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Single miRNAs are not specific and accurate enough to meet the strict diagnosis requirements in practice. Therefore, simultaneous monitoring of multiplexed miRNA in biological samples can not only improve the accuracy and specificity of bioassays but also avoid the squandering of valuable biological specimens. Herein, we designed a metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade strategy for the sensitive and simultaneous multiplex miRNA detection in a single assay. Firstly, the capture probes (H1, H2) specifically recognize targets (miRNA-21, miRNA-141), exposing the stem of H1 and H2. Then, with the introduction of a detection probe (CuS-H3, ZnS-H4), the exposed H1 and H2 catalyze the hairpin assembly (CHA) reaction, realizing target miRNA recycling, and forming H1/H3-CuS and H2/H4-ZnS complexes. Subsequently, the formed H1/H3-CuS and H2/H4-ZnS complexes are encoded on magnetic beads through the biotin/streptavidin interaction. The CuS and ZnS nanoparticles captured by magnetic beads release thousands of Cu2+and Zn2+viathe cation exchange reaction. Finally, the released Cu2+and Zn2+specially activate the DNAzyme of the catalytic and molecular beacon (CAMB) system. The CAMB system affords an amplified fluorescence signal output by cycling and regenerating the metal ion-dependent DNAzyme to realize multiple enzymatic turnovers. Benefiting from target recycling, nanoparticle amplification, and catalytic and molecular beacon amplification, there is substantial amplification and the target miRNAs can be detected at 0.06 fM (miRNA-21) and 0.048 fM (miRNA-141) in a single assay. Furthermore, the high selectivity and accuracy of the assay were proved by practical analysis of different cancer cells, which exhibited good practicability in multiplex miRNA detection in clinical sera. The results indicate that the proposed strategy holds great potential for the sensitive detection of multiplex cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.

A key challenge for the discrete distribution-based Förster resonance energy transfer system (D-FRET) is the reduced intensity and stability of signal probes in complex biological matrices. Here, we present a spatially confined FRET (SC-FRET) probe with a stable structure and strong signal output. It consists of multivalent FRET pairs labeled with FAM or TAMRA. In this assay, p53 DNA was chosen as a model hairpin probe (HP), and two kinds of branched DNA probes (ssDNA-FAM, ssDNA-TAMRA) were involved. Under the action of p53 DNA, the unfolded HP acts as a primer to initiate polymerization extension of KFP polymerase and cleavage of Nb.BbvCI endonuclease, which produces plenty of ssDNA (primer-DNA). The branched DNA is designed to have the same binding core and different sticky ends, the core part of which can self-assemble to form X-shaped branched DNA (X-FAM or X-TAMRA), and the sticky ends of which are complementary to the primer-DNA. Therefore, the primer-DNAs released during the polymerization cleavage process will combine a large number of X-FAM and X-TAMRA in a limited space through complementary base pairing. Fluorescence was transferred from FAM to TAMRA, and a strong FRET response was generated by the locational effects. The proposed SC-FRET system based on the multivalent assembly of branched DNA exhibited a strong FRET response with an LOD of 0.01394 pM. Importantly, it also showed a high-contrast and stable FRET response in HeLa cells. Its superior biological stability is attributed to the large steric hindrance of the compact and rigid frame of the SC-FRET probe, which helps prevent intracellular degradation and provides a powerful tool for biomedical research.

Abstract: Melodinus henryiis a good source of terpenoid indole alkaloids, and traditionally used as a folk medicine in the treatment of meningitis and fracture. In order to further exploit their potential uses, its anti-inflammatory and immunosuppressive activities, safety evaluations and chemical profiles have been illustrated. Compared to the crude methanol extract from M. henryiand its non-alkaloidal fraction, the total alkaloidal fraction (MHTA) had the strongest anti-inflammatory and immunosuppressive activities. In the acute oral toxicity assay, the half lethal dose (LD50) of MHTA was more than 2000 mg/kg. The sub-acute toxicity assay for consecutive 28 days exhibited MHTA at a lower concentrations of less than 500 mg/kg might be regarded as safe, and might damage spleen, liver, kidney, and heart when the dose is higher than 1000 mg/kg. In addition, a phytochemical investigation on MHTA led to the isolation of 15 monoterpenoid indole alkaloids. Thus, in regard with the potent side effects of MHTA, it should be used with caution in the development of phytomedicine. Graphic Abstract:

Developing a sensitive, low-cost and general sensing platform for the analysis of a DNA biomarker and its mutation is important for early cancer screening. In our work, the tumor suppressor gene–p53 DNA was chosen as the model DNA biomarker due to its vital role in preventing oncogene cancer-inhibiting activity through mediating cellular proliferation and apoptosis. Compared with tumor biopsy, the quantification of p53 DNA and its mutation in biofluids (such as urine) is more convenient due to its simple operation and non-invasiveness. Herein, a label-free amplified fluorescence assay has been developed for p53 DNA in urine samples through the KFP polymerase-driven double strand displacement reactions and a magnetic nanoprobe. First, the ssDNA probe (RP) was designed with antisense sequences for p53 DNA and the Nb.BbvCI endonuclease recognition site. In the presence of p53 DNA, the formed dsDNA between RP and p53 DNA served as an engaging primer to initiate the first strand displacement reaction (SDA) under the action of KFP DNA polymerase and Nb.BbvCI, generating abundant short ssDNA (primer). Subsequently, the resulting primers will initiate the downstream SDA through the primer–hairpin DNA (HPa) binding, opening up, and extension of HPb and HPc under the action of KFP DNA polymerase. In the process of this final DNA polymerization reaction, the primer hybridized on HPa is released and goes on to initiate another round, forming plenty of duplex Y-shaped DNA. With the integration of SYBR Green I (SG I) into these duplex DNA, the amplified label-free fluorescence detection platform for p53 DNA can be achieved. Moreover, a biotin modified nanoprobe (bio-CP) was used to capture the superfluous HP. By performing the separation function, the binding of superfluous HP and SG could be avoided and a low background can be acquired. Benefiting from the abundant SG intercalation sites of Y-shaped DNA and low background signals, this method showed excellent sensitivity with a detection limit of 0.012 nM, and the p53 DNA in urine samples was evaluated, offering a powerful tool for biomedical research and clinical diagnosis.

A novel, homing peptide-based ELISA-like method for the determination of fibrin is presented. Compared to the antibody, homing peptides have lower antigenicity, better biodegradability, and higher penetration ability. In this study, fibrin was coated on the surface of a 96-well plate. Then, the homing peptide (CREKA), which was labeled by horseradish peroxidase (HRP), was added and allowed to react with fibrin. After washing, the TMB–H2O2colorimetric system was utilized for the quantitative analysis of fibrin. The assay showed a linear response toward fibrin concentration in the range of 0.1–10 nM with a correlation coefficient of 0.9976. The limit of detection for fibrin was experimentally determined to be 0.04 nM, based on a signal-to-noise ratio (S/N) of 3. The sensing system has been used for the determination of fibrin in human serum samples. Relative to the conventional methods, this method offers the advantages of high-throughput, stability, simplicity, sensitivity, low cost and selectivity, showing great potential for applications in clinical diagnosis.