N- and B-Codoped Graphene: A Strong Candidate To Replace

3 days ago - (8−14) Among them, transition metal oxide-based nanozymes have a .... higher ID/IG intensity ratio with a D band wider than that of NB-...
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N- and B‑Codoped Graphene: A Strong Candidate To Replace Natural Peroxidase in Sensitive and Selective Bioassays Min Su Kim,†,∥ Seongyeon Cho,‡ Se Hun Joo,§ Junsang Lee,∥ Sang Kyu Kwak,*,§ Moon Il Kim,*,‡ and Jinwoo Lee*,∥ ACS Nano Downloaded from pubs.acs.org by OCCIDENTAL COLG on 03/27/19. For personal use only.

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Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk 37673, Republic of Korea ‡ Department of BioNano Technology, Gachon University, Seongnam, Gyeonggi 13120, Republic of Korea § Department of Energy Engineering, School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea ∥ Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea S Supporting Information *

ABSTRACT: The work describes a carbon-based peroxidase mimic, N- and B-codoped reduced graphene oxide (NB-rGO), which shows high peroxidase-like activity without oxidase-like activity and has a catalytic efficiency nearly 1000-fold higher than that of undoped rGO. The high catalytic activity of NB-rGO is explained by density functional theory by calculating Gibbs free energy change during the peroxide decomposition reaction. Acetylcholine and C-reactive protein are successfully quantified with high sensitivity and selectivity, which were comparable to or better than those obtained using natural peroxidase. Furthermore, NB-rGO, which does not have oxidase-like activity, is proven to have higher sensitivity toward acetylcholine than Pt nanoparticles having oxidase-like activity. This work will facilitate studies on development, theoretical analysis for rational design, and bioassay applications of enzyme mimics based on nanomaterials. KEYWORDS: graphene, enzyme mimic, density functional theory, biosensors, immunoassays

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including extraction from horseradish roots and subsequent purifications, which makes HRP expensive. Nanomaterials that have enzyme-like characteristics (nanozymes) may provide alternatives to natural peroxidases.4−7 Nanozymes are superior to their counterpart natural enzymes in some ways, such as high stability and low cost; however, known nanozymes have catalytic activity and selectivity lower than that of natural enzymes. Therefore, an increase in the catalytic activity of nanozymes and improvement of selectivity are the main research topics in this field. After discovery of the first peroxidase-like nanozymes, Fe3O4 nanoparticles (NPs), many different inorganic nanomaterials including those made of transition metal oxides (e.g., CeO2,

eroxidases have been one of the main subjects of biochemical research for centuries.1,2 They catalyze oxidative reactions of various substrates by transferring electrons to peroxide species to produce hydroxyl radicals which may have important functions in biological systems.3 These natural enzymes have been found in most living organisms and are involved in various biological oxidoreductive processes. Among them, horseradish peroxidase (HRP) is representatively used in bioassays; it is generally produced from a plant and is commonly used as a signaling enzyme for the detection of important molecules in immunoassays, including enzyme-linked immunosorbent assay (ELISA) and oxidase-coupled biosensors. HRP has many merits as a signaling enzyme, such as small size, high turnover rate, and facile conjugation with other biological receptors. However, it still has several unresolved drawbacks. First, natural instability of HRP often limits its utilization during operation or after storage. Also, production of HRP needs many procedures © XXXX American Chemical Society

Received: December 17, 2018 Accepted: March 25, 2019 Published: March 25, 2019 A

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ACS Nano Scheme 1. Schematic Illustration of NB-rGO Reactions in Bioassays

Figure 1. (a) TEM image, (b−d) elemental mappings, and (e) XPS survey with high-resolution spectra of N, B, and C 1s core levels of NBrGO.

toward H2O2, which would be produced from the catalysis of oxidase toward the target molecules.5,17 In this case, noble metals having both peroxidase as well as oxidase-like activity are not suitable due to their inability to selectively detect H2O2. For this reason, peroxidase-like nanozymes having negligible oxidase-like activity are critically important. Carbonbased nanomaterials are the cheapest to produce but have catalytic activity lower than that of transition-metal- or noblemetal-based nanozymes. Carbon nanomaterials such as graphene oxide (GO) and carbon nanodots also have abundant carboxyl functionalities on their surface, which is highly beneficial to conjugate bioreceptors including antibody molecules.18−21 Therefore, if the peroxidase-like activity of

