Construction Of Indicator-free Biosensor Based On Electrocatalytic Hydrogen Evolution Performance Of Pt-based Nanomaterials

Pt-based noble metal catalysts are still the most effective catalysts for oxygen reduction reaction（ORR）and hydrogen evolution reaction（HER）.Because of its good biocompatibility,excellent electrical conductivity,excellent chemical stability and unique enzyme-like activity,it has been widely used in catalysis,sensing and medical fields.On the other hand,traditional electrochemical biosensors usually require labeling electroactive molecules on biological probes or using exogenous electroactive molecules to signal the biosensing process,which greatly increases the sensor preparation process and cost.In order to solve this defect,based on the excellent HER electrocatalytic activity of Pt in neutral media,three ultra-high sensitivity and excellent specific indicator-free electrochemical biosensors were constructed for the detection of cancer markers mi RNA Let-7a,mi RNA-21 and antibiotic chloramphenicol（CAP）.The main research work is as follows:（1）The commercially available HER reference catalyst Pt@C was immobilized on the surface of glassy carbon electrode（GCE）,and the ampere-level current density was used as the electrocatalytic interface of HER.Then,the thiolated probe DNA（aptamer）was chemically immobilized on Pt@C by self-assembly of Pt-S bonds to construct a simple biosensor interface.Electrochemical experiments show that the Pt@C modified electrode can produce ampere-level HER catalytic current density in neutral buffer.The effective electroactive area and conductivity of the catalyst modified electrode surface were characterized by cyclic voltammetry and impedance method in non-faradic interval.When the probe DNA or aptamer binds to the target mi RNA Let-7a or CAP,a non-conductive complex is formed,which reduces the conductivity of the electrode interface and the reduction of catalytic active sites,resulting in a decrease in HER catalytic current.Based on this,a sensitive detection of mi RNA Let-7a and CAP can be achieved with a detection limit of 8.6f M and 0.47 p M,respectively.This study provides a new idea for the construction of multifunctional universal indicator-free biosensor interface.（2）A novel indicator-free ultrasensitive micro RNA detection technology was constructed by combining the HER electrocatalytic performance of Pt-based catalyst with Exo III enzyme cleavage cycle amplification technology.Firstly,nano-Pt was in-situ grown on the surface of carboxylated multi-walled carbon nanotubes（COOH-MWCNT）modified GCE by electrodeposition method,and thenβ-cyclodextrin（β-CD）with thiol group was self-assembled on nano-Pt by Pt-S bond to obtain an interface with HER catalytic performance.Secondly,the probe DNA（p DNA）was hybridized with the target mi RNA Let-7a in a homogeneous solution to obtain a double-helical nucleic acid structure.Then Exo III enzyme was introduced to hydrolyze the probe DNA and release the target mi RNA Let-7a at the same time.Further hybridization with p DNA and cleavage by Exo III enzyme to achieve cyclic reaction and release a large number of nucleotide（NT）monomers.A large amount of NT in aqueous solution was captured byβ-CD on the surface of the electrode,which inhibited the HER catalytic performance of nano-Pt/COOH-MWCNT,thus achieving highly sensitive and indicator-free detection of mi RNA Let-7a.According to the signal change of electrocatalytic HER,the biosensor can detect the target mi RNA Let-7a in the concentration range of 1 f M to100 n M with a detection limit of 0.36 f M.This work provides a new electrochemical biosensor construction strategy and shows great application prospects in practical applications.（3）The nano-platinum and fullerenol complex（Pt@FLN）was obtained by ultrasonic-assisted reduction of chloroplatinic acid with sodium borohydride（Na BH₄）in water-soluble fullerenol（FLN）solution,and the recognition element streptavidin（SA）was self-assembled on the surface of the material to obtain a bifunctional material（SA@Pt@FLN）with HER electrocatalysis and nucleic acid conformation recognition.At the same time,a layer of gold nanoparticles was electrodeposited on the surface of GCE,and the thiol-modified hairpin DNA probe was immobilized on the surface of gold nanoparticles modified electrode by Au-S bond.When the target mi RNA-21 is present,it undergoes a base pairing reaction with the loop structure part of the hairpin DNA probe,opens the DNA stem,and extends to form a SA-binding aptamer structure.In this way,SA@Pt@FLN can be adsorbed to the electrode surface through aptamer chemical and biological affinity,thereby exhibiting HER electrochemical catalytic activity.Electrochemical sensing experiments show that based on this sensing strategy,the HER catalytic current has a good linear relationship with the logarithm of mi RNA-21 target concentration in the concentration range of 1 f M to 100 n M,and the detection limit is as low as 20 a M.At the same time,the sensor showed excellent specificity,which provided a new indicator-free analysis method for high sensitivity and high specificity micro RNA detection.