Optimization of on-bead emulsion polymerase chain reaction based on single particle analysis.

Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies. Despite these advantages, ePCR remains underused due to the lack of optimal starting conditions, straightforward methods to evaluate success, and guidelines for tuning the reaction. This knowledge has been elusive for bulk emulsion generation methods, such as stirring and vortexing, the only methods that can emulsify libraries of ≥108 sequences within minutes, because these emulsions have not been characterized in ways that preserve the heterogeneity that defines successful ePCR. Our study quantifies the outcome of ePCR from conditions specified in the literature using single particle analysis, which preserves this heterogeneity. We combine ePCR with magnetic microbeads and quantify the amplification yield via qPCR and the proportion of clonal and saturated beads via flow cytometry. Our single particle level analysis of thousands of beads resolves two key criteria that define the success of ePCR: 1) whether the target fraction of 20% clonal beads predicted by the Poisson distribution is achieved, and 2) whether those beads are partially or maximally covered by amplified DNA. We found that among the two concentrations of polymerase tested, only the higher one, which is 20-fold more than the concentration recommended for conventional PCR, could yield sufficient PCR products. Dramatic increases in the concentrations of reverse primer and nucleotides recommended in literature gave no measurable change in outcome. We thus provide evidence-based starting conditions for effective and economical ePCR for real DNA libraries and a straightforward workflow for evaluating the success of tuning ePCR prior to downstream applications.

Gelatin Microcartridges for Onboard Activation and Antioxidant Protection of Sperm.

Sperm-driven micromotors are promising devices for developing autonomous, noninvasive diagnostic and therapeutic tools, specifically in reproductive biology and medicine. In this article, we present template-based microstructures fabricated from gelatin for sperm capture, activation, and antioxidant protection. The cartridge-like structures with one closed end-capture single bovine sperm cells and are propelled by the sperm's flagellum. We demonstrate the pH-dependent release of heparin from the gelatin microcartridges to induce capacitation, a crucial maturation process prior to fertilization. Further, the gelatin cartridges exhibit a protection against oxidative stress, one of the main causes for sperm damage. Finally, we investigate the stability and degradability of the microstructures in various physiological environments, such as endometrial cell culture, different pH, and in the presence of proteases. Overall, we prove that these gelatin spermbots display great potential for the development of noninvasive theranostic tools in reproductive biology and medicine for the protection and activation of sperm, especially useful for studying sperm migration. We present their beneficial features which comprise not only biocompatibility and biodegradability but also pH response, loading stability, and antioxidant protection.

The motility-based swim-up technique separates bull sperm based on differences in metabolic rates and tail length.

Swim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. However, the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms that the upper fraction of bull sperm has not only improved motility compared to the cells in the lower fraction but also shows higher metabolic rates and longer flagella. This pattern was consistent across media of two different levels of viscosity. We conclude that the motility-based separation of the swim-up technique is also reflected in underlying metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality.