Introductory Chapter: Endoplasmic Reticulum-Knowledge and Perspectives

The endoplasmic reticulum is one of the most studied and fascinating organelles. It is found in all eukaryotic cells and performs a variety of functions. The organelle was designated by this name by Keith Porter in 1953 on the basis of studies carried out with the electron microscope in cells in tissue culture. Porter was able to differentiate the exoplasm, an adjacent region devoid of organelles, from the neighboring endoplasm. In the endoplasm, he examined a system of interrelated tubules, a reticulum, for this reason, the name “endoplasmic reticulum” (ER). The collaboration between Keith Porter and George Palade showed that ER exists in all eukaryotic cells and that it consists of different but continuous domains, the smooth and rough ER, whose abundance fluctuates between different types of cells. Palade observed on the surface of the rough ER the ribosomes that synthesized secretory proteins. The secretory proteins would cross an intracellular membrane, instead of the plasma membrane. The verification of this concept led to the discovery of the secretion pathway and the conception of intracellular protein binding to various organelles.Fil: Catala, Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentin

Introductory Chapter: Liposomes - Advances and Perspectives - My Point of View

Liposomes are vesicular arrangements composed of one or more phospholipid bilayers surrounding an aqueous core. Liposomes were discovered almost six decades ago. Due to its versatility, liposomes are now analyzed for their applicability both in laboratory techniques and in medical studies. Its interest lies in its ability to traverse cell membranes and to transport certain types of molecules to defined places in the human body. Liposomes can carry both hydrophilic and hydrophobic molecules. The preparation of the liposomes results in different properties for these systems. There are several factors involved in the preparation of liposomes that can modify their structures. Due to its biological compatibility, nonimmunogenicity, greater solubility of chemotherapeutic agents, and its ability to encapsulate a wide variety of drugs, the supply of drugs using liposomes has meant a great advance. The purpose of this book is to focus on recent developments in liposomes. The chapters selected in this book are contributions from invited researchers with long experience in different areas of research. This book offers expert and updated reviews of the field of liposomes.Fil: Catala, Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentin

Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection

[EN] Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands.The authors acknowledge the financial support received from EU FEDER, the Spanish Ministry of Economy and Competitiveness (PID2019-110713RB-I00), and the Generalitat Valenciana (PROMETEO/2020/094 and GVA-FPI-2017 Ph.D. grant).Lázaro-Zaragozá, A.; Maquieira Catala, A.; Tortajada-Genaro, LA. (2022). Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection. ACS Sensors. 7(3):758-765. https://doi.org/10.1021/acssensors.1c02220S7587657

