A reversible fluorescent probe for directly monitoring protein-small molecules interaction utilizing vibration-induced emission

Article abstract of DOI:10.1016/j.dyepig.2018.12.027

The interactions between proteins and small molecules play an important role in the regulation of various cellular processes and modern drug discovery. Herein, we have developed a novel strategy for real-time monitoring the protein-ligand interaction based on vibration-induced emission (VIE) mechanism. These results demonstrated that the probe DPAC-DB could directly visualize the binding process in biotin-avidin system, which was undisturbed to other environmental stimuli, such as pH, interference species and temperature. Furthermore, the unique VIE effect caused by dramatic and reversible intramolecular vibrations enabled our strategy possible to build up the bio-logic gate. This method to efficiently control the emission of VIE-based probe through the reversible noncovalent protein-small molecules interactions opens new avenues for the development of potent protein pharmaceuticals and small molecule drugs.

Full text of DOI:10.1016/j.dyepig.2018.12.027

DyesandPigments163(2019)425–432
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A reversible fluorescent probe for directly monitoring protein-small
molecules interaction utilizing vibration-induced emission
Na Wanga, Chenqi Xina, Zheng Lia, Gaobin Zhanga, Lei Baia, Qiuyu Gonga, Chenchen Xua,
Xu Hanb, Changmin Yua,∗∗, Lin Lia,∗, Wei Huanga,b
a Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing,
211800, PR China
b Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), 127 West Youyi Road, Xi'an, 710072, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The interactions between proteins and small molecules play an important role in the regulation of various
cellular processes and modern drug discovery. Herein, we have developed a novel strategy for real-time mon-
itoring the protein-ligand interaction based on vibration-induced emission (VIE) mechanism. These results de-
monstrated that the probe DPAC-DB could directly visualize the binding process in biotin-avidin system, which
was undisturbed to other environmental stimuli, such as pH, interference species and temperature. Furthermore,
the unique VIE effect caused by dramatic and reversible intramolecular vibrations enabled our strategy possible
to build up the bio-logic gate. This method to efficiently control the emission of VIE-based probe through the
reversible noncovalent protein-small molecules interactions opens new avenues for the development of potent
protein pharmaceuticals and small molecule drugs.
Vibration-induced emission (VIE)
Biotin-avidin system
Bio-logic gate
Reversible
1. Introduction
specialized equipment. Another problem is that they are unable to
realize real-time visualization during the protein-ligand interaction
High affinity protein interactions are ubiquitous that has played a
key role in many spatiotemporal cellular structures, such as regulatory
multiprotein complexes, immunological recognition processes, and the
quaternary architecture of ligand-gated ion channels [1,2]. Amongst
them, noncovalent reversible binding between small molecules and
proteins, in particular, is of central importance in the regulation of
various cellular processes, including transcription regulations, enzy-
matic activity, protein synthesis and degradation [3,4]. Furthermore,
such interactions are also important in modern drug discovery as small
molecule drugs are designed as potent protein pharmaceuticals to alter
protein functions upon binding [5–7]. Thus, it is of substantial interest
to accurately monitor the binding affinity between biomolecules and
target proteins.
process. Fluorescence technology has been proven to be a promising
tool because of its unique advantages, such as excellent sensitivity,
simplicity and real-time visual detection of analytes both in vitro and in
vivo [15–21]. So far, fluorescence assays based on conventional spec-
troscopic manners such as fluorescence anisotropy (FA) and fluores-
cence resonance energy transfer (FRET) have also been designed to
screen interactions between proteins and ligands [22–25].
Recently, a class of specific fluorophores with unique VIE effect
have been reported by Tian's group [26–30], which intrinsically exhibit
orange-red fluorescence in the free state, but abnormally display blue
fluorescence in the constrained state. This fantastic phenomenon that
undergoes an extremely distinct fluorescence color change has been
demonstrated to be attributed to the limitation of intramolecular vi-
brations in disubstituted hydrophenazine derivatives [28]. Obviously,
this novel VIE mechanism differs from conventional photophysical or
photochemical processes in several aspects [31,32], such as distinctive
fluorescence change caused by the intrinsic change in intramolecular
planarity, large Stokes' shift up to over 250 nm, and instantaneously
fluorescence response within femtosecond scale that could capture the
Biophysical techniques such as mass spectrometry [8], micro-
biological method [9], atomic force microscopy [10], electrochemical
[11], nuclear magnetic resonance (NMR) [12], isothermal titration
calorimetry (ITC) [13] and surface plasmon resonance (SPR) [14] have
been developed to determine protein-ligand binding affinities. The
major shortcoming, however, is that these methods typically require
multistep sample processing, large amounts of sample and/or very
dynamic process, etc. Furthermore, such
a dramatic fluorescent
∗ Corresponding author.
