Knowledge and regulation on fungal contamination of sand and water: Progress report and perspectives.

Fungal flora in coastal/inland beach sand and recreational water is a neglected field of study, despite its potential impact on human health. A joint International Society for Human and Animal Mycology/European Confederation for Medical Mycology (ISHAM/ECMM) working group was formed in 2019 with the task to set up a vast international initiative aimed at studying the fungal contamination of beaches and bathing waters. Here we review the importance of the topic, and list the main results and achievements from 12 scientific publications. Fungal contamination exists at different levels, and the genera most frequently found were Aspergillus spp., Candida spp., Fusarium spp., and Cryptococcus spp., both in sand and in water. A site-blind median was found to be 89 colony-forming units of fungi per gram of sand in coastal/inland freshwaters. This threshold has been used for the sand quality criterion of the blue flag in Portugal. Additionally, our data were considered pivotal and therefore used for the first inclusion of fungi as a biological taxon of interest in water quality and sand monitoring recommendations of the World Health Organization's new guidelines on recreational water quality (Vol.1-Chap7). The findings of the consortium also suggest how environmental conditions (climate, salinity, soil pH, nitrogen, etc.) influence microbial communities in different regions, and that yeast species like Candida glabrata, Clavispora lusitaniae, and Meyerozyma guilliermondii have been identified as potential fungal indicators of fecal contamination. Climate change and natural disasters may affect fungal populations in different environments, and because this is still a field of study under exploration, we also propose to depict the future challenges of research and unmet needs.

3D-printed microfluidics integrated with optical nanostructured porous aptasensors for protein detection.

Microfluidic integration of biosensors enables improved biosensing performance and sophisticated lab-on-a-chip platform design for numerous applications. While soft lithography and polydimethylsiloxane (PDMS)-based microfluidics are still considered the gold standard, 3D-printing has emerged as a promising fabrication alternative for microfluidic systems. Herein, a 3D-printed polyacrylate-based microfluidic platform is integrated for the first time with a label-free porous silicon (PSi)-based optical aptasensor via a facile bonding method. The latter utilizes a UV-curable adhesive as an intermediate layer, while preserving the delicate nanostructure of the porous regions within the microchannels. As a proof-of-concept, a generic model aptasensor for label-free detection of his-tagged proteins is constructed, characterized, and compared to non-microfluidic and PDMS-based microfluidic setups. Detection of the target protein is carried out by real-time monitoring reflectivity changes of the PSi, induced by the target binding to the immobilized aptamers within the porous nanostructure. The microfluidic integrated aptasensor has been successfully used for detection of a model target protein, in the range 0.25 to 18 µM, with a good selectivity and an improved limit of detection, when compared to a non-microfluidic biosensing platform (0.04 µM vs. 2.7 µM, respectively). Furthermore, a superior performance of the 3D-printed microfluidic aptasensor is obtained, compared to a conventional PDMS-based microfluidic platform with similar dimensions.

Aptamers vs. antibodies as capture probes in optical porous silicon biosensors.

Over the past decade aptamers have emerged as a promising class of bioreceptors for biosensing applications with significant advantages over conventional antibodies. However, experimental studies comparing aptasensors and immunosensors, under equivalent conditions, are limited and the results are inconclusive, in terms of benefits and limitations of each bioreceptor type. In the present work, the performance of aptamer and antibody bioreceptors for the detection of a his-tagged protein, used as a model target, is compared. The bioreceptors are immobilized onto a nanostructured porous silicon (PSi) thin film, used as the optical transducer, and the target protein is detected in a real-time and label-free format by reflective interferometric Fourier transform spectroscopy. For the antibodies, random-oriented immobilization onto the PSi nanostructure results in a poor biosensing performance. Contrary, Fc-oriented immobilization of the antibodies shows a similar biosensing performance to that exhibited by the aptamer-based biosensor, in terms of binding rate, dynamic detection range, limit of detection and selectivity. The aptasensor outperforms in terms of its reusability and storability, while the immunosensor could not be regenerated for subsequent experiments.

Neuroprotective Effect of Nerve Growth Factor Loaded in Porous Silicon Nanostructures in an Alzheimer's Disease Model and Potential Delivery to the Brain.

