Innovative Detection of Biomarkers Based on Chemiluminescent Nanoparticles and a Lensless Optical Sensor.

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.

Design of a Portable Microfluidic Platform for EGOT-Based in Liquid Biosensing.

In biosensing applications, the exploitation of organic transistors gated via a liquid electrolyte has increased in the last years thanks to their enormous advantages in terms of sensitivity, low cost and power consumption. However, a practical aspect limiting the use of these devices in real applications is the contamination of the organic material, which represents an obstacle for the realization of a portable sensing platform based on electrolyte-gated organic transistors (EGOTs). In this work, a novel contamination-free microfluidic platform allowing differential measurements is presented and validated through finite element modeling simulations. The proposed design allows the exposure of the sensing electrode without contaminating the EGOT device during the whole sensing tests protocol. Furthermore, the platform is exploited to perform the detection of bovine serum albumin (BSA) as a validation test for the introduced differential protocol, demonstrating the capability to detect BSA at 1 pM concentration. The lack of contamination and the differential measurements provided in this work can be the first steps towards the realization of a reliable EGOT-based portable sensing instrument.

3D Cell Culture: Recent Development in Materials with Tunable Stiffness.

It is widely accepted that three-dimensional cell culture systems simulate physiological conditions better than traditional 2D systems. Although extracellular matrix components strongly modulate cell behavior, several studies underlined the importance of mechanosensing in the control of different cell functions such as growth, proliferation, differentiation, and migration. Human tissues are characterized by different degrees of stiffness, and various pathologies (e.g., tumor or fibrosis) cause changes in the mechanical properties through the alteration of the extracellular matrix structure. Additionally, these modifications have an impact on disease progression and on therapy response. Hence, the development of platforms whose stiffness could be modulated may improve our knowledge of cell behavior under different mechanical stress stimuli. In this review, we have analyzed the mechanical diversity of healthy and diseased tissues, and we have summarized recently developed materials with a wide range of stiffness.

Functional 3D printing: Approaches and bioapplications.

3D printing technology has become a mature manufacturing technique, widely used for its advantages over the traditional methods, such as the end-user customization and rapid prototyping, useful in different application fields, including the biomedical one. Indeed, it represents a helpful tool for the realization of biodevices (i.e. biosensors, microfluidic bioreactors, drug delivery systems and Lab-On-Chip). In this perspective, the development of 3D printable materials with intrinsic functionalities, through the so-called 4D printing, introduces novel opportunities for the fabrication of "smart" or stimuli-responsive devices. Indeed, functional 3D printable materials can modify their surfaces, structures, properties or even shape in response to specific stimuli (such as pressure, temperature or light radiation), adding to the printed object new interesting properties exploited after the fabrication process. In this context, by combining 3D printing technology with an accurate materials' design, functional 3D objects with built-in (bio)chemical functionalities, having biorecognition, biocatalytic and drug delivery capabilities are here reported.

Laser-Triggered Writing and Biofunctionalization of Thiol-Ene Networks.

The light responsivity of ortho-nitrobenzyl esters (o-NBE) is exploited to inscribe µ-scale 2.5D patterns in thiol-ene networks by direct laser writing. For this purpose, a multifunctional thiol and a photosensitive alkene with an o-NBE chromophore are cured upon visible light exposure without inducing a premature photocleavage of the o-NBE links. Once the network is formed, a laser beam source with a wavelength of 375 nm is used for selectively inducing the photocleavage reaction of the o-NBE groups. Positive tone patterns are directly inscribed onto the sample surface without the requirement of a subsequent development step (removing soluble species in an appropriate organic solvent). Along with the realization of dry-developable micropatterns, the chemical surface composition of the exposed areas can be conveniently adjusted since different domains with a tailored content of carboxylic groups are obtained simply by modulating the laser energy dose. In a following step, those are activated and exploited as anchor points for attaching an Alexa-546 conjugated Protein A. Thus, the laser writable thiol-ene networks do not only provide a convenient method for the fabrication of positive tone patterns but also open future prospectives for a wide range of biosensing applications.

A modular 3D printed lab-on-a-chip for early cancer detection.

A functional polymeric 3D device is produced in a single step printing process using a stereolithography based 3D printer. The photocurable formulation is designed for introducing a controlled amount of carboxyl groups (-COOH), in order to perform a covalent immobilization of bioreceptors on the device. The effectiveness of the application is demonstrated by performing an immunoassay for the detection of protein biomarkers involved in angiogenesis, whose role is crucial in the onset of cancer and in the progressive metastatic behavior of tumors. The detection of angiogenesis biomarkers is necessary for an early diagnosis of the pathology, allowing the employment of a less invasive therapy for the patient. In particular, vascular endothelial growth factor and angiopoietin-2 biomarkers are detected with a limit of detection of 11 ng mL-1 and 0.8 ng mL-1, respectively. This study shows how 3D microfabrication techniques, material characterization, and device development could be combined to obtain an engineered polymeric chip with intrinsic tuned functionalities.

