Biosensors and Bioelectronics 71 (2015) 396–400

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

An ultrasensitive electrochemical immunosensor for apolipoprotein E4 based on fractal nanostructures and enzyme amplification Yibiao Liu a,b, Li-Ping Xu a,b,n, Shuqi Wang a,b, Weizhao Yang a,b, Yongqiang Wen a,b, Xueji Zhang a,b,n a b

Research Centre for Bioengineering and Sensing Technology, University of Science & Technology Beijing, Beijing 100083, PR China School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 12 February 2015 Received in revised form 18 April 2015 Accepted 21 April 2015 Available online 23 April 2015

Human apolipoprotein E4 (APOE4) is a major risk factor for Alzheimer's disease (AD) and can greatly increase the morbidity. In this work, an ultrasensitive sandwich-type electrochemical immunosensor for the quantitative detection of APOE4 was designed based on fractal gold (FracAu) nanostructures and enzyme amplification. The FracAu nanostructures were directly electrodeposited by hydrogen tetrachloroaurate (HAuCl4) on polyelectrolytes modified indium tin oxide (ITO) electrode. The sensing performances of the modified interface were investigated by cyclic voltammetry (CV). After functionalization with HRP-labeled APOE4 antibody, the human APOE4 could be detected quantitatively by current response. The current response has a linear relationship with the logarithm of human APOE4 concentrations in a range from 1.0 to 10,000.0 ng/mL, with a detection limit of 0.3 ng/mL. The fabricated APOE4 electrochemical immunosensor exhibits strong specificity, high sensitivity, low detection limit and wide linear range. The detection of human APOE4 provides a strong support for the prevention of AD and early-stage warning for those susceptible populations. & 2015 Elsevier B.V. All rights reserved.

Keywords: Immunosensor APOE4 Fractal Au nanostructure Alzheimer's disease

1. Introduction Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly, and the main symptom of AD is characterized by cerebral extracellular amyloid plaques and intracellular neurofibrillary tangles (Selkoe, 2003; Kosik, 1992; Jakob-Roetne and Jacobsen, 2009; Scott and Orvig, 2009; Gaggelli et al., 2006). Although the molecular mechanisms of AD pathogenesis have not been clearly understood, recent studies have demonstrated that apolipoprotein E4 (APOE4) is a key factor related to AD (Bu, 2009). Human apolipoprotein E (APOE), a 34-kDa protein, mediates the binding of lipoproteins to the low density lipoprotein receptor. There are three isoforms: APOE2, APOE3 and APOE4. Of these three isoforms, APOE4 is a mainly causative factor for AD (Mahley and Innerarity, 1983; Kim et al., 2009; Verghese et al., 2011; Utermann et al., 1977). As early as 1993, it was reported that APOE4 allele frequency was about 15% in general populations but was more than 50% in AD patients. Individuals with heterozygosity n Corresponding authors at: Research Centre for Bioengineering and Sensing Technology, University of Science & Technology Beijing, Beijing 100083, PR China. Fax: þ86 10 82375840. E-mail addresses: [email protected] (L.-P. Xu), [email protected] (X. Zhang).

http://dx.doi.org/10.1016/j.bios.2015.04.068 0956-5663/& 2015 Elsevier B.V. All rights reserved.

of APOE4 allele were three to four times more likely to suffer from developing AD than those without APOE4 allele, and APOE4 homozygosity allele increased the risk more than tenfold (Corder et al., 1993; Bertram and Tanzi, 2008; Roses and Saunders, 1994). It was reported that APOE4 protein promoted the formation of cerebral extracellular amyloid plaques and intracellular neurofibrillary tangles. APOE4 can also increase the cytotoxicity of βamyloid (Aβ) peptides oligomers, and the binding between APOE4 and Aβ-degrading enzyme can inhibit the clearance of Aβ aggregations. Moreover, APOE4 also increase blood–brain barrier (BBB) susceptibility to injury and finally lead to BBB breakdown (Nishitsuji et al., 2011; Bell et al., 2012). The key role of APOE4 in the pathogenesis of AD prompts APOE4 as a potential biomarker for AD diagnosis. Up till now, the detection of APOE4 achieves very few progresses. In previous works, only some standard techniques such as enzyme-linked immunosorbent assays (ELISA) and western-blot analysis (Vincent-Viry et al., 1998; Taddeia et al., 1997), microarray technology (Morales-Narváez et al., 2012) were used to detect the total APOE level. They found that the levels of APOE were typically in the μg/mL range for cerebrospinal fluid (CSF) and serum. Recently, Sullivan et al. (2011) and Martínez-Morillo et al. detected APOE by ELISA and mass spectrometry (MS) (MartínezMorillo et al., 2014). In 2012, Wang et al. (2012) detected APOE4 in brain tissue by MS through constructing 15N-Labeled APOE4 as an internal standard. Nevertheless, these conventional detection

