Last Month's Most Accessed Feature: More than the sum of its parts: Detecting endotoxins with SPE, CE and LIF

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  • Published: Dec 1, 2017
  • Categories: Sample Preparation
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More than the sum of its parts: Detecting endotoxins with SPE, CE and LIFStrong immune response

Endotoxins are unusual toxins. Unlike most other toxins, they don’t produce their toxic effect by acting directly on tissues or cells within the body, but by eliciting an immune response. For endotoxins, also known as lipopolysaccharides, are a major constituent of the outer membrane of gram-negative bacteria, and so the human immune system has evolved to attack them at the first chance it gets.

The problem is that gram-negative bacteria tend to shed endotoxins all the time, which means endotoxins get everywhere. Thus we can be exposed to endotoxins from sources such as drinking water without actually encountering any bacteria. What is more, because endotoxins can build up in environmental sources like drinking water, they sometimes elicit a dangerously strong immune response, which can occasionally prove fatal.

Although endotoxins are generally removed during water treatment, the ubiquity of bacteria mean endotoxins can still find their way into drinking water even after treatment, such as if bacteria colonize a clean water pipe. Hence the need for regular monitoring of drinking water to ensure endotoxin concentrations don’t exceed safe levels.

Same but different

The current standard method for monitoring endotoxins is known as the Limulus amoebocyte lysate (LAL) test and is based on the fact that cells known as Limulus amoebocytes derived from horseshoe crabs will form a yellow gel when exposed to endotoxins. Although sensitive, with a detection limit as low as 100pg/mL, this method doesn’t lend itself to accurate measurements of endotoxin concentrations and is not very selective, as other biomolecules can also trigger gel formation.

So there’s a pressing need for a new monitoring method. Unfortunately, there are a number of challenges involved in detecting endotoxins that make developing a new method rather tricky. For a start, different bacteria produce slightly different endotoxins and any method would need to be able to detect all of them. Endotoxins are made up of three main components: a lipid molecule known as lipid A, a core oligosaccharide and a long polysaccharide tail known as the O-antigen. Whereas the lipid and core oligosaccharide are similar in all endotoxins, the O-antigen varies in both its composition and length. In addition, as endotoxins can get everywhere, any monitoring method should be able to detect them in a wide variety of samples, not just water.

A team of chemists from the National University of Singapore led by Hua-tao Feng has now shown that a combination of solid-phase extraction (SPE), capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection can meet all these challenges. Indeed, this potential replacement for the LAL test actually turned out to be greater than the sum of its parts.

Polysaccharides and lipids

The SPE step neatly solves the challenge of monitoring endotoxin concentrations in different samples, because SPE can work with lots of them. Feng and his team chose to try a silica-based aminopropyl sorbent as the extraction material, as this is known to bind with hydrophilic polysaccharides. By contrast, on the face of it, CE and LIF detection would seem to be curious choices for a monitoring method, because endotoxins are uncharged and don’t naturally fluorescence.

To get around this slight problem, Feng and his team used a borate buffer, because borate anions will readily bind with the polysaccharides in endotoxins, giving them a negative charge. To make the endotoxins fluoresce, they used a fluorescent label that readily binds to lipids.

This turned out to by an inspired combination because it made the method highly selective. Although the silica-based aminopropyl sorbent will extract all the polysaccharides from a sample, no other polysaccharides will show up because they don’t contain a lipid for the fluorescent label to bind with. Only the endotoxins will be both extracted and labelled.

Feng and his team first showed that this novel method worked with a selection of 13 endotoxin standards, all of which had similar migration times, allowing their overall concentration to be determined. The chemists then showed that their method was equally effective at detecting endotoxins in extracts from the bacterium Escherichia coli, and samples of wastewater and spiked tap water, which is just what you want from a monitoring method.

Scientific Reports, 2017, 7, 10774: "Extraction, separation and characterization of endotoxins in water samples using solid phase extraction and capillary electrophoresis-laser induced fluorescence"

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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