Photo-click chemistry to create nucleic acids dextran-based microarrays

The final publication is available at link.springer.com[EN] In the literature, there are reports of the utilization of various hydrogels to create generic platforms for protein microarray applications. Here, a novel strategy was developed to obtain high-performance microarrays. In it, a dextran hydrogel is used to covalently immobilize oligonucleotides and proteins. This method employs aqueous solutions of dextran methacrylate (Dx-MA), which is a biocompatible photopolymerizable monomer. Capture probes are immobilized inside the hydrogel via a light-induced thiol-acrylate coupling reaction at the same time as the dextran polymer is formed. Hydrogel microarrays based on this technique were prepared on different surfaces, such as a Blu-ray Disk and polycarbonate or alkene-functionalized glass slides, and these systems showed high probe-loading capabilities and good biorecognition yields. This methodology presents advantages such as a low cost, a short analysis time, a low limit of detection, and multiplexing capabilities, among others. Confocal fluorescence microscopy analysis demonstrated that in these hydrogel-based microarrays, receptor immobilization and the biorecognition event occurred within the hydrogel and not merely on the surface.Funding from MINECO through the project BIHOLOG CTQ/2016/75749-R is acknowledged.Díaz-Betancor, Z.; Bañuls Polo, M.; Maquieira Catala, A. (2019). Photo-click chemistry to create nucleic acids dextran-based microarrays. Analytical and Bioanalytical Chemistry. 411(25):6745-6754. https://doi.org/10.1007/s00216-019-02050-3S6745675441125Heller MJ. DNA microarray technology: devices, systems, and applications. Annu Rev Biomed Eng. 2002;4:129–53. https://doi.org/10.1146/annurev.bioeng.4.020702.153438 .Sassolas A, Leca-Bouvier BD, Blum LJ. DNA biosensors and microarrays. Chem Rev. 2008;108:109–39. https://doi.org/10.1021/cr0684467 .Uttamchandani M, Neo JL, Ong BNZ, Moochhala S. Applications of microarrays in pathogen detection and biodefence. Trends Biotechnol. 2009;27:53–61. https://doi.org/10.1016/J.TIBTECH.2008.09.004 .Yu X, Schneiderhan-Marra N, Joos TO. Protein microarrays for personalized medicine. Clin Chem. 2010;56:376–87. https://doi.org/10.1373/clinchem.2009.137158 .North SH, Taitt CR. Immobilization of biomolecular probes for arrays and assay: critical aspects of biointerfaces. In: Chemoselective and bioorthogonal ligation reactions. Weinheim: Wiley-VCH; 2017. p. 459–95.Nimse S, Song K, Sonawane M, Sayyed D, Kim T. Immobilization techniques for microarray: challenges and applications. Sensors. 2014;14:22208–29. https://doi.org/10.3390/s141222208 .Cardenas-Benitez B, Djordjevic I, Hosseini S, Madou MJ, Martinez-Chapa SO. Review: Covalent functionalization of carbon nanomaterials for biosensor applications: an update. J Electrochem Soc. 2018;165:B103–17. https://doi.org/10.1149/2.0381803jes .Qu Z, Xu H, Gu H. Synthesis and biomedical applications of poly((meth)acrylic acid) brushes. ACS Appl Mater Interfaces. 2015;7:14537–51. https://doi.org/10.1021/acsami.5b02912 .Oh SJ, Hong BJ, Choi KY, Park JW. Surface modification for DNA and protein microarrays. OMICS. 2006;10:327–43. https://doi.org/10.1089/omi.2006.10.327 .Luderer F, Walschus U. Immobilization of oligonucleotides for biochemical sensing by self-assembled monolayers: thiol–organic bonding on gold and silanization on silica surfaces. In: Immobilisation of DNA on chips I. Berlin: Springer; 2005. p. 37–56.Caminade A-M. Dendrimers as biological sensors. In: Dendrimers. Chichester: Wiley; 2011. p. 375–92.Kiat NJ, Simeon F, Phon TH, Ajikumar PK. DNA-directed assembly microarray for protein and small molecule inhibitor screening. Totowa, NJ: Humana; 2011. p. 127–40.Basinska T. Reactions leading to controlled hydrophilicity/hydrophobicity of surfaces. Curr Org Chem. 2017;21(24):2479–501. https://doi.org/10.2174/1385272821666170428123013 .Weinrich D, Köhn M, Jonkheijm P, Westerlind U, Dehmelt L, Engelkamp H, et al. Preparation of biomolecule microstructures and microarrays by thiol-ene photoimmobilization. ChemBioChem. 