Human serum albumin (HSA) is the most abundant protein in blood plasma; generally, its concentration is maintained at levels 35–55 g/L in normal serum and 30 mg/L in normal urine. HSA has multiple functions in the human body: it plays a major role in maintaining the oncotic pressure and in the transport of various drugs and metabolites [1
]. Thus, an abnormal HSA level in a body fluid can be closely associated with many health problems. Hypoalbuminemia, defined as a very low HSA concentration in serum, is frequently observed in patients with malnutrition and cirrhosis [7
]. In addition, microalbuminuria (high level of HSA in urine) is related to kidney disease in diabetes mellitus and hypertension [9
]. HSA concentration is a key health-related indicator and its quantitation in biological fluids is of great significance for diagnosis [11
To date, various HSA detection methods have been developed, such as immunoassay, LC-MS/MS-based proteomics, capillary electrophoresis, colorimetric, and fluorescent probes [12
]. In comparison with other detection methods, fluorescent probes are favored because of advantages including a rapid, non-invasive, and sensitive response. Accordingly, several fluorescent probes have been reported using many different types of fluorophore such as tetraphenylethylene, squaraine dye, and indolium derivative, which is based on aggregation-induced emission (AIE), molecular rotor containing donor-π-acceptor (D-π-A) structured fluorophore, and self-assembled nanoparticles [22
]. However, there remain some problems to be solved, such as the low sensitivity and selectivity toward HSA. They could successfully detect high levels of HSA in serum but were not applicable to detect trace amounts of HSA in urine. Furthermore, most could hardly distinguish HSA from bovine serum albumin (BSA), which has a similar structure but different functional level.
To develop a novel fluorescent probe having high sensitivity and selectivity for HSA, we utilized the fluorescence-based, high-throughput screening of a set of novel fluorophore derivatives against plasma proteins (Scheme 1
). Recently, we have reported novel fluorophore scaffolds: thieno[3,2-b
)-one derivatives [hereafter, collectively referred to as KIOST-Fluor (KF)], synthesized via a series of reactions including the Suzuki-Miyaura cross-coupling reaction and a regioselective aza-[3+3] cycloaddition of 3-aminothiophenes with α,β-unsaturated carboxylic acids [38
]. By varying the functional group on the KF scaffold, the fluorophore showed different photophysical properties; in particular, they exhibited remarkable environmental sensitivities.
Herein, we report a new fluorescent probe for the highly selective and sensitive detection of HSA using the environment-sensitive properties of the KF system. We have demonstrated the HSA-sensing performance of this probe in terms of sensitivity, selectivity, binding properties, and applicability; this fluorescent probe exhibited superior selectivity over BSA with a 160-fold fluorescence intensity increase. In addition, we explored the application of this fluorescent probe to the detection of trace amounts of HSA spiked in artificial urine. A rapid-response and highly sensitive probe for HSA using this novel fluorophore scaffold would greatly contribute to clinical diagnosis.
2. Materials and Methods
2.1. Materials and Instruments
All the biological analytes, HSA, BSA, γ-globulin from human blood, fibrinogen from human plasma, transferrin from human, hemoglobin from human, haptoglobin from human, chymotrypsin from bovine pancreas, trypsin from bovine pancreas, lysozyme from pooled human plasma, and butyrylcholinesterase (BChE) from equine serum were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. High-throughput screening results were recorded by a Cytation 3 Multi-Mode Reader (BioTek, Winooski, VT, USA) using a 96-well plate. UV-vis spectra were recorded by a JASCO V-630 Spectrophotometer (JASCO Inc., Easton, MD, USA), and fluorescence spectra were recorded by a Cary Eclipse Fluorescence Spectrophotometer (Agilent, Santa Clara, CA, USA) using a quartz cell of path length 1 cm.
