Single-type reporter multiplexing with A single droplet through bead-based digital microfluidics.

With the limited sample volume, the droplet-based microfluidic becomes attractive in biomedical diagnosis, especially for measuring multiple analytes. Usually, for multiplexing by parallel processing, a larger sample volume is required. In our previous study, simultaneously detecting two analytes from a single droplet was first achieved by measuring different fluorescence wavelengths for different analytes. However, the number of detectable analytes could be limited by the spectral resolution of fluorescence. Here a different approach is proposed for multiplexing by sharing a single droplet in multiple sub-assays. Therefore, only a single-type reporter, i.e., the fluorescence with the same wavelength, is needed for detection of different analytes from a single sample droplet, called single-type reporter multiplexing (STRM). The standard curves of two analytes, human IL-1ß and human TNF-α, are demonstrated. The required sample volume for one measurement is only 520 nL; the total duration of the on-chip process is less than 50 min. The limits of detection (LOD) of human IL-1ß and human TNF-α are about 1.14 and 0.97 pg/mL, respectively. It is shown that the proposed bead-based digital microfluidic immunoassay can achieve multiple analytes detection with low LOD from a single sample droplet using the single-type reporter, which has never been achieved before.

Taurine homeostasis requires de novo synthesis via cysteine sulfinic acid decarboxylase during zebrafish early embryogenesis.

Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos.