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Printed sensors, a low cost alternative for clinical detection

In today’s society there is a great interest in developing new technologies that allow low-cost mass manufacturing, also called “rapid prototyping” or “additive manufacturing”. Rapid prototyping includes technologies such as sterolithography, 3D printing, laser sintering or printed electronics, among others. All of these share digital design and manufacturing from the consecutive addition of layers, that is, techniques that allow creating almost any shape or geometric feature in a very fast time.
Printed electronics consists of printing inks on different types of substrates such as textiles, plastics, papers or films to make them “smart”. This technique is presented as an alternative to traditional silicon that is being implemented in sectors as varied as health and wellness, automotive and transport, professional sports, safety and protection, packaging, architecture and construction, and technical textiles. Printed electronics is one of the new technologies that will have a long history throughout the functional electronic device manufacturing space, with a wide range of applications, electronic designs, processes and materials, compared to conventional electronic and microelectronics based manufacturing technology in silicon. It is expected that in the next decade it will be part of everyday life, with products such as electronic skin, electronic tissues and organs or architectural elements that respond to external stimuli.
Among the many areas of interest of these technologies, one of them is the development of low-cost sensors for the medical or environmental area. For example, in these disciplines, it is essential to get devices that can be very economical or even single-use to promote sustainable environmental control and personalized medicine. Specifically with inkjet technology, researchers from NANBIOSIS Unit 8 Micro-Nano Technology Unit belonging to the Institute of Microelectronics of Barcelona (IMB-CNM, CSIC /and CIBER-BBN), have developed over the last few years multiple materials, inks, substrates and sensors for the development of electrochemical sensors in micrometric dimensions.

  • Inks: Most commercial inks are composed of a solvent that contains some material with insulating, conductive or semiconductor properties. As a general rule, an ink must be stable, with a particle size of several orders of magnitude smaller than the injector orifice, have a viscosity of less than 20 mPa s and a surface tension of less than 80 mN m-1. Although these values ​​may depend on the system in question. The final devices are obtained by selectively depositing in previously drawn areas, layer by layer, thin or thick structures on the substrates. They have worked with multiple commercial metallic inks such as gold, silver and platinum.
    Normally in an electrochemical system a noble material that is electrochemically stable is needed to be used in the working electrode and in the counter electrode, and for this gold or platinum are a good alternative. To make measurements with any electrochemical sensor, a reference electrode is essential since it is one that has a stable and constant potential over time and that allows us to reference our voltage value. We use the impression of an Ag / AgCl bilayer since it is one of the interfaces most used as a reference electrode. One of the main problems faced by miniaturization, however, is the rapid loss of the small volume of internal reference solution that these electrodes must have, which has a direct impact on their useful life and stability. For this, a polymeric membrane that can be printed was formulated, which allows the reference to have high performance compared to other commercial miniaturized reference electrodes (Ref1).
  • Substrates: a wide range of rigid, flexible, porous, plastic, fabric, etc. substrates can be used. However, the interaction of the ink with the substrate is crucial in determining the good quality of the printed pattern. For this reason, the properties of the inks are adapted for the different substrates with their own properties. For this reason, it is common practice to pretreat the substrate surface to improve hydrophobicity and adhesion issues mainly. It is common to use plastic substrates with a thickness of the order of microns that provide them with great flexibility and that are already specially treated to obtain excellent printing qualities. The deposition of uniform gold and silver conductive inks on porous substrates can be achieved by using a primer layer to seal the porosity of the membrane in specific and defined areas, with the aim of building a sensor device over the sealed area and leaving the rest of the intact substrate (Ref.2). With a paper substrate, alternatively we can print a silane ink, as a strategy that allows a monolayer of hydrophobic material to grow on the substrate and thus be able to obtain uniform lines of ink on its surface. (Ref.3)
  • Sensors developed: Dissolved oxygen (DO) (Ref.4) and pH (Ref.5) sensors have been developed using gold and platinum inks respectively, commercially available on plastic substrates. The inks have a specially designed formulation that allows their sintering at temperatures as low as 150 and 180 ° C for Au and Pt respectively. This is a key point in the development of low-cost sensors made on polymeric substrates or paper that cannot withstand high temperatures. These sensors integrate in a single platform all the basic elements for the registration of pH and DO, allowing measurements without any external electrode. DO is measured directly with a gold working electrode and pH sensors are achieved after electroplating an iridium oxide film on the platinum working electrode. In addition, this water-based platinum ink has another unique feature, it provides the electrode surface with high roughness, which promotes adhesion of the deposited sensor material, in this case iridium oxide. Long-term stability tests for more than 1 year demonstrate excellent stability of the mechanical sensor layer, and that it correlates perfectly with the different roughness of the printed platinum layer. Along the same lines and in relation to the development of inks, it has been possible to obtain a fully printed pH sensor based on a conductive polymer specially formulated to be printed by IJP . The measurements obtained with this ink have a good response in a wide pH range (pH 3 to 10) and the response in the physiological zone (pH 7-7.5) is well resolved, one of the main drawbacks of conductive polymers. We also present an IJP-printed electrochemical sensor for enzyme-free glucose analysis on flexible PEN substrate (Ref.6). In this case, CuO microparticles were used to modify the electrodes, and the detection of glucose was validated in concentrations that coincide with those of the tear fluid, which allows us to foresee applications in ocular diagnosis, where a painless control can be achieved and not invasive of diabetes by analyzing the glucose contained in tears.

