Mini-Cases of Professional-Inspired Activities in E-Learning Platforms: An Experience for the Formative Assessment

[EN] The formative assessment is a strategy that allows students to be aware of the state of their learning and to establish ways to correct the identified deficiencies; at the same time, it provides information to the instructor about those issues where misunderstandings occur. In the present study, we study its application to the solving of problems in university subject of scientific field. The activity involved short extension cases, coming from professional situations combined to self-learning tasks and supported on e-learning platform (Sakai software). The methodology was designed to achieve deep learning and a continuous evaluation of the process, not only the final product. The evidences collected (student's documents, interviews and smartphone-based surveys) informed about the impact of the presented approach. The participants positively valued the initiative against the more traditional methodologies. In conclusion, the study demonstrates that students can actively participate in the learning-oriented assessment, maintaining the guarantees of the educational process, mainly suitable for large classes, limited schedule or on-line classes.Tortajada-Genaro, LA. (2022). Mini-Cases of Professional-Inspired Activities in E-Learning Platforms: An Experience for the Formative Assessment. Multidisciplinary Journal of Educational Research. 12(1):38-59. https://doi.org/10.447/remie.6070S385912

Collaborative activities for solving multi-step problems in general chemistry

[EN] The learning of solving mult-step problems is a relevant aim in chemical educaton for engineering students. In these questons, afer analyzing inital data, a complex reasoning and an elaborated mathematcal procedure is needed to achieve the correct numerical answer. However, many students are able to efectvely use algorithms even with a lack of meaningful understanding of involved chemical concepts. This paper reports the applicaton of some collaboratve actons in order to induce a complete acquisiton of problem solving skills. The studied approaches, performed inside and outside the classroom, are classifed in low-collaboratve and highcollaboratve actvites, depending on the relatve partcipaton of instructor/students in their development. The critcal descripton of the proposed methodology and the produced outcomes are exposed. The contributon of each major reasoning mode (model, rule or case based) employed in these actvites is analyzed. Also, the percepton of students is evaluated on the basis on the data provided by direct observaton and a specifc survey with Likert-scale and open-ended questons. The results indicate that the changes of teaching to a more conceptual orientaton lead a deeper understanding, minimizing misconceptons or a rote learning.Tortajada-Genaro, LA. (2014). Collaborative activities for solving multi-step problems in general chemistry. Journal of Technology and Science Education. 4(4):250-259. doi:10.3926/jotse.119S2502594

Multiple recombinase polymerase amplification and low-cost array technology for the screening of genetically modified organisms

[EN] The identification of different transgenic products that are potentially present in foods is a priority given their impact on environmental safeness and health care. In this context, reliable, fast and inexpensive detection methods are demanded to screen the compliance of product labelling and traceability regulations. We herein developed a method that combines recombinase polymerase amplification (RPA) in a multiple format and a hybridisation assay. This system was optimised for the simultaneous amplification/detection of the 35S promoter, the NOS terminator and taxa, soya (Glycine max), corn (Zea mays) and potato (Solanum tuberosum), to denote the transgenic ingredients present in samples and to help to identify their source. As proof-of-concept, compact disc technology automated the optical sensing of RPA products. Discs worked as an analytical platform in the microarray format, and the reader/recorder as a detector. The analysis of the food mixtures containing genetically modified organisms up to 0.2 % showed excellent selectivity (no false-positives), reproducibility (relative error <20 %) and sensitivity (0.04 ng). The isothermal method was validated using certified reference materials and successfully compared to PCR-ELISA. The results of food products also confirmed it as an effective high-throughput tool for supporting simple, low-cost food safety controls, which makes it ideal for laboratories with limited resources.MINECO Project PID2019-110713RB-I00 and GVAPROMETEO/2020/094.Tortajada-Genaro, LA.; Maquieira Catala, Á. (2021). Multiple recombinase polymerase amplification and low-cost array technology for the screening of genetically modified organisms. Journal of Food Composition and Analysis. 103:1-7. https://doi.org/10.1016/j.jfca.2021.104083S1710

