Glycopeptide nanofiber platform for Aβ-sialic acid interaction analysis and highly sensitive detection of Aβ

Article abstract of DOI:10.1021/acs.analchem.9b00377

The variation of amyloid β peptide (Aβ) concentration and Aβ aggregation are closely associated with the etiology of Alzheimer’s diseases (AD). The interaction of Aβ with the monosialoganglioside-rich neuronal cell membrane has been suggested to influence Aβ aggregation. Therefore, studies on the mechanism of Aβ and sialic acids (SA) interaction would greatly contribute to better understanding the pathogenesis of AD. Herein, we report a novel approach for Aβ?SA interaction analysis and highly sensitive Aβ detection by mimicing the cell surface presentation of SA clusters through engineering of SA-modified peptide nanofiber (SANF). The SANF displayed well-ordered 1D nanostructure with high density of SA on surface. Using FAM-labeled Aβ fragments of Aβ₁?16, Aβ₁₆?23, and Aβ₂₄?40, the interaction between Aβ and SA was evaluated by the fluorescence titration experiments. It was found that the order of the SA-binding affinity was Aβ₁?₁₆ > Aβ₂₄?₄₀ > Aβ₁₆?23. Importantly, the presence of full-length Aβ₁?40 monomer triggered a significant fluorescence enhancement due to the multivalent binding of Aβ₁?₄₀ to the nanofiber. This fluorescent turn-on response showed high selectivity and sensitivity for Aβ₁?40 detection and the method was further used for Aβ aggregation process monitoring and inhibitor screening. The results suggest the proposed strategy is promising to serve as a tool for mechanism study and the early diagnosis of Alzheimer’s disease.

Full text of DOI:10.1021/acs.analchem.9b00377

Article
pubs.acs.org/ac
Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Glycopeptide Nanofiber Platform for Aβ-Sialic Acid Interaction
Analysis and Highly Sensitive Detection of Aβ
Li Lei, Rui Geng, Zhiai Xu,* Yijing Dang, Xianli Hu, Lingling Li, Ping Geng, Yang Tian,
and Wen Zhang*
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China
S
* Supporting Information
ABSTRACT: The variation of amyloid β peptide (Aβ) concentration and Aβ
aggregation are closely associated with the etiology of Alzheimer’s diseases (AD).
The interaction of Aβ with the monosialoganglioside-rich neuronal cell membrane
has been suggested to influence Aβ aggregation. Therefore, studies on the
mechanism of Aβ and sialic acids (SA) interaction would greatly contribute to
better understanding the pathogenesis of AD. Herein, we report a novel approach
for Aβ−SA interaction analysis and highly sensitive Aβ detection by mimicing the
cell surface presentation of SA clusters through engineering of SA-modified peptide
nanofiber (SANF). The SANF displayed well-ordered 1D nanostructure with high
density of SA on surface. Using FAM-labeled Aβ fragments of Aβ1−16, Aβ16−23, and
Aβ24−40, the interaction between Aβ and SA was evaluated by the fluorescence
titration experiments. It was found that the order of the SA-binding affinity was
Aβ1−16 > Aβ24−40 > Aβ16−23. Importantly, the presence of full-length Aβ1−40
monomer triggered a significant fluorescence enhancement due to the multivalent
binding of Aβ1−40 to the nanofiber. This fluorescent turn-on response showed high selectivity and sensitivity for Aβ1−40
detection and the method was further used for Aβ aggregation process monitoring and inhibitor screening. The results suggest
the proposed strategy is promising to serve as a tool for mechanism study and the early diagnosis of Alzheimer’s disease.
significant effect on the conformation of Aβ peptides, then it
lzheimer’s disease (AD) is a chronic neurodegenerative
Adisease which brings huge burden to the patients and would accelerate or inhibit oligomerization and fibrillation in
the following processes depending on the ganglioside
concentration.25−30 To exactly decipher the role of GM1 in
Aβ aggregation, fully understanding the Aβ-GM1 interaction at
the molecular level is essential. Recently, molecular dynamics
(MD) simulations were performed to analyze the physico-
chemical interaction of Aβ1−40 with GM1 and it showed that
Aβ1−40 inserted into the lipid membrane through hydrogen
bonds between His 13 or His 14 and Leu 34 of Aβ and the
membrane.31,32 Nevertheless, versatile and sensitive methods
to explore the interaction of Aβ with the SA epitopes
experimentally are limited and highly desired.
SA epitopes are present as clusters on membrane, which
display multivalent interaction with proteins or other
receptors.33,34 To better understand the nature of interactions,
mimicking the cell surface that display multivalent ligands is a
rational option.35,36 Many researchers have attempted to
synthesize biomimetic nanostructures using polymers, in-
organic nanomaterials and peptides that display multiple
ligands for the applications such as enhanced targeting, protein
activation and inhibition of biological pathogens.37−40 Among
these materials, the self-assembled peptide nanostructures hold
their family. Up to now, there are no effective strategies to cure
AD, but an early diagnosis and treatment would be beneficial
to ameliorate the symptoms of this terrible disease. Amyloid β
peptide (Aβ) is the major component of senile plaques in AD
and it is believed to be linked with the pathogenesis of AD.1−5
Aβ is produced from the proteolytic process of amyloid
precursor protein (APP) by β- and γ-secretases.6,7 Under
aberrant conditions, extracellular Aβ would aggregate into
oligomers, protofibrils and fibrils, and Aβ accumulation in the
brain are thought to be associated with the neurotoxicity.8−12
Aβ aggregation disrupts the homeostasis and results in the
variation of Aβ content in the cerebrospinal fluid (CSF).
Importantly, the changes of the Aβ level occur in the early
stage of AD development.13−15 Therefore, monitoring Aβ
concentration alteration is critical and could provide a
promising way for preclinical AD diagnosis.
Aβ aggregation could impair neurons by interaction with cell
membranes. Since monosialoganglioside GM1-bound Aβ was
discovered in brains of AD patients,16,17 it has also been
suggested that Aβ interaction with neuronal lipid membranes
would greatly influence the aggregation behavior and contrib-
ute to the neurotoxicity.18−22 GM1 gangliosides are sialic acid
(SA)-containing glycosphingolipids which are abundant in
neuronal membranes in the form of clusters.16,23−25 A series of
in vitro investigations have demonstrated that GM1 has a
Received: January 22, 2019
Accepted: June 4, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
unique features including well-defined morphologies, tailorable
functionality, and good biocompatibility.41−43
MΩ·cm) from a laboratory water purification system (Hitech
Instruments Co., Ltd.) was used throughout the experiment.
