An ultrasensitive rapid-response fluorescent probe for highly selective detection of HSA

Article abstract of DOI:10.1016/j.tetlet.2018.02.065

A fluorescent probe HCAB based on twisted intramolecular charge transfer (TICT) mechanism has been designed and synthesized by introducing benzoyl moiety to 4-dimethylamino-2′-hydroxychalcone. HCAB showed excellent selectivity towards human serum albumin

Full text of DOI:10.1016/j.tetlet.2018.02.065

Accepted Manuscript
An ultrasensitive rapid-response fluorescent probe for highly selective detection
of HSA
Pengbo Li, Yiru Wang, Shufang Zhang, Liang Xu, Gongchen Wang, Jingnan
Cui
PII:
S0040-4039(18)30260-0
https://doi.org/10.1016/j.tetlet.2018.02.065
TETL 49750
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 December 2017
7 February 2018
23 February 2018
Please cite this article as: Li, P., Wang, Y., Zhang, S., Xu, L., Wang, G., Cui, J., An ultrasensitive rapid-response
fluorescent probe for highly selective detection of HSA, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/
j.tetlet.2018.02.065
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
An ultrasensitive rapid-response fluorescent
probe for highly selective detection of HSA
Pengbo Lia,b1, Yiru Wangc,1, Shufang Zhangb, Liang Xud, Gongchen Wanga,*, Jingnan Cuib,*

1
Tetrahedron Letters
An ultrasensitive rapid-response fluorescent probe for highly selective detection of
HSA
Pengbo Lia,b,1, Yiru Wangc,1, Shufang Zhangb, Liang Xud, Gongchen Wanga,*, Jingnan Cuib,*
a
Dalian sixth people hospital, Dalian 116031 ,China
b
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
c
Dalian Medical University, Dalian 116044, China
d
School of Chemistry, Dalian University of Technology, Dalian 116024, China.
1
These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A fluorescent probe HCAB based on twisted intramolecular charge transfer (TICT)
mechansim has been designed and synthesized by introducing benzoyl moiety to 4-
dimethylamino-2’-hydroxychalcone. HCAB showed excellent selectivity towards human serum
albumin (HSA) among different proteins with a remarkable 160-fold fluorescence enhancement
and a wider linear range 0~100 mg/L of HSA. Job’s Plot analysis suggested that the formation
of HCAB-HSA complex followed a 1:1 stoichiometry. Molecular docking and the displacement
assay demonstrated the binding site of HCAB was subdomain IIA and IB of HSA. We also
tested HSA levels in human plasma for practical application, the results obtained from HCAB
method were similar to those obtained from the standard clinical method.
Available online
Keywords:
human serum albumin
fluorescent probe
2’-hydroxychalcone
TICT
2009 Elsevier Ltd. All rights reserved.
rapid detection
1. Introduction
Up to now, various methods including dye-binding assays,
immunoassays, capillary electrophoresis, as well as LC-MS/MS
Human serum albumin (HSA), the most abundant protein in
human blood plasma (60% of total plasma proteins), plays an
important role in maintaining osmotic pressure of blood
compartment. HSA is synthesized exclusively in liver, the
average daily synthesis is about 12~25 g, and it is secreted into
the portal circulation as soon as it is manufactured. The
concentration of albumin is approximately 35~50 g/L in serum,
based proteomic techniques, have been utilized for the detection
of HSA. Pertaining to the scope of clinical practice and analytical
assays, HSA measurement should meet the following
requirements: 1) high selectivity, sensitivity and rapid-response;
2) high accuracy and repeatability; 3) high throughput and low
cost. Dye-binding based assays, especially fluorescent probe, can
meet the above-mentioned requirements. However, only a few of
these fluorescent probes were developed to be selective for HSA,
and some reported probes exhibited fluorescence response only
under high concentration of probes or with a narrow linear
determination range of HSA. [19~23] Thus, the development of
selective fluorescent probe with a wider linear range in the
presence of low concentration of probe for monitoring HSA is
imperious.
1
and normally below 30 mg/L in urine. The levels of serum
albumin in biofluids is a useful health indicator,2-4 which is
associated with hypertension, kidney disease, diabetes mellitus,
and liver diseases such as hepatitis and liver cirrhosis.5-8 On the
other hand, HSA serves as the main carrier for endogenous and
exogenous molecules including many drugs, glycolipids, fatty
acids and metabolites,9 there are two major and structurally
selective binding sites in HSA, namely, site I and site II, which
are located in the subdomain IIA and IIIA of the hydrophobic
region of HSA.[10-15]. In addition, HSA is also available for
therapeutic use, such as replacing lost fluid and helping restore
blood volume in surgery patients, trauma and burns. Therefore,
the selective detection and precise quantification of serum
albumin have gained much more interest from biological and
medical researchers. [16-18]
In this work, we designed a probe based on twisted
intramolecular charge transfer (TICT) mechanism and it may
detect HSA through inhibition of its TICT behaviour upon
interaction with the hydrophobic domain of HSA (Scheme 1).
The probe was synthesized by introducing benzoyl moiety to 4-
dimethylamino-2’-hydroxyhalcone with high yield (55 %) and
characterized by 1H NMR, 13C NMR and HRMS.

