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A Highly Sensitive Gold-Nanoparticle-Based Assay for Acetylcholinesterase in Cerebrospinal Fluid of Transgenic Mice with Alzheimers Disease
Dingbin Liu, Wenwen Chen, Yue Tian, Sha He, Wenfu Zheng, Jiashu Sun, Zhuo Wang,* and Xingyu Jiang*

This study provides a highly sensitive and selective Rhodamine B-modied gold nanoparticle (RB-AuNP)-based assay with dual readouts (colorimetric and uorometric) for monitoring the levels of acetylcholinesterase (AChE) in the cerebrospinal uid (CSF) of transgenic mice suffering from Alzheimers disease (AD). The use of AuNPs as colorimetric assays has recently become an attractive system because molecular events can be easily transformed into color changes, which corresponds well with the change of absorption spectum or surface plasmon resonance.[1] The past few years have witnessed a variety of AuNP-based colorimetric sensors for various analytes such as metal ions,[2] anions,[3] small organic compounds,[4] proteins,[5] and DNA.[6] However, most reported colorimetric detection schemes still lack sufcient sensitivity; a problem that limits the broad application of this kind of assay in complex samples for practical applications. In addition, many components such as biothiols in real samples may interfere with the detection, generating false positive results. Therefore, it is necessary to create a scheme to enhance the sensitivity and selectivity of this detection so that complex samples could be diluted many times and tiny amounts of analyte could still be effectively detected while interferents can be avoided.[7] AChE is an enzyme that can catalytically break down acetylcholine at cholinergic synapses, resulting in the termination of synaptic transmission. Much research has provided evidence that the AChE level in the CSF of individuals suffering from AD is signicantly reduced, and a reduced level of AChE correlates well with a certain degree of the cognitive impairment.[8] The low level of AChE in the CSF may indicate a brain at risk or that the person is in the preclinical stage of AD. This information could be useful for early prevention and treatment of the disease due to the fact that the current diagnosis of AD requires
D. Liu, W. Chen, Y. Tian, S. He, Prof. W. Zheng, Prof. J. Sun, Prof. Z. Wang, Prof. X. Jiang CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology 11 Beiyitiao, Zhongguancun, Beijing, 100190, P. R. China E-mail: wangz@nanoctr.cn; xingyujiang@nanoctr.cn D. Liu, W. Chen, Y. Tian Graduate University of Chinese Academy of Sciences Shijingshan, Yuquan Road, 19(A), Beijing, 100049, P. R. China

DOI: 10.1002/adhm.201100002

not only the presence of severe cognitive decits but also postmortem conrmation of the presence of the typical AD histopathologic changes in the brain.[9] There is still no accurate method for identifying AD in an early or asymptomatic stage of the disease.[10] More and more evidence has shown that the use of CSF biomarkers (levels of -amyloid protein, total tau protein, and phosphorylated tau protein, etc.) on their own or in combination with neuroimaging or/and biomarkers may provide useful complementary information and thus improve the accuracy of the clinical diagnosis of AD.[11] The AChE levels in the CSF, which correlate well with the degrees of the cognitive progression of AD, could be applied as an important parameter to monitor the progression of AD and the effects of treatment, especially in conjugation with other CSF biomarkers as well as neuroimaging techniques. It is therefore important to develop reliable tools for measuring the AChE level in the CSF.[8,12] Traditional methods to measure the level of AChE include the colorimetric method by using Ellmans reagent[13] and the detection of hydrogen peroxide produced by oxidation of the AChE-induced choline.[14] Both methods lack sufcient sensitivity and require time-consuming procedures. In order to enhance the sensitivity, advanced chemical methods such as electrochemical probes[15] and chemiluminescent or uorescent assays for AChE, based on organic compounds[16] and quantum dots,[17] have played important roles. These approaches, however, still lack sufcient sensitivity and require a tedious chemical synthesis. Two kinds of AuNP-based colorimetric assays for AChE have been reported: one is based on the aggregation of AuNPs, which causes the red-shift of the absorption band along with a color change from red to blue;[18] the other relies on an AChEcatalyzed enlargement of the size of the monodispersed AuNPs, which leads to a change of the maximum absorbance.[19] However, researchers still face the challenge of improving sensitivity and accuracy of the assays and, more importantly, no report has assessed their utility in detection of analytes in real samples of complex mixtures such as the CSF or serum. Therefore, the development of sensitive and selective assays for AChE in real samples is of high demand. Herein, we present a highly sensitive assay for AChE by using RB-AuNPs and demonstrate its utility in sensing AChE levels in the CSF of transgenic mice suffering from AD (Scheme 1). RB is an ideal ligand for this assay, because it is water-soluble, photostable, strongly uorescent, and readily

