Femtomolar Detection of Lipopolysaccharide in Injectables and Serum Samples Using Aptamer-Coupled Reduced Graphene Oxide in a Continuous Injection-Electrostacking Biochip

A method for microfluidic sample preconcentration to detect femtomolar level of lipopolysaccharide (LPS) is introduced, enabled by 6-carboxyfluorescein (6-FAM) labeled aptamer-LPS binding along with reduced graphene oxide (rGO). The free FAM-aptamers can be adsorbed onto the surface of rGO, resulting in fluorescence quenching of background signals. Conversely, the aptamer LPS complex cannot be adsorbed by rGO, so the fluorescence is maintained and detected. When an electric field is applied across the microchannel with Nafion membrane in the chip, only the fluorescence of aptamer-LPS complex can be detected and stacked by continuous injection-electrostacking (CI-ES). The method shows a high selectivity (in the presence of pyrophosphate, FAD(+), NAD(+), AMP, ADP, ATP, phosphatidylcholine, LTA, and beta-D-glucans which respond positively to LAL) to LPS and an extreme sensitivity with the limit of detection (LOD) at 7.9 fM (7.9 X 10(-4) EU/mL) and 8.3 fM (8.3 X 10(-4) EU/mL) for water sample and serum sample, respectively. As a practical application, this method can detect LPS in injections and serum samples of human and sepsis model mouse and quickly distinguish Gram-negative bacteria Escherichia coli (E. coli) from Gram-positive bacteria Staphylococcus aureus (S. aureus) and fungus Candida albicans (C. albicans). More importantly, by changing the aptamers based on different targets, we can detect different analytes. Therefore, aptamer-coupled rGO in a CI-ES biochip is a universal, sensitive, and specific method. For TOC only.