A facile dual-mode immunosensor based on speckle Ag-doped nanohybrids for ultrasensitive detection of Ochratoxin A.

Ochratoxin A (OTA) is a potent carcinogen, and is among the most dangerous mycotoxins in agricultural products. In this study, an ultrasensitive dual-mode immunosensor was developed for naked-eye and fluorescence detection of OTA based on Ag-doped core-shell nanohybrids (Ag@CSNH). Complete antigen-labeled Ag@CSNH (CA-Ag@CSNH) were used as a competitive bind and dual-mode probe. The diffused doping structure of CA-Ag@CSNH provided improved stability, color and fluorescence quencher performance. Antibodies modified magnetic beads were used as a capture probe. The competitive binding between OTA and CA-Ag@CSNH produced both color change and fluorescence quenching. Ultraviolet and fluorescence intensitie correlated linearly with OTA concentration ranges of 0.03-3 ng/mL and 10-10000 pg/mL, and limits of detection of 0.0235 ng/mL and 0.9921 pg/mL, respectively. The practical applicability of proposed strategy was demonstrated by analysis of OTA in spiked corn, soybean and flour samples. This study offers a new insight on multi-mode platforms for various applications.

The development of a fluorescence/colorimetric biosensor based on the cleavage activity of CRISPR-Cas12a for the detection of non-nucleic acid targets.

Nano-biosensors are of great significance for the analysis and detection of important biological targets. Surprisingly, the CRISPR-Cas12a system not only provides us with excellent gene editing capabilities, it also plays an important role in biosensing due to its high base resolution and high levels of sensitivity. However, most CRISPR-Cas12a-based sensors are limited by their recognition and output modes, are therefore only utilized for the detection of nucleic acids using fluorescence as an output signal. In the present study, we further explored the potential application of CRISPR-Cas12a and developed a CRISPR-Cas12a-based fluorescence/colorimetric biosensor (UCNPs-Cas12a/hydrogel-MOF-Cas12a) that provides an efficient targeting system for small molecules and protein targets. These two sensors yield multiple types of signal outputs by converting the target molecule into a deoxyribonucleic acid (DNA) signal input system using aptamers, amplifying the DNA signal by catalyzed hairpin assembly (CHA), and then combining CRISPR-Cas12a with various nanomaterials. UCNPs-Cas12a/hydrogel-MOF-Cas12a exhibited prominent sensitivity and stability for the detection of estradiol (E2) and prostate-specific antigen (PSA), and was successfully applied for the detection of these targets in milk and serum samples. A major advantage of the hydrogel-MOF-Cas12a system is that the signal output can be observed directly. When combined with aptamers and nanomaterials, CRISPR-Cas12a can be used to target multiple targets, with a diverse array of signal outputs. Our findings create a foundation for the development of CRISPR-Cas12a-based technologies for application in the fields of food safety, environmental monitoring, and clinical diagnosis.

The orphan nuclear receptor Nur77 plays a vital role in BPA-induced PC12 cell apoptosis.

Bisphenol A (BPA) is a typical environmental endocrine disruptor that can migrate into organisms through skin contact, breathing, diet and various other approaches. The reproductive toxicity and neurotoxicity of BPA has been confirmed by several toxicological studies. However, the neurotoxicity of BPA is still controversial. In the present study, we used PC12 cells as a model to investigate the mechanism of BPA-induced neuronal apoptosis. BPA exposure reduced cell viability, altered cell morphology and aggravated intracellular Lactate dehydrogenase (LDH) release, intracellular Ca2+ concentration, Reactive oxygen species (ROS) levels, apoptosis and the reduction in the mitochondrial transmembrane potential (ΔΨm). Moreover, the results of the Western blot (WB) and Real-time quantitative polymerase chain reaction (RT-qPCR) assays indicated that the expression levels of Nur77 in the BPA group were down-regulated and accompanied by the downregulation of the NF-κb/Bcl-2 proteins and the upregulation of cleaved-caspase 3, which is a marker of apoptosis. However, these changes were significantly reversed with the upregulation of the Nur77 protein by introducing plasmids carrying the nur77 gene. These results indicated that BPA-induced apoptosis was closely related to Nur77-mediated inhibition of the NF-κb/Bcl-2 pathway.

