Ji Nie

Novel Homogeneous Label-Free Electrochemical Aptasensor Based on Functional DNA Hairpin for Target Detection

We first developed a label-free and immobilization-free homogeneous electrochemical aptasensor, which combined a smart functional DNA hairpin and a designed miniaturized electrochemical device. Cocaine was chosen as a model target. The anticocaine aptamer and peroxidase-mimicking DNAzyme were integrated into one single-stranded DNA hairpin. Both aptamer and G-quadruplex were elaborately blocked by the stem region. The conformation switching induced by the affinity interaction between aptamer and cocaine released G-quadruplex part and turned on DNAzyme activity. The designed electrochemical device, constructed by a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode, was applied to the detection of homogeneous DNAzyme catalytic activity at the microliter level. The aptasensor realized the quantification of cocaine ranging from 1 to 500 μM with high specificity. The clever combination of the functional DNA hairpin and the novel device achieved an absolutely label-free electrochemical aptasensor, which showed excellent performance like low cost, easy operation, rapid detection, and high repeatability.

G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection

A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNA in the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in the second stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readout element. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidase-mimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Taking advantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermal cycling . As no complex synthesis or extra downstream operation is needed, the whole easy handling procedure can be finished in no more than 1h. This assay allows the sensing of the model DNA with the limit of detection to be 2.5pM. Moreover, it demonstrates good discrimination of mismatched sequences. The strategy has also been successfully implemented to sensitively detect Tay-Sachs genetic disorder mutant.

Methylene blue as a G-quadruplex binding probe for label-free homogeneous electrochemical biosensing.

Herein, G-quadruplex sequence was found to significantly decrease the diffusion current of methylene blue (MB) in homogeneous solution for the first time. Electrochemical methods combined with circular dichroism spectroscopy and UV-vis spectroscopy were utilized to systematically explore the interaction between MB and an artificial G-quadruplex sequence, EAD2. The interaction of MB and EAD2 (the binding constant, K ≈ 1.3 × 10(6) M(-1)) was stronger than that of MB and double-stranded DNA (dsDNA) (K ≈ 2.2 × 10(5) M(-1)), and the binding stoichiometry (n) of EAD2/MB complex was calculated to be 1.0 according to the electrochemical titration curve combined with Scatchard analysis. MB was proved to stabilize the G-quadruplex structure of EAD2 and showed a competitive binding to G-quadruplex in the presence of hemin. EAD2 might mainly interact with MB, a positive ligand of G-quadruplex, through the end-stacking with π-system of the guanine quartet, which was quite different from the binding mechanism of dsDNA with MB by intercalation. A novel signal read-out mode based on the strong affinity between G-quadruplex and MB coupling with aptamer/G-quadruplex hairpin structure was successfully implemented in cocaine detection with high specificity. G-quadruplex/MB complex will function as a promising electrochemical indicator for constructing homogeneous label-free electrochemical biosensors, especially in the field of simple, rapid, and noninvasive biochemical assays.

Micropipet Tip-Based Miniaturized Electrochemical Device Combined with Ultramicroelectrode and Its Application in Immobilization-Free Enzyme Biosensor

In this study, a simple miniaturized microliter electrochemical device was constructed using a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode. The novel electrochemical device set the electrochemical reaction in a micropipet tip containing an ultramicroelectrode. We investigated the feasibility of the designed electrochemical device by cyclic voltammetric measurements of redox probe. Its application in an immobilization-free enzyme electrochemical biosensor was also evaluated. Horseradish peroxidase and glucose oxidase were selected to test sensor feasibility. Our results showed that the micropipet tip-based electrochemical device could detect low substrate or enzyme concentration or enzymatic reaction rate. The electrochemical device was applied to analyze the glucose content in human blood samples. With the advantages of low cost, easy operation, rapid detection and high reproducibility, this design provides a new approach in immobilization-free enzyme biosensor construction. Integrated with an ultramicroelectrode, our micropipet tip-based electrochemical device could replace most normal electrodes and electrochemical cells in common laboratories for electroanalysis.

A cupric ion triggered DNA diode based on a tandem linkage–cleavage reaction

Based on click chemistry and DNAzyme, we constructed for the first time a cupric ion triggered DNA diode using a tandem linkage-cleavage process, which could occur in order in a programmable and autonomous manner. This DNA diode is a new DNA functional element and can precisely control the translation direction of information.