Spectral Properties of Fluorogenic Thiophene-derived Triarylmethane Dyes

Article abstract of DOI:10.1111/php.13037

Spectral properties and fluorogenic behaviors of five novel thiophene variants of malachite green (MG), termed MGTs, were determined. Appreciable changes as a function of homologation and substitution pattern, including absorption band positions and inten

Full text of DOI:10.1111/php.13037

DR. BRUCE ARMITAGE (Orcid ID : 0000-0003-0109-1461)
Article type : Special Issue Research Article
Spectral Properties of Fluorogenic Thiophene-Derived
Triarylmethane Dyes†
Kenneth F. Stensrud,1,3 Kimberly J. Zanotti,1 Alan S. Waggoner,2,3 and Bruce A. Armitage*,1,3
Departments of Chemistry1 and Biological Sciences,2 and Molecular Biosensor and Imaging
Center,3 Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213
Corresponding author’s email: army@cmu.edu (Bruce Armitage)
†This article is part of a Special Issue celebrating Photochemistry and Photobiology's
55th Anniversary.
Abstract. Spectral properties and fluorogenic behaviors of five novel thiophene variants of
Malachite Green (MG), termed MGTs, were determined. Appreciable changes as a function
of homologation and substitution pattern, including absorption band positions and intensities
and fluorescence quantum yields were observed. In particular, the shorter wavelength y-
band absorption was found to shift over a nearly 200 nm range based on aryl group
variation, allowing fine-tuning of the excitation wavelength for these dyes. In addition, the
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doi: 10.1111/php.13037
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fluorescence intensity of some MGTs increased significantly (up to 4,600-fold) when the dye
was bound to a cognate protein partner, which is potentially useful for cell imaging studies.
Introduction
Triarylmethane salts (TAMs) comprise a unique genre of organic compounds used,
classically, as commercial dyes owing to the variety of blue and green colors (1), favorable
lightfastness traits, particularly on acrylic fibers (2), and restricted photoreactivities in
matrices of low viscosity (3). Furthermore, the near absence of radiative emission in TAMs,
stemming from rapid torsional movements of triaryl “propellers” around the central
carbenium atom, has provided an impetus for researchers to study both the ground and
excited state molecular kinetics and dynamics of these species for decades (4).
More recently, the application of TAMs has extended into the realm of molecular
biology, e.g., as enzyme inhibitors (5-6), fluorogens for RNA aptamers (7-8), and recognition
of DNA G-quadruplexes (9). Our group has exploited the fluorogenic nature of a specific
TAM, Malachite Green (MG, λmax = 427, 621 nm, λem = 652 nm) in binding studies with
fluorescence activating proteins.
We previously observed striking fluorescence
enhancements (>10,000-fold) and nanomolar binding constants (Kd) of a modified MG when
bound to a single chain variable fragment antibody (scFv) selected from a randomized library
(10).
Over the past few decades, much attention has been devoted to the development of
fluorescent probes that allow in vivo imaging (11). From this perspective, near infra-red (NIR)
emission is ideal, on account of negligible autoabsorption, autoemission, and background
scattering, as well as a deep penetrating capacity imparted by photons in this region (12-13).
The aforementioned discovery of MG aptamers has prompted us to seek out novel MG
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variants that possess NIR fluorescence maxima while preserving the absorbance band(s),
which are aligned with the primary emission bands of commonly used diode lasers.
TAMs typically exhibit two distinct absorption bands (Figure 1) (1). The primary x-
band absorption is dominated by polarization of π electron density across the rings mediated
by the dimethylamino moieties and, for the secondary y-band, an attenuated polarization
across the orthogonal, pendant ring. Electronic transitions are believed to occur from the
nonbonding molecular orbital (NBMO) to lowest unoccupied molecular orbital (LUMO) for the
x-band and highest occupied molecular orbital (HOMO) to LUMO for the y-band (14). LUMO
stabilization (and hence, red-shifted absorption/emission) is expected to be conferred by
heteroarene substitution of the pendant group, particularly by groups having improved
electron donating capacity relative to phenyl since this will provide greater stabilization of the
central carbenium ion (15).