2010;11:235–47. https://doi.org/10.1002/cbic.200900559 .Wendeln C, Rinnen S, Schulz C, Kaufmann T, Arlinghaus HF, Ravoo BJ. Rapid preparation of multifunctional surfaces for orthogonal ligation by microcontact chemistry. Chem Eur J. 2012;18:5880–8. https://doi.org/10.1002/chem.201103422 .Makaraviciute A, Ramanaviciene A. Site-directed antibody immobilization techniques for immunosensors. Biosens Bioelectron. 2013;50:460–71. https://doi.org/10.1016/j.bios.2013.06.060 .Bañuls M-J, Jiménez-Meneses P, Meyer A, Vasseur J-J, Morvan F, Escorihuela J, et al. Improved performance of DNA microarray multiplex hybridization using probes anchored at several points by thiol–ene or thiol–yne coupling chemistry. Bioconjug Chem. 2017;28:496–506. https://doi.org/10.1021/acs.bioconjchem.6b00624 .Neumann K, Conde-González A, Owens M, Venturato A, Zhang Y, Geng J, et al. An approach to the high-throughput fabrication of glycopolymer microarrays through thiol–ene chemistry. Macromolecules. 2017;50:6026–31. https://doi.org/10.1021/acs.macromol.7b00952 .Gupta N, Lin BF, Campos LM, Dimitriou MD, Hikita ST, Treat ND, et al. A versatile approach to high-throughput microarrays using thiol-ene chemistry. Nat Chem. 2010;2:138–45. https://doi.org/10.1038/nchem.478 .Rubina AY, Dementieva EI, Stomakhin AA, Darii EL, Pan’kov SV, Barsky VE, et al. Hydrogel-based protein microchips: manufacturing, properties, and applications. Biotechniques. 2003;34:1008–22. https://doi.org/10.2144/03345rr01 .Varshosaz J. Dextran conjugates in drug delivery. Expert Opin Drug Deliv. 2012;9:509–23. https://doi.org/10.1517/17425247.2012.673580 .Desmet C, Blum LJ, Marquette CA. High-throughput multiplexed competitive immunoassay for pollutants sensing in water. Anal Chem. 2012;84:10267–76. https://doi.org/10.1021/ac302133u .Moschallski M, Evers A, Brandstetter T, Rühe J. Sensitivity of microarray based immunoassays using surface-attached hydrogels. Anal Chim Acta. 2013;781:72–9. https://doi.org/10.1016/j.aca.2013.04.013 .Beyer A, Pollok S, Berg A, Weber K, Popp J. Easy daylight fabricated hydrogel Array for colorimetric DNA analysis. Macromol Biosci. 2014;14:889–98. https://doi.org/10.1002/mabi.201300487 .Alonso R, Jiménez-Meneses P, García-Rupérez J, Bañuls M-J, Maquieira Á. Thiol–ene click chemistry towards easy microarraying of half-antibodies. Chem Commun. 2018;54:6144–7. https://doi.org/10.1039/C8CC01369A .Escorihuela J, Bañuls MJ, Grijalvo S, Eritja R, Puchades R, Maquieira Á. Direct covalent attachment of DNA microarrays by rapid thiol-ene “click” chemistry. Bioconjug Chem. 2014;25:618–27.Escorihuela J, Bañuls M-J, Puchades R, Maquieira Á. Site-specific immobilization of DNA on silicon surfaces by using the thiol–yne reaction. J Mater Chem B. 2014;2:8510–7. https://doi.org/10.1039/C4TB01108B .Escorihuela J, Bañuls MJ, Puchades R, Maquieira Á. Development of oligonucleotide microarrays onto Si-based surfaces via thioether linkage mediated by UV irradiation. Bioconjug Chem. 2012;23:2121–8.Dondoni A. The emergence of thiol–ene coupling as a click process for materials and bioorganic chemistry. Angew Chem Int Ed. 2008;47:8995–7. https://doi.org/10.1002/anie.200802516 .Mira D, Llorente R, Morais S, Puchades R, Maquieira A, Marti J. High-throughput screening of surface-enhanced fluorescence on industrial standard digital recording media. Proc SPIE. 2004;5617:364–73.van Dijk-Wolthuis WNE, Franssen O, Talsma H, van Steenbergen MJ, Kettenes-van den Bosch JJ, Hennink WE. Synthesis, characterization, and polymerization of glycidyl methacrylate derivatized dextran. Macromolecules. 1995;28:6317–22. https://doi.org/10.1021/ma00122a044 .Pirrung MC. How to make a DNA chip. Angew Chem Int Ed. 2002;41:1276–89. https://doi.org/10.1002/1521-3773(20020415)41:83.0.CO;2-2 .Wang C, Jia X-M, Jiang C, Zhuang G-N, Yan Q, Xiao S-J. DNA microarray fabricated on poly(acrylic acid) brushes-coated porous silicon by in situ rolling circle amplification. Analyst. 2012;137:4539. https://doi.org/10.1039/c2an35417a .Casanova-Salas I, Rubio-Briones J, Calatrava A, Mancarella C, Masiá E, Casanova J, et al. Identification of miR-187 and miR-182 as biomarkers of early diagnosis and prognosis in patients with prostate cancer treated with radical prostatectomy. J Urol. 2014;192:252–9. https://doi.org/10.1016/j.juro.2014.01.107

