Method to discriminate amyloids using fluorescent probes

Article abstract of DOI:10.1016/bs.mie.2020.04.010

The aggregation of misfolded proteins into amyloids is a common characteristic of many neurodegenerative and non-neurologic diseases. Fluorescent amyloid-targeting probes that discriminate amyloids based on differences in protein composition can provide rapid information to aid in disease diagnosis. In this chapter, we present protocols for the synthesis and use of ANCA-11 as an environmentally-sensitive amyloid-targeting probe that can fluorescently discriminate between amyloids with different disease origin. We also present a protocol for preparing amyloid samples of synthetic Amyloid-β(1-42), as problems with amyloid preparations can be a large driver of time and cost for research. The methods presented here can be generalized for evaluation of other amyloid-targeting fluorescent probes with different aggregates of amyloidogenic proteins in solution or in tissue.

Full text of DOI:10.1016/bs.mie.2020.04.010

CHAPTER FIVE
Method to discriminate amyloids
using fluorescent probes
Kristine L. Teppang, Rachel S. Ehrlich, Jerry Yang∗
Department of Chemistry and Biochemistry, University of California, San Diego, CA, United States
∗Corresponding author: e-mail address: jerryyang@ucsd.edu
Contents
1. Introduction
92
2. Methods
94
94
2.1 General chemical methods and instrumentation for analysis
2.2 Synthesis of ANCA-11
95
2.3 Preparation and aggregation of synthetic amyloid-β(1–42) peptides
2.4 Fluorescence detection of amyloids in solution using ANCA-11
99
104
2.5 Determining the binding constant of ANCA-11 to aggregated Aβ (1–42)
peptides
106
107
2.6 Staining and fluorescence discrimination of amyloid plaques in tissue using
ANCA-11
2.7 Determining the relative polarity of the binding pocket on amyloids
by estimation of relative permittivity
3. Summary
Acknowledgments
108
112
113
113
References
Abstract
The aggregation of misfolded proteins into amyloids is a common characteristic of
many neurodegenerative and non-neurologic diseases. Fluorescent amyloid-targeting
probes that discriminate amyloids based on differences in protein composition can
provide rapid information to aid in disease diagnosis. In this chapter, we present pro-
tocols for the synthesis and use of ANCA-11 as an environmentally-sensitive amyloid-
targeting probe that can fluorescently discriminate between amyloids with different dis-
ease origin. We also present a protocol for preparing amyloid samples of synthetic
Amyloid-β(1-42), as problems with amyloid preparations can be a large driver of time
and cost for research. The methods presented here can be generalized for evaluation of
other amyloid-targeting fluorescent probes with different aggregates of amyloidogenic
proteins in solution or in tissue.
#
Methods in Enzymology, Volume 639
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2020.04.010
2020 Elsevier Inc.
All rights reserved.
91

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Kristine L. Teppang et al.
1. Introduction
One of the hallmarks of many neurodegenerative and systemic diseases
is the abnormal accumulation of insoluble protein aggregates (amyloids)
(Soto, 2003; Takalo, Salminen, Soininen, Hiltunen, & Haapasalo, 2013).
On a macroscopic level, these amyloids share the characteristic of having
high β-sheet content in their secondary structure, despite having vastly
different protein compositions associated with specific diseases. The pres-
ence of degradation-resistant amyloids in the brain are highly prevalent
in patients with neurodegenerative diseases such as Alzheimer’s disease
(AD) and Creutzfeldt Jakob Disease (CJD) (Luers et al., 2010; Murphy &
Levine III, 2010). Accordingly, methods to identify the presence of these
amyloid deposits in vitro and in vivo play an essential role in basic patholog-
ical studies and are increasingly used in clinical research studies to aid in
early diagnosis of disease (Lee, Kim, Hong, & Kim, 2019; Maezawa
et al., 2008).
While the development of amyloid-targeting radioligands for positron
emission tomography (PET) imaging of amyloids in the brain has aided in
the identification of symptomatic and asymptomatic AD patients (Iaccarino,
Sala, Caminiti, & Perani, 2017; Thompson et al., 2010), helping differentiate
diagnosis between various neurodegenerative disorders remains a difficult
challenge since these ligands target the high β-sheet content regions charac-
teristic of all amyloids. Alternatively, amyloidogenic protein biomarkers can
be detected in cerebral spinal fluid (CSF) or blood (Nabers et al., 2018;
Palmqvist, Mattsson, & Hansson, 2016), which can provide information
on the nature and disease state of affected individuals. While the total con-
centration of such biomarkers can be quantified in these biofluids with the
aid of protein-specific antibodies, information on the aggregation state of
the amyloidogenic protein is difficult to ascertain using conventional
methods, which can result in misdiagnosis (Pawlowski, Meuth, & Duning,
2017; Perret-liaudet, Pelpel, Tholance, & Dumont, 2012). A method that
can provide information on the extent and type of aggregation species
(i.e., amyloid-like aggregates with high β-sheet content) as well as the pro-
tein composition present may lead to increased confidence in diagnosing
specific diseases.
The development of amyloid-targeting fluorescent probes has emerged
as potential cost-effective tools that can distinguish different types of amyloids
in tissue (Cao & Yang, 2018). For example, Condello et al. (2018) reported