Secondary organic aerosol formation from the photo-oxidation of benzene

The production of condensate compounds from the degradation of benzene by OH radical chemistry was studied. Secondary organic aerosol (SOA) formation was investigated in the EUPHORE (European Photoreactor) simulation chambers. Experiments were performed under different OH-production conditions - addition of H 2O 2, NO or HONO -, in a high-volume reactor, with natural light and in the absence of seed aerosols. The consumption of precursor/reagents, the formation of gas-phase and particulate-phase products and the temporal evolution of aerosol were monitored. Several aerosol physical properties - mass concentration, overall aerosol yield, particle size distribution and density - were determined and found to be clearly dependent on OH radical production and NO x concentrations. Furthermore, the use of one and/or two products gas-particle partitioning absorption models allowed us to determine the aerosol yield curves. The SOA yield ranged from 1.6 to 9.7 %, with higher SOA formation under low-NO x conditions. Chemical characterization of the SOA was carried out, determining multi-oxygenated condensed organic compounds by a method based on the gas chromatography-mass spectrometry technique. Several ring-retaining and ring-cleavage products were identified and quantified. The compounds with the highest percentage contribution to the total aerosol mass were 4-nitrobenzene-1,2-diol, butenedioic acid, succinic acid and trans-trans-muconic. In addition, a multigenerational study was performed comparing with the photo-oxidations of phenol and catechol. The results showed that although the mass concentration of SOA produced was different, the physical and chemical properties were quite similar. Finally, we suggest a general mechanism to describe how changes in benzene degradation pathways - rate of OH generation and concentration of NO x - could justify the variation in SOA production and properties. © 2011 Elsevier Ltd.The authors wish to thank J.T.B, Martin-Reviejo, the Spanish Ministry of Science and Technology IMSEUR project (Project no REN2002-01484/CLI). The Instituto Universitario CEAM-UMH is partly supported by Generalitat Valenciana, Fundacion Bancaja, and the projects GRACCIE (Consolider-Ingenio 2010) and FEEDBACKS (Prometeo-GVA).Borrás García, EM.; Tortajada Genaro, LA. (2012). Secondary organic aerosol formation from the photo-oxidation of benzene. Atmospheric Environment. 47:154-163. https://doi.org/10.1016/j.atmosenv.2011.11.020S1541634

Determination of oxygenated compounds in secondary organic aerosol from isoprene and toluene smog chamber experiments

