Precise functionalization of NPs towards chiral NP dimers

In the ChiroSense project we are developing a new chiroptical biosensor based on a dimer of gold and silicon nanoparticles (NPs), utilizing plasmonically enhanced circular dichroism (CD) measurements to probe molecule conformation in solution with increased sensitivity [1]. The development will strongly be guided by the use of consensus tetratricopeptide repeat (CTPR) proteins as a highly modular and well-defined structural probe, while simultaneously being employed as structural linker of the nano-particle dimer [2]. To deconvolute the structural and molecular components of the CD signal, the dimer will also be formed using single stranded DNA (ssDNA), which is also modular but lacks the distinct CD signal of CTPR [3]. Precisely controlling the loading ratio of molecules per NP is crucial for both approaches to achieve stable NPs that do not aggregate but are still able to form dimers. Here we present our initial results on the development of these chiral NP dimers. To achieve functionalized NPs that enable dimer formation, it is important to achieve low loading ratios, to avoid aggregation, while maintaining the NP stability in solution. We developed a PEG based approach that allows us to precisely control the NP loading ratio, ranging from a few to several hundred molecules per NP. These particles were then used to synthesize chiral NP dimers with high yields

Protein-based (bio)materials: a way toward high-performance graphene enzymatic biosensors

Enzymes are ideal receptors for biosensors since they offer excellent selectivity and high catalytic activity. However, once removed from their native environment, enzymes present a short lifespan determining a huge drawback for their application in bio-analytical systems. The use of appropriate immobilization matrices is an effective strategy to preserve enzymatic activity. In this work, an enzymatic amperometric biosensor is designed by entrapping lactate oxidase into a protein-based immobilization matrix, formed by the self-assembly of engineered repeat proteins. Electrochemically exfoliated graphene, functionalized with cobalt phthalocyanine, is employed as electroactive material and transducer of the sensor. Due to the extraordinary enzymatic stabilization provided by the engineered protein film, the device sensitivity is preserved for more than 6 months at room temperature. Furthermore, the presented biosensor can detect lactate with outstanding performance in terms of sensitivity, repeatability, and reproducibility

Proteins Are Solitary! Pathways of Protein Folding and Aggregation in Protein Mixtures

We present a computational and experimental study on the folding and aggregation in solutions of multiple protein mixtures at different concentrations. We show how in protein mixtures, each component is capable of maintaining its folded state at desensitises higher then the one at which they would precipitate in single species solutions. We demonstrate the generality of our observation over many different proteins using computer simulations capable of fully characterising the cross-aggregation phase diagram of all the mixtures. Dynamic light Scattering experiments were performed to evaluate the aggregation of two proteins, the bovine serum albumin (BSA) and the consensus tetratricopeptide repeat (CTPR), in solutions of one or both proteins. The experiment confirm our hypothesis and the simulations. These findings elucidate critical aspects on the cross-regulation of expression and aggregation of proteins exerted by the cell and on the evolutionary selection of folding and not-aggregating protein sequences, paving the way for new experimental tests

An Electroactive and Self-Assembling Bio-Ink, based on Protein-Stabilized Nanoclusters and Graphene, for the Manufacture of Fully Inkjet-Printed Paper-Based Analytical Devices

Hundreds of new electrochemical sensors are reported in literature every year. However, only a few of them makes it to the market. Manufacturability, or rather the lack of it, is the parameter that dictates if new sensing technologies will remain forever in the laboratory in which they are conceived. Inkjet printing is a low-cost and versatile technique that can facilitate the transfer of nanomaterial-based sensors to the market. Herein, an electroactive and self-assembling inkjet-printable ink based on protein-nanomaterial composites and exfoliated graphene is reported. The consensus tetratricopeptide proteins (CTPRs), used to formulate this ink, are engineered to template and coordinate electroactive metallic nanoclusters (NCs), and to self-assemble upon drying, forming stable films. The authors demonstrate that, by incorporating graphene in the ink formulation, it is possible to dramatically improve the electrocatalytic properties of the ink, obtaining an efficient hybrid material for hydrogen peroxide (H2O2) detection. Using this bio-ink, the authors manufactured disposable and environmentally sustainable electrochemical paper-based analytical devices (ePADs) to detect H2O2, outperforming commercial screen-printed platforms. Furthermore, it is demonstrated that oxidoreductase enzymes can be included in the formulation, to fully inkjet-print enzymatic amperometric biosensors ready to use

Engineering Iron Oxide Nanoparticles for Clinical Settings

Iron oxide nanoparticles (IONPs) occupy a privileged position among magnetic nanomaterials with potential applications in medicine and biology. They have been widely used in preclinical experiments for imaging contrast enhancement, magnetic resonance, immunoassays, cell tracking, tissue repair, magnetic hyperthermia and drug delivery. Despite these promising results, their successful translation into a clinical setting is strongly dependent upon their physicochemical properties, toxicity and functionalization possibilities. Currently, IONPs-based medical applications are limited to the use of non-functionalized IONPs smaller than 100 nm, with overall narrow particle size distribution, so that the particles have uniform physical and chemical properties. However, the main entry of IONPs into the scene of medical application will surely arise from their functionalization possibilities that will provide them with the capacity to target specific cells within the body, and hence to play a role in the development of specific therapies. In this review, we offer an overview of their basic physicochemical design parameters, giving an account of the progress made in their functionalization and current clinical applications. We place special emphasis on past and present clinical trials

