Ultrabright Terbium Nanoparticles for FRET Biosensing and in Situ Imaging of Epidermal Growth Factor Receptors**

Article abstract of DOI:10.1002/chem.202002007

Lanthanide-doped nanoparticles (LnNPs) have become an important class of fluorophores for advanced biosensing and bioimaging. LnNPs that are photosensitized by surface-attached antenna ligands can possess exceptional brightness. However, their functional

Full text of DOI:10.1002/chem.202002007

Accepted Article
Accepted Article
Title: Ultrabright terbium nanoparticles for FRET biosensing and in-situ
imaging of epidermal growth factor receptors
Authors: Loic Joanny Charbonnière, Cyrille Charpentier, Vjona Cifliku,
Joan Goetz, Aline Nonat, Clémence Cheignon, Marcelina
Cardoso Dos Santos, Laura Francés-Soriano, Ka-Leung
Wong, and Niko Hildebrandt
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10.1002/chem.202002007
Chemistry - A European Journal
Ultrabright terbium nanoparticles for FRET biosensing and in-
situ imaging of epidermal growth factor receptors
Cyrille Charpentier,a‡ Vjona Cifliku,b,c‡ Joan Goetz,a,d Aline Nonat,a Clémence Cheignon,a
Marcelina Cardoso Dos Santos,b Laura Francés-Soriano,c Ka-Leung Wong,d Loïc J.
Charbonnièrea,* Niko Hildebrandt,b,c*
Addresses
a) Equipe de synthèse pour l’analyse (SynPA), Institut Pluridisciplinaire Hubert Curien (IPHC),
UMR 7178, CNRS, Université de Strasbourg, 67087 Strasbourg Cedex, France.
b) Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Univeristé Paris-
Sud, CNRS, CEA, 91405 Orsay Cedex, France.
c) nanoFRET.com, Laboratoire COBRA (Chimie Organique, Bioorganique, Réactivité et
Analyse), Université de Rouen Normandie, CNRS, INSA, 76821 Mont-Saint-Aignan Cedex,
France.
d) Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, Hong Kong
‡ : These authors contributed equally.
* : niko.hildebrandt@univ-rouen.fr, l.charbonn@unistra.fr
Abstract. Lanthanide-doped nanoparticles (LnNPs) have become an important class of
fluorophores for advanced biosensing and bioimaging. LnNPs that are photosensitized by
surface-attached antenna ligands can possess exceptional brightness. However, their functional
bioconjugation remains an important challenge for their translation into bioanalytical
applications. To solve this problem, we designed a ligand that can be simultaneously applied as
efficient light harvesting antenna for Tb surface ions and strong linker of biomolecules to the
LnNPs surfaces. To demonstrate generic applicability of the photosensitized TbNP-
bioconjugates, we applied them in two prototypical applications for biosensing and bioimaging.
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First, in-solution biorecognition was shown by time-resolved Förster resonance energy transfer
(FRET) between streptavidin-functionalized TbNPs to biotinylated dyes (ATTO 610). Second,
in-situ detection of ligand-receptor binding on cells was accomplished with TbNP-antibody
(Matuzumab) conjugates that could specifically bind to transmembrane epidermal growth
factor receptors (EGFR). High specificity and sensitivity were demonstrated by time-gated
imaging of EGFR on both strongly (A431) and weakly (HeLa and Cos7) EGFR-expressing cell
lines, whereas non-expressing cell lines (NIH3T3) and EGFR-passivated A431 cells did not
show any signals. Despite the relatively large size of TbNP-antibody conjugates, they could be
internalized by A431 cells upon binding to extracellular EGFR, which showed their potential
as bright and stable luminescence markers for intracellular signalling.
Keywords: Lanthanide nanoparticles, brightness, time-resolved detection, bio-labeling, FRET,
luminescence microscopy, fluorescence, terbium, EGFR, bioconjugation,
Introduction
With the ever increasing needs for early detection, improved sensitivity and
multiplexing capacity, luminescent nanoparticles (NPs) have become tools of choice in the pool
of probes for bioanalysis.[1] Semiconductor quantum dots (QDs), have played a crucial role in
that field, revealing excellent brightness properties applicable to numerous analytical issues. [2]
Beyond QDs, the family of luminescent NPs continues to grow with the advents of, e.g., dye-
doped polymeric NPs,[3] nanopolymers[4] or upconverting NPs.[5] Lanthanide (Ln) based NPs,
displaying line like spectral emission signatures, very long excited state lifetimes for auto-
fluorescence free time-gated detection, and emission throughout almost all the UV-Vis-NIR
domain, were shown to be particularly appealing for various bioanalytical applications,[6]
including in-solution assays[7] as well as in-vitro and in-vivo imaging.[3,8] However, in contrast
to QDs, for which the quantum confinement impeded them with very high brightness, the
Laporte forbidden character of f-f electronic transitions of Ln[9] severely impact the absorption
properties, and concomitantly their brightness. To overcome this issue, the antenna effect,
consisting in embedding Ln3+ cations in the cavity of photosensitizing ligands for Ln molecular
complexes,[10] was extended to Ln based NPs[11] and we recently demonstrated that the rational
design of surface capping antenna ligands can afford ultra-luminescent LnNPs that combine
exceptional spectroscopic properties, with brightness exceeding those of classical QDs.[12]
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While such surface-photosensitized LnNPs were shown to be bright, stable, and biocompatible,
their bioanalytical application has been limited to proof of concept studies, in which the LnNPs
were used for optical imaging without bioconjugation.[3,12] However, stable and robust labeling
of biomolecules to NP surfaces (functional nanobioconjugates)[13] and efficient biological
recognition for biosensing in solution and imaging on and in cells are among the most important
prerequisites to implement NPs into actual bioanalytical applications.
To address the challenge of biologically functional surface-photosensitized LnNPs in a
most straightforward manner, we synthesized and characterized ligands that can be
simultaneously used for both bioconjugation and photosensitization. Activated functional
groups were used for covalent binding of the ligands to amine or sulfhydryl groups on
biomolecules. These antenna-ligand-labeled biomolecules were then attached to Tb-doped LaF3
NPs (TbNPs) via coordination between the chelating antenna functions of the ligand and Tb3+
ions on the TbNP surface. Thus, surface attachment of biomolecules directly resulted in
photosensitization of the TbNPs, such that the biomolecules could “switch on” the TbNP
photoluminescence (PL) and lead to ultra-bright luminescent TbNPs for specific biological
recognition (Figure 1). To demonstrate the functionality of the TbNP-bioconjugates, we
applied them to two prototypical biosensing approaches. First, time-resolved and separation-
free FRET (Förster resonance energy transfer) bioassays showed that biotinylated organic dyes
(ATTO 610) could efficiently bind to streptavidin (sAv)-functionalized TbNPs and that the
biotin-sAv recognition resulted in efficient FRET from photosensitized Tb3+ ions to the
biotinylated dyes. Second, TbNPs conjugated with Matuzumab antibodies (ABs) against the
epidermal growth factor receptor (EGFR) were used to visualize EGFR expression and
internalization by different cell lines with time-gated PL microscopy. Careful PL analysis in
both applications showed that these TbNP-bioconjugates can be efficiently implemented into
real bioanalytical applications in both biosensing and bioimaging.
