Inflammatory biomarker detection in milk using label-free porous SiO2 interferometer.

N-acetyl-ß-d-glucosaminidase (NAGase) is an established indicative biomarker released upon damage or necrosis of tubular epithelial cells in both humans and animals, indicating severe nephrological disorders and bovine mastitis (BM), respectively. The latter is the most common and costly disease in dairy cattle associated with production losses, elevated somatic cell counts and deteriorated health status. Herein, we report on a reflective based assay for early diagnosis of BM through the analysis of NAGase inherent content found in whole milk samples using a miniaturized optical transducer. Gelatin functionalized porous Si Fabry-Pérot interferometers are employed for monitoring the lysosomal activity in various stages of the inflammation (healthy, subclinical and clinical). The enzymatic reaction products precipitate and accumulate within the porous nanostructure, thus alter the average refractive index monitored using reflectometric interference spectroscopy. The optical assay is calibrated within the clinically relevant concentrations of BM while presenting a dynamic range of 1.04-16.7 µM min-1 and the detection limit of 0.49 µM min-1. The specific optical performance of the biosensor correlates with a gold standard laboratory-based approach, in which escalated somatic cell counts reveal augmented NAGase levels and thus severe pathogenesis. Overall, our study provides new opportunities to develop a convenient bio-diagnostic sensing system for BM detection and classification by addressing the limitations of conventional practices.

Porous Silicon Fabry-Pérot Interferometer for N-Acetyl-ß-d-Glucosaminidase Biomarker Monitoring.

Bovine mastitis (BM) is a prominent inflammatory disease affecting the dairy industry worldwide, originated by pathogenic agent invasion onto the mammary gland. Early detection of new BM cases is of high importance for infection control within the herd. Conventional analytical techniques lack the ability to detect BM-predicting biomarkers, used as analytical indicators for health status evaluation, in real time or outside the laboratory boundaries. Herein, we describe a biosensing platform for label-free detection and identification of BM onset through targeting N-acetyl-ß-d-glucosaminidase (NAGase) for potential evidence-based therapy. The lysosomal activity in dissimilar milk qualities was monitored by a gelatin-functionalized porous Si Fabry-Pérot interferometer, while estimating the biochemical reaction precipitating products within the nanostructure. The optical response was proportional to the inherent NAGase concentration found in real milk samples, influenced by two dominant BM causative pathogens (i.e., Escherichia coli and Streptococcus dysgalactiae) at various somatic cell counts. Quantitative analysis of NAGase levels within the entire inflammatory spectrum (healthy, subclinical, and clinical BM) was obtained within the range of 1.0-4.2 µM/min (enzymatic activity per volume unit), while presenting a detection limit of 0.51 µM/min. The optical performances correspond with standardized biochemical activity assay in dissimilar milk qualities. Overall, the presented sensing concept exhibits the potential of BM-predicting biomarker detection using a simple and portable experimental setup for convenient early biodiagnostics and health status evaluation.

Milk haptoglobin detection based on enhanced chemiluminescence of gold nanoparticles.

The suggested research specifically addresses the major source of economic loss of the dairy industry, the bovine mastitis (BM), an inflammatory disease of mammary gland caused by bacterial intramammary infection. During udder inflammation, the concentrations of acute phase proteins (APP) in both plasma and milk are escalated, which can be distinctively utilized as predicting diagnostic biomarkers of cattle's BM clinical status. Herein, we demonstrate 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 CL intensity is linearly proportional to Hb-Hp complex formation, resulting in peroxidase-like activity inhibition of luminol-H2O2-Hb CL system. 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., CNS 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.