Y. Liu et al. / Biosensors and Bioelectronics 71 (2015) 396–400

methods often suffer from low sensitivity, narrow linear range, time-consuming and the requirement of expensive instruments. Therefore, exploring simple, sensitive and low cost diagnostic methods for the detection of APOE4 is needed. Electrochemical immunosensors have attracted considerable interest due to their advantages including ease of operation, rapid detection, low-cost and high sensitivity (Drummond et al., 2003; Jarocka et al., 2014; Wang et al., 2014). Due to their unique physical/chemical properties, good biocompatibility and large effective electroactive surface area, nanomaterials have been widely used in many fields including the electrochemical immunosensor field. The introduction of nanomaterials greatly enhances the sensitivity of the sensor (Soleymani et al., 2009). So far, a great variety of nanomaterials with interesting morphologies have been used to construct electrochemical sensors and biosensors; such as nanoparticles (Xiao et al., 2012; Shu et al., 2015; Kwak et al. 2014), nanodiamonds (Zhang et al., 2014), nanowires (Li et al., 2013), carbon nanotubes (Münzer et al., 2013; Zamolo et al., 2012), quantum dots (Dong et al., 2010; Sarkar et al., 2014) and graphene (Tian et al., 2013; An et al. 2013; Zeng et al., 2010). Recently, our group reported a novel electrode modified with FracAu on ITO surface by mimicking the nasal membrane in humans (Xu et al. 2012). Compared to traditional two-dimensional electrodes, FracAu electrode has a poriferous surface, which greatly increases the electrochemical active surface area and accelerates electron transfer efficiency. Enzyme amplification is another popular method employed for signal enhancement in electrochemical immunosensors (Yu et al., 2006; Zhao et al., 2014). The combination of FracAu nanostructures and enzyme amplification will be helpful in fabricating high sensitive sensor. In this work, we construct a novel APOE4 electrochemical immunosensor on the basis of FracAu nanostructure and enzyme amplification. The combination of fractal nanostructure and enzyme-catalyzed signal amplification enables the sensor to obtain a better sensitivity and lower detection limit. As shown in Fig. 1, the fabricated APOE4 electrochemical immunosensor includes a FracAu electrode and electrochemical detection system. In the electrochemical detection system triggered by horse reddish peroxidase (HRP), hydroquinone (HQ) is added to the detection solution as a mediator to transfer the electron between HRP and hydrogen peroxide (H2O2). In the presence of H2O2, the HRP molecules catalyze the oxidation of hydroquinone (HQ) into quinine (Q), during which the reductive current can be monitored (Yu et al., 2006; Dai et al., 2003; Ionescu et al., 2007). The detection limit of the APOE4 electrochemical immunosensor is 0.3 ng/mL. The results presented here make a progress in the detection of APOE4 and may pave the way for early-stage warning and diagnosis of AD.