∗∗ Corresponding author.
E-mail addresses: iamcmyu@njtech.edu.cn (C. Yu), iamlli@njtech.edu.cn (L. Li).
https://doi.org/10.1016/j.dyepig.2018.12.027
Received 8 November 2018; Received in revised form 15 December 2018; Accepted 15 December 2018
Availableonline18December2018
0143-7208/©2018ElsevierLtd.Allrightsreserved.

N. Wang et al.
DyesandPigments163(2019)425–432
Scheme 1. Schematic illustration of the ratiometric visualization of the binding process between avidin and VIE-based probe DPAC-DB.
response to environmental change is ratiometric and visible by naked
eyes, which combines with the above features enable VIE with the ca-
pacity of real-time monitoring the changed stimuli, such as environ-
mental response sensors (including viscosity and soluble cryogenic
thermometer) [29], ion detection [30], hole transport materials [33],
low-molecular-weight gelator [34] and mechanosensitive membrane
probes [35].
purchased from Sigma. Petroleum ether (PE, 60–90 °C), DCM, ethyl
acetate (EA) and methanol (MeOH) were used as eluents for flash
column chromatography with Merck silica gel (0.040–0.063). Reaction
progress was monitored by TLC on pre-coated silica plates (250 μM
thickness) and spots were visualized by UV light or iodine. Distilled
water was used throughout the experiments. Absorption spectra were
recorded using Synergy HTX microplate reader or a Shimadzu UV-3600
UV–Vis–NIR spectrophotometer. Photoluminescent spectra were re-
corded using BioTek Cytatio5 Cell Imaging Multi-Mode Reader. All the
measurements were performed at room temperature. 1H and 13C NMR
spectra were collected in CDCl3 or DMSO‑d6 using an Avance AV-500
spectrometer and Avance AV-300 spectrometer. 1H NMR coupling
constants (J) were reported in Hertz (Hz) and multiplicity was indicated
as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
dd (doublet of doublet). Mass spectra were recorded on Finnigan LCQ
mass spectrometer and Shimadzu LC-IT-TOF spectrometer.
Herein, as a proof of concept, we developed a novel probe to detect
the binding interaction between protein and small molecule based on
the VIE-active fluorogen N, N′-diphenyl-dihydrodibenzo[a,c]phenazine
(DPAC). In this study, we chose the biotin-avidin system (BAS) as the
model to evaluate the protein-ligand interaction. The capture of biotin
with avidin is a powerful tool in biology, as well as an ideal model
system for the study on interactions of high-affinity protein-ligand,
based on the strong specific binding (Ka ≈ 1015 M−1) between avidin
and biotin [36]. As shown in Scheme 1, the biotin moieties were cou-
pled to the DPAC core through a nonconjugated spacer [37]. In this
rationally designed layout, configuration change and the sequential VIE
of DPAC could be modulated when the biotin moieties bind with the
coordination site of avidin. In the absence of avidin, the free in-
tramolecular vibrations bring the hydrophenazine unit to a stable
planar state, resulting in the orange-red emission. However, upon
binding of the biotin molecules with avidin, the restriction of the in-
tramolecular vibrations in hydrophenazine unit will be achieved caused
by the steric hindrance between the N, N′-disubstituents, just like the
blocking of the butterflies wings. Thus, the fluorescence color changes
from orange-red to blue emission due to the reduced electronic con-
jugation. Based on the VIE principle, the fluorescence response of this
probe is from the intrinsic change in planarity of the molecular excited
state caused by the binding interaction between biotin and avidin,
which is essentially irrelevant to the physical formation and inter-
molecular relationships of the probes, thus enabling our strategy quite
suitable to detect the interaction process between small molecule and
its targeted protein. It is noted that such a dramatic and reversible
fluorescent ratiometric response to the interaction between protein and
small molecule could make our probe to be VIE-based bio-logic gate
with different “input”.
2.2. Synthesis and characterization
2.2.1. Synthesis of the N9,N10-diphenylphenanthrene-9,10-diamine (1)
TiCl4 (4 mL) was dropwise added through an injector to a cooled
solution (0 °C) of phenanthroquinone (5.0 g, 24.0 mmol) and aniline
(8 mL, 84.3 mmol) in toluene (150 mL). The solution was stirred at
room temperature for 12 h. The product was isolated with solvent under
reduced pressure and dissolved in THF/EtOH (1/1, v/v) (100 mL).
