Lab on a bead with oscillatory centrifugal microfluidics for fast and complete mixing enables fast and accurate biomedical assays.

Rapid mixing and precise timing are key for accurate biomedical assay measurement, particularly when the result is determined as the rate of a reaction: for example rapid immunoassay in which the amount of captured target is kinetically determined; determination of the concentration of an enzyme or enzyme substrate; or as the final stage in any procedure that involves a capture reagent when an enzyme reaction is used as the indicator. Rapid mixing and precise timing are however difficult to achieve in point-of-care devices designed for small sample volumes and fast time to result. By using centrifugal microfluidics and transposing the reaction surface from a chamber to a single mm-scale bead we demonstrate an elegant and easily manufacturable solution. Reagents (which may be, for example, an enzyme, enzyme substrate, antibody or antigen) are immobilised on the surface of a single small bead (typically 1-2 mm in diameter) contained in a cylindrical reaction chamber subjected to periodically changing rotational accelerations which promote both mixing and uniform mass-transfer to the bead surface. The gradient of Euler force across the chamber resulting from rotational acceleration of the disc, dΩdisc/dt, drives circulation of fluid in the chamber. Oscillation of Euler force by oscillation of rotational acceleration with period, T, less than that of the hydrodynamic relaxation time of the fluid, folds the fluid streamlines. Movement of the bead in response to the fluid and the changing rotational acceleration provides a dynamically changing chamber shape, further folding and expanding the fluid. Bead rotation and translation driven by fluid flow and disc motion give uniformity of reaction over the surface. Critical parameters for mixing and reaction uniformity are the ratio of chamber radius to bead radius, rchamber/rbead, and the product Trchamber(dΩdisc/dt), of oscillation period and Euler force gradient across the fluid. We illustrate application of the concept using the reaction of horse radish peroxidase (HRP) immobilised on the bead surface with its substrate tetramethylbenzidine (TMB) in solution. Acceleration from rest to break a hydrophobic valve provided precise timing for TMB contact with the bead. Solution uniformity from reaction on the surface of the bead in volumes 20-50 uL was obtained in times of 2.5 s or less. Accurate measurement of the amount of surface-bound HRP by model fitting to the measured kinetics of colour development at 10 s intervals is demonstrated.

Non-Invasive Pregnancy Diagnosis in Big Cats using the PGFM (13,14-dihydro-15-keto-PGF2α) Assay.

Non-invasive monitoring of hormones using feces has become a vital tool for reproductive management and reliable pregnancy diagnosis in big cats. Previous studies described the PGF2α metabolite (PGFM) as an indicator of pregnancy in various feline species. The present study aimed to standardize pregnancy detection in big cats like the tiger (Panthera tigris), jaguar (Panthera onca) and lion (Panthera leo) using fecal samples. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM in feces. An EIA developed against 9α,11α-dihydroxy-15-oxo-prost-5-en-1-oic acid-BSA was used to assay PGFM in fecal samples of the Bengal tiger, Asiatic lion and jaguar. The PGFM levels increased after 9 weeks of pregnancy and remained elevated until parturition. All animals showed elevated levels of PGFM in the last trimester of pregnancy, thus making PGFM a reliable tool for pregnancy diagnosis during this period that can be useful in captive breeding programs in these species.

Glucocorticoid Stress Responses of Reintroduced Tigers in Relation to Anthropogenic Disturbance in Sariska Tiger Reserve in India.

Tiger (Panthera tigris), an endangered species, is under severe threat from poaching, habitat loss, prey depletion and habitat disturbance. Such factors have been reported causing local extermination of tiger populations including in one of the most important reserves in India, namely Sariska Tiger Reserve (STR) in northwestern India. Consequently, tigers were reintroduced in STR between 2008 and 2010, but inadequate breeding success was observed over the years, thus invoking an investigation to ascertain physiological correlates. In the present study, we report glucocorticoid stress responses of the reintroduced tigers in relation to anthropogenic disturbance in the STR from 2011 to 2013. We found anthropogenic disturbance such as encounter rates of livestock and humans, distance to roads and efforts to kill domestic livestock associated with an elevation in fecal glucocorticoid metabolite (fGCM) concentrations in the monitored tigers. In this regard, female tigers seem more sensitive to such disturbance than males. It was possible to discern that tiger's fGCM levels were significantly positively related to the time spent in disturbed areas. Resulting management recommendations include relocation of villages from core areas and restriction of all anthropogenic activities in the entire STR.