2
Tetrahedron Letters
transferrin, CRP, lysozyme, CA, pepsin, trypsin, AMG,
myoglobin and BSA 10 mg/L or 10 U/L. (b) Chemical structures
of compound 1, 2 and HCAB.
In order to investigate the structure–signaling relationship, two
HCAB derivatives were synthesized. Compared with the 160-
fold fluorescence enhancement showed by the HCAB-HSA
complex, compound 1 and 2 displayed fluorescence response
with 1.5-fold and 25-fold respectively (Fig S1). The difference in
the results indicated that the ester moiety was important to induce
high fluorescence enhancement which may be caused by the ester
group participation in the binding with HSA and the increased
hydrophobic interaction between the compound and HSA.
Scheme 1. Schematic illustration of the mechanism for the
interaction of probe HCAB with HSA.
2. Results and discussion
The optical property of HCAB toward HSA was first
investigated in PBS (pH 7.4, 1% DMSO). HCAB exhibited a
weak emission intensity at 524 nm (Φ = 0.006) when excited at
426 nm, upon the addition of HSA, the fluorescence intensity at
524 nm increased significantly (Φ = 0.199) (Table S1). However,
the presence of various enzymes such as haptoglobin,
hemoglobin, fibrinogen, globulins, immunoglobulin G (IgG), α-
chymotrypsin (AC), human carboxylesterase 1 (hCE1), human
carboxylesterase
2
(hCE2), acetylcholinesterase (AChE),
butyrylcholine esterase (BChE), lipase, transferrin, C-reactive
protein (CRP), lysozyme, carbonic anhydrase (CA), pepsin,
trypsin, α-macroglobulin (AMG), myoglobin and bovine serum
albumin (BSA) did not cause an observable fluorescence
changes (Fig 1a). With increasing of HSA, an excellent linear
correlation (R2 = 0.9970) between fluorescence intensity (524
nm) and HSA concentration was found in the range of HSA
concentration from 0 to 100 mg/L (Figure 2). The limit of
detection (LOD) of HCAB was determined to be 1.91 mg/L
(Supporting Information), indicating that HCAB would be a
potential tool for selective and sensitive detection HSA.
Fig. 2. (a) Fluorescence emission spectra of HCAB (2.0 µM) in
the presence of increasing concentration of HSA (0 ~ 100 mg/L)
in PBS buffer. (b) The relationship of fluorescence intensity at
524 nm and the concentration of HSA (0 ~ 100 mg/L). (pH 7.4,
1% DMSO). λex = 426 nm.
The fluorescence enhancement upon the addition of HSA is
due to the binding of HCAB to HSA's hydrophobic pocket,
which could impose restricted bond rotation on the probe and
block TICT. To confirm the TICT phenomena, we studied the
fluorescence behavior of HCAB in different water-glycerol
fractions. The results showed that, upon increasing the ratio of
glycerol from 0% to 99%, the fluorescence emission of HCAB
enhanced remarkably (Fig. 3a). The increase in fluorescence
intensity was attributed to the fact that glycerol increased the
viscosity of the solvent which restricted the rotational modes of
the molecule and ultimately suppressed the TICT leading to
fluorescence enhancement. To find out the origin of fluorescence
enhancement, we also carried out fluorescence lifetime studies of
HCAB in the presence of HSA. The free HCAB exhibited a
diexponential decay with a lifetime of 0.495 ns (74.89%), and
2.890 ns (25.11%). Upon the addition of 100 mg/L of HSA,
HCAB-HSA exhibited a diexponential decay with lifetimes of
0.844 ns (68.17%), and 2.737 ns (31.83%). The increase in the
Fig.1. (a) Fluorescence response of HCAB (2.0 µM) towards
various species of proteins in PBS (pH 7.4, 1% DMSO). λex
=
426 nm. HSA: 100 mg/L, haptoglobin, hemoglobin, fibrinogen,
globulins, IgG, AC, hCE1, hCE2, AChE, BChE, lipase,

