A novel hydrazide Schiff base self-assembled nanoprobe for selective detection of human serum albumin and its applications in renal disease surveillance

Article abstract of DOI:10.1039/d0tb01411g

Human serum albumin (HSA) is considered as a biomarker for the early diagnosis of renal disease, therefore identifying and detecting HSA in biological fluids (especially urine) with an easy method is of great importance. Herein, we report a novel hydrazide Schiff base fluorescent probe N′-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)pyrazine-2-carbohydrazide (NPC), which self-assembled into nanoparticles in aqueous solution. Based on disassembly-induced emission and the site-specific recognition mechanism, the binding of NPC with HSA resulted in a fluorescence "turn-on"response. Probe NPC exhibited superior selectivity and sensitivity toward HSA with a detection limit of 0.59 mg L-1 in PBS and 0.56 mg L-1 in the urine sample. The site-binding mechanism of NPC with HSA was explored by fluorescence quenching study, Job's plot analysis, HSA destruction, site marker displacement and molecular docking. Fluorescence imaging of HSA in MCF-7 cells was achieved by using a non-toxic NPC probe, suggesting that NPC could be applied to visualize the level of HSA in vivo. More importantly, further practical applications of probe NPC in human urine samples were achieved with satisfactory results by using a fluorometer or test paper, which could provide extensive application in clinical diagnosis.

Full text of DOI:10.1039/d0tb01411g

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A novel hydrazide Schiff base self-assembled
nanoprobe for selective detection of human
serum albumin and its applications in renal
disease surveillance†
Cite this:DOI: 10.1039/d0tb01411g
Zhi-Gang Wang,‡a Xiao-Jing Yan,‡a Hai-Bo Liu,b De-Long Zhang,c Wei Liu,d
e
a
Cheng-Zhi Xie, *a Qing-Zhong Li
and Jing-Yuan Xu
*
Human serum albumin (HSA) is considered as a biomarker for the early diagnosis of renal disease,
therefore identifying and detecting HSA in biological fluids (especially urine) with an easy method is of great
importance. Herein, we report a novel hydrazide Schiff base fluorescent probe N0-((7-(diethylamino)-2-oxo-
2H-chromen-3-yl)methylene)pyrazine-2-carbohydrazide (NPC), which self-assembled into nanoparticles in
aqueous solution. Based on disassembly-induced emission and the site-specific recognition mechanism, the
binding of NPC with HSA resulted in a fluorescence ‘‘turn-on’’ response. Probe NPC exhibited superior
selectivity and sensitivity toward HSA with a detection limit of 0.59 mg Lꢀ1 in PBS and 0.56 mg Lꢀ1 in the
urine sample. The site-binding mechanism of NPC with HSA was explored by fluorescence quenching study,
Job’s plot analysis, HSA destruction, site marker displacement and molecular docking. Fluorescence imaging
of HSA in MCF-7 cells was achieved by using a non-toxic NPC probe, suggesting that NPC could be applied
to visualize the level of HSA in vivo. More importantly, further practical applications of probe NPC in human
urine samples were achieved with satisfactory results by using a fluorometer or test paper, which could
provide extensive application in clinical diagnosis.
Received 3rd June 2020,
Accepted 28th July 2020
DOI: 10.1039/d0tb01411g
rsc.li/materials-b
kidney damage. In normal individuals, the concentration of
HSA is about 35–50 g Lꢀ1 in serum and less than 30 mg Lꢀ1 in
1. Introduction
Chronic kidney disease is characterized by high morbidity and urine.8,9 When the kidney is damaged, an obvious enhance-
mortality, whereas shows little symptoms in the early stage. ment of the HSA level is found in urine of patients, which
Accordingly, the early diagnosis of chronic kidney disease is increased along with the severity of kidney disease. If
crucial for timely medical treatment, thereby improving the untreated, 50% of them will develop to kidney disease within
survival of patients.1 Human serum albumin (HSA), the most 5 to 10 years.10,11 Urine detection is more convenient and timely
abundant biomacromolecule in human blood plasma, plays than blood detection. Hence, it is very important to detect
important roles in many biological functions, such as regula- the HSA level in urine for the early diagnosis and prevention of
tion of colloid osmotic pressure,2 delivery of hormones,3 and chronic kidney disease, as well as monitoring the disease
transportation of nutrients, metabolites and drugs.4–7 More- progression of patients during the medical procedure. How-
over, it is the routine clinical biomarker for the assessment of ever, relatively low concentration of HSA and the existence of
biological interferents in urine enhance the difficulty of its
a Department of Chemical Biology and Tianjin Key Laboratory on Technologies
Enabling Development of Clinical Therapeutics and Diagnostics, School of
Pharmacy, Tianjin Medical University, Tianjin 300070, P. R. China.