Method to discriminate amyloids using fluorescent probes
93
the use of commercially available fluorescent dyes: (E,E) 1-Fluoro-2,5-bis
(3-carboxy-4-hydroxystyryl)benzene (FSB, 1), Curcumin (2), and benz-
imidazole derivative BF-188 (3) to distinguish patients with familial AD
and cerebral amyloid angiopathy (CAA) from patients with sporadic AD
by inspection of fluorescent spectral fingerprints with intensity and emission
that was distinct for each patient cohort (Fig. 1). In addition, Nilsson et al.
(2006) reported that conformation-sensitive fluorescent conjugated poly-
electrolyte dyes such as polythiophene acetic acid (PTAA, 4, Fig. 1) can dis-
criminate conformationally distinct strains of prion deposits in an ex vivo
rodent brain.
We previously reported the ability of Amino Naphthalenyl-2-Cyano-
Acrylate (ANCA) probes to label amyloid-β (Aβ) plaques in human brain tis-
sue sections from AD patients (Chang et al., 2011). ANCA-11 (5, Fig. 1), for
instance, exhibits a large fluorescence enhancement upon binding to amy-
loids compared to its fluorescence as a free probe when in solution. This
enhancement in fluorescence intensity arises from its molecular architec-
ture, which contains an electron donor moiety in conjugation with an
electron acceptor (D-π-A); the donor and acceptor moieties are separated
by σ bonds that are capable of freely rotating in solution, leading to low
fluorescence properties due to non-radiative mechanical de-excitation of
the photon-induced excited state. Upon binding of these fluorescent dyes
to an amyloid surface, however, the σ bonds separating the donor and
Fig. 1 Examples of fluorescent probes that bind to amyloids.

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Kristine L. Teppang et al.
acceptor become restricted leading to better electronic coupling and an
increase in fluorescence intensity.
In addition to amyloids comprised of Aβ peptides, we found that
ANCA-11 could fluorescently label amyloids comprised of 10 other
protein compositions (Cao et al., 2018). In many cases, we found that the
observed fluorescence emission wavelength of ANCA-11 was distinctive
of specific amyloid compositions, providing a method to fluorescently dis-
criminate amyloids associated with different diseases. We attribute the capa-
bility of ANCA probes to discriminate amyloids to their environmentally
sensitive solvatochromic properties, where the polarity of their surrounding
environment (e.g., the binding pocket on the amyloid) strongly influences
the excited state energies of the molecule and affects fluorescence emission
wavelength (Cao et al., 2018). While many fluorophores can be used to
detect amyloids, not all probes have the capability to discriminate amyloids
fluorescently. For example, while ANCA-11 exhibits different fluorescence
emission wavelengths when bound to Aβ versus PrPSc (scrapie isoform of
the prion protein) deposits in tissue, Congo Red (6, Fig. 1) showed no
observable difference in fluorescence wavelength when bound to amy-
loid plaques of different composition (Cao et al., 2018). In cases where
ANCA-11 was not able to discriminate amyloid compositions with similar
binding pocket polarities, a fluorescence fingerprint assay using multiple
members of the ANCA family of dyes can be used to distinguish different
amyloids (Cao et al., 2018). The capability of the ANCA family of probes
to specifically target amyloids in complex biological samples such as bio-
fluids (Do et al., 2018) and tissue (Cao et al., 2012, 2018; Chang et al.,
2011) by recognizing features common to all amyloids (e.g., high β-sheet
content) yet also retain the spectral sensitivity to fluorescently distinguish
different amyloid compositions demonstrate that these probes could serve
as powerful tools for both basic research studies and for aiding in diagnosis
of disease.
2. Methods
2.1 General chemical methods and instrumentation
for analysis
All chemical reagents and anhydrous solvents purchased were used without
further purification. All non-aqueous reactions were performed under

Method to discriminate amyloids using fluorescent probes
95
argon using oven-dried or flame-dried glassware. Anhydrous solvents such
as tetrahydrofuran (THF) and dichloromethane (CH2Cl2) were purified
through a commercial activated alumina column solvent delivery system.
Column chromatography was performed on silica gel (Merck Kiesel gel 60,
230–400 mesh) using a mixture of ethyl acetate:hexanes or pure diethyl
ether as the eluent. Thin-layer chromatography (TLC) was executed using
glass precoated with silica gel-60 F254 to a thickness of 0.5mm (Merck)
and the compounds were visualized by irradiating with ultra-violet (UV) light
and/or use of TLC stains such as ceric ammonium molybdate (CAM) or
potassium permanganate (KMnO4) in water followed by heating with a
heat gun.
13C NMR and 1H NMR spectra were taken on either a 400 or 500MHz
Varian instrument or a 500MHz JEOL instrument. NMR spectra were
taken by dissolving compounds in deuterated chloroform (CDCl3) that
was dried with anhydrous potassium carbonate. A VG 7070 HS mass spec-
trometer under electron spray ionization or electron impact conditions was
used for high resolution mass spectrometry (HRMS). Permittivity values
(i.e., dielectric constants and refractive index values) of solvents used for
estimating environmental sensitivity were taken from published values
available from Sigma and CRC Press.
2.2 Synthesis of ANCA-11
While many members of the ANCA family of compounds can be used as
environmentally-sensitive amyloid discriminating fluorescent probes,
ANCA-11 is the most well characterized ANCA probe for these types of
studies. We, therefore, provide detailed descriptions of the preparation
(Fig. 2) and use of ANCA-11 only for the detection and fluorescence dis-
crimination of amyloids. We previously reported the preparation of ANCA-
11 and other ANCA probes (Cao et al., 2012, 2018; Chang et al., 2011), and
the procedures described here can be generalized for other probes for anal-
ysis of amyloid samples.
2.2.1 Synthesis of 6-bromo-2-(hydroxymethyl)naphthalene