3
lifetime of HCAB upon the addition of HSA was due to the
interaction of the probe with the hydrophobic pocket of HSA
which resulted in the inhibition of TICT. Due to the inhibition of
TICT, the energy dissipated by non-radiative pathway became
suppressed leading to an increase in the lifetime of HCAB in the
excited state and hence favouring the fluorescence emission
enhancement in the presence of HSA.
subdomain IIA & IB), digitoxin (a site marker of subdomain
IIIA), indomethacin (a site marker of subdomain IIA& IB). As
can be seen in Fig 4b, upon the addition of indomethacin, about
60% of the probe was displaced, while warfarin and salicylic acid
caused about 30% of the fluorescence intensity decrease, while
other drugs showed almost no significant change in the
fluorescence intensity (Fig. S14). Molecular docking were
carried out to gain a deep insight into the binding mode of
HCAB on HSA, as shown in Fig. 5, the preferred binding site
was similar to that of indomethacin. All of these results
confirmed that the binding site of HCAB was subdomain IIA and
IB of HSA.
Fig 3. (a) The fluorescence intensity of HCAB in different
volume ratio of glycerol and PBS at 524 nm. (b) Fluorescence
lifetime decay profiles of HCAB in HSA solution (100 mg/L at
pH 7.4). Blue line represents free HCAB and red line represents
HCAB-HSA complex, while black line represents instrument
response function (IRF).
Furthermore, our study on HCAB's pH responses confirmed
the suitable pH range for HSA detection. It was found that the
fluorescence intensity of HCAB was quite stable in the pH range
2-11, while the fluorescence intensity of HCAB-HSA could
reach a plateau at pH 7.4-11 (Fig.S2). These results demonstrated
that HCAB could function properly under physiological
conditions (pH 7.4). The response of HCAB to HSA has short
detection time. The fluorescence intensity at 524 nm exhibited a
sharp increase once HSA was added (Fig S3), meaning that the
HCAB could perform a rapid measurement.
Fig 4. (a) Job’s plot analysis. HCAB was mixed with HSA at
different molar ratios in PBS (pH 7.4). (b) Displacement of
HCAB from the HCAB-HSA complex by the addition of
different site-specific drugs (50 μM).
Job's plot analysis was performed to investigate the
stoichiometry of the complex formed between HCAB and HSA,
which was found to be 1:1 (Fig. 4a), indicating that HCAB
bound mainly at one site of the pocket. The dissociation constant
(Kd) was 1.3 ± 0.1 µM, which suggested that HCAB had high
binding affinity with HSA. To verify the binding site of HSA for
HCAB, the displacement assay was designed with six site-
specific drugs, namely warfarin (a site marker of subdomain IIA),
propofol (a site marker of subdomain IIIA & IIIB), ibuprofen (a
site marker of subdomain IIIA), salicylic acid (a site marker of