397

2. Experiment section 2.1. Apparatus and reagents Scanning electron microscopy analysis was performed using a JEOL JSM-6701F (JEOL, Japan). Electrochemical measurements were carried out on a 660D Electrochemical Analyzer (CH Instrument Inc., USA). Human APOE4 antibody, HRP labeled human APOE4 antibody, Human APOE4 and bovine serum albumin (BSA) were obtained from Abcam (Hong Kong) Ltd. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4  3H2O) was obtained from Alfa Aesar. The buffer of 0.01 M sodium phosphate-buffered saline (PBS) of pH 7.4 was used as an incubating and washing buffer, and 0.01 M PBS of pH 7.4 containing 0.1 M KCl was used as supporting electrolyte for enzymatic substrates. All other reagents were of analytical reagent grade and commercially available. All solutions were prepared with ultrapure water (Milli-Q, 18.2 MΩ cm). 2.2. Fabrication of FracAu electrode The indium tin oxide (ITO) glass substrate was firstly cleaned successively by sonication in acetone and ethanol for 20 min successively, and then thoroughly rinsed with ultrapure water. The clean ITO was then immersed in diluted ammonia solution for 10 min. Subsequently, the positively charged substrate was successively immersed in an aqueous solution of PSS (1 mg/mL) and PDDA (1 mg/mL) for 10 min. This process was repeated six times to produce the (PSS/PDDA)6 film. Every deposition was followed by rinsing with distilled water for 2 min and drying with a stream of nitrogen (N2) for 1 min. The FracAu electrode was prepared by potentiostatic electrodeposition on the (PSS/PDDA)6 modified ITO electrode. The working electrode (ITO glass) was placed facing a platinum slice (1  2 cm2) counter electrode with a 2 cm gap between them. An Ag/AgCl electrode was employed as the reference electrode. The electrodeposition was proceeded by 1800 s in a solution of HAuCl4 (10 mg/mL) and H2SO4 (0.5 M) at  1800 mV. 2.3. Fabrication of sensing interface After the electrodeposition process, the FracAu electrode was rinsed with ultrapure water for 10 min. The sensing interface was constructed on the FracAu electrode surface through a series of self-assembling processes. Firstly, the FracAu electrode was modified with 100 μg/mL human APOE4 antibody for 2 h. Subsequently, BSA (1 mg/mL) was used to block the non-specific binding sites of the FracAu electrode. Then the modified electrode was incubated in APOE4 solution with specific concentration for 1.5 h. Finally, 50 μg/mL HRP-labeled human APOE4 antibodies were added on the electrode surface and allowed to react for 1.5 h. Every step was followed by rinsing with 0.01 M PBS for 10 min. Each incubation procedure was performed at 37 °C. 2.4. Electrochemical measurements

Fig. 1. The schematic illustration of the APOE4 electrochemical immunosensor.

All electrochemical measurements were performed in a conventional three-electrode system with FracAu electrode modified by human APOE4 antibody as the working electrode, an Ag/AgCl electrode as a reference electrode and a platinum slice (1  2 cm2) electrode as the counter electrode. Amperometric measurements of electrochemical immunoassay were performed in 30 mL PBS buffer solution containing 1 mM hydroquinone by applying a working potential of  0.08 V under constant stirring. When transient currents decayed to a steady-state value, 200 μL of 1 mM H2O2 was quickly added to the solution. The selectivity of the fabricated sensor was studied using the same protocol in the

398

Y. Liu et al. / Biosensors and Bioelectronics 71 (2015) 396–400

presence of the proteins BSA, APOE2 and APOE3. All experiments were carried out at room temperature.

3. Results and discussion 3.1. Characterization of the FracAu nanostructure modified electrode The FracAu nanostructures were directly electrodeposited by hydrogen tetrachloroaurate (HAuCl4) on the indium tin oxide (ITO) electrode surface. Before the electrodeposition, the ITO electrode was coated with (PSS/PDDA)6 film via layer by layer assembly, which could make the FracAu nanostructures more firm and stable on the ITO surface (Zhang et al., 2004). To evaluate the stability of the FracAu electrode, electrochemical measurements were carried out in 10 mM PBS (pH 7.4) containing 0.1 M KCl and 5 mM [Fe (CN)6]3  /[Fe(CN)6]4  , as shown in Fig. S1. The results show that there is no noticeable change of currents over a period of 72 h in water at room temperature, which indicates that the FracAu nanostructures are very stable on the ITO surface. The typical SEM images of the FracAu nanostructures are shown in Fig. 2(A) and (B). The FracAu nanostructures exhibit a multidimensional branched structure on the ITO surface in the scale of micrometers. The length of these micro-trees is about 5– 10 μm. The micro-trees could be further divided into small parts, each of which is approximately a smaller version of the whole. The micro-trees are comprised of several gold nanoparticles and the smallest part is just a few tens of nanometers. Fig. 2B is the sideview image of the FracAu nanostructures, which indicates that the height of FracAu nanostructure is about 15–26 μm. Moreover, the surface roughness and three dimensional (3D) morphology of the FracAu nanostructures were also characterized by atomic force microscope (AFM). The results show that the surface roughness of the FracAu nanostructures is about 400 nm (scan size: 10 μm  10 μm). From the high resolution of AFM images, some small particles can be resolved (Fig. S2). 3.2. Fabrication of sensing interface The fabrication process of the sensing interface was investigated by CV measurements. As can be seen from Fig. 3A, the peak current gradually decreases with the increase of assembly procedure. The bare FracAu electrode shows a large peak current in 5 mM [Fe(CN)6]3  /[Fe(CN)6]4  (Fig. 3A, black line). After the FracAu electrode is modified with human APOE4 antibody, the peak current exhibits sharp decline (Fig. 3A, red line), which suggests that the assembly of human APOE4 antibody introduces a barrier to the interfacial electron transfer. Then BSA is used to block the non-specific binding sites. After the addition of BSA, the peak current decreases remarkably (Fig. 3A, green line), which

