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Matrix metalloproteinases (MMPs) have been proposed as markers of many pathological conditions for their ability to degrade extracellular matrix components and remodel tissues. This review presents an overview of biosensors for the measurement of MMPs in serum, sputum and cell cultures. Major benefits and limitations of these technologies are discussed with respect to zymography and immunoassays, which are the reference methods to measure MMP activity and concentration. The paper also compares the analytical performances of sensors to the specific requirements for the application in clinical research, and comments on future trends in this field.

Here we report on a high-sensitivity interferometric technique, namely Interferogram Averaged over Wavelength - IAW-reflectance spectroscopy, enabling sub-nanomolar detection (both nonspecific and specific) of proteins to be accomplished in direct (without any amplification) label-free mode using porous silicon (PSi) interferometers. The IAW reflectance spectroscopy is based on both calculation and elaboration of spectral interferograms. Interferograms are obtained by subtracting (intensity, wavelength by wavelength) reflectance spectra acquired on PSi interferometers in buffer solution after protein injection (at different concentrations) from a reference reflectance spectrum acquired in buffer solution before protein injection. The output signal, namely IAW value, is eventually obtained as the average value, calculated over a given wavelength interval, of each interferogram. Using the IAW technique nonspecific adsorption of Bovine Serum Albumin (BSA) down to 0.15 nM (DL=20 pM) and specific detection of Tumor Necrosis Factor (TNF?) down to 3 nM (DL=0.2 nM) was reliably achieved in direct label-free mode using PSi interferometers. Both the results represent a 10000-fold reduction in DL with respect to the current literature on the use of PSi for direct label-free measurements.

Porous silicon (PSi) is a promising nanostructured material for label-free interferometric biosensing, though it currently suffers of restricted “real” applications in medical diagnostics. This is usually ascribed to PSi interferometer limited sensitivity due to mass-limited diffusion of (bio)molecules inside its nanopores. Here we report on a high-sensitivity label-free interferometric technique (i.e. Interferogram Average over Wavelength, IAW, reflectance spectroscopy) for, though not limited to, PSi interferometers. The IAW reflectance spectroscopy is based on the calculation of spectral interferograms obtained by subtraction of reflectance spectra acquired after interaction of target biomolecules with the PSi interferometer inner surface, from a reference reflection spectrum acquired in buffer before injection of target molecules. The IAW reflectance spectroscopy was set up studying nonpecific adsorption of Bovine Serum Albumin (BSA) onto oxidized PSi interferometers and then validated through the development of an aptasensor using PSi interferometers for the specific detection of Tumor Necrosis Factor a (TNFa, a sepsis biomarker). In both the cases we demonstrated that sub-nanomolar detection limits (DLs) can be achieved, which are 4 orders of magnitude lower than DLs achieved using Fast Fourier Transform (FFT) reflectance spectroscopy (usually in the micromolar range), in direct label-free operation. These results open exciting perspectives for point of care (POC) analysis based on PSi interferometers, for which increasingly lower detection limits are required.

In-field analysis (e.g., clinical and diagnostics) using nanostructured porous silicon (PSi) for label-free optical biosensing has been hindered so far by insufficient sensitivity of PSi biosensors. Here we report on a label-free PSi interferometric aptasensor able to specifically detect tumor necrosis factor alpha (TNFα, a protein biomarker of inflammation and sepsis) at concentration down to 3.0 nM with signal-to-noise ratio (S/N) of 10.6 and detection limit (DL) of 200 pM. This represents a 10 000-fold improvement with respect to direct (i.e., nonamplified) label-free PSi biosensors and pushes PSi biosensors close to the most sensitive optical and label-free transduction techniques, e.g., surface plasmon resonance (SPR) for which a lowest DL of 100 pM in aptasensing has been reported. A factor 1000 in improvement is achieved by introducing a novel signal-processing technique for the optical readout of PSi interferometers, namely, interferogram average over wavelength (IAW) reflectance spectroscopy. The...

