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.

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.

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.

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.

A 3D-printed microfluidic gradient generator with integrated photonic silicon sensors for rapid antimicrobial susceptibility testing.

With antimicrobial resistance becoming a major threat to healthcare settings around the world, there is a paramount need for rapid point-of-care antimicrobial susceptibility testing (AST) diagnostics. Unfortunately, most currently available clinical AST tools are lengthy, laborious, or are simply inappropriate for point-of-care testing. Herein, we design a 3D-printed microfluidic gradient generator that automatically produces two-fold dilution series of clinically relevant antimicrobials. We first establish the compatibility of these generators for classical AST (i.e., broth microdilution) and then extend their application to include a complete on-chip label-free and phenotypic AST. This is accomplished by the integration of photonic silicon chips, which provide a preferential surface for microbial colonization and allow optical tracking of bacterial behavior and growth at a solid-liquid interface in real-time by phase shift reflectometric interference spectroscopic measurements (PRISM). Using Escherichia coli and ciprofloxacin as a model pathogen-drug combination, we successfully determine the minimum inhibitory concentration within less than 90 minutes. This gradient generator-based PRISM assay provides an integrated AST device that is viable for convenient point-of-care testing and offers a promising and most importantly, rapid alternative to current clinical practices, which extend to 8-24 h.

Gold Nanorod-Incorporated Halloysite Nanotubes Functionalized with Antibody for Superior Antibacterial Photothermal Treatment.

The global spread of antibiotic-resistant strains, and the need to protect the microflora from non-specific antibiotics require more effective and selective alternatives. In this work, we demonstrate for the first time a superior antibacterial photothermal effect of plasmonic gold nanorods (AuNRs) via their incorporation onto natural clay halloysite nanotubes (HNTs), which were functionalized with anti-E. coli antibodies (Ab-HNTs). AuNRs were incorporated onto the Ab-HNTs through a facile freeze-thaw cycle, and antibody integrity following the incorporation was confirmed via infrared spectroscopy and fluorescence immunolabeling. The incorporation efficiency was studied using UV-Vis absorption and transmission electron microscopy (TEM). Mixtures of E. coli and AuNR-Ab-HNTs hybrids or free AuNRs were irradiated with an 808 nm laser at 3-4 W cm-2, and the resulting photothermal antibacterial activity was measured via plate count. The irradiated AuNR-Ab-HNTs hybrids exerted an 8-fold higher antibacterial effect compared to free AuNR under 3.5 W cm-2; whereas the latter induced a 6 °C-higher temperature elevation. No significant antibacterial activity was observed for the AuNR-Ab-HNTs hybrid against non-target bacteria species (Serratia marcescens and Staphylococcus epidermidis). These findings are ascribed to the localization of the photothermal ablation due to the binding of the antibody-functionalized clay to its target bacteria, as supported through TEM imaging. In the future, the HNTs-based selective carriers presented herein could be tailored with other antibacterial nanoparticles or against another microorganism via the facile adjustment of the immobilized antibody.

Shining light in blind alleys: deciphering bacterial attachment in silicon microstructures.

With new advances in infectious disease, antifouling surfaces, and environmental microbiology research comes the need to understand and control the accumulation and attachment of bacterial cells on a surface. Thus, we employ intrinsic phase-shift reflectometric interference spectroscopic measurements of silicon diffraction gratings to non-destructively observe the interactions between bacterial cells and abiotic, microstructured surfaces in a label-free and real-time manner. We conclude that the combination of specific material characteristics (i.e., substrate surface charge and topology) and characteristics of the bacterial cells (i.e., motility, cell charge, biofilm formation, and physiology) drive bacteria to adhere to a particular surface, often leading to a biofilm formation. Such knowledge can be exploited to predict antibiotic efficacy and biofilm formation, and enhance surface-based biosensor development, as well as the design of anti-biofouling strategies.

Aptasensors versus immunosensors-Which will prevail?

Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element.

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.

Design considerations of aptasensors for continuous monitoring of biomarkers in digestive tract fluids.

We present a porous Si (PSi)-based label-free optical biosensor for sensitive and continuous detection of a model target protein biomarker in gastrointestinal (GI) tract fluids. The biosensing platform is designed to continuously monitor its target protein within the complex GI fluids without sample preparation and washing steps. An oxidized PSi Fabry-Pérot thin films are functionalized with aptamers, which are used as the capture probes. The optical response of the aptamer-conjugated PSi is studied upon exposure to unprocessed GI fluids, originated from domestic pigs, spiked with the target protein. We investigate biological and chemical surface passivation methods to stabilize the surface and reduce non-specific adsorption of interfering proteins and molecules within the GI fluids. For the passivated PSi aptasensor we simulate continuous in vivo biosensing conditions, demonstrating that the aptasensor could successfully detect the target in a continuous manner without any need for surface washing after the target protein binding events, at a clinically relevant range. Furthermore, we simulate biosensing conditions within a smart capsule, in which the aptasensor is occasionally exposed to GI fluids in flow or via repeated cycles of injection and static incubation events. Such biosensor can be implemented within ingestible capsule devices and used for in situ biomarker detection in the GI tract.

