# Lipo-oligo/polysaccharide biorecognition at the nano-bio interface: insights into LA27 aptamer binding to biomimetic bacterial membranes

Alessandro Cangiano, Noemi Gallucci, Cristina Di Carluccio, Luke A Clifton, Nicolas Martin, Apostolos Vagias, Alba Silipo, Luigi Paduano, Giuseppe Vitiello

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Alessandro Cangiano, Noemi Gallucci, Cristina Di Carluccio, Luke A Clifton, Nicolas Martin, et al.. Lipo-oligo/polysaccharide biorecognition at the nano-bio interface: insights into LA27 aptamer binding to biomimetic bacterial membranes. Journal of Colloid and Interface Science, 2026, 704 (1), pp.139292. 10.1016/j.jcis.2025.139292. hal-05369080

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# Lipo-oligo/polysaccharide biorecognition at the nano-bio interface: insights into LA27 aptamer binding to biomimetic bacterial membranes

<figure><img src="image_3.png" /></figure>

Alessandro Cangiano<sup>a,b</sup>, Noemi Gallucci<sup>a,b</sup>, Cristina Di Carluccio<sup>a</sup>, Luke A. Clifton<sup>c</sup>, Nicolas Martin<sup>d,e</sup>, Apostolos Vagias<sup>f</sup>, Alba Silipo<sup>a</sup>, Luigi Paduano<sup>a,b</sup>, Giuseppe Vitiello<sup>b,g,*</sup>

## GRAPHICAL ABSTRACT

### The LOS and LPS architecture tunes the biorecognition ability of LA27 aptamer

<figure><img src="image_4.png" /></figure>

LOS favors LA27 penetration and interaction with acyl chains

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

LPS O-antigen moiety hinders LA27 penetration into hydrophobic region

<figure><img src="image_7.png" /></figure>

## ARTICLE INFO

**Keywords:**

Bacterial endotoxin

Lipopolysaccharide

Aptamer

Molecular recognition

Neutron technique

## ABSTRACT

Gram-negative bacteria feature an asymmetric outer membrane, composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) or lipooligosaccharides (LOS) in the outer leaflet, which underpins virulence and antibiotic resistance. Conventional LPS assays like the Limulus amebocyte lysate test face ethical and practical challenges, motivating the development of aptamer-based detection. Here, we probe the interaction of the LA27 DNA aptamer ($K_d \approx 46$ nM) with biomimetic membranes incorporating LOS/LPS from *Akkermansia muciniphila*, *Flavobacterium* sp. *Root935*, and *Paenalcaligenes hominis* strains. Asymmetric supported lipid bilayers (SLBs) and symmetric large unilamellar vesicles (LUVs) were characterized by neutron reflectometry (NR), small-angle neutron scattering (SANS), and dynamic light scattering (DLS). NR confirmed preserved bilayer asymmetry and revealed that LA27 deeply penetrates *Akkermansia* LOS bilayers, evidenced by increased core hydration and structural disruption, whereas interactions with *Flavobacterium* LOS are moderate, and those with *Paenalcaligenes*

<footnote><sup>a</sup> Department of Chemical Sciences, University of Naples Federico II, Complesso Monte S. Angelo, 80126 Naples, Italy</footnote>

<footnote><sup>b</sup> CSGI, Center for Colloid and Surface Science, via della Lastruccia 3, Sesto Fiorentino, FI, Italy</footnote>

<footnote><sup>c</sup> ISIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 OQX, UK.</footnote>

<footnote><sup>d</sup> Laboratoire Léon Brillouin, UMR12 CEA-CNRS, Gif-sur-Yvette, 91191, France</footnote>

<footnote><sup>e</sup> Laboratoire Rhéologie et Procédés, UMR5520 CNRS-UGA, Gières, 38610, France</footnote>

<footnote><sup>f</sup> Institut Laue Langevin (ILL), rue des Martyrs, 38000 Grenoble, France.</footnote>

<footnote><sup>g</sup> Department of Chemical, Materials and Production Engineering, University of Naples Federico II, Piazzale V. Tecchio 80, 80125 Naples, Italy</footnote>

<footnote>* Corresponding author at: Department of Chemical, Materials and Production Engineering, University of Naples Federico II, Piazzale V. Tecchio 80, 80125 Naples, Italy - CSGI, Center for Colloid and Surface Science, via della Lastruccia 3, Sesto Fiorentino, FI, Italy.

