An NIR fluorescent probe of uric HSA for renal diseases warning

Article abstract of DOI:10.1016/j.dyepig.2016.05.039

Diseases of the kidney, including acute kidney injury, nephritic syndromes, chronic kidney disease, and renal cysts, are accompanied by a series of characteristic clinical features. Human Serum Albumin (HSA) is regarded as an important biomarker for early warning of kidney diseases among them. To the best of our knowledge, although some fluorescent probes of HSA had reported in blood plasma under high concentrations of HSA, most of them could not reach the detection sensitivity of low concentrations of HSA in urine. In this work, an NIR fluorescent probe NIR-HSA for HSA based on TICT mechanism is reported, which can used in solution detection with 8.83-fold fluorescence enhancement, rapid response (completed within 5 s), excellent sensitivity (DL 26.16 nM), and excellent stability. In human urine detection, NIR-HSA clearly demonstrated perfect recovery property, and good potential to be applied to detect trace HSA for warning of critical diseases.

Full text of DOI:10.1016/j.dyepig.2016.05.039

Accepted Manuscript
An NIR fluorescent probe of uric HSA for renal diseases warning
Haidong Li, Qichao Yao, Jiangli Fan, Jianjun Du, Jingyun Wang, Xiaojun Peng
PII:
S0143-7208(16)30229-7
10.1016/j.dyepig.2016.05.039
DYPI 5271
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 7 April 2016
Revised Date: 20 May 2016
Accepted Date: 21 May 2016
Please cite this article as: Li H, Yao Q, Fan J, Du J, Wang J, Peng X, An NIR fluorescent probe of uric
HSA for renal diseases warning, Dyes and Pigments (2016), doi: 10.1016/j.dyepig.2016.05.039.
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An NIR Fluorescent Probe of Uric HSA for Renal Diseases
Warning
Haidong Lia, Qichao Yaoa, Jiangli Fana, Jianjun Dua, Jingyun Wangb, Xiaojun Peng*a
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road,
Dalian 116024, P.R. China.
bSchool of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road,
Dalian 116024, P.R. China.
Email:Pengxj@dlut.edu.cn.
ABSTRACT
Diseases of the kidney, including acute kidney injury, nephritic syndromes, chronic kidney
disease, and renal cysts, are accompanied by a series of characteristic clinical features. Human
Serum Albumin (HSA) is regarded as an important biomarker for early warning of kidney
diseases among them. To the best of our knowledge, although some fluorescent probes of HSA
had reported in blood plasma under high concentrations of HSA, most of them could not reach
the detection sensitivity of low concentrations of HSA in urine. In this work, an NIR fluorescent
probe NIR-HSA for HSA based on TICT mechanism is reported, which can used in solution
detection with 8.83-fold fluorescence enhancement, rapid response (completed within 5 s),
excellent sensitivity (DL 26.16 nM), and excellent stability. In human urine detection, NIR-HSA
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clearly demonstrated perfect recovery property, and good potential to be applied to detect trace
HSA for warning of critical diseases.
KEYWORDS: NIR fluorescent probe; Human Serum Albumin; ultra-low detection limit; urine
1．Introduction
Human Serum Albumin (HSA) is known to play vital roles in various physiological and
pathological processes[1, 2]. Normally, HSA functions as an omnipresent transporter to deliver
hormones, fatty acids, drugs, and metabolites and maintains plasma colloid osmotic pressure[3,
4]. Generally, it is generated in the liver as preproalbumin with an N-terminal peptide. After a
series of manufacture from rough endoplasmic reticulum to Golgi, it is fleetly secreted into the
portal cirulation[5]. The pioneering investigated shown that serum albumin exhibits a three-
dimensional structure consisting of several binding pockets owing to as much as 67% of the
secondary structure formed via α-helix of six turns[6]. There are two major and structurally
selective binding sites located in subdomains IIA and IIIA of HSA, namely, site I and site II[7].
When the kidneys are working as they should, there may be less than 30 mg/L of serum albumin
in healthy urine, which the reference range for albumin contents is approximately 35-50 g/L in
serum[8]. But, mounting evidence studies have showed that larger amounts of albumin leak into
the urine after the kidneys damaged (e.g. glomerular filtration function injury), which could not
only attack body fluid osmotic pressure but is also relevant to serious dieases such as chronic
kidney disease, acute kidney injury, nephritic syndromes, and protein-losing enteropathy[9-15].
