The Construction Of Light-gated Cascade DNA Circuit Under The Encapsulation Of A Protein-derived Facilitate Nanocarrier For Imaging In Living Cells

With the development of fluorescent biosensor technology,a series of breakthroughs and achievements have been made in the imaging research of cancer-related biomarkers.Meantime,the biological imaging still has many bottlenecks and difficulties to be solved.Therefore,the sound evolution of bioimaging can be effectively promoted by continuously optimizing fluorescence biosensing schemes and technologies.Objective Accurate detection of mi RNA provides a crucial basis for tumor screening,differential diagnosis,evaluation of efficacy and prognosis.Cascade DNA circuits are widely used in fluorescent biosensors for mi RNA detection due to their outstanding design flexibility and efficient signal amplification capabilities.In order to further explore its potential in imaging mi RNA in living cancer cells,we propose corresponding solutions to the existing problems of non-specific triggering and low cell uptake efficiency.Methods In this study,a protein-derived facilitate nanocarrier encapsulated photo-controlled cascade DNA circuit sensor was constructed for controllable and targeted imaging of let-7a in living cancer cells.Firstly,a light-gated cascade DNA circuit is constructed,and the amplification strategy of entropy-driven strand displacement and hybridization chain reaction is integrated to form a cascade DNA circuit.By embedding a photocleavage linker(PC-linker),the light-gated mode is introduced into the cascade DNA circuit to realize the controllable sensing under the irradiation of ultraviolet light,so as to overcome the non-specific activation of intracellular sensing process.The protein-derived facilitate nanocarriers were further synthesized to encapsulate the above-constructed light-gated cascade DNA circuit to achieve effective targeted delivery of the sensing system to enhance its cell uptake efficiency.For the constructed fluorescent biosensor,the morphological structure,element distribution and charge properties were characterized by transmission electron microscopy,energy dispersive spectroscopy and Zeta potential analysis,and further experiments were designed to verify the functional characteristics of protein-derived facilitate nanocarriers.Then the optical control feasibility of the sensing system is analyzed,including polyacrylamide gel electrophoresis experiment and fluorescence spectrum analysis.After optimizing the ultraviolet irradiation time,temperature and p H involved in the sensing,the performance of the sensing system for detecting and imaging the target let-7a in buffer,live cancer cells and animals was investigated in turn.Results Transmission electron microscopy,energy dispersive spectroscopy and Zeta potential analysis proved the successful synthesis of cascaded DNA circuits encapsulated by protein-derived facilitate nanocarriers.Further experiments verified that the sensing system had high encapsulation efficiency,enzyme resistance and weak acid-responsive dissociation characteristics.The feasibility of ultraviolet light control was confirmed by polyacrylamide gel electrophoresis and fluorescence spectrum analysis.The sensing conditions were further optimized,ultraviolet irradiation time of 15 minutes,sensing temperature of 37℃,p H 7.5(cascade DNA circuit)and 6.0(protein-derived facilitate nanocarrier encapsulation cascade DNA circuit)After optimization,the performance of the sensing system to detect the target let-7a in the buffer was analyzed.It was found that the recovery rate of Cy3 signal was linearly correlated with the logarithm of the target concentration(R~2=0.9962),and the limit of detection was 4.32 p M.Furthermore,the sensing system was applied to cell imaging.The sensing system can not only qualitatively distinguish normal cells from cancer cells,but also different cancer cells,and quantitatively analyze the content of target let-7a in MCF-7 cells and He La cells.The analysis results were consistent with the real-time RT-PCR,which is the gold standard method.Finally,the exploratory in vivo imaging also depicts the favorable performance of the sensor in complex biological environments.Conclusion The fluorescent biosensor constructed in this study realized the activation of sensing with ultraviolet light through introducing light-gated manner.In the meantime,the synthesized protein-derived facilitate nanocarrier has the characteristics of biocompatibility and targeted delivery.The integrated sensing system not only demonstrated excellent sensitivity in the buffer detection,but also showed specificity in distinguishing single-base mutations,and further cell and in vivo imaging also demonstrated its targeting accuracy and biocompatibility.Therefore,this sensing method can be used as a templated imaging platform to identify cancer cells and respond to intratumoral targets,providing fresh notion for cancer-related biomarkers analysis.