Subscriber access provided by Gothenburg University Library
Article
Donor Engineering for NIR-II Molecular Fluorophores with Enhanced Fluorescent Performance Qinglai Yang, Zhubin Hu, Shoujun Zhu, Rui Ma, Huilong Ma, Zhuoran Ma, Hao Wan, Tong Zhu, Zhengyan Jiang, Weiqiang Liu, Liying Jiao, Haitao Sun, Yongye Liang, and Hongjie Dai J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10334 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society
page 1 of 26
Journal of the American Chemical Society
Donor Engineering for NIR-II Molecular Fluorophores with Enhanced Fluorescent Performance
Qinglai Yang†,||,§,‡, Zhubin Hu#,‡, Shoujun Zhuǂ,‡, Rui Ma†, Huilong Ma†, Zhuoran Maǂ, Hao Wan†,ǂ, Tong Zhuψ, Zhengyan Jiang†, Weiqiang Liu||, Liying Jiao§, Haitao Sun#,*, Yongye Liang†,*, and Hongjie Daiǂ,*
†
Department of Materials Science & Engineering, Shenzhen Key Laboratory of Printed Organic
Electronics, South University of Science & Technology of China, Shenzhen 518055, China #
State Key Laboratory of Precision Spectroscopy, School of Physics and Materials Science, East China
Normal University, Shanghai 200062, China ǂ
Department of Chemistry, Stanford University, Stanford, California 94305, USA
||
Research Center for Advanced Materials and Biotechnology, Research Institute of Tsinghua University
in Shenzhen, Shenzhen 518057, China §
Department of Chemistry, Tsinghua University, Beijing 100084, China.
ψ
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062,
China ‡
These authors contributed equally to this work.
*Correspondence should be addressed to Y.L. (
[email protected]), H.S. (
[email protected]) and H.D. (
[email protected]).
ACS Paragon Plus Environment
Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
page 2 of 26
Page 2 of 26
Journal of the American Chemical Society
KEYWORDS: molecular fluorophores, second near-infrared window, biological imaging, donor engineering. ABSTRACT:
Organic fluorophores have been widely used for biological imaging in the visible and
the first near-infrared windows. However, their application in the second near-infrared window (NIR-II, 1000-1700 nm) is still limited mainly due to low fluorescence quantum yields (QYs). Here, we explore molecular engineering on the donor unit to develop high performance NIR-II fluorophores. The fluorophores are constructed by a shielding unit-donor(s)-acceptor-donor(s)-shielding unit structure. Thiophene is introduced as the second donor connected to the shielding unit, which can increase the conjugation length and red-shift the fluorescence emission. Alkyl thiophene is employed as the first donor connected to the acceptor unit. The bulky and hydrophobic alkyl thiophene donor affords larger distortion of the conjugated backbone and less interactions with water molecules compared to other donor units studied before. The molecular fluorophore IR-FTAP with octyl thiophene as the first donor and thiophene as the second donor exhibits fluorescence emission peaked at 1048 nm with a QY of 5.3% in aqueous solutions, one of the highest for molecular NIR-II fluorophore reported so far. Superior temporal and spatial resolutions have been demonstrated with IR-FTAP fluorophore for NIR-II imaging of the blood vessels of a mouse hindlimb. 1
■ INTRODUCTION
2
Organic fluorophores have been widely used in biological imaging in the visible (400-650 nm) and the
3
first near-infrared (NIR-I, 650-900 nm) windows,1-4 as well as photoacoustic imaging and photothermal
4
therapy applications.5-10 Structural engineering on organic molecules can afford powerful tunability on
5
the optical properties of the fluorophores and versatile conjugation methods with target
6
biomolecules.11-13 Further, organic fluorophores generally exhibit good biocompatibility. For example,
ACS Paragon Plus Environment
Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
page 3 of 26
Journal of the American Chemical Society Journal of the American Chemical Society
1
CyX series14-17 and boron-dipyrromethene (BODIPY)18-19 dyes have been well studied for cellular and
2
tissue applications. Indocyanine green (ICG)20-22 and methylene blue (MB)23-25 have been approved by
3
the US Food and Drug Administration (FDA) for in vivo imaging. However, there are very few organic
4
fluorophores reported so far in the second near-infrared (NIR-II, 1000-1700 nm) window, which has
5
recently demonstrated superior imaging performance in tissue penetration and signal/noise ratio
6
compared to the visible and the NIR-I windows.26-32 It should be pointed out that very recent studies
7
revealed that some NIR-I fluorophores, such as ICG and IR800, could exhibit fluorescence tail in
8
NIR-II window.33 However, it is important to pursue new fluorophores with fluorescence peaked at
9
NIR-II window for high fluorescence quantum yield (QY).
10
Through increase of conjugation length and heteroatom substitutions, a few polymethine molecules,
11
such as IR-26, IR-1048, IR-1051, IR-1061, can emit fluorescence peaked over 1000 nm.34-37 Another
12
type of NIR-II organic fluorophores is based on donor-acceptor-donor (D-A-D) structure.38-42 Strong
13
acceptors with stabilized quinoidal structure (such as benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole, BBTD)
14
are often employed to drive the fluorescence emission to the NIR-II window. Most of these NIR-II
15
organic fluorophores are hydrophobic and cannot be used directly for biological imaging. 43-45 To make
16
them water soluble, these molecules are either encapsulated in hydrophilic polymer matrixes or
17
functionalized with polyethylene glycol (PEG) or ionic groups in the side chains. 38-42,
18
problem of NIR-II organic fluorophores is their low fluorescence quantum yields (QYs), especially in
19
aqueous solutions. A D-A-D molecule, CH1055-PEG, with BBTD as the acceptor, triphenyl amine as
20
the donor and PEG as side chains, only exhibited QY of < 0.3% in aqueous solutions. 38 In another case,
21
the QY of a quaternary ammonium salt molecule, Q-FITBBTTFI, is less than 0.1% in water.49 Such low
22
QYs present a major bottleneck for NIR-II organic fluorophores to achieve ultrafast and high resolution
23
biological imaging.