NaBH4 (1.0 g, 26.4 mmol) was added in portion at room temperature
and refluxed for 2 h. Then water was added, and the product was ex-
tracted with DCM. After drying with MgSO4 and removal of DCM, the
resulting precipitate was refluxed in ethanol, filtrated, washed with
ethanol and dried under vacuum to give the compound 1, a pale yellow
solid (Yield: 4.1 g, 47%). 1H NMR (300 MHz, DMSO‑d6) δ: 8.87 (d,
J = 3.0 Hz, 2H), 7.94 (d, J = 6.0 Hz, 2H), 7.66 (t, J = 9.0 Hz, 2H), 7.61
(s, 2H), 7.56 (t, J = 9.0 Hz, 2H), 7.01 (t, J = 9 Hz, 4H), 6.62 (t,
J = 9.0 Hz, 2H), 6.52 (d, J = 3 Hz, 4H).
2.2.2. Synthesis of the 9,14-diphenyl-9,14-dihydrodibenzo[a,c]phenazine
(DPAC)
To a solution of compound 1 (500 mg, 1.4 mmol) and iodo-aryl
compound (428 mg, 2.1 mmol) in trichlorobenzene (10 mL), K2CO3
(387 mg, 2.8 mmol) and Cu(OTf)2 (127 mg, 2.8 mmol) were added. The
mixture was stirred at 210 °C for 6 h. Most trichlorobenzene was re-
moved by vacuum distillation, and black residue was obtained. After
cooling to room temperature, the residue was washed with water and
extracted with dichloromethane. The residue was purified by column
chromatography (eluent: 5% dichloromethane in petroleum ether) to
2. Experimental section
2.1. Materials and instrumentation
All reagents and solvents were purchased from commercial sup-
pliers and used without further purification, unless otherwise noted.
Biotin (67896B) was purchased from Adamas and avidin (A9275) was
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N. Wang et al.
DyesandPigments163(2019)425–432
give pure product N, N′-diaryl-dihydrodibenzo[a, c]phenazines (DPAC)
(Yield:160 mg, 27%). 1H NMR (300 MHz, CDCl3) δ: 8.76 (d, J = 9 Hz,
2H), 8.16 (d, J = 6 Hz, 2H), 7.76 (s, 2H), 7.65 (d, J = 6 Hz, 2H), 7.56
(d, J = 6 Hz, 2H), 7.35 (s, 2H), 7.01 (s, 8H), 6.80 (s, 2H).
2.2.6. Synthesis
of
the
(dibenzo[a,c]phenazine-9,14-diylbis(4,1-
phenylene))bis(methylene)bis((4-
(2-oxohexahydro-1H-thieno[3,4-d]
imidazol-4-yl)butyl)carbamate) (DPAC-DB)
DPAC-OH (250 mg, 0.5 mmol), biotin (489 mg, 2.0 mmol), DPPA
(413 mg, 2.0 mmol), and Et3N (141 mg, 2.0 mmol) were dissolved with
anhydrous CH3CN (10 mL), and the solution was continuously stirred
overnight in nitrogen at 55 °C. The reaction mixture was cooled before
vacuum dry. The resulting residue was reconstituted in DCM (50 mL)
and sequentially washed with 5% aq citric acid (2 × 50 mL), water
(50 mL), saturated aq NaHCO3 (50 mL) and brine (50 mL). The organic
phase was isolated and then dried with Na2SO4. Purification by flash
chromatography (eluent: 10% CH3OH in DCM) gave DPAC-DB as a
white solid (Yield: 200 mg, 41%). 1H NMR (500 MHz, DMSO‑d6) δ: 8.95
(d, J = 5 Hz, 2H), 7.92-7.90 (m, 4H), 7.72 (t, J = 15 Hz, 2H), 7.62 (t,
J = 15 Hz, 2H), 7.44-7.42 (m, 2H), 7.14 (t, J = 10 Hz, 2H), 7.09 (d,
J = 10 Hz, 4H), 7.00 (d, J = 10 Hz, 4H), 6.43 (s, 2H), 6.38 (s, 2H), 4.80
(s, 4H), 4.26-4.25 (m, 2H), 4.09-4.07 (m, 2H), 3.06-3.02 (m, 2H), 2.93-
2.92 (m, 4H), 2.78-2.76 (m, 2H), 2.56 (d, J = 10 Hz, 2H), 1.59-1.54 (m,
2H), 1.45-1.41 (m, 6H), 1.36-1.33 (m, 4H); 13C NMR (125 MHz,
DMSO‑d6) δ: 162.73, 156.10, 143.67, 136.86, 129.55, 129.29, 128.19,
127.03, 125.81, 123.76, 116.26, 64.89, 60.91, 59.20, 55.48, 29.20,
24.50, 22.10. HRMS (m/z): calcd [M+Na]+ for C54H54N8O6S2Na:
999.3764; found, 999.3721.
