Nanopore-based Tetracycline Detection

Tetracycline(Tet) is one kind of the antibiotics, which is widely used in both human and animal medicine. The use of antibiotics may bring many environment problems. For example, the antibiotics residue in environment may mobile into the ground and surface water, and then cause the development of antibiotics-resistant bacteria and some unknown chronic disease. There are many approaches have been developed for antibiotics residue: enzyme-linked immunosorbent assay, liquid chromatography(LC), surface plasmon resonance biosensor, and liquid chromatography-tandem mass spectrometry(LCMS/MS). But each of them has its own disadvantages. For instance, high cost, time consuming, simple operating, and so on. These shortcoming prevent all these kinds of methods developing. So it is important to find a simple, efficient method.Nanopore sensor has been studied for several decades since the development of coulter counter. Because the development of nano technology, nanopore-based analysis attract more and more attention. Nanopore sensor could detect biomolecule at single molecular level, which is not only a lab-free recognition approach needing low sample volume, but also more direct, clear and sensitive. The development of nanopore sensor include two stages: biological nanopore and solid-state nanopore. Both two kinds of nanpore has its own advantage and disadvantage. For example, the smaller biological channel size improve the spatial resolution, but their size is hard to adjust. The size of solid-state nanopore is tunable, but they do not have high spatial resolution. Nanopores are often fabricated in Si3N4 or SiO2 membranes by using focused ion beam(FIB), transmission electron microscope(TEM) or dielectric breakdown method. Nanopore sensor is often used in sensing nucleic acid polymer structure, ion detection, DNA sequencing, biomolecule identification, and so on.In this article, we adapt nanopore technique combined with biological mechanism to monitor Tet. rtTA(reverse tetracycline-controlled transactivator) and TRE(tetracycline responsive element) could bind with each other and form a complex under the action of Tet. When this complex passes through a nanopore with appropriate size, it will generate a block ionic current. Then we can identify rtTA, TRE and their complex through detection of their ionic current. We used two different method to detect rtTA, TRE fragment, and their complex in vitro, one is electrophoretic mobility shift assays(EMSA), the other is nanopore sensor. From EMSA, we successfully identify these different kinds of biomolecules. In the nanopore experiment, we firstly fabricate solid-state nanopore at sub-10 nm diameter in Si3N4 membranes with 10 nm thick by using dielectric breakdown. Then we test rtTA and TRE at the single molecule level, which identified them successfully. Moreover, we detected that there is some interaction even with the absence of Tet, which their ionic signals were different from both rtTA and TRE fragment. To further study how the rtTA interact with Tet, we detected this interaction under different concentration of Tet and visualized the process that rtTA bind to TRE fragment. Compare to EMSA, the namopore sensor could distinguish these different kinds of molecule and their interactions at the level of single molecule, which is more direct, clear and sensitive.