As a vital marker for early warning of critical disease[16], although numerous previous research
works was about the physiological functions of HSA, accurate molecular mechanisms of action
and trace recognition in body fluids insufficiently overcame. Therefore, to fully understand
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chemical biology of HSA, it is highly needed to develop efficient methods for accurate
monitoring of HSA with a low detection limit in complex biological samples, especially in urine,
which is vital for biological research as well as clinical diagnoses.
To date, there are several methods to detect serum albumin in vitro or vivo including LC-
MS/MS on a basis of proteomic techniques[17], radioimmunoassay method commonly in clinical
laboratories[8], electrochemistry[18], and fluorescence-based assays[19-23]. Among these
techniques, dye-binding technology has been paid special attention owing to simple operation,
high sensitivity, sensitivity, excellent high spatial and temporal resolution and especially
nondestructive characteristics[24-30]. In recent years, several of fluorescent probes have been
developed for the detection of serum albumin in vitro or vivo. Unfortunately, we found that most
of reported probes have quite shorter excitation wavelength (< 500 nm), emission wavelength (<
600 nm) and poor detection limit (> 30 mg/L), accompanied by negative effects such as
photobleaching, and auto-fluorescence interference which serious limit their biological
applications for detecting trace HSA in complex body fluids. Furthermore, the thermal stability
of most probes need to be improved on withstanding high temperature in long-distance
transportation. Therefore, it inspires us to develop a near-infrared (> 650 nm) fluorescent probe
with high selectivity, sensitivity, stability and ultra-low detection limit for the recognition of
HSA in biological samples.
In our previous work, by introducing conjugated vinyl group to increase its emission
wavelength to the NIR region[22, 31], we designed and synthesized a series of fluorescent
probes employing molecular-rotor based twisted intramolecular charge transfer (TICT)
mechanism, which exhibited specific serum albumin detection with NIR emission in vitro and
vivo. Inspired by forenamed studies, in this work, we synthesized an NIR fluorescent probe NIR-
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HSA using hemicyanine dye, as shown in Scheme 1. Cyanine/hemicyanine is an idiomatical
chromophore with long wavelength emission in NIR region via the extension vinyene moiety of
the chromophore backbone[32-36]. When NIR-HSA was added into mixing system of HSA, the
near-infrared (NIR) fluorescence emission was obtained via ultrafast restricting twisted
intramolecular charge transfer (TICT) effect with large Stokes shift (ꢀλ = 165 nm) and 8.83-fold
enhancement in the emission of around 680 nm. The displacement experimental assay clearly
indicated that the binding of NIR-HSA on HSA was site I. Importantly, the detection limit (3σ/k)
toward HSA was calculated to be 26.16 nM (1.73 mg/L), which is superior to the most reported
fluorescent probes. This probe exhibited excellent heat stability in PBS buffer (0.01 M, pH 7.4)
under 60 oC. Significantly, good recoveries were obtained in range of 107.23-108.79% with RSD
from 0.09% to 1.00%, clearly indicating that probe NIR-HSA could be used to monitor the
concentrations of HSA in pure urine without interference.
2．Experimental section
2.1. General information and materials
All reagents used were obtained from commercial suppliers and were used without further
purification unless otherwise stated. Solvents used were purified via standard methods. Twice-
1
distilled purified water used in all experiments was from Milli-Q systems. H NMR and 13C
NMR spectra were recorded on a VARIAN INOVA-400 (or a Bruker Avance II 400 MHz or 500
MHz) spectrometer. Chemical shifts (δ) were reported as ppm (in MeOD or DMSO, with TMS
as the internal standard). Fluorescence spectra were performed on a VAEIAN CARY Eclipse
fluorescence spectrophotometer (Serial No.MY15210003). Excitation and emission slit widths
were modified to adjust the fluorescence intensity to a suitable range. Absorption spectra were
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measured on a Agilent Technologies CARY 60 UV-Vis spectrophotometer (Serial No.
MY1523004). Mass spectrometric data were achieved with HP1100LC/MSD MS and an LC/Q-
TOF-MS instruments. All pH measurements were performed using a Model PHS-3C meter. The
fluorescence quantum yields for compounds with Absolute PL Quantum Yield Spectrometer
(HAMAMATSU C11347). Fluorescence lifetime decay profiles were recorded via
Luminescence lifetime spectrometer (Edinburgh Instruments, UK). Slight pH variations in the
solutions were achieved by adding the minimum volumes of HCl or NaOH (1 M). Flash column
chromatography was performed using silica gel (100-200 mesh) and aluminum oxide (neutral.
