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.

A Novel Hybrid Platform for Live/Dead Bacteria Accurate Sorting by On-Chip DEP Device.

According to the World Health Organization (WHO) forecasts, Antimicrobial Resistance (AMR) will be the leading cause of death worldwide in the next decades. To prevent this phenomenon, rapid Antimicrobial Susceptibility Testing (AST) techniques are required to drive the selection of the most suitable antibiotic and its dosage. In this context, we propose an on-chip platform, based on a micromixer and a microfluidic channel, combined with a pattern of engineered electrodes to exploit the di-electrophoresis (DEP) effect. The role of the micromixer is to ensure the proper interaction of the antibiotic with the bacteria over a long time (≈1 h), and the DEP-based microfluidic channel enables the efficient sorting of live from dead bacteria. A sorting efficiency of more than 98%, with low power consumption (Vpp = 1 V) and time response of 5 s, within a chip footprint of ≈86 mm2, has been calculated, which makes the proposed system very attractive and innovative for efficient and rapid monitoring of the antimicrobial susceptibility at the single-bacterium level in next-generation medicine.

Advances in Plant Disease Detection and Monitoring: From Traditional Assays to In-Field Diagnostics.

Human activities significantly contribute to worldwide spread of phytopathological adversities. Pathogen-related food losses are today responsible for a reduction in quantity and quality of yield and decrease value and financial returns. As a result, "early detection" in combination with "fast, accurate, and cheap" diagnostics have also become the new mantra in plant pathology, especially for emerging diseases or challenging pathogens that spread thanks to asymptomatic individuals with subtle initial symptoms but are then difficult to face. Furthermore, in a globalized market sensitive to epidemics, innovative tools suitable for field-use represent the new frontier with respect to diagnostic laboratories, ensuring that the instruments and techniques used are suitable for the operational contexts. In this framework, portable systems and interconnection with Internet of Things (IoT) play a pivotal role. Here we review innovative diagnostic methods based on nanotechnologies and new perspectives concerning information and communication technology (ICT) in agriculture, resulting in an improvement in agricultural and rural development and in the ability to revolutionize the concept of "preventive actions", making the difference in fighting against phytopathogens, all over the world.

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.

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.

High Stability and Low Power Nanometric Bio-Objects Trapping through Dielectric-Plasmonic Hybrid Nanobowtie.

Micro and nano-scale manipulation of living matter is crucial in biomedical applications for diagnostics and pharmaceuticals, facilitating disease study, drug assessment, and biomarker identification. Despite advancements, trapping biological nanoparticles remains challenging. Nanotweezer-based strategies, including dielectric and plasmonic configurations, show promise due to their efficiency and stability, minimizing damage without direct contact. Our study uniquely proposes an inverted hybrid dielectric-plasmonic nanobowtie designed to overcome the primary limitations of existing dielectric-plasmonic systems, such as high costs and manufacturing complexity. This novel configuration offers significant advantages for the stable and long-term trapping of biological objects, including strong energy confinement with reduced thermal effects. The metal's efficient light reflection capability results in a significant increase in energy field confinement (EC) within the trapping site, achieving an enhancement of over 90% compared to the value obtained with the dielectric nanobowtie. Numerical simulations confirm the successful trapping of 100 nm viruses, demonstrating a trapping stability greater than 10 and a stiffness of 2.203 fN/nm. This configuration ensures optical forces of approximately 2.96 fN with an input power density of 10 mW/µm2 while preserving the temperature, chemical-biological properties, and shape of the biological sample.

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.

Ultrasensitive qPCR platform for rapid detection of bacterial contamination of raw biological samples at the point of care.

Contamination of cell cultures can result in a significant loss of precious biological material, particularly in long-term processes including amplification of chimeric antigen receptors (CAR)-T cells and differentiation of patient-derived stem cells, for therapeutic purposes. Bacterial contamination can also lead to more complex conditions such as sepsis which can cause morbidity and mortality, despite strict controls and good laboratory/manufacturing practices in the manipulation of complex biological samples such as blood used in autologous and allogeneic stem cells transplantation. The current standard method to identify biological risk is the set-up of microbial cultures, which can be time consuming with the likelihood of wasting large amounts of reagents in the event of contamination. Real-Time Polymerase Chain Reaction (qPCR) is a molecular method able to detect biological agents in a highly sensitive and specific way and in a short time. However, qPCR assays require complex DNA/RNA purification steps and expensive benchtop instruments, which may not always be available. This paper reports an extraction-free and low-volume protocol for qPCR in a standard instrument, which has been demonstrated to be effective on both Gram-positive (Gram+) and Gram-negative (Gram-) bacteria. Detection has been obtained from spiked cell culture samples, reaching a limit of detection (LOD) of 1 colony forming unit (CFU)/ml. To demonstrate the high potential of this optimized procedure, the same samples were also tested on a Point-Of-Care platform, which includes a cartridge with micro-chambers and a compact instrument, capable of performing qPCR with the same efficiency. Staphylococcus aureus (Gram+) was selected as the target for a proof of concept, achieving a LOD of 1 CFU/ml also on the portable device. The availability of these results paves the way for a simplified protocol for DNA extraction and amplification.

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 [...].

Miniaturized Real-Time PCR systems for SARS-CoV-2 detection at the Point-of-Care.

Over the past two years, SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) infection has spread globally causing multi-organ disease and severely impacting the healthcare systems of all countries. Accordingly, the development of easy-to-access diagnostic devices has become essential to limit the effect of the virus worldwide. Real-Time PCR is considered the gold standard to identify SARS-CoV-2 infection due to high sensitivity, affordability, and capacity to detect low viral loads at early disease stage. Advances in lab on a chip technology has led to the development of some Point-of-Care (POC) devices using Real-Time PCR and approved by the United States Food and Drug Administration. We provide an overview on recently developed POC tests for the rapid diagnosis of COVID-19 infection. Practical applications of miniaturized devices based on viral genome amplification as well as favorable features such as reduced sample processing time, ease of use by non-specialized personnel, and the potential of PCR-based POC technologies will be highlighted and reviewed.

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.

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.

Innovative 3D Microfluidic Tools for On-Chip Fluids and Particles Manipulation: From Design to Experimental Validation.

Micromixers are essential components in lab-on-a-chip devices, of which the low efficiency can limit many bio-application studies. Effective mixing with automation capabilities is still a crucial requirement. In this paper, we present a method to fabricate a three-dimensional (3D) poly(methyl methacrylate) (PMMA) fluidic mixer by combining computer-aided design (CAD), micromilling technology, and experimental application via manipulating fluids and nanoparticles. The entire platform consists of three microfabricated layers with a bottom reservoir-shaped microchannel, a central serpentine channel, and a through-hole for interconnection and an upper layer containing inlets and outlet. The sealing process of the three layers and the high-precision and customizable methods used for fabrication ensure the realization of the monolithic 3D architecture. This provides buried running channels able to perform passive chaotic mixing and dilution functions, thanks to a portion of the pathway in common between the reservoir and serpentine layers. The possibility to plug-and-play micropumping systems allows us to easily demonstrate the feasibility and working features of our device for tracking the mixing and dilution performances of the micromixer by using colored fluids and fluorescent nanoparticles as the proof of concept. Exploiting the good transparency of the PMMA, spatial liquid composition and better control over reaction variables are possible, and the real-time monitoring of experiments under a fluorescence microscope is also allowed. The tools shown in this paper are easily integrable in more complex lab-on-chip platforms.

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).

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.

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.

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.