E-mail: xiechengzhi@tmu.edu.cn, xujingyuan@tmu.edu.cn
application in disease surveillance.
At present, several traditional analytical techniques have been
used to detect HSA, including immunoassays,12 liquid chromato-
graphy mass spectrometry proteomic,13 and electrochemistry,14
but they generally require expensive materials and equipment,
complicated sample preparation processes, toxic reagents,
and time-consuming procedures. Colorimetric methods15
could overcome the shortcoming mentioned above, but often
suffer from low sensitivity and selectivity. Alternatively, fluores-
cence methods have recently attracted great attention due to
their advantages of convenience, low-cost, real-time detection
b Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences,
Peking Union Medical College, Beijing 100193, P. R. China
c Department of Pharmacy, Tianjin Santan Hospital, Tianjin 300193, P. R. China
d The Second Hospital of Tianjin Medical University, Tianjin 300211, P. R. China
e The Laboratory of Theoretical and Computational Chemistry, School of Chemistry
and Chemical Engineering, Yantai University, Yantai 264005, P. R. China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0tb01411g
‡ These authors contributed equally.
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and good biocompatibility.16–19 The current fluorescent
probes toward HSA are based on the following mechanisms:
Green Fluorescent Protein (GFP) simulate strategy,20 nano-
particle based enhancement,21 dye binding imaging,22 and
site-binding interactions.23 Unfortunately, most of the probes
still suffered from high detection limits, poor anti-interference
ability, insolubility in water, and lower excitation and emission
wavelengths.24–28 Moreover, specific detection was always
demanded for the HSA probe, since some proteins have similar
structures to HSA. For example, the amino acid sequence of
BSA (Bovine Serum Albumin, which is always served as a
substitute of HSA in many physiological experiments) possesses
high similarity with HSA, consequently displaying similar phy-
sicochemical properties and fluorescence response.29 There-
Scheme 1 Fluorescence detection mechanism of nanoprobe NPC
toward HSA.
fore, the development of simple, rapid, stable, sensitive, highly nanoprobe NPC exhibited excellent detection capability for HSA
selective and environmental friendly fluorescent probes for in urine, by which we could establish a simple and efficient
identifying HSA is desirable.
method to monitor the health conditions for normal people
The rational combination of chemical functional groups is and the progress of kidney disease in patients.
an effective strategy for designing excellent fluorescent probe
molecules. Coumarin compounds exhibit diverse biological
2. Experimental section
activities (antimicrobial, antitumor, and antioxidant) and can
strongly bind to HSA through site-binding interactions with
2.1. Materials and instrumentations
obvious fluorescence signals.30,31 Accordingly, they have been
All commercially available chemical reagents and biological
widely used as components of fluorescent probe molecules
which always possess admirable optical properties like good
analytes were used without any purification. H-NMR and 13C-
1
NMR spectra of NPC were measured with a Bruker AVANCE III-
photostability, visible excitation and emission wavelengths,
400 spectrometer. The absorption spectra were tested by using
high fluorescence quantum yield and so on.32,33 The different
a JASCO V-770 UV-vis spectrophotometer. The fluorescence
functional groups of hydrazide Schiff base compounds exhibit
spectra were obtained using a HITACHI F-7000 fluorescence
distinct hydrophilicity and lipophilicity, and the amide group
spectrophotometer. Mass spectra were achieved with a LCQ
could increase the binding affinity with HSA, which are all
electron spray mass spectrometer (ESMS, Finnigan). The bio-
beneficial to work as a chemical sensor for HSA.34,35 Owing to
imaging tests were taken using an Olympus FV-1000 microscope
the sensitivity to the surrounding environment, site-binding
(Japan). The particle sizes were measured by dynamic light
interaction is useful to improve the selectivity of the probe to
scattering (DLS) with a Nano ZS90 instrument. The transmission
HSA.7,8,31,36–38 In addition, several organic-molecule self-
electron microscopy (TEM) image was obtained from a HT7700
assembled nanoparticles have also received great attention in
transmission electron microscope. Steady-state fluorescence spectra
protein sensing due to their optical stability, good biocompat-
were acquired on an Edinburgh FS5 spectrofluorometer.
