Preparation Of Biosensors Based On The Mesoporous Carbon Materials Immobilized With Sortase A And The Detection Of Staphylococcus Aureus

Sortase A is one of the important members of the sorting enzyme family and belongs to a membrane bound thiol transpeptidase,widely presenting in Gram-positive bacteria,the key enzyme of anchoring the bacterial surface protein to the cell wall.In this paper,two kinds of electrochemical enzyme biosensors were prepared by using the catalytic mechanism of Sortase A.The biosensors were applicable to the detection of Staphylococcus aureus.In this study,two kinds of mesoporous carbon materials,CMK-3 and Hollow carbon sphere,were prepared.Then the functionalization of carboxyl groups by the wet oxidation method.The immobilization of Sortase A by using carboxyl group and the amino group.The morphology and crystal structure of the two materials were observed by SEM,TEM and XRD.N2 adsorption-desorption experiments shows that both materials belong to the mesopore range,and the specific surface area is large,which is beneficial to the immobilization of the enzyme.The characterization of carboxyl functionalization of two materials was demonstrated by Fourier transform infrared spectroscopy.The analysis of the Sortase A before and after immobilization was carried out by the infrared spectrum fitting,found that the concentration of?-sheet in the enzyme A was stained 70% after immobilization,then it can be seen the enzyme activity was ensured preliminarily.Raman,XPS and other means proved the successful combination of carrier material and Sortase A.In the process of immobilization,the optimum conditions were explored.Under these conditions,the fixation amount was 85±3.1 mg/g and 91±3.2 mg/g respectively.The enzyme activity was retained more than 85% of the original.Two kinds of materials immobilized Sortase A were combined with graphite and paraffin respectively into powder to make carbon paste electrode.Using the electrochemical three electrode detection system to detect the electrode properties,the electrical signal was detected by changing the concentration of different strains of Staphylococcus aureus,discovered that the O-CMK-3-M biosensors has a good linear relationshipin the range of 0.125×102~2.5×102CFU/m L(r=0.9852),and theO-CMK-3-M biosensors has a good linear relationshipin the range of 0.125×102~3×102CFU/m L(r=0.9867)detection limits were 10 CFU/m L and 9 CFU/m L,respectively.