Ultrasensitive detection of 25-hydroxy vitamin D3 in real saliva using sandwich-type electrochemical aptasensor

We developed a novel sandwich-type electrochemical aptasensor to detect 25-hydroxy vitamin D3 (25OHVD3), which has high related to various disorders, such as osteoporosis, mental illness, and coronavirus disease 2019. We fabricated heterogeneous nanostructures comprising molybdenum disulfide and an electrochemically reduced graphene oxide composite for amplifying the electrochemical signal. To enhance the sensing performance by applying the sandwich-type strategy, methylene blue-labeled 1,2,4-triazole-3,5-dione (MB-TAD) was functionalized on an electrochemical aptasensor, wherein, MB as a redox mediator in the electrochemical aptasensor is responsible for electron production, and TAD can combine with 25OHVD3 to form aptamer/25OHVD3/MB-TAD complexes. The binding events of 25OHVD3 were studied by monitoring differential pulse voltammetry signals. Moreover, 1,6-hexanedithiol was functionalized on the electrochemical aptasensor to prevent nonspecific binding with proteins present in the saliva and to reduce the steric hindrance between aptamers. The detection capability of the electrochemical aptasensor exhibited a linear detection range of 0.1–150 ng/mL, with a limit of detection of 0.02 ng/mL. Furthermore, the electrochemical aptasensor selectively detected 25OHVD3 from other vitamins and further revealed the possibility of detecting 25OHVD3 in the saliva. Our results suggest that the electrochemical aptasensor based on the sandwich-type method is a highly selective and ultrasensitive method with immense potential for the quantitative detection of vitamins in saliva. •Developed a sandwich-type electrochemical aptasensor for detection of 25OHVD3.•From the sandwich-type strategy, we enhanced the sensing performance of the sensor.•The electrochemical aptasensor could selectively detect 25OHVD3 from other vitamins.•The limit of detection was 0.6 ng/mL in real saliva sample.