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

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

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)

Holographic biosensors made of DNA-functionalised hydrogels for in vitro diagnostic

Tesis por compendio[ES] La tesis doctoral se centra en el desarrollo de un hidrogel sensible a analitos, funcionalizado con sondas de ADN, con estructura difractiva como transductor óptico para aplicaciones de diagnóstico in vitro. El primer capítulo incluye una visión general de los diferentes conceptos relacionados con el biosensado, los desarrollos recientes en el mercado del diagnóstico in vitro y, en particular, los biosensores de ADN. Además, se presenta la síntesis y caracterización de hidrogeles, su papel como matriz de soporte en biosensado y las estrategias de inmovilización. Por último, se explican los conceptos básicos de la holografía como nueva estrategia de detección y el papel de las diferentes redes de difracción en la biosensación. A continuación, en el Capítulo 2, se discuten los objetivos de este proyecto. El objetivo de esta investigación es desarrollar hidrogeles que incorporen sondas de ADN y dotarlas de una estructura difractiva para que actúen como transductores ópticos sin etiquetas. Se consideran dos tipos de estructuras difractivas: redes holográficas de relieve superficial (SRG) y redes de transmisión de volumen (VTG). La fase inicial de este trabajo se centró en la optimización de hidrogeles, ajustando su composición para que actuaran como biosensores holográficos. Se seleccionaron acrilamida y bisacrilamida para la preparación del hidrogel mediante reacción de polimerización por radicales libres. Además, para introducir la respuesta del analito en la red de hidrogeles 3D, hubo que investigar y poner a punto diferentes estrategias de inmovilización de biorreceptores. En el capítulo 3, la estrategia optimizada consiste en incorporar directamente sondas de ADN modificadas con acridita mediante copolimerización con monómeros de acrilamida durante la formación del hidrogel. Los hidrogeles funcionalizados con ADN se caracterizaron mediante imágenes de fluorescencia y se exploró su versatilidad mediante la fabricación de microarrays. Por último, el hidrogel optimizado sensible a los analitos se utilizó como plataforma para la preparación de SRG. El capítulo 4 describe otro enfoque adoptado para la funcionalización del hidrogel con sondas de ADN. Se añadió un comonómero de acrilato de propargilo al hidrogel de acrilamida, con el fin de introducir la presencia de residuos alcínicos y facilitar una mayor incorporación de las sondas de ADN. Las sondas de ADN utilizadas tenían grupos terminales tiol y se incorporaron mediante química de clic tiol-eno/tiol-yo, debido a la presencia de enlaces C-C dobles y triples. Con esta estrategia, se demostraron dos enfoques de inmovilización de sondas de ADN: durante y después de la síntesis del hidrogel. Los resultados preliminares mostraron que los SRGs tienen potencial para detectar directamente la hibridación de oligonucleótidos en un formato libre de etiquetas. En el capítulo 5, se optimizó el proceso de grabación de VTGs no inclinados en capas de hidrogel para mejorar el rendimiento del transductor. Tras una cuidadosa evaluación de los parámetros de grabación holográfica, las composiciones de las soluciones de incubación y los tiempos de incubación, las estructuras VTG se grabaron con una buena reproducibilidad, logrando una excelente eficiencia de difracción. Además, se estudió su estabilidad en agua para bioensayos. Por último, se observó que los VTG, modificados con oligonucleótidos, respondían selectivamente hibridándose sólo con la diana complementaria, a la vez que conservaban sus propiedades de difracción. El trabajo de investigación demostró la viabilidad de utilizar redes difractivas en capas de hidrogel como biosensores libres de etiquetas, capaces de detectar sondas de ADN, complementarias a la secuencia inmovilizada, en un medio acuoso. Por último, en el capítulo 6, se analizan comparativamente el rendimiento y la aplicabilidad de los distintos enfoques estudiados y se discuten las perspectivas futuras de los hidrogeles de ácidos nucleicos para la detección holográfica.