DNA-Oligonucleotide Encapsulating Liposomes as a Secondary Signal Amplification Means

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• 作者：Katie A. Edwards ; Antje J. Baeumner
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：March 1, 2007
• 年：2007
• 卷：79
• 期：5
• 页码：1806 - 1815
• 全文大小：300K
• 年卷期：v.79,no.5(March 1, 2007)
• ISSN：1520-6882

文摘

A novel liposome-based signal amplification system wasdeveloped by encapsulating DNA oligonucleotides withinantibody-tagged liposomes and subsequently detecting theoligonucleotide with dye-encapsulating liposomes fordouble signal enhancement. In this sandwich immunoassay, the model analyte, protective antigen protein fromB. anthracis, was captured by one set of antibodiesimmobilized in microtiter plate wells and detected usinga second antibody conjugated to oligonucleotide-encapsulating liposomes. Bound liposomes were lysed releasingthe encapsulated fluorescein-tagged DNA 25-mer probe,which was then permitted to hybridize with its complementary sequence immobilized in a second plate. Finally,the amount of oligonucleotide was detected through theaddition of anti-fluorescein antibody tagged dye-encapsulating liposomes. These secondary liposomes allowed fora ~400× lower LOD than detection of the fluorescein-labeled probe alone. Several aspects were investigated,including the encapsulation of various oligonucleotideconcentrations within liposomes; oligonucleotide hybridization times and buffers; degree of anti-fluoresceinantibody coverage on the liposomes; and immobilizedanti-protective antigen antibody concentration. We foundthat the encapsulation efficiency increased with the starting oligonucleotide concentration. As many as 4000 DNA25-mers were successfully entrapped in the liposome, andminimal leakage was observed over the course of 8months. When used in the sandwich immunoassay, a limitof detection of 4.1 ng/mL protective antigen was observedwith an upper limit of 5000 ng/mL. Due to the endlesscombination of DNA oligonucleotide sequences, this assaylends itself perfectly for multiplexing on the order of tensto hundreds of analytes.