2.2.3. Synthesis
of
the
4,4'-(dibenzo[a,c]phenazine-9,14-diyl)
dibenzaldehyde (DPAC-CHO)
Phosphorus oxychloride (3.5 g, 22.8 mmol) was injected into N, N′-
dimethylformamide (5.0 mL) under a nitrogen atmosphere at 0 °C, and
the mixture was vigorously stirred for 1 h to obtain the Vilsmeier re-
agent. The solution of DPAC (1.0 g, 2.3 mmol) in N, N′-di-
methylformamide (10 mL) was then added by dropwise. The mixture
was heated to 85 °C and stirred for 12 h. After cooling to room tem-
perature, the reaction mixture was poured into ice water, followed by
the adjustment of pH to neutral. The deep yellow precipitate was then
filtered and purified by column chromatography eluting with 20%
petroleum ether in dichloromethane. A light yellow product 4, 4'-(di-
benzo[a,c]phenazine-9, 14-diyl)dibenzaldehyde (DPAC-CHO) was ob-
tained (Yield: 0.9 g, 80%). 1H NMR (300 MHz, CDCl3) δ: 9.69 (s, 2H),
8.81 (d, J = 6 Hz, 2H), 8.07 (d, J = 9.0 Hz, 2H), 7.85 (dd, J1 = 3 Hz,
J2 = 6 Hz, 2H), 7.76 (t, J = 15 Hz, 2H), 7.63 (t, J = 15 Hz, 2H), 7.54 (d,
J = 9 Hz, 4H), 7.49 (dd, J1 = 3 Hz, J2 = 6 Hz, 2H), 7.01 (d, J = 9 Hz,
4H). 13C NMR (125 MHz, DMSO‑d6) δ: 190.72, 150.78, 142.89, 137.06,
131.04, 129.70, 129.18, 127.91, 127.72, 127.69, 127.56, 126.77,
123.95, 123.72, 114.72. HRMS (m/z): [M+H]+ calcd for C34H23N2O2,
491.1754; found, 491.1754.
2.3. Preparation of probes and analytes
Concentrated liquor of probe (1.0 mM) was prepared in DMSO. Test
solution of probe (2 μM) in DMSO/H2O was obtained by diluting con-
centrated liquor. All solutions was stored in 1.5 mL centrifuge tube, and
then shaken well and incubated at room temperature. Avidin stock
solution was prepared by diluting commercial avidin solid with purified
2.2.4. Synthesis
of
the
(dibenzo[a,c]phenazine-9,14-diylbis(4,1-
phenylene))dimethanol (DPAC-OH)
DPAC-CHO (1.0 g, 2.0 mmol) was dissolved in 30 mL methanol, and
sodium borohydride (0.8 g, 21 mmol) was then added in an ice bath.
The mixture was stirred overnight at room temperature. The reaction
mixture was poured into ice water, followed by the filtration of beige
precipitate to give the DPAC-OH (Yield: 0.9 g, 89%). 1H NMR
(300 MHz, DMSO‑d6) δ: 8.92 (d, J = 6.0 Hz, 2H), 7.96 (d, J = 6.0 Hz,
2H), 7.88 (s, 2H), 7.69 (s, 2H), 7.59 (s, 2H), 7.40 (s, 2H), 7.03 (m, 8H),
4.95 (s, 2H), 4.30 (d, J = 3.0 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6) δ:
145.95, 143.88, 136.76, 135.57, 129.41, 128.82, 128.24, 128.12,
127.37, 127.12, 126.71, 125.46, 123.74, 123.55, 116.91, 62.36. HRMS
(m/z): [M+H]+ calcd for C34H27N2O2, 495.2067; found, 495.2063.
water with the concentration of 10 μg μL−1
.
2.4. The spectroscopic testing procedures
The equilibrium and kinetics of the reaction between avidin and
biotin were investigated at different feed avidin concentration with
DPAC-SB/DPAC-DB (Final concentration: 2 μM) and the experiments
were carried out at 298 K and incubation time of 1 min. For the effect of
avidin concentration on avidin-biotin interaction, 1.5 mL centrifuge
tube containing a uniform DPAC-SB/DPAC-DB concentration (2 μM)
and a variable avidin concentration were incubated at 298 K for 1 min,
which was then measured at 355 nm excitation and 450 nm emission
wavelength (10 nm slit for standard experiments). The effect of pH on
avidin-biotin interaction was investigated at different pH values (from
4.0 to 8.0). The fluorescence emission spectrum of the probe DPAC-DB
(2 μM) and the DPAC-DB (2 μM) with avidin (1.03 μg μL−1) were
measured after incubating for 1 min in various pH solutions In all the
measurements, the effect of dilution was corrected being repeated each
measurements by triplicate and the mean and standard deviation were
calculated.
