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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 demonstrate for the first time the development of a universal DNAzyme-based bioluminescent sensor for label-free detection of various biomolecules including DNAzyme and DNA. The presence of DNAzyme may induce the cyclic cleavage of riboadenosine (rA)-containing substrates, and the subsequent digestion of the cleaved substrates by exonuclease III (Exo III) releases abundant AMPs to initiate cyclic AMP pyrophosphorylation-ATP depyrophosphorylation for the generation of an enhanced bioluminescence signal. This sensor can real-time monitor the DNAzyme activity with a detection limit of 3.16 × 10−12 M. Moreover, the DNAzyme may be divided into two subunits for sensitive detection of target DNA. In the presence of target DNA, the two separated subunits may assemble into an active DNAzyme which can catalyze the cyclic cleavage of substrates and initiate the digestion of cleaved substrates by Exo III for the generation of an enhanced bioluminescence signal. This sensor can sensitively detect target DNA with a detection limit of 3.31 × 10−12 M. Importantly, this bioluminescent sensor can achieve a zero-background signal, and its output signal originates from the release of AMP for the generation of self-illuminating light emission without the requirement of either the external labels or the reporting reagents. Graphical abstract We develop a universal DNAzyme-based bioluminescent sensor for label-free detection of various biomolecules including DNAzyme and DNA. Image 1 Highlights • We develop a universal DNAzyme-based bioluminescent sensor for label-free detection of various biomolecules. • DNAzyme can cleave its substrate and release AMPs for the generation of self-illuminating light emission. • This sensor does not require either external labels or the reporting reagents. • This sensor can generate an enhanced bioluminescence signal with zero background. [ABSTRACT FROM AUTHOR]