A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test.
A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test.
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适用于多种常见生化试剂的多功能过渡金属盐反应：即时且可量化的颜色测试。
DOI：
10.1016/0003-2697(86)90048-5
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发表时间：
1986
影响因子：
2.9
通讯作者：
Nixon,RA
中科院分区：
文献类型：
--
作者：
Fleming,N;Nixon,RA
利用过渡金属盐氯化铜及其大量相关有色化合物，开发了一种适合目视或分光光度定量的快速、灵敏的斑点测试，适用于各种生化试剂。该测定法可能是一种广泛适用的多用途测试，用于快速检测未知物质的存在。测试样品与工作试剂的组合导致立即形成可沉淀的独特颜色的产物。有些化合物需要进一步添加氢氧化钠才能产生独特颜色的产品。使用以下试剂会发生独特的反应，其视觉检测极限在括号中标明：碳酸氢铵 (12.5 mm)、乙酸铵 (25 mm)、氢氧化铵 (0.1%)、硫酸铵 (2%)、过硫酸铵 (0.02 mm)、l-(+)-半胱氨酸 (0.07 mM)、 二硫苏糖醇 (DTT) (1.25 mM)、EDTA (0.6 mM)、乙二醇双(β-氨基乙基醚) N,N'-四乙酸 (5 mm)、d-葡萄糖 (6 mm)、甘油 (0.3%)、咪唑 (12.5 mm)、dl-蛋氨酸 (100 mm)、巯基乙醇 （0.05％），叠氮化钠（19毫米，0.1％），连二亚硫酸钠（0.25％），焦亚硫酸钠（25毫米），亚硝酸钠（6.2毫米），高碘酸钠（3.1毫米），亚硫酸钠（12.5毫米），硫代亚硫酸钠（12.5毫米），蔗糖（6毫米），和 N,N,N',N'-四甲基乙二胺（0.05%）。与过氧化氢发生独特的放热反应，但没有颜色变化。反应不显着的化合物包括 50 mm Tris 缓冲液、尿素、N,N'-亚甲基双丙烯酰胺、十二烷基硫酸钠、异丙醇、氟化钠、三氯乙酸、苯酚、甘露糖、K2HPO4、盐酸胍、氯胺-T、氯化镁和硼酸，其中固体在大约 10 毫克/毫升。利用这些反应产生的澄清上清液开发了 DTT 和叠氮化钠的分光光度标准曲线。至少四种试剂的组合可以被区分，如葡萄糖、叠氮化钠、EDTA 和 DTT 的混合物所证明的。此外，在 50 mm Tris 缓冲液中存在蛋白质、DTT 和 EDTA 时，硫酸铵的检测限可达 4%。开发了斑点测试，该测试利用试剂浸渍的滤纸，并在添加 5 μl 测试样品后产生独特的有色产物。
A rapid and sensitive spot test amenable to visual or spectrophotometric quantitation has been developed for a wide variety of biochemical reagents by utilizing the transition metal salt cupric chloride and its large number of related colored compounds. This assay is potentially a widely applicable multipurpose test for rapidly detecting the presence of unknown substances. Combination of the test sample with the working reagent results in the immediate formation of a distinctive colored product that may be precipitable. Some compounds require the further addition of sodium hydroxide in order to generate the distinctively colored product. Distinctive reactions occur with the following reagents, and their limit of visual detection is indicated in parentheses: ammonium bicarbonate (12.5 mm), ammonium acetate (25 mm), ammonium hydroxide (0.1%), ammonium sulfate (2%), ammonium persulfate (0.02 mm), l-(+)-cysteine (0.07 mM), dithiothreitol (DTT) (1.25 mM), EDTA (0.6 mM), ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (5 mm), d-glucose (6 mm), glycerol (0.3%), imidazol (12.5 mm), dl-methionine (100 mm), mercaptoethanol (0.05%), sodium azide (19 mm, 0.1%), sodium dithionite (0.25%), sodium metabisulfite (25 mm), sodium nitrite (6.2 mm), sodium periodate (3.1 mm), sodium sulfite (12.5 mm), sodium thiosulfite (12.5 mm), sucrose (6 mm), and N,N,N′,N′-tetramethylethylenediamine (0.05%). A distinctive exothermic reaction occurs with hydrogen peroxide, but without color change. Compounds reacting insignificantly include 50 mm Tris buffer, urea, N,N′-methylene bisacrylamide, sodium dodecyl sulfate, isopropyl alcohol, sodium fluoride, trichloroacetic acid, phenol, mannose, K2HPO4, guanidine HCl, chloramine-T, magnesium chloride, and boric acid, where the solids were tested at approximately 10 mg/ml. Spectrophotometric standard curves were developed for DTT and sodium azide utilizing the clear supernatants resulting from these reactions. Combinations of at least four reagents could be discriminated, as demonstrated with mixtures of glucose, sodium azide, EDTA, and DTT. In addition ammonium sulfate could be detected to a limit of 4% in the presence of protein, DTT, and EDTA in a 50 mm Tris buffer. Spot tests were developed which utilized reagent-impregnated filter paper and gave distinetive colored products on addition of 5 μl of test sample.