Optical Nanosensing
Nanoparticle and nanomaterial technologies in the biomedical field are significantly impacting the development of diagnostic tools.
In particular, the advent of optical nanoprobes for the quantitative determination of bioanalytes at intracellular level is one of the most fascinating challenges offered by the new developments in these technologies. With the advent of new nanostructured materials, such as magnetic and gold nanoparticles, quantum dots and carbon nanomaterials, new perspectives were opened to drive molecules inside cells.
The overall strategy for the detection of intracellular bioanalytes, with optical nanoprobes inside the cell, is not a trivial problem but relies on the conjugation of nanosized structures with different molecules capable:
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to address the nanostructures to the unhealthy cells,
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to bind marker molecules differentially expressed by these cells or interact with the chemicals of interest ,
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to improve the cell membrane penetration,
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to provide optical signals related to the amount of investigated analyte.
The development of nanosized optical fibre tips is an alternative solution to monitor intracellular parameters.
Activity in the last years has been concentrated on the use of molecular beacons and pH fluorescent indicators either immobilised on nanoparticles, more specifically either carbon nanotubes or PMMA nanoparticle, or fixed at the extremity of optical fibre nanotips.
Molecular beacons are formed by oligonucleotidic sequences labeled at the two side ends, with a fluorophore FA on one side and either a quencher Q or a second fluorophore FB on the other side, characterized by an absorption band overlapping with the FA emission band (FB λem>FA λem). These structures are generally designed to form a hairpin structure (stem-loop) when the complementary sequence is not present. In this condition, the fluorescence of FA is inhibited because of its closeness to Q or FB. In fact, if excited, FA does not emit, but transfers its energy to Q or FB along a nonradiative pathway, since the condition for this energy transfer—a distance between the two molecules on the order of a few angstroms—is well satisfied in the hairpin structure of the MB. When the hybridization with the complementary sequence occurs, the stem-loop structure opens up and FA and Q (or FB) become sufficiently spaced that the nonradiative transfer cannot occur and FA emits once it is excited. The interaction with the complementary sequence is consequently accompanied by the emission of light (in the case from the FA/Q pair)—and from this behavior these molecules are often denoted as molecular beacons—or by the shift of the emitted light at FA λem.
In collaboration with the MIPLAB of our same Institute, the use of optical fibre tips with the molecular beacon immobilised on them was also investigated as tools for the intracellular detection of mRNA messenger.
Projects
Main Publications on the subject
B.Adinolfi, M.Pellegrino, A.Giannetti, S.Tombelli, C.Trono, G.Sotgiu, G.Varchi, M.Ballestri, T.Posati, S.Carpi, P.Nieri, F.Baldini: Molecular beacon-decorated Polymethylmethacrylate Core-shell Fluorescent Nanoparticles for the Detection of Survivin mRNA in Human Cancer Cells, Biosens Bioelectron., 2016, published on line DOI 10.1016/j.bios.2016.05.102
Abstract:One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60 nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.9070.40 with a mean area of 1.0470.20 mm2), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.5072.30 fluorescent spots per cell with a mean area of 2.3570.16 mm2). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation.
B.Adinolfi. M.Pellegrino, F.Baldini, Human Dermal Fibroblasts HDFa can be used as an appropriate healthy control for PMMA nanoparticles-survivin molecular beacon cellular uptake studies, Biomedicine & Pharmacotherapy, 69, 228-232, 2015
Abstract :Survivin is a member of the inhibitor of apoptosis protein family (IAPs); besides its inhibitory action on apoptosis, it is also involved in the regulation of cell division. The protein expression is up-regulated in several cancers, being involved in the tumor progression and evasion of apoptosis. In line with its physiological roles, it is expressed also in several healthy tissues. The high expression of survivin in cancer cells correlates to poor prognosis and resistance to chemotherapeutic treatment, thus making this protein an attractive target in anticancer therapy. The dual role of survivin in cells, regulation of cell division and inhibition of apoptosis, combined with controversial data concerning the expression in normal tissues, emphasize the need to have an appropriate healthy control for in vitro studies. Aim of this study is to highlight this problem and to clarify the experimental conditions in which HDFa fibroblasts represent a negative control for survivin mRNA expression while ensuring the NPs-MB uptake. In this paper, by using confocal microscopy analysis supported by RT- and real-time-PCR experiments studies, we showed that HDFa fibroblasts represent a healthy negative control for survivin mRNA expression, only at very low cell density or at confluence. At the same time, we demonstrated that HDFa at any cell density are able to internalize NPs-MB after 6 h of treatment.
