Hypocalcemia is caused by a sharp decline in blood calcium concentration after dairy cow calving, which can lead to various diseases or even death. It is necessary to develop an inexpensive, easy-to-operate, reliable sensor to diagnose hypocalcemia. The cellulose-paper-based microfluidic field-effect biosensor is promising for point-of-care, but it has poor mechanical strength and a short service life after exposure to an aqueous solution. Octadecyltrichlorosilane (OTS), as a popular organosilane derivative, can improve the hydrophobicity of cellulose paper to overcome the shortage of cellulose paper. In this work, OTS was used to produce the superhydrophobic cellulose paper that enhances the mechanical strength and short service life of MFB, and a microfluidic field-effect biosensor (MFB) with semiconducting single-walled carbon nanotubes (SWNTs) and DNAzyme was then developed for the Ca²⁺ determination. Pyrene carboxylic acid (PCA) attached to SWNTs through a non-covalent π-π stacking interaction provided a carboxyl group that can bond with an amino group of DNAzyme. Two DNAzymes with different sensitivities were designed by changing the sequence length and cleavage site, which were functionalized with SPFET/SWNTs-PCA to form Dual-MFB, decreasing the interference of impurities in cow blood. After optimizing the detecting parameters, Dual-MFB could determine the Ca²⁺ concentration in the range of 25 μM to 5 mM, with a detection limit of 10.7 μM. The proposed Dual-MFB was applied to measure Ca²⁺ concentration in cow blood, which provided a new method to diagnose hypocalcemia after dairy cow calving.

An electrochemical biosensor for detecting Ca²⁺ concentration was proposed using glass carbon electrodes (GCEs) modified with nitrogen-doped graphene (NGR), gold nanoparticles (AuNPs) and DNAzyme. The resistance signal was amplified through two methods: electrochemical reduction of AuNPs on the NGR surface to increase the specific surface area of the electrode and strengthen the adsorption of DNAzyme; and increasement of the DNAzyme base sequence. The process of electrode modification was characterized by scanning electron microscopy, Raman spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Experimental parameters’ influence, such as the deposition time of gold nanoparticles and the detection time, were assessed by electrochemical methods. The linear ranges of the electrochemical biosensor were in the range from 5 × 10⁻⁶ to 5 × 10⁻⁵ and 5 × 10⁻⁵ to 4 × 10⁻⁴ M, with a detection limit of 3.8 × 10⁻⁶ M. The concentration of Ca²⁺ in the serum of dairy cows was determined by the biosensor with satisfactory results, which could be potentially used to diagnose subclinical hypocalcemia.

This study aimed to investigate the inhibitory effects of rottlerin, a plant-derived polyphenol compound, on methane (CH₄) production in dairy cow rumen fermentation by targeting methyl-coenzyme M reductase (MCR). Molecular docking and virtual screening revealed that rottlerin exhibited a high binding affinity (-8.300 kcal/mol) with key residues in the catalytic site of MCR (GLY-142 and GLN-230). In vitro rumen fermentation showed that rottlerin significantly reduced CH₄ production in a dose-dependent manner (up to 32.4%), while maintaining stable pH, dry matter digestibility, and VFA concentration. Microbial community analysis revealed a reduction in the relative abundance of Methanobrevibacter, a dominant hydrogenotrophic methanogen, and a simultaneous increase in Candidatus_Methanomethylophilus (P < 0.05), a methylotrophic methanogen, indicating a metabolic shift favoring methylotrophic rather than hydrogenotrophic methanogenesis. The in vitro MCR inhibition assay showed a concentration dependent inhibition of MCR activity (half-maximal inhibitory concentration [IC₅₀] = 23.72 μM), suggesting a direct interaction between rottlerin and MCR, consistent with the docking results. These findings highlight the potential of rottlerin as a naturally occurring, MCR-targeted feed additive for sustainable methane mitigation in dairy cows.

The A-to-G mutation (FecB) in the BMPR1B gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the FecB gene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting the FecB mutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detecting FecB mutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.