ibility, diversity and tunable architectures.21,38–40 Based on the
characteristics of functional groups and detection mechanisms
mentioned above, we designed and prepared a unique
2.2. Synthesis and characterization of NPC
self-assembled nanoparticle probe, N0-((7-(diethylamino)-2-
oxo-2H-chromen-3-yl)methylene)pyrazine-2-carbohydrazide
(NPC) by condensation of pyrazine-2-carbohydrazide and
7-diethylaminocoumarin-3-aldehyde in methanol, which could
detect HSA in aqueous media by fluorescence response and
achieve rapid visual recognition based on obvious color
changes. The ‘‘turn-on’’ fluorescence is attributed to the site-
specific protein–ligand recognition and disassembly of nano-
particles (Scheme 1). Specific advantages of the probe could be
listed as follows: (1) compared with most of the HSA probes
with CQC bonds, a Schiff base fluorescent probe with a CQN
bond was beneficial to combine different functional groups
and easy to synthesize; (2) the combination of disassembly-
induced emission and the site-specific recognition mechanism
is favorable to improve the selectivity and sensitivity of the
probe; and (3) the fluorescence response of NPC to HSA is very
quick (less than 0.5 min). Most strikingly, the self-assembled
The synthetic process of probe NPC is shown in Scheme 2. Pyrazine-
2-carbohydrazide41 and 7-diethylaminocoumarin-3-aldehyde42 were
synthesized according to the published procedures. Then
30 mL absolute methanol solution of pyrazine-2-carbohydrazide
(69.06 mg, 0.5 mmol) and 7-diethylaminocoumarin-3-aldehyde
Scheme 2 Synthetic route of probe NPC.
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(122.64 mg, 0.5 mmol) was refluxed at 80 1C for 6 h. After cooling And the following formula was used to calculate the quantum
the reaction solution to room temperature, the resulting precipi- yields:
tate was filtered and washed sufficiently with absolute methanol.
ꢀ
ꢁ
2
The solid was dried under reduced pressure to yield a red product
Gradx Zx
Grads Zs
1
Fx ¼ Fs
(155.28 mg, 85%). H-NMR (400 MHz, DMSO-d6): d (ppm): 12.45
(s, 1H), 9.26 (s, 1H), 8.93 (s, 1H), 8.80 (s, 1H), 8.68 (s, 1H), 8.41
(s, 1H), 7.68 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 9.0 Hz, 1H), 6.59 (s, 1H),
3.48 (q, J = 6.9 Hz, 4H), 1.14 (t, J = 7.0 Hz, 6H) (Fig. S1, ESI†).
13C-NMR (101 MHz, DMSO-d6): d (ppm): 160.68, 159.34, 156.65,
151.41, 147.71, 144.73, 144.44, 144.09, 143.31, 139.10, 131.00,
112.40, 109.71, 108.01, 96.34, 44.23, 12.34 (Fig. S2, ESI†). ESI-MS
(m/z) calcd for [NPC + H]+: 366.1566, found: 366.1566; calcd. for
[NPC + Na]+: 388.1386, found: 388.1385 (Fig. S3, ESI†).
where the subscripts x and s represent the test sample and
standard reference, respectively. F denotes the fluorescence
quantum yield, Grad is the slope from the integral area of the
fluorescence spectra vs. absorbance, and Z stands for the
solvent refractive index.
2.6. Molecular docking
2.3. Spectral analysis
Molecular docking studies were performed with the MOE2019
program to explore the binding mode of NPC with HSA. The 3D
geometry of probe NPC was optimized at the B3LYP/6-31+G(d,p)
level by using Gaussian 09 programs.44 The crystal structure of
HSA (PDB code: 2BXG) was obtained from the RSCB protein
database and further optimized through UCSF Chimera 1.8.1.
Chain A of 2BXG was selected for docking calculation. After the
docking of probe NPC with HSA, five probe–protein conforma-
tions were generated. And the configuration with the lowest
energy was used for detailed studies. The final docking
configuration diagram was rendered and visualized using
PyMoL 1.5 software.
The stock solution of probe NPC was prepared in DMF. The
stock solutions of various cations and anions were prepared in
ultrapure water using their related chloride salts and sodium
salt, respectively, except Pb2+ (from Pb(OAc)2) and Ag+ (from
AgNO3). The stock solutions of biological analytes were pre-
pared in ultrapure water. All stock solutions were prepared at
1 mM concentration and stored at 4 1C. The spectral measure-
ments were conducted in a PBS buffer system (pH 7.4, 10 mM)
by mixing stock solutions with appropriate proportion. In
titration experiments, aliquots of HSA solution were gradually
added to 2 mL PBS solution containing 5 mM NPC. Then the
solution was mixed by pipetting 20 times with a micropipette.
