Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection

[EN] The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 mu M. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents.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. Lucío acknowledges her Juan de la Cierva-Incorporación grant (IJC 2018-035355-I) funded by MCIN/AEI/10.13039/501100011033. P. Zezza acknowledges Generalitat Valenciana for her Grisolia fellowship grant.Zezza, P.; Lucío, MI.; Fernández, E.; Maquieira Catala, A.; Bañuls Polo, M. (2023). Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection. Biosensors. 13(3). https://doi.org/10.3390/bios1303031213

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

Time-stable holograms stored in hydrogel matrix

Storing holograms in hydrogel matrices and its temporal stability is real challenge when the material is immersed in aqueous medium. Hydrogels are 3D polymer networks capable of undergoing reversible volume changes. Since they can be synthesized to be sensitive to a range of relevant analytes, one of the most important applications of this type of system are holographic sensors. These sensors have great potential to be used in different areas such as environmental detection, veterinary testing, pharmaceutical bioassays, and medical diagnosis. Therefore, a key factor in the manufacture of holographic sensors is to develop and optimize hydrogel matrices in which stable holograms can be stored over time and in aqueous media where analytes are present.Thanks to the government of Algeria for the scholarship offered to develop my thesis in the Holography and Optical Processing Group of the University of Alicante, to the Spanish Ministerio de Ciencia e Innovación under project PID2019-106601RB-I00, to Generalitat Valenciana (Spain) CDIAGENT/2018/024, to IDIFEDER/2021/014 (potencial FEDER funding

Label-free detection of C-Reactive protein using bioresponsive hydrogel-based surface relief diffraction gratings

[EN] Responsive hydrogel-based surface relief gratings have demonstrated great performances as transducers in optical sensing. However, novel and smart designs of hydrogels are needed for the appropriate detection of analytes and biomolecules since the existing materials are very limited to specific molecules. In this work, a biosensing system based on surface relief gratings made of bioresponsive hydrogels has been developed. In particular, the hydrogel contains phosphocholine moieties to specifically recognize C-Reactive protein (CRP). The CRP-Sensing hydrogel capacity to selectively detect CRP was fully demonstrated. Using Direct Laser Interference Patterning, micro-gratings were created on CRP-Sensing hydrogel substrates and applied for the label-free sensing of CRP using a simple laser-based homemade optical setup. Limits of detection (LOD) and quantification (LOQ) in human serum dilutions of 1.07 and 8.92 mg L-1, respectively, were reached. These results demonstrate that the biosensing system allows the selective label-free detection of CRP within concentration ranges around those related to risks of cardiovascular diseases and sepsis. Besides, amplification strategies have been carried out improving the sensitivity, widening the linear range, and reaching better LOD and LOQ (0.30 mg L-1 and 4.36 mg L-1). Finally, all the approaches were tested for the quantification of CRP in certified human serum with recoveries of around 100%.This work was financially supported by the E.U. FEDER, the Spanish Ministry of Economy and Competitiveness MINECO (BiHolog-CTQ2016- 75749-R and AdBiHol-PID2019-110713RB-I00) and Generalitat Valenciana (PROMETEO/2020/094). M. I. Lucío acknowledges MINECO for her Juan de la Cierva Formacion and Incorporacion grants (FJCI-2016-29593, IJC 2018-035355-I). The authors acknowledge the assistance and advice of the Electron Microscopy Service of the Universitat Politècnica de València.Lucío, MI.; Hernández-Montoto, A.; Fernández-Sánchez, ME.; Alamri, S.; Kunze, T.; Bañuls Polo, M.; Maquieira Catala, Á. (2021). Label-free detection of C-Reactive protein using bioresponsive hydrogel-based surface relief diffraction gratings. Biosensors and Bioelectronics. 193:1-10. https://doi.org/10.1016/j.bios.2021.11356111019

Enhanced docetaxel-mediated cytotoxicity in human prostate cancer cells through knockdown of cofilin-1 by carbon nanohorns delivered siRNA.

