Alberto M. Hernández‐Pinto

Centro de Investigaciones Biológicas Margarita Salas

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Enrique J. de la Rosa

Menéndez Pelayo International University

L. Puebla‐Jiménez

Universidad de Alcalá

E. Arilla

Universidad de Alcalá

Catalina Hernández‐Sánchez

Centro de Investigaciones Biológicas Margarita Salas

Fàtima Bosch

Universitat Autònoma de Barcelona

Miguel Marchena

Instituto Cajal

Pedro de la Villa

Universidad de Alcalá

Patricia Boya

Centro de Investigaciones Biológicas Margarita Salas

Patricia Vázquez

Universidad Nacional de La Plata

J.A. Plaza

Institut de Microelectrònica de Barcelona

Cooperative Institutions

Consejo Superior de Investigaciones Científicas

Centro de Investigaciones Biológicas Margarita Salas

Instituto de Salud Carlos III

Centro de Investigación Biomédica en Red

Universidad de Alcalá

Universidad Autónoma de Madrid

Centre for Biomedical Network Research on Rare Diseases

Institut de Microelectrònica de Barcelona

Universitat Autònoma de Barcelona

Hospital Universitario Ramón y Cajal

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Additional Neuroprotective Effects Of Proinsulin On Vision And Retinal Structure In The Rd10 Mouse Model Of Retinitis Pigmentosa

Investigative Ophthalmology & Visual Science (2012)

Enrique J. de la Rosa Núria Forns Miguel Marchena Alberto M. Hernández‐Pinto Ryan W. J. Steel

Proinsulin

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Suspended Planar‐Array Chips for Molecular Multiplexing at the Microscale

Advanced Materials (2015)

Núria Torras Juan Pablo Agusil Patricia Vázquez Marta Duch Alberto M. Hernández‐Pinto

