Enhanced Detection Specificity and Sensitivity of Alzheimer's Disease

Feb 26, 2016 - (D, E) Heat maps showing the staining patterns and signal intensities for (D) QD-PEG-BTA and (E) ThT in the regions highlighted by yell...
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Enhanced Detection Specificity and Sensitivity of Alzheimer’s Disease Using Amyloid-beta Targeted Quantum Dots Li Quan, Jiangxiao Wu, Lucas A. Lane, Jianquan Wang, Qian Lu, Zheng Gu, and Yiqing Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00019 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on March 1, 2016

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Bioconjugate Chemistry

Enhanced Detection Specificity and Sensitivity of Alzheimer’s Disease Using Amyloid-beta Targeted Quantum Dots Li Quan,† Jiangxiao Wu,† Lucas A. Lane,‡ Jianquan Wang,† Qian Lu,† Zheng Gu,‡ Yiqing Wang*,†,‡ †

Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu Province 210093, China. ‡ Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, Georgia 30322, USA. ABSTRACT: Diagnostics of Alzheimer’s disease (AD) commonly employ the use of fluorescent thioflavin derivatives having affinity for the amyloid-β (Aβ) proteins associated with AD progression. However thioflavin probes have limitations in their diagnostic capabilities arising from a number of undesireable qualities including poor photostaility, weak emission intensitites, and high emission overlap with the backgound tissue autofluorescence. In an aim to overcome such limitations, we have developed nanoformulated probes consisting of red-emitting fluorescent quantum dot (QD) cores encapsulated in a pegylated shell with benzotriazole (BTA) targeting molecules on the surface (QD-PEG-BTA). The combination of strong red-emitting fluorescence, multivalence binding, decreased backgound signal and nonspecific binding provided the ability of the QD-PEG-BTA probes to achieve detection sensitivites four orders of magnitude greater than conventional thioflavin derivatives. This study opens the use of QDs in AD detection applications.

By recent advancements in medicine, many developed countries around the world are experiencing significant growths in aging populations. With age there are increasing chances of developing Alzheimer’s disease (AD), reaching near 50% once reaching the age of 85.1 Despite the growing prevalence of AD,2,3 diagnosis is quite difficult at the early stages of onset due to the symptoms of lapses in short-term memory and the behavioral changes are often associated with normal signs of aging.4 Early diagnosis of AD can have profound impact in treatment strategies limiting neuronal loss or even aid in future strategies aimed at halting or even reversing the progression of the disease. The main contributor to the pathogenesis of AD is the accumulation of neurotoxic forms of amyloid-β (Aβ) proteins aggregates consisting of oligomers or longer fibril structures.5-7 Therefore, the development of detection methods for AD are towards the ability to label Aβ proteins in brain tissues, cerebral spinal fluid (CSF), or the blood.8-12 To date, the development of probes for imaging of Aβ has been very extensive,13-15 where the activity of Aβ is typically assessed by spectrophotometric assays using Aβ-binding dyes.16,17 Typical employed Aβ-binding dyes include thioflavin-T (ThT) and derivatives such as benzotriazole (BTA). Though such dyes have high affinity for Aβ, they exhibit low signal-to-noise ratios resulting from weak fluorescent signals along with high emission overlap with the tissue autofluorescence. Additionally ThT and its derivatives can only be imaged in short time frames due to rapid photobleaching.18 Here we have developed targeted quantum dot (QD) probes for enhanced detection of Aβ(1-40) which are devoid of the previously mentioned shortcomings of ThT based dyes. In order to create a multivalent fluorescent probe to target Aβ analytes, we conjugated BTA molecules to the distal amine ends of PEGylated quantum dots. BTA is able to target to the β2 position of the Aβ fibers with high affinity,19,20 which enable the final QD to detect amyloid fibril. To our knowledge, this is the first example demonstrating QDs as an amyloid targeted probe to detect Aβ aggregation within biological tissues and show its potential for the future diagnosis of AD (Scheme 1).

