Article pubs.acs.org/ac
Photoluminescent C‑dots@RGO Probe for Sensitive and Selective Detection of Acetylcholine Chen-I Wang, Arun Prakash Periasamy, and Huan-Tsung Chang* Department of Chemistry, National Taiwan University, Taipei, Taiwan S Supporting Information *
ABSTRACT: We have developed a sensitive and selective photoluminescence (PL) quenching assay for the detection of acetylcholine (ACh) that uses reduced graphene oxide decorated with carbon dots (C-dots@RGO). The highly stable C-dots@RGO synthesized from catechin and graphene oxide through a hydrothermal reaction displays excitation-wavelength dependence of PL. Acetylcholinesterase (AChE) converts ACh to choline, which in turn is oxidized by choline oxidase (ChOx) to produce betaine and H2O2 that generates the reactive oxygen species (ROS). The as-produced ROS induces PL quenching of the C-dots@RGO through an etching process. With respect to sensitivity, the optimal reaction/sensing temperature and pH are 37 °C and 9.0, respectively, using C-dots@RGO (0.4 mg·mL−1) and AChE and ChOx at the activities of 0.5 and 0.1 unit·mL−1, respectively. The PL intensity (excitation/emission wavelengths 365/440 nm) of the C-dots@RGO is inversely proportional to the concentration of ACh over a range of 0.05−10 nM (r = 0.997), with a limit of detection (signal-to-noise ratio 3) of 30 pM. We have validated this assay by determination of concentrations of ACh in plasma and blood samples, with results of 2.6 ± 0.8 nM (n = 5) and 6.8 ± 0.4 nM (n = 5), respectively. Our study opens an avenue for the detection of various analytes by use of C-dots@RGO in conjunction with different enzymes, substrates, and/or inhibitors.
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(TMB), and luminol,4−8 for the H2O2-mediated reaction are used in highly sensitive and selective optical sensors for the detection of ACh.4−8 Although these sensing systems are sensitive, interference, high cost, short shelf life, and/or tedious processes of preparation of the ACh biosensors are problematic. Many nanoparticle-based approaches have been developed for determining the activity of AChE and its inhibitors, based on the enzymatic conversion of acetylthiocholine to thiocholine, which induces the growth of gold nanoparticles (Au NPs) and subsequent optical changes.9−11 Although the above-stated approaches are sensitive and selective for the tested analytes, they cannot be used for the detection of ACh, mainly because the product (choline) cannot induce the growth of Au NPs. Recently we have demonstrated Au/Ag bimetallic NPs possessing peroxidase-like activity for the fluorescence detection of ACh, using AUR as a substrate.4 AChE converts ACh to choline, which in turn is oxidized by ChOx to produce betaine and H2O2 that reacts with AUR to form a fluorescent product catalyzed by the Au/Ag NPs. Although the sensing system is selective and sensitive (with a limit of detection, LOD, of 0.21 nM), stability and loss in the catalytic activity of Au/Ag NPs due to adsorption of matrices are problematic.
cetylcholine (ACh) is a neurotransmitter that can be found in the peripheral nervous system and central nervous system.1 In cholinergic neurons, ACh is synthesized from choline by choline acetyltransferase and acetyl-coenzyme A.1 Increases in the level of ACh cause a decreased heart rate and increased production of saliva. On the other hand, decreases in the level of ACh are associated with motor dysfunction, as well as Parkinsonʼs and Alzheimerʼs diseases.1 Thus, determination of the concentration of ACh in biological samples such as blood is important for cholinergic research. This is a difficult task, mainly because its normal concentration in human blood is quite low (8.66 ± 1.02 nM) and the matrix is rather complicated.1 There is no electro-active, chromophore, or fluorophore group in ACh, which makes it difficult to detect. Although gas chromatography and high-performance liquid chromatography have been applied for the detection of ACh, tedious sample pretreatment, long separation time, use of large volumes of sample and eluate, and an experienced operator are required.2 To overcome these disadvantages, many sensitive sensing systems have been demonstrated.1,2 ACh biosensors are fabricated by taking advantage of high catalytic activities of acetylcholinesterase (AChE) and choline oxidase (ChOx) for converting nonelectroactive or nonfluorescent substrates to an electrochemically active product (H2O2) or fluorescent products.3 Common substrates, including Amplex UltraRed (AUR), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3,3,5,5-tetramethylbenzidine © 2013 American Chemical Society
Received: December 12, 2012 Accepted: February 11, 2013 Published: February 11, 2013 3263
dx.doi.org/10.1021/ac303613d | Anal. Chem. 2013, 85, 3263−3270
Analytical Chemistry
Article
Recently, reduced graphene oxide (RGO), with its flat network of sp2-hybridized carbon atoms and two-dimensional (2D) honeycomb lattices, has become a very popular sensing material for the detection of DNA, proteins, and small molecules, mainly because it has large planar surface and high photoluminescence (PL) quenching efficiency of fluorophores (e.g., organic dyes, quantum dots).12,13 Although these detection strategies can be sensitive, expensive fluorophores and tedious processes are required for preparation of the sensing probes. In addition, as-prepared RGO is usually hydrophobic and nonphotoluminescent, leading to irreversible agglomeration and precipitation, which limit its direct use for biological application (e.g., biosensing and cell imaging).14 Herein, we developed a simple, green, and cost-effective onepot synthetic strategy for the synthesis of hydrophilic, photoluminescent carbon dots on RGO (C-dots@RGO) from graphene oxide (GO) through a hydrothermal reduction route using catechin as a reductant, based on our previous study.15,16 On the basis of reactive oxygen species (ROS) -induced PL quenching of C-dots@RGO, we demonstrated a sensitive and selective sensing system for the detection of ACh. The ROS was generated through the formation of H2O2 from ACh in an AChE/ChOx system. We evaluated several important factors such as the concentrations of C-dots@ RGO, AChE/ChOx, and NaCl, temperature, reaction time, and solution pH values to optimize the sensing conditions. The practicality of the H2O2−C-dots@RGO sensing system was validated through the detection of ACh in blood samples. To the best of our knowledge, C-dots@RGO probes have not been demonstrated. In addition, no tradition fluorophores are required in our sensing system.
