A cellulose-based bioassay for the colorimetric detection of pathogen DNA

Cellulose-paper-based colorimetric bioassays may be used at the point of sampling without sophisticated equipment. This study reports the development of a colorimetric bioassay based on cellulose that can detect pathogen DNA. The detection was based on covalently attached single-stranded DNA probes and visual analysis. A cellulose surface functionalized with tosyl groups was prepared by the N,N-dimethylacetamide-lithium chloride method. Tosylation of cellulose was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and elemental analysis. Sulfhydryl-modified oligonucleotide probes complementary to a segment of the DNA sequence IS6110 of Mycobacterium tuberculosis were covalently immobilized on the tosylated cellulose. On hybridization of biotin-labelled DNA oligonucleotides with these probes, a colorimetric signal was obtained with streptavidin-conjugated horseradish peroxidase catalysing the oxidation of tetramethylbenzamidine by H2O2. The colour intensity was significantly reduced when the bioassay was subjected to DNA oligonucleotide of randomized base composition. Initial experiments have shown a sensitivity of 0.1 μM. A high probe immobilization efficiency (more than 90 %) was observed with a detection limit of 0.1 μM, corresponding to an absolute amount of 10 pmol. The detection of M. tuberculosis DNA was demonstrated using this technique coupled with PCR for biotinylation of the DNA. This work shows the potential use of tosylated cellulose as the basis for point-of-sampling bioassays.Peer reviewedFinal Accepted Versio

Fabrication of DNA microarrays on Poly (methylmethacrylate) substrates for biomolecular reporting

DNA microarrays require the use of substrates with well-established surface modification / probe attachment chemistries. Glass/quartz have been widely adopted as typical support materials since their surface modification chemistries which involve the use of siloxane –based chemistries have been widely studied however; these chemistry is susceptible to hydrolytic cleavage especially at high or low pH values. Recently, polymers have been sought as alternative microarray support materials but their surface modification strategies are not well characterized compared to glass. This report will entail surface photo-modification of PMMA polymer substrates by UV irradiation which produces functional scaffolds of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes onto these surfaces via carbodiimide coupling chemistries. The photo-modification process for microarray fabrication involves only three steps; (1) broadband UV exposure of the polymer surface; (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. Since microfluidics offer several advantages such as reduction in reagent cost, reduction in hybridization assay times and parallel processing of samples; we incorporate them in the microarray construction by using poly (dimethylsiloxane) microchannels that are reversibly sealed to the photoactivated PMMA substrates. Parallel sample processing minimizes contamination effects that can give rise to false positives which can be a significant issue especially for diagnostic applications. We demonstrate use of these protocols with linear oligonucleotide probes for screening multiple KRAS 2 mutations possessing high diagnostic value for colorectal cancers whereby a Ligase Detection Reaction/universal zipcode array assays was carried out using parallel detection of two different low abundant DNA point mutations in KRAS 2 oncogenes with allelic composition evaluated at one locus. The same covalent attachment protocols were utilized for immobilizing hairpin probes (molecular beacons) in a microarray format that were used to report on the analysis of complementary DNA (cDNA) specific for fruitless (fru) and ods-site homeobox (OdsH) genes extracted from Drosophila Melanogaster fruit flies. To further improve the analytical sensitivities exhibited by these hairpin probes; we used phthalocyanine dyes for dual labeling of oligonucleotide probes that will be used for reporting on biomolecular association events

Solid‐Phase Supports for Oligonucleotide Synthesis

This unit begins with a discussion of the advantages and disadvantages of oligonucleotide synthesis using solid supports. The physical and chemical properties of solid‐phase supports are discussed in terms of their suitability for oligonucleotide synthesis. In addition, the unit outlines the properties of linkers used for transient or permanent attachment of properly protected nucleosides to the derivatized support, as well as strategies for coupling nucleosides to linkers and conditions for the release of synthetic oligonucleotides from specific supports.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143613/1/cpnc0301.pd

Thiol-click photochemistry for surface functionalization applied to optical biosensing