ACS Paragon Plus Environment
46-49
A vital
3
page 4 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 4 of 26
1
NIR-II organic fluorophores generally have large conjugated backbones in order to emit
2
fluorescence in the NIR-II window. As a result, the molecules tend to have strong intermolecular
3
interactions, and the excited states of the fluorophore molecules could be easily attacked and then
4
quenched. Particularly, it seems the low QYs of NIR-II organic fluorophores in aqueous media is highly
5
related to the interactions with water molecules.41,50 To address these problems, we introduced shielding
6
unit (S) to construct S-D-A-D-S type fluorophores and 2,6-dialkoxy substituted benzene or 9,9’-dialkyl
7
substituted fluorene was used as the shielding units.40-42 The side chains on the shielding units extend
8
out of the conjugated backbone and are able to reduce intermolecular interactions. Further,
9
3,4-ethoxylene dioxythiophene (EDOT) or 3-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy) (TEG) substituted
10
thiophene was employed as the donor to distort the conjugated backbones, which could also reduce
11
intermolecular interactions and energy transfer from the excited states of the molecules to the
12
surrounding water.41-42 The organic fluorophore IR-FE, which is constructed with BBTD as the
13
acceptor, EDOT as the donor and fluorene as the shielding units, exhibits fluorescence peaked at 1013
14
nm with a QY of 31% in toluene.41The analogue IR-FEP conjugated with PEG chains exhibits
15
fluorescence QY of 2.0% in aqueous solution. In contrast, the counterpart with thiophene as donor,
16
IR-FTP, exhibits a low QY of 0.02%.41
17
Donor units play important roles on the fluorescence performance of D-A-D fluorophores. They are
18
not only coupled with the acceptor units to afford low bandgap (accurately speaking, the gap between
19
the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO))
20
and fluorescence emission in the NIR-II window, but also able to significantly affect the fluorescence
21
QYs as revealed by previous studies.40-42Here, we explore donor engineering to develop high
22
performance NIR-II molecular fluorophores and reveal the relationship between the donor structures
23
and fluorescence properties. Since the photon scattering in tissue is reduced with increasing wavelength,
24
fluorescence emission at longer wavelength is favored.51-54 As a result, a thiophene ring is added as the
25
second donor next to the shielding unit to increase the conjugation length. Such thiophene insertion ACS Paragon Plus Environment
4
Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
page 5 of 26
Journal of the American Chemical Society Journal of the American Chemical Society
1
results in redshift of the absorption and fluorescence emission, but decrease of QYs. The first donor
2
connected to the acceptor is further tuned. Besides oxygen containing EDOT and TEG substituted
3
thiophene, alkyl chain substituted thiophene is also introduced as the first donor. It is found that alkyl
4
thiophene can increase the dihedral angle between acceptor and donor and hydrophobicity for improved
5
QY, but results in blue-shift of the absorption and emission spectra. The fluorophore IR-FTAP with
6
octylthiophene as the first donor and thiophene as the second donor shows the optimized performance
7
with fluorescence peaked at 1048 nm and a high QY of 5.3% in aqueous solutions. The high fluorescent
8
performance of IR-FTAP allows for ultrafast (>25 frames s-1) biological imaging in the NIR-II window
9
with superior spatial resolution for molecular fluorophores.
10
■ RESULTS AND DISCUSSION
11
Donor engineering on the molecular fluorophores
12
As illustrated in Scheme 1, the molecular fluorophores employ an S-D2-D1-A-D1-D2-S structure with
13
BBTD as the acceptor and dialkyl fluorene as the shielding unit. To increase the conjugation length for
14
redshift of fluorescence emission, a thiophene ring is inserted as the second donor (D2) connected to the
15
shielding unit. To investigate donor engineering, thiophene (T), EDOT (E), 3-TEG substituted
16
thiophene (G) and 3-alkyl (octyl) substituted thiophene (A) are used as the first donor (D1) conjugated
17
to the acceptor to afford the molecular fluorophores IR-FTXs: IR-FTT, IR-FTE, IR-FTG and IR-FTA,
18
respectively (Scheme 1). Compared with the oxygen containing EDOT and TEG thiophene, alkyl
19
thiophene is more hydrophobic and can afford larger dihedral distortion considering the bulkier CH2
20
linked on the thiophene ring. Molecular fluorophores without the second donor (IR-FXs: IR-FT, IR-FE,
21
IR-FG and IR-FA) are also included for comparison (Scheme 1).
22
The molecular fluorophores were feasibly synthesized by coupling reactions. Generally, the donor
23
units were conjugated to the shielding unit first, and then coupled to the acceptor to yield the molecules. ACS Paragon Plus Environment
5
page 6 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 6 of 26
1
The synthetic procedures and structural characterizations of the new fluorophores IR-FA, IR-FTT,
2
IR-FTE, IR-FTG, IR-FTA are detailed in Supporting Information (SI). To enable water solubility, the
3
molecular fluorophores were first functionalized with azide on the termini of the side chains and then
4
conjugated with 2 equivalences of PEG-alkyne (Mn = 600) to afford the PEGylated molecules IR-FTTP,
5
IR-FTEP, IR-FTGP, IR-FTAP (Scheme 1). The successful PEGylation was confirmed by comparing
6
the molecular weight difference before and after reaction from SEC measurements (Figure S1 and
7
Table S1). Most of the PEGylated fluorophores exhibit good water solubility. However, the PEG600
8
version of IR-FTAP is not water soluble due to the more hydrophobic nature of the alkyl chain on the
9
thiophene donor. As a result, the PEG5000 (Mn = 5000) version of IR-FTAP was prepared.
10
Scheme 1. Schematic illustration of the design of molecular fluorophores.
11 12
Frontier molecular orbitals and geometric structures
13
Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations were performed to
14
analyze the electronic properties and geometries of the molecular fluorophores at the optimally-tuned ACS Paragon Plus Environment
6
Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
page 7 of 26
Journal of the American Chemical Society Journal of the American Chemical Society
1
ωB97XD*/6-31G(d) level55-60 using the Gaussian 09 software61 (see SI for computational details).
2
Figure 1 shows the HOMOs and LUMOs of the molecular fluorophores. All the HOMOs are
3
delocalized along the conjugated backbone in the molecular fluorophores. With the added thiophene
4
donor unit, IR-FTX series show higher HOMO energy levels than IR-FX series. The LUMOs in
5
IR-FTX series are mainly localized on the D1-A-D1 core, thus the LUMO energy levels are similar to
6
those of the IR-FX series. The LUMO distribution for each fragment in the molecule was quantified
7
using Hirshfeld method by Multiwfn software62 (see Table S2). The results confirm that the proportion
8
of LUMO localized on the BBTD acceptor is the largest in the conjugated backbone. IR-FTA with
9
alkyl thiophene as D1 has the highest value of 87.8%, while IR-FTT with thiophene as D1 has the
10
lowest value of 73.1% (Table S2), indicating that the LUMO distribution is more localized on the
11
BBTD moiety of IR-FTA than that of IR-FTT. It should be noticed that there is a significant portion in
12
D2 in the IR-FTX fluorophores. The D2 portion decreases following the order: IR-FTT (1.4%) >
13
IR-FTE (0.9%) > IR-FTG (0.8%) > IR-FTA (0.4%). The extension of LUMOs can increase the
14
probability of intermolecular interactions and is not favorable for the energy maintenance of excited
15
states.
16
The optimized ground-state (S0) and first singlet excited-state (S1) geometries of the molecular
17
fluorophores are shown in Figure 2. For the S0 geometries, the dihedral angle between A and D1
18
gradually enlarges with the increase of side-chain steric hindrance on D1: 2° for IR-FTT, 42° for
19
IR-FTE, 45°for IR-FTG, and 58°for IR-FTA. IR-FTE exhibits a small dihedral angle between D1
20
and D2 due to the oxygen (EDOT) – sulfur (D2 thiophene) interaction.63-65 The dihedral angles between
21
D2 and fluorene shielding unit (S) in the IR-FTX fluorophores are similar with values of 30-33°. By
22
adding up all the three dihedral angles (A-D1, D1-D2 and D2-S), it yields the twist between shielding
23
unit and acceptor, which follows the order: IR-FTA > IR-FTG > IR-FTE > IR-FTT. The larger the
24
value, the side chains on the shielding units stretch out the conjugated backbone to a larger extent,
25
which can reduce the intermolecular interactions. For the S1 geometries, all the dihedral angles decrease ACS Paragon Plus Environment
7
page 8 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 8 of 26
1
compared to those in the S0 geometries, indicating a geometric reorganization in the excited-state to
2
allow for planarization of the π-conjugated backbone and more delocalized character of the S1 states.
3
Interestingly, IR-FTA (S0 vs S1 = 58°vs 40°), IR-FTG (45°vs 33°), and IR-FTE (42°vs 31°) exhibit
4
much larger decrease of D1-A dihedral angles than IR-FTT (2°vs 0.5°).
