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Abstract Human telomerase is responsible for the maintenance of chromosome end structures and is a valuable biomarker for malignant growth. However, the accurate measurement of telomerase activity at the single-cell level has remained a great challenge. Here we develop a simple quantum dot (QD)-based electrochemical biosensor for stripping voltammetric detection of telomerase activity at the single-cell level. We designed a thiol-modified capture DNA which may be immobilized on the gold electrode by the gold-sulfur bond. The presence of telomerase enables the addition of the telomere repeats of (TTAGGG) n to the 3′ end of the primer, accompanied by the incorporation of abundant biotins in the extension product with the assistance of the biotin-tagged dATP. The subsequent hybridization of extension product with the capture DNA and the addition of streptavidin-coated QDs induce the assembly of large amounts of QDs onto the electrode via specific biotin-streptavidin binding. After the acidic dissolution of QDs, the released Cd (II) can be simply quantified by anodic stripping voltammetry (ASV). Due to the introduction of large amounts of QDs by telomerase-induced primer extension reaction and the synergistic signal amplification induced by the release of Cd (II) from the QDs, this biosensor can detect telomerase activity at the single-cell level without the involvement of any thermal cycling and extra enzymes for signal amplification. Moreover, this assay exhibits a large dynamic range over four orders of magnitude and it is very simple without the involvement of specific hairpin probe design and complicated labelling, holding great potential in point-of-need testing. Highlights • A simple quantum dot-based electrochemical biosensor is developed for sensitive detection of telomerase activity. • This biosensor can detect telomerase activity at the single-cell level. • This biosensor can be used for the discrimination of cancer cells from normal cells. • This biosensor can be used for the screening of telomerase inhibitors. [ABSTRACT FROM AUTHOR]

Uracil-DNA glycosylase (UDG) is essential to the maintenance of genomic integrity due to its critical role in base excision repair pathway. However, existing UDG assays suffer from laborious procedures, poor specificity, and limited sensitivity. In this research, we construct a catalytic single-molecule Föster resonance energy transfer (FRET) biosensor for in vitro and in vivo biosensing of UDG activity. Target UDG can remove uracil base from the detection probe and cause the cleavage of detection probe by apurinic/apyrimidinic endonuclease (APE1), which exposes its toehold domain and initiates catalytic assembly of two fluorescently labeled hairpin probes via toehold-meditated strand displacement reaction (SDA) to generate abundant DNA duplexes with amplified FRET signal. In this assay, target UDG signal is amplified via enzyme-free catalytic reaction and the whole reaction may be completed in one step, which greatly simplifies the assay procedure, reduces the assay time, and facilitates the cellular imaging. This biosensor enables specific and sensitive measurement of UDG down to 0.00029 U/mL, and it is suitable for analyzing kinetic parameters, screening inhibitors, and even imaging endogenous UDG in live cells. Importantly, this biosensor can visually quantify various DNA repair enzymes by rationally altering DNA substrates. [ABSTRACT FROM AUTHOR]

Abstract We develop for the first time a new electrochemical biosensor for signal-on detection of T4 polynucleotide kinase (PNK) based on the enhanced quasi-reversible redox signal of prussian blue generated by self-sacrificial label of iron metal-organic framework (FeMOF). The DNA hairpin probe modified with FeMOF@AuNPs at the 3'-thiol end acts as the substrate of PNK. The presence of PNK enables the 5'-phosphorylation of hairpin probe, which may subsequently function as the substrate of lambda exonuclease. Lambda exonuclease removes the 5' mononucleotides from the stem, unfolding the hairpin structure and releasing the single-stranded DNA (ssDNA). The resultant FeMOF@AuNP-modified ssDNA may specifically hybridize with the capture probe to form the double-strand DNA (dsDNA) duplex, enabling the immobilization of FeMOF on the electrode surface. The reaction of Fe3+ in the MOF with K 4 Fe(CN) 6 leads to the formation of prussian blue on the electrode surface and consequently the generation of a high electrochemical signal. This assay is very simple without the involvement of the pre-synthesized prussian blue which is unstable in neutral buffer, and the self-sacrificial label of FeMOF can achieve great signal amplification without the involvement of any extra functional materials. Due to the introduction of self-sacrificial label of FeMOF and the formation of prussian blue nanolayers with good quasi-reversible cyclic voltammetric electrochemical activity, this biosensor exhibits high sensitivity and a large dynamic range. Moreover, this biosensor can be used to screen the PNK inhibitors, holding great potential in disease diagnosis and drug discovery. Highlights • We develop a new electrochemical biosensor for signal-on detection of T4 polynucleotide kinase (PNK). • This electrochemical biosensor is based on the enhanced quasi-reversible redox signal of prussian blue generated by self-sacrificial label of iron metal-organic framework (FeMOF). • This assay is very simple without the involvement of the pre-synthesized prussian blue which is unstable in neutral buffer. • The self-sacrificial label of FeMOF can achieve great signal amplification without the involvement of any extra functional materials. [ABSTRACT FROM AUTHOR]

Hybridization chain reaction (HCR) based enzyme-free amplification techniques have recently been developed for the visualization of intracellular messenger RNA (mRNA). However, the slow kinetics and potential interference with the intricate biological environments hinder its application in the clinic and in vivo. Herein, we designed a nanofirecracker probe-based strategy using i ntramolecular h ybridization c hain r eaction (IHCR) amplifier for rapid, efficient, sensitive, specific detection and imaging of survivin mRNA both in vitro and vivo. Two probes, HP1 and HP2, in IHCR were simultaneously incorporated into a DNA nanowire scaffolds to bring HP1 and HP2 to close proximity on the assembled nanowire scaffolds. Empowered by the DNA nanowire scaffolds and spatial confinement effect, the nanofirecracker probe-based IHCR sensing system exhibited improved biostability, accelerated reaction kinetics, and enhanced signal amplification. This new strategy has been successfully applied to imaging mRNA in both cultured cells and in mice. Importantly, this novel sensing method was capable of detecting survivin mRNA in clinical blood samples from subjects with colorectal cancer. Thus, this novel nanofirecracker probe-based IHCR strategy holds great potential in advancing both biomedical research and in molecular diagnostics. [ABSTRACT FROM AUTHOR]