Ag Nanoparticles for Fluorescent Detection of

Oct 26, 2012 - ... Xiaowei Ma , Jing Jin , Wei Ji , Xu Wang , Wei Song , Bing Zhao , and Chengyan He ..... Nano-Gold as Artificial Enzymes: Hidden Tal...
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Enzyme Mimics of Au/Ag Nanoparticles for Fluorescent Detection of Acetylcholine Chen-I Wang, Wen-Tsen Chen, and Huan-Tsung Chang* Department of Chemistry, National Taiwan University, Taipei, Taiwan S Supporting Information *

ABSTRACT: We have developed a highly sensitive and selective fluorescent assay for the detection of acetylcholine (ACh) based on enzyme mimics of Au/ Ag nanoparticles (NPs). These NPs were prepared via a one-step solution phase reaction between 13 nm Au NPs and Ag+ ions in the presence of stabilizing agents such as adenosine triphosphate (ATP) and polyethylene glycol (PEG). Our sensing strategy involves reacting ACh with acetylcholinesterase (AChE) to form choline that is in turn oxidized by choline oxidase (ChOx) to produce betaine and H2O2, which reacts with Amplex UltraRed (AUR) in the presence of bimetallic NPs catalyst to form a fluorescent product. The fluorescence intensity (excitation/emission wavelengths of 540/592 nm) is proportional to the concentration of ACh over a range of 1−100 nM (R2 = 0.998), with a limit of detection of 0.21 nM (signal/noise = 3). When compared with Au NPs and horseradish peroxidase, the Au/Ag NPs provide 150- and 115-fold higher catalytic activity toward the H2O2-mediated AUR reaction. The practicality of the assay has been validated by determining the concentrations of ACh in plasma and blood samples, with results of 2.69 ± 0.84 nM (n = 5) and 6.75 ± 1.42 nM (n = 5), respectively. Thus, the present assay holds great potential for the analysis of ACh in biological samples.


To date, peroxidase- or oxidase-like activities have been demonstrated at various types of nanoparticles (NPs) such as Fe3O4 NPs, sheet-like FeS nanostructures, spherical CeO2 NPs, single-walled carbon nanotubes, graphene oxide, AgM (M = Au, Pd, Pt) NPs, and metallic nanocomposites.12−17 It is worth noting that these activities have been used to develop highly sensitive and selective sensors for H2O2, glucose, melamine, proteins, and DNA through their catalytic oxidation of various substrates including 2,2′-azino-bis(3-ethylbenzo-thiazoline-6sulfonic acid) diammonium salt (ABTS) and 3,3,5,5-tetramethylbenzidine (TMB).17−20 We have recently reported the peroxidase-like activity at Au NPs for the fluorescent detection of Hg2+ and Pb2+ ions using Amplex UltraRed (AUR) as a substrate, based on the analyte-induced enhancement of their catalytic activity.21 We found that the formation of Au/Hg and Au/Pb NPs enhanced the catalytic activity toward the reaction of AUR with H2O2. However, using potentially toxic metal ions such as Hg2+ and Pb2+ may limit their biological applications. Therefore, in this study, we attempt to explore the catalytic activity of the biocompatible Au/Ag bimetallic NPs toward the H2O2-mediated oxidation of AUR. We have used AChE and ChOx as model enzymes to catalyze the substrate, ACh, in order to produce H2O2. The as-produced H2O2 was made to react with AUR in the presence of Au/Ag bimetallic NPs. Since the fluorescence intensity of the AUR product was proportional

