POROUS SILICON BASED OPTICAL DEVICES FOR BIOCHEMICAL SENSING

This thesis summarizes a three years scientific research investigation on the design and fabrication of porous silicon based optical devices for applications in the field of biochemical sensing. Porous silicon is an ideal transducer material due to its sponge-like morphology, characterized by a specific surface area up to 500 m2 cm-3, which assures an effective interaction with gas and liquid substances. Moreover, porous silicon is a low cost material, completely compatible with standard microelectronic processes. In this work, different porous silicon structures such as Fabry-Perot interferometer, Bragg mirror, optical microcavity, Thue-Morse sequences and optical waveguide have been realized and characterized as optical transducers for the monitoring of chemical and biological interactions. The selectivity, reversibility and sensitivity of these devices as optical sensors have been discussed. The porous silicon surface has been modified in order to gain chemical stability, proper wettability, and specific features such as biomolecules immobilization. Standard chemical functionalizations, but also an innovative pure biological passivation method based on selfassembled biofilms of the Hydrophobins proteins, have been successfully experimented. Some standard micromachining techniques, such as HF wet etching and anodic bonding, have been optimized to integrate the porous silicon sensing element into a Lab-on-Chip prototype. The integrated devices have been characterized as fast sensors of chemical compounds and response times shorter than 100 ms have been demonstrated. The Direct-Laser-Writing of the porous silicon surface, as alternative process to the photolithographic patterning in the device miniaturization has been also exploited. Finally, a bottom-up approach in microoptics has been developed by using the silica shells of some marine Diatoms, microalgae which show impressive morphological and physical analogies with porous silicon

Peptide functionalization of silicon for detection and classification of prostatic cells

The development of simple, rapid, and low costmethods for early detection, identification, andmeasurement ofmultiple biomarkers remains a challenge to improve diagnosis, treatment monitoring, and prognosis of cancer. Biosensing technology, combining the properties of biological systems with functional advanced materials, guarantees rapid, reproducible, and highly sensitive cell detection. In this study, we developed silicon-based biochips for prostate cancer PC3 cells detection by using cytokeratin 8/18 and Urotensin Receptor (UTR) as markers in order to obtain a biochip-based diagnostic system. Spectroscopic ellipsometry and fluorescence microscopy were used to characterize surface homogeneity and chemical properties. Cell detection was investigated by optical microscopy.Moreover, synthetic fluorescently labeled peptides were prepared and used for developing faster and lowercost identification assay compared with classic ELISA immunoassay. Results showed an effective immobilization of PC3 cells on silicon surface and the specific recognition of these cells by fluorescent Urotensin II (4-11). In conclusion, this strategy could be really useful as diagnostic system for prostate cancer

Surface bioengineering of diatomite based nanovectors for efficient intracellular uptake and drug delivery

Diatomite is a natural porous silica material of sedimentary origin. Due to its peculiar properties, it can be considered as a valid surrogate of synthetic porous silica for nano-based drug delivery. In this work, we exploit the potential of diatomite nanoparticles (DNPs) for drug delivery with the aim of developing a successful dual-biofunctionalization method by polyethylene glycol (PEG) coverage and cell-penetrating peptide (CPP) bioconjugation, to improve the physicochemical and biological properties of the particles, to enhance the intracellular uptake in cancer cells, and to increase the biocompatibility of 3-aminopropyltriethoxysilane (APT) modified-DNPs. DNPs-APT-PEG-CPP showed hemocompatibility for up to 200 mu g mL(-1) after 48 h of incubation with erythrocytes, with a hemolysis value of only 1.3%. The cytotoxicity of the modified-DNPs with a concentration up to 200 mu g mL(-1) and incubation with MCF-7 and MDA-MB-231 breast cancer cells for 24 h, demonstrated that PEGylation and CPP-bioconjugation can strongly reduce the cytotoxicity of DNPs-APT. The cellular uptake of the modified-DNPs was also evaluated using the above mentioned cancer cell lines, showing that the CPP-bioconjugation can considerably increase the DNP cellular uptake. Moreover, the dual surface modification of DNPs improved both the loading of a poorly water-soluble anticancer drug, sorafenib, with a loading degree up to 22 wt%, and also enhanced the drug release profiles in aqueous solutions. Overall, this work demonstrates that the biofunctionalization of DNPs is a promising platform for drug delivery applications in cancer therapy as a result of its enhanced stability, biocompatibility, cellular uptake, and drug release profiles.Peer reviewe