[EN] In the field of biosensing, suitable procedures for efficient probes immobilization are of outmost importance. Here we present different light-based strategies to promote the covalent attachment of thiolated capture probes (oligonucleotides and proteins) on different materials and working formats. One strategy employs epoxylated surfaces and uses the light to accomplish the ring opening by a thiol moiety present in a probe. However, most of this work lies on the use of thiol-ene photocoupling chemistry to covalently attach probes to the supports. And thus, both alkenyl and thiol derivatized surfaces are assayed to immobilize thiol or alkene ended probes, respectively, and their performances are compared. Also, the effect of the number of thiols carried by the probe is analyzed comparing single-point and multi-point attachment. The performance of the analogous tethering, but onto alkynylated surfaces is also carried out, and the sensing response is related to the surfaces hydrophobicity. A newly developed reaction is also discussed where a fluorinated surface undergoes the covalent immobilization of thiolated probes activated by light, creating small hydrophilic areas where the probes are attached, and leaving the rest of the surface highly hydrophobic and repellent against protein unspecific adsorption. These mixed surfaces confine the sample (aqueous) uniquely on the hydrophilic spots lowering the background signal and thus increasing the sensitivity. These probe immobilization approaches are applied to fluorescence microarray and label-free nanophotonic biosensing. All the exposed reactions have in common the photoactivation of the thiol moieties, and give rise to quick, clean, versatile, orthogonal and biocompatible reactions. Water is the only solvent used, and light the only catalyzer applied. Thus, all of them can be considered as having the attributes of click-chemistry reactions. For these reasons we named them as thiol-click photochemistry, being a very interesting pool of possibilities when building a biosensor.This work was supported by the European Union program Horizon 2020, projects H2020-PHC-634013 and H2020-ICT-644242, and by the Spanish Ministry of Economy and Competitiveness, project CTQ2016-75749-R. Authors thank the whole "Signal and Measurement" research group, from the IDM, UPV, for sharing space, research and life. Special thanks to Pilar Jimenez-Meneses, Rafael Alonso, Daniel Gonzalez-Lucas, Pilar Aragon and Patricia Noguera for their contribution to the development of thiol photoattaching chemistry and surface wettability modulation.Bañuls Polo, M.; González Martínez, MÁ.; Sabek, J.; García-Rupérez, J.; Maquieira Catala, Á. (2019). Thiol-click photochemistry for surface functionalization applied to optical biosensing. Analytica Chimica Acta. 1060:103-113. https://doi.org/10.1016/j.aca.2019.01.055S103113106

Study of substrate modulation and bioreceptor anchoring for the development of high performance microarrays