Nerve growth factor (NGF) plays a vital role in reducing the loss of cholinergic neurons in Alzheimer's disease (AD). However, its delivery to the brain remains a challenge. Herein, NGF is loaded into degradable oxidized porous silicon (PSiO2 ) carriers, which are designed to carry and continuously release the protein over a 1 month period. The released NGF exhibits a substantial neuroprotective effect in differentiated rat pheochromocytoma PC12 cells against amyloid-beta (Aß)-induced cytotoxicity, which is associated with Alzheimer's disease. Next, two potential localized administration routes of the porous carriers into murine brain are investigated: implantation of PSiO2 chips above the dura mater, and biolistic bombardment of PSiO2 microparticles through an opening in the skull using a pneumatic gene gun. The PSiO2 -implanted mice are monitored for a period of 8 weeks and no inflammation or adverse effects are observed. Subsequently, a successful biolistic delivery of these highly porous microparticles into a live-mouse brain is demonstrated for the first time. The bombarded microparticles are observed to penetrate the brain and reach a depth of 150 µm. These results pave the way for using degradable PSiO2 carriers as potential localized delivery systems for NGF to the brain.

Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light.

The online monitoring of recombinant protein aggregate inclusion bodies during microbial cultivation is an immense challenge. Measurement of scattered and reflected light offers a versatile and non-invasive measurement technique. Therefore, we investigated two methods to detect the formation of inclusion bodies and monitor their production: (1) online 180° scattered light measurement (λ = 625 nm) using a sensor platform during cultivation in shake flask and (2) online measurement of the light reflective interference using a porous Si-based optical biosensor (SiPA). It could be shown that 180° scattered light measurement allows monitoring of alterations in the optical properties of Escherichia coli BL21 cells, associated with the formation of inclusion bodies during cultivation. A reproducible linear correlation between the inclusion body concentration of the non-fluorescent protein human leukemia inhibitory factor (hLIF) carrying a thioredoxin tag and the shift ("Δamp") in scattered light signal intensity was observed. This was also observed for the glutathione-S-transferase-tagged green fluorescent protein (GFP-GST). Continuous online monitoring of reflective interference spectra reveals a significant increase in the bacterium refractive index during hLIF production in comparison to a non-induced reference that coincide with the formation of inclusion bodies. These online monitoring techniques could be applied for fast and cost-effective screening of different protein expression systems.

Label-free optical biosensors based on aptamer-functionalized porous silicon scaffolds.

A proof-of-concept for a label-free and reagentless optical biosensing platform based on nanostructured porous silicon (PSi) and aptamers is presented in this work. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensor design. Here we describe the fabrication and characterization of aptamer-conjugated PSi biosensors, where a previously characterized his-tag binding aptamer (6H7) is used as model system. Exposure of the aptamer-functionalized PSi to the target proteins as well as to complex fluids (i.e., bacteria lysates containing target proteins) results in robust and well-defined changes in the PSi optical interference spectrum, ascribed to specific aptamer-protein binding events occurring within the nanoscale pores, monitored in real time. The biosensors show exceptional stability and can be easily regenerated by a short rinsing step for multiple biosensing analyses. This proof-of-concept study demonstrates the possibility of designing highly stable and specific label-free optical PSi biosensors, employing aptamers as capture probes, holding immense potential for application in detection of a broad range of targets, in a simple yet reliable manner.

Tethered Lipid Bilayers within Porous Si Nanostructures: A Platform for (Optical) Real-Time Monitoring of Membrane-Associated Processes.

The importance of cell membranes in biological systems has prompted the development of artificial lipid bilayers, which can mimic the cellular membrane structure. Supported lipid bilayers (SLBs) have emerged as a promising avenue for studying basic membrane processes and for possible biotechnological applications. Conventional methods for SLB formation involve the spreading of lipid vesicles on hydrophilic solid supports. Herein, a facile approach for the construction of tethered SLB within an oxidized porous Si (pSiO2) nanostructure, avoiding liposome preparation, is presented. We employ a two-step lipid self-assembly process, in which a first lipid layer is tethered to the pore walls resulting in a highly stable monolayer. A subsequent solvent exchange step induces the self-assembly of the unbound lipids into a robust SLB. Formation of pSiO2-SLB is confirmed by fluorescence resonance energy transfer (FRET), and the properties of the confined SLB are characterized by environment-sensitive fluorophores. The unique optical properties of the pSiO2 support are employed to monitor in real time the partitioning of a model amphiphilic molecule within the SLB via reflective interferometric Fourier transform spectroscopy (RIFTS) method. These self-reporting SLB platforms provide a highly generic approach for bottom-up construction of complex lipid architectures for performing biological assays at the micro- and nanoscale.