Paving the Way to Overcome Antifungal Drug Resistance: Current Practices and Novel Developments for Rapid and Reliable Antifungal Susceptibility Testing.

The past year has established the link between the COVID-19 pandemic and the global spread of severe fungal infections; thus, underscoring the critical need for rapid and realizable fungal disease diagnostics. While in recent years, health authorities, such as the Centers for Disease Control and Prevention, have reported the alarming emergence and spread of drug-resistant pathogenic fungi and warned against the devastating consequences, progress in the diagnosis and treatment of fungal infections is limited. Early diagnosis and patient-tailored therapy are established to be key in reducing morbidity and mortality associated with fungal (and cofungal) infections. As such, antifungal susceptibility testing (AFST) is crucial in revealing susceptibility or resistance of these pathogens and initiating correct antifungal therapy. Today, gold standard AFST methods require several days for completion, and thus this much delayed time for answer limits their clinical application. This review focuses on the advancements made in developing novel AFST techniques and discusses their implications in the context of the practiced clinical workflow. The aim of this work is to highlight the advantages and drawbacks of currently available methods and identify the main gaps hindering their progress toward clinical application.

Porous Silicon-Based Aptasensors: Toward Cancer Protein Biomarker Detection.

The anterior gradient homologue-2 (AGR2) protein is an attractive biomarker for various types of cancer. In pancreatic cancer, it is secreted to the pancreatic juice by premalignant lesions, which would be an ideal stage for diagnosis. Thus, designing assays for the sensitive detection of AGR2 would be highly valuable for the potential early diagnosis of pancreatic and other types of cancer. Herein, we present a biosensor for label-free AGR2 detection and investigate approaches for enhancing the aptasensor sensitivity by accelerating the target mass transfer rate and reducing the system noise. The biosensor is based on a nanostructured porous silicon thin film that is decorated with anti-AGR2 aptamers, where real-time monitoring of the reflectance changes enables the detection and quantification of AGR2, as well as the study of the diffusion and target-aptamer binding kinetics. The aptasensor is highly selective for AGR2 and can detect the protein in simulated pancreatic juice, where its concentration is outnumbered by orders of magnitude by numerous proteins. The aptasensor's analytical performance is characterized with a linear detection range of 0.05-2 mg mL-1, an apparent dissociation constant of 21 ± 1 µM, and a limit of detection of 9.2 µg mL-1 (0.2 µM), which is attributed to mass transfer limitations. To improve the latter, we applied different strategies to increase the diffusion flux to and within the nanostructure, such as the application of isotachophoresis for the preconcentration of AGR2 on the aptasensor, mixing, or integration with microchannels. By combining these approaches with a new signal processing technique that employs Morlet wavelet filtering and phase analysis, we achieve a limit of detection of 15 nM without compromising the biosensor's selectivity and specificity.

Morlet Wavelet Filtering and Phase Analysis to Reduce the Limit of Detection for Thin Film Optical Biosensors.

The ultimate detection limit of optical biosensors is often limited by various noise sources, including those introduced by the optical measurement setup. While sophisticated modifications to instrumentation may reduce noise, a simpler approach that can benefit all sensor platforms is the application of signal processing to minimize the deleterious effects of noise. In this work, we show that applying complex Morlet wavelet convolution to Fabry-Pérot interference fringes characteristic of thin film reflectometric biosensors effectively filters out white noise and low-frequency reflectance variations. Subsequent calculation of the average difference in extracted phase between the filtered analyte and reference signals enables a significant reduction in the limit of detection (LOD). This method is applied on experimental data sets of thin film porous silicon sensors (PSi) in buffered solution and complex media obtained from two different laboratories. The demonstrated improvement in the LOD achieved using wavelet convolution and average phase difference paves the way for PSi optical biosensors to operate with clinically relevant detection limits for medical diagnostics, environmental monitoring, and food safety.

Antibody-Functionalized Halloysite Nanotubes for Targeting Bacterial Cells.