Nanogravimetric and Optical Characterizations of Thrombin Interaction with a Self-Assembled Thiolated Aptamer

Efficient biorecognition of thrombin (TB), a serine protease with crucial role in physiological and pathological blood coagulation, is a hot topic in medical diagnostics. In this work, we investigate the ability of synthetic thrombin aptamer (TBA), immobilized on a gold substrate, to bind thrombin by two different label-free techniques: the quartz crystal microbalance (QCM) and the spectroscopic ellipsometry (SE). By QCM characterization in the range from 20 to 110 nM, we demonstrate high specificity of TBA-TB interaction and determine affinity constant (Kd) of 17.7 ± 0.3 nM, system sensitivity of 0.42 ± 0.03 Hz nM-1, and limit of detection (LOD) of 240 ± 20 pM. The interaction between TBA and TB is also investigated by SE, an all-optical method, by quantifying the thickness increase of the TBA film assembled on gold substrate. AFM characterization of TBA and TB molecules deposited on flat silicon surface is also supplied

silicon based technology for ligand receptor molecular identification

One of the most important goals in the fields of biology and medicine is the possibility to dispose of efficient tools for the characterization of the extraordinary complexity of ligand-receptor interactions. To approach this theme, we explored the use of crystalline silicon (cSi) technology for the realization of a biotechnological device in which the ligand-receptor interactions are revealed by means of optical measurements. Here, we describe a chemical procedure for the functionalization of microwell etched on silicon wafers, and the subsequent anchoring of biological molecules like an antibody anti-A20 murine lymphoma cell line. The optical analysis of the interaction on the biochips between the bound biomolecule and their corresponding ligand indicated that the functionalized cSi is suitable for this application

Bioengineered Surfaces for Real-Time Label-Free Detection of Cancer Cells

Biosensing technology is an advancing field that benefits from the properties of biological processes combined to functional materials. Recently, biosensors have emerged as essential tools in biomedical applications, offering advantages over conventional clinical techniques for diagnosis and therapy. Optical biosensors provide fast, selective, direct, and cost-effective analyses allowing label-free and real-time tests. They have also shown exceptional potential for integration in lab-on-a-chip (LOC) devices. The major challenge in the biosensor field is to achieve a fully operative LOC platform that can be used in any place at any time. The choice of an appropriate strategy to immobilize the biological element on the sensor surface becomes the key factor to obtain an applicable analytical tool. In this chapter, after a brief description of the main biofunctionalization procedures on silicon devices, two silicon-based chips that present an (i) IgG antibody or (ii) an Id-peptide as molecular probe, directed against the B-cell receptor of lymphoma cancer cells, will be presented. From a comparison in detecting cells, the Id-peptide device was able to detect lymphoma cells also at low cell concentrations (8.5 × 10−3 cells/μm2) and in the presence of a large amount of non-specific cells. This recognition strategy could represent a proof-of-concept for an innovative tool for the targeting of patient-specific neoplastic B cells during the minimal residual disease; in addition, it represents an encouraging starting point for the construction of a lab-on-a-chip system for the specific recognition of neoplastic cells in biological fluids enabling the follow-up of the changes of cancer cells number in patients, highly demanded for therapy monitoring applications

Microfluidic-Assisted Production of Gastro-Resistant Active-Targeted Diatomite Nanoparticles for the Local Release of Galunisertib in Metastatic Colorectal Cancer Cells (Adv. Healthcare Mater. 6/2023)

Nanoparticle Drug DeliveryThe encapsulation of nanoparticles in hydroxypropyl methyl cellulose is key to treat colorectal cancer through oral administration. In article 2202672 by Ilaria Rea, Hélder A. Santos, and co-workers, gelatin-coated diatomite nanoparticles loaded with galunisertib are functionalized with an anti-L1-CAM antibody that targets metastatic colon cancer cells. These nanoparticles are then encapsulated in a gastro-resistant matrix using microfluidics. When the nanoparticles interact with the targeted cells, the gastro-resistant coating dissolves and galunisertib is released