**5. Conclusions**

Several assays have been developed using aptamers as the detection ligand. Su et al. [160, 186, 187] used aptamers attached to gold nanoparticles to detect LPS using EIS, with an impressive detection limit of 0.1 pg/mL [10]. Aptamers have also been used in a magnetic aptasensor to detect LPS in medias containing BSA, sucrose, glucose, or RNA [163], and provide a detection

152 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

developed an EC sensor where aptamers that bind LPS were hybridized with capture probes, which were hybridized to complementary DNA sequences on gold nanoparticles with a very sensitive range (10 fg/mL up to 50 ng/mL). However, multiple aptamer libraries against O‐ag would be essential before this method could be implemented for serogroup discrimination. Modifications to improve sensitivity include use of SAMs to functionalize sensors with peptides [189], PmB [162], antibodies [10], and aptamers [159]. Despite optimal surface capture methods,

some of these assays suffer from poor detection limits or range of performance [10, 159].

and filter LPS from blood using piezoelectric quartz discs functionalized with PmB.

Other methods have taken advantage of the amphipathic nature of LPS. Harmon et al. [197] demonstrated that disrupting the hydrophobic association of LPS with liposomes increases the sensitivity of the LAL assay. Stromberg et al. [74, 198] were able to detect 4.20 µg/mL of amphiphilic LPS O157 in beef lysates on a waveguide biosensor using a technique called membrane insertion, which has previously been applied to other amphiphiles such as LAM and phenolic glycolipids [8, 9, 199]. Membrane insertion uses the natural association of amphiphiles with a lipid bilayer to facilitate detection and fluorescent detection of a labeled antibody is performed within an evanescent field [168, 199]. Many biosensors report exquisite sensitivity, even down to the picogram [164] and femtomolar [9, 168, 199] range, but very few are capable of physiological presentation of amphiphiles to facilitate discriminative detection

Cell systems are ideal for recognizing endotoxin, although interpreting the signal response can be challenging. Bouafsoun et al. [169] functionalized the surface of an impedance biosen‐ sor with endothelial cells, and measured the decrease in impedance with LPS binding, with a sensitivity of 500 ng/mL. Veiseh et al. [200] patterned macrophage cells onto gold electrodes to detect LPS concentrations of 0.1–10 µg/mL. However, cells were concurrently stained with necrosis and apoptosis markers in parallel studies, and no staining effect could be seen in cells using concentrations less than 10 µg/mL. This is an interesting effect, as in many *in vitro* studies,

Investigators have utilized the interaction of LPS with synthetic systems such as copoly‐ thiophene copolymers [164] and polydiacetylene liposomes [190, 191]. Johnson et al. [192] demonstrated an endotoxin capture technique by functionalizing a bead matrix with proan‐ thocyanidins and binding with fluorescein isothiocyanate‐labeled LPS [192]. Pyrenyl‐derived quaternary ammonium probes, developed by Zeng et al. [193] exhibited fluorescence when bound to LPS and detected nanomolar concentrations, while fluorescently labeled CD14 syn‐ thetic peptides demonstrated an increase in Förster resonance energy transfer when bound to LPS, but were only able to detect µM concentrations [194]. Lim et al. [195] used a functionalized graphene oxide to develop a fluorescence quench‐recovery method for LPS, targeting the lipid A component. Thompson et al. [196] designed a tandem system to both detect (LoD = 1.0 ng/mL)

ng/mL (LPS O55:B5) by flow cytometry within 1 minute. Bai et al. [188]

range of 0.01–1.0 × 106

of O‐ag groups [74, 167, 198].

**4.4. Cell‐based LPS detection systems**

Many novel approaches have been used for the detection of amphiphilic LPS, not all of which are functional in physiological matrices or have the required sensitivity or ease of use. One major reason for this is the failure to incorporate the amphiphilic properties of the antigen into assay design. The presentation, conformation, and host‐interactions of the antigens should be considered for the development of effective assays. While both LAL and EC assays are the most sensitive for testing endotoxicity, identifying O‐ag with a high degree of selec‐ tivity remains elusive, and limited to methods that use specific recognition ligands, such as membrane insertion and ELISAs. By far, the greatest limitation has been the lack of sensitive and selective ligands for the serogroup‐specific detection of the antigen. Thus, as repositories of these necessary recognition molecules expand to include more serogroups, so too will our ability to selectively detect LPS.