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Figure 1. Principle of biofunctionalized ultra-bright Ln nanoparticles. Without antenna-ligand-
functionalized biomolecules (in this case antibodies - AB), TbNPs do not emit significant PL
upon UV excitation (left). Efficient coordination of the AB-ligand conjugates to Ln3+ ions on
the TbNP surface leads to bright photosensitized Tb PL and biofunctional NP surfaces.
Results and discussion
Synthesis of functionalizable surface capping photosensitizing ligands and formation of
TbNPs. Based on prior works in the field of luminescent Tb complexes,[14] we have
demonstrated that hydroxy-isophthalic acid can be an excellent surface photosensitizing ligand
for Tb doped LaF3 NPs.[11] Additionally, the introduction of a linker between two such units
afforded a supplementary stabilization of the coordination at the surface. In order to render such
bis-chelating units linkable to any compound of interest, the linker between the two units was
modified to introduce an activated function (noted by the symbol * after the ligand) that should
further react with amine (L1*) or thiol functions (L2*). The synthetic methodology for the
preparation of the activated ligands is depicted in Scheme 1.
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Scheme 1. Synthesis of the activated ligands for the surface functionalization of LnNPs.
a) KOH, KMnO4, H2O, 110°C. b) H2SO4, EtOH, 90°C, 79% (a+b). c) KOH, EtOH, 90°C, 76% d) EDCI, HOBt,
Et3N, 0°C, 74%. e) t-BuOK, THF, r.t., 51%. f) TFA, DCM, 50°C, 70%. g) EDCI, HOBt, Et3N, 0°C, 61%. h) BBr3,
DCM, r.t.. j) NaOH, EtOH, 90°C, 80% (h+j). k) bis-isothiocyanate, Et3N, DMF, r.t., 88%. l) N-methylmorpholine,
DMF, r.t., 73%.
Briefly, 2,6-dimethyl-anisol was oxidized by KMnO4, followed by a sulphuric acid
catalyzed esterification of the formed acid with ethanol to afford the diester 1, which was
subjected to a controlled single saponification using substoechiometric amounts of KOH. A
double peptide coupling reaction was performed using EDCI in the presence of HOBt to couple
two chromophoric units to a 2-hydroxy-1,3-propylene diamine spacer. The alcohol function of
compound 3 was deprotonated to react in a Williamson nucleophilic substitution with t-butyl-
bromoacetate, leading to the intermediate 4, which, after deprotection of the t-butyl protecting
group affording compound 5, was coupled to a mono-protected hexylenediamine,[15] in a second
peptidic coupling reaction. The Boc protection of the amine function of the as-obtained
intermediate compound 6 was removed by acidic treatment with trifluoroacetic acid and the
amine was engaged into a coupling reaction with 1,4-bis-benzene-isothiocyanate to afford the
amine reactive ligand L1* ligand or with the NHS ester of N-propionic-maleimide to give the
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thiol reactive ligand L2*. The UV-Vis absorption spectra and excitation and emission spectra
of L1* and L2* are presented in Figures S24 and S25 (Supplementary Information).
TbNPs were synthesized according to previously published procedures and their shape
and composition were characterized by TEM (Figure S26, Supplementary Information), DLS
and ICP-MS analysis.[12] The concentration of the NPs was determined (following the
previously published method)[12] using the average size of the NPs as determined by TEM
measurement, the density of La0.9Tb0.1F3 calculated as the percentage weighted density of LaF3
and TbF3, and the total amount of La (or Tb) amount in the solution, as determined by ICP-MS
analysis.
Spectrofluorimetric titration of TbNPs by the activated ligand L1*. In a following step, the
surface coordination of the NPs by the activated ligand was investigated by a combination of
UV-Vis absorption spectroscopy and PL titration experiments. Briefly, increasing quantities of
ligands were added to a nanomolar solution of the TbNPs. Figure S27 (Supplementary
Information) represents the evolution of the absorption spectra of the solution as a function of
added ligands while Figure 2 shows the evolution of the emission spectra and the variations of
the Tb centred emission at 545 nm upon excitation into the ligand absorption band at 337 nm
as a function of the number of ligands per NPs.
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Figure 2. a) Evolution of the PL spectra (exc = 337 nm) of a 1.7 nM solution of La0.9Tb0.1F3
(Tris/HCl 0.01 M, pH = 7.0) upon addition of L1* (the number of L1* per NP is indicated by
7
the color code). Inset: Evolution of the Tb centred emission intensity at 545 nm (5D4 → F5
transition) as a function of the [L1*]/[NP] ratio.
As can be observed in Figure 2, the addition of L1* to the NPs’ solution immediately
leads to the observation of a strong PL arising from Tb emission with the typical emission bands
corresponding to the 5D4 → 7FJ transitions at 485, 545, 590 and 620 nm, respectively for J = 6
to 3. One can also notice the presence of weak bands above 630 nm corresponding to J = 2, 1
and 0. Interestingly, the presence of a residual fluorescence band, observed between 450 and
500 nm, points to an imperfect L1* to Tb energy transfer. The evolution of the Tb PL intensity
as a function of the [L1*]/[NP] ratio (Inset of Figure 2), shows a strong initial increase followed
by a plateau region. This final part corresponds to the saturation of the TbNP surface by a layer
of ligands and additional ligands can no longer coordinate at the surface and do not result in
further photosensitization of the Tb centred emission. Using the intercept of the lines plotted
for the rising and the plateau parts of the Tb intensity curves, we determine full surface coverage
at ~1700 L1*/NP. The luminescence quantum yield of a solution of Tb NPs coated with 2030
L1* per NP was determined using an integration sphere and the brightness of the sample could
be determined (see Figures S28 to S30 and full experimental details in the supplementary in
formation section). A maximum brightness of 1.8×106 M-1.cm-1 was measured at ca 310 nm,
which makes these nanoparticles among the brightest lanthanide containing NPs,12 comparable
to doped polymeric nanoparticles containing Ln complexes (brightness of 4×107 M-1.cm-1)3
with the unique additional feature that they can be bio-functionalized.
