentally might be attributed to nonuniform disk size fabricated by imperfect micro-processing. Our experimental results (Figure 3d) exhibit that the resonance peak of BG-800 nanodisk array is located at 543 cm-1, with a blue-shift of 33 cm-1 as compared to that of BG-950 nanodisk array (510 cm-1), which is in agreement with the tendency of the numerical simulations (Figure 3b), indicating that graphene plasmonic resonance frequency is 12


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tunable via chemical doping. For practical application of the graphene plasmonic resonance in real-time and in situ analysis, the IR absorptions of the unstructured and nanostructured BG samples at total reflection mode were measured. As shown in Figure S9, a prominent peak at about 625 cm-1 is observed for the BG nanodisk array, which could stem from the localized surface plasmonic resonance; while no obvious peak appears for the unstructured BG. Simulation was further carried out to explore the optical property of the nanodisk (D=60 nm, Ef= -0.2 eV) on ATR mode (Figure S10). Comparing the numerical simulation with the experimental results (Figure S9), their resonance frequencies are similar, but the resonance linewidth of the experimental result is broadened due to imperfect micro-fabrication.

Figure 3. (a) FTIR spectra of the BG-950 nanodisk array with the diameters of 60 and 100 nm on the ZnSe substrates using 110 s (red line) and 90 s (black line) plasma etching of the monolayer PS colloidal crystal template. (b) Calculated plasmonic resonances of individual BG nanodisk with different Ef. (The 13



disk diameter is set to be 100 nm). The resonance frequency locates at 418, 503, 561, 597, 640 and 680 cm-1 corresponding to Ef at -0.15, -0.20, -0.25, -0.30, -0.35 and -0.40 eV. (c) Calculated plasmonic resonances of individual BG nanodisk with different disk diameters (Ef is set to be -0.2 eV). The resonance frequency locates at 740, 617, 540, 503 and 440 cm-1 corresponding to the diameters of 40, 60, 80, 100 and 120 nm. (d) FTIR spectra of the BG-800 and BG-950 nanodisk array with the diameter of 100 nm. The resonance peak locates at 543 and 510 cm-1, respectively. Further numerical simulations were carried out to theoretically investigate the optical response of the BG nanodisk. As shown in Figure 4, a large electric field enhancement occurs near the edges of BG nanodisk. In addition, the electric field intensity obviously decreases as the mode order increases. Distributions of the z-component of the electric field are shown in Figure S11. As the bond vibrational absorption is proportional to the square of the near-field intensity, the electromagnetic field from the fundamental and higher-order harmonic modes could contribute to the IR absorption enhancement.25

Figure 4. Simulation of electric field distribution of the BG nanodisk. (a) Normalized electric field distribution of the fundamental (left, 617 cm-1) and second-order (right, 1234 cm-1) harmonic modes of a BG nanodisk with the diameter of 60 nm. (b) Normalized electric field distribution of the fundamental

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(left, 503 cm-1) and second-order (right, 1006 cm-1) harmonic modes of a BG nanodisk with the diameter of 100 nm. (Ef is set to be -0.2 eV) Design and fabrication of graphene based IR sensor. As simulation reveals (Figure S12), the enhanced electromagnetic field around the graphene antenna decays rapidly within several tens of nanometers,16 thus extreme subwavelength detection distance can be achieved to improve the sensitivity and detection limit. Herein, we take the BG nanodisk array on top of a ZnSe prism as the enhanced IR sensing interface to monitor the immune-recognition process for protein bioassays at ATR mode in aqueous solution shown in Scheme 1. The model system consists of L-selectin and its aptamer. Aptamers are single stranded DNA or RNA molecules with a small size and high stability toward external change, and have been extensively applied in protein bioassays. The targeted L-selectin protein captured by the immobilized aptamer can be drawn onto the vicinity of the sensing platform.39 The detailed preparation of graphene-based IR sensor has been described in Methods. The band of P=O vibration located at 1260 cm-1 in DNA with a large IR absorption cross-section provides us a feasible way to monitor the interaction between DNA and the sensing interface of BG nanodisk array. The peak intensity increases with the immobilizing time (from bottom to top, Figure S13a), demonstrating that the DNA molecules are captured on the structured BG-ZnSe sensing interface. In order to estimate the enhancement effect of the unstructured and structured BG modified ZnSe substrates, the same amount of DNA was added to the solution and the IR spectra were collected. It can be seen in Figure S13b that much larger P=O signal is observed on the structured BG-ZnSe sensing interface as compared to the one on the unstructured BG-ZnSe sensing interface. From the peak intensity ratio of these two sensing interfaces, an apparent enhancement factor of 5 is estimated. This indicates that BG nanodisk array is an ideal sensing interface for enhanced IR detection. On the other hand, the water molecules at the sensing interface will be expelled because of the adsorption of DNA molecules, which also enables to investigate the molecular interaction by observing the change of interfacial water due to its extremely large extinction coefficient in the mid-infrared region.40 As shown in Figure S13b, the emergency of a remarkably negative peak (ν (OH) band around 3000-3500 cm-1) of water molecules indicates that DNA molecules have been successfully deposited on the sensing interface via the π-π 15