Tesis por compendio[EN] The present PhD thesis is focused on the study of new approaches able to improve the performance of microarrays. Aspects such as the nature of the surfaces and the probes, functionalization of the substrates, probe printing, immobilization and target detection were considered in the fabrication process. Within all these features, modulation of the surface behavior and probe anchoring were the most challenging aspects, as the interface is key for the immobilization of the receptors and the later detection, which will determine the performance of the final device. In this work, two microarray types have been developed, one for oligonucleotides and another one for antibodies. Then, a characterization of the reached achievements is done. All the routes have in common the use of light to catalyze the attachment of bioreceptors on the surface substrates, employing click-chemistry reactions. In the first chapter, the state of the art of microarray technology is overviewed, with special focus in the main aspects of microarray design. In the second chapter, the goals for this PhD thesis are settled. These general objectives are addressed in the following experimental chapters. In the third chapter, the effect of hydrophobicity and probe multi-point attachment on the microarray performance are studied. Thus, modulation of glass slide surfaces with alkenyl and alkynyl motifs for the anchoring of mono and multithiolated oligonucleotide probes by thiol-ene and thiol-yne photocoupling reactions, respectively, was accomplished. Surfaces modified with the most hydrophobic silane (alkynyl), or anchoring polythiolated probes, revealed better performances. These microarray systems were applied to the discrimination of SNPs and to detect bacterial genome PCR products. In the fourth chapter, a rational design for the preparation of microarrays of antibodies, is done. The immobilization approach displays the oriented anchoring of thiol-bearing antibody fragments to alkenylated glass slides by thiol-ene photocoupling reaction. Multiplexed detection of cardiac biomarkers is demonstrated. The designed microarray shows higher recognition capacity in comparison to whole antibody microarrays. In the fifth chapter, improvement of a novel methodology for the anchoring of thiolated oligonucleotides has been developed. Due to the interest on modifying highly hydrophobic surfaces, a new photoinduced reaction is set up. Thanks to the features of the named "fluor-thiol photocoupling reaction", immobilization of thiolated probes to surfaces containing C-F bonds in a fast, easy and biocompatible with aqueous media way, was achieved. Hydrophobicity of the surfaces was controlled to get successful hybridizations. Because of the high hydrophobicity of the surfaces, a huge confinement of the probes is accomplished, which allows the approximation of the analytes only where the probe is linked, keeping a high repulsion in the remaining surface. The perfluorinated glass slides improved the immobilization densities and detection capacity, regarding to the alkenylated and alkynylated surfaces, and allowed the discrimination of SNPs and detection of bacterial PCR products, as well. In the sixth chapter, other surfaces different than glass are explored. Thus, polyvinylidene fluoride membranes were employed as substrates for the development of oligonucleotide microarrays. Therefore, a fast, easy and mild functionalization process by UV irradiation and organosilane chemistry, was developed. Then, alkenyl functionalized and non-functionalized membranes were applied to microarray technology by covalent anchoring through thiol-ene and fluor-thiol photocoupling reactions, respectively. Promising results were obtained with both surfaces.[ES] La presente tesis tesis doctoral se centra en el estudio de nuevas aproximaciones capaces de mejorar el rendimiento de los microarrays. Aspectos como la naturaleza de las superficies y las sondas, la funcionalización de los sustratos, la impresión, la inmovilización y la detección de las sondas se consideraron en el proceso de fabricación. Dentro de todas estas características, la modulación de la superficie y el anclaje de la sonda fueron los aspectos más desafiantes, ya que la interfaz es clave para la inmovilización de los receptores y la posterior detección, lo que determinará el rendimiento del dispositivo final. En este trabajo, se han desarrollado dos tipos de microarrays, uno para oligonucleótidos y otro para anticuerpos. Luego, se ha realizado una caracterización de los logros alcanzados. Todas las rutas tienen en común el uso de la luz para catalizar la unión de los biorreceptores en los sustratos de la superficie, empleando reacciones de la química clic. En el primer capítulo, se facilita una visión general del estado del arte de la tecnología de microarrays con un enfoque especial en los aspectos principales del diseño de microarrays. En el segundo capítulo, se establecen los objetivos de esta tesis doctoral. Estos objetivos generales se abordan en los siguientes capítulos experimentales. En el tercer capítulo, se estudia el efecto de la hidrofobia y el uso de sondas con múltiples puntos de unión, en el rendimiento del microarray. De este modo, se llevó a cabo la modulación de superficies vidrio con grupos alquenilo y alquinilo para el anclaje de sondas de oligonucleótidos mono y multitioladas mediante las reacciones de foto anclaje del tiol-eno y tiol-ino, respectivamente. Las superficies modificadas con el silano más hidrofóbico (alquinilo) y las sondas politioladas ancladas, revelaron mejores rendimientos. Estos sistemas de microarrays se aplicaron a la discriminación de SNPs y a la detección de productos de PCR de bacterias. En el cuarto capítulo, se realiza un diseño racional para la preparación de microarrays de anticuerpos. El enfoque de inmovilización muestra el anclaje orientado de los fragmentos de anticuerpos