In order to bioconjugate the TbNPs, the activated ligands can be labeled to various
biological molecules of interest via their amine (L1*) or thiol (L2*) functionalities. The
preparation of functional NP-bioconjugates can be challenging when the biomolecules are
relatively large, i.e. similar size to the NPs, and therefore we selected two prominent examples,
namely streptavidin (sAv) and antibodies (AB). SAv has a very strong interaction with biotin,
which is frequently exploited for the design of biosensors.[16] Matuzumab is a humanized
monoclonal AB that binds with strong affinity to EGFR, which is known to be upregulated in
different types of cancers.[17]
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NP bioconjugation and FRET biosensing. An important method for biomolecular
quantification in solution is FRET, which exploits the proximity between an energy donor and
an energy acceptor upon biological recognition.[18] We therefore challenged our TbNP
bioconjugates with a prototypical FRET assay, in which the biomolecular recognition between
TbNP-sAv and the biotinylated dye ATTO 610 (A610B) was monitored by TbNP-to-A610B
FRET. While A610B was commercially available (cf. Scheme S1, Supplementary Information
for the chemical structure of A610B), we prepared TbNP-sAv bioconjugates as detailed in the
Experimental Section (Supplementary Information). Labeling of sAv with the photosensitizing
ligands L1* (determined by UV-Vis absorption spectroscopy, Figure 3) resulted in an average
value of 1.0 ± 0.1 L1 per sAv. Addition of L1-sAv to TbNPs resulted in a maximum labeling
ratio of ~66 sAv per TbNP, as determined by increasing Tb PL intensities with increasing
amounts of L1-sAv, which showed an inflection point corresponding to the presence of ~66 L1-
sAv per TbNP (Figure S31, Supplementary Information). Considering the size of the tetrameric
streptavidin protein (ca. 5 nm in diameter),[19] it is not surprising that the number of L1-sAv per
NP is largely inferior to that observed for the activated ligand alone.
Figure 3. UV-Vis absorption spectra of sAv (green), L1* (blue), L1 labeled sAv (L1-sAv, black)
and the best fit for a linear combination of sAv and L1* (dotted line).
For the FRET experiments, we used a conjugation ratio of 33 L1-sAv per TbNPs (half
of the maximum conjugation ratio) to obtain many sAv on the TbNP surface without steric
hindrance between the sAv, allowing for efficient biotin binding. Under these conditions, the
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PL lifetime of the Tb emission was composed of two main components (Figure 5), namely 1
= 0.66 ms (amplitude fraction of 18%) and 2 = 2.07 ms (amplitude fraction of 82%), which we
attributed to Tb ions at the very interface of surface and environment (1 is shorter due to PL
quenching by the aqueous solvent) and Tb ions close to the surface but not in direct contact
with the environment (2). As all of these ions were sensitized by the surface ligands, we
assumed that they can also engage in FRET to surface-attached dyes, and we used the
amplitude-averaged lifetime (<> = 1.8  0.1 ms) as the Tb-donor lifetime. Taking into account
a radiative lifetime of Tb of 4.75 ms,[19] this value resulted in a donor PL quantum yield of Tb
= 1.8/4.75 = 0.38  0.03. Figure S31 (Supplementary Information) shows the spectral overlap
of TbNP-sAv emission and A610B absorption, which is an important requirement for FRET.
Using equation 1,[18] with the orientation factor 2 = 2/3 (dynamic averaging as justified by the
isotropic emission and long lifetime of Tb), the refractive index n = 1.33 (aqueous solution),
the wavelength , the normalized (to unity over the integral) TbNP-sAv PL spectrum with
intensity ID(), and the A610B absorption spectrum with molar extinction coefficient (in
M-1cm-1) we obtained a Förster distance (donor-acceptor distance for 50 % FRET efficiency)
of R0 = 5.4  0.3 nm.
4
1/6
푅0 = 0.021(휅2Φ푇푏푛−4
퐼
퐷
(1)
( ) ( )
휆 휀퐴 휆 휆 푑휆)
∫
The FRET experiment consisted in adding increasing amounts of A610B to a solution
of the TbNP-sAv and record the time-gated (from 0.08 to 1.08 ms after the excitation pulse) PL
spectra after excitation into the L1 absorption band at 337 nm. Notably, A610B was also excited
at this wavelength. However, the time-gated detection mode suppressed the short-lived
(nanosecond range) fluorescence of directly excited A610B and detected only the long-lifetime
PL components of Tb donor PL and FRET-sensitized A610 acceptor PL, which possessed the
same PL decay time as the FRET-quenched Tb donor.[18a,21] Interaction of TbNP-sAv with
A610B resulted in a close proximity between Tb donors and A610 acceptors and concomitant
quenching of Tb PL and sensitization of A610 PL (in the 620 to 700 nm region), as shown in
Figure 4. TbNP PL decreased almost linearly until A610B/sAv = 3 and remained constant for
higher ratios (Figure S33b, Supplementary Information). Although sAv can bind up to four
biotins, sAv was attached to the TbNP surface, which limited the maximum amount of bound
A610B to three and additional A610B did not result in further quenching. For FRET-sensitized
A610B PL, the picture was slightly different because there was a linear intensity increase up to
A610B/sAv = 1.5 followed by a decrease between 1.5 and 3, after which A610 PL remained at
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a minimum level (Figure S33a, Supplementary Information). The strong PL quenching of
A610B after only 1.5 A610B per sAv and the low PL intensity of A610B in general (compared
to the strong PL quenching of Tb) was most probably related to self-quenching, which is a
frequently encountered phenomenon when high numbers of dyes are attached to the surface of
a NP (1.5 A610B/sAv corresponds to ~50 A610B per TbNP, which contain ~33 sAv on their
surface) and is caused by an increased probability of homo-FRET between the dyes in
combination with the formation of non-fluorescent dimers of acceptor dyes that serve as energy
trap states.[22] To confirm that the FRET signals resulted from A610B-sAv binding, the same
experiment was performed with TbNPs coated with the same number of L1* ligands alone
(without sAv). Neither Tb FRET-quenching nor A610B sensitization could be found in these
control experiments (Figure S35, Supplementary Information).
Figure 4. Time-gated PL spectra of the TbNP-sAv ([NP] = 1.6 nM and A610B upon addition
of different equivalents of A610B (number of equivalents per sAv are indicated by different
colors, exc = 337 ± 5 nm, delay time of 80 µs, integration time of 1 ms). Inset: zoom of the 600
– 700 nm range. (Figure S34 in Supplementary information, shows the deconvoluted TbNP
and A610B PL spectra).
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In parallel to spectroscopic measurements, time-resolved PL decays of the TbNP donor
(measured at 545 ± 2.5 nm) and of the A610 acceptor (measured at 637.0 ± 2.5 nm) were
analyzed (Figure 5). Similar to the time-gated intensity results (Figure 4), the Tb donor curves
(Figure 5a) decreased in both intensity and decay time until circa 3 A610B/sAv (and were
more or less constant for higher ratios) and the A610 acceptor curves (Figure 5b) increased in
intensity until ca. 1.5 A610B/sAv (and decreased to background levels for higher ratios). In
addition to the background of Tb PL (black curve in Figure 5b), which decreased with
increasing A610B/sAv ratios, the acceptor decay curves showed FRET-sensitized PL of A610
(faster decay contribution until ca. 1.5 ms, which is clearly above the Tb background PL). This
A610 PL in the hundreds of microsecond range is clear evidence for FRET-sensitization via
TbNPs as the intrinsic PL lifetime of A610 is in the nanosecond range.
