| Background and Purpose:Human albumin(HA)is the most abundant plasma protein in the human body with a plasma concentration of 35-55 g/L.HA is synthesized exclusively in hepatocytes,and normal individuals are synthesized at a rate of 12-25 g/d.Multiple studies have clearly shown that the levels of HA in body fluids are closely associated with the progression of many human diseases,including post-menopausal obesity,kidney disease,diabetes mellitus,hypertension and liver injury.Hence,the precise quantitative determination of HA in biological fluids is of importance in both clinical diagnosis and biomedical researches.In this paper,a specific fluorescent probe substrate was constructed by using HA as target molecule,and the corresponding fluorescence analysis methods were established for the rapid detection of target molecule in complex system.The newly developed probes and the proposed fluorescent-based methods provide foundation for exploring the in vivo functionsof target molecules,metabolic regularity and the role of related diseases to provide.Methods:1.The spectral changes of the reaction between the probe and the albumin were investigated by using fluorescence analysis and ultraviolet spectrophotometry.2.The selectivity of the probe was examinedby phenotyping assays,the sensitivity was evaluated by measuring the limit of detection(LOD)value,and the biological stability of probe was investigated by the interference experiment of small molecules.3.The liquid mass spectrometry based proteomics method was used to examine the detection mechanism of probe.4.The fluorescence analysis method constructed by the probe was used to detect the albumin in cell supernatant and human plasma,and compared with other detection methods.5.The biological process of uptake of albumin in renal cells was visualized by fluorescence imaging.Results:1.After the probe DDABP was metabolized by albumin,a significant fluorescence signal is generate at 662 nm.Accompanied with the fluorescence turn on response,the addition of HA could also trigger a dramatic change in UV absorption spectrum.2.The probe substrate DDABP displayedhigh selectivity,and could only be hydrolyzed byalbumin,while other plasma hydrolases and protein molecules do not cause fluorescence signal generation;and the probe hasvery high sensitivity,with a detection limit of 6.51g/m L;theperformance of probe display high antiinterferenceability and could not be influenced by the addition of common biological metallic ions or amino acids.3.The detection of HA was based on the pseudo-esterase activity of HA,after hydrolyzed by albumin,the ester bond of DDABP was disconnected and the alcohol group dissociates,and the carboxylic acid part is covalent to the lysine residue of the albumin molecule.4.The probe DDABP can measure trace albumin secreted by hepatocytes,and the results are very close to proteomics.Meanwhile,the plasma albumin concentration measured by the new developed method was in close agreement with that determined by the BCGmethod.5.The probe DDABP can be hydrolyzed by the uptake albumin in cells,brings significant fluorescence signal changes,and can be used for visual analysis of renal cell endocytosis of albumin dynamic process.Conclusion:The probe DDABP displays high selectivity,sensitivity and anti-interference ability,can be used as a promising tool for the study of HA-mediated biological processes and is of great significance in exploring basic research and clinical diagnosis of HA-related diseases. |
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