Petit and coworkers independently corroborated this theory in their measurements of
cation electrophilicities via equilibrium constants, pKR+, in hydrolysis reactions of
triphenylmethyl (trityl) cations and heterocyclic analogs (16). Conveyed in their reports were
substantial declines of pKR+ (augmented cation stability) when 2- and 3-thiophenes replaced
one of the phenyl groups (-6.6, -3.4, -5.0 respectively). Correspondingly, they reported a
good linear fit between the determined pKR+ values and calculated Δπ bonding energies
represented as Huckel M.O. resonance integral β coefficients; the thiophene cations showed
lower overall β values than the trityl parent. They inferred, from their findings, that the
electron-rich sulfur was responsible for the sharp declines in the π bonding energy of the
trityl cation system.
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Observations consonant with this were made previously with MG derivatives bearing
ortho and para methoxy substituents on the phenyl appendage, each revealing considerable
y-band red shifts relative to MG (17). Additional reports by Nord (18-19) and Bengtsson (20-
21) illustrated y-band red-shifts for two thiophene TAMs (1 and 2).
These reports of y-band red-shifting are potentially significant in terms of biological
imaging applications. If the y-band can be repositioned near 488 nm, the dye can be excited
with widely available Argon ion lasers which are less likely to induce cellular photodamage
than occurs with shorter wavelength lasers. However, emission should still be observed in
the far red/near infra-red, corresponding to a red-shift of up to 200 nm relative to the
excitation wavelength, virtually eliminating background fluorescence due to direct excitation
of endogenous cellular fluorophores. These factors motivated our studies on thiophene-
based analogues of MG.
Herein, we report the synthesis, optical properties, and fluorogenic behaviors of
seven highly colored, thiophene-derived MGs, collectively termed MGTs, including a
terthiophene, 3, that exhibits radiative emission deep in the NIR region. Synthesis of
compounds 1 and 2 was previously reported (18-21).
Materials and Methods
All reagents were purchased from Sigma-Aldrich or Alfa Aesar and were used as received.
Products of all reactions were assessed for purity using the following instrumental
1
techniques: For H, 13C, and 19F NMR, Bruker Avance™ 300 and 500 MHz spectrometers
were utilized with samples dissolved in corresponding deuterated solvents. Low-resolution
and high-resolution masses were determined using a Thermo-Fisher LCQ ESI/APCI Ion
Trap Mass Spectrometer. UV/Vis data was obtained on a Cary Bio200 instrument using 1.5
mL quartz cuvettes. Fluorescence data were acquired on a Cary Eclipse Fluorescence
Spectrophotometer using a 10 mm quartz cell. Spectral data were processed using Origin
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6.0 software. Relative fluorescence quantum yields, ΦF, were determined in triplicate and
averaged using absorption and emission data from three independent experiments of 1 μM
samples of MGTs dissolved in glycerol at 25oC, and appliance of the following equation: Φ =
2
ΦR*(I*AR*η2)/(IR*A*ηR ), R represents the reference (Malachite Green), I integrated
fluorescence intensity, A absorption, and η solvent viscosity of the medium. The
fluorescence quantum yield of Malachite Green has been previously reported (Reference in
Table 1) and the value used as the ΦR in the above relation. Micromolar concentrations were
appropriate for maintaining absorptions at less than 0.1 a.u., thus minimizing autoabsorption
events and background interferences.
Product separation was achieved by flash
chromatography using EM Science silica gel and gradient hexanes/ethyl acetate (leuco
MGs) and methylene chloride/methanol (MGs) as eluting solvents. All reactions were
performed under ambient conditions unless otherwise stated. Full synthesis details and
spectra are provided in Supporting Information.
