Elisabetta Primiceri

Automatic transwell assay by an EIS cell chip to monitor cell migration

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.

Biosensors for the Detection of Food Pathogens

Food pathogens frequently cause foodborne diseases. There is a need to rapidly identify the source of the bacteria in order to contain their spread and epidemics. A pre-enrichment culture or a direct culture on agar plate are standard microbiological methods. In this review, we present an update on alternative molecular methods to nucleic acid-based detection for species identification. Biosensor-based methods rely on the recognition of antigen targets or receptors by antibodies, aptamers or high-affinity ligands. The captured antigens may be then directly or indirectly detected through an antibody or high-affinity and high-specificity recognition molecule. Various different detection methods are discussed, from label-free sensors and immunosensors to fluorescence-based ones. Each method shows advantages and disadvantages in terms of equipment, sensitivity, simplicity and cost-effectiveness. Finally, lab-on-a-chip (LOC) devices are introduced briefly, with the potential to be fast, sensitive and useful for on-site bacteria detection in food processing laboratories to check potential contamination by sample monitoring combined with a rapid pre-enrichment step.

Portable gliadin-immunochip for contamination control on the food production chain

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.

Real-time monitoring of copper ions-induced cytotoxicity by EIS cell chips

An important goal of biomedical research is the development of tools for high-throughput evaluation of drug effects and cytotoxicity tests. Here we demonstrate EIS cell chips able to monitor cell growth, morphology, adhesion and their changes as a consequence of treatment with drugs or toxic compounds. As a case study, we investigate the uptake of copper ions and its effect on two cell lines: B104 and HeLa cells. For further understanding, we also carried out in parallel with EIS studies, a complete characterization of cell morphology and changes induced by copper ions through complementary methodologies (including state-of-the-art AFM, viability test and Western blot). Our results reveal a strong correlation between EIS data and both MTT test and AFM characterization so our chip can be used as powerful tools in all biology lab in combination with other standard methods giving additional information that can be useful in a complete and deep investigation of a biological process. This chip can be used even alone replacing in vitro drug tests based on conventional biochemical methods, being very cheap and reusable and allowing to perform cytotoxicity tests without using any expensive reagent or equipment.

A multipurpose biochip for food pathogen detection

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.

Simultaneous detection of multiple lower genital tract pathogens by an impedimetric immunochip

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.

Sample preparation for the quick sizing of metal nanoparticles by atomic force microscopy

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs. Microsc. Res. Tech., 2008.

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.

Development of a lab-on-a-chip method for rapid assay of Xylella fastidiosa subsp. pauca strain CoDiRO

Xylella fastidiosa subsp. pauca strain CoDiRO, a pathogen responsible for Olive Quick Decline Syndrome (OQDS), is strongly threatening the agricultural-based economy of South Italy and making its typical landscape collapse. The bacteria can also infect more than other twenty woody or shrub species and quarantine programs are carried out in Italy. Since symptoms of OQDS like leaf scorching and wilting of canopy may appear several months after infection and some hosts are asymptomatic, a tool for the rapid and early screening of plants is desirable, in order to plan a sudden control strategy and apply programs for pest management. X. fastidiosa detection is usually performed by ELISA and PCR methods. In this work, the two standard methods are compared with an innovative on-chip detection strategy for X. fastidiosa assay from leaves samples, based on an electrochemical transduction method. The realized lab-on-chip includes also a microfluidic module and its performances are competitive with conventional diagnostic methods in terms of reliability, but with further advantages of portability, low-costs and ease of use. Thus, the proposed technology has the potential to provide a useful assay method for large-scale monitoring programs.