Co3O4, V2O5, and Mn3O4) and noble metals (e.g., Pt, Pd, and Au) or carbon having sp2 configuration have been studied.8−14 Among them, transition metal oxide-based nanozymes have a moderate level of catalytic activities and prices but require surface functionalization to conjugate antibody molecules before they can be useful in immunoassays.4,15 Noble metals have relatively high peroxidase activities but are expensive and generally also have oxidase-like activities, so they may oxidize substrate even if H2O2 is absent.12,16 Because the selective detection of H2O2 is of crucial importance in many biosensing systems, the dual enzyme-like activities critically hinder their usability. For example, peroxidase involved in oxidase− peroxidase-mediated cascade reactions should react only B

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treated in a single step, 27 atom % of N and 31.2 atom % of B were present in the synthesized sample; this result means that h-BN was formed during pyrolysis (Figure S6 and Table S1). Furthermore, in the Raman spectra, h-BN-rGO yielded higher ID/IG intensity ratio with a D band wider than that of NB-rGO; these traits are further support for the hypothesis that h-BN formed when the rGO was doped with B before it was doped with N (Figure S7).22 Although there were reducing conditions during the reduction steps, carboxylic groups of NB-rGO for antibody conjugation and for dispersion in aqueous solution were preserved (Figure 1e and Table S2). Also, it was confirmed that ζ-potential of NB-rGO was −39.3 ± 1.7 mV in DI water (Figure S8). The ζ-potential values, which are more positive than +30 mV or more negative than −30 mV, are generally considered to represent sufficient electrostatic repulsion to ensure the dispersion stability, as is generally known from colloidal science.28 Investigation for the Peroxidase-like Activity of NBrGO. We next investigated the catalytic activities of the prepared NB-rGO by performing the peroxidase-mediated oxidation reactions of 3,3′,5,5′-tetramethylbenzidine (TMB) and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in the presence of H2O2. The peroxidase activity of NB-rGO was compared with that of B-rGO, N-rGO, P-doped graphene (P-rGO), S-doped graphene (S-rGO), N- and Pcodoped graphene (NP-rGO), N- and S-codoped graphene (NS-rGO), GO, and undoped rGO (Figure S9). The NB-rGO had highly increased oxidation rates for both TMB and ABTS and, consequently, much more intense colorimetric responses than the other rGO species (Figure S10). This result confirms that coupling N and B with rGO significantly increases its peroxidase activity. Preliminary studies of the effects of pH, kinds of buffer, H2O2 concentration, and temperature T on the catalytic activity of NB-rGO determined that pH 4.0, sodium acetate buffer, 100 mM H2O2, and 37 °C were ideal assay conditions (Figure S11). Incubation at temperature T = 37 °C resulted in the highest peroxidase activity, but incubation was conducted at room temperature rather than 37 °C because of the practical convenience; the catalytic performance of NB-rGO at room temperature was >70% of that at 37 °C. NB-rGO was stable at 2 ≤ pH ≤ 9 and 20 ≤ T ≤ 90 °C. In contrast, HRP was not stable to lose most of its activity at pH < 5 and T > 37 °C. This stability of NB-rGO suggests that it may be a viable replacement for HRP in diverse fields of biotechnology including bioassays (Figure S12). To further demonstrate the catalytic mechanism, steadystate kinetic parameters of NB-rGO were calculated and compared with those of B-rGO, N-rGO, BN-rGO, h-BN-rGO, and rGO. Plots of initial reaction velocities against diverse concentrations of TMB and H2O2 showed typical Michaelis− Menten curves (Figure S13). The curves were then used to obtain Lineweaver−Burk plots to determine kinetic parameters (Figure S13 and Table S3). The apparent Km values of B-rGO and N-rGO for TMB were about 800% higher and 8% lower, respectively, than that of rGO; this comparison indicates that B-doping significantly decreases the affinity of rGO for TMB, whereas N-doping increases its affinity. Consequently, NBrGO or BN-rGO resulted in ∼230% higher Km value to that of rGO, presumably as a result of the combined effects of N- and B-doping. Interestingly Km of h-BN-rGO was ∼60% of Km of rGO; this difference means that hexagonal additives on the

