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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.

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.

Phytopathological adversities are often attributable to human activities (as a consequence of the globalization of trade or tourism mass, changes in common agricultural practices and climate change), adding food losses due to pathogens such as fungi, bacteria, viruses etc. For this, we are developing a lab-on-chip as a diagnostic approach to phytopathological problems caused by infectious agents capable of spreading in agro-ecosystems, such as the Xylella fastidiosa epidemic in Puglia (Chiriaco et al., 2018) or other bacteriosis and virosis such as Grapevine leafroll-associated virus 3 (GLRaV-3). In particular, grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases, affecting grapevines worldwide. Several viruses from the family Closteroviridae are associated with it and Grapevine leafroll-associated virus 3(GLRaV-3) is considered as the most important causative agent. Symptoms of GLD can vary greatly with the season, grape cultivar, and climatic conditions and some varieties can be completely symptomless. (Maree et al., 2013). There is no cure for the virus but only preventive actions. In fact, fighting strategy is based exclusively on the use of plant material free from virus, such as the use of certified material. These pathogens can have serious economic and environmental repercussions on two of the major cultivated woody plant of Mediterranean basin, due to the absence of therapeutic techniques and the need of rapid, in-field and low-cost detection methods. Here we present a lab-on-chip platform coupled with microfluidic module, based on an electrochemical transduction method, able to recognize serial dilutions of Grapevine leafroll-associated virus 3. LOC represents smart and versatile devices due to their miniaturization. They require small sample volumes, allowing a rapid detection of the targets, offering also the opportunity to study biomechanical properties of plants (Nezhad et al., 2013) and other plant cells studies (Nezhad et al., 2014; Julich et al., 2011). In particular, thanks the aid of a microfluidic component, such as polydimethylsiloxane (PDMS), is possible to realize biochemistry conventional laboratories functions such as sample preparation, reaction, separation and detection (McDonald et al, 2000). This device can show competitive performances with conventional diagnostic methods in terms of reliability, with further advantages of portability, low costs and ease of use, making the difference in real time detection of the pathogens.

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.

Aerosol particulate matter (PM) is a known risk factor for lung and liver cancer. In particular, PM under 1 μm in diameter (PM1) is very dangerous for health, affecting the respiratory system with several heavy outcomes and reaching other human districts like liver, where foreign substances are processed. Today PM1 is still difficult to be detected on-field with standard techniques and this paper reports a comparison between two sensing approaches for environmental particulate monitoring, which are suitable for miniaturization and integration into portable and low-cost devices. Specifically, transducers based on surface acoustic waves (SAW) and electrochemical impedance spectroscopy (EIS) were comparatively investigated as alternatives to more common but also expensive optical methods. Moreover, their performances were evaluated for the detection of nanoparticles of different dimensions (1 μm, 200 nm and 40 nm) to mimic different compositions of PM1 and reach a size scale which is an order of magnitude below present literature. Notably, both transduction methods were able to detect the presence of PM1 particulate. The SAW-based approach showed better performances in terms of sensitivity and detection of nanoscale particles, although the EIS strategy has a competitive sensitivity considering also its even lower cost.

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.

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 &times, g for 5&rsquo, 300 &times, g for 10&rsquo, 1500 &times, and 1500 &times, 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.

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.

A computational study of the electromechanical response of micro-structure engineered two port surface acoustic wave delay lines on gallium arsenide is presented. The influence on the results of geometrical, material, and mesh parameters is also discussed. Furthermore, experimental results are provided to validate the numerical study. The device consists of two interdigital transducers composed of 40, 80, and 120 pairs of electrodes, respectively, with a pitch pidt=8μmpidt=8μm and distant didt=1502μmdidt=1502μm. In particular, a microwave burst of surface acoustic waves propagating on gallium arsenide is fully characterized including multiple transit effects. These results are of major interest for understanding the dynamical behavior of complex systems such as surface acoustic wave–based sensors or energy harvesting devices at the nano and microscale.