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Kristine L. Teppang et al.
Fig. 2 Scheme for the synthesis of ANCA-11.
2.2.1.1. Dissolve methyl-6-bromo-2-naphthoate (3.0g, 11mmol) in 34mL
anhydrous THF at 0°C in a round bottom flask under argon atmo-
sphere and add DIBAL-H (1.0M in heptane, 34mL, 34mmol)
dropwise. Stir overnight while letting the reaction warm to room
temperature.
2.2.1.2. Quench the reaction with 5mL of MeOH and add 30mL saturated
sodium potassium tartrate solution and 10mL of ethyl acetate.
2.2.1.3. Separate the organic and aqueous phases using a separatory funnel,
collect the organic phase and add a saturated solution of ammo-
nium chloride followed by a brine solution. Dry the organic layer
over MgSO4, filter over celite to remove the MgSO4 drying
agent, and remove the solvent using a rotary evaporator to yield
6-bromo-2-(hydroxymethyl)naphthalene as a crude mixture.
2.2.1.4. Take the crude product to the next step without further purifica-
tion. The retention value for the 6-bromo-2-(hydroxymethyl)
naphthalene for TLC using a 3:7 mixture of EtOAc: Hexanes
as eluent is Rf ¼0.33.
2.2.1.5. The NMR spectrum of crude 6-bromo-2-(hydroxymethyl)naph-
thalene reveals the following peaks: 1H NMR (400MHz, CDCl3):
δ 7.99 (bs, 1H), 7.77 (bs, 1H), 7.74 (d, J ¼8.5Hz, Br H O S1 S3
1H), 7.69 (d, J ¼8.7Hz, 1H), 7.55 (dd, J ¼1.7, 8.7Hz, 1H), 7.49
(dd, J ¼1.7, 8.5Hz, 1H), 4.84 (bs, 2H). 13C NMR (100MHz,
CDCl3) δ 138.8, 133.9, 131.7, 129.7, 129.5, 129.5, 127.4,
126.1, 125.2, 119.8, 65.2.

Method to discriminate amyloids using fluorescent probes
97
2.2.2 Synthesis of 6-bromo-2-naphthaldehyde
2.2.2.1. Dissolve the above crude product of 6-bromo-2-(hydroxymethyl)
naphthalene in anhydrous CH2Cl2 (60mL), and add pyridinium
chlorochromate (2.4g, 11mmol) and heat under reflux for 5h.
2.2.2.2. Cool the reaction to room temperature and pour into a flask con-
taining diethyl ether. Filter the solution through a small pad of
silica, and remove the organic solvents using a rotary evaporator
to yield 6-bromo-2-naphthaldehyde as a white solid.
2.2.2.3. Purify the compound by silica gel chromatography using a 3:7
mixture of EtOAc: Hexanes as eluent. Isolated Yield¼95% (over
2 steps). The retention value for TLC using this solvent mixture as
eluent is Rf ¼0.67.
2.2.2.4. The NMR spectrum of 6-bromo-2-naphthaldehyde reveals the
1
following peaks: H NMR (400MHz, CDCl3) δ 10.15 (s, 1H),
8.31 (bs, 1H), 8.08 (bs, 1H), 7.98 (dd, J ¼1.5, 8.5Hz, 1H), 7.86
(m, 2H), 7.67 (dd, J ¼1.5, 8.5Hz, 1H). 13C NMR (100MHz,
CDCl3) δ 191.8, 137.3, 134.3, 134.1, 131.0, 131.0, 130.6,
130.2, 128.2, 124.0, 123.6.
2.2.3 Synthesis of 6-(piperidin-1-yl)naphthalene-2-carbaldehyde
2.2.3.1. Add dry and degassed toluene (0.8mL) to a dry round bottom flask
and add Pd(OAc)2 (0.022mmol) and P(tBu)3 (0.078mmol). Let
stir for 20min.
2.2.3.2. Add 6-bromo-2-naphthaldehyde (0.208mmol), piperidine
(0.249mmol), and Cs2CO3 (0.280mmol) to the reaction flask
and reflux for 72h.
2.2.3.3. Cool the reaction to room temperature and dilute with 5mL of
CH2Cl2. Filter the organic solution over celite and remove the
solvent using a rotary evaporator to give 6-(piperidin-1-yl)naph-
thalene-2-carbaldehyde as a yellow solid.

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Kristine L. Teppang et al.
2.2.3.4. Purify the compound by silica gel chromatography using a gradi-
ent of 0 to 10% EtOAc in Hexane as eluent. Isolated yield¼70%.
The retention value for TLC using a 3:7 mixture of EtOAc:
Hexanes as eluent is Rf ¼0.61.
2.2.3.5. The NMR spectrum of 6-(piperidin-1-yl)naphthalene-2-
1
carbaldehyde reveals the following peaks: H NMR (400MHz,
CDCl3) δ 10.03 (s, 1H), 8.15 (s, 1H), 7.83 (m, 2H), 7.68 (d, J ¼
8.6Hz, 1H) 7.32 (dd, J ¼2.4, 9.1Hz, 1H), 7.08 (d, J ¼2.4Hz,
1H), 3.38 (m, 4H), 1.78–1.63 (m, 6H). 13C NMR (100MHz,
CDCl3) δ 191.9, 151.9, 138.5, 134.4, 131.3, 130.4, 127.2,
126.3, 123.4, 119.5, 108.8, 49.6, 25.5, 24.3.
2.2.3.6. High resolution mass spectrum (HRMS) analysis reveals the fol-
lowing: Calculated for C16H18NO (M+H+) 240.1383. Found
240.1381.
2.2.4 Synthesis of 2-(2-(2-methoxyethoxy)ethoxy)ethyl-2-cyanoacetate
2.2.4.1. Dissolve 2-cyanoacetic acid (230mg, 2.72mmol) and triethylene
glycol monomethyl ether (0.4mL, 2.27mmol) in CH2Cl2
(2.5mL) and cool to 0°C. Add 4-Dimethylaminopyridine
(DMAP) (2mg, 0.013mmol). After addition of DMAP, add
N,N0-dicyclohexylcarbodiimide (DCC) (560mg, 2.72mmol)
and stir at 0°C for 6h.
2.2.4.2. Dilute the reaction with 10mL of CH2Cl2 and filter the solution
over celite to remove the dicyclohexylurea byproduct. Dry the
organic layer over MgSO4, filter over celite to remove the
MgSO4, and remove the solvent using a rotary evaporator to
yield 2-(2-(2-methoxyethoxy)ethoxy)ethyl-2-cyanoacetate as a
clear liquid.
2.2.4.3. Purify the compound by silica gel chromatography using diethyl
ether as eluent. Isolated yield¼86%. The retention value for TLC
using diethyl ether as eluent is Rf ¼0.45.
2.2.4.4. The NMR spectrum of 2-(2-(2-methoxyethoxy)ethoxy)ethyl-2-
1
cyanoacetate reveals the following peaks: H NMR (400MHz,
CDCl3) δ 4.29 (m, 2H), 3.67, (m, 2H), 3.59 (m, 6H), 3.50

