Detection and imaging the expression of the trans-membrane protein CD44 in RT112 cells by use of enzyme-labeled antibodies and SECM.
Roberts WS., Davis F., Holmes JL., Collyer SD., Larcombe LD., Morgan SL., Higson SP.
The deposition of human RT112 cells in a patterned fashion onto glass substrates and subsequent imaging of the expression of the trans-membrane protein CD44 have been studied using scanning electrochemical microscopy (SECM). Patterns of RT112 cells derived from a transitional cell carcinoma of the bladder could be deposited on amino-modified glass substrates by cytospinning. These were then treated with horseradish peroxidase (HRP) labeled secondary antibodies to the trans-membrane protein CD44. Expression of CD44 protein by the cells directly leads to immobilisation of the labeled antibodies. The presence of the enzyme substrate (hydrogen peroxide) along with a hydroquinone mediator then allowed an enzymatic reaction to proceed, generating benzoquinone. Reduction of benzoquinone gave rise to positive feedback between the substrate and the SECM microelectrode tip. Control samples such as blank slides or slides not treated with HRP-labeled antibody showed negative feedback effects. Patterns of RT112 cells could be assembled and their expression of the target protein imaged whereas control samples showed minimal activity.