Biosensors Developed For MicroRNA Detection Based On Signal Amplification Strategies

Micro RNAs(mi RNAs)are a type of short,single-stranded,non-coding RNAs with the length of about 22 nucleotides,which play important roles in gene expression,cell development,cell differentiation and cell apoptosis.Due to its short length,high sequence homology,low cellular abundance and easy degradability,it is a challenge to realize the sensitive detection of mi RNAs.Therefore,an effective method to achieve sensitive detection of mi RNAs is necessary.Up to now,various analysis methods have been developed,such as northern blotting,microarrays,electrochemistry and fluorescence.Although most of them are sensitive,it is still challenging for mi RNA detection due to the unique properties of mi RNA,including short lengths,low abundance and susceptibility to degradation.Therefore,it is urgent to develop a rapid,convenient,reliable and ultrasensitive analytical method for mi RNAs detection.(1)Herein,a signal-on electrochemiluminescence biosensor was fabricated for mi RNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction(ISDPR).In the presence of target mi RNA,amounts of trigger DNA could be generated by the first ISDPR.Then,the trigger DNA and the primer hybridized simultaneously with the hairpin probe to open the stem of the probe,and then the ECL signal will be emitted.In the presence of phi29 DNA polymerase and d NTPs,the trigger DNA could be displaced to initiate a new cycle which was the second ISDPR.Due to the two-stage amplification,this method presented excellent detection sensitivity with a low detection limit of 0.18 f M.Moreover,the applicability of the developed method was demonstrated by detecting the change of mi RNA-319a content in the leaves of rice seedlings after the rice seeds were incubated with chemical mutagen of ethyl methanesulfonate.(2)A photoelectrochemical biosensor is described for sensitive detection of mi RNA-162a.By using these amplification strategies,the detection limit is as low as 0.14 f M of mi RNA,and the photocurrent increases linearly with the concentration of mi RNA-162 in the range from 0.5 f M to 1 p M.The method was successfully applied to quantify the expression level of mi RNA-162a in total RNA extracted from the leaves of maize seedlings after incubation with the chemical mutagen ethyl methanesulfonate.The results confirmed the applicability of the method to the analysis of practical biological samples.(3)Herein,a novel photoelectrochemical(PEC)biosensor was developed for the ultrasensitive detection of mi RNA-396a based on a Mo S₂/g-C₃N₄/black Ti O₂ heterojunction as the photoactive material and gold nanoparticles carrying Histostar antibodies(Histostar@Au NPs)for signal amplification.The developed biosensor could detect mi RNA-396a at concentrations from 0.5 f M to 5000 f M,with a detection limit of 0.13 f M.Further,the proposed method can also be used to investigate the effect of heavy metal ions on the expression level of mi RNAs.Results suggest that the biosensor developed herein offers a promising platform for the ultrasensitive detection of mi RNA.(4)Herein,a novel photoelectrochemical(PEC)biosensor was developed for the ultrasensitive detection of dual mi RNAs(mi RNAs),with the detection being based on energy transfer(ET)between carbon quantum dots(CQDs)and gold nanoparticles(Au NPs).Two hairpin probes(H1 and H2)carrying the Au NPs were used“switch off”and“switch on”the PEC signal of the CQDs,with a close approach of the tagged Au NPs to the CQDs quenching the PEC signal.This approach allowed the highly sensitive detection of both mi RNA-159b and mi RNA-166a,with low detection limits of 0.15 f M and 0.21 f M,respectively.To our knowledge,this is the first reported CQDs-based ET biosensor for the PEC detection of dual mi RNAs.Results suggest that this approach offers a promising platform for the ultrasensitive detection of multiple mi RNAs.(5)An ultrasensitive electrochemiluminescence biosensor was fabricated for mi RNA detection by using Ag NCs@Mo S₂ as electrochemiluminescence material,peroxodisulfate(S₂O₈^2-)as co-reactant,and semicarbazide(Sem)as co-reaction accelerator.The Cu-Mo Fs loaded with a large amount of Sem was linked to the modified electrode,which can promote the ECL reaction rate.With the introduction of the new co-reaction accelerator,this biosensor exhibits excellent sensitivity with a detection limit of 0.03 f M,indicating that the introduction of co-reaction accelerators can provide a simple and effective method for signal amplification.For the development of biosensing,this will be a promising method of signal amplification.