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Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface functionalization of biosensors is a crucial aspect that influences their overall performance, resulting in the accurate, sensitive, and specific detection of target molecules. Here, the surface of a microring resonator (MRR)-based biosensor was functionalized for the detection of protein biomarkers. Among the several existing functionalization methods, a strategy based on aptamers and mercaptosilanes was selected as the most highly performing approach. All steps of the functionalization protocol were carefully characterized and optimized to obtain a suitable protocol to be transferred to the final biosensor. The functionalization protocol comprised a preliminary plasma treatment aimed at cleaning and activating the surface for the subsequent silanization step. Different plasma treatments as well as different silanes were tested in order to covalently bind aptamers specific to different biomarker targets, i.e., C-reactive protein, SARS-CoV-2 spike protein, and thrombin. Argon plasma and 1% v/v mercaptosilane were found as the most suitable for obtaining a homogeneous layer apt to aptamer conjugation. The aptamer concentration and time for immobilization were optimized, resulting in 1 µM and 3 h, respectively. A final passivation step based on mercaptohexanol was also implemented. The functionalization protocol was then evaluated for the detection of thrombin with a photonic biosensor based on microring resonators. The preliminary results identified the successful recognition of the correct target as well as some limitations of the developed protocol in real measurement conditions.

The identification and quantification of biomarkers with innovative technologies is an urgent need for the precise diagnosis and follow up of human diseases. Body fluids offer a variety of informative biomarkers, which are traditionally measured with time-consuming and expensive methods. In this context, lateral flow tests (LFTs) represent a rapid and low-cost technology with a sensitivity that is potentially improvable by chemiluminescence biosensing. Here, an LFT based on gold nanoparticles functionalized with antibodies labeled with the enzyme horseradish peroxidase is combined with a lensless biosensor. This biosensor comprises four Silicon Photomultipliers (SiPM) coupled in close proximity to the LFT strip. Microfluidics for liquid handling complete the system. The development and the setup of the biosensor is carefully described and characterized. C-reactive protein was selected as a proof-of-concept biomarker to define the limit of detection, which resulted in about 0.8 pM when gold nanoparticles were used. The rapid readout (less than 5 min) and the absence of sample preparation make this biosensor promising for the direct and fast detection of human biomarkers.

The presence of residual antibiotics in food is increasingly emerging as a worrying risk for human health both for the possible direct toxicity and for the development of antibiotic-resistant bacteria. In the context of food safety, new methods based on microfluidics could offer better performance, providing improved rapidity, portability and sustainability, being more cost effective and easy to use. Here, a microfluidic method based on the use of magnetic microbeads specifically functionalized and inserted in polymeric microchambers is proposed. The microbeads are functionalized either with aptamers, antibodies or small functional groups able to interact with specific antibiotics. The setup of these different strategies as well as the performance of the different functionalizations are carefully evaluated and compared. The most promising results are obtained employing the functionalization with aptamers, which are able not only to capture and release almost all tetracycline present in the initial sample but also to deliver an enriched and simplified solution of antibiotic. These solutions of purified antibiotics are particularly suitable for further analyses, for example, with innovative methods, such as label-free detection. On the contrary, the on-chip process based on antibodies could capture only partially the antibiotics, as well as the protocol based on beads functionalized with small groups specific for sulfonamides. Therefore, the on-chip purification with aptamers combined with new portable detection systems opens new possibilities for the development of sensors in the field of food safety.

Platelets are emerging as a promising source of blood biomarkers for several pathologies, including cancer. New automated techniques for easier manipulation of platelets in the context of lab-on-a-chips could be of great support for liquid biopsy. Here, several polymeric materials were investigated for their behavior in terms of adhesion and activation of human platelets. Polymeric materials were selected among the most used in microfabrication (PDMS, PMMA and COC) and commercial and home-made resins for 3D printing technology with the aim to identify the most suitable for the realization of microdevices for human platelets isolation and analysis. To visualize adherent platelets and their activation state scanning, electron microscopy was used, while confocal microscopy was used for evaluating platelets’ features. In addition, atomic force microscopy was employed to further study platelets adherent to the polymeric materials. Polymers were divided in two main groups: the most prone to platelet adhesion and materials that cause few or no platelets to adhere. Therefore, different polymeric materials could be identified as suitable for the realization of microdevices aimed at capturing human platelets, while other materials could be employed for the fabrication of microdevices or parts of microdevices for the processing of platelets, without loss on surfaces during the process.

Advanced materials could bring about fundamental improvements in the evolution of innovative analytical devices, i.e., biosensors or lab-on-a-chip devices, in particular in the context of liquid biopsies. Here, plasma deposition processes were tested for the introduction of primary amines on silicon surfaces by tuning the amounts and availability of amino-charged residues. Different binary (CH4/NH3) and ternary (CH4/NH3/H2 and CH4/NH3/N2) mixtures of gases were used as feeds for the plasma treatments. The obtained surfaces were fully characterized for their chemical and physical properties before their use as capture materials in a functional test. Synthetic and fluorescently conjugated microRNA-21 (miR-21) was selected as the target molecule. The capture of miR-21 increased linearly with the increase in amino nitrogen measured on surfaces. The surface showing the most promising performance was further analyzed in different conditions, i.e., varying pH and time of incubation, incubation with different microRNAs, and possible elution of captured microRNAs. The apparent pH range of primary amines present on the surfaces was around 3.5–4. Positively charged surfaces prepared via PE-CVD were, therefore, demonstrated as being suitable materials for the capture of microRNA biomarkers, paving the way for their inclusion in biomedical devices for the purification and analysis of circulating biomarkers.