indicates that a large number of non-specific binding sites have been blocked by BSA. As can be seen from the turquoise line and blue line in Fig. 3A, the slight decrease in the peak current is observed after introducing APOE4 and the HRP labeled human APOE4 antibody onto the electrode surface. Based on the analysis above, it can be inferred that the sandwich structure is formed successfully. Thereby, the subsequent quantification of APOE4 can be realized by electrochemical detection in the presence of the enzyme catalytic substrates. Fig. 3B shows the typical CVs of as-prepared electrodes in enzymatic substrate solution containing 1 mM HQ and 1 mM H2O2. A well-defined reduction peak at  0.08 V is observed in the CVs. Notably, the response current increases with the increase of APOE4 concentration. In other words, the HRP labeled human APOE4 antibody is captured at the electrode surface and results in the increase of the reduction peak current in the presence of HQ and H2O2. The higher the APOE4 concentration is, the more HRP labeled human APOE4 antibody is captured and a subsequently higher peak current is obtained. 3.3. The characterization of analytical performance The deposited FracAu nanostructures serve as the carrier of human APOE4 antibody, APOE4 and the HRP labeled APOE4 antibody. In the presence of H2O2, the HRP molecules catalyze the oxidation of hydroquinone (HQ) into quinine (Q) and the reductive current in this process can be recorded. According to this principle, the changes in the electrochemical signal (reductive current) could be used to detect APOE4 quantitatively. The changes in the current (ΔI) were monitored by chronoamperometry versus the concentration of human APOE4. Before that, the concentrations of APOE4 antibody and HRP-labeled APOE4 antibody were optimized on an immobilized efficiency. The optimized concentrations of APOE4 antibody and HRP-labeled APOE4 antibody were 100 μg/mL and 50 μg/mL respectively. Fig. 4A shows that the amperometric current from the electrochemical immunosensor based on the FracAu nanostructures reaches a steady state response rapidly. The corresponding calibration plot of the amperometric response vs. logarithm of human APOE4 concentration is shown in Fig. 4C. There is a linear relationship between ΔI and the logarithm of APOE4 concentration from 1 ng/mL to 10,000 ng/mL. Excellent sensor reproducibility is illustrated by the small error bars in Fig. 4C. The reproducibility of the method shows a relative standard deviation (RSD) of 6.6% ([APOE4]: 10,000 ng/mL). The detection limit for human APOE4 based on the FracAu electrochemical immunosensor is 0.3 ng/mL (S/N ¼3, Fig. 4C red line). In physiological conditions, the concentration of APOE4 is typically in the μg/mL range for cerebrospinal fluid (CSF) in the individuals that do have APOE4 allele (Lefranc et al., 1996). Hence, the detection limit of the as-fabricated

Fig. 2. The top-view (A) and side-view (B) SEM images of the FracAu nanostructure electrode synthesized by electrodeposition in 10 mM HAuCl4 at  1.8 V for 1800 s.

Y. Liu et al. / Biosensors and Bioelectronics 71 (2015) 396–400

399

Fig. 3. Characteristics of sensing interface: (A) Cyclic voltammogram of the FracAu electrode (a) modified with human APOE4 antibody, (b) human APOE4 antibody þ BSA, (c) human APOE4 antibody þBSAþ APOE4, (d) human APOE4 antibody þ BSAþAPOE4 þHRP-labeled human APOE4 antibody (e). CVs are performed in 5 mM [Fe(CN)6]3  /[Fe (CN)6]4  at scan rates of 0.05 V s  1. (B) CVs in 0.01 M PBS solution (pH 7.4) containing 1 mM HQ and 1 mM H2O2 of the as-prepared immunosensor for APOE4 at different concentrations. The red dash line represents the reduction potential of H2O2 at  0.08 V. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

