Sensitive Detection Of MicroRNA-221 And Myoglobin By Using Multiple Amplifications

The detection of biomarkers has become one of the hotspots.However,most of biomarkers existed at very low concentrations.For example,circulating microRNAs(miRNAs)are present at femtomolar or even lower levels in blood.Thus,it is necessary to construct high sensitive biosensors.Given that single amplification strategy cannot meet the requirements for detecting low concentrations of biomarkers,the development of multiple amplification technology has become a trend.In this thesis,miRNA-221 and myoglobin were chosen as model target,high sensitive biosensors for the detection of biomarker were proposed based on the combination of catalyzed hairpin assembly,supersandwich amplification and enzyme catalytic amplification strategies.In addition,a portable biosensor was prepared to simplify the operation steps and equipment.The details were as follows:1.A novel isothermal electrochemical biosensor was proposed for the sensitive detection of miRNA-221 based on the ingenious combination of the target-catalyzed hairpin assembly(CHA)and supersandwich amplification strategies.On the one hand,CHA can achieve the cyclicality of the target miRNA,which led to signal amplification;on the other hand,the signal probe was modified with horseradish peroxidase(HRP)to form a supersandwich multiplex HRP-DNA label on the electrode,which could achieve an amplified electrochemical signal.Using the isothermal dual signal amplification strategies,as low as 0.6 pM(S/N=3)miRNA-221 could be detected.In addition,this biosensor showed high selectivity and could discriminate miRNA-221 from the homologous miRNAs.Note that human miRNA from cancer cells could also be detected and the results were in excellent agreement with those obtained using qRT-PCR.That it is to say,the proposed biosensor has great potential for broad applications in the field of clinical analysis.Given that the biosensor avoided the introduction of nanoparticles,the limitation of using the nanoparticles was overcome.2.A simple and portable colorimetric aptasensor was proposed for sensitive myoglobin detection.On the one hand,the colorimetric aptasensor achieved the signal enhancement by using supersandwich amplification and HRP-DNA catalytic amplification strategies.On the other hand,the entire process of the colorimetric aptasensor can be done directly in the Ep tube,requiring a few simple steps and simple operation.Moreover,with a portble fiber optic sensor as a detector,it did not need large equipment and professional technical personnel.More importantly,the colorimetric aptasensor could detect as low as 3 nM(S/N=3)myoglobin,which was lower than clinical cutoff for myoglobin in healthy patients.Since the light source was modulated light,fiber optic sensor was not affected by external light.This work was expected to develop into a novel point of care testing.