the vial was capped well. The mixture was heated in an oil bath at 95 ± 2 °C for 3 h. The yellow color of the GO solution gradually changed to black, indicating the formation of RGO. The resulting RGO solution was washed with ultrapure water (ca. 50 mL) to remove excess hydrazine monohydrate and ammonium hydroxide after filtration through an inorganic membrane filter paper (pore size 0.02 μm). Washing was continued until the pH of the filtrate reached approximately 7.0. The product was carefully collected from the filter paper and dried overnight in an oven at 50 °C. The RGO was then dispersed in N,N-dimethylformamide (DMF). Experimental details can be found in the Supporting Information. By controlling reaction time (2−12 h), we prepared different sizes of C-dots on the surfaces of RGO. Using the same amount of catechin, we separately prepared C-dots@RGO with three different sizes of RGO from GO sheets (0.3−0.5, 0.7−0.9, and 1−2 μm) that had been prepared according to the method of Hummers and Richard.18 Each of the as-prepared C-dots@ RGO solutions were subjected to centrifugation at different rotation speeds (5000g and 45000g for the first two and the last, respectively) for 30 min. We extracted the C-dots from the surface of RGO by ultrasonicating the C-dots@RGO sample for 1 h and further subjecting it for centrifugation at 45000g for 30 min. A similar approach has also been demonstrated for determining the concentration of single-walled carbon nanotubes (SWCNTs) in aqueous dispersions containing both surfactants and SWCNTs.19 On the basis of the fact that C-dots provided a defined PL peak at 440 nm, but RGO did not, we determined the concentration of as-extracted C-dots using a calibration plot of the PL intensities at 440 nm as a function of pure C-dots concentration. There was about 0.32 mg of C-dots in 1 mg of C-dots@RGO. Detection of H2O2 and ACh by Use of Photoluminescent C-dots@RGO. To detect H2O2, aliquots (300 μL) of 25 mM Tris-HCl buffer (pH 9.0) containing C-dots@ RGO (0.4 mg·mL−1), and H2O2 (10 pM−15 μM) were equilibrated and reacted under gentle shaking at 25−70 °C for 1 h. In order to compare the sensitivity of pure C-dots and Cdots@RGO toward H2O2, they were used to detect H2O2 under the same condition (pH 9.0, 37 °C). Pure C-dots (0.13 mg·mL−1) and C-dots@RGO (0.4 mg·mL−1, about 0.13 mg·mL−1 C-dots) were separately mixed with H2O2 (0.1 nM−15 μM), which were reacted under gentle shaking for 1 h. The slope of (I PL0 − I PL )/I PL0 ratios against H 2 O 2 concentration were used to compare the sensitivity of the pure C-dots and C-dots@RGO in 25 mM Tris-HCl buffers for H2O2, where IPL0 and IPL represent the PL intensities of carbon nanomaterials (pure C-dots and C-dots@RGO) in the absence and presence of H2O2, respectively. For the detection of ACh, aliquots (300 μL) of 25 mM Tris-HCl buffer (pH 9.0) containing C-dots@RGO (0.4 mg·mL −1 ), AChE (0.5 unit·mL−1), ChOx (0.1 unit·mL−1), and ACh (0.05−50 nM) were equilibrated and reacted under gentle shaking at 37 °C for 2 h. To optimize sensitivity in a short analysis time, the enzyme reaction and sensing were conducted at temperatures of 25−50 and 25−70 °C, respectively, and over a pH range 4.0−10.0, both for 10 min. Prior to PL measurement, aliquots (200 μL) of the product solutions were transferred separately into 96well microtiter plates. Their fluorescence spectra were recorded on a microplate reader (μ-Quant Biotek Instruments, Winooski, VT). The excitation and emission wavelengths were set at 365
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EXPERIMENTAL SECTION Synthesis of Photoluminescent C-dots@RGO and RGO. Catechin-mediated reduction of GO was applied to prepare photoluminescent C-dots@RGO. Catechin (21.6 mg) was added to a diluted GO solution (0.34 mg·mL−1, 5 mL). The resulting mixture was gently stirred for 10 min before it was transferred to a Teflon-lined stainless steel autoclave, which was then placed in a muffle furnace and subjected to hydrothermal treatment at 300 °C for 2 h. During the hydrothermal reaction process, C-dots were produced and deposited on the RGO sheets. Upon completion of the reaction, the resulting black solution of C-dots@RGO was cooled to room temperature. It was then subjected to filtration through an inorganic membrane filter paper (pore size 0.02 μm) and the filtrate was washed with ultrapure water (ca. 50 mL) to remove the unreacted excess catechin. The product was dried overnight at 50 °C in an oven and then carefully collected from the filter paper. The as-purified C-dots@RGO product was redispersed in distilled water (4 mg·mL−1) with the aid of ultrasonication for 1 h, and then filtered again to remove the large chunks and other graphitic impurities by use of cotton cellulose filter paper (pore size