(Ref.1): Moya A, Pol R, Martínez-Cuadrado A, Villa R, Gabriel G, Baeza M. Stable Full Inkjet-Printed Solid-State Ag/AgCl Reference Electrode. Analytical Chemistry 91 (2019) 15539-15546

(Ref.2): M. Ortega-Ribera; X. Guimerà; E. Sowade; M. Zea; X. Illa; E. Ramon; R. Villa; J. Gracia-Sancho; G. Gabriel. Online oxygen monitoring using integrated inkjet-printed sensors in a liver-on-a-chip system. Lab on a Chip. 18 – 14, pp. 2023 – 2035. 2018

(Ref.3): All Inkjet Printing Sensor Device on Paper: for Immunosensors Applications M Zea, A Moya, I Abrao-Nemeir, J Gallardo-Gonzalez, N Zine, A Errachid, … 2019 20th International Conference on Solid-State Sensors, Actuators and 

(Ref.4): Moya A, Sowade E, del Campo FJ, Mitra KY, Ramon E, Villa R, Baumann RR, Gabriel G. All-inkjet-printed dissolved oxygen sensors on flexible plastic Organic Electronics 39 (2016) 168-176

(Ref.5): Zea M, Moya A, Fritsch M, Ramon E, Villa R, Gabriel G Enhanced performance stability of iridium oxide based pH sensors fabricated on rough inkjet-printed platinum ACS Applied Materials & Interfaces 11 (2019) 15160-15169

(Ref.6): Romeo A, Moya A, Leung TS, Gabriel G, Villa R, Sánchez S. Inkjet printed flexible non-enzymatic glucose sensor for tear fluid analysis Applied Materials Today 10 (2018) 133-141

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Fabry disease & Smart4Fabry project

The Fabry disease (FD) is a lysosomal storage disorder (LSD) that currently lacks an effective treatment. Lysosomes are spherical vesicles, which contain hydrolytic enzymes found in nearly all animal cells. LSDs are caused by lysosomal dysfunctions, usually because of the deficiency of a single enzyme required for the metabolism of macromolecules such as lipids, glycoproteins and mucopolysaccharides. Fabry disease is a progressive, X-linked inherited disorder caused by deficiency or absence of the α-galactosidase A (GLA) activity, an enzyme involved in the glycosphingolipid metabolism. The substrates of GLA are glycosphingolipids, being the primary substrate the globotriaosylceramide (Gb3). Therefore, the failure of GLA activity leads to progressive intracellular accumulation of Gb3, in many cells, particularly in renal epithelial cells, endothelial cells, pericytes, vascular smooth muscle cells, cardiomyocytes, and neurons of the autonomic nervous system, leading to multisystemic clinical symptoms. First clinical signs of FD occur during childhood and, over time, microvascular lesions of the affected organs progress leading to early death. It affects mostly men but serious cases have also been reported in women.

