Studies On Construction Of DNA Sensor Based On Different Nanotracer And Detection Of Single-gene Genetic Diseases Related Gene Mutations

There are many kinds of genetic diseases and it is difficult to treat.It not only seriously harms the health of patients,but also brings a heavy burden on patients,their families and society.In many genetic diseases,single-gene genetic diseases are caused by single-gene mutations.Although the incidence is not high and disease types are relatively rare,due to its hereditary nature,that is,the risk of disease recurrence is high.Achondroplasia?ACH?and Rett syndrome?RTT?are two of common single-gene genetic diseases.Fibroblast growth factor receptor 3?FGFR3?gene mutations and the methyl Cp G-binding protein 2?MECP2?gene mutations can cause ACH and RTT,respectively.For now,nucleic acid molecular hybridization,PCR and DNA sequencing are common detection techniques for single-gene genetic diseases.However,this technology requires operators has excellent experimental operation skills and professional background knowledge.Therefore,the development of a new precise and rapid gene mutation detection technology has important practical significance for the timely clinical diagnosis of genetic diseases.Electrochemical sensing technology is a combination of biomolecular recognition technology and electrochemical sensing system.It has the advantages of low cost,high sensitivity,good stability,and easy operation.Therefore,it has attracted wide attention in the detection of disease-related biomolecules.In order to provide a new approach for the detection of clinical genetic disease genes,a novel electrochemical DNA sensor was constructed by using different nanobeacons to achieve accurate and rapid detection of ACH and RTT-related pathogenic genes mutations?FGFR3 and MECP2?.The main research contents and results are as follows:1.An electrochemical DNA sensor based on hemin-MOFs/PtNPs nanotracer for detection of FGFR3 mutationIn this study,encapsulation of hemin in metal-organic frameworks-based materials?hemin-MOFs?and platinum nanoparticles?PtNPs?were used to prepare hemin-MOFs/PtNPs composites.Hemin-MOFs/PtNPs as a novel nanotracer and streptavidin functionalized gold nanoparticles?streptavidin/AuNPs?as a sensor surface modification material to construct an electrochemical DNA sensor for the detection of FGFR3 gene mutation in serum.The sensor surface was first modified with reduced graphene oxide-tetraethylene pentaamine?rGO-TEPA?,and then Au NPs was deposited on the rGO-TEPA.After that,the streptavidin was introduced to immobilize more biotin-modified capture probe?Bio-CP?.At the same time,using hemin-MOFs as a carrier,a large number of PtNPs were immobilized,and the synthesized hemin-MOFs/Pt NPs nanobeacons have excellent catalytic performance.Under the optimal experimental conditions,the electrochemical DNA sensor exhibits high sensitivity?low detection limit is0.033 fM?,good reproducibility and stability,good specificity and wide linear range from 0.1 fM to 1 nM.Finally,the recovery rate is from 97.4%to 105.2%.The results show that the sensor can quickly and accurately identify the FGFR3 mutations in serum,which has potential clinical application value.2.Construction of electrochemical DNA sensor based on PdPt-melamine network for detection of MECP2 mutationAn electrochemical DNA sensor was constructed by palladium platinum nanodendrites?PdPt NDs?integrated with melamine?PdPt-melamine network?serving as a nanobeacon and electrodeposited gold nanoflowers?AuNFs?serving as electrode surface modification materials for the detection of MECP2 gene mutation in serum.In this study,based on signal probe coupled PdPt-melamine networks?SP-PdPt-melamine networks?,the electron transfer of electrochemically active substances was accelerated by catalyzing the hydrogen peroxide?H₂O₂?in the substrate solution to achieve signal amplification.On the one hand,electrodeposition of AuNFs on the electrode surface not only increases the conductivity of the sensor,but also can immobilize a large number of capture probes?CPs?on the surface of the electrode because of its good biocompatibility,and thus can detected different concentrations of the target DNA sequence.On the other hand,through the combination of SP with the target DNA sequence,PdPt-melamine networks was introduced and finally an electrochemical DNA sensor was constructed to realize the sensitive detection of MECP2gene mutation.Based on this signal amplification strategy,the electrochemical DNA sensor has a wide linear range from 1 fM to1 nM and a low detection limit of 0.33 fM.In addition,the recovery rate of this method is between 95.7-100.3%,indicating that the sensor can be applied to the detection of MECP2 mutation in serum.In summary,this study provides a new idea for the detection of single gene mutation with high sensitivity and selectivity.The main innovations are:1)It is first time that use of biotin-avidin system,hemin-MOFs/PtNPs to construct electrochemical DNA sensors to achieve accurate and ultrasensitive detection of FGFR3 mutations.The sensor provides a new idea for the detection of genetic mutation sequences with extremely low abundance.2)It is first time that use of AuNFs as a sensor interface modification material,PdPt/melamine network structure as a signal amplification nanobeacon to build a simple and sensitive electrochemical DNA sensor for the detection of MECP2 mutation.The simple constructed DNA sensor have high sensitivity and strong specificity,and provides a new solution for rapid and accurate detection of gene mutation.