2.2. Synthesis of KF Derivatives (Thieno[3,2-b]pyridine-5(4H)-one Derivatives)
General synthetic procedure for KF derivatives can be found in our previous report [38
]. The detailed synthetic procedures of KF 1
were previously reported and that of KF 13
and KF 14
were described in Supplementary Material
2.3. Fluorescence-Based High-Throughput Screening of KF Derivatives against Major Plasma Proteins
Stock solutions of all KFs, 1–14, (50 μM) were prepared in dimethyl sulfoxide (DMSO). Samples containing each fluorophore (5 μM, 10% DMSO) under different buffer conditions (pH 5 acetate, pH 7 Tris, pH 9 Tris; 20 mM) were prepared in 96-well plates and the absorption wavelengths of the fluorophores were recorded by the Cytation 3 Multi-Mode Reader.
Stock solutions of five major plasma proteins (100 μM) including HSA, BSA, γ-globulin, fibrinogen, and transferrin were prepared in distilled water. Fluorophores-containing samples (5 μM in 10% DMSO), with and without proteins (10 μM), were prepared under various buffer conditions (pH 5 acetate, pH 7 Tris, pH 9 Tris; 20 mM) in a 96-well plate; fluorescence spectra were recorded by the Cytation 3 Multi-Mode Reader at different excitation wavelengths (λex) (370, 400, 420, and 450 nm).
2.4. Comparison of Fluorophores as Human Serum Albumin (HSA) Probe Candidates in Terms of Selectivity
Stock solutions of KF 4 and 11 were prepared in DMSO, and stock solutions of HSA and BSA were prepared in distilled water. Firstly, UV-vis spectra of the samples containing each KF 4 and 11, (5 μM in 10% DMSO) with and without HSA (1, 5, 10, 20, 50, and 100 μM) or BSA (50, 100 μM) were recorded in pH 9 buffer (Tris, 20 mM) using an UV-vis spectrophotometer. Then, blanks, each containing only one fluorophore (5 μM), and samples, containing each fluorophore (5 μM, 10% DMSO) with different concentrations of albumin (HSA or BSA, 5, 10, and 20 μM), were prepared in a pH 9 buffer solution (Tris, 20 mM). The fluorescence spectra were recorded by the fluorescence spectrophotometer under excitation at 455 nm for 4 and at 440 nm for 11.
2.5. The Effects of pH and DMSO on the HSA-Sensing Behavior of 4
To investigate the effect of pH on the HSA-sensing performance using 4, the fluorescence intensity at 546 nm of samples containing fluorophore 4 (5 μM, 10% DMSO) in absence and presence of albumin (HSA or BSA, 10 μM), in different pH buffers (pH 5, 6 acetate, pH 7, 7.4, 8, 9 Tris; 20 mM), were recorded by the fluorescence spectrophotometer under excitation at 455 nm. To investigate the effect of DMSO on the HSA-sensing performance using 4, the fluorescence intensity at 546 nm of samples containing fluorophore 4 (5 μM) in absence and presence of albumin (HSA or BSA, 10 μM) with different ratios of DMSO (1, 5, 10%) were recorded under excitation at 455 nm. The experiment was repeated three times.
2.6. Determination of the Limit of Detection (LOD)
The fluorescence spectra of 4 (5 μM, 10% DMSO) with various concentrations of HSA (0, 0.05, 0.1, 0.3, 0.5, 0.8, 1, 3, 5, 10, 20, and 50 μM), in pH 9 buffer (Tris, 20 mM), were recorded by the fluorescence spectrophotometer under excitation at 455 nm; the titration experiment was repeated three times. The LOD was calculated by using the 3σ/slope rule based on the titration experiments, in which σ is the standard deviation of the blank measurements.