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thiocholine and the residual RB molecules attached to different AuNP surfaces may be able to interact via electrostatic interaction between the quaternary ammonium group on thiocholine and the acidic group on RB and cause the aggregation of AuNPs. This process resulted in a rapid change of the absorption band as well as the color change of the AuNPs solution from red to blue (Figure 1a). In addition, if AChE inhibitors were present, AChE fails to catalyze ATC and thus to generate the thiocholine that is required to either cause aggregation of RB-AuNPs or recover the uorescence of RB molecules. We thus anticipate that both the color change of the AuNP solution and the uorescence recovery of RB molecules can be applied as an effective tools to detect AChE in complex samples and screen its inhibitors. Furthermore, the two simultaneous outputs can also be used to avoid a -positive signal. As a proof of concept, we rst synthesized AuNPs using citrate as the reducting agent as Scheme 1. The detection (colorimetric and uorometric) of AChE based on RB-AuNPs. The well as the stabilizer. The as-prepared citratewell-dispersed RB-AuNPs (red) are induced to aggregate (purple) via electrostatic interaction AuNPs were red in color and showed a typin the presence of thiocholine derived from the hydrolysis of ATC catalyzed by AChE in the CSF of transgenic mice, accompanied with the uorescence recovery of RB (the color of the stars ical absorption band at 520 nm, which was attributed to the surface plasmon resonance changed from gray to green). of AuNPs, which are about 13 nm in size.[1] We prepared RB-AuNPs by allowing various adsorbs onto surfaces of AuNPs to result in quenched uoresnal concentrations of RB (02.0 M) to mix with a xed concence. The positively charged amino groups on RB molecules centration of AuNPs (5 nM) under mild stirring, the mixtures are capable of recognizing the negatively charged citrate-AuNPs were prepared in 2.5 mM NaHCO3-NaOH buffer (pH 10.0). The via electrostatic interactions, thus attaching onto surfaces of uorescence spectra of the RB-AuNPs solutions were recorded AuNPs. Simutaneously the uorescence of RB molecules is after 2 h of equilibration. We found the optimal concentration quenched by AuNPs.[20] Upon the addition of both acetylthiof RB to be 1.2 M, when very weak uorescence was observed, ocholine (ATC, an analogue of acetylcholine) and AChE into indicating that no excess RB was free in the solution (Figure S1 a RB-AuNPs solution, AChE could hydrolyze ATC to generate in the Supporting Information). We reasoned that RB molethiocholine. Thiocholine strongly binds onto surfaces of AuNPs cules are positively charged, thus can readily attach onto the via the formation of AuS bond to replace RB molecules,[21] negatively charged citrate-AuNPs via electrostatic interactions, resulting in the desorption of RB molecules from Au surfaces which results in the formation of RB-AuNP assemblies and to recover the uorescence of RB (Figure 1b). At the same time, the quenched uorescence of RB by AuNPs.[20] The absorption band of RB-AuNPs was similar to that of the citrate-capped AuNPs, and the color of AuNPs solution remained red after modifying with RB molecules (Figure 1a). To conrm the proposed mechanism, we conducted zeta-potential measurements to investigate the change of the surface charge of RB-AuNPs before and after incubation with both AChE and ATC. The charge on RB-AuNPs is negative because of the acidic groups on RB. The zeta potential of well-dispersed RB-AuNPs was about 38 mV, while that of the AChEinduced aggregates of RB-AuNPs increased to be 0.27 mV (Figure S2), most likely due to Figure 1. a) UVvis absorption and b) uorescence spectra of RB-AuNPs (red curve) and RBthe presence of positively charged quaternary 1 AuNPs that were incubated with AChE (1 U mL , U is the abbreviation of units) and ATC (20 M) (blue curve). a) Inset: Color change of the AuNP solution from red to purple accompanied ammonium groups in the thiocholines derived from the AChE-catalyzed hydrolysis of ATC. with the formation of aggregates. b) Inset: Fluorescence recovery after the incubation of AChE The aggregation process was also supported and ATC.