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Figure 5. PL intensity decays (in Log units) of TbNP donors (a, em = 545.0 ± 2.5 nm) and
A610 acceptors (b, em = 637.0 ± 2.5 nm) for different ratios of A610B per sAv (ratios and
color code shown on the right of graph a) upon pulsed excitation (exc = 337 ± 5 nm).
Owing to the relatively weak and self-quenched FRET-sensitized PL of A610, its time-
gated intensities and time-resolved decays could not be used for distance estimations by FRET.
However, the FRET-quenched Tb PL intensities and decays provided very useful data for
quantifying Tb-donor to A610-acceptor distances. The FRET-efficiency (EFRET) of an emitting
Tb3+ ions (sensitized by the sAv-attached ligand L1) depends on the number of acceptors (a)
and can be calculated by equation 2, which assumes the same donor-acceptor distance (R) for
all acceptors:16a
6
푎푅
0
퐸퐹푅퐸푇
=
(2)
6
6
푎푅 +푅
0
EFRET can also be calculated by donor PL intensities (I) and decay times () in the
absence (subscript D) and in the presence (subscript DA) of the acceptor:
퐼
퐷퐴
퐸퐹푅퐸푇 = 1 −
퐸퐹푅퐸푇 = 1 −
(3a)
(3b)
퐼
퐷
휏
퐷퐴
휏
퐷
Combining equations 2 and 3 allows for calculating the donor-acceptor distance R by
the Tb PL intensities (Figure 4) and decays (Figure 5 and Table S1 Supporting Information):
1/6
)
퐼
퐷퐴
푅 = 푅0 (푎
푅 = 푅0 (푎
(4a)
(4b)
퐼
−퐼
퐷퐴
퐷
1/6
)
휏
퐷퐴
휏
−휏
퐷퐴
퐷
Equation 4 must take into account that a maximum of three A610B can bind to one sAv
on the TbNP surface (a  3), which means that a = 3 is constant for all A610B/sAv  3. For a
correct determination of R, a few other considerations are necessary. PL intensity measurements
quantify both FRET-quenched and non-quenched Tb PL (they cannot be distinguished in the
spectra). Thus, equation 3a determines an apparent FRET efficiency that takes into account
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donor-acceptor pairs as well as free donors (unquenched by FRET to the acceptor). For low
A610B/sAv ratios with a significant fraction of unquenched Tb3+ ions, the apparent FRET
efficiency is significantly lower than the actual FRET efficiency for each donor-acceptor pair,
which, in turn, is the correct FRET efficiency for calculating the donor-acceptor distance. The
best solution for determining a donor-acceptor distance with Tb PL intensities is to use
A610B/sAv ratios  3 because for these cases the TbNP surface is saturated with A610
acceptors and both EFRET and R are constant (Figure S36, Supplementary Information). Under
these conditions, the average donor-acceptor distance was R = 4.5 ± 0.3 nm, which is in very
good agreement with the dimensions of sAv in the solid state (5.5 x 5.8 x 4.8 nm3)[19] and the
structure (including the linker between biotin and A610) of A610B (Scheme S1, Supplementary
Information).
The advantage of PL lifetime data is the concentration independence and that decay
times from non-quenched and FRET-quenched donors can be distinguished. Therefore, the
FRET-quenched decay times provide a FRET efficiency solely for donor-acceptor pairs, which
can then be used for calculating the correct average donor-acceptor distance R. Using a
previously developed and tested method for fitting multiexponential FRET decays for distance
analysis,[23] we fitted the TbNP PL decays from Figure 5 to obtain average Tb donor decay
times (D) and FRET decay time (DA) that could be used to calculate the average donor-
acceptor distance R using equation 4b (Figure S37 and Table S1, Supplementary Information).
Because the PL decay curves of 0.5 and 1 A610B/sAv are very similar to the unquenched TbNP
decay curve, these were not taken into account for calculating the average distance (fit results
are nevertheless shown in Figure S37 and Table S1, Supplementary Information). All the other
seven decay curves were analyzed and resulted in an average donor-acceptor distance of R =
4.6 ± 0.3 nm, which is in excellent agreement with the FRET-saturated results from time-gated
PL intensities (vide supra). Both time-gated PL intensity and time-resolved PL decays clearly
demonstrate that this prototypical biotin-streptavidin binding system could be successfully used
for analyzing biomolecular binding by FRET from TbNPs to dyes.
Fluorescence microscopy imaging of EGFR. Our second prototypical bioanalytical
application consisted in the specific imaging of EGFR on cell membranes. Targeting EGFR and
EGFR signaling are important for cancer research and many different cell lines have been
investigated concerning their EGFR content and function.[24] Bright and non-photobleaching
nanoprobes for specific imaging of single, multiple, or clustered receptors from the epidermal
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growth factor family of receptor tyrosine kinases (EGFR, HER2, HER3, HER4) have the
potential to significantly advance such cancer-related studies.[25] One paramount requirement
for a successful application of NP-based imaging agents is the functional conjugation with
biological ligands (against the cell membrane receptor) that allow for specific targeting and the
suppression of non-specific adsorption of the NPs to the cell, which often results in non-specific
endocytosis of the NPs.[3,26] We selected the AB Matuzumab,[27] which is not only a very
specific ligand against EGFR but can also serve as a prototypical system for demonstrating
functional TbNP-AB conjugation in general. Another important prerequisite is a good
dispersion of the AB-conjugated NPs during incubation with cells, such that they do not interact
with each other (formation of NP clusters) but can accomplish specific EGFR imaging without
deposition of NP aggregates around or on the cell surface. Finally, a highly interesting
photophysical property of our TbNPs is their long PL lifetime (millisecond range), which can
be exploited in time-gated imaging to suppress all autofluorescence background (which is in
the nanosecond range).[28] All time-gated images were taken on live cells and did not require
delicate fixation protocols.
