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Gram-type Differentiation of Bacteria with 2D Hollow Photonic Crystal Cavities

Abstract : Fast and label-free techniques to analyze viruses and bacteria are of crucial interest in biological and bio-medical applications. For this purpose, optofluidic systems based on the integration of photonic structures with microfluidic layers were shown to be promising tools for biological analysis, thanks to their small footprint and to their ability to manipulate objects using low powers. In this letter, we report on the optical trapping of living bacteria in a 2D Silicon hollow photonic crystal cavity. This structure allows for the Gram-type differentiation of bacteria at the single cell scale, in a fast, label-free and non-destructive way. During the last decade, optical resonators integrated with microfluidic layers arose as suitable structures for biological analysis 1 , thanks to their small footprint and especially thanks to their ability to trap objects with low powers 2-5 , beneath the damage threshold of biological entities. The trapping of biomolecules 6-8 , viruses 9 and bacteria 10,11 was reported. Moreover, the resonant nature of the optical cavities enables for the simultaneous acquisition of information on the trapped object such as size, refractive index and morphology, thanks to a feedback effect induced by the trapped specimen on the trapping field itself 11-17. In parallel, the massive and inappropriate use of antibiotics since the 1950s has led to antimicrobial resistance 18. Because of multidrug resistant pathogens, in a near future, common infections and minor injuries could kill once again. This misuse of antibiotherapy is partly due to long, compelling and / or expensive diagnostic tools based on the analysis of a large number of bacteria (typically 10 6 to 10 8). The initial step in this diagnostic consists in determining the bacterium responsible for the infection and the rapidity in its identification is of crucial importance. Currently, the first test performed in hospital environment is the Gram staining procedure of the specimen under study, so as to yield a very first characterization of the pathogen to be identified. This differential staining allows for the classification of bacteria in two groups, Gram-positive and Gram-negative, depending on the chemical and physical properties of the cell wall 19,20. Gram staining method is widely used 21 but it is a restrictive and destructive technique that requires carcinogen dyes 22 and a large number of bacteria 23. Other Gram-type identification techniques were suggested, based on KOH for marine bacteria 24 , on pyrolisis-mass spectrometry 25 or on the reaction between polymyxin B and lipolysaccharides 26. Gram negative bacteria were also identified through a functionalized porous silicon microcavity detecting lipolysaccharides 27. Here we propose a method based on resonant trapping in a 2D hollow photonic crystal (PhC) cavity. With this structure, we implemented a fast, label-free and nondestructive technique to distinguish the Gram-type of bacteria at the single-cell level. The photonic crystal structures are fabricated on Silicon-On-Insulator substrates with conventional electron beam lithography techniques and inductively coupled plasma etching 12,28,29. The silica sacrificial layer is then removed via wet etching. The PhC cavity is designed to have a resonant frequency around 1550 nm and is evanescently excited via a W1 waveguide in an end-fire setup. The lattice holes measure 250 nm in diameter and they are hexagonally arranged with a lattice constant of 420 nm; the defect hole is 700 nm in diameter. More details on the photonic crystal cavity can be found in Ref. [12]. The entire set of measurements was performed on the same optical cavity, featuring a Q factor of 4500 in water. To enable the transport of bacteria in the vicinity of the PhC structures, a polydimethylsiloxane (PDMS) frame (100 µm in thickness) is placed on the sample, and it acts as a container for a drop of the bacteria suspension in deionized water. A glass coverslip, 170 µm thick, is then attached to avoid evaporation. Light from a tunable laser is injected with a polarization maintaining lensed fiber, and the transmitted power through the waveguide is collected with a microscope objective and detected by a photodiode. A visible camera placed on the top of the sample allows for imaging and visual checking of the trapping events. The device and the optical structure we developed are shown in Fig. 1a.
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R. Therisod, M. Tardif, P. Marcoux, E. Picard, J.-B. Jager, et al.. Gram-type Differentiation of Bacteria with 2D Hollow Photonic Crystal Cavities. Applied Physics Letters, American Institute of Physics, 2018, 113 (11), pp.111101. ⟨10.1063/1.5037849⟩. ⟨cea-02529379⟩

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