HL4-F104L was selected through an affinity maturation of HL4 (unpublished work by
Carol Woolford). Expression of the protein on the yeast cell surface or as a soluble protein
was as described previously (10). Microscopy images were taken on a Carl Zeiss LSM 510
Meta Confocal Microscope with a photomultiplier tube. Samples containing 1 µM dye were
excited with a 633 nm laser and a 650 nm longpass filter. Cells were imaged without
washing away unbound dye. False color appropriate for the emission wavelength was
applied using Image J software (Rasband, W.S., Image J, U.S. National Institutes of Health,
Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2010). Fluorescence emission
spectra of the dyes bound to soluble protein was obtained on a Tecan Safire2 96-well plate
reader, λex = 633 nm. Spectra were processed with Origin 6.0 software.
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Results
Synthesis
The synthesis of MGTs adhered to a relatively straightforward, two-step process that was
adapted from a previous report (Scheme 1) (19). In the initial step, temperatures above
130°C and/or reaction times >12 h generated a number of side products as evidenced by
discrete bands on a TLC plate. This phenomenon has been noted previously (19) and can
perhaps be attributed to known facile polymerizations (oxidations) of mono and
oligothiophenes at high temperatures, especially in the presence of an electron-rich metal
catalyst (22).
UV-vis studies
The spectral properties of 1-7 in assorted solvents are summarized in Table 1 and revealed
some striking trends. Our results with 1 and 2 are in agreement with previous reports
(17,19), with red-shifted y-bands, relative to MG. Notably, the y-band shifted progressively to
the red within the homologous series 1, 3 and 5, ultimately leading to near perfect overlap of
the x and y bands for terthiophene 5 (Figure 2). The ability of the electron-donating
thiophene rings to stabilize the central carbenium ion and consequent reduction in the
HOMO-LUMO energy gap for the secondary band transition likely explains this trend.
Intriguingly, 3, with sulfur atoms at the 2 and 2’ loci of the dimers, exhibited a much greater
y-band repositioning than isomer 4, which places sulfurs in the 2 and 3’ loci (Δλy = 86 nm vs
28 nm, relative to monothiophene 1). Future theoretical studies on these compounds should
help to explain these trends.
Appreciable y-band hyperchromism was observed with 3 (Δε = 24,000 M-1 cm-1) and
especially 5 (Δε = 54,400 M-1 cm-1) compared with 1 (Figure 2); along with negligible
changes displayed by the monophenes, these were congruent with previous studies that
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established a direct correlation of the extinction coefficient with the degree of π-bond
elongation in the y-direction (transverse conjugation) and/or coplanarity (23-24). Again, the
isomeric bithiophene 4 exhibits a much smaller effect (Δε = 3,400 M-1 cm-1), in contrast to the
near doubling for 3. Greater transverse conjugation in 3 aptly explains the evolution of this
phenomenon, but a decline in ring coplanarity could also contribute (sulfur electron pair vs.
C-H crowding) and would account for both the smaller red-shift and weaker hyperchromism
of 4, relative to 3.
[Insert Figure 2 here]
Motivated by the significant spectral differences between isomers 3 and 4, derivatives with a
distal ring exchange of phenyl for thiophene at the 5’ position (6) and substitution at the 4’
position (7) respectively, were synthesized and spectrally evaluated (Table 1). As
anticipated, bithiophene 3 exhibited a more red-shifted y-band than thiophene-phenyl 6 (Δλy
= 32 nm) although the effect is attenuated relative to the comparison of 1 and MG (Δλy = 43
nm), presumably due to the more remote location of the substitution relative to the central
carbenium ion. Finally, in comparing the two thiophene-phenyl isomers, 6 exhibits a
substantially greater y-band red-shift and hyperchromicity owing to the phenyl-derived
increase in the number of resonance contributors (none in 7), whereas the y-band for 7 is
much better suited for excitation at 488 nm.