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by dynamic light scattering (DLS) data (Figure S3). The average hydrodynamic diameter of well-dispersed RB-AuNPs was 21 nm, while that of the AChE-induced aggregates of RB-AuNPs increased to 400 nm, congruent with transmission electron microscopy (TEM) analysis (Figure S4a, b). In addition, we tried to analyze the ligands on surfaces of welldispersed RB-AuNPs and their aggregates, respectively, by using high-resolution TEM. However, the organic layers on surfaces of RB-AuNPs were too thin to be observed, similar to previous reports (Figure S4c,d).[22] The aggregation process was further conrmed by UVvis absorption. UVvis spectroscopy showed that with the formation of aggregates, the absorption band of RB-AuNPs at 520 nm decreased, accompanied by the emergence of a new absorption peak between 600 and 800 nm (blue curve, Figure 1a). At the same time, the detached RB molecules from Au surfaces were detected by uorescence spectroscopy (Figure 1b). The very weak uorescence of the attached RB molecules on AuNPs increased signicantly after adding AChE and ATC. All the above-mentioned characterizations supported our proposed mechanism for Figure 2. The sensitivity of this assay for AChE by color change and uorescence recovery. this detection system. a) Color change with the increase of concentrations of AChE from left to right (05.0 mU mL1); We next investigated the detection limit b) Fluorescent images of RB molecules released by incubating with various concentrations of of this assay for AChE in aqueous solutions. AChE (03.0 mU mL1) and ATC. c) Absorbance responses for (a). d) Fluorescence spectra for To a mixture of ATC (20 M) and RB-AuNPs b). e) ratio A/D values versus different concentrations of AChE and (f) their corresponding solutions (5 nM) we added various amounts of F/F0 values versus different concentrations of AChE. The concentration of ATC is 20 M, and the AChE to nal concentrations of 0, 1.0, 2.0, 3.0, incubation time is 20 min. The excitation wavelength was 550 nm. The uorescence intensities were collected at 575 nm. 4.0, and 5.0 mU mL1 (U is the abbreviation of units). We allowed the solutions to incuintegrals for region D, (D, dispersion, absorption area spanning bate at room temperature for 21 min. The absorption spectra from 450 to 570 nm) and region A, (A, aggregation, absorpwere recorded every 3 min during the hydrolysis of ATC catation area spanning from 580 to 750 nm) under the curve were lyzed by AChE. We note that the aggregation of RB-AuNPs was computed for all samples (Figure S5). Measuring the plots of a linearly dynamic process. The degree of aggregation of AuNPs the ratio A/D versus the concentrations of AChE shows that depended on the concentration of AChE, where higher concenthe detection limit can reach 1.0 mU mL1 (Figure 2e). Moretrations of AChE induced more complete aggregation. Figure 2a over, the aggregation is a time-dependent process. Figure S6 shows the concentration-dependent color change, which was shows the plots of the ratio A/D versus the incubation time further demonstrated by the change of UVvis absorption after (021 min) for various concentrations of AChE. Larger ratios 20 min of incubation time. With the increase of the concentraA/D indicate higher degrees of aggregation. Almost all ratios tion of AChE, the absorbance at 520 nm decreased gradually, A/D increased with the incubation time, except that of the blank along with an increase in the absorption band between 600 nm sample (0 mU mL1), which showd a negligible change in the and 800 nm (Figure 2c). It is worth noting that with the addi 1 ratio A/D. We conclude that the degree of aggregation of RBtion of such low concentrations of AChE (05.0 mU mL ), the AuNPs is in proportion to the concentrations of AChE and the absorption peaks spanning from 600 to 800 nm were unable incubation time. The detection limit can be further improved to be observed clearly. We reasoned that the aggregation of by monitoring the uorescence of the detached RB to as low as AuNPs was incomplete, and thus the color of the solutions 0.1 mU mL1 (Figure 2b,d,f), which, to our knowledge, is much changed from red to purple gradually. As a result, the new peak lower than most AuNP-based probes for AChE.