Optical biosensors for bacteria detection by a peptidomimetic antimicrobial compound.

In this work we present a label-free optical biosensor for rapid bacteria detection using a novel peptide-mimetic compound, as the recognition element. The biosensor design is based on an oxidized porous silicon (PSiO2) nanostructure used as the optical transducer, functionalized with the sequence K-[C12K]7 (referred to as K-7α12), which is a synthetic antimicrobial peptide. This compound is a member of a family of oligomers of acylated lysines (OAKs), mimicking the hydrophobicity and charge of natural antimicrobial peptides. The OAK is tethered to the PSiO2 film and the changes in the reflectivity spectrum are monitored upon exposure to Escherichia coli (E. coli) bacterial suspensions and their lysates. We show that capture of bacterial cell fragments induces predictable changes in the reflectivity spectrum, proportional to E. coli concentrations, thereby enabling rapid, sensitive and reproducible detection of E. coli at concentrations as low as 10(3) cells per mL. While for intact bacterial cells, the K-7α12-tethered PSiO2 shows a poor capturing ability, resulting in an insignificant optical response. The biosensor performance is also studied upon exposure to model Gram positive and negative bacterial lysates, suggesting preferential capture of E. coli cell fragments in the presented scheme. These OAK-based biosensors offer significant advantages in comparison with conventional antibody-based assays, in terms of their simple and cost-effective production, while providing numerous possible sequence combinations for designing new detection schemes.

Detection of trace heavy metal ions in water by nanostructured porous Si biosensors.

A generic biosensing platform, based on nanostructured porous Si (PSi), Fabry-Pérot thin films, for label-free monitoring of heavy metal ions in aqueous solutions by enzymatic activity inhibition, is described. First, we show a general detection assay by immobilizing horseradish peroxidase (HRP) within the oxidized PSi nanostructure and monitor its catalytic activity in real time by reflective interferometric Fourier transform spectroscopy. Optical studies reveal the high specificity and sensitivity of the HRP-immobilized PSi towards three metal ions (Ag(+) > Pb(2+) > Cu(2+)), with a detection limit range of 60-120 ppb. Next, we demonstrate the concept of specific detection of Cu(2+) ions (as a model heavy metal) by immobilizing Laccase, a multi-copper oxidase, within the oxidized PSi. The resulting biosensor allows for specific detection and quantification of copper ions in real water samples by monitoring the Laccase relative activity. The optical biosensing results are found to be in excellent agreement with those obtained by the gold standard analytical technique (ICP-AES) for all water samples. The main advantage of the presented biosensing concept is the ability to detect heavy metal ions at environmentally relevant concentrations using a simple and portable experimental setup, while the specific biosensor design can be tailored by varying the enzyme type.

Trap and track: designing self-reporting porous Si photonic crystals for rapid bacteria detection.

The task of rapid detection and identification of bacteria remains a major challenge in both medicine and industry. This work introduces a new concept for the design of self-reporting optical structures that can detect and quantify bacteria in real-time. The sensor is based on a two-dimensional periodic structure of porous Si photonic crystals in which the pore size is adjusted to fit the target bacteria cells (Escherichia coli). Spontaneous bacteria capture within the pores induces measurable changes in the zero-order reflectivity spectrum collected from the periodic structure. Confocal laser microscopy and electron microscopy confirm that the Escherichia coli cells are individually imprisoned within the porous array. A simple model is suggested to correlate the optical readout and the bacteria concentration and its predictions are found to be in good agreement with experimental results. In addition, we demonstrate that sensing scheme can be easily modified to potentially allow monitoring of concentration, growth and physiological state of bacteria cells. This generic platform can be tailored to target different microorganisms by tuning the array periodicity and its surface chemistry for rapid and label-free detection outside the laboratory environment.

Enhancing the performance of porous silicon biosensors: the interplay of nanostructure design and microfluidic integration.