E-mail address: giuseppe.vitiello@unina.it (G. Vitiello).</footnote>
LPS are confined to surface adhesion due to extensive LPS O-antigen chains. SANS and DLS of LUVs corroborated these modes: *Akkermansia* vesicles showed negligible changes in lamellar thickness and hydrodynamic radius, consistent with insertion, while those containing *Flavobacterium* LOS and *Paenalcaligenes* LPS exhibited significant increases in both size-related metrics, indicative of surface binding for those latter cases. These results elucidate how polysaccharide composition and supramolecular architecture of LOS/LPS govern aptamer affinity and specificity, providing a rigorous framework for nano-bio interface studies and laying the groundwork for selective, aptamer-based endotoxin sensing platforms.

# 1. Introduction

Bacteria are broadly classified as either Gram-positive (+) or Gram-negative (−) (Gram (+) and (−)) based on differences in their cell wall thickness. In particular, Gram(−) bacteria are a wide class of microorganisms which encompasses numerous common pathogens of medical and economic significance, such as *Escherichia coli*, *Salmonella enterica*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii* and *Klebsiella pneumoniae* [1]. These organisms are implicated in various diseases, ranging from urinary tract infections to life-threatening conditions like sepsis and pneumonia [2–4]. Moreover, Gram(−) bacteria are prominent agents of foodborne illnesses, contributing to acute health risks and chronic contamination challenges in food safety [5–7]. Crucial for Gram (−) pathogenicity is the role played by the bacterial envelope, which is characterized by a complex and dynamic architecture [8,9]. This consists of a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane, which is a peculiar feature of the Gram(−) strains [10–12] and is responsible for many processes, including the enhanced resistance to antibiotics and environmental stresses that make them particularly challenging to face in clinical and industrial contexts [13,14].

From a chemical and structural point of view, the outer membrane has an asymmetric character, where the inner leaflet is predominantly composed of phospholipids while the outer leaflet is enriched with lipopolysaccharides (LPS) molecules, which are crucial for membrane stability and host-pathogen interactions [15]. LPS has a complex molecular organization, consisting of three portions: i) the lipid A, ii) the core oligosaccharide, and iii) the O-Antigen polysaccharide chain [16]. The lipid A is embedded in the membrane and is primarily responsible for the LPS endotoxic activity, triggering huge immune responses in hosts. The core oligosaccharide stabilizes the overall structure, while the O-Antigen, highly variable among bacterial species, acts as a shield against host immune defences and antimicrobial agents. This structural complexity makes LPS a key player in bacterial virulence and survival [17,18].

Considering their crucial role, LPS can be considered valid analytes for the efficient and specific detection of the presence of bacterial contaminants in food and water supplies, or for diagnosing bacterial infections from a biomedical point of view. Conventional LPS detection tests, such as the Limulus Amebocyte Lysate (LAL) assay, face some limitations, such as high cost and ethical and environmental concerns regarding animal-derived reagents [19,20].

Recently, aptamer-based detection systems have emerged as highly specific and versatile alternatives [21]. Indeed, aptamers are synthetic oligonucleotides capable of binding their targets with exceptional affinity and specificity [22]. Their modularity and adaptability allow for the design of highly efficient detection of several molecules, including LPS. For this study, we selected the LA27 aptamer, which was developed by truncating a parent sequence (LA80) originally selected for its broad-spectrum recognition of LPS from various Gram-negative bacteria. In detail, LA27 has demonstrated a great binding affinity towards LPS (Kd = 46.2 ± 9.5 nM) [21,23].

The high binding affinity and ease of synthesis make LA27 a promising candidate for specific biorecognition and biosensing applications, both in clinical diagnostics and environmental monitoring [24]. However, to date, the molecular mechanisms through which this aptamer can

recognize and interact with LPS are not yet well defined.

In this context, the present study aims to investigate the molecular interaction between the LA27 aptamer and LPS/LOS-containing asymmetric bilayers designed to mimic the outer membrane of Gram(−) bacteria through the use of neutron scattering techniques, which have become the first-choice tools for the investigation of biomolecular interactions at the nanoscale [25]. We focused on the LPS isolated from three distinct bacterial species *Akkermansia muciniphila*, *Paenalcaligenes hominis* and *Flavobacterium* sp. Root935, selected due to the significant variations in the structure, length, and properties of their LPS, providing a broad scope for comparative analysis. The intestinal mucosal symbiont *Akkermansia muciniphila* is known to confer beneficial effects on the host and the resulting LOS displayed unprecedented structural and signaling properties [26]. The LOS consists of a complex glycan chain bearing two distinct undeca- and hexadecasaccharide units each and a mixture of differently phosphorylated and acylated lipid A species with either linear or branched acyl moieties. *Paenalcaligenes hominis*, increasingly prevalent in elderly individuals, associated with cognitive decline and gut-brain axis dysfunction, produces an LPS with an O-antigen characterized by a trisaccharide repeating unit containing rhamnose and glucosamine, displaying nonstoichiometric O-acetylation and a terminal methylated rhamnose capping the saccharide chain and a Lipid A composed of penta- to tetra-acylated species [27]. *Flavobacterium* sp. Root935, a member of the *Arabidopsis thaliana* root microbiota, producing a short-chain LPS, whose structure is ongoing, and mainly pentaacylated lipid A species.

