Enhanced Detection Specificity and Sensitivity of Alzheimer's Disease Using Amyloid-β-Targeted Quantum Dots

Article abstract of DOI:10.1021/acs.bioconjchem.6b00019

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 capabilit

Full text of DOI:10.1021/acs.bioconjchem.6b00019

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Article
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
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Bioconjugate Chemistry
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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,‡ Yiqing Wang*,†,‡
†Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiang-
su Province 210093, China.
‡Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, Georgia 30322,
USA.
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ABSTRACT: Diagnostics of Alzheimer’s disease (AD) commonly employ the use of fluorescent thioflavin derivatives
having affinity for the amyloidꢀβ (Aβ) proteins associated with AD progression. However thioflavin probes have limitaꢀ
tions 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 redꢀemitting fluorescent quantum dot (QD)
cores encapsulated in a pegylated shell with benzotriazole (BTA) targeting molecules on the surface (QDꢀPEGꢀBTA).
The combination of strong redꢀemitting fluorescence, multivalence binding, decreased backgound signal and nonspecifꢀ
ic binding provided the ability of the QDꢀPEGꢀBTA 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
OH
K3PO4, CuI
OH
OHC
NH
OHC
MeO
Br
+
H2N
1
L-proline
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 shortꢀterm
memory and the behavioral changes are often associated
with normal signs of aging.4
OH
SH
NH2
MeO
S
N
NH
2
BTA
DMSO
O
O
MeO
S
N
O
O
O
3
N
OH
Early diagnosis of AD can have profound impact in treatꢀ
ment 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 amyloidꢀβ
(Aβ) proteins aggregates consisting of oligomers or longer
fibril structures.5ꢀ7 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.8ꢀ12
H
O
Pyridine
BTA-COOH
O
MeO
O
NH2
S
N
O
O
O
m
N
H
N
O
m
H
DCC/DMAP
O
PEG-BTA
O
BTA-COOH
EDC, sulf-NHS
O
N
H
NH2
O
n
n
QD-PEG
To date, the development of probes for imaging of Aβ has
been very extensive,13ꢀ15 where the activity of Aβ is typically
assessed by spectrophotometric assays using Aβꢀbinding
dyes.16,17 Typical employed Aβꢀbinding dyes include thioflaꢀ
vinꢀT (ThT) and derivatives such as benzotriazole (BTA).
Though such dyes have high affinity for Aβ, they exhibit low
signalꢀtoꢀnoise ratios resulting from weak fluorescent sigꢀ
nals along with high emission overlap with the tissue autoꢀ
fluorescence. Additionally ThT and its derivatives can only
be imaged in short time frames due to rapid photobleachꢀ
ing.18
O
N
O
O
N
N
H
N
H
H
S
O
OMe
QD-PEG-BTA
Here we have developed targeted quantum dot (QD) probes
for enhanced detection of Aβ(1ꢀ40) 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 diagꢀ
nosis of AD (Scheme 1).
Scheme 1. Top：Synthesis of QDꢀPEGꢀBTA. Bottom：
Illustration of Benzotriazole (BTA) modified QD binding
onto amyloidꢀbeta (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 enveꢀ
lopes, narrow emission bands, tunable fluorescent waveꢀ
lengths from the visible to the redꢀemitting, and being exꢀ
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ceptionally photostable for long periods of excitation.21ꢀ26
and QDꢀPEGꢀBTA (E). FITC excitation filter was used (465ꢀ
495 nm) and emission spectra were collected from 500ꢀ720
nm. Scale bar: (A), (B) and (C) 10 ꢀm; (D) and (E) 100 ꢀm.
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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’ polyꢀ
ethylene glycol (PEG) coatings with BTA (QDꢀPEGꢀBTA).
Here BTA molecules are modified with carboxyl groups and
conjugated to the distal amines of the PEGylated QDs (Figꢀ
ure 1 & Figure S1). BTA is chosen for its affinity for amyloid
aggregates (Ki = 11 nM for Aβ(1ꢀ40),27 and PEG coatings are
used as they are well known for preventing the nonspecific
binding of proteins to nanoparticles.28,29 The resulting QDꢀ
PEGꢀBTA 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 QDꢀPEGꢀBTA
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 signalꢀtoꢀnoise ratios.
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Figure 3. AD tissue specimen stained by a cocktail of BTAꢀ
QD (30 nM) and THT (1 ꢁM). (A) Deconvolved image showꢀ
ing the staining pattern by QDꢀPEGꢀBTA (with pseudo color
of red); (B) Deconvolved image showing the staining pattern
by ThT (with pseudo color of green); (C) Merged image of
QDꢀPEGꢀBTA and ThT staining; (D) and (E) are heat maps
showing the staining pattern and signal intensity by QDꢀ
PEGꢀBTA or ThT in a region highlighted by yellow dashed
lines in (A) and (B). Scale bar: (A), (B) and (C) 100 ꢀm; (D)
and (E) 5 ꢀm.