[EN] We synthesized a non-viral delivery system (f-CNH3) for small interfering RNA (siRNA) by anchoring a fourth-generation polyamidoamine dendrimer (G4-PAMAM) to carbon nanohorns (CNHs). Using this new compound, we delivered a specific siRNA designed to knockdown cofilin-1, a key protein in the regulation of cellular cytoskeleton, to human prostate cancer (PCa) cells. The carbon nanohorn (CNH) derivative was able to bind siRNA and release it in the presence of an excess of the polyanion heparin. Moreover, this hybrid nanomaterial protected the siRNA from RNAse-mediated degradation. Synthetic siRNA delivered to PCa cells by f-CNH3 decreased the cofilin-1 mRNA and protein levels to about 20% of control values. Docetaxel, the drug of choice for the treatment of PCa, produced a concentration-dependent activation of caspase-3, an increase in cell death assessed by lactate dehydrogenase release to the culture medium, cell cycle arrest and inhibition of tumor cell proliferation. All of these toxic effects were potentiated when cofilin-1 was down regulated in these cells by a siRNA delivered by the nanoparticle. This suggests that knocking down certain proteins involved in cancer cell survival and/or proliferation may potentiate the cytotoxic actions of anticancer drugs and it might be a new therapeutic approach to treat tumors.F.C.P-M. and B.C. are recipients of Torres Quevedo contracts from Ministerio de Ciencia e Innovacion (Spain) and NanoDrugs, S.L. This work has been supported, in part, by grants BFU2011-30161-C02-01 from Ministerio de Ciencia e Innovacion; PII1I09-0163-4002 and POII10-0274-3182 from Consejeria de Educacion, JCCM to V.C.Pérez-Martínez, FC.; Carrión, B.; Lucío, MI.; Rubio, N.; Herrero, MA.; Vázquez, E.; Ceña, V. (2012). Enhanced docetaxel-mediated cytotoxicity in human prostate cancer cells through knockdown of cofilin-1 by carbon nanohorns delivered siRNA. Biomaterials. 33(32):8152-8159. https://doi.org/10.1016/j.biomaterials.2012.07.03881528159333

Bending in the holograms stored in hydrogel matrices

The study of the optical properties and behaviour of holograms stored in hydrogel matrices when the material is immersed in liquid medium represent a very important challenge currently. Hydrogels are 3D polymer networks capable of undergoing reversible volume changes. These hydrogels can be chemically modified to obtain materials with different properties such as to be sensitive to a range of relevant analytes. Emergent applications require that the holograms stored in hydrogels be time-stable in a liquid medium. One of the most important applications of this type of system are holographic sensors. Holographic sensors have advantages over other types of sensors such as the possibility of miniaturization due to the use of holographic techniques, the ability to produce three-dimensional images, real-time quantification, possibility of low-cost mass manufacturing and label-free analyte- responsive. Due to these advantages, these sensors have great potential to be used in different areas such as environmental detection, veterinary testing, pharmaceutical bioassays and medical diagnosis. Therefore, the optical behaviour of the holograms and the optimization of the hydrogel’s matrices must be well studied. When volume phase holograms are stored in hydrogels matrices in liquid medium, the holographic planes can undergo a bending process that give rise to asymmetries in the lateral lobes around the Bragg angle. This bending affect to the diffraction efficiency, wavelength of maximum diffraction efficiency and the angular sensibility. These parameters are used as signal transducers in holographic sensors in transmission mode. The general aim of this work has been study the bending that is produced in the holographic planes when unslanted transmission volume phase holographic grating with a frequency of 1200 lines/mm have been stored in hydrogel matrices based on acrylamide (AA) and N,N'-methylenebisacrylamide (MBA) using different solvents (dimethyl sulfoxide, water and buffer solution) during the manufacturing process. Considering previous works on bending, and grating attenuated hologram.This work was supported by the by “Generalitat Valenciana” of Spain (projects PROMETEO/2021/006 and IDIFEDER/2021/014, cofunded by European Union through the FEDER Programme) and by the “Ministerio de Ciencia e Innovación” of Spain (projects FIS2017-82919-R and PID2019-106601RB-I00)