LUMO
HOMO
5 6
IR-FTT
-5.68 eV
-4.41 eV
IR-FTE
-5.69 eV
-4.24 eV
IR-FTG
-5.82 eV
-4.34 eV
IR-FTA
-6.20 eV
-4.50 eV
IR-FE
-5.85 eV
-4.29 eV
IR-FT
-5.86 eV
-4.46 eV
Figure 1. Calculated HOMOs and
LUMOs of the molecular fluorophores
at the
7
tuned-ωB97XD*/6-31G(d) level. The HOMO and LUMO energy levels are also presented in the figures.
8
To reduce the computational requirements, side chains on the fluorene units are replaced by methyl
9
groups. Note that the LUMO levels are obtained by subtracting the optical gap from the HOMO levels.
ACS Paragon Plus Environment
8
Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
page 9 of 26
Journal of the American Chemical Society Journal of the American Chemical Society
IR-FE
IR-FT
1 2
Figure 2. Optimized ground-state (S0) and first singlet excited state (S1) geometries of the molecular
3
fluorophores at the optimally-tuned ωB97XD*/6-31G(d) level. To reduce the computational
4
requirements, side chains on the fluorene units are replaced by methyl groups. The dihedral angles are
5
shown in the figures.
6 7
ACS Paragon Plus Environment
9
page 10 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 10 of 26
1
Optical properties of the fluorophores
2
The absorption and emission spectra of the molecular fluorophores were measured in toluene (Figure 3
3
and Figure S3). The optical properties data are summarized in Table 1 and Table S4. Compared to
4
IR-FX fluorophores, IR-FTX fluorophores exhibit substantial redshift of the absorption, which could
5
be attributed to the raising of HOMO energy levels in IR-FTX. For example, the absorption peaks of
6
IR-FTE and IR-FTA locate at 810 nm and 721 nm, while the ones of IR-FE and IR-FA locate at 767
7
nm and 680 nm, respectively. In the IR-FTX series, the absorption peaks are gradually blue-shifted
8
following the order: IR-FTT (889 nm), IR-FTE (810 nm), IR-FTG (757 nm), IR-FTA (721 nm),
9
owing to the more torsion of conjugated backbones and more localized LUMOs from IR-FTT to
10
IR-FTA. The extinction coefficient of IR-FTX decreases after PEGylation (IF-FTXP) (Table 1), which
11
could be due to the interaction of the polar ethylene glycol units (-OCH2CH2O-) of PEG chains with the
12
conjugated backbones, reducing the probability of an electronic transition from ground state S 0 to
13
excited state S1 (absorption S0-S1). The fluorescence emission spectra (excited at 808 nm) of both the
14
IR-FTX and IR-FX fluorophores are located at 900-1400 nm (Figure 3b). The IR-FTX emission
15
spectra are slightly red-shifted compared to the ones of IR-FX. For example, the emission peak of
16
IR-FTE (1052 nm) is longer than the one of IR-FE (1013 nm). As IR-FA exhibits very weak
17
absorption at 808 nm, its emission spectrum was not measured. In the IR-FTX fluorophores, the
18
emission peaks are gradually blue-shifted in the order: IR-FTT (1070 nm), IR-FTE (1052 nm),
19
IR-FTG (1036 nm), IR-FTA (1005 nm). The emission peak difference is smaller than absorption peak
20
difference, mainly due to the increasing reorganization energies from IR-FTT to IR-FTA: 0.24 eV for
21
IR-FTT, 0.35 eV for IR-FTE, 0.44 eV for IR-FTG, 0.49 eV for IR-FTA (Figure S2 and Table S3).
22
Theoretical calculations confirm that all the S1 excitations arise from the dominated HOMO to LUMO
23
transitions (Table S3). Therefore, building a hydrophobic environment around the BBTD acceptor
24
where the LUMO mainly distributes could efficiently prevent the intermolecular interactions with water,
25
reduce non-radiative energy transfer and afford a high fluorescence QY in water. From the analysis of ACS Paragon Plus Environment
10
page 11 of 26
Journal of the American Chemical Society Journal of the American Chemical Society
Page 11 of 26
1
electronic configurations (Table S3), the simulated results of E01/E10 (eV) and corresponding
2
absorption/emission peaks (nm) are 1.27/1.01, 1.45/1.08, 1.48/1.06, 1.70/1.21 and 977/1227, 855/1150,
3
839/1166, 730/1024 for IR-FTT, IR-FTE, IR-FTG, IR-FTA respectively.
1.4
Absorbance
1.2 1.0 0.8 0.6
IR-FE IR-FA IR-FTT IR-FTE IR-FTG IR-FTA
(b) Normalized Fluorescence
(a)
In Toluene
0.4 0.2 0.0
808 nm 600
1.2 1.0 0.8
800
900
1000
1100
Wavelength (nm)
(c) 1.4
700
IR-FEP IR-FTTP IR-FTEP IR-FTGP IR-FTAP
(d) Normalized Fluorescence
500
Absorbance
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
In Water
0.6 0.4 0.2 0.0 500
4
600
700
800
900
1000
1100
1.2
IR-FE QY=31% IR-FTT QY=12% IR-FTE QY=15% IR-FTG QY=17% IR-FTA QY=27%
1.0 0.8 0.6
In Toluene
0.4 0.2 0.0 900
1000
1100
1200
1300
1400
1500
Wavelength (nm) 1.2
IR-FEP QY=2.0% IR-FTTP QY=0.1% IR-FTEP QY=1.0% IR-FTGP QY=1.4% IR-FTAP QY=5.3%
1.0 0.8 0.6
In Water
0.4 0.2 0.0 900
Wavelength (nm)
1000
1100
1200
1300
1400
1500
Wavelength (nm)
5
Figure 3. (a) Absorption spectra and (b) Fluorescence emission spectra of the molecular fluorophores in
6
toluene solutions. The absorption spectra in (a) were measured with 50 mmol/L solutions. The emission
7
spectra in (b) were measured with optical density (OD) of 0.08 at 808 nm and the fluorescence was
8
normalized with IR-FE peak intensity. (c) Absorption spectra and (d) Fluorescence emission spectra of
9
the PEGylated fluorophores in aqueous solutions. The absorption spectra in (c) were measured with 150
10
mmol/L solutions. The emission spectra in (d) were measured with OD of 0.08 at 808 nm and the
11
fluorescence was normalized with IR-FTAP peak intensity. (An 808 nm laser was used for excitation
12
with exposure time of 50 ms.)
ACS Paragon Plus Environment
11
page 12 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 12 of 26
1
The fluorescence QYs (determined with emission > 900 nm) were determined with the
2
single-walled carbon nanotube (HIPCO SWCNT) as the reference fluorophore (QY = 0.40%, see SI).38,
3
66
4
IR-FTT show lower QY values of 15% and 12% in toluene, respectively. It could be due to the increase
5
of intermolecular interactions with the conjugation length increase. The emission QY of IR-FTA is 27%
6
in toluene, substantially higher than those of IR-FTG (17%), IR-FTE (15%) and IR-FTT (12%). As
7
revealed by our previous work,41 the less delocalization of the LUMO in IR-FTA could weaken
8
interactions between excited state (S1) and external stimuli to reduce non-radiative energy transfer.