OH OHC

MeO

MeO

1

S NH

NH2

2

N

BTA O

O

MeO

S

O

N H

N Pyridine

O

NH

OH

SH

O

OHC

L-proline

DMSO O

K3 PO4, CuI

OH

Br + H 2N

OH

3

O

BTA-COOH O

MeO

NH2

O m

S N H

N

DCC/DMAP

O

O

N H

O

O m

PEG-BTA O N H

BTA-COOH

O n

NH2

n

N H

EDC, sulf-NHS

QD-PEG O

O N H

O

O O

N N H

S

OMe

QD-PEG-BTA

Scheme 1. Top ： Synthesis of QD-PEG-BTA. Bottom ： Illustration of Benzotriazole (BTA) modified QD binding onto amyloid-beta (Aβ) fibrils.

■ RESULTS AND DISCUSSION QDs are semiconductor nanocrystals which offer a number of advantages over conventional fluorophores in biological imaging applications such as having wide excitation envelopes, narrow emission bands, tunable fluorescent wavelengths from the visible to the red-emitting, and being ex-

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ceptionally photostable for long periods of excitation.21-26 Due to the exceptional fluorescent properties of QDs, we set out to demonstrate their ability to enhance the detection of Aβ. Using QDs with 605 nm emission, we developed Aβ targeted probes by functionalizing the nanoparticles’ polyethylene glycol (PEG) coatings with BTA (QD-PEG-BTA). Here BTA molecules are modified with carboxyl groups and conjugated to the distal amines of the PEGylated QDs (Figure 1 & Figure S1). BTA is chosen for its affinity for amyloid aggregates (Ki = 11 nM for Aβ(1-40),27 and PEG coatings are used as they are well known for preventing the nonspecific binding of proteins to nanoparticles.28,29 The resulting QDPEG-BTA showed no significant changes in the size (HD = 28 nm) and optical properties (λab = 592 nm, λem =605 nm) of the unmodified PEGylated QDs. It is important to note that the BTA molecules on the surface of the QD-PEG-BTA probes serve only as the targeting ligands as the fluorescent signals from the QDs are stronger and have less overlap with the emission from tissue autofluorescence enabling greater signal-to-noise ratios.

Figure 1. DLS data of (A) QD-PEG and (B) QD-PEGBTA in H2O. UV-vis (C) and fluorescent spectra (D) of QD-PEG and QD-PEG-BTA in aqueous solution, excitation wavelength was 400 nm.

Figure 2. Amyloid fibrils stained by (A) none, (B) QD-PEG, (C) QD-PEG-BTA (all were 50 nM in PBS, incubation time was 30 min). (A) and (B): fibrils show auto fluorescence only. A drop of QD-PEG was intentionally left in the image to demonstrate that no binding between the fibril and QDPEG; (C): fibrils were stained by QD-PEG-BTA into red. Samples from Alzheimer’s patients stained by QD-PEG (D)

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and QD-PEG-BTA (E). FITC excitation filter was used (465495 nm) and emission spectra were collected from 500-720 nm. Scale bar: (A), (B) and (C) 10 µm; (D) and (E) 100 µm.

