Endotoxin Aggregate Zeta Potential and Direct TEM Observation with Zinc Complex Fluorescence

Clever research from Japan detects Picomolar Endotoxin in the presence of chemicals known to inhibit the Horseshoe Crab Blood LAL test.

Thanks to my friend OpenVAET who found a recent paper on a much improved, fast and effective test for bacterial Endotoxin with a detection limit of 11 Picomoles per Litre.

Takahashi Hayashita and coworkers¹ have developed a method using a Zinc complex Zn-dpa-C2OPy where the Zinc atom combines with an organic ligand made from 2,2'-dipicolylamine and 1-hydroxypyrene. The Zinc complex binds a number of Endotoxin (Lipopolysaccharide, LPS) strands together, affecting their Zeta Potential and enabling direct observation of the aggregates via Transmission Electron Microscopy. They experimented with a number of Zinc compounds.

The supplementary file for their paper shows the effect of Zinc ions altering the Zeta Potential of the Endotoxin (Fig. 7) and at (Fig. 8c) the 10 micromolar Endotoxin aggregates become easily visible. Note the scale bar is 500 nanometres.

They successfully performed this analysis in the presence of 5 millimolar HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid), which is known to interfere with the LAL test.²

Weak response to Lipid A

The main paper shows in Figure 3 that this new technique has low sensitivity for the most toxic component of the jabs, Lipid A.

The authors state:

A selectivity assay was conducted by adding biologically important compounds that could coexist with LPS in water or are similar in structure to LPS (i.e., phospholipids, negatively charged molecules, or polysaccharides), instead of LPS (Figure 3a). Phosphate (Pi), pyrophosphate (PPi), triphosphate (Tri), adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) are representative phosphate derivatives existing ubiquitously in biological samples.29−31 Zndpa-C2OPy showed the highest selectivity for LPS among all the examined compounds. The enhancement of F477/F384 by lipid A was expected because lipid A is a toxic phosphate lipid component of LPS; this also meant that the chemosensor was able to recognize the toxic component of LPS. In addition, lipid A alone was not sufficient to obtain a strong response like LPS alone, suggesting that the unique structure of LPS, which is composed of sugar chains and lipid A, was selectively recognized, judging from the results of galactose, curdlan, and lipid A.

The new method is currently not as sensitive as Mass Spectrometry³ or electronic sensors⁴ under development, however it might benefit from advanced photon detectors in the future.

1. Hiroshi Kimoto, Moeka Takahashi, Masakage Masuko, Kai Sato, Yuya Hirahara, Masamitsu Iiyama, Yota Suzuki, Takeshi Hashimoto and Takashi Hayashita. 2023. High-Throughput Analysis of Bacterial Toxic Lipopolysaccharide in Water by Dual-Wavelength Monitoring Using a Ratiometric Fluorescent Chemosensor. https://pubs.acs.org/doi/10.1021/acs.analchem.3c01870

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