Universal Ratiometric Electrochemical Biosensing Platform Based On Mesoporous Platinum Nanocomposite And Nicking Endonuclease Assisted Dna Walking Strategy

Objective The occurrence and development of many complex diseases are associated with various molecules,including small molecules at the metabolic level,proteins at the functional level,and DNAs at the genetic level.However,the contents of them are rarely in the early stage of the disease.Many biomolecular detection platforms,including enzyme-linked immunosorbent assay(ELISA),polymerase chain reaction(PCR),have been developed to determine individual types of molecular targets.Although these platforms are well suited for the selective detection of single-level biomarkers,they usually need costly apparatus,complicated pretreatment procedure,and trained personnel.Most importantly,they exhibit a limited detection ability at other molecular levels.Currently available single biomarkers cannot usually predict and evaluate the occurrence and prognosis of diseases accurately.Advances in genomics,proteomics,and metabolomics has been shown that complex diseases such as cancer are characteristics of multiple complex molecular events in disease development and prognosis.Thus,a rapid,inexpensive,durable,ultrasensitive,and easy-operation platform should be developed to detect multiple-level biomarkers,which would facilitate the development of early diagnosis and precision medicineMethods In this work,DNA modified and DOX encapsulated MPNs(MPNs@DOX@DNA)were first synthesized and utilized.In this study,the MPNs were not only employed as nanocarriers for electroactive signal reporter(DOX)and thiol-functionalized DNA,and as electrocatalytic substances for remarkably enhanced sensitivity of the devices.DNA walkers(DWs)were powered by nicking endonuclease(Nt.BbvCI),and they exhibited autonomous cleavage behavior on the surface of the substrate DNA-modified electrode.we introduced an electrochemical biosensing system on nicking endonuclease(Nt.BbvCI)assisted DNA walking strategy.We successfully constructed a universal signal-off-on ratiometric electrochemical biosensor for various biomolecules,including small molecules,nucleic acids,and proteins,by progressively optimizing the schematics.The MB-hairpin probes(MB-HPs)acted as a signal-off probe,and nanocomposites(MPNs@DOX@DNA2)acted as a conventional signal-on probe.With the aid of the MPNs@DOX@DNA2 and Nt.BbvCI assisted DNA walking mechanism,the designed ratiometric electrochemical biosensor showed a high sensitivity and broad detection range.Results Under the optimum experimental conditions,the proposed universal ratiometric electrochemical biosensor showed a high sensitivity with a broad detection range to different biomolecules,including small molecules,nucleic acids,and proteins.This biosensor also realized well-accepted signal response in identifying serum samples.Conclusions By progressively optimizing the schematics,two types of DNA probes were simultaneously immobilized on the Au electrode.The terminally protected DWs were powered by Nt.BbvCI,and they exhibited autonomous cleavage behavior,which required no repeated manual operations and cumbersome thermal cycling.Numerous nonspecific signals were reduced,which rendered the proposed method cost efficient and practical for clinical applications.the MPNs were employed as nanocarriers for electroactive signal reporter(DOX)and thiolfunctionalized DNA and as electrocatalytic activity substances for remarkably enhanced sensitivity of devices.This biosensor also realized well-accepted signal response in identifying serum samples,thereby resulting in a wide prospect for bioanalysis and clinical diagnosis.