Figure 3. AD tissue specimen stained by a cocktail of BTAQD (30 nM) and THT (1 µM). (A) Deconvolved image showing the staining pattern by QD-PEG-BTA (with pseudo color of red); (B) Deconvolved image showing the staining pattern by ThT (with pseudo color of green); (C) Merged image of QD-PEG-BTA and ThT staining; (D) and (E) are heat maps showing the staining pattern and signal intensity by QDPEG-BTA or ThT in a region highlighted by yellow dashed lines in (A) and (B). Scale bar: (A), (B) and (C) 100 µm; (D) and (E) 5 µm. To confirm that the modified BTA derivatives conjugated to PEG maintain their ability to target Aβ, aqueous BTA-PEG solutions of different concentrations were incubated with Aβ(1–40) peptides for 30 min followed by purification by centrifugation, then evaluated using the UV-vis and fluorescent spectra of the purified solutions. The spectra revealed increases in intensities of both UV absorption and fluorescent emission with BTA-PEG concentration which provided strong evidence that the BTA-PEG conjugates did bind to Aβ(1–40) peptides (Figure S2). To demonstrate the QD-PEG-BTA probe’s binding to Aβ, we incubated the nanoparticles with Aβ(1–40) fibrils, which were prepared from Aβ(1–40) peptides according to previously reported procedures.30,31 QD-PEG-BTA exhibited high levels of Aβ binding which was implied by the strong red emission seen within the images (Figure 2C & S3). When samples from Alzheimer’s patients were stained by QD-PEG and QD-PEG-BTA, the same phenomenon was observed (Figure 2D & 2E). Incubation of the fibrils with the nontargeted QD-PEG probes exhibited almost no signal (Figure 2B), along with normal tissues incubated with the targeted QD-PEG-BTA probes (Figure S4). These results imply that QD-PEG-BTA binds Aβ with high specificity, which inspired us to use them as a postmortem correlate for amyloid imaging. Using a solution containing a mixture of 3×10-8 mol/L QD-PEG-BTA and 1×10-6 mol/L ThT probes, we stained AD tissues and compared the signal intensities obtained by fluorescence microscopy (Figure 3). Fluorescence imaging of AD tissue revealed that the spatial positions of ThT and QD signals overlapped in the images indicating that both probes are targeting the Aβ proteins. However, from the corresponding heat maps of the intensities obtained within the fluorescent images (Figure 3D & E), we found the QDs provided much brighter signals. This is believed to be due to a combination of the greater extinction of QDs compared to organic dyes, and the higher affinity of the nanoparticle probes to their targets compared to molecular probes as a result of the multivalence effect.22

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Bioconjugate Chemistry complex were proportional to the concentration of amyloid fibrils.

Figure 5. Fluorescence intensity as a function of amyloid fibril concentration obtained from our cerebral spinal fluid assay employing QD-PEG-BTA probes.

■ CONCLUSIONS

Figure 4. Comparison of Aβ screening using QD-PEGBTA and BTA at diverse concentrations: (A) 10 nM of QD-PEG-BTA, (B) 2 nM of QD-PEG-BTA, (C) 0.4 nM of QD-PEG-BTA, (D) 0.08 nM of QD-PEG-BTA, (E) 50 µM of BTA, (F) 50 nM of BTA. Scale bar: 100 µm. A comparison of Aβ targeting specificity between QD-PEGBTA and BTA was performed by measuring fluorescence intensities with a range of concentrations. As shown in Figure 4, 0.08 nM of QD-PEG-BTA still showed a high response to Aβ screening and is on par with that achieved using 50 µM concentrations of BTA, while 50 nM of BTA exhibited no observable signal. According to the applied concentrations and fluorescence signals, the sensitivity of QD-PEG-BTA was 4.5×10-11 mol/L/a.u., 4.4×104 more times than BTA (2.0×10-6 mol/L/a.u.). These results indicated that QD-PEG-BTA was a highly efficient probe for Aβ detection, much more so than the traditionally employed thioflavin derivative molecular probes. As the QD-PEG-BTA probes were highly sensitive probes in labelling amyloid fibrils in postmortem tissue samples, we then sought to perform a proof of concept study on evaluating the effectiveness of the probes to detect amyloid fibrils within the cerebral spinal fluid as a means of early AD detection. We tested the effectiveness of our assay by QD probes mixed with artificial 34 cerebral spinal fluid (ACSF) spiked with fixed concentrations of amyloid protein. After mixing, excess QD probes were removed by centrifugation and the remaining sediment consisting of the tagged amyloid proteins were resuspended in PBS and analysed by fluorescence spectroscopy. As shown in Figure 5, we found that the fluorescent intensities of QD-PEG-BTA/amyloid fibrils

In conclusion, we have developed a highly sensitive probe for the detection of the Aβ proteins associated with AD. The strong red-emitting fluorescence and high avidity of the QDPEG-BTA probes have great potential for future AD diagnostic applications, especially in cases where the analytes concentration may be too low in the sample for conventional thioflavin-based fluorescent detection methods to be useful. Future studies will focus on the use of QD-PEG-BTA probes in the detection of AD associated analytes, such as soluble Aβ（1-40） (and Aβ（1-42） proteins, in clinical cerebr0spinal fluid samples.