Measurements. Transmission electron microscopy
(TEM) measurements were collected with a HT7700
transmission electron microscope (Hitachi, Tokyo, Japan).
Atomic force microscopy (AFM) images were obtained on a
Dimension FastScan (Bruker, Germany) using tapping mode
under ambient condition. Infrared spectra were performed on a
Nicolet 500 Fourier Transform Infrared (FT-IR) spectrometer
(Madison, WI). Circular dichroism (CD) spectra were
obtained using a Chirascan spectropolarimeter (Applied
Photophysics, UK). Proton nuclear magnetic resonance (1H
NMR) and carbon nuclear magnetic resonance spectra (13C
NMR) were measured by using a Bruker 500 MHz NMR
spectrometer at 25 °C. The ultraviolet−visible (UV−vis)
absorption and fluorescence spectra were obtained by a Cary
60 UV−vis spectrophotometer and a Cary Eclipse spectro-
fluorophotometer, respectively (Agilent Technologies, Palo
Alto, CA, USA). The zeta potential measurement was
conducted at 25 °C using a Malvern Zetasizer Nanoseries
(Malvern, England). The mass spectra were measured with a
LC-MS spectrometer (Bruker, Germany).
Herein, we have prepared the glycopeptide nanofiber for
Aβ−SA interaction analysis based on the self-assembly of SA-
modified peptide and developed it for highly sensitive Aβ
detection. SA moieties were covalently conjugated onto the
Ac-KLVFFAL-NH2 (KL-7) peptide using one alkyne linker by
click chemistry (named as SAKL-7, Scheme 1). The self-
Scheme 1. Preparation of PAKL-7 and SAKL-7 and Their
Self-Assembly into Nanofibers
Synthesis of SA-ME. A 500.0 mg amount of Neu5Ac (1.6
mmol) and 30 μL of TFA were added to 10 mL of anhydrous
MeOH in the flasks. Then the solution reacted at room
temperature with stirring until it was clarified. The solvent was
removed by evaporation, and SA-ME was obtained as a white
solid (484.5 mg, 1.5 mmol) in 93.8% yield. The solid product
1
was dried over P2O5 in vacuo overnight for further use. H
NMR (500 MHz, CD3OD) δ 4.07−3.98 (m, 2H), 3.82 (dd, J
= 13.3, 6.6 Hz, 2H), 3.80 (m, 3H), 3.72 (ddd, J = 8.8, 5.7, 2.9
Hz, 1H), 3.64 (dd, J = 11.3, 5.7 Hz, 1H), 3.50 (dd, J = 9.2, 1.4
Hz, 1H), 3.33 (s, 3H), 2.24 (m, 1H), 2.03 (s, 3H), 1.91 (t, iJ =
12.9, 11.4 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ 175.11
(s), 171.77 (s), 96.66 (s), 72.07 (s), 71.64 (s), 70.18 (s), 67.85
(s), 64.83 (s), 54.31 (s), 53.14 (s), 40.69 (s), 22.65 (s). MS
calcd for C12H21O9N, 323.20; found, 346.11 (M + Na+).
assembly of SAKL-7 resulted in the formation of nanofiber
(SANF), which was characterized by a high density of SA
decorated on the surface. The SANF were then evaluated for
their ability to recognize different FAM-labeled Aβ fragments
of Aβ1−16, Aβ16−23, and Aβ24−40. The experimental studies
based on the fluorescence quenching effect clarified that Aβ
could bind tightly to the SANF surface. The binding affinity of
SA and Aβ followed the order of Aβ1−16 > Aβ24−40 > Aβ16−23
.
The full-length Aβ1−40 monomer induced the fluorescence
recovery of the SANF−Aβ24−40 system owing to the specific
multivalent interaction between Aβ1−40 and SA. A fluorescent
turn-on strategy with high sensitivity and selectivity for
detecting Aβ and Aβ aggregation study was successfully
proposed.
Synthesis of Ts-SA. A 500.0 mg amount of solid SA-ME
(1.5 mmol) was coevaporated with dry pyridine twice to
remove a trace amount of water and then dissolved in 6 mL of
pyridine. The solution was cooled to 0 °C, and p-TsCl (600.0
mg, 3.2 mmol) was added. The mixture was allowed to stay at
room temperature and stirred overnight. After removing
pyridine, the residue was purified by column chromatography
(EtOAc:MeOH = 15:1) to gain Ts-SA as a white solid (572.0
mg, 1.2 mmol) in 80.0% yield. 1H NMR (500 MHz, CD3OD)
δ 7.81 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 4.29 (dd, J
= 10.0, 2.2 Hz, 1H), 4.10−4.02 (m, 2H), 3.97 (d, J = 10.5 Hz,
1H), 3.90−3.86 (m, 1H), 3.81 (d, J = 4.6 Hz, 1H), 3.79−3.74
(m, 4H), 3.45 (t, J = 9.7 Hz, 1H), 3.34 (dd, J = 11.2, 9.7 Hz,
3H), 2.47 (s, 3H), 2.23 (dd, J = 12.9, 4.9 Hz, 1H), 2.04 (s,
3H), 1.80 (m, 1H). 13C NMR (100 MHz, CD3OD) δ 175.16
(s), 171.88 (s), 146.42 (s), 134.15 (s), 131.02 (s), 129.05 (s),
129.02 (d, J = 9.2 Hz), 126.95 (s), 96.61 (s), 73.63 (s), 71.70
(s), 69.93 (s), 69.28 (s), 67.64 (s), 54.09 (s), 53.29 (s), 40.71
(s), 22.67 (d, J = 19.4 Hz), 21.62 (d, J = 13.7 Hz). MS calcd
for C19H27O11NS; 477.12, found, 500.12 (M + Na+).
EXPERIMENTAL SECTION
■
Chemicals and Reagents. The peptide Ac-KLVFFAL-
NH2 (KL-7) and the FAM-labeled Aβ fragments were
purchased from GL Biochem, Ltd. (Shanghai, China). Aβ1−40
(DAEFR HDSGY EVHHQ KLVFF AEDVG SNKGA IIGLM
VGGVV) was obtained from American Peptide Co. (CA,
USA). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), trifluoro-
acetic acid (TFA), and p-toluenesulfonyl chloride (p-TsCl)
were bought from Tokyo Chemical Industry (TCI) Co., Ltd.
