One of the great challenges in metal-enhanced fluorescence (MEF) technology is the achievement of distance modulation with nanometer accuracy between the fluorophore and metal surface to obtain maximum enhancement. We propose an MEF-based core-shell Ag@SiO2 nanoflare for distance control via the thickness of silica shell with cooperation of DNA hybridization. The nanoflare contains a 50 nm spherical silver nanoparticle (Ag NP) core, a 8 nm silica shell, and cyanine (Cy5)-labeled aptamer hybridized with a complementary DNA (cDNA) immobilized onto the shell surface. The formation of the Cy5-labeled aptamer/cDNA duplex on the Ag@SiO2 NP surface results in the confinement of Cy5 to the shell surface and an increase in the fluorescence of Cy5 with a 32-fold enhancement factor in bulk solution (signal-on). In the presence of affinity-binding targets, the Cy5-labeled aptamers confined onto the Ag@SiO2 NP surface dissociate from their cDNA into the solution because of structure switching. The target-induced release of aptamer leads to a reduction in the enhanced fluorescence signal of the labeled Cy5 moiety (signal-off). Thus, the nanoflare can be used as a sensor for target recognition. Using adenosine-5'-triphosphate (ATP) aptamer, detection of ATP has a linear response from 0 to 0.5 mM and a detection limit of 8 μM. With various types of DNA probes immobilized onto the core-shell Ag@SiO2 NPs, the MEF-based nanoflare has provided an effective platform for the detection and quantification of a broad range of analytes, such as mRNA regulation and detection, cell sorting, and gene profiling.
We have developed a fluorescence turn-on assay using DNA-templated silver nanoclusters (Ag NCs) (i.e., 12 polycytosine-templated silver nanoclusters, dC12-Ag NCs), which is amenable to rapid, ultrasensitive assay of acetylcholinesterase (AChE). The detection mechanism is based on the concept, that is, AChE hydrolyzes the acetylthiocholine (ATCh) chloride to produce thiocholine (TCh). Subsequently, TCh sensitively and rapidly reacts with dC12-Ag NCs via Ag-S bond forming and enhances the fluorescence of dC12-Ag NCs. Using dC12-Ag NCs, detection of TCh has a linear concentration range of 2.0 nM to 16.0 nM and a detection limit of 0.3 nM. Due to the sensitive and rapid fluorescence turn-on response of dC12-Ag NCs to TCh, AChE with activity as low as 0.5 × 10(-4) U/mL (signal/noise = 3) can be analyzed with a dynamic range of 0.1 to 1.25 × 10(-3) U/mL. The promising application of the proposed method in AChE inhibitor screening was demonstrated. AChE concentrations were determined in human blood red cell (RBC) membranes from clinical specimens using dC12-Ag NCs, and the quantitative results were validated with Ellman's method. Aside from the ease of manufacture, reduction of matrix effect, and low background noise, the continuous detection format and detection sensitivity can open up to wider applications to AChE activity assay in neurobiology, toxicology, and pharmacology, among other fields.
The purpose of the present study was to investigate whether catalpol exhibited neuroprotective effects in chronic unpredictable mild stress (CUMS) mice through oxidative stress-mediated nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin-domain-containing 3 (NLRP3) inflammasome and neuroinflammation. Deficits in behavioral tests, including open field test (OFT), forced swim test (FST), and elevated plus-maze test (EPM), were ameliorated following catalpol administration. To study the potential mechanism, western blots, quantitative real-time PCR (qRT-PCR) analysis and immunofluorescence imaging were performed on the hippocampus samples. We found that the defects of behavioral tests induced by CUMS could be reversed by the absence of NLRP3 and NLRP3 inflammasome might be involved in the antidepressant effects of catalpol on CUMS mice. Similar to the NLRP3 inflammasome, the expression of interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and inducible nitride oxide synthase (iNOS) were increased after CUMS. The current study demonstrated that catalpol possessed anti-inflammatory effect on CUMS mice and inhibited microglial polarization to the M1 phenotype. In addition, the activity of mitochondrial oxidative stress might be involved in the NLRP3 activation, which was proved by the downregulation of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and cleaved IL-1β, after the administration of mitochondrion-targeted antioxidant peptide SS31. Taken together, we provided evidence that catalpol exhibited antidepressive effects on CUMS mice possibly via the oxidative stress-mediated regulation of NLRP3 and neuroinflammation.
A highly sensitive and rapid biosensor test based on disposable screen-printed thick-film electrodes was developed, which is suitable for monitoring organophosphate and carbamate residues in foods of animal origin with increased fat contents such as milk. The wild-type enzyme was combined with three engineered variants of Nippostrongylus brasiliensis acetylcholinesterase (NbAChE), to obtain enhanced sensitivity. The sample pretreatment could be reduced to a minimum. There was no extraction or fat removal necessary. With the biosensor test paraoxon concentrations down to 1 microg/L could be detected in milk. The detection limit for carbaryl was 20 microg/L. Recovery rates for paraoxon and carbaryl in milk samples lay between 89 and 107%. Ten milk samples from local markets were tested both with the biosensor test and with standard chromatographic multiresidue methods. Two milk samples caused AChE inhibition rates of >50%. Accordingly, 4 microg/L tebufenpyrad, 4 microg/L tetraconazole, and 2 microg/L bifenthrin were detected in one of these milk samples. The other milk sample contained 2 microg/L tebufenpyrad.
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