The combination of Neutron Reflectometry (NR), Small-Angle Neutron Scattering (SANS), and Dynamic Light Scattering (DLS) analyses leads to a build a detailed characterisation of the structural and dynamic changes occurring upon LA27 aptamer interaction with the bacterial model membranes. The insights gained from this study extend beyond biosensing applications, contributing to a deeper understanding of membrane biophysics and the principles overriding molecular bio-recognition at the nano-bio-interface level.

# 2. Materials and methods

## 2.1. Materials

Deuterated water (99.9 atom% D) and chloroform (≥99.8 %, contains ethanol as stabiliser) solvents were purchased from Merck (Germany). The phospholipids 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine-1,2,1,2,2-d4-N,N,N-trimethyl-d9 (d-DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (h-DPPC), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and were used without further purification. *Akkermansia* LOS (COC), *Flavobacterium* LOS (FLAVO), and *Paenalcaligenes* LPS (pH) were isolated as previously described [18]. The LA27 aptamer (3'-CGATAGATTGTTGTAAGACAATCTTCC-5'-NH) was purchased from DBA s.r.l. (Milan, Italy) as HPLC-purified oligonucleotide.

## 2.2. Preparation of the biomimicking bilayers of bacterial outer membranes

### 2.2.1. Supported lipid bilayers (SLBs)

Asymmetric bilayers mimicking the bacterial outer membrane were
<figure><img src="image_8.png" /><figcaption>**Fig. 1.** NR profiles and corresponding best fits (A), SLD profiles (B) for *Akkermansia* LOS-containing bilayers, Volume fraction occupancy (C) of *Akkermansia* LOS moieties: silicon (black), silicon dioxide (grey), inner head groups (light green), PC tails (orange), LPS tails (magenta), Core region (mid-green), solvent (blue).</figcaption></figure>

prepared on silicon substrates. The process involved sequential mono-layer depositions using a KSV-Nima Langmuir-Blodgett (LB) trough [28].

First, the inner leaflet was formed by LB deposition. A solution of d-DPPC in chloroform was spread onto a pure water subphase containing 5 mM CaCl₂ and cooled to 10 °C. The resulting phospholipid monolayer was compressed to a target surface pressure of 35 mN m⁻¹. A submerged silicon wafer was then vertically withdrawn through this monolayer at a constant speed of 4 mm/min, transferring the d-DPPC film onto the substrate. Next, after thoroughly cleaning the through, the outer leaflet was prepared. A monolayer of the three endotoxins (*Akkermansia* LOS, *Flavobacterium* LOS, or *Paenalcaligenes* LPS) was formed by spreading it from a 2:5:8 (v/v) phenol:chloroform:petroleum ether solution onto a fresh aqueous subphase containing 2 mM CaCl₂ at 10 °C and compressing to 35 mN m⁻¹.

The asymmetric bilayer was completed using the Langmuir-Schaeffer (LS) technique. The d-DPPC-coated silicon wafer was positioned horizontally above the LOS/LPS monolayer and then lowered through the interface at 4 mm/min. This allowed the deposition of the outer leaflet onto the inner leaflet, finalising the biomimetic membrane structure within a purpose-built sample cell. For the interaction studied, a 20 µM solution of the LA27 aptamer was subsequently injected into the sample cell.