Method to discriminate amyloids using fluorescent probes
99
(m, 2H), 3.49 (s, 2H), 3.32 (s, 3H). 13C NMR (100MHz, CDCl3)
δ 163.0, 113.0, 71.7, 70.4, 70.3, 68.3, 65.5, 58.8, 24.5.
2.2.4.5. HRMS analysis reveals the following: Calculated for C10H17NO5
(M+H+) 232.1185. Found 232.1199.
2.2.5 Synthesis of ANCA-11
2.2.5.1 Add piperidine (0.02mmol) in a solution containing 6-(piperidin-
1-yl)naphthalene-2-carbaldehyde (0.21mmol) and 2-(2-(2-
methoxyethoxy)ethoxy)ethyl-2-cyanoacetate (0.23mmol) in
THF (0.8mL). Stir at 50°C for 21h.
2.2.5.2 Remove the solvent using a rotary evaporator to yield ANCA-11
as a slightly yellow solid.
2.2.5.3 Purify the compound by silica gel chromatography using a gradient
of 10 to 30% ethyl acetate in hexanes as eluent. Isolated yield¼90%.
The retention value for TLC using a 1:1 mixture of EtOAc:
Hexanes as eluent is Rf ¼0.44.
2.2.5.4 The NMR spectrum of ANCA-11 reveals the following peaks: 1H
NMR (400MHz, CDCl3) δ 8.31 (s, 1H), 8.22, (bs, 1H), 8.10
(d, J ¼8.8Hz, 1H), 7.76 (d, J ¼9.2Hz, 1H), 7.65 (d, J ¼8.8Hz,
1H), 7.30 (dd, J ¼2.1, 9.2Hz, 1H), 7.05 (d, J ¼2.1Hz, 1H),
4.47 (m, 2H), 3.83 (m, 2H), 3.74–3.66 (m, 6H), 3.56 (m, 2H),
3.42–3.38 (m, 4H), 3.37 (s, 3H), 1.74 (m, 6H). 13C NMR
(100MHz, CDCl3) δ 163.3, 155.4, 151.9, 137.7, 130.6, 127.2,
126.4, 125.9, 125.6, 119.2, 116.4, 108.3, 71.8, 70.7, 70.5, 68.7,
65.3, 58.9, 49.3, 25.4, 24.3.
2.2.5.5 HRMS analysis reveals the following: Calculated for
C26H32N2O5Na (M+Na+) 475.2203. Found 475.2197.
2.3 Preparation and aggregation of synthetic amyloid-β(1–42)
peptides
Amyloid-β(Aβ)(1–42) is the major component of amyloid plaques in AD.
As such, it is considered one of the most important amyloidogenic peptides

100
Kristine L. Teppang et al.
or proteins to study. It should be noted, however, that this peptide is
notoriously difficult to work with for obtaining reproducible data
(Finder, Vodopivec, Nitsch, Glockshuber, & Forel-strasse, 2010; Kasim,
Kavianinia, Harris, & Brimble, 2019; Zagorski et al., 1999). Here, we
describe one general method that we commonly use to generate amyloids
from synthetic Aβ(1–42).
Tip: The commercial source of Aβ(1–42) peptides is an important con-
sideration for your studies. We have examined a number of different
commercial sources and have settled on two sources that have made it pos-
sible to generate reproducible data in our lab over many years: (1) Custom
synthesis of Aβ(1–42) peptides contracted to PL laboratories (Port Moody,
British Columbia) and delivered as a lyophilized powder. (2) Aβ(1–42)
peptides purchased from Biopeptide Co., Inc. (San Diego, CA) and deliv-
ered as a lyophilized powder.
2.3.1 Pre-treatment of Aβ(1–42) peptides
Equipment and reagents
1. Aβ(1-42)
2. 1,1,1,3,3,3, hexafluoro-2-propanol (Sigma Aldrich)
3. Orbital Shaker
4. Deionized Water
5. Dry Ice and Acetone Bath
6. Vacuum
7. PBS
8. Lyophilizer
9. Sonicator
10. Vortex mixer
Protocol
1. Dissolve synthetic Aβ(1–42) in pure 1,1,1,3,3,3, hexafluoro-2-propanol
(HFIP) to a final concentration of 1mM and place on an orbital shaker
for 21h at room temperature.
2. Sonicate the solution and then vortex to make sure any particulates are
dissolved as much as possible.
3. Dilute the HFIP solution in cold deionized water to a final ratio of 2:1
H2O: HFIP.
4. Fraction into aliquots if necessary (e.g., if the volume is too large) and
freeze the solution by placing the fractions in a bath containing dry
ice in acetone. Lyophilize for at least 2 days and store lyophilized
fractions in a ꢀ80°C freezer until ready to use.