■ EXPERIMENTAL SECTION Materials. Mili-Q deionized water (Millipore, 18.2 MΩ cm1) was used throughout the experiments. The chemicals were obtained from commercial sources and were used without further purification: SuperBlock Blocking Buffer in PBS solution (The Shanghai Biological Technology Co. Ltd., IL); maleic anhydride activated clear strip plate (Beijing green orange blue Biological Technology Co., Ltd., IL); Amyloid-β (Aβ) [Glu11] (1-40) (Shanghai Haoran Biological Technology Co. Ltd.). Qdot® 605 ITK™ amino (PEG) quantum dots (8 µM solution) was purchased from Invitrogen Inc. PD-10 desalting column was purchased from Sigmaaldrich. Brain tissue samples (frontal lobe from human Alzheimer’s Diease or human adult normal) were obtained from Biochain Institute Inc. All other reagents were obtained from Sigma-Aldrich (St Louis, MO) at the highest purity available. Instrumentation. Mass spectra determinations were obtained using a LTQ-Orbitrap XL (Thermofisher) mass spectrometer. Analytical thin layer chromatography (TLC) was performed on Whatman Reagent 250µm layer silica gel aluminum backed plateswith visualization by ultraviolet (UV) irradiation at 254 nM and/or staining with potassium permanganate, phosphomolybdic acid, or ninhydrin. Chromatography solvents were used without distillation. The fluorescence microscope was an ARL-9800 X-ray fluorescence spectrometer. 1H-NMR spectra were recorded on a Bruker NMR 400 DRX Spectrometer at 400 MHz and ref-

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erenced to the proton resonance resulting from incomplete deuteration of deuterated chloroform (δ 7.26). UV-Vis absorption spectra were obtained using a Shimadzu UV-2450 PC UV-Vis Spectrophotometer. Fluorescence microscope was performed using Hitachi Fluorescence spectro-photometer-F-7000. Imaging was performed with a wide field fluorescent microscope (Olympus IX 70) using a 100 W mercury lamp excitation source (Osram HBO 103w/1) with 435 ± 40 nm and 610 excitation bandpass and emission long pass filters, respectively. Synthesis of 4-(2-(4-(6-methoxybenzo[d]thiazol-2yl)phenylamino)ethoxy)-4-oxobutanoic acid (3, BTA-COOH). Compound 1 and 2 were synthesized according to the references. See Supporting Information for more details. A mixture of compound 2 (0.784 g, 2.61 mmol) and succinic anhydride (0.785 g, 7.85 mmol) in of pyridine (12 mL) was reacted at room temperature for 3 h, then evaporated to dryness in vacuo. The residue was extracted with water and ethyl acetate. The combined organic layers were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was further purified by flash chromatography (silica gel, ethyl acetate) to give compound 3 (0.815 g, 78.1 %). 1H NMR (d-DMF, 400 MHz) δ: 7.86 (d, 1H, J = 8.8 Hz), 7.84 (d, 2H, J = 9.2 Hz), 7.67 (d, 1H, J = 2.4 Hz), 7.11 (dd, 1H, J = 2.4 Hz, J = 8.8 Hz), 6.85 (d, 2H, J = 8.8 Hz), 6.49 (t, 1H, J = 4.8 Hz, NH), 4.29 (t, 2H, J = 5.6 Hz), 3.90 (s, 3H, OCH3), 3.45 (m, 2H), 2.62 (m, 2H), 2.56 (m, 2H). 13C NMR (d-DMF, 100 MHz) δ: 173.9, 171.2, 166.4, 157.1, 151.6, 148.5, 135.2, 128.7(2), 122.8, 120.7, 115.4, 112.4(2), 105.1, 63.1, 55.8, 41.9, 29.1, 28.9. HRMS (ESI) m/z Calcd for [(C20H20N2O5S) + H]+: 401.1165. Found: 401.1166. Synthsis of BTA-PEG (4). Excess compound 3 (20 mg) in DMSO (2 mL) was pre-activated by NHS (2 mg), and EDC (1 mg) at RT for 30 min, then MeO-PEG-NH2 was added. The mixture was reacted at RT overnight. DMSO was removed under reduced pressure (8 mm Hg) at 65℃.The mixture was dissolved in DI water, and unreacted compound 3 was removed by PD-10 column. Synthsis of QD-PEG-BTA (5, QD-PEG-BTA).32 Excess compound 3 (1.0 mM, 10 µL, 10 nmol), sulfo-NHS (10 mM, 0.25 mL, 2.5 µmol), and EDC (40 mM, 1.25 mL, 50 µmol) were mixed with PBS buffer (pH = 6.5). The mixture was gently stirred for 30 min at room temperature. Then, Qdot 605 (QDs-NH2, coated with PEG-NH2, average 40 amine functional groups per particle, 8.4 µM, 50 µL, 0.42 nmol,) was added and stirred overnight at room temperature. The resulting solution was loaded on a pre-equilibrated disposable PD-10 desalting gel column and purified with the PBS buffer to remove excess unreacted small molecular weight impurities such as 3, EDC and sulfo-NHS. QD-PEG-BTA binding amyloid fibrils. Amyloid fibrils were prepared from Aβ(1–40) were prepared according to the literature reported procedure.33 The formed amyloid fibrils were sedimented upon centrifugation at 16,000 rpm for 15 min. Amyloid fibril in PBS (2×10-5 mol/L) was immobilized in the maleic anhydride modified 96-well plate (20 µL each well, RT, overnight). Then, 200 µL SuperBlock Blocking Buffer Solution was added for 2 h, followed by rinsing with PBS buffer 3 times. The fibrils were incubated with QD at different concentrations for 30 min. The excess QD was removed by rinsing wells with PBS buffer (3 ×5 min) and DI water (2×5 min). QD-PEG-BTA binding amyloid fibrils in artificial cerebrospinal fluid (ACSF). ACSF which was prepared according to a previously reported procedure.34 Amyloid fibrils were re-suspended in ACSF (100 µL) to obtain 1.00,