Processing of Holographic Hydrogels in Liquid Media: A Study by High-Performance Liquid Chromatography and Diffraction Efficiency

The storage of time-stable holographic gratings in hydrogel matrices when the material is immersed in aqueous media is a real challenge at present. The optimization of the storage stages of the holograms must be properly investigated to identify the most suitable development processes. For this reason, this work is focused on the study of the optimization of the washing stages of the hydrogels based on acrylamide and N,N’-methylenebis(acrylamide) once unslanted transmission holograms have been stored. High-performance liquid chromatography and UV-visible measurements have been employed in our system to analyze the composition of the washing solutions. PBST and DMSO:H2O are used as solvents in the washing stages. The diffraction efficiencies are measured during the washing stages and after the storing of the holograms during several days in PBST. Maximum diffraction efficiencies of 38 and 27.6% are reached when PBST and DMSO:H2O are employed, respectively, for the washing process. Holograms show temporal stability after being stored immersed in PBST at 4 °C for 4 days.This research was funded by Ministerio de Ciencia, Innovación y Universidades, Spain, under project PID2019-106601RB-I00 and AdBiHol-PID2019-110713RB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; Generalitat Valenciana, Spain, under projects CDEIGENT/2018/024, GRISOLIAP/2019/143, PROMETEO/2021/006, PROMETEO/2020/094 and IDIFEDER/2021/014 (co-funded by European Union through the FEDER Program). M. I. Lucío acknowledges her Juan de la Cierva Incorporación grant (IJC 2018-035355-I) funded by MCIN/AEI/10.13039/501100011033. M. I. Lucío and A. Beléndez acknowledge the Programa Propio para el fomento de la I + D + I del Vicerrectorado de Investigación y Transferencia de Conocimiento of the Universidad de Alicante for the grant “Ayudas para estancias de personal investigador invitado“ (INVA20-02). K. Berramdane thanks to the government of Algeria for her scholarship offered to develop her thesis in the Holography and Optical Processing Group at the University of Alicante (Spain)

High-performance liquid chromatography and UV-visible measurements to optimize the storage of volume holograms in hydrogels

The storage of time-stable holographic gratings in photohydrogels when the material is immersed in liquid media represents a great challenge at present. A very important stage in the process of storing holograms in photohydrogels are the washing stages to eliminate the remains of the components that have not reacted in the photochemical reaction. The main goal of this work is focusing on the study of the optimization of the washing stages of the photohydrogels based on acrylamide and N,N’-methylenebis(acrylamide) once unslanted transmission holograms have been stored. For the purpose of determining the compositions of the wash solutions, High-Performance Liquid Chromatography and UV-visible measurements have been employed in our system. PBST and DMSO:H2O 6:4 (v/v) are used as solvents in the washing stages. The diffraction efficiencies are measured during the washing stages and after the storing of the holograms during several days in PBST. Maximum diffraction efficiencies of 38.0 and 27.6% are reached when PBST and DMSO:H2O 6:4 are employed, respectively.Work funded by Ministerio de Ciencia e Innovación, Spain, under project PID2019-106601RB-I00; by Generalitat Valenciana under projects GRISOLIAP/2019/143, CIDEXG/2022/60, PROMETEO/2021/006, PROMETEO/2020/094 and IDIFEDER/2021/014 (co-funded by European Union through the FEDER Program), Spain. K. Berramdane thanks to the government of Algeria for her scholarship offered to develop her thesis in the Holography and Optical Processing Group at the University of Alicante (Spain). M. I. Lucío acknowledges her Juan de la Cierva Incorporación grant (IJC 2018-035355-I) funded by MCIN/AEI/10.13039/501100011033

