A microtiter modification of the anthrone-sulfuric acid colorimetric assay for glucose-based carbohydrates
A microtiter modification of the anthrone-sulfuric acid colorimetric assay for glucose-based carbohydrates
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DOI：
10.1016/s0003-2697(02)00704-2
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发表时间：
2003-04-01
影响因子：
2.9
通讯作者：
Edwards, CA
中科院分区：
文献类型：
--
作者：
Laurentin, A;Edwards, CA
1955年，Roe [1]描述了一种测量血液和尿液中葡聚糖的方法的改进[2]，用于估计血液和脊髓液中的糖。在这些方法中，使用蒽酮硫酸反应测量总碳水化合物。在这里，热和强酸性环境产生糖苷键的水解和单体的脱水以产生糠醛衍生物。这些化合物与蒽酮反应并产生有色产物。然而，大量样品的常规测量需要使用大量的浓硫酸，并且需要严格的安全预防措施以及许多试管。以前的自动化尝试受到该反应放热性质的阻碍（G。Palacios，personal communication）.微量滴定板技术的使用提供了一种克服这些问题并降低成本、时间和危险的方法。因此，我们的目标是采用众所周知的蒽酮-硫酸测定法，使用96孔微量滴定板对葡萄糖基碳水化合物进行可靠的定量测定，该测定法需要每个板总共12 mL蒽酮硫酸试剂（2g/L蒽酮浓硫酸溶液）和0.5 mL标准溶液（0.4 g/L葡萄糖水溶液）。蒽酮-硫酸试剂必须在临用前制备。将24 mg蒽酮的等分试样批量制备至具有防漏盖的30 mL通用塑料容器中，并避光保存直至使用。就在向板中加入试剂之前，将12 mL储存在4 ° C下的浓硫酸加入到板中。
In 1955, Roe [1] described a modification of a method to measure dextran in blood and urine [2] for estimation of sugar in blood and spinal fluid. In these methods, total carbohydrates are measured using the anthronesulfuric acid reaction. Here, heat and a strong acidic environment produce both hydrolysis of glycosidic bonds and dehydration of monomers to produce furfuraldehyde derivatives. These compounds react with anthrone and produce colored products. However, routine measurement of a large number of samples requires the use of a significant amount of concentrated sulfuric acid and needs strict safety precautions as well as many test tubes. Previous attempts of automation have been hampered by the exothermic nature of this reaction (G. Palacios, personal communication). The use of microtitration plate technology offers a way to overcome these problems and reduce cost, time, and hazard. Therefore, our aim was to adapt the well-known anthrone-sulfuric acid assay for reliable quantification of glucose-based carbohydrates using 96-well microtitration plates.This assay requires a total of 12mL of anthronesulfuric acid reagent (2g/L anthrone solution in concentrated sulfuric acid) and 0.5 mL standard solution (0.4 g/L glucose solution in water) per plate. The anthrone-sulfuric acid reagent must be prepared just before use. Aliquots of 24mg of anthrone were made in bulk into 30-mL universal plastic containers with leakproof caps and kept protected from light until used. Just before addition of the reagent to the plate, 12mL of concentrated sulfuric acid, stored at 4 C, was added to