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5.00, 10.0, and 20.0 µM solutions. The amyloid fibrils-ACSF solutions were incubated with QD-PEG-BTA (5.00 µM, 1.00 mL) for 30 min. The excess QD was removed by centrifugation at 16,000 rpm, and rinsing with ACSF (3 ×). Brain tissue Staining.35 The literature reported staining procedure was used with slight modifications. Briefly, frozen tissue slides were put into 4℃ fridge for 1 h, then washed with ice cold acetone. After drying in the hood for 0.5 h, those slides were rinsed gently with PBS buffer 3 times (5 min each). To minimize nonspecific binding, slides were treated with super blocking buffer (30 min). After rinsing with PBS (3 times, 5 min each time), slides were incubate QD-NH2 and QD-PEG-BTA with tissue slides (RT, 2h). The excess QD were removed by rinsing with PBS (3×5min) and water (1×5 min).

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

■ AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Phone: +86-2583593263. Fax: +86-25-83593263.

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENT This work was supported by grants from the NCI Centers of Cancer Nanotechnology Excellence (CCNE) Program (U54CA119338), the Start-up fund from Nanjing University and the Fundamental Research Funds for the Central Universities. Prof. Yiqing Wang gratefully acknowledges “Jiangsu Specially Appointed Professor” award. We thank Dr. Jian Liu for the help of plotting the heat map in Figure 3.

■ REFERENCES (1) Evans, D. A., Funkenstein, H. H., Albert, M. S., Scherr, P. A., Cook, N. R., Chown, M. J., Hebert, L. E., Hennekens, C. H., and Taylor, J. O. (1989) Prevalence of Alzheimer's disease in a community population of older persons higher than previously reported. JAMA 262(18), 2551-2556. (2) Kalaria, R. N., Maestre, G. E., Arizaga, R., Friedland, R. P., Galasko, D., Hall, K., Luchsinger, J. A., Ogunniyi, A., Perry, E. K., and Potocnik, F. (2008) Alzheimer's disease and vascular dementia in developing countries: prevalence, management, and risk factors. Lancet Neurol. 7, 812-826. (3) Brookmeyer, R., Johnson, E., Ziegler-Graham, K., and Arrighi, H. M. (2007) Forecasting the global burden of Alzheimer's disease. Alzheimers Dement. 3, 186-191. (4) McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D., and Stadlan, E. M. (1984) Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of the department of health and human services task force on alzheimer's disease. Neurology 34, 939944. (5) Glabe, C. C. (2005) Amyloid accumulation and pathogensis of Alzheimer's disease: significance of monomeric, oligomeric and fibrillar Aβ. Subcell. Biochem. 38, 167-177. (6) Li, S. L., Liu, Z. H., Ji, F. T., Xiao, Z. J., Wang, M. J., Peng, Y. J., Zhang, Y. L., Liu, L., Liang, Z. B., and Li, F. (2012) Delivery of quantum dot-siRNA nanoplexes in SK-N-SH cells for BACE1 gene silencing and intracellular imaging. Mol. Ther. Nucleic. Acid. 1(4), e20.