Type (II) diabetes is one of the major threats to mankind as it causes insulin resistance in human body and Retinol Binding Protein 4 (RBP4) is currently considered as a potential biomarker for early management of this disease. Hence a low-level detection of RBP4 is a very important task and for this purpose, a novel RBP4 biosensor has been developed using homemade plastic chip electrodes (PCEs) as a platform for self-assembled monolayer (SAM) of 4-ATP and further functionalization with glutaraldehyde. Anti RBP4 is used as biorecognition species and electrochemical impedance spectroscopy has been performed to detect different RBP4 concentrations plotted against charge transfer resistance. A wide concentration range from 100 fg/mL to 1 ng/mL has been tested and a low limit of detection (LOD) of 100 fg/mL has been achieved. This is the first report for fabrication of electrochemical biosensor of RBP4 using Ag-Ab interaction having such low LOD. The sensor is characterized by various physico-chemical techniques. Excellent reproducibility and quick measurement make this biosensor extremely useful for the biomedical industry.

A great improvement in food safety and quality controls worldwide has been achieved through the development of biosensing platforms. Foodborne pathogens continue to cause serious outbreaks due to the ingestion of contaminated food. The development of new, sensitive, portable, high-throughput, and automated platforms is a primary objective to allow detection of pathogens and their toxins in foods. Listeria monocytogenes is one common foodborne pathogen. Major outbreaks of listeriosis have been caused by a variety of foods, including milk, soft cheeses, meat, fermented sausages, poultry, seafood and vegetable products. Due to its high sensitivity and easy setup, electrochemical impedance spectroscopy (EIS) has been extensively applied for biosensor fabrication and in particular in the field of microbiology as a mean to detect and quantify foodborne bacteria. Here we describe a miniaturized, portable EIS platform consisting of a microfluidic device with EIS sensors for the detection of L. monocytogenes in milk samples, connected to a portable impedance analyzer for on-field application in clinical and food diagnostics but also for biosecurity purposes. To achieve this goal microelectrodes were functionalized with antibodies specific for L. monocytogenes. The binding and detection of L. monocytogenes was achieved in the range 2.2 x 103 cfu/ml to 1 x 102 with a Limit of Detection (LoD) of 5.5 cfu/ml.

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.

Lab-on-chip devices are revolutionizing life science research and methodologies permitting sensible molecular and cellular assays, drug screening and large-scale monitoring. Here the optimization of a multipurpose and cheap impedance-based biochip platform is reported, consisting of sensors and microfluidic components for sample delivery and detection. Such biochips were demonstrated to be suitable for medical diagnostics in both flow immunoassays for biomarker detection and migration assays on cell populations. With small modifications, similar biochips were also applied for quality control by the detection of allergens (gliadin) and pathogens.

In this work, we demonstrate the excitation of surface acoustic waves (SAW) harmonics up to GHz regime in photolitographed devices fabricated on gallium arsenide (GaAs) by acting on the IDT metallization ratio among the finger width and pitch. Specifically, we observed up to the 13th harmonic, which corresponds to a frequency of about 1.7 GHz. Moreover, we employed time-resolved spectroscopy for isolating the shape of the SAW bandpass-filter response (for each harmonic) eliminating the interference between acoustic and electromagnetic waves. Notably, the extracted SAW spectra are characterized by a bandwidth which remains constant for the different harmonic modes, unlike the case of traditional SAW filters (having a 0.5 metallization ratio) where the pass band Delta f = f(0)/n(p) np increases with the working frequency. These results are relevant for applications where high frequencies and multiple harmonics excitation are desirable, or where quantitative measurements of the direct SAW signal are required.

