Aptasensor Based On Isothermal Enzyme-free Nucleic Acid Signal Amplification

Small biomolecules,including small nucleic acids,organic molecules,and low molecular proteins,can regulate various biochemical reactions and cellular metabolism processes in organisms and cells.Thus these biomolecules have been attracted substantial attentions in the field of analytical chemistry and clinical researches since their abnormal expressions can indicate a variety of diseases.However,the expression levels of small biomolecules are relatively low in cells and tissues.Traditional one target triggering one readout method can not meet the need of high sensitivity.Therefore,the nucleic acid signal amplification technique hold great potential for highly sensitive detection of small biomolecules.Aptamers,single-strand oligonucleotides(DNA orRNA)obtained by in vitro selection procedure,are able to bind to their targets with high affinity and specificity.Compared to traditional antibodies,aptamers are applicable to a wide range of recognized targets,easiness to synthesize and modify,low cost,and chemical stability,making them promising recognition elements for biomolecules detection.The integration of facile aptamer recognitions and nucleic acid amplifications facilitates the high performance bioanalytical platfroms.Nevertheless,the introduction of enzymes or nanomaterials to the aptasensors compromised their simplicity and sensitivity.Herein,we constructed two kinds of isothermal enzyme-free(non-protease)cascaded nucleic acid amplification techniques(HCR-HCR and CHA-DNAzyme).By designing a“plug and play”module,a versatile sensing platform was developed.The research work is carried out based on the following two aspects:(1)We constructed an aptamer-based concatenated hybridization chain reaction(C-HCR)amplifier by integrating two successive HCR circuit for analysing adenosine triphosphate(ATP)and thrombin.The concatenated hybridization chain reaction is consisting of the upstream HCR-1 and the downstream HCR-2.The aptamer was grafted into the trigger of upstream HCR-1,and the corresponding inhibitor was introduced to hybridize with aptamer-trigger strand for avoiding signal leakage.The target molecule can specifically bind to the aptamer and release the trigger to initiate the C-HCR,achiving N²-amplified“signal-on”readout signal.In addition,the present aptasensor can be employed as a versatile sensing platform for more other biomolecules by introducing their corresponding aptamers.(2)We developed an isothermal enzyme-free CHA-DNAzyme system for the analysis of nucleic acids by integrating catalytic hairpin assembly(CHA)circuit and DNAzyme biocatalyst.The target catalyzes the hybridization of two functional hairpins to form a dumbbell-shaped double-stranded complex to generate numerous DNAzyme units,which are activated to cyclically cleave the substrates,producing an amplified readout signal.In addition,a universal sensing platform for various microRNA(miRNA)analysis is facilely implemented by introducing a“help”hairpin as a modular.We further designed the label-free CHA-DNAzyme system by using another hemin/G-quadruplex HRP-mimicking DNAzyme to replace Mg²⁺-dependent DNAzyme,which can catalyze the H₂O₂oxidation of ABTS^2-to produce colorimetric signal.What's more,the CHA-DNAzyme fluorescence system enables the imaging of miRNA in living cells for achieving more bioanalytical and biomedical applications.