Mercury/Homocysteine Ligation-Induced ON/OFF-Switching of a T–T Mismatch-Based Oligonucleotide Molecular Beacon

A molecular beacon (MB) with stem-loop (hairpin) DNA structure and with attached fluorophore–quencher pair at the ends of the strand has been applied to study the interactions of Hg²⁺ ions with a thymine–thymine (T–T) mismatch in Watson–Crick base-pairs and the ligative disassembly of MB·Hg²⁺ complex by Hg²⁺ sequestration with small biomolecule ligands. In this work, a five base-pair stem with configuration 5′-GGTGG...CCTCC-3′ for self-hybridization of MB has been utilized. In this configuration, the four GC base-pair binding energy is not sufficient to hybridize fully at intermediate temperatures and to form a hairpin MB conformation. The T–T mismatch built-in into the stem area can effectively bind Hg²⁺ ions creating a bridge, T–Hg–T. We have found that the T–Hg–T bridge strongly enhances the ability of MB to hybridize, as evidenced by an unusually large MB melting temperature shift observed on bridge formation, Δ<i><i>T</i></i>_m = +15.1 ± 0.5 °C, for 100 nM MB in MOPS buffer. The observed Δ<i><i>T</i></i>_m is the largest of the Δ<i><i>T</i></i>_m found for other MBs and dsDNA structures. By fitting the parameters of the proposed model of reversible MB interactions to the experimental data, we have determined the T–Hg–T bridge formation constant at 25 °C, <i>K</i>₁ = 8.92 ± 0.42 × 10¹⁷ M^–1 from mercury­(II) titration data and <i>K</i>₁ = 1.04 ± 0.51 × 10¹⁸ M^–1 from the bridge disassembly data; Δ<i>G</i>° = −24.53 ± 0.13 kcal/mol. We have found that the biomarker of oxidative stress and cardiovascular disease, homocysteine (Hcys), can sequester Hg²⁺ ions from the T–Hg–T complex and withdraw Hg²⁺ ions from MB in the form of stable Hg­(Hcys)₂H₂ complexes. Both the model fitting and independent ¹H NMR results on the thymidine–Hg–Hcys system indicate also the high importance of 1:1 complexes. The high value of <i>K</i>₁ for T–Hg–T bridge formation enables analytical determinations of low concentrations of Hg²⁺ (limit of detection LOD = 19 nM or 3.8 ppb, based on 3σ method) and Hcys (LOD = 23 nM, 3σ method). The conditional stability constants for Hg­(Hcys)­H₂²⁺ and Hg­(Hcys)₂H₂ at 52 °C have been determined, β₁₁₂ = 5.37 ± 0.3 × 10⁴⁶ M^–3, β₁₂₂ = 3.80 ± 0.6 × 10⁶⁸ M^–4, respectively

Surface Enhanced Raman Scattering Detection of Cancer Biomarkers with Bifunctional Nanocomposite Probes

This report describes new findings of an investigation of a bifunctional nanocomposite probe for the detection of cancer biomarkers, demonstrating the viability of magnetic focusing and SERS detection in a microfluidic platform. The nanocomposite probe consists of a magnetic nickel–iron core and a gold shell. Upon bioconjugation, the nanoprobes are magnetically focused on a specific spot in a microfluidic channel, enabling an enrichment of “hot spots” for surface enhanced Raman scattering detection of the targeted carcinoembryonic antigen. The detection sensitivity, with a limit of detection of ∼0.1 pM, is shown to scale with the magnetic focusing time and the nanoparticle size. The latter is also shown to exhibit an excellent agreement between the experimental data and the theoretical simulation. Implications of the findings to the development of rapid and sensitive microfluidic detection of cancer biomarkers are also discussed