A great improvement in food safety and quality controls worldwide has been achieved through the development of biosensing platforms. Foodborne pathogens continue to cause serious outbreaks, due to the ingestion of contaminated food. The development of new, sensitive, portable, high-throughput, and automated platforms is a primary objective to allow detection of pathogens and their toxins in foods. Listeria monocytogenes is one common foodborne pathogen. Major outbreaks of listeriosis have been caused by a variety of foods, including milk, soft cheeses, meat, fermented sausages, poultry, seafood and vegetable products. Due to its high sensitivity and easy setup, electrochemical impedance spectroscopy (EIS) has been extensively applied for biosensor fabrication and in particular in the field of microbiology as a mean to detect and quantify foodborne bacteria. Here we describe a miniaturized, portable EIS platform consisting of a microfluidic device with EIS sensors for the detection of L. monocytogenes in milk samples, connected to a portable impedance analyzer for on-field application in clinical and food diagnostics, but also for biosecurity purposes. To achieve this goal microelectrodes were functionalized with antibodies specific for L. monocytogenes. The binding and detection of L. monocytogenes was achieved in the range 2.2 &times, 103 cfu/mL to 1 &times, 102 with a Limit of Detection (LoD) of 5.5 cfu/mL.

Lower genital tract infections caused by both sexually and not-sexually transmitted pathogens in women are a key public health priority worldwide, especially in developing countries. Since standard analyses are time-consuming, appropriate therapeutic intervention is often neglected or delayed. Lab-on-chips and biosensors open new perspectives and offer innovative tools to simplify the diagnosis by medical staff, especially in countries with inadequate resources. Here we report a biosensing platform based on Electrochemical Impedance Spectroscopy (EIS) that allows multiplexed detection of Candida albicans, Streptococcus agalactiae and Chlamydia trachomatis with a single biochip, enabling a quick screening thanks to the presence of different immobilized antibodies, each specific for one of the different target pathogens.

The development of new microfabrication techniques is attracting more and more interest because of the increasing demand for three-dimensional tools with features of biocompatibility, flexibility and low-costs in the lab-on-chip field. Photolithographic techniques involving the molding of organic polymers, like SU-8, allow for short fabrication times and simplicity in devices prototyping. In this paper we used LOR and SU-8 resists in combination within an innovative lithographic approach. LOR resist was employed not as a typical sacrificial layer for the production of free-standing structures but as a three-dimensional solid resist, which can be patterned and embedded in a SU-8 monolith. After dissolution it can form cavities to provide a final multilevel structure. A detailed description of the optimization process required to obtain the final structure and to overcome issues related to the employ of LOR is reported. In the end, a network of working interconnected multilevel microchannels, useful for biological applications, has been realized through a new, cheap and time-saving method. Display Omitted A process to obtain multilevel microchannels in a monolithic SU8 structure was optimized.We used LOR and SU8 resists in combination for an innovative lithographic approach.A tool for biological applications in the lab-on-chip field was realized.

Foodborne illnesses caused by the ingestion of foods contaminated with pathogens and/or their toxins are still one of the major public health threats worldwide. Disposable devices, allowing the on-site, early and multiplexed quantitative detection of pathogenic bacteria are therefore highly sought. Herein, we report biochips that are able to quantitatively detect two of the most common food-associated pathogens, namely Listeria monocytogenes and Staphylococcus aureus from the suspensions of bacteria stationary-phase broth culture. With a detection limit as low as 5.00 CFU ml(-1) for L. monocytogenes and 1.26 CFU ml(-1) for S. aureus, our platform may be a promising point-of-care device not only for clinical and food diagnostics but also for biosecurity purposes.

Celiac disease (CD) is one of the most common digestive disorders caused by an abnormal immune reaction to gluten. So far there are no available therapies, the only solution is a strict gluten-free diet, which however could be very challenging as gluten can be hidden in many food products. Furthermore an additional problem is related to cross-contamination of nominal gluten-free foods with gluten-based ones during manufacturing. Here we propose a lab on chip platform as a powerful tool to help food manufacturers to evaluate the real amount of gluten in their products by an accurate in-situ control of the production chain and maybe to specify the real gluten content in packages labeling. Our portable gliadin-immunochips, based on an electrochemical impedance spectroscopy transduction method, were first calibrated and then validated for both liquid and solid food matrixes by analyzing different beers and flours. The high specificity of our assay was also demonstrated by performing control experiments on rice and potatoes flours containing prolamin-like proteins. We achieved limit of quantification of 0.5 ppm for gliadin that is 20 times lower than the worldwide limit established for gluten-free food while the method of analysis is faster and cheaper than currently employed ELISA-based methods. Moreover our results on food samples were validated through a mass spectrometry standard analysis. (C) 2015 Elsevier B.V. All rights reserved.