The operation time is less than 0.5 min. The fluorescence
spectra were obtained immediately and the fluorescence emis-
sion reached a maximum signal. Based on titration study, the
detection limit was obtained from 3s/k (s is the standard
deviation of blank measurements, and k is the slope of fluores-
cence intensity changes versus HSA concentrations). The solution
of NPC (5 mM) was measured 10 times to obtain the standard
deviation. Job’s plot was recorded by using the fluorescence
method. The fluorescence intensity of various mixtures (con-
taining different volume ratios of NPC with HSA, while the
overall concentration remained at 5 mM) was measured to
ascertain the stoichiometric point.
2.7. MTT assay
The MCF-7 cells were grown in DMEM culture medium and
maintained under appropriate conditions (under 95% air and
5% CO2 at 37 1C). The cytotoxicity of NPC was evaluated with
MCF-7 cells by MTT assay. Firstly, the cells with better vitality
were transferred to 96-microplates at 3 ꢁ 103 cells each well and
continuously cultivated in a 5% CO2 incubator at 37 1C for 24 h.
NPC samples with different concentrations were added into the
cells for another 6 h. Then the cells were treated with 10 mL
MTT solution (5 mg mLꢀ1) under the same conditions for 4 h.
Finally, 100 mL DMSO was added to dissolve the formed
formazan crystals after removing the supernatant, and an
Infinite 200-Pro Nano Quant Microplate Reader was used to
measure the absorbance at 570 nm. The cell viability was
calculated based on six parallel experiments.
2.4. Preparation of self-assembled nanoparticles
The stock solution of NPC (10 mL) was added into 2 mL PBS
buffer solution (pH 7.4) under stirring. Nanoparticles were
formed and distributed uniformly in solution. At room tem-
perature, the size distributions of NPC nanoparticles were
2.8. Intracellular imaging
analyzed by DLS. For morphology observation, nanoparticle MCF-7 cells (about 1 ꢁ 105 cells) were propagated in two
solution was dripped on the copper mesh, dried in vacuum and confocal cell culture dishes respectively and maintained in
analyzed by TEM.
5% CO2 incubator at 37 1C overnight. After being washed with
DMEM medium, one dish was incubated only with probe NPC
(10 mM) for 30 min in the 5% CO2 incubator, and the other dish
2.5. Quantum yield measurements
Rhodamine B (F = 0.69) was chosen as a standard sample was treated with NPC (10 mM, 30 min) following HSA (10 mM,
to determine the quantum yields of probe NPC and the probe– 30 min) at 37 1C in an incubator. After eliminating the free NPC
protein complex.43 The absorption and fluorescence spectra and HSA with PBS, the cells were observed and imaged using an
of rhodamine B and NPC were measured in ethanol and Olympus FV-1000 fluorescence microscope at an excitation
PBS buffer systems, respectively. The fluorescence spectra wavelength of 488 nm. Quantified fluorescence intensities of
were obtained under the excitation wavelength of 450 nm. cells were measured by Image J software.
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2.9. HSA detection in human urine samples
The urine samples of healthy human were collected from
authors, and the urine samples of patients with kidney disease
were obtained from Tianjin Santan hospital, which were all
mixed with two-fold amounts of PBS solution (15 mM). After
removing the precipitate by centrifugation (10 000 rpm, room
temperature, 3 minutes), a clear supernatant extract of the
urine sample was determined. The standing times of the samples
were all within 2 hours before measurement. The contents of
HSA were tested via the immune scatter turbidimetry method in
the Tianjin Santan hospital as a reference. All samples were
collected obeying ethic and academic principles. All experiments
were performed in compliance with the ethical committee of
Tianjin Santan Hospital’s policy on human subject use and ethics
(case number IRB2019-YX-001) and conform to international
ethical guidelines for biomedical research involving human subjects
(2002) that were developed by the Council for International
Organizations of Medical Sciences in collaboration with the
World Health Organization. The HSA test papers were made
by dipping filter papers into the PBS solution of NPC, which is
stored at 4 1C after natural air-dry. For sample detection, the
HSA test paper was immersed in pure urine for 2 minutes then
observed under 365 nm UV light.
3. Results and discussion
3.1. Self-assembly behavior of NPC and disassembly induced
by HSA
Self-assembly of organic small-molecules can significantly
change their optical properties such as absorbance and fluores-
cence spectra.45 In order to prove the aggregation behavior of
NPC, we investigated the changes of photophysical properties
based on absorption (Fig. S4a, ESI†) and fluorescence spectra
(Fig. S4b, ESI†). NPC in pure DMF solution showed an intrinsic
absorption peak at 454 nm and a strong fluorescence emission
with a maximum peak at 513 nm. With the gradual change of
solvent composition from pure DMF to PBS buffer (pH 7.4), the
absorption peak of NPC shifted from 454 nm to 461 nm,
meanwhile, the fluorescence intensity was markedly decreased.