Polycrystalline silicon–germanium (poly-SiGe) is a promising structural material for the post-processing of micro electro-mechanical systems (MEMS) on top of complementary metal-oxide-semiconductor (CMOS) substrates. Combining MEMS and CMOS allows for the development of high-performance devices. We present for the first time selective protein immobilization on top of poly-SiGe surfaces, an enabling technique for the development of novel poly-SiGe based MEMS biosensors. Active regions made of 3-aminopropyl-triethoxysilane (APTES) were defined using silane deposition onto photoresist patterns followed by lift-off in organic solvents. Subsequently, proteins were covalently bound on the created APTES patterns. Fluorescein-labeled human serum albumin (HSA) was used to verify the immobilization procedure while the binding capability of the protein layer was tested by an antigen-labeled antibody pair. Inspection by fluorescence microscopy showed protein immobilization inside the desired bioactive areas and low non-specific adsorption outside the APTES pattern. Furthermore, the quality of the silane patches was investigated by treatment with 30 nm-diameter gold nanoparticles and scanning electron microscope observation. The developed technique is therefore a promising first step towards the realization of poly-SiGe based biosensors.

The Nuclear Factor kappa B (NF-kB), a transcription factor regulating a battery of inflammatory genes, has been implicated as playing a fundamental role in the development of numerous pathological states. The NF-kB dimer is located in the cytoplasm of cells in an inactive form that can be activated by many stimuli (e.g. cytokines, bacterial or viral products, UV-radiation, etc.). It has been demonstrated that anti-inflammatory agents, both steroid and non steroid, besides their principal mechanism of action, have a secondary mechanism with anti-NF-kB effects. Therefore, when anti-inflammatory therapy is applied to an inflammatory disease, for which detection of the efficacy of the therapeutic agent is particularly important, monitoring of NF-kB-active form concentration (for example in cellular lysate) is an important target. The critical need for a simple and direct method to evaluate the presence of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. In order to evaluate FRET due to the DNA/protein binding interaction taking place between dsDNA immobilized in a capillary wall and p50 proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used.

A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab′)-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10−8 M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen–antibody complex and hence to regenerate the immunosensor surface was also evaluated.

Smart, MEMS-based biosensors are a promising new platform for the delivery of diagnostic services, but inclusion of on-chip CMOS electronics requires the definition a CMOS compatible techniques for the bio-activation of the sensor surface. In this work, a comparison between a sensor functionalization procedure by complete immersion of the sensor chip or by exposure to a single drop of the reagents (the latter approach being more compatible with the presence of on-chip CMOS components) is presented.

A simple and fast methodology for protein patterning on silicon substrates is presented, providing an insight into possible issues related to the interaction between biological and microelectronic technologies. The method makes use of standard photoresist lithography and is oriented towards the implementation of biosensors containing Complementary Metal-Oxide-Semiconductor (CMOS) conditioning circuitry. Silicon surfaces with photoresist patterns were prepared and hydroxylated by means of resist- and CMOS backend-compatible solutions. Subsequent aminosilane deposition and resist lift-off in organic solvents resulted into well-controlled amino-terminated geometries. The discussion is focused on resist- and CMOS-compatibility problems related to the used chemicals. Some samples underwent gold nanoparticle (Au NP) labeling and Scanning Electron Microscopy (SEM) observation, in order to investigate the quality of the silane layer. Antibodies were immobilized on other samples, which were subsequently exposed to a fluorescently labeled antigen. Fluorescence microscopy observation showed that this method provides spatially selective immobilization of protein layers onto APTES-patterned silicon samples, while preserving protein reactivity inside the desired areas and low non-specific adsorption elsewhere. Strong covalent biomolecule binding was achieved, giving stable protein layers, which allows stringent binding conditions and a good binding specificity, really useful for biosensing.

A novel optical platform for point of care testing application was developed. The core of the platform is a miniaturised polymethylmetacrylate (PMMA) chip through which the analysed sample flows. Thanks to the fluorescence anisotropy exhibited by any dipole emitting at a distance from a medium interface of the order of the emitted wavelength, a large fraction of the fluorescence emitted by the sensing layer immobilized onto the internal flow-channel travels along the thickness of the PMMA up to its end-face where it is collected by a plastic optical fibre connected to a spectrum analyser. The potentiality of the optical chip was investigated with a sandwich assay for the C-reactive protein, one of the markers for inflammatory diseases.