<figure><img src="image_9.png" /><figcaption>**Fig. 2.** NR profiles and corresponding best fits (A), SLD profiles (B) for *Flavobacterium* LOS-containing bilayers, Volume fraction occupancy (C) of *Flavobacterium* LOS moieties: silicon (black), silicon dioxide (grey), inner head groups (light green), PC tails (orange), LPS tails (magenta), Core region (mid-green), solvent (blue).</figcaption></figure>

### 2.2.2. Large unilamellar vesicles (LUV)

For SANS and DLS studies, LUVs were prepared as model bacterial membranes. Two compositions were used: DPPC:LOS/LPS at molar ratios of 90:10 and 80:20. The required quantities of phospholipids and LOS/LPS were co-dissolved in chloroform in a round-bottom flask. A thin lipid film was created by removing the solvent with a nitrogen stream, followed by a minimum of 3 h under vacuum to eliminate residual solvent traces. The film was then hydrated with 1 mL of water or D₂O and vortexed vigorously to form a homogeneous suspension of Multi-Lamellar Vesicles (MLVs). To produce a uniform LUV population, this suspension was extruded at least 21 times through a 100 nm polycarbonate membrane using an Avanti Polar Lipids extruder. DLS titrations were performed by varying the LUV-to-LA27 aptamer molar ratios (1:20, 1:5, and 1:3).

## 3. Bacterial bilayers/LA27 aptamer molecular recognition by neutron techniques

### 3.1. Specular neutron reflectometry (NR)

NR experiments were conducted on the INTER reflectometer at the ISIS spallation Neutron Source (UK). The instrument used a white
<figure><img src="image_10.png" /><figcaption>Fig. 3. NR profiles and corresponding best fits (A), SLD profiles (B) for *Paenkaligenes hominis* LPS-containing bilayers, Volume fraction occupancy (C) of *Paenkaligenes* LPS moieties: silicon (black), silicon dioxide (grey), inner head groups (light green), PC tails (orange), LPS tails (blue), Core region (yellow), O-Antigen Region (light green), solvent (blue).</figcaption></figure>

neutron beam (λ = 1–16 Å) at two glancing angles (0.7° and 2.3°) to probe a wavevector transfer range of 0.01 Å⁻¹ < q < 0.32 Å⁻¹. The absolute reflectivity R(q) was expressed as a function of the wavevector transfer:

$$q = \frac{4\pi\sin(\theta/2)}{\lambda} \qquad (1)$$

where θ was the angle of the incident beam and λ its wavelength.

The SLBs, housed in custom-built liquid flow cells, were mounted on a variable-angle sample stage. A liquid chromatography pump (Knaur Smartline 1000) was used to exchange the bulk solvent within the cell in situ. To obtain multiple contrast datasets, the cell (3 mL volume) was flushed with 15 mL of 20 mM HEPES buffer at 1.5 mL/min for each new solvent condition. The three contrasts used were D₂O, H₂O, and silicon-matched water (SMW; 38 % D₂O in H₂O). Data analysis was performed with the RasCal software, which models the interface as a series of slabs defined by thickness, roughness, and scattering length density (SLD). The reflectivity curves from all three contrasts for a single sample were co-refined against a unified structural model. This global fitting approach allowed for robust determination of the bilayer’s structural parameters, including layer-specific hydration. Parameter uncertainties were quantified using RsCal’s Bayesian error estimation routines, which employ a Delayed Rejection Adaptive Metropolis algorithm [29–31].

## 3.2. Small-angle neutron scattering (SANS)

SANS experiments were performed on the SAM instrument, operated by the Laboratoire Léon Brillouin at ILL - Institute Laue Langevin (Grenoble, France) [32,33]. Samples were filled into flat quartz cells [34] with 1 mm path length and thermally regulated at T = (37.0 ± 0.1) °C using a refrigerated bath circulator. A range of scattering vectors q = 4.10⁻³–0.35 Å⁻¹ was covered using neutrons with mean wavelength λ₀ = 7.5 Å, selected using a helical velocity selector with resolution Δλ/λ₀ ≈ 0.1, and two different sample-to-detector distances (0.9 and 6.7 m). The incoming beam was collimated through a square source (30 × 30 mm²) and a circular sample aperture (φ = 7.6 mm) over collimation lengths of 2.5 and 9 m, respectively. Two-dimensional raw patterns were recorded using a 64 × 64 cm²³He position-sensitive detector (256 × 128 pixels). They were then corrected for electronic + ambient background, empty cell scattering, detector uniformity and beam transmissions, before being scaled to absolute cross sections, via the attenuated direct beam intensity, measured for each instrument configuration, and radially-averaged using the GRASP software [35]. The structural information was extrapolated by choosing the appropriate model to fit the experimental data. In the present work, the obtained data were fitted with the SASView v5.0.6 Software [36] using the Lamellar model. With this model, the scattering intensity is calculated as:

$$I(q) = \text{scale} \frac{2\pi F(q)}{q^2 \delta} + \text{background} \qquad (2)$$

where:

$$F(q) = \frac{2\Delta\rho^2}{q^2} (1 - \cos(q\delta)) = \frac{4\Delta\rho^2}{q^2} \sin^2\left(\frac{q\delta}{2}\right) \qquad (3)$$

where δ is the total layer thickness and Δρ is the scattering length density difference [37,38].

