Toehold-mediated strand displacement reaction-propelled cascade DNAzyme amplifier for microRNA let-7a detection

DNAzyme amplifiers have been extensively explored as a useful sensing platform,but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity.Herein,a cascade DNAzyme amplifier was designed by exploiting concurrent amplification cycle principles of toehold-mediated strand displacement reaction(TSDR)and Zn^(2+)-assisted DNAzyme cycle with lower cost and simpler procedures.Compared with single DNAzyme amplifier,the proposed TSDR-propelled cascade DNAzyme amplifier exhibited higher sensitivity by releasing more DNAzyme through TSDR to cleave substrate strand during the DNAzyme cycle.Base on this,let-7a could be sensitively detected in the range of 5-50 nmol/L with a detection limit of 64 pmol/L.Furthermore,the dual signal amplification strategy of the cascade DNAzyme amplifier exhibited excellent selectivity to distinguish single-base mismatched DNA strands,which has been successfully applied to the determination of let-7a in blood serum,showing high promise in early cancer diagnosis.

Entropy-driven strand displacement reaction for ultrasensitive detection of circulating tumor DNA based on upconversion and Fe_(3)O_(4) nanocrystals

Early detection of cancer biomarkers applied in real-time disease diagnosis and therapies can increase the survival rate of patients.Circulating tumor DNA(ct DNA)as a typical cancer biomarker plays a great role in the process of tumor disease monitoring,especially in early diagnosis.Unfortunately,most ct DNA detection systems have not been widely used due to their low sensitivity,poor specificity,and high cost.Herein,we developed an alternative ct DNA detection system to present the levels of ct DNA by recording the fluorescence signals of the system containing upconversion nanoparticles(UCNPs),Fe_(3)O_(4),and entropy-driven strand displacement reaction.The method has a practical sensitivity with a wide linear range from 100 amol L^(-1)to 1 nmol L^(-1)and a low detection limit of 1.6 amol L^(-1).Furthermore,the system demonstrates a practical application in mouse blood serum samples and meets the requirements for rapid,sensitive,specific,and economical diagnosis of cancers.Thus,this ct DNA detection system may have great potential for ct DNAdetection and clinical diagnosis.

Selective Enrichment of Low-Abundance DNA Variants Based on Programmable Peptide Nucleic Acid Probes

Single-nucleotide variants(SNVs)are crucial in disease development,but their accurate detection is challenging due to their low abundance and interference from wild-type targets.Although nucleic acid analogs like peptide nucleic acids(PNAs)have been used for SNV detection,they often lack programmable sensitivity and specificity due to poorly calculated thermodynamics and kinetics.Here,we present a computational method for calculating the stacking energy of PNA and DNA hybrids,leveraging nearest neighbor parameters.Validation against experimental data from 16 sequences under varied hybridization conditions yielded good agreement using Bland-Altman analysis,with all data points falling within the confidence interval.Our findings indicate that PNA-DNA hybridization is thermodynamically more stable and exhibits kinetics 140-fold faster than DNA-DNA hybridization for identical sequences.Utilizing this computational framework,we designed PNA toehold probes,which were screened via simulations and experiments.This combined approach facilitated the identification of highly sensitive and specific PNA toehold probes for single point mutation detection via strand displacement reaction.Our results demonstrate the successful application of PNA toehold probes for detecting point mutations with high sensitivity and specificity,achieving a selective amplification of approximately 200-fold for variants with a variant allele frequency(VAF)of 0.5%using quantitative polymerase chain reaction.

Target Recycling Transcription of Lighting-Up RNA Aptamers for Highly Sensitive and Label-Free Detection of ATP

We describe here a target recycling transcription of lighting-up aptamer strategy for detecting ATP in human serums in a label-free means with high sensitivity.ATP molecules specifically recognize the binding aptamer and result in the structure switching of the DNA assembly probes to imitate the target ATP molecule recycling cycles through the toehold-mediated strand displacement reaction,which causes the formation of many dsDNAs containing the RNA promoter sequences for subsequent transcription generation of large amounts of lighting-up aptamers.The organic dye,malachite green,then associates with these lighting-up aptamers to produce significantly enhanced fluorescence signals,which can sensitively detect ATP within a dynamic range from 10 to 500 nM in a label-free way.The sensing approach shows a detection limit of 7.3 nM and also has an excellent selectivity for ATP analogue molecules.In addition,this method can detect ATP molecules in diluted human serum samples sensitively,which proves the promising potential to diagnose ATP-related diseases.