100-200 mesh) obtained from Qingdao Ocean Chemicals. All the interferential reagents were
prepared based on published literature[31].
2.2. Determination of the detection limit
The detection limit (DL) was calculation based on the fluorescence titration of NIR-HSA in
the presence of HSA (0-1.0 ꢁM). The fluorescence intensity of NIR-HSA was measured by three
times and standard deviation of the blank measurement was achieved. The detection limit was
calculated by using detection limit was calculated with the following equation:
Detection limit = 3σ/k
Where σ is the standard deviation of the blank measurement, k is the slope between the NIR
fluorescence emission intensity (F695 nm) versus different HSA concentrations.
2.3. Determination of the quantum yield
The fluorescence quantum yields for compounds with Absolute PL Quantum Yield
Spectrometer (HAMAMATSU C11347). Operating this system is simple. Load a sample and
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press the start button to measure the photoluminescence quantum yields, excitation wavelength
dependence, PL excitation spectrum and other properties in a short time. The PL Quantum Yield
(Φ) is expressed as the ratio of the number of photons emitted from molecules (PNem) to that
absorbed by molecules (PNabs).
Φ= PNem/ PNabs
2.4. The solubility of probe NIR-HSA in PBS buffer (0.01 M, pH 7.4)
In order to explore the solubility of probe NIR-HSA, the UV-Vis absorbance at the maximum
absorbance was adopted via Agilent Technologies CARY 60 UV-Vis spectrophotometer (Serial
No. MY1523004). Following, the intensity of maximum absorbance was plotted with different
concentrations of HSA.
2.5. The detection of NIR-HSA for HSA in urine
The urine samples were taken from the healthy volunteers, and the content of serum albumin
was tested via urinary albumin assay in the Second Hospital of Dalian Medical University. The
testing samples were prepared through adding different spiked HSA concentrations in urine.
Fluorescence signal was recorded on a VAEIAN CARY Eclipse fluorescence spectrophotometer
(Serial No. MY1523004).
2.6. Synthesis of probe NIR-HSA
Compound 3 were synthesized from 2-Methylbenzothiazole and ethyl iodide by the procedure
published in literature[37]. 2-Methylbenzothiazole (500 mg, 3.93 mmol), ethyl iodide (620 mg, 4
mmol), 15 ml acetonitrile were refluxed for 8 h under N2. After cooling to room temperature and
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then 20 ml of diethyl ether poured into the reaction mixture, vacuum filter with 5 ml diethyl ether
washed three times. Yield: 840 mg (70 %). The purity of compound 3 enough to the next step.
A mixture of 3 (310 mg, 1 mmol), 4 (180 mg, 1 mmol), and acetic anhydride (10 ml) were
refluxed for 30 min. After cooling to room temperature and then 15 ml of deionized water
poured into the reaction mixture, and the product was extracted with ethyl acetate, and the orgnic
layer was washed by sodium chloride solution. The organic layer was dried with anhydrous
Na2SO4 followed by evaporation of solvent, and the resulting was yellow crude product solid.
The crude product was purified by silica gel column chromatography to afford NIR-HSA as a
black-blue solid. Yield: 300 mg (65 %) 1H NMR (400 MHz, DMSO), δ: δ 8.34 (d, J = 8.1, 1 H),
8.19 (d, J = 8.4, 1 H), 8.02 (dd, J = 14.4, 11.0, 1 H), 7.81 (t, J = 7.7, 1 H), 7.71 (t, J = 7.7, 1 H),
7.55 (d, J = 8.8, 2 H), 7.47 (d, J = 15.0, 1 H,), 7.37 (d, J = 14.5, 1 H), 7.22 (dd, J = 14.9, 11.0, 1
H), 6.80 (d, J = 8.8, 2 H), 4.74 (q, J = 7.2, 2 H), 3.05 (s, 6 H), 1.44 ( t, J = 7.1, 3 H). 13C NMR
(101 MHz, DMSO): δ 170.13, 152.28, 151.73, 148.37, 140.87, 130.61, 129.26, 127.74, 127.63,
124.23, 122.94, 122.29, 115.92, 112.25, 112.01, 111.62, 43.77, 13.76. TOF HRMS: m/z calcd
for C21H23N2S+ [M]+: 335.15760, found: 335.1582.
