Development of an MIP based electrochemical sensor for TGF-ß1 detection and its application in liquid biopsy.

Oral cancer is one of the most common types of cancer in Europe and its large diffusion requires, together with prevention, the development of low-cost and reliable portable platforms for its diagnosis, with features of high selectivity and sensitivity. In this study, the development and characterization of a molecularly imprinted polymer (MIP)-based electrochemical sensor for TGF-ß1 detection are reported. The optimized biosensor is a potential tool for the early screening of oral cancer. A biomimetic surface has been obtained by electropolymerization of o-phenylenediamine (o-PD) on platinum electrodes, in the presence of TGF-ß1 as a template molecule. MIP synthesis, template removal and TGF-ß1 rebinding have been monitored by Differential Pulse Voltammetry (DPV). Atomic Force Microscopy (AFM) has been performed to investigate and characterize the surface morphology and the influence of the washing step on MIP and NIP (non-imprinted polymer as the control) while the thickness of the polymer layer has been measured by Scanning Transmission Electron Microscopy (STEM) analysis. The MIP sensor performance has been tested in both buffer solution and saliva samples with TGF-ß1, showing a linear response in the considered range (from 20 ng ml-1 down to 0.5 ng ml-1), an outstanding LOD of 0.09 ng mL-1 and affinity and selectivity to TGF-ß1 also in the presence of interfering molecules. The sensor was used also for the detection of target molecules in spiked saliva samples with good recovery results suggesting the possibility of the use of the proposed system for large scale fast screening in oral cancer diagnosis.

Femtomolar Biodetection by a Compact Core-Shell 3D Chiral Metamaterial.

Advanced sensing tools, detecting extremely low concentrations of circulating biomarkers, can open unexplored routes toward early diagnostics and diseases progression monitoring. Here, we demonstrate the sensing capabilities of a chip-based metamaterial, combining 3D chiral geometry with a functional core-shell nanoarchitecture. The chiral metamaterial provides a circular polarization-dependent optical response, allowing analysis in a complex environment without significant background interferences. The functional nanoarchitecture, based on the conformal coating with a polymer shell, modifies the chiral metamaterial near- and far-field optical response because of the energy transfer between dielectric shell polarization charges and plasmonic core free electrons, leading to efficient interaction with biomolecules. The system sensitivity slope is 27 nm/pM, in the detection of TAR DNA-binding protein 43, clinically relevant for neurodegenerative diseases. Measurements were performed in spiked solution and in human serum with concentrations from 1 pM down to 10 fM, which is a range not accessible with common immunological assays, opening new perspectives for next-generation biomedical systems.

Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization.

Interest in extracellular vesicles and in particular microvesicles and exosomes, which are constitutively produced by cells, is on the rise for their huge potential as biomarkers in a high number of disorders and pathologies as they are considered as carriers of information among cells, as well as being responsible for the spreading of diseases. Current methods of analysis of microvesicles and exosomes do not fulfill the requirements for their in-depth investigation and the complete exploitation of their diagnostic and prognostic value. Lab-on-chip methods have the potential and capabilities to bridge this gap and the technology is mature enough to provide all the necessary steps for a completely automated analysis of extracellular vesicles in body fluids. In this paper we provide an overview of the biological role of extracellular vesicles, standard biochemical methods of analysis and their limits, and a survey of lab-on-chip methods that are able to meet the needs of a deeper exploitation of these biological entities to drive their use in common clinical practice.

Key Enabling Technologies for Point-of-Care Diagnostics.

A major trend in biomedical engineering is the development of reliable, self-contained point-of-care (POC) devices for diagnostics and in-field assays. The new generation of such platforms increasingly addresses the clinical and environmental needs. Moreover, they are becoming more and more integrated with everyday objects, such as smartphones, and their spread among unskilled common people, has the power to improve the quality of life, both in the developed world and in low-resource settings. The future success of these tools will depend on the integration of the relevant key enabling technologies on an industrial scale (microfluidics with microelectronics, highly sensitive detection methods and low-cost materials for easy-to-use tools). Here, recent advances and perspectives will be reviewed across the large spectrum of their applications.

Advancing amyotrophic lateral sclerosis disease diagnosis: A lab-on-chip electrochemical immunosensor for ultra-sensitive TDP-43 protein detection and monitoring in serum patients'.

The global increase in population aging has led to a rise in neurodegenerative diseases (NDs), posing significant challenges to public health. Developing selective and specific biomarkers for early diagnosis and drug development is crucial addressing the growing burden of NDs. In this context, the RNA-binding protein TDP-43 has emerged as a promising biomarker for amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and TDP-43-associated proteinopathies. However, existing detection methods suffer from limitations such as cost, complexity, and operator dependence. Here, we present a novel electrochemical biosensor integrated into a lab-on-chip (LoC) platform to detect TDP-43. The sensor utilizes electrosynthesized polypyrrole derivatives with carboxylic groups for transducer functionalization, enabling targeted immobilization of TDP-43 antibodies. Differential pulsed voltammetry (DPV) is used for the indirect detection and quantification of TDP-43. The chip exhibits rapid response, good reproducibility, a linear detection range, and sensitivity from 0.01 ng/mL to 25 ng/mL of TDP-43 protein concentration with a LOD = 10 pg/mL. Furthermore, successful TDP-43 detection in complex matrices like serum of ALS patients and healthy individuals demonstrates its potential as a point-of-care diagnostic device. This electrochemical biosensor integrated into a chip offers good sensitivity, rapid response, and robust performance, providing a promising avenue for advancing neurodegenerative disease diagnostics and therapeutic development.

On-chip screening for prostate cancer: an EIS microfluidic platform for contemporary detection of free and total PSA.

Prostate cancer affects a large part of the western male population. The need for an early and accurate detection is thus a great challenge in common clinical practice, but the lack of specificity of the serum marker PSA (Prostate Specific Antigen) is a serious problem since its increased concentration can be related to several abnormalities. PSA, however, is found in serum in both a free and a complexed form with other proteins and the percentage amount of unbound PSA (the free-to-total PSA ratio) can be employed to distinguish prostate cancer from benign prostatic conditions, and also to predict the future risk of prostate cancer. To improve the operating characteristics of current PSA tests and to provide a clinical tool able to run label-free and sensitive analysis, we thus developed a biosensing platform based on Electrochemical Impedance Spectroscopy (EIS), which allows the contemporary detection of free and total PSA on a single biochip, enabling a quick screening for the risk of prostate cancer thanks to the presence of two different immobilized antibodies specific for the different antigens researched.

Synthesis and Characterization of SPIONs Encapsulating Polydopamine Nanoparticles and Their Test for Aqueous Cu2+ Ion Removal.

The removal of pollutants, such as heavy metals, aromatic compounds, dyes, pesticides and pharmaceuticals, from water is still an open challenge. Many methods have been developed and exploited for the purification of water from contaminants, including photocatalytic degradation, biological treatment, adsorption and chemical precipitation. Absorption-based techniques are still considered among the most efficient and commonly used approaches thanks to their operational simplicity. In recent years, polydopamine-coated magnetic nanoparticles have emerged for the uptake of heavy metals in water treatment, since they combine specific affinity towards pollutants and magnetic separation capacity. In this context, this work focuses on the synthesis of polydopamine (PDA)-coated Super Paramagnetic Iron Oxide Nanoparticles (PDA@SPIONs) as adsorbents for Cu2+ ions, designed to serve as functional nanostructures for the removal of Cu2+ from water by applying a magnetic field. The synthetic parameters, including the amount of SPIONs and PDA, were thoroughly investigated to define their effects on the nanostructure features and properties. Subsequently, the ability of the magnetic nanostructures to bind metal ions was assessed on Cu2+-containing solutions. A systematic investigation of the prepared functional nanostructures was carried out by means of complementary spectroscopic, morphological and magnetic techniques. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) measurements were performed in order to estimate the Cu2+ binding ability. The overall results indicate that these nanostructures hold great promise for future bioremediation applications.

Biofluids manipulation methods for liquid biopsy in minimally-invasive assays.