2.2.5. Synthesis of the 4-(14-(4-(hydroxymethyl)phenyl)dibenzo[a,c]
phenazin-9(14H)- yl)benzyl(4-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-
4-yl)butyl)carbamate (DPAC-SB)
DPAC-OH (250 mg, 0.5 mmol), biotin (244 mg, 1.0 mmol), DPPA
(206 mg, 1.0 mmol), and Et3N (71 mg, 1.0 mmol) were dissolved with
anhydrous CH3CN (10 mL), and the solution was continuously stirred
overnight in nitrogen at 55 °C. The reaction mixture was cooled before
vacuum dry. The resulting residue was reconstituted in DCM (50 mL)
and sequentially washed with 5% aq citric acid (2 × 50 mL), water
(50 mL), saturated aq NaHCO3 (50 mL) and brine (50 mL). The organic
phase was isolated and then dried with Na2SO4. Purification by flash
chromatography (eluent: 5% CH3OH in DCM) gave DPAC-SB as a white
solid (Yield: 140 mg, 38%). 1H NMR (500 MHz, DMSO‑d6) δ: 8.94 (d,
J = 10 Hz, 2H), 7.94-7.90 (m, 4H), 7.72-7.68 (m, 2H), 7.64-7.57 (m,
2H), 7.42-7.40 (m, 2H), 7.13 (t, J = 15 Hz, 1H), 7.07-6.98 (m, 8H),
6.42 (s, 1H), 6.36 (s, 1H), 4.81 (s, 2H), 4.31 (s, 2H), 4.28-4.25 (m, 1H),
4.10-4.06 (m, 1H), 3.05-3.03 (m, 1H), 2.95-2.90 (m, 2H), 2.79-2.75 (m,
1H), 2.59-2.53 (m, 1H), 1.60-1.54 (m, 1H), 1.45-1.41 (m, 3H), 1.39-
1.34 (m, 2H); 13C NMR (125 MHz, DMSO‑d6) δ: 162.74, 156.11,
146.74, 145.75, 137.12, 135.68, 129.28, 128.21, 127.49, 126.91,
123.89, 123.74, 116.24, 64.89, 62.40, 60.90, 59.19, 55.49, 29.83,
27.96, 25.77. HRMS (m/z): calcd [M+Na]+ for C44H40N5O4SNa:
758.2801; found, 758.2792.
2.5. The method for determining the limit of detection (DPAC-DB)
First the calibration curve was obtained from the plot of fluores-
cence intensity ratio (I450/I600), as a function of the avidin concentra-
tion. The regression curve equation was then obtained for the lower
concentration part.
The detection limit = 3 × S.D. / k
Where k is the slope of the curve equation, and S.D. represents the
standard deviation for the probe DPAC-DB solution's fluorescence in-
tensity ratio (I450/I600) in the absence of avidin [38].
I
450/I600 = 0.65442 + 14.1773[avidin] (R2 = 0.9928)
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DyesandPigments163(2019)425–432
LOD = 3 × 0.0250 / 14.1773 = 5.3 ng μL−1
subscript s stands for the sample, Φ is the fluorescence quantum yield, A
is the absorbance of the solution, n is the refractive index of the solvent,
and F is fluorescence intensity of the solution at the excitation wave-
length. The absorption maximum was kept during 0.02–0.05 [40].
Thus, the fluorescence quantum yields of DPAC-DB in the absence and
presence of avidin at 298 K in DMSO/H2O mixtures (5/5, v/v, 2.0 μM,
2.6. The method for determining the reversibility (DPAC-DB)
Fluorescence intensity of DPAC-DB (2 μM) was investigated upon
the alternate addition of avidin/biotin (Cycle 0: 0 μM, 0.5: avidin
0.26 μg μL−1, 1: biotin 45 μM, 1.5: avidin 0.61 μg μL−1, 2: biotin 80 μM,
2.5: 0.65 μg μL−1, 3: 150 μM) in DMSO/water mixture (5/5, v/v).
λ
ex = 355 nm) were calculated to be 1.4% and 21.6%, respectively. The
fluorescence quantum yields of DPAC-SB in the absence and presence
of avidin at 298 K in DMSO/H2O mixtures (4/6, v/v, 2.0 μM,
λ
ex = 355 nm) were calculated to be 1.8% and 7.8%, respectively.
2.7. The SEM/TEM of DPAC-DB
3. Results and discussion
Prior to the TEM, SEM, the samples of DPAC-DB (2.0 μM), avidin
(1.03 μg μL−1), and DPAC-DB (2.0 μM) incubated with avidin
(1.03 μg μL−1) in the DMSO/H2O (5/5, v/v) were dropping onto the
carbon-coated carbon support copper grids, Si, respectively, and then
dried at 40 °C. TEM was operated at an acceleration voltage of 120 kV
(JEM-1400). SEM images were obtained by using JSM-7800.