Method to discriminate amyloids using fluorescent probes
101
Tip: While synthetic Aβ(1–42), when purchased, is assumed to exist in a
monomeric form as a lyophilized powder, this assumption is not always
correct and varies between commercial sources and batches of peptide.
HFIP is widely used as a solvent to dissolve Aβ and other amyloidogenic
proteins to help disrupt hydrogen bonds and convert aggregates back into
monomers. Without this pretreatment, we found large variability in repro-
ducibility in studies of aggregated Aβ(1–42). Some literature methods for
pretreatment of Aβ(1–42) using HFIP suggest diluting a solution of Aβ
(1–42) in HFIP with an aqueous solution and simply removing HFIP by
allowing it to evaporate overnight open to air or by blowing a stream of
nitrogen over the top of the sample (Sokolov et al., 2006). However, this
method of removing HFIP leaves significant amounts of residual HFIP in
the Aβ(1–42) sample (Capone et al., 2009). This issue can be resolved by
lyophilizing the solution for at least 2 days to remove HFIP.
2.3.2 Aggregation of Aβ(1–42) peptides
Protocol
1. In an Eppendorf tube, dissolve HFIP pre-treated Aβ(1–42) peptides in
sterile phosphate buffered saline (PBS) (pH 7.4) or in distilled water
to a final concentration of 100 μM and place on an orbital shaker at
1200rpm for 3 days at room temperature.
2.3.3 Confirmation of aggregation of Aβ(1–42) peptides
Several traditional and modern techniques exist to monitor and confirm
aggregation of proteins into amyloids in vitro. Methods that we have used
to confirm aggregation of proteins include: a thioflavin-T fluorescence
(ThT) aggregation assay, atomic force microscopy, and western blots.
2.3.4 Thioflavin-T fluorescence assay
Equipment and reagents
1. 96-well black microtiter plate (Nunc, Roskilde, Denmark)
2. Fluorescence Microplate Reader (We used a SpectraMax M2
Microplate Reader from Molecular Devices, Sunnyvale, CA but any
microplate reader should work similarly)
3. Thioflavin-T (ThT)
4. Dimethyl sulfoxide (DMSO)
5. Distilled water
6. Aggregated Aβ(1–42)

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Kristine L. Teppang et al.
Protocol
1. In an Eppendorf tube, dissolve 4μL of 100μM aggregated Aβ(1–42)
peptides in 72μL of distilled water.
2. Add 4μL of 80μM ThT in 100% DMSO (Final concentrations: 5μM Aβ
(1–42) and 4μM ThT, 5% DMSO in water). Vortex and centrifuge
to mix.
3. Pipette 80μL of solution into a microtiter plate.
4. Measure the fluorescence of ThT using a microplate reader with an exci-
tation of 440nm and emission of 482nm.
5. The observed fluorescence signal should increase as the concentration of
amyloid increases. Repeat measurements twice a day until aggregation is
complete and fluorescence signal no longer further increases.
2.3.5 Atomic force microscopy (AFM)
Equipment and reagents
1. Metrology ProbeTM, Tap 300 probe tips (Ted Pella, Inc., Redding,
CA, USA)
2. Mica (SPI, Westchester, PA, USA)
3. DI Nanoscope-IV Multimode AFM (Veeco, Santa Barbara, CA, USA)
4. Distilled water
5. Filter paper
6. Aβ(1–42)
7. Vacuum
Protocol
1. Pipette a 10μL volume of an Aβ(1–42) solution in distilled water
(0.33mg/mL) onto freshly cleaved mica for 2min.
2. Wick the solution using the corner of a piece of filter paper until the mica
surface looks wet but most of the excess aqueous solution has been
removed.
3. After the Aβ(1–42) solution has dried, wash the mica surface twice by
applying 10μL of water followed by wicking the water off with a corner
of filter paper.
4. Dry the sample under vacuum.
5. Scan the surface by AFM in tapping mode under ambient conditions to
reveal images of amyloid fibrils (Fig. 3A).
2.3.6 Western blot analysis
Equipment and reagents
1. Aggregated Aβ(1–42)
2. NuPAGE® Novex® 12% Tris-Bis Mini Gel

Method to discriminate amyloids using fluorescent probes
103
Fig. 3 Analysis of amyloid content from aggregation of Aβ(1–42) peptides. (A) Atomic
force microscopy image of Aβ(1–42) fibrils deposited on a mica surface. Scale
bar¼200nm. (B) Western blot image of Aβ(1–42) monomers, oligomers, and fibrils.
Lane 1, HFIP treated Aβ(1–42) without incubation. Lane 2, HFIP treated Aβ(1–42) incu-
bated at 4°C in deionized water for 3 days (ꢁ18% oligomers, no fibrils). Lane 3, HFIP
treated Aβ(1–42) incubated at 37°C in deionized water for 3 days (ꢁ76% fibrils).
Panel (A): Adapted with permission from Inbar, P., Bautista, M. R., Takayama, S. A., &
Yang, J. (2008). Assay to screen for molecules that associate with Alzheimer’s related
β-amyloid fibrils. Analytical Chemistry, 80(9), 3502–3506. Copyright 2008 American
Chemical Society. Panel (B): Adapted with permission from Zhao, X., & Yang, J. (2010).
Amyloid-Β peptide is a substrate of the human 20S proteasome. ACS Chemical
Neuroscience, 1(10), 655–660. Copyright 2010 American Chemical Society.
3. XCell IITM Blot Module CE Mark (Invitrogen SKU# EI9051)
4. Nitrocellulose membrane
5. Antibody raised against residues 3-8 of Aβ(1–42) (clone 6E10,
Convance, Cat# SIG-39320)
6. Polyclonal horseradish peroxidase conjugated goat anti-mouse antibody
(Invitrogen, Cat # A10551)
7. GE Healthcare ECL Plus™ Western Blotting Detection System
8. Typhoon 9400 Variable Mode Imager (GE Healthcare Bio-Sciences
Corp., Piscataway, NJ)
9. ImageQuant 5.2 Software (Molecular Dynamics, Sunnyvale, CA)
Protocol
1. Load a solution of aggregated Aβ(1–42) on a NuPAGE® Novex® 12%
Tris-Bis Mini Gel.
2. After separation of aggregates by SDS-PAGE, electroblot onto ani-
trocellulose membrane.

