Magnetic micromixing for highly sensitive detection of glyphosate in tap water by colorimetric immunosensor.

Glyphosate is the most widely used herbicide in the world and, in view of its toxicity, there is a quest for easy-to-use, but reliable methods to detect it in water. To address this issue, we realized a simple, rapid, and highly sensitive immunosensor based on gold coated magnetic nanoparticles (MNPs@Au) to detect glyphosate in tap water. Not only the gold shell provided a sensitive optical transduction of the biological signal - through the shift of the local surface plasmon resonance (LSPR) entailed by the nanoparticle aggregation -, but it also allowed us to use an effective photochemical immobilization technique to tether oriented antibodies straight on the nanoparticles surface. While such a feature led to aggregates in which the nanoparticles were at close proximity each other, the magnetic properties of the core offered us an efficient tool to steer the nanoparticles by a rotating magnetic field. As a result, the nanoparticle aggregation in presence of the target could take place at higher rate (enhanced diffusion) with significant improvement in sensitivity. As a matter of fact, the combination of plasmonic and magnetic properties within the same nanoparticles allowed us to realize a colorimetric biosensor with a limit of detection (LOD) of 20 ng∙L-1.

Multifunctional Core@Satellite Magnetic Particles for Magnetoresistive Biosensors.

Magnetoresistive (MR) biosensors combine distinctive features such as small size, low cost, good sensitivity, and propensity to be arrayed to perform multiplexed analysis. Magnetic nanoparticles (MNPs) are the ideal target for this platform, especially if modified not only to overcome their intrinsic tendency to aggregate and lack of stability but also to realize an interacting surface suitable for biofunctionalization without strongly losing their magnetic response. Here, we describe an MR biosensor in which commercial MNP clusters were coated with gold nanoparticles (AuNPs) and used to detect human IgG in water using an MR biochip that comprises six sensing regions, each one containing five U-shaped spin valve sensors. The isolated AuNPs (satellites) were stuck onto an aggregate of individual iron oxide crystals (core) so that the resulting core@satellite magnetic particles (CSMPs) could be functionalized by the photochemical immobilization technique-an easy procedure that leads to oriented antibodies immobilized upright onto gold. The morphological, optical, hydrodynamic, magnetic, and surface charge properties of CSMPs were compared with those exhibited by the commercial MNP clusters showing that the proposed coating procedure endows the MNP clusters with stability and ductility without being detrimental to magnetic properties. Eventually, the high-performance MR biosensor allowed us to detect human IgG in water with a detection limit of 13 pM (2 ng mL-1). Given its portability, the biosensor described in this paper lends itself to a point-of-care device; moreover, the features of the MR biochip also make it suitable for multiplexed analysis.

Advances and emerging challenges in MXenes and their nanocomposites for biosensing applications.

Two-dimensional materials have unique properties and their better functionality has created new paradigms in the field of sensing. Over the past decade, a new family of 2D materials known as MXenes has emerged as a promising material for numerous applications, including biosensing. Their metallic conductivity, rich surface chemistry, hydrophilicity, good biocompatibility, and high anchoring capacity for biomaterials make them an attractive candidate to detect a variety of analytes. Despite such notable properties, there are certain limitations associated with them. This review aims to present a detailed survey of MXene's synthesis; in particular, their superiority in the field of biosensing as compared to other 2D materials is addressed. Their low oxidative stability is still an open challenge, and recent investigations on MXene's oxidation are summarized. The hexagonal stacking network of MXenes acts as a distinctive matrix to load nanoparticles, and the embedded nanoparticles can bind an excess number of biomolecules (e.g., antibodies) thereby improving biosensor performance. We will also discuss the synthesis and corresponding performance of MXenes nanocomposites with noble metal nanoparticles and magnetic nanoparticles. Furthermore, Nb and Ti2C-based MXenes, and Ti3C2-MXene sandwich immunoassays are also reviewed in view of their importance. Different aspects and challenges associated with MXenes (from their synthesis to final applications) and the future perspectives described give new directions to fabricate novel biosensors.

Loading of Polydimethylsiloxane with a Human ApoB-Derived Antimicrobial Peptide to Prevent Bacterial Infections.