Development of lab-on-chip devices attracted large interest for detection of specific analyte/markers, cellular studies and drug screening. In the case of DNA, one of the biggest problems is the low concentration of target sequences in biological samples, which is the reason for typical use of amplification steps. In this paper, we discuss our results in developing DNA sensors integrated with microfluidic components in biochips for DNA recognition. Two different approaches are investigated and compared which are based respectively on impedance and magnetoresistive transduction.

Cell culture technologies were initially developed as research tools for studying cell functions, but nowadays they are essential for the biotechnology industry, with rapidly expanding applications requiring more and more advancements with respect to traditional tools. Miniaturization and integration of sensors and microfluidic components with cell culture techniques open the way to the development of cellomics as a new field of research targeting innovative analytic platforms for high-throughput studies. This approach enables advanced cell studies under controllable conditions by providing inexpensive, easy-to-operate devices. Thanks to their numerous advantages cell-chips have become a hotspot in biosensors and bioelectronics fields and have been applied to very different fields. In this review exemplary applications will be discussed, for cell counting and detection, cytotoxicity assays, migration assays and stem cell studies. © 2013 The Royal Society of Chemistry.

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.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers in Europe and the United States. It has a very low 5 years-survival rate and its diagnosis is often late and imprecise due to the lack of specificity of currently used markers for PDAC. As previously demonstrated PDAC patients’ sera may contain autoantibodies towards phosphorylated a-enolase (ENOA), which in combination with other standard markers can increase specificity in diagnosis of PDAC. In this context we realized a microfluidic platform with integrated EIS biosensors. We achieved a specific antibodies detection by immobilizing onto electrodes peptides corresponding to a portion of ENOA. Phosphorylation of peptides was found to influence the recognition of antibodies in PDAC patients’ sera detected by the developed biochip thus validating the EIS technique as a strong tool for quick, cost-saving and label-free analysis of serum samples. Biochip results are in agreement with those from traditional techniques, such as ELISA and western blot, but measurements are much more sensitive and specific, increasing the possibility of PDAC diagnosis. In addition this approach is faster and more reproducible compared to traditional techniques making the developed biochips ideal for a quick, cost-saving and label-free analysis of serum samples.

Here an EIS (electrochemical impedance spectroscopy) biochip to detect cell migration is demonstrated. This biochip has been inspired by a traditional transwell assay/modified Boyden chamber and consists of two compartments separated by a porous membrane. This structure (PDMS-based) is aligned to EIS sensors. Cells are seeded in the upper chamber through microfluidic channels. During migration cells go through the pores of the membrane and get in touch with the electrodes that detect migrated cells. The performance of our cell-chip was tested by investigating the migratory ability of hepatocellular carcinoma (HCC) cells as a function of microenvironment. For this purpose we challenged HCC cells to migrate on different extra-cellular matrix (ECM) components including laminin 1, collagen IV and laminin 5. The results reveal that our cell chip provides reliable results that consistently overlap with those obtained with traditional standardized Boyden chambers. Thus, we demonstrate a new, easy tool to study cell migration and to perform automatic assays. This approach is easier and faster than traditional transwell assays and can be suitable for high-throughput studies in drug discovery applications.

A flow-injection impedimetric immunosensor for the sensitive, direct and label-free detection of cholera toxin is reported. A limit of detection smaller than 10 pM was achieved, a value thousands of times lower than the lethal dose. The developed chips fulfil the requirement of low cost and quick reply of the assay and are expected to enable field screening, prompt diagnosis and medical intervention without the need of specialized personnel and expensive equipment, a perspective of special relevance for use in developing countries. Since the chip layout includes two sensing areas each one with a 2 × 2 sensor array, our biochips can allow statistical or (alternatively) multiplex analysis of biorecognition events between antibodies immobilized on each working electrode and different antigens flowing into the chamber.