Fluorescence Resonance Energy Transfer (FRET) Based Sensors for Bioanalysis

The objective of my PhD study was to develop and characterize new methods and sensors based on fluorescence resonance energy transfer (FRET) for bioanalysis. Chapter 3 describes the use of FRET between donor fluorophores and acceptor labeled murine macrophage cells. FRET microscopy was used to determine whether the donor molecules truly permeate through the cell membrane or only adsorb to the cell surface. This method was found to be partially successful since the donor red tail fluorescence overlapped with the sensitized acceptor fluorescence and led to false reading of FRET. We found that is easier to monitor delivery of acceptor molecules into donor-labeled cells. Using donor labeled cells it was possible to determine whether the acceptor molecules were actually delivered into cells. However, a relatively high acceptor concentration in the hundreds of micromolar level was needed to obtain measurable FRET signals in the 3-D cellular system. The results underscored the need to reduce the dimensionality of FRET systems in order to increase the FRET efficiency between donor and acceptor molecules. Chapter 4 describes the development of FRET sensing lipobeads labeled with donors and their use to evaluate the interactions of acceptor molecules with the phospholipid membrane of FRET sensing lipobeads. The change in the dimensionality of the system in which FRET occurs, improved the sensitivity of our measurements by 3-folds compared to FRET measurements in solution. We concluded that a molecular recognition component had to be added to the sensing particles to further increase their selectivity and sensitivity. Chapter 5 describes the development of FRET trap sensing beads and their use for screening nonfluorescent carbohydrates and glycoproteins. The FRET sensing technique was based on binding between dextran molecules labeled with Texas Red (Dextran-TR) and polystyrene microparticles labeled with Fluorescein tagged Concanavalin A (FITC-ConA). It was found that carbohydrates and glycoproteins inhibit the binding between dextran-TR and FITC-ConA labeled particles. The inhibition effect was concentration dependent thus enabled screening carbohydrates and glycoproteins based on their inhibition potency. The dissertation critically evaluates the performance of FRET microscopy and FRET based sensors in delivery and screening applications

Fluorescence Resonance Energy Transfer (FRET) Based Sensors for Bioanalysis

The objective of my PhD study was to develop and characterize new methods and sensors based on fluorescence resonance energy transfer (FRET) for bioanalysis. Chapter 3 describes the use of FRET between donor fluorophores and acceptor labeled murine macrophage cells. FRET microscopy was used to determine whether the donor molecules truly permeate through the cell membrane or only adsorb to the cell surface. This method was found to be partially successful since the donor red tail fluorescence overlapped with the sensitized acceptor fluorescence and led to false reading of FRET. We found that is easier to monitor delivery of acceptor molecules into donor-labeled cells. Using donor labeled cells it was possible to determine whether the acceptor molecules were actually delivered into cells. However, a relatively high acceptor concentration in the hundreds of micromolar level was needed to obtain measurable FRET signals in the 3-D cellular system. The results underscored the need to reduce the dimensionality of FRET systems in order to increase the FRET efficiency between donor and acceptor molecules. Chapter 4 describes the development of FRET sensing lipobeads labeled with donors and their use to evaluate the interactions of acceptor molecules with the phospholipid membrane of FRET sensing lipobeads. The change in the dimensionality of the system in which FRET occurs, improved the sensitivity of our measurements by 3-folds compared to FRET measurements in solution. We concluded that a molecular recognition component had to be added to the sensing particles to further increase their selectivity and sensitivity. Chapter 5 describes the development of FRET trap sensing beads and their use for screening nonfluorescent carbohydrates and glycoproteins. The FRET sensing technique was based on binding between dextran molecules labeled with Texas Red (Dextran-TR) and polystyrene microparticles labeled with Fluorescein tagged Concanavalin A (FITC-ConA). It was found that carbohydrates and glycoproteins inhibit the binding between dextran-TR and FITC-ConA labeled particles. The inhibition effect was concentration dependent thus enabled screening carbohydrates and glycoproteins based on their inhibition potency. The dissertation critically evaluates the performance of FRET microscopy and FRET based sensors in delivery and screening applications

