Real-time nanoplasmonic sensing of three-dimensional morphological changes in a supported lipid bilayer and antimicrobial testing applications

Abstract

We report the development of a real-time localized surface plasmon resonance (LSPR) biosensing strategy to detect three-dimensional morphological changes in a supported lipid bilayer (SLB) on a plasmonic substrate. The sensing concept advances on past efforts to detect subtle conformational changes in adsorbed biomacromolecules by demonstrating the capability to track large-scale, complex adsorbate shape changes and to classify different types of shape changes based on specific, multi-step measurement signatures. To validate this concept, we tested the addition of antimicrobial fatty acids, monoglycerides, and surfactants in micellar form to the SLB platform, which triggered specific three-dimensional membrane morphological changes such as tubule or bud formation along with solubilization. Experimentally, the different remodeling events were detected by distinct measurement signatures related to the shape and size of lipid protrusions that formed and evolved over time, which agreed well with a newly developed theoretical model. Our conceptual approach and formalism are applicable to various biosensing techniques, including not only LSPR but also surface plasmon resonance (SPR) and total internal reflection fluorescence (TIRF) microscopy. These sensing capabilities are advantageous for evaluating the mechanisms of antimicrobial drug candidates and other membrane-active compounds, and the measurement strategy is extendable to a wide range of biomimetic lipid compositions.

Introduction

There is broad interest in developing real-time biosensing strategies to characterize cell-membrane-mimicking supported lipid bilayer (SLB) interactions with a wide range of biomacromolecules and biological nanoparticles, such as peptides, proteins, micelles, exosomes, and virus particles, as well as with drug delivery vehicles, e.g., liposomes and lipid nanoparticles (Buck et al., 2019; Han et al., 2019; Nishio et al., 2020; Park et al., 2019). One of the most challenging measurement aspects is to track SLB morphological changes, which result from biomacromolecular interactions and are relevant to various biological phenomena and pharmaceutical drug testing applications. Key examples include cholesterol-induced membrane remodeling and three-dimensional crystallization (Lee et al., 2020; Rahimi et al., 2016; Varsano et al., 2015), and antiviral drug development to inhibit membrane-associated viral genome replication (Cho et al., 2010, 2016). Currently used biosensing techniques include surface plasmon resonance (SPR) (Ryu et al., 2019; Soler et al., 2018), total internal reflection fluorescence (TIRF) microscopy (Mapar et al., 2018), evanescent light scattering (Agnarsson et al., 2016), and quartz crystal microbalance with dissipation (QCM-D) (Di Iorio et al., 2020), which all have penetration depths of about 100–250 nm while the SLB thickness is much shorter (~5 nm). On the other hand, nanoplasmonic sensors can exhibit penetration depths of ~20 nm or less that are more comparable to the SLB thickness and thus potentially more sensitive to membrane morphological changes (Bruzas et al., 2016; Jose et al., 2013; Oh and Altug, 2018).

While nanoplasmonic sensing experiments are typically conducted on metal nanoparticle surfaces, indirect nanoplasmonic sensing (INPS) platforms enable the use of silica-coated gold nanodisk arrays on which SLBs can readily form (Langhammer et al., 2010). The embedded nanodisks exhibit localized surface plasmon resonance (LSPR), whereby light extinction induces the collective oscillation of conduction-band electrons within the nanodisk that gives rise to an enhanced electromagnetic field in close proximity to the silica coating, which is the active sensing interface (Dahlin et al., 2013; Jackman et al., 2017a; Unser et al., 2015). This field is scattered by adsorbate molecules that induce changes in collective electromagnetic oscillations. Physically, this sensing concept is similar to SPR and the main difference is in the type of evanescent field that is generated by the sensor (exponential in the SPR case vs. dipole-like in the LSPR case) and the degree of surface sensitivity. Experimentally, the LSPR sensing approach has been widely utilized to detect subtle conformational changes involving adsorbed liposomes, SLBs, and proteins that can be modeled as a uniform film or spherically shaped objects (Ferhan et al., 2018; Jackman et al., 2017b, Jackman et al., 2017c). However, various types of biologically relevant biomacromolecular interactions involve more complex, non-uniform adsorbate shape changes and there is an outstanding need to develop advanced nanoplasmonic sensing approaches to track such changes.

Herein, using a combination of experimental and theoretical approaches, we developed a real-time LSPR biosensing strategy to detect and classify complex adsorbate shape changes based on proof-of-concept experiments involving three-dimensional membrane morphological changes in an SLB platform. Specifically, we evaluated the LSPR measurement responses that occur when membrane-active, antimicrobial compounds interact with an SLB platform on a silica-coated gold nanodisk array and give rise to complex, dynamic changes in three-dimensional membrane morphology. The test compounds included two of the most biologically active fatty acids and monoglycerides termed lauric acid (LA) and glycerol monolaurate (GML), respectively, along with a related surfactant, sodium dodecyl sulfate (SDS), all of which demonstrate potent antimicrobial effects by disrupting the lipid membranes surrounding bacteria and enveloped viruses (Yoon et al., 2018). The mechanistic details of how each compound disrupts phospholipid membranes remain under investigation and we demonstrate how the LSPR technique is well-suited to address this measurement need as well as its compelling advantages compared to other biosensing techniques used in past works. Furthermore, the sensing concepts developed in our work and the underlying theoretical formalism are broadly useful for not only LSPR measurements but also readily extendable to the SPR and TIRF microscopy techniques as well.