Construction Of Ratiometric Electrochemical Biosensor And Its Application

Electrochemical biosensor is one kind of sensors which can quickly detect biomolecules such as proteins and miRNA,and inorganic ions by conversion into electrochemical signal output with some devices.Due to the simple operation,low cost and high sensitivity of electrochemical sensors,researchers in various countries have used electrochemical biosensors to build different types of analysis platforms in the past decades to perform multiple analysis targets.The conventional electrochemical analysis usually uses single-signal output mode,which is susceptible to the system background interference.The application of single signal electrochemical sensors have been limited by background interference,microenvironmental pollution,and slower electron transfer kinetics.Therefore,the ratiometric biosensor with two probe signals have been developed to contrast as the final signal output.Compared with the conventional sensor,dual-signal output of the ratiometric electrochemical biosensor with internal correction would reduce the interference of electrical signals generated by the background,and effectively improve the detection sensitivity and reliability.Owing to its remarkable advantages,the ratiometric electrochemical biosensor has been aroused considerable interest.In this paper,we propose a ratiometric detecting platform based on the conformational change of the aptamer for Staphylococcus aureus and miRNA detection.At first,the hemin double-labeled molecular beacon（Hs-MB）and the anthracycline antibiotic epirubicin are used as the output electrochemical signals to detect staphylococcus aureus.The sensor constructed from it has simple assembly steps and high sensitivity.And on this basis,we have designed a DNA Walker-based signal amplification scheme to improve the sensitivity of the sensor.Chapter 1.IntroductionIn this chapter,the basic principle of electrochemical biosensor and different signal detection strategies have been briefly presented firstly.And then the basic knowledge,development background and application prospect of dual-signal electrochemical biosensor is introduced.At the last,we elaborate the working mechanism and application of the ratiometric biosensor which constructed in our laboratory.Chapter 2.A electrochemical ratiometric biosensor for ultrasensitive detection of Staphylococcus aureus based on conformation conversion of aptamers with switch of electrochemical signalIn this chapter,a reusable ratiometric electrochemical biosensor for highly sensitive and selective detection of Staphylococcus aureus had been designed.A DNA strand（H₁）immobilized on the electrode surface through Au-S bond was hybridizes with the linear shaped Hs-MB containing a piece of anti-staphylococcus aureus aptamer sequence..And then,epirubicin as the second signal tag intercalated into H1-H2duplex.In the presence of S.aureus,H₂ binds to bacteria.The Hemin signal is quenched by the tags at both ends of H₂ strand are close to each other.Epirubicin diffuses in solution by destruction of the double-stranded complex structure.Above changes result in an increase in the reduction peak current of epirubicin（I₁）with a decrease in the reduction peak current of Hemin（I₂）.The linear relationship range from 4 to 4×10⁶ cfu/mL with the detection limit as low as 2 cfu/mL.In this work,we successfully develop the ratiometric biosensor,which has great potential for rapid testing.Chapter 3.A ratiometric biosensor based on DNA Walker to amplify the signal to detect miRNA-21.In this chapter,the signal amplification strategy based on DNA Walker is introduced firstly.A highly selective and sensitive ratio metric biosensor for detecting miRNA-21 was designed.The DNA Walker can be fixed to the surface of the electrode with the presence of target.Then the triple-strand complex structure destroyed,causing the single DNA,which modified the group of Fc,detached from the surface of the electrode.At the meantime,the hairpin DNA was opened to activate the Hemin.During the detection,the peak current value of Hemin（I₁）increased,while the peak current value of Fc（I₂）decreased.The output signal can be amplified in multiples,because a single DNA Walker can destroy multiple triple-stranded complexes.The dual-signal output mode of the sensor can improve the accuracy of detection.Experimental results show that the detection limit of miRNA-21 is as low as 0.088 nmol/L By changing the acquisition probe sequence,the detection target category of the detection platform can be expanded,which has a good development prospect.