9
Table 1. Optical Data of the NIR-II fluorophores. Dye ε (103 L/mol·cm) λabs(nm)
Compared to IR-FE (31%) and IR-FT (19%), the thiophene inserted fluorophores IR-FTE and
λex (nm)
Φf%
IR-FE
19
767
1013
Stokes shift (nm/cm-1) 246/3170
IR-FTT
24
889
1070
181/1900
12±0.4
IR-FTE
23
810
1052
242/2840
15±0.4
IR-FTG
18
757
1036
279/3560
17±0.2
IR-FTA
17
721
1005
284/3920
27±0.4
IR-FEP
5.7
780
1047
267/3270
2.0±0.04
IR-FTTP
7.9
895
1112
217/2180
0.10
IR-FTEP
7.1
820
1100
280/3100
1.0±0.04
IR-FTGP
5.5
784
1068
284/3390
1.4±0.03
IR-FTAP
5.0
733
1048
315/4100
5.3±0.02
31±0.3
10 11
*The data were measured in toluene solutions for IR-FE and IR-FTX series and in aqueous solutions for IR-FEP and IR-FTXP series.
12
In aqueous solution, the IR-FTXP fluorophores also exhibit significant redshifts in absorption and
13
fluorescence emission compared to the counterparts in the IR-FXP series, suggesting that the added
14
thiophene for extended conjugation can drive the absorption and emission to longer wavelength
15
effectively (Figure 3c-d). The emission peak of IR-FTTP, IR-FTEP, IR-FTGP and IR-FTAP locates
16
at 1112, 1100, 1068, 1048 nm, respectively. It follows the trend of the molecular fluorophores in toluene.
17
However, the measured fluorescence QYs of IR-FTEP (1.0%) and IR-FTGP (1.4%) are lower than
18
IR-FEP (2.0%) and IR-FGP (1.9%). IR-FTTP is similar to IR-FTP, showing little QYs in aqueous ACS Paragon Plus Environment
12
page 13 of 26
Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
1
solution. It confirms our previous finding that thiophene as a donor next to the acceptor is detrimental to
2
the fluorescence emission in aqueous solutions.41 Although IR-FTA exhibits a lower QY than IR-FE in
3
toluene, the water soluble analogue IR-FTAP exhibits a higher QY of 5.3% than the previous champion
4
IR-FEP (2.0%) in aqueous solution. We also prepared IR-FTEP and IR-FTGP with PEG5000 and find
5
that their fluorescence QYs is similar to the PEG600 ones (Figure S3e-f). It can exclude the PEG
6
molecular weight as a major contributor for the high QYs of IR-FTAP. The better maintenance of the
7
fluorescence QY from IR-FTA in toluene to IR-FTAP in water should be attributed to the hydrophobic
8
alkyl thiophene as D1. We also tested the photostability of IR-FTAP in water, phosphate buffer saline
9
(PBS), and fetal bovine serum (FBS). As shown in Figure S4, there is a small decay (< 10%) on the
10
fluorescence signal under continuous laser exposure (808 nm, 0.33 W/cm2) for over 150 minutes. In
11
contrast, the fluorescence signals of ICG decay very fast, and almost disappear after 30 minutes. It
12
indicates that IR-FTAP possesses good photostability for bio-imaging. Long pass filters with
13
wavelength >1100 nm are often applied to improve the imaging quality.38,
14
proportions of longer fluorescence emission (>1100 nm) in the molecular fluorophores are further
15
quantitatively analyzed, and the data are as followed: IR-FTEP (56%) vs IR-FEP (32%), IR-FTGP
16
(43%) vs IR-FGP (28%), IR-FTAP (33%). And the fluorescence QY (>1100 nm) of the molecular
17
fluorophores are 0.56% for IR-FTEP vs 0.64% for IR-FEP, 0.60% for IR-FTGP vs 0.54% for
18
IR-FGP, 1.7% for IR-FTAP, respectively. It is clearly that the thiophene insertion as the second donor
19
can increase the portion of fluorescence emission at longer wavelength. Such increase can compensate
20
or even outweigh the loss of fluorescence QY in the whole window. The combination of thiophene as
21
the second donor and alkyl thiophene as the first donor makes IR-FTAP to exhibit the highest >1100
22
nm QY.
66-67
As a result, the
23 24
ACS Paragon Plus Environment
13
page 14 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 14 of 26
1
Decay effects for fluorescence quantum yield
2
In general, QY = kr / kr + knr, where kr is the radiative decay rate and knr is the nonradiative decay rate.
3
68,69
4
by Einstein spontaneous emission equation kr = ƒΔE2/1.499 for a two-level system. And ƒ is the
5
dimensionless oscillator strength and ΔE is the energy gap in units of cm−1.The values of kr for the four
6
fluorophores are then calculated to be in the range of 3~4.5 x 107 s-1 (Table S3). As shown in Figure S5,
7
since the kr values are close for the four fluorophores, the suppression of knr is more important to obtain
8
high QYs. There are generally three major pathways for nonradioactive decay: (i) internal conversion
9
(kIC), (ii) intersystem crossing (kISC) from singlet excited state to triplet one, and (iii) the charge/energy
10
transfer (kqh) during the production of excited state, often induced by solvents. So the fluorescence QYs
11
are determined by the competition of these decay rates.
The kr constant is mainly determined by the electronic structures of molecules and can be calculated
12 13
Figure 4. Torsional potential of the bond (in red) bridging the central BBTD and the first donor unit
14
based on the optimized excited-state geometries of the IR-FTT, IR-FTE, IR-FTG and IR-FTA
15
molecules.
16
The internal conversion (kIC) decay rate can be greatly suppressed with a more rigid structure since
17
the rotation of functional groups contributes largely to the internal conversion processes.68,70 The 14 ACS Paragon Plus Environment
page 15 of 26
Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
1
calculated torsional potential as a function of dihedral angle between BBTD acceptor and the first
2
thiophene donor is shown in Figure 4. The steep potential energy barrier and narrow distribution of
3
torsion angles indicate that the π-conjugated skeleton of IR-FTA is more rigid than other molecules. As
4
a result, the kIC decay rate should be in the order: IR-FTA < IR-FTG < IR-FTE < IR-FTT, that are
5
consistent with the trend of torsional potential.
6
For a singlet−triplet (S−T) conversion process, kISC can be typically increased through the
7
heavy-atom substitution or significantly small singlet-triplet gap.71,72 As shown in Table S5, the S−T
8
gaps (ΔEST) of the four molecules are calculated to be around 0.9~1.0 eV. Neither the small
9
singlet-triplet gap nor heavy-atom substitution exists in the molecular systems studied herein.
Thus, a
10
large kISC value should not be expected. However, it has been pointed out that the dissolved molecular
11
oxygen could be a mediator for this decay process. To study the effects of the molecular oxygen, we
12
measured the fluorescence behaviors of IR-FTAP in aqueous solutions after continuously feeding
13
nitrogen or oxygen, respectively. As shown in Figure S6, the feeding of nitrogen to remove oxygen in
14
solutions can cause an increase of the fluorescence intensity of 13%, while the continuous feeding of
15
oxygen greatly decreases the fluorescence intensity. The T1 energies of the four molecular fluorophores
16
are all below the energy gap between triplet ground-state oxygen and singlet oxygen and the intersystem
17
crossing between S1 and T1 of the fluorophores cannot be efficient due to the relatively large ΔEST.
18
Therefore, the direct energy transfer from the S1 state of molecular fluorophore to the ground-state 3O2
19
with the subsequent generation of singlet 1O2 is more possible.72 The relatively large ΔEST gaps of the
20
four molecular fluorophores can also allow for sufficiently long singlet state lifetimes to effectively
21
contact with oxygen molecules. In addition, the activated singlet oxygen is capable of reacting with the
22
molecular fluorophores to induce oxidative effects that deteriorate the fluorophores.