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[Fluorescence studies
As mentioned previously, TAMs possess efficient non-radiative decay pathways (ΦF = 7.9 x
10-5 in methanol) (7), owing to a near barrierless excited state rotational motion of the three
arene moieties in solvents of low viscosity. In highly viscous glycerol, however, radiative
emissions can be readily detected. Compared with MG, red-shifted emission spectra were
observed for most of the MGTs, except for 2 (Table 1). The homologous series 1, 3 and 5
again exhibited a progressive red-shift (Δλem = 16, 48 and 71 nm) relative to MG. The
quantum yields for these dyes were all lower than MG, but the values again increased with
the number of thiophene rings. (Table 1 gives relative ΦF values with MG = 1.00.) Another
interesting observation was that the shift of the emission band, when calculated from the
absorption x-band, exhibits a progressive red-shift due to the insensitivity of the long-
wavelength absorption to the nature of the arene substituent, whereas the shift relative to the
absorption y-band actually gets smaller as the number of thiophene rings increases because
the absorption band red-shift is greater than the emission band red-shift. Moreover, the NIR
emission of 3 (λem = 700 nm) and 5 (λem = 723 nm) could be particularly useful for
bioimaging applications.
Interestingly, of all the MGTs, the 3’-linked monothiophene 2 exhibited the greatest
quantum yield, which was 24% greater than MG and more than 5-fold greater than isomer 1.
This observation held for the bithiophenes, where the 3’-linked isomer 4 exhibited 2.3-fold
greater fluorescence than the 2’-linked 3. Meanwhile, thiophenyl-phenyl dye 7 exhibited 60%
greater fluorescence than isomer 6.
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The excitation spectra of MGTs were consistent with the absorption spectra (Figure 3 with
MGT 6). As shown in Supporting Information, red and near-IR fluorescence is effectively
sensitized by excitation into the y-band, leading to large (>200 nm) shifts for these dyes.
Protein-Binding Studies
The MGTs were also examined in the presence of HL4-F104L protein, an scFv
antibody fragment that was previously selected to activate the fluorescence of an MG
derivative (unpublished data). We tested binding of the MGTs to this protein in two contexts,
either displayed on the surface of yeast cells or in homogeneous solution. Figure 4 shows
fluorescence of MGT 7 in the presence of scFv-expressing yeast, demonstrating that the dye
effectively labels only the surface of the cell, i.e. where the corresponding protein is
expressed.
Emission spectra were recorded for each of the MGT dyes and the parent MG in the
presence of soluble, purified HL4-F104L protein (Figure 5). Six of the seven MGT dyes are
activated by the protein, the lone exception being terthiophenyl analogue 5. (Since 5 has a
higher φf than several of the other analogues in glycerol, it is likely that the lack of
fluorescence in the presence of protein to failure of 5 to bind the protein rather than binding
without activation, although we cannot rule out the latter.) Monothiophene 2 is notable for its
comparable intensity to MG.
Fluorescence enhancement factors were determined for the MGT dyes in the
presence of soluble, purified protein (Figure 5). The enhancement factors correspond to the
ratio of the fluorescence in the presence and absence of the protein. The fluorescence
increases by 2-3 orders of magnitude in all cases, with the largest enhancement for the
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thiophenyl dyes being exhibited by 3’-thiophene 2, which also exhibited the highest quantum
yield in glycerol.
These are preliminary results and have not been corrected for possible differences in
affinity for the various dyes with this protein. In future work, we will determine the equilibrium
binding constants and fluorescence quantum yields for these new fluorogen-protein
complexes. In addition, a straightforward affinity maturation procedure can be used to create
mutant libraries of the HL4-F104L protein which are then subjected to fluorescence-activated
cell sorting to obtain higher affinity, higher quantum yield complexes (10).
Conclusion
In conclusion, five novel and two known thiophene MG derivatives were synthesized,
spectral properties were evaluated, and fluorogenic capacities were probed. Appreciable
changes in y-band absorption positions and intensities were manifest as a function of the
extent and nature of arene substitution. Furthermore, correlations between emission
wavelengths and quantum yields were found to be proportional to the degree of 2-thiophene
homologation. Substantial fluorescence enhancements of MGTs were observed when bound
to free or yeast surface expressed HL4-F104L protein, results that intimate future use as
biological imaging agents.
Acknowledgements
This work was supported by the U.S. National Institutes of Health (grant RR022241). K.J.Z.
gratefully acknowledges funding from a DOD, Air Force Office of Scientific Research,
NDSEG Fellowship 32 CFR 168a. NMR instrumentation is partially funded by NSF (CHE-
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0130903). Mass spectrometers are supported by NSF (DVI-9729351). HL4-F104L was a
generous gift from Dr. Carol Woolford and Dr. Christopher Szent-Gyorgyi.