interaction and van der Waals forces through the replacement of surface water molecules. 41 From the absorption band for replaced water molecules, it is clear that more DNA molecules are deposited on the unstructured BG film due to the larger surface area. Thus, the apparent enhancement factor of 5 is certainly underestimated. If the surface area of the sensing interface is considered, the enhancement factor becomes larger (~60). To further explore the effect of different surfaces, ZnSe also serves as the sensing platform. The results in the Figure S13b (blue curve) show that the weakest signal is observed on the bare ZnSe because DNA molecules mainly exists in the bulk solution which are far away from the field of evanescent wave. As depicted in the preceding paragraphs, signal enhancement is extremely confined to immediate vicinity of the enhanced sensing interface in ATR-SEIRAS. Thus, only molecules specifically associating with the sensing interface can be examined. The species from the surrounding (e.g., bulk molecules and water) in the ATR-IR analysis are substantially eliminated. When the target protein is introduced, it will be attracted to the sensing interface via the specific recognition reaction with the immobilized aptamer. The real-time ATR-SEIRAS spectra (Figure 5a) reveal the immunoreaction kinetics of L-selectin to its aptamer at the sensing interface. It can be seen that the characteristic amide bands at 1640 and 1548 cm-1 gradually increase as the recognition occurs. The amide II vibration at 1548 cm-1 is attributed to the C-C and C-N stretching vibrations, which can be employed for protein quantitation.39 The peak intensity of the amide II band is plotted as a function of binding time, which is displayed in Figure 5b, showing a typical character of interfacial immunoreaction. The amide II band intensity increases rapidly within the early 10 min; then, it immediately reaches an equilibrium state after 20 min, implying a fast kinetics for the aptamer-protein recognition reaction. Hence, spectra were recorded at 30 min after the addition of target protein, which ensures all the IR signals measured at the equilibrium state of the immunoreaction.

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Figure 5. (a) Evolution of SEIRA spectra as a function of immunoreaction time in 50 nM L-selectin + 50 mM PBS solution (pH = 7.4) on the aptamer modified BG nanodisk array (D= 60 nm). (b) The intensity of amide II band (blue dots) calculated from the spectra versus immunoreaction time. The red line is a fitting curve.

In order to evaluate the specific identification, control experiment was carried out. When adding the nonspecific hemoglobin sample, no detectable IR absorption signal is observed (Figure S14), suggesting the target protein could be discriminated from the other proteins.

A further investigation on the immunoreaction with different concentrations of L-selectin was performed (Figure 6a). Fitting the intensity of amide II bands (red line in Figure 6b), a nonlinear increase in amide II band intensity as a function of target protein concentration is observed. The detection limit reaches down to 0.5 nM. The association equilibrium constant was calculated to be (3.1±0.5) x108 M-1 via simulating the Langmuir adsorption isotherm,42 indicating strong affinity of the aptamer-protein interaction.

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Figure 6. (a) ATR-SEIRA spectra of protein recognized by the immobilized aptamer with different Lselectin concentrations. The concentrations are: 33.33, 28.58, 23.08, 16.67, 6.98, 4.77, 1.96, 0.99, 0.50, and 0 nM (from top to bottom). All the spectra were collected after 30 min of immunoreaction. (b) The intensity of amide II band (black square) calculated from the spectra in (a) versus L-selectin concentration. The red line is the fitting curve. The BG nanodisk array with the diameter of 60 nm was used as the platform. ■CONCLUSIONS As demonstrated in our experiments, boron doping level, to some extent, can regulate the electronic structure and tune plasmonic resonances of graphene nanodisks. However, two issues should be considered for chemical doping. On one hand, homogeneous doping is still a challenge for synthesis. On the other hand, the boron atoms incorporated into graphene lattice may serve as strong scattering centers, leading to deterioration of the carrier mobility. These two factors may affect the plasmonic properties of graphene. Proper amount and distribution of dopant could contribute to the improvement of the quality factor. Thus, further optimization of the synthesis procedures should be performed in the next stage. Graphene plasmonic detection is usually investigated when the graphene plasmonic resonance matches with the frequency of the molecular vibrations, exhibiting plasmon-induced transparency features.7,15,16 Recently, Loh’s group25 has demonstrated that the IR enhancement could be achieved for the molecular vibration mode far from the plasmonic resonance. They ascribed this phenomenon to the electric field enhancement of higher order harmonic resonances. In our work, the ultrahigh 18


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electromagnetic field confinement of the fundamental and higher-order harmonic plasmonic resonances of boron-doped graphene nanodisk array could be responsible for the spectral signal amplification. This will be helpful to deepen the understanding of the mechanism of surface enhanced infrared spectroscopy. In conclusion, we propose a low-cost and efficient graphene plasmon enhanced IR biosensor based on boron-doped graphene nanodisk array. The advantage lies in that biomolecules in aqueous solution can be in situ and label-free identified by the near-field enhancement effect of chemical doped-graphene plasmon combined with ATR-SEIRAS technique. Our work presents a novel application of graphene plasmonics in aqueous solution and offers a promising sensing platform for investigating interfacial biorecognition reactions and structure-function relationship of biomolecules. ■ASSOCIATED CONTENT Supporting Information The supplementary information is available free of charge via the Internet at http://pubs.acs.org. ■AUTHOR INFORMATION Corresponding Author: *E-mail: [email protected] ORCID Xing-Hua Xia: 0000-0001-9831-4048 Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (21327902, 21635004), Nanjing University graduate student research innovation fund (2015CL6)，the State Key Program for Basic Research of China (SKPBRC, 2013CB632703) and the Open Research Fund of State Key Laboratory of Bioelectronics of Southeast University. ■REFERENCES (1) Huck, C.; Neubrech, F.; Vogt, J.; Toma, A.; Gerbert, D.; Katzmann, J.; Hartling, T.; Pucci, A. ACS Nano 2014, 8, 4908-4914.

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Graphic abstract 333x161mm (72 x 72 DPI)


Graphene Plasmon-Enhanced IR Biosensing for in Situ Detection of

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