All the above spectral change indicated the aggregation of
NPC in aqueous solution, which is due to hydrophobic forces
between organic molecules.39
To further ascertain the self-assembly behavior of NPC, the
PBS buffer solution of NPC was studied by DLS analysis. As
shown in Fig. 1a, an average grain diameter of 258 nm for NPC
was observed, which confirmed the formation of NPC nano-
particles in aqueous solution. Moreover, the TEM image exhib-
ited the size of NPC nanoparticles (Fig. 1b). The nanoparticle
self-assembly process in aqueous solution would result in
fluorescence-silent due to the aggregation-caused quenching
(ACQ) effect,46 which provided a basis for fluorescence ‘‘turn-
on’’ sensing toward HSA. The disassembly of nanoprobe NPC
was also explored by DLS. After adding equal amounts of HSA
Fig. 1 (a) The DLS analysis of NPC. (b) TEM image of NPC nanoparticles,
scale bar = 200 nm. (c) The DLS analysis of NPC, HSA, and the mixture of
NPC and HSA in PBS buffer solution. [NPC] = [HSA] = 5 mM.
of HSA induced the disassembly of nanoparticles, and NPC
molecules may be encapsulated in the hydrophobic pockets of
HSA. The smaller average grain diameter may be due to the
aggregates of the NPC–HSA inclusion complex.45
3.2. Selectivity studies
into the solution of NPC nanoparticles, average grain diameter For practical sample testing, high selectivity of the fluorescent
changed to 157 nm (Fig. 1c), which validated that the addition probe toward the analyte is necessary. Therefore, the selectivity
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of NPC toward HSA from various potential interferents as well 513 nm, respectively. Broadened selectivity research of NPC
as some typical coexisting substances in blood and urine was toward various cations (Fig. S6, ESI†) and anions (Fig. S7, ESI†)
evaluated in a PBS buffer system. As shown in Fig. 2a and was also conducted, resulting in negligible influence on the
Fig. S5 (ESI†), NPC exhibited almost no fluorescence in PBS fluorescence emission of NPC. This indicates that NPC can act
buffer solution (F = 0.004) at an excitation wavelength of as a good analytical tool for independently recognizing and
450 nm, whereas with the addition of HSA, the solution showed detecting HSA.
significant fluorescence enhancement (F = 0.034) as well as
Urine or blood contains a wide variety of components, and
a blue-shift effect. Notably, the response time was less than thus high anti-interference ability of NPC is required for target
0.5 min. The turn-on fluorescence could be attributed to the recognition in these complex environments. The emission
site-specific recognition mechanism and disassembly-induced intensities of NPC towards HSA in the presence of other
effect induced by HSA. Other proteins (pepsin, lipase, lysozyme, ingredients of urine/blood as well as common proteins were
ovalbumin, fibrinogen, hemoglobin, and lactoglobulin) and measured to explore the anti-interference ability of NPC. As
amino acids didn’t display such results. At the same time, a shown in Fig. 2c, the fluorescence response of NPC to HSA at
corresponding naked-eye visible color change from colorless 513 nm didn’t significantly change when other interferents
to green could be observed (Fig. 2b). As a homologous protein existed in the detection system. Furthermore, interference
of HSA, BSA displayed weak fluorescence response for the experiments also proceeded in the presence of other species
NPC nanoprobe. The fluorescence intensity increased 14-fold (Fig. S8–S10, ESI†). All of them had no obvious interference for
for 1 equiv. HSA and 4-fold for 5 equiv. BSA at a wavelength of the detection of HSA in the PBS buffer system. Therefore, HSA
fluorescence detection could be achieved by the NPC platform
in complicated biological and environmental samples.