electrochemical immunosensor can meet the requirements of clinical application. Compared to the immunosensor based on FracAu, signals corresponding to the immunosensors based on the plane Au electrode decreases to 60% (Fig. 4B). The detection limit of the sensor based on the plane Au electrode is 1 ng/mL (S/N ¼ 3, Fig. 4C black line). It is obvious that the electrochemical immunosensor based on FracAu is much more sensitive than the one based on the plane Au electrode. The electroactive surface area was measured by CV in 0.5 M H2SO4 according to previous studies (Nagaraju and Lakshminarayanan, 2009; Trasatti and Petrii, 1992; Xu et al., 2012). The electroactive surface area for FracAu electrode was 56.28 cm2, which is almost 60 times as much as its projected area (Fig. S3). Compared to the plane Au electrode, FracAu electrodes have larger electroactive surface area, which greatly enhances the active sites for recognition. Besides the increase in surface area, FracAu electrodes also exhibit multidirectional structures, which shorten the diffusion distance from solution to electrode surface and enhance the binding efficiency remarkably. The 3D porous nanostructures of FracAu facilitate the accessibility of analytes, leading to faster and more efficient binding (Xu et al., 2012). Moreover, the introduction of enzyme amplification based on HRP greatly enhances electrochemical signals. Ultralow concentration of APOE4 could be detected successfully by employing the as-fabricated electrochemical sensing platform. Based on the combination of FracAu nanostructures and enzyme amplification, a highly sensitive method to the novel APOE4 detection was achieved. In the last two years, some biosensors based on nanomaterials were used to detect APOE or APOE4 (Table S1). Merkoçi's group

designed on-chip magneto-immunoassay based on commercial quantum dot labels for detecting the APOE, achieving a limit of detection of 12.5 ng/mL with a linear range from 10 to 200 ng/mL (Medina-Sánchez et al., 2014). Recently, this group developed a new nanobiosensor based on porous magnetic microspheres (PMM) as an efficient capturing platform and electrocatalytic gold nanoparticles as tags for the detection of APOE. By benefiting from this PPM preconcentrating platform, the limit of detection for APOE was lowered to 0.08 ng/mL (De La Escosura-Muñiz et al., 2015). However, these biosensors cannot distinguish different isoforms of APOE. Our FracAu electrodes are simple and have easy fabrication. The limit of detection of APOE4 electrochemical immunosensor is 0.3 ng/mL with a wide linear range from 1 to 10,000 ng/mL. Once an additional preconcentrating platform is incorporated into the fabricated immunosensor, the limit of detection may be further lowered. Remarkably, the electrochemical immunosensor in this work can distinguish APOE4 from different isoforms of APOE well. 3.4. The specificity of the APOE4 electrochemical immunosensor The specificity of the novel APOE4 electrochemical immunosensor was also tested. Fig. 5 presents the histogram of the current response in the presence of 1 μg/mL BSA, 1 μg/mL human APOE2, 1 μg/mL human APOE3 and 1 ng/mL human APOE4. Only human APOE4 gave an obvious current response even though the concentration of other proteins was 1000 times the concentration of APOE4. The ΔI for BSA, APOE2 and APOE3 are 6.3%, 9.0%, and 9.8% of the ΔI for human APOE4, respectively, which indicates that

Fig. 4. Amperometric results for the APOE4 electrochemical immunosensor incubated with different concentrations of human APOE4 protein in the range of 1 ng/mL to 10,000 ng/mL: steady state amperometric current at  0.08 V and 1000 rpm after placing the sensor based on the FracAu electrode (A) or plane Au electrode (B) in 0.01 M PBS containing 1 mM hydroquinone and 1 mM H2O2. (C) The calibration plots for the detection of APOE4 by the electrochemical immunosensor. The linear equation: y¼ 33.1xþ 311.7, R2 ¼ 0.9931 (Fractal Au electrode); y ¼9.6x þ87.2, R2 ¼ 0.9847 (Plane Au electrode). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

400

Y. Liu et al. / Biosensors and Bioelectronics 71 (2015) 396–400

Fig. 5. Current responses of the APOE4 electrochemical immunosensor. The ΔI represents the current change of the sensor to different analytes. The concentration of APOE4 is 1 ng/mL; the concentration of BSA, APOE2 and APOE3 was 1 μg/mL. The error bars represent the standard deviation from three independent experiments.

the as-prepared electrochemical immunosensor has very good specificity for human APOE4.