Novel and rapid activation of polyvinylidene fluoride membranes by UV light

[EN] Polyvinylidene fluoride (PVDF) membranes have become essential because of their huge applicability to the industry; however, they still present some limitations. This study focuses on the modification of PVDF membrane properties such as hydrophobicity, wettability, and functionality. To obtain a stable grafting, the surface of the membrane is hydroxylated using UV light at 254¿nm, followed by covalent immobilization of (3-aminopropyl)triethoxysilane (APTES) and vinyltriethoxysilane (VTES). The physicochemical and morphological properties of modified and raw PVDF membranes were analyzed by spectroscopy, microscopy, and goniometry. Finally, nucleic acid microarray technology results showed that PVDF and PVDF-VTES membranes had probe immobilization densities of 5 and 11¿pmol/cm2 and hybridization limits of detection of 1 and 5¿nM, respectively.Financial support from Spanish Ministry of Economy and Competitiveness (BIHOLOG Project CTQ2016-75749-R) and FEDER is acknowledged. P.J.-M. acknowledges the Spanish Ministry of Economy, Industry and Competitiveness for the public FPI grant (Project CTQ2013-45875-R) and cofinancing by the European Social Fund.Jimenez-Meneses, P.; Bañuls Polo, M.; Puchades, R.; Maquieira Catala, A. (2019). Novel and rapid activation of polyvinylidene fluoride membranes by UV light. Reactive and Functional Polymers. 140:56-61. https://doi.org/10.1016/j.reactfunctpolym.2019.04.012S566114

Fluor-thiol Photocoupling Reaction for Developing High Performance Nucleic Acid (NA) Microarrays

[EN] Spatially controlled anchoring of NA probes onto microscope glass slides by a novel fluor-thiol coupling reaction is performed. By this UV-initiated reaction, covalent immobilization in very short times (30 s at 254 nm) is achieved with probe densities of up to 39.6 pmol/cm(2). Modulating the surface hydrophobicity by combining a hydrophobic silane and a hydrophilic silane allows the fabrication of tuned surfaces where the analyte approaches only the anchored probe, which notably reduces nonspecific adsorption and the background. The generated substrates have proven clear advantages for discriminating single-base-pair mismatches, and for detecting bacterial PCR products. The hybridization sensitivity achieved by these high-performance surfaces is about 1.7 pM. Finally, this anchoring reaction is demonstrated using two additional surfaces: polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) membranes. This provides a very interesting pathway for anchoring thiolated biomolecules onto surfaces with C-F motifs via a quick clean UV reaction.Financial support from INTERBOINTER (project CTQ2013-45875-R) and BIHOLOG (Project CTQ2016-75749-R), FEDER, and GVA PROMETEO II 2014/040 is acknowledged. The authors also thank Dr. Tortajada-Genaro and Dr. Ninoles Rodenes for providing the Salmonella and Campylobacter PCR-amplified products. P.J.-M. acknowledges the Spanish Ministry of Economy, Industry and Competitiveness for the public FPI grant (Project CTQ2013-45875-R) and the cofinancing by, the European Social Fund. Dr. Miguel Angel Gonzalez-Martinez, Dr. Sergio Navalon, and Dr. Patricia Concepcion from Universitat Politecnica de Valencia are acknowledged for their help in the XPS analysis.Jimenez-Meneses, P.; Bañuls Polo, M.; Puchades, R.; Maquieira Catala, A. (2018). Fluor-thiol Photocoupling Reaction for Developing High Performance Nucleic Acid (NA) Microarrays. Analytical Chemistry. 90(19):11224-11231. https://doi.org/10.1021/acs.analchem.8b00265S1122411231901

Digital versatile discs as platforms for multiplexed genotyping based on selective ligation and universal microarray detection

