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

Abstract

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 10 5 ng/ml.

A new colorimetric aptasensor for paraquat detection based on the designed aptamer with multiple paraquat binding sites in combination with gold nanoparticles

“…However, upon the addition of paraquat, the aptamer predominantly binds to paraquat, resulting in the release of SYBR Green I from the aptamer binding, thus reducing the fluorescence signal. 25 The change of fluorescence intensity can indicate the binding capability of the aptamer to paraquat. Fig.…”

“…The optimized pH was dened as the pH value that yielded the maximum absorbance ratio between A 650 and A 525 . Additionally, the optimization of temperature was determined by incubating AuNPs (7 nM) and aptamer (25 nM) at different temperatures (20,25,30,35, and 40 °C) for 30 min. Then, paraquat (50 nM) was added and incubated for an additional 30 min.…”

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation

“…Although the method is simple and rapid, it relies mainly on enzymatic reaction, and the results are greatly affected by factors such as pH and temperature. In addition, the presence of many other components such as thromboplastin, thrombin, glucans, and polynucleotides can lead to a false-positive result. , Recently, a number of methods for detecting LPS have been developed, such as electrochemical sensor, DNA-modified gold nanoparticles (AuNPs), fluorescence assay, electrochemical aptasensor, ,, collaborative amplification of dual enzymes, ratio-metric fluorescence probe, − and microfluidic chip . However, these methods require expensive equipment/reagents and specialized personnel to operate.…”

Surfactant-Assisted Label-Free Fluorescent Aptamer Biosensors and Binding Assays

“…Developing assays to study aptamer binding is of great importance to validate newly selected aptamers, characterize aptamer binding, and design biosensors [ 1 , 2 , 3 , 4 , 5 ]. Among the numerous aptamer binding assays [ 6 , 7 , 8 , 9 ], those based on DNA staining dyes such as SYBR Green I, thioflavin T, and thiazole orange have been very popular [ 10 , 11 , 12 , 13 , 14 , 15 ] due to their label-free nature and cost-effectiveness. In a typical assay, a dye and an aptamer are mixed, and their fluorescence is monitored as a function of the target analyte concentration.…”