Study On Protein Biosensor Based On G-quadruplex Probe

The G-quadruplex is a kind of secondary structure of DNA.It is a special structure formed by guanine(G)-rich nucleic acids that form a G-quadron by four Hoogsteen hydrogen bonds and then form a π-π stack.Some proteins,metal ions and organic dyes can induce the formation of Gquadruplexes and combine with them to form stable complexes.Thioflavin T(ThT)is a highly specific G-quadruplex fluorescent probe that can be embedded in the G-quadruplex and emit strong fluorescence,which providing a new idea for the design of highly sensitive biosensors.Water-soluble conjugated polymers have strong light absorption,high fluorescence quantum yield and good light stability,which have been widely used in the fields of chemical/biological sensing and fluorescence imaging.Proteins are the most abundant and important biological macromolecules in organisms.Many diseases are related to the abnormal expression of proteins.Therefore,the detection of specific proteins has important significance in both biological and medical fields.Based on the G-quadruplex probe ThT and fluorescent conjugated polymers with high brightness,two highly sensitive optical biosensing strategies were designed to achieve simple and quick detection of γ-interferon and telomerase activity.Interferon-gamma(INF-γ)is an important cytokine protein with antiviral,antiproliferative,differentiation-inducing and immunoregulatory properties.The aptamer of INF-γ is a special G-rich sequence that can form a G-quadruplex structure induce by ThT,while ThT is embedded in the Gquadruplex structure and emits fluorescence.However,in the presence of the target protein INF-γ,since the specific affinity between the aptamer and INF-γ is much greater than the interaction with ThT,the INF-γ/aptamer complex is formed,releasing ThT in a free state,so the fluorescence of the system is weak.By detecting changes the fluorescence of the system,specific detection of INF-γ can be achieved.The method is simple to operate and can be tested in minutes,with a detection limit of 5 nM for INF-γ.Telomerase is a special ribonucleoprotein reverse transcriptase that can use its own RNA as a template for telomere DNA elongation.At present,telomerase has been used as a broad tumor marker,and its activation is closely related to the cancerous process of cells.First,an in vitro telomerase detection strategy based on ThT was designed.ThT serves as a fluorescence reporter group.Under the catalysis of telomerase,the primer TS-primer can be extended with TTAGGG as a repeating unit to form a longer G-rich sequence.In this case,ThT can be embedded and emits strong fluorescence.However,the primer is short and the G-quadruplex structure could not be formed in the absence of telomerase,so the fluorescence of the system is weak.This method is simple and rapid in the detection of telomerase in vitro,but there is still a problem of low brightness for imaging of telomerase in living cells.Further,in order to improve the resolution and brightness of telomerase imaging in living cells,a water-soluble conjugated polymer PPE-PLL having excellent optical properties was introduced.Since both the excitation wavelength and the fluorescence emission wavelength of ThT are very close each other,a significant amplification of the original ThT signal can be achieved by the enhanced fluorescence of aggregated polymer and the fluorescence superposition between PPE-PLL and ThT.Thus,high-brightness imaging of telomerase activity was achieved.