carbon nanomaterials can be increased to the level of nanozymes based on metal oxides or noble metals, or even to that of HRP, these carbon nanomaterials will be a promising candidate to replace HRP for practical uses. We have developed N- and B-codoped reduced graphene oxide (NB-rGO) as a peroxidase-mimicking carbon nanomaterial that shows catalytic activity much higher than that of conventional peroxidase-mimicking carbon-based nanozymes and is even comparable to that of HRP. Carbon nanomaterials codoped with heteroatoms constitute a class of materials used in electrochemical applications including fuel cells, energy storage devices, etc., but these materials have not been successfully applied as nanozymes in bioassays.22−24 Here, we describe fabrication of NB-rGO, demonstrate its peroxidase-like activity, and evaluate applications to detect important biomarkers in bioassays. We also used density functional theory (DFT) analysis to explain theoretically why NB-rGO has peroxidase activity higher than that of several related types of doped rGO (Scheme 1).

RESULTS AND DISCUSSION Synthesis and Characterization of NB-rGO. We synthesized NB-rGO from GO that had been produced using a modified Hummers method.25 N-doped graphene (NrGO) was prepared (Figure S1) with a solid-state reaction of GO with melamine by heat treatment under Ar, and boron (B) was then introduced by pyrolysis of the N-rGO with boric acid under Ar. As controls, rGO, N-rGO, B-doped graphene (BrGO), N- and B-codoped graphene with reverse doping order (BN-rGO), and a hexagonal boron nitride−graphene composite (h-BN-rGO) were also prepared. Graphene doped with N followed by B was the most efficient peroxidase mimic due to their synergistic effects. The prepared samples were characterized using transmission electron microscopy (TEM), high-resolution TEM (HRTEM), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). As shown in TEM and AFM images, nanosheet morphology with a thickness of 1 nm or less was well preserved after solid-state reactions (Figures S2 and S3). N and B were homogeneously distributed in the graphene framework (Figure 1a−d). High-resolution spectra of C, N, and B 1s from XPS analysis show the state of doping contents in NB-rGO (Figure 1e and Table S1). The analysis showed that the heteroatoms in the graphene frameworks synthesized by sequential incorporation of the N and B were bonded to the surrounding C atoms. N atoms are dispersed in pyridinic N (1.83 atom %, binding energy (BE) = 398.1 eV), pyrrolic N (1.38 atom %, BE = 399.4 eV), and quaternary N (0.52 atom %, BE = 401.0 eV).26 The states of N in NB-rGO were close to those in the N-rGO (Figure S3a); that is, B-doping did not displace N atoms. Furthermore, the peak in the B 1s spectrum was symmetrical, which reveals that most of the B atoms were dispersed in the form of a BC3 structure (2.25 atom %, BE = 190.7 eV), not a BC2O structure (0.05 atom %, BE = 192.5 eV).27 The C−N, B−C, and CC chemical bonds have also been observed in the corresponding Fourier transform infrared spectra (FTIR) spectrum (Figure S4). In BN-rGO (i.e., reverse doping order) >30% of B atoms were dispersed in the form of BC2O, which may generate during the B-doping step due to oxygen species in GO (Figure S5). XPS spectra of N 1s and B 1s indicate that sequential doping replaced C species in the graphene framework without forming the byproduct, h-BN. However, when boric acid, melamine, and GO were heatC

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Figure 2. Comparison of the catalytic activities of various nanozymes (GO, Fe3O4 NPs, Co3O4 NPs, Pt NPs, and NB-rGO) and HRP toward (a) TMB and (b) H2O2.4,5,8,29 The values of relative activity per molecules are plotted versus relative activity per mass (see Table S3 for detailed comparison). Note that both axes are logarithmic. (c) Comparison of the catalytic activities of diverse sets of N- or B-doped graphene and HRP toward TMB and H2O2. The values of relative activity toward H2O2 are plotted versus relative activity toward TMB. (d) Absorbance intensities of the blue signal after 3 min oxidation of TMB by Fe3O4 NP (MNP), Pt NP, BN-rGO, and NB-rGO in the presence or absence of H2O2 and (e,f) corresponding photographs. For this reaction, the same amount of MNP, Pt NPs, BN-rGO, and NB-rGO (10 μg/mL) were added into a reaction buffer (sodium acetate, 0.1 M, pH 4.0) containing TMB (500 μM) with or without H2O2 (100 mM). Three independent measurements were performed to obtain error bars showing standard deviations.