23
Considering the significantly different QYs between IR-FTXP in toluene (12 ~ 27 %) and in water
24
(0.1~ 5.3 %), the quenching process owing to the existence of water should be considered as an
ACS Paragon Plus Environment
15
page 16 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 16 of 26
1
important nonradiative decay path. In order to better understand the interactions between fluorophores
2
and water molecules, the molecular dynamics (MD) simulations on the designed NIR-II fluorophores
3
were performed. Both the PEG chains and the shielding units of substituted fluorene are included in the
4
models. The simulations were initiated by solvating the geometry-optimized molecules in a cubic water
5
box of 10 Å side lengths and equilibration runs of 60 ns durations were then conducted (see
6
computational details in SI). The MD results show that both the PEG chains (in blue) and the side
7
chains of substituted fluorene (in grey) can twine around the π-conjugated skeletons to some extent. The
8
number of water molecules around the fluorophore core region (i.e., BBTD acceptor) is revealed by the
9
radial distribution functions (RDFs) of oxygen atoms in water molecules around BBTD (Figure S7a). It
10
shows that the water molecules are very likely to exist around the BBTD acceptor center when the
11
sphere radii R > 3.5 Å, although most water molecules are excluded outside the π-conjugated skeletons
12
(Figure S7b). As shown in Figure 5 and Figure S7b, the water molecules are obviously less structured
13
around the BBTD acceptor of IR-FTAP compared to that of IR-FTTP. This can be mainly attributed to
14
the existence of two octyl chains (in yellow) of IR-FTAP, favoring to excluding water molecules due to
15
their steric hindrance and hydrophobicity.
16
In addition, a computational model based on DFT calculations was also introduced to evaluate the
17
probability of water molecules presenting in the fluorophore core region (see SI). The model including a
18
water molecule and a molecular fluorophore is constructed as shown in Figure 6 (top left). The
19
interaction energy (in kcal/mol) on each position is calculated based on the equation: ΔE =
20
E(FTT…H2O) – E(FTT) – E(H2O). The larger the ΔE values, the more unstable the water molecule will
21
exist therein. The red region corresponding to lower energy indicates where the water molecule can
22
easily exist. The almost red region in IR-FTTP indicates that its surrounding of the D1-A-D1 core has a
23
great chance to interact with water molecules. As a comparison, IR-FTEP, IR-FTGP, and IR-FTAP
24
whose D1 units are substituted thiophene possess significant non-red areas, indicating water molecules
25
are more difficult to enter into the D1-A-D1 core region of these molecules than that of IR-FTTP. ACS Paragon Plus Environment
16
page 17 of 26
Journal of the American Chemical Society Journal of the American Chemical Society
Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
IR-FTAP possesses the minimum red area, suggesting that the D1-A-D1 core of IR-FTAP has the least
2
interactions with water molecules compared to other molecular fluorophores (Figure 6). This
3
computational model provides a direct schematic description of the interactions between molecular
4
fluorophore core region and a water molecule, which is in line with the MD simulations. Such
5
computations confirmed the role of the alkyl thiophene due to the obvious steric hindrance and
6
hydrophobic nature, which can account for the highest fluorescence QY of IR-FTAP in aqueous
7
solutions.
R
IR-FTTP
IR-FTAP
8 9
Figure 5. Representative schematic diagram of water molecules surrounding the BBTD acceptor of the
10
IR-FTAP and IR-FTTP. The π-skeleton of molecular fluorophore is shown in grey; the PEG chain in
11
blue; the alkyl chain in yellow; the water molecules around the BBTD center in the effective contact
12
distance (R = 6 ~ 7 Å) are shown as explicit water model. The water molecules outside the whole
13
backbone of fluorophore are shown as implicit water model (transparent water).
ACS Paragon Plus Environment
17
page 18 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
Page 18 of 26
IR-FTT/IR-FTTP
1
IR-FTE/IR-FTEP
IR-FTG/IR-FTGP
IR-FTA/IR-FTAP
2
Figure 6. Schematic illustration of the molecular fluorophores’ interactions with a water molecule. The
3
model including a water molecule and a molecular fluorophore is constructed (top left). The water
4
molecule is placed 2 Å above the BBTD plane and it is further located on the circular plane as a
5
function of radius (2Å, 4Å, 6Å) and angle (0°~360°). All the energies are calculated at the
6
ωB97XD*/6-31G (d) level of theory.
7
Further, to confirm the influence of water molecules on the excited states of the molecular
8
fluorophores. We thus performed the time-dependent density functional theory (TDDFT) calculation at
9
the ωB97XD*/6-31G(d) level on the IR-FTT molecule which is surrounded by six water molecules.
10
Interestingly, as shown in Figure S9, the water molecules do have certain contribution to the LUMO,
11
which is closely related to the excited-state property.
12 13 ACS Paragon Plus Environment
18
page 19 of 26
Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
1
Ultrafast imaging of blood vessels
2
High fluorescence QY of the NIR-II fluorophores can benefit biological imaging with longer
3
wavelength emission to reduce light scattering and shorter exposure time to increase imaging rate. The
4
high brightness of IR-FTAP allows for ultrafast NIR-II imaging (>25 frames s-1) at >1200 nm with
5
small-molecule organic fluorophore for the first time. We performed an ultrafast video (Supplement
6
Video 1) rate imaging of the mouse hindlimb with tail vein injection of IR-FTAP PBS solution.
7
Immediately after injection, the superior imaging of blood vessels of hindlimb is clearly observed from
8
the surrounding background tissue, which allows us to identify the change in blood flow within a single
9
cardiac cycle (≈118-143 ms) (Figure 7a-d). The signal to surrounding tissue ratio (S/T) (obtained from
10
the red line) is 4.9±0.3, which is higher than that of IR-FEP (3.8±0.2) with the same injection dose
11
and imaging conditions. In addition, the NIR-II fluorescence images of hindlimb at sequential long pass
12
filters (4 ms exposure time for 1100 nm LP, 20 ms exposure time for 1200 nm LP, 100 ms exposure
13
time for 1300 nm LP, respectively) were performed and the results are shown in Figure 7a, e-g. The
14
line profile of the fluorescence intensity with 1100, 1200 and 1300 nm LP filter shows vessel to
15
surrounding tissue ratio of 3.6±0.3, 4.9±0.3 and 6.7±0.4, respectively. IR-FTAP demonstrates
16
significantly higher spatial and temporal resolution than the reported NIR-II molecular fluorophores
17
CQS1000 (1000 nm LP, 200 ms exposure time) and CH1055 (1200 nm LP, 200 ms exposure time and
18
1300 nm LP, 1s exposure time) applied in biological imaging.38,
19
principal component analysis (PCA)73 was applied to observe behavior of organs/vessels that are not
20
easily seen or resolved from nearby features in the raw images. The vessels in hindlimb position, mixed
21
with vein and artery were distinguished by PCA through the group images of ultrafast video (Figure 7h).
22
All the results indicate the promising potentials of IR-FTAP as an NIR-II fluorophore for in vivo
23
imaging.
ACS Paragon Plus Environment
67
Dynamic contrast imaging by
19
page 20 of 26
(a)
(b)
IR-FTAP, 1200 nm LP, 20 ms
Page 20 of 26
IR-FTEP, 1200 nm LP, 20 ms
5 mm
(d)150
180 150
S/T=4.9±0.3
PL intensity / a.u.