3.1. Design and synthesis
In this study, two biotin moieties were respectively coupled to the
“wings” of DPAC core through nonconjugated linker, namely DPAC-
DB. As a comparison, another probe named DPAC-SB is further de-
signed by incorporating one biotin moiety to the DPAC core as shown in
Fig. 1. Their characterizations were confirmed by NMR and LCMS.
2.8. Determination of the affinity constant (Ka) of avidin-DPAC-DB
binding
3.2. Photophysical properties
The affinity constant (Ka) of DPAC-DB with avidin in DMSO/H2O
solution (5/5, v/v) was determined [39]. The fluorescence intensity
ratio (I450/I600) was obtained by changing the concentration of DPAC-
DB from 0.1 nM to 50 μM in the presence of avidin (0.13 μg μL−1).
When the ratio was plotted against the concentration of DPAC-DB, a
curve was obtained. The EC50 was calculated to be 48.05 nM, thus,
First, the photophysical properties of DPAC-SB and DPAC-DB were
studied in organic solvents with various polarities. As depicted in Fig. 2,
both of DPAC-SB and DPAC-DB exhibited a maximum absorption at
around 350 nm, which is virtually insensitive to the solvent polarity.
Notably, the fluorescence spectra of DPAC-SB displayed a main orange-
red emission around 600 nm and a negligible blue emission in various
solvents with extremely large Stokes’ shift (∼250 nm). However, the
main emission peaks of DPAC-DB appeared in blue band around
450–470 nm in less polar solvents, while with the polarity of the sol-
vents increasing, it gradually showed red shifted (∼600 nm), which is
due to the reduced solubility of DPAC-DB in less polar solvents after the
modification of two biotin moieties (see Fig. 2D).
Ka = 1/EC50 = 0.21 × 108 M−1
.
2.9. Quantum yield measurement
The fluorescence quantum yields were measured in DMSO by using
quinine sulfate at 365 nm (ФF = 56%). as a standard. The fluorescence
quantum yield of DPAC-DB was calculated in terms of the following
equation (eq (1)):
We could control the vibration of the phenazine unit by simply
changing the portions between the good solvent (e.g. DMSO) and poor
solvent (e.g. H2O) to achieve the obvious emission color-tunability.
According to VIE mechanism, the phenazine unit vibration would be
restricted as probe molecules aggregate in the high water fraction,
ns2
F
F
r
Ar (
As (
)
)
s
r
s
=
S
r
nr2
(1)
Where the subscript r stands for the reference molecule and
Fig. 1. Synthetic routes for DPAC-SB and DPAC-DB. Reagents and conditions. a) TiCl4, toluene 12 h; NaBH4, THF/EtOH (1/1, v/v) 2 h; 47%; b) Cu(OTf)2, K2CO3,
trichlorobenzene, 6 h, 210 °C, 27%; c) POCl3, DMF, 12 h, 85 °C, 80%; d) NaBH4, MeOH, overnight, 89%; e) biotin (1.0 mmol), DPPA (1.0 mmol), Et3N (1.0 mmol)
DPPA, Et3N, 55 °C, overnight, 38%; f) biotin (2.0 mmol), DPPA (2.0 mmol), Et3N (2.0 mmol) DPPA, Et3N, 55 °C, overnight, 41%.
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Fig. 2. The absorption spectra of DPAC-SB (A) and DPAC-DB (B) in various solvents. The fluorescent spectra of DPAC-SB (C) and DPAC-DB (D) in various solvents
(10 μM, λex = 355 nm).
subsequently resulting in the blue-shift of fluorescence emission. As
shown in Fig. S1, the fluorescence spectrum of DPAC-SB and DPAC-DB
were investigated with increasing proportion of water in DMSO at the
excitation of 355 nm. The fluorescence intensities in both colors showed
weakly changes when the water content was increased from 0 to 70 vol
%. However, an obvious blue emission band around 450 nm was ob-
served with a further increase of the water fraction (fw). Thus, to
completely dissolve the probes and ensure a strong ratiometric color
change, we next detect the biotin-avidin interaction in the H2O/DMSO
mixture below fw = 70% (60% for DPAC-SB and 50% for DPAC-DB).
avidin and reached a plateau within seconds due to the high binding
affinity between avidin and biotin moiety (Fig. S3). To further de-
monstrate the VIE response, we have performed the negative control
experiments with the DPAC-OH (no biotin moieties). As shown in Fig.
S4, due to the lack of binding with avidin protein, DPAC-OH showed
negligible fluorescence changes in comparison with DPAC-DB.
