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Updated: Oct 10

Opinion Review Article | Open Access | Published 11th October 2024

Image designed by Tim Sandle


Drop by drop: A new method for endotoxin detection?


 Tim Sandle, Ph.D., CBiol, FIScT | EJPPS | 293 (2024) | Click to download pdf   


Pharmaceutical Microbiologist & Contamination Control Consultant and Expert. Author, journalist, lecturer, editor, and scientist. 

A new, portable method for assessing endotoxin in water could emerge from new research that looks at how droplets interact with microstructures, taking advantage of the amphiphilic nature of endotoxin. Is this a suitable replacement for LAL? ¹ This method could be of importance for assessing water purification processes to determine endotoxin levels ². Endotoxin is an important marker for water system control, not only in terms of the relative toxic risk but also as an indicator for viable microbial contamination ³. 

Endotoxin is a complex of lipopolysaccharide (LPS) and other cellular material derived from the cell walls of Gram-negative bacteria. LPS comprises a relatively constant proportion of a Gram-negative bacterial cell (and Gram-negative bacteria account for the vast majority of prokaryotes found in water) ⁴. 


Researchers have developed a new method to evaluate liquids for a ubiquitous family of chemical compounds known as amphiphiles, which are used to detect diseases and to detect toxins in medicines and water supplies. 

In this article, the focus is on this thermodynamic model and its potential for improving endotoxin detection in water. 


Endotoxin: amphiphilic 

All endotoxin molecules share a general structural pattern, which usually consists of three major portions: lipid A, core oligosaccharide and O-antigen ⁵. The O-antigen portion is stretched outwards to the outer environment of the cell, whereas lipid A is anchored on the cell wall membrane, with the Lipid-A component being pyrogenic. 



Endotoxins are amphiphilic molecules with a variable chemical composition, as shown across bacterial strains. The net negative charge of the lipid A component and its distribution within the hydrophilic headgroup strongly influence the molecular conformation and, therefore, also the biologic activity ⁶. 

An amphiphile is a chemical compound possessing both hydrophilic (water-loving, polar) and lipophilic (fat-loving) properties ⁷. 


A property for detection 

Bacteria in water. Image designed by Tim Sandle.  


A property of amphiphiles enables scientists to detect measurable changes of their surface properties at concentrations close to their critical micelle concentrations. [Note : A micelle is a small spherical structure formed by amphiphilic molecules in water, with a hydrophobic core and a hydrophilic outer shell ⁸. This property is taken advantage of in drug delivery in order to protect hydrophobic drugs until they reach their target. This shape formation is also essential for the absorption of fat-soluble vitamins and complicated lipids within the human body]. 

Endotoxin monomers form micelles as aggregates with a size between 100 and 1000 nm ⁹,¹⁰. In relation to ‘critical micelle concentrations’ (CMC), the consumption of water containing endotoxin at concentrations as low as 2 ng/mL – which is four orders of magnitude below its CMC – can be sufficient to trigger ill-health effects, like sepsis (should the endotoxin enter the bloodstream or perhaps be inhaled), in humans ¹¹ . 


New research 

Scientists based at the Harvard John A. Paulson School of Engineering and Applied Sciences developed a rapid and low-cost wettability-based assay method, intended to evaluate liquids for amphiphiles. The initial research findings were published in PNAS ¹². 

Collection of bacteria. Image created by Tim Sandle.  


Rolling droplets method 

The standard method for testing for endotoxins is Limulus Amoebocyte Lysate (LAL) or a recombinant cascade /recombinant factor C method. To improve sensitivity, the new method uses rolling droplets on microstructured surfaces to detect amphiphiles at ultralow concentrations. 

The researchers demonstrated the method’s capability to detect levels of c amphiphilic endotoxins in water. These are bacterial-derived compounds that remain toxic, even at extremely low concentrations. 

The microstructured surface is composed of thousands of circular micropillars. These are evenly coated, self-assembled long-tailed molecules. The surface appears as a smooth, friction-free interface (with the exception of the edges of the micropillars). 

Wettability. Image by Tim Sandle. 


At these junctures there are gaps in the coating much like molecular potholes. When a droplet rolls down the surface, where there are no amphiphile molecules the droplet will contact a pothole and proceed to stop. This is because it is unable to overcome the friction of the disordered edge ¹³. 

However, a droplet with higher levels of amphiphiles will continue to roll. This occurs since amphiphile molecules co-assemble with long-tailed molecules and proceed to fill any gaps existing in the surface. This is described by the researchers as something much like a road paver smoothing the surface of a road. 

Water production. Image created by Tim Sandle 

 

With bacterial endotoxins, as amphiphile molecules come into contact onto the surface the endotoxic components connect with other amphiphile molecules within the droplet. Consequently, this interconnection creates larger and larger compounds which eventually slow and stop the droplet. 

