- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe
摘要： The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5′ hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL?1), a low detection limit (0.01 U·mL?1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.
关键词： DNA-AgNCs,Cell extracts,Clinical diagnostics,Lambda exonuclease,Proximity effect,T4 PNK,Inhibitor,ATP,Na2HPO4,Phosphorylation
更新于2025-09-23 15:23:52
-
In-line whole blood fractionation for Raman analysis of blood plasma
摘要： Blood plasma evaluation has high significance in clinical diagnostics. Current schemes involve the preparation of blood plasma by centrifugation of whole blood followed by electrochemical or spectroscopic analysis. However, centrifugation is often too time-consuming for application in clinical emergency and point-of-care settings. We propose to combine microfluidic, instantaneous plasma fractionation with localized spectroscopic methods for in-line analysis. As an example, we present confocal Raman spectroscopy in fractionated plasma domains at two different Raman excitation wavelengths. Resonance Raman spectroscopy with laser excitation at 408 nm allows the specific detection of free hemoglobin in blood plasma at concentrations above 22 mg dl?1 (level of detection). Consequently, we are able to accurately resolve the range of clinical relevance regarding hemolysis. At near-infrared excitation (785 nm) we furthermore demonstrate the acquisition of characteristic Raman spectra of fractionated blood plasma in the microfluidic setting. These spectra can serve as starting point for a multi-parameter regression analysis to quantify a set of blood plasma parameters from a single Raman spectrum. The combined microfluidics and Raman spectroscopy method is non-destructive and has a whole blood consumption of less than 100 μl per hour. It thus allows for continuous in-line blood plasma monitoring.
关键词： microfluidics,hemoglobin,Raman spectroscopy,blood plasma,clinical diagnostics
更新于2025-09-04 15:30:14