Construction Of Highly Sensitive Electrochemical Cytosensor Based On Enzyme Cascade Reaction

Circulating tumor cells（CTCs）are cancer cells that fall off from the primary site of tumor and enter the blood circulation.The detection of CTCs is of great significance for the early diagnosis and treatment monitoring of cancer patients.Electrochemical biosensors are widely used because of their advantages such as timely response,high sensitivity,low cost and convenient use.In this paper,based on the enzyme cascade reaction,a super-sensitive electrochemical biosensor is constructed for the detection of CTCs.The details are as follows:（1）In this study,an electrochemical biosensor based on multi-walled carbon nanotubes（MWCNTs）and chitosan（CS）composite modified materials loaded with glucose oxidase（GOD）and polystyrene microspheres（PS）loaded with horseradish peroxidase（HRP）was constructed for the detection of CTCs.The composites of MWCNTs and chitosan CS were used as both the modified materials of electrode and the carrier of GOD,and PS supported by HRP were as the signal probe.Through self-assembly of layer by layer,MWCNTs-CS,aptamer,CTCs and PS-HRP were constructed onto the working electrode successively.In the electrolyte solution containing glucose,GOD catalyzed glucose to form gluconic acid and H₂O₂,and HRP catalyzed the decomposition of H₂O₂promoting the electron transfer of sensing system.The current change in the system was positively correlated with the concentration of CTCs,which realized the electrochemical biosensing detection of CTCs based on the efficient enzyme cascade reaction system.The electrochemical biosensor had good sensitivity.The linear concentration range was 10 cells m L^-1to 10⁶cells m L^-1,and the detection limit was 3 cells m L^-1.In addition,the sensor was applied to CTCs detection in spiked blood serum,showing great potential for clinical detection.（2）In this study,an electrochemical biosensor based on GOD,HRP,and G-quadruplex hemin multi-enzyme co-immobilization was constructed for indirect detection of CTCs.PS-GOD-HRP/G-quadruplex-hemin probe was prepared by GOD,HRP and G-quadruplex-hemin polyenzyme co-fixed on PS;Meanwhile,Au NPs were prepared by the reduction of sodium citrate for chloraurate,and the MWCNTs-Au NPs composite was used for electrode material.In the incubation solution,the signal probe was captured by CTCs with some concentration and the uncaptured probe was assembled on the electrode surface by capturing DNA.GOD catalyzed glucose into gluconic acid,H₂O₂and hemin in the electrolyte solution containing glucose,and HRP catalyzed the decomposition of H₂O₂to promote electron transfer.The current change in the system was negatively correlated with the concentration of CTCs,and the indirect detection of CTCs was obtained.The electrochemical biosensor had good sensitivity,selectivity and specificity.The linear concentration range was 10 cells m L^-1to 10⁶cells m L^-1,and the detection limit was 2 cells m L^-1.