[CA] La tesi doctoral se centra en el desenvolupament d'un hidrogel sensible a anàlits, funcionalitzat amb sondes d'ADN, amb estructura difractiva com a transductor òptic per a aplicacions de diagnòstic in vitro. El primer capítol inclou una visió general dels diferents conceptes relacionats amb el biosensado, els desenvolupaments recents en el mercat del diagnòstic in vitro i, en particular, els biosensores d'ADN. A més, es presenta la síntesi i caracterització d'hidrogels, el seu paper com a matriu de suport en biosensado i les estratègies d'immobilització. Finalment, s'expliquen els conceptes bàsics de l'holografia com a nova estratègia de detecció i el paper de les diferents xarxes de difracció en la biosensación. A continuació, en el Capítol 2, es discuteixen els objectius d'este projecte. L'objectiu d'esta investigació és desenvolupar hidrogels que incorporen sondes d'ADN i dotar-les d'una estructura difractiva perquè actuen com a transductors òptics sense etiquetes. Es consideren dos tipus d'estructures difractivas: xarxes hologràfiques de relleu superficial (SRG) i xarxes de transmissió de volum (VTG). La fase inicial d'este treball es va centrar en l'optimització d'hidrogels, ajustant la seua composició perquè actuaren com biosensores hologràfics. Es van seleccionar acrilamida I bisacrilamida per a la preparació de l'hidrogel mitjançant reacció de polimerització per radicals lliures. A més, per a introduir la resposta de l'anàlit en la xarxa d'hidrogels 3D, va caldre investigar i posar a punt diferents estratègies d'immobilització de biorreceptores. En el capítol 3, l'estratègia optimitzada consisteix a incorporar directament sondes d'ADN modificades amb acridita mitjançant copolimerización amb monòmers d'acrilamida durant la formació de l'hidrogel. Els hidrogels funcionalitzats amb ADN es van caracteritzar mitjançant imatges de fluorescència i es va explorar la seua versatilitat mitjançant la fabricació de bioxips. Finalment, l'hidrogel optimitzat sensible als anàlits es va utilitzar com a plataforma per a la preparació de SRG. El capítol 4 descriu un altre enfocament adoptat per a la funcionalització de l'hidrogel amb sondes d'ADN. Es va afegir un comonómero de acrilato de propargilo a l'hidrogel d'acrilamida, amb la finalitat d'introduir la presència de residus alcínicos i facilitar una major incorporació de les sondes d'ADN. Les sondes d'ADN utilitzades tenien grups terminals tiol i es van incorporar mitjançant química de clic tiol-eno/tiol-ino, a causa de la presència d'enllaços C-C dobles i triples. Amb esta estratègia, es van demostrar dos enfocaments d'immobilització de sondes d'ADN: durant i després de la síntesi de l'hidrogel. Els resultats preliminars van mostrar que els SRGs tenen potencial per a detectar directament la hibridació de oligonucleótidos en un format lliure d'etiquetes. En el capítol 5, es va optimitzar el procés de gravació de VTGs no inclinats en capes d'hidrogel per a millorar el rendiment del transductor. Després d'una acurada avaluació dels paràmetres de gravació hologràfica, les composicions de les solucions d'incubació i els temps d'incubació, les estructures VTG es van gravar amb una bona reproducibilidad, aconseguint una excel·lent eficiència de difracció. A més, es va estudiar la seua estabilitat en aigua per a bioensayos. Finalment, es va observar que els VTG, modificats amb oligonucleótidos, responien selectivament hibridant-se només amb la diana complementària, alhora que conservaven les seues propietats de difracció. El treball de recerca va demostrar la viabilitat d'utilitzar xarxes difractivas en capes d'hidrogel com biosensores lliures d'etiquetes, capaces de detectar sondes d'ADN, complementàries a la seqüència immobilitzada, en un medi aquós. Finalment, en el capítol 6, s'analitzen comparativament el rendiment i l'aplicabilitat dels diferents enfocaments estudiats i es discuteixen les perspectives futures dels hidrogels d'àcids nucleics per a la detecció hologràfica.