[EN] The determination of multifunctional oxygenated compounds in secondary organic aerosols (SOA) usually requires a derivatisation protocol prior to gas chromatography-mass spectrometry analysis (GC-MS). Our proposed protocol, a combination of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) plus diluted N-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA) without catalyst, has improved the determination of carbonyls, polyhydroxyl-compounds, hydroxyl- carbonyls, hydroxyl-carboxylic acids and di-carboxylic acids. The optimised derivatisation protocol has been successfully used for blanks, standard mixtures and photo-oxidation products from isoprene and toluene generated in a high-volume simulation chamber (European Photoreactor, EUPHORE). Some previously identified degradation products for isoprene including tetrols such as threitol, erythritol; 2-methyltetrols and 2-methylglyceric acid; and for toluene including nitrophenols, methyl-nitrophenols, benzaldehyde, p-cresol, benzoic acid, glyoxylic acid and methyl-glyoxylic acid, have been identified in our aerosol samples, thus confirming the successful applicability of the proposed derivatisation protocol. Moreover, the reduction of artefacts and enhanced signal-to-noise ratio, have allowed us to extend the number of multifunctional compounds determined. These findings have demonstrated the validity of this analytical strategy, which will contribute to a better understanding of the atmospheric degradation chemistry of biogenic and anthropogenic pollutants. © 2012 Taylor & Francis.We gratefully acknowledge the Generalitat Valenciana, Fundacion Bancaja and the GRACCIE CBS2007-00067 project in the CONSOLIDER-INGENIO 2010 program for supporting this study.Borrás García, EM.; Tortajada-Genaro, LA. (2012). Determination of oxygenated compounds in secondary organic aerosol from isoprene and toluene smog chamber experiments. International Journal of Environmental Analytical Chemistry. 92(1):110-124. doi:10.1080/03067319.2011.572164S110124921Hallquist, M., Wenger, J. C., Baltensperger, U., Rudich, Y., Simpson, D., Claeys, M., … Wildt, J. (2009). The formation, properties and impact of secondary organic aerosol: current and emerging issues. Atmospheric Chemistry and Physics, 9(14), 5155-5236. doi:10.5194/acp-9-5155-2009Yu, J., Jeffries, H. E., & Le Lacheur, R. M. (1995). Identifying Airborne Carbonyl Compounds in Isoprene Atmospheric Photooxidation Products by Their PFBHA Oximes Using Gas Chromatography/Ion Trap Mass Spectrometry. Environmental Science & Technology, 29(8), 1923-1932. doi:10.1021/es00008a009Yu, J., Flagan, R. C., & Seinfeld, J. H. (1998). Identification of Products Containing −COOH, −OH, and −CO in Atmospheric Oxidation of Hydrocarbons. Environmental Science & Technology, 32(16), 2357-2370. doi:10.1021/es980129xSpaulding, R., & Charles, M. (2002). Comparison of methods for extraction, storage, and silylation of pentafluorobenzyl derivatives of carbonyl compounds and multi-functional carbonyl compounds. Analytical and Bioanalytical Chemistry, 372(7-8), 808-816. doi:10.1007/s00216-002-1252-8Edler, M., Metze, D., Jakubowski, N., & Linscheid, M. (2002). Quantification of silylated organic compounds using gas chromatography coupled to ICP-MS. Journal of Analytical Atomic Spectrometry, 17(10), 1209-1212. doi:10.1039/b207227kJaoui, M., Kleindienst, T. E., Lewandowski, M., & Edney, E. O. (2004). Identification and Quantification of Aerosol Polar Oxygenated Compounds Bearing Carboxylic or Hydroxyl Groups. 