Role of probe design and bioassay configuration in surface enhanced Raman scattering based biosensors for miRNA detection.

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100-10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays.

Endosperm structure and Glycemic Index of Japonica Italian rice varieties.

Introduction: Given that rice serves as a crucial staple food for a significant portion of the global population and with the increasing number of individuals being diagnosed with diabetes, a primary objective in genetic improvement is to identify and cultivate low Glycemic Index (GI) varieties. This must be done while ensuring the preservation of grain quality. Methods: 25 Italian rice genotypes were characterized calculating their GI "in vivo" and, together with other 29 Italian and non-Italian genotypes they were studied to evaluate the grain inner structure through Field Emission Scanning Electron Microscopy (FESEM) technique. Using an ad-hoc developed algorithm, morphological features were extracted from the FESEM images, to be then inspected by means of multivariate data analysis methods. Results and Discussion: Large variability was observed in GI values (49 to 92 with respect to glucose), as well as in endosperm morphological features. According to the percentage of porosity is possible to distinguish approximately among rice varieties having a crystalline grain (< 1.7%), those intended for the preparation of risotto (> 5%), and a third group having intermediate characteristics. Waxy rice varieties were not united by a certain porosity level, but they shared a low starch granules eccentricity. With reference to morphological features, rice varieties with low GI (<55) seem to be characterized by large starch granules and low porosity values. Our data testify the wide variability of Italian rice cultivation giving interesting information for future breeding programs, finding that the structure of the endosperm can be regarded as a specific characteristic of each variety.

Real-Time Monitoring of the In Situ Microfluidic Synthesis of Ag Nanoparticles on Solid Substrate for Reliable SERS Detection.

A sharpened control over the parameters affecting the synthesis of plasmonic nanostructures is often crucial for their application in biosensing, which, if based on surface-enhanced Raman spectroscopy (SERS), requires well-defined optical properties of the substrate. In this work, a method for the microfluidic synthesis of Ag nanoparticles (NPs) on porous silicon (pSi) was developed, focusing on achieving a fine control over the morphological characteristics and spatial distribution of the produced nanostructures to be used as SERS substrates. To this end, a pSi membrane was integrated in a microfluidic chamber in which the silver precursor solution was injected, allowing for the real-time monitoring of the reaction by UV-Vis spectroscopy. The synthesis parameters, such as the concentration of the silver precursor, the temperature, and the flow rate, were varied in order to study their effects on the final silver NPs' morphology. Variations in the flow rate affected the size distribution of the NPs, whereas both the temperature and the concentration of the silver precursor strongly influenced the rate of the reaction and the particle size. Consistently with the described trends, SERS tests using 4-MBA as a probe showed how the flow rate variation affected the SERS enhancement uniformity, and how the production of larger NPs, as a result of an increase in temperature or of the concentration of the Ag precursor, led to an increased SERS efficiency.

Rational engineering of the lccß T. versicolor laccase for the mediator-less oxidation of large polycyclic aromatic hydrocarbons.

Laccases are among the most sought-after biocatalyst for many green applications, from biosensors to pollution remedial, because they simply need oxygen from the air to oxidize and degrade a broad range of substrates. However, natural laccases cannot process large and toxic polycyclic aromatic hydrocarbons (PAHs) except in the presence of small molecules, called mediators, which facilitate the reaction but are inconvenient for practical on-field applications. Here we exploited structure-based protein engineering to generate rationally modified fungal laccases with increased ability to process bulky PAHs even in a mediator-less reaction. Computational simulations were used to estimate the impact of mutations in the enzymatic binding pocket on the ability to bind and oxidize a selected set of organic compounds. The most promising mutants were produced and their activity was evaluated by biochemical assays with phenolic and non-phenolic substrates. Mutant laccases engineered with a larger binding pocket showed enhanced activity (up to ~ 300% at pH 3.0) in a wider range of pH values (3.0-8.0) in comparison to the wild type enzyme. In contrast to the natural laccase, these mutants efficiently degraded bulky and harmful triphenylmethane dyes such as Ethyl Green (up to 91.64% after 24 h), even in the absence of mediators, with positive implications for the use of such modified laccases in many green chemistry processes (e.g. wastewater treatment).

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.

Electrospun Nanofibers: from Food to Energy by Engineered Electrodes in Microbial Fuel Cells.

