Doctor of Philosophy
University of Wollongong. Dept. of Chemistry
Campbell, Toni E., Development of a biosensor for determination of human blood groups, Doctor of Philosophy thesis, University of Wollongong. Dept. of Chemistry, University of Wollongong, 1999. http://ro.uow.edu.au/theses/1136
The incorporation, for the first time, of whole intact human erythrocytes, containing viable antigens, into an electroactive polypyrrole matrix, from which signals, direct and indirect, were induced provides encouragement for the development of an immunobiosensor for the detection of binding of blood group antibodies to their respective antigens. The detection of binding of blood group antibodies to their respective antigens is an integral part of determining a blood group and in antibody detection. The interest in blood grouping and antibody detection corresponds to the need by the medical profession to administer or transfuse human blood and blood products to other humans in order to improve the quality of life and longevity. Antibodies to ABO and Rhesus (Rh) antigens are also important indicators in screening for haemolytic disease of the new-born (HON) and autoimmune haemolytic anaemia (AIHA).
A prototype of an immuno-biosensor surface for detecting antibodies recognising the 'A' and Rh(O) antigens was constructed. The biosensor surface was synthesised to incorporate intact erythrocytes into the polymer (plastic), comprising polypyrrole, polyelectrolyte and a significant proportion of the mass as erythrocytes. Polymers were synthesised with a constant electrical current (1.5 mAcm-2 for 10 secs), where the polyanion of polyvinyl sulphonate (PVS) and the human erythrocytes (approximately 1.0 x 106 erythrocytes cm-2) were incorporated during synthesis to balance the positive charge on the polymer backbone. The process was followed by using oximetry and light microscopy to demonstrate the integrity of the erythrocytes in the polymerisation solution and in the polymer matrix. The majority of erythrocytes were incorporated with even distribution as intact discs, as shown by light and atomic force microscopy (AFM).
Electrochemical characterisation of the polymer using cyclic voltammetry (CV), Electrochemical Quartz Crystal Microgravimetry (EQCM) and resistometry showed that incorporating erythrocytes and some erythrocyte membrane (upto 70-72% of the surface area of the polymer) into an electrically conducting polymer did not compromise the ability of the matrix to be continuously oxidised and reduced which was so important for the direct signal phase of this work.
ELISA techniques were developed, to accommodate polymer discs containing erythrocytes, then used to determine that the antigens in the polymer matrix remained functional. Indirect electrical signals were obtained electrochemically from an enzyme amplified immunoassay (ELISA) and validated spectrophotometrically. Indirect signals showed results where test cases were twice the blanks whether determined electrochemically or spectrophotometrically after addition of the enzymelinked purified monoclonal antibody.
Use of human sera in the test system showed differentiation between positive and negative binding responses (assay sensitivity 90%). The number of false positive reactions was too high (specificity of 63%). Further optimisation of the system with greater numbers of donor sera could realise a novel and reliable indirect electrochemical technique for the determination of reverse blood groups in human sera.
Direct electrical signals were obtained by resistance changes when purified antibody bound to the corresponding antigen in the erythocyte membrane, using resistometry techniques. Resistometry studies showed altered polymer resistance with the addition of purified IgG monoclonal antibody (Anti-Rh(D) and Anti-A) when the polymer was cycled between +0.35V and -0.70V (vs Ag/ Agel). After the addition of antibody (100-250 !lg/mL), the change in resistance during the resistogram decreased by 1.1 Ohms (p