Incorporation of Erythrocytes into Polypyrrole to Form the Basis of a Biosensor to Screen for Rhesus (D) Blood Groups and Rhesus (D) Antibodies

Abstract

Antibodies to Rhesus (Rh) antigens are important indicators in screening for haemolytic disease of the new-born (HDN) and autoimmune haemolytic anaemia (AIHA). Identification of the Rh antibodies formed by immune stimulation is also essential in order to maximize the in vivo survival time of transfused erythrocytes. Currently this is performed by agglutination based assays that are time consuming. A prototype of an immuno-biosensor for detecting antibodies recognizing the Rhesus blood group antigen, Rh (D), was constructed. Human erythrocytes were incorporated into a conducting polypyrrole, polyelectrolyte matrix. The process was followed by using oximetry and light microscopy to demonstrate the integrity of the erythrocytes in the polymerization solution and in the polymer matrix; cyclic voltammetry and resistometry for electrochemical characterization of the polymer and then agglutination, ELISA techniques and cyclic resistometry for analysis of the immuno response from antigen/antibody binding. Antigen/antibody binding could be detected qualitatively by using resistometry while cycling the polymer between +0.35 V and –0.7 V (vs. Ag/AgCl). A characteristic cyclic change in resistance (a resistogram) was recorded. After addition of Anti-Rh (D) antibody (250 µg/mL), the change in resistance during the resistogram decreased by 1.1 Ω (p<0.0008) in polymers containing Rh (D) positive erythrocytes, whereas polymers without erythrocytes showed no significant change.