4. Conclusions This work reported a novel APOE4 electrochemical immunosensor based on FracAu nanostructure and enzyme amplification. Due to good biocompatibility and large electroactive surface area of FracAu electrodes, the binding sites and the binding efficiency for the antigen–antibody interaction are increased. And the introduction of enzyme amplification further enhances the sensitivity of sensor. The detection limit of the proposed APOE4 electrochemical immunosensor is 0.3 ng/mL with a wide linear range from 1 to 10,000 ng/mL. Moreover, the fabricated electrochemical immunosensor also exhibits high specificity, sensitivity, good reproducibility, fast response and long stability. Meanwhile, the presented system is able to well distinguish APOE4 among the three APOE isoforms. Therefore, it could be applied for the detection of APOE4 for prevention and diagnosis of AD in clinical, which holds great significance for the health of the elderly.

Acknowledgements The work was supported by National Natural Science Foundation of China (NSFC Grant nos. 21475009, 21475008, 21073203, 2127007, 21275017), the Fundamental Research Funds for the Central Universities (FRF-TP-14-065A2) and Beijing Higher Education Young Elite Teacher Project (YETP0424).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.068.

References An, J.H., Park, S.J., Kwon, O.S., Bae, J., Jang, J., 2013. ACS Nano 7, 10563–10571. Bell, R.D., Winkler, E.A., Singh, I., Sagare, A.P., Deane, R., Wu, Z.H., Holtzman, D.M.,