PL intensity / a.u.
(c) Vessel
120 90
Tissue
60 30 0 0
50
100
150
200
250
Position / pixel
(e)
90 60 30 0 0
300
(f)
(g)
50
100
150
200
250
300
Position / pixel
IR-FTAP, 1300 nm LP, 100 ms
(h)
250 200
S/T=3.8±0.2
120
IR-FTAP, 1100 nm LP, 4 ms
PL intensity / a.u.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
vein artery
1100 nm LP 1200 nm LP 1300 nm LP
150 100 50 120
1
140
160
180
200
Position / pixel
2
Figure 7. The hindlimb vessel imaging of IR-FTAP with 1100, 1200 and 1300 nm long pass filters,
3
respectively. IR-FTAP suspended in PBS (200 uL, OD = 6 at 808 nm) was injected intravenously into
4
an six-weeks-old C57BL/6 mouse (n = 3). (a) An NIR-II fluorescence image of the left hindlimb of a
5
mouse by IR-FTAP, (b) by IR-FEP with an ultrafast frame rate of 25.6 frames per second. (c-d) The
6
line profile of the fluorescence intensity in (a) and (b). Image from IR-FTAP possesses an improved
7
S/T ration compared with IR-FEP. The S/T ratio was calculated according to the value of vessel signal
8
divided by surrounding normal tissue signal. (e-f) NIR-II fluorescence images of hindlimb at sequential
9
long pass filters (4 ms exposure time for 1100 nm LP, 100 ms exposure time for 1300 nm LP,
10
respectively). (g) The line profile of the fluorescence intensity in (a, e, f) with vessel to surrounding
11
tissue ratio 3.6±0.3, 4.9±0.3 and 6.7±0.4, respectively. (h) Time-dependent PCA images of hindlimb
12
vessels in a C57BL/6 mouse. ACS Paragon Plus Environment
20
page 21 of 26
Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
1
■ CONCLUSIONS
2
In summary, molecular engineering on the donor units is performed to develop high performance NIR-II
3
molecular fluorophores with BBTD as the acceptor and fluorene as the shielding unit. Thiophene is
4
introduced as the second donor connected to the shielding unit, which increases the conjugation length
5
of the molecular fluorophore and red-shifts the absorption and emission spectra, but results in the
6
decrease of the fluorescence QY. Alkyl thiophene is used as the first donor connected to the acceptor in
7
NIR-II molecular fluorophores. Theoretical calculations reveal that it affords larger distortion of the
8
conjugated backbone and less interactions with water molecules than EDOT or TEG thiophene donor
9
studied before. As a result, IR-FTAP with octylthiophene as the first donor and thiophene as the second
10
donor exhibits fluorescence peaked at 1048 nm with a QY of 5.3% in aqueous solutions, one of the
11
highest for molecular fluorophore reported so far. IR-FTAP can be superior to the inorganic
12
fluorophores like HIPCO SWCNT, quantum dots and rare earth nanoparticles with its straightforward
13
and well-controlled synthesis and conjugation with biomolecules.74, 26, 75-76 The QY of IR-FTAP is
14
about 13 times of HIPCO SWCNT, though it is still inferior to some bright quantum dot or rare earth
15
nanoparticle systems.77-78 Preliminary imaging studies reveal the great potential of IR-FTAP as an
16
NIR-II fluorophore with superior spatial and temporal performance. With the powerful tunability of
17
optical properties by molecular engineering, organic fluorophores can become promising probes for
18
biological imaging in the NIR-II window.
19
■ ASSOCIATED CONTENT
20
Supporting Information.
21 22 23
This material is available free of charge via the Internet at http://pubs.acs.org. General information about materials and methods, synthesis and characterizations, NMR spectra of the compounds, Figure S1-S9 and tables S1-S5, etc.
24
■ AUTHOR INFORMATION
25
Corresponding Author ACS Paragon Plus Environment
21
page 22 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
1 2 3
* Y.L. (
[email protected]), H.S. (
[email protected]), H.D. (
[email protected]).
4
Author Contributions
5 6
‡Q.Y., Z.H. and S.Z. contributed equally. All authors have given approval to the final version of the manuscript.
7 8
Notes The authors declare no competing financial interest
9
■ ACKNOWLEDGMENT
10 11 12 13 14 15 16 17 18 19 20 21
Page 22 of 26
Y.L. acknowledges financial supports from the National Science Foundation of China (21772084), "The Recruitment Program of Global Youth Experts of China", the Shenzhen Key Lab funding (ZDSYS201505291525382), the Shenzhen Technical Research funding (JSGG20160301095829250), and the Shenzhen peacock program (KQTD20140630160825828). Q.Y acknowledges financial supported by China Postdoctoral Science Foundation (2017M612752), Shenzhen Basic Research funding (JCYJ20170307151634428). H.S. thanks the National Science Foundation of China (21603074 and 11474096), “Chenguang Program” by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (16CG25) and Shanghai-International Scientific Cooperation Fund (16520721200) for funding support. This study was partially supported by grants from the Calbrain Program to H.D. Animal experiments were approved by Stanford University's administrative panel on Laboratory Animal Care. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
22 23
REFERENCES
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
1. Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W., Chem. Soc. Rev. 2013, 42, 622-661. 2. Haque, A.; Faizi, M. S. H.; Rather, J. A.; Khan, M. S., Biorg. Med. Chem. 2017, 25, 2017-2034. 3. Hong, G.; Antaris, A. L.; Dai, H., Nat. Bio. Eng. 2017, 1, 0010. 4. Xu, C.; Shi, P.; Li, M.; Ren, J.; Qu, X., Nano Research 2015, 8 (7), 2431-2444. 5. Wu, C.; Chiu, D. T., Angew. Chem. Int. Ed. 2013, 52 (11), 3086-3109. 6. Jin, Y.; Ye, F.; Zeigler, M.; Wu, C.; Chiu, D. T., Acs Nano 2011, 5 (2), 1468-1475. 7. Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J., Nat. Nanotech. 2014, 9 (3), 233-239. 8. Lyu, Y.; Xie, C.; Chechetka, S. A.; Miyako, E.; Pu, K., J. Am. Chem. Soc. 2016, 138 (29), 9049-9052. 9. Zhen, X.; Zhang, C.; Xie, C.; Miao, Q.; Lim, K. L.; Pu, K., ACS nano. 2016, 10 (6), 6400-6409. 10. Jiang, Y.; Upputuri, P. K.; Xie, C.; Lyu, Y.; Zhang, L.; Xiong, Q.; Nano Lett. 2017, 17 (8), 4964-4969. 11. Gonçalves, M. S. T., Chem. Rev. 2008, 109, 190-212. 12. Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C., Biomaterials. 2011, 32, 7127-7138. 13. Huang, S.; Peng, S.; Li, Y.; Cui, J.; Chen, H.; Wang, L., Nano Research 2015, 8 (6), 1932-1943. 14. Choi, H. S.; Gibbs, S. L.; Lee, J. H.; Kim, S. H.; Ashitate, Y.; Liu, F.; Hyun, H.; Park, G.; Xie, Y.; Bae, S., Nat. Biotechnol. 2013, 31, 148-153. 15. Zaheer, A.; Lenkinski, R. E.; Mahmood, A.; Jones, A. G.; Cantley, L. C.; Frangioni, J. V., Nat. ACS Paragon Plus Environment
22
page 23 of 26
Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Journal of the American Chemical Society Journal of the American Chemical Society
Biotechnol. 2001, 19, 1148-1154. 16. Hyun, H.; Owens, E. A.; Wada, H.; Levitz, A.; Park, G.; Park, M. H.; Frangioni, J. V.; Henary, M.; Choi, H. S., Angew. Chem. Int. Ed. 2015, 54, 8648-8652. 17. Cruz, L. J.; Que, I.; Aswendt, M.; Chan, A.; Hoehn, M.; Löwik, C.,. Nano Research 2016, 9 (5), 1276-1289. 18. Quan, L.; Liu, S.; Sun, T.; Guan, X.; Lin, W.; Xie, Z.; Huang, Y.; Wang, Y.; Jing, X., ACS. Appl. Mater. Inter. 2014, 6, 16166-16173. 19. Watanabe, H.; Ono, M.; Matsumura, K.; Yoshimura, M.; Kimura, H.; Saji, H., Mol. Imag. 2013, 12, 338-347. 20. Frangioni, J. V., Curr. Opin. Chem. Biol. 2003, 7, 626-634. 21. Troyan, S. L.; Kianzad, V.; Gibbs-Strauss, S. L.; Gioux, S.; Matsui, A.; Oketokoun, R.; Ngo, L.; Khamene, A.; Azar, F.; Frangioni, J. V., Ann. Surg. Oncol. 2009, 16, 2943-2952. 22. Vinegoni, C.; Botnaru, I.; Aikawa, E.; Calfon, M. A.; Iwamoto, Y.; Folco, E. J.; Ntziachristos, V.; Weissleder, R.; Libby, P.; Jaffer, F. A., Sci. Transl. Med. 2011, 84, 84-45. 23. Verbeek, F. P.; van der Vorst, J. R.; Schaafsma, B. E.; Swijnenburg, R.-J.; Gaarenstroom, K. N.; Elzevier, H. W.; van de Velde, C. J.; Frangioni, J. V.; Vahrmeijer, A. L., J. Urology. 2013, 190, 574-579. 24. Ashitate, Y.; Vooght, C. S.; Hutteman, M.; Oketokoun, R.; Choi, H. S.; Frangioni, J. V., Mol. Imag. 2012, 11, 7290-2011. 25. Tummers, Q. R.; Schepers, A.; Hamming, J. F.; Kievit, J.; Frangioni, J. V.; van de Velde, C. J.; Vahrmeijer, A. L., Surgery. 2015, 158, 1323-1330. 26. Welsher, K.; Liu, Z.; Sherlock, S. P.; Robinson, J. T.; Chen, Z.; Daranciang, D.; Dai, H., Nat. Nanotech. 2009, 4, 773-780. 27. Hong, G.; Wu, J. Z.; Robinson, J. T.; Wang, H.; Zhang, B.; Dai, H., Nat. Commun. 2012, 3.700. 28. Hong, G.; Lee, J. C.; Robinson, J. T.; Raaz, U.; Xie, L.; Huang, N. F.; Cooke, J. P.; Dai, H., Nat. Med. 2012, 18, 1841-1846. 29. Naczynski, D.; Tan, M.; Zevon, M.; Wall, B.; Kohl, J.; Kulesa, A.; Chen, S.; Roth, C.; Riman, R.; Moghe, P., Nat. Commun. 2013, 4, 2119 30. Dang, X.; Gu, L.; Qi, J.; Correa, S.; Zhang, G.; Belcher, A. M.; Hammond, P. T., P. Natl. Acad. Sci. USA. 2016, 113, 5179-5184. 31. Diao, S.; Hong, G.; Antaris, A. L.; Blackburn, J. L.; Cheng, K.; Cheng, Z.; Dai, H., Nano research 2015, 8 (9), 3027-3034. 32. Antaris, A. L.; Chen, H.; Diao, S.; Ma, Z.; Zhang, Z.; Zhu, S.; Wang, J.; Lozano, A. X.; Fan, Q.; Chew, L. Nat. Commun. 2017, 8, 15269. 33. Starosolski, Z.; Bhavane, R.; Ghaghada, K. B.; Vasudevan, S. A.; Kaay, A. l.; Annapragada, A. PloS One, 2017, 12(11): e0187563. 34. Cheng, K.; Cheng, Z., Fluorescent Probes. In Imaging and Visualization in The Modern Operating Room, Springer: 2015; pp 29. 35. Kanofsky, J. R.; Sima, P. D., Photochem. Photobiol. 2000, 71, 361-368. 36. Prosposito, P.; Casalboni, M.; De Matteis, F.; Quatela, A.; Glasbeek, M.; Van Veldhoven, E.; Zhang, H., J. Sol-Gel Sci. Technol. 2003, 26, 909-913. 37. Casalboni, M.; De Matteis, F.; Prosposito, P.; Quatela, A.; Sarcinelli, F., Chem. Phys. Lett. 2003, 373, 372-378. 38. Antaris, A. L.; Chen, H.; Cheng, K.; Sun, Y.; Hong, G.; Qu, C.; Diao, S.; Deng, Z.; Hu, X.; Zhang, B.; Zhang, X.; Yaghi, O. K.; Alamparambil, Z. R.; Hong, X.; Cheng, Z.; Dai, H., Nat. Mater. 2016, 15, 235-242. 39. Sun, Y.; Qu, C.; Chen, H.; He, M.; Tang, C.; Shou, K.; Hong, S.; Yang, M.; Jiang, Y.; Ding, B., Chem. Sci. 2016, 7, 6203-6207. 40. Zhu, S.; Yang, Q.; Antaris, A. L.; Yue, J.; Ma, Z.; Wang, H.; Huang, W.; Wan, H.; Wang, J.; Diao, S.; Zhang, B.; Li, X.; Zhong, Y.; Yu, K.; Hong, G.; Luo, J.; Liang, Y.; Dai, H., P. Natl. Acad. Sci. USA. 2017, 114, 962-967. ACS Paragon Plus Environment
23
page 24 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Journal of the American Chemical Society Journal of the American Chemical Society
Page 24 of 26
