Optimization of Surface Functionalizations for Ring Resonator-Based Biosensors.

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.

Human Blood Platelets Adsorption on Polymeric Materials for Liquid Biopsy.

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.

Tuning Surface Properties via Plasma Treatments for the Improved Capture of MicroRNA Biomarkers.

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.

On-Chip Purification of Tetracyclines Based on Copper Ions Interaction.

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.

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.

AFM1 Detection in Milk by Fab' Functionalized Si3N4 Asymmetric Mach-Zehnder Interferometric Biosensors.

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.

Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.

BACKGROUND: Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. METHODS: We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. FINDINGS: From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. INTERPRETATION: We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).

Asymmetric Mach-Zehnder Interferometer Based Biosensors for Aflatoxin M1 Detection.

In this work, we present a study of Aflatoxin M1 detection by photonic biosensors based on Si3N4 Asymmetric Mach-Zehnder Interferometer (aMZI) functionalized with antibodies fragments (Fab'). We measured a best volumetric sensitivity of 104 rad/RIU, leading to a Limit of Detection below 5 × 10(-7) RIU. On sensors functionalized with Fab', we performed specific and non-specific sensing measurements at various toxin concentrations. Reproducibility of the measurements and re-usability of the sensor were also investigated.

Bio-hybrid interfaces to study neuromorphic functionalities: New multidisciplinary evidences of cell viability on poly(anyline) (PANI), a semiconductor polymer with memristive properties.

The interfacing of artificial devices with biological systems is a challenging field that crosses several disciplines ranging from fundamental research (biophysical chemistry, neurobiology, material and surface science) to frontier technological application (nanotechnology, bioelectronics). The memristor is the fourth fundamental circuit element, whose electrical properties favor applications in signal processing, neural networks, and brain-computer interactions and it represents a new frontier for technological applications in many fields including the nanotechnologies, bioelectronics and the biosensors. Using multidisciplinary approaches, covering surface science, cell biology and electrophysiology, we successfully implemented a living bio-hybrid system constituted by cells adhering to films of poly(aniline) (PANI), a semiconductor polymer having memristive properties assembled with polyelectrolytes. Here we tested whether the PANI devices could support survivor, adhesion and differentiation of several cell lines, including the neuron-like SHSY5Y cells. Moreover, we performed electrophysiology on these cells showing that the biophysical properties are retained with differences occurring in the recorded ion currents. Taken together, the cell viability here reported is the key requirement to design and develop a reliable functional memristor-based bio-hybrid able to mimic neuronal activity and plasticity.

Design and Optimization of SiON Ring Resonator-Based Biosensors for Aflatoxin M1 Detection.

In this article, we designed and studied silicon oxynitride (SiON) microring-based photonic structures for biosensing applications. We designed waveguides, directional couplers, and racetrack resonators in order to measure refractive index changes smaller than 10-6 refractive index units (RIU). We tested various samples with different SiON refractive indexes as well as the waveguide dimensions for selecting the sensor with the best performance. Propagation losses and bending losses have been measured on test structures, along with a complete characterization of the resonator's performances. Sensitivities and limit of detection (LOD) were also measured using glucose-water solutions and compared with expected results from simulations. Finally, we functionalized the resonator and performed sensing experiments with Aflatoxin M1 (AFM1). We were able to detect the binding of aflatoxin for concentrations as low as 12.5 nm. The results open up the path for designing cost-effective biosensors for a fast and reliable sensitive analysis of AFM1 in milk.

An easy way to realize SPR aptasensor: A multimode plastic optical fiber platform for cancer biomarkers detection.

The introduction of new compact systems for sensitive, fast and simplified analysis is currently playing a substantial role in the development of point-of-care solutions aimed to assist both prognosis and diagnosis. Here we report a simple and low cost biosensor based on Surface Plasmon Resonance (SPR) taking advantage of a plastic optical fiber (POF) for the detection of Vascular endothelial growth factor (VEGF), selected as a circulating protein potentially associated with cancer. Our system is based onto two crucial aspects. By one hand, the functional layer which allows the transduction signal is based on DNA aptamers, short oligonucleotide sequences that bind to non-nucleic acid targets with high affinity and specificity. By the other hand, the light guiding structure is based on a POF with a planar gold layer as the sensing region, which is particularly suitable for bioreceptors implementation. The sensor revealed to be really useful in the interface characterization. The developed system is relatively easy to realize and could well address the development of a rapid, portable and low cost diagnostic platform, with a sensitivity in the nanomolar range.

Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes.

Translation is increasingly recognized as a central control layer of gene expression in eukaryotic cells. The overall organization of mRNA and ribosomes within polysomes, as well as the possible role of this organization in translation are poorly understood. Here we show that polysomes are primarily formed by three distinct classes of ribosome assemblies. We observe that these assemblies can be connected by naked RNA regions of the transcript. We show that the relative proportions of the three classes of ribosome assemblies reflect, and probably dictate, the level of translational activity. These results reveal the existence of recurrent supra-ribosomal building blocks forming polysomes and suggest the presence of unexplored translational controls embedded in the polysome structure.

SPAD aptasensor for the detection of circulating protein biomarkers.

The need for decentralized clinical tests together with the concept of time and cost saving are pushing the development of portable, miniaturized, compact biosensors with diagnostic and prognostic purpose. Here, we propose an innovative detection system based on a Single Photon Avalanche Diode (SPAD) with high sensitivity and low noise, crucial features for an efficient chemiluminescence biosensor. The SPAD detector, having 60 µm diameter, has a Photon Detection Efficiency higher than 55% at 425 nm and a Dark Count Rate lower than 100 Hz at room temperature. Our design allows a good optical coupling efficiency between sample and detector. A specific biofunctional surface was implemented taking advantage of aptamers, short DNA sequences having high selectivity and affinity toward their targets. We successfully detected physiological levels of Vascular Endothelial Growth Factor (VEGF), a circulating protein biomarker highly correlated with cancer. The SPAD aptasensor showed a Limit of Detection (LoD) in the pM range, stability (up to 42 days) and re-usability (up to seven cycles). This compact biosensor is therefore a promising step toward the actual use of portable microdevices in diagnostics.

OncomiR detection in circulating body fluids: a PDMS microdevice perspective.

There is an increasing interest in circulating microRNAs (miRNAs) as potential minimally invasive diagnostic biomarkers in oncology. Considerable efforts are being made in the development of lab-on-a-chip devices for biomedical applications to purify and detect miRNAs from biological fluids. Here, we report the development of an innovative polydimethylsiloxane (PDMS)-based parallel device whose internal surface can opportunely be functionalized with positively charged 3-aminopropyltriethoxysilane (APTES) alone or mixed with two different neutral poly(ethylene glycol) silanes (PEG-s). The differently functionalized internal surfaces of the PDMS chip were characterized with s-SDTB (sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) and the portion of the surface able to adsorb a synthetic fluorescently labeled miRNA was determined. Interestingly, the adsorbed miRNA (both synthetic and cell supernatant-derived) was found mainly on the bottom surface of the chip and could be reverse transcribed into cDNA directly on the same PDMS chip used for its purification, saving hours with respect to the use of standard purification kits. We identified 0.1% APTES/0.9% PEG-silane as the most efficient PDMS functionalization to capture both synthetic and extracellular miRNA. Moreover, the amount of captured miRNA was increased by treating the cell supernatant with a commercially available lysis buffer for RNA extraction. We assessed that the available miRNA binding sites on the functionalized surface were efficiently saturated with only one incubation, shortening the time and greatly simplifying the protocol for miRNA purification from biological samples. Finally, the extracellular miRNA purification efficiency of the PDMS functionalized multichip determined via real-time quantitative polymerase chain reaction (RT-qPCR) was confirmed by droplet digital PCR (ddPCR) quantification. This work shows an innovative, rapid and easy to use microdevice for the purification and reverse transcription of circulating miRNAs, approaching the realization of diagnostic and prognostic oncomiR-based assays.

Whispering gallery mode aptasensors for detection of blood proteins.

Whispering gallery mode resonators (WGMR), as silica microspheres, have been recently proposed as an efficient tool for the realisation of optical biosensors. In this work we present a functionalization procedure based on the DNA-aptamer sequence immobilization on WGMR, able to recognize specifically thrombin or VEGF protein, preserving a high Q factor. The protein binding was optically characterized in terms of specificity in buffer solution or in 10% diluted human serum. Simulation of the protein flow was found in good agreement with experimental data. The aptasensor was also chemically regenerated and tested again, demonstrating the reusability of our system.

Solid phase DNA extraction on PDMS and direct amplification.