1. Method Development. Analytical Chemistry, 76(16), 4765-4778. doi:10.1021/ac049919hWang, W., Vas, G., Dommisse, R., Loones, K., & Claeys, M. (2004). Fragmentation study of diastereoisomeric 2-methyltetrols, oxidation products of isoprene, as their trimethylsilyl ethers, using gas chromatography/ion trap mass spectrometry. Rapid Communications in Mass Spectrometry, 18(16), 1787-1797. doi:10.1002/rcm.1553Claeys, M., Wang, W., Ion, A. C., Kourtchev, I., Gelencsér, A., & Maenhaut, W. (2004). Formation of secondary organic aerosols from isoprene and its gas-phase oxidation products through reaction with hydrogen peroxide. Atmospheric Environment, 38(25), 4093-4098. doi:10.1016/j.atmosenv.2004.06.001Szmigielski, R., Surratt, J. D., Vermeylen, R., Szmigielska, K., Kroll, J. H., Ng, N. L., … Claeys, M. (2007). Characterization of 2-methylglyceric acid oligomers in secondary organic aerosol formed from the photooxidation of isoprene using trimethylsilylation and gas chromatography/ion trap mass spectrometry. Journal of Mass Spectrometry, 42(1), 101-116. doi:10.1002/jms.1146Surratt, J. D., Murphy, S. M., Kroll, J. H., Ng, N. L., Hildebrandt, L., Sorooshian, A., … Seinfeld, J. H. (2006). Chemical Composition of Secondary Organic Aerosol Formed from the Photooxidation of Isoprene. The Journal of Physical Chemistry A, 110(31), 9665-9690. doi:10.1021/jp061734mOrtiz, R., Enya, K., Sekiguchi, K., & Sakamoto, K. (2009). Experimental testing of an annular denuder and filter system to measure gas–particle partitioning of semivolatile bifunctional carbonyls in the atmosphere. Atmospheric Environment, 43(2), 382-388. doi:10.1016/j.atmosenv.2008.09.074Healy, R. M., Wenger, J. C., Metzger, A., Duplissy, J., Kalberer, M., & Dommen, J. (2008). Gas/particle partitioning of carbonyls in the photooxidation of isoprene and 1,3,5-trimethylbenzene. Atmospheric Chemistry and Physics, 8(12), 3215-3230. doi:10.5194/acp-8-3215-2008Kleindienst, T. E., Lewandowski, M., Offenberg, J. H., Jaoui, M., & Edney, E. O. (2009). The formation of secondary organic aerosol from the isoprene + OH reaction in the absence of NOx. Atmospheric Chemistry and Physics, 9(17), 6541-6558. doi:10.5194/acp-9-6541-2009Forstner, H. J. L., Flagan, R. C., & Seinfeld, J. H. (1997). Secondary Organic Aerosol from the Photooxidation of Aromatic Hydrocarbons:  Molecular Composition. Environmental Science & Technology, 31(5), 1345-1358. doi:10.1021/es9605376(s. f.). doi:10.1021/es010676Kleindienst, T. E., Conver, T. S., McIver, C. D., & Edney, E. O. (2004). Determination of Secondary Organic Aerosol Products from the Photooxidation of Toluene and their Implications in Ambient PM2.5. Journal of Atmospheric Chemistry, 47(1), 79-100. doi:10.1023/b:joch.0000012305.94498.28HAMILTON, J., WEBB, P., LEWIS, A., & REVIEJO, M. (2005). Quantifying small molecules in secondary organic aerosol formed during the photo-oxidation of toluene with hydroxyl radicals. Atmospheric Environment, 39(38), 7263-7275. doi:10.1016/j.atmosenv.2005.09.006Orzechowska, G. E., Nguyen, H. T., & Paulson, S. E. (2005). Photochemical Sources of Organic Acids. 2. Formation of C5−C9Carboxylic Acids from Alkene Ozonolysis under Dry and Humid Conditions. The Journal of Physical Chemistry A, 109(24), 5366-5375. doi:10.1021/jp050167kClaeys, M., Szmigielski, R., Kourtchev, I., Van der Veken, P., Vermeylen, R., Maenhaut, W., … Edney, E. O. (2007). Hydroxydicarboxylic Acids:  Markers for Secondary Organic Aerosol from the Photooxidation of α-Pinene. Environmental Science & Technology, 41(5), 1628-1634. doi:10.1021/es0620181Claeys, M. (2004). Formation of Secondary Organic Aerosols Through Photooxidation of Isoprene. Science, 303(5661), 1173-1176. doi:10.1126/science.1092805Jaoui, M., Kleindienst, T. E., Lewandowski, M., Offenberg, J. H., & Edney, E. O. (2005). Identification and Quantification of Aerosol Polar Oxygenated Compounds Bearing Carboxylic or Hydroxyl Groups. 