Liposome-assisted Chemical Redox Cycling Amplification For New Optical Sensing Method

Highly sensitive detection of low-abundance biomarkers is important for early diagnosis of diseases.Fluorescent assays are of great interest due to their high sensitivity,selectivity,simplicity and rapidity.To improve the sensitivity of fluorescent detection,they often combine with a variety of signal amplification techniques,such as chain substitution reaction signal amplification,polymerase chain reaction signal amplification,ring-mediated isothermal amplification signal amplification and rolling loop amplification signal amplification.Although these signal amplification strategies can significantly improve sensitivity,they often suffer from cumbersome procedures,high biomolecule consumption,the need for fluorescent label and high costs.Therefore,it is important to develop simple,cost-effective and sensitive signal amplification strategies for the ultra-sensitive fluorescence analysis of low-abundance tumor markers.Therefore,utilizing simple and economical redox chemical cycle reaction as signal amplification technique,a series of new rapid and highly sensitive fluorescent biosensing methods were constructed by combining to liposome signal amplification and enzyme-catalyzed signal amplification techniques and using Ru@SiO₂@MnO₂ nanocomposites and terephthalic acid molecules as a fluorescent probe.The main research and innovations are as follows:1.An universal ultra-sensitive fluorescent biosensing platform was constructed for DNA methylation detection using chemical-chemical（CC）redox cycle reaction as signal amplification strategy and a manganese dioxide nanosheet-encapsulated Ru@SiO₂nanoparticle（Ru@SiO₂@MnO₂）quench system as fluorescent probe.In the chemical-chemical redox cycle system,ascorbic acid（AA）and tris（2-carboxyethyl）phosphine（TCEP）were used as reducing agents and the MnO₂nanosheets acted as oxidizing agents to react with AA to produce Mn²⁺and dehydroascorbic acid（DHA）.The DHA could be re-reduced to AA by TCEP and continue to react with the MnO₂nanosheets.Thus,the continuously regenerated AA etched a large amount of MnO₂nanosheets,resulting in great recovery of the fluorescence of Ru@SiO₂ nanoparticles.Compared with fluorescent detection method in the absence of chemical redox cycle signal amplification strategy,the sensitivity of AA detection was improved almost 52times by using chemical redox cycling signal amplification strategy.With the help of synergistic signal amplification of liposomes through encapsulating AA molecules inside liposomes,the proposed concept was further used to detect DNA methylation.Highly specific capture of methylated DNA was achieved using 5m C antibody-modified liposomes to specifically recognize the DNA methylation sites.Subsequently,AA molecules were released from liposomes through the emulsion breaking by Triton X-100.The released AA molecules participated in chemical-chemical redox cycle reaction to amplify the detection signal,enabling highly sensitive detection of methylated DNA with a detection limit of 16.2 fmol/L.2.To further improve the detection sensitivity based on the chemical-chemical redox cycling signal amplification,an ultrasensitive fluorescent sensing platform based on multiple signal amplification was constructed for the ultrasensitive detection of DNA methylation by combining enzyme-catalyzed signal amplification technology.After binding to methylation sites through alkaline phosphatase（ALP）encapsulated liposomes（Liposome@ALP）,the released ALP catalyzed AA to form ascorbyl-2-phosphate（AAP）to participate in chemical-chemical redox cycle reaction,which continuously etched the MnO₂ nanosheets on Ru@SiO₂ surface.Thus,the fluorescence of Ru@SiO₂nanomaterials was greatly recovered,which realized the multiple signal amplification detection of DNA methylation with the detection limit as low as 2.9 fmol/L.The sensitivity of this work was further improved compared to signal amplification strategy constructed in the previous chapter.At the same time,in order to improve the accuracy of the detection,a fluorescent-colorimetric dual-mode detection platform was constructed for methylation detection based on the reaction of Fe³⁺with K₄[Fe（CN）₆]and AAP to respectively generate orange AAP-Fe³⁺chelate and Prussian blue as multi-color indicators.In the absence of methylated DNA,there was no liposome-encapsulated ALP and the AAP cannot be catalyzed by ALP to produce AA.Thus,the Fe³⁺reacted with AAP to form orange chelates.In the presence of methylated DNA,the released ALP from liposome catalyzed AAP to generate AA,which reduced K₃[Fe（CN）₆]to K₄[Fe（CN）₆].Then,the generated K₄[Fe（CN）₆]self-assembled with Fe³⁺to produce Prussian blue.At the same time,the produced DHA could be reduced by TCEP to re-generate AA,making large amount of K₃[Fe（CN）₆]to form K₄[Fe（CN）₆].Thus,colorimetric detection of methylated DNA was achieved through the color change from orange to blue.The detection limit for colorimetric detection of methylated DNA is 25 pmol/L.3.Fenton reaction accelerated by tannic acid-mediated Fe²⁺/Fe³⁺cycling was firstly utilized to construct ultra-sensitive fluorescent biosensing method for the detection of DNA methylation with low background.In the Fenton reaction,Fe²⁺reacted with H₂O₂to produce hydroxyl radicals（·OH）and Fe³⁺.The generated·OH oxidized the non-fluorescent terephthalic acid（TA）to form hydroxy terephthalic acid（TAOH）with fluorescent emitting.Tannic acid was selected as a reducing agent to reduce Fe³⁺to Fe²⁺in the Fenton reaction.The Fe²⁺/Fe³⁺cycle mediated by tannic acid greatly improved the reactivity of the Fenton reaction to generate a large amount of·OH,which oxidized a large amount of TA to fluorescent TAOH molecules.Thus,the fluorescent signal was amplified.The results showed that the sensitivity of the fluorescent detection was improved almost 116 times based on Fenton reaction accelerated by tannic acid-mediated Fe²⁺/Fe³⁺cycling.With the help of synergistic signal amplification of liposomes through encapsulating tannic-Fe³⁺complex molecules inside liposomes,the developed amplification strategy based on Fenton reaction accelerated by tannic acid-mediated Fe²⁺/Fe³⁺cycling was applied to the ultra-sensitive detection of DNA methylation.The method showed good analytical performance for the detection of DNA methylation with a detection limit of 1.4 fmol/L.