| Biosensor is a kind of analysis method based on biomolecule specific identification and its downstream signal transitions to achieve the detection of target.The nucleotide probe is a probe based on the principle of nucleic acid molecular hybridization.Nucleic acid probe has the advantages of strong specificity,high sensitivity,rapid and accurate measurement results,and it has been widely used in the detection of biomolecule,drug analysis,clinical disease diagnosis and treatment and so on.Nanomaterials due to their unique quantum size effect,surface effect and volume effect in the field of biosensing,especially fluorescence sensing has been vigorously developed.In recent years,the application of nucleic acid probes and nanomaterials in biosensing and detection has been widely concerned and developed.Based on functional nucleic acid probes and nanomaterials,two nucleic acid fluorescent probes were used for detection of extracellular pH in hypoxic environment and imaging of intracellular mRNAs.(1)In situ imaging analysis of extracellular membrane pH in cells under hypoxia based on FRET DNA-lipidIn this chapter,diacyl conjugates are used to interact with the cell membrane,and the diacyl liposomes with double labeled i-motif sequences are inserted into the surface of the cell membrane to achieve the ratio of extracellular pH detection in hypoxia and normoxia environment.Firstly,two kinds of fluorophores cy3 and cy5 were labeled at both ends of the i-motif sequence.Under the condition of high pH,the fluorophores at both ends of the i-motif chain were separated and the FRET effect was weak.Under the condition of low pH,The i-motif chain forms a tetrameric structure on the surface of the cell membrane,and the fluorescent groups at both ends are close to each other,resulting in a strong FRET effect.Quantitative detection of pH can be achieved by measuring the fluorescence intensity ratio of the two fluorophores.In the absence of oxygen,intracellular glycolysis produces a large amount of acid,and the dynamic equilibrium of pH is broken.Tumor cells are up-regulated by the H+transporters of the cytoplasmic membrane,such as sodium hydrogen exchange protein-1(NHE-1),the H+ excretion of cells,and ultimately cause extracellular environment acidic.This change in pH affects the adhesion,migration,and drug resistance of the tumor.Therefore,the development of accurate detection of extracellular pH in the detection of hypoxic conditions is of great significance in pathology.The sensitive response of this probe to pH can quantitatively detect the pH outside the cell under hypoxic conditions and in normal oxygen.(2)Programmable DNA triple-helix molecular switch(THMS)in biosensing:from homogenous solution to living cellsIn this chapter,we construct a new programmable triple-helix molecular switch(THMS)based on gold nanoparticle for simultaneous imaging of multiple mRNAs in living cells.In general,under neutral conditions,CGC parallel triplets can not be stably present due to the protonation of the C base,and the deprotonation of the T bases in the TAT triple chains keeps them relatively stable,so we designed the ratio of TAT/CGC in the sequence,the triplets molecule is designed to overcome its dependence of the pH and to be stable in neutral and even weak alkaline environment,and then expand its application in living cells.On the other hand,taking full advantage of the superiority of the AuNPs-nucleic acid configuration,such as the unique anti-digestion ability and high endocytosis efficiency,we constructed a programmable designed THMS on the surface of AuNPs to achieve intracellular simultaneous imaging of tumor-associated mRNAs.When there are multiple kinds of mRNAs,we can achieve a variety of analytes detection only need to connect with different fluorophores labeled capture probe,greatly avoiding the tedious AuNPs particles modification process.As far as we know,this is the first time that AuNPs-mediated THMS has been applied to live cells for imaging of multiple analytes. |
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