To confirm that the modified BTA derivatives conjugated to
PEG maintain their ability to target Aβ, aqueous BTAꢀPEG
solutions of different concentrations were incubated with
Aβ(1–40) peptides for 30 min followed by purification by
centrifugation, then evaluated using the UVꢀvis and fluoresꢀ
cent spectra of the purified solutions. The spectra revealed
increases in intensities of both UV absorption and fluoresꢀ
cent emission with BTAꢀPEG concentration which provided
strong evidence that the BTAꢀPEG conjugates did bind to
Aβ(1–40) peptides (Figure S2).
To demonstrate the QDꢀPEGꢀBTA probe’s binding to Aβ, we
incubated the nanoparticles with Aβ(1–40) fibrils, which
were prepared from Aβ(1–40) peptides according to previꢀ
ously reported procedures.30,31 QDꢀPEGꢀBTA 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 QDꢀPEG
and QDꢀPEGꢀBTA, the same phenomenon was observed
(Figure 2D & 2E). Incubation of the fibrils with the nonꢀ
targeted QDꢀPEG probes exhibited almost no signal (Figure
2B), along with normal tissues incubated with the targeted
QDꢀPEGꢀBTA probes (Figure S4). These results imply that
QDꢀPEGꢀBTA binds Aβ with high specificity, which inspired
us to use them as a postmortem correlate for amyloid imagꢀ
ing. Using a solution containing a mixture of 3×10ꢀ8 mol/L
QDꢀPEGꢀBTA and 1×10ꢀ6 mol/L ThT probes, we stained AD
tissues and compared the signal intensities obtained by fluꢀ
orescence 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 correꢀ
sponding heat maps of the intensities obtained within the
fluorescent images (Figure 3D & E), we found the QDs proꢀ
vided 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
Figure 1. DLS data of (A) QDꢀPEG and (B) QDꢀPEGꢀ
BTA in H2O. UVꢀvis (C) and fluorescent spectra (D) of
QDꢀPEG and QDꢀPEGꢀBTA in aqueous solution, excitaꢀ
tion wavelength was 400 nm.
Figure 2. Amyloid fibrils stained by (A) none, (B) QDꢀPEG,
(C) QDꢀPEGꢀBTA (all were 50 nM in PBS, incubation time
was 30 min). (A) and (B): fibrils show auto fluorescence
only. A drop of QDꢀPEG was intentionally left in the image
to demonstrate that no binding between the fibril and QDꢀ
PEG; (C): fibrils were stained by QDꢀPEGꢀBTA into red.
Samples from Alzheimer’s patients stained by QDꢀPEG (D)
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Bioconjugate Chemistry
complex were proportional to the concentration of amyꢀ
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Figure 5. Fluorescence intensity as a function of amyloid
fibril concentration obtained from our cerebral spinal fluid
assay employing QDꢀPEGꢀBTA probes.
■ CONCLUSIONS
In conclusion, we have developed a highly sensitive probe
for the detection of the Aβ proteins associated with AD. The
strong redꢀemitting fluorescence and high avidity of the QDꢀ
PEGꢀBTA probes have great potential for future AD diagꢀ
nostic applications, especially in cases where the analytes
concentration may be too low in the sample for conventionꢀ
al thioflavinꢀbased fluorescent detection methods to be useꢀ
ful. Future studies will focus on the use of QDꢀPEGꢀBTA
probes in the detection of AD associated analytes, such as
soluble Aβ（1ꢀ40） (and Aβ（1ꢀ42） proteins, in clinical
cerebr0spinal fluid samples.
Figure 4. Comparison of Aβ screening using QDꢀPEGꢀ
BTA and BTA at diverse concentrations: (A) 10 nM of
QDꢀPEGꢀBTA, (B) 2 nM of QDꢀPEGꢀBTA, (C) 0.4 nM of
QDꢀPEGꢀBTA, (D) 0.08 nM of QDꢀPEGꢀBTA, (E) 50
ꢁM of BTA, (F) 50 nM of BTA. Scale bar: 100 ꢀm.
A comparison of Aβ targeting specificity between QDꢀPEGꢀ
BTA and BTA was performed by measuring fluorescence
intensities with a range of concentrations. As shown in Figꢀ
ure 4, 0.08 nM of QDꢀPEGꢀBTA still showed a high reꢀ
sponse to Aβ screening and is on par with that achieved
using 50 ꢂM concentrations of BTA, while 50 nM of BTA
exhibited no observable signal. According to the applied
concentrations and fluorescence signals, the sensitivity of
QDꢀPEGꢀBTA was 4.5×10ꢀ11 mol/L/a.u., 4.4×104 more times
than BTA (2.0×10ꢀ6 mol/L/a.u.). These results indicated
that QDꢀPEGꢀBTA was a highly efficient probe for Aβ detecꢀ
tion, much more so than the traditionally employed thioflaꢀ
vin derivative molecular probes.