Microbial fuel cells (MFCs) are bio-electrochemical devices able to directly transduce chemical energy, entrapped in an organic mass named fuel, into electrical energy through the metabolic activity of specific bacteria. During the last years, the employment of bio-electrochemical devices to study the wastewater derived from the food industry has attracted great interest from the scientific community. In the present work, we demonstrate the capability of exoelectrogenic bacteria used in MFCs to catalyze the oxidation reaction of honey, employed as a fuel. With the main aim to increase the proliferation of microorganisms onto the anode, engineered electrodes are proposed. Polymeric nanofibers, based on polyethylene oxide (PEO-NFs), were directly electrospun onto carbon-based material (carbon paper, CP) to obtain an optimized composite anode. The crucial role played by the CP/PEO-NFs anodes was confirmed by the increased proliferation of microorganisms compared to that reached on bare CP anodes, used as a reference material. A parameter named recovered energy (Erec) was introduced to determine the capability of bacteria to oxidize honey and was compared with the Erec obtained when sodium acetate was used as a fuel. CP/PEO-NFs anodes allowed achieving an Erec three times higher than the one reached with a bare carbon-based anode.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.

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.

Advanced ELISA-like Biosensing Based on Ultralarge-Pore Silica Microbeads.

In this work, functionalized porous silica-based materials, widely used in the literature as drug and biomolecule nanocarriers, were innovatively used as an effective three-dimensional (3D) substrate for the development of a specific biomolecular assay showing great versatility in terms of detection performance. One-pot synthesis of ultralarge-pore silica microbeads was optimized to develop an enzyme-linked immunosorbent (ELISA)-like DNA detection assay. Cocondensation synthesis enabled introducing thiol functionalities into the silica framework while preserving both the high specific surface area (560 m2/g) and large pore size (17 nm average diameter), which are essential to guaranteeing high loading capability. Indeed, the bead-capturing ability was proved by developing an ELISA-like assay for the detection of short DNA sequences (≈20 bp), both in labeled and label-free configurations. In particular, the suppression of unspecific binding on the bead surface by testing two different blocking agents was a matter of interest. The detection performances were evaluated and compared to the ones obtained by following the same detection protocol on a standard flat surface (two-dimensional, 2D), which is most commonly used for this purpose. The bead-based assay showed a limit of detection two times lower than the flat-surface assay, confirming the promising capturing ability due to the larger active surface area. Furthermore, compared to traditional ELISA, the bead-based assay showed an intrinsic larger dynamic range that can be tailored depending on the final amount of beads used for the colorimetric quantification.

Photofabrication of polymeric biomicrofluidics: New insights into material selection.

We propose a versatile method to evaluate the suitability of polymers for the fabrication of microfluidic devices for biomedical applications, based on the concept that the selection and the design of convenient materials should involve different properties depending on the final microfluidic application. Here polymerase chain reaction (PCR) is selected as biological model and target microfluidic reaction. A class of photocured siloxanes is introduced as device building polymers and copolymerization is adopted as strategy to finely tune and optimize the final material properties. All-polymeric flexible devices are easily fabricated exploiting the rapidity of the photopolymerization reaction: they resist to thermal cycles without leakage or de-bonding (i.e., without separation of different chip parts made of the same material bonded together), show very limited water swelling and permeability, are bioinert and prevent the inhibition of the biochemical reaction. PCR is thus successfully conducted in the photocured microfluidic devices made with a specifically designed siloxane copolymer.

Label-Free SERS Discrimination and In Situ Analysis of Life Cycle in Escherichia coli and Staphylococcus epidermidis.

Surface enhanced Raman spectroscopy (SERS) has been proven suitable for identifying and characterizing different bacterial species, and to fully understand the chemically driven metabolic variations that occur during their evolution. In this study, SERS was exploited to identify the cellular composition of Gram-positive and Gram-negative bacteria by using mesoporous silicon-based substrates decorated with silver nanoparticles. The main differences between the investigated bacterial strains reside in the structure of the cell walls and plasmatic membranes, as well as their biofilm matrix, as clearly noticed in the corresponding SERS spectrum. A complete characterization of the spectra was provided in order to understand the contribution of each vibrational signal collected from the bacterial culture at different times, allowing the analysis of the bacterial populations after 12, 24, and 48 h. The results show clear features in terms of vibrational bands in line with the bacterial growth curve, including an increasing intensity of the signals during the first 24 h and their subsequent decrease in the late stationary phase after 48 h of culture. The evolution of the bacterial culture was also confirmed by fluorescence microscope images.