There are currently three products authorized in the EU for the treatment of FD. Two products available in EU since 2001 for Enzymatic Replacement Therapy (ERT), Replagal (Shire Human Genetic Therapies AB) and Fabrazyme (Genzyme Europe B.V.), which have to be i.v. administered every other week. The ERT strategy is based on supplying recombinant GLA to cells, reversing several of the metabolic and pathologic abnormalities. There is a third product in the EU market since 2016, which is based on the chaperone migalastat hydrochloride (Galafold Amicus Therapeutics UK Ltd), designed to selectively and reversibly bind with high affinity to the active sites of certain mutant forms of GLA, facilitating proper protein folding and allowing for correct trafficking of the mutant enzyme. However, it is a genotype-specific treatment (only one-third to one-half of mutations may be amenable).

To date, no direct comparisons exist between Fabrazyme and Replagal but significant clinical benefits compared with placebo, however, have been demonstrated with ERT, with positive effects on the heart, kidneys, nervous system and quality of life. Of note, a stabilization of renal function was only observed at an early phase of FD.

ERT success with free GLA is limited mainly due to the instability and low efficacy of the exogenously administered therapeutic enzyme. Furthermore, some patients can develop immune responses after receiving the infused recombinant enzyme. Clinical data has confirmed that the immunological consequences of ERT may impair efficacy in some patients. Furthermore, the short elimination t1/2 of the enzyme and the need for repeated administration of large amounts of enzyme are other limitations of current ERT. In addition, GLA does not cross of the Blood Brain Barrier (BBB), which prevents the product for reducing the Gb3 deposits in the central nervous system (CNS). Moreover, it is a lifelong treatment which becomes a burden for the health system due to its extremely high cost.

Therefore, there is a need for other therapeutic strategies, which can either serve as primary or supplemental treatments. Gene and substrate reduction therapies constitute alternative therapies which are at present under investigation.

The European “Smart-4-Fabry” project aims to develop a new nanoformulation based on the encapsulation of the GLA enzyme in nanoliposomes, to improve the current ERT of FD. A Consortium formed by ten partners, including private companies and public institutions in Europe and Israel, has been granted (July 2017) with a Horizon2020 financial programme by the European Commission (H2020-NMBP-2016-2017; call for nanotechnologies, advanced materials, biotechnology and production; Proposal number: 720942-2).

The project is expecting to last for 48 months and contemplates the necessary activities to advance a nanoliposome formulation of GLA enzyme, i.e., nano-GLA, from an experimental proof of concept up to an advanced nonclinical stage of development. The S4F should complete an advanced regulatory safety and toxicology package supporting future nano-GLA clinical development in patients with FD.

To the best of S4F knowledge, there is no previous experience on the encapsulation of a GLA for treating FD patients following an ERT approach.

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Diabetes mellitus and pancreatic islet transplant: DRIVE

Today in the World Diabetes Day it is a good moment to remember The Patient Panel of the European project DRIVE- Diabetes Reversing Implants, held in Vitoria (Alava) on June 2 where researches and doctors met with patients and families.

The Panel was organized by the NanoBioCel group of CIBER BBN and NANBIOSIS Unit 10 Drug Formulation, with the purpose of letting know the DRIVE project to patients and concect patients with the project scientists and experts in pancreas and stem cell transplantation. Thus, one of the most important health objectives of the European Union was met: to promote direct access for patients and families, in this case patients with diabetes mellitus, to information on the cutting-edge research being carried out at European level on this disease in order to empower them, so that they are aware of their rights and responsibilities and so that through patient organisations cooperation with research groups is promoted.