smaller Km for TMB (0.149 mM) than does HRP (0.43 mM); that is, NB-rGO has an affinity higher than that of HRP toward TMB, presumably because NB-rGO has higher surface-tovolume ratio than HRP, and high affinity to organic substrates by π−π and hydrophobic interactions than HRP does.4 Among the B- or N-doped rGO, NB-rGO had the highest catalytic activity for both TMB and H2O2 (Figure 2c). Particularly, NB-rGO also had negligible ability to oxidize the TMB substrate in the absence of H2O2. In contrast, Pt NP nanozymes, which are recognized to have the highest peroxidase activity, have considerable catalytic activity to oxidize TMB without H2O2.31 Even Fe3O4 NPs induce color change without H2O2, although they have extremely low activity compared to those of NB-rGO and Pt NPs; oxidaselike activity impairs their selectivity for detection of H2O2. Therefore, considering the high peroxidase activity of NB-rGO for selective detection of H2O2, this nanozyme is a promising signaling probe for bioassays (Figure 2d−f). DFT Analyses To Theoretically Identify Increased Activity of NB-rGO. To understand the microscopic origin of the peroxidase activity of NB-rGO, we performed DFT calculations on rGO, B-rGO, N-rGO, and NB-rGO models (Experimental Section, models and computational details sections; Figure S14). To identify the active sites of each

surface of rGO could facilitate TMB binding, presumably due to increased π−π and hydrophobic interactions.5 Km values of all kinds of doped rGOs for H2O2 were significantly lower than that of rGO. This difference confirms that doping with N or B is effective to increase their affinity to H2O2. B-rGO had the lowest Km, only ∼9% of that of rGO. Although NB-rGO did not have the highest affinity toward TMB or H2O2, its kcat was the highest: ∼700 and ∼320 times higher than those of rGO for TMB and H2O2, respectively; this is the highest increase in kcat that has been reported so far without addition of noble metals.12,29,30 We propose that this increase in kcat of NB-rGO may occur because codoping of N and B on rGO has a synergistic effect, which may significantly increase the electron transfer rate during the peroxidasemediated reaction. BN-rGO resulted in similar Km but ∼30% lower kcat compared with NB-rGO. This comparison confirms that the sequence of doping affects the catalytic efficiency, as previously reported.22 Overall, NB-rGO has a catalytic efficiency (kcat/Km) ∼300 and ∼1000 times higher than that of rGO for TMB and H2O2, respectively; this increase ensures the usefulness of NB-rGO in many applications. In a plot of relative activity kcat per molecule versus relative activity per mass, NB-rGO clusters with Pt NPs and HRP for both TMB and H2O2 (Figure 2a,b). Moreover, NB-rGO has D

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Figure 3. Comparison of peroxidase activity of rGO, B-rGO, N(q)-rGO, N(p)-rGO, and NB-rGO. (a) Gibbs free energy diagram of peroxidase reaction on the basal plane. (b) Gibbs free energy diagram of peroxidase reaction at the edge. (c) Optimized structure of the reaction intermediate (i.e., two adsorbed OH*) in the rate-determining step. Asterisk: adsorbed state of reaction intermediate. Gray, carbon; white, hydrogen; red, oxygen; pink, boron; blue, nitrogen.

