Research On The Construction Of Highly Sensitive Fluorescence Detection Methods For Protein And MicroRNA

Highly sensitive and rapid detection of biomarkers can significantly facilitate scientific research,disease prevention,drug analysis and so on.Especially in the screening of biomarkers of early disease,advances in cancer control are greatly aided by early detection.With the continuous development of science and technology,fluorescent biosensor has attracted more and more scientists attention for their advantages of high selectivity,simple operation,high sensitivity and homogeneous system.It has been widely used in the fields of disease diagnosis,efficacy evaluation,scientific research and food analysis.In order to further improve the level of biomarker detection,three fluorescent biosensors were constructed in combination with different signal amplification methods for sensitive detection MUC1 and microRNA-21.The specific research work can be divided into the following parts:(1)Target-induced autonomous synthesis of G-quadruplex sequences for label-free and amplified fluorescent aptasensing of mucin 1.Highly sensitive and simple detection of biomarkers can significantly facilitate early disease diagnosis and treatment.Using a new target-triggered autonomous synthesis of G-quadruplex sequences via primer exchange signal amplification strategy,we developed an ultrasensitive and simple label-free aptamer-based method for sensing MUC1,a protein biomarker,in this work.The target molecules bind and cause the change in conformation of the aptamer region in the hairpin probes to expose the primer complementary sequences,which subsequently hybridize with the primers to initiate the ssDNA synthesis process with the aid of the DNA polymerase.By encoding the G-quadruplex complementary sequence in the template,numerous G-quadruplex sequences with various lengths can therefore be produced.The thioflavin T further binds these G-quadruplex sequences to yield substantially enhanced fluorescence for ultrasensitively detecting MUC1 within the linear range from 5 to 1000 pM with the 1.9 pM detection limit in a non-label approach.Showing high sensitivity with convenient signal amplification,such an aptamer-based protein sensing strategy thus exhibits promising potentials for establishing various platforms for detecting different cancer markers at trace levels.(2)Target-initiated bidirectional enzyme-free catalytic signal amplification strategy for MUC1 detection.Rapid and sensitive detection of biomarkers is significance for the early diagnosis of disease.Here,we developed a bidirectional enzyme-free catalytic signal amplification strategy for detecting MUC1,using a long DNA track strand to limit probe components to a compact space.The target binds to the aptamer to release the initiation strand,which can open the detection probe in both directions.After a series of strand displacement processes,the short strand in the probe are released and participate in the cycle as indirect initiation strands.Significantly enhanced fluorescence signals are available due to the fast opening of all quenched double strands.The method for MUC1 detection demonstrated excellent linear relationship to concentrations varying from 0.5 pM to 1000 pM and the detection limit of 0.18 pM.It provides a promising detection platform for biomarker detection and has a potential application prospects in disease diagnosis and treatment.We expect that this design can be widely used in basic biomedical research and disease diagnosis.(3)Target-triggered chain replacement to form RCA signal amplification strategy for highly sensitive detection of microRNA.microRNA is an important biomarker for cancer diagnosis.We report a sensing strategy for sensitive and selective detection of microRNA using a new target-induced chain replacement to form RCA signal amplification strategy in this work.This method initiates the chain replacement reaction in the presence of miRNA-21 to form the DNA quadruplex structure and form two rolling rings under the action of T7 RNA polymerase and T4 DNA ligase.Two rolling rings produce two long RNA strands and hybridize to form Broccoli aptamers.The fluorescent dye DFHBI further combines with the Broccoli to show significantly enhanced fluorescence signal for sensitive detection of miRNA-21.Under the optimal experimental conditions,the method for miRNA-21 detection has excellent linear relationship to concentrations varying from 10 fM to 10 nM and the detection limit of 4.1 fM.More importantly,the method has high selectivity and sensitivity.With the demonstration of this detection method,this detection strategy can be potentially extended to the detection of different types of biomolecules.