Generally, the interaction between protein and ligands was affected
by different pH values. We next measured the fluorescence signal
changes of DPAC-DB response to avidin over a wide pH range of
4.0–8.0. Due to the emission arises intrinsically from the intramolecular
conformational transitions, the fluorescence intensity of DPAC-DB itself
was completely irrelevant to the changes of pH values, which is shown
in Fig. S5. Meanwhile, significant fluorescence intensity changes were
still observed after binding with avidin. In addition, slightly enhanced
fluorescence ratio I450/I600 of the probe was observed with the increase
of pH values, indicating the binding capacity between avidin and biotin
was increased from pH 4.0 to 8.0, which is consistent with previous
studies [41]. Furthermore, the excellent anti-interference performance
of DPAC-DB that interacted with/without avidin was also observed in
other potentially biological interference species, including ions, amino
acids and other nontargeted proteins (Fig. S6). Notably, the thermal
stability in the biotin-avidin system could be easily performed by de-
tecting the fluorescence intensity of DPAC-DB upon addition of avidin
in different temperature. As shown in Fig. S7, the remarkable fluor-
escent intensity remained from 0 to 65 °C, while reduced vastly up to
75 °C, which indicated the interaction between biotin and avidin was
disrupted at such high temperature [42]. These above results clearly
demonstrated that this VIE-based ratiometric system could be suitable
to conveniently and accurately monitor the binding process between
biotin and avidin.
3.3. The principle for spectral response of DPAC-DB with avidin
The principle for spectral response of DPAC-DB with different
concentrations of avidin was first carried out. As shown in Fig. 3A, the
UV–vis absorption peak around 350 nm remained unchanged upon
addition of avidin. However, a significant turn on fluorescence intensity
at 450 nm appeared and enhanced gradually accompanied by further
additions of avidin (Fig. 3B). Correspondingly, the changed ratio I450
/
I600 of the probe showed a good linear relationship with the con-
centration of avidin ranged from 0 to 1.03 μg μL−1 and the response
limit of avidin was calculated to be 5.3 ng μL−1 (Fig. 3C). In addition,
the binding process between biotin and avidin was also directly vi-
sualized by fluorescence color changes. As shown in Fig. 3D, the color
of probe DPAC-DB could pass from orange-red to blue through the
white-light emission region with corresponding chromaticity co-
ordinates (CIE) coordinates changing from (0.4, 0.38) to (0.2, 0.15) as
the avidin concentration increased. Furthermore, the same fluorescence
color changes could be observed by naked eyes with a handheld UV
lamp (365 nm) (Fig. 3E). And, more remarkable, another probe DPAC-
SB exhibited the same performance in the response to avidin (Fig. S2),
which demonstrated the diversity in our designed probes based on VIE
mechanism. However, due to only one binding site for the DPAC-SB
with avidin, the probe still has weak vibrational freedom, and hence
exhibiting relatively less fluorescence change upon avidin binding. It is
noted that both of DPAC-DB and DPAC-SB showed a rapid response for
3.4. The response mechanism of DPAC-DB with avidin
Herein, in order to directly visualize the interaction between avidin
and biotin, we introduced the VIE-based fluorophore as the indicator.
The proposed mechanism of DPAC-DB response to avidin is that the
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DyesandPigments163(2019)425–432
Fig. 3. (A) The absorption spectra of DPAC-
DB (2.0 μM) in DMSO/H2O solution without/
with avidin (1.03 μg μL−1); (B) The fluores-
cence spectra of DPAC-DB (2.0 μM) with
0–1.29 μg μL−1 of avidin in DMSO/H2O
mixture (λex = 355 nm); (C) Linear fit of
fluorescence intensity ratio (I450/I600
) of
DPAC-DB (2.0 μM) versus avidin concentra-
tion (0–1.29 μg μL−1); (D) The corresponding
chromaticity coordinates (CIE) of DPAC-DB
(2.0 μM)
in
DMSO/H2O
solution
(λex = 355 nm) with avidin (0–1.29 μg μL−1);
(E) The corresponding fluorescence photo-
graphs of DPAC-DB (2.0 μM) in DMSO/H2O
mixture with avidin (0–1.29 μg μL−1, from
left to right) under 365 nm UV light illumi-
nation.
biotin moieties coupled on the “wings” of DPAC core specifically bind
with avidin, subsequently resulting in the restriction of the in-
tramolecular vibrations in hydrophenazine unit. It is noted that there
are four identical subunits for each avidin, which could crosslink with
DPAC-DB and thus tends to further aggregate as depicted in Scheme 1.
We next performed the scanning electron microscope (SEM) and
transmission electron microscope (TEM) analysis to explore the detail
mechanism. As shown in Fig. S8, both of the SEM and TEM images
revealed that nanoscopic aggregates were formed in the solution of
DPAC-DB containing avidin (8.0 equiv.), while no aggregation was
observed in DPAC-DB solution and avidin solution, respectively.