L2-AB conjugates were prepared by coupling the maleimide-activated ligand L2* to
thiols (reduced cysteines) on the AB and resulted in a labeling ratio of ~7 L2 per AB (see
Experimental Section, Supplementary Information) to assure efficient attachment of the ABs to
the TbNP surface. To investigate the influence of AB-conjugation conditions for optimized
EGFR imaging performance, we prepared TbNP-AB conjugates with i) different amounts of
L2-AB conjugates, ii) L2-AB and additional photosensitizing ligands (to entirely cover the
TbNP surface with photosensitizing ligands), and iii) under different conjugation conditions
(different conjugation buffers). TbNP-AB conjugates were prepared by simple mixture of L2-
AB conjugates with TbNPs at ratios of ca 20, 100, and 200 ABs per TbNP (see Experimental
Section, Supplementary Information). In initial titration experiments (addition of L2-AB to
TbNPs), we could show that up to ca 200 L2-AB could be added to TbNPs to accomplish
saturation of PL increase due to surface-attached photosensitizing ligands (Figure S38,
Supplementary Information). We therefore assumed that L2-AB can efficiently bind to TbNPs
until a maximum ratio of ~200 and that this value corresponded to ~100 % surface coverage.
Relatively high ratios of ABs per TbNP were used because previous experiments (not shown)
resulted in strong clustering of TbNP-AB conjugates at lower labeling ratios. For the
20AB/TbNP samples we also added non-activated photosensitizing ligands (LCN, Scheme S2,
Supplementary Information) to investigate the influence of a complete coverage of the TbNP
14
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10.1002/chem.202002007
Chemistry - A European Journal
surface with photosensitizers on brightness and stability. While 20AB/TbNP and 100AB/TbNP
samples were prepared in Tris buffer (10 mM, pH 7.2) to match the conditions of the in-solution
FRET experiments (vide supra), we used HEPES buffer (10 mM, pH 7.0) for the highest
labeling ratio of 200AB/TbNP. HEPES was selected because DLS measurements of bare
TbNPs showed less aggregation compared to Tris. While TbNPs were stable over time (up to
60 days) and pH from 4 to 8 in H2O, Tris resulted in significant aggregation (Figure S39).
Sonicating TbNPs in HEPES for one hour resulted in small monodisperse TbNPs (Figure S40)
and therefore, we used freshly sonicated TbNPs in HEPES for AB conjugation and performed
imaging experiments directly afterwards.
To also evaluate the aggregation of the different TbNP-AB conjugates, we first
performed DLS measurements in solution (Figure S41, Supplementary Information). These
analyses showed that the hydrodynamic diameters of TbNP-AB conjugates became smaller for
higher labeling ratios, that the 20AB/TbNP and 100AB/TbNP samples showed some clustering
(additional species with very large hydrodynamic diameters above 4 µm), and that the addition
of non-conjugated ligands (20AB/TbNP+LCN) could not significantly reduce aggregation or the
size of the main NP species. Time-gated PL imaging of A431 cells (Figure S42, Supplementary
Information) confirmed strong clustering of the 20AB/TbNP samples on the cell surfaces.
While the addition of LCN resulted in higher PL intensities and reduced clustering (smaller areas
of PL spots), significantly better results were accomplished for the samples with higher labeling
ratios. Previous studies showed that EGFR forms clusters of ~150 nm in diameter on HeLa cells
and that EGFR on highly EGFR-overexpressing A431 cells results in even stronger clustering
and favors the assembly of NPs into larger clusters.[29] Thus, we also expected a clustered
fluorescence EGFR staining pattern for higher AB/TbNP ratios but with significantly smaller
clusters.
To investigate the possibility of detecting EGFR on cells with high (A431)[30] and low
(HeLa and COS-7)[31] EGFR expression, we incubated those cells for 30 minutes with
20AB/TbNP or 100AB/TbNP. Similar to non-targeted (pure TbNPs without bioconjugation)
imaging,[26] we required only low nanomolar concentrations (1 nM or 2 nM) of TbNP-AB
conjugates to acquire sufficient PL signals for imaging EGFR on the cell membranes (Figures
S43 and S44, Supplementary Information). Additionally, the stability of the TbNPs-AB in
culture media was investigated by luminescence measurements, showing a gradual loss of ca
75% intensity over the course of 15h, but with still remarkable intensities (Figure S45,
Supplementary Information). For the lower EGFR-expressing HeLa and COS-7 cell lines,
15
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10.1002/chem.202002007
Chemistry - A European Journal
20AB/TbNP signals were below the detection threshold and could not image EGFR.,both
conjugation ratios could be used for EGFR-targeted imaging on A431 cells. However, the
20AB/TbNP conjugates showed significant clustering, whereas the 100AB/TbNP conjugates
clearly demonstrated the advantages of higher AB-conjugation ratios in terms of sensitivity.
The different PL spot sizes in the 100AB/TbNP PL images (smaller for HeLa and COS-7 and
larger for A431) also provided a good indication of the different EGFR expression levels in
these cell lines.
To further investigate sensitivity, specificity (A431 cells vs EGFR-passivated A431 and
EGFR-deleted NIH3T3 cell lines), and endocytosis of EGFR-bound TbNP-AB conjugates, we
used the highest possible conjugation ratio of 200AB/TbNP. Time-gated PL imaging of A431
cells showed that different concentrations of the TbNP-AB conjugates resulted in different
immunostaining patterns (Figure 6). Although the signals were relatively weak, 0.5 nM TbNPs
could still image EGFR on the cell membranes with high specificity. Using the same TbNP
concentrations on NIH3T3 cells that did not express EGFR or on A431 cells that were
passivated with non-conjugated AB (to saturate EGFR binding sites on the cell membrane) did
not result in any PL signals (Figure S46, Supplementary Information). At higher
concentrations, the EGFR on the cell membranes became more visible. For 2nM of TbNP-AB
conjugates, almost the entire membrane could be stained, which showed the very high EGFR
expression of A431 cells (Figure 6 bottom). These results demonstrated the importance of
optimizing the AB-conjugation conditions and that a maximum coverage of AB on the TbNP
surface leads to TbNP-AB conjugates that can be applied for bright and targeted EGFR imaging
with stable lanthanide emitters.
16
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10.1002/chem.202002007
Chemistry - A European Journal
Figure 6. Time-gated PL imaging of EGFR on the membranes of A431 cells that were
incubated for 30 minutes with different concentrations of 200AB/TbNP. The representative
images show DIC transmission (left), time-gated PL (centre), and their overlay (right). PL of
TbNPs is shown in green. GPE: Gates per exposure. Additional images are shown in Supporting
Figure S47 (Supplementary Information).