The droplet stops on the surface indicates how many holes it has filled, and thus this measure can be used to assess the concentration of amphiphiles and hence an indicator of the level of endotoxin. 


A closer look at the science 

The mobility of water droplets on surfaces is influenced by the friction that exists around a droplet’s contact line. This friction is a property of the surface tension of the droplet and the local roughness of the surface. 

The researchers reasoned that when amphiphiles become locally concentrated around the contact line of a droplet, this presents the means to detect the amphiphiles based on the droplet’s pinning forces and sliding behaviour. 

The method relies upon concentrating amphiphiles into a droplet’s contact lines to allow for their ultrasensitive detection. At self-assembled monolayer (SAM) it is possible to selectively concentrate amphiphiles inside water droplets at the disordered, loosely packed SAM regions to generate pinning forces ¹⁴. 

Capillary action. Image by Tim Sandle. 


By introducing a spatial gradient of the contact line’s length allows the developed surfaces to sort aqueous droplets into discrete groups based on their amphiphile concentrations. 

A thermodynamic model can consequently be developed. The model describes the essential change that occurs in the concentration-dependent cooperative association with the loosely packed molecules in an SAM. 

 

Bacterium. Image by Tim Sandle.  


Specificity 

In terms of fine tuning the method for bacterial endotoxin, the researchers developed a model to predict how different amphiphile compounds at different concentrations would interact with the structured surface. The scientists discovered that by changing the size, shape and distance between pillars together with the molecular coating, the surface can be fine-tuned to detect specific types of amphiphiles at specific concentrations (such as endotoxin). 


Rapid microbiology? 

A key advantage of the method is its potential to be both rapid and portable. 


Summary 

While alternative endotoxin assays are being developed, such as those based on liquid crystals and fluorescent proteins, these require specialized equipment. The wettability approach discussed could lay the foundations for a rapid, portable and sensitive test method to assess water supplies ¹⁵. 

 

References


01.  Sandle, T. (2015) Bacterial Endotoxin Testing using the Limulus Amebocyte Lysate Assay. In Kőszegi, T. and Chesca, A. (Eds.) Laboratory Techniques with Applicability in Medical Practice, Lambert Academic Publishing, pp19-32

 

02. Rapala J, Lahti K, Räsänen LA, Esala AL, Niemelä SI, Sivonen K. Endotoxins associated with cyanobacteria and their removal during drinking water treatment. Water Res. 2002 May;36(10):2627-35

 

03. Sattar AA, Good CR, Saletes M, Brandão J, Jackson SK. Endotoxin as a Marker for Water Quality. Int J Environ Res Public Health. 2022 Dec 9;19(24):16528

 

04. Anwar M.A., Choi S. Gram-negative marine bacteria: Structural features of lipopolysaccharides and their relevance for economically important diseases. Mar. Drugs. 2014;12:2485–2514. doi: 10.3390/md12052485

 

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07. Schubert, D; Behl, C; Lesley, R; Brack, A; Dargusch, R; Sagara, Y; Kimura, H (1995). Amyloid peptides are toxic via a common oxidative mechanism. Proceedings of the National Academy of Sciences. 92 (6): 1989–93

 

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10. Anderson WB, Slawson RM, Mayfield CI. A review of drinking-water-associated endotoxin, including potential routes of human exposure. Can J Microbiol. 2002 Jul;48(7):567-87

 

11. Cohen J. The immunopathogenesis of sepsis. Nature. 2002 Dec 19-26;420(6917):885-91

 

12. Yuxing Yao, Robert K. A. Bennett, Yang Xu, et al. Wettability-based ultrasensitive detection of amphiphiles through directed concentration at disordered regions in self-assembled monolayers. Proceedings of the National Academy of Sciences, 2022; 119 (43) DOI: 10.1073/pnas.2211042119

 

13. D. K. Schwartz, Mechanisms and kinetics of self-assembled monolayer formation. Annu. Rev. Phys. Chem. 52, 107–137 (2001).

 

14. J. Aizenberg, A. J. Black, G. M. Whitesides, Controlling local disorder in self-assembled monolayers by patterning the topography of their metallic supports. Nature 394, 868–871 (1998)

 

15. Lin IH, Miller DS, Bertics PJ, Murphy CJ, de Pablo JJ, Abbott NL. Endotoxin-induced structural transformations in liquid crystalline droplets. Science. 2011 Jun 10;332(6035):1297-300

 

Author Information

Corresponding Author: Tim Sandle, Head of Microbiology

                                         Bio Products Laboratory ,  

UK Operations,                                           England                                                                                 













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