the active center for strong adsorption of OH* (ΔGOH*+OH* = −0.49 eV) (Figure 3a,c and Figures S15b and S17), and the peroxidase activity of the C atom at the edge remains intact (Figure 3b,c). Therefore, B-doping increases the number of catalytically active sites and thus increases the catalytic activity of the B-rGO. In N-rGO, N atoms are doped predominantly at the edge in the pyridinic form (N(p)-rGO), but a few occur on the basal plane in the quaternary form (N(q)-rGO). The quaternary N atom activates the C atoms bonded to it (Figure 3a,c and Figures S15c and S18), while preserving the peroxidase activity of the edge C atoms (Figure 3b,c). The quaternary N atom has the same function in increasing the number of active sites as the B atom in B-rGO, even though the adsorption of OH* on the C atoms next to the quaternary N atom (ΔGOH*+OH* = −0.02 eV) is weaker than that on the B atom. In contrast, the pyridinic N atom has little effect on the catalytic activity of the graphene basal plane (Figure 3a,c and Figures S15d and S19) but instead suppresses the peroxidase activity of the doped edge site (Figure 3b,c); that is, the quaternary N rather than the pyridinic N has the greatest effect on the increase in catalytic reactivity, as has been noted previously.22 Interestingly, the N- and B-codoping (i.e., incorporation of B atom next to the pyridinic N atom in BC3 structure) turns the inert pyridinic N atom into a catalytically active center (ΔGOH*+OH* = −0.29 eV) (Figure 3b,c), while preserving the peroxidase activity of the single B atom (Figure 3a,c and Figures S15e and S20). Considering that most of the doped N atoms are in the pyridinic form, the B dopant has an important function in substantially increasing the active site density. Moreover, adsorption of OH* on the B atom next to the pyridinic N atom became much stronger (ΔGOH*+OH* = −0.81 eV) than in any other cases. Overall, the synergistic effect of Nand B-codoping further increased the peroxidase activity over the undoped or singly doped (N or B) graphene; this result

model and evaluate their peroxidase activity, the Gibbs free energy diagram of peroxidase-mediated reaction (Figures 3 and S15) was constructed for several basal plane and edge sites that are potential catalytic active sites (Figures S16−S20). Here, the peroxidase reaction was considered to consist of five thermochemical reaction steps that entail decomposition of H2O2 and electrochemical reaction steps that involve electron−proton pair transfer:32,33 H 2O2 (aq) → OH* + OH*

OH* + OH* → O* + H 2O* O* + H 2O* → O* + H 2O(aq)

O* + H 2O(aq) + (H+ + e−) → OH* + H 2O(aq) OH* + H 2O(aq) + (H+ + e−) → 2H 2O(aq)

The Gibbs free energy diagram of control graphene without any doping (i.e., rGO) showed that the C atoms on the basal plane have a high energy barrier and therefore are catalytically inert (Figures 3a and S15a). The highest Gibbs free energy change (ΔGOH*+OH* = 0.83 eV) occurs during the formation of two adsorbed OH* by chemisorption of H2O2 on the basal plane (Figures 3c and S16), so this is the rate-determining step of the overall reaction. In contrast, adsorption of OH* by the C atom at the edge was thermodynamically favored (ΔGOH*+OH* = −0.73 eV), so this is the active site to facilitate the formation of two adsorbed OH* (Figure 3b,c). However, given that the surface of graphene is mostly occupied by the basal plane C atoms, the catalytic inertness of the graphene basal plane contributes significantly to the low peroxidase activity of rGO. In B-rGO, the B atom is doped onto the basal plane in the form of a BC3 structure; in this case, the B atom itself becomes E

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Figure 4. Dose−response curve for detection of (a) choline and (b) ACh+ by NB-rGO and their corresponding linear calibration plots. (c) Absorption intensities of the blue color signal and their corresponding well plate photos to specifically and quantitatively detect CRP using NB-rGO-based immunoassays. Three independent measurements were performed to obtain error bars showing standard deviations.

the selective detection of H2O2, peroxidase-like nanozymes having negligible oxidase-like activity should be employed. As we discussed in the previous section, NB-rGO having such catalytic property would be one of the best enzyme mimetic catalysts for selective detection of H2O2. Because ACh+ in body fluid typically exists in a very tiny amount in the nanomolar range, we employed another substrate, Amplex UltraRed (AUR), which could be transformed to a highly sensitive fluorescent product through the peroxidase-mediated oxidation. Using AUR as a substrate, the catalytic activity of NB-rGO was observed near pH 7, so this nanozyme can be used in physiological conditions (Figure S21). Under typical assay conditions, the fluorescence intensity upon oxidation of AUR increased sharply as H2O2 concentrations increased (Figure S22). The relationship between fluorescence intensity and [H2O2] was highly linear (R2 > 0.99) from 0.5 to 30 μM with a low detection limit of ∼100 nM, which implies that NB-rGO could be used in analytical systems for the sensitive detection of H2O2. This level of sensitivity is sufficiently high to enable coupling with AChE and ChOx to realize an ACh+ biosensor.38 Before detection of ACh+, we performed a detection experiment for choline, which is an intermediate molecule of our cascade enzymatic reaction and is also the main precursor in synthesis of ACh+.39 Choline in the sample was first hydrolyzed by ChOx to generate H2O2 that can be utilized in the catalytic oxidation of AUR by the peroxidase activity of NB-rGO to produce highly fluorescent oxidized AUR. Choline was detected by the generation of fluorescence intensity by oxidized AUR (Figure 4a); the relationship between the fluorescence intensity and [choline] was linear (R2 > 0.99) from 0.05 to 5 μM with the limit of detection of ∼10 nM. Then, we added AChE to the above enzymatic reaction to determine the level of ACh+ in a sample. In a one-step reaction