SUPPORTING INFORMATION
Additional Supporting Information is available in the online version of this article.
References
1.
Duxbury, D. F., The Photochemistry and Photophysics of Triphenylmethane Dyes in Solid and
Liquid Media. Chem. Rev. 1993, 93 (1), 381-433.
2.
Allen, N. S.; McKellar, J. F.; Mohajerani, B., Lightfastness and Spectroscopic Properties of
Basic Triphenylmethane Dyes: Effect of the Substrate. Dyes Pigments 1980, 1, 49-57.
3.
Baptista, M.; Indig, G., Mechanism of Photobleaching of Ethyl Violet Non-covalently Bound
to Bovine Serum Albumin Chem. Commun. 1997, 1791-1792.
4.
Maruyama, Y.; Magnin, O.; Satzono, H.; Ishikawa, M., Ground- and Excited-State
Isomerization of Triphenylmethane Dyes in the Femtosecond Regime. J. Phys. Chem. A 1999, 103,
5629-5635.
5.
Liao, L. L.; Horwitz, S. B.; Huang, M. T.; Grollman, A. P.; Steward, D.; Martin, J., J. Med. Chem.
1975, 18, 117-120.
6.
Chen, C.-S.; Chiou, C.-T.; Chen, G. S.; Chen, S.-C.; Hu, C.-Y.; Chi, W.-K.; Chu, Y.-D.; Hwang, L.-
H.; Chen, P.-J.; Chen, D.-S.; Liaw, S.-H.; Chern, J.-W., Structure-Based Discovery of Triphenylmethane
Derivatives as Inhibitors of Hepatitis C Virus Helicase. J. Med. Chem. 2009, 52, 2716-2723.
This article is protected by copyright. All rights reserved.

7.
Babendure, J. R.; Adams, S. R.; Tsien, R. Y., Aptamers Switch on Fluorescence of
Triphenylmethane Dyes. J. Am. Chem. Soc. 2003, 125, 14716-14717.
8.
Kolpashchikov, D. M., Binary Malachite Green Aptamer for Fluorescent Detection of Nucleic
Acids. J. Am. Chem. Soc. 2005, 127, 12442-12443.
9.
Bhasikuttan, A.; Mohanty, J.; Pal, H., Interaction of Malachite Green with Guanine-Rich
Single-Stranded DNA: Preferential Binding to a G-Quadruplex. Angewandte Chemie International
Edition in English 2007, 46, 9305-9307.
10.
Szent-Gyorgyi, C.; Schmidt, B. F.; Creeger, Y.; Fisher, G. W.; Zakel, K. L.; Adler, S.; Fitzpatrick,
J. A.; Woolford, C. A.; Yan, Q.; Vasilev, K. V.; Berget, P. B.; Bruchez, M. P.; Jarvik, J. W.; Waggoner, A.,
Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 2007,
26, 235-240.
11.
12.
Lavis, L. D., Chemistry is Dead. Long Live Chemistry! Biochemistry 2017, 56, 5165-5170.
Ding, F.; Zhan, Y.; Lu, X.; Sun, Y., Recent Advances in Near-Iinfrared II Fluorophores for
Multifunctional Biomedical Imaging. Chem. Sci. 2018, 9, 4370-4380.
13.
Umezawa, K.; Citterio, D.; Suzuki, K., New Trends in Near-Iinfrared Fluorophores for
Bioimaging. Anal. Sci. 2014, 30, 327-349.
14.
Hallas, G.; Potts, R., J. Chem. Soc. Perk. Trans. 2 1974, Extended Conjugation in Di- and
Triarylmethane Dyes. IV. Electronic Absorption Spectra of Phenanthrene Analogs of Malachite
Green, 59-60.
15.
Mason, C. D.; Nord, F. F., Heterocyclics. XIII. Triarylmethane Dyes Containing a Thiophene
Ring. J. Org. Chem. 1951, 16, 722-727.
