Amperometric immunosensor using Tc85-11 protein and investigations of the protein interaction with anti-T. Cruzi antibody by atomic force microscopy

A. P. Ferreira, A.

Chagas" disease is an American trypanosomiasis caused by the hemoflagellate Trypanosoma cruzi (T. cruzi). In Brazil, the control measures to eradicate the populations of native triatomine species have significantly reduced the risk of T. cruzi transmission by contaminated hematophagous insects but contamination by blood transfusion is still a serious problem, particularly in remote regions. The detection of antigen in blood sera could be useful just for the acute phase of the Chagas" disease while the detection of anti-T. cruzi antibodies in the serologic analysis is the chosen method for the etiological diagnosis of Chagas" disease in the chronic phase.The present work reports the development of an amperometric immunosensor for the diagnosis of Chagas" disease and the preliminary investigation on atomic force microscopy (AFM) by using a specific glycoprotein of the trypomastigote surface which belongs to the Tc85-11 protein family of Trypanosoma cruzi Tc85-11 was expressed as polyhistidine fusion protein using the vector pCR T7/NT TOPO® Cloning (Invitrogen) and Escherichia coli strain BL21 (DE3) plysS (Invitrogen). Tc85-11 protein was purified from inclusion bodies, solubilized in 50 mM Tris-HCl, pH 6.0, 100 mM NaCl and 1 M imidazole and purified with a Ni2+ (nitrilotriacetic acid)-column (Chelating Sepharose Fast Flow resin/Amersham-Pharmacia Biotech). Approximately 0.08 mg m