A Novel Molecularly Imprinted Polymer for the Selective Removal of Interfering Hemoglobin Prior to Whole Blood Analysis

Abstract

A heavy red globular protein, hemoglobin, responsible for whole blood red pigmentation often interferes with the identification and quantification of disease associated biomarkers from whole blood, in field of molecular diagnosis. The main challenge is the direct introduction of whole blood as a sample into analyzing instruments because of its physiological complexity and `dirty` nature. For example, the red pigment in whole blood, which is characterized as ‘dirt’, usually co-elute with the biomarkers and masks them from easy chromatographic separation prior to their final detection. It also clogs the instrument’s components such as the separating columns which are known to be sensitive, hence leading to imprecise and inaccurate results during bio-assaying. To address these challenges, our lab synthesised a novel, selective, effective and a robust hemoglobin imprinted polymer, in the form of a powder, through bulk, freeradical polymerization employing molecular imprinting technology, to selectively remove interfering hemoglobin from whole blood samples prior to instrumental analysis of disease associated biomarkers. From the results, the polymer powder effectively removed hemoglobin from whole blood sample as demonstrated by the ultraviolet-visible absorbance reduction from as high as 0.794 Au to lower values of 0.193 Au before and after polymer powder application, respectively. Experimentally, the powder had a high binding ability towards the targeted hemoglobin as demonstrated by the high percentage removal efficiency of 76% from hemoglobin standard solutions, when compared to its low binding ability towards an analogous species, (chlorophyll), at 32% from chlorophyll standard solutions. Furthermore, the polymer powder proved to be robust as it removed hemoglobin interference from the ‘dirty’ complex matrix of real human whole blood samples by up to 74% hemoglobin removal which was comparable to 76% hemoglobin removal from neat standards, thus, the polymer powder demonstrated that it can work effectively in diverse environments of clean and dirty matrix. Furthermore, the polymer powder presented itself as an efficient, selective and non-destructive whole blood clean-up pre-analytical tool that with further research may replace the destructive and non-selective conventional whole blood clean-up strategies such as the commonly employed centrifugation.