A fluorescent amplification strategy for high-sensitive detection of 17 ß-estradiol based on EXPAR and HCR.

Environmental endocrine disruptors in the environment and food, especially 17 ß-estradiol (E2), are important factors affecting the growth and development of organisms. In this research, we constructed a fluorescence strategy for two-step amplification that combined two currently popular methods, exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR). E2 competed with the complementary DNA (cDNA) to bind the aptamer modified on the magnetic beads. The free complementary strand in the supernatant was used as a trigger sequence to activate EXPAR, producing a large amount of short single-stranded DNA (ssDNA). The amplified ssDNA can trigger the second HCR amplification, producing many long double-stranded DNA (dsDNA) analogues. According to the principle of fluorescence resonance energy transfer, the carboxyfluorescein (FAM) signals in H1 and H2 hairpins were quenched by black hole quencher (BHQ-1). After the addition of E2 and initiation of amplification, the initially quenched fluorescent signal would be restored. This strategy with a detection limit of 0.37 pg mL-1 (S/N = 3) showed a good linear relationship in the range of 0.4-800 pg mL-1. In addition, the recovery rates of the method for milk and water samples were 98.55%-116.95% and 92.32%-107.00%, respectively. This is the first report of the combined detection of EXPAR and HCR, providing a reference for rapid and highly sensitive detection using multiple isothermal amplification methods.

Rapid and sensitive detection of prostate-specific antigen via label-free frequency shift Raman of sensing graphene.

The sensitive and accurate detection of cancer biomarkers is critically important to early clinical diagnosis, disease monitoring, and successful cancer treatment. Here, we first demonstrate an aptamer-based frequency shift Raman approach via sensing of graphene. This biosensor allows the rapid, sensitive, and label-free detection of the acknowledged protein cancer biomarker, prostate-specific antigen (PSA). Monolayer graphene is employed as the Raman substrate, which is highly sensitive to its electronic structure and interface properties. The PSA aptamer can be adsorbed strongly on the surface of substrates through π-π stacking interactions. The vibrational frequency of the G peak of graphene shifted upon the specific binding between the PSA and its aptamer. The corresponding frequency shifts of the G peak are directly correlated with PSA concentrations. The limit of detection is as low as 0.01 ng/mL, with a wide linear range from 0.05 ng/mL to 25 ng/mL. The analytic samples can be detected directly without any extensive preparation and label process. The whole detection is completed in only 30 min. Furthermore, excellent recoveries are acquired to validate the feasibility of this assay in human serum samples. The proposed technology could provide a selective, versatile, and user-friendly strategy for the early detection of cancer biomarkers.

The extracellular domain of Staphylococcus aureus LtaS binds insulin and induces insulin resistance during infection.

Insulin resistance is a risk factor for obesity and diabetes and predisposes individuals to Staphylococcus aureus colonization; however, the contribution of S. aureus to insulin resistance remains unclear. Here, we show that S. aureus infection causes impaired glucose tolerance via secretion of an insulin-binding protein extracellular domain of LtaS, eLtaS, which blocks insulin-mediated glucose uptake. Notably, eLtaS transgenic mice (eLtaS trans ) exhibited a metabolic syndrome similar to that observed in patients, including increased food and water consumption, impaired glucose tolerance and decreased hepatic glycogen synthesis. Furthermore, transgenic mice showed significant metabolic differences compared to their wild-type counterparts, particularly for the early insulin resistance marker α-hydroxybutyrate. We subsequently developed a full human monoclonal antibody against eLtaS that blocked the interaction between eLtaS and insulin, which effectively restored glucose tolerance in eLtaS trans and S. aureus-challenged mice. Thus, our results reveal a mechanism for S. aureus-induced insulin resistance.