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Vitamin D is an essential metabolite, and its deficiency causes many skeletal and chronic diseases. 25-hydroxyvitamin D 3 (25(OH)D 3) is known as a vitamin D biomarker and is widely used in clinical diagnosis to determine vitamin D status in human bodies. Herein, we propose a novel electrochemical aptasensor for the sensitive detection of 25(OH)D 3 via the employment of DNA tetrahedron and catalytic hairpin assembly (CHA) amplification strategy. The electrochemical aptasensor was fabricated by the immobilization of the DNA tetrahedron on the Au surface, followed by assembling the hairpin DNA probe-25(OH)D 3 complex (HP1-D). The HP1-D could be replaced by the HP2 hairpin and released for initiating CHA cycles. Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) had been employed to study the electrochemical properties of the developed sensor. The designed electrochemical aptasensor showed a wide linear range of 0.1–1000 nM with a low detection limit (LOD) of 0.026 nM for the detection 25(OH)D 3 with high selectivity, stability, and reproducibility. Furthermore, the proposed sensor was demonstrated in human serum to detect 25(OH)D 3 , indicating that it has a promising potential application in the clinical diagnosis of vitamin D deficiency. • Aptamer of 25(OH)D 3 was applied to establish novel electrochemical biosensor. • Catalytic hairpin assembly strategy and DNA tetrahedron were used for enhancing the aptasensor performances. • The developed aptasensor had good specificity and sensitivity towards target, suggesting potential application in clinic. [ABSTRACT FROM AUTHOR]

The cancer diagnosis with single level of biomarkers suffers from limitation of insufficient accuracy. Hence, developing sensitive, rapid and adaptative analytical strategies for double-level biomarkers are essential for improving the accuracy of clinical cancer diagnosis at early stage. Herein, a DNA biosensor was established based on the catalytic hairpin assembly-mediated Y-junction nicking enzyme assisted signal amplification (CHA-YNEASA) circuits, where the two circuits were concatenated by molecular beacon (MB). In absence of target, both the CHA and YNEASA circuits were effectively hindered because of MB's outstanding ability to control signal background. In presence of target, the initiated CHA circuits made enzyme recognition sequences in close proximity to the assisted sequences to open MB, leading to further trigger the YNEASA circuits. Due to the unique design of dual signal amplification strategies, CHA-YNEASA circuits significantly shorten the reaction time, and improve signal-to-background ratio as well as facilitate the analysis process. It was demonstrated that a high sensitivity with limit of detection (LOD) of 0.9 pM for p53 gene detection was obtained just within 23 min by the proposed DNA biosensor. Moreover, mismatched p53 gene at nucleic acid level was effectively discriminated and strong anti-interference capability was achieved. Noticeably, the DNA biosensor was adaptative for designing a cytosensor at cell level using hairpin DNA, containing MUC1 aptamer and initiation strand of CHA-YNEASA circuits, as switch based on modularity principle. The cytosensor is able to measure MUC1 positive breast cancer cells (MCF-7) with the LOD as low as 100 cells/mL. Excellent specificity for MUC1 negative cells, and good anti-interference capability in 10% fetal bovine serum (FBS) were observed by the cytosensor. Therefore, the proposed DNA biosensor is a sensitive, rapid, adaptative platform for detection of double-level biomarkers, offering novel strategy applied for clinical cancer diagnosis. Image 1 1. DNA biosensor was developed based on catalytic hairpin assembly-mediated Y-junction nicking enzyme assisted signal amplification. 2. The DNA biosensor shows high efficiency of reaction leading to rapid analysis just in one-step. 3. High sensitivity, strong selectivity and anti-interference were achieved for p53 gene and cancer cell detection. 4. The DNA biosensor is adaptative for the detection of multilevel biomarkers using modularity principle. [ABSTRACT FROM AUTHOR]