Peptide nucleic acid modified magnetic beads for intercalator based electrochemical detection of DNA hybridization
Peptide nucleic acid modified magnetic beads for intercalator based electrochemical detection of DNA hybridization
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DOI：
10.1016/j.stam.2004.01.009
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发表时间：
2004-01
影响因子：
5.5
通讯作者：
K. Kerman;Y. Matsubara;Y. Morita;Y. Takamura;E. Tamiya
中科院分区：
文献类型：
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作者：
K. Kerman;Y. Matsubara;Y. Morita;Y. Takamura;E. Tamiya
肽核酸（PNA）与其互补DNA靶的特异性结合与磁性分离相结合，以能够区分单核苷酸多态性（SNP）。将在5 '端具有生物素标记的PNA探针连接到链霉亲和素包被的超顺磁性氧化铁珠。然后用非互补的、含SNP的和完全匹配的DNA靶标攻击PNA修饰的珠。PNA探针对非互补DNA无亲和力。通过在空白缓冲液中使用十二烷基硫酸钠进行珠的洗涤步骤，抑制了含有SNP的DNA靶的非特异性结合。然后，将电活性嵌入剂7-二甲基-氨基-1，2-二苯并恶嗪鎓盐（Meldola’s blue，MDB）引入到珠粒中。MDB插入珠粒上的杂化分子的双螺旋之间。除去过量的MDB后，通过将生物素修饰的碳糊电极浸入溶液中从溶液中收集珠粒。通过监测MDB的伏安峰来确定PNA探针与靶DNA之间的特异性杂交。考察了靶DNA浓度、嵌入剂浓度、积累时间等因素对MDB信号的影响。MDB信号指示与20分钟杂交时间相关的2pM的检测限。
The specific binding of peptide nucleic acid (PNA) to its complementary DNA target is combined with magnetic separation to enable discrimination against single nucleotide polymorphisms (SNP). PNA probes with biotin label at 5'-end were attached to strepavidin coated superparamagnetic iron oxide beads. PNA modified beads were then challenged with non-complementary, SNP containing and perfect-match DNA targets. PNA probe showed no affinity towards non-complementary DNA. The non-specific binding of SNP containing DNA target was suppressed by the washing step of the beads by using sodium dodecylsulfate in blank buffer solution. Then, an electro-active intercalator, 7-dimethyl-amino-1, 2-benzophenoxazinium salt (Meldola's blue, MDB) was introduced to the beads. MDB intercalated between the double-helix of the hybrid molecules on the beads. After removing the excessive MDB, the beads were collected from the solution by immersing a biotin modified carbon paste electrode into the solution. Specific hybridization between PNA probe and DNA target was determined by monitoring the voltammetric peak of MDB. Numerous factors affecting the MDB signal, such as target DNA concentration, intercalator concentration and accumulation time were investigated. MDB signal indicated a detection limit of 2 pM in connection with 20 min hybridization time.