Garzaforrest1803

The maximum sampling rate was 6 Hz for dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at full width at half-maximum (FWHM) of 105 ms and a relative standard deviation (%RSD) of 7.7/7.5% without internal standard correction. Enzyme assay buffer and crude dog plasma caused signal suppression of 51/73% at %RSD of 5.7/6.7% (n = 120). JTC-801 concentration An HTS endurance buffer was used for >25 000 injections with minor OPI pollution and constant signals (%RSD = 8.5%, FWHM of 177 ms ± 8.5%, n = 10 557). The optimized hardware and method setup resulted in high-throughput performance and enables further implementation in a fully automated platform for ESI-MS-based high-throughput screening.Here, using human metallothionein (MT2) as an example, we describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity utilizing iodoacetamide (IAM) and N-ethylmaleimide reagents. We concluded that an SN2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissociation. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addition reaction after separation by reverse-phase C18 at acidic pH. Finally, we evaluated the efficiency of the method by mapping metal-binding sites of Zn7-xMT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resolution. The methodology presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-containing proteins.Nanopore analysis based on a resistive-pulse technique is an attractive tool for single-molecule detection in different fields, but it suffers a great drawback in selectivity. A common solution to this challenge is to add extra sensing aptamers and labels to analytes by improving the sensitivity of their pulses for distinguishing. Compared to the labeling methods, we alternatively develop and demonstrate a novel data process for label-free nanopore analysis that enables the conversion of resistive current signals to more specific frequency domain phase angle features with the contribution from both sinusoidal voltage excitation and Fourier transform. In particular, we find that the transmural capacitance induced by nanoparticle translocations under a sinusoidal voltage plays an important role in this process, making phase angle features more pronounced. In practical applications, the method is successfully applied to directly distinguish the translocation events through a nanopipette by their unique phase angles for similarly sized SiO2, Ag, and Au nanoparticles and soft living organisms of HeLa and LoVo and even in a more complicated case of a SiO2, Ag, and Au nanoparticle mixture.Human microbiome contains billions of microorganisms that play important roles in the biological system and different diseases. Due to its complexity, conventional culture-independent technology may underestimate the value of low-abundance bacteria, which calls for a highly efficient method for its enrichment and comprehensive analysis. In this study, we developed a recycling free-flow isoelectric focusing (RFFIEF) method-based electrophoresis method to separate salivary microbiome. First, we used Escherichia coli (DH5α) as a model for RFFIEF method development, which was focused in a narrow pH range (0.38 pH unit). The recovery rate was 80.81% with 5.85% relative standard deviation (n = 5). The optimized method was then adopted to separate the human salivary microbiome into 32 fractions, followed by 16S rRNA gene sequencing and metaproteomics analysis. After RFFIEF fractionation, we identified 508 bacterial genera, which increased by 225% on average (n = 3) when compared to the results before fractionation. We further compared the compositional change of microbiome in the saliva of lung cancer group (n = 22) and control group (n = 21) through RFFIEF. Quantitative results demonstrated that six bacterial genera were upregulated dramatically in the lung cancer group, while two genera were downregulated. Through qPCR verification in an independent sample set (n = 48), we confirmed that genus Granulicatella was significantly upregulated in the lung cancer group, whereas Pseudomonas was remarkably downregulated (p less then 0.001). RFFIEF is an efficient and reproducible technology to fractionate the microbiome for its comprehensive analysis, which can be further applied to the in-depth study of the complex microbiomes and contribute to the discovery of disease-associated bacteria.Electrochemical impedance spectroscopy (EIS) is a versatile tool for electrochemistry, particularly when applied locally to reveal the properties and dynamics of heterogeneous interfaces. A new method to generate local electrochemical impedance spectra is outlined, by applying a harmonic bias between a quasi-reference counter electrode (QRCE) placed in a nanopipet tip of a scanning ion conductance microscope (SICM) and a conductive (working electrode) substrate (two-electrode setup). The AC frequency can be tuned so that the magnitude of the impedance is sensitive to the tip-to-substrate distance, whereas the phase angle is broadly defined by the local capacitive response of the electrical double layer (EDL) of the working electrode. This development enables the surface topography and the local capacitance to be sensed reliably, and separately, in a single measurement. Further, self-referencing the probe impedance near the surface to that in the bulk solution allows the local capacitive response of the working electrode substrate in the overall AC signal to be determined, establishing a quantitative footing for the methodology. The spatial resolution of AC-SICM is an order of magnitude larger than the tip size (100 nm radius), for the studies herein, due to frequency dispersion. Comprehensive finite element method (FEM) modeling is undertaken to optimize the experimental conditions and minimize the experimental artifacts originating from the frequency dispersion phenomenon, and provides an avenue to explore the means by which the spatial resolution could be further improved.