This work presents the development and design of aptasensor employing porous silicon (PSi) Fabry‒Pérot thin films that are suitable for use as optical transducers for the detection of lactoferrin (LF), which is a protein biomarker secreted at elevated levels during gastrointestinal (GI) inflammatory disorders such as inflammatory bowel disease and chronic pancreatitis. To overcome the primary limitation associated with PSi biosensors-namely, their relatively poor sensitivity due to issues related to complex mass transfer phenomena and reaction kinetics-we employed two strategic approaches: First, we sought to optimize the porous nanostructure with respect to factors including layer thickness, pore diameter, and capture probe density. Second, we leveraged convection properties by integrating the resulting biosensor into a 3D-printed microfluidic system that also had one of two different micromixer architectures (i.e., staggered herringbone micromixers or microimpellers) embedded. We demonstrated that tailoring the PSi aptasensor significantly improved its performance, achieving a limit of detection (LOD) of 50 nM-which is >1 order of magnitude lower than that achieved using previously-developed biosensors of this type. Moreover, integration into microfluidic systems that incorporated passive and active micromixers further enhanced the aptasensor's sensitivity, achieving an additional reduction in the LOD by yet another order of magnitude. These advancements demonstrate the potential of combining PSi-based optical transducers with microfluidic technology to create sensitive label-free biosensing platforms for the detection of GI inflammatory biomarkers.

Photonic Si microwell architectures for rapid antifungal susceptibility determination of Candida auris.

We present the application of a photonic silicon chip-based optical sensor system for expeditious and phenotypic antifungal susceptibility testing. This label-free diagnostic assay optically monitors the growth of Candida auris at varying antifungal concentrations on a microwell-structured silicon chip in real-time, and antifungal susceptibility is detected within 6 h, four times faster than in the current gold standard method.

Dual-functionalized porous Si/hydrogel hybrid for label-free biosensing of organophosphorus compounds.

A multifunctional porous Si (PSi) nanostructure is designed to combine a responsive PSi/hydrogel hybrid interfaced with a biorecognition element to selectively recognize small model molecules, organophosphorus compounds (OPCs), of high biological importance. A pH-responsive poly(2-dimethylaminoethyl methacrylate) [poly(DMAEMA)] hydrogel is synthesized and patterned in situ within an oxidized PSi Fabry-Pérot thin film. The resulting new hybrid displays a well-defined, rapid, and reversible optical response to pH changes. We employ this hybrid as an optical transducer element in a biosensing scheme by integrating it with organophosphorus hydrolase (OPH), capable of selective OPC hydrolysis. The enzyme is immobilized onto the pore walls of the oxidized PSi scaffold, resulting in an array of catalytic nanoscale chambers for the degradation of OPCs. Thus, the biosensor function relies on diffusion of the OPCs hydrolysis products from the catalytic chambers, through the interconnected pores network, to the hybrid region triggering its optical response. Exposure to the model target analyte results in a rapid and reproducible change in the optical reflectivity spectrum of the hybrid, allowing for label-free detection and quantification of OPCs in a simple and reliable manner.

Picking up the pieces: a generic porous Si biosensor for probing the proteolytic products of enzymes.

A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry-Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL(-1), in comparison to previous reports, 1 mg mL(-1)), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.

Rising to the surface: capturing and detecting bacteria by rationally-designed surfaces.

Analytical microbiology has made substantial progress since its conception, starting from potato slices, through selective agar media, to engineered surfaces modified with capture probes. While the latter represents the dominant approach in designing sensors for bacteria detection, the importance of sensor surface properties is frequently ignored. Herein, we highlight their significant role in the complex process of bacterial transition from planktonic to sessile, representing the first and critical step in bacteria detection. We present the main surface features and discuss their effect on the bio-solid interface and the resulting sensing capabilities for both flat and particulate systems. The concepts of rationally-designed surfaces for enhanced bacterial detection are presented with recent examples of sensors (capture probe-free) relying solely on surface cues.

Self-assembled fatty acid crystalline coatings display superhydrophobic antimicrobial properties.