2.7. Determination of Quantum Yield of 4
To determine the quantum yield (Φ) of 4
in absence or presence of HSA, fluorescein in 0.1 M NaOH (Φref
= 0.95) was used as a reference. The quantum yield was calculated according to the following equation:
is the slope of the line obtained from the plot of the integrated fluorescence intensity versus absorbance and n
is the refractive index of solvent [27
2.8. Selectivity Test of 4 toward HSA over Plasma Proteins
The fluorescence intensity at 546 nm of a blank containing only 4 (5 μM, 10% DMSO), in pH 9 buffer (Tris, 20 mM), and of samples containing 4 with each plasma protein (HSA, BSA 200 mg/L; γ-globulin, fibrinogen, and transferrin 50 mg/L; hemoglobin, haptoglobin, chymotrypsin, trypsin, and lysozyme 10 mg/L; and BChE 10 U/L) were recorded by the fluorescence spectrophotometer under excitation at 455 nm. The experiment was repeated three times.
2.9. Job’s Plot Analysis
Samples were prepared by mixing 4
with HSA at different ratios in pH 9 buffer (Tris, 20 mM), while maintaining the overall concentration ([4
] + [HSA]) at 10 μM. Then, the fluorescence intensities of the samples at 546 nm were recorded by the fluorescence spectrophotometer under excitation at 455 nm. The experiment was repeated three times [32
2.10. Determination of the Dissociation Constant (Kd) of HSA and 4
The dissociation constant was calculated based on the method presented in previous literature reports based on the HSA titration results using 4
(5 μM, 10% DMSO) in pH 9 buffer (Tris, 20 mM) [27
2.11. Assignment of the Binding Site of 4 on HSA
A stock solution of 4 in DMSO (100 μM) was prepared as well as those of site-specific binding drugs (Ibuprofen, Digitoxin, Warfarin, salicylic acid, 5 mM) in DMSO. Mixtures of HSA (10 μM) with each drug (500 μM) in pH 9 buffer (Tris, 20 mM) were incubated for 3 min, then 4 (5 μM, 10% DMSO) was added to the mixture and the fluorescence intensities at 546 nm were recorded by the fluorescence spectrophotometer under excitation at 455 nm. The experiment was repeated three times.
2.12. Identification of the Sensing Mechanism of 4 toward HSA
Using the Gaussian 09 program, calculations were performed to investigate the electronic transition of 4. The identification of molecular orbitals (MOs) was accomplished by optimizing the geometry of 4 via a density functional theory (DFT) method, using the B3LYP hybrid functional and the 6-311G(d) basis set in the conductor-like polarizable continuum model (CPCM) solvent model. To demonstrate the viscosity-dependence of the fluorescence enhancement of 4, the fluorescence intensity of 4 at 546 nm upon increasing the fraction of glycerol (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%), in pH 9 buffer (Tris, 20 mM), were recorded by the fluorescence spectrophotometer under excitation at 455 nm.
Molecular docking is the widely adopted computational technique to identify the binding and intermolecular interactions between small molecules and biological macromolecules. The three dimensional structure of human and bovine serum albumin was downloaded from Protein DataBank database from RCSB website (PDB Id: 2BXG and 6QS9, respectively). The protein structures and probe 4
was preprocessed using AutoDockTools1.7.6 package with standard protocol prior to docking in AutoDock4.2 [40
]. The docking was carried out with only polar hydrogens and Gasteiger charges. The grid box was constructed (90 × 90 × 90 points with grid spacing of 0.375 Å) to accommodate both primary and secondary binding site of human serum albumin because we expected that probe 4
would bind at Ibuprofen binding site. Molecular docking was carried out with lamarkian genetic algorithm for 100 runs with initial population size of 300 and number of evaluations of 1,000,000. The docking results were visually inspected with the help of Discovery Studio Visualizer [42
2.13. Quantitative Detection of HSA in Artificial Urine
Artificial urine was prepared according to previous literature reports [43
], and HSA stock solutions of various concentrations (5, 10, 25, 50, 80, 100, and 200 mg/L) were prepared both in artificial urine and in distilled water. To quantify the amount of HSA, the samples were prepared by mixing 4
(5 μM, 10% DMSO) with spiked HSA (20% artificial urine) in pH 9 buffer (Tris, 20 mM), and the fluorescence intensities of the samples were recorded by the fluorescence spectrophotometer under excitation at 455 nm. Then, the results were compared with those obtained in distilled water. The experiment was repeated three times.