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Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes

机译:使用脂质体在萤火虫生物发光法中检测ATP萃取剂的存在

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Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an AT? extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) Cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes.
机译:在阳离子表面活性剂的存在下,将含有磷脂酰胆碱(PC)和胆固醇(Chol)的脂质体用于萤火虫生物发光(BL)ATP增强剂,用作从活细胞中释放ATP的萃取剂。苯扎氯铵(BAC)用作AT?萃取剂。但是,BAC严重抑制了萤光素酶的活性,从而导致BL分析对ATP的敏感性显着下降。另一方面,我们发现BAC与脂质体结合形成含有BAC的阳离子脂质体。 BAC与脂质体的缔合速率要快于BAC与荧光素酶的缔合速率。结果,在脂质体存在下消除了BAC对荧光素酶的抑制作用。另外,阳离子脂质体因此形成增强的BL发射。根据脂质体的电荷类型,脂质体大小以及PC和Chol的总浓度优化了BL测量条件。使用脂质体无需稀释分析样品即可灵敏地确定ATP。在含0.06%BAC的ATP水溶液中,有和没有脂质体的ATP的检出限分别为100 amol和25 fmol。该方法用于大肠埃希菌提取物中ATP的测定。在没有脂质体的情况下,BL强度从4 x 10(4)到1 x 10(7)细胞mL(-1)是线性的。另一方面,在脂质体存在下,BL强度从4 x 10(3)到4 x 10(6)细胞mL(-1)是线性的。通过使用脂质体,大肠杆菌提取物中ATP的检出限提高了10倍。

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