3．Results and discussion
Insert Scheme 1
3.1. Synthesis of fluorescent probe NIR-HSA
The probe NIR-HSA could be smoothly synthesized through three steps similar to the
previously reported literature[38], which was displayed in detail and was carefully characterized
by nuclear magnetic resonance (1H, 13C NMR) and high resolution mass spectrum (HRMS) in
the Supporting Information section. In addition, proposed sensing mechanism of probe NIR-
HSA was also demonstrsted in Scheme 1.
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3.2. Spectroscopic studies
The UV-Vis spectrum of NIR-HSA in PBS buffer (0.01 M, pH 7.4) showed that with the
concentration of HSA, the intensities of maximum absorption peak appeared linearly increasing
trend in range of 2~70 ꢁM (Figure S1), which demonstrated the probe NIR-HSA has a good
solubility in aqueous solution. Then, we investigated the photophysical properties of NIR-HSA
(10 ꢁM) in different solutions. As shown in Figure S2 and Table 1, there was basically the same
absorbance spectrum in most common organic solvents except dichloromethane as well as
fluorescence emission spectroscopy. This type of solvatochromic dye could serve as a clever
chemosensor to distinguish the chlorinated solvent from common organic solvents (Figure S3)
due to the solvatochromic effect and the electronic states of hemicyanine dye reported in
detail[39, 40].
Insert Table 1
NIR-HSA contains a strong “ pull-push” structure (with the positive charge on the nitrogen
atom as electron acceptor and a alkylamino group as electron donor). It may means that probe
NIR-HSA is particularly sensitive to polarity changes in the microenvironment of protein.
Therefore, we deduced that the probe might interact with the protein’s binding sites by
noncovalent bonding leading to great changes in the fluorescence spectrum. Free NIR-HSA (10
ꢁM) probe displayed almost nonfluorescent in PBS buffer (0.01 M, pH 7.4) possibly owing to
the effective twisted intramolecular charge transfer (TICT) mechanism of unrestricted torsional
rotation part. After 50 ꢁM HSA was added, however, a remarkable NIR fluorescence signals
gradually enhanced at around 680 nm upon excitation at 580 nm (Figure S4), as expected.
Insert Figure 1
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Insert Figure 2
Upon the addition of HSA, the emission peak at 680 nm was promptly enhanced (Figure 1a).
When 100 ꢁM of HSA was added, these changes were found to reach a plateau with the 8.83-
fold fluorescence intensity (680 nm) enhancement (Figure 1b). Moreover, in order to evaluate
the sensitivity of NIR-HSA probe (10 ꢁM) to HSA in PBS buffter (0.01 M, pH 7.4), titration
experiments of HSA at low concentration were carried out (Figure S5). Satisfactorily, the NIR
fluorescent intensities of NIR-HSA probe at centered 695 nm increased excellent linearly
(R2=0.9978) with the concentration of HSA ranging from 0.1 to 1.0 ꢁM and the detection limit
(3σ/k) was calculated as low as 26.16 nM (1.73 mg/L) (Figure 2), which implied that the probe
NIR-HSA possesses potentially ability for quantitative determination of HSA concentrations in
urine.
Insert Figure 3
Insert Figure 4
3.3. Response speed and pH influence
The time-dependent (0-450 s) fluorescence responses of probe NIR-HSA (5 ꢁM) in the
presence of HSA (10 equiv) showed that the recognition was almost completed within 5 s
(Figure 3). It is a rapid-response. Subsequently, we investigated the effects of pH on the
recognition of NIR-HSA for HSA through pH titration. As shown in Figure S6, probe NIR-HSA
displayed little changes in emission in the range of pH 4.0-9.0, which implies the detection and
fluorescence imaging would not be disturbed by humoral pH micro-environment. By comparison,
NIR-HSA/HSA complex exhibited obvious fluorescence intensity in alkaline solutions (pH 7.0-
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9.0), which is also applicable to the detection of serum albumin in urine (pH 5.0-8.0). We
speculated that the net negative charge of HSA was increased by pH of its surrounding
environment above isoelectric point (pI) of protein, strengthening the electrostatic interaction
between host and guest. Furthermore, the probe NIR-HSA and NIR-HSA/HSA complex could
remain stable as long as 70 min in PBS buffer (0.01 M, pH 7.4) (Figure 4), clearly indicating the
possibility capability of this probe for bioassay.
