Detection Of Biomolecules Based On A New Method Of Fluorescence Resonance Energy Transfer Principle

At present,in the field of life analysis chemistry,the detection methods for biomolecules are becoming more and more demanding,and the construction of a sensitive,efficient and economical detection strategy,which has become the focus and hotspot of research in the field of life analysis science.Fluorescence resonance energy transfer?FRET?is widely used in biomedical detection as a comparatively advanced analytical technique.Fluorescence resonance energy transfer refers to the phenomenon that energy between two fluorophores are transmitted from the donor to the acceptor in a non-radiative manner through dipole-dipole coupling.Two different emissions can be recorded simultaneously in different wavelength ranges.The changes of many biomarkers?DNA,miRNA-21,PLA₂?in humans are a critical step in the study of biological functions,disease diagnosis and related gene drug development.DNA biosensors are recognized as a basis for the use of nucleic acid base pairing.Gene fragments enable new methods for continuous,rapid,sensitive and selective detection.miRNA-21,as an endogenous small RNA,is widely found in human and almost all model organisms,and it is a series of life processes such as cell proliferation,development,growth and differentiation,apoptosis,metabolism and carcinogenesis.Phospholipase A₂ is a rate-limiting enzyme that catalyzes the hydrolysis of free fatty acids and lysophospholipids by the phosphodiglycerol diacyl group.Its downstream products are involved in gene expression,energy metabolism,plasma membrane remodeling,signal transduction,and various basic physiology such as inflammation and injury.In this paper,three kinds of biomolecules were detected by fluorescence resonance energy transfer technique and biomolecular signal amplification technology,which includes DNA,miRNA-21 and phospholipase A_2.A new signal amplification detection technology platform was constructed and its quantitative analysis was realized.The main contents include:1.Fluorescence-enhanced Protein p19-Conjugated Quantum Dot with multiplex antenna for One-Step,Specific and Sensitive miRNAs DetectionThis chapter mainly designs a fluorescence resonance energy transfer sensing system based on the modified p19 protein-modified quantum dots for the one-step,rapid and quantitative detection of miRNA-21 in tumor cells.In this method,the viral p19 protein is first used to modify quantum dots,and the p19 protein-modified quantum dots can specifically bind to double-stranded RNA.After the Cy-3-modified RNA detection probe hybridizes with the target miRNA-21,the double-stranded RNA can be specifically adsorbed onto the surface of the quantum dot,thereby generating fluorescece resonance energy transfer and realizing rapid quantification of miRNA-21 Detection.In the absence of the target miRNA-21,the Cy-3 modified RNA detection probe is remote from p19-QD,thereby preventing the occurrence of fluorescence resonance energy transfer.The method does not require washing,avoids separation,and simplify the experimental procedure;the method has high specificity by the selectivity of p19 protein,and the detection limit can reach 0.6 fM.In addition,we further tested the activity of miRNA-21 in breast cancer cells and compared it with traditional RT-qPCR.Excellent results were obtained.2.Assembly of self-replicating catalytic hairpin probes based on fluorescence resonance transfer technique?FRET?for DNA detection in serum.This chapter is based on a self-replicating catalytic hairpin probe self-assembly signal amplification system for the detection of sustained,rapid,sensitive and selective detection of DNA.In this method,the hairpin probes H1 and H2,respectively labeled with fluorescent dyes,are the specially designed sequence which has a sequence in the stem segment that is identical to the target.The target DNA acts as a primer and can be cycled in this system to generate a H1-H2 complex,which undergoes fluorescence resonance energy transfer.At the same time,the two replicas generated identical to the target are reused as new primers,triggering a new round of catalytic hairpin assembly reaction?CHA?.Thereby achieving the effect of signal amplification,improving the sensitivity,the detection limit can reach 0.01 pM,and the method is successfully used for detecting the small molecule DNA.3.A new method for sensitive and accurate detection of serum?PLA₂?based on target-controlled liposome"on-off"fluorescence resonance transfer technique?FRET?Based on the liposome-encapsulated primer DNA/hairpin probe H1/H2,this paper constructs a new method for the detection of PLA₂ with fluorescence spectrum as the detection tool with simple operation,high sensitivity and specificity.First,we bind the encapsulated DNA liposomes to the hairpin probes H1 and H2.When PLA₂ is present,the liposomes are lysed to release the target DNA,which can be circulated in this system.When the H2-H1 complex is formed,the fluorescence resonance energy transfer occurs.The new method has achieved signal amplification,improved sensitivity,and the detection limit can reach 0.0001 U L-1.The method is successfully used for detecting organisms and small molecule PLA₂.