A rapid aptamer-based fluorescence assay for the detection of lipopolysaccharides using SYBR Green I.

Lipopolysaccharides (endotoxins), found on Gram-negative bacteria, can trigger a severe immune response in humans leading to septic shock and in extreme cases, even death. Therefore, the detection and neutralization of lipopolysaccharides (LPS) is of utmost importance in the pharmaceutical and medical industries. The United States Food and Drug Administration (US FDA) recommended detection method for LPS, the Limulus amebocyte lysate (LAL) assay, is expensive, time consuming, complex, and is prone to interference from proteases. As an alternative, this paper proposes a rapid, label-free fluorescence-based assay using LPS-specific aptamers and the SYBR Green DNA stain. The proposed method has a detection limit of 0.1 ng/ml, which is sufficient to detect the permissible levels of LPS in many pharmaceutical drugs and medical products. The fluorescence signal was found to be a linear function of the concentration of LPS in the range from 0.1 ng/ml to 105 ng/ml.

dsDNA-templated fluorescent copper nanoparticles for the detection of lipopolysaccharides.

The introduction of lipopolysaccharides (LPS) or endotoxins that originate from Gram-negative bacteria into the human blood stream induces a severe immune response that can lead to septic shock, and even death. Hence, the accurate detection of LPS is of great importance in the medical and pharmaceutical sectors. This paper proposes a novel label-free fluorescence assay for the detection of LPS utilizing aptamers and the interference synthesis of dsDNA-templated copper nanoparticles. The assay can be performed at room temperature and does not require expensive reagents. The proposed assay has a limit of detection of 0.95 ng ml-1 of LPS, and the fluorescence emission from the copper nanoparticles was found to vary linearly with the concentration of LPS over a wide range (1 to 105 ng ml-1) with R2 = 0.9877.

Enhancement of the fluorescence properties of double stranded DNA templated copper nanoparticles.

The weak fluorescence emission from dsDNA templated copper nanoparticles necessitates the use of high-end detectors like photomultiplier tubes for their detection. This sets limitations on their applicability to in-situ analyte detection and point-of-care applications which utilize comparatively low cost and less sensitive detectors. In this article, a technique to improve the fluorescence properties of copper nanoparticles templated on dsDNA is reported. The fluorescence enhancement is achieved by introducing a modification in the conventional synthesis technique by using a combination of sodium ascorbate and Taq buffer. When compared to the existing methods, the proposed method achieves 11 times higher fluorescence signal intensity from the dsDNA templated copper nanoparticles and 4 times faster attainment of maximum fluorescence signal. The effect of the ionic strength of the individual constituent components of Taq buffer on the fluorescence emission from the copper nanoparticles is also studied here. The utility of this enhancement strategy for analyte measurement is demonstrated with the example of melamine detection from milk samples. A linear relationship was observed between the fluorescence intensity from the copper nanoparticles and the concentration of melamine in the range from 0.5 ppm to 100 ppm (R2 = 0.9919), with a limit of detection of 0.1 ppm. The reported fluorescence enhancement technique also results in 2.95 times improved sensitivity of detection when compared to the conventional technique.