Development Of Screen Printed Electrode-based Electrochemical Sensor For Bacterial 16S RRNA Gene Detection

In recent years,the global public health problem caused by bacterial infections is increasing,leading to as high as billions of dollars economic losses every year.Bacterial infection not only impedes the development of social economy,but also threatens the safety of human life.Therefore,how to find the source of bacterial infection and quickly determine the type of bacteria has become an effective way to deal with bacterial infection.As an effective means to prevent bacterial infection,bacterial detection has received extensive attention in the fields of environmental monitoring,food safety,medical diagnosis and treatment,etc.Traditional detection methods have the disadvantages of long detection period,complex detection procedures,and high requirements for samples,which seriously restrict the rapid prevention of bacterial infections.Therefore,it is an urgent need to develop new,fast,low-cost,and high-precision detection technologies to achieve early detection of bacterial infections,thereby effectively curbing the occurrence and spread of bacterial infections.Electrochemical biosensor plays a vital role in bacterial infection detection due to its low cost,simplicity,rapidity,high sensitivity and easy deviceization,which is favored by researchers.In summary,this thesis aims to construct electrochemical biosensors for sensitive and specific detection of bacteria by selecting miniaturized,portable,easy-to-functionalize,mass-produced screen-printed electrodes as electrode materials and choosing 16S rRNA gene as the research object for distinguishing different bacterial species.This thesis is mainly divided into the following two aspects:1.An electrochemical sensor for the detection of Escherichia coli 16S rRNA gene was constructed.First,gold flower-like microstructure-functionalized screen-printed electrodes（SPCE-Au NFs）were fabricated by electrochemical deposition to improve the conductivity of the screen-printed electrodes.Meanwhile,gold nanoparticles and thionine co-functionalized molybdenum disulfide nanocomposites（Mo S₂-Au NPs-Thi）were synthesized as nanoprobe construction materials by one-step chemical synthesis method.Two thiol-labeled DNA1 and DNA2strands were assembled onto SPCE-Au NFs electrode and Mo S₂-Au NPs-Thi nanocomposite through Au-S bonds,respectively.In the presence of E.coli 16S rRNA gene,a classic DNA1-16S rRNA-DNA2 sandwich structure is formed on the surface of the electrode,resulting in the specific recognition and detection of E.coli 16S rRNA gene.Under the optimal experimental conditions,the sensor can detect E.coli 16S rRNA gene concentration in the range of 10 f M-100 p M with a detection limit of 2.8 f M.Experimental results have shown that the designed sensor also has good stability and reproducibility.Therefore,this sensor can not only be used for the efficient detection of16S rRNA in milk samples,but also for the highly sensitive detection of 16S rRNA in bacterial lysate,further proving that the developed sensor has a promising potential application in real sample detection.2.An electrochemical sensor for the detection of Staphylococcus aureus 16S rRNA gene was constructed.With the introduction of a new gene editing technology（CRISPR/Cas system）,CRISPR-electrochemical sensing platform was developed to detect 16S rRNA gene with high sensitivity and selectivity.A stem-loop DNA structure labeled with both a sulfhydryl group and a ferrocene signal molecule was assembled on the surface of the gold electrode to construct an electrochemical sensing interface.With the addition of the Staphylococcus aureus 16S rRNA gene,a 16S rRNA-crRNA-Cas12 ternary complex was formed and the cleavage activity of Cas12 protein was activated.As a result,stem-loop DNA structure was cut.The labelled ferrocene signal molecule is released from the surface of the electrode,resulting in the decrease of electrochemical signal.The detection of Staphylococcus aureus 16S rRNA gene can be achieved by monitoring the changes of the electrochemical signal.Under the optimal experimental conditions,the peak current of the sensor showed a good linear relationship with the 16S rRNA gene of Staphylococcus aureus in the range of 10 p M-100 n M with a detection limit of 2.0 p M.The sensor also has good stability and reproducibility,which can be used for highly sensitive detection of Staphylococcus aureus 16S rRNA gene in milk samples.