Abstract
Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10−18 mol l−1 for t-DNA has been achieved.
Highlights
IntroductionSome diseases like cancers are induced by genetic disorders; specific genes usually play important roles in the diagnosis of these diseases [10, 11]
It has been demonstrated that 350 nm diameter Magnetic nanoparticles (MNPs) can be against the washing operation after they are conjugated to single t-deoxyribo nucleic acid (DNA) molecules immobilized on the substrate [15]
In order to reduce the steric hindrance between the biotinylated DNAp (B-DNAp) and the matched APDNAd, and between the B-DNAp and the matched target DNA (t-DNA) immobilized on the substrate, a sequence with 20 thymines as a spacer was inserted to the B-DNAp between biotin and sequence 1, and between sequence 1 and sequence 2
Summary
Some diseases like cancers are induced by genetic disorders; specific genes usually play important roles in the diagnosis of these diseases [10, 11]. Sensitive and selective amplification methods for DNA detection are desirable and represent an important advance in these fields. Magnetic nanoparticles (MNPs) [1, 13,14,15,16] and noble metal
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