Bifunctional Au@Pt/Au nanoparticles as electrochemiluminescence signaling probes for SARS-CoV-2 detection.

A novel immunosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) for the sensitive determination of N protein of the SARS-CoV-2 coronavirus is described. For this purpose, bifunctional core@shell nanoparticles composed of a Pt-coated Au core and finally decorated with small Au inlays (Au@Pt/Au NPs) have been synthesized to act as ECL acceptor, using [Ru (bpy)3]2+ as ECL donor. These nanoparticles are efficient signaling probes in the immunosensor developed. The proposed ECL-RET immunosensor has a wide linear response to the concentration of N protein of the SARS-CoV-2 coronavirus with a detection limit of 1.27 pg/mL. Moreover, it has a high stability and shows no response to other proteins related to different virus. The immunosensor has achieved the quantification of N protein of the SARS-CoV-2 coronavirus in saliva samples. Results are consistent with those provided by a commercial colorimetric ELISA kit. Therefore, the developed immunosensor provides a feasible and reliable tool for early and effective detection of the virus to protect the population.

Sensitive SARS-CoV-2 detection in wastewaters using a carbon nanodot-amplified electrochemiluminescence immunosensor.

Given the great utility that having fast, efficient and cost-effective methods for the detection of SARS-CoV-2 in wastewater can have in controlling the pandemic caused by this virus, the development of new dependable and specific SARS-CoV-2 coronavirus sensing devices to be applied to wastewater is essential to promote public health interventions. Therefore, herein we propose a new method to detect SARS-CoV-2 in wastewater based on a carbon nanodots-amplified electrochemiluminescence immunosensor for the determination of the SARS-CoV-2 Spike S1 protein. For the construction of the immunosensor, N-rich carbon nanodots have been synthetized with a double function: to contribute as amplifiers of the electrochemiluminescent signal in presence of [Ru(bpy)3]2+ and as antibody supports by providing functional groups capable of covalently interacting with the SARS-CoV-2 Spike S1 antibody. The proposed ECL immunosensor has demonstrated a high specificity in presence of other virus-related proteins and responded linearly to SARS-CoV-2 Spike S1 concentration over a wide range with a limit of detection of 1.2 pg/mL. The immunosensor has an excellent stability and achieved the detection of SARS-CoV-2 Spike S1 in river and urban wastewater, which supplies a feasible and reliable sensing platform for early virus detection and therefore to protect the population. The detection of SARS-CoV-2 Spike S1 in urban wastewater can be used as a tool to measure the circulation of the virus in the population and to detect a possible resurgence of COVID-19.

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

High glucose enhances inducible nitric oxide synthase expression. Role of protein kinase C-betaII.

The aim was to determine whether high glucose levels interfere with nitric oxide (NO) production and inducible NO synthase (iNOS) protein expression in interleukin-1beta-stimulated vascular smooth muscle cells from normotensive Wistar Kyoto and spontaneously hypertensive rats. Cells were incubated with either normal (5.5 mM) or high (22 mM) d-glucose for 72 h and with interleukin-1beta (10 ng/ml) for the last 24 h. High glucose increased nitrite levels, iNOS expression and protein kinase C activity in cells from normotensive rats and had no effect in cells from hypertensive rats. High glucose effects on nitrite production and iNOS expression was abolished by the selective inhibitor for the protein kinase C-betaII, 5,21:12,17-dimetheno-18H-dibenzo[i,o]pyrrolo[3,4-1] [1,8]diacyclohexadecine-18,20 (19H)-dione, 8-[(dimethylamino) methyl]-6,7,8,9,10,11-hexahydro-monomethanesulfonate (LY379196, 30 nM). Calphostin C (1 microM) and LY379196 (10 microM) reduced nitrite levels and iNOS expression only in cells from normotensive rats treated with both media. These results suggest that high glucose increases inducible nitric oxide synthase induction and subsequent NO production by activating the protein kinase C-betaII; this mechanism seems to be altered in hypertension.