In this paper we report an innovative use of Poly(DiMethyl)Siloxane (PDMS) to design a microfluidic device that combines, for the first time, in one single reaction chamber, DNA purification from a complex biological sample (blood) without elution and PCR without surface passivation agents. This result is achieved by exploiting the spontaneous chemical structure and nanomorphology of the material after casting. The observed surface organization leads to spontaneous DNA adsorption. This property allows on-chip complete protocols of purification of complex biological samples to be performed directly, starting from cells lysis. Amplification by PCR is performed directly on the adsorbed DNA, avoiding the elution process that is normally required by DNA purification protocols. The use of one single microfluidic volume for both DNA purification and amplification dramatically simplifies the structure of microfluidic devices for DNA preparation. X-Ray Photoelectron Spectroscopy (XPS) was used to analyze the surface chemical composition. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) were employed to assess the morphological nanostructure of the PDMS-chips. A confocal fluorescence analysis was utilized to check DNA distribution inside the chip.

Effect of materials for micro-electro-mechanical systems on PCR yield.

In this study we analyzed the surface properties of different silicon-based materials used for micro-electro-mechanical systems (MEMS) production, such as thermally grown silicon oxide, plasma-enhanced chemical vapor deposition (PECVD)-treated silicon oxide, reactive-ion etch (RIE)-treated silicon oxide, and Pyrex. Substrates were characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) to define the surface chemical and morphological properties, and by fluorescence microscopy to directly assess the absorption of the different polymerase chain reaction (PCR) components. By using microchips fabricated with the same materials we investigated their compatibility with PCR reactions, exploiting the use of different enzymes and reagents or proper surface treatments. We established the best conditions for DNA amplification in silicon/Pyrex microdevices depending on the type of device and fabrication method used and the quality of reagents, rather than on the passivation treatment or increment in standard Taq polymerase concentration.

Human plasma protein adsorption on carbon-based materials.

In this study the initial reactions of different carbon-based materials with human blood were investigated by short-time exposure to platelet poor plasma (PPP). Extent of protein adsorption and conformational changes of proteins adsorbed on material surfaces are known to be keys factors affecting further biological reactions. Plasma protein adsorption on multi-walled carbon nanotubes (MWCNTs), highly oriented pyrolytic graphite (HOPG) and nanocrystalline graphite (NG) were investigated and the results obtained on these materials were compared with those obtained studying pyrolytic carbon (PyC), a material showing good anti-trombogenic properties. The quantification of adsorbed plasma proteins on sample surfaces was obtained by Micro BCA Protein Assay, while immunofluorescence analysis was employed to monitor the surface density and distribution of two selected proteins, namely fibrinogen (Fg) and Hageman factor (FXII), proteins playing a leading role in mediating platelet adhesion. The dependence of the biological response on the surface chemical and morphological properties were also investigated and data obtained using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and atomic force microscopy (AFM) are presented. After PPP incubation PyC is characterized by the presence of low level of whole proteins and FXII adsorption, in contrast to a high adhesion of Fg. Compared to PyC the analysis of the other carbon-based materials result in a higher whole protein adsorption with an increasing trend moving from MWCNTs, NG and HOPG respectively. The Fg surface density on PyC, NG and MWCNTs is about four times higher than on HOPG while only HOPG show a detectable fluorescent signal of FXII. If AFM data indicate that surface morphology does not play a crucial role in protein adhesion, XPS analysis show chemical differences that can be correlated with this biological response.

Covalently anchored lipid structures on amine-enriched polystyrene.

The study of the adhesion of lipid vesicles on surfaces is of increasing interest in the field of medical implants and tissue engineering (protein-resistant surfaces), drug delivery, biosensors, and biochips. In this work, lipid coverage was developed from PEG-coated vesicles (with sizes from 100 to 300 nm) by covalently binding poly(ethylene glycol)-alpha-disteroylphosphatidylethanolamine-omega-benzotriazole carbonate (DSPE-PEG-BTC) molecules onto the surface amine groups by carbamate chemistry. Lipid surface density and the surface structure of multilamellar (MLVs) and extruded unilamellar (LUVs) vesicles deposited on three types of polystyrene (PS) well-plates were probed by fluorescence and atomic force microscopy (AFM) imaging. A significant difference in the vesicle surface coverage of PS substrates was observed with a substantial increase in lipid multilayers on the amine-enriched PS surface using both unilamellar and multilamellar vesicles.