Elsevier

Optics Communications

Volume 294, 1 May 2013, Pages 420-426
Optics Communications

SPR based immunosensor for detection of Legionella pneumophila in water samples

https://doi.org/10.1016/j.optcom.2012.12.064Get rights and content

Abstract

Detection of legionellae by water sampling is an important factor in epidemiological investigations of Legionnaires' disease and its prevention. To avoid labor-intensive problems with conventional methods, an alternative, highly sensitive and simple method is proposed for detecting L. pneumophila in aqueous samples. A compact Surface Plasmon Resonance (SPR) instrumentation prototype, provided with proper microfluidics tools, is built. The developed immunosensor is capable of dynamically following the binding between antigens and the corresponding antibody molecules immobilized on the SPR sensor surface. A proper immobilization strategy is used in this work that makes use of an important efficient step aimed at the orientation of antibodies onto the sensor surface. The feasibility of the integration of SPR-based biosensing setups with microfluidic technologies, resulting in a low-cost and portable biosensor is demonstrated.

Introduction

Legionella pneumophila, the causative agent of Legionnaires' disease and Pontiac fever, was first recognized in 1977 following an outbreak of acute pneumoniae in Philadelphia [1]. Since then, the bacterium has been isolated from numerous sources in the environment [2] and was revealed to be a ubiquitous freshwater inhabitant. It is believed that bacterial transmission to humans occurs through droplets generated from environmental sources such as cooling towers [2], [3], [4], [5], showerheads [4], [6], [7], whirlpools [8], [9], and other human-made devices that generate aerosols [10], [11]. Since legionellae widely inhabit many water environments, the significance of isolating the bacteria from water reservoirs of aerosol-producing devices is often uncertain [12]. Legionella pneumophila is responsible for more than 90% of cases of Legionnaires' disease [13]. Outbreaks of community-acquired and nosocomial L. pneumophila infection have been previously described [14], [15]. Therefore, detection of legionellae by water sampling is important in epidemiological investigations of Legionnaires' disease and its prevention. Actually, “the gold standard” for identification of L. pneumophila is the culture method. A selective medium is used for isolation; however, several problems are encountered with this method, including the presence of viable but non-culturable cells, loss of viability of bacteria after collection, difficulties in isolation from biocontaminated samples, and the long time (about one week) required for culture and confirmation [16]. In order to avoid the problems of conventional methods, an alternative, highly sensitive and easy method with a short detection time is needed for detecting L. pneumophila.

Immuno-analytical methods could be a proper choice for sensitive detection of Legionella [17]. Traditionally, immunoanalytical methods rely on labels that covalently bind to one of the molecules to display the molecular recognition event with high sensitivity. However, methods requiring labeling impose extra cost due to the need to develop labeled reagents that perform appropriately in the assay (i.e., due to their hydrophobic nature, some fluorescent compounds produce significant background signal).

Research therefore developed label-free immunosensor devices to improve sensitivity, speed and analytical efficiency. An optic biosensor, based on imaging ellipsometry (IE), has been developed for the multiple detection of various pathogens such as E. coli O157:H7, S. typhimurium, Y. enterocolitica, and L. pneumophila, with a detection limit of 103–107 CFU/ml. Oh demonstrated the use of an optical biosensor based on Surface Plasmon Resonance (SPR) for the detection of L. pneumophila in artificially contaminated waters with a sensibility of 105 cells/ml [18].

The most widespread label-free detection systems found in the market are based on surface-plasmon resonance (SPR) of a gold film [19] or localized SPR (LSPR) of gold nanoparticles [20]. SPR provides a good compromise between sensitivity threshold and measurement throughput. Indeed SPR-based optical biosensors have been widely reported in literature in recent years, especially in the field of life sciences and drug discovery.

Due to optical transduction based on the monitoring of thin film refractive index (RI), SPR enables one to avoid time-consuming labeling steps and thus obtain all reaction kinetics constants within minutes. To produce the SPR effect, p-polarized light is passed through a prism and reflected from a gold covered prism facet contacting a liquid medium. A resonant transfer of energy from incident light to a plasmon wave over the gold film leads to a dip in the angular (spectral) dependence of the reflected intensity. Since the position of this dip is directly linked to RI of the liquid medium, its control can be used to follow the course of biomolecular binding events on the gold surface.

The advantage of this sensing approach is that part of the propagating guided light penetrates into the sensing area (where target and analyte interact) and is affected by optical changes occurring only in this region.

Recently, SPR immunosensors have been developed to measure the binding of antigens to antibody molecules immobilized on the SPR sensor surface [21].

These immunosensors are capable of detecting analytes in complex biological media with high specificity and sensitivity [22]. The sensitivity of SPR immunosensors is influenced by the surface property available for binding to antibody. Thus, a highly oriented antibody molecular layer on a solid surface can provide ultimate sensitivity in immunosensing.

In this study, a compact SPR instrumentation prototype is realized, provided with miniaturized biochips and proper microfluidics channels for ensuring an on-site characterization of water samples. After testing the immunoassay by a traditional ELISA technique, the feasibility of the assay in the developed prototype is tested for the detection of L. pneumophila in environmental water. To this purpose, a preliminary step aimed at the orientation of antibody molecules on the SPR sensor surface is required. The study demonstrates the possibility of the integration of SPR-based biosensing setups with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments. Thus, a contribution to the advancement of this technology in the field of optics and photonics is outlined.

A key factor in the success of the immunosensor is the choice of a proper immobilization approach and the use of microfluidics supports. The first ensures highly efficient immunoreactions and enhances detection system performance, the latter is essential for reduction in volume samples without losing important information concerning the biomolecular interactions.

Section snippets

Method of analysis

Plasmons, or plasma waves, are charge–density oscillations propagating in a plasma; in the case of a metallic solid, the plasma consists of a ‘gas’ of free electrons. Plasma oscillations that can propagate at the interface of a metal and a dielectric medium are called surface plasmons (SPs) or surface plasma waves (SPWs). They can be excited by a transverse magnetic (TM) optical wave (the magnetic vector is perpendicular to the direction of propagation of the SPW and to the plane of incidence).

Results and discussion

An important aspect to be investigated in the development of an immunosensor assay, is the identification of highly specific Pab (polyclonal antibody) for pathogen detection [26]. Therefore, we preliminarily verified the specificity of a commercial Pab against L. pneumophila for its potential uses in the immunosensor for the detection of this pathogen. In order to investigate Pab specificity, the cross-sensitivity of the Pab against several environmental pathogens, i.e., E. coli, Salmonella spp

Conclusions

A protein chip based on the SPR immunosensor was developed to detect L. pneumophila in contaminated water. After a proper characterization of the proposed immunoassay by a traditional technique such as ELISA, an SPR prototype instrumentation was realized by using proper mechanics and electronics tools, with the purpose to test similar immuno–chemical approaches in the compact setup able to perform in situ measurements. The prototype allows recording SPR curves and dynamic reflectivity

Acknowledgments

This work has been funded by the European Commission (No. NMP3-SL-2008-214107-Nanomagma), FIRB-Futuro in ricerca (Nanoplasma project) and was supported by grants from: POR PUGLIA 2007–2013 – Asse I Linea 1.1 – Azione 1.1.2 Bando “Aiuti agli Investimenti in Ricerca per le PMI” of the area Development Policies for The Work and Innovation of Regione Puglia.

P. Poltronieri of CNR-ISPA is supported with funding by the Italian project C.I.S.I.A. Conoscenze Integrate per Sostenibilità ed Innovazione

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