Electrochemical Immunoassay And SiRNA Specific Delivery Based On DNA Assembly

DNA has recently emerged as a versatile material for molecular imaging and disease diagnosis and therapy in chemistry and biology.The proximity induced DNA dynamic assembly immunoassay that combines DNA dynamic assembly with immunoreaction has many advantages such as washing free,easily operation and fast.By coupling this technology with electrochemical biosensors,the developed immunoassay can achieve high sensitivity and selectivity with easy and simple operation,which displays great potential for convenient point-of-care testing.DNA nano structure is a kind of good biocompatibility and low cytotoxicity nanomaterials with controllable morphology and size.Based on the assembly of DNA nanostructure,this dissertation develops a series of fast and sensitive immunoassay methods,and a double parameters controlled high efficient and specific siRNA delivery method.It includes the following five parts:1.Ratiometric electrochemical proximity assay for sensitive one-step protein detectionThis work proposes the concept of ratiometric electrochemical proximity assay(REPA),which can be used for one-step,highly sensitive and selective detection of protein.The assay strategy was achieved on a sensing interface that was formed by hybridization of methylene blue(MB)-labeled antibody-DNA probe(MB-DNA1-Abl)with ferrocene(Fc)-labeled DNA capture probe(Fc-P)modified gold electrode.On the interface the target protein could trigger the formation of immunocomplex between MB-DNA1-Abl and detection antibody-DNA probe(Ab2-DNA2)and subsequently the proximity hybridization of DNA1-DNA2,which led to the departure of MB-DNA1-Abl from the interface.The remained Fc-P could form a hairpin structure to take Fc group to electrode surface.Therefore,the recognition of target protein to Ab1 and Ab2 resulted in both the "signal-off" of MB and the "signal-on"of Fc for dual-signal electrochemical ratiometric readout.The proposed REPA could be carried out in one-step with 40-min duration and showed a wide detection range from 0.05 to 100 ng/mL with pg/mL limit of detection,displaying great potential for convenient point-of-care testing and commercial application.2.Immunoreaction-triggered DNA assembly for one-step sensitive ratiometric electrochemical biosensing of protein biomarkerA sensitive ratiometric electrochemical readout was designed with an immunoreaction-triggered DNA assembly for one-step,fast and flexible assay of protein biomarker.The sensing interface was prepared by immobilizing a ferrocene(Fc)-labeled hairpin DNA on a gold electrode.In the presence of DNA2-antibody2(Ab2)and methylene blue(MB)-labeled DNAl-Abl probes,the addition of target protein could induce the sandwich immunoreaction among two probes and the protein to trigger the hybridization of DNA1 and DNA2,which subsequently unfolded the hairpin DNA to form a three-arm DNA structure on the sensing interface.The DNA assembly caused the departure of Fc from the electrode and the approach of MB to the electrode,resulting in the electrochemical signal off and on of Fc and MB respectively for ratiometric readout.Using prostate specific antigen(PSA)as a model target,the ratiometric electrochemical assay showed a linear detection range from 0.01 to 200 ng/mL with a detection limit(the signal mean of blank measures + 3?)of 4.3 pg/mL.By changing the affinity probe pairs this method could be easily expanded for other protein analytes.The assay feature with easy operation,high detection sensitivity and good flexibility,showed promising potential for point-of-care testing and extensive applications in bioanalysis.3.Target-driven triple-binder assembly of MNAzyme for amplified electrochemical immunosensing of protein biomarkerA simple electrochemical immunosensing method is presented for highly sensitive and selective detection of protein biomarker.This method uses a newly designed assembly of Mg2+_dependent MNAzyme via target-driven triple-binder proximity hybridization to catalyze the cleavage of methylene blue(MB)-labeled hairpin,which leads to the departure of MB from the electrode surface and thus an amplified decrease of electrochemical signal for immunoassay of the target protein.The MNAzyme assembly is achieved by the simultaneous recognition of target protein with three DNA-labeled antibodies in the presence of Mg2+.which greatly improves the detection sensitivity and selectivity.As a proof of concept,this strategy can detect carcinoembryonic antigen(CEA)ranging from 0.002 to 500 ng/mL with a detection limit of 1.5 pg/mL.The whole assay including the target-driven MNAzyme formation and subsequent cleavage of hairpin can be completed with one step in 40 min.The immunosensor,prepared with a hairpin DNA,possesses good extensibility for large protein biomarkers as CEA by using corresponding antibodies,though the protein target size dependence was not investigated in this work.The proposed immunoassay method shows the advantages of easy operation,high sensitivity,wide concentration range,good selectivity,and excellent versatility,displaying potential application for protein analysis.4.Proximity hybridization regulated DNA biogate for sensitive electrochemical immunoassayAn electrochemical DNA biogate was designed for highly sensitive homogeneous electrochemical immunoassay by combining target-induced proximity hybridization with mesoporous silica nanoprobe(MSN).The electroactive methylene blue(MB)was sealed in the inner pores of MSN with single strand DNA.In the presence of target protein and two DNA-labeled antibodies,the formed proximate complex could hybridize with the DNA strand to form a rigid double-strand structure and thus open the biogate,which led to the release of MB entrapped in the MSN.The target protein-depended amount of released MB could be conveniently monitored with a screen-printed carbon electrode.Moreover,the detachment process of MB could be further amplified with an in situ enzymatic recycling binding of the proximate complex with the single strand DNA.Using prostate specific antigen as a model target,the proposed assay showed a wide detection range from 0.002 to 100 ng/mL with a detection limit of 1.3 pg/mL.This strategy was simple and universal for various analytes with different affinity ligands.This method possessed great potential for convenient point-of-care testing and commercial application.5.A structure switchable "lock-smart key" for cell-subtype specific siRNA deliveryA "lock-smart key" switch was developed to perform precise siRNA delivery and specific gene silencing in target cells with the incorporation of dual cell membrane receptors and their respective aptamers sgc4f and sgc8c.A self-assembled oligonucleotide nano vehicle with a hairpin structure opening was used as the delivery carrier.The hairpin structure opening could be auto-cleaved to single strand on site at target cell surface by reacting with sgc4f and acts as a "smart key" to open sgc8c"lock" and subsequently to achieve siRNA delivery.The success of this strategy demonstrated the precise delivery of siRNA to target cell with the participation of multiple cell surface markers,thus paved the way for further application of RNAi for accurate diagnosis and intervention.