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. 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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. 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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

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

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

Allele-specific ligation and recombinase polymerase amplification for the detection of single nucleotide polymorphisms

[EN] A novel multiplex detection of single nucleotide polymorphisms (SNPs), with point-of-care testing as its aim, is reported for supporting pharmacogenetic-based decisions. The strategy relies on allele-specific ligation to discriminate base sequence variations at the SNP site and the extension of generated products by isothermal amplification and recombinase polymerase amplification (RPA). Having demonstrated the assay principle, the variables for the adequate integration of the ligation-amplification process were studied and compared to a conventional PCR approach. One key result was the development of RPA in a universal format using short-length primers, which enabled detection based on selective hybridisation on a barcode-DNA chip and a low-cost optical sensor. As proof of concept, we successfully discriminated genetic variants related to cardiovascular diseases and the adequate prescription of oral anticoagulant antagonists of vitamin K (genes CYP2C9 and VKROC1).The authors acknowledge the financial support received from the Generalitat Valenciana (GRISOLIA/2014/024PhD Grant and GVA-FPI-2017PhD Grant) and the Spanish Ministry of Economy and Competitiveness (MINECO Project CTQ2016-75749-R).Lázaro-Zaragozá, A.; Yamanaka, E.; Maquieira Catala, A.; Tortajada-Genaro, LA. (2019). Allele-specific ligation and recombinase polymerase amplification for the detection of single nucleotide polymorphisms. Sensors and Actuators B Chemical. 298. https://doi.org/10.1016/j.snb.2019.126877S29

Holographic Recording of Unslanted Volume Transmission Gratings in Acrylamide/Propargyl Acrylate Hydrogel Layers: Towards Nucleic Acids Biosensing

[EN] The role of volume hydrogel holographic gratings as optical transducers in sensor devices for point-of-care applications is increasing due to their ability to be functionalized for achieving enhanced selectivity. The first step in the development of these transducers is the optimization of the holographic recording process. The optimization aims at achieving gratings with reproducible diffraction efficiency, which remains stable after reiterative washings, typically required when working with analytes of a biological nature or several step tests. The recording process of volume phase transmission gratings within Acrylamide/Propargyl Acrylate hydrogel layers reported in this work was successfully performed, and the obtained diffraction gratings were optically characterized. Unslanted volume transmission gratings were recorded in the hydrogel layers diffraction efficiencies; up to 80% were achieved. Additionally, the recorded gratings demonstrated stability in water after multiple washing steps. The hydrogels, after functionalization with oligonucleotide probes, yields a specific hybridization response, recognizing the complementary strand as demonstrated by fluorescence. Analyte-sensitive hydrogel layers with holographic structures are a promising candidate for the next generation of in vitro diagnostic tests.This work was financially supported by the E.U. FEDER, the Spanish Ministry of Science and Innovation (ADBIHOL-PID2019-110713RB-I00/AEI/10.13039/501100011033) and Generalitat Valenciana (PROMETEO/2020/094). M. I. Lucio acknowledges MINECO for her Juan de la Cierva-Incorporacion grants (IJC 2018-035355-I). P. Zezza acknowledges the Generalitat Valenciana for her S. Grisolia grant and the UPV for the mobility grant (BEFPI 2022). Funding was also received from Aid for First Research Projects (PAID-06-22) and the Vice-rectorate for Research of the Universitat Politecnica de Valencia (UPV). Partially funded by the European Space Agency, through PEA4000129503 collaborative project: Wound Healing In Space: Key challenges towards Intelligent and Enabling Sensing platforms.Zezza, P.; Lucío, MI.; Naydenova, I.; Bañuls Polo, M.; Maquieira Catala, A. (2023). Holographic Recording of Unslanted Volume Transmission Gratings in Acrylamide/Propargyl Acrylate Hydrogel Layers: Towards Nucleic Acids Biosensing. Gels. 9(9). https://doi.org/10.3390/gels90907109

Enzyme Inhibition Microassays on Blu-Ray Disks for Drug Discovery

[EN] An enzyme inhibition-based assay for drug discovery developed by microarraing on Blu-ray disks is presented. As a proof-of-concept, the system screens a selected molecule library of potential chemical inhibitors against the glycoenzyme peroxidase, identifying the promising lead compounds with high selectivity using standard Blu-ray disks and drives. In order to face the first drug discovery stages, we establish the bases for a high-throughput screening assay and a methodology based on hypersurfaces suitable to manage a high number of data as well.This work was supported by the Spanish Ministry of Economy and Competitiveness and the European Region Development Fund under award reference: CTQ2016-75749-R.Sancho-Fornes, G.; Peris Chanzá, EJ.; Giménez-Romero, D.; Morais, S.; Maquieira Catala, A. (2019). Enzyme Inhibition Microassays on Blu-Ray Disks for Drug Discovery. ACS Omega. 4(3):5595-5600. https://doi.org/10.1021/acsomega.8b03537S559556004

A label-free diffraction-based sensing displacement immunosensor toquantify low molecular weight organic compounds

[EN] Herein we present a diffractometric immunosensor to quantify low molecular weight organic compounds in a label-free, simple, and sensitive fashion. The approach is based on patterning analyte analogues (haptens) on solid surfaces according to a diffractive structure, and then loading specific antibodies on them to be subsequently displaced by free analytes in solution. This displacement generates a measurable change in the diffractive response that enables to quantify the analyte concentration. In this study we address the fabrication, optimization, and assessment of these diffractive structures of biological probes and their application to the analysis of atrazine, an organic compound extensively used as pesticide. This immunosensor displays well-correlated dose-response curves that reach a detection limit of 1.1¿ng¿mL¿1 of atrazine in label-free conditions. From a general viewpoint, this study also aims to provide insights into exploiting this approach towards prospective in-field analysis and screening strategies to sense multiple low molecular weight compounds in label-free conditions.This work was supported by the Spanish Ministry of Economy and Competitiveness (CTQ2013-45875-R and FIS2011-23175), FEDER, and the Generalitat Valenciana (PROMETEO II/2014/040 and PROMETEO II/2014/072). Special thanks go to Richard A. McAloney and M. Cynthia Goh for hosting M.A.-O. as visiting researcher, sharing their expertise, and offering their valuable support. M.A.-O. also acknowledges the FPI program of the Spanish Ministry of Economy and Competitiveness for a PhD and an EEBB mobility grant.Avella-Oliver, M.; Ferrando Martín, V.; Monsoriu Serra, JA.; Puchades, R.; Maquieira Catala, A. (2018). A label-free diffraction-based sensing displacement immunosensor toquantify low molecular weight organic compounds. Analytica Chimica Acta. 1033:173-179. https://doi.org/10.1016/j.aca.2018.05.060S173179103