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(7) Ishigaki, Y., Tanaka, H., Akama, H., Ogara, T., Uwai, K., and Tokuraku, K. (2013) A Microliter-scale high-throughput screening system with quantum-dot nanoprobes for amyloid-β aggregation inhibitors. Plos. One 8, e72992. (8) Klunk, W. E., Engler, H., Nordberg, A., Wang, Y., Blomqvist, G., Holt, D. P., Bergström, M., Savitcheva, I., Huang, G. F., and Estrada, S. (2004) Imaging brain amyloid in Alzheimer's disease with pittsburgh compound-B. Ann. Neurol. 55, 306-319. (9) Vallabhajosula, S. (2011) Positron emission tomography radiopharmaceuticals for imaging brain beta-amyloid. Semin. Nucl. Med. 41, 283-299. (10) Kawarabayashi, T., Younkin, L. H., Saido, T. C., Shoji, M., Ashe, K. H., and Younkin, S. G. (2001) Age-dependent changes in brain, CSF, and plasma amyloid β protein in the Tg2576 transgenic mouse model of Alzheimer's disease. J. Neurosci. 21, 372-381. (11) DeMattos, R. B., Bales, K. R., Parsadanian, M., O'Dell, M. A., Foss, E. M., Paul, S. M., and Holtzman, D. M. (2002) Plaque‐associated disruption of CSF and plasma amyloid-β (Aβ) equilibrium in a mouse model of Alzheimer's disease. J. Neurochem. 81, 229-236. (12) Vandermeeren, M., Mercken, M., Vanmechelen, E., Six, J., Voorde, A., Martin, J. J., and Cras, P. (1993) Detection of Proteins in Normal and Alzheimer's disease cerebrospinal fluid with a sensitive sandwich enzyme-linked immunosorbent assay. J. Neurochem. 61, 1828-1834. (13) Jakob-Roetne, R., and Jacobsen, H. (2009) Alzheimer's disease: from pathology to therapeutic approaches. Angew. Chem. Int. Edit. 48, 3030-3059. (14) Klunk, W. E. (2011) Amyloid imaging as a biomarker for cerebral β-amyloidosis and risk prediction for Alzheimer dementia. Neurobiol. Aging 32, S20-S36. (15) Li, Q. A., Lee, J. S., Ha, C., Park, C. B., Yang, G., Gan, W. B., and Chang, Y. T. (2004) Solid-phase synthesis of styryl dyes and their application as amyloid sensors. Angew. Chem. Int. Edit. 43, 6331-6335. (16) Singer, O., Marr, R. A., Rockenstein, E., Crews, L., Coufal, N. G., Gage, F. H., Verma, I. M., and Masliah, E. (2005) Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model. Nat. Neurosci. 8, 13431349. (17) Laird, F. M., Cai, H. B., Savonenko, A. V., Farah, M. H., He, K. W., Melnikova, T., Wen, H. J., Chiang, H. C., Xu, G. L., Koliatsos, et al (2005) BACE1, a major determinant of selective vulnerability of the brain to amyloid-β amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J. Neurosci. 25, 11693-11709. (18) Escosura-Muniz, A. de la, Parolo, C., and Merkoci, A. (2010) Immunosensing using nanoparticles. Mater. Today 13, 24-34. (19) Wolfe, L. S., Calabrese, M. F., Nath, A., Blaho, D. V., Miranker, A. D., and Xiong Y. (2010) Protein-induced photophysical changes to the amyloid indicator dye thioflavin T. Proc. Natl. Acad. Sci. USA 107, 16863-16868. (20) Gu, L., Tran, J., Jiang, L., Guo Z. F. (2016) A new structural model of Alzheimer’s Ab42 fibrils based on electron paramagnetic resonance data and Rosetta modeling. J. Struct. Biol. http://dx.doi.org/10.1016/j.jsb.2016.01.013. (21) Medintz, I. L., Uyeda, H. T., Goldman, E. R., and Mattoussi, H. (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435-446.

