Elsevier

Biophysical Chemistry

Volume 254, November 2019, 106262
Biophysical Chemistry

Research Article
Label-free, real-time on-chip sensing of living cells via grating-coupled surface plasmon resonance

https://doi.org/10.1016/j.bpc.2019.106262Get rights and content

Highlights

  • Surface Plasmon Resonance (SPR) keeps moving from biochemistry to cell biology.

  • Grating-coupled SPR is amenable for integration in microfluidic chamber.

  • Grating-coupled SPR has been successfully used for live cell investigation.

Abstract

The application of nanotechnologies to address biomedical questions is a key strategy for innovation in biomedical research. Among others, a key point consists in the availability of nanotechnologies for monitoring cellular processes in a real-time and label-free approach. Here, we focused on a grating-coupled Surface Plasmon Resonance (GC-SPR) sensor exploiting phase interrogation. This sensor can be integrated in a microfluidic chamber that ensures cell viability and avoids cell stress. We report the calibration of the sensor response as a function of cell number and its application to monitor cell adhesion kinetics as well as cell response to an external stimulus. Our results show that GC-SPR sensors can offer a valuable alternative to prism-coupled or imaging SPR devices, amenable for microfluidic implementation.

Introduction

Biomedical research is continuously evolving and the need for more sophisticated and innovative tools is rising mainly due to the contamination of different knowledge.

Cell-based analysis constitutes a fundamental step in life science research, where it is used to assess cell behaviour and response to external stimuli [1]. A gold standard approach to visualize cell response is offered by fluorescence microscopy, which usually requires cell labelling and post-detection analysis, this latter causing the complete loss of information about the kinetic of the process [2]. Moreover, cell adhesion to specific substrates is of increasing interest in the field of 3D culture models aiming to recapitulate in vivo complexity with the ease of in vitro culture [3].

Surface Plasmon Resonance (SPR) based sensors are of growing interest because of the promising label- and enzyme-free, as well as real-time detection approach for the biological specimens [4,5].

SPR refers to the collective oscillation of conduction electrons at the interface between a conductor material (typically gold or silver) and a dielectric (i.e. air or water, but also nucleic acids or proteins) upon light interaction [6]. Nanostructured plasmonic biosensors have been developed to improve miniaturization, integration and multiplexing in a lab-on-a-chip approach, while providing high sensitivity and specificity [7,8].

SPR biosensing is widely used to investigate the binding affinity and dynamics of two ligands and has been successfully applied to immunoassays demonstrating the suitability of the technique for molecular biology studies [9,10]. It allows monitoring molecular interactions occurring within the first few hundreds of nm over a gold surface, thanks to the plasmon evanescent field, that exponentially decays as it penetrates the material [6].

There are two major classes of SPR coupling technique that are based, respectively, on prism- and grating-coupling. In the first configuration, the light is totally reflected at the interface between the prism and the thin metal layer, making it particularly suitable for SPR sensing and the most used in commercial systems [4]. The interrogation system is usually based on angular interrogation, but some systems work through intensity modulation, the so-called SPR-imaging [11,12]. However, the recent interest in lab-on-a-chip brought the need for further integration of the SPR sensors, which is difficult to obtain with prism-coupling, because of the cumbersome optical system required for measurement. Grating-coupled SPR (GC-SPR) utilizes a metallic nanofabricated grating combined with excitation and detection system azimuthally rotated, that has been shown to reach performances close to prism-coupled systems [13,14]. In this configuration, the sensitivity of the resonance to incident light polarization allows an approach called “phase-interrogation”, which minimize moving parts during the scan. The system used in this work has been previously optimized for phase-interrogation in water-based environment [14,15]. This approach was found to be particularly suitable for integration of the plasmonic sensor in microfluidic Polydimethylsiloxane (PDMS) circuits, to allow sensing procedures and tightly control volumes, including multiplexing [7,16].

Recently, applications of SPR sensors have been reported for dynamic cellular analysis exploiting their label-free and real-time approach, with promising results [17]–[19]. Although most applications to cell biology are based on SPR imaging [10,20], live cell dynamic reactions to chemical stimuli are detectable as a change of the angle of resonance. Thanks to the spatial confinement of the evanescent optical field, SPR sensing ensures the precise monitoring of phenomena occurring on the cell membrane (like adhesion or rearrangement), regardless of changes in the intracellular compartments, that are far from the SPR sensitive volume. To date, several challenges need to be addressed to promote the application of SPR-based label-free cell analyses, such as the maintenance of cell integrity during the experimental process [9,17,21]. Most of commercially available instruments use glass slides coated with gold, that should be installed on the prism before analysis with a careful handling process to reduce cell damage [17]. Moreover, some instruments can tolerate in the microfluidic components only particulates under few microns, thus cells would cause clogging [17].

Here, we performed an experimental application of our GC-SPR integrated in a PDMS microfluidic chamber to label-free monitor cell adhesion capability and cell-surface interaction, while scaling down medium volume and maintaining cell integrity during manipulation. For this study we focused on SHI-1, a human acute myeloid leukemia (AML) cell line, that in vitro grows in suspension. Although this is a highly specific cell line, most of the results can be transferred to any other cell type as will be discussed. We used this cell line in the context of a wider research project aimed at evaluating the role of the bone marrow microenvironment on leukemia cells [22]. Indeed, in vivo AML cells home to the bone marrow, where they interact with stroma components and extracellular matrix proteins [23,24]. AML cells derive their proliferative capacity from this interaction and the following adhesion. The method we show here poses the basis for further in vitro studies of cell adhesion dynamics and affinity to different substrates, that currently rely mainly on microscopy-based analysis.

Section snippets

Numerical simulations

We performed numerical simulations using the commercial software COMSOL Multiphysics® [25]. The design of the structure replicates the physical dimensions of the grating (duty cycle, linewidth and height) and the field is simulated at a fixed azimuth (as done experimentally) at the resonance angle. Both gold and medium are considered bulk. The same simulation was performed using two refractive indexes: 1.33 for water and 1.38 for cells. The colour code adopted to visualize the field represents

GC-SPR in conical mounting characterization

The system used in this work has been developed by our group and optimized for water-based environment. Briefly, the sensitive area consists of a gold nano-grating embedded in a PDMS circuit positioned on a rotating stage. The adoption of azimuthal rotation of the grating with respect to the incidence plane (see representation in Fig. 1a) allowed to obtain the best performances. Incident light crosses a rotating half-wave plate, and this is the only moving part during the scan, thus making the

Conclusions

In this proof-of-principle paper we have shown that grating-coupled SPR biosensors can be used for live cell dynamics investigation. Our GC-SPR system is based on phase interrogation, that means a sinusoidal behavior of the reflectance as a function of incident light polarization. The SPR signal, in terms of phase shift, is proportional to the surface coverage over the grating, allowing a calibration for cell number quantification.

Most of the applications of SPR to whole cell studies relies on

Acknowledgement

G.B. acknowledges the support from Fondazione Istituto di Ricerca Pediatrica (Grant: MyFirstIRP 19/06IRP). The authors declare no conflict of interest.

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