(Shanghai, China). Thioflavin T (ThT), (−)-epigallocatechin
gallate (EGCG), glutathione (GSH), bovine serum albumin
(BSA), N-acetylneuraminic acid (Neu5Ac), pyridine, resorci-
nol, and 4-pentynoic acid (PA) were purchased from Sigma-
Aldrich Ltd. (Shanghai, China). Dialysis bags with a molecular
weight cut off (MWCO) of 500 Da were obtained from
Yuanye Biotechnology Co., Ltd. (Shanghai, China). All other
chemicals are of analytical grade except those noted otherwise
and used directly without further purification. Water (18.2
B
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
Synthesis of N3−SA. An 800 mg mount of sodium azide
(12.0 mmol), 1400 mg of Ts-SA (3.0 mmol), and deionized
water (5 mL) were added to acetone (15 mL). The mixture
was heated under reflux overnight. Then the solvent was
completely removed, and the residues were purified by column
chromatography to afford N3−SA as a pale yellow solid (798
mg, 2.4 mmol, 80.1%). 1H NMR (500 MHz, D2O) δ 3.93 (d, J
= 6.5 Hz, 1H), 3.89 (d, J = 12.8 Hz, 1H), 3.85 (ddd, J = 14.6,
10.1, 8.2 Hz, 1H), 3.82 (d, J = 3.5 Hz, 1H), 3.54 (d, J = 2.7 Hz,
1H), 3.51−3.43 (m, 2H), 3.41−3.36 (m, 1H), 3.27 (s, 1H),
2.19−2.04 (m, 2H), 1.99 (s, 3H), 1.81 (d, J = 20.0 Hz, 1H),
1.73 (d, J = 11.7 Hz, 1H). 13C NMR (100 MHz, D2O) δ
176.24 (s), 174.29 (s), 96.01 (s), 69.61 (s), 68.67 (s), 68.43
(s), 66.78 (s), 53.38 (s), 51.80 (s), 38.92 (s), 21.64 (s). MS
calcd for C11H18N4O8, 334.10; found, 357.10 (M + Na+).
Peptide Self-Assembly. Briefly, 3.5 mM SAKL-7 and
PAKL-7 peptide were dissolved in a 2:1 (v/v) mixture of
DMSO/water, respectively. Then they were allowed to
assemble for approximately 24 h in a 37 °C water bath and
ready for use.
Quantitative Estimation of SA on Nanofiber Surface.
SA amount modified on the nanofiber surface was determined
according to the literature.44 Briefly, 20 μL of Neu5Ac (5−40
μg/mL) or 20 μL of SANF (1 mg/mL) was added to the
solution containing 0.1 M CuSO4, 4% resorcinol reagent, and
hydrochloric acid. Then the mixture was heated for 15 min in a
boiling water bath. After heating, the solutions were
immediately cooled in running water, and the absorbance
was detected by a UV−vis spectrophotometer.
Preparation of Aβ1−40 Samples. Lyophilized Aβ1−40
peptides powder was dissolved in HFIP, and then HFIP
solution containing Aβ1−40 (1 mg/mL) was put in the ice−
water bath for 20 min for further dissolution. HFIP was
evaporated by treatment of N2 flow to get a thin film and
stored at −20 °C before use. The Aβ1−40 monomer solution
(10 μM) was prepared by dissolving the Aβ1−40 film in 10 mM
Tris-HCl buffer (pH 7.4) in the dark and used at once. The
Aβ1−40 oligomer and fibril were obtained by incubating Aβ1−40
solution at 37 °C for 6 and 48 h, respectively. The Aβ1−40
aggregation study was performed as follows: Aβ1−40 solution
was mixed with 10 mM GSH or EGCG, and then the mixture
was incubated at 37 °C for different times.
Synthesis of PAKL-7. Briefly, PA were dissolved in DMSO
and then activated with EDCI, HOBT, and DMAP
(PA:EDCI:HOBT:DMAP = 1:3:3:3) for 2 h at room
temperature under N2 atmosphere. Then the peptide KL-7
(dissolved in DMSO) solution was dropwise added to the PA
solutions with gentle agitation, and the mixture was reacted for
24 h at room temperature. Then it was purified with dialysis
bags (MWCO, 500) to remove unreacted PA and other ions.
Later, a white solid was obtained under reduced pressure and
stored at 4 °C for further use. MS calcd for C51H75N9O9,
957.65; found, 980.56 (M + Na+).
SA and Aβ Interaction Analysis. For the assay, the
mixture comprising a fixed concentration (7 μM) of FAMDK16
(FAM-DAEFRHDSGYEVHHQK), FAMKD8 (FAM-
K L V F F A E D ) ,
o r
F A M V V
( F A M -
1 7
VGSNKGAIIGLMVGGVV) and varying concentrations of
SANF in 10 mM Tris-HCl buffer (pH 7.4) was prepared. The
mixing solutions were allowed to react further for 30 min at
room temperature in the dark. The fluorescence was then
detected by a fluorescence spectrophotometer. For the contrast
assay, the SANF was replaced by PANF.
Fluorescence Turn-on Assay. The fluorescence turn-on
assay was fabricated by incubating 350 μM SANF and 7 μM
FAMVV17 aqueous solution at room temperature for 30 min.
Then Aβ1−40 of different concentrations in the 10 mM Tris-
HCl buffer (pH 7.4) were added into the above mixture. The
change of FAM fluorescence was detected by a fluorescence
spectrophotometer by excitation with 462 nm light. For
comparison, BSA samples of different concentrations were also
examined.
Synthesis of SAKL-7. A 140 mg amount of N3−SA (0.150
mmol) and 89.1 mg of sodium ascorbate (0.450 mmol) were
added to a stirred solution of 100 mg of PAKL-7 (0.104 mmol)
in DMF (6.00 mL), followed by the addition of 1.00 M CuSO4
(45.0 μL). The reaction mixture was stirred at room
temperature for 24 h, and then it was purified with dialysis
bags (MWCO, 500). Later, a white solid was obtained under
reduced pressure and stored at 4 °C for further use. MALDI-
TOF MS calcd for C62H93N13O17, 1291.68; found, 1290.67 (M
− H).
C
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
Determination of Aβ1−40 in Artificial Cerebrospinal
Fluid and Fetal Bovine Serum. The artificial cerebrospinal
fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl
(2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), CaCl2 (1.1
mM), NaHCO3 (27.5 mM), and Na2SO4 (0.5 mM) into
doubly distilled water, and the solution pH was adjusted to 7.4.