Halloysite nanotubes (HNTs) are naturally occurring tubular clay particles which have emerged in recent years as a promising nanomaterial for numerous applications. Specifically, HNTs' large pore volume and high specific surface area in combination with their biocompatibility make them ideal nanocarriers for bioactive compounds. This research aims to design and synthesize functionalized HNTs, which could selectively bind to target bacterial cells in suspension. Such a system can allow us to treat target cells within a challenging heterogeneous population, such as contaminated ecosystems or gut flora. HNTs functionalization is achieved by immobilizing specific antibodies onto the nanotube surface. The synthetic route is realized by the following subsequent steps: acidic etching of the HNTs, silanization of reactive surface hydroxyls, conjugation of protein A, and oriented immobilization of the antibody. HNT functionalization is studied by a set of analytical techniques including attenuated total reflectance Fourier-transform infrared spectroscopy, zeta potential measurements, thermal gravimetric analysis, scanning and transmission electron microscopy, as well as fluorescence microscopy. The selective binding of the functionalized HNTs to their target bacteria is observed upon incubation with live homogenous and heterogeneous cultures using fluorescence microscopy and high-throughput flow cytometry. Plate count and live/dead staining experiments demonstrate the biocompatibility of the antibody-HNT hybrid with its target bacteria. The suggested HNT-based smart carrier constitutes a generic platform for targeted delivery that could be selectively tailored against any microorganism by facile antibody adjustment.

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.

Halloysite nanotubes - the nano-bio interface.

The numerous biological applications of nanoparticles in general and nano-clays in particular are rooted in understanding and harnessing their dynamic nano-bio interface. Among clays, the intrinsically-mesoporous halloysite nanotubes (HNTs) have emerged in recent years as promising nanomaterials. The diverse interactions of these nanotubes with living cells, encompassing electrostatic, van der Waals, and ion exchange, along with cellular response, are crucial in determining the behaviour of HNTs in biological systems. Thus, rational engineering of the nanotube properties allows for vast applications ranging from bacteria encapsulation for bioremediation, through algae flocculation for aquaculture, to intracellular drug delivery. This review summarizes the many aspects of the nano-bio interface of HNTs with different cell types (bacteria, algae and fungi, and mammalian cells), highlighting biocompatibility/bio-adverse properties, interaction mechanisms, and the latest cutting-edge technologies.

Antifungal Susceptibility Testing of Aspergillus niger on Silicon Microwells by Intensity-Based Reflectometric Interference Spectroscopy.

There is a demonstrated and paramount need for rapid, reliable infectious disease diagnostics, particularly those for invasive fungal infections. Current clinical determinations for an appropriate antifungal therapy can take up to 3 days using current antifungal susceptibility testing methods, a time-to-readout that can prove detrimental for immunocompromised patients and promote the spread of antifungal resistant pathogens. Herein, we demonstrate the application of intensity-based reflectometric interference spectroscopic measurements (termed iPRISM) on microstructured silicon sensors for use as a rapid, phenotypic antifungal susceptibility test. This diagnostic platform optically tracks morphological changes of fungi corresponding to conidia growth and hyphal colonization at a solid-liquid interface in real time. Using Aspergillus niger as a model fungal pathogen, we can determine the minimal inhibitory concentration of clinically relevant antifungals within 12 h. This assay allows for expedited detection of fungal growth and provides a label-free alternative to broth microdilution and agar diffusion methods, with the potential to be used for point-of-care diagnostics.

Mass Transfer Limitations of Porous Silicon-Based Biosensors for Protein Detection.

Porous silicon (PSi) thin films have been widely studied for biosensing applications, enabling label-free optical detection of numerous targets. The large surface area of these biosensors has been commonly recognized as one of the main advantages of the PSi nanostructure. However, in practice, without application of signal amplification strategies, PSi-based biosensors suffer from limited sensitivity, compared to planar counterparts. Using a theoretical model, which describes the complex mass transport phenomena and reaction kinetics in these porous nanomaterials, we reveal that the interrelated effect of bulk and hindered diffusion is the main limiting factor of PSi-based biosensors. Thus, without significantly accelerating the mass transport to and within the nanostructure, the target capture performance of these biosensors would be comparable, regardless of the nature of the capture probe-target pair. We use our model to investigate the effect of various structural and biosensor characteristics on the capture performance of such biosensors and suggest rules of thumb for their optimization.

Aptasensors for Point-of-Care Detection of Small Molecules.

Aptamers, a group of nucleic acids which can specifically bind to a target molecule, have drawn extensive interest over the past few decades. For analytics, aptamers represent a viable alternative to gold-standard antibodies due to their oligonucleic nature combined with advantageous properties, including higher stability in harsh environments and longer shelf-life. Indeed, over the last decade, aptamers have been used in numerous bioanalytical assays and in various point-of-care testing (POCT) platforms. The latter allows for rapid on-site testing and can be performed outside a laboratory by unskilled labor. Aptamer technology for POCT is not limited just to medical diagnostics; it can be used for a range of applications, including environmental monitoring and quality control. In this review, we critically examine the use of aptamers in POCT with an emphasis on their advantages and limitations. We also examine the recent success of aptasensor technology and how these findings pave the way for the analysis of small molecules in POCT and other health-related applications. Finally, the current major limitations of aptamers are discussed, and possible approaches for overcoming these challenges are presented.