A novel suspended planar-array chips technology is described, which effectively allows molecular multiplexing using a single suspended chip to analyze extraordinarily small volumes. The suspended chips are fabricated by combining silicon-based technology and polymer-pen lithography, obtaining increased molecular pattern flexibility, and improving miniaturization and parallel production. The chip miniaturization is so dramatic that it permits the intracellular analysis of living cells. Advances in biomolecular studies benefit enormously from the miniaturization of biological assays.1, 2 The identification, quantification, and determination of biochemical and physiological changes in small volumes3 has been revolutionized by the use of planar arrays (PA).4, 5 PAs consist of a collection of multiple independent and ordered sensing features on a single device, allowing parallel assays. Progress towards size-reduction was continued by the emergence of suspended arrays (SA) of particles, whose advantages come from their fluid-phase kinetics, faster detection, and sample reduction.1, 6, 7 However, they lose the critical advantage posed by the PA chips regarding the parallel assays using a single device.1, 2 In SA technologies, the tracking of individual assays6 is guaranteed by subpopulations of particles each bearing a different molecular probe on their surfaces (array element), whereas each subpopulation is identified by a unique attribute (code).6, 7 Therefore, in recent years considerable effort has been focused on developing novel fabrication methods to encode microparticles.8-12 Considering that particle size remains a critical property for many biological applications,13-15 there is a continuous need to miniaturize monodisperse particles for further sample reduction and higher throughput.3, 9, 16 One promising alternative is to achieve multiplexed biomolecular probes in a single suspended particle (i.e., patchy particles).17, 18 However, these particles are strongly limited by the impossibility of identifying each probe by its x-y position, their large volume compared with that of living cells,19, 20 and/or difficult multimaterial synthesis and chemical functionalization due to the necessary restrictive orthogonal-chemistry, which must allow different types of compatible chemical reactions to occur without interference on the same particle.12, 18, 21 Here, we introduce suspended planar-array (SPA) chips (Figure 1a) whose in-situ capabilities with a spatial molecular-probe arrangement combine the advantages of both SA and PA. This novel technology opens the way towards the multiplexed detection of intracellular biological parameters using a single device in dramatically reduced volumes, such as a living HeLa cell. Silicon technologies based on photolithographic processes, such as those used for chip manufacturing, offer superb capabilities to produce complex 3D structures.22 Thus, to produce SPA we developed a fabrication process (Figure 1b and S1) that started with the growth of a 1 μm thick SiO2 layer as the chip-material on a silicon wafer. This material was selected due to its high transmission of light in the visible region of the spectrum, thus obtaining transparent chips. A subsequent photolithographic step followed by a dry etching process allowed the parallel arrangement, batch fabrication, and high miniaturization of the chips. We fixed the dimensions of the chips to (3 × 3 × 1) μm3 anticipating their use inside living cells, as the volume of the chips (9 μm3) represents only ca. 0.35% of the total volume of a typical HeLa cell.23, 24 In addition, chips with similar dimensions have been shown to be easily internalized inside living HeLa cells and to affect neither the cell viability nor the cell division.25 Using this highly-reproducible technology we produced 2.7 × 106 chips cm−2. Chip-release from the wafer was a main issue as it must respect the eventual printed molecule pattern. We developed a release method based on controlled nanofracture. Consequently, anchors with non-uniform cross-sections were nanomachined underneath the chips (Figure 1c) by time-controlled anisotropic silicon etching. The anchor dimensions were optimized with the finite element method (FEM) (Figure 1d) to bear an initial molecular-printing process and on-demand fracture to collect the chips. The mechanical stresses concentrate on the narrowest part of the silicon anchor for vertical and lateral forces. Lateral rather than vertical forces induce larger stresses and the maximum stress-concentration was dependent on the width of the narrowest part of the anchor (Figure 1e). Accordingly, the width of the narrowest part of the anchor was designed to be ca. 450 nm, expecting a fracture with a lateral force of above 85 μN or a vertical force higher than 3.3 × 103 μN. Our experimental tests confirmed the fracture on the predicted anchor region without damaging the chip (Figure 1f). Soft lithographic techniques allow the direct placement of molecules adsorbed on a stamp on a desired substrate. Among these techniques, polymer pen lithography (PPL),26 uses sharp elastomeric pens to print localized and spatially distributed features, offering the possibility to create 2D grids of motifs.27 This patterning approach combines the large-area printing capabilities of microcontact printing with the potential nanometric resolution of Dip-Pen nanolithography.28 We used PPL to pattern molecules directly on the area-restricted surfaces of the anchored chips (Figure 2a). This parallel printing method uses the same chemistry and immobilization protocol for each molecular probe overcoming the required orthogonal chemistry. Briefly, the top surface of the anchored chips was selectively modified with an epoxy-silane coupling agent.29, 30 Then, a polydimethylsiloxane (PDMS) PPL stamp (Figure S2 and S3), whose spatial distribution of elastomeric pens matched that of the anchored chips was used to print an identical and precise molecular pattern on every single anchored chip. A glass slide attached to the back of the stamp prevented any contraction.31 The stamp was loaded onto our PPL apparatus where the printing force and the precise alignment between the stamp and the substrate were monitored (Figure S4), assuring that all the pens were localized