## 3.3. Dynamic light scattering (DLS)

The hydrodynamic radius (R_h) of the vesicles was determined via DLS experiments on an LSI spectrometer featuring a 638 nm fibre-coupled laser. Measurements were performed at a 90° scattering angle, with the temperature stabilised at (37.0 ± 0.1)°C using a thermostatic bath. For statistical reliability, at least three independent samples were analysed for each experimental condition. The raw correlation functions (See Supplementary Material) were processed with the CORENN algorithm to determine diffusion coefficients. These coefficients were then used to calculate the R_h using the Stokes-Einstein equation:

$$R_h = \frac{kT}{6\pi\eta_0\langle D \rangle} \qquad (4)$$

where k is the Boltzmann constant, T is the absolute temperature, and η₀ is the solvent viscosity. This experimental approach allowed us to obtain the hydrodynamic radius of the scattering object [39–41].

# 4. Results and discussion

## 4.1. Interaction of LA27 aptamer with SLBs by neutron reflectometry

To understand the nanoscale structural changes induced by the LA27 aptamer, we employed NR on asymmetric bilayers composed of a d-DPPC inner leaflet and an outer leaflet containing one of the three LOS/LPS. The experiments, conducted at 37 °C, used three distinct solvent contrasts (D₂O, H₂O, and SMW), providing a robust dataset for structural refinement. Co-refining the data against a single structural model ensured reliable determination of layer thickness, roughness, and hydration levels [42].
<figure><img src="image_11.png" /><figcaption>Fig. 4. Experimental NR curves with best fits and SLD profiles of Akkermansia LOS (A, B), *Flavobacterium* LOS (C, D), and *Flavobacterium* LOS (E, F) bilayers before and after the interaction with LA27 aptamer molecules.</figcaption></figure>

<caption>**Table 1**</caption>

Structural parameters obtained by best fitting of experimental NR profiles of SLBs. Fitting errors are generated by the Bayesian error estimation routines described in the Methods section.

<table><caption>SLBs containing Akkermansia LOS</caption><thead><tr><th>Layer</th><th>Thickness [Å]</th><th>Roughness [Å]</th><th>Hydration %</th></tr></thead><tbody><tr><td>Silicon oxide</td><td>17.8 ± 0.2</td><td>5.5 ± 0.3</td><td>12.5 ± 1.0</td></tr><tr><td>Inner Headgroups</td><td>6.3 ± 0.5</td><td>–</td><td>75.0 ± 1.5</td></tr><tr><td>Inner tails</td><td>18.5 ± 0.3</td><td>–</td><td>–</td></tr><tr><td>Outer tails</td><td>12.5 ± 0.6</td><td>–</td><td>–</td></tr><tr><td>Whole lipid bilayer</td><td>–</td><td>4.6 ± 0.6</td><td>16 ± 2</td></tr><tr><td>Core region</td><td>45.0 ± 0.4</td><td>–</td><td>87 ± 2</td></tr></tbody></table><table><caption>SLBs containing Flavobacterium LOS</caption><thead><tr><th>Layer</th><th>Thickness [Å]</th><th>Roughness [Å]</th><th>Hydration %</th></tr></thead><tbody><tr><td>Silicon oxide</td><td>17.9 ± 0.8</td><td>7.6 ± 0.6</td><td>5.0 ± 0.2</td></tr><tr><td>Inner Headgroups</td><td>6.1 ± 0.4</td><td>–</td><td>82.6 ± 1.4</td></tr><tr><td>Inner tails</td><td>19.2 ± 0.5</td><td>–</td><td>–</td></tr><tr><td>Outer tails</td><td>10.6 ± 0.2</td><td>–</td><td>–</td></tr><tr><td>Whole lipid bilayer</td><td>–</td><td>10.0 ± 1.0</td><td>10.2 ± 0.2</td></tr><tr><td>Core region</td><td>24.4 ± 0.3</td><td>–</td><td>55.0 ± 1.5</td></tr></tbody></table><table><caption>SLBs containing Paenalcaligenes LPS</caption><thead><tr><th>Layer</th><th>Thickness [Å]</th><th>Roughness [Å]</th><th>Hydration %</th></tr></thead><tbody><tr><td>Silicon oxide</td><td>18.1 ± 0.1</td><td>2.5 ± 0.5</td><td>25.0 ± 1.3</td></tr><tr><td>Inner Headgroups</td><td>6.3 ± 0.3</td><td>–</td><td>80 ± 3</td></tr><tr><td>Inner tails</td><td>18.8 ± 0.2</td><td>–</td><td>–</td></tr><tr><td>Outer tails</td><td>16.5 ± 0.3</td><td>–</td><td>–</td></tr><tr><td>Whole lipid bilayer</td><td>–</td><td>4.1 ± 0.1</td><td>17.2 ± 1.0</td></tr><tr><td>Core region</td><td>20.2 ± 0.6</td><td>–</td><td>28.5 ± 0.9</td></tr><tr><td>O-antigen region</td><td>89.1 ± 0.5</td><td>42 ± 3</td><td>85 ± 2</td></tr></tbody></table>

