新近红外染料5,5′-二羧基-1,1′-二磺丁基-3,3,3,′3′-四甲基吲哚三碳菁分光光度法直接测定.pdf

- 1 -Spectrophotometric Determination of Albumin in Serum Using a Novel Near-Infrared Cyanine Dye, 5,5’-Dicarboxy-1,1’-Disulfobutyl-3,3,3’,3’- Tetramethylindotricarbocyanine Wei-Rong Li, Xiao-Feng Guo, Hua-Shan Zhang, Hong Wang*** Department of Chemistry, Wuhan University, Wuhan 430072, P.R.C E-mail：wanghong@ Abstract Near-infrared (NIR) dyes (λem > 650 nm) are very promising for the analysis of biological samples, because their long wavelengths allow for minimization of interferences from biomolecule matrices. In this paper, a novel NIR dye, 5,5’-dicarboxy-1,1’-disulfobutyl-3,3,3’,3’-tetramethylindotricarbocyanine (DCDSTCY) has been developed for the spectrophotometric assay of total proteins in serum. Under acidic conditions, the binding of DCDSTCY to proteins caused a new peak at 878 nm, the height of which was proportionable to the concentration of proteins. The linear range was found to be 0.04-0.5 µg mL-1 for bovine serum albumin (BSA) and human serum albumin (HAS) with the detection limits of 5 ng mL-1, respectively. The maximum binding number of BSA with DCDSTCY was measured as 133. The proposed method has been applied to the quantitation of total proteins in serum with recoveries of 96.6-104%. Keywords: Near infrared (NIR) dye; 5,5’-dicarboxy-1,1’-disulfobutyl-3,3,3’,3’- tetramethylindotricarbocyanine (DCDSTCY); Spectrophotometry; bovine serum albumin (BSA); human serum albumin (HAS); Serum 1. Introduction Proteins are foundation of the life, the most characteristic chemical compound in the living cell, and an important research object of biochemical. Some proteins are also biomarkers for diseases. Therefore, determination of proteins with rapid and reliable methods is of great importance in analytical biochemistry, clinical diagnosis, nutritional chemistry and food research. Commonly used methods for determining total proteins are developed quickly. At first, total proteins are determined by the examination of total nitrogen in samples, for example, Kjeldahl digestion follows by microtitration[1, 2], Dumas combustion [3, 4], and modified Lassaigne procedures [5] with their modifications, which are based on nitrogen liberation as ammonia or elementary. Later, it has been found that proteins can combine with some dyes by static attraction or hydrogen bond in the solution because they are biomacromolecules and carry O and N atoms which have strong electronegativity. Thereby, many spectrophotometric methods based on dyes, such as bromphenol blue [6, 7], bromcresol-green method [8, 9], Coomassie brilliant blue [10-13], alizarin red S [14, 15] and arsenazo III [16, 17] for direct determination of total proteins have been developed. Recently, the near-infrared (NIR) dyes attracted more and more attention in the biological and medical fields because of their unique properties and a series of NIR dyes have been synthesized for analytical applications, including Cy5 [18, 19], Cy7 [20-22], phthalocyanine [23], etc. The maximal absorptions of them are in the NIR region, where biomolecules have no absorption. Therefore, the NIR spectral region is clean of complex biological interferences and the NIR dyes have features of high sensitivity and selectivity for biological samples [24, 25]. In spite of advantages of good reproducibility, convenient operation, and low cost for spectrophotometric method, the application of NIR dyes in spectrophotometry is few. In our previous work, 1,1’-disulfobutyl-3,3,3’,3’-tetramethylindotricarbocyanine (DSTCY) has been used as a new NIR probe for the successful spectrophotometric analysis of proteins and DNA [22, 26]. In order to further improve the sensitivity and lengthen the absorbance of DSTCY, two carboxyl groups were introduced and 5, 5’ -dicarboxy-1,1’-disulfobutyl-3,3,3’,3’-tetramethylindotricarbocyanine (DCDSTCY) has been synthesized accordingly. As expected, compared with DSTCY [22], its absorption maximum was red shifted (λmax=755 nm) and maximum binding number to bovine serum albumin (BSA) was larger. Using DCDSTCY as a spectrophotometric probe, total proteins in serum have been quantitated rapidly and sensitively. 2. Experimental - 2 -2.1. Apparatus A UV-1601 spectrophotometer (Shimadzu, Japan) and a 722 spectrophotometer (Shanghai, China) equipped with 1 cm × 1 cm cells were used for recording spectra. A DF-801 pH meter (Zhongshan University, China) was used for pH measurement. 2.2. Reagents 2.0×10-4 mol L-1 DCDSTCY (synthesized in our lab) solution: 0.0146 g DCDSTCY was dissolved in 100 mL water and stored in refrigerator in a brown flask. BSA and HSA solution: 0.0010 g BSA and HAS (Chemicals Co., Shanghai, China) were dissolved in water to prepare 1.0 mg mL-1 solutions and stored in refrigerator. The solutions were 10-fold diluted for use. BSA and HAS solutions were prepared again after a week. Walpole buffer: 1.0 mol L-1 sodium acetate solution and 1.。