■ EXPERIMENTAL SECTION
Materials. MiliꢀQ deionized water (Millipore, 18.2 Mꢃ cmꢀ
1) 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);
Amyloidꢀβ (Aβ) [Glu11] (1ꢀ40) (Shanghai Haoran Biological
Technology Co. Ltd.). Qdot® 605 ITK™ amino (PEG) quanꢀ
tum dots (8 ꢂM solution) was purchased from Invitrogen
Inc. PDꢀ10 desalting column was purchased from Sigmaꢀ
aldrich. Brain tissue samples (frontal lobe from human Alzꢀ
heimer’s Diease or human adult normal) were obtained
from Biochain Institute Inc. All other reagents were obꢀ
tained from SigmaꢀAldrich (St Louis, MO) at the highest
purity available.
As the QDꢀPEGꢀBTA 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 effectiveꢀ
ness of our assay by QD probes mixed with artificial
cerebral spinal fluid34 (ACSF) spiked with fixed concenꢀ
trations of amyloid protein. After mixing, excess QD
probes were removed by centrifugation and the remainꢀ
ing 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 QDꢀPEGꢀBTA/amyloid fibrils
Instrumentation. Mass spectra determinations were obꢀ
tained using a LTQꢀOrbitrap XL (Thermofisher) mass specꢀ
trometer. Analytical thin layer chromatography (TLC) was
performed on Whatman Reagent 250ꢁm 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 ARLꢀ9800 Xꢀray fluoꢀ
1
rescence spectrometer. HꢀNMR spectra were recorded on a
Bruker NMR 400 DRX Spectrometer at 400 MHz and refꢀ
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erenced to the proton resonance resulting from incomplete
5.00, 10.0, and 20.0 µM solutions. The amyloid fibrilsꢀACSF
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deuteration of deuterated chloroform (δ 7.26). UVꢀVis abꢀ
sorption spectra were obtained using a Shimadzu UVꢀ2450
PC UVꢀVis Spectrophotometer. Fluorescence microscope
was performed using Hitachi Fluorescence spectroꢀphotoꢀ
meterꢀFꢀ7000. 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.
solutions were incubated with QDꢀPEGꢀBTA (5.00 ꢁM, 1.00
mL) for 30 min. The excess QD was removed by centrifugaꢀ
tion at 16,000 rpm, and rinsing with ACSF (3 ×).
Brain tissue Staining.35 The literature reported staining
procedure was used with slight modifications. Briefly, froꢀ
zen 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 rinsꢀ
ing with PBS (3 times, 5 min each time), slides were incuꢀ
bate QDꢀNH2 and QDꢀPEGꢀBTA with tissue slides (RT, 2h).
The excess QD were removed by rinsing with PBS (3×5min)
and water (1×5 min).
9
Synthesis of 4-(2-(4-(6-methoxybenzo[d]thiazol-2-
yl)phenylamino)ethoxy)-4-oxobutanoic acid (3,
BTA-COOH). Compound 1 and 2 were synthesized accordꢀ
ing 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 concentratꢀ
ed in vacuo. The residue was further purified by flash chroꢀ
matography (silica gel, ethyl acetate) to give compound 3
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■ ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website.
1
(0.815 g, 78.1 %). H NMR (dꢀDMF, 400 MHz) δ: 7.86 (d,
■ AUTHOR INFORMATION
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 (dꢀDMF, 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.
Corresponding Author
* Eꢀmail: wangyiqing@nju.edu.cn. Phone: +86ꢀ25ꢀ
83593263. Fax: +86ꢀ25ꢀ83593263.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENT
Synthsis of BTA-PEG (4). Excess compound 3 (20 mg)
in DMSO (2 mL) was preꢀactivated by NHS (2 mg), and
EDC (1 mg) at RT for 30 min, then MeOꢀPEGꢀNH2 was addꢀ
ed. 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 comꢀ
pound 3 was removed by PDꢀ10 column.
Synthsis of QD-PEG-BTA (5, QD-PEG-BTA).32 Excess
compound 3 (1.0 mM, 10 ꢁL, 10 nmol), sulfoꢀNHS (10 mM,
0.25 mL, 2.5 ꢁmol), and EDC (40 mM, 1.25 mL, 50 ꢁmol)
were mixed with PBS buffer (pH = 6.5). The mixture was
gently stirred for 30 min at room temperature. Then, Qdot
605 (QDsꢀNH2, coated with PEGꢀNH2, average 40 amine
functional groups per particle, 8.4 ꢁM, 50 ꢁL, 0.42 nmol,)
was added and stirred overnight at room temperature. The
resulting solution was loaded on a preꢀequilibrated disposaꢀ
ble PDꢀ10 desalting gel column and purified with the PBS
buffer to remove excess unreacted small molecular weight
impurities such as 3, EDC and sulfoꢀNHS.
This work was supported by grants from the NCI Centers of
Cancer Nanotechnology Excellence (CCNE) Program
(U54CA119338), the Startꢀup fund from Nanjing University
and the Fundamental Research Funds for the Central Uniꢀ
versities. Prof. Yiqing Wang gratefully acknowledges “Jiangꢀ
su Specially Appointed Professor” award. We thank Dr. Jian
Liu for the help of plotting the heat map in Figure 3.
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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 (2×10ꢀ5 mol/L) was immoꢀ
bilized in the maleic anhydride modified 96ꢀwell plate (20
ꢂL each well, RT, overnight). Then, 200 ꢁL 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
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