BACKGROUND: medical device-induced infections affect millions of lives worldwide and innovative preventive strategies are urgently required. Antimicrobial peptides (AMPs) appear as ideal candidates to efficiently functionalize medical devices surfaces and prevent bacterial infections. In this scenario, here, we produced antimicrobial polydimethylsiloxane (PDMS) by loading this polymer with an antimicrobial peptide identified in human apolipoprotein B, r(P)ApoBLPro. METHODS: once obtained loaded PDMS, its structure, anti-infective properties, ability to release the peptide, stability, and biocompatibility were evaluated by FTIR spectroscopy, water contact angle measurements, broth microdilution method, time-killing kinetic assays, quartz crystal microbalance analyses, MTT assays, and scanning electron microscopy analyses. RESULTS: PDMS was loaded with r(P)ApoBLPro peptide which was found to be present not only in the bulk matrix of the polymer but also on its surface. ApoB-derived peptide was found to retain its antimicrobial properties once loaded into PDMS and the antimicrobial material was found to be stable upon storage at 4 °C for a prolonged time interval. A gradual and significant release (70% of the total amount) of the peptide from PDMS was also demonstrated upon 400 min incubation and the antimicrobial material was found to be endowed with anti-adhesive properties and with the ability to prevent biofilm attachment. Furthermore, PDMS loaded with r(P)ApoBLPro peptide was found not to affect the viability of eukaryotic cells. CONCLUSIONS: an easy procedure to functionalize PDMS with r(P)ApoBLPro peptide has been here developed and the obtained functionalized material has been found to be stable, antimicrobial, and biocompatible.

A Combinatorial Neural Network Analysis Reveals a Synergistic Behaviour of Multiparametric Magnetic Resonance and Prostate Health Index in the Identification of Clinically Significant Prostate Cancer.

BACKGROUND: The widespread use of prostate specific antigen (PSA) caused high rate of overdiagnosis. Overdiagnosis leads to unnecessary definitive treatments of prostate cancer (PCa) with detrimental side effects, such as erectile dysfunction and incontinence. The aim of this study was to evaluate the feasibility of an artificial neural network-based approach to develop a combinatorial model including prostate health index (PHI) and multiparametric magnetic resonance (mpMRI) to recognize clinically significant PCa at initial diagnosis. METHODS: To this aim we prospectively enrolled 177 PCa patients who underwent radical prostatectomy and had received PHI tests and mpMRI before surgery. We used artificial neural network to develop models that can identify aggressive PCa efficiently. The model receives as an input PHI plus PI-RADS score. RESULTS: The output of the model is an estimate of the presence of a low or high Gleason score. After training on a dataset of 135 samples and optimization of the variables, the model achieved values of sensitivity as high as 80% and 68% specificity. CONCLUSIONS: Our preliminary study suggests that combining mpMRI and PHI may help to better estimate the risk category of PCa at initial diagnosis, allowing a personalized treatment approach. The efficiency of the method can be improved even further by training the model on larger datasets.

Solid-state optical properties of self-assembling amyloid-like peptides with different charged states at the terminal ends.

The self-assembling of small peptides not only leads to the formation of intriguing nanoarchitectures, but also generates materials with unexpected functional properties. Oligopeptides can form amyloid-like cross-ß assemblies that are able to emit intrinsic photoluminescence (PL), over the whole near-UV/visible range, whose origin is still largely debated. As proton transfer between the peptide chain termini within the assembly is one of the invoked interpretations of this phenomenon, we here evaluated the solid state PL properties of a series of self-assembled hexaphenylalanine peptides characterized by a different terminal charge state. Overall, our data indicate that the charge state of these peptides has a marginal role in the PL emission as all systems exhibit very similar multicolour PL associated with a violation of the Kasha's rule. On the other hand, charged/uncharged ends occasionally produce differences in the quantum yields. The generality of these observations has been proven by extending these analyses to the Aß16-21 peptide. Collectively, the present findings provide useful information for deciphering the code that links the spectroscopic properties of these assemblies to their structural/electronic features.

Use of some cost-effective technologies for a routine clinical pathology laboratory.