Development of a versatile biotinylated material based on SU-8

[EN] The negative epoxy-based SU-8 photoresist has a wide variety of applications within the semiconductor industry, photonics and lab-on-a-chip devices, and it is emerging as an alternative to silicon-based devices for sensing purposes. In the present work, biotinylation of the SU-8 polymer surface promoted by light is reported. As a result, a novel, effective, and low-cost material, focusing on the immobilization of bioreceptors and consequent biosensing, is developed. This material allows the spatial discrimination depending on the irradiation of desired areas. The most salient feature is that the photobiotin may be directly incorporated into the SU-8 curing process, consequently reducing time and cost. The potential use of this substrate is demonstrated by the immunoanalytical detection of the synthetic steroid gestrinone, showing excellent performances. Moreover, the naked eye biodetection due to the transparent SU-8 substrate, and simple instrumental quantification are additional advantages.Financial support from Ministerio de Economia y Competitividad (MINECO TEC2012-31145) is acknowledged.Ortega Higueruelo, FJ.; Bañuls Polo, M.; Sanza, FJ.; Laguna, MF.; Holgado Bolaños, M.; Casquel Del Campo, R.; Angulo Barrios, C.... (2013). Development of a versatile biotinylated material based on SU-8. Journal of Materials Chemistry B. 1:2750-2756. https://doi.org/10.1039/c3tb20323aS275027561Liu, C. (2007). Recent Developments in Polymer MEMS. Advanced Materials, 19(22), 3783-3790. doi:10.1002/adma.200701709Bêche, B., Papet, P., Debarnot, D., Gaviot, E., Zyss, J., & Poncin-Epaillard, F. (2005). Fluorine plasma treatment on SU-8 polymer for integrated optics. Optics Communications, 246(1-3), 25-28. doi:10.1016/j.optcom.2004.10.081Chung, C. K., & Hong, Y. Z. (2006). Surface modification of SU8 photoresist for shrinkage improvement in a monolithic MEMS microstructure. Journal of Micromechanics and Microengineering, 17(2), 207-212. doi:10.1088/0960-1317/17/2/004Lorenz, H., Despont, M., Fahrni, N., LaBianca, N., Renaud, P., & Vettiger, P. (1997). SU-8: a low-cost negative resist for MEMS. Journal of Micromechanics and Microengineering, 7(3), 121-124. doi:10.1088/0960-1317/7/3/010Ribeiro, J. C., Minas, G., Turmezei, P., Wolffenbuttel, R. F., & Correia, J. H. (2005). A SU-8 fluidic microsystem for biological fluids analysis. Sensors and Actuators A: Physical, 123-124, 77-81. doi:10.1016/j.sna.2005.03.032Sikanen, T., Tuomikoski, S., Ketola, R. A., Kostiainen, R., Franssila, S., & Kotiaho, T. (2005). Characterization of SU-8 for electrokinetic microfluidic applications. Lab on a Chip, 5(8), 888. doi:10.1039/b503016aLee, J., Shin, H., Kim, S., Hong, S., Chung, J., Park, H., & Moon, J. (2003). Fabrication of Atomic Force Microscope Probe with Low Spring Constant Using SU-8 Photoresist. Japanese Journal of Applied Physics, 42(Part 2, No. 10A), L1171-L1174. doi:10.1143/jjap.42.l1171Genolet, G., Despont, M., Vettiger, P., Anselmetti, D., & de Rooij, N. F. (2000). All-photoplastic, soft cantilever cassette probe for scanning force microscopy. Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures, 18(2), 617. doi:10.1116/1.591248Koerner, T., Brown, L., Xie, R., & Oleschuk, R. D. (2005). Epoxy resins as stamps for hot embossing of microstructures and microfluidic channels. Sensors and Actuators B: Chemical, 107(2), 632-639. doi:10.1016/j.snb.2004.11.035Schneider, A., Su, B., Button, T. W., Singleton, L., Wilhelmi, O., Huq, S. E., … Lawes, R. A. (2002). Comparison of PMMA and SU-8 resist moulds for embossing of PZT to produce high-aspect-ratio microstructures using LIGA process. Microsystem Technologies, 8(2-3), 88-92. doi:10.1007/s00542-001-0141-yLinan Jiang, Gerhardt, K. P., Myer, B., Zohar, Y., & Pau, S. (2008). Evanescent-Wave Spectroscopy Using an SU-8 Waveguide for Rapid Quantitative Detection of Biomolecules. Journal of Microelectromechanical Systems, 17(6), 1495-1500. doi:10.1109/jmems.2008.2006814Chang-Yen, D. A., & Gale, B. K. (2003). An integrated optical oxygen sensor fabricated using rapid-prototyping techniques. Lab on a Chip, 3(4), 297. doi:10.1039/b305358jWang, Y., Bachman, M., Sims, C. E., Li, G. P., & Allbritton, N. L. (2006). Simple Photografting Method to Chemically Modify and Micropattern the Surface of SU-8 Photoresist. Langmuir, 22(6), 2719-2725. doi:10.1021/la053188eMarie, R., Schmid, S., Johansson, A., Ejsing, L., Nordström, M., Häfliger, D., … Dufva, M. (2006). Immobilisation of DNA to polymerised SU-8 photoresist. Biosensors and Bioelectronics, 21(7), 1327-1332. doi:10.1016/j.bios.2005.03.004Blagoi, G., Keller, S., Johansson, A., Boisen, A., & Dufva, M. (2008). Functionalization of SU-8 photoresist surfaces with IgG proteins. Applied Surface Science, 255(5), 2896-2902. doi:10.1016/j.apsusc.2008.08.089Joshi, M., Pinto, R., Rao, V. R., & Mukherji, S. (2007). Silanization and antibody immobilization on SU-8. Applied Surface Science, 253(6), 3127-3132. doi:10.1016/j.apsusc.2006.07.017Joshi, M., Kale, N., Lal, R., Ramgopal Rao, V., & Mukherji, S. (2007). A novel dry method for surface modification of SU-8 for immobilization of biomolecules in Bio-MEMS. Biosensors and Bioelectronics, 22(11), 2429-2435. doi:10.1016/j.bios.2006.08.045Deepu, A., Sai, V. V. R., & Mukherji, S. (2008). Simple surface modification techniques for immobilization of biomolecules on SU-8. Journal of Materials Science: Materials in Medicine, 20(S1), 25-28. doi:10.1007/s10856-008-3471-9Qvortrup, K., Taveras, K. M., Thastrup, O., & Nielsen, T. E. (2011). Chemical synthesis on SU-8. Chem. Commun., 47(4), 1309-1311. doi:10.1039/c0cc03876hCavalli, G., Banu, S., Ranasinghe, R. T., Broder, G. R., Martins, H. F. P., Neylon, C., … Roach, P. L. (2007). Multistep Synthesis on SU-8:  Combining Microfabrication and Solid-Phase Chemistry on a Single Material. Journal of Combinatorial Chemistry, 9(3), 462-472. doi:10.1021/cc060079pSethi, D., Kumar, A., Gandhi, R. P., Kumar, P., & Gupta, K. C. (2010). New Protocol for Oligonucleotide Microarray Fabrication using SU-8-Coated Glass Microslides. Bioconjugate Chemistry, 21(9), 1703-1708. doi:10.1021/bc100262nBroder, G. R., Ranasinghe, R. T., She, J. K., Banu, S., Birtwell, S. W., Cavalli, G., … Morgan, H. (2008). Diffractive Micro Bar Codes for Encoding of Biomolecules in Multiplexed Assays. Analytical Chemistry, 80(6), 1902-1909. doi:10.1021/ac7018574Birtwell, S. W., Broder, G. R., Roach, P. L., & Morgan, H. (2012). Multiplexed suspension array platform for high-throughput protein assays. Biomedical Microdevices, 14(4), 651-657. doi:10.1007/s10544-012-9641-zKim, H.-N., Kang, J.-H., Jin, W.