The Liquid Biopsy (LB) is an opportunity for non-invasive diagnosis and prognosis of various diseases. To date, it isn't possible to consider that tissue biopsy can represent a pathology entirety. Then, body fluids are rich in a large number and variety of biomarkers and they can provide information about several diseases.•Recently, other biological fluids, easy to be collected are rising for their significant content of biomarkers and for the possibility to collect and manipulate them without the intervention of medical staff.•The management of biological fluids requires suitable storage methods. Temperature, storage time and physical stresses due to sample handling can lead to chemical and physical changes that may induce sample degradation and incorrect analysis.•The reliability of a diagnostic or screening test depends on its sensitivity and specificity. As the liquid biopsy is a 'snapshot' of a pathophysiological condition, it is crucial that its components do not degrade due to the improper handling of the body fluid.In this review, some handling methods of Saliva, Urine, Stool, Seminal Fluid, Tears and Sweat samples will be described, as well as protocols to facilitate the analysis of metabolites, nucleic acids, proteins and Extracellular Vesicles (EVs) from those unusual body fluids.

Editorial for the Special Issue on Emerging and Disruptive Next-Generation Technologies for POC: Sensors, Chemistry and Microfluidics for Diagnostics.

Recently, the attention paid to self-care tests and the need for easy and large-scale screenings of a high number of people has dramatically increased [...].

Beyond liquid biopsy: Toward non-invasive assays for distanced cancer diagnostics in pandemics.

Liquid biopsy technologies have seen a significant improvement in the last decade, offering the possibility of reliable analysis and diagnosis from several biological fluids. The use of these technologies can overcome the limits of standard clinical methods, related to invasiveness and poor patient compliance. Along with this there are now mature examples of lab-on-chips (LOC) which are available and could be an emerging and breakthrough technology for the present and near-future clinical demands that provide sample treatment, reagent addition and analysis in a sample-in/answer-out approach. The possibility of combining non-invasive liquid biopsy and LOC technologies could greatly assist in the current need for minimizing exposure and transmission risks. The recent and ongoing pandemic outbreak of SARS-CoV-2, indeed, has heavily influenced all aspects of life worldwide. Ordinary tasks have been forced to switch from "in presence" to "distanced", limiting the possibilities for a large number of activities in all fields of life outside of the home. Unfortunately, one of the settings in which physical distancing has assumed noteworthy consequences is the screening, diagnosis and follow-up of diseases. In this review, we analyse biological fluids that are easily collected without the intervention of specialized personnel and the possibility that they may be used -or not-for innovative diagnostic assays. We consider their advantages and limitations, mainly due to stability and storage and their integration into Point-of-Care diagnostics, demonstrating that technologies in some cases are mature enough to meet current clinical needs.

Nanoarchitectonics of highly sensitive and with large working range 3D piezoresistive microporous foam based on carbon nanotubes and elastomer.

HYPOTHESIS: Nanocarbon/polymeric 3D porous composites have been widely developed as piezoresistive sensors due to their improved performances. Functionalized nanocarbon is usually used to allow its adsorption on the surface of porous polymeric material. However, both the functionalization and the surface localized distribution of the nanomaterial can limit the nanocarbon effect on conductivity and mechanical stability of the material thus affecting piezoresistive performances. EXPERIMENTS: A novel nanoarchitectonics strategy to prepare an elastomeric/carbon nanotubes (CNTs) 3D porous piezoresistive nanocomposite is developed. The fabrication route does not require complex apparatus and CNTs chemical functionalization. Moreover, foams of any shape and dimensions can be produced with neither complex machinery and procedures nor wastes production. FINDINGS: The obtained material is characterized by the presence of well dispersed pristine CNTs on both surface and bulk of the polymeric matrix. The foam exhibited improved piezoresistive properties with excellent compressive stress (>150 kPa), sensitivity at low displacement (29 kPa-1) and limit of detection for both pressure (2 Pa) and extension (130 nm). These excellent features could allow the use of the as prepared nanocomposite in different applications ranging from wearable devices to robotic or infrastructure monitoring with outstanding flexibility.

Does Size Matter? The Case of Piezoresistive Properties of Carbon Nanotubes/Elastomer Nanocomposite Synthesized through Mechanochemistry.