3.3. Fluorescence detection study
The fluorescence properties of NPC were firstly studied under
different polarity and viscosity conditions. The mixed solutions
of ethanol/water, glycerol/water and ethanol–glycerol at differ-
ent ratios were used to simulate the corresponding microenvir-
onment, respectively. As shown in Fig. S11 (ESI†), in a low
polarity or high viscosity system, the fluorescence emission of
NPC increased significantly accompanied by wavelength blue-
shifts. A similar fluorescence enhancement and blue-shift of
NPC with HSA can be clearly explained by the fact that HSA
cavities can provide a microenvironment with high viscosity
and low polarity for NPC.7 In this kind of system, the dimethyl-
amino group often acts as a rotatable tail to trigger twisted
intramolecular charge transfer behavior,8,36,37 meanwhile caus-
ing more ICT and thus emitting more strongly in organic
solvents.47 Therefore, the lifetime decay of NPC before and
after binding with HSA was measured (Fig. S12, ESI,† lex
=
450 nm, lem = 513 nm). The decay associated parameters are
listed in Table S1 (ESI†). The fluorescence lifetime of NPC was
1.17 ns in the absence of HSA and increased to 1.80 ns after
binding with HSA, which are consistent with the fact that
fluorescent molecules with rotatable tails usually show longer
fluorescence lifetimes in a restricted environment.48
The fluorescence properties of probes are always correlated
with the pH environment. Consequently, we investigated the
fluorescence behavior of NPC with or without HSA in the pH
range of 4–12. As shown in Fig. S13 (ESI†), the fluorescence
intensity of NPC remained almost unchanged under different
pH conditions, which implied that NPC was comparatively
stable in a different micro-environment. In contrast, the
NPC–HSA system displayed significant fluorescence enhance-
ment under neutral and weak alkaline conditions, while acid
and alkaline conditions could affect the protonated state of
NPC and amino acids in HSA, thus destructing the combi-
nation of NPC and HSA, and further inducing fluorescence
Fig. 2 (a) The fluorescence emission of NPC (5 mM) toward 1 equiv. HSA
and other biological analytes in PBS buffer solution (5 equiv. for BSA only).
(b) The corresponding photographs of NPC with 1 equiv. HSA, BSA and
other biological analytes under UV light. (c) Fluorescence responses of
NPC (5 mM) toward HSA (5 mM) in the presence of other ingredients
(1 equiv.) of urine/blood and common proteins (5 equiv. for BSA only).
For (a and c), lex = 450 nm, lem = 513 nm.
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weakening. Considering the application of NPC in biosystems,
The binding stoichiometry of NPC with HSA was further
we chose physiological pH 7.4 for the sensing studies. The deduced by Job’s plot analysis. As shown in Fig. 3a, the
fluorescence intensity of NPC and NPC–HSA remained nearly maximum fluorescence intensity appeared at concentration
unchanged for 70 min in PBS buffer solution (Fig. S14, ESI†), ratio 1 : 1, suggesting that NPC was mainly binding with one
suggesting the excellent stability and practicability of NPC.
site of HSA. In addition, the site-binding mechanism can be
To further evaluate the sensitivity and get a quantitative determined by the HSA denaturation experiment (Fig. 3b).
appraisal of nanoprobe NPC to HSA, titration experiment was When trypsin was gradually added into the NPC–HSA system,
performed (Fig. S15a, ESI†). The fluorescence of NPC was the fluorescence intensity decreased linearly (over the range of
gradually triggered by the addition of HSA, and significant 0–0.6 mg mLꢀ1 for trypsin) as trypsin could cleave the primary
fluorescence enhancement at 513 nm was found with 1 equiv. structure of HSA (Fig. S19, ESI†).7 This phenomenon could also
HSA participation. As shown in Fig. S15b (ESI†), the fluores- be used to indirectly detect trypsin due to the good linearity.
cence linear response of NPC toward HSA in the concentration Generally, HSA mainly possesses two binding sites (site I and
range of 0–5 mM was observed (R2 = 0.99169). The detection site II). Site-binding displacement experiments can provide
limit (DL) for HSA was determined to be 0.59 mg Lꢀ1 (8.97 nM), important information for the binding site of the probe mole-
which is rather lower than those of most of the reported HSA cule in HSA.38 To further elucidate the binding location of NPC,
fluorescent probes23,26,45,49 and much lower than the clinical two site-specific drugs, warfarin (site I marker) and ibuprofen
value of urine for kidney patients. Benefited from the low DL, (site II marker), were chosen for fluorescence experiments. As
good linearity and high anti-interference, NPC is suitable for shown in Fig. S20 (ESI†), when two drugs were separately mixed
HSA quantitative detection in biological samples, especially for with the NPC–HSA complex, the fluorescence intensity at
urine samples of kidney disease patients. Similarly, the quantita- 513 nm significantly decreased in the warfarin group, whereas
tive detection of BSA with NPC could also be achieved, in which the influence of ibuprofen was relatively less. It clearly indi-
the linear response was observed in the concentration range of cated that the replacement of NPC from the NPC–HSA system
0–4.0 mM with a DL of 2.36 mg Lꢀ1 (35.6 nM) (Fig. S16, ESI†).
by warfarin is easier than ibuprofen, and further illustrated that
NPC is primarily located at site I of HSA.
