Construction Of Dual-mode Aptamer Biosensors And The Application In Toxin And Disease Marker Detection

The detection of mycotoxins and disease markers is key to food safety and early disease diagnosis.Current food safety and disease issues have created the need for a rapid,sensitive and reliable mycotoxin and disease marker detection platform.Ochratoxin A（OTA）is a common mycotoxin that can affect commodities for human consumption directly or indirectly through various pathways.Therefore,rapid and highly sensitive OTA detection is a key component to ensure food safety and is now a worldwide concern.p53 gene is a common marker for oncological diseases and is important for early diagnosis of diseases.However,the levels of these mycotoxins and disease markers are low,and conventional assays usually require expensive instruments,cumbersome processes,high costs and low sensitivity.To this end,in this study,a series of highly sensitive,selective and accurate bimodal detection systems for mycotoxin and disease marker detection were constructed using metal-organic backbone materials（MOFs）as electrochemical signal groups,along with their catalytic properties of peroxidase-like and phosphatase-like enzymes,combined with various signal amplification strategies.The following four research works are to be carried out:1.Dual-signal ratiometric electrochemical aptamer sensor based on DNA tetrahedral nanomaterials for ochratoxin A detectionOchratoxin A（OTA）is a highly toxic food contaminant that seriously harms human health.In this paper,a ratiometric electrochemical aptamer sensor based on DNA tetrahedral nanomaterials（NTH）combined with a zirconium metal organic backbone（Ui O-66）signal tag was developed for the detection of OTA.In this sensor,Ui O-66 and[Fe（CN）₆]^3-/4-electrolyte solutions are used as signal probe and internal reference probe,respectively.In the presence of OTA,the OTA aptamer is released from the electrode due to the specific binding of the OTA aptamer to the OTA.Thus,the signal probe P1-labeled Ui O-66（P1-Ui O-66）is assembled on the electrode surface by hybridization with the DNA NTH terminal sequence.The proximity of the signal probe P1-Ui O-66 to the electrode surface leads to an increase in the current signal of Ui O-66 at+0.9 V;while the current signal of[Fe（CN）₆]^3-/4-at+0.2 V decreases as the conductivity of the electrode decreases.The proposed ratiometric electrochemical sensor can effectively eliminate the effects of external environmental and electrochemical background signals.The sensor showed good specificity for OTA with a linear range of 1 pg/m L-100 ng/m L and a detection limit of 280 fg/m L.The developed electrochemical aptamer biosensor effectively detected OTA in corn,melon seed and peanut samples,validating its practical application for OTA determination in real samples.2.Biotinylated scaffold-based bimodal aptamer biosensor for ochratoxin A detectionOTA is highly toxic and seriously contaminates food and crops.In this paper,a biotinylated scaffold-based biochemical and colorimetric bimodal aptamer biosensor was constructed for OTA detection.In the sensor,a biotinylated linker strand（Bio-linker）is hybridized with an OTA aptamer strand（OTA aptamer）to form a biotinylated DNA scaffold containing multiple OTA recognition sites,and the scaffold with a rigid structure is attached to the surface of the affinylated magnetic beads by biotin on the surface.In the presence of OTA,the scaffold will lose its stable structure and dismember into primitive DNA fragments due to OTA aptamer-specific binding of OTA.The affinylated cerium metal-organic backbone material（Strep-Au NPs@Ce-FMA）and alkaline phosphatase（Strep-ALP）can be bound to the biotinylated scaffold under alkaline conditions,catalyzing the production of p-nitrophenol（PNP）from disodium nitrobenzene phosphate hexahydrate（PNPP）,which can be detected at UV-Vis（400nm）and DPV（+1.0 V）.Thus,multifunctional scaffolds with high recognition and capture efficiency as well as significant signal amplification can be constructed.The electrochemical and colorimetric methods of the proposed dual-modal aptamer sensor can achieve excellent performance in the range of 100 fg/m L-150 ng/m L and 1 pg/m L-50 ng/m L,respectively,for short time detection of OTA with detection limits of 26fg/m L and 0.23 pg/m L,which is much better than most previous methods.By detecting OTA in maize and wheat samples,the biotinylated scaffold-based aptamer sensor works well and is comparable to the detection results of commercial ELISA kits.Thus,the scaffold promises to be a powerful analytical tool for a wide range of analyte assays.3.A bimodal aptamer biosensor based on a rolling cycle amplification signal amplification strategy for the detection of ochratoxin AIn this paper,a bimodal aptamer biosensor based on rolling loop amplification signal amplification was developed for ochratoxin A detection.In the sensor,magnetic beads are used as a sensing platform to anchor the linker chain（m DNA）and the OTA aptamer to the surface of the beads.In the presence of OTA,the aptamer is released from the electrode due to the specific binding of the OTA aptamer to the OTA.Simultaneously,signal amplification is achieved by preparing a long strand of DNA through a rolling loop amplification reaction（RCA）,the ends of which can be hybridized to m DNA and DNA1-labeled Fe-MIL-88,respectively.Fe-MIL-88 has excellent electrochemical activity and can detect a response signal at+1.0 V;meanwhile Fe-MIL-88 is a peroxidase that catalyzes H₂O₂ oxidation of TMB to generate oxidized TMB（ox TMB）.Under acidic conditions,the blue ox TMB was converted to yellow ox TMB,and the maximum UV absorption of ox TMB was detected at 450 nm.The electrochemical and colorimetric methods can achieve quantitative detection of OTA in the range of 1 fg/m L-250 ng/m L and 1 fg/m L-100 ng/m L,respectively,with detection limits of 0.22 fg/m L and 0.25 fg/m L,respectively.by detecting OTA in maize and wheat samples,the roll-loop amplification-based aptamer sensor can work well and is comparable to commercial ELISA The detection results of the kit were comparable to those of commercial ELISA kits.And the sensor has high sensitivity,high specificity and good stability.4.Detection of p53 gene by bimodal biosensor based on DNA Walker signal amplification strategyThe p53 gene is important for the early diagnosis and treatment of cancer.Herein,a bimodal sensor based on DNA Walker with enzyme-free signal amplification strategy is developed for the detection of p53 gene.In the sensor,the hairpin structure of the walker（DNA walker）DH and the track DA/DB are anchored on the magnetic bead surface.The target molecule p53 gene binds to the neck of the hairpin structure DH,initiating an autonomous walk of the DNA Walker DH,which performs strand replacement of the DB strand on the track.In the presence of free replacement hairpin DF in the solution,it can compete for the DB chains displaced by DNA Walker,prompting DNA Walker to restore its initial state and perform the same task toward another track,and achieve signal amplification by repeated cyclic oscillation of DNA Walker.The DB strand labeled with biotin on the orbital is connected with the affinylated Ui O-66 and Fe-MIL-88.Ui O-66 has excellent electrochemical activity and can detect the response signal at+1.0 V;Fe-MIL-88 has peroxidase-like activity and can catalyze H₂O₂ to oxidize TMB to generate ox TMB,and under acidic conditions,blue ox TMB is converted to Under acidic conditions,blue ox TMB was converted into yellow ox TMB,and the maximum UV absorption of ox TMB was detected at 450 nm.The electrochemical method and colorimetric method can achieve quantitative detection of p53 gene in the range of 50 fmol/L-50 nmol/L and 50 fmol/L-10 nmol/L,respectively,with detection limits of 16.65 fmol/L and 12.5 fmol/L,and the sensor is sensitive,specific and stable.