Membrane Insertion for Direct Detection of Lipoteichoic Acid

Abstract

The incorrect diagnosis of bacterial pathogens results in the misuse of antibiotics, a major contributor to the evolution of antibiotic resistance. The ability to rapidly identify a pathogen could facilitate more effective treatment strategies, reducing the unnecessary use of antibiotics. Current diagnostics of bacterial infections often require cell growth, and are therefore less capable of informing timely treatment options. Here, we describe the development of a rapid assay (<45 min) to identify Gram-positive bacteria directly in human serum without any growth or isolation required. We have developed a fluorescence-based immunoassay specifically targeting the Gram-positive bacterial biomarker lipoteichoic acid (LTA). LTA is an amphiphilic molecule with both hydrophobic and hydrophilic portions. Our approach, termed membrane insertion, exploits the amphiphilic biochemistry of the molecule by allowing it to passively insert itself into a lipid bilayer, thereby trapping it for subsequent probing with a fluorescently labeled α-LTA antibody. Using this assay, we have successfully detected less than 10 µg/ml of LTA directly in spiked human serum. We intend to combine this rapid assay with others currently in development into a diagnostic tool for point-of-care detection of bacterial pathogens.

A centrifugal microfluidic cross-flow filtration platform to separate serum from whole blood for the detection of amphiphilic biomarkers

“…The design was optimized to purify serum to commercial standards, as validated by performing cell counts on all samples. Materials used for the fabrication of the centrifugal platform were compatible with our sample processing criteria, as confirmed by testing samples from the device on our waveguide-based optical biosensor for the retention of amphiphilic biomarkers of interest 6 , 8 , 10 , 35 , 36 . This development greatly simplifies the sample processing requirements for our biosensing assays and facilitates the transition of such technologies to the point of need.…”

“…We validated the ability of our microfluidic device to retain serum biomarkers of interest by comparing the efficacy of the process against the benchtop method developed by our team. The benchtop sample processing method consists of two major steps: (1) the separation of serum from blood on a microcentrifuge; and (2) the isolation of amphiphilic biomarkers from serum using a chloroform/methanol extraction 5 , 6 , 8 , 10 , 36 , 41 , 42 . In this work, we sought to automate the first step of sample processing on a microfluidic chip, and ensure that biomarkers of interest were retained in the sample and not adsorbed to the plastic device materials.…”

“…Initial biomarker retention experiments were performed on the Los Alamos National Laboratory’s waveguide-based optical biosensor, which is used for the detection of biomarkers from Gram-negative, -positive, and -indeterminate bacteria 6 , 8 , 10 , 36 . The technology is described in detail elsewhere 6 , 8 , 10 , 35 , 36 , 41 .…”

“…The bacterial biomarkers targeted by our assays are the same as those targeted by our human innate immune response, and are most often lipidated sugars (lipoglycans or glycolipids, such as lipopolysaccharide, lipoteichoic acid, and lipoarabinomannan). Previous work from our laboratory and others has shown that the amphiphilic biochemistry of these biomarkers causes them to be sequestered by host lipoprotein carriers, including high- and low-density lipoproteins (HDL and LDL) 4 , 6 , 7 , 9 , 10 . Because of this sequestration, it is necessary to liberate the amphiphilic biomarkers from their lipoprotein carriers in order to enable sensitive measurement.…”

“…Because of this sequestration, it is necessary to liberate the amphiphilic biomarkers from their lipoprotein carriers in order to enable sensitive measurement. Detection can then occur via enzyme-linked immunosorbent assays (ELISA’s), waveguide-based biosensors 5 , 6 , 8 , 10 , or other methods 9 .…”