Classically, the need for highly sophisticated instruments with important economic costs has been a major limiting factor for clinical pathology laboratories, especially in developing countries. With the aim of making clinical pathology more accessible, a wide variety of free or economical technologies have been developed worldwide in the last few years. 3D printing and Arduino approaches can provide up to 94% economical savings in hardware and instrumentation in comparison to commercial alternatives. The vast selection of point-of-care-tests (POCT) currently available also limits the need for specific instruments or personnel, as they can be used almost anywhere and by anyone. Lastly, there are dozens of free and libre digital tools available in health informatics. This review provides an overview of the state-of-the-art on cost-effective alternatives with applications in routine clinical pathology laboratories. In this context, a variety of technologies including 3D printing and Arduino, lateral flow assays, plasmonic biosensors, and microfluidics, as well as laboratory information systems, are discussed. This review aims to serve as an introduction to different technologies that can make clinical pathology more accessible and, therefore, contribute to achieve universal health coverage.

Fluorescence Emission of Self-assembling Amyloid-like Peptides: Solution versus Solid State.

Analysis of the intrinsic UV-visible fluorescence exhibited by self-assembling amyloid-like peptides in solution and in solid the state highlights that their physical state has a profound impact on the optical properties. In the solid state, a linear dependence of the fluorescence emission peaks as a function of excitation wavelength is detected. On the contrary, an excitation-independent emission is observed in solution. The present findings constitute a valuable benchmark for current and future explanations of the fluorescence emission by amyloids.

Reply to Jue et al. Value of MRI to Improve Deep Learning Model That Identifies High-Grade Prostate Cancer. Comment on "Gentile et al. Optimized Identification of High-Grade Prostate Cancer by Combining Different PSA Molecular Forms and PSA Density in a Deep Learning Model. Diagnostics 2021, 11, 335".

In their comment "Value of MRI to Improve Deep Learning Model That Identifies High-Grade Prostate Cancer [...].

Optimized Identification of High-Grade Prostate Cancer by Combining Different PSA Molecular Forms and PSA Density in a Deep Learning Model.

After skin cancer, prostate cancer (PC) is the most common cancer among men. The gold standard for PC diagnosis is based on the PSA (prostate-specific antigen) test. Based on this preliminary screening, the physician decides whether to proceed with further tests, typically prostate biopsy, to confirm cancer and evaluate its aggressiveness. Nevertheless, the specificity of the PSA test is suboptimal and, as a result, about 75% of men who undergo a prostate biopsy do not have cancer even if they have elevated PSA levels. Overdiagnosis leads to unnecessary overtreatment of prostate cancer with undesirable side effects, such as incontinence, erectile dysfunction, infections, and pain. Here, we used artificial neuronal networks to develop models that can diagnose PC efficiently. The model receives as an input a panel of 4 clinical variables (total PSA, free PSA, p2PSA, and PSA density) plus age. The output of the model is an estimate of the Gleason score of the patient. After training on a dataset of 190 samples and optimization of the variables, the model achieved values of sensitivity as high as 86% and 89% specificity. The efficiency of the method can be improved even further by training the model on larger datasets.

Randomly positioned gold nanoparticles as fluorescence enhancers in apta-immunosensor for malaria test.

A plasmon-enhanced fluorescence-based antibody-aptamer biosensor - consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly - is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies - covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) - and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10-15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays.

The Union Is Strength: The Synergic Action of Long Fatty Acids and a Bacteriophage against Xanthomonas campestris Biofilm.

Xanthomonas campestris pv. campestris is known as the causative agent of black rot disease, which attacks mainly crucifers, severely lowering their global productivity. One of the main virulence factors of this pathogen is its capability to penetrate and form biofilm structures in the xylem vessels. The discovery of novel approaches to crop disease management is urgent and a possible treatment could be aimed at the eradication of biofilm, although anti-biofilm approaches in agricultural microbiology are still rare. Considering the multifactorial nature of biofilm, an effective approach against Xanthomonas campestris implies the use of a multi-targeted or combinatorial strategy. In this paper, an anti-biofilm strategy based on the use of fatty acids and the bacteriophage (Xccφ1)-hydroxyapatite complex was optimized against Xanthomonas campestris mature biofilm. The synergic action of these elements was demonstrated and the efficient removal of Xanthomonas campestris mature biofilm was also proven in a flow cell system, making the proposed approach an effective solution to enhance plant survival in Xanthomonas campestris infections. Moreover, the molecular mechanisms responsible for the efficacy of the proposed treatment were explored.