4
Tetrahedron Letters
Fig 5. The distribution of 100 binding conformations, with the
Acknowledgments
most frequency and lowest binding energy highlighted with red
rectangle.
This work was supported by the National Natural Science
Foundation of China (21572029).
Encouraged by the above-mentioned results, we estimated the
HSA levels in the human blood serum for the practical
application. All the serum samples were collected from Dalian
sixth people hospital. The addition of blood samples from
different peoples to the solution of HCAB in PBS (pH 7.4, 1%
DMSO) showed an enhancement of fluorescence and the HSA
level was quantified using the fluorescence calibration curve. A
comparative plot of the level of HSA in different blood samples
using HCAB method and those from the same clinical test is
shown in Fig. 6. The results obtained from our method were
almost similar to those obtained from the standard clinical
methods, no matter whether the samples were derived from a
healthy person (Fig. 6a) or patients with liver disease (Fig. 6b).
Thus, HCAB may act as an efficient tool for detecting the level
of HSA in clinical setting.
References and notes
1. Peters, T. All About Albumin; Elsevier, 1995, 319−413.
2. Chung, C. Y. S.; Yam, V. W.W. J. Am. Chem. Soc.
2011, 133, 18775-18784.
3. Yudkin, J. S.; Forrest, R. D.; Jackson, C. A. Lancet
1988, 2, 530-533.
4. Viberti, G. C.; Hill, R. D.; Jarrett, R. J.; Argyropoulos,
A.; Mahmud, U.; Keen, H. Lancet 1982, 1, 1430-1432.
5. Alino, V. J.; Yang, K.-L. Analyst 2011, 136, 3307-
3313.
6. Hoogenberg, K.; Sluiter, W. J.; Dullaart, R. P. Acta
Endocrinol-buch 1993, 129, 151-7.
7. Mogensen, C. E.; Christensen, C. K. N. Engl. J. Med.
1984, 311, 89-93.
8. Murch, S. H.; Winyard, P. J.; Koletzko, S.; Wehner, B.;
Cheema, H. A.; Risdon, R. A.; Phillips, A. D.;
Meadows, N.; Klein, N. J.; Walker-Smith, J. A. Lancet
1996, 347, 1299-1301.
9. Kumar, C. V.; Buranaprapuk, A.; Opiteck, G. J.;
Moyer, M. B.; Jockusch, S.; Turro, N. J. Proc. Natl.
Acad. Sci. U. S. A. 1998, 95, 10361-10366.
10. Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol.
Pharmacol. 1975, 11, 824-832.
11. Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol.
Pharmacol. 1976, 12, 1052-1061.
12. He, X. M.; Carter, D. C. Nature 1992, 358, 209-215.
13. Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Nat.
Struct. Boil. 1998, 5, 827-835.
14. Zhu, L.; Yang, F.; Chen, L.; Meehan, E. J.; Huang, M.
J. Struct. Biol. 2008, 162, 40-49.
15. Green, A. M.; Abelt, C. J. J. Phys. Chem. B 2015, 119,
3912-3919.
16. Ward, K. M. Anal. Chem. 1995, 67, 383-391.
17. Er, J. C.; Tang, M. K.; Chia, C. G.; Liew, H.; Vendrell,
M.; Chang, Y. T. Chem. Sci. 2013, 4, 2168-2176.
18. Er, J. C.; Vendrell, M.; Tang, M. K.; Zhai, D.; Chang,
Y. T. Acs Comb. Sci. 2013, 15, 452-457.
19. Wu, Y. Y.; Yu, W. T.; Hou, T. C.; Liu, T. K.; Huang,
C. L.; Chen, I. C.; Tan, K. T. Chem. Commun. 2014, 50,
11507-11510.
20. Fan, J.; Sun, W.; Wang, Z.; Peng, X.; Li, Y.; Cao, J.
Chem. Commun. 2014, 50, 9573-9576.
21. Li, W.; Chen, D.; Wang, H.; Luo, S.; Dong, L.; Zhang,
Y.; Shi, J.; Tong, B.; Dong, Y. ACS Appl. Mater.
Interfaces. 2015, 7, 26094-26100.
22. Dey, G.; Gaur, P.; Giri, R.; Ghosh, S. Chem. Commun.
Fig 6. Data obtained for HSA level in ten different samples
comprising of normal (a) and liver disease blood samples (b)
from HCAB assay as well was clinical data.
2016, 52, 1887-1890.
23. Wang, Y. R.; Feng, L.; Xu, L.; Hou, J.; Jin, Q.; Zhou,
N.; Lin, Y.; Cui, J. N.; Ge, G. B. Sensor. Actuat. B-
Chem. 2017, 245, 923-931.
3. Conclusion
In this work, a TICT-based fluorescent probe HCAB was
designed and synthesized, which exhibited a remarkable 160-fold
fluorescence enhancement upon the addition of 100 mg/L HSA.
HCAB can act as an efficient probe for its sensitive detection
under low-concentration of probe HCAB with the wider linear
range and precise quantification of HSA. The probe HCAB could
be used to quantify trace HSA in blood serum. Based on the
above advantages, we hope to present HCAB as a low-cost tool
for the clinical analysis of HSA in blood serum.
Supplementary Material
Supplementary material that may be helpful in the review
process should be prepared and provided as a separate electronic
file. That file can then be transformed into PDF format and
submitted along with the manuscript and graphic files to the
appropriate editorial office.
Click here to remove instruction text...

5
 Corresponding author. Tel.: +86-0411-84986229; fax: +86-
Tel: +86-0411-39728555; fax: +86-0411-39728555; e-mail:
15909862690@139.com
0411-84986229; e-mail: jncui@dlut.edu.cn

6
Tetrahedron Letters
1. A fluorescent probe HCAB based on TICT
mechanism has been synthesized.
2. HCAB could detect HSA in a wider linear
range with low concentration.
3. HCAB showed excellent selectivity towards
HSA among different proteins.
4. HCAB could detect HSA in blood samples
from healthy person or hepatopath.