A sensitive "off-on" carbon dots-Ag nanoparticles fluorescent probe for cysteamine detection via the inner filter effect.

In this study, we describe the construction of an "off-on" fluorescent probe based on carbon dots (CDs) and silver nanoparticles (AgNPs) mixture for sensitive and selective detection of cysteamine. By mixing AgNPs with CDs solution, the fluorescence of CDs was significantly decreased due to the inner filter effect (IFE). Upon addition of cysteamine to the mixed aqueous of CDs and AgNPs, the silver-sulfur bond between cysteamine and AgNPs caused AgNPs to aggregate, and the quenched fluorescence of CDs could in turn be recovered. The probe was employed to quantitatively detect cysteamine, and the results showed that it could detect cysteamine in a concentration range of 2-16 µM with the detection limit of 0.35 µM (signal-to-noise ratio of 3). The detection of cysteamine spiked into bovine serum samples showed high recovery rates ranging from 95.5 to 111.7%. More importantly, the developed probe had low cytotoxicity and was successfully used for in vivo imaging of HepG2 cells.

Lysosome-targeted ratiometric fluorescent sensor for monitoring pH in living cells based on one-pot-synthesized carbon dots.

A hydrothermal method has been employed to synthesize a green and one-pot carbon dots-based sensor for ratiometric monitoring and imaging lysosomal pH in living cells. The carbon dots were directly functionalized by abundant amino groups during synthesis and exhibited dual emission bands at 439 and 550 nm under single-wavelength excitation of 380 nm without any additional modification. In addition to its small size, the established sensor had good biocompatibility. Owing to its abundant amino groups and good hydrophilicity, the sensor is able to target lysosome with high Pearson's colocalization coefficients (0.935 and 0.924) and responds to change of lysosomal pH in living cells. It also had excellent pH sensitivity and reversibility, and anti-interference capability, thus enabling sensing pH change in intracellular environment in real time, as demonstrated by successful monitoring of lysosomal pH changes during lysosomal alkalization, dexamethasone-induced stimulation, and stress in Michigan Cancer Foundation-7 cells (blue channel, excitation = 405 nm and emission = 419-459 nm bandpass; and yellow channel, excitation = 405 nm and emission = 530-570 nm bandpass). Graphical abstract.

A novel fluorescent probe for the localization of nucleoli developed via a chain reaction of endogenous cysteine in cells.

Nucleolus imaging is important for the understanding of gene expression, proliferation, and growth of cells. Traditional nucleoli localization mainly relies on the use of RNA fluorescent probes which are required in large amounts. These probes also have low selectivity, thus causing the generated images to have high background noise and the localization of nucleoli to become vague. In the present paper, a novel probe for nucleoli localization, BEB-A, which can specifically bind to RNA via the chain reaction of endogenous cysteine (Cys), was designed and developed. In addition to its mitochondria-targeting ability, the BEB-A probe could be used in the imaging of Cys in the cytoplasm, and its product, BEB-OH, could quickly penetrate into the cell nucleus to combine with nucleolar RNA to generate strong red fluorescence signals. The luminescence property and RNA-binding capability of the probe were also investigated via theoretical calculations and molecular docking simulations. This work presents a tool that can be applied to analyze the variation of Cys in mitochondria and RNA in cells.

One-step fabrication of boronic-acid-functionalized carbon dots for the detection of sialic acid.

Typically, sialic acids (SA) with a nine-carbon backbone are found at the glycan chain termini on the cell membranes, which play crucial roles in various physiological and pathological processes. The expression level of SA in the blood serum has been reported to correlate with various disease states among cancer. In this study, a novel approach for preparing fluorescent boronic-acid-modified carbon dots (C-dots) for the detection of SA was developed. The functionalized C-dots were synthesized by a facile, one-step hydrothermal method using 3-pyridineboronic acid as the sole carbon source. The added SA selectively recognized the C-dots, leading to the fluorescence quenching of the C-dots in a linear range of 80-4000 µM with a detection limit of 54 µM. The as-developed boronic-acid nanoprobe was successfully applied for the detection of SA in human serum samples with satisfactory results. In addition, this method afforded results within 4 min. Compared to other methods, this new proposed approach was simpler and exhibited excellent sensitivity and selectivity, demonstrating immense potential as an alternative for SA detection.

Selective and sensitive fluorescence detection method for pig IgG based on competitive immunosensing strategy and magnetic bioseparation.

A novel fluorescence detection method based on competitive immunoassay and magnetic bioseparation technique was developed and applied to the determination of pig immunoglobulin G (IgG) in serum samples. Core-shell structured Fe3O4@SiO2 nanoparticles were synthesized by chemical coprecipitation, followed by functionalization with amino groups and immobilization of pig IgG antibodies. The synthesized Fe3O4@SiO2-antibody nanoparticles were employed as the probe for the competitive immune recognition of the target antigens in samples and the antigens labeled with fluorescein isothiocyanate (FITC). After the magnetic separation of probes binding with these two types of antigens, fluorescence of the free FITC-labeled antigens was measured for the quantification of the target antigens, since the ratio of the FITC-labeled antigens in supernatant before and after the competitive immune recognition depends on the amount of the target antigens in sample, due to the competitive nature of the binding of the antibody for these two types of antigens. Under the optimal conditions, a linear relationship was obtained between the change of fluorescence intensity and the concentration of pig IgG in a range from 0.75 to 23.50 µg L-1, with a detection limit (LOD) of 0.031 µg L-1. With the facile-prepared probes, this fluorescence competitive method can provide a rapid, specific and highly sensitive immunoassay protocol for the determination of target proteins in complex matrix samples.

Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum.

The dominant mechanism to repair double-stranded DNA breaks in filamentous fungi is the non-homologous end joining (NHEJ) pathway, and not the homologous recombination (HR) pathway that operates in the mutation of genes by replacement of target DNA for selection cassettes. The key to improve HR frequency is the inactivation of the NHEJ pathway by eliminating components of its Ku70/80 heterodimeric complex. We have obtained ku70 mutants of Penicillium digitatum, the main citrus postharvest pathogen. The increased efficiency of HR in Δku70 strains was demonstrated by the generation of mutants in two different chitin synthase genes (PdchsII and PdchsV). P. digitatum Δku70 strains showed no differences from the parental strain in vegetative growth, asexual development or virulence to citrus fruit, when experiments were conducted at the optimal temperature of 24°C. However, growth of Δku70 strains at temperatures higher than 24°C demonstrated a detrimental effect in axenic growth and conidia production. These observations are in agreement with previous studies describing differences between ku70 mutants and their parental strains in some fungal species, and must be taken into account for future applications of the Δku approach to increase HR efficiency in fungi.