A Cyanine Dye for Highly Specific Recognition of Parallel G-Quadruplex Topology and Its Application in Clinical RNA Detection for Cancer Diagnosis.

G-quadruplex (G4), an unconventional nucleic acid structure, shows polymorphism in its topological morphology. The parallel G4 topology is the most prevalent form in organisms and plays a regulatory role in many biological processes. Designing fluorescent probes with high specificity for parallel G4s is important but challenging. Herein, a supramolecular assembly of the anionic cyanine dye SCY-5 is reported, which selectively identifies parallel G4 topology. SCY-5 can clearly distinguish parallel G4s from other G4s and non-G4s, even including hybrid-type G4s with parallel characteristics. The high specificity mechanism of SCY-5 involves a delicate balance between electrostatic repulsion and π-π interaction between SCY-5 and G4s. Using SCY-5, cellular RNA extracted from peripheral venous blood was quantitatively detected, and a remarkable increase in RNA G4 content in cancer patients compared to healthy volunteers was confirmed for the first time. This study provides new insights for designing specific probes for parallel G4 topology and opens a new path for clinical cancer diagnosis using RNA G4 as a biomarker.

Determination of ligand selectivity to G-tetrad through an AMCA fluorescence quenching approach.

The selective binding of ligand molecules towards the 5' and 3' ends of G-quadruplex (G4) may differentially affect the physiological function of G4s. However, there is still a lack of sensitive and low-cost approaches to accurately measure the binding preference of ligands on G4s, although multiple ways have been developed to evaluate the interaction between ligands and G4s. Here, we propose a new protocol named G4-AFQ to test the selectivity of ligands towards the two terminal G-tetrads of G4s. In this protocol, the fluorophore AMCA is respectively modified at the 5' or 3' end of G4, and which end of AMCA fluorescence is quenched means that the ligand binds to the G-tetrad at that end. Through G4-AFQ, the affinity constant of ligands towards the binding site can also be obtained. Compared with the commonly used nuclear magnetic resonance (NMR) method, G4-AFQ is more convenient, sensitive, cost-effective, and suitable for the measurement of the vast majority of G4 ligands, with a great potential for widespread application.

Molecularly imprinted solid-phase extraction combined with non-ionic hydrophobic deep eutectic solvents dispersed liquid-liquid microextraction for efficient enrichment and determination of the estrogens in serum samples.

Hormonal drugs in biological samples are usually in low concentration and highly intrusive. It is of great significance to enhance the sensitivity and specificity of the detection process of hormone drugs in biological samples by utilizing appropriate sample pretreatment methods for the detection of hormone drugs. In this study, a sample pretreatment method was developed to effectively enrich estrogens in serum samples by combining molecularly imprinted solid-phase extraction, which has high specificity, and non-ionic hydrophobic deep eutectic solvent-dispersive liquid-liquid microextraction, which has a high enrichment ability. The theoretical basis for the effective enrichment of estrogens by non-ionic hydrophobic deep eutectic solvent was also computed by simulation. The results showed that the combination of molecularly imprinted solid-phase extraction and deep eutectic solvent-dispersive liquid-liquid microextraction could improve the sensitivity of HPLC by 33∼125 folds, and at the same time effectively reduce the interference. In addition, the non-ionic hydrophobic deep eutectic solvent has a relatively low solvation energy for estrogen and possesses a surface charge similar to that of estrogen, and thus can effectively enrich estrogen. The study provides ideas and methods for the extraction and determination of low-concentration drugs in biological samples and also provides a theoretical basis for the application of non-ionic hydrophobic deep eutectic solvent extraction.

Dummy-template Pickering emulsion imprinted microspheres online pretreatment and analysis for the estrogens in cosmetics.

Estrogens are a class of steroid hormone with strong physiological activity. Due to the pronounced beauty effect, such drugs are highly susceptible to illegal addition and cause other adverse effects. To avoid template leakage and the negative impacts on the environment caused by the estrogens, diosgenin was selected as the dummy template due to its similar skeleton structure. The Pickering emulsion polymerization was used to obtain the dummy-template molecularly imprinted polymers (dt-MIPs). Scanning electron microscopy, optical microscopy, specific surface area testing, Fourier transform infrared spectroscopy and adsorption experiments were used to characterize the apparent morphology and the recognition performance of the microspheres. Then, the prepared microspheres and commercial fillers were used to construct an on-line solid phase extraction (on-line SPE) analytical system coupled with HPLC via a two-position switching valve. On-line solid phase extraction-HPLC analytical methods were established and verified, for the simultaneous determination of four estrogens in cosmetic samples. The accuracy and precision RSDs for the established methods using the imprinted sorbents were 92.00-104.02% and less than 9.12%, respectively. All four estrogens exhibited good linearity in the range of 0.05 to 5 µg/mL with a coefficient of determination R2 greater than 0.9810. The method comparison results suggest that the established analytical method is simple in pre-treatment, easy to automate, and has excellent sensitivity to meet the analytical requirements of complex samples.

Thermosensitive molecularly imprinted polymer coupled with HPLC for selective enrichment and determination of matrine in traditional Chinese medicine.

In this study, a temperature-sensitive molecularly imprinted polymer was prepared by using the bifunctional monomer with the critical phase transition characteristics. Infrared spectrometry, scanning electron microscopy, and specific surface area testing were used to characterize the polymers. Then, the recognizing properties of the polymers were studied. Based on the prepared smart polymers, an SPE-HPLC analytical method for the determination of quinolizidine alkaloids in the extracts of Sophora flavescens was established and verified. Finally, the smart polymers were applied to the enrichment of quinolizidine alkaloids in plant extracts. By changing the temperature and solvents of the solid phase extraction conditions, the extraction process can increase the concentration of quinolizidine alkaloids by 4.3 to 5.2 folds. The extraction process has mild conditions and less time consumption, avoiding the use of a large number of toxic reagents, which indicate that the extraction process are more efficient and environmentally friendly.