[16,18,24] between 600 nm and 800 nm was not so distinct as that shown We evaluated the interference of this assay from other spein Figure 1a, where the concentration of AChE was 1U mL1. cies such as biothiols and proteins, particularly those that exist The changes in UVvis absorption spectra were quantied by in human body. Biothiols (0.1 mM for each biothiol) such as calculating the changes of the ratio A/D (aggregated/dispersed cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) area) for the area under the absorption peak,[23] while their corcontain thiol groups. They are similar to thiocholine that can responding changes in uorescence spectra were monitored by readily adsorb onto surfaces of AuNPs via AuS bonds, thus calculating the changes of uorescence intensity (F/F0).[20] The

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potentially removing RB molecules from Au surfaces. Howknown drug for the treatment of AD in the clinic) by high (5.0 ever, biothiols, unlike ATC, were unable to cause aggregation mg kg1 day1) and low (0.05 mg kg1 day1) doses respectively of AuNPs, because they lacked the positively charged quaterfor 10 days, and chose those treated with the equal volumes of nary ammonium groups that were capable of interacting with sodium phosphate buffer (pH 7.4) as controls. the acidic groups on RB. Certain proteins such as human Because this assay is highly sensitive and selective, a very serum albumin (HSA) containing thiol groups exhibit funcsmall amount of sample is required for the detection. We just tions similar to biothiols, i.e., they can abstract RB molecules need to add 0.5 L of each CSF sample into a 1.0 mL mixture of from Au surfaces. They however also failed to induce aggregaATC (20 M) and RB-AuNPs (5 nM), and incubate them for 20 tion of AuNPs due to the lack of the quaternary ammonium min. The addition of such a little amount of CSF sample can groups. We also investigated interferences from other proteins not only avoid the possible interference from biothiols in CSF, (1 U mL1 for each protein) that are commonly used in bioanalbut also make the color changes of AuNP solutions and their corresponding uorescence recovery amongst the four samysis, such as phosphatase alkaline (P. Alka), phosphatase acid ples more apparent than those with a large amount of added (P. Acid), acylase I (Acyl. I), -galactosidase (Galac), and glucose CSF samples. Upon the addition of a large amount of CSF the oxidase (Glu. O). All these proteins had negligible capability in changes in color and uorescence recovery amongst the four changing the color of RB-AuNPs solutions, although some of samples were similar, i.e., the color of the four samples changed them, such as P. Alka, P. Acid and Glu. O could turn on the uto purple, and, correspondingly, their uorescence recovered orescence. Each biothiol and protein was incubated with ATC in a similar level. Therefore, after adding 0.5 L of each CSF (20 M) and RB-AuNPs (5 nM), and we set the sample with only sample into the mixture of ATC and RB-AuNPs, the degrees of ATC as the blank and the presence of both AChE (0.1 U mL1) color change amongst the CSF samples were different (top in and ATC (20 M) in RB-AuNPs solution as positive comparison. Figure 3a). For the SAM-R1 sample (sample 1), the color of the None of the biothiols and proteins were able to cause aggresolution changed from red to purple within 20 min, while that gation of RB-AuNPs (Figure S7), but those containing thiol of the SAM-P8 (sample 2) with the same treatments remained groups could trigger the recovery of uorescence of RB molered. For the SAM-P8 samples treated with neostigmine, the cules (Figure S8), which fully agreed with our design. degrees of aggregation of AuNPs for the high dose (sample 3) The use of dual readouts in this assay is a remarkable advanwas higher than that of the low dose (sample 4; sample 3 was tage over most reported assays that only have one output signal. more purple in color than sample 4), both of which are, however, As mentioned above, biothiols have the similar function as that more purple than that of the SAM-P8 treated with sodium phosof thiocholine in the uorescence assay, i.e., removing RB from phate buffer (pH7.4), and less purple than that of the SAM-R1. surfaces of AuNPs, thus leading to recovery of uorescence, but We ascribed the color change to the concentrations