Microwave-assisted functionalization of carbon nanohorns with oligothiophene units with SERS activity

[EN] Carbon nanohorns have been functionalized with oligothiophene unitsviathe 1,3-dipolar cycloaddition reaction under microwave irradiation and solvent-free conditions. A dramatic Raman enhancement was found for one of the synthesized derivatives. Experimental andin silicostudies helped to understand the enhancement, attributed to the modification of electromagnetic fields upon functionalization at the tip of the nanostructures.This work was supported by the Iberdrola Foundation (CONV120313), the Spanish Ministerio de Economia y Competitividad (CTQ2017-88158-R), the Junta de Comunidades de Castilla-La Mancha (SBPLY/17/180501/000204) and FEDER-JCCM (UNCM13-1E-1663). The work at the University of Malaga was funded by the MICINN (PID2019-110305GB-I00) and Junta de Andalucia (P09FQM-4708) projects. M. I. L. acknowledges MINECO for her Juan de la Cierva-formacion grant (FJCI-2016-29593). The authors acknowledge the computer resources, technical expertise, and assistance provided by the Supercomputing and Bioinformatics centre of the University of Malaga.Iglesias, D.; Guerra, J.; Lucío, MI.; González-Cano, RC.; Lopez Navarrete, JT.; Ruiz Delgado, MC.; Vázquez, E.... (2020). Microwave-assisted functionalization of carbon nanohorns with oligothiophene units with SERS activity. Chemical Communications. 56(63):8948-8951. https://doi.org/10.1039/D0CC03496G89488951566

A Prato Tour on Carbon Nanotubes: Raman Insights

[EN] The functionalisation of carbon nanotubes has been instrumental in broadening its application field, allowing especially its use in biological studies. Although numerous covalent and non-covalent functionalisation methods have been described, the characterisation of the final materials has always been an added challenge. Among the various techniques available, Raman spectroscopy is one of the most widely used to determine the covalent functionalisation of these species. However, Raman spectroscopy is not a quantitative technique, and no studies are reported comparing its performance when the same number of functional groups are added but using completely different reactions. In this work, we have experimentally and theoretically studied the functionalisation of carbon nanotubes using two of the most commonly used reactions: 1,3-dipolar cycloaddition of azomethylene ylides and diazonium-based radical addition. The number of groups introduced onto the tubes by these reactions has been determined by different characterisation techniques. The results of this study support the idea that data obtained by Raman spectra are only helpful for comparing functionalisations produced using the same type of reaction. However, they should be carefully analysed when comparing functionalisations produced using different reaction types.The authors acknowledge financial support from the Spanish government (project PID2020-113080RB-I00) and the Junta de Comunidades de Castilla-La Mancha (project SBPLY/21/180501/000135/1). This study forms part of the Advanced Materials program and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by the Junta de Comunidades de Castilla-La Mancha. The work at the University of Malaga was funded by the MICINN (PID202-139548NB-I00) and by the Junta de Andalucia (P18-FR-4559) projects. The authors thankfully acknowledge the computer resources, technical expertise, and assistance provided by the SCBI (Supercomputing and Bioinformatics) centre of the University of Malaga. M. I. L. acknowledges her Juan de la Cierva Incorporacion grant (IJC 2018-035355-I) funded by MCIN/AEI/10.13039/501100011033. Funded with Aid for First Research Projects (PAID-06-22), Vice-rectorate for Research of the Universitat Politecnica de Valencia (UPV).Lucío, MI.; Giacalone, F.; La Parola, V.; Gámez-Valenzuela, S.; Muñoz-Alba, F.; Ruiz Delgado, MC.; Herrero, MA.... (2023). A Prato Tour on Carbon Nanotubes: Raman Insights. Chemistry - A European Journal. 29. https://doi.org/10.1002/chem.2023024762