Scientists Develop New Electrochemical/fluorescent Dual-mode Biosensor

Researchers at the Suzhou Institute of Biomedical Engineering and Technology (SIBET) of the Chinese Academy of Sciences recently proposed a structured DNA assembly strategy and developed an electrochemical/fluorescent dual-mode biosensor for circulating tumor DNA based on methylene blue. and red emission carbon nanodots.

The new sensors, combining the characteristics of electrochemical and fluorescent sensors, whose signal sources and construction methods are usually very different, according to the researchers.

Electrochemical sensor is a qualitative or quantitative method based on the correlation between targets caused by changes in electrical signals and concentrations or other physical parameters. Fluorescence sensor is a qualitative or quantitative detection method by transmitting a specific combination of target elements and introduction to the fluorescence element, causing a change in the fluorescence intensity or emission wavelength.

“Integrating two technologies for synchronous detection in one unique system can not only effectively improve detection accuracy, but also reduce the influence of background signals, instrument fluctuations, and other factors on the acquired signal,” said MIAO Peng, the lead researcher of the study. and also a scientist from SIBEBT.

Specifically, Probe A in this sensor is immobilized at the electrode surface via a thiol group labeled on its 5′ terminal. In the presence of the target, a complete double strand is formed between the target and its binding domain in Probe A.

Meanwhile, the rod region is opened and a single strand region is released which is responsible for opening the second hairpin of Probe B. Previously conjugated to a red emission carbon nanodot via the 3′ NH terminal₂. Next, the hairpin structure of Probe C was opened with the single strand region of Probe B released.

In addition, Probe C is able to replace the target sequence to form a complete three-way junction structure. The targets are thus recycled and aid in the formation of multiple three-way junctions. Since the abundant methylene blue molecules at the 3′ terminal of Probe C are located near the electrode interface, a significant electrochemical response can be recorded to reveal the target.

The released single-stranded swing arm formed by the 3 terminal of Probe A and the terminal 5′ of Probe B at the three-way junction acts as the DNAzyme-coupling terminal 3 of Probe B at the adjacent three-way junction as a substrate. Thus, a hand-in-hand structured DNA monolayer was created.

In the presence of Mg²⁺, the substrate sequence can be cleaved and the conjugated carbon nanodots released into the solution. “By measuring the increase in fluorescence emission, the original target level can also be evaluated by fluorescence techniques,” MIAO said.

Theoretical calculations and gel electrophoresis imaging confirmed the feasibility of the reaction. The synthesized carbon nanodot has strong anti-interference and can maintain high fluorescence stability in physiological environments.

Through a series of condition optimizations and quantitative tests, MIAO and his team established a linear calibration curve of electrochemical/fluorescence intensity and target concentration, which can achieve a six-fold wide linear range.

At the same time, the target contents can also be easily distinguished by fluorescence imaging. The dual mode sensor developed in this work is a novelty with high sensitivity and strong scalability, which can provide a powerful tool for nucleic acid analysis and clinical diagnosis.

The sensor is expected to be widely used in basic research, environmental detection, clinical trials and other fields.

The results of the study entitled “Hand-in-hand structured DNA monolayer for dual mode analysis of circulating tumor DNA” published in the latest issue of the Journal of Chemical Engineering.

Figure 1. Standard free energy (ΔG) of the reaction process and PAGE analysis. (Image by SIBET)

Figure 2. Fluorescence response of red emission carbon nanodots to potential interference

ion. (Image by SIBET)

Figure 3. Square wave voltammogram and fluorescent response after target DNA triggers DNA assembly. (Image by SIBET)

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