Encouraged by the excellent EGFR imaging conditions, we aimed at investigating a
possible cellular uptake (endocytosis) for intracellular trafficking of EGFR.[29a,32] To localize
the TbNP-AB conjugates on the cell envelope, we used co-staining with the plasma membrane
marker MemBright-640 (MB-640).[33] Several PL images were acquired at different heights of
the cells (so called “z-stacking” from the ventral (bottom) to the dorsal (top) plasma membrane
with z-steps of 0.22 µm). Movie S1 (Supplementary Information) shows the entire z-stack of
PL images with TbNP-AB conjugates at distinct locations across three different cells. Figure 7
17
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10.1002/chem.202002007
Chemistry - A European Journal
shows representative sections of the z-stack with TbNP-AB inside the membrane, inside the
cell, and outside the cell.
z = 2.86 µm
z = 8.58 µm
z = 9.90 µm
A3
A4
A5
A2
A1
D1
C3
C1
C6
C5
C4
B2
B3
C2
B1
B4
B5
z = 11.0 µm
z = 13.2 µm
z = 14.3 µm
A9
A11
A8
A7
A10
C8
A6
C7
Figure 7. PL image sections (cf. Movie S1, Supplementary Information for the entire z-stack)
taken at different heights (bottom: z = 0) of three representative cells (A, B, and C). The plasma
membrane is colored in red and TbNP-AB in green. Colocalization of membrane and TbNP-
AB is represented in yellow. Numbered areas show TbNP-AB at different locations within the
cells. All areas were also analyzed with lateral sections (xz and yz), which are shown in Figure
S48, (Supplementary Information). Scale bar: 10 µm.
In the lowest z-section image (z = 2.86 µm) the two 2 yellow (colocalized TbNP-AB and MB-
640 PL) points (A1 and B1) indicate that TbNP-AB are localized in the ventral membrane. In
the second image (z = 8.58 µm) almost all TbNP-AB (with the exception of C1 and D1) are
colocalized with the membrane (yellow spots). Taking into account that this section is close to
the center of the cell, the TbNP-ABs are most probably localized in the side membranes of the
cells. In contrast, C1 is not colocalized with the membrane (green), indicating that the TbNP-
AB has not yet adhered to the plasma membrane. D1 (also green) is a cluster of TbNP-AB
18
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10.1002/chem.202002007
Chemistry - A European Journal
outside the cell (floating in the medium). Slightly higher in the cells (z = 9.90 µm) TbNP-AB
A4 and A5 (yellow) are probably colocalized with side membrane, C3 is outside the cell but
close to the membrane (bound to extracellular EGFR) and C4, C5, and C6 appear to be inside
the cell (internalized very recently and located just below the membrane), which confirms
findings that EGFR already internalizes after 15 to 20 minutes at 37°C.[32a] Similar to the above
interpretation we can assign A6 and C7 (z = 11.0 µm) to the side membrane, A7, A8 and C8 (z
= 13.2 µm) to the dorsal membrane, and A9, A10, and A11 (z = 14.3 µm) to be outside and
close to the dorsal membrane (bound to extracellular EGFR). This analysis of only 25 PL spots
and in only three cells can merely provide a broad picture of the different possible localizations
of TbNP-AB in and on the cells (64% colocalized with the plasma membrane, 20% on the
extracellular side of the membrane, 12% internalized inside the cells, and 4% in the medium
outside the cells). However, it suggests that TbNP-AB conjugates are capable of specifically
recognizing EGFR without significantly hindering its internalization. Indeed, incubation of
A431 cell lines with TbNP-AB (200AB/TbNP) for 5 hours revealed clearly different staining
patterns (Figure 8) with PL signals originating from inside the cells (beyond the membranes
and around the nuclei), which is another good evidence that the binding of TbNP-AB conjugates
to the transmembrane EGFR leads to endocytosis and internalization of the entire EGFR-TbNP-
AB complexes. While our results are only a proof-of-concept and did not aim at the
investigation of intracellular signalling, the results demonstrate that TbNP-AB conjugates may
become useful for intracellular live cell bioanalysis. Deeper investigation with many more cells,
higher spatial resolution (confocal or super resolution), and at different TbNP-AB incubation
times (kinetic study) would be necessary to fully understand the dynamic cellular uptake
behaviour of EGFR-attached TbNP-AB.
19
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10.1002/chem.202002007
Chemistry - A European Journal
Figure 8. Time-gated PL imaging of EGFR on and in A431 cells that were incubated for 5
hours with 2 nM of 200AB/TbNP. The representative images show DIC transmission (left),
time-gated PL (center), and their overlay (right). PL of TbNPs is shown in green. GPE: Gates
per exposure. Additional images are shown in Figure S49 (Supplementary Information).
Supplementary Information. Full synthetic details for the ligands and conjugation
experiments, details of the spectroscopic titrations, FRET experiments, cell culture and
microscopy can be found in the supplementary information section.
Conclusion
Lanthanide-doped NPs combine photophysical advantages, such as high brightness, high
photostability, line-like spectra, long PL lifetimes, and biocompatibility, with large surfaces for
the attachment of various biomolecules. One of the most important prerequisites for
implementing such NPs into actual bioanalytical applications is a straightforward design of
functional NP-bioconjugates. In our present study, we developed a new type of ligand that can
be used for both photosensitization of Tb surface ions and bioconjugation. This simultaneous
photosensitization and bioconjugation approach resulted in extremely bright and stable TbNP
bioconjugates, which we investigated in two prototypical bioanalytical applications. Time-
resolved PL spectroscopy was used to show that streptavidin-coated TbNPs could specifically
recognize biotinylated ATTO610 dyes in aqueous solution, which resulted in efficient Tb-to-
dye FRET, demonstrating that these bright Tb surface emitters are capable of quantitatively
analyzing ligand-receptor binding in solution, which is a frequently applied concept in clinical
diagnostics. Time-gated PL microscopy was used to demonstrate the application of TbNP-
antibody conjugates for ligand-receptor binding on cell membranes and a possible
implementation into the investigation of transmembrane receptor trafficking. Different cell
lines with strong, weak, and no expression of EGFR as well as EGFR-passivated cells were
incubated with Matuzumab-functionalized TbNPs and the antibody-TbNP labeling ratio was
optimized for specific recognition of extracellular EGFR. TbNP PL images revealed the
formation of EGFR clusters and time-dependent internalization of TbNP-Matuzumab-EGFR
complexes that started after only 30 minutes of incubation and demonstrated the capability of
TbNP-antibody conjugates to be specifically endocytosed by A431 cells.
20
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10.1002/chem.202002007
Chemistry - A European Journal
The two representative applications for in-solution FRET bioassays and in-situ live cell imaging
of ligand-receptor binding and endocytosis demonstrate that surface-photosensitized
lanthanide-doped NPs can be efficiently bioconjugated and used in actual biological
applications. While UV excitation of the surface-photosensitized TbNPs will certainly limit the
penetration depth for in-vivo or tissue studies, the irradiation doses used for our live-cell time-
gated PL imaging approach are extremely low (~0.5 µJ/cm2, which is more than 106 times lower
than conventional UV irradiation doses used for cellular investigations) and the TbNPs were
found to be non-cytotoxic over at least 72 h.[26] Time-gated PL imaging for autofluorescence-
background detection, extremely high brightness, photophysical stability, and the use of very
low concentrations for high signal-to-background ratios are other specific benefits of surface-
photosensitized LnNPs. Our bifunctional ligands accomplished the highly important first step
of translating surface-photosensitized LnNPs into useful nanobioconjugates for efficient
biological recognition with bright PL signals for in-solution sensing and receptor-targeted cell
imaging, which is one of the most critical prerequisites for implementing bright and stable
lanthanide-NPs as standard PL probes into diverse clinical and bioanalytical research studies.