The calibration curve was first established by detecting the
fluorescence intensity of FAM in the mixture containing 7 μM
FAMVV17 and 350 μM SANF solutions upon addition of
Aβ1−40 at a series of concentrations. Then the aCSF samples
containing three Aβ1−40 concentrations were detected.
To test the applicability of Aβ1−40 detection in serum, a
series of fetal bovine serum (FBS) samples containing Aβ1−40
in FBS were prepared by spiking Aβ1−40 into FBS. Then 20 μL
of FBS with different concentrations of Aβ1−40 were added to
180 μL of mixed solution, which consisted of 350 μM SANF
and 7 μM FAMVV17. After incubating for 30 min, the change
of FAM fluorescence was detected. The results were validated
by using human Aβ1−40 ELISA kits (Elabscience Biotechnol-
ogy, China) following the manufacturer’s instructions.
seen from Figure S16, the 1H NMR spectrum at 3.9−3.5 ppm
of SAKL-7 revealed the presence of SA by comparing that of
free N3−SA (Figure S10) and PAKL-7 (Figure S1).
Furthermore, the absence of a peak at 2127 cm−1 of the
CC stretching of PAKL-7 in the FT-IR spectra of SAKL-7
(Figure S17) as well as HRMS at 1290.67 (M − H) (Figure
S18) confirmed the successful preparation of SAKL-7.
Glycopeptide Self-Assembly. We first studied the
capacity of SAKL-7 and PAKL-7 to self-assemble into
supramolecular nanostructures by transmission electron
microscopy (TEM). As shown in Figure 1A and 1B, TEM
Aβ1−40 Aggregation Assay. To test the applicability of the
method for Aβ1−40 aggregation assay, 350 μM SANF and 7 μM
FAMVV17 were mixed with (i) 10 μM Aβ1−40, (ii) 10 μM
Aβ1−40 with EGCG, or (iii) 10 μM Aβ1−40 with GSH incubated
in 10 mM Tris-HCl buffer (pH 7.4) for a certain time. The
fluorescence spectra were monitored by a fluorescence
spectrophotometer.
RESULTS AND DISCUSSION
■
Design and Synthesis of Glycosylated Peptide. The
peptide sequence containing the FF nucleating core is known
to have a high propensity to self-assemble into diverse
nanostructures through electrostatic, π−π stacking, and
hydrophobic interactions.45−48 Recently, much attention has
been focused on their use in material fields and biotechnology
because of their high stability and biocompatibility.48−50 To
obtain glycan clusters in a highly ordered array based on the
supramolecular self-assembly, the KL-7 peptide containing FF
with the ability to form β-sheets and a terminal K residue for
further glycosylation is selected. Subsequently, 4-pentynoic
acid (PA) was introduced by covalent coupling with lysine to
serve as the conjugation site for saccharide (PAKL-7). Neu5Ac
(a representative of SA) was then covalently linked to the
peptide PAKL-7 by click chemistry, forming an amphiphilic
glycosylated peptide (SAKL-7) (Scheme 1).
To verify the successful coupling of PA on KL-7 peptide, the
chemical structure of the resultant PAKL-7 was characterized
by nuclear magnetic resonance (NMR), Fourier transform
infrared (FT-IR) spectra, and high-resolution mass spectra
(HRMS). New characteristic peaks of CH2 assigned to PA
appeared at 2.4−2.2 ppm in the 1H NMR spectrum of PAKL-7
(Figures S1 and S2), suggesting the successful covalent
attachment of PA. Moreover, the FT-IR spectra showed the
characteristic absorption bond at 2127 cm−1 assigned to CC
stretching in the PAKL-7 (Figure S3). Meanwhile, the
molecular ion peak at 980.56 (M + Na+) in HRMS spectra
revealed the successful synthesis of PAKL-7 (Figures S4 and
S5).
Figure 1. TEM images of SANF (A) and PANF (B). (C) FT-IR
spectra of PANF and SANF. (D) CD spectra of PANF and SANF.
images after negative staining obviously show that the SANF
and PANF have similar fibril morphology. SAKL-7 and PAKL-
7 formed high-aspect-ratio filaments with a diameter of
approximately 12 nm and length up to 300 nm in the mixed
solvent of DMSO and water. Zeta potential measurements
were carried out to characterize the surface charge of these
assembled nanostructures. The conversion of positively
charged PANF (4.8 0.5 mV) to negatively charged SANF
(−19.6
1.2 mV) as shown in Figure S19 suggested the
covalent grafting of SA onto the surface of peptide nanofiber.
The SA grafting ratio was determined according to a previous
method, and it showed that 73.6% of the KL-7 peptides were
modified with SA (Figure S20),44 indicating that plenty of
sialic acids were exposed on the surface of nanofiber. FT-IR
and circular dichroism (CD) spectroscopy were utilized to
characterize the secondary structure of the peptide nanofiber.
Both SANF and PANF showed a strong amide I absorption
band at 1629 cm−1, which indicated H-bonded β-sheet
structures. Weak absorption bands observed at 1685 cm−1
could be attributed to the antiparallel configuration (Figure
1C).51,52 Amide II bands at 1546 cm−1 were observed for
SANF and PANF. Both of the self-assembled supramolecular
structures exhibited negative peaks near 218 nm in the CD
spectra (Figure 1D), indicating the presence of β-sheet
conformation.52,53
Next, SA was conjugated on PAKL-7 via copper(I)-catalyzed
alkyne−azide cycloaddition (CuAAC) with N3−SA to obtain
SAKL-7. N3−SA was synthesized and characterized by carbon
nuclear magnetic resonance spectroscopy (13C NMR), 1H
NMR, FT-IR spectra, and HRMS (Figures S6−15). As can be
Interaction of SA with Different Aβ Fragments. To
clearly investigate the interaction profile of Aβ1−40 with SA, the
Aβ1−40 peptides were separated into three parts: the N-
terminal residues of Aβ1−16, the core residues of Aβ16−23, and
the C-terminal residues of Aβ24−40, each of which was labeled
D
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
with fluorophore FAM and designated as FAMDK16,
FAMKD8, and FAMVV17, respectively (Figure 2A). Fluo-
investigate the binding ability of SA with KD8. Figure 2C
describes that the fluorescence of FAMKD8 was slightly
quenched by various concentrations of SANF or PANF,
indicating that KD8 may interact with SA mildly. FAMVV17
was further used to explore the interaction between the SA on
the surface of nanofiber and the C-terminal residues of Aβ
peptide. Upon addition of SANF into the FAMVV17 solution,
the fluorescence signal of FAMVV17 gradually decreased as
shown in Figure 2D; however, the contrast group in the
presence of PANF displayed insignificant fluorescence change
(Figure S21), demonstrating the strong interaction of SANF
with VV17. From the data we can see the binding affinity
followed the order of DK16 > VV17 > KD8. The results suggest
that N-terminal and C-terminal residues may contribute greatly
to the high-affinity binding by hydrogen bonds and electro-
static interaction, which is consistent with the results of
molecular dynamic simulations.54,55
Detection of Aβ1−40 Based on SANF and FAMVV17.
