A Visual Detection For Microrna Based On Ratiometric Fluorescent Probes

MicroRNAs（miRNA）are a class of small non-coding RNAs that play critical regulatory role in cell differentiation,biological development and disease development by targeting mRNAs for translational repression or cleavage.Many literatures have indicated that abnormal expression of miRNA is associated with the occurrence and development of cancers,so miRNAs may become the potential biomarkers of disease diagnosis and prognosis.Therefore,the detection of miRNA is very important to further reveal its function and its role in disease diagnosis.Herein,three strategies for the detection of miRNA are established based on the amplification methods including rolling circle amplification（RCA）,hybridization chain reaction（HCR）and catalytic hairpin assembly（CHA）and the ratiometric fluorescent probes.The details are as follows:Firstly,a label-free detection of let-7a is developed based on the rolling circle amplification（RCA）and cationic conjugated polymer（CCP）-mediated fluorescence resonance energy transfer（FRET）.In this strategy,the cationic poly[（9,9-bis（6′-N,N,N-triethylammonium）hexyl）fluorenylenephenylene dibromide]（PFP）serves as the donor of FRET and SYBR GreenⅠ（SG）serves as the acceptor.A padlock probe matched with let-7a and a DNA probe,a complementary sequence to RCA product,are designed.When let-7a is present,the padlock probe hybridizes with target sequences bringing its two ends close to each other and can be covalently ligated into a loop in the catalyzing of T4DNA ligase.In the present of phi29 DNA polymerase and dNTPs,the rolling circle amplification of the circularized padlock probe is initiated from the target molecules and then a long single strand DNA with a lot of repetitive sequences is produced.When the complementary sequence of the repetitive sequence and SG are added,a long dsDNA is formed by hybridization and SG binds to it.The dsDNA-SG-complex and PFP combine with each other through electrostatic interaction and the strong FRET from PFP to SG occurs due to the overlapping between the fluorescent emitting spectrum of the PFP and the absorption spectrum of SG.On the contrary,when let-7a is absent,the padlock probe is not circularized leading to the inhibition of rolling circle amplification and hybridization process.Due to the weak interaction between SG and dsDNA,SG is far away from PFP and the FRET efficiency is greatly inhibited.As a result,the let-7a can be quantitatively determined by monitoring the change of FRET signal.The results show that the detection signals are linearly proportional to the let-7a concentrations in the range of 50 pmol·L^-15 nmol·L^-1.The specific of this strategy is investigated and the most of tested interfering substances have no influence to the test result of let-7a except let-7b and let-7c.Additionally,through the detection of let-7a concentration in the extract solution of Hela cells,it is proved that the method can be applied to the practical samples analysis.Secondly,a visual detection of let-7a is developed based on the dual-emission fluorescent probe and hybridization chain reaction（HCR）.In this strategy,the biotin-modified hairpin DNA probe（bio-H）is enriched on the surface of the Dynabeads modified with streptavidin.When let-7a is present,it hybridizes with the sticky end of bio-H and then the hairpin is opened by unbiased strand-displacement interaction.5’end of bio-H as the newly released sticky sequence can induce the hybridization chain reaction between two special sequence hairpin probes（H1 and H2）labeled with glucose oxidase（GOD）and then a long nicked dsDNA with GOD was produced on the surface of Dynabeads.In theory,each target molecule could trigger a HCR reaction.When glucose was added into these Dynabeads,hydrogen peroxide（H₂O₂）was generated by the glucose/GOD reaction.The fluorescence of CdTe in the shell of the dual-emission fluorescent probe will be sensitively responsive to H₂O₂ while the inner dye-embedded microbead is inert,which enables ratiometric and visual detection of let-7a.The results show that the linear range of let-7a is 100 pmol·L^-110 nmol·L^-1.The other sequences of the let 7 family,miR-143,miR-21 and miR-221 are used as interfering substances to investigate the specific of this strategy.The results show that these substances except let-7f have little interference on let-7a detection.Therefore,this proposed assay shows excellent selectivity for let-7a.Thirdly,a visual detection of let-7a using the dual-emission fluorescent probe is developed based on catalytic hairpin assembly（CHA）and chain hybridization（HCR）.In this method,a biotin-labeled hairpin DNA probe（P1）is immobilized on the surface of Dynabeads by the affinity of biotin and streptavidin and then its hairpin is opened by unbiased strand-displacement interaction between it and targeting molecule let-7a.The newly released sticky sequence of P1 would hybridize with P2 to open the hairpin of P2 and release another new sticky end for initiating HCR.Then,the let-7a hybridized with P1 is released.The released let-7a continues to hybridize with other P1 and is re-released by the hybridization of P1 and P2.Thus,hybridization and release can be repeated continuously and result in the linear cyclic amplification.After 1 h reaction,Dynabeads was separated from un-hybridized P2 by magnetic separation,and hybridization products of P1 and P2 were retained on the surface of Dynabeads.Subsequently,GOD-labeled H3 and H4（H3-GOD and H4-GOD）are added to the reaction system,the sticky end of P2 initiates chain hybridization reaction between H3-GOD and H4-GOD and then a long nicked dsDNA with GOD was produced on the surface of Dynabeads.When glucose was added into these Dynabeads,hydrogen peroxide（H₂O₂）is generated by oxidizing glucose with GOD.H₂O₂ could quench the outer green fluorescence of the dual-emission fluorescent probes,but the red emission of the dual-emission fluorescent probes would not be affected,so the dual-emission fluorescent probes become red colored.Therefore,miRNAs can be easily determined by identifying the color changes of the dual-emission fluorescent probes under a UV lamp only by naked eye.The results show that the linear ranges of let-7a are 2 pmol·L^-1200 pmol·L^-1 and 200 pmol·L^-12 nmol·L^-1.The sequences of miR-143,miR-21 and miR-221 as potential interfering biomolecules are investigated for evaluating the selective of this strategy.The results show that the interferences are negligible.In addition,it is proved that the method can be applied to the practical samples analysis by the detection of let-7a concentration in the extract solution of Hela cells.