For all samples, NR experiments highlighted the asymmetry of the lipid bilayers. The inner leaflet consisted mainly of d-DPPC, while the outer leaflet primarily comprised LOS or LPS, as shown in Figs. 1–3 and Tables S1–S3 for Akkermansia LOS, *Flavobacterium* LOS, and Paenalcaligenes LPS, respectively. Similar values in the thickness of the PC headgroups region were observed across the three systems. Interestingly, the high hydration percentages, which were equal to 75.0 %, 82.6 %, and 78.0 % for Akkermansia LOS, *Flavobacterium* LOS, and Paenalcaligenes LPS systems, respectively, indicated that the hydrophilic PC regions were highly penetrable to solvent molecules. Looking at the whole bilayer, composed of d-DPPC and LOS/LPS acyl chains regions, comparable thickness values were obtained for all systems. Notably, the roughness of the bilayers was significantly different as a function of the composition of the external leaflet: Akkermansia LOS and Paenalcaligenes LPS exhibited roughness values ~4 Å, suggesting a well-ordered, thigh packing of the acyl chains. In contrast, the *Flavobacterium* LOS system showed a greater roughness of ~10 Å, indicative of a more disordered and looser packing arrangement between the phospholipid and LOS acyl chains.

At the same time, significant differences were also observed in the core region and hydration level. In particular, the thickness values were estimated to be ~45 Å in the case of Akkermansia LOS and ~24 Å in the case of *Flavobacterium* LOS, respectively. At the same time, a hydration percentage of 86.6 % was obtained for the Akkermansia LOS bilayer compared to 55.0 % for *Flavobacterium* LOS bilayer, implying a
<figure><img src="image_12.png" /><figcaption>**Fig. 5.** Experimental SANS curves and best fitting related to LUVs with DPPC:LOS molar ratio of 90:10 and 80:20 before and after interaction with LA27 aptamer containing *Akkermansia* LOS (A and B), *Flavobacterium* LOS (C and D), *Paenalcaligenes* LPS (E and F).</figcaption></figure>

significative reduced affinity for water. On the other hand, the O-Antigen region of *Paenalcaligenes* LPS was ~87 Å thick and ~41 Å rough, suggesting loose packing of the O-Antigen chains. Indeed, this layer showed a hydration content of 81.0 %, consistent with a loosely packed, hydrophilic chain structure [28]. The microstructural characterisation of pure bilayers was preparatory to detecting the effects induced by the LA27 aptamer and its biorecognition behavior. In Fig. 4, the NR profiles obtained after the addition of the LA27 aptamer were simultaneously fitted with the pre-interaction data to assess the perturbation of the asymmetric membrane. For all the investigated systems, a significant perturbation of the bilayers mimicking the bacterial membranes was observed, thus suggesting an effective interaction with the LA27 aptamer, as observable by the fitting parameters values reported in

**Table 1.** In particular, SLD profiles revealed that the asymmetric bilayer composed of *Akkermansia* LOS experienced the most significant perturbation. Comparing the LOS and LPS results, we observed a lower degree of perturbation in the *Paenalcaligenes* asymmetric membrane, likely due to the screening effect of the O-Antigen moiety, which prevents the LA27 aptamer from reaching the inner part of the asymmetric bilayer.

## 4.2. Interaction of LA27 aptamer with LUVs by small-angle neutron scattering and dynamic light scattering

The interaction of LA27 aptamer with symmetric bilayers of LUVs as mimicking models of the bacterial membranes, was investigated by SANS and DLS to understand the effect of curvature and meso-structural
<figure><img src="image_13.png" /><figcaption>**Fig. 6.** Trends of the hydrodynamic radius, $R_h$, values as a function of the LUVs:aptamer molar ratio for DPPC: *Akkermansia* LOS 90:10 (green dots) and 80:20 (blue dots)(A), DPPC: *Flavobacterium* LOS 90:10 (green dots) and 80:20 (blue dots) (B), and DPPC: *Paenalcaligenes* *Hominic* LPS 90:10 (green dots) and 80:20 (blue dots) (C). Data points and error bars represent the mean and standard deviation from at least three independent sample preparations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<caption>**Table 2**</caption>

Structural parameters obtained by best fitting of the experimental SANS profiles. Errors are calculated by the SASView software from the covariance matrix of the least-squares fit.