Inspired by the high binding affinity of SA with Aβ fragments
of DK16 and VV17, we expected that the binding affinity of the
full-length Aβ1−40 might be greatly higher than the Aβ
fragments. Therefore, a competitive fluorescent turn-on
method for Aβ1−40 detection was set up by employing the
SANF-FAMVV17 or SANF-FAMDK16 system. Figure 3A
shows that the fluorescence intensity of FAMVV17 increases
Figure 2. (A) Chemical structures of the peptides: DK16, KD8, and
VV17. (B−D) Fluorescence spectra (left) and fluorescence intensity−
concentration relationship (right) of the FAM-modified (B) DK16,
(C) KD8, and (D) VV17 (7 μM) after being treated with different
concentrations of SANF or PANF (0−350 μM) in 10 mM Tris-HCl
buffer (pH 7.4) (λex = 462 nm; F and F0 represent the fluorescence
intensity of FAM-modified DK16, KD8, or VV17 with and without
SANF or PANF, respectively).
rescence titration experiments were performed by preparing
SANF with a series of concentrations in Tris-HCl buffer and
used to investigate the fluorescence behavior of FAMDK16,
FAMKD8, and FAMVV17 in the presence of SANF. PANF
without SA on the surface was used as a control. Noticeably,
the fluorescence intensity of FAMKD16 was greatly quenched
along with the increase of SANF concentration as shown in
Figure 2B. However, almost no FAMKD16 fluorescence
decrease in the presence of PANF was observed (Figure
S21), indicating that the SA moiety of SANF was responsible
for the binding due to the SA interaction with DK16. It can be
speculated that the N-terminal Aβ made a great contribution
to the affinity between Aβ and SA. Next, we sought to
Figure 3. (A) Fluorescence spectra of FAMVV17 (7 μM) in the
presence of SANF (350 μM) and increasing concentration of Aβ
monomer in 10 mM Tris-HCl buffer (pH 7.4). Aβ concentration: 0, 2
× 10−5, 1 × 10−4, 5 × 10−4, 2 × 10−3, 1 × 10−2, 5 × 10−2, 2 × 10−1, 1,
5, and 20 nM. (B) Fluorescence intensity change vs the concentration
of Aβmonomer or BSA; F and F0 represent the fluorescence intensity
of the SANF-FAMVV17 system with and without Aβ monomers or
BSA, respectively. (C) Fluorescence spectra of FAMVV17 in the
presence of SANF (350 μM), Aβ (20 nM), and increasing
concentration of Cu2+ (0, 1, 5, 10, 12, 14, 16, 18, 20, 24, and 25
μM). (D) Fluorescence intensity change vs Cu2+ concentration. (E)
Proposed mechanisms of Aβ/SA and Aβ/Cu2+ interactions.
E
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
with the increase of Aβ1−40 concentration in the range from 2
× 10−5 to 2 × 101 nM. SANF-FAMDK16 system responded to
Aβ1−40 in a similar way (Figure S22). The linear relationship
thus spans 3 orders of magnitude before reaching a plateau at
higher concentration (Figure 3B). The limit of detection was
3.8 × 10−4 nM approximately (three times the standard
deviation of the blank, 3s), which is similar or superior to that
reported for other assays as summarized in Table 1.
(Table S1). The simplicity, high sensitivity, and selectivity
make the fluorescence turn-on method promising as a new
platform for the detection of Aβ in real samples.
Aβ Aggregation Monitoring and Inhibitor Screening.
Aβ aggregation and plaque formation are implicated in AD and
development of inhibitors for Aβ aggregation is regarded as a
potential therapy. Therefore, determination of Aβ fibrillation is
important for mechanism study and drug screening. For this
purpose, methods with high specificity to discriminate Aβ
monomers and fibrils are required. Figure 4A and 4D depict
Table 1. Comparison of Aβ1−40 Detection Performance with
Other Methods
methods
detection limit
linear range
1−550 nM
10.5−313.5 nM
20−50 nM
ref
LMOCP sensor
Cu2+ AuNP assay
STM-based
immunosensor
SANF-FAMVV17
system
0.3 nM
0.6 nM
0.23 fM
56
57
58
3.8 × 10−4 nM
2 × 10−5−2 × 101 nM this
work
To evaluate the selectivity of SANF and Aβ1−40 interaction,
bovine serum albumin (BSA) comprised of 583 amino acid
residues with an isoelectric point of 4.7 was tested as the
possible interference. The result displayed that only a slight
change in the fluorescence intensity of the SANF-FAMVV17
system in the presence of BSA under the same conditions
(Figure 3B), suggesting good selectivity for Aβ1−40 detection.
In addition, it has been well recognized that copper ions
(Cu2+) could bind to Aβ through interaction with Asp 1, His 6,
His 13, or His 14.59 Therefore, we investigated the influence of
Cu2+ on the fluorescence recovery ability of Aβ1−40. As shown
in Figure 3C and 3D, the fluorescence intensity decreased with
the increase of the Cu2+ concentration compared to that
without Cu2+. The concentrations of Cu2+ added were
controlled to ensure Cu2+ had a negligible effect on
FAMVV17 fluorescence (Figure S23). Considering that Cu2+
may bind to SA on the nanofiber and influence the response,
control experiments of adding different concentrations of Cu2+
into the SANF-FAMVV17 system in the absence of Aβ1−40 were
also performed. Cu2+ could induce slight fluorescence
enhancement (Figure S24). Therefore, the decreased fluo-
rescence recovery of Aβ1−40 may be attributed to the fact that
the binding of Cu2+ to Aβ influenced the binding of Aβ1−40 to
SANF. From the results we could deduce that His 13 or His 14
was involved in the interaction between Aβ1−40 and SA (Figure
3E).
