Indirect Detection of Bacteria on Optically Enhanced Porous Silicon Membrane-Based Biosensors Using Selective Lytic Enzymes

Abstract

In this work, we developed a biosensor for the indirect detection of bacteria via their lysate. The developed sensor is based on porous silicon membranes, which are known for their many attractive optical and physical properties. Unlike traditional porous silicon biosensors, the selectivity of the bioassay presented in this work does not rely on bio-probes attached to the sensor surface; the selectivity is added to the analyte itself, by the addition of lytic enzymes that target only the desired bacteria. The resulting bacterial lysate is then able to penetrate into the porous silicon membrane and affects its optical properties, while intact bacteria accumulate on top of the sensor. The porous silicon sensors, fabricated using standard microfabrication techniques, are coated with TiO 2 layers using atomic layer deposition. These layers serve as passivation but also enhance the optical properties. The performance of the TiO 2 -coated biosensor is tested for the detection of Bacillus cereus, using the bacteriophage-encoded PlyB221 endolysin as the lytic agent. The sensitivity of the biosensor is much improved compared to previous works, reaching 10 3 CFU/mL, with a total assay time of 1 h 30 min. The selectivity and versatility of the detection platform are also demonstrated, as is the detection of B. cereus in a complex analyte.

Design of a Porous Silicon Biosensor: Characterization, Modeling, and Application to the Indirect Detection of Bacteria

“…Based on the simulation results, the PSi biosensor was a Fabry-Perot interferometer, with a double-layered structure. The fabrication of the PSi biosensor is fully detailed elsewhere [21]. The fabrication process started with a 3 in double-etched p++ wafer (<100>, 0.8-0.9 mΩ•cm, 380-400 µm) (Sil'tronix Silicon Technologies).…”

“…The membranes were then opened using dry etching. To assure the stability of the sensor, a thin layer of TiO 2 was coated inside the PSiM using atomic layer deposition [21].…”

“…For this study, it was decided to use highly doped p-type silicon substrates in order to reach pores size ranging from 10 to 100 nm. With this pore range, most bacterial fragments, as measured in [21], can penetrate into the porous matrix. The possible morphologies of PSi samples were mapped using different fabrication parameters; the main parameter studied here is the current density, as it has been shown that pore dimensions increase with current density [33].…”

“…Based on these design parameters, a PSi biosensor was fabricated using a combination of electrochemical etching and standard microfabrication techniques. The full wafer manufacturing process has been used and optimized in several studies [21,28,39], and has been observed to be both robust and repeatable, reaching a success yield of 75%.…”

“…These experimental parameters were analyzed using varying fabrication conditions while ensuring that the bacterial fragments were able to penetrate into the porous layer. The sizes of the bacterial fragments were based on a model of Bacillus cereus lysate, as previously studied [21]. Two PSi nanostructures were compared in this diffusion study: (i) a double-layer, relying on reflective interferometric Fourier transform spectroscopy (RIFTS) [11] for the optical read-out, and (ii) a photonic microcavity, relying on the monitoring of its spectral features and/or on its structural color [22].…”