Editorial for Special Issue on Biosensors for Biomedical and Environmental Applications.

A sensor is typically defined as a device able to transform a physical quantity of interest into a different kind of signal that can be easily measured and recorded [...].

Additive Manufacturing of Wet-Spun Polysulfone Medical Implants.

Research on additive manufacturing (AM) of high-performance polymers provides novel materials and technologies for advanced applications in different sectors, such as aerospace and biomedical engineering. The present article is contextualized in this research trend by describing a novel AM protocol for processing a polysulfone (PSU)/N-methyl-2-pyrrolidone (NMP) solution into medical implant prototypes. In particular, an AM technique involving the patterned deposition of the PSU/NMP mixture in a coagulation bath was employed to fabricate PSU implants with different predefined shape, fiber diameter, and macropore size. Scanning electron microscopy (SEM) analysis highlighted a fiber transversal cross-section morphology characterized by a dense external skin layer and an inner macroporous/microporous structure, as a consequence of the nonsolvent-induced polymer solidification process. Physical-chemical and thermal characterization of the fabricated samples demonstrated that PSU processing did not affect its macromolecular structure and glass-transition temperature, as well as that after post-processing PSU implants did not contain residual solvent or nonsolvent. Mechanical characterization showed that the developed PSU scaffold tensile and compressive modulus could be changed by varying the macroporous architecture. In addition, PSU scaffolds supported the in vitro adhesion and proliferation of the BALB/3T3 clone A31 mouse embryo cell line. These findings encourage further research on the suitability of the developed processing method for the fabrication of customized PSU implants.

Biosensing Systems Based on Graphene Oxide Fluorescence Quenching Effect.

Graphene oxide (GO) is a versatile material obtained by the strong oxidation of graphite. Among its peculiar properties, there is the outstanding ability to significantly alter the fluorescence of many common fluorophores and dyes. This property has been exploited in the design of novel switch-ON and switch-OFF fluorescence biosensing platforms for the detection of a plethora of biomolecules, especially pathological biomarkers and environmental contaminants. Currently, novel advanced strategies are being developed for therapeutic, diagnostic and theranostic approaches to widespread pathologies caused by viral or bacterial agents, as well as to cancer. This work illustrates an overview of the most recent applications of GO-based sensing systems relying on its fluorescence quenching effect.

Towards an Innovative Sensor in Smart Capsule for Aerial Drones for Blood and Blood Component Delivery.

Aerial drone technology is currently being investigated worldwide for the delivery of blood components. Although it has been demonstrated to be safe, the delivered medical substances still need to be analyzed at the end of the flight mission to assess the level of haemolysis and pH prior to the use in a patient. This process can last up to 30 min and prevent the time saved using drone delivery. Our study aims to integrating an innovative sensor for the haemolysis and pH detection into the Smart Capsule, an already demonstrated technology capable of managing transfusion transport through drones. In the proposed scenario, the haemolysis is evaluated optically by a minilysis device using LED-photodetector combination. The preliminary validation has been demonstrated for both the thermal stability of the Smart Capsule and the haemolysis detection of the minilysis device prototype. Firstly, the onboard temperature test has shown that the delivery system is capable of maintaining proper temperature, even though the samples have been manipulated to reach a higher temperature before inserting into the Smart Capsule. Then, in the laboratory haemolysis test, the trend of linear regression between the outputs from the spectrophotometer and the minilysis prototype confirmed the concept design of the minilysis device.

Fluorescence Lifetime Imaging Microscopy of Porphyrins in Helicobacter pylori Biofilms.

Bacterial biofilm constitutes a strong barrier against the penetration of drugs and against the action of the host immune system causing persistent infections hardly treatable by antibiotic therapy. Helicobacter pylori (Hp), the main causative agent for gastritis, peptic ulcer and gastric adenocarcinoma, can form a biofilm composed by an exopolysaccharide matrix layer covering the gastric surface where the bacterial cells become resistant and tolerant to the commonly used antibiotics clarithromycin, amoxicillin and metronidazole. Antimicrobial PhotoDynamic Therapy (aPDT) was proposed as an alternative treatment strategy for eradicating bacterial infections, particularly effective for Hp since this microorganism produces and stores up photosensitizing porphyrins. The knowledge of the photophysical characteristics of Hp porphyrins in their physiological biofilm microenvironment is crucial to implement and optimize the photodynamic treatment. Fluorescence lifetime imaging microscopy (FLIM) of intrinsic bacterial porphyrins was performed and data were analyzed by the 'fit-free' phasor approach in order to map the distribution of the different fluorescent species within Hp biofilm. Porphyrins inside bacteria were easily distinguished from those dispersed in the matrix suggesting FLIM-phasor technique as a sensitive and rapid tool to monitor the photosensitizer distribution inside bacterial biofilms and to better orientate the phototherapeutic strategy.

The in vitro Photoinactivation of Helicobacter pylori by a Novel LED-Based Device.