2. Organic Tracer Compounds from Monoterpenes. Environmental Science & Technology, 39(15), 5661-5673. doi:10.1021/es048111bBöge, O., Miao, Y., Plewka, A., & Herrmann, H. (2006). Formation of secondary organic particle phase compounds from isoprene gas-phase oxidation products: An aerosol chamber and field study. Atmospheric Environment, 40(14), 2501-2509. doi:10.1016/j.atmosenv.2005.12.025Kourtchev, I., Warnke, J., Maenhaut, W., Hoffmann, T., & Claeys, M. (2008). Polar organic marker compounds in PM2.5 aerosol from a mixed forest site in western Germany. Chemosphere, 73(8), 1308-1314. doi:10.1016/j.chemosphere.2008.07.011Pio, C., Alves, C., & Duarte, A. (2001). Organic components of aerosols in a forested area of central Greece. Atmospheric Environment, 35(2), 389-401. doi:10.1016/s1352-2310(00)00135-7Little, J. L. (1999). Artifacts in trimethylsilyl derivatization reactions and ways to avoid them. Journal of Chromatography A, 844(1-2), 1-22. doi:10.1016/s0021-9673(99)00267-8Ruppert, L., & Heinz Becker, K. (2000). A product study of the OH radical-initiated oxidation of isoprene: formation of C5-unsaturated diols. Atmospheric Environment, 34(10), 1529-1542. doi:10.1016/s1352-2310(99)00408-2Martín-Reviejo, M., & Wirtz, K. (2005). Is Benzene a Precursor for Secondary Organic Aerosol? Environmental Science & Technology, 39(4), 1045-1054. doi:10.1021/es049802aVolkamer, R., Klotz, B., Barnes, I., Imamura, T., Wirtz, K., Washida, N., … Platt, U. (2002). OH-initiated oxidation of benzene. Physical Chemistry Chemical Physics, 4(9), 1598-1610. doi:10.1039/b108747aHurley, M. D., Sokolov, O., Wallington, T. J., Takekawa, H., Karasawa, M., Klotz, B., … Becker, K. H. (2001). Organic Aerosol Formation during the Atmospheric Degradation of Toluene. Environmental Science & Technology, 35(7), 1358-1366. doi:10.1021/es0013733Paulsen, D., Dommen, J., Kalberer, M., Prévôt, A. S. H., Richter, R., Sax, M., … Baltensperger, U. (2005). Secondary Organic Aerosol Formation by Irradiation of 1,3,5-Trimethylbenzene−NOx−H2O in a New Reaction Chamber for Atmospheric Chemistry and Physics. Environmental Science & Technology, 39(8), 2668-2678. doi:10.1021/es0489137Baltensperger, U., Kalberer, M., Dommen, J., Paulsen, D., Alfarra, M. R., Coe, H., … Zenobi, R. (2005). Secondary organic aerosols from anthropogenic and biogenic precursors. Faraday Discussions, 130, 265. doi:10.1039/b417367hMcMurry, P. H., & Rader, D. J. (1985). Aerosol Wall Losses in Electrically Charged Chambers. Aerosol Science and Technology, 4(3), 249-268. doi:10.1080/02786828508959054Halket, J. M., & Zaikin, V. G. (2003). Derivatization in Mass Spectrometry—1. Silylation. European Journal of Mass Spectrometry, 9(1), 1-21. doi:10.1255/ejms.527Clements, A. L., & Seinfeld, J. H. (2007). Detection and quantification of 2-methyltetrols in ambient aerosol in the southeastern United States. Atmospheric Environment, 41(9), 1825-1830. doi:10.1016/j.atmosenv.2006.10.056Rogge, W. F., Mazurek, M. A., Hildemann, L. M., Cass, G. R., & Simoneit, B. R. T. (1993). Quantification of urban organic aerosols at a molecular level: Identification, abundance and seasonal variation. Atmospheric Environment. Part A. General Topics, 27(8), 1309-1330. doi:10.1016/0960-1686(93)90257-yEdney, E. O., Driscoll, D. J., Weathers, W. S., Kleindienst, T. E., Conver, T. S., McIver, C. D., & Li, W. (2001). Formation of Polyketones in Irradiated Toluene/Propylene/NO x /Air Mixtures. Aerosol Science and Technology, 35(6), 998-1008. doi:10.1080/027868201753306769Temime, B., Healy, R. M., & Wenger, J. C. (2007). A Denuder-Filter Sampling Technique for the Detection of Gas and Particle Phase Carbonyl Compounds. Environmental Science & Technology, 41(18), 6514-6520. doi:10.1021/es070802

Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection

[EN] The costs of current genotyping methods limit their application to personalized therapy. The authors describe an alternative approach for the detection of single-point-polymorphisms using recombinant polymerase amplification as an allele-specific technique. The use of short and chemically modified primers and locked nucleic acids allowed for a selective isothermal amplification of wild-type or mutant variants at 37 °C within 40 min. An amplification chip platform containing 100 wells was manufactured with a 3D printer and using thermoplastic polylactic acid. The platform reduces reagent consumption and allows parallelization. As a proof of concept, the method was applied to the genotyping of four SNPs that are related to the treatment of tobacco addiction. The target polymorphisms included rs4680 (COMT gene), rs1799971 (OPRM1 gene), rs1800497 (ANKK1 gene), and rs16969968 (CHRNA5 gene). The genotype populations can be well discriminated.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project).Yamanaka, E.; Tortajada-Genaro, LA.; Maquieira, A. (2017). Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection. Microchimica Acta. 184(5):1453-1462. https://doi.org/10.1007/s00604-017-2144-0S145314621845Manolio TA, Chisholm RL, Ozenberger B, Roden DM, Williams MS, Wilson R et al (2013) Implementing genomic medicine in the clinic: the future is here. Genitourin Med 15:258–267Scott SA (2013) Clinical pharmacogenomics: opportunities and challenges at point-of-care. Clin Pharmacol Ther 93:33Limaye N (2013) Pharmacogenomics, Theranostics and personalized medicine-the complexities of clinical trials: challenges in the developing world. Appl Transl Genomics 2:17–21Abul-Husn NS, Owusu Obeng A, Sanderson SC, Gottesman O, Scott SA (2014) Implementation and utilization of genetic testing in personalized medicine. Pharmacogenomics Pers Med 7:227–240Knez K, Spasic D, Janssen KP, Lammertyn J (2014) Emerging technologies for hybridization based single nucleotide polymorphism detection. Analyst 139:353–370Shen W, Tian Y, Ran T, Gao Z (2015) Genotyping and quantification techniques for single-nucleotide polymorphisms. TrAC Trends Anal Chem 69:1–13Milbury CA, Li J, Makrigiorgos GM (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55:632–640Asari M, Watanabe S, Matsubara K, Shiono H, Shimizu K (2009) Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal Biochem 386:85–90Taira C, Matsuda K, Yamaguchi A, Sueki A, Koeda H, Takagi F, Kobayashi Y, Sugano M, Honda T (2013) Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms. Clin Chim Acta 424:39–46Tortajada-Genaro LA, Mena S, Niñoles R, Puigmule M, Viladevall L, Maquieira A (2016) Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Anal Bioanal Chem 408:2339–2345Woolley CF, Hayes MA (2014) Emerging technologies for biomedical analysis. Analyst 139:2277–2288Craw P, Balachandran W (2012) Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 12:2469–2486Zhang L, Zhang Y, Wang C, Feng Q, Fan F, Zhang G, Kang X, Qin X, Sun J, Li Y, Jiang X (2014) Integrated microcapillary for sample-to-answer nucleic acid pretreatment, amplification, and detection. Anal Chem 86:10461–10466Chen F, Zhao Y, Fan C, Zhao Y (2015) Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification. Anal Chem 87:8718–8723Li J, Macdonald J (2015) Advances in isothermal amplification: novel strategies inspired by biological processes. Biosens Bioelectron 64:196–211Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira A (2014) One-pot isothermal DNA amplification–hybridisation and detection by a disc-based method. Sens Actuator B-Chem 204:273–281Santiago-Felipe S, Tortajada-Genaro LA, Puchades R, Maquieira Á (2016) Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchim Acta 183:1195–1202Tortajada-Genaro LA, Santiago-Felipe S, Amasia M, Russom A, Maquieira A (2015) Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs). RSCAdv 5:29987–29995Li Z, Liu Y, Wei Q, Liu Y, Liu W, Zhang X, Yu Y (2016) Picoliter well Array Chip-based digital recombinase polymerase amplification for absolute quantification of nucleic acids. PLoS One 11:e0153359Daher RK, Stewart G, Boissinot M, Boudreau DK, Bergeron MG (2015) Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Mol Cell Probes 29:116–121Shin Y, Perera AP, Kim KW, Park MK (2013) Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers. Lab Chip 13:2106–2114NgePN RCI, Woolley AT (2013) Advances in microfluidic materials, functions, integration, and applications. Chem Rev 113:2550–2583Bhattacharjee N, Urrios A, Kang S, Folch A (2016) The upcoming 3D-printing revolution in microfluidics. Lab Chip 16:1720–1742Waheed S, Cabot JM, Macdonald NP, Lewis T, Guijt RM, Paull B, Breadmore MC (2016) 3D printed microfluidic devices: enablers and barriers. Lab Chip 16:1993–2013Bierut LJ, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau OF, Swan GE, Rutter J, Bertelsen S, Fox L, Fugman D, Goate AM, Hinrichs AL, Konvicka K, Martin NG, Montgomery GW, Saccone NL, Saccone SF, Wang JC, Chase GA, Rice JP, Ballinger DG (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum MolGen 16:24–35Carpenter MJ, Jardin BF, Burris JL, Mathew AR, Schnoll RA, Rigotti NA, Cummings KM (2013) Clinical strategies to enhance the efficacy of nicotine replacement therapy for smoking cessation: a review of the literature. Drugs 73:407–426Moody C, Newell H, Viljoen H (2016) FA mathematical model of recombinase polymerase amplification under continuously stirred conditions. Biochem Eng J 112:193–201Dimitrov RA, Zuker M (2004) Prediction of hybridization and melting for double-stranded nucleic acids. Biophys J 87:215–226Zhang C, Xing D (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res 35:4223–4237Liu B, Huang PJJ, Zhang X, Wang F, Pautler R, IpACF LJ (2013) Parts-per-million of polyethylene glycol as a non-interfering blocking agent for homogeneous biosensor development. Anal Chem 85:10045–10050Wu J, Kodzius R, Cao W, Wen W (2014) Extraction, amplification and detection of DNA in microfluidic chip-based assays. Microchim Acta 181:1611–1631Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14:579–584Shen R, Fan JB, Campbell D, Chang W, Chen J, Doucet D, Yeakley J, Bibikova M, Garcia EW, McBride C, Steemers F, Garcia F, Kermani BG, Gunderson K, Oliphant A (2005) High-throughput SNP genotyping on universal bead arrays. Mut Res Fund Mol M 573:70–82David SP, Strong DR, Leventhal AM, Lancaster MA, McGeary JE, Munafò MR, Bergen AW, Swan GE, Benowitz NL, Tyndale RF, Conti DV, Brown RA, Lerman C, Niaura R (2013) Influence of a dopamine pathway additive genetic efficacy score on smoking cessation: results from two randomized clinical trials of bupropion. Addiction 108:2202–221

Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection

[EN] Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands.The authors acknowledge the financial support received from EU FEDER, the Spanish Ministry of Economy and Competitiveness (PID2019-110713RB-I00), and the Generalitat Valenciana (PROMETEO/2020/094 and GVA-FPI-2017 Ph.D. grant).Lázaro-Zaragozá, A.; Maquieira Catala, A.; Tortajada-Genaro, LA. (2022). Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection. ACS Sensors. 7(3):758-765. https://doi.org/10.1021/acssensors.1c02220S7587657