The European DRIVE project aims to improve pancreatic islet transplantation therapy for diabetes mellitus. Transplantation of purified insulin-producing pancreatic islets from the donor pancreas can restore strict natural blood sugar control and eliminate the need for daily insulin injections, improving the patient’s quality of life. However, despite their proven efficacy among current treatments for type 1 diabetes in the transplantation therapy of insulin-producing pancreatic islets, there is a need to increase the survival of transplanted islet graft. There are also risks associated with immunosuppressive medication to be taken by islet transplant recipients. These factors limit the use of this therapy to a small percentage of patients with “fragile” type 1 diabetes for whom daily insulin injections are not sufficient to control their diabetes.

DRIVE’s significant contribution to improving the quality of life of a patient with diabetes mellitus is to achieve a better outcome in the management of this disease through the development of technologies to increase the survival rate of islet graft transplanted and waive the need for lifelong immune suppression. DRIVE’s vision is to extend the application of islet transplant therapy to more insulin-dependent diabetic patients (T1D and T2D).

Drive Pannel Diabetes - Nanbiosis
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Mechanical Characterization of soft biological tissues

The experimental study of the mechanical properties of biological tissues is of vital importance. Thorough research into themechanical response of biomaterials is the basis for the creation ofmodels which can accurately reproduce the mechanical behavior ofsuch materials. Adequate mechanical characterization of tissue materials is of paramount interest to the clinical simulations, diagnostic and tissue engineering fields – where the tissue structure, in contrast to classical mechanics application fields, is just simply a biological tissue.

In order to obtain their material properties, classical engineering testing techniques have been applied to biologicalmaterials. To reproduce the mechanical behavior of these kinds ofmaterial, many constitutive models have been proposed for softtissues (. In some cases, the validation of these models basedon only uniaxial test data is inappropriate because biological andbiomaterial membranes generally develop multiaxial stress statesduring real-life loading conditions.Although a large number of experiments have been conductedover the years on measuring the structural and functional properties of biological tissues, the standardization of such measurementsand the interpretation of results are difficult to establish. The geometry of the sample, the method of gripping the sample edges andthe method used to determine the strain may have profound effectson any measured mechanical properties since they directly influence how the load is transferred to the membrane.

In typical (hard) engineering materials like metals and composites, strain may be measuredat any instance during the mechanical test. Some techniques require a close contact with the samplesduring the test. The use of extensometers attached directly (glued with cyanoacrylateadhesives, for instance) to the gage section of the test specimen, and strain gages thatare mounted directly to the specimen. Sincestrain gages have knife edges to define the exact points of contact with the specimenthey are also incompatible with soft tissues.Imaging techniques such as video extensometry or digital image correlation may be an alternative to the contact methods. Digitalimage correlation (DIC) is based on a process called imagematching. It is possible to use this technique to measure surface displacement and buildup full field 2D and 3D deformation vector fields and in-plane strain maps. It requiresa set of markings, known as speckle, on the analyzed surface(s).Video extensometers performing principle is based on tracking i.e., on following theposition of a given number of markings or regions of interest (ROI).

For the particular case of tension tests (uniaxial or biaxial), the physical quantitiesdirectly measurable are the load, F and the elongation, l.These quantities are normalizedto account for the test specimen’sgeometry. The normalizedengineering parameters arethe engineering stress, σ and engineering strain or stretch λ. Engineeringstress is the ratio between theload F and the sample’s crosssection i.e., σ = F/A. Engineeringstrain is given by=l/lo and engineering stretch λ=1+.

Mechanical Characterization of soft biological tissues

Mechanical Characterization of soft biological tissues - Nanbiosis

Creep and relaxation tests are sometimes performed to determine the viscoelastic properties ofsoft tissues; specimen deformation is measured under a constant force in creep testing, whereasin relaxation tests, change in load is determined under constant specimen length. In these tests,stress (= load divided by undeformed specimen cross-sectional area) and strain (= change inspecimen length divided by reference length) are commonly used to represent the data.

Mechanical Characterization of soft biological tissues - Nanbiosis
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Characterization of nanomedicines

The quick development of nanotechnology and its application in medicine have generated new alternatives for the diagnosis and treatment of diseases thanks to the novel methods of preparation, modification and characterization of nanomaterials. The knowledge about the behavior of matter at the atomic and molecular level has allowed the creation of tools and processes to observe manipulate and control biological structures on a scale between 100 and 10,000 times smaller than a mammalian cell.