Experimental evidence of Fano resonances in nanomechanical resonators.

Fano resonance refers to an interference between localized and continuum states that was firstly reported for atomic physics and solid-state quantum devices. In recent years, Fano interference gained more and more attention for its importance in metamaterials, nanoscale photonic devices, plasmonic nanoclusters and surface-enhanced Raman scattering (SERS). Despite such interest in nano-optics, no experimental evidence of Fano interference was reported up to now for purely nanomechanical resonators, even if classical mechanical analogies were referred from a theoretical point of view. Here we demonstrate for the first time that harmonic nanomechanical resonators with relatively high quality factors, such as cantilevers vibrating in vacuum, can show characteristic Fano asymmetric curves when coupled in arrays. The reported findings open new perspectives in fundamental aspects of classical nanomechanical resonators and pave the way to a new generation of chemical and biological nanoresonator sensors with higher parallelization capability.

Functionalized ZnO nanowires for microcantilever biosensors with enhanced binding capability.

An efficient way to increase the binding capability of microcantilever biosensors is here demonstrated by growing zinc oxide nanowires (ZnO NWs) on their active surface. A comprehensive evaluation of the chemical compatibility of ZnO NWs brought to the definition of an innovative functionalization method able to guarantee the proper immobilization of biomolecules on the nanostructured surface. A noteworthy higher amount of grafted molecules was evidenced with colorimetric assays on ZnO NWs-coated devices, in comparison with functionalized and activated silicon flat samples. ZnO NWs grown on silicon microcantilever arrays and activated with the proposed immobilization strategy enhanced the sensor binding capability (and thus the dynamic range) of nearly 1 order of magnitude, with respect to the commonly employed flat functionalized silicon devices. Graphical Abstract An efficient way to increase the binding capability of microcantilever biosensors is represented by growing zinc oxide nanowires (ZnO NWs) on their active surface. ZnO NWs grown on silicon microcantilever arrays and activated with an innovative immobilization strategy enhanced the sensor binding capability of nearly 1 order of magnitude, with respect to the commonly employed flat functionalized silicon devices.

Succinic anhydride functionalized microcantilevers for protein immobilization and quantification.

Microcantilever-based systems have been proposed as sensing platforms owing to their high sensitivity when used as mass sensors. The controlled immobilization on a surface of biomolecules used as recognition elements is fundamental in order to realize a highly specific and sensitive biosensor. Here, we introduce for the first time the application to a microcantilever-based system of a reliable chemical functionalization consisting of silanization with an aminosilane followed by a modification resulting in a carboxylated thin film. This chemical functionalization was tested for reproducibility of molecule deposition and for its protein grafting ability. Finally, this system was employed for the quantification of grafted proteins on the microcantilever surface. Moreover, a theoretical surface density of immobilized proteins estimated with bioinformatics tools was compared with the experimental surface density data, providing information about the orientation that the biomolecules assumed with respect to the sensing surface.

Optimization and characterization of a homogeneous carboxylic surface functionalization for silicon-based biosensing.

A well-organized immobilization of bio-receptors is a crucial goal in biosensing, especially to achieve high reproducibility, sensitivity and specificity. These requirements are usually attained with a controlled chemical/biochemical functionalization that creates a stable layer on a sensor surface. In this work, a chemical modification protocol for silicon-based surfaces to be applied in biosensing devices is presented. An anhydrous silanization step through 3-aminopropylsilane (APTES), followed by a further derivatization with succinic anhydride (SA), is optimized to generate an ordered flat layer of carboxylic groups. The properties of APTES/SA modified surface were compared with a functionalization in which glutaraldehyde (GA) is used as crosslinker instead of SA, in order to have a comparison with an established and largely applied procedure. Moreover, a functionalization based on the controlled deposition of a plasma polymerized acrylic acid (PPAA) thin film was used as a reference for carboxylic reactivity. Advantages and drawbacks of the considered methods are highlighted, through physico-chemical characterizations (OCA, XPS, and AFM) and by means of a functional Protein G/Antibody immunoassay. These analyses reveal that the most homogeneous, reproducible and active surface is achieved by using the optimized APTES/SA coupling.