Preparation And Application Of Homogeneous Electrochemical And Electrochemiluminesence Aptasensor Enhanced By Silica Isoporous Membrane

In recent years,electrochemical biosensors play an important role in clinical diagnosis,environmental monitoring,food safety and other fields due to their advantages of high sensitivity,good selectivity,low preparation cost,and fast analysis speed.As a key component of electrochemical biosensors,biometric elements are involved in the specific recognition and capture of target analytes.Compared with traditional recognition elements（such as enzymes,antibodies,cells,etc.）,aptamers as new recognition elements are widely used in the detection of proteins or small molecules because of their advantages of low cost,good stability,and wide application range.It is one of the common construction strategies to fix aptamers on the surface of electrodes as recognition elements.However,cumbersome immobilization procedures will cause problems such as high background signal and poor reproducibility,which limits the practical analysis and application of sensors to a certain extent.However,the homogeneous strategy can effectively avoid the complicated electrode fixation and modification process,thus allowing the recognition reaction to occur with the highest efficiency in a homogeneous solution.Homogeneous electrochemical biosensors,a subcategory of electrochemical biosensors,have emerged as powerful and promising electrochemical tools for simple,effective,and practical detection of target analytes in real samples and have attracted much attention worldwide.The working electrode,as the core component of the electrochemical biosensor,determines the analytical performance of the sensor.Conventional electrodes that are not modified with functional materials have limited the detection of low abundance targets in complex samples due to their insufficient sensitivity.Therefore,in order to improve the detection sensitivity,it is essential to select functional nanomaterials with excellent performance and suitable signal probes to achieve signal amplification.Vertically-ordered mesoporous silica films（VMSF）are a kind of porous material with vertically oriented nanochannels.VMSF with a diameter of about 2-11 nm displays excellent size selectivity;VMSF has high pore density(10⁵μm^-2)and facilitates analyte transport,showing high permeability;Moreover,VMSF exhibits good cationic permselectivity due to the deprotonation of silanol groups of VMSF（p K_a～2）,which could electrostatically preconcentrate positively charged electroactive molecules and electrochemiluminescence probes.Above characteristics of VMSF are very suitable for construction of electrochemical and electrochemiluminescence sensors with anti-fouling and highly sensitive abilities.This thesis explored three aptamer-based homogeneous label-free electrochemical and electrochemiluminescence sensing strategies.Combined with the signal amplification and anti-fouling properties of VMSF,the specific and highly sensitive detection of heavy metal ions,tumor markers and toxins in complex samples was achieved.The specific research content is as follows:（1）Based on the allosteric induction of Pb²⁺and the electrochemical signal amplification ability of VMSF/ITO on oxidized substrates,a novel homogeneous electrochemical sensor was constructed for the specific and highly sensitive detection of Pb²⁺.In the presence of Pb²⁺,the G-quadruplex substructure with high peroxidase activity can be induced to alter,which reduces the enzyme activity and limits the formation process of oxidized substrates,thereby reducing the electrochemical signal of oxidized substrates.Based on the correlation between the reduction degree of the electrical signal of oxidized substrates and the content of Pb²⁺,the indirect determination of Pb²⁺can be realized.In addition,the VMSF/ITO electrode can significantly amplify the electrochemical signal to the positive oxidation state substrate,effectively improving the detection sensitivity.Compared with the traditional colorimetric method,the constructed electrochemical sensing strategy can also avoid the interference of colored samples and avoid false positive results.Combined with the anti-fouling and anti-interference ability of VMSF,the indirect detection of Pb²⁺in tap water and colored lake water was realized.（2）Based on the aptamer recognition induced probe release combined with VMSF/ITO electrode-assisted signal amplification strategy,a novel homogeneous ECL sensing platform with high sensitivity and low background signal was constructed to realize the universal detection of tumor markers.Ru（bpy）₃²⁺-aptamer-magnetic graphene oxide（M-GO）-ECL nanocomposite probe was prepared by using theπ-πstacking interaction between aptamer and M-GO and their electrostatic adsorption ability for positive ECL probe Ru（bpy）₃²⁺.Transmission electron microscopy（TEM）,X-ray photoelectron spectroscopy（XPS）and hysteresis curve were used to characterize the morphology,composition and magnetic properties of M-GO.The successful preparation of the composite probe was demonstrated by the electrochemiluminescence method as well as the Zeta potential.VMSF has a strong electrostatic enrichment effect on the positive ECL probe Ru（bpy）₃²⁺,which effectively improves the ECL detection sensitivity.In the presence of the target,the aptamer binds to the target with high affinity,and desorbs from the surface of M-GO.At the same time,Ru（bpy）₃²⁺is induced to desorb from the composite probe and dissociate in the solution,which could be separated by magnetic separation technology and effectively reduce the background signal.Free Ru（bpy）₃²⁺in solution could be preconcentrated and then detected by VMSF/ITO electrodes using ECL method,eventually allowing the indirect detection of cancer markers.The proposed universal ECL sensing platform utilizes the magnetic separation technology to reduce the background signal.By simply changing the sequence of the aptamer,the proposed ECL sensing platform is able to realize the specific and highly sensitive detection of two tumor markers,alpha-fetoprotein（AFP）and prostate antigen（PSA）.Combined the anti-fouling and anti-interference ability of VMSF,the sensitive detection of AFP and PSA in human serum samples was realized.（3）Based on the unique reaction mechanism of Ru（bpy）₃²⁺-DNA system on VMSF modified electrodes,a novel ECL dual-strategy homogeneous aptamer sensing platform was established for the detection of marine toxin okadaic acid（OA）.Both strategies are based on the G base in DNA acting as a co-reactive agent of Ru（bpy）₃²⁺,enhancing the ECL signal.The first strategy:in the presence of the target,the aptamer binds to the target to form a complex,and its catalytic reaction to Ru（bpy）₃²⁺is inhibited with the partial shielding of the G base,resulting in a decrease in the ECL luminescence signal.The second strategy was to fabricate M-MWCNTs/DNA composite probes based on theπ-πconjugation between magnetic multi-walled carbon nanotubes（M-MWCNTs）and DNA.The successful preparation of the M-MWCNTs/DNA composite probes was confirmed by Zeta potential.In the presence of the target,the target binds specifically to the aptamer,resulting in the desorbed aptamer from the surface of M-MWCNTs.The G base in the exposed fraction still promoted the electrochemiluminescence of Ru（bpy）₃²⁺,leading to the so the enhanced ECL signal.Combined with the anti-fouling and anti-interference ability of VMSF,the detection of OA in seawater samples was realized.VMSF used in the proposed sensing platform showed a strong electrostatic enrichment effect for cationic Ru（bpy）₃²⁺,resulting in an enhanced ECL signal.Furthermore,the magnetic separation function of M-MWCNTs was further employed to reduce the background signal and improve the detection sensitivity.By simply changing the sequence of the aptamer,it is expected to realize the universal detection of other toxins.