Haptoglobin biomarker detection based on enhanced chemiluminescence of gold nanoparticles

Bovine mastitis (BM) is one of the most frequent diseases in dairy cattle, with staggering adverse effects on farm economics in terms of increased treatment costs, decreased milk yield, escalation of somatic cell counts (SCC), and increased risk of removal from the herd or even death. BM is an inflammatory disease of mammary gland caused by bacterial intramammary infection. During udder inflammation, elevated levels of acute phase proteins (APP) are secreted onto plasma and milk, which can be distinctively utilized as predicting diagnostic biomarkers of cattle’s BM clinical status. Herein, we have developed a liquid-phase luminol chemiluminescence (CL) system for sensitive detection of haptoglobin (Hp), a predictive APP of BM, by utilizing the binding capacity of hemoglobin (Hb). The inhibition of peroxidase-like activity luminol-H₂O₂-Hb CL system due to Hb-Hp complex formation is linearly proportional to CL intensity. Enhanced CL, at least 10-fold effect within real samples, is attained by the addition of catalytically active cross-linked gold nanoparticles (GNPs) onto the luminol-H₂O₂ solution. Moreover, the influence of different somatic cell counts (representing subclinical and clinical BM status) and pathogen types (i.e., Coagulase-negative Staphylococci and Streptococcus dysgalactiae) on the secreted milk Hp levels obtained from Holstein cows are established. The analyzed Hp concentrations are in agreement with a commercial enzyme-linked immunosorbent assay kit. The proposed CL sensing concept offers cost-effective, simple, label-free and reliable systematic analysis of Hp biomarker for BM, potentially initiating a positive effect on animals’ health and overall economy of the dairy farms.

Keywords: Gold nanoparticles; Chemiluminescence; Haptoglobin; Pathogens; Somatic cell counts