Insert Figure 5
3.4. Selectivity of NIR-HSA towards HSA and stability
In order to examine whether NIR-HSA probe could specifically recognize HSA, we
investigated its ability to discriminate between HSA and various bioanlytes, such as various
proteins, amino acids, and biologically relevant ions. As shown in Figure 5 and Figure S7, upon
addition of HSA, the NIR fluorescence intensity at 680 nm of NIR-HSA/HAS complex
exhibited significant fluorescence enhancement accompanied by NIR-HSA/BSA (bovine serum
albumin) complex interference because of their closely related structure and function. By
contrast, other proteins including histone, proteinaes K, collage, lysozyme, hemoglobin, and
chymotrypsinogen A induced only slight fluorescence variations under the same conditions,
which caused by microstructure and microenvironment of serum albumin inside their
hydrophobic binding pockets. We also studied the fluorescence responses towards various amino
acids, such as histidine, glycine, tryptophan, phenylalanine, serine, asparaginate, glutamine,
asparaginic acid, and cysteine (Figure S7). No distinct interference was found as well as various
biologically relevant ions (Figure S8 and S9) (cations: Na+, Mg2+, Ca2+, Zn2+, K+, Pb2+, Mn2+,
+
-
-
-
2-
2-
2-
-
NH4 , Ni2+; anions: F-, Cl-, Br-, I-, NO3 , NO2 , ClO4 , SO4 , SCN-, OAc-, HPO4 , CO3 , HCO3 ).
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Therefore, the excellent selectivity for HSA over other relative analytes demonstrated that probe
NIR-HSA has potential applications for the detection of HSA in complex biological system.
Insert Figure 6
3.5. Recognition mechanism
To gain insights into the binding properties of NIR-HSA on HSA is very essential to better
understand the working principle of this fluorescent probe. The Job’s plot assay was carried out
to confirm the maximum number of probe NIR-HSA combining with HSA. As shown in Figure
6a, the changes in fluorescence intensity occured up to 1:1 [HSA]/[NIR-HSA] ratio,
demonstrating that the stoichiometry of NIR-HSA/HSA complexes is 1:1. These results
foreboded that probe NIR-HSA was mainly combined with one site of HAS.
The previous research work revealed that serum albumin has two major selective binding sites,
namely, site I and II. The binding affinity of site I is mainly via hydrophobic interactions. On the
contrary, site II always involves a series of combination process, such as hydrogen bonding,
hydrophobic, and electrostatic effects[41]. We used the unambiguous site-selective drug (e.g.
warfarin for site I and ibuprofen for site II) to further investigated which the binding site of NIR-
HSA on HSA through the ligand displacement strategy[42]. As shown in Figure S15, the
fluorescence intensity of NIR-HSA/HSA complex was induced obvious decrease via the
addition of warfarin known site I specific drugs, while with the addition of ibuprofen there was
almost no effect. So, the displacement experimental assay clearly indicated that the binding of
NIR-HSA on HSA was site I.
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Due to unrestricted torsional rotation part existing in probe NIR-HSA, TICT emission is very
weak in PBS buffer (0.01 M, pH 7.4), which could be rationalised considering the nature of
TICT state[43]. When HSA was added into mixing system, probe NIR-HSA entered into the
hydrophobic cavity of HSA with low polarity and strong space steric hindrance. With the
increase of the space steric hindrance, the rotation of C-C single bond in conjugated chain and C-
N single bond in doner moiety was hindered, which restrained the transition from the locally
exicted state to the TICT state. Therefore, the fluorescence emission intensity of probe NIR-
HSA/HSA complex was obtained obvious enhancement. This phenomenon is well explained our
above test results and the TICT mechanism is also confirmed by temperature (Figure S10-12)
and viscosity (Figure S13-14) effects. In addition, the lifetimes of NIR-HSA before and after
binding with HSA were carried out. In the absence of HSA, probe NIR-HSA shown the
fluorescence lifetime of τ1 = 0.104 ns (95.47%) and τ2 = 0.104 ns (95.47%). After adding HSA
into PBS buffter (0.01 M, pH 7.4), the NIR-HSA/HSA complex displayed longer fluorescence
lifetime of τ1 =0.7690 ns (46.037 %) and τ2 =2.6485 ns (53.97 %) (Figure 6b). These results
further verified the binding of NIR-HSA on HSA caused by restricting the rotation of NIR-HSA.