Figure 4. (A) Fluorescence spectra of FAMVV17 (7 μM) in the
presence of SANF (350 μM) upon addition of 20 nM Aβ1−40
monomer, oligomer or fibril. (B) Fluorescence spectra of FAMVV17
(7 μM) in the presence of SANF (350 μM) upon addition of 20 nM
Aβ1−40 incubated for different times. (C) Time-dependent fluo-
rescence recovery of 20 nM Aβ1−40, Aβ1−40 in the presence of GSH, or
Aβ1−40 in the presence of EGCG. (D) Selectivity of the fluorescent
turn-on assay in 10 mM Tris-HCl buffer (pH 7.4) containing 20 nM
samples: (1) control, (2) Aβ1−40 monomer, (3) Aβ1−40 + GSH (24 h),
(4) Aβ1−40 + EGCG (24 h); (5) Aβ1−40 oligomer (6 h); (6) Aβ1−40
fibril (48 h).
the response of SANF-FAMVV17 to Aβ1−40 monomers,
oligomers (incubated at 37 °C for 6 h) and fibrils (incubated
at 37 °C for 48 h). The Aβ1−40 samples incubated for 48 h
showed slight fluorescence recovery compared to monomers,
indicating that fibrils have less exposed interaction sites with
SA. The fluorescence recovery of Aβ1−40 sample incubated for
6 h which may contain monomers and oligomers was between
that of monomers and fibrils. Then, we used the method to
monitor the fibrillation of Aβ1−40 as a function of time and the
process of Aβ1−40 aggregation was also studied by CD,
thioflavin T (ThT) and atomic force microscopy (AFM)
assay accordingly. As shown in Figure 4B, the silenced
fluorescence signal of FAMVV17 was rapidly recovered by
Aβ1−40 that incubated for a short time. The extent of FAMVV17
fluorescence recovery became weaker with the prolonging of
Aβ1−40 incubation time, and after 48 h the fluorescence
intensity does not show an obvious difference with the
background. CD spectra showed that the typical peaks of β-
sheet-rich aggregates with a maximum at 195 nm and a
minimum at 215 nm increased with the increase of incubation
time as well as the enhancement of ThT fluorescence (Figure
S26). AFM was also used to directly visualize Aβ of different
morphologies by incubating Aβ1−40 monomer in Tris-HCl for
different times (Figure 5). The transitions from monomer to
Determination of Aβ1−40 in Biological Samples.
Encouraged by the low detection limit and high selectivity of
the method, Aβ1−40 monomer detection was also carried out in
samples of artificial cerebrospinal fluid (aCSF). As illustrated in
Table 2, the standard addition method analyzed by the
calibration curve (Figure S25) showed acceptable recovery,
which indicated the feasibility of the fluorescence analysis for
Aβ monomer in CSF samples using this method. Application
of the method to detect Aβ1−40 in FBS was also performed.
Good recoveries were obtained and validated by ELISA assay
Table 2. Results of Aβ1‑40 Detection in aCSF
samples
added (nM)
detected (nM)
recovery (%)
RSD (%)
1
2
3
0.5
3.0
9.0
0.48 0.1
3.1 0.4
8.4 0.6
95.9
104.4
97.1
3.8
2.9
4.7
F
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
of SA-ME; 1H NMR spectra of Ts-SA; 13C NMR spectra
of Ts-SA; 1H NMR spectra of N3-SA; 13C NMR spectra
of N3-SA; FT-IR spectra of SA, SA-ME, Ts-SA, and N3-
1
SA; HRMS spectrum of SA-ME, Ts-SA, and N3-SA; H
NMR spectra of SAKL-7; FT-IR spectra of N3-SA,
PAKL-7, and SAKL-7; HRMS spectrum of SAKL-7; zeta
potential of PANF and SANF; UV−vis absorbance
spectra of Neu5Ac at different concentrations in the
solution containing 0.1 M CuSO4, 4 % resorcinol
reagent, and hydrochloric acid, corresponding calibra-
tion curve, and absorbance spectra of SANF obtained at
the same condition; fluorescence spectra of FAMDK16,
FAMKD8, and FAMVV17 in the presence of different
concentrations of PANF; fluorescence spectra of
FAMDK16 in the presence of SANF upon addition of
Aβ monomers in 10 mM Tris-HCl buffer (pH 7.4), and
fluorescence intensity change as a function of Aβ
monomer concentration; fluorescence spectra of
FAMVV17 in the presence of Cu2+ with different
concentrations in 10 mM Tris-HCl buffer (pH 7.4);
fluorescence spectra and fluorescence intensity of
FAMVV17 system in the presence of SANF and Cu2+
with different concentrations in 10 mM Tris-HCl buffer
(pH 7.4); fluorescence spectra of FAMVV17 in the
presence of SANF and Aβ1−40 with different concen-
trations in aCSF, and corresponding calibration curve;
CD spectra and ThT assay of Aβ1−40 solutions incubated
at 37 °C for different times; fluorescence spectra of
FAMVV17 in the presence of SANF upon addition of
Aβ1−40 incubated with GSH or EGCG for different
times; results of Aβ1−40 detection in FBS (PDF)
Figure 5. AFM images of Aβ samples incubated for different times in
10 mM Tris-HCl buffer (pH 7.4) with or without the presence of
inhibitors: (A) 0 h, (B) 6 h, (C) 12 h, (D) 24 h, (E) 36 h, (F) 48 h,
(G) Aβ1−40+ EGCG 24 h, and (H) Aβ1−40 + GSH 24 h.
oligomer, protofibrils, and long fibrils were clearly seen. The
results suggest that the fibrillation of Aβ could be monitored by
this fluorescence method based on the specific interaction
between Aβ and SA. Compared to the routinely used reference
method, this strategy showed the advantage being sensitive,
having fast response, and being suitable for high-throughput
assay.
Furthermore, we continued to investigate its applicability for
inhibitor screening. (−)-Epigallocatechin gallate (EGCG) and
glutathione (GSH), two compounds reported to inhibit the
on-pathway Aβ oligomerization and fibrillation, were chos-
en.60,61 The processes of Aβ1−40 fibrillation in the presence of
EGCG or GSH were also monitored by AFM (Figure 5G and
5H), CD, and ThT assay (Figure S26). We found that the
fluorescence intensity remained high with the extension of Aβ
incubation time in the presence of EGCG or GSH (Figures
4C, 4D, and S27). AFM images display that no aggregates are
present in the view, implying that EGCG or GSH could exert
an inhibition effect on Aβ1−40 aggregation. The strategy based
on the specific and high-affinity binding of Aβ to SA-modified
nanofiber is efficient for inhibitor screening.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: zaxu@chem.ecnu.edu.cn, Tel/Fax: +86-21-54340053.