concurs with the experimental observation of significantly improved peroxidase activity of NB-rGO. We believe that this DFT-mediated strategy can be extended to rationally design nanozymes that have superior catalytic activity because DFT enables calculation of the required activation energy during catalytic reactions mediated by the expected active sites; this energy can be estimated by appropriate modeling of structure of the nanozymes. NB-rGO-Based Bioassays for the Detection of Acetylcholine and C-Reactive Protein. As a proof of principle for the potent utilization of highly active and stable NB-rGO as an alternative to HRP in bioassays, we performed experiments to detect two important model biomolecules, acetylcholine (ACh+) and C-reactive protein (CRP). ACh+ is a key neurotransmitter and involved in brain functions such as alertness, learning, and memory. Its low level is closely associated with diverse neural disorders such as Alzheimer’s disease, Parkinson’s disease, schizophrenia, and progressive dementia, and thus reliable and sensitive determination of ACh+ level in biological fluids is required.34−36 CRP is also an important class of biomarker, found in blood, and nowadays widely used as a reliable indicator of inflammation, tissue damage, and can be used to predict cardiovascular diseases.37 Several commercialized methods to quantify CRP are available, but increase in sensitivity, selectivity, and speed would allow prioritization of cardiovascular disease patients. ACh+ levels in samples were measured using cascade enzymatic reactions composed of acetylcholine esterase (AChE) and choline oxidase (ChOx), followed by the peroxidase-mediated reaction catalyzed by NB-rGO. In the presence of target ACh+, AChE catalyzes hydrolysis of ACh+ to generate choline, which is subsequently cleaved by ChOx; one of the products is H2O2. NB-rGO can use this H2O2 to oxidize a substrate to develop a chromogenic response. In this case, for F

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ACS Nano performed at pH 7, ACh+ only induced a vivid increase in fluorescence intensity of AUR (Figure S23); this result indicates that this NB-rGO-based assay is highly selective toward target ACh+ in the presence of typical interfering molecules even at 10 times higher concentrations than those of ACh+. Consequently, a highly linear fluorescence enhancement was verified when the concentration of ACh+ was increased from 0.05 to 5 μM with a low detection limit of ∼30 nM, which is among lowest recently reported for ACh + determination.17,40−42 Our NB-rGO-based ACh+ assay system had higher sensitivity for both choline and ACh+ than did other control system assays based on HRP or Pt NPs due to high peroxidaselike activity without any oxidase-like activity (Figure S24 and Table 1); this result confirms the superiority of the NB-rGO-

ELISA for CRP; this low limit confirms the superiority of the current method.43

CONCLUSION This study has led to the development of NB-rGO, a carbonbased nanozyme that shows significant peroxidase-like activity without oxidase-like activity. The catalytic efficiency of NBrGO was up to 3 orders of magnitude higher than that of undoped rGO, showing the synergistic effect of dual-doped elements to increase electron transfer during the catalytic reaction. DFT analyses demonstrated that the peroxidase activity of NB-rGO increases after sequential doping by N and B as a consequence of modification of active sites on graphene with the dopants. NB-rGO was used in bioassays for ACh+ and CRP and showed high selectivity, sensitivity, and linearity. The technology confirms the viability of the strategy of using NBrGO for rapid, robust, and convenient identification of biomolecules. The approach may have applications in pointof-care clinical diagnostics and in environmental monitoring.