G.Tuci, L.Luconi, A.Rossin, F. Baldini, S. Tombelli, C. Trono, A. Giannetti, S. Cicchi, I. Manet, S. Fedeli, A. Brandi, G. Giambastiani: A Hetero-bi-Functional Spacer for the Smart Engineering of Carbon-based Nanostructures, Chem Plus Chem, 80, 704-714, 2015
Abstract :Efforts have been made in recent years to develop novel functionalisation protocols aimed at imparting multimodality and improved properties to complex carbon-based nanostructures. The incorporation of cleavable bonds to the nanomaterial sur-face for the controlled release (or exchange) of specific molecules under appropriate chemical and biological settings is relatively unexplored. The design and synthesis of a hetero bifunctional linker joining a “cleavable” disulfide moiety for the covalent anchoring of a wide range of thiol end-capped (bio)-molecules and a “clickable” terminal acetylene group is de-scribed. The strategy is based on the well-established copper-mediated acetylene–azide coupling reaction between the acetylene linker and single-walled carbon nanotubes decorated with phenylazido pendant arms. As a result, easily “post-derivatisable” and traceable nanostructured platforms containing a linking group potentially available for a wide range of biological probes are prepared and completely characterised.
A.Giannetti, A.Barucci, F.Cosi, S.Pelli, S.Tombelli, C.Trono, F.Baldini: Optical Fiber Nanotips Coated with Molecular Beacons for DNA Detection, Sensors, 15, 9666-9680, 2015
Abstract :Optical fiber sensors, thanks to their compactness, fast response and real-time measurements, have a large impact in the fields of life science research, drug discovery and medical diagnostics. In recent years, advances in nanotechnology have resulted in the development of nanotools, capable of entering the single cell, resulting in new nanobiosensors useful for the detection of biomolecules inside living cells. In this paper, we provide an application of a nanotip coupled with molecular beacons (MBs) for the detection of DNA. The MBs were characterized by hybridization studies with a complementary target to prove their functionality both free in solution and immobilized onto a solid support. The solid support chosen as substrate for the immobilization of the MBs was a 30 nm tapered tip of an optical fiber, fabricated by chemical etching. With this set-up promising results were obtained and a limit of detection (LOD) of 0.57 nM was reached, opening up the possibility of using the proposed nanotip to detect mRNAs inside the cytoplasm of living cells.
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S.Carpi, S.Fogli, A.Giannetti, B.Adinolfi, S.Tombelli, E.Da Pozzo, A.Vanni, E. Martinotti, C.Martini, M.C.Breschi, M.Pellegrino, P.Nieri, F. Baldini: Theranostic properties of a survivin-directed molecular beacon in human melanoma cells, PLoS ONE, 9(12): e114588 1-16, 2014
Abstract :Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment.
A. Giannetti, S. Tombelli, F. Baldini: Oligonucleotide optical switches for intracellular sensing, Anal Bioanal Chem 405, pp. 6181–6196, 2013
Abstract :Fluorescence imaging coupled with nanotechnology is making possible the development of powerful tools in the biological field for applications such as cellular imaging and intracellular messenger RNA monitoring and detection. The delivery of fluorescent probes into cells and tissues is currently receiving growing interest because such molecules, often coupled to nanodimensional materials, can conveniently allow the preparation of small tools to spy on cellular mechanisms with high specificity and sensitivity. The purpose of this review is to provide an exhaustive overview of current research in oligonucleotide optical switches for intracellular sensing with a focus on the engineering methods adopted for these oligonucleotides and the more recent and fascinating techniques for their internalization into living cells. Oligonucleotide optical switches can be defined as specifically designed short nucleic acid molecules capable of turning on or modifying their light emission on molecular interaction with well-defined molecular targets. Molecular beacons, aptamer beacons, hybrid molecular probes, and simpler linear oligonucleotide switches are the most promising optical nanosensors proposed in recent years. The intracellular targets which have been considered for sensing are a plethora of messenger-RNA-expressing cellular proteins and enzymes, or, directly, proteins or small molecules in the case of sensing through aptamer-based switches. Engineering methods, including modification of the oligonucleotide itself with locked nucleic acids, peptide nucleic acids, or L-DNA nucleotides, have been proposed to enhance the stability of nucleases and to prevent false-negative and high background optical signals. Conventional delivery techniques are treated here together with more innovative methods based on the coupling of the switches with nano-objects.
G.Ghini, C. Trono, A.Giannetti, G.L. Puleo, L. Luconi, J. Amadou, G. Giambastiani, F.Baldini: Carbon Nanotubes modified with fluorescein derivatives for pH nanosensing, Sens Actuat B, 179, pp. 163–169, 2013
Abstract :Multi-walled carbon nanotubes (MWCNTs) functionalized with carboxylic acid (MW-COOH) were investigated as macromolecular carriers for pH nanosensors. The activation of carboxylic groups with thionyl chloride (SOCl2) followed by reaction with a family of fluorescein ethylene glycol derivatives led to the covalent anchoring of the dyes to the CNT sidewall. This type of functionalization was found suitable for preserving the fluorescence properties of the dye, while providing at the same time higher water solubility to the modified macromolecular systems as compared with pristine carbon nanotubes. The use of polyether spacers between the dye and the nanotube surface was used to reduce fluorescence quenching effects. Fluorescence measurements conducted on fluorescein functionalized CNTs dispersed in water showed pH dependence in the 4.5–8 pH range with good sensitivities. Dye-excitation was obtained by means of a laser diode with a peak emission at 488 nm. An optical fiber orthogonally placed in relation to the excitation beam and connected to a spectrum analyzer, made it possible to record the fluorescence spectra of the nanostructured material.