Antibiotics are widely used to both prevent and treat bacterial diseases as well as promote animal growth. This massive use leads to the presence of residual antibiotics in food with severe consequences for human health. Limitations and regulations on the tolerated amount of antibiotics in food have been introduced and analytical methods have been developed. The bioanalytical methods usually employed to detect antibiotic residues, however, are time-consuming, expensive and laboratory-based. Novel methods with improved rapidity, portability and cost that are easy-to-use and sustainable are therefore highly desirable. In the attempt to fulfill this need, a microfluidic system was set up herein for the purification and pre-concentration of tetracyclines from raw milk selected as the case-study. The system includes a polymeric microfluidic chip containing magnetic beads loaded with copper to exploit the preferential interaction of tetracycline with divalent ions. The microfluidic system was demonstrated to efficiently pre-concentrate tetracycline, oxytetracycline and chlortetracycline with similar performances and efficiently purify tetracycline from raw milk without any pre-treatment. The simplified method described in this paper could be easily integrated in a compact and portable device for the in-field detection of tetracyclines, with the economic advantage of preventing food wastes and guaranteeing food safety.

Protein synthesis is a central process in all cells, crucial for cell development and maintenance. Translational dysregulation, in fact, is associated with cancer or neurodegenerative diseases. Active protein synthesis occurs on a supramolecular complex, named polyribosome or polysome, formed by a mRNA associated with multiple ribosomes. Polysomes therefore can be considered as a privileged molecular platform to obtain information about the physiological or pathological state in cells. The classical methods for purifying the mRNAs associated with polysomes mainly rely on ultracentrifugation in sucrose gradient followed by standard RNA extraction. This method present several drawbacks, among all it is a time-consuming procedure, which requires a fairly large amounts of starting material. New methods offering an efficient, rapid and user-friendly alternative to standard methods are therefore highly desirable. Here, a panel of surfaces and surface functionalizations were screened for their ability to entrap polysomes with the ultimate aim to set up smart biofunctional surfaces for the purification of nonlabelled polysomes and their associated mRNAs. As a proof-of-concept, prepurified ribosomes and polysomes were incubated on multiple functional surfaces and characterized by atomic force microscopy to assess number and morphology of entrapped polysomes. Surfaces able to efficiently capture polysomes were then included in a microdevice with promising results, opening the future perspective of developing protocols and devices based on biofunctional surfaces.

The isolation and analysis of circulating biomarkers, the main concern of liquid biopsy, could greatly benefit from microfluidics. Microfluidics has indeed the huge potentiality to bring liquid biopsy into the clinical practice. Here, two polydimethylsiloxane (PDMS)-based microdevices are presented as valid tools for capturing microRNAs biomarkers from clinically-relevant samples. After an extensive study of functionalized polydimethylsiloxane (PDMS) properties in adsorbing/eluting microRNAs, the best conditions were transferred to the microdevices, which were thoroughly characterized. The channels morphology and chemical composition were measured, and parameters for the automation of measures were setup. The best working conditions were then used with microdevices, which were proven to capture microRNAs on all channel surfaces. Finally, microfluidic devices were successfully validated via real-time PCR for the detection of a pool of microRNAs related to non-small cell lung cancer, selected as proof-of-principle. The microfluidic approach described here will allow a step forward towards the realization of an efficient microdevice, possibly automated and integrated into a microfluidic lab-on-a-chip with high analytical potentialities.

Aflatoxins (AF) are naturally occurring mycotoxins, produced by many species of Aspergillus. Among aflatoxins, Aflatoxin M1 (AFM1) is one of the most frequent and dangerous for human health. The acceptable maximum level of AFM1 in milk according to EU regulation is 50 ppt, equivalent to 152 pM, and 25 ppt, equivalent to 76 pM, for adults and infants, respectively. Here, we study a photonic biosensor based on Si 3 N 4 asymmetric Mach–Zehnder Interferometers (aMZI) functionalized with Fab’ for AFM1 detection in milk samples (eluates). The minimum concentration of AFM1 detected by our aMZI sensors is 48 pM (16.8 pg/mL) in purified and concentrated milk samples. Moreover, the real-time detection of the ligand-analyte binding enables the study of the kinetics of the reaction. We measured the kinetic rate constants of the Fab’-AFM1 interaction.

The demand of miniaturized, accurate and robust micro-tools for minimally invasive surgery or in general for micro-manipulation, has grown tremendously in recent years. To meet this need, a new-concept comb-driven microgripper was designed and fabricated. Two microgripper prototypes differing for both the number of links and the number of conjugate surface flexure hinges are presented. Their design takes advantage of an innovative concept based on the pseudo-rigid body model, while the study of microgripper mechanical potentialities in different configurations is supported by finite elements’ simulations. These microgrippers, realized by the deep reactive-ion etching technology, are intended as micro-tools for tissue or cell manipulation and for minimally invasive surgery; therefore, their biocompatibility in terms of protein fouling was assessed. Serum albumin dissolved in phosphate buffer was selected to mimic the physiological environment and its adsorption on microgrippers was measured. The presented microgrippers demonstrated having great potential as biomedical tools, showing a modest propensity to adsorb proteins, independently from the protein concentration and time of incubation.