of AChE in they are unable to cause aggregation of AuNPs, which was monitored by the naked eye. Both the condition of recovery of uorescence and the condition of color change of solutions have to be met to indicate the presence of AChE-catalyzed thiocholine. This requirement effectively avoids interference from biothiols, thus guaranteeing the accuracy of the detection results. Encouraged by the above-mentioned investigations, we evaluated if the probe we described here could be utilized to monitor AChE in complex samples such as CSF. CSF, an ideal source that reects the metabolic and pathological states of the central nervous system more directly than any other bodily uids, is more accessible and less costly than neuroimaging that is sometimes used for clinical diagnosis of AD.[25] In order to demonstrate the practical potential of this assay for AChE in CSF, we obtained transgenic senescence-accelerated mouse (SAM), which is an accelerated aging model for studying age-related learning and memory decits.[26] Figure 3. Monitoring the level of AChE in CSF of transgenic mice. a) Top: color change SAM prone/8 (SAM-P8) is a common model after adding four samples (1, 2, 3, 4), to each of which (0.5 L) was added into 1.0 mL mixof age-related dementia of the Alzheimer ture of RB-AuNPs (5 nM) and ATC (20 M). Bottom: the four CSF samples were pretreated phenotype, in whose CSF the level of AChE is with 100 nM of galantamine, and then RB-AuNPs (5 nM) and ATC (20 M) was added. b) Fluorescent images of (a). c) Absorption responses of (a). d) The corresponding changes of much lower than that in the SAM resistant/1 uorescence intensity of (b). 1: SAM-R1 sample treated with the equal volume of sodium phos(SAM-R1), which is another species of SAM phate buffer (pH 7.4); 2: SAM-P8 sample treated with the equal volume of sodium phosphate [ 27 ] that shows normal aging characteristics. buffer (pH 7.4); 3: SAM-P8 sample treated with high dose (5.0 mg kg1 day1) of neostigmine; We treated SAM-P8 mice with neostigmine (a 4: SAM-P8 sample treated with low dose (0.05 mg kg1 day1) of neostigmine.

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different samples. As published results showed that the level of AChE in CSF of AD brain is signicantly decreased,[8] we reasoned that the level of AChE in SAM-P8 sample without treatments of neostigmine is much lower than that of the SAM-R1 sample, thus the former remained red while the latter caused the complete aggregation of AuNPs. After 10 days of persistent treatment with neostigmine, SAM-P8 mice treated with a high dose of neostigmine become more active in their behaviors and their CSF also contain a higher level of AChE than those treated with low dose of neostigmine. This result is exciting because the levels of color change of solutions correlate well with the doses of treated drug. In order to further conrm the results of color changes, the ratios A/D were used to measure the degrees of aggregation of AuNPs (Figure 3c, black bars). To ensure that the aggregation was specically caused by AChE in CSF, we pretreated the four CSF samples with 100 nM of galantamine (another kind of commercial AD drug) to inhibit the activity of AChE, the color of RB-AuNPs solutions indeed remained red (bottom images in Figure 3a) and the ratios A/D displayed negligible difference (Figure 3c, red bars). Moreover, the recovery of RB uorescence for the four CSF samples was still different, higher levels of AChE in SAM-R1 CSF induced stronger uorescence recovery (top images in Figure 3b), and pretreatment of the CSF samples with AD drug could prevent the turn-on of uorescence (bottom images in Figure 3b). The uorescence variations were conrmed by comparing the F/F0 values of the CSF samples (Figure 3d). To establish calibration curves to quantitatively measure the levels of AChE in CSF using this present assay, we need to nd the smallest volume of SAM-P8 CSF sample as the background where the AChE level is undetectable. The background samples were then spiked with various concentrations of AChE. As shown in Figure S9, this smallest volume of CSF sample was determined to be 0.125 L (incubated with a 1.0 mL mixture of ATC (20 M) and RB-AuNPs (5 nM)) and then used as the background to establish the calibration curves. With a standard addition method, we established