Detection of stealthy small amphiphilic biomarkers

Abstract

Pathogen-specific biomarkers are secreted in the host during infection. Many important biomarkers are not proteins but rather small molecules that cannot be directly detected by conventional methods. However, these small molecule biomarkers, such as phenolic glycolipid-I (PGL-I) of Mycobacterium leprae and Mycobactin T (MbT) of Mycobacterium tuberculosis, are critical to the pathophysiology of infection, and may be important in the development of diagnostics, vaccines, and novel therapeutic strategies. Methods for the direct detection of these biomarkers may be of significance both for the diagnosis of infectious disease, and also for the laboratory study of such molecules. Herein, we present, for the first time, a transduction approach for the direct and rapid (30min) detection of small amphiphilic biomarkers in complex samples (e.g. serum) using a single affinity reagent. To our knowledge, this is the first demonstration of an assay for the direct detection of PGL-I, and the first single-reporter assay for the detection of MbT. The assay format exploits the amphiphilic chemistry of the small molecule biomarkers, and is universally applicable to all amphiphiles. The assay is only the first step towards developing a robust system for the detection of amphiphilic biomarkers that are critical to infectious disease pathophysiology.

Keywords: Amphiphiles; Biomarkers; Lipid bilayer; Membrane insertion assay; Mycobactin; Phenolic glycolipid.

Fig. 1.

Schematic illustration of the structure of (A) lipoarabinomannan, (C) MbT, (D) carboxymycobactin T from Mycobacterium tuberculosis, and (B) PGL-I from Mycobacterium leprae (not drawn to scale).

Fig. 2.

Schematic representation of the membrane-based assay for amphiphilic PAMPs and virulence factors on the waveguide-based optical biosensor platform.

Fig. 3.

Detection of PGL-I in bovine or human serum using the insertion assay on the optical biosensor. Left: 6 μM of PGL-I detection in bovine serum. Black triangles indicate positive signal for detection of PGL-I spiked in bovine serum. Dark gray line indicates measure of non-specific binding of the reporter antibody with control serum and light gray line indicates the waveguide-associated background. Right: Specific signals with different concentrations of PGL-I (13 μm [black lines] and 20 μm [gray line]) spiked in human serum with the insertion assay. The non- specific binding associated with the reporter antibody was measured and subtracted from the specific data before plotting.

Fig. 4.

Concentration dependence of the insertion of MbT. Left: Prototype measurement of 50 μM of MbT in human serum using the membrane insertion assay. Light gray line indicates the waveguide-associated background. Dark gray circles indicate non-specific background and black triangles show specific detection of MbT. Right: Standard curve showing concentration dependent increase in signal for the detection of MbT in PBS.