cetylcholine (ACh) is a neurotransmitter that appears in both the peripheral and central nervous system of many organisms. In neurons, ACh is produced from choline in the presence of choline acetyltransferase and acetyl-coenzyme A.1 The ACh concentration in human blood is approximately 8.66 ± 1.02 nM.2 By binding to its receptors, ACh regulates muscle contraction at the neuromuscular junction; thus, its concentration is related to behavioral activities, learning, sleep, etc.3 In addition, metabolic abnormalities of ACh in the brain are associated with neuropsychiatric disorders such as Parkinson’s and Alzheimer’s diseases.3 Thus, determining the concentration of ACh in biological samples such as blood is of paramount importance. Nevertheless, ACh detection is a difficult task to achieve because it lacks not only electro-active, chromophore, and fluorophore groups but also functional groups for ready conjugation. Though the conventional approaches such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) provide sensitive ACh detection,4,5 these techniques require large sample volume, tedious sample pretreatment, long separation time, and skilled personnel. Alternatively, biosensors are fruitfully applied for the highly sensitive ACh detection.6−11 In such ACh based biosensors, acetylcholinesterase (AChE) or choline oxidase (ChOx) is commonly used to catalyze substrates in order to form an electrochemically active product (H2O2) or fluorescent products.3 Although these products are sensitive, their selectivity and the tedious process of preparing the ACh biosensors are problematic. In addition, the high cost and short shelf life of the biosensors are also of great concern. © 2012 American Chemical Society

Received: March 29, 2012 Accepted: October 26, 2012 Published: October 26, 2012 9706 | Anal. Chem. 2012, 84, 9706−9712

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μL) were separately mixed with the Au/Ag NP solution (50 μL). Then, tris-acetate (50 mM, 50 μL, pH 7.0), AUR (100 μM, 50 μL), and ultrapure water (150 μL) were added to the aforementioned mixture containing Au/Ag NPs, which were then allowed to react for another 2 h. Before fluorescence measurements, the product solutions were transferred into 96well microtiter plates. Their fluorescence spectra were recorded in conjunction with the use of a microplate reader (μ-Quant Biotek Instruments, Winooski, VT, USA) at an excitation wavelength of 540 nm. Blood Sampling. The whole blood and plasma samples collected from five anonymous female donors were used in this study. The whole blood samples were collected immediately into glass vials containing 4% sodium citrate in PBS to prevent pore clogging. It is notable that 1 part sodium citrate was mixed with 9 parts of each blood sample. To each blood samples, 8% EDTA was added and immediately centrifuged (4000 rpm, 10 min, 4 °C). Each of the supernatants, i.e., plasma, was transferred to a polypropylene test tube. The whole blood and plasma samples were stored at −20 °C before analysis. Determination of ACh from Blood Samples. Prior to analysis, the blood samples (200 μL) were treated with acetonitrile (300 μL, 99.5 w/w %) for 30 min to denature the protein and release ACh from the protein complexes. After centrifugation (9000 rpm, 10 min), the supernatant was evaporated by gentle heating and finally cooled down to room temperature. The remaining sample was then passed through a filter having a cutoff of 3 KDa (membrane nominal pore size ca. 0.3 nm). Thus, purified samples were diluted with Tris−acetate buffer (2 mL, 5 mM, pH 7.0). Aliquots of the diluted samples (100 μL) were spiked with standard ACh solution (50 μL, final concentrations of 0−100 nM). Before analysis, aliquots of Tris−acetate (1.5 mL, 5 mM, pH 7.0) containing samples spiked with AChE (0.5 U/mL), ChOx (0.5 U/mL), GSH (100 μM), BSA (100 μM), and ACh were equilibrated under gentle shaking at 37 °C for 15 min. Subsequently, aliquots of the mixtures (200 μL) were separately mixed with the Au/Ag NP solution (50 μL). Each of the mixtures containing Au/Ag NPs was mixed with Tris− acetate (50 mM, pH 7.0, 50 μL), AUR (100 μM, 50 μL), and ultrapure water (150 μL). The mixtures were allowed to react for 2 h before fluorescence measurement.

to the concentration of ACh, subsequently, we were able to detect ACh with high sensitivity and selectivity. Several important factors such as the solution pH, the concentrations of Au/Ag NPs, AChE/ChOx, and stabilizing agents have been evaluated to optimize the sensing conditions. The practicality of the H2O2−AUR−Au/Ag NP sensing system has also been validated by detecting ACh in blood samples.