3.6. Quantitative detection of HSA in urine
To further assess the performance of NIR-HSA for the actual sample testing, we investigated
the applicability of this probe for HSA in urine, subsequently. It had been reported that the
concentrations of serum albumin in body fluids could be considered as a reliable healthy
indicator in clinical diagnosis. In the normal population, the content of serum albumin less than
30 mg/L in urine, which is lower than the detection limit of fluorescence probes previously
reported. In view of the excellent performance of NIR-HSA, we tried to use this probe for the
detection of serum albumin in urine. The urine samples were taken from the healthy volunteers,
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and the concentrations of serum albumin was tested as 27.00 mg/L (0.41 ꢁM, much higher than
the detection limit of 26.16 nM of NIR-HSA) through urinary albumin assay in the Second
Hospital of Dalian Medical University. On the contrary, for patients with kidney disease, the
concentrations of serum albumin are more than 200 mg/L in urine. In our titration experiment,
with the addition of HSA in pure urine, the fluorescence signal of NIR-HSA/HSA complex
gradually enhanced (Figure 7a). Gratifyingly, as shown in Figure 7b, an excellent linear
correlation (R2 = 0.9994) was obtained with a large range of 0.41- 41.41 ꢁM, which indicated
that probe NIR-HSA possessed quantitative detection of serum albumin in low concentration
range. For detecting HSA in real urine samples, two urine samples were prepared via spiking
HSA artificially in healthy urine of a volunteer. Satisfactory recoveries were obtained, as seen
from Table 2, which demonstrated that probe NIR-HSA could effective and accurate for
quantitative detection of trace HSA in biosystem without hampered by interference of other
biological substances.
Insert Figure 7
Insert Table 2
4．Conclusions
In conclusion, through the introduction of N,N-dimethyl arylamine part into the classical
structure of hemicyanine as specificity recognition site based on TICT mechanism, we have
synthesized a NIR fluorescent probe NIR-HSA for the recognition of HSA with rapid response,
high selectivity, stability and excellent sensitivity. This probe exhibited an outstanding
thermostability under 60 oC as long as 2 h, which indicated the probe has good heat resistance in
PBS buffer (0.01 M, pH 7.4). Furthermore, the detection of HSA in real human urine via
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fluorometric method further validated that the probe can function as an efficient reporter for
early warning of critical diseases including acute renal failure, diabetic nephropathy, liver injury
and sepsis.
Acknowledgments
This work was financially supported by National Natural Science Foundation of China
(21136002, 21421005, 21376037, 21422601 and 21406028),973 Program of China (2013CB733
702), and 863 program of China (2013AA020202).
Supporting Information Available
Solubility, additional spectroscopic, fluorescence response, HSA titration, pH titration, selectitiy
1
of probe, temperature and viscosity test, displacement experimental, HRMS spectra , H-NMR
13
and C-NMR spectra are available in Supporting Information. This material is available free of
charge.
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Table of Contents
1. Scheme 1 Synthetic procedures of probe NIR-HSA and proposed sensing mechanism for
HSA.
2. Table 1 Photophysical properties of NIR-HSA (10 ꢁM) in different solvents.
3. Figure 1 (a) Fluorescence spectra changes of probe NIR-HSA (10 ꢁM) in PBS buffer (0.01
M, pH 7.4) after incubation with different concentrations of HSA (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 15.0, 20.0, 30.0, 50.0, 70.0, 90.0, 95.0, 100.0,
105.0, and 110.0 ꢁM). (b) Fluorescence intensity at 680 nm enhancement of NIR-HSA (10 ꢁM)
as a function of HSA (0-120 ꢁM) with excitation at 580 nm in PBS buffer (0.01 M, pH 7.4).
4. Figure 2 Fluorescence intensity at 695 nm enhancement of NIR-HSA (10 ꢁM) as a function
of HSA (0-1.0 ꢁM) with excitation at 580 nm in PBS buffer (0.01 M, pH 7.4).
5. Figure 3 Reaction-time profiles of NIR-HSA (5 ꢁM) in the presence of 10 equiv HSA in PBS
buffer (0.01 M, pH 7.4). Time range: 0-90 min, fluorescence signal was collected at 680 nm with
excitation wavelength at 580 nm.
6. Figure 4 The stability of time dependence of NIR-HSA (10 ꢁM) with 10 equiv HSA (black
line) or not (red line) in PBS buffer (0.01 M, pH 7.4) were measured with a spectrophotometer
every 5 min from 0 to 70 min with excitation at 580 nm.
7. Figure 5 The selectivity of probe NIR-HSA (10 ꢁM) toward various various analytes. Data
shown are for 5 equiv HSA and 0.5 mg/mL for all other analytes. All data were obtained after
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incubation in PBS buffer (0.01 M, pH 7.4) with the analytes at room temperature. Collected
emission at 680 nm with excitation at 580 nm.
8. Figure 6 (a) Job’s plot of probe NIR-HSA for HSA with [NIR-HSA] + [HSA] =10 ꢁM. (b)
Fluorescence lifetime decay profiles of NIR-HSA (10 ꢁM) with 10 equiv HSA in PBS buffter
(0.01 M, pH 7.4). Blue line represents free probe NIR-HSA and red line represents probe NIR-
HSA/HSA complex in PBS buffter (0.01 M, pH 7.4).
9. Figure 7 (a) Fluorescence responses of NIR-HSA (10 ꢁM) toward various additional HSA in
urine. (b) Fluorescence signal of NIR-HSA (10 ꢁM) was collected at 700 nm with excitation
wavelength at 580 nm in pure urine.
10. Table 2 The capacity detection of NIR-HSA (10 ꢁM) for HAS in pure urine.
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Scheme 1 Synthetic procedures of probe NIR-HSA and proposed sensing mechanism for
HSA.
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Table 1 Photophysical properties of NIR-HSA (10 ꢁM) in different solvents.
[a]
[b]
[c]
Solvent
λabs(nm)
λem(nm)
ε(×105 L/mol cm
)
Φ[d]
△λ(nm)
DCM
EtOH
621
556
555
561
554
551
553
522
702
702
701
708
706
705
714
701
81
0.538
0.392
0.411
0.367
0.376
0.386
0.362
0.285
0.12
0.04
0.02
0.09
0.05
0.02
0.03
0.01
146
146
148
152
154
161
179
MeOH
1,4-Dioxane
EA
ACN
DMF
PBS buffer[e]
[a, b] λmax of the absorption and emission spectra in nm. [c] Molar extinction coefficient. [d] Fluorescence quantum yield. [e]
PBS buffer (0.01M, pH = 7.4).
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(a)
(b)
Figure 1 (a) Fluorescence spectra changes of probe NIR-HSA (10 ꢁM) in PBS buffer (0.01 M,
pH 7.4) after incubation with different concentrations of HSA (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 15.0, 20.0, 30.0, 50.0, 70.0, 90.0, 95.0, 100.0, 105.0,
and 110.0 ꢁM). (b) Fluorescence intensity at 680 nm enhancement of NIR-HSA (10 ꢁM) as a
function of HSA (0-120 ꢁM) with excitation at 580 nm in PBS buffer (0.01 M, pH 7.4).
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Figure 2 Fluorescence intensity at 695 nm enhancement of NIR-HSA (10 ꢁM) as a function of
HSA (0-1.0 ꢁM) with excitation at 580 nm in PBS buffer (0.01 M, pH 7.4).
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Figure 3 Reaction-time profiles of NIR-HSA (5 ꢁM) in the presence of 10 equiv HSA in PBS
buffer (0.01 M, pH 7.4). Time range: 0-90 min, fluorescence signal was collected at 680 nm with
excitation wavelength at 580 nm.
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Figure 4 The stability of time dependence of NIR-HSA (10 ꢁM) with 10 equiv HSA (black line)
or not (red line) in PBS buffer (0.01 M, pH 7.4) were measured with a spectrophotometer every 5
min from 0 to 70 min with excitation at 580 nm.
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Figure 5 The selectivity of probe NIR-HSA (10 ꢁM) toward various various analytes. Data
shown are for 5 equiv HSA and 0.5 mg/mL for all other analytes. All data were obtained after
incubation in PBS buffer (0.01 M, pH 7.4) with the analytes at room temperature. Collected
emission at 680 nm with excitation at 580 nm.
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