[EN] The PhD thesis focuses on the development of an analyte-sensitive hydrogel, functionalised with DNA probes, with a diffractive structure as an optical transducer for in vitro diagnostic applications. The first chapter includes an overview of the different concepts related to biosensing, recent developments in the in vitro diagnostics market and, in particular, DNA biosensors. Furthermore, the synthesis and characterisation of hydrogels, their role as a support matrix in biosensing and immobilisation strategies are presented. Finally, the basic concepts of holography as a new detection strategy and the role of different diffraction gratings in biosensing are explained. Then, in Chapter 2, the objectives of this project are discussed. The aim of this research is to develop hydrogels that incorporate DNA probes and provide them with a diffractive structure to act as label-free optical transducers. Two types of diffractive structures are considered: surface-relief holographic gratings (SRGs) and volume transmission gratings (VTGs). The initial phase of this work focused on the optimisation of hydrogels, adjusting their composition to act as holographic biosensors. Acrylamide and bisacrylamide were selected for hydrogel preparation by free radical polymerisation reaction. Furthermore, in order to introduce the analyte response into the 3D hydrogel network, different bioreceptor immobilisation strategies had to be investigated and fine-tuned. In chapter 3, the optimised strategy is to directly incorporate acridite-modified DNA probes by copolymerisation with acrylamide monomers during hydrogel formation. The DNA-functionalised hydrogels were characterised by fluorescence imaging and their versatility was explored by microarray fabrication. Finally, the optimised analyte-responsive hydrogel was used as a platform for SRG preparation. Chapter 4 describes another approach adopted for functionalisation of the hydrogel with DNA probes. A propargyl acrylate comonomer was added to the acrylamide hydrogel in order to introduce the presence of alkyl residues and facilitate further incorporation of the DNA probes. The DNA probes used had thiol end-groups and were incorporated by thiol-ene/thiol-yo click chemistry, due to the presence of double and triple C-C bonds. With this strategy, two approaches to DNA probe immobilisation were demonstrated: during and after hydrogel synthesis. Preliminary results showed that SRGs have the potential to directly detect oligonucleotide hybridisation in a label-free format. In chapter 5, the recording process of unslanted VTGs in hydrogel layers was optimised to improve transducer performance. After careful evaluation of holographic recording parameters, incubation solution compositions and incubation times, the VTG structures were recorded with good reproducibility, achieving excellent diffraction efficiency. In addition, their stability in water for bioassays was studied. Finally, oligonucleotide-modified VTGs were found to respond selectively by hybridising only to the complementary target, while retaining their diffraction properties. The research work demonstrated the feasibility of using diffractive networks in hydrogel layers as label-free biosensors, capable of detecting DNA probes, complementary to the immobilised sequence, in an aqueous medium. Finally, in chapter 6, the performance and applicability of the different approaches studied are comparatively analysed and future prospects of nucleic acid hydrogels for holographic detection are discussed.I would like to acknowledge the government of Valencia to for the PhD fellowship “Santiago Grisolia” and the BEFPI/2022 grant for a 4-months doctoral stay and also the Spanish Ministry of Economy and Competitiveness MINECO (ADBIHOL national project) for their financial support.Zezza, P. (2023). Holographic biosensors made of DNA-functionalised hydrogels for in vitro diagnostic [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202597Compendi