(22) Kairdolf, B. A., Smith, A. M., Stokes, T. H., Wang, M. D., Young, A. N., and Nie, S. (2013) Semiconductor quantum dots for bioimaging and biodiagnostic applications. Annu. Rev. Anal. Chem. 6, 143-162. (23) Maestro, L., Ramirez-Hernandez, J., Bogdan, N., Capobianco, J., Vetrone, F., Solé, J. G., and Jaque, D. (2012) Deep tissue bio-imaging using two-photon excited CdTe fluorescent quantum dots working within the biological window. Nanoscale 4, 298-302. (24) Smith, A. M., Lane, L. A., and Nie, S. (2014) Gene disruption methodologies for drug target discovery. Nat. Commun. 5, 4506-5506. (25) Chen, O., Zhao, J., Chauhan, V. P., Cui, J., Wong, C., Harris, D. K., Wei, H., Han, H. S., Fukumura, D., and Jain, R. K. (2013) Compact high-quality CdSe–CdS core–shell nanocrystals with narrow emission linewidths and suppressed blinking. Nat. Mater. 12, 445-451. (26) Lane, L. A., Smith, A. M., Lian, T., and Nie, S. (2014) Compact and blinking-suppressed quantum dots for singleparticle tracking in live cells. J. Phys. Chem. B 118, 14140-14147. (27) Levine, H. (2005) Multiple ligand binding sites on Aβ (140) fibrils. Amyloid 12, 5-14. (28) Lane, L. A., Qian, X., Smith, A. M., and Nie, S. (2015) Physical Chemistry of Nanomedicine: Understanding the Complex Behaviors of Nanoparticles in Vivo. Annu. Rev. Phys. Chem. 66, 521-547. (29) Quan, L., Liu, S., Sun, T. T., Guan, X. G., Lin, W. H., Xie, Z. G., Huang, Y. B., Wang, Y. Q., and Jing, X. B. (2014) Nearinfrared emitting fluorescent BODIPY nanovesicles for in vivo molecular imaging and drug delivery. ACS Appl. Mater. Inter. 6, 16166-16173. (30) Maya, Y., Ono, M., Watanabe, H., Haratake, M., Saji, H., and Nakayama, M. (2009) Novel radioiodinated aurones as probes for SPECT imaging of β-Amyloid plaques in the brain. Bioconjugate Chem. 20 (1), 95-101. (31) Ouberai, M., Dumy, P., Chierici, S., and Garcia J. (2009) Synthesis and biological evaluation of clicked curcumin and clicked KLVFFA conjugates as inhibitors of β-Amyloid fibril formation. Bioconjugate Chem. 20 (11), 2123-2132. (32) Susumu, K., Uyeda, H. T., Medintz, I. L., Pons, T., Delehanty, J. B., and Mattoussi, H. (2007) Enhancing the stability and biological functionalities of quantum dots via compact multifunctional ligands. J. Am. Chem. Soc. 129, 13987-13996. (33) Wang, Y. M., Klunk, W. E., Debnath, M. L., Huang, G. F., Holt, D. P., Li, S., and Mathis, C. A. (2004) Development of a PET/SPECT agent for amyloid imaging in Alzheimer's disease. J. Mol. Neurosci. 24(1), 55-62. (34) Gilbert, E, Tang, J. M., Ludvig, N. and Bergold, P. J. (2006) Elevated lactate suppresses neuronal firing in vivo and inhibits glucose metabolism in hippocampal slice cultures. Brain Res 1117, 213-223. (35) Yun, X., Chaudry, Q., Shen, C., Kong, K. Y., Zhua, H. E., Chung, L. W., Petros, J. A., Regan, R. M., Yezhelyev, M. V., Simons, et al (2007) Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nat. Protoc. 2, 1152-1165.

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