Betsholtz, C., Armulik, A., Sallstrom, J., Berk, B.C., Zlokovic, B.V., 2012. Nature 485, 512–516. Bertram, L., Tanzi, R.E., 2008. Nat. Rev. Neurosci. 9, 768–778. Bu, G.J., 2009. Nat. Rev. Neurosci. 10, 333–344. Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines, J.L., Pericak-Vance, M.A., 1993. Science 261, 921–923. Dai, Z., Yan, F., Chen, J., Ju, H.X., 2003. Anal. Chem. 75, 5429–5434. De La Escosura-Muñiz, A., Plichta, Z., Horák, D., Merkoçi, A., 2015. Biosens. Bioelectron. 67, 162–169. Dong, H.F., Yan, F., Ji, H.X., Wong, D.K.Y., Ju, H.X., 2010. Adv. Funct. Mater. 20, 1173–1179. Drummond, T.G., Hill, M.G., Barton, J.K., 2003. Nat. Biotechnol. 21, 1192–1199. Gaggelli, E., Kozlowski, H., Valensin, D., Valensin, G., 2006. Chem. Rev. 106, 1995–2044. Ionescu, R.E., Cosnier, S., Herrmann, S., Marks, R.S., 2007. Anal. Chem. 79, 8662–8668. Jakob-Roetne, R., Jacobsen, H., 2009. Angew. Chem. Int. Ed. 48, 3030–3059. Jarocka, U., Sawicka, R., Góra-Sochacka, A., Sirko, A., Zagórski-Ostoja, W., Radecki, J., Radecka, H., 2014. Biosens. Bioelectron. 55, 301–306. Kim, J., Basak, J.M., Holtzman, D.M., 2009. Neuron 63, 287–303. Kosik, K.S., 1992. Science 256, 780–783. Kwak, K., Kumar, S.S., Pyo, K., Lee, D., 2014. ACS Nano 8, 671–679. Lefranc, D., Vermersch, P., Dallongeville, J., Daems-Monpeurt, C., Petit, H., Delacourte, A., 1996. Neurosci. Lett. 212, 91–94. Li, B.R., Hsieh, Y.J., Chen, Y.X., Chung, Y.T., Pan, C.Y., Chen, Y.T., 2013. J. Am. Chem. Soc. 135, 16034–16037. Mahley, R.W., Innerarity, T.L., 1983. Biochim. Biophys. Acta 737, 197–222. Martínez-Morillo, E., Nielsen, H.M., Batruch, I., Drabovich, A.P., Begcevic, I., Lopez, M.F., Minthon, L., Bu, G.J., Mattsson, N., Portelius, E., Hansson, O., Diamandis, E. P., 2014. J. Proteome Res. 13, 1077–1087. Medina-Sánchez, M., Miserere, S., Morales-Narváez, E., Merkoçi, A., 2014. Biosens. Bioelectron. 54, 279–284. Morales-Narváez, E., Montón, H., Fomicheva, A., Merkoçi, A., 2012. Anal. Chem. 84, 6821–6827. Münzer, A.M., Michael, Z.P., Star, A., 2013. ACS Nano 7, 7448–7453. Nagaraju, D.H., Lakshminarayanan, V., 2009. J. Phys. Chem. C 113, 14922–14926. Nishitsuji, K., Hosono, T., Nakamura, T., Bu, G.J., Michikawa, M., 2011. J. Biol. Chem. 286, 17536–17542. Roses, A.D., Saunders, A.M., 1994. Curr. Opin. Biotechnol. 5, 663–667. Sarkar, D., Liu, W., Xie, X.J., Anselmo, A.C., Mitragotri, S., Banerjee, K., 2014. ACS Nano 8, 3992–4003. Scott, L.E., Orvig, C., 2009. Chem. Rev. 109, 4885–4910. Selkoe, D.J., 2003. Harvey Lect. 99, 23–45. Shu, T., Su, L., Wang, J.X., Li, C.Z., Zhang, X.J., 2015. Biosens. Bioelectron. 66, 155–161. Soleymani, L., Fang, Z., Sargent, E.H., Kelley, S.O., 2009. Nat. Nanotechnol. 4, 844–848. Sullivan, P.M., Han, B., Liu, F., Mace, B.E., Ervin, J.F., Wu, S., Koger, D., Paul, S., Bales, K. R., 2011. Neurobiol. Aging 32, 791–801. Taddeia, K., Clarnette, R., Gandy, S.E., Martins, R.N., 1997. Neurosci. Lett. 223, 29–32. Tian, J., Deng, S.Y., Li, D.L., Shan, D., He, W., Zhang, X.J., Shi, Y., 2013. Biosens. Bioelectron. 49, 466–471. Trasatti, S., Petrii, O.A., 1992. J. Electroanal. Chem. 327, 353–376. Utermann, G., Hees, M., Steinmetz, A., 1977. Nature 269, 604–607. Verghese, P.B., Castellano, J.M., Holtzman, D.M., 2011. Lancet Neurol. 10, 241–252. Vincent-Viry, M., Schiele, F., Gueguen, R., Bohnet, K., Visvikis, S., 1998. Clin. Chem. 44, 957–965. Wang, M.Y., Chen, J.J., Turko, I.V., 2012. Anal. Chem. 84, 8340–8344. Wang, Y.L., Li, X.J., Cao, W., Li, Y.Y., Li, H., Du, B., Wei, Q., 2014. Biosens. Bioelectron. 61, 618–624. Xiao, F., Song, J.B., Gao, H.C., Zan, X.L., Xu, R., Duan, H.W., 2012. ACS Nano 6, 100–110. Xu, L.P., Wang, S.Q., Dong, H.F., Liu, G.D., Wen, Y.Q., Wang, S.T., Zhang, X.J., 2012. Nanoscale 4, 3786–3790. Yu, X., Munge, B., Patel, V., Jensen, G., Bhirde, A., Gong, J.D., Kim, S.N., Gillespie, J., Gutkind, J.S., Papadimitrakopoulos, F., Rusling, J.F., 2006. J. Am. Chem. Soc. 128, 11199–11205. Zamolo, V.A., Valenti, G., Venturelli, E., Chaloin, O., Marcaccio, M., Boscolo, S., Castagnola, V., Sosa, S., Berti, F., Fontanive, G., Poli, M., Tubaro, A., Bianco, A., Paolucci, F., Prato, M., 2012. ACS Nano 6, 7989–7997. Zeng, Q., Cheng, J.S., Tang, L.H., Liu, X.F., Liu, Y.Z., Li, J.H., Jiang, J.H., 2010. Adv. Funct. Mater. 20, 3366–3372. Zhang, W.L., Patel, K., Schexnider, A., Banu, S., Radadia, A.D., 2014. ACS Nano 8, 1419–1428. Zhang, X., Shi, F., Yu, X., Liu, H., Fu, Y., Wang, Z.Q., Jiang, L., Li, X.Y., 2004. J. Am. Chem. Soc. 126, 3064–3065. Zhao, Y., Liu, L.Q., Kong, D.Z., Kuang, H., Wang, L.B., Xu, C.L., 2014. ACS Appl. Mater. Interfaces 6, 21178–21183.