Laura Pasquardini

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.

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.

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.

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.

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.

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.

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.

Innovative microRNA purification based on surface properties modulation.

The increasing interest in circulating microRNAs (miRNAs) as potential non-invasive cancer biomarkers has prompted the rapid development of several extraction techniques. However, current methods lack standardization and are costly and labor intensive. In light of this, we developed a microRNA solid-phase extraction strategy based on charge and roughness modulation on substrate surfaces. PECVD treated silicon oxide (PECVD-SO) and thermally grown silicon oxide (TG-SO) surfaces were functionalized with positively charged 3-aminopropyltriethoxysilanes (APTES) and neutral poly(ethylene glycol) silanes (PEG-s) mixed in different proportions to modulate the density of net positive charges and the roughness of the substrate. Characterization of the surfaces was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and s-SDTB (sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) assay in order to investigate the surface morphology and chemical composition, respectively. Adsorption and elution efficiency were assessed by fluorescence microscopy by means of synthetic fluorescently labeled microRNAs. We identified PECVD-SO functionalized with 0.1% APTES and 0.9% 21-24 units long PEG-s as a promising surface able to selectively bind microRNAs and release them in the presence of a basic buffer (pH=9) compatible with downstream analyses. MicroRNA integrity was assessed by reverse transcription and real-time PCR and confirmed by electrophoresis (Agilent 2100 Bioanalyzer), while binding competition from circulating DNA and proteins was excluded by fluorescence analyses and real-time PCR. On the contrary, total RNA slightly decreased miRNA adsorption. In conclusion, we showed an innovative and easy solid-state purification method for circulating miRNAs based on charge interaction, which could pave the path to future diagnostic and prognostic assays feasible as a routine test.

Smart detection of microRNAs through fluorescence enhancement on a photonic crystal

The detection of low abundant biomarkers, such as circulating microRNAs, demands innovative detection methods with increased resolution, sensitivity and specificity. Here, a biofunctional surface was implemented for the selective capture of microRNAs, which were detected through fluorescence enhancement directly on a photonic crystal. To set up the optimal biofunctional surface, epoxy-coated commercially available microscope slides were spotted with specific anti-microRNA probes. The optimal concentration of probe as well as of passivating agent were selected and employed for titrating the microRNA hybridization. Cross-hybridization of different microRNAs was also tested, resulting negligible. Once optimized, the protocol was adapted to the photonic crystal surface, where fluorescent synthetic miR-16 was hybridized and imaged with a dedicated equipment. The photonic crystal consists of a dielectric multilayer patterned with a grating structure. In this way, it is possible to take advantage from both a resonant excitation of fluorophores and an angularly redirection of the emitted radiation. As a result, a significant fluorescence enhancement due to the resonant structure is collected from the patterned photonic crystal with respect to the outer non-structured surface. The dedicated read-out system is compact and based on a wide-field imaging detection, with little or no optical alignment issues, which makes this approach particularly interesting for further development such as for example in microarray-type bioassays.

Highly parallel SPAD detector for time-resolved lab-on-chip

Fluorescence lifetime detection is widely used in molecular biology to monitor many cell parameters (such as pH, ion concentrations, etc.) and for an early diagnosis of many pathologies. In a typical fluorescence lifetime experiment a pulsed laser is used to excite the fluorescent dyes and the emitted light is revealed by means of high sensitivity detectors, typically: intensified CCD, PMTs or Single-Photon Avalanche Diodes (SPADs).In this contribute we present a SPAD detector module fabricated in a 0.35μm High Voltage CMOS technology to be used within a lab-on-chip system consisting of a micro-reactor array for bioaffinity assays based on fluorescence markers. The detector module, having a total area of 600 x 900 μm2, can be arranged to build a small pixel array to be directly coupled to the micro-reactors. No emission filters are needed, since the ultra-short laser pulse is cut off in the time domain. The module consists of a 10x10-SPAD array, where each SPAD cell is equipped with dedicated active quenching and recharging circuit. Each cell has a pitch of 26μm with a fill factor of 48%. The SPADs have been binned in order to realize a large photosensitive area detector exhibiting a reasonably low dark count rate (DCR) and reduced dead time, as required in a fast measurement system. A memory has also been implemented in order to enable only low DCR SPADs, so that a total DCR of about 100kHz can be achieved for the whole photosensitive area. The digital output generated by the SPAD array is sent to a time-discriminator stage which allows a time-gated detection of the incident light. Two time-windows have been implemented in this architecture. Their time width is controlled by an on-chip digital PLL locked to the external laser clock whereas the width of the time-windows can be set within the range 500ps-10ns with a resolution of 500ps. Photons detected within each time window are then counted by two 10-bits digital counters. Time-interleaved operation has been implemented to read out the pixel data in parallel with the photon detection phase.