[EN] The development of a high-performance assay readout using integrated detectors is a current challenge in the implementation of DNA tests in diagnostic laboratories, particularly for supporting pharmacogenetic tests. A method for allelic discrimination, associated with single nucleotide polymorphisms (SNPs), is presented. Genomic DNA is extracted from blood and buccal swab samples. The procedure comprises fast multiplex ligation-dependent probe amplification, PCR amplification using universal primers and subsequent barcode hybridization. In this last step, each product is recognized by the specific probes immobilized on the surface of an optical disc. Assay results can be obtained with a disc reader. The optical sensing method in a DNA microarray format was optimized and evaluated for the simultaneous identification of 28 polymorphisms associated with psychiatric pharmacogenomics. The target biomarkers were located in the genes related to drug-metabolizing enzymes and drug transporters. The multiplexing capability and assay selectivity strongly depended on correct design (ligation probes, tails and barcodes). The discriminant analysis of reader outputs (spot intensities) led to patients being classified into different allelic populations. The obtained assignations correlated properly with the results provided by the reference technique (bead arrays), and the assay ended in an 8-fold shorter time using affordable equipment. The combination of a highly selective genotyping reaction as array-MLPA and the compact disc technology provides a reliable point-of-care approach. This genotyping tool is useful for the selection of personalized drug therapies in decentralized clinical laboratories.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 Project) through the FEDER funds and the Spanish Ministry of Economy and Competitiveness (the MINECO CTQ2016-75749- R Project)Tortajada-Genaro, LA.; Niñoles Rodenes, R.; Mena-Mollá, S.; Maquieira Catala, A. (2019). Digital versatile discs as platforms for multiplexed genotyping based on selective ligation and universal microarray detection. The Analyst. 144:707-715. https://doi.org/10.1039/C8AN01830HS70771514

A group-based wireless body sensors network using energy harvesting for soccer team monitoring

[EN] In team-based sports, it is difficult to monitor physical state of each athlete during the match. Wearable body sensors with wireless connections allow having low-power and low-size devices, that may use energy harvesting, but with low radio coverage area but the main issue comes from the mobility. This paper presents a wireless body sensors network for soccer team players' monitoring. Each player has a body sensor network that use energy harvesting and each player will be a node in the wireless sensor network. This proposal is based on the zone mobility of the players and their dynamism. It allows knowing the physical state of each player during the whole match. Having fast updates and larger connection times to the gateways, the information can be routed through players of both teams, thus a secure system has been added. Simulations show that the proposed system has very good performance in high mobility.This work has been partially supported by the Instituto de Telecomunicacoes, Next Generation Networks and Applications Group (NetGNA), Portugal, by Government of Russian Federation, Grant 074-U01, by National Funding from the FCT - Fundacao para a Ciencia e a Tecnologia through the PEst-OE/EEI/LA0008/2013 Project.Lloret, J.; García Pineda, M.; Catala Monzo, A.; Rodrigues, JJPC. (2016). A group-based wireless body sensors network using energy harvesting for soccer team monitoring. International Journal of Sensor Networks. 21(4):208-225. https://doi.org/10.1504/IJSNET.2016.079172S20822521

Polymorphism genotyping based on loop-mediated isothermal amplification and smartphone detection

[EN] The genotyping of a single-nucleotide polymorphism (SNP) is addressed through methods based on loop-mediated isothermal amplification (LAMP) combined with user-friendly optical read-outs to cover the current demand for point-of-care DNA biomarker detection. The modification of primer design and reaction composition improved the assay selectivity yielding allele-specific results and reducing false-positive frequency. Furthermore, the reduced cost, ease of use and effectiveness of calorimetric detection (solution and hybridisation chip formats) were availed for the image capture by a smartphone, reching high sensitivity. In order to evaluate their discriminating capacities, LAMP-based methods were applied to human samples to genotype a SNP biomarker (rs1954787) located in the GRIK4 gene and related to the treatment response to anti-depressants drugs. Sensitive (limit of detection: 100 genomic DNA copies), reproducible ( < 15% error), fast (around 70 min) and low-cost assays were accomplished. Patient subgroups were correctly discriminated, agreeing with reference sequencing techniques. The achieved analytical performances using the developed amplification-detection principles confirmed the approach potential for point-of-care optical DNA testing.The authors acknowledge the financial support received from the Generalitat Valenciana (Project GVA-PROMETEOII/2014/040 and GRISOLIA/2014/024 Ph.D. grant) and from the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R Project).Yamanaka, E.; Tortajada-Genaro, LA.; Pastor Navarro, N.; Maquieira Catala, A. (2018). Polymorphism genotyping based on loop-mediated isothermal amplification and smartphone detection. Biosensors and Bioelectronics. 109:177-183. https://doi.org/10.1016/j.bios.2018.03.008S17718310