-M., & Moon, J. H. (2011). Surface modification of 2D/3D SU-8 patterns with a swelling–deswelling method. Soft Matter, 7(6), 2989. doi:10.1039/c0sm01006eShew, B. Y., Cheng, Y. C., & Tsai, Y. H. (2008). Monolithic SU-8 micro-interferometer for biochemical detections. Sensors and Actuators A: Physical, 141(2), 299-306. doi:10.1016/j.sna.2007.08.029Holgado, M., Barrios, C. A., Ortega, F. J., Sanza, F. J., Casquel, R., Laguna, M. F., … Maquieira, A. (2010). Label-free biosensing by means of periodic lattices of high aspect ratio SU-8 nano-pillars. Biosensors and Bioelectronics, 25(12), 2553-2558. doi:10.1016/j.bios.2010.04.042Sanza, F. J., Holgado, M., Ortega, F. J., Casquel, R., López-Romero, D., Bañuls, M. J., … Maquieira, A. (2011). Bio-Photonic Sensing Cells over transparent substrates for anti-gestrinone antibodies biosensing. Biosensors and Bioelectronics, 26(12), 4842-4847. doi:10.1016/j.bios.2011.06.010Sanza, F. J., Laguna, M. F., Casquel, R., Holgado, M., Barrios, C. A., Ortega, F. J., … Puchades, R. (2011). Cost-effective SU-8 micro-structures by DUV excimer laser lithography for label-free biosensing. Applied Surface Science, 257(12), 5403-5407. doi:10.1016/j.apsusc.2010.10.010Ortega, F. J., Bañuls, M.-J., Sanza, F. J., Casquel, R., Laguna, M. F., Holgado, M., … Puchades, R. (2012). Biomolecular Interaction Analysis of Gestrinone-anti-Gestrinone Using Arrays of High Aspect Ratio SU-8 Nanopillars. Biosensors, 2(3), 291-304. doi:10.3390/bios2030291A. Fleming, S. (1995). Chemical reagents in photoaffinity labeling. Tetrahedron, 51(46), 12479-12520. doi:10.1016/0040-4020(95)00598-3Blagoi, G., Keller, S., Persson, F., Boisen, A., & Jakobsen, M. H. (2008). Photochemical Modification and Patterning of SU-8 Using Anthraquinone Photolinkers. Langmuir, 24(18), 9929-9932. doi:10.1021/la800948wWilchek, M., & Bayer, E. A. (1988). The avidin-biotin complex in bioanalytical applications. Analytical Biochemistry, 171(1), 1-32. doi:10.1016/0003-2697(88)90120-0Dontha, N., Nowall, W. B., & Kuhr, W. G. (1997). Generation of Biotin/Avidin/Enzyme Nanostructures with Maskless Photolithography. Analytical Chemistry, 69(14), 2619-2625. doi:10.1021/ac9702094Wilde, L. M., Farace, G., Roberts, C. J., Davies, M. C., Sanders, G. H. W., Tendler, S. J. B., & Williams, P. M. (2001). Molecular patterning on carbon based surfaces through photobiotin activation. The Analyst, 126(2), 195-198. doi:10.1039/b008475lChoi, H. J., Kim, N. H., Chung, B. H., & Seong, G. H. (2005). Micropatterning of biomolecules on glass surfaces modified with various functional groups using photoactivatable biotin. Analytical Biochemistry, 347(1), 60-66. doi:10.1016/j.ab.2005.08.015Brun, E. M., Hernández-Albors, A., Ventura, R., Puchades, R., & Maquieira, Á. (2010). Enzyme-linked immunosorbent assays for the synthetic steroid gestrinone. Talanta, 82(4), 1581-1587. doi:10.1016/j.talanta.2010.07.067Benlarbi, M., Blum, L. J., & Marquette, C. A. (2012). SU-8-carbon composite as conductive photoresist for biochip applications. Biosensors and Bioelectronics, 38(1), 220-225. doi:10.1016/j.bios.2012.05.026Aung, K. M. M., Ho, X., & Su, X. (2008). DNA assembly on streptavidin modified surface: A study using quartz crystal microbalance with dissipation or resistance measurements. Sensors and Actuators B: Chemical, 131(2), 371-378. doi:10.1016/j.snb.2007.11.058Malainou, A., Petrou, P. S., Kakabakos, S. E., Gogolides, E., & Tserepi, A. (2012). Creating highly dense and uniform protein and DNA microarrays through photolithography and plasma modification of glass substrates. Biosensors and Bioelectronics, 34(1), 273-281. doi:10.1016/j.bios.2012.02.02

