Porous silicon (PSi) has been widely used as a biosensor in recent years due to its large surface area and its optical properties. Most PSi biosensors consist in close-ended porous layers, and, because of the diffusion-limited infiltration of the analyte, they lack sensitivity and speed of response. In order to overcome these shortcomings, PSi membranes (PSiMs) have been fabricated using electrochemical etching and standard microfabrication techniques. In this work, PSiMs have been used for the optical detection of Bacillus cereus lysate. Before detection, the bacteria are selectively lysed by PlyB221, an endolysin encoded by the bacteriophage Deep-Blue targeting B. cereus. The detection relies on the infiltration of bacterial lysate inside the membrane, which induces a shift of the effective optical thickness. The biosensor was able to detect a B. cereus bacterial lysate, with an initial bacteria concentration of 10⁵ colony forming units per mL (CFU/mL), in only 1 h. This proof-of-concept also illustrates the specificity of the lysis before detection. Not only does this detection platform enable the fast detection of bacteria, but the same technique can be extended to other bacteria using selective lysis, as demonstrated by the detection of Staphylococcus epidermidis, selectively lysed by lysostaphin.

Paper substrates are promising for development of cost-effective and efficient point-of-care biosensors, essential for public healthcare and environmental diagnostics in emergency situations. Most paper-based biosensors rely on the natural capillarity of paper to perform qualitative or semi-quantitative colorimetric detections. To achieve quantification and better sensitivity, technologies combining paper-based substrates and electrical detection are being developed. In this work, we demonstrate the potential of electrical measurements by means of a simple, parallel-plate electrode setup towards the detection of whole-cell bacteria captured in nitrocellulose (NC) membranes. Unlike current electrical sensors, which are mostly integrated, this plug and play system has reusable electrodes and enables simple and fast bacterial detection through impedance measurements. The characterized NC membrane was subjected to (i) a biofunctionalization, (ii) different saline solutions modelling real water samples, and (iii) bacterial suspensions of different concentrations. Bacterial detection was achieved in low conductivity buffers through both resistive and capacitive changes in the sensed medium. To capture Bacillus thuringiensis, the model microorganism used in this work, the endolysin cell-wall binding domain (CBD) of Deep-Blue, a bacteriophage targeting this bacterium, was integrated into the membranes as a recognition bio-interface. This experimental proof-of-concept illustrates the electrical detection of 10⁷ colony-forming units (CFU) mL⁻¹ bacteria in low-salinity buffers within 5 min, using a very simple setup. This offers perspectives for affordable pathogen sensors that can easily be reconfigured for different bacteria. Water quality testing is a particularly interesting application since it requires frequent testing, especially in emergency situations.

Endolysins are phage-encoded enzymes implicated in the breaching of the bacterial cell wall at the end of the viral cycle. This study focuses on the endolysins of Deep-Blue (PlyB221) and Deep-Purple (PlyP32), two phages preying on the Bacillus cereus group. Both enzymes exhibit a typical modular organization with an enzymatically active domain (EAD) located in the N-terminal and a cell wall binding domain (CBD) in the C-terminal part of the protein. In silico analysis indicated that the EAD domains of PlyB221 and PlyP32 are endowed with peptidase and muramidase activities, respectively, whereas in both proteins SH3 domains are involved in the CBD. To evaluate their antimicrobial properties and binding specificity, both endolysins were expressed and purified. PlyB221 and PlyP32 efficiently recognized and lysed all the tested strains from the B. cereus group. Biochemical characterization showed that PlyB221 activity was stable under a wide range of pHs (5–9), NaCl concentrations (up to 200 mM), and temperature treatments (up to 50 °C). Although PlyP32 activity was less stable than that of PlyB221, the endolysin displayed high activity at pH 6–7, NaCl concentration up to 100 mM and the temperature treatment up to 45 °C. Overall, PlyB221 and PlyP32 display suitable characteristics for the development of biocontrol and detection tools.

The rapid detection of hazardous bacteria is important for healthcare situations, where such identification can lead to substantial gains for patient treatment and recovery and a reduced usage of broad-spectrum antibiotics. Potential biosensors must be able to provide a fast, sensitive and selective response with as little sample preparation as possible. Indeed, some of these pathogens, such as Staphylococcus aureus, can be yet harmful at very low concentrations in the blood stream, e.g., below 10 colony forming units per mL (CFU/mL). These stringent requirements limit the number of candidates, especially for point-of-care applications. Amongst several biosensing techniques, optical sensing using porous silicon (PSi) substrate has been widely suggested in recent years thanks to unique features such as a large surface area, tunable optical characteristics, and above all relatively easy and affordable fabrication techniques. In most configurations, PSi optical biosensors are close-ended porous layers; this limits their sensitivity and responsiveness due to diffusion-limited infiltration of the analytes in the porous layer. Also, PSi is a reactive material, its oxidation in buffer solutions results in time-varying shifts. Despite its attractive properties, several challenges must still be overcome in order to reach practical applications. Our work addresses three main improvement points. The first one is the stability over time in saline solutions helped by atomic layer deposition of metal oxides inside the pores. Besides a better stability, our solution is helping with an increase of the optical signal to noise ratio, thus reducing the limit of detection. The second one is to perform the lysis of the bacteria prior to its exposure to the sensor, such that the selective detection is based upon the percolation of bacterial residues inside the pores rather than the bacteria themselves. The third one is to remove the bulk silicon below a PSi layer to create a membrane, that allows for flow-through of the analytes, thus enhancing the interactions between the lysate and the sensor’s surface. This approach allows us to avoid the step of surface functionalization used in classical biosensors. We tested thanks to these improvements the selective detection of Bacillus cereus lysate with concentrations between 10³ and 10⁵ CFU/mL. Future works are dedicated to further improvements, including optical signal enhancement techniques and dielectrophoretic assisted percolation in the porous silicon membrane.