16.
Turnbo, R. G.; Sullivan, D. L.; Pettit, R., The Stabilities of Heteroaromatic Sulfur-Containing
Cations. J. Am. Chem. Soc. 1964, 86, 5630-5632.
This article is protected by copyright. All rights reserved.

17.
18.
Waring, D. R., Adv. Colour Sci. Technol. 1999, 1, 87-92.
Mason, C. D.; Nord, F. F., Heterocyclics. XX. The Absorption Spectra of Some Triarylmethane
Dyes Containing a Thiophene Ring. J. Org. Chem. 1952, 18, 778-785.
19.
Ghaisas, V. V.; Kane, B. J.; Nord, F. F., Studies on the Chemistry of Heterocyclics. XXXII.
Preparation and Absorption Spectra of Triarylmethane Dyes Containing a Thiophene or a
Thianaphthene Ring. J. Org. Chem. 1958, 23, 560-565.
20.
Cigen, R.; Bengtsson, G., Thiophene Analogs of Malachite Green. I. Protolytic Equilibriums
and Reaction Rate Constants of Thiophene Green in Aqueous Solutions. Acta Chem. Scand. 1962, 16,
1837-1848.
21.
Bengtsson, G., Thiophene Analogs of Malachite Green. II. Protolytic Equilibrums and
Reaction Rate Constants of 3-Thiophene Green in Aqueous Solutions. Acta Chem. Scand. 1963, 17,
2165-2172.
22.
Schwab, P. F. H.; Smith, J. R.; Michl, J., Synthesis and Properties of Molecular Rods. 2. Zig-zag
Rods. Chem. Rev. 2005, 105, 1197-1279.
23.
Lewis, G. N.; Bigeleisen, J., The Y-Bands in Absorption Spectra. J. Am. Chem. Soc. 1943, 65,
2102-2106.
24.
Murrell, J. N., The Theory of Electronic Spectra of Organic Molecules. Mehtheun & Co:
London, 1963.
25.
Baptista, M. S.; Indig, G. L., Effect of BSA Binding on Photophysical and Photochemical
Properties of Triarylmethane Dyes. J. Phys. Chem. B 1998, 102, 4678-4688.
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Table 1. Spectral data of MGTs and MG.
b,c
λx (nm)a
εx (M-1 cm-1) λy (nm)a εy (M-1 cm-1)
λ
em (nm)b
ΦF
(λem - λx) (nm)d
MGT
1
2
630
619
639
632
641
637
633
621
78,000
94,000
73,400
87,700
83,600
82,400
95,100
81,300
470
448
556
498
610
524
490
427
25,200
24,500
ND
668
653
700
665
723
693
667
652
0.23
1.24
0.50
1.15
0.83
0.52
0.83
1.00f
38
34
61
33
82
56
34
31
3
4
28,700
ND
5
6
49,400
35,500
17,800
7
MGe
a Measured in EtOH. b Measured in glycerol. c Fluorescence quantum yield relative to MG. The errors
d
e
were < 10%. Difference between excitation and emission maxima measured in glycerol. Purchased
from Sigma Aldrich. fDetermined as 0.0037 in glycerol (25).
Figure Legends
Scheme 1. General synthetic scheme for thiophene-containing triarylmethane dyes.
Figure 1. x and y band designations of Malachite Green.
Figure 2. Progressive y-band red-shifts and hyperchromism for the homologous series 1, 3, and 5
compared with MG.
Figure 3. Overlay of the excitation and emission spectra of 6, measured in glycerol.
Figure 4. Yeast cell surface displayed HL4-F104L protein stained with 1 µM MGT7. Fluorescence
enhancement for samples containing 1 µM dye and excess soluble protein was calculated by dividing
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the maximum emission signal of the protein-dye complex in PBS buffer by the corresponding signal
of the dye in buffer. λex = 633 nm.
Figure 5. Fluorescence spectra of MGTs in the presence of HL4-F104L protein, 1 μM dye, 3 μM
protein, λex = 633 nm.
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