SERS-active metal-dielectric nanostructures integrated in microfluidic devices for label-free quantitative detection of miRNA.

In this work, SERS-based microfluidic PDMS chips integrating silver-coated porous silicon membranes were used for the detection and quantitation of microRNAs (miRNAs), which consist of short regulatory non-coding RNA sequences typically over- or under-expressed in connection with several diseases such as oncogenesis. In detail, metal-dielectric nanostructures which provide noticeable Raman enhancements were functionalized according to a biological protocol, adapted and optimized from an enzyme-linked immunosorbent assay (ELISA), for the detection of miR-222. Two sets of experiments based on different approaches were designed and performed, yielding a critical comparison. In the first one, the labelled target miRNA is revealed through hybridization to a complementary thiolated DNA probe, immobilized on the silver nanoparticles. In the second one, the probe is halved into shorter strands (half1 and half2) that interact with the complementary miRNA in two steps of hybridization. Such an approach, taking advantage of the Raman labelling of half2, provides a label-free analysis of the target. After suitable optimisation of the procedures, two calibration curves allowing quantitative measurements were obtained and compared on the basis of the SERS maps acquired on the samples loaded with several miRNA concentrations. The selectivity of the two-step assay was confirmed by the detection of target miR-222 mixed with different synthetic oligos, simulating the hybridization interference coming from similar sequences in real biological samples. Finally, that protocol was applied to the analysis of miR-222 in cellular extracts using an optofluidic multichamber biosensor, confirming the potentialities of SERS-based microfluidics for early-cancer diagnosis.

Polymeric 3D Printed Functional Microcantilevers for Biosensing Applications.

In this study, we show for the first time the production of mass-sensitive polymeric biosensors by 3D printing technology with intrinsic functionalities. We also demonstrate the feasibility of mass-sensitive biosensors in the form of microcantilever in a one-step printing process, using acrylic acid as functional comonomer for introducing a controlled amount of functional groups that can covalently immobilize the biomolecules onto the polymer. The effectiveness of the application of 3D printed microcantilevers as biosensors is then demonstrated with their implementation in a standard immunoassay protocol. This study shows how 3D microfabrication techniques, material characterization, and biosensor development could be combined to obtain an engineered polymeric microcantilever with intrinsic functionalities. The possibility of tuning the composition of the starting photocurable resin with the addition of functional agents, and consequently controlling the functionalities of the 3D printed devices, paves the way to a new class of mass-sensing microelectromechanical system devices with intrinsic properties.

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.

Immobilization of Oligonucleotides on Metal-Dielectric Nanostructures for miRNA Detection.

The development of nanostructured metal-dielectric materials, suitable for biodetection based on surface plasmon resonance and surface enhanced Raman scattering (SERS), requires the refinement of proper biological protocols for their effective exploitation. In this work, the immobilization of DNA probes on nanostructured metal-dielectric/semiconductor substrates has been optimized, to develop a bioassay for the detection of miRNA. To ensure a broad relevance, the proposed biological protocol was applied to different silver-decorated functional supports: porous silicon (pSi), TiO2 nanotube arrays, and polydimethylsiloxane (PDMS). The efficiency and the stability of the substrates were carefully analyzed by Raman spectroscopy and electron microscopy after the incubation in buffers with the appropriate combination of pH, ionic strength, and surfactant content. The customized protocol, initially developed on multiwell plates, was transferred and refined on the nanostructured substrates. The nonspecific interaction of the biological species with the surface was evaluated and reduced thanks to a tailored surface pretreatment. SERS analysis was applied to check the immobilization of DNA probes on pretreated samples. Silvered PDMS-supported pSi membranes, the most promising substrates in terms of stability, were subjected to further optimizations. Concentrations, volume, and duration of incubations were finely adapted with respect to the surface probe density and to the corresponding hybridization of the complementary miRNA. The optimized ELISA-like assay shows sensitivities comparable to those of commercial plates for the detection of miRNA222 (LOD: 485 pM), paving the way for the application of the developed protocol on metal-dielectric/semiconductor nanostructures for ultrasensitive SERS biosensing applications.

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.

Rational modification of estrogen receptor by combination of computational and experimental analysis.

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor-ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an half maximal inhibitory concentration (IC50) value of 2 nM for the 17α-ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated estrogen receptor instead of the wild type as bio-recognition element would be beneficial in an assay or biosensor.