EXPERIMENTAL SECTION Chemicals. Acetic acid, acetonitrile, ACh, adenosine triphosphate (ATP), 4-aminobutyric acid, aspartic acid, bovine serum albumin (BSA), dopamine, ethylenediaminetetraacetic acid (EDTA), glutamic acid, glutathione (GSH), glycine, horseradish peroxidase (HRP), phosphate buffered saline (PBS), polyethylene glycol (PEG, MW 2000), serotonin, silver chloride, sodium chloride, sodium citrate, sodium tetrachloroaurate (III) dehydrate, tris(hydroxymethyl)aminomethane (Tris), and trisodium citrate were purchased from SigmaAldrich (St. Louis, MO, USA). AUR (structure is not provided) and ACh/AChE assay kits were purchased from Invitrogen (Eugene, OR, USA). Ultrapure water was obtained from MilliQ ultrapure system. Synthesis of Au and Au/Ag NPs. We prepared 13 nm spherical Au NPs from NaAuCl4 by citrate-mediated reduction.22 Aqueous NaAuCl4 solution (1 mM, 250 mL) was brought to a vigorous boil by stirring in a round-bottom flask fitted with a reflux condenser. Trisodium citrate (38.8 mM, 25 mL) was then added rapidly to the solution. The whole solution was refluxed for another 3 min, during which its color changed from pale yellow to deep red. After the synthesis, the solution was cooled to 27 °C. To prepare Au/Ag bimetallic NPs, aliquots of Au NP solutions (0.15 nM, 1 mL) containing ATP (10 mM), PEG (0.01%), and Ag+ ions (10 μM) were equilibrated under gentle shaking at 27 °C for 30 min. Characterization of Au/Ag Alloys. The sizes of the asprepared Au NPs and Au/Ag NPs were measured by counting 100 NPs from their corresponding transmission electron microscopy (TEM) images (H7100, Hitachi High-Technologies Corporation, Tokyo, Japan). The Au and Au/Ag NPs appeared to be nearly monodisperse, with average diameters of 13.0 ± 0.5 and 25 ± 1 nm, respectively. A Cintra 10e doublebeam UV−Vis spectrophotometer (GBC, Victoria, Australia) was used to record the extinction of the as-prepared Au NP solution. According to Beer’s law, the concentration of Au NPs (ca. 15 nM) was determined using an extinction coefficient of ca. 108 M−1 cm−1 at 520 nm for the 13.0 nm Au NPs.23−25 In order to confirm the composition of the Au/Ag bimetallic NPs, energy-dispersive X-ray (EDX) spectroscopy was conducted using a Philips Tecnai 20 G2 S-Twin microscope (Philips, Holland), operated at 200 kV. X-ray photoelectron spectroscopy (XPS) was performed using a PHI 5000 VersaProbe (Physical Electronics, Minnesota, USA). The zeta potentials of the Au NPs and Au/Ag NPs were measured using a Zetasizer 3000HS analyzer (Malvern Instruments, Malvern, UK). Surface-assisted laser desorption/ionization time-of-flight ionization mass spectrometry (SALDI−TOF MS) was employed to examine the Au/Ag NPs. Mass spectra were recorded in the positive ion mode using a Microflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Assays of ACh. Aliquots of tris-acetate buffer (5 mM, pH 7.0, 1.5 mL) containing AChE (0.5 U/mL), ChOx (0.5 U/mL), and ACh (0.005−1 μM) were equilibrated under gentle shaking at 37 °C for 15 min. Subsequently, aliquots of this mixture (200

