It remains a huge challenge to integrate the sensitivity, stability, reproducibility, and anti-fouling ability of electrochemical biosensors for practical applications. Herein, we propose a self-assembled electrode combining hexanethiol (HT), poly-adenine (poly-A), and cholesteryl-modified DNA to meet this challenge. HT can tightly pack at the electrode interface to form a hydrophobic self-assembled monolayer (SAM), effectively improving the stability and signal-to-noise ratio (SNR) of electrochemical detection. Cholesteryl-modified DNA was immobilized at the electrode through the hydrophobic interaction with HT to avoid the competition between the SAM and the DNA probe on the gold site. Thus, the assembly efficiency and uniformity of the DNA probe as well as the detection reproducibility were increased remarkedly. Poly-A was added on the HT assembled electrode to occupy the unreacted sites of gold to further enhance the antifouling ability. The combination of HT and poly-A allows the electrode to ensure favorable anti-fouling ability without sacrificing the detection performance. On this basis, we proposed a dual-signal amplification electrochemical biosensor for the detection of exosomal microRNAs, which showed excellent sensitivity with a detection limit down to 1.46 aM. Importantly, this method has been successfully applied to detect exosomal microRNA-21 in cells and human serum samples, proving its potential utility in cancer diagnosis.
Understanding the functions of enzymes in various physiological processes is important, but the design of signaling probes for fast analysis of enzymatic activity is particularly challenging. Herein, a fluorescence-enhanced probe, 10-methyl-2-nitro-acridone (MNA), was synthesized and applied to analyze nitroreductase (NTR) activity in vitro and in vivo. The detection mechanism is based on the nitro group in MNA reacting toward NTR with high reactivity and generating 10-methyl-2-amino- acridone (MAA) accompanied by an obvious fluorescence signal enhancement at 525 nm emission. The probe shows low cytotoxicity, fast response, and high selectivity and sensitivity with a limit of detection as low as 150 ng·mL−1. The probe was also employed for two-photon fluorescence imaging of NTR in zebrafish in vivo revealing the distribution of NTR. Versus existing NTR probes, the proposed probe shows favorable analytical performance including near-infrared light excitation with no other byproducts produced after the reaction. The superior properties of this signaling probe allow it to become a fluorescence imaging candidate in other biosystems.
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