Repeat protein scaffolds: ordering photo- and electroactive molecules in solution and solid state

The precise control over the organization of photoactive components at the nanoscale is one of the main challenges for the generation of new and sophisticated macroscopically ordered materials with enhanced properties. In this work we present a novel bioinspired approach using protein-based building blocks for the arrangement of photo and electroactive porphyrin derivatives. We used a designed repeat protein scaffold with demonstrated unique features that allow for the control of their structure, functionality, and assembly. Our designed domains act as exact biomolecular templates to organize porphyrin molecules at the required distance. The hybrid conjugates retain the structure and assembly properties of the protein scaffold and display the spectroscopic features of orderly aggregated porphyrins along the protein structure. Finally,we achieved a solid ordered bio-organic hybrid thin film with anisotropic photoconductivity

Tailored functionalized magnetic nanoparticles to target breast cancer cells including cancer stem-like cells

Nanotechnology-based approaches hold substantial potential to avoid chemoresistance and minimize side effects. In this work, we have used biocompatible iron oxide magnetic nanoparticles (MNPs) called MF66 and functionalized with the antineoplastic drug doxorubicin (DOX) against MDA-MB-231 cells. Electrostatically functionalized MNPs showed effective uptake and DOX linked to MNPs was more efficiently retained inside the cells than free DOX, leading to cell inactivation by mitotic catastrophe, senescence and apoptosis. Both effects, uptake and cytotoxicity, were demonstrated by different assays and videomicroscopy techniques. Likewise, covalently functionalized MNPs using three different linkers—disulfide (DOX-S-S-Pyr, called MF66-S-S-DOX), imine (DOX-I-Mal, called MF66-I-DOX) or both (DOX-I-S-S-Pyr, called MF66-S-S-I-DOX)—were also analysed. The highest cell death was detected using a linker sensitive to both pH and reducing environment (DOX-I-S-S-Pyr). The greatest success of this study was to detect also their activity against breast cancer stem-like cells (CSC) from MDA-MB-231 and primary breast cancer cells derived from a patient with a similar genetic profile (triple-negative breast cancer). In summary, these nanoformulations are promising tools as therapeutic agent vehicles, due to their ability to produce efficient internalization, drug delivery, and cancer cell inactivation, even in cancer stem-like cells (CSCs) from patientsThis research was funded by the European Seventh Framework Program (grant agreement number 262943); the European Union’s Horizon 2020 research and innovation programme (grant agreement number 685795); Ministerio de Economía y Competitividad, Spain (grants CTQ2016-78454-C2-2-R, BIO2016-77367-C2-1-R and SAF2017-87305-R); Basque Government Elkartek KK- 2017/00008; Comunidad de Madrid (IND2017/IND-7809; S2017/BMD-3867 RENIM-CM and S2018/NMT-4321 NANOMAGCOST-CM); NIHR Manchester Biomedical Research Centre (IS-BRC-1215-20007) and Breast Cancer Now (MAN-Q2); co-financed by European Structural Cancers 2020, 12, 1397 17 of 19 and Investment Fund, Asociación Española Contra el Cáncer (Singulares 2014) and IMDEA Nanociencia. CIC biomaGUNE acknowledges Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant MDM-2017-0720). IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686

Protein-directed crystalline 2D fullerene assemblies

Water soluble 2D crystalline monolayers of fullerenes grow on planar assemblies of engineered consensus tetratricopeptide repeat proteins. Designed fullerene-coordinating tyrosine clamps on the protein introduce specific fullerene binding sites, which facilitate fullerene nucleation. Through reciprocal interactions between the components, the hybrid material assembles into two-dimensional 2 nm thick structures with crystalline order, that conduct photo-generated charges. Thus, the protein-fullerene hybrid material is a demonstration of the developments toward functional materials with protein-based precision control of functional elements

Engineering conductive protein films through nanoscale self-assembly and gold nanoparticles doping

Protein-based materials are usually considered as insulators, although conductivity has been recently shown in proteins. This fact opens the door to develop new biocompatible conductive materials. While there are emerging efforts in this area, there is an open challenge related to the limited conductivity of protein-based systems. This work shows a novel approach to tune the charge transport properties of protein-based materials by using electron-dense AuNPs. Two strategies are combined in a unique way to generate the conductive solid films: (1) the controlled self-assembly of a protein building block; (2) the templating of AuNPs by the engineered building block. This bottom-up approach allows controlling the structure of the films and the distribution of the AuNPs within, leading to enhanced conductivity. This work illustrates a promising strategy for the development of effective hybrid protein-based bioelectrical materialsThis work was partially supported by the European Research Council ERC-CoG-648071-ProNANO, ERC-PoC-841063-NIMM, Agencia Estatal de Investigación, Spain (PID2019- 111649RB-I00; and MAT2017-88693-R), and the Basque Government (Elkartek KK-2017/00008), E.L-M thanks the Spanish Ministry of Science and Innovation for the FPI grant (BES-2017-079646). This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720 (CIC biomaGUNE) and SEV-2016-0686 (IMDEA Nanociencia