RESULTS AND DISCUSSION Sensing Strategy. Scheme 1 illustrates the general pathway involved in the detection of ACh using enzyme mimics based on Au/Ag bimetallic NPs. It is obvious from Scheme 1 that AChE converts ACh to choline, which is in turn oxidized by ChOx to produce betaine and H2O2. The as-produced H2O2 reacts with AUR in the presence of Au/Ag bimetallic NPs catalyst to produce a highly fluorescent AUR product (quantum yield (Φ) of 70% at an excitation/emission wavelength of 540/ 592 nm).26 The H2O2 concentrations are directly proportional to ACh concentrations, and therefore, the fluorescence increases with increasing ACh concentrations. It has been known that metal ions catalyze the reactions of H2O2 with some organic compounds such as AUR and luminol.27−30 The selectivity of the ACh sensor is based on the specific activity of the enzymes AChE/ChOx that convert ACh to betaine and H2O2. When the Au NPs reacted with Ag+ ions, Au/Ag NPs were formed on their surfaces, leading to an increased catalytic activity for the H2O2-mediated oxidation of AUR.31 Eqs 1−4 present a possible reaction process of AUR (OH group) with 9707 | Anal. Chem. 2012, 84, 9706−9712

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Scheme 1. Schematic Representation of the Sensing Strategy of the Fluorescent Assay Using Au/Ag NPs to Detect ACh

H2O2 to form AUR product (CO) on the surfaces of the Au/ Ag NPs. Au 0 + Ag + → Au/Ag +


ACh + AChE + ChOx → betaine + H 2O2


Au/Ag + + 2H 2O2 + 2e− → Au/Ag + 2•OH + 2OH− (3) •

2 OH + 2OH + AUR → AUR product + 2H 2O + O2 + 2e−


The as-formed Au/Ag NPs have similar enzymatic activity to that of horseradish peroxidase (HRP), which catalyzed the conversion of nonfluorescent AUR to a fluorescent product. Although AUR itself is weakly fluorescent, its oxidation product is highly fluorescent. As a result, the fluorescence of the solutions at 592 nm increased when they were excited at 540 nm. Characterization of Au/Ag NPs. We found that Tris− acetate (pH 7.0) is superior to phosphate, tetraborate, and ammonia-acetate, since they assisted the ease of Au/Hg and Au/Pb bimetallic NPs formation and enhanced their catalytic activity.21 In particular, the hydroxyl groups of Tris, acting as electron donors, facilitated the formation of bimetallic NPs.32 However, Au NPs are relatively unstable in Tris−acetate solutions containing high concentrations of salts such as NaCl. To stabilize Au NPs in Tris−acetate (5 mM, pH 7.0), ATP and PEG were used (Figure 1a).33,34 The maximum absorption wavelengths of the surface plasmon resonance (SPR) bands of 0.15 nM Au NPs in Tris−acetate (5 mM, pH 7.0) containing ATP and PEG in the absence and presence of Ag+ ions (0−100 μM) were almost identical (around 518 nm). The SPR band undergoes slight red shifts with increasing Ag+ ion concentration, mainly because of increase in the size and changes in the refractive index and composition of the Au NP surfaces.35 The zeta potentials of the Au NP surfaces in the presence and absence of Ag+ (10 μM) were −32.8 and −42.6 mV, respectively. TEM, SALDI-MS, and XPS measurements were conducted to further characterize the Au NPs in the presence and absence of Ag+ ions (10 μM). The TEM images revealed that the Au NPs in the presence (Figure 1b) and absence (inset to Figure 1b) of Ag+ ions differed markedly in size and shape. In the presence of Ag+ ions, some of the Au NPs changed from

Figure 1. (a) UV−vis absorption spectra of Au NPs in Tris−acetate solutions (5 mM, pH 7.0) with/without ATP and PEG in the absence and presence of Ag+ ions (0, 10, 50, and 100 μM). Au NP, ATP, and PEG concentrations are 0.15 nM, 10 mM, and 0.01%, respectively. Absorbance values are plotted in arbitrary units (a. u.). (b) TEM images and (c) EDX results of Au/Ag NPs prepared in the presence of 10 μM Ag+ ions, 100 μM ATP, and 0.01% PEG. Insets to (b) and (c) are the results obtained for Au NPs.