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Kristine L. Teppang et al.
3. Incubate blot with 6E10 anti-Aβ antibody followed by the
horseradishperoxidase conjugated goat anti-mouse antibody according
to the manufacturer’s recommended protocol.
4. Develop the blot using GE Healthcare ECL Plus™ reagent.
5. Image the blot using a Typhoon 9400 Variable Mode Imager or other
suitable imager to reveal gel bands corresponding to monomer, oligo-
mers, and large aggregates (including fibrils) (Fig. 3B).
6. For estimating the percentage of different aggregate species (e.g., mono-
mers oligomers, and fibrils) in the sample, quantification of the gel
images can be performed using software such as ImageQuant 5.2.
Tip: Estimation of the percentage of each aggregated species can be obtained
by taking the intensity of a band of interest and dividing by the total intensity
of bands in the same gel lane. Note: The extent of aggregation is highly
dependent on both time and temperature of aggregation (Fig. 3B).
2.4 Fluorescence detection of amyloids in solution using
ANCA-11
There are two main characteristics of fluorescence of ANCA-11 that sup-
port the presence of amyloids in solution: (1) a large increase in fluorescence
intensity in amyloid-containing solutions compared to in amyloid-free so-
lutions; and (2) a blue shift in the maximum wavelength of fluorescence
emission when ANCA-11 is bound to an amyloid compared to the maxi-
mum fluorescence emission wavelength of the free probe in aqueous solu-
tion. The blue shift in emission wavelength is presumably a consequence of
the probe being in a more non-polar environment within the binding
pocket of the amyloid (Chang et al., 2011) compared to when the probe
is in an aqueous environment in the absence of amyloid in solution.
Tip: While we consistently find a large fold increase in fluorescence emis-
sion intensity of ANCA-11 when bound to an amyloid compared to free
probe in solution, the degree of fluorescence enhancement can vary greatly
(e.g., by over an order of magnitude) between experiments. We attribute
this variability in fluorescence enhancement at least in part to inconsistency
in the distribution of aggregates from different amyloid preparations and to
inconsistency in the degree of precipitation of large aggregates from the
solution (which can sometimes look cloudy).
Equipment and reagents
1. Distilled, deionized water
2. Dimethyl sulfoxide (DMSO)

Method to discriminate amyloids using fluorescent probes
105
3. ANCA-11
4. Aggregated Aβ(1–42)
5. Fluorometer (we used an instrument from Photon Technology
International (currently owned by Horiba), but any fluorometer should
work similarly)
Protocol
1. Mix 15μL of aggregated amyloid solution (100μM) and 285μL of
ANCA probe (in 5% DMSO in deionized water) to attain 300μL of a
final concentration of 5μM of aggregated protein and 4μM of the probe.
As a control, add 15μL of deionized water to 285μL of ANCA probe (in
5% DMSO in deionized water) to attain 300μL of a 4μM solution of
the probe
2. Transfer the solution to a 300μL cuvette suitable for fluorescence
measurements.
3. Measure fluorescence at 22.5°C using an excitation wavelength of
410nm (Fig. 4).
Fig. 4 Fluorescence emission spectra of ANCA-11 in 5% DMSO in water in the presence
or absence of aggregated Aβ(1–42) peptides. Adapted with permission from Cao, K.,
Farahi, M., Dakanali, M., Chang, W. M., Sigurdson, C. J., Theodorakis, E. A., & Yang, J.
(2012). Aminonaphthalene 2-cyanoacrylate (ANCA) probes fluorescently discriminate
between amyloid—β and prion plaques in brain. Journal of the American Chemical
Society, 3(134), 17338–17341. Copyright 2012 American Chemical Society.

106
Kristine L. Teppang et al.
2.5 Determining the binding constant of ANCA-11
to aggregated Aβ (1–42) peptides
Another method to evaluate the quality of an aggregated Aβ(1–42) prepa-
ration for reproducibility purposes is to measure its binding to a fluorophore
such as ANCA-11. In estimation of the dissociation constant (Kd) between
ANCA-11 and aggregated Aβ using a fluorescence binding assay, we make
the following three assumptions: (1) The probe is dissolved completely and
there are no competing processes such as self-aggregation. (2) The concen-
tration of unbound probe is sufficiently large such that the free probe con-
centration can be approximated as the total concentration of added probe.
(3) The binding sites along the amyloid surface do not become saturated
over the entire range of concentrations of probe used in the assay. By using
a fixed concentration of amyloid peptide/protein, measuring the fluores-
cence intensity (at λmax) for a suitable range of concentrations of fluorescent
amyloid-binding probe (e.g., ANCA-11), and plotting the fluorescence
intensity (Y) as a function of the total concentration of added probe (X),
the Kd can be estimated fitting the data to the following one-site specific
Hill-binding equation:
Bmax
Kd + X
X
Y ¼
(1)
where Bmax corresponds to the apparent maximum fluorescence signal
upon binding of the probe to the amyloid (estimated from the fitting the
data with Eq. 1).
Equipment and reagents
1. Distilled, deionized water
2. DMSO
3. ANCA-11
4. Aggregated Aβ(1–42)
5. Fluorometer
Protocol
1. Make solutions containing aggregated Aβ (1–42) peptides at a final con-
centration of 5μM and ANCA-11 at final concentrations ranging from
0 to 10μM in 5% DMSO in distilled, deionized water.
2. Transfer solutions to a 300μL cuvette suitable for fluorescence
measurements.
3. Measure fluorescence at 22.5°C using an excitation wavelength of
410nm.
4. Estimate the Kd value by fitting the data to Eq. (1) (Fig. 5).