To gain insight into the binding of NPC with HSA, the computa-
tional calculations were performed using a Molecular Operating
Environment (MOE). As shown in Fig. 3c, the optimal confor-
3.4. HSA binding studies
Fluorescence quenching is an effective method to investigate
the interaction of a compound with HSA. HSA itself can emit
strong fluorescence at an excitation wavelength of 295 nm
corresponding to tryptophan residues, which is located in
Sudlow site I of HSA. The microenvironment of the site would
affect the emission of Trp-214. If the probe molecule binds with
HSA in site I, the intensity of emission will decrease.50 As
shown in Fig. S17a (ESI†), the intrinsic fluorescence peak of
HSA at 332 nm decreased gradually with increasing concentra-
tions of NPC, which was induced by the interaction of HSA with
NPC and suggest that NPC binds preferentially in site I. The
binding parameters (Table 1) were obtained by using the
classical Stern–Volmer equation and Scatchard equation (see
the ESI,† Fig. S17a inset and S17b). The quenching rate con-
stant kq is much higher than the limiting diffusion constant 2 ꢁ
mation with the lowest binding free energy (ꢀ51.2 kcal molꢀ1
)
was obtained, in which probe NPC was embedded into site I of
HSA. Residue Leu-327 could form hydrogen bond interaction
with the imino group of the NPC molecule, while Phe-206, Ala-
210, Ala-213, Ala-350, Val-482, Lys-351, and Arg-209 interacted
with the hydrophobic parts of NPC by hydrophobic forces
(Fig. 3d). Evidently, the constrained cavity of HSA and the
interaction of residues with NPC restricted the rotation of the
N–N bond and C–N bond in free receptor NPC, thus leading to
obvious fluorescence enhancement. Molecular docking studies
agreed with experimental results and further corroborated the
binding site of NPC in HSA. The results also showed that the
functional groups in NPC played an important role in binding
HSA, which was consistent with our design idea.
1010 Mꢀ1 ꢀ1, indicating that the quenching mechanism was
s
static quenching.51 The fluorescence lifetimes of HSA (Fig. S18, 3.5. Application of NPC in kidney disease diagnosis
ESI†) in the absence and presence of NPC were determined to
evaluate the quenching process. The amplitude–weighted life-
Serum albumin concentration in urine is a diagnostic indicator
for kidney damage, while most reported fluorescent probes
time of HSA–NPC (t = 5.26 ns) was close to that of HSA (t =
and diagnostic methods are inappropriate for convenient
5.06 ns), confirming the static quenching mechanism.52 The
detection.49,53 To assess the applicability of NPC in kidney
binding constant Kb (2.13 ꢁ 105
M
ꢀ1) indicated a moderate
disease diagnosis, we used probe NPC to quantitatively detect
the concentration of HSA in human urine. Firstly, the titration
experiment was carried out in the urine–PBS system with the
addition of HSA. As shown in Fig. 4a, the turn-on fluorescence
of NPC in the urine sample gradually increased upon the addition
of HSA. Fortunately, the fluorescence intensity increased linearly
with HSA in the concentration range of 0–5 mM (Fig. 4b) and the
DL was 0.56 mg Lꢀ1 (8.42 nM). Then three urine samples with
known HSA concentrations (0.5, 1.0, and 2.5 mM) were separately
combination between NPC and HSA.31 Besides, n (the number
of binding sites) is close to 1, implying that the binding site of
probe NPC with HSA is one.
Table 1 The binding parameters of NPC with HSA
Complex
T (K)
KSV (10ꢀ5
)
Kq (10ꢀ14
)
Kb (10ꢀ5
)
n
NPC–HSA
298
1.69
1.69
2.13
0.80
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Fig. 4 (a) The fluorescence emission spectra of NPC (5 mM) with the
addition of HSA (0–1 equiv.) in treated urine sample. (b) The linear
relationship between fluorescence intensity changes at 513 nm and HSA
concentrations in the range of 0–5 mM (lex = 450 nm, lem = 513 nm). (c)
Color changes of HSA test papers after dipping into pure urine of healthy
human and kidney disease patient. (d) Color changes of HSA test
papers with the addition of different concentrations of HSA (0, 10, 20,
40, 80 mg Lꢀ1). For (c) and (d), lex = 365 nm.
prepared by using healthy human urine. The samples were
measured by the fluorescence method, and the calibration
equation (Fig. 4b) was used for calculating the HSA content.
