Ion Input Logic Gates Fluorescence-based DNA Biosensor Research

DNA has the advantages of easy to synthetize, high stability, specificity over antibody or enzymes that are molecular recognition element in antibody or enzyme sensors. The research on biosensor using DNA as molecular recognition element has become one of the most important area in the field of life science. Among them, fluorescence methods have been extensively explored because it is rapid, highly sensitivive and flexible in signal transfer. Therefore, it is of important theoretical and practival significance to develope cost-effective and universal DNA biosensor. This thesis proposed new signal principles of molecular recognition based on conformation transfer using DNA and DNAzyme as molecule recognition probe. Novel DNA fluorescence biosensors have been established for detection of heavy metal ions and protein. Concrete research content include the following three parts:1. Research on Pb-K ion-input based AND Logic Gate for the Detection of Lead ionA label-free fluorescent AND logic gate has been developed utilizing ion-tuned configuration conversion of DNA probe with K+and Pb2+as two inputs. A well-designed hairpin DNA which is composed of a poly-G loop (PS2.M) and a Pb2+DNAzyme (GR-5DNAzyme) stem serves as a recognition probe. The signal molecule aloe-emodin derivative (AED) was designed and synthesized by our group. In the presence of Pb2+, the substrate strand of DNAzyme is irreversibly and specifically cleaved by the DNAzyme strand, which made PS2.M form G-quadruplex in the presence of K+. AED could insert into the G-quadruplex resulting in fluorescence decreased. Such a structural change significantly affects the spectral behaviors of AED. By combing the high specificity of hairpin DNA and GR-5DNAzyme, Pb2+can be highly selectively detected even when coexisted with other metal ions. Circular dichroism (CD), UV-Vis absorption spectrometry and fluorescence polarization (FP) measurements further verified the sensing mechanism. This method can be explored to ultra-sensitively detect Pb2+with a limit of detection of23pM, the linear range was form50pM to50nM. Therefore, it provides a solid sensing platform for the detection of targets by altering the specific sequence of nucleic acid probe. 2. Research on Pb-Hg ions-input OR logic gate by GNRs-based fluorescence resonance energy transfer assayA rapid and simple metal ions OR logic gate has been developed based on the difference in fluorescence resonance energy transfer (FRET) efficiency between gold nanorods (GNRs) and DNA with different configuration. Fluorescein labeled DNA aptamer was used as molecular recongnition probe. When the positively charged gold nanorods mixed with DNA, the fluorescent intensity is decreased due to the FRET between GNRs and DNA. Input Pb2+or Hg2+, the negative charge density of increased due to the configuration switching of DNA, which greatly strengthened the electrostatic interaction between GNRs and DNA and led to a further quench of the fluorescence. UV-Vis absorption spectrometry and circular dichroism (CD) measurements further verified the reliability and reasonability of the sensing mechanism. The research results showed that detection linear range of lead (Ⅱ) and mercury (Ⅱ) range from5pM to10nM, the detection limits were2pM and3pM, respectively. It is a simple, cost-effective, high sensitivive method for detection of heavy metal ions.3. Research on Detection of Thrombin by Gold Nanorods-based FRET AssayA fluorescence resonance energy transfer (FRET) aptasensor has been developed to specifically and sensitively detect thrombin based on the difference in FRET efficiency between gold nanorods and DNAs. Carboxyfluorescein-labeled thrombin-binding aptamer (F-TBA) was used as a recognition probe. It is based on the difference in electrostatic interaction between GNRs and F-TBA in the absence and presence of thrombin. When the positively charged gold nanorods mixed with DNA, the fluorescence intensity of F-TBA decreased due to the FRET between GNRs and DNA. In the presence of thrombin, F-TBA formed G-quadruplex and the charge density of increased, leading to a further quench of F-TBA. Therefore, thrombin can be quantitatively detected by monitoring the change of fluorescence intensity induced by G-quadruplex formation. Sensing mechanism has been further verified by circular dichroism (CD), transmission electron microscope (TEM) and UV-vis absorption measurements, indicating that the fluorescence change is owing to the specific binding of F-TBA with thrombin. Under the optimum conditions, the method exhibits a dynamic response range from1pM to5nM with a detection limit of0.3pM. Therefore, it is a simple, sensitive and cost-effective method for homogeneous proteins assay.