que contienen tiol sobre superficies de vidrio alqueniladas mediante reacción de foto anclaje del tiol-eno. De esta forma, se demuestra la detección multiplexada de biomarcadores cardíacos. El microarray diseñado muestra una mayor capacidad de reconocimiento en comparación con los microarrays de anticuerpos completos. En el quinto capítulo, se ha desarrollado una nueva metodología para mejorar el anclaje de oligonucleótidos tiolados. Dado el interés en modificar superficies altamente hidrófobas, se establece una nueva reacción fotoinducida. Gracias a las características de la llamada "reacción de fotoacoplamiento de fluor-tiol", se logró la inmovilización de sondas tioladas a superficies que contienen enlaces C-F de una manera rápida, fácil y biocompatible con medios acuosos. La hidrofobicidad de las superficies se controló para obtener hibridaciones exitosas. Debido a la alta hidrofobicidad de las superficies, se logra un gran confinamiento de las sondas, lo que permite la aproximación de los analitos solo donde está unida la sonda, manteniendo una alta repulsión en la superficie restante. Las superficies de vidrio perfluoradas mejoraron las densidades de inmovilización y la capacidad de detección, con respecto a las superficies alqueniladas y alquiniladas, y también, permitieron la discriminación de SNPs y la detección de productos de PCR bacterianos. En el sexto capítulo, se exploran otras superficies diferentes al vidrio. Por lo tanto, membranas de fluoruro de polivinilideno se emplearon como sustratos para el desarrollo de microarrays de oligonucleótidos. Para ello, se desarrolló un proceso de funcionalización rápido, fácil y suave, mediante el empleo de irradiación UV y la química de los organosilanos.[CA] La present tesi doctoral es centra en l'estudi de noves aproximacions capaces de millorar el rendiment dels microarrays. Aspectes com ara la naturalesa de les superfícies i les sondes, la funcionalització dels substrats, la impressió, la immobilització i la detecció de les sondes es van considerar en el procés de fabricació. Dins de totes aquestes característiques, la modulació de la superfície i l'ancoratge de la sonda van ser els aspectes més desafiadors, ja que la interfície és clau per a la immobilització dels receptors i la posterior detecció, la qual cosa determinarà el rendiment del dispositiu final. En aquest treball, s'han desenvolupat dos tipus de microarrays, un per a oligonucleòtids i un altre per a anticossos. Després, s'ha realitzat una caracterització dels resultats aconseguits. Totes les rutes tenen en comú l'ús de la llum per a catalitzar la unió dels biorreceptores en els substrats de la superfície, emprant reaccions de la química clic. En el primer capítol, es facilita una visió general de l'estat de l'art de la tecnologia de microarrays amb un enfocament especial en els aspectes principals del disseny de microarrays. En el segon capítol, s'estableixen els objectius d'aquesta tesi doctoral. Aquests objectius generals s'aborden en els següents capítols experimentals. En el tercer capítol, s'estudia l'efecte de la hidrofòbia i l'ús de sondes amb múltiples punts d'unió, en el rendiment del microarray. D'aquesta manera, es va dur a terme la modulació de superfícies de vidre amb grups alquenil i alquinil per a l'ancoratge de sondes de oligonucleòtids mono i multitiolades mitjançant les reaccions de foto ancoratge del tiol-doble enllaç i tiol-triple enllaç, respectivament. Les superfícies modificades amb el silà més hidrofòbic (alquinil) i les sondes politiolades ancorades, van revelar els millors rendiments. Aquests sistemes de microarrays es van aplicar a la discriminació de SNPs i a la detecció de productes de PCR de bacteris. En el quart capítol, es realitza un disseny racional per a la preparació de microarrays d'anticossos. L'enfocament d'immobilització mostra l'ancoratge orientat dels fragments d'anticossos que contenen el grup tiol sobre superfícies de vidre alquenilades mitjançant reacció de foto ancoratge del tiol-doble enllaç. D'aquesta forma, es demostra la detecció multiplexada de biomarcadors cardíacs. El microarray dissenyat mostra una major capacitat de reconeixement en comparació amb els microarrays d'anticossos complets. En el cinqué capítol, s'ha desenvolupat una nova metodologia per a millorar l'ancoratge de oligonucleòtids tiolats. Donat l'interés de modificar superfícies altament hidròfobes, s'estableix una nova reacció fotoinduïda. Gràcies a les característiques de l'anomenada "reacció de fotoacoplament de fluor-tiol", es va aconseguir la immobilització de sondes tioladas a superfícies que contenen enllaços C-F d'una manera ràpida, fàcil i biocompatible amb medis aquosos. La hidrofobicitat de les superfícies es va controlar per a obtindre bones hibridacions reeixides. A causa de l'alta hidrofobicidad de les superfícies, s'aconsegueix un gran confinament de les sondes, la qual cosa permet l'aproximació dels anàlits únicament on està unida la sonda i manté una alta repulsió en la superfície restant. Les superfícies de vidre perfluorades van millorar les densitats d'immobilització i la capacitat de detecció, respecte a les superfícies alquenilades i alquinilades, i també van permetre la discriminació de SNPs i la detecció de productes de PCR bacterians. En el sisé capítol, s'exploren altres superfícies diferents al vidre. Per tant, membranes de fluorur de polivinilidé es van emprar com a substrats per al desenvolupament de microarrays d'oligonucleòtids. Per a això, es va desenvolupar un procés de funcionalització ràpid, fàcil i suau, mitjançant l'ús d'irradiació UV i la química dels organosilanAgradecer al Ministerio de Economía y Competitividad de España, por su programa de becas doctorales FPIJiménez Meneses, P. (2020). Study of substrate modulation and bioreceptor anchoring for the development of high performance microarrays [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137993TESISCompendi

