A rapid and sensitive aptamer-based biosensor for beta-lactoglobulin in milk.

Beta-lactoglobulin (ß-Lg), a prominent milk protein, is a major contributor to milk allergies. The quantitative assessment of ß-Lg is a valuable method for assessing the allergenic potential of dairy products. In this study, a specific aptamer, ß-Lg-01, with an affinity constant (KD) of 28.6 nM for ß-Lg was screened through seven rounds of magnetic bead SELEX (MB-SELEX). A novel bio-layer interferometry (BLI)-based aptasensor was developed, which had a limit of detection (LOD) of 0.3 ng mL-1, a linear range of 1.5 ng mL-1-15 µg mL-1, and a recovery rate of 102-116% among the milk samples. This aptasensor provides a potential tool for the detection and risk assessment of ß-Lg within 10 min.

Effect of Elamipretide on the Vitrification of Mouse Ovarian Tissue by Freezing.

The importance of ovarian cortical cryopreservation in fertility preservation is receiving increasing attention from reproductive specialists, and mitochondrial dysfunction is an important cause of reduced ovarian tissue cryopreservation. Elamipretide (SS-31) is a novel mitochondria-targeted antioxidant. However, whether it has a protective effect on mouse ovarian tissue cryopreservation remains to be studied. In this study, we examined follicular morphology and viability, mitochondrial function and oxidative stress levels, apoptosis, and culture in vitro after vitrification cryoresuscitation operation by treating ovarian tissues with SS-31 in cryoprotectant resuscitation solution. At the end of the experiment, the addition of 100 µmol/L SS-31 significantly improved follicle quality and oocyte maturation rate in vitro (p < 0.05) and significantly reduced apoptosis (p < 0.05) and oxidative stress levels (superoxide dismutase, catalase, malondialdehyde, p < 0.05). Meanwhile, mitochondrial respiratory chain complex enzyme activity, mtDNA copy number (p < 0.05), and adenosine triphosphate (p < 0.05) content were significantly increased in the 100 µmol/L SS-31-treated group. In addition, the mRNA expression levels of mitochondrial energy metabolism- and biosynthesis-related genes (STRT1, PGC-1a, PPAR-a, TFAM, p < 0.05) were markedly upregulated (p < 0.05) in the 100 µmol/L SS-31 group. In conclusion, SS-31 improved the cryopreservation of ovarian tissues, and 100 µmol/L SS-31 was found to be the most effective.

The effect of the mitochondria-targeted antioxidant Mito-tempo during sperm ultra-rapid freezing.

During the freeze-thaw process, human spermatozoa are susceptible to oxidative stress, which may cause cryodamage and reduce sperm quality. As a novel mitochondria-targeted antioxidant, Mito-tempo has been used for sperm cryopreservation. However, it is currently unknown what role it will play in the process of sperm ultra-rapid freezing. The purpose of this study was to investigate whether Mito-tempo can improve sperm quality during ultra-rapid freezing. In this study, samples with the addition of Mito-tempo (0, 5, 10, 20, and 40 µM) to sperm freezing medium were selected to evaluate the changes in sperm quality, antioxidant capacity and ultrastructure after ultra-rapid freezing. After ultra-rapid freezing, the quality and antioxidant function of the spermatozoa were significantly reduced and the spermatozoa ultrastructure was destroyed. The addition of 10 µM Mito-tempo significantly increased post thaw sperm motility, viability, plasma membrane integrity and mitochondrial membrane potential (P < 0.05). Moreover, the DNA fragmentation index (DFI), ROS levels and MDA content were reduced, and the antioxidant enzyme (CAT and SOD) activities were enhanced in the 10 µM Mito-tempo group (P < 0.05). Moreover, Mito-tempo protected sperm ultrastructure from damage. In conclusion, Mito-tempo improved the quality and antioxidant function of sperm after ultra-rapid freezing while reducing freezing-induced ultrastructural damage.

A sensitive label-free biosensor based on Ag2S-sensitived Bi2WO6/BiOBr heterojunction for photoelectrochemical immunoassay of prostate specific antigen.

Prostate cancer is one of the most common cancers in the world, and its early diagnosis can effectively reduce mortality. A new label-free photoelectrochemical (PEC) immunosensor on the basis of Bi2WO6/BiOBr nanocomposite materials has been successfully prepared for the test of prostate-specific antigen (PSA) in human serum in this work. The Ag2S-sensitized Bi2WO6/BiOBr heterojunction was used as a photosensitive material, which effectively improved the photocurrent response. On Bi2WO6/BiOBr surface, dopamine immobilized PSA antibody by self-polymerizing to form polydopamine membrane. Antigen and antibody are specifically combined to achieve quantitative detection of PSA according to the current changes at different concentrations of antigen. Under the optimal experimental conditions, the PEC immunosensor has an ideal linear relationship between 1 pg/mL - 50 ng/mL, and the detection limit is 0.084 pg/mL. In addition, the prepared immunosensor has good stability, reproducibility and selectivity, providing a new method for the detection of PSA in actual sample analysis.

A novel photoelectrochemical immunosensor based on TiO2@Bi2WO6 hollow microspheres and Ag2S for sensitive detection of SARS-COV-2 nucleocapsid protein.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) is a cluster of ß coronaviruses. The 2019 coronavirus disease (COVID-19) caused by SARS-COV-2 is emerging as a global pandemic. Thus, early diagnosis of SARS-COV-2 is essential to prevent severe outbreaks of the disease. In this experiment, a novel label-free photoelectrochemical (PEC) immunosensor was obtained based on silver sulfide (Ag2S) sensitized titanium dioxide@bismuth tungstate (TiO2@Bi2WO6) nanocomposite for quantitative detection of SARS-COV-2 nucleocapsid protein. The constructed TiO2@Bi2WO6 hollow microspheres had large specific surface area and could produce high photocurrent intensity under visible light illumination. Ag2S was in-situ grown on the surface of thioglycolic acid (TGA) modified TiO2@Bi2WO6. In particular, TiO2@Bi2WO6 and Ag2S formed a good energy level match, which could effectively enhance the photocurrent conversion efficiency and strength the photocurrent response. Ascorbic acid (AA) acted as an effective electron donor to effectively eliminate photogenerated holes. Under optimal experimental conditions, the constructed immunosensor presented a supersensitive response to SARS-COV-2 nucleocapsid protein, with a desirable linear relationship ranged from 0.001 to 50 ng/mL for nucleocapsid protein and a lower detection limit of 0.38 pg/mL. The fabricated sensor exhibited a wide linear range, excellent selectivity, specificity and stability, which provided a valuable referential idea for the detection of SARS-COV-2.

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media.

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca(2+) and Mg(2+) for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl(2) and MgCl(2) concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca(2+)/Mg(2+) feeding to 117 h with Ca(2+)/Mg(2+) feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 x 10(6) and 9.12 x 10(6) cells ml(-1), respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.

A High-throughput End-point Assay for Viable Mammalian Cell Estimation.

A single wavelength colorimetric microplate-based assay was developed using non-cytotoxic dye resazurin for the estimation of viable cell concentrations of Chinese hamster ovary (CHO) and hybridoma cells. Experimental results showed variations in pH and temperature caused by cell cultivation and assay operations were well tolerated. Cell concentrations can be effectively determined in the range of 10(5)-10(7) cells ml(-1) using a microplate reader at the wavelength of 605 nm. This assay can be performed in a high-throughput manner such that a large number of cell culture samples can be screened within a relatively short time frame. When used together with a cell culture system of high-throughput format, it may have potential utilities in applications such as cell culture medium formulation and optimization.