*E-mail: wzhang@chem.ecnu.edu.cn, Tel/Fax: +86-21-
62235761.
■
CONCLUSIONS
■
ORCID
In summary, we constructed a glycopeptide nanofiber platform
by using a self-assembly method. The peptide nanofiber
provided multiple SA epitopes exposed on the surface for Aβ−
SA interaction analysis. By dividing Aβ1−40 into three
fragments, we found that glycopeptide nanofiber had a high
binding affinity to the N- and C-terminal domains of Aβ. We
developed the system to be a fluorescent turn-on sensor to
Aβ1−40. It exhibited high specificity and selectivity to recognize
Aβ1−40 monomer. It was also successfully applied to Aβ
aggregation process monitoring and inhibitor screening.
Further studies such as optimization of fluorophores to lower
the background or developing a ratiometric response would
greatly improve the analytical performace of this strategy.
Therefore, this engineered carbohydrate-modified 1D nano-
fiber may have great potential in applications of Aβ1−40
detection and the early diagnosis of Alzheimer’s disease.
Zhiai Xu: 0000-0002-4391-2507
Yang Tian: 0000-0001-8850-0349
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support by the National Natural Science Foundation
of China (Nos. 21675055, 21775046, 21635003) is greatly
appreciated.
REFERENCES
■
(1) McLaurin, J.; Franklin, T.; Fraser, P.; Chakrabartty, A. J. Biol.
Chem. 1998, 273, 4506−4515.
(2) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353−356.
(3) Gorman, P. M.; Yip, C. M.; Fraser, P. E.; Chakrabartty, A. J. Mol.
Biol. 2003, 325, 743−757.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.9b00377.
(4) Andreasen, N.; Blennow, K. Peptides 2002, 23, 1205−1214.
(5) LaFerla, F. M.; Green, K. N.; Oddo, S. Nat. Rev. Neurosci. 2007,
8, 499−509.
■
S
(6) Benilova, I.; Karran, E.; De Strooper, B. Nat. Neurosci. 2012, 15,
349−357.
(7) Hamley, I. W. Angew. Chem., Int. Ed. 2007, 46, 8128−8147.
(8) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353−356.
(9) Butterfield, S. M.; Lashuel, H. A. Angew. Chem., Int. Ed. 2010, 49,
5628−5654.
1H NMR spectra of PAKL-7 and KL-7; FT-IR spectra of
KL-7, PA, and PAKL-7; HRMS spectrum of KL-7 and
PAKL-7; 1H NMR spectra of SA-ME; 13C NMR spectra
G
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
̈
(10) Hard, T. J. Phys. Chem. Lett. 2014, 5, 607−614.
(43) Qi, G. B.; Gao, Y. J.; Wang, L.; Wang, H. Adv. Mater. 2018, 30,
1703444.
(11) Zhu, L.; Zhang, J.; Wang, F.; Wang, Y.; Lu, L.; Feng, C.; Xu, Z.;
Zhang, W. Biosens. Bioelectron. 2016, 78, 206−212.
(12) Zhang, J.; Liu, J.; Zhu, Y.; Xu, Z.; Xu, J.; Wang, T.; Yu, H.;
Zhang, W. Chem. Commun. 2016, 52, 12044−12047.
(13) Mehta, P. D.; Pirttila, T.; Mehta, S. P.; Sersen, E. A.; Aisen, P.
S.; Wisniewski, H. M. Arch. Neurol. 2000, 57, 100−105.
(14) Hardy, J.; Higgins, G. Science 1992, 256, 184−185.
(15) Zhou, Y.; Liu, L.; Hao, Y.; Xu, M. Chem. - Asian J. 2016, 11,
805−817.
(44) Svennerholm, L. Biochim. Biophys. Acta 1957, 24, 604−611.
(45) Safaryan, S.; Slabov, V.; Kopyl, S.; Romanyuk, K.; Bdikin, I.;
Vasilev, S.; Zelenovskiy, P.; Shur, V. Y.; Uslamin, E. A.; Pidko, E. A.;
Vinogradov, A. V.; Kholkin, A. L. ACS Appl. Mater. Interfaces 2018,
10, 10543−10551.
̈
(46) Lee, J. H.; Heo, K.; Schulz-Schonhagen, K.; Lee, J. H.; Desai,
M. S.; Jin, H. E.; Lee, S. W. ACS Nano 2018, 12, 8138−8144.
(47) Almohammed, S.; Oladapo, S. O.; Ryan, K.; Kholkin, A. L.;
Rice, J. H.; Rodriguez, B. J. RSC Adv. 2016, 6, 41809−41815.
(48) Rengifo, R. F.; Li, N. X.; Sementilli, A.; Lynn, D. G. Org.
Biomol. Chem. 2017, 15, 7063−7071.
(16) Yanagisawa, K.; Odaka, A.; Suzuki, N.; Ihara, Y. Nat. Med.
1995, 1, 1062−1066.
(17) Yanagisawa, K. Glycoconjugate J. 2015, 32, 87−91.
(18) Mori, K.; Mahmood, M. I.; Neya, S.; Matsuzaki, K.; Hoshino,
T. J. Phys. Chem. B 2012, 116, 5111−5121.
(49) Kapil, N.; Singh, A.; Das, D. Angew. Chem. 2015, 127, 6592−
6595.
(50) Omosun, T. O.; Hsieh, M. C.; Childers, W. S.; Das, D.; Mehta,
A. K.; Anthony, N. R.; Pan, T.; Grover, M. A.; Berland, K. M.; Lynn,
D. G. Nat. Chem. 2017, 9, 805−809.
(19) Matsuzaki, K. Biochim. Biophys. Acta, Biomembr. 2007, 1768,
1935−1942.
(20) Kreutz, F.; Frozza, R. L.; Breier, A. C.; de Oliveira, V. A.; Horn,
A. P.; Pettenuzzo, L. F.; Netto, C. A.; Salbego, C. G.; Trindade, V. M.
T. Neurochem. Int. 2011, 59, 648−655.
(51) Miyazawa, T. J. Chem. Phys. 1960, 32, 1647−1652.
