Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Twisted intramolecular charge transfer (TICT) based fluorescent probe for lighting up serum albumin with high sensitivity in physiological conditions
Graphical abstract
Introduction
Human serum albumin (HSA), accounting for 50–60% of total plasma protein, is the most abundant protein in human blood [1,2]. With the special heart-shaped three-dimensional structure, HSA exerts a great influence on regulation of physiological activities, including balancing plasma osmotic pressure [3,4], modulating clotting system [5] and vascular permeability [6]. The crystal structure of albumin shows that this protein contains an equilateral triangle with sides of ~8 nm and a depth of 3 nm [7]. HSA possesses excellent water-solubility and stability [[8], [9], [10]], arising from a large content of water-soluble amino acids, enabling it to be the main carrier of many endogenous bio-molecules (i.e., fatty acids, thyroxine, ferroheme and bilirubin) [[11], [12], [13], [14]] in human body. Additionally, many therapeutic drugs (i.e., paclitaxel and all-trans retinoic acid) [15] or probes could also be loaded into the cavity of HSA by the non-covalent sorption and hydrophobic interaction [[16], [17], [18], [19], [20]], which makes a great improvement to the uptake efficiency [[21], [22], [23]].
HSA is mainly synthesized by hepatocytes and plays a significant role in blood circulation [24]. The normal level of HSA in human serum is in the range of 35–50 g/L, whereas it is below 30 mg/L in urine because of the kidney with dialysis function [25]. HSA with excessive low concentration in serum indicates the possibility of cirrhosis or chronic hepatitis [3], and the presence of HSA in urine can be considered as a bio-marker for kidney and cardiovascular diseases [26]. Hence, HSA level in body fluids can be considered as an indicator for health evaluation. It is challenging and highly desired to prepare effective probes for quantitative detection of HSA with high sensitivity [[27], [28], [29], [30], [31]].
To date, enormous methods for HSA detection have been developed, including LC-MS/MS based proteomics, immunoassay, capillary electrophoresis and colorimetry [[32], [33], [34], [35]]. However, applications of proteomics and immunoassay are quite limited because of the requirement for expensive instruments, complex operation process and high labor-cost, and colorimetry is unsuitable for quantitative trace detection. Fluorescent sensing assay with high sensitivity and handy operation is therefore considered to be promising for quantitative analysis of bio-analytes, and fluorescent probes for HSA detection have been extensively studied [24,36]. Most of the reported probes were lack of sensitivity [[37], [38], [39]], including bromocresol green (BCG), a worldwide dye probe for clinical diagnosis, which could only be applied for bio-samples with high-level HSA and is unworkable for samples of trace amounts of HSA. It is therefore of great demand to develop a fluorescence probe for HSA sensing with high sensitivity and selectivity to monitor HSA level in complex cellular environment.
With these considerations in mind, we herein put forward a novel probe 1-ethyl-4-[2-[4-(diethylamino)-2-hydroxyphenyl]ethenyl]]-pyridinium salt (DEHP) for HSA by a handy one-step reaction, accompanied by a control compound DMP without a hydroxyl group. The inherent fluorescence of probe DEHP is essentially negligible under physiological conditions assigned to the well-developed twisted intramolecular charge transfer (TICT) protocol [[40], [41], [42]]. An intriguing fluorescence enhancement is triggered as the addition of HSA, on account of the inhibited TICT procedure when DEHP enters the hydrophobic cavity of protein HSA. This probe claims detection of limit as low as 4.8 nM, which is lower than that reported in most literatures. The job-plot experiments confirmed the binding ratio of HSA and DEHP to be 1:1. Under an overall study in terms of site competition with drugs, temperature-dependent spectral monitor and enzyme-hydrolysis investigation, the sensing pattern of HSA by probe DEHP is determined to be related to the hydrophobic interaction inside the HSA cavity and the well-known specific site-binding. Moreover, this protocol was proved to govern good biocompatibility, and was readily achievable in cell imaging.
Section snippets
Reagents and materials
All the reagents and solvents applied for synthesis were purchased from commercial suppliers (J & K Scientific Ltd., Energy Chemical and Aladdin reagent co., Ltd.) and used as received without further purification unless otherwise stated. The water used for optical spectra was deionized water produced from Milli-Q Advantage A10 with a conductivity of 0.055 μS/cm. HSA, bovine serum albumin (BSA), warfarin, alkaline phosphatase (ALP), glucose (Glu), urea, telomerase, inorganic pyrophosphatase
General properties of probe DEHP
The fluorescence quenching procedure, arising from the forbidden emission transformation from the zero vibrational level of TICT state, was employed to build up a “light-on” probe for HSA with depressed background. According to the reported TICT molecules, a structural model of “D-π-A” is required for the construction. N, N-diethylamino group, well-known as an electron-rich unit, was employed as a strong electron donor (D) in this TICT probe DEHP (Scheme 2). As for the pyridinium cation, which
Conclusion
In summary, a TICT-based probe DEHP for HSA has been built up herein, with a hydroxyl group to promote the optical properties, which was readily synthesized by a condensation reaction. The initial fluorescence quenching of this probe in aqueous medium was clarified to originate from the TICT procedure, leading to a depressed optical background. The fluorescence response was lighted on upon the additional HSA by restricting the free intramolecular rotation, producing a low detection limit of
Acknowledgements
We gratefully acknowledge financial support from the National Natural Science Foundation of China (21375070, 21422504), the Fund Project for Shandong Province Key Research and Development Program of China (2017GGX20121), and the support from Key Laboratory of Spectrochemical Analysis & Instrumentation (Xiamen University), Ministry of Education (SCAI1702, SCAI1703).
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