Superhydrophobicity is a well-known wetting phenomenon found in numerous plants and insects. It is achieved by the combination of the surface's chemical properties and its surface roughness. Inspired by nature, numerous synthetic superhydrophobic surfaces have been developed for various applications. Designated surface coating is one of the fabrication routes to achieve the superhydrophobicity. Yet, many of these coatings, such as fluorine-based formulations, may pose severe health and environmental risks, limiting their applicability. Herein, we present a new family of superhydrophobic coatings comprised of natural saturated fatty acids, which are not only a part of our daily diet, but can be produced from renewable feedstock, providing a safe and sustainable alternative to the existing state-of-the-art. These crystalline coatings are readily fabricated via single-step deposition routes, namely thermal deposition or spray-coating. The fatty acids self-assemble into highly hierarchical crystalline structures exhibiting a water contact angle of ∼165° and contact angle hysteresis lower than 6°, while their properties and morphology depend on the specific fatty acid used as well as on the deposition technique. Moreover, the fatty acid coatings demonstrate excellent thermal stability. Importantly, this new family of coatings displays excellent anti-biofouling and antimicrobial properties against Escherichia coli and Listeria innocua, used as relevant model Gram-negative and Gram-positive bacteria, respectively. These multifunctional coatings hold immense potential for application in numerous fields, ranging from food safety to biomedicine, offering sustainable and safe solutions.

Accurate Prediction of Antimicrobial Susceptibility for Point-of-Care Testing of Urine in Less than 90 Minutes via iPRISM Cassettes.

The extensive and improper use of antibiotics has led to a dramatic increase in the frequency of antibiotic resistance among human pathogens, complicating infectious disease treatments. In this work, a method for rapid antimicrobial susceptibility testing (AST) is presented using microstructured silicon diffraction gratings integrated into prototype devices, which enhance bacteria-surface interactions and promote bacterial colonization. The silicon microstructures act also as optical sensors for monitoring bacterial growth upon exposure to antibiotics in a real-time and label-free manner via intensity-based phase-shift reflectometric interference spectroscopic measurements (iPRISM). Rapid AST using clinical isolates of Escherichia coli (E. coli) from urine is established and the assay is applied directly on unprocessed urine samples from urinary tract infection patients. When coupled with a machine learning algorithm trained on clinical samples, the iPRISM AST is able to predict the resistance or susceptibility of a new clinical sample with an Area Under the Receiver Operating Characteristic curve (AUC) of ∼ 0.85 in 1 h, and AUC > 0.9 in 90 min, when compared to state-of-the-art automated AST methods used in the clinic while being an order of magnitude faster.

Advancing nanostructured porous si-based optical transducers for label free bacteria detection.

Optical label-free porous Si-based biosensors for rapid bacteria detection are introduced. The biosensors are designed to directly capture the target bacteria cells onto their surface with no prior sample processing (such as cell lysis). Two types of nanostructured optical transducers based on oxidized porous Si (PSiO(2)) Fabry-Pérot thin films are synthesized and used to construct the biosensors. In the first system, we graft specific monoclonal antibodies (immunoglobulin G's) onto a neat electrochemically-machined PSiO(2) surface, based on well-established silanization chemistry. The second biosensor class consists of a PSiO(2)/hydrogel hybrid. The hydrogel, polyacrylamide, is synthesized in situ within the nanostructured PSiO(2) host and conjugated with specific monoclonal antibodies to provide the active component of the biosensor. Exposure of these modified-surfaces to the target bacteria results in "direct-cell-capture" onto the biosensor surface. These specific binding events induce predictable changes in the thin-film optical interference spectrum of the biosensor. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations, in the range of 10(3)-10(5) cell/ml, within minutes. The sensing performance of the two different platforms, in terms of their stability in aqueous media and sensitivity, are compared and discussed. This preliminary study suggests that biosensors based on PSiO(2)/hydrogel hybrid outperform the neat PSiO(2) system.

Lab-on-a-Chip Devices for Point-of-Care Medical Diagnostics.

The recent coronavirus (COVID-19) pandemic has underscored the need to move from traditional lab-centralized diagnostics to point-of-care (PoC) settings. Lab-on-a-chip (LoC) platforms facilitate the translation to PoC settings via the miniaturization, portability, integration, and automation of multiple assay functions onto a single chip. For this purpose, paper-based assays and microfluidic platforms are currently being extensively studied, and much focus is being directed towards simplifying their design while simultaneously improving multiplexing and automation capabilities. Signal amplification strategies are being applied to improve the performance of assays with respect to both sensitivity and selectivity, while smartphones are being integrated to expand the analytical power of the technology and promote its accessibility. In this chapter, we review the main technologies in the field of LoC platforms for PoC medical diagnostics and survey recent approaches for improving these assays.