Method to discriminate amyloids using fluorescent probes
107
Fig. 5 Plot of the fluorescence intensity at λmax versus concentration of ANCA-11 in the
presence of a fixed 5μM total concentration of Aβ (1–42) peptides (in aggregated form).
Adapted with permission from Cao, K., Farahi, M., Dakanali, M., Chang, W. M.,
Sigurdson, C. J., Theodorakis, E. A., & Yang, J. (2012). Aminonaphthalene 2—cyanoacrylate
(ANCA) probes fluorescently discriminate between amyloid—β and prion plaques in brain.
Journal of the American Chemical Society, 3(134), 17338–17341. Copyright 2012
American Chemical Society.
2.6 Staining and fluorescence discrimination of amyloid
plaques in tissue using ANCA-11
Equipment, samples, and reagents
1. AD mouse brain sections were from male and female transgenic
mice transgenic mice (199959) that expressed the mutant human amy-
loid precursor protein 695 APPSwelnd, harboring both the Indiana
(V717F) and the Swedish (K670N/M671L) mutations. Prion
plaque-containing mouse brain sections came from wild type mice that
were inoculated with mouse-adapted prion strain mCWD (Cao
et al., 2018)
2. ANCA-11
3. Ethanol
4. PBS
5. Microscope slides and cover slips
6. DAKO fluorescence mounting media

108
Kristine L. Teppang et al.
7. For spectral scanning, Olympus FluoView FV1000 spectral decon-
volution confocal microscope with 5% power under 10ꢂ magnification
8. For true color imaging, Olympus MVX10 Macroview equipped with a
MWB2 (Japan) long-pass filter and a 2ꢂ MVX (PF) Plan Apchromat
lens with a 0.5 numerical aperture
Protocol
1. Adhere dry frozen, unfixed brain sections (10–20μm thick) to a micro-
scope slide for 1h and then treat with 100%, 95%, and 70% ethanol for
5min each.
2. Rinse the tissue with deionized water.
3. Rinse with 1ꢂPBS for 30min.
4. Prepare a stock solution containing 3mM ANCA-11 in DMSO and
dilute 1:50 in 11ꢂPBS to a final concentration of 60μM.
5. Add enough of the ANCA-11 solution to cover the brain section and
incubate at room temperature for 30min.
6. After incubation with the probe, wash the tissue with 1ꢂ PBS.
7. Cover the section with DAKO fluorescent mounting media and affix
a coverslip.
8. To obtain a spectral scan and reveal the λmax of fluorescence emission of
ANCA-11 bound to different amyloid plaques, place the stained neural
tissue slices in a confocal or deconvolution microscope and excite with
a 488nm laser. Collect the emission spectra of ANCA-11 bound to
Aβ plaques, PrPSc plaques, and background tissue by measuring the
emission intensity in 1nm increments from 450 to 650nm. Collect a
minimum of 10 fluorescence emission measurements for ANCA-11
bound to plaque and non-plaque regions as a background control.
The wavelength with the maximum relative fluorescence intensity
was taken as the emission λmax
.
9. To capture real color fluorescence images of brain sections stained with
ANCA-11, place the stained neural tissue slices in a Microscope equipped
with a long-pass filter. Illuminate with epifluorescence and image with an
exposure time of 0.2–0.8s, depending on the sample brightness.
2.7 Determining the relative polarity of the binding pocket on
amyloids by estimation of relative permittivity
The observed difference in color of fluorescence emission of ANCA-11
when bound to amyloids of different protein composition (e.g., Aβ-
containing plaques associated with AD versus PrPSc-containing plaques
associated with prion disease, Fig. 6) is largely due to differences in the polar-
ity of the binding pocket on the amyloid, which can affect the stability of

Method to discriminate amyloids using fluorescent probes
109
Fig. 6 Spectral scan and real color imaging of amyloid deposits in tissue stained with
ANCA-11. (A) Aβ deposits in the hippocampal of an AD mouse model brain stained with
ANCA-11. (B) PrPSc deposits in the corpus callosum of a prion-infected mouse stained
with ANCA-11. (C) Fluorescence spectral scan of amyloid deposits stained with ANCA-
11. Note the distinct emission maxima for Aβ and PrPSc. N.F.I.¼Normalized Fluorescence
Intensity. Scale bar¼100μm. Adapted with permission from Cao, K., Farahi, M.,
Dakanali, M., Chang, W. M., Sigurdson, C. J., Theodorakis, E. A., & Yang, J. (2012).
Aminonaphthalene 2—cyanoacrylate (ANCA) probes fluorescently discriminate between
amyloid—β and prion plaques in brain. Journal of the American Chemical Society,
3(134), 17338–17341. Copyright 2012 American Chemical Society.
the photoexcited state and, thus, energy of photon emission (Cao et al.,
2018). A relationship between spectroscopy properties (i.e., λmax of ab-
sorption and emission) and inherent and environmental parameters of a
fluorophore can be approximated by Lippert-Mataga equation (Mataga,
Kaifu, & Koizumu, 1955) (2):
νabsorption ꢀ νemission ¼ Δν
ꢀ
ꢁ
2
ꢄ
ꢂ
ꢃꢅ
ꢀ
ꢁ
2 μe ꢀ μg
ε ꢀ 1
η2 ꢀ 1
¼
ꢀ
+ C0 (2)
2η2 + 1
hca3
2ε + 1
where Δν is the average frequency of the observed Stokes shift, μe and μg
are the ground and excited state dipole moments, η is Plank’s constant, a
is the Onsager radius of the fluorophore, η is the refractive index of the
environmental medium, and ε is the relative permittivity (or dielectric
constant) of the local environment surrounding the fluorophore. Eq. (2)
can be used to extrapolate the environmental sensitivity of a fluorophore