and exerted the same force on each anchored chip. To demonstrate the multiplexing capability, we initially printed three individually-labelled proteins on top of the anchored chips. Specifically, three areas of a stamp were independently inked with Texas Red wheat germ agglutinin (WGA), Oregon Green 488 WGA, and AMCA (7-amino-4-methylcoumarin-3-acetic acid) rabbit anti-Goat IgG (H+L), and consecutively printed (Figure S5, Movie S8). The three fluorophores had different absorption and emission maxima, λAbs = (596, 501, 344) nm and λEmi = (615, 526, 446) nm, respectively. Fluorescence characterization showed a 2D spatial arrangement of printed molecules (Figure 2b-c). We decided to print spots of Ø ∼ 1 μm to allow a feasible inspection on fluorescence and confocal laser scanning microscopes (CLSM). Mainly, two parameters contribute to the size of the printed spots, the apex diameter of the PDMS pyramidal pens before compression, Ltop, and the applied printing force, F,32 which deforms the pens during writing. By applying an experimental total F of 480 mN ± 2 mN (0.17 μN per pen), with an average pen Ltop of 212 nm ± 23 nm (Figure 2d), we predicted a spot diameter of 742 nm ± 62 nm. This value agrees with the experimentally obtained spot mean value of 962 nm ± 207 nm. The printed spots showed similar aspect ratios and fluorescence emissions across the fluorescent channels (Figure 2e). These results demonstrate a theoretical density < 1 spot μm−2. To release the printed chips from the wafer, we developed a simple but highly effective peel-off method (Figure 3a). A drop of an aqueous mounting medium was first placed directly on top of the anchored chips. A subsequent manual force was used to peel the solidified, flexible membrane encircling the chips. The chips were collected by centrifugation after dissolving the water-soluble membrane (Figure 3b). Liberated chips were structurally intact as predicted by mechanical simulations (Movie S9), and fluorescence microscopy showed that the printed molecular-spots remained on the chips (Figure 3c) showing spot size, aspect ratios and fluorescence emissions (Figure 3d) as those before being released. Furthermore, an antibody sandwich assay, using a primary goat anti-WGA and a secondary AMCA rabbit anti-Goat IgG (H+L), confirmed the integrity of the printed proteins (Figure S6). In order to demonstrate the major achievement of these devices, we decided to detect, as a proof of concept, externally induced pH changes towards living HeLa cells. Three commercially available probes were multiplexed on a new set of previously aminofunctionalized SPA chips: two pH-sensitive fluorescent dyes (Oregon Green 488 and pHrodo Red), for a ratiometric detection, and one control dye (Alexa 647) (Figure 4a). The SPA chips were successful internalized by lipofection into HeLa cells, as previously demonstrated.23 After lipofection, the cells were stained with MitoTracker Red and examined via CLSM to confirm the internalization of multifunctionalized SPA chips (Figure 4b). In order to test whether the chips affected cell viability, in vivo DiOC6 staining was used to analyze mitochondrial membrane potential, finding no differences between cells with or without internalized chips (Figure 4c) in agreement with previous results.23, 25 After internalization, CLSM images showed that the three printed fluorescent probes remained clearly visible inside the cells (Figure 4d and S7), even after 72 h of culture. Next, the external pH of the medium was changed from physiological pH 7.4 to pH 5. Nigericin was added to force the cell-membrane exchange of H+ ions, therefore altering the intracellular pH and, consequently, changing the fluorescence emitted by the two pH-sensitive multiplexed dyes on the internalized SPA chips (Figure 4d). The fluorescence intensity of the pHrodo Red and Oregon Green 488 spots, after normalization to the Alexa 647 intensity, increased and decreased, respectively. Finally, the ability of this new multiplexed device allowed us a ratiometric detection (Oregon Green 488/pHrodo Red fluorescence ratio) that certainly detected the pH change, even when the SPA chips inside HeLa cells appeared tilted (Figure S7). We have proved SPA chips as a new concept for molecular probes at microscale allowing 2D arranged multiplexed assays in a single device. An extraordinary high density spotting in a single SPA chip is anticipated, as PPL can reach a sub-100 nm printing resolution.28 Our technology allows the batch fabrication of SPA with high potential anisotropy-attributes,33 together with the dramatic miniaturization of the PA chips offering in situ, high-throughput and new potential applications. We demonstrated the first PA chip inside a living cell and the potential use of molecular printing techniques for intracellular applications. Living-arrays3, 16 have been developed to observe from the exterior the response of living cells to external stimuli. SPA chips allow single-cell intracellular analysis. Emerging intracellular arrays would allow the identification of physicochemical intracellular parameters related to genetic determinants of diseases, cellular-function modulators or dynamic responses of the cell to its local environment, allowing researchers to attain their goal of analyzing multiple bioparameters inside single living cells.2, 13, 16 Materials: All reagents and materials were purchased from different providers and used without further purification. The complete protocols are detailed in the Supporting Information. Fabrication: Details of the SPA fabrication process along with the numerical simulations and optimization by the FEM, the PPL patterning protocol, stamp and apparatus, together with cell culture conditions are fully described in the Supporting Information. Characterization: All characterization tools and protocols used in this work are presented in the Supporting Information. This work was supported by the EU ERDF (FEDER) funds and the Spanish Government grants TEC2011-29140-C03-01/02 and TEC2014-51940-C2-1/2. PV was supported by a JAE-DOC contract (FSE funding). The authors also thank the cleanroom staff of IMB-CNM for fabrication of the chips and the confocal microscopy service of the CIB. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Microscale chemistry