Ultrasensitive antibody-aptamer plasmonic biosensor for malaria biomarker detection in whole blood.

Development of plasmonic biosensors combining reliability and ease of use is still a challenge. Gold nanoparticle arrays made by block copolymer micelle nanolithography (BCMN) stand out for their scalability, cost-effectiveness and tunable plasmonic properties, making them ideal substrates for fluorescence enhancement. Here, we describe a plasmon-enhanced fluorescence immunosensor for the specific and ultrasensitive detection of Plasmodium falciparum lactate dehydrogenase (PfLDH)-a malaria marker-in whole blood. Analyte recognition is realized by oriented antibodies immobilized in a close-packed configuration via the photochemical immobilization technique (PIT), with a top bioreceptor of nucleic acid aptamers recognizing a different surface of PfLDH in a sandwich conformation. The combination of BCMN and PIT enabled maximum control over the nanoparticle size and lattice constant as well as the distance of the fluorophore from the sensing surface. The device achieved a limit of detection smaller than 1 pg/mL (<30 fM) with very high specificity without any sample pretreatment. This limit of detection is several orders of magnitude lower than that found in malaria rapid diagnostic tests or even commercial ELISA kits. Thanks to its overall dimensions, ease of use and high-throughput analysis, the device can be used as a substrate in automated multi-well plate readers and improve the efficiency of conventional fluorescence immunoassays.

Core-Shell Magnetic Nanoparticles for Highly Sensitive Magnetoelastic Immunosensor.

A magnetoelastic (ME) biosensor for wireless detection of analytes in liquid is described. The ME biosensor was tested against human IgG in the range 0-20 µg∙mL-1. The sensing elements, anti-human IgG produced in goat, were immobilized on the surface of the sensor by using a recently introduced photochemical immobilization technique (PIT), whereas a new amplification protocol exploiting gold coated magnetic nanoparticles (core-shell nanoparticles) is demonstrated to significantly enhance the sensitivity. The gold nanoflowers grown on the magnetic core allowed us to tether anti-human IgG to the nanoparticles to exploit the sandwich detection scheme. The experimental results show that the 6 mm × 1 mm × 30 µm ME biosensor with an amplification protocol that uses magnetic nanoparticles has a limit of detection (LOD) lower than 1 nM, works well in water, and has a rapid response time of few minutes. Therefore, the ME biosensor is very promising for real-time wireless detection of pathogens in liquids and for real life diagnostic purpose.

Time-gated luminescence imaging of positively charged poly-l-lysine-coated highly microporous silicon nanoparticles in living Hydra polyp.

The development of non-toxic fluorescent agents alternative to heavy metal-based semiconductor quantum dots represents a relevant topic in biomedical research and in particular in the bioimaging field. Herein, highly luminescent Si─H terminal microporous silicon nanoparticles with µs-lived photoemission are chemically modified with a two step process and successfully used as label-free probes for in vivo time-gated luminescence imaging. In this context, Hydra vulgaris is used as model organism for in vivo study and validity assessment. The application of time gating allows to pursue an effective sorting of the signals, getting rid of the most common sources of noise that are fast-decay tissue autofluorescence and excitation scattering within the tissue. Indeed, an enhancement by a factor ~ 20 in the image signal-to-noise ratio can be estimated.

Screen Printed Based Impedimetric Immunosensor for Rapid Detection of Escherichia coli in Drinking Water.

