Abstract
Bovine mastitis (BM) is a frequent disease in the dairy industry that causes staggering economical losses due to decreased milk production and increased health care costs. Traditionally, BM detection depends on the efficacy and reliability of analytical techniques that measure somatic cell counts (SCC), detect pathogens, and reveal inflammatory status. Herein, we demonstrate the detection of bovine haptoglobin, a well-documented acute phase protein for evaluating BM clinical status, by utilizing hemoglobin-binding capacity within luminol chemiluminescence (CL) system. The resulting haptoglobin–hemoglobin complex reduces the CL signal proportionally to inherent haptoglobin concentrations. Different sizes of cross-linked gold nanoparticles (GNPs) were examined for enhanced CL (eCL) signal amplification, presenting over 30-fold emitted radiation enhancement for optimized size within real milk samples with respect to nanoparticle-free assay. The eCL values were proportionally related to nanoparticle size and content, influenced by SCC and pathogen type (e.g., Escherichia coli and coagulase-negative staphylococci). The optimized bioassay showed a broad linear response (1 pg mL−1–10 µg mL−1) and minute detection limit of 0.19 pg mL−1, while presenting quantitative performance in agreement with commercial ELISA kit. Finally, the resulting optimized eCL concept offers an efficient label-free detection of haptoglobin biomarker, offering means to diagnose the severity of the associated diseases.
Highlights
Chemiluminescence (CL) is widely applied chemically driven electromagnetic radiation for real-life applications, from clinical monitoring, food and pharmaceutical safety control, and up to trace levels of environmental pollutants analysis, utilizing its high sensitivity toward the assessment of the limiting target reactant involved in the luminol reaction [1,2,3,4,5,6,7,8,9,10,11]
Different sizes of Gold nanoparticles (GNPs) were produced according to a modified procedure of Brown et al [12,25]
The colloidal GNPs showed a correlative surface plasmon resonance (SPR) peak position which red-shifted with nanoparticle size enhancement (505, 507, 519, 522, and 534 nm for 2.6, 8, 13, 25, and 38 nm, respectively) [13,17]
Summary
Chemiluminescence (CL) is widely applied chemically driven electromagnetic radiation for real-life applications, from clinical monitoring, food and pharmaceutical safety control, and up to trace levels of environmental pollutants analysis, utilizing its high sensitivity toward the assessment of the limiting target reactant involved in the luminol reaction [1,2,3,4,5,6,7,8,9,10,11]. Numerous case studies and reports employed GNPs for enhanced CL (eCL) presenting a repertoire of novel ultrasensitive bioassays [4,5,6,12,13,14,15,16,17,18,19]. Singh et al have reported the use of sequence-specific single-stranded DNA probes anchored on the exterior of GNPs surface for the hybridization with the complementary DNA target regions of Ceratocystis fagacearum [15]. The molecular recognition of the target-specific hybridization resulted in instantaneous GNPs aggregation into cluster formation, which directly induced an eCL signal. The label-free strategy has presented a limit of detection (LOD)
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