The rise of antibiotic resistance is the main cause for the failure of conventional antibiotic therapy of Helicobacter pylori infection, which is often associated with severe gastric diseases, including gastric cancer. In the last years, alternative non-pharmacological approaches have been considered in the treatment of H. pylori infection. Among these, antimicrobial PhotoDynamic Therapy (aPDT), a light-based treatment able to photoinactivate a wide range of bacteria, viruses, fungal and protozoan parasites, could represent a promising therapeutic strategy. In the case of H. pylori, aPDT can exploit photoactive endogenous porphyrins, such as protoporphyrin IX and coproporphyrin I and III, to induce photokilling, without any other exogenous photosensitizers. With the aim of developing an ingestible LED-based robotic pill for minimally invasive intragastric treatment of H. pylori infection, it is crucial to determine the best illumination parameters to activate the endogenous photosensitizers. In this study the photokilling effect on H. pylori has been evaluated by using a novel LED-based device, designed for testing the appropriate LEDs for the pill and suitable to perform in vitro irradiation experiments. Exposure to visible light induced bacterial photokilling most effectively at 405 nm and 460 nm. Sub-lethal light dose at 405 nm caused morphological changes on bacterial surface indicating the cell wall as one of the main targets of photodamage. For the first time endogenous photosensitizing molecules other than porphyrins, such as flavins, have been suggested to be involved in the 460 nm H. pylori photoinactivation.

A 4,4'-bis(2-benzoxazolyl)stilbene luminescent probe: assessment of aggregate formation through photophysics experiments and quantum-chemical calculations.

A combination of experimental and quantum mechanical investigations is applied to the study of the optical features of 4,4'-bis(2-benzoxazolyl)stilbene (BBS) dissolved in solution or in a poly(l-lactic acid) (PLA) thermoplastic matrix at different concentrations. The experimental analyses allow the characterization of BBS solutions and dispersions in terms of absorption and emission features, along with the collection of some key parameters such as fluorescence quantum yield, anisotropy and lifetime, while the computational approach gives a detailed description of the photophysical behavior of BBS in the different environments. For the 10-5 M BBS solution, the fluorescence spectra show the expected peaks at 425 and 455 nm of the non-interacting BBS molecules with a single fluorescence lifetime of 0.85 ns without revealing any aggregation phenomena, prevented by the short lifetime and fast diffusion rate of the monomer. Moreover, the calculated spectra are in excellent agreement with the experiments, thus showing the reliability of the computational approach. In time-resolved emission experiments (TRES) on more concentrated solutions (10-4 M) and on BBS crystals, the presence of an excimer is revealed by the appearance of a broad peak around 540 nm, followed by the disappearance of the two main peaks at 460 nm on a time scale of about 10 ns. The computational analysis attributes this behavior to the formation of aggregates of different geometries. The BBS dispersions in PLA reveal the presence of different BBS architectures depending on the fluorophore content. Even at low concentrations, BBS is mainly dispersed as a monomer in the matrix, spheroid aggregates of about 800-900 nm in diameter are also present and the relevant fluorescence spectra arise from the combination of monomer and aggregate contributions. At higher concentrations, BBS starts forming crystals of a peculiar helicoidal shape, with a diameter of about 2 µm, variable length up to several hundreds of µm and emission spectra similar to those of isolated BBS crystals.

Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins.

Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells.

Organization of inner cellular components as reported by a viscosity-sensitive fluorescent Bodipy probe suitable for phasor approach to FLIM.

According to the recent developments in imaging strategies and in tailoring fluorescent molecule as probe for monitoring biological systems, we coupled a Bodipy-based molecular rotor (BoMe) with FLIM phasor approach to evaluate the viscosity in different intracellular domains. BoMe rapidly permeates cells, stains cytoplasmic as well as nuclear domains, and its optical properties make it perfectly suited for widely diffused confocal microscopy imaging setups. The capability of BoMe to report on intracellular viscosity was put to the test by using a cellular model of a morbid genetic pathology (Hutchinson-Gilford progeria syndrome, HGPS). Our results show that the nucleoplasm of HGPS cells display reduced viscosity as compared to normal cells. Since BoMe displays significant affinity towards DNA, as demonstrated by an in vitro essay, we hypothesize that genetic features of HGPS, namely the misassembly of lamin A protein within the nuclear lamina, modulates chromatin compaction. This hypothesis nicely agrees with literature data.

Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime.

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress.

Light-Responsive Polystyrene Films Doped with Tailored Heteroaromatic-Based Fluorophores.

We describe a simple but effective strategy for imparting light-responsive peculiarity to polystyrene films. A pH-sensitive fluorescent dye having the electron-poor pyridine nucleus as a key structural feature was synthesized and dispersed at low loadings (0.2-0.5 wt %) in a PS matrix. Once light irradiation in the near-UV range was sent to PS/dye films, PS photooxidation likely occurred at the film surface with the formation of carboxylic compounds. These species locally promoted dye protonation, thus, yielding a clear change of the film emission from blue to green. This study opens the door to a wide range of light-responsive materials from easily accessible polymers, enabling the use of UV light as an effective trigger for smart materials and devices.

Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging.

A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy.