Oxidative Cleavage-Based Three-Dimensional DNA Biosensor for Ratiometric Detection of Hypochlorous Acid and Myeloperoxidase

Abstract

Methods to detect and quantify disease biomarkers with high specificity and sensitivity in biological fluids play a key role in enabling clinical diagnosis, including point-of-care testing. Myeloperoxidase (MPO) is an emerging biomarker for the detection of inflammation, neurodegenerative diseases, and cardiovascular disease, where excess MPO can lead to oxidative damage to biomolecules in homeostatic systems. While numerous methods have been developed for MPO analysis, most techniques are challenging in clinical applications due to the lack of amplification methods, high cost, or other practical drawbacks. Enzyme-linked immunosorbent assays are currently used for the quantification of MPO in clinical practice, which is often limited by the availability of antibodies with high affinity and specificity and the significant nonspecific binding of antibodies to the analytical surface. In contrast, nucleic acid-based biosensors are of interest because of their simplicity, fast response time, low cost, high sensitivity, and low background signal, but detection targets are limited to nucleic acids and non-nucleic acid biomarkers are rare. Recent studies reveal that the modification of a genome in the form of phosphorothioate is specifically sensitive to the oxidative effects of the MPO/H 2 O 2 /Cl − system. We developed an oxidative cleavage-based threedimensional DNA biosensor for rapid, ratiometric detection of HOCl and MPO in a "one-pot" method, which is simple, stable, sensitive, specific, and time-saving and does not require a complex reaction process, such as PCR and enzyme involvement. The constructed biosensor has also been successfully used for MPO detection in complex samples. This strategy is therefore of great value in disease diagnosis and biomedical research.

Rolling-Circle-Amplification-Assisted DNA Biosensors for Sensitive and Specific Detection of Hypochlorous Acid and Myeloperoxidase

“…As shown in Figure 4a,b, the fluorescence intensity of the sensing system increased monotonically with the MPO concentration, and a linear relationship (R 2 = 0.9857) was obtained in the MPO concentration range of 1~400 ng/mL, providing a linear regression equation of F = 1.04 C MPO (ng/mL) + 15.23 (F is the fluorescence value at 485 nm, and C MPO is the MPO concentration). Based on the 3σ/S rule, the LOD was calculated to be 0.33 ng/mL, which is comparable to or better than reported fluorescent sensors of MPO (Table S3) [19,20,23,24,32,35,36].…”

“…In the range of 5 nM ~ 5 μM, the fluorescence intensity was linear with the concentration of HClO (Figure 2c), with a linear relationship (R 2 = 0.9915) of F = 76.31CHClO (μM) + 14.45 (F was the fluorescence intensity at 485 nm, and CHClO was the HClO concentration). This RCA−assisted HClO biosensor showed a good sensitivity, with the limit of detection (LOD) calculated as 1.67 nM, based on the 3σ/S rule, which is superior to or equivalent to other fluorescent detection methods of HClO (Table S1) [11,19,24,[30][31][32].…”

“…They named this HClO-dependent DNA cleavage behavior "DNA scissors" and thus developed an MPO biosensor based on the hybridization chain reaction (HCR), providing a detection limit of 8.35 ng/mL. Furthermore, our group used HClO to trigger DNA-tetrahedron-mediated hyperbranched HCR [24,25], realizing a further improvement of detection sensitivities (0.8 nM for HClO and 3.75 ng/mL for MPO). All these methods are developed on the basis of enzyme-free DNA amplification techniques.…”

An Oxidative Cleavage-Based Cruciform DNA Nanostructure for In Vivo Hypochlorous Acid Visualization to Monitor Intestinal Inflammation

“…As a kind of inflammatory disease, it has been documented that immune cells, specifically neutrophils and monocytes, infiltrate and accumulate in the colon of UC patients. − These immune cells are known to secrete the heme-containing enzyme myeloperoxidase (MPO). − Through MPO catalysis, HClO is generated via the peroxidation reaction between the elevated hydrogen peroxide and chlorine ions (Cl – ) at sites of inflammation. , Therefore, the direct detection of HClO could provide useful information for the early diagnosis of UC. There has been a range of research for the detection of HClO, encompassing colorimetry, electrochemistry, fluorescence, and chromatography. − Notably, fluorescence techniques have emerged as a prominent approach for HClO detection owing to their precise spatiotemporal resolution, noninvasive nature, heightened sensitivity, and exceptional selectivity. − …”