The growing interest in piezoresistive sensors has favored the development of numerous approaches and materials for their fabrication. Within this framework, carbon nanotubes (CNTs) are often employed. However, CNTs are a heterogeneous material with different morphological characteristics in terms of length and diameter, and, so far, experimental studies have not usually considered the effect of these parameters on the final sensor performances. Here, we observe how, by simply changing the CNTs length in a solvent-free mechanochemistry fabrication method, different porous 3D elastomeric nanocomposites with different electrical and mechanical properties can be obtained. In particular, the use of longer carbon nanotubes allows the synthesis of porous nanocomposites with better mechanical stability and conductivity, and with a nine-times-lower limit of detection (namely 0.2 Pa) when used as a piezoresistive sensor. Moreover, the material prepared with longer carbon nanotubes evidenced a faster recovery of its shape and electrical properties during press/release cycles, thus allowing faster response at different pressures. These results provide evidence as to how CNTs length can be a key aspect in obtaining piezoresistive sensors with better properties.

Polydopamine-Coated Magnetic Iron Oxide Nanoparticles: From Design to Applications.

Magnetic iron oxide nanoparticles have been extensively investigated due to their applications in various fields such as biomedicine, sensing, and environmental remediation. However, they need to be coated with a suitable material in order to make them biocompatible and to add new functionalities on their surface. This review is intended to give a comprehensive overview of recent advantages and applications of iron oxide nanoparticles coated by polydopamine film. The synthesis method of magnetic nanoparticles, their functionalization with bioinspired materials and (in particular) with polydopamine are discussed. Finally, some interesting applications of polydopamine-coated magnetic iron oxide nanoparticles will be pointed out.

Femtosecond Laser Fabrication of Microporous Membranes for Biological Applications.

The possibility of fabricating micrometric pore size membranes is gaining great interest in many applications, from studying cell signaling, to filtration. Currently, many technologies are reported to fabricate such microsystems, the choice of which depends strictly on the substrate material and on the final application. Here, we demonstrate the capability with a single femtosecond laser source and experimental setup to fabricate micromembranes both on polymeric and multilayer metallic substrate, without the need for moulds, mask, and complex facilities. In particular, the flexibility of laser drilling was exploited to obtain microfilters with pore size of 8 and 18 µm in diameter, on metallic and polymeric substrate, respectively, and controlled distribution. For evaluating the possibility to use such laser-fabricated membranes into biological assay, their biocompatibility has been investigated. To this aim, as a proof of concept, we tested the two materials into viability tests. The culture of mammalian cells on these microfabricated membranes were studied showing their compatibility with cells.

SMILE Platform: An Innovative Microfluidic Approach for On-Chip Sample Manipulation and Analysis in Oral Cancer Diagnosis.

Oral cancer belongs to the group of head and neck cancers, and, despite its large diffusion, it suffers from low consideration in terms of prevention and early diagnosis. The main objective of the SMILE platform is the development of a low-cost device for oral cancer early screening with features of high sensitivity, specificity, and ease of use, with the aim of reaching a large audience of possible users and realizing real prevention of the disease. To achieve this goal, we realized two microfluidic devices exploiting low-cost materials and processes. They can be used in combination or alone to obtain on-chip sample preparation and/or detection of circulating tumor cells, selected as biomarkers of oral cancer. The realized devices are completely transparent with plug-and-play features, obtained thanks to a highly customized architecture which enables users to easily use them, with potential for a common use among physicians or dentists with minimal training.

Pectobacterium atrosepticum Biosensor for Monitoring Blackleg and Soft Rot Disease of Potato.

Pectobacterium atrosepticum (Pba) is a quarantine and threatening phytopathogen known as the causal agent of blackleg and soft rot disease of potatoes in many areas. Its early detection is then important to have healthy potato tubers and reduce economic losses. Today, conventional methods such as enzyme-linked immunosorbent-assay (ELISA) and polymerase chain reaction (PCR) are typically used for Pba detection, but they are expensive and time-consuming. Here we report on the optimization of an alternative approach based on an electrochemical impedance immunosensor combining a microfluidic module and a microelectrodes array, and having advantages in terms of low cost, ease of use and portability. For validation and for assessing its performance, the lab-on-chip platform has been compared with two standard methods (ELISA and PCR).