spherical to irregular and chain-like structures, and their sizes increased from 13 nm (diameter) to 16−30 nm (length). The EDX spectrum of bimetallic NPs (Figure 1c) confirmed the existence of Ag on the Au NPs, while the purity of Au NPs used in this work were revealed from their EDX spectrum provided in the inset to Figure.1c. The peaks at m/z 107.91, 196.95, 393.42, and 394.87 in the SALDI-MS spectrum (Figure S-1a, Supporting Information) are assigned to Ag+, Au+, Au−Ag+, and Au2+ ions, respectively. The binding energies (BEs) of Au 4f7/2 electrons in the Au and Au/Ag NPs were 83.6 and 83.8 eV, respectively (Figure S-2a, Supporting Information). The peak at a BE of 368.12 eV, which is assigned to Ag 3d5/2 electrons, was detected only in the Au/Ag NPs (Figure S-2b, Supporting Information).36 The XPS spectra of the Au and Au/ Ag NPs reveal two important features in the Au 3d region. The main peak (Au 4f7/2) of Au NPs (83.6 eV) shifted toward a lower BE (ca. 0.4 eV) relative to that of metallic Au (84.0 eV),37,38 suggesting the electrons transfer from the COO− of the citrate ions to the Au surface. On the other hand, the main peak (Au 4f7/2) of Au NPs shifted toward 83.8 eV in the presence of Ag+, which indicates increased screening, attributed primarily to the increased coordination number of the alloying 9708 | Anal. Chem. 2012, 84, 9706−9712

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leading to reduced catalytic activity and poor reproducibility. In addition, free Ag+ catalyzed the reaction between AUR and H2O2, leading to higher fluorescence background (IF0).50 As a result, the sensitivity for ACh decreased upon further increasing the concentration of Ag+ greater than 10 μM. We also observed that only Ag+ ions at low concentrations exhibit negligible catalytic activity toward the H2O2-mediated oxidation of AUR. On the other hand, compared to HRP, the Au/Ag NPs provided 115-fold higher activity toward the H2O2-mediated oxidation of AUR. We then investigated the effect of pH (Tris solution) and the AChE and ChOx concentrations on the detection of ACh. To minimize the effect of pH on the fluorescence of the AUR product, IF − IF0 is plotted versus pH (5.0−11.0) as depicted in Figure 2, where IF0 and IF represent the fluorescence intensities

Au atom and secondarily to the charge transfer from Ag to Au.39 Furthermore, the electronegativity of Au (2.4) is higher than Ag (1.9), which also helps for the charge transfer from Ag to Au, leading to the formation of alloys.40,41 In addition, the Ag 3d spectrum displays two spin−orbit components. The Ag 3d5/2 peak appearing at an binding energy of 368.12 eV corresponds to Au/Ag alloy-type.39 This data confirms the formation of Au/Ag NPs attributed to the formation of strong bonds between Au and Ag. Stability and Activity of Au/Ag NPs. We investigated the effects of ATP and PEG concentrations on the stability and catalytic activity of the Au/Ag NPs in solutions containing NaCl (15 mM), BSA (10 μM), and GSH (10 μM). In the absence of ATP and/or PEG, the as-prepared Au/Ag NPs were unstable and exhibited no catalytic activity. However, the adsorption of BSA and GSH molecules onto the surfaces of the Au/Ag NPs reduced their catalytic activity, mainly because it is more difficult for H2O2 to access the surfaces of Au/Ag NPs. To minimize BSA and GSH adsorption, we used ATP and PEG that have been commonly used to minimize the matrix effect caused by the interfering species,42,43 whereas the fluorescence of the AUR product increased as the ATP concentration was increased from 1 to 100 μM and thereafter it decreased (Figure S-3a, Supporting Information). On the other hand, it increased notably as the PEG concentration was increased from 0.001% to 0.01% (Figure S-3b, Supporting Information). The Au/Ag NPs exhibited increased catalytic activity in both cases (Figure S-3a,b, Supporting Information), mainly because the matrix effect caused by the interfering species decreased. Upon further increasing the ATP and PEG concentrations, it is more difficult for H2O2 to access the surfaces of Au/Ag NPs, thereby reducing the decomposition of H2O2 to reactive oxygen species (ROS) such as superoxide (O2•−) and hydroxyl radicals (•OH).44 We also investigated the effect of the concentration of Ag+ ions on the catalytic activity of Au/Ag NPs under the optimal conditions (10 μM ATP and 0.01% PEG). Figure S-3c, Supporting Information, reveals that the fluorescence of the AUR product increased as the Ag+ ion concentration increased from 1 to 10 μM. The activity of the as-prepared Au/Ag NPs prepared in the presence of 10 μM Ag+ ions was about 150-fold higher than that of the Au NPs, mainly because of the synergistic and electronic effects (Figure S-3c, Supporting Information).21 The as-prepared Au/Ag NPs were well dispersed in the solutions when the concentrations of Ag+ were lower than 10 μM (Figure 1a). The improved catalytic activity of bimetallic NPs could be due to the electronic charge transfer effect among the different components along with geometric effects that leads to appropriate modification of electronic structure.45 Moreover, the ionization potentials for Ag and Au are 7.58 and 9.22 eV, respectively; thus, electron transfers could occur from Ag to Au NPs, leading to a decrease in electronic density on the shell surfaces of Ag.46,47 Those electron-deficient surfaces promote the adsorption of H2O2, which leads to the facile electron transfer from H2O2 to the surface of bimetallic NPs, resulting in the efficient oxidation of H2O2. The synergistic effect of bimetallic NPs, mainly due to the strong metal−metal interactions, might be ascribed to the distinct binding properties of the alloy with reactants.48 It has been shown that Au−Ag NPs provided greater catalytic activities for CO oxidation as compared to Au or Ag alone.49 Our results revealed that pure Au NPs did not exhibit any catalytic activity toward H2O2. Further increases in the Ag+ concentration (>10 μM) caused instability to the Au/Ag NPs,