Acknowledgements. PolyDTech SA (Strasbourg, France) is gratefully acknowledged for the
gift of TbNPs. We thank A. Klymchenko and M. Collot for providing MemBright-640. The
French Agence Nationale de la Recherche is acknowledged for financial support (ANR project
Neutrinos n°16-CE09-0015-02). We thank the Imagerie‐Gif core facility (supported by ANR-
11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging, and ANR‐11‐IDEX‐
0003‐02/ Saclay Plant Sciences) for TEM measurements.
References
1/
a) P.D. Howes, R. Chandrawati, M. M. Stevens, Science, 2014, 346, 1247390. b) A. M.
Coto-García, E. Sotelo-González, M. T. Fernández-Argüelles, R. Pereiro, J. M. Costa-
Fernández, A. Sanz-Medel, Anal. Bioanal. Chem. 2011, 399, 29. c) N. Hildebrandt, O. Tagit.
J. Nanosci. Nanotech. 2018, 18, 6671.
21
This article is protected by copyright. All rights reserved.

10.1002/chem.202002007
Chemistry - A European Journal
2/
a) W. R. Algar, A. J. Tavares, U. J. Krull, Anal. Chim. Acta 2010, 673, 1. b) R.C. Somers,
M.G. Bawendi, D.G. Nocera, Chem. Soc. Rev. 2007, 36, 579. c) N. Hildebrandt, C. M.
Spillmann, W. R. Algar, T. Pons, M. H. Stewart, E. Oh, K. Susumu, S. A. Díaz, J. B. Delehanty,
I. L. Medintz. Chem. Rev. 2017, 117, 536. d) D. Geißler, N. Hildebrandt, Anal. Bioanal. Chem.
2016, 408, 4475. e) K.D. Wegner, N. Hildebrandt, Chem. Soc. Rev. 2015, 44, 4792. e) Z. Lia,
J.-J. Zhu, Analyst 2013, 138, 2506. f) I.V. Martynenko, A.P. Litvin, F. Purcell-Milton, A.V.
Baranov, A.V. Fedorov, Y.K. Gun'ko, J. Mater. Chem. B 2017, 5, 6701. g) J. Zhou, Y. Yang,
C. Zhang, Chem. Rev. 2015, 115, 11669.
3/
M. Cardoso Dos Santos, A. Runser, H. Bartenlian, A. M. Nonat, L.J. Charbonnière, A.
S. Klymchenko, N. Hildebrandt, A. Reisch, Chem. Mater. 2019, 31, 4034.
4/
a) K. Li, B. Liu, B. Chem. Soc. Rev. 2014, 43, 6570. b) A. Kaeser, A.P.H. Schenning,
Adv. Mater. 2010, 22, 2985.
5/
a) Y.I. Park, K. T. Lee, Y. D. Suh, T. Hyeon, Chem. Soc. Rev., 2015, 44, 1302. b) S. F
Himmelstoß, T. Hirsch, Methods Appl. Fluoresc. 2019, 7, 022002. c) F. Wang, X. Liu, Chem.
Soc. Rev. 2009, 38, 976. c) U. Resch-Genger, H.H. Gorris, Anal. Bioanal. Chem. 2017, 409,
5855. d) U. Resch-Genger, H.H. Gorris, Anal. Bioanal. Chem. 2017, 409, 5875.
6/
a) A. Escudero, A. I. Becerro, C. Carrillo-Carrión, N. O. Núñez, M. V. Zyuzin, M.
Laguna, D. González-Mancebo, M. Ocaña, W. J. Parak, Nanophotonics, 2017, 6, 881. b) D. Tu,
W. Zheng, P. Huang, X. Chen, Coord. Chem. Rev. 2019, 378, 104. c) H. Dong, S.-R. Du, X.-
Y. Zheng, G.-M. Lyu, L.-D. Sun, L.-D. Li, P.-Z. Zhang, C. Zhang, C.-H. Yan Chem. Rev. 2015,
115, 10725. d) C. Bouzigues, T. Gacoin, A. Alexandrou. ACS Nano 2011, 5, 8488. e) M. Sy,
A. Nonat, N. Hildebrandt, L.J. Charbonnière, Chem. Commun. 2016, 52, 5080−5095.
7/ a) S. Tang, M. Moayeri, Z. Chen, H. Härmä, J. Zhao, H. Hu, R.H. Purcell, S.H. Leppla, I.K.
Hewlett, Clin. Vaccine Immunol. 2009, 16, 408. b) H. Hꢀrmꢀ, T. Soukka, T. Lꢁvgren, Clin.
Chem. 2001, 47, 561. c) Y. Chen, Y.M. Chi, H.M. Wen, Z.H. Lu, Anal. Chem. 2007, 79, 960.
8/ a) A. Davies, D.J. Lewis, S.P. Watson, S.G. Thomas, Z. Pikramenou, Proc. Natl. Acad. Sci.
U.S.A. 2012, 109, 1862. b) K.L. Wong, G.L. Law, M.B. Murphy, P.A. Tanner, W.T. Wong,
P.K.S. Lam, M.H.W. Lam, Inorg. Chem. 2008, 47, 5190. c) W. Di, J. Li, N. Shirahata, Y. Sakka,
M. Willinger, N. Pinna, Nanoscale 2011, 3, 1263. d) V. Vꢀisꢀnen, H. Hꢀrmꢀ, H. Lilja, A.
Bjartell, Luminescence 2000, 15, 389. e) K. Kattel, J.Y. Park, W. Xu, H.G. Kim, E.J. Lee, B.A.
22
This article is protected by copyright. All rights reserved.

10.1002/chem.202002007
Chemistry - A European Journal
Bony, W.C. Heo, Y. Chang, T.J. Kim, J.Y. Do, K.S. Chae, Y.W. Kwak, G.H. Lee, Colloids
Surf. A 2012, 394, 85. f) Q. Zhao, Y.H. Liu, Y.F. Cao, W. Lv, Q. Yu, S.J. Liu, X.M. Liu, M.
Shi, W. Huang, Adv. Opt. Mater. 2015, 3, 233. g) W. Sun, J.B. Yu, R.P. Deng, Y. Rong, B.
Fujimoto, C.F. Wu, H.J. Zhang, D.T. Chiu, Angew. Chem., Int. Ed. 2013, 52, 11294.
9/
J.-C. G. Bünzli, Chem. Rev. 2010, 110, 2729.
10/
11/
S. I. Weissman, J. Chem. Phys. 1942, 10, 214.
a) S. Li, X. Zhang, Z. Hou, Z. Cheng, P. Ma, J. Lin, Nanoscale 2012, 4, 5619. b) J.
