Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles

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.

Fluorometric and Colorimetric Dual-Readout Immunoassay Based on an Alkaline Phosphatase-Triggered Reaction

“…pNPP as a common chromogenic substrate for ALP is frequently utilized in the standard colorimetric assay of ALP activity, which can be converted to p -nitrophenol (pNP) together with an absorption spectrum red-shift (from 312 to 405 nm). On the other hand, MUP as a commercially fluorescent substrate can be dephosphorylated by ALP to produce 4-methylumbelliferone (MU, i.e., 7-hydroxy-4-methylcoumarin), accompanied by blue fluorescence emission. , The colorimetric and fluorometric assay of ALP was carried out with the same concentration of pNPP and MUP (100 μM) as substrate, respectively. The results in Figure S11 indicated that the analytical sensitivity for ALP with m -HPP as the substrate was superior to that of pNPP (linear range of 0.2 to 20 mU/mL) and comparable with that of MUP (linear range of 0.02 to 2 mU/mL).…”

Highly Chemiluminescent Magnetic Beads for Label-Free Sensing of 2,4,6-Trinitrotoluene

“…Over the past decade, magnetic materials have attracted considerable attention due to their excellent performance in the fields of bioassays, − drug delivery, − electronics, , magnetic resonance imaging, − and environmental remediation. − As typical magnetic materials, magnetic beads (MBs) can be concentrated, trapped, or used in rapid separation by applying a magnetic field. Owing to their outstanding properties, such as high loading capacity, high magnetic susceptibility, and good biocompatibility, MBs have extensively been used for label-based bioassays such as immunoassays − and DNA assays. − Particularly, label-based chemiluminescence (CL) technology with MBs has been successfully commercialized in immunoassays. However, they still suffer from complicated labeling, purification, and washing procedures and need secondary antibody, which makes them labor-intensive, time-consuming, and expensive.…”

Optical-Based (Bio) Sensing Systems Using Magnetic Nanoparticles

“…Magneto-fluorometric methods for sensing of numerous materials such as heavy metals [111,112], toxins [113], drugs [114,115], pesticide [116], biological materials i.e. DNA [117,118], cell [119,120], bacteria [121], etc. have been investigated.…”