Planar array

10.1002/adma.201504164

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Citations (23)

CHAPTER 7. PEDF Peptides in Retinal Degenerations

RSC drug discovery/RSC drug discovery series (2018)

Alberto M. Hernández‐Pinto S. Patricia Becerra

Pigment epithelium-derived factor (PEDF) is a multifunctional member of the serine proteinase inhibitor (serpin) superfamily. This widely studied protein is considered an ocular guardian because it protects the retina from degeneration induced by cell death, pathological neovascularization, tumorigenesis and inflammation. Studies of the independent activities of PEDF are challenged by the presence of other properties of the same molecule. This chapter summarizes approaches for such investigations using peptide synthesis, protein chemistry and recombinant DNA technologies based on the three-dimensional structure of PEDF to separate and alter them individually. The major focus is to discuss relevant applications of PEDF mimetics for protection against retinal degenerations.

PEDF

10.1039/9781788013666-00099

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Lysosomal membrane permeabilization and autophagy blockade contribute to photoreceptor cell death in a mouse model of retinitis pigmentosa

Cell Death and Differentiation (2014)

Natalia Rodríguez‐Muela Alberto M. Hernández‐Pinto Ana Serrano‐Puebla Lucía García‐Ledo Sergio Latorre

Photoreceptor cell

10.1038/cdd.2014.203

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Citations (121)

Silicon chips detect intracellular pressure changes in living cells

Nature Nanotechnology (2013)

Rodrigo Gómez Alberto M. Hernández‐Pinto Marta Duch Patricia Vázquez Kirill Zinoviev

Hydrostatic pressure

Pipette

HeLa

Osmotic pressure

10.1038/nnano.2013.118

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Citations (77)

Adalimumab Reduces Photoreceptor Cell Death in A Mouse Model of Retinal Degeneration

Scientific Reports (2015)

Cristina Martínez-Fernández de la Cámara Alberto M. Hernández‐Pinto Lorena Olivares‐González Carmen Cuevas-Martín María Sánchez‐Aragó

Abstract Growing evidence suggests that inflammation is involved in the progression of retinitis pigmentosa (RP) both in patients and in animal models. The aim of this study was to investigate the effect of Adalimumab, a monoclonal anti-TNFα antibody, on retinal degeneration in a murine model of human autosomal recessive RP, the rd10 mice at postnatal day (P) 18. In our housing conditions, rd10 retinas were seriously damaged at P18. Adalimumab reduced photoreceptor cell death, as determined by scoring the number of TUNEL-positive cells. In addition, nuclear poly (ADP) ribose (PAR) content, an indirect measure of PAR polymerase (PARP) activity, was also reduced after treatment. The blockade of TNFα ameliorated reactive gliosis, as visualized by decreased GFAP and IBA1 immunolabelling (Müller cell and microglial markers, respectively) and decreased up-regulation of TNFα gene expression. Adalimumab also improved antioxidant response by restoring total antioxidant capacity and superoxide dismutase activity. Finally, we observed that Adalimumab normalized energetic and metabolic pattern in rd10 mouse retinas. Our study suggests that the TNFα blockade could be a successful therapeutic approach to increase photoreceptor survival during the progression of RP. Further studies are needed to characterize its effect along the progression of the disease.

Gliosis

Photoreceptor cell

10.1038/srep11764

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Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa

Cell Death and Disease (2022)

Alonso Sánchez‐Cruz Alberto M. Hernández‐Pinto Concepción Lillo Carolina Isiegas Miguel Marchena

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.

Proinsulin

Photoreceptor cell

10.1038/s41419-022-04839-0

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Citations (6)

Vitamin E deficiency impairs the somatostatinergic receptor–effector system and leads to phosphotyrosine phosphatase overactivation and cell death in the rat hippocampus

The Journal of Nutritional Biochemistry (2012)

Alberto M. Hernández‐Pinto L. Puebla‐Jiménez Aránzazu Perianes‐Cachero E. Arilla

10.1016/j.jnutbio.2012.05.005

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Oxidative Stress and Lymphocyte Alterations in Chronic Relapsing Experimental Allergic Encephalomyelitis in the Rat Hippocampus and Protective Effects of an Ethanolamine Phosphate Salt