Zhang, C. M. Shade, D. A. Chengelis, S. Petoud, J. Am. Chem. Soc. 2007, 129, 14834. c) L. J.
Charbonnière, J.-L. Rehspringer, R. Ziessel, Y. Zimmermann, New J. Chem. 2008, 32, 1055.
12/
J. Goetz, A. Nonat, A. Diallo, M. Sy, I. Sera, A. Lecointre, C. Lefevre, C. F. Chan, K.-
L. Wong, L. J. Charbonnière, ChemPlusChem 2016, 81, 526.
13/
K. Sapsford, W.R. Algar, L. Berti, K. Boeneman Gemmill, B.J. Casey, E. Oh, M.H.
Stewart, I.L. Medintz, Chem. Rev. 2013, 113, 1904.
14/
a) A. D’Aléo, J. Xu, K. Do, G. Muller, K. N. Raymond, Helv. Chim. Acta, 2009, 92,
2439. b) A. P. S. Samuel, E. G. Moore, M. Melchior, J. Xu, K. N. Raymond, Inorg. Chem.
2008, 47, 7535.
15/
B. Horstmann, M. Korbus, T. Friedmann, C. Wolff, C. M. Thiele, F.-J. Meyer-Almes,
Bioorg. Chem. 2014, 57, 155.
16/
17/
C.M. Dundas, D. Demonte, S. Park, Appl. Microbiol. Biotech. 2013, 97, 9343.
S. Sebastian, J. Settleman, S. J. Reshkin, A. Azzariti, A. Bellizzi, A. Paradiso, Biochim.
Biophys. Acta - Rev. Canc. 2006, 1766, 120.
18/ a) I. Medintz, N. Hildebrandt, editors: FRET – Förster Resonance Energy Transfer.
From Theory to Applications”, Wiley-VCH, Germany 2014, ISBN 978-3-527-32816-1; b)
W.R. Algar, N. Hildebrandt, S.S. Vogel, I.L. Medintz, Nature Methods 2019, 16, 815.
19/
W.A. Hendrickson, A. Pahler, J. L. Smith, Y. Satow , E.A. Merritt, R. P. Phizackerley,
Proc. Nat. Acad. Sci. 1989, 86, 2190.
20/
G. Stein, E. Würzberg, J. Chem. Phys. 1975, 62, 208.
23
This article is protected by copyright. All rights reserved.

10.1002/chem.202002007
Chemistry - A European Journal
21/
22/
23/
N. Hildebrandt, K. D. Wegner, W. R. Algar, Coord. Chem. Rev. 2014, 273–274, 125.
C. Chen, B. Corry, L. Huang, N. Hildebrandt, J. Am. Chem. Soc. 2019, 141, 11123.
a) N. Hildebrandt. How to apply FRET - From experimental design to data analysis. In:
I. Medintz and N. Hildebrandt, editors: FRET – Förster Resonance Energy Transfer. From
Theory to Applications”, Wiley-VCH, Germany 2014, ISBN 978-3-527-32816-1; b) K. D.
Wegner, F. Morgner, E. Oh, R. Goswami, K. Susumu, M. H. Stewart, I. L. Medintz, N.
Hildebrandt Chem. Mater. 2014, 26, 4299. c) C. Chen, C. Midelet, S. Bhuckory, N. Hildebrandt,
M. H. V. Werts, J. Phys. Chem. C 2018, 122, 17566. d) J. Guo, X. Qiu, C. Mingoes, J.R.
Deschamps, K. Susumu, I.L. Medintz, N. Hildebrandt, ACS Nano 2019, 13, 505.
24/
a) N. Normanno, A De Luca, C. Bianco, L. Strizzi, M. Mancino, M.R. Maiello, A.
Carotenuto, G. De Feo, F. Caponigro, D.S. Salomon, Gene 2006, 366, 2. b) O.M. Fischer, S.
Hart, A. Gschwind, A. Ullrich, Biochem. Soc. Trans. 2003, 31, 1203. c) J. D. Orth, E. W.
Krueger, S. G. Weller, M. A. McNiven. Canc. Res. 2006, 66, 3603.
25/
H. S. Afsari, M. Cardoso dos Santos, S. Lindén, T. Chen, X. Qiu, P. M. P. van Bergen
en Henegouwen, T. L. Jennings, K. Susumu, I. L. Medintz, N. Hildebrandt, L. W. Miller,
Science Adv. 2016, 2, e1600265.
26/
a) M. Cardoso Dos Santos, J. Goetz, H. Bartenlian, K.-L. Wong, L.J. Charbonnière, N.
Hildebrandt, Bioconjugate Chem. 2018, 29, 1327.
27/
a) C. Kollmannsberger, M. Schittenhelm, F. Honecker, J. Tillner, D. Weber, K. Oechsle,
L. Kanz, C. Bokemeyer, Ann. Oncol. 2006, 17, 1007. b) K. Kuester, A. Kovar, C. Lüpfert, B.
Brockhaus, C. Kloft, Brit. J. Canc. 2008, 98, 900.
28/
a) M. Cardoso Dos Santos, N. Hildebrandt, Trends Anal. Chem. 2016, 84, 60-71; b) J.
Zwier, N. Hildebrandt. Time-gated FRET detection for multiplexed biosensing. In: Chris D.
Geddes, editor: Reviews in Fluorescence 2016. Springer International 2017, ISBN 978-3-319-
48259-0.
29/
a) A. Abulrob, Z. Lu, E. Baumann, D. Vobornik, R. Taylor, D. Stanimirovic, L.J.
Johnston, J. Biol. Chem. 2010, 285, 3145. b) J. Wang, X. Yu, S. V. Boriskina, B. M. Reinhard,
Nano Letters 2012, 12, 3231.
24
This article is protected by copyright. All rights reserved.

10.1002/chem.202002007
Chemistry - A European Journal
30/
31/
R. B. Lichtner, V. Schirrmacher, J. Cell. Physiol. 1990, 44, 303.
a) A. Sorkin, L. K. Goh, Exp. Cell Res. 2009, 315, 683. b) F. Zhang, S. Wang, L. Yin,
Y. Yang, Y. Guan, W. Wang, H. Xu, N. Tao, Anal. Chem. 2015, 87, 9960.
32/ a) A. D. Sorkin, L. V. Teslenko, N. N. Nikolsky, Exp. Cell Res. 1988, 175, 192. b) A.
Sorkin, E. Kornilova, L. Teslenko, A. Sorokin, N. Nikolsky, Biochim. Biophys. Acta - Mol.
Cell Res. 1989, 1011, 88.
33/
M. Collot, P. Ashokkumar, H. Anton, E. Boutant, O. Faklaris, T. Galli, Y. Mély, L.
Danglot, A.S. Klymchenko, Cell Chem. Biol. 2019, 26, 600.
25
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