Application of equilibrium binding model for analysis of conformational transitions in poly(rA)poly(rU) complexes with metal ions

Aim. The study is aimed at generalization of the previous experimental results on the metal ion (Mg²⁺, Ni²⁺, Cd²⁺) effects on conformation transitions in poly(rA)poly(rU). The objective was to find out how the type of a metal ion-polynucleotide complex influences the phase transitions and to estimate the constants (K) of ions binding to polymers of different structures. Methods. The K values were obtained upon theoretical and experimental transition temperature fitting by the least-square method with the root mean square deviation minimized through the procedure of the gradient descent in the multidimensional space. Results. Calculations of diagrams with Mg²⁺ are shown to permit obtaining satisfactory results if concentration-independent, mean values of constants are used. For Ni²⁺ and Cd²⁺ the concentration dependence of K must be taken into account, especially for high ion contents at which compaction of single-stranded poly(rA) emerges. It was revealed that the main factor responsible for the differences in diagrams with Ni²⁺ and Cd²⁺ is a significant distinction of their constants of binding to poly(rA) and poly(rU). Conclusions. The model theory of equilibrium binding is capable to describe adequately conformation transitions in polynucleotides in the presence of metal ions.Роботу присвячено узагальненню отриманих раніше експериментальних результатів щодо впливу іонів металів (Mg²⁺, Ni²⁺, Cd²⁺) на конформаційні переходи в poly(rA)poly(rU). Мета роботи полягала у з’ясуванні впливу типу комплексу іона металу з полінуклеотидом на вид фазового переходу і визначенні констант їхнього зв’язування (К) з полімерами різної структури. Методи. Величини К визначали підгонкою теоретично розрахованих значень температур переходів до експериментальних за методом найменших квадратів з мінімізацією середньоквадратичного відхилення методом градієнтного спуску в багатовимірному просторі. Результати. Розрахунки фазових діаграм за присутності іонів Mg²⁺ показали можливість одержання задовільних результатів при використанні постійних, незалежних від концентрації середніх значень констант. У разі Ni²⁺ і Cd²⁺ такі залежності мають бути враховані, особливо в межах великих концентрацій іонів, де відбувається компактизація однонитчастої poly(rA). Встановлено, що відмінність діаграм з Ni²⁺ і Cd²⁺ обумовлена значною різницею у їхніх константах зв’язування з poly(rA) та poly(rU). Висновки. Отримані результати свідчать про можливість успішного опису конформаційних переходів у полінуклеотидах за присутності іонів металів при використанні модельної теорії рівноважного зв’язування.Работа посвящена обобщению полученных ранее экспериментальных результатов по влиянию ионов металлов (Mg²⁺, Ni²⁺, Cd²⁺) на конформационные переходы в poly(rA)poly(rU). Цель работы – выяснить, как тип комплекса иона металла с полинуклеотидом влияет на вид фазового перехода, и определить константы связывания (К) ионов с полимерами различной структуры. Методы. Значения К определяли подгонкой теоретически вычисленных значений температур переходов к экспериментальным по методу наименьших квадратов с минимизацией среднеквадратичного отклонения методом градиентного спуска в многомерном пространстве. Результаты. Расчеты фазовых диаграмм в присутствии Mg²⁺ показали возможность получения удовлетворительных результатов при использовании постоянных, не зависящих от концентрации средних значений констант. В случае Ni²⁺ и Cd²⁺ эту зависимость нужно учитывать, особенно в области больших концентраций ионов, где происходит компактизация однонитчатой poly(rA). Установлено, что отличие в диаграммах с Ni²⁺ и Cd²⁺обусловлено существенным различием их констант связывания с poly(rA) и poly(rU). Выводы. Полученные результаты свидетельствуют о возможности успешного описания конформационных переходов в полинуклеотидах в присутствии ионов металлов с помощью модельной теории равновесного связывания

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

Ultra-Sensitivity Glucose Sensor Based on Field Emitters

A new glucose sensor based on field emitter of ZnO nanorod arrays (ZNA) was fabricated. This new type of ZNA field emitter-based sensor shows high sensitivity with experimental limit of detection of 1 nM glucose solution and a detection range from 1 nM to 50 μM in air at room temperature, which is lower than that of glucose sensors based on surface plasmon resonance spectroscopy, fluorescence signal transmission, and electrochemical signal transduction. The new glucose sensor provides a key technique for promising consuming application in biological system for detecting low levels of glucose on single cells or bacterial cultures