Moreover, the above hypothesis could be further proved by the time-
resolved experiments as shown in Figure S9 and Table S1. After adding
the avidin into the DPAC-DB solution, the emission monitored at
450 nm exhibited a prolonged decay from 2.36 to 9.47 ns, manifesting
the suppression of the vibrational motions of DPAC core by the inter-
action between biotin moieties and avidin. The prolonged decay time of
the red emission at 600 nm correlated well with the observation from
the blue region. Thus, the fluorescence changes could be observed by
the suppression of the vibration along NeN axis in DPAC-DB even if
there were only the biotin-avidin interactions without aggregation of
DPAC-DB molecules [40].
anomalous photophysical phenomenon in VIE-based fluorophores is
caused by the dramatic and reversible intramolecular vibrations, which
makes our probe possible to build the bio-logic gate. The affinity con-
stant between DPAC-DB and avidin obtained by measuring fluores-
cence intensity ratio (I450/I600) of DPAC-DB response to avidin was
calculated to be 0.21 × 108 M−1 (Fig. S10). The equilibrium constant
value was very small in comparison with the affinity constant of the
avidin-biotin binding (Ka ≈ 1015 M−1) [36], which is possibly due to
the labeling with DPAC and introduction of DMSO solvent [43]. Thus, a
biotin elution assay can be achieved by the competition reaction of free
biotin and DPAC-DB with the limited binding sites of avidin [44]. As
shown in Fig. 4A, upon addition of avidin, the fluorescence emission
was changed from orange-red to blue with a remarkable increase in the
fluorescence intensity around 450 nm. Notably, as excess free biotin
molecules was added to the solution, the fluorescence spectra of the
solution recovered to that of initial DPAC-DB. Such reversible change
could be attributed to competition between the free biotin and DPAC-
DB with avidin. Several cycles were conducted, and interestingly, this
probe exhibited cycling stability without obvious changes in fluores-
cence ratio I450/I600 during the state transition (Fig. 4B). Thus, de-
pending on the two biological inputs (avidin and biotin), this probe
DPAC-DB could switch between two different fluorescence emission
states, orange-red and blue, which could display “Write-Read-Erase-
Read” behavior in binary logic. As shown in Table S2, in this system,
the sequential logic operations are represented by two inputs: InA
(avidin input) and InB (biotin input) as a function of a memory element.
On the other hand, the orange-red emission in initial DPAC-DB solution
3.5. The reversible response and molecular logic gates
Recently, a significant development has been achieved in the bio-
logical system which behaves as molecular logic gates. In principle, the
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N. Wang et al.
DyesandPigments163(2019)425–432
Fig. 4. Fluorescence spectra of DPAC-DB
(black line), DPAC-DB with of avidin (red line)
and re-adding biotin (blue line) in DMSO/
water mixture (2.0 μM,
λex = 355 nm).
Repetitive write-erase cycles based on fluor-
escence emission of DPAC-DB obtained in the
presence of avidin (Off state) and biotin (On
state). (B) Fluorescence intensity of DPAC-DB
(2.0 μM) upon the alternate addition of avdin/
biotin (Cycle 0: 0 μM, 0.5: avidin 0.26 μg μL−1
,
1: biotin 45 μM, 1.5: avidin 0.61 μg μL−1, 2:
biotin 80 μM, 2.5: 0.65 μg μL−1, 3: 150 μM) in
DMSO/water mixture. (For interpretation of
the references to color in this figure legend,
the reader is referred to the Web version of this
article.)
was considered as “write” and “logic state 0“. Upon addition of avidin
(InA = 1), the blue-shift emission was “read” out and saved as “logic
state 1“. Finally, the blue fluorescence emission was “erased” by adding
biotin (InB = 1).
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In summary, we have developed a novel strategy for monitoring the
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strategy possible to build the bio-logic gate. However, there are still
several obstacles to be suffered in this study, such as the poor solubility
of the probe in the water, the addition of organic solvent and chemical
modification of small molecule ligand. Future research will be focused
on these issues. Notwithstanding, we believe our strategy will provide a
promising way to investigate the protein-ligand interaction for the de-
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This work was financially supported by the National Natural Science
Foundation of China (81672508, 61505076), Jiangsu Provincial
Foundation for Distinguished Young Scholars (BK20170041), China-
Sweden Joint Mobility Project (51811530018), Postgraduate Research
& Practice Innovation Program of Jiangsu Province and Natural Science
Foundation of Guangdong Province (2017A030313299). We also
warmly thank Prof. He Tian from East China University of Science and
Technology for donating sample and experimental support, and Prof.
Yao Shao Q. from National University of Singapore for insightful
comments.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
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