| As a certain characteristic biochemical index,biomarkers can be objectively measured and evaluated in disease research.We can understand the current biological process of the organism by detecting biomarkers.The determination of a disease-specific biomarker can play an important role in disease identification,early diagnosis and prevention,especially in the prevention and control of chronic diseases and complex diseases?such as cardiovascular disease,diabetes,tumor and other complex diseases?.With the continuous improvements of people's health and living standards,modern medicine and analytical techniques are also changing with each passing day.It is imperative to rely on some simple,sensitive and efficient detection methods to detect disease markers.Among them,fluorescent biosensor is a kind of device which can specifically recognize biomolecules specifically and transform this recognition into detectable fluorescent signals.Therefore,in this paper,we imply a variety of signal amplification techniques,including rolling circle amplification,toehold-mediated strand displacement and multiple recycling amplification,respectively,to achieve highly selective and sensitive detection of miRNA let-7a,telomerase and platelet-derived growth factor BB.The specific research content of this paper is as follows:Part 1.Hairpin/DNA ring ternary probes for highly sensitive detection and selective discrimination of microRNA among family members.The detection and quantification of microRNA?miRNA?plays essential roles in clinical and biomedical research.Yet,it is of major challenge to sense miRNA with high degree of selectivity and sensitivity due to its unique characteristics of short length,similarity of sequence among family members and low abundance.Here,with the design of a new hairpin/DNA ring ternary probe,we describe the development of a rolling circle amplification?RCA?method for sensitively and selectively sensing miRNA from cancer cells.The target mi RNA binds the hairpin/DNA ring probes through toehold-mediated strand displacement?TSD?to form the ternary structures,in which the bound miRNA and DNA ring are respectively used as the primer and template to realize RCA,leading to the generation of many repeated metal ion-dependent DNAzyme sequences.The fluorescently quenched hairpin signal probes can be cyclically cleaved by these DNAzyme sequences with co-existence of the corresponding metal ions in buffer to show drastically enhanced fluorescence recovery for highly sensitive sensing of miRNA in the range between 10fM and 10 nM with a detection limit of 1.51 fM.Besides,owing to the high base variation discrimination ability of TSD,selective detection of the target miRNA among the corresponding family members can be achieved by this method.Moreover,such a method can also be employed to differentiate miRNA expression variations in cancer cells for screening potential therapeutic drugs.Part 2.A ternary probe for target-triggered autonomous multi-branch rolling circle amplification for highly sensitive colorimetric sensing of platelet-derived growth factor BB.Highly sensitive detection of protein biomarkers has an important role in clinical diagnosis and disease treatment.On the basis of a newly designed ternary probe containing the aptamer,primer and circular DNA sequences,we have established an ultrasensitive and label-free colorimetric approach for detecting platelet-derived growth factor BB protein biomarkers via a target-triggered autonomous multi-branch rolling circle amplification?mbRCA?strategy.The target molecules specifically bind the aptamer sequences in the ternary probes and causes the configuration transition of the primers to trigger mbRCA generation of numerous of G-quadruplex sequences with the presence of the assistance hairpin.Hemin further associates with these G-quadruplexes to yield many hemin/G-quadruplex DNAzymes with the horseradish peroxidase-mimicking function,which catalyze the substrate solution to induce intense color transition for ultrasensitive and label-free colorimetric determination of the target molecules with a 0.055 pg mL-11 of detection limit in the range of 0.1 to 1000 pg mL-1.This aptamer-based sensing method exhibits high selectivity and can achieve the detection of low levels of protein biomarkers in diluted serum samples,which offers the developed approach new opportunities for detecting other trace protein biomarkers for the purpose of early disease diagnosis.Part 3.A multi-recycling amplification-based sensor for label-free and highly sensitive detection of telomerase from cancer cells.The development of methods that can detect telomerase with high selectivity and sensitivity is critical for early diagnosis and treatment of telomerase-related cancers.In this regard,we describe in this work the establishment of a telomerase-initiated and nicking endonuclease-assisted cascade recycling signal amplification approach for non-label and highly sensitive fluorescent detection of telomerase from cancer cells.The target telomerase triggers the elongation of one strand in a partial dsDNA duplex with a pre-designed sequence to induce the release of a ssDNA,which can initiate three cascaded recycling cycles for the generation of many G-quadruplex sequences by cleaving two hairpin signal probes with the assistance of the Nt.AlwI endonuclease.The thioflavin dye further binds these G-quadruplex sequences to exhibit substantial fluorescence enhancement for sensitive detection of telomerase at 8.93×10-1111 IU.Moreover,this developed method is capable of differentiating telomerase activity among different cancer cells and screening telomerase inhibitors for anticancer drugs. |
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