two calibration curves: one was based on the ratios A/D, the other was based on the F/F0 values. As shown in Figure S10, the sensitivity of this assay based on the F/F0 values was higher than that based on the ratios A/D and, moreover, the detection range of this assay based on the F/F0 values was wider than that based on the ratios A/D. Thus, we chose the calibration curve based on the F/F0 values to measure the AChE levels in the CSF samples. Owing to the F/F0-valuebased calibration curve (Figure S10b) and the changes of the F/F0 values (Figure 3d) for CSF samples, the AChE levels in the four CSF samples can be calculated approximately to be 3.1 U mL1, 0.56 U mL1, 1.1 U mL1, and 0.85 U mL1. These results indicate that the treatment of AChE inhibitors could cause a dosedependent variation of the AChE levels in CSF, which agreed with the reported results.[28] To the best of our knowledge, this is the rst report in which an AuNP-based assay was utilized to monitor AChE in CSF samples of clinical relevance. In conclusion, we present a highly sensitive and selective assay with dual readouts (colorimetric and uorometric) for AChE based on RB-AuNPs. The detection limit is 0.1 mU mL1, much lower than that of reported assays for AChE.[16,18,24] We demonstrate the practical application of this assay by measuring the levels of AChE in CSF of transgenic mice and monitoring

the disease progression and drug treatment effects for AD. We believe that this RB-AuNP-based assay could be useful for monitoring AChE in human CSF for early diagnostics and prognostics of AD, especially in combination with other currently existing neuroimaging techniques and CSF biomarkers as well as other settings such as lab-on-a-chip systems.[29]

Experimental Section
Preparation of Transgenic Senescence-Accelerated Mouse CSF Samples: SAM-P8 and SAM-R1 mice at 3 months of age were obtained from the Department of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). All mice were maintained in an AAALACaccredited facility and the use of animals was approved by the Animal Care and Use Committee of National Center for Nanoscience and Technology (2010-0013). SAM-P8 mice were treated with high doses (5.0 mg kg1 day1) and low doses (0.05 mg kg1 day1) of neostigmine for 10 days, and meanwhile both SAM-P8 and SAM-R1 treated with sodium phosphate buffer (pH 7.4) were used as controls. The positive and negative control groups were randomly chosen. After anaesthetising each mouse with 0.8 mL of 3.5% chloral hydrate dissolved in normal saline, the CSF samples were collected using a microinjector, whose needle was connected with a plastic pipe terminated in another needle. The CSF samples were centrifugalized at 1000 rpm for 5 min, the resulting supernatants were collected for the following detection experiments. Other experimental details are provided in the Supporting Information.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements
We thank the Chinese Academy of Sciences (KJCX2-YW-M15), the National Science Foundation of China (21105018, 21025520, 90813032, 20890020), and the Ministry of Science and Technology (2009CB930001, 2011CB933201) for nancial support. Received: September 25, 2011 Published online: November 29, 2011 [1] a) S. K. Ghosh, T. Pal, Chem. Rev. 2007, 107, 4797; b) M. C. Daniel, D. Astruc, Chem. Rev. 2004, 104, 293. [2] a) S. P. Song, Y. Qin, Y. He, Q. Huang, C. H. Fan, H. Y. Chen, Chem. Soc. Rev. 2010, 39, 4234; b) D. B. Liu, W. S. Qu, W. W. Chen, W. Zhang, Z. Wang, X. Y. Jiang, Anal. Chem. 2010, 82, 9606; c) S. He, D. Li, C. Zhu, S. Song, L. Wang, Y. Long, C. H. Fan, Chem. Commun. 2008, 40, 4885. [3] W. L. Daniel, H. S. Han, J. S. Lee, C. A. Mirkin, J. Am. Chem. Soc. 2009, 131, 6362. [4] Y. Jiang, H. Zhao, N. N. Zhu, Y. Q. Lin, P. Yu, L. Q. Mao, Angew. Chem. Int. Ed. 2008, 47, 8601. [5] J. M. Nam, A. R. Wise, J. T. Groves, Anal. Chem. 2005, 77, 6985. [6] S. S. Agasti, S. Rana, M. H. Park, C. K. Kim, C. C. You, V. M. Rotello, Adv. Drug. Deliver. Rev. 2010, 62, 316. [7] a) Y. Zhang, Y. Li, X. P. Yan, Anal. Chem. 2009, 81, 5001; b) F. Gao, Q. Q. Ye, P. Cui, X. X. Chen, M. G. Li, L. Wang, Anal. Methods 2011, 3, 1180; c) B. Kowalczyk, D. A. Walker, S. Soh, B. A. Grzybowski, Angew. Chem. Int. Ed. 2010, 49, 5737; d) D. G. Georganopoulou,

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