Ultrasensitive Detection and Discrimination of Cancer-Related Single Nucleotide Polymorphisms Using Poly-Enzyme Polymer Bead Amplification

We report the development of a new ultrasensitive approach for label-free DNA detection using magnetic nanoparticle (MNP)-assisted rapid target capture/separation in combination with signal amplification using poly-enzyme tagged polymer nanobead. The sensor uses a MNP linked capture DNA and a biotin modified signal DNA to sandwich bind the target followed by ligation to provide high single-nucleotide polymorphism discrimination. Only the presence of a perfect match target DNA yields a covalent linkage between the capture and signal DNAs for subsequent binding to a neutravidin-modified horseradish peroxidase (HRP) enzyme via the strong biotin–neutravidin interaction. This converts each captured full match DNA target into a HRP which can convert millions of copies of a non-fluorescent substrate (amplex red) to a highly fluorescent product (resorufin) for great signal amplification. The use of polymer nanobead each tagged with thousands of copies of HRPs as the signal amplifier greatly improves the signal amplification power, leading to greatly improved sensitivity. This biosensing approach can specifically detect an unlabelled DNA target down to 10 aM with a wide dynamic range of 5 orders of magnitude (from 0.01 fM to 1000 fM). Furthermore, our approach has a high discrimination between a perfectly matched gene and its cancer-related single-base mismatch targets (SNPs): it can positively detect the perfect match DNA target even in the presence of 100-fold excess of co-existing SNPs. This sensing approach also works robustly in clinical relevant media (e.g., 10% human serum) and gives almost the same SNP discrimination ratio as that in clean buffers. Therefore, this ultrasensitive SNP biosensor appears to be well-suited for potential diagnostic applications of genetic diseases

A simple magnetic nanoparticle-poly-enzyme nanobead sandwich assay for direct, ultrasensitive DNA detection

A simple magnetic nanoparticle (MNP)-poly-enzyme nanobead sandwich assay for direct detection of ultralow levels of unlabeled target-DNA is developed. This approach uses a capture-DNA covalently linked to a dense PEGylated polymer encapsulated MNP and a biotinylated signal-DNA to sandwich the target-DNA. A DNA ligation is then followed to offer high discrimination between the perfect-match and single-base mismatch target-DNAs. Only the presence of a perfect-match target can covalently link the biotinylated signal-DNA onto the MNP surface for subsequent binding to a polymer nanobead tagged with thousands of copies of high-activity neutravidin-horseradish peroxidase (NAV-HRP) for great enzymatic signal amplification. Combining the advantages of the dense MNP surface PEGylation to reduce non-specific adsorption (assay background) and the powerful signal amplification of poly-enzyme nanobead, this assay can directly quantify the target-DNA down to single digit attomolar with a large linear dynamic range of 5 orders of magnitude (from 10− 18 to 10− 13 M)

In situ scanning tunneling microscopy imaging of electropolymerized poly(3,4-ethylenedioxythiophene) on an iodine-modified Au(111) single crystal electrode

The electrochemical polymerization of 3,4-ethylenedioxythiophene (EDOT) on an iodine-modified Au(1 1 1) single crystal electrode in aqueous 0.10 M HClO4 was investigated by cyclic voltammetry (CV) and electrochemical scanning tunneling microscopy (EC-STM). The cyclic voltammetric and EC-STM data revealed the stability of the iodine adlayer provided a suitable potential range for EDOT electropolymerization was controlled at 1.20V (vs. the reversible hydrogen electrode). EC-STM was used to examine the formation of the PEDOT adlayer on the iodine-modified Au(1 1 1) electrode. In situ electropolymerization of EDOT was carried out by slowly increasing the electrode potential to 1.20V. This process resulted in the formation of single molecular chains of PEDOT with a diameter of 0.90 nm and lengths of 5–7 nm. Higher resolution STM images further revealed PEDOT nanostructures with bent polymer backbones at angles of 105◦, 144◦ and 180◦. The growth of PEDOT multi-layers was observed when the potential was held for a longer time