BACKGROUND:Kanamycin is an antibiotic that can easily cause adverse side effects if used improperly. Due to the extremely low concentrations of kanamycin in food, quantitative detection of kanamycin becomes a challenge. As one of the DNA self-assembly strategies, entropy-driven strand displacement reaction (EDSDR) does not require enzymes or hairpins to participate in the reaction, which greatly reduces the instability of detection results. Therefore, it is a very beneficial attempt to construct a highly sensitive and specific fluorescence detection method based on EDSDR that can detect kanamycin easily and quickly while ensuring that the results are effective and stable. RESULTS:We created an enzyme-free fluorescent aptamer sensor with high specificity and sensitivity for detecting kanamycin in milk by taking advantage of EDSDR and the high specific binding between the target and its aptamer. The specific binding can result in the release of the promoter chain, which then sets off the pre-planned EDSDR cycle. Fluorescent label modification on DNA combined with the fluorescence quenching-recovery mechanism gives the sensor impressive fluorescence response capabilities. The research results showed that within the concentration range of 0.1 nM-50 nM, there was a good relationship between the fluorescence intensity of the solution and the concentration of kanamycin. Specificity experiments and actual sample detection experiments confirmed that the biosensor could achieve highly sensitive and specific detection of trace amounts of kanamycin in food, with a detection limit of 0.053 nM (S/N = 3). SIGNIFICANCE:To our knowledge, this is the first strategy to combine EDSDR with fluorescence to detect kanamycin in food. Accurate results can be obtained in as little as 90 min with no enzymes or hairpins involved in the reaction. Furthermore, our enzyme-free biosensing method is straightforward, highly sensitive, and extremely specific. It has many possible applications, including monitoring antibiotic residues and food safety.

Herein, we report a facile method for cholesterol detection by coupling the peroxidase-like activity of polypyrrole nanoparticles (PPy NPs) and cholesterol oxidase (ChOx). ChOx can catalyze the oxidation of cholesterol to produce H2O2. Subsequently, PPy NPs, as a nanozyme, induce the reaction between H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the increase is proportional to cholesterol with concentrations from 10 to 800 μM in absorbance of TMB at 652 nm. The linear range for cholesterol is 10-100 μM, with a detection limit of 3.5 μM. This reported method is successfully employed for detection of cholesterol in human serum. The recovery percentage is ranged within 96-106.9%. Furthermore, we designed a facile and simple portable assay kit using the proposed system, realizing the on-site semiquantitative and visual detection of cholesterol in human serum. The cholesterol content detected from the portable assay kit were closely matching those obtained results from solution-based assays, thereby holding great potential in clinical diagnosis and health management.

MicroRNAs play a crucial role in regulating gene expression and cellular function. Reliable detection of miRNA is highly demanded in clinical diagnosis and therapy. Herein, we designed a structure-convertible DNA switch and constructed a novel switch-conversional ratiometric fluorescence biosensor (SCRF biosensor) for highly sensitive miRNA detection by the use of amplicon fragments to convert the structure of the switch. The DNA switch was a sophisticated designed single-strand DNA with a stem-loop structure and modified with two fluorophores (Cy3 and Cy5) and one quencher at specific sites of the switch. Amplicon fragments (c*) were produced by an exponential amplification reaction. When the c* hybridized to the loop of a DNA switch, the structure of the switch would convert, and fluorescence resonance energy transfer occurred between Cy5 and Cy3. Then two fluorescence signals with different trends would be observed. As a result, by the ratio of the two signals, we can quantitatively and quickly detect the target miRNA with the concentration range from 100 fM to 100 nM and the excellent detection limit down to 70.9 fM, providing this new SCRF biosensor broad application prospects.

Nucleic acid nanoswitches have a status that cannot be ignored in the field of biosensing due to the excellent biocompatibility and flexibility of design. In our current research, we have constructed a new electrochemical platform based on self-assembled pH-sensitive continuous circular DNA nanoswitch for miRNA-21 detection. We elaborately designed an inside ring probe (IRP) which could form a circle when complemented with an outside ring probe (ORP). Under the weakly acidic condition, IRPs and ORPs are self-assembled into continuous annular DNA, meanwhile, the nanoswitch is activated. However, if it is not a weakly acidic environment with a pH equal to 6, these circles are separated and the nanoswitch cannot be triggered. Therefore, the biosensor doesn't work. Only when the pH is 6, can the nanoswitch be activated. Consequently, a large number of RuHex will accumulate on the continuous annular DNA, which leads to highly sensitive detection of miRNA-21, with concentration ranged from 10-15 to 10-8 M and limit of detection down to 0.84 fM. More importantly, this nanoswitch-based biosensor can directly detect the target microRNA in human serum without pretreatment. Therefore, the proposed novel electrochemical DNA nanoswitch will have broad application prospects in biomarker detection and clinical diagnosis.