Figure 2. Effect of pH (5.0−11.0) on the catalytic activity of Au/Ag NPs in 5 mM Tris−acetate solutions toward H2O2-mediated reaction of AUR. ACh concentration is 100 nM. Other conditions are the same as those described in Figure 1.

of the mixtures in the absence and presence of ACh, respectively. This data clearly reveals that the optimal pH value was 7.0. The fluorescence of the AUR product was constant at pH > 6.0, whereas the activities of AChE/ChOx increased from pH 4.0 to 7.0 and then decreased significantly at pH > 8.0.51 The activities of AChE/ChOx decreased at pH values higher than 8.0, mainly due to pH-induced denaturation. In addition, Ag+ ions formed various species, such as Ag2O, through reactions with OH− ions (2Ag+ + 2OH− → Ag2O + H2O), thereby minimizing the formation of Au/Ag NPs.52,53 Therefore, we optimized the concentrations of AChE and ChOx at pH 7.0. Although the reaction became more rapid at high AChE and ChOx concentrations, adsorption of the enzymes on the surfaces of the Au/Ag NPs became problematic, thereby decreasing the catalytic activity of Au/ Ag particles. From the above experimental results, the optimal concentrations of the two enzymes were both explicated as 0.5 U/mL. Selectivity and Sensitivity. We investigated the selectivity of the assay toward ACh (10 nM) over some possible interfering substances (100 nM each), including dopamine, serotonin, glutamic acid, 4-aminobutyric acid, glycine, and aspartic acid. Remarkably, only ACh induced a significant increase in the fluorescence, revealing that the assay is highly selective toward ACh, mainly due to the high specificity of AChE and ChOx toward ACh and choline, respectively (Figure 3). Although the as-prepared Au/Ag NPs have the enzyme catalytic ability for H2O2-mediated oxidations of many organic compounds, only rare fluorescent products can be generated. Thus, interference from other organic compounds can be 9709 | Anal. Chem. 2012, 84, 9706−9712

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Figure 3. Selectivity of the fluorescent assay using Au/Ag NPs for ACh. ACh concentration is 10 nM, whereas those for other solutes are all 100 nM. Au/Ag NPs were prepared in the presence of 10 μM Ag+ ions. Other conditions are the same as those described in Figure 1.

