Target-promoted autocatalytic hairpin assembly of bivalent DNAzymes for sensitive and label-free electrochemical metallothionein assay.

Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg2+-stabilized DNA duplex (Hg2+-dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg2+ in Hg2+-dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios.

An aptamer triple helix molecular switch for sensitive electrochemical assay of lipocalin 1 biomarker via dual signal amplifications.

Sensitively monitoring the concentration change of lipocalin 1 (LCN1) can provide data support for accurately diagnosing diabetic retinopathy and efficacy of treatment. Using a new aptamer triplex switch (ATS) probe and catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) dual signal amplifications, a highly sensitive electrochemical and enzyme-free biosensor for detecting LCN1 is reported. The ATS probes recognize and bind LCN1 to change its structure and release ssDNA sequences, which trigger the capture of methylene blue (MB)-tagged DNA on the sensor electrode via the CHA and HCR reactions. These MB tags are then subjected to electrochemical measurements to obtain highly amplified current outputs to sensitively detect LCN1 from 5 pM to 1 nM with a detection limit of 0.85 pM. This sensor also exhibits high interfering protein discrimination capability and can be employed to monitor low levels of LCN1 in diluted tear samples. Our established sensor significantly outperforms current major LCN1 detection methods based on immunoassays and thus holds promising potential for evaluating the severity of diabetic retinopathy and monitoring treatment response.

Lighting-up aptamer transcriptional amplification for highly sensitive and label-free FEN1 detection.

Specific and sensitive detection of flap endonuclease 1 (FEN1), an enzyme biomarker involved in DNA replications and several metabolic pathways, is of high values for the diagnosis of various cancers. In this work, a fluorescence strategy based on transcriptional amplification of lighting-up aptamers for label-free, low background and sensitive monitoring of FEN1 is developed. FEN1 cleaves the 5' flap of the DNA complex probe with double flaps to form a notched dsDNA, which is ligated by T4 DNA ligase to yield fully complementary dsDNA. Subsequently, T7 RNA polymerase binds the promoter region to initiate cyclic transcriptional generation of many RNA aptamers that associate with the malachite green dye to yield highly amplified fluorescence for detecting FEN1 with detection limit as low as 0.22 pM in a selective way. In addition, the method can achieve diluted serum monitoring of low concentrations of FEN1, exhibiting its potential for the diagnosis of early-stage cancers.

Programmable bidirectional dynamic DNA nano-device for accurate and ultrasensitive fluorescent detection of trace MUC1 biomarker in serums.

Accurate and ultrasensitive detection of biomarkers is significance for the diagnosis of diseases at early stage. For this purpose, we herein develop a bidirectional dynamic DNA nano-device for amplified fluorescent detection of tumor marker of mucin 1 (MUC1). The nano-device is constructed by immobilizing two sets of DNA cascade catalytic probes on two opposite directions of a single-stranded DNA tracker to limit probe reactants to a compact space. Once target MUC1 binds to the aptamer sequence, the initiator DNA locked in the duplex DNA substrate can be released to induce DNA-initiated cascade hybridization reactions (DCHRs) simultaneously in two opposite directions along the tracker DNA, accompanying the displacement of two quencher labeled-DNA intermediate initiators to facilitate successively execution of DCHRs on the DNA nano-devices, which results in the separation of fluorophore (FAM) and quencher (Dabycl) to produce substantially recovered fluorescent signals for rapid and sensitive detection of MUC1 with a detection limit down to 0.18 pM. In addition, this strategy also exhibits high selectivity against other interfering proteins and potential application capacity in real serum samples, indicating its promising application prospects in disease diagnosis and treatment.

CRISPR/Cas12a-derived sensitive electrochemical biosensing of NF-κB p50 based on hybridization chain reaction and DNA hydrogel.

Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.

The reference genome and transcriptome of the limestone langur, Trachypithecus leucocephalus, reveal expansion of genes related to alkali tolerance.

BACKGROUND: Trachypithecus leucocephalus, the white-headed langur, is a critically endangered primate that is endemic to the karst mountains in the southern Guangxi province of China. Studying the genomic and transcriptomic mechanisms underlying its local adaptation could help explain its persistence within a highly specialized ecological niche. RESULTS: In this study, we used PacBio sequencing and optical assembly and Hi-C analysis to create a high-quality de novo assembly of the T. leucocephalus genome. Annotation and functional enrichment revealed many genes involved in metabolism, transport, and homeostasis, and almost all of the positively selected genes were related to mineral ion binding. The transcriptomes of 12 tissues from three T. leucocephalus individuals showed that the great majority of genes involved in mineral absorption and calcium signaling were expressed, and their gene families were significantly expanded. For example, FTH1 primarily functions in iron storage and had 20 expanded copies. CONCLUSIONS: These results increase our understanding of the evolution of alkali tolerance and other traits necessary for the persistence of T. leucocephalus within an ecologically unique limestone karst environment.