41. Yang, Q.; Ma, Z.; Wang, H.; Zhou, B.; Zhu, S.; Zhong, Y.; Wang, J.; Wan, H.; Antaris, A.; Ma, R.; Zhang, X.; Zhang, X.; Liu, W.; Liang, Y.; Sun, H.; Dai, H., Adv. Mater. 2017, 29. 1605497. 42. Zhang, X. D.; Wang, H.; Antaris, A. L.; Li, L.; Diao, S.; Ma, R.; Nguyen, A.; Hong, G.; Ma, Z.; Wang, J.; Liang, Y.; Dai, H., Adv. Mater. 2016, 32, 6872-6879. 43. Qian, G.; Dai, B.; Luo, M.; Yu, D.; Zhan, J.; Zhang, Z.; Ma, D.; Wang, Z. Y., Chem. Mater. 2008, 20, 6208-6216. 44. Qian, G.; Wang, Z. Y., Can. J. Chem. 2010, 88, 192-201. 45. Qian, G.; Gao, J. P.; Wang, Z. Y., Chem. Commun. 2012, 48, 6426-6428. 46. Huang, S.; Upputuri, P. K.; Liu, H.; Pramanik, M.; Wang, M., J. Mater. Chem. B. 2016, 4, 1696-1703. 47. Jana, A.; Bai, L.; Li, X.; Agren, H.; Zhao, Y., ACS. Appl. Mater. Inter. 2016, 8, 2336-2347. 48. Hong, G.; Zou, Y.; Antaris, A. L.; Diao, S.; Wu, D.; Cheng, K.; Zhang, X.; Chen, C.; Liu, B.; He, Y.; Yuan, J.; Zhang, B.; Tao, Z.; Fukunaga, C.; Dai, H., Nat. Commun. 2014, 5, 4206. 49. Woo, S. J.; Park, S.; Jeong, J. E.; Hong, Y.; Ku, M.; Kim, B. Y.; Jang, I. H.; Heo, S. C.; Wang, T.; Kim, K. H., ACS. Appl. Mater. Inter. 2016, 8, 15937-15947. 50. Singha, S.; Kim, D.; Roy, B.; Sambasivan, S.; Moon, H.; Rao, A. S.; Kim, J. Y.; Joo, T.; Park, J. W.; Rhee, Y. M., Chem. Sci. 2015, 6, 4335-4342. 51. Pansare, V. J.; Hejazi, S.; Faenza, W. J.; Prud’homme, R. K., Chem. Mater. 2012, 24 (5), 812-827. 52. Ntziachristos, V., Annu. Rev. Biomed. Eng. 2006, 8, 1-33. 53. Monici, M., Biotechnol. Ann. Rev. 2005, 11, 227-256. 54. Villa, I.; Vedda, A.; Cantarelli, I. X.; Pedroni, M.; Piccinelli, F.; Bettinelli, M.; Speghini, A.; Nano Research 2015, 8 (2), 649-665. 55. Chai, J. D.; Head Gordon, M., Phys. Chem. 2008, 10, 6615-6620. 56. Sun, H.; Autschbach, J., Chemphyschem. 2013, 14, 2450-2461. 57. Sun, H.; Autschbach, J., J. Chem. Theory. Comput. 2014, 10, 1035-1047. 58. Sun, H.; Zhang, S.; Zhong, C.; Sun, Z., J. Comput. Chem. 2016, 37, 684-693. 59. Sun, H.; Zhong, C.; Bredas, J. L., J. Chem. Theory. Comput. 2015, 11, 3851-3858. 60. Moore, B.; Sun, H.; Govind, N.; Kowalski, K.; Autschbach, J., J. Chem. Theory. Comput. 2015, 11, 3305-3320. 61. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G E.; Robb, M.A.; Cheeseman, J.R.; Montgomery Jr., J.A.; Vreven, T.; Kudin, K.N.; Burant, J.C.; Millam, J.M.; Iyengar, S.S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;Petersson, G.A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J.E.; Hratchian, H.P.; Cross, J.B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R.E.; Yazyev, O.; Austin, A.J.; Cammi, R.; Pomelli, C.; Ochterski, J.W.; Ayala, P.Y.; Morokuma, K.; Voth, G.A.; Salvador, P.; Dannenberg, J.J.; Zakrzewski, V.G.; Dapprich, S.; Daniels, A.D.; Strain, M.C.; Farkas, O.; Malick, D.K.; Rabuck, A.D.; Raghavachari, K.; Foresman, J.B.; Ortiz, J.V.; Cui, Q., Baboul, A.G., Clifford, S., Cioslowski, J., Stefanov, B B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I.; Martin, R.L.; Fox, D.J.; Keith, T.; Al-Laham, M.A.; Peng, C.Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.M.W.; Johnson, B.; Chen, W.; Wong, M.W.; Gonzalez, C. and Pople, J.A. Gaussian 09, Revision D.01, Gaussian, Inc.: Wallingford, CT, 2013. 62. Lu, T.; Chen, F., J. Comput. Chem. 2012, 33, 580-592. 63. Huang, J.; Tang, Y.; Gao, K.; Liu, F.; Guo, H.; Russell, T. P.; Yang, T.; Liang, Y.; Cheng, X.; Guo, X., Macromolecules. 2017, 50, 137-150. 64. Pelletier, M.; Brisse, F.; Cloutier, R.; Leclerc, M., Acta Crystallogr. Sect. C: Cryst. Struct. Commun. 1995, 51, 1394-1397. 65. Shi, W.; Zhao, T.; Xi, J.; Wang, D.; Shuai, Z., J. Am. Chem. Soc. 2015, 137, 12929-12938. 66. Diao, S.; Hong, G.; Robinson, J. T.; Jiao, L.; Antaris, A. L.; Wu, J. Z.; Choi, C. L.; Dai, H., J. Am. Chem. Soc. 2012, 134, 16971-16974. 67. Shou, K.; Qu, C.; Sun, Y.; Chen, H.; Chen, S.; Zhang, L.; Xu, H.; Hong, X.; Yu, A.; Cheng, Z., Adv. ACS Paragon Plus Environment
24
page 25 of 26
Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Journal of the American Chemical Society Journal of the American Chemical Society
Funct. Mater. 2017.27, 1700995 68. Peng, Q.; Yi, Y.; Shuai, Z.; Shao, J. J. Am. Chem. Soc. 2007, 129, 9333-9339. 69. Shuai, Z.; Wang, D.; Peng, Q.; Geng, H. Acc. Chem. Res. 2014, 47, 3301-3309. 70. Cooper, M.; Ebner, A.; Briggs, M.; Burrows, M.; Gardner, N.; Richardson, R.; West, R. J. Fluoresc. 2004, 14, 145-150. 71. Peceli, D.; Hu, H.; Fishman, D. A.; Webster, S.; Przhonska, O. V.; Kurdyukov, V. V.; Slominsky, Y. L.; Tolmachev, A. I.; Kachkovski, A. D.; Gerasov, A. O. J. Phys. Chem. A. 2013, 117, 2333-2346. 72. Cekli, S.; Winkel, R. W.; Alarousu, E.; Mohammed, O. F.; Schanze, K. S. Chem. Sci. 2016, 7, 3621-3631. 73. Welsher, K.; Sherlock, S. P.; Dai, H. P. Natl. Acad. Sci. USA. 2011, 108, 8943-8948. 74. Cherukuri, P.; Bachilo, S. M.; Litovsky, S. H.; Weisman, R. B. J. Am. Chem. Soc. 2004, 126, 15638. 75. Naczynski, D.; Tan, M.; Zevon, M.; Wall, B.; Kohl, J.; Kulesa, A.; Chen, S.; Roth, C.; Riman, R.; Moghe, P. Nat. Commun. 2013, 4, 2199. 76. Wang, R.; Zhou, L.; Wang, W.; Li, X.; Zhang, F. Nat. Commun. 2017, 8, 14702. 77. Hong, G.; Robinson, J. T.; Zhang, Y.; Diao, S.; Antaris, A. L.; Wang, Q.; Dai, H. Angew. Chem. Int. Ed. 2012, 124, 9956. 78. Bruns, O. T.; Bischof, T. S.; Harris, D. K.; Franke, D.; Shi, Y.; Riedemann, L.; Bartelt, A.; Jaworski, F. B.; Carr, J. A.; Rowlands, C. J. Nat. Bio. Eng. 2017, 1, 0056.
21 22
ACS Paragon Plus Environment
25
page 26 of 26
1
20 ms exposure
1.0 0.8 0.6 0.4
5 mm
5 mm S/B=4.9
0.2 0.0
2
Page 26 of 26
Table of Contents artwork
Normalized FE
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society Journal of the American Chemical Society
900
QY = 5.3% in water 1000
1100
1200
1300
1400
1500
Wavelenght /nm
3
ACS Paragon Plus Environment
26