Nanomedicine is defined as the application of nanotechnology in view of making a medical diagnosis or treating or preventing diseases. It exploits the improved and often novel physical, chemical, and biological properties of materials at nanometer scale.

One of the applications in nanomedicine is diagnostic. Nanodiagnosis consists of developing systems and image analysis techniques both in vivo and in vitro for the early detection of disease, at cellular or molecular level. One of the detection systems developed to date is based on nanoparticles (semiconductor, or magnetic metal) such as quantum dots that are used as cell labeling, identification of tumors or diseased areas. Another line of action within this field is the diagnosis with biosensors or nanobiosensors. These biosensors integrated nanoscale devices for a biological receptor (proteins, DNA, cells) are prepared to specifically detect a substance and a transducer or sensor, capable of measuring biomolecular recognition reaction and translate it into a measurable signal.

On the other hand, nanomedicine is being investigated as a way to improve the properties of medicines, such as their solubility or stability, and to develop medicines that may provide new ways to target medicines in the body more accurately and to support the Regeneration of cells and tissues. Therapeutic nanosystems research line includes both the development of pharmacological release systems optimized to traverse the blood-brain barrier, and the specific release of enzymes, proteins or gene inhibition strategies by means of siRNA. The development of therapeutic nanoconjugates and of local and controlled release systems for these nanoconjugates, would allow guiding the treatment to the area of action, in the attempt to achieve perfect control of the therapy, preventing the action of the drug or therapeutic particle in areas that might entail a potential risk for the patient.

It is widely accepted that the use of nanotechnology offers impressive potential in the development of innovative pharmaceuticals with enhanced therapeutical properties. For example, it is known that the integration of therapeutically active molecules in nanoparticulate materials (polymer nanoparticles, micelles, nanosuspensions, nanovesicles), with well-defined structural characteristics, has shown to be a very effective strategy to increase the efficacy and reduces toxicity of drugs . However, producing such nanoparticulate materials at large scale with the narrow structural variability, high reproducibility, purity and cost required to meet the high-performance requirements and regulatory demands dictated by the EMA and US FDA agencies is a challenge.
However, knowledge about the toxicology of nanoparticles is limited. At the environmental level for example, Nanotoxicology has revolutionized toxicology, since nanoparticles can reach unsuspected sites due to their small size. Toxicological studies have shown an increase in the toxicity of nanoparticles ( In addition to that, the processes by which the organism will remove the nanoparticles are not well understood. Nanodrugs can show complex action mechanisms that combine mechanical, chemical, pharmacological and immunological properties. It is necessary necessary a depth knowledge and expertise to assure the quality and to determine the safety efficacy and make a correct risk analysis of nantotechnoligy based products; a complete preclinical validation with correct nanomedicine animal models.

According to EMEA, altyhough some Medicinal products containing nanoparticles in the form of liposomes (i.e. Caelyx, Myocet), polymer protein conjugates (i.e. PegIntron, Somavert), polymeric substances (i.e. Copaxone) or suspensions (i.e. Rapamune, Emend) have already been granted Marketing Authorisations within the Community under the existing regulatory framework, there is insufficient knowledge and data concerning characterization of nanomedicines, their detection and measurement, the fate (and especially the persistence) of nanoparticles in humans and in the environment, and all aspects of toxicology and environmental toxicology related to nanoparticles, to allow for satisfactory risk assessments for humans and ecosystems to be performed. Although the existing toxicological and ecotoxicological methods are appropriate to assess many of the hazards associated with the products and processes involving nanoparticles, they may not be sufficient to address all the hazards.EMEA. A cascade characterization based methodology to carry out a preclinical validation based on milestones and adapting existing methodologies and methods and managed by expert staff like the offered by NANBIOSIS is essential to market new nanomedicines.

U12-Nanostructured liquid characterization unit
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