110
Kristine L. Teppang et al.
by measurement of absorbance and emission of the fluorophore in differ-
ence solvents of known permittivity and refractive index (Cao et al., 2012).
We have shown that the absorption spectrum of ANCA-11 is not signif-
icantly affected by solvent polarity (Cao et al., 2012), allowing simplifica-
tion of Eq. (2) to Eq. (3), which relies only on the observed fluorescence
emission, λmax(em), to analyze the effects of the polar environment on the
fluorescence properties of ANCA-11:
ꢄ
ꢂ
ꢃꢅ
ꢀ
ꢁ
1
ε ꢀ 1
η2 ꢀ 1
νemission
¼
¼ C
ꢀ
+ C0
(3)
2η2 + 1
λmax ðemÞ
2ε + 1
where C and C0 are constants that represent inherent properties of the
fluorophore.
Fig. 7 (open squares) shows the linear relationship between the fluores-
cence emission of ANCA-11 and the published permittivity and refractive
(ε0-1)/(2ε0+1)-(n2-1)/(2n2+1)
0.0
2.0
0.1
0.2
0.3
500
550
600
1.9
1.8
1.7
Ab
PrPSc
1
0.2
0.3
0.4
0.5
(ε0-1)/(2ε0+1)
Fig. 7 Dependence of fluorescence emission of ANCA-11 on environmental polarity.
The frequency (1/λmax) of the observed fluorescence emission of ANCA-11 is plotted
as a function of the relative permittivity (ε) of different solvents, with (□) or without
(ꢃ) inclusion of the contribution of refractive index (η) of the solvent. The linear fit
according to Eq. (4) is shown in red. The relative permittivity of the binding pocket
for Aβ and PrPSc can be extrapolated using this fit and the observed fluorescence
emission of ANCA-11 bound to plaques in brain samples. Adapted with permission from
Cao, K., Farahi, M., Dakanali, M., Chang, W. M., Sigurdson, C. J., Theodorakis, E. A., & Yang, J.
(2012). Aminonaphthalene 2—cyanoacrylate (ANCA) probes fluorescently discriminate
between amyloid—β and prion plaques in brain. Journal of the American Chemical
Society, 3(134), 17338–17341. Copyright 2012 American Chemical Society.

Fig. 8 Using ANCA-11 as a spectroscopic ruler for fluorescence discrimination of amyloids. (A) The observed fluorescence emission (λmax) of
ANCA-11 bound to different amyloids in solution or tissue and extrapolated permittivity values of the binding pocket on each amyloid. (B) Plot
of extrapolated permittivity values of amyloid binding pockets and common solvent dielectric constants (εo). (C) Tabulated emission and
extrapolated permittivity values for ANCA-11 bound to different amyloid aggregates. See Cao et al. (2018) for detailed description of amyloid
species used for this analysis. Adapted with permission from Cao, K. J., Elbel, K. M., Cifelli, J. L., Cirera, J., Sigurdson, C. J., Paesani, F., et al. (2018).
Solvation-guided design of fluorescent probes for discrimination of amyloids. Scientific Reports, 8(1), 1–13. Copyright 2018 shared under CC By 4.0.

112
Kristine L. Teppang et al.
index of selected solvents according to Eq. (3), supporting that this equation
represents a reasonable model for the dependence of fluorescence emission
of ANCA-11 on environmental polarity. Interestingly, when we exclude
the refractive index of the solvent and replot the relationship between fluo-
rescence emission and relative permittivity of the solvent only (Fig. 7, open
circles), we found the linear fit was improved (Cao et al., 2018) compared to
when we included the refractive index. This observation suggests that the
relative permittivity (i.e., dielectric constant) dominates over the refractive
index in terms of the effect of solvent polarity on fluorescence emission of
ANCA-11. Thus, the relationship between the fluorescence properties of
ANCA-11 and polarity of its environment can be approximated by Eq. (4):
ꢀ
ꢁ
1
ε ꢀ 1
¼ C1
+ C2
(4)
λmax ðemÞ
2ε + 1
where C1 and C2 are constants that represent inherent properties of the fluo-
rophore. This simple relationship between λmax (emission) and ε (Eq. (4))
make it possible to extrapolate estimates of relative permittivity of the bind-
ing pockets on 11 different amyloids in tissue or in solution (Fig. 8) (Cao
et al., 2018).
3. Summary
A major challenge in developing new chemical tools to study amyloids
in native or complex environments is the capability to target or otherwise
identify aggregates selectively and unambiguously. This chapter describes
one method for using an environmentally-sensitive amyloid-targeting fluo-
rescent probe that is capable of spectroscopically discriminating amyloids
related to different diseases in solution and in tissue. While environmentally-
sensitive fluorophores other than the ANCA-11 probe described here may
also be capable of binding to amyloids, discriminating amyloids spectro-
scopically requires that the fluorescent properties of the probe be sensi-
tive to small differences in otherwise mostly hydrophobic environments.
Nevertheless, amyloid-discriminating fluorescent probes can relatively
quickly provide a substantial amount of information regarding the abun-
dance, location, and protein content of amyloids in biological samples,
which can serve as valuable tools for both basic studies and for aiding in
disease diagnosis.

Method to discriminate amyloids using fluorescent probes
113
Acknowledgments
Research reported in this publication was supported by the National Institute on Aging of the
National Institutes of Health under Award Number RF1AG062362. J.Y. is a founder, equity
interest holder, and advisor of Amydis, Inc.
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