Photoattachment of thiolated DNA probes on SU-8 spin-coated Blu-ray disk surfaces for biosensing

[EN] A methodology to modify Blu-ray disk (BD) surfaces by spin-coating an SU-8 epoxy photoresist is evaluated, to create a hybrid material with new functionalities. The optical performance of the new film surface disk is analyzed. The light-mediated activation of epoxy-to-thiol chemistry is applied to the site-specific and covalent binding of thiol-ended oligonucleotide probes to the SU-8 disk surface. For the optimized hybridization conditions, a limit of detection of 1 pM is reached employing enzymatic development. PCR products, from a Salmonella typhimurium serotype, are detected at an amplicon concentration of 20 pM. The reading of the assays is performed using a commercial BD-player.This work has been funded by the projects FEDER CTQ2010-15943 (CICYT, Spain), PROMETEO 2010/008 (Generalitat Valenciana) and an interdisciplinary UPV PAID 05 - 10 grant.Peris Chanzá, EJ.; Bañuls Polo, M.; Puchades, R.; Maquieira Catala, Á. (2013). Photoattachment of thiolated DNA probes on SU-8 spin-coated Blu-ray disk surfaces for biosensing. Journal of Materials Chemistry B. 1(45):6245-6253. https://doi.org/10.1039/c3tb21026jS6245625314

Attachment of oligonucleotide probes to polymer biochips and its application for the detection of point mutations

This dissertation is on the fabrication of polymer-based microfluidic arrays for the detection of genetic mutations. Poly(methyl methacrylate) was chosen as one of the polymer substrate materials due to its low background noise, low adsorption of biomolecules, and low assembly temperature. The surface modification of polymer substrates for covalent attachment of oligonucleotide probes, the construction of fluidic channels/arrays, and hybridization kinetics will be covered. As an example of the application, point mutation detection using immobilized arrays constructed in microfluidic devices will be demonstrated. The PMMA surface was derivatized with N-lithioethylenediamine solution to introduce amine groups, which were utilized for the covalent immobilization of terminal amino modified oligonucleotide probes via a homo-bi-functional linker molecule. The coupling bonds formed were stable enough to withstand multiple denaturation/rehybridization cycles. To overcome the drawbacks associated with conventional 2-D flat microarrays, such as long hybridization times and large sample consumption, oligonucleotide arrays were constructed into the microfluidic channels hot embossed into PMMA substrate. With the use of these fluidic channels we observed increased hybridization kinetics as compared to that on the flat arrays. Another benefit is that the channel-attached oligonucleotide probes allow the detection of target concentrations down to pM levels. As such, the specially designed oligonucleotide probes, which have similar melting temperatures, were constructed in microfluidic channels. Low-abundance point mutations in K-ras genes were successfully detected by using a ligase detection reaction (LDR) combined with the microfluidic hybridization. Near-IR laser induced fluorescence technique was used for the detection of surface conducted bioanalytical reactions and high detection sensitivity was obtained. In addition, preliminary work was also conducted on direct photo-patterning of deep ultraviolet (UV) light for immobilizing oligonucleotides on poly(methyl methacrylate) and polycarbonate substrates. Deep UV patterning using a through-hole mask indicated that more oligonucleotide molecules were immobilized on the UV-exposed areas than the non-exposed area in the presence of EDC conjugating reagent. However, the proper dose of UV-exposure and the appropriate EDC concentration need to be optimized in future work to increase the contrast on the immobilization efficiency between the exposed regions and un-exposed regions

Improved Performance of DNA Microarray Multiplex Hybridization Using Probes Anchored at Several Points by Thiol-Ene or Thiol-Yne Coupling Chemistry

[EN] Nucleic acid microarray-based assay technology has shown lacks in reproducibility, reliability, and analytical sensitivity. Here, a new strategy of probe attachment modes for silicon-based materials is built up. Thus, hybridization ability is enhanced by combining thiol-ene or thiol-yne click chemistry reactions with a multipoint attachment of polythiolated probes. The viability and performance of this approach was demonstrated by specifically determining Salmonella PCR products up to a 20 pM sensitivity level.The authors thank Dr. Elena Pinilla for her helpful discussion about AFM results. This work was funded by EU’s program Horizon 2020 ICT-26-2014-644242, Spanish Ministry MINECO CTQ/2013/45875-R FEDER, and local administration GVA PROMETEO II 2014/40. The authors acknowledge Luis Tortajada-Genaro and Regina Niñoles Rodenes for kindly providing the Salmonella and Campylobacter PCR products. F.M. is member of Inserm.Bañuls Polo, M.; Jimenez-Meneses, P.; Meyer, A.; Vasseur, J.; Morvan, F.; Escorihuela Fuentes, J.; Puchades, R.... (2017). Improved Performance of DNA Microarray Multiplex Hybridization Using Probes Anchored at Several Points by Thiol-Ene or Thiol-Yne Coupling Chemistry. Bioconjugate Chemistry. 28(2):496-506. https://doi.org/10.1021/acs.bioconjchem.6b00624S49650628