(52) Mehta, A. K.; Lu, K.; Childers, W. S.; Liang, Y.; Dublin, S. N.;
Dong, J.; Snyder, J. P.; Pingali, S. V.; Thiyagarajan, P.; Lynn, D. G. J.
Am. Chem. Soc. 2008, 130, 9829−9835.
(21) Rushworth, J. V.; Hooper, N. M. Int. J. Alzheimer's Dis. 2011,
2011, 603052.
(53) Lu, K.; Jacob, J.; Thiyagarajan, P.; Conticello, V. P.; Lynn, D. G.
J. Am. Chem. Soc. 2003, 125, 6391−6393.
(22) Kakio, A.; Nishimoto, S. L.; Yanagisawa, K.; Kozutsumi, Y.;
Matsuzaki, K. Biochemistry 2002, 41, 7385−7390.
(23) Wang, B. Annu. Rev. Nutr. 2009, 29, 177−222.
(24) Ariga, T.; Yu, R. K. Neurochem. Res. 1999, 24, 219−226.
(25) Wakabayashi, M.; Okada, T.; Kozutsumi, Y.; Matsuzaki, K.
Biochem. Biophys. Res. Commun. 2005, 328, 1019−1023.
(26) Schladitz, C.; Vieira, E.; Hermel, H.; Mohwald, H. Biophys. J.
1999, 77, 3305−3310.
(54) Vahed, M.; Neya, S.; Matsuzaki, K.; Hoshino, T. J. Phys. Chem.
B 2018, 122, 3771−3781.
(55) Hoshino, T.; Mahmood, M. I.; Mori, K.; Matsuzaki, K. J. Phys.
Chem. B 2013, 117, 8085−8094.
(56) Liu, B.; Shen, H.; Hao, Y.; Zhu, X.; Li, S.; Huang, Y.; Qu, P.;
Xu, M. Anal. Chem. 2018, 90, 12449−12455.
(57) Zhou, Y. L.; Dong, H.; Liu, L. T.; Xu, M. T. Small 2015, 11,
2144−2149.
(27) Williams, T. L.; Serpell, L. C. FEBS J. 2011, 278, 3905−3917.
(28) Matsuzaki, K.; Noguch, T.; Wakabayashi, M.; Ikeda, K.; Okada,
T.; Ohashi, Y.; Hoshino, M.; Naiki, H. Biochim. Biophys. Acta,
Biomembr. 2007, 1768, 122−130.
(58) Prabhulkar, S.; Piatyszek, R.; Cirrito, J. R.; Wu, Z. Z.; Li, C. Z. J.
Neurochem. 2012, 122, 374−381.
(59) Faller, P.; Hureau, C.; La Penna, G. Acc. Chem. Res. 2014, 47,
2252−2259.
́
(29) Amaro, M.; Sachl, R.; Aydogan, G.; Mikhalyov, I. I.; Vacha, R.;
Hof, M. Angew. Chem., Int. Ed. 2016, 55, 9411−9415.
(30) Matsubara, T.; Nishihara, M.; Yasumori, H.; Nakai, M.;
Yanagisawa, K.; Sato, T. Langmuir 2017, 33, 13874−13881.
(31) Vahed, M.; Neya, S.; Matsuzaki, K.; Hoshino, T. J. Phys. Chem.
B 2018, 122, 3771−3781.
(60) Ehrnhoefer, D. E.; Bieschke, J.; Boeddrich, A.; Herbst, M.;
Masino, L.; Lurz, R.; Engemann, S.; Pastore, A.; Wanker, E. E. Nat.
Struct. Mol. Biol. 2008, 15, 558−566.
(61) More, S. S.; Vartak, A. P.; Vince, R. ACS Chem. Neurosci. 2013,
4, 330−338.
(32) Williamson, M. P.; Suzuki, Y. U.; Bourne, N. T.; Asakura, T.
Biochem. J. 2006, 397, 483−490.
(33) Kelm, S.; Schauer, R. Int. Rev. Cytol. 1997, 175, 137−240.
(34) Crocker, P. R.; Paulson, J. C.; Varki, A. Nat. Rev. Immunol.
2007, 7, 255−266.
(35) Wittmann, V.; Pieters, R. J. Chem. Soc. Rev. 2013, 42, 4492−
4503.
(36) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.;
Kiessling, L. L. J. Am. Chem. Soc. 2002, 124, 14922−14933.
(37) Battigelli, A.; Kim, J. H.; Dehigaspitiya, D. C.; Proulx, C.;
Robertson, E. J.; Murray, D. J.; Rad, B.; Kirshenbaum, K.;
Zuckermann, R. N. ACS Nano 2018, 12, 2455−2465.
́
(38) Lee, S. S.; Fyrner, T.; Chen, F.; Alvarez, Z.; Sleep, E.; Chun, D.
S.; Weiner, J. A.; Cook, R. W.; Freshman, R. D.; Schallmo, M. S.;
Katchko, K. M.; Schneider, A. D.; Smith, J. T.; Yun, C.; Singh, G.;
Hashmi, S. Z.; McClendon, M. T.; Yu, Z.; Stock, S. R.; Hsu, W. K.;
Hsu, E. L.; Stupp, S. I. Nat. Nanotechnol. 2017, 12, 821.
(39) Munoz, A.; Sigwalt, D.; Illescas, B. M.; Luczkowiak, J.;
̃
́
Rodríguez-Perez, L.; Nierengarten, I.; Holler, M.; Remy, J. S.; Buffet,
K.; Vincent, S. P.; Rojo, J.; Delgado, R.; Nierengarten, J. F.; Martín, N.
Nat. Chem. 2016, 8, 50−57.
(40) Dai, B.; Li, D.; Xi, W.; Luo, F.; Zhang, X.; Zou, M.; Cao, M.;
Hu, J.; Wang, W.; Wei, G.; Zhang, Y.; Liu, C. Proc. Natl. Acad. Sci. U.
S. A. 2015, 112, 2996−3001.
(41) Lock, L. L.; Reyes, C. D.; Zhang, P.; Cui, H. J. Am. Chem. Soc.
2016, 138, 3533−3540.
(42) Sun, H. L.; Chen, Y.; Han, X.; Liu, Y. Angew. Chem. 2017, 129,
7168−7171.
H
DOI: 10.1021/acs.analchem.9b00377
Anal. Chem. XXXX, XXX, XXX−XXX