The development of a simple and low cost electrochemical impedance immunosensor based on screen printed gold electrode for rapid detection of Escherichia coli in water is reported. The immunosensor is fabricated by immobilizing anti-E. coli antibodies onto a gold surface in a covalent way by the photochemical immobilization technique, a simple procedure able to bind antibodies upright onto gold surfaces. Impedance spectra are recorded in 0.01 M phosphate buffer solution (PBS) containing 10 mM Fe(CN)63-/Fe(CN)64- as redox probe. The Nyquist plots can be modelled with a modified Randles circuit, identifying the charge transfer resistance Rct as the relevant parameter after the immobilization of antibodies, the blocking with BSA and the binding of E. coli. The introduction of a standard amplification procedure leads to a significant enhancement of the impedance increase, which allows one to measure E. coli in drinking water with a limit of detection of 3 × 101 CFU mL-1 while preserving the rapidity of the method that requires only 1 h to provide a "yes/no" response. Additionally, by applying the Langmuir adsorption model, we are able to describe the change of Rct in terms of the "effective" electrode, which is modified by the detection of the analyte whose microscopic conducting properties can be quantified.

A multi-scale time-resolved study of photoactivated dynamics in 5-benzyl uracil, a model for DNA/protein interactions.

We combine fluorescence up-conversion and time correlated single photon counting experiments to investigate the 5-benzyl uracil excited state dynamics in methanol from 100 fs up to several ns. This molecule has been proposed as a model for DNA/protein interactions. Our results show emission bands at about 310 and 350 nm that exhibit bi-exponential sub-ps decays. Calculations, including solvent effects by a mixed discrete-continuum model, indicate that the Franck Condon region is characterized by significant coupling between the excited states of the benzyl and the uracil moieties, mirrored by the short-lived emission at 310 nm. Two main ground state recovery pathways are identified, both contributing to the 350 nm emission. The first 'photophysical' decay path involves a ππ* excited state localized on the uracil and is connected to the ground electronic state by an easily accessible crossing with S0, accounting for the short lifetime component. Simulations indicate that a possible second pathway is characterized by exciplex formation, with partial benzene → uracil charge transfer character, that may lead instead to photocyclization. The relevance of our results is discussed in view of the photoactivated dynamics of DNA/protein complexes, with implications on their interaction mechanisms.

Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy.

Surface functionalization is a key step in biosensing since it is the basis of an effective analyte recognition. Among all the bioreceptors, antibodies (Abs) play a key role thanks to their superior specificity, although the available immobilization strategies suffer from several drawbacks. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has been shown to be a quick, easy-to-use and very effective method to tether Abs oriented upright by means of thiols produced via tryptophan mediated disulphide bridge reduction. Although the molecular mechanism of this process is quite well identified, the detailed morphology of the immobilized antibodies is still elusive due to inherent difficulties related to the microscopy imaging of Abs. The combination of Mass Spectrometry, Surface-Enhanced Raman Spectroscopy and Ellman's assay demonstrates that Abs irradiated under the conditions in which PIT is realized show only two effective disulphide bridges available for binding. They are located in the constant region of the immunoglobulin light chain so that the most likely position Ab assumes is side-on, i.e. with one Fab (i.e. the antigen binding portion of the antibody) exposed to the solution. This is not a limitation of the recognition efficiency in view of the intrinsic flexibility of the Ab structure, which makes the free Fab able to sway in the solution, a feature of great importance in many biosensing applications.

Effects of human antimicrobial cryptides identified in apolipoprotein B depend on specific features of bacterial strains.

Cationic Host Defense Peptides (HDPs) are endowed with a broad variety of activities, including direct antimicrobial properties and modulatory roles in the innate immune response. Even if it has been widely demonstrated that bacterial membrane represents the main target of peptide antimicrobial activity, the molecular mechanisms underlying membrane perturbation by HDPs have not been fully clarified yet. Recently, two cryptic HDPs have been identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, and with anti-biofilm, wound healing and immunomodulatory properties. Moreover, ApoB derived HDPs are able to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, by using a multidisciplinary approach, including time killing curves, Zeta potential measurements, membrane permeabilization assays, electron microscopy analyses, and isothermal titration calorimetry studies, the antimicrobial effects of ApoB cryptides have been analysed on bacterial strains either susceptible or resistant to peptide toxicity. Intriguingly, it emerged that even if electrostatic interactions between negatively charged bacterial membranes and positively charged HDPs play a key role in mediating peptide toxicity, they are strongly influenced by the composition of negatively charged bacterial surfaces and by defined extracellular microenvironments.

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

PURPOSE: The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute - at the same time - to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on. MATERIALS AND METHODS: Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen. RESULTS: Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions. CONCLUSION: Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.