Real-Time Tracking the Synthesis and Degradation of Albumin in Complex Biological Systems with a near-Infrared Fluorescent Probe

Abstract

In this study, a novel fluorescent detection system for biological sensing of human albumin (HA) was developed on the basis of the pseudoesterase activity and substrate preference of HA. The designed near-infrared (NIR) fluorescent probe (DDAP) could be effectively hydrolyzed by HA, accompanied by significant changes in both color and fluorescence spectrum. The sensing mechanism was fully investigated by fluorescence spectroscopy, NMR, and mass spectra. DDAP exhibited excellent selectivity and sensitivity toward HA over a variety of human plasma proteins, hydrolases, and abundant biomolecules found in human body. The probe has been successfully applied to measure native HA in diluted plasma samples and the secreted HA in the hepatocyte culture supernatant. DDAP has also been used for fluorescence imaging of HA reabsorption in living renal cells, and the results show that the probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. Furthermore, DDAP has been successfully used for real-time tracking the uptaking and degradation of albumin in ex vivo mouse kidney models for the first time. All these results clearly demonstrated that DDAP-based assay held great promise for real-time sensing and tracking HA in complex biological systems, which would be very useful for basic researches and clinical diagnosis of HA-associated diseases.

Human carboxylesterases: a comprehensive review

“…Based on the substrate specificities of both CES1 and CES2, some optical probe substrates have been recently developed for assessing the real activities of CES1 or CES2 in complex biological systems ( Supplementary Information Table S2 ) 49 , 51 , 58 , 59 , 60 , 61 , 62 , 63 , 64 . These optical probes provide practical and efficient tools for high-throughput screening (HTS) of CES modulators in cell/tissue preparations or even in living cells, due to the inherent advantages including non-destructive, highly sensitive, easily managed, and applicable to HTS assay 49 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 .

Figure 3 Substrate specificity of CES1.

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A near-infrared excitation/emission fluorescent probe for imaging of endogenous cysteine in living cells and zebrafish

“…The reaction progress was monitored by thin-layer chromatography (TLC) on silica gel plates (60F-254) that were observed by means of UV light. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were obtained on a Bruker AVB-500 spectrometer taking tetramethylsilane (TMS) as an internal standard (Bruker Co., Switzerland). High-resolution mass spectra (HRMS) were acquired on a 6500 Accurate-Mass Q-TOF spectrometer coupled to an Agilent HPLC 6500 series (Agilent Co., America).…”

“…The synthetic routes of DXM are shown in Scheme 2 and Scheme S1 (see Electronic Supplementary Material, ESM), and the products were characterized by 1 H NMR and 13 C NMR (Figs. S1-S4, see ESM).…”

“…Near-infrared (NIR) fluorescent probe with long emission wavelength at 650-900 nm has obvious advantages, such as low autofluorescence interference and minimum photodamage to biological samples [9][10][11][12][13][14]. Recently, numerous NIR emission fluorescent probes have been developed [15][16][17][18][19][20].…”

“…) 13. C NMR (126 MHz, DMSO-d 6 ) δ 169.80, 160.89, 157.74, 153.69, 142.04, 141.70, 130.94, 128.75, 128.59, 127.02, 126.48, 125.54, 123.62, 115.50, 113.91, 113.53, 111.73, 105.29, 101.85, 34.99, 28.48, 24.23, 20.06.…”