Gated nanoprobe utilizing metal-organic frameworks for identifying and distinguishing between the wild strains and the vaccine strains of brucella.

In the assay for Brucella, the identification and differentiation of wild strains and vaccine strains present a significant challenge. Currently, there aren't any commercially available product to address this issue. In this study, we have developed a novel gated nanoprobe by utilizing Metal-Organic Frameworks (MOFs) as a scaffold and hairpin DNA as a "gating switch". Specifically, Probe 1 with hairpin structure (P1h) targets a gene that is present in both wild strains Y3 (B. melitensis biovar 3) and vaccine strains A19 (Brucella abortus strains A19). We successfully applied this probe to screen positive samples of Brucella without any cross-reactivity with other substances. Additionally, we identified another specific gene exclusively found in wild strains, which serves as Probe 2 with hairpin structure (P2h) to confirm the strain type. Simultaneous detachment of both P1h and P2h from the MOFs leads to the release of Rhodamine 6G (Rho 6G) and Fluorescein (Flu), specifically indicating the presence of wild strains. If only P1h detaches and the Flu signal is detected, it suggests the presence of vaccine strains. Importantly, this method offers high accuracy, with a detection rate of 90% and a recovery rate of 94.71% to 107.65%, while avoiding cross-reactions with MO and TB. This one-step experiment provides reliable identification and differentiation of Y3 and A19, addressing concerns related to long periodicity, interference from individual variations, and the complex design of primers in existing laboratory methods. Furthermore, our approach successfully detects target 1 (T1) and target 2 (T2) at concentrations ranging from 10-6 M to 10-9 M, with a detection limit of 6.7 × 10-10 M and 6.4 × 10-10 M, respectively. Importantly, our strategy is cost-effective (around $1) and offers higher detection efficiency compared to traditional laboratory methods.

Dual carminic acid/hemin-marked DNA probes for simultaneously detecting CV-A16 and EV-A71 based on the mechanism of dimer to monomer transition.

This study aimed to prepare two hairpin-structure DNA probes by conjugating carminic acid (CA) or hemin into two ends of specific genes of coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) (probeCV-A16-CA and probeEV-A71-hemin). Then, probeCV-A16-CA and probeEV-A71-hemin as the signal molecules were adsorbed onto NH2-MIL-53 (Al) (MOF). Based on these biocomposites, an electrochemical biosensor with dual-signal outputs for simultaneous assay of CV-A16 and EV-A71 was constructed. The stem-loops of probes switched both CA and hemin monomer to dimer, reducing the electrical activity of both CA and hemin. Subsequently, the target-induced opening of the stem-loop switched both CA and hemin dimers to monomers, resulting in two nonoverlapping increasing electrical signals. This sensitively reflected the concentration of targetCV-A16 and targetEV-A17 ranging from 10-10 to 10-15 M with a detection limit of 0.19 and 0.24 fM. This strategy was mainly applied to the simultaneous determination of targetCV-A16 and targetEV-A17 in 100% serum with satisfactory results. The MOF combined with the high loading capacity broke through the intrinsic limitation on sensitivity using the traditional methods. An increase of three orders of magnitude was observed. This study involved simple one-step detection, and only a simple replacement of a gene could trigger its potential in clinical and diagnostic applications.