<table><thead><tr><th>System</th><th>Lamellar thickness/Å</th></tr></thead><tbody><tr><td>DPPC:COC LOS 90:10</td><td>104.3 ± 0.4</td></tr><tr><td>DPPC:COC LOS 90:10 + 20 µM LA27</td><td>106.5 ± 1.0</td></tr><tr><td>DPPC:COC LOS 80:20</td><td>117.0 ± 0.5</td></tr><tr><td>DPPC:COC LOS 80:20 + 20 µM LA27</td><td>118.7 ± 0.6</td></tr><tr><td>DPPC:FLAVO LOS 90:10</td><td>94.5 ± 0.7</td></tr><tr><td>DPPC:FLAVO LOS 90:10 + 20 µM LA27</td><td>105.4 ± 0.6</td></tr><tr><td>DPPC:FLAVO LOS 80:20</td><td>105.0 ± 1.0</td></tr><tr><td>DPPC:FLAVO LOS 80:20 + 20 µM LA27</td><td>120.3 ± 0.5</td></tr><tr><td>DPPC:PH LPS 90:10</td><td>125.1 ± 0.6</td></tr><tr><td>DPPC:PH LPS 90:10 + 20 µM LA27</td><td>133.5 ± 1.0</td></tr><tr><td>DPPC:PH LPS 80:20</td><td>131.2 ± 0.4</td></tr><tr><td>DPPC:PH LPS 80:20 + 20 µM LA27</td><td>143.3 ± 0.7</td></tr></tbody></table>

organization in modulating the aptamer-lipids interaction.

Two different phospholipid:LOS/LPS molar ratios (90:10 and 80:20) were considered to study the interaction with LA27. SANS experiments were performed in the same conditions of NR analysis in terms of LA27 concentration to study the effect on the bilayers microstructure, while, for DLS experiments, a titration was conducted by increasing the lipo-some:aptamer ratio to evaluate the effect on the vesicles' size. As reported in Figs. 5 and 6 and Table 2, LUVs containing *Akkermansia* LOS did not show any increase in the lamellar thickness and the

hydrodynamic radius, $R_h$, after interaction with the LA27 aptamer. In contrast, both LUVs including *Flavobacterium* LOS and *Paenalcaligenes* LPS showed an increase in the lamellar thickness of 10–15 nm and $R_h$ of ~10 nm, respectively. However, in the case of *Flavobacterium* LOS-containing LUVs, the increase in $R_h$ value was only observed at the higher LOS concentration. Meanwhile, for the *Paenalcaligenes* LPS-containing LUVs, this increment was observed in both samples, but a greater effect was detected at a higher LPS content. These results seem to suggest that the LA27 aptamer, when interacting with the *Akkermansia* LOS-containing LUVs, likely penetrated the lipid bilayer thus locating in the inner aqueous region. This hypothesis was directly related to the high level of bilayer perturbation as observed by NR experiments. In the case of *Flavobacterium* LOS and *Paenalcaligenes* LPS-containing LUVs, the observed increase in lamellar thickness and $R_h$ values was probably due to the adhesion of LA27 on the vesicles' surface. These findings were in good agreement with the NR results, which indicated a less pronounced perturbation of the asymmetric membrane and suggested a greater tendency of the aptamer to locate on the more external region of the bilayers. These results confirmed the significant tendency of the LA27 aptamer to interact with the LOS/LPS molecules included in the bilayers, although through different molecular mechanisms, which were presumably influenced by the chemical nature of these molecules as well as by their supramolecular organization at the nanoscale.