The dynamics of international competitiveness: first results from an analysis at the industry level

ntroduction Over the past two decades a growing number of theoretical contributions and empirical verifications have led to the recognition of the key role of technology in determining trade flows and international competitiveness at the firm and country level (for a survey see These results are related both to the new trade and growth theories based on imperfect competition models One particular feature of technological innovation is its sectoral differentiatio

DNA -based hydrogels for high-performance optical biosensing application

[EN] Analyte-sensitive DNA-based hydrogels find multiple applications in the field of biosensors due to their adaptable nature. Here, the design of DNA-based hydrogel and its application as sensing platform for the detection of a specific target sequence are presented. DNA-functionalized hydrogel structures were formed via a free radical co-polymerization process. A simple one-step probe immobilization procedure is reported: DNA probe molecules are added to the photoactive polymer mixture, dispensed onto a solid support, or a mold, and covalently attached while the hydrogel is formed through UV light exposure. Such hydrogels can be synthesized with desired recognition ability through the selection of a certain nucleotide sequence. Here we show the application of DNA -based hydrogel to detect the target with high performance in fluorescence microarray format and, additionally, to fabricate holographic surface relief gratings for label-free sensing assays.This work was financially supported by the E.U. FEDER, the Spanish Ministry of Economy and Competitiveness MINECO (ADBIHOLPID2019-110713RB-I00) and Generalitat Valenciana (PROMETEO/2020/094). M. I. Lucio acknowledges MINECO for her Juan de la Cierva Formacion and Incorporacion grants (FJCI-2016-29593, IJC 2018035355-I). Also, P. Zezza acknowledges Generalitat Valenciana for her Grisolia fellowship grant. The authors acknowledge the assistance and advice of the Electron Microscopy Service of the Universitat Politecnica de Valencia.Zezza, P.; Lucío, MI.; Maquieira Catala, A.; Bañuls Polo, M. (2022). DNA -based hydrogels for high-performance optical biosensing application. Talanta. 244. https://doi.org/10.1016/j.talanta.2022.12342712342724

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

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

Spinal Cord Ischemia Prevention Through Hypogastric and Inferior Mesenteric Artery Preservation During Complex Endovascular Aortic Procedures

Objectives: Endovascular procedures have been widely accepted as safe treatment in patients with thoracoabdominal aortic aneurysms (TAAAs). Spinal cord ischemia (SCI) is the most dreaded complication. Its incidence is estimated in 3.9% of patients undergoing thoracoabomi- nal procedures. As reported in American and European guidelines, spinal fluid drainage, mean arterial pressure above 100 mm Hg, hemoglobin above 10 g/dL, transcutaneous oxygen measurement above 95%, staged procedures, and collateral networks preservation are recognized as possible preventive strategies. The aim of the study is to describe the re- sults of distal aortic collateral network preservation (internal iliac artery [IIA], inferior mesenteric artery [IMA]) with different endovascular tech- niques during elective or urgent endovascular procedures for thoracoab- dominal aortic diseases. Methods: Between December 2016 and January 2021, 50 complex aortic endovascular procedures were performed. The following inclusion criteria were identified: IIA occlusion or patent IMA with minimum diam- eter of 5 mm combined with compromised IIA patency. Nine patients matched the inclusion criteria. Staged procedures and spinal fluid drainage were employed in all elective settings (seven patients). Accord- ing to TAAA Crawford classification, 3 were type III, 2 post-dissection type II, 2 type IV, and 2 type V. Early and late mortality, aortic-related mortality, incidence of transient or permanent SCI, and target vessel patency were evaluated during follow-up. Follow-up was performed at 1, 6, and 12 months with computed tomography scan. IMA was preserved in one patient using an iliac branch proximally combined with a branched stent graft. In the second case, a parallel graft technique was employed, whereas in the third patient, a bell bottom graft was deployed above the IMA ostium. In five patients, at least one of the two IIA was recanal- ized using a parallel grafts technique, whereas in one patient, an iliac branch was implanted. Results: None of our patients experienced SCI. One of the two patients treated in the urgent setting died from cardiorespiratory complications 1 week after the procedure. Among the patients treated in the elective setting, no complications were observed. At follow-up, 100% target vessel patency was detected. Conclusions: According to our data, prevention of SCI through distal aortic collateral network preservation is safe and feasible in both the ur- gent and elective settings. This strategy may be an ulterior tool to pre- vent SC

Study of ‘Cona degli Ordini’ by Colantonio with IR and XRF Analyses

Two paintings by the Neapolitan Renaissance painter Colantonio were studied with two non-invasive techniques to enrich the technical–scientific documentation. Infrared reflectography (IR) and x-ray fluorescence (XRF) analyses were performed on Saint Jerome in the studio and Saint Francis delivering the Rule, paintings preserved in the Museo e Real Bosco di Capodimonte. The IR scanning was performed to look beyond the visible layers of the paint for the preparatory drawings and pentimenti, or changes made during the painting process. The XRF technique was applied in many points to determine the elemental composition and enable the identification of pigments and materials used in paint and in the preparatory layers. Elemental XRF mapping was also carried out on a region of particular interest. Results provide an initial overview and hypothesis of color palette and techniques used by the artist