ignored. Under the optimal conditions, the fluorescence intensity increased with increasing ACh concentration, with a linear response (R2 = 0.998) over an ACh concentration range of 1−100 nM (Figure 4a). Here, IF0 and IF represent the fluorescence intensities of the mixtures in the absence and presence of ACh, respectively. The limit of detection (LOD) for ACh (signal-to-noise ratio = 3) was 0.21 nM, revealing that the assay is comparable to or better than most reported optical sensors.54,55 In comparison with optical and electrochemical methods (Table 1), our assay for ACh detection is relatively simple, cost-effective, selective, and sensitive. In addition, the stability of the low-cost Au/Ag NPs (stable for at least two months) is much better than that of enzymes such as HRP. The enzyme mimics property of Au/Ag NPs holds a great potential for practical applications. Detection of ACh in Blood Samples. To validate the practicality of the present assay, we determined the concentration of ACh in blood samples by applying a standard addition method. The fluorescence responses against the concentrations of ACh spiked into representative samples of whole blood and plasma were both linear (R2 > 0.98) in the concentration ranges of 0−100 nM (Figure 4b,c). We obtained ACh concentrations in the plasma and blood samples as 2.69 ± 0.84 nM (n = 5) and 6.75 ± 1.42 nM (n = 5), respectively. The ACh concentrations determined using a commercial assay kit were 2.50 ± 0.29 nM (456 ± 53 pg/mL) and 6.95 ± 0.82 nM (1264 ± 149 pg/mL) in the plasma and whole blood samples, respectively. Student’s t test performed at the 95% confidence level demonstrated that the two approaches did not provide significantly different results.

Figure 4. (a) Fluorescence spectra of AUR product at various ACh concentrations. Inset: linearity of the expression (IF − IF0) versus the ACh concentration. The AChE/ChOx and AUR concentrations were 0.5 U/mL/0.5 U/mL and 10 μM, respectively. Reaction times for the enzyme and enzyme mimetic reactions at 30 °C are 30 min and 2 h, respectively. Analyses of (b) blood and (c) plasma samples using Au/ Ag NP based fluorescent assay. Each sample was spiked with ACh (final concentrations of 0−100 nM). Other conditions are the same as those described in Figure 1.

Table 1. Comparison of Various Approaches to ACh Detectiona

CONCLUSION We have developed a sensitive, selective, and cost-effective approach for ACh detection. When compared to Au NPs and HRP, Au/Ag bimetallic NPs display greater catalytic activity toward the oxidation of AUR with H2O2. Our results reveal that the catalytic activity of the Au/Ag NPs depends on the number of active sites on their surfaces. To prevent adsorption of interfering substances and to stabilize the Au/Ag NPs, ATP and PEG are required to cap their surfaces. The present approach provides an LOD of 0.21 nM for ACh and has been validated by analysis of blood and plasma samples; it offers the advantages of low cost, simplicity, selectivity, and sensitivity. Owing to the great catalytic activity of Au/Ag NPs toward


LOD (nM)

detection mode


LC/MS/MS LC-ED biosensor biosensor biosensor biosensor biosensor FIA-CLD biosensor

0.01 0.2 0.1 10 49.3 10 000 50 2 0.2

mass spectrometry amperometry amperometry amperometry amperometry potentiometry chemiluminescence chemiluminescence fluorescence

56 3 2 11 57 58 54 55 this study

a LC-ED: liquid chromatography−electrochemical detection; LC/MS/ MS: liquid chromatography−tandem mass spectrometry; FIA: flow injection analysis; CLD: chemiluminometric detection; LOD: limit of detection.

H2O2-mediated oxidation of AUR, this approach can be extended to the detection of various analytes such as glucose, cholesterol, and uric acid using suitable enzymes. 9710 | Anal. Chem. 2012, 84, 9706−9712

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S Supporting Information *

Figures S-1 to S-3, as noted in the text. This material is available free of charge via the Internet at


Corresponding Author

*Address: Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan. Tel/Fax: 011-886-2-33661171. E-mail: [email protected] Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was supported by the National Science Council of Taiwan under contracts NSC 101-2113-M-002-002-MY3.


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