As shown in Table 2, satisfactory recoveries demonstrated that
probe NPC possessed excellent advantages on HSA detection in
urine samples, which are favor to examine the health status of
kidneys.
Next, we evaluated the clinical application of NPC by mea-
suring five urine samples from patients with kidney disease.
The HSA contents in the urine–PBS system were determined
using the same calibration equation. As shown in Table 3, the
Fig. 3 (a) Job’s plot based on fluorescence intensity at 513 nm, in which
the total concentration of NPC and HSA was 5 mM. (b) The fluorescence
emission spectra of the HSA–NPC system ([NPC] = [HSA] = 5 mM) with the
addition of trypsin. (c) Molecular docking image for the binding pocket of
HSA toward NPC. (d) Detailed interaction of NPC with residues in site I of
HSA. The blue dotted line represents the hydrogen bond of NPC with the
corresponding residue in HSA.
Table 2 The determination of HSA content in urine samples (n = 3)
Sample Added (mM) Measured (mM) SD
Recovery (%) RSD (%)
1
2
3
0.500
1.000
2.500
0.556
0.966
2.281
0.024 111.2
0.021 96.6
0.063 91.24
4.32
2.17
2.76
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Table 3 The determination of HSA levels in urine samples from patients
with kidney disease by a clinical method and this fluorescence method
(NPC) (n = 3)
behaviors of probe NPC stimulated us to use it for HSA imaging in
living cells. Before imaging, the cytotoxicity of NPC was evaluated
in MCF-7 cells by MTT assay. As shown in Fig. 5a, even when
treating cells with 50 mM NPC for 6 h, the cell viability remained
almost unchanged, which suggested that NPC is safe and suitable
for intracellular imaging under experimental conditions. There-
fore, we cultured MCF-7 cells with NPC (10 mM) in the absence
and presence of HSA (10 mM) for 30 min, respectively. As shown in
Fig. 5b, the cells cultured only with NPC didn’t exhibit obvious
fluorescence signals. In contrast, in the presence of HSA, a
significant fluorescence enhancement was observed in cells.
These results proved that NPC has good cell permeability and
could visually detect HSA in living cells.
Clinical method
Fluorescence method
Samples
(mg Lꢀ1
)
(NPC) (mg Lꢀ1
)
1
2
3
4
5
41.53
47.75 ꢂ 1.06
181.62 ꢂ 2.15
351.27 ꢂ 2.93
242.83 ꢂ 3.09
206.07 ꢂ 3.71
185.05
338.83
232.28
186.22
detection results are in good agreement with the clinical
results, in which the relative variations are all less than 15%.
Thus, NPC suggested an effective tool for monitoring HSA levels
for patients and sub-healthy people. For conveniently detecting
urine samples, the HSA test papers were prepared by PBS
solution of NPC. After being dipped into pure urine of kidney
disease patients, the test papers showed obvious fluorescence
under UV irradiation (Fig. 4c), while this phenomenon will not
appear in the urine of normal human. For this HSA test paper,
the minimum detection concentration is 20 mg Lꢀ1 (Fig. 4d),
which will be very useful for the fast detection of HSA in urine.
4. Conclusions
In this work, a nanoprobe NPC, which possessed self-assembly
behavior in aqueous solution, was designed for sensing HSA.
Nanoprobe NPC showed ‘‘turn-on’’ fluorescence response to
HSA with high selectivity and sensitivity, which is due to the
disassembly of nanoparticles and the subsequent site-specific
recognition. The 1 : 1 site-binding (site I) interaction of probe
NPC with HSA was confirmed by fluorescence quenching
studies, Job’s plot analysis, HSA destruction, site marker drug
competition and molecular docking. The non-toxic NPC was
successfully applied for HSA imaging in living cells. More
importantly, probe NPC could be successfully applied in HSA
detection in human urine samples, which is expected to be a
promising tool for kidney disease diagnosis and health status
monitoring. Using a simple test paper, the over expression of HSA
in urine could be easily picked up for household measurement. In
addition, this efficient platform combining site-binding and the
disassembly-induced detection strategy will provide a new avenue
to develop excellent biosensors for clinical diagnosis.
3.6. Application in cell imaging
Studying the distribution and content of HSA in cells is of great
pharmacological significance.54 The outstanding fluorescence
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Natural Science Founda-
tion of China (No. 21977080), the Tianjin Municipal Natural
Science Foundation (No. 17JCZDJC33100 and 18JCYBJC91300)
and the CAMS Innovation Fund for Medical Sciences (CIFMS)
(2016-I2M-3-015).
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