Advances in the Development of Innovative Sensor Platforms for Field Analysis.

Sustainable growth, environmental preservation, and improvement of life quality are strategic fields of worldwide interest and cornerstones of international policies. Humanity health and prosperity are closely related to our present choices on sustainable development. The main sources of pollution concern industry, including mining, chemical companies, and refineries, wastewater treatment; and consumers themselves. In order to guide and evaluate the effects of environmental policies, diffuse monitoring campaigns and detailed (big) data analyses are needed. In this respect, the development and availability of innovative sensor platforms for field analysis and remote sensing are of crucial relevance. In this review, we provide an overview of the area, analyzing the major needs, available technologies, novel approaches, and perspectives. Among environmental pollutants that threaten the biosphere, we focus on inorganic and organic contaminants, which affect air and water quality. We describe the technologies for their assessment in the environment and then draw some conclusions and mention future perspectives opened by the integration of sensing technologies with robotics and the Internet of Things. Without the ambition to be exhaustive in such a rapidly growing field, this review is intended as a support for researchers and stakeholders looking for current, state-of-the-art, and key enabling technologies for environmental monitoring.

Sperm selection in assisted reproduction: A review of established methods and cutting-edge possibilities.

Male infertility often involves idiopathic or unknown causes, leading to an increasing demand for assisted reproduction technologies (ART). Conventional sperm sorting techniques rely on centrifugation steps that are known to cause oxidative stress and consequently damage cells. Alternative novel techniques have been introduced but offer disadvantages that need to be overcome. These techniques are also employed to increase the number and the quality of subjects in the animal breeding industry, to obtain purebred subjects or to preserve endangered animal species. Microfluidics deals with the manipulation of small amounts of volume within a microdevice known as lab-on-a-chip (LOC), which offers rapid analyses, ease of use, small reagent sample volumes, high-throughput processing and wide reproducibility owing to automation and standardization. As the LOC allows gamete handling within a microenvironment that strictly mimics physiological in vivo conditions and avoids centrifugation steps and long processing time, the use of microfluidics for sperm sorting and selection have been proposed during the last 15 years and is currently under investigation. Moreover, LOC technologies to sort, identify and analyse other kinds of cells could be transferred to sperm selection and analysis, thus opening the way to a novel approach to the sperm cell selection and manipulation. This review describes the techniques routinely performed in human and animal clinical practice for sorting good-quality sperm for in vitro fertilization procedures, and focuses on the positive and negative aspects of each method. Emerging microfluidic devices, recently proposed for sperm selection, are also described and, when possible, compared with standard methods.

Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm.

Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300 ×g for 5', 300 ×g for 10', 1500 ×g for 5' and 1500 ×g for 10' vs no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantly altered, as observed by computer-assisted sperm analysis. A polarographic assay of oxygen consumption showed a progressive decrease in mitochondria respiration from the gentlest to the strongest protocol. By laser scanning confocal microscopy, significant reduction of mitochondrial membrane potential, at any tested protocol, and time-dependent effects, at the same centrifugal force, were found. Increased DNA fragmentation index at any tested protocol and time-dependent effects at the same centrifugal force were found, whereas increased protein carbonylation was observed only at the strongest centrifugal force. These results provide more comprehensive understandings on centrifugation-induced effects on cryopreserved stallion sperm and suggest that, even at a weak force for a short time, centrifugation impairs different aspects of equine sperm metabolism and functionality.

A nanobiosensor to detect single hybridization events.

An economical nanoarray method to electrically detect hybridization events is demonstrated. As a proof of concept, we fabricated a sensor for DNA sequencing, in which targets are oligonucleotides conjugated to gold nanoparticles. As a consequence of target-probe binding events, a conductive bridge forms between two electrodes, resulting in a quantized change in conductivity. This enables a robust detection of a few (down to single) hybridization events and can be potentially applied also to other binding events (like specific interactions between proteins, antibodies, ligands and receptors). Moreover, target amplification techniques (such as PCR) are no longer necessary.