Chun-yang Zhang

An ultrasensitive electrochemical biosensor for polynucleotide kinase assay based on gold nanoparticle-mediated lambda exonuclease cleavage-induced signal amplification

Polynucleotide kinase (PNK) plays an essential role in cellular nucleic acid metabolism and the cellular response to DNA damage. However, conventional methods for PNK assay suffer from low sensitivity and involve multiple steps. Herein, we develop a simply electrochemical method for sensitive detection of PNK activity on the basis of Au nanoparticle (AuNP)-mediated lambda exonuclease cleavage-induced signal amplification. We use [Ru(NH3)6]3+ as the electrochemically active indicator and design two DNA strands (i.e., strand 1 and strand 2) to sense PNK. The assembly of strand 2 on the AuNP surface leads to the formation of AuNP-strand 2 conjugates which can be subsequently immobilized on the gold electrode through the hybridization of strand 1 with strand 2 for the generation of a high electrochemical signal. The presence of PNK induces the phosphorylation of the strand 2-strand 1 hybrid and the subsequent cleavage of double-stranded DNA (dsDNA) by lambda exonuclease, resulting in the release of AuNP-strand 2 conjugates and [Ru(NH3)6]3+ from the gold electrode surface and consequently the decrease of electrochemical signal. The PNK activity can be simply monitored by the measurement of [Ru(NH3)6]3+ peak current signal. This assay is very sensitive with a detection limit of as low as 7.762 × 10-4UmL-1 and exhibits a large dynamic range from 0.001 to 10UmL-1. Moreover, this method can be used to screen the PNK inhibitors, and it shows excellent performance in real sample analysis, thus holding great potential for further applications in biological researches and clinic diagnosis.

Development of quantum dot-based biosensors: principles and applications

Development of efficient biosensors for sensitive and selective detection of specific biomolecules is crucial to both fundamental biomedical research and clinical diagnosis. Due to their unique and superior optical and electronic properties, such as high brightness, good photostability, broad absorption spectrum, narrow and size-tunable emission spectrum, large Stokes shift, versatile surface modification, and distinctive photoelectrochemical activity, semiconductor quantum dots (QDs) have been regarded as promising and attractive building blocks for the development of efficient biosensors with high sensitivity, good selectively, rapidity and simplicity. In this review, we summarize the progress of QD-based biosensors in the last 5 years (2013-2018) including QD-based fluorescent, bioluminescent, chemiluminescent, photoelectrochemical biosensors, and focus on their basic principles and their applications for the detection of DNAs, microRNAs, proteins, enzymes, and living cells. Moreover, we give new insight into the future direction and challenges in the development of QD-based biosensors.

Mimic Peroxidase- and Bi2S3 Nanorod-Based Photoelectrochemical Biosensor for Signal-On Detection of Polynucleotide Kinase

We demonstrate for the first time the development of a mimic peroxidase- and bismuth sulfide (Bi2S3) nanorod-based photoelectrochemical (PEC) biosensor for signal-on detection of polynucleotide kinase (PNK) on the basis of manganese-based mimic enzyme (MnME) catalytic precipitation. We use the hybrid film which consists of Bi2S3 nanorods and Au nanoparticles (AuNPs) as the immobilization matrix of capture probe. The capture probe on the modified electrode can specifically hybridize with the MnME@AuNPs-labeled signal probe to form the double-stranded DNA (dsDNA), generating a PEC biosensor. In the absence of PNK, MnME may stimulate the mimic enzyme catalytic precipitation onto the electrode surface, blocking the interfacial electron transfer and eventually leading to a low PEC signal. While in the presence of PNK, the dsDNA is phosphorylated and subsequently cleaved by lambda exonuclease to release the MnME@AuNPs conjugates from the electrode, leading to the decrease of catalytic precipitation on the surface of electrode and consequently the production of a high PEC signal. Notably, the MnME can be easily synthesized and possesses higher catalytic activity than the manganese-based mimic enzyme. This signal-on PEC biosensor exhibits high sensitivity with a detection limit of 1.27 × 10-5 U mL-1 and an extrembly large dynamic range of 5 orders of magnitude. Moreover, it can be applied for the screening of PNK inhibitors and accurate quantification of PNK activity in cancer cell extracts.

A universal DNAzyme-based bioluminescent sensor for label-free detection of biomolecules

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.