Table 1. Linear Range and Limit of Detection (LOD) of NB-rGO-, HRP-, and Pt NP-Based Assay for Detection of Choline and Acetylcholine choline

acetylcholine

catalyst

linear range (nM)

LOD (nM)

linear range (nM)

LOD (nM)

NB-rGO HRP Pt NPs

50−5000 50−2000 150−2500

10 35 85

100−5000 100−2500 250−5000

30 50 115

EXPERIMENTAL SECTION Preparation of GO and Doped Graphene. GO was prepared using modified Hummers method.25 B-rGO was synthesized by heating solid mixture of GO (100 mg) and H3BO3 (500 mg) to 900 °C in flowing Ar for 1 h. The sample was cooled to room temperature (RT), and the byproduct (B2O3) was removed by washing with hot water (∼70 °C). N-rGO was synthesized by heating a solid mixture of GO (100 mg) and melamine (500 mg) to 900 °C in flowing Ar for 1 h. BN-rGO was synthesized by heating a solid mixture of B-rGO (30 mg) and melamine (150 mg) to 900 °C in flowing Ar for 1 h. NBrGO was synthesized by heating a solid mixture of N-rGO (30 mg) and H3BO3 (150 mg) to 900 °C in flowing Ar for 1 h. The sample was cooled to RT, and then the byproduct (B2O3) was removed by washing with hot water (∼70 °C). h-BN-rGO was synthesized by directly annealing a mixture of GO (100 mg), melamine (500 mg), and H3BO3 (500 mg) in Ar at 900 °C for 1 h. The sample was cooled to RT, and then the byproduct (B2O3) was removed by washing with hot water (∼70 °C). Determination of Peroxidase-like Activities of rGO-Based Nanozymes. Peroxidase-like activity of diverse rGO samples was evaluated through the catalytic oxidation of TMB in the presence of H2O2. Typically, rGO samples (80 μg/mL) were added into a reaction buffer (0.1 M sodium acetate, pH 4.0) containing TMB (500 μM) and H2O2 (100 mM), followed by the 3 min incubation at RT. The catalytic materials were subsequently removed by centrifugation at 10 000g for 3 min. After separation, images of the supernatant were captured. The absorption intensity was measured in scanning mode or at 652 nm using a microplate reader (Synergy H1, BioTek, VT). For the HRP-mediated TMB oxidation reaction, HRP (20 ng/mL) and 10 mM H2O2 in sodium phosphate buffer (0.1 M, pH 6.0) were used, with 100 mM H2O2 and sodium acetate buffer (0.1 M, pH 4.0), and subjected to the procedures described above. Models. The peroxidase activity of rGO, N-rGO, B-rGO, and NBrGO was investigated using a graphene cluster model (Figure S14a), which has been used in previous studies on the oxygen reduction reaction activity of heteroatom-doped graphene.22,44 On the basis of experimental XPS results, we considered the doping state in which heteroatoms were bonded to the surrounding C atoms in the graphene. In the B-doped graphene, most B atoms are doped in the form of a BC3 structure rather than BC2O structure (Figure S14b), so only B atoms in BC3 form were considered. In the N-doped graphene, most N atoms doped at the edge are in the pyridinic form, but on the basal plane, they are all in the quaternary state. Therefore, we focused on pyridinic N and quaternary N and as the representative doping states at the edge and on the basal plane, respectively (Figure S14c,d). In N- and B-codoped graphene, we considered the BC3 structures

based assay. We demonstrated the practical utility of our assay by determining the level of ACh+ in serum (10%) by using a standard addition method. The obtained recovery was 99− 103% (Table S4), which agrees well with the added amount of ACh+; therefore, the developed NB-rGO-based assay is applicable for quantification of ACh+ in real serum samples. CRP was detected by first conjugating the surface of the NBrGO with antibody molecules to CRP. The process used EDCNHS chemistry to exploit the abundant carboxyl groups (Experimental Section). The resulting antibody-conjugated NB-rGO specifically binds to the CRP molecules in a sample during conventional sandwich-type immunoassay. By adding H2O2 and TMB, NB-rGO catalyzes oxidation of TMB to produce a blue-colored solution. We detected CRP molecules by using NB-rGO as an alternative to HRP in sandwich-type immunoassays. Antibody molecules to CRP were easily conjugated to the surface of NBrGO by exploiting the abundant carboxyl residues, and the resulting antibody-conjugated NB-rGO efficiently retained >80% of its original activity (Figure S25). Specific blue colors corresponding to the oxidized TMB were rapidly (