Supramolecular Nucleic Acid-Based Organosilica Nanoparticles Responsive to Physical and Biological Inputs.

Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol-gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson-Crick-Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics.

Folding-upon-Repair DNA Nanoswitches for Monitoring the Activity of DNA Repair Enzymes.

We present a new class of DNA-based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding-upon-repair DNA nanoswitches are synthetic DNA sequences containing O6 -methyl-guanine (O6 -MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of the O6 -MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding-upon-repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.

Rapid, Cost-Effective Peptide/Nucleic Acid-Based Platform for Therapeutic Antibody Monitoring in Clinical Samples.

We demonstrate here a homogeneous assay, named NanoHybrid, for monoclonal antibody quantification directly in serum samples in a single-step format. NanoHybrid is composed of both synthetic peptide nucleic acids (PNAs) and nucleic acid strands conjugated to recognition elements and optical labels and is designed to allow fast fluorescence quantification of a therapeutic antibody. More specifically, we have characterized our analytical assay for the detection of trastuzumab (Herceptin), a monoclonal antibody (mAb) drug used for breast cancer treatment and for tumors overexpressing the HER2/neu protein. We show here that NanoHybrid is capable of performing fast drug quantification directly in blood serum. The results obtained with a pool of samples from breast cancer patients under trastuzumab treatment are compared with CE-IVD ELISA (enzyme-linked immunosorbent assay) showing a good agreement (Cohen's K = 0.729). Due to the modular nature of the NanoHybrid platform, this technology can be programmed to potentially detect and quantify any antibody for which a high-affinity recognition element has been characterized. We envision the application of NanoHybrid in a point-of-care (POC) drug monitoring system based on disposable kits for therapeutic drug management.

Integrated chemiluminescence-based lab-on-chip for detection of life markers in extraterrestrial environments.

The detection of life markers is a high priority task in the exploration of the Solar System. Biochips performing in-situ multiplex immunoassays are a very promising approach alternative to gas chromatography coupled with mass spectrometry. As part of the PLEIADES project, we present the development of a chemiluminescence-based, highly integrated analytical platform for the detection of biomarkers outside of the Earth. The PLEIADES device goes beyond the current lab-on-chip approaches that still require bulky external instrumentation for their operation. It exploits an autonomous capillary force-driven microfluidic network, an array of thin-film hydrogenated amorphous silicon photosensors, and chemiluminescence bioassays to provide highly sensitive analyte detection in a very simple and compact configuration. Adenosine triphosphate was selected as the target life marker. Three bioassay formats have been developed, namely (a) a bioluminescence assay exploiting a luciferase mutant with enhanced thermal and pH stability and (b and c) binding assays exploiting antibodies or functional nucleic acids (aptamers) as biospecific recognition elements and peroxidase or DNAzymes as chemiluminescence reporters. Preliminary results, showing limits of detection in the nanomolar range, confirm the validity of the proposed approach.

Photoinduced electron transfer through peptide-based self-assembled monolayers chemisorbed on gold electrodes: directing the flow-in and flow-out of electrons through peptide helices.

Photoinduced electron transfer (PET) experiments have been carried out on peptide self-assembled monolayers (SAM) chemisorbed on a gold substrate. The oligopeptide building block was exclusively formed by C(α)-tetrasubstituted α-aminoisobutyric residues to attain a helical conformation despite the shortness of the peptide chain. Furthermore, it was functionalized at the C-terminus by a pyrene choromophore to enhance the UV photon capture cross-section of the compound and by a lipoic group at the N-terminus for linking to gold substrates. Electron transfer across the peptide SAM has been studied by photocurrent generation experiments in an electrochemical cell employing a gold substrate modified by chemisorption of a peptide SAM as a working electrode and by steady-state and time-resolved fluorescence experiments in solution and on a gold-coated glass. The results show that the electronic flow through the peptide bridge is strongly asymmetric; i.e., PET from the C-terminus to gold is highly favored with respect to PET in the opposite direction. This effect arises from the polarity of the Au-S linkage (Au(δ+)-S(δ-), junction effect) and from the electrostatic field generated by the peptide helix.

Photocurrent generation through peptide-based self-assembled monolayers on a gold surface: antenna and junction effects.

The photocurrent generation properties of mono- and bi-component peptide-based self-assembled monolayers (SAMs) immobilized on a gold surface were studied by electrochemical and spectroscopic techniques. The peptides investigated comprised almost exclusively C-tetrasubstituted -amino acids. These non-coded residues, because of their unique conformational properties, forced the peptide backbone to attain a helical conformation, as confirmed by X-ray crystal structure and CD determinations in solution. The peptide helical structure promoted the formation of a stable SAM on the gold surface, characterized by an electric macrodipole directed from the C(δ−) to the N(δ+) terminus, that remarkably affected the electron transfer (ET) process through the peptide chain. The peptides investigated were derivatized with chromophores strongly absorbing in the UV region to enhance the efficiency of the photocurrent generation (antenna effect). The influence of the nature of the peptide­gold interface on the ET process (junction effect) was analyzed by comparing the photocurrent generation process in peptide SAMs immobilized on a gold surface through AuS linkages with that in a bi-component SAM embedding a photoactive peptide into the linked palisade formed by disulfide-functionalized peptides.