The structural properties of the LUVs containing the LOS and LPS before and after the interaction with the LA27 aptamer were summarized in Fig. 7.
<figure><img src="image_14.png" /><figcaption>**Fig. 7.** Hydrodynamic radius and bilayer thickness variation upon the interaction of LA27 aptamer with the LUVs composed of DPPC and *Akkermansia* LOS, *Flavobacterium* LOS, and *Paenalcaligenes* LPS.</figcaption></figure>

## 5. Conclusions

In this work, a detailed nanoscale characterisation was carried out on how the LA27 aptamer interacts with lipid bilayers that contain LOS and LPS from Gram-negative bacterial strains. Our results show that the key factor controlling this interaction is the endotoxin in the membrane. Specifically, for bilayers with *Akkermansia* LOS, which lacks an O-antigen, the LA27 aptamer deeply penetrates the membrane, causing notable structural changes and hydration alterations. In contrast, the long O-antigen chain in *Paenalcaligenes* LPS acts as a steric barrier, preventing penetration and restricting the interaction to surface adhesion, while *Flavobacterium* LOS presents an intermediate case. These different binding modes, whether penetration or adhesion, were consistently observed in both planar, asymmetric supported lipid bilayers and curved, symmetric LUVs, confirming the reliability of our findings.

The central innovation of our study lies in the application of advanced neutron scattering techniques to probe these biorecognition events at a level of detail not previously achieved. While prior work successfully identified the LA27 aptamer and confirmed its high affinity for LPS in solution using methods like fluorescence polarization [23], the actual binding mechanism at a realistic membrane interface remained undefined. Our approach, utilizing asymmetric bilayers that more accurately mimic the outer membrane of Gram(−) bacteria, represents a significant methodological advance. The creation of such biomimetic systems is a current frontier in membrane biophysics, presenting considerable technical challenges [43,44]. This advanced model allowed us to move beyond the homogenous, solution-based approaches that characterize most of the aptamer-based sensor development [45,46] and reveal the fundamental biophysical principles governing aptamer recognition at a functionally relevant interface.

Future work should expand this methodology to investigate a broader library of LPS/LOS structures from other clinically relevant pathogens, thereby establishing a more comprehensive map of aptamer-glycolipid interaction rules. Furthermore, the influence of environmental factors, such as the concentration of divalent cations ($Ca^{2+}$,

$Mg^{2+}$), should be investigated. The crucial role of these cations in modulating the packing, fluidity, and stability of the LPS layer is well-documented, and their removal or displacement is known to profoundly impact outer membrane integrity (Clifton et al., 2015; Lam et al., 2014; Ginez et al., 2022; Savenko et al., 2025) [47–50]. Understanding how these ions affect the aptamer-LPS interaction is, therefore, a critical next step. Ultimately, this fundamental understanding will be crucial for translating these findings into applied technologies, such as the development of highly selective, aptamer-based biosensor platforms for the rapid and specific detection of bacterial endotoxins in clinical and environmental samples.

## Fundings and acknowledgments

The work was supported by Fondo per il Programma Nazionale della Ricerca (PNR) and Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN 2022), project title "Glycolipids-coated colloidal quantum dots as optical biosensing platform for selective molecular recognition - GLY-BIOSENS", funded by the Italian Ministry of University and Research (MUR) and European Union - Next Generation EU, Mission 4, Component 2, Investment 1.1 - D.D. n. 104 del 02.02.2022, C.I. 2022PJP24F; Piano Nazionale di Ripresa e Resilienza (PNRR) - Cascade Call, project title "nanoBIOSensori per la Rilevazione di Endotossine Batteriche: dalla ricerca alla formazione avanzata - BIOREB", Innovation Ecosystem "Rome Technopole", funded by European Union - Next Generation EU, Mission 4, Component 2, Investment 1.5 - D.D. n. 3277 30-12-2021, CUP: F63C24000230006.

The authors gratefully acknowledge the financial support of Consiglio Nazionale delle Ricerche within CNR-STFC Agreement 2021–2027 (N 0065606), concerning collaboration in scientific research experiment RB2210267 at the ISIS Neutron and Muon Source (UK) of Science and Technology Facilities Council (STFC).

The authors gratefully acknowledge the Institut Laue Langevin (ILL) for provision of beamtime on SAM instrument, proposal number: 8-03-1108.
CRediT authorship contribution statement

**Alessandro Cangiano:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. **Noemi Gallucci:** Writing – original draft, Investigation, Formal analysis, Data curation. **Cristina Di Carluccio:** Writing – review & editing, Investigation, Formal analysis. **Luke A. Clifton:** Writing – review & editing, Validation, Methodology, Investigation, Data curation. **Nicolas Martin:** Writing – review & editing, Methodology, Formal analysis. **Apostolos Vagias:** Writing – review & editing, Visualization, Methodology, Data curation. **Alba Silipo:** Writing – review & editing, Visualization, Validation, Methodology, Data curation. **Luigi Paduano:** Writing – review & editing, Visualization, Validation, Supervision, Methodology, Conceptualization. **Giuseppe Vitiello:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2025.139292.

Data availability

Data will be made available on request.

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