Sesión de posters apoyada en plataformas de e-learning

[EN] The poster sessions are a teaching-learning strategy, that simulates working scenarios in the university classroom. The success of this exercice is related to how the previous tasks have been addressed. In this communication, the results of a teaching action focused on the elaboration and exhibition of a poster as materialization of a learning process by inquiry are presented. Given its complexity, the proposed approach has been a colaborative activity and supported by a virtual educational space that facilitates an active and flexible interaction between teachers and students and between the students themselves. Furthermore, a system of continuous formative assessment has been set up so that the feedback can be highlighted during its development. The student results achieved show the excellent combination of quality and satisfaction for the academic work carried out. In addition to inducing the acquisition of knowledge in specialized topics, it has been possible to promote skills related to communication, critical thinking, creativity, and cooperation.[ES] Las sesiones de posters son una estrategia de enseñanza-aprendizaje que permiten simular escenarios laborales en el aula universitaria. El éxito de esta actividad está relacionado con el modo con que se han abordado tareas anteriores. En esta comunicación, se presentan los resultados de una acción docente dirigida a la elaboración y exposición de un póster como materialización de un proceso de aprendizaje por indagación. Dada su complejidad, el enfoque propuesto ha consistido en una actividad colaborativa apoyada por un espacio virtual educativo que facilite una interacción activa y flexible entre los profesores y los alumnos, y entre los propios alumnos. Al mismo tiempo, se ha instaurado un sistema de evaluación continua formativa para que la retro-alimentación fuera una acción destacada durante su desarrollo. Los resultados alcanzados por los alumnos ponen de manifiesto la excelente combinación de calidad y satisfacción por el trabajo académico realizado. Además de inducir la adquisición de conocimiento en temáticas especializadas, se ha logrado fomentar habilidades relacionadas con la comunicación, pensamiento crítico, creatividad, trabajo en equipo y cooperación.Tortajada Genaro, LA.; Puchades Pla, R. (2017). Sesión de posters apoyada en plataformas de e-learning. En In-Red 2017. III Congreso Nacional de innovación educativa y de docencia en red. Editorial Universitat Politècnica de València. 1183-1193. https://doi.org/10.4995/INRED2017.2017.6783OCS1183119

Primer design for SNP genotyping based on allele-specific amplification Application to organ transplantation pharmacogenomics

[EN] Diagnostic methods based on single nucleotide polymorphism (SNP) biomarkers are essential for the real adoption of personalized medicine. Allele specific amplification in a homogeneous format or combined to microarray hybridization are powerful approaches for SNP genotyping. However, primers must be properly selected to minimize cross-reactivity, dimer formation and nonspecific hybridization. This study presents a design workflow diagram for the selection of required oligonucleotides for multiplex assays. Based on thermodynamic restrictions, the oligonucleotide sets are chosen for a specific amplification of wild- and mutant-type templates. Design constraints include the structural stability of primer-template duplexes, template-probe duplexes and self-annealing complexes or hairpins for each targeted gene. The performance of the design algorithm was evaluated for the simultaneous genotyping of three SNPs related to immunosuppressive drugs administered after solid organ transplantation. The assayed polymorphisms were rs1045642 (ABCS] gene), rs1801133 (MTHFR gene) and rs776746 (CYP3A5 gene). Candidates were confirmed by discriminating homozygote and heterozygote populations using a fluorescence solution method and two colorimetric microarray methods on polycarbonate chips. The analysis of patient samples provided excellent genotyping results compared to those obtained by a reference method. The study demonstrates that the development of the allele-specific methods as pharmacogenetic tools can be simplified. (C) 2016 Elsevier B.V. All rights reserved.Tortajada-Genaro, LA.; Puchades Pla, R.; Maquieira Catala, Á. (2017). Primer design for SNP genotyping based on allele-specific amplification Application to organ transplantation pharmacogenomics. Journal of Pharmaceutical and Biomedical Analysis. 136:14-21. doi:10.1016/j.jpba.2016.12.030S142113

Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene

[EN] An accurate genetic diagnostic is key for adequate patient management and the suitability of healthcare systems. The scientific challenge lies in developing methods to discriminate those patients with certain genetic variations present in tumor cells at low concentrations. We report a method called enhanced asymmetric blocked qPCR (EAB-qPCR) that promotes the blocker annealing against the primer-template hybrid controlling thermal cycling and reaction conditions with nonmodified oligonucleotides. Real-time fluorescent amplification curves of wild-type alleles were delayed by about eight cycles for EAB-qPCR, compared to conventional blocked qPCR approaches. This method reduced the amplification of native DNA variants (blocking percentage 99.7%) and enabled the effective enrichment of low-level DNA mutations. Excellent performance was estimated for the detection of mutated alleles in sensitivity (up to 0.5% mutant/total DNA) and reproducibility terms, with a relative standard deviation below 2.8%. The method was successfully applied to the mutational analysis of metastatic colorectal carcinoma from biopsied tissues. The determined single-nucleotide mutations in the KRAS oncogene (codon 12¿13) totally agreed with those obtained from next-generation sequencing. EAB-qPCR is an accurate cheap method and can be easily incorporated into daily routine to detect mutant alleles. Hence, these features are especially interesting to facilitate the diagnosis and prognosis of several clinical diseases.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-FPI-2017 PhD grant), the Spanish Ministry of Economy and Competitiveness (MINECO project CTQ2016-75749-R), and European Regional Development Fund (ERDF)Lázaro-Zaragozá, A.; Tortajada-Genaro, LA.; Maquieira Catala, Á. (2021). Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene. Analytical and Bioanalytical Chemistry. 413(11):2961-2969. https://doi.org/10.1007/s00216-021-03229-3S2961296941311Chandler NJ, Ahlfors H, Drury S, Mellis R, Hill M, McKay FJ, et al. Noninvasive prenatal diagnosis for cystic fibrosis: implementation, uptake, outcome, and implications. Clin Chem. 2020;66:207–16.Schmidt RLJ, Simon A, Popow-Kraupp T, Laggner A, Haslacher H, Fritzer-Szekeres M, et al. A novel PCR-based point-of-care method facilitates rapid, efficient, and sensitive diagnosis of influenza virus infection. Clin Microbiol Infect. 2019;25:1032–7.Reck M, Rabe KF. Precision diagnosis and treatment for advanced non–small-cell lung cancer. N Engl J Med. 2017;377:849–61.Vargas DY, Kramer FR, Tyagi S, Marras SAE. Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer. PLoS One. 2016;11:e0156546.Allegra CJ, Rumble RB, Hamilton SR, Mangu PB, Roach N, Hantel A, et al. Extended RAS gene mutation testing in metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy: American Society of Clinical Oncology provisional clinical opinion. J Clin Oncol. 2016;34:179–85.Das V, Kalita J, Pal M. Predictive and prognostic biomarkers in colorectal cancer: a systematic review of recent advances and challenges. Biomed Pharmacother. 2017;87:8–19.Khodakov D, Wang C, Zhang DY. Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches. Adv Drug Deliv Rev. 2016;105:3–19.Kim S, Misra A. SNP genotyping: technologies and biomedical applications. Annu Rev Biomed Eng. 2007;9:289–320.Tsiatis AC, Norris-Kirby A, Rich RG, Hafez MJ, Gocke CD, Eshleman JR, et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagnostics. 2010;12:425–32.Milbury CA, Li J, Makrigiorgos GM. PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem. 2009;55:632–40.Lin CC, Huang WL, Wei F, Su WC, Wong DT. Emerging platforms using liquid biopsy to detect EGFR mutations in lung cancer. Expert Rev Mol Diagn. 2015;15:1427–40.Strohmeier O, Laßmann S, Riedel B, Mark D, Roth G, Werner M, et al. Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment. Microchim Acta. 2014;181:1681–8.Markou A, Tzanikou E, Ladas I, Makrigiorgos GM, Lianidou E. Nuclease-assisted minor allele enrichment using overlapping probes-assisted amplification-refractory mutation system: an approach for the improvement of amplification-refractory mutation system-polymerase chain reaction specificity in liquid biopsies. Anal Chem. 2019;91:13105–11.Zhang H, Song J, Ren H, Xu Z, Wang X. Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions. PLoS One. 2013;8:54510.Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods. 2013;10:1003–5.Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med. 2008;14:579–84.Castellanos-Rizaldos E, Milbury CA, Guha M, Makrigiorgos GM. COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing. Molecular Diagnostics for Melanoma: Methods and Protocols, Methods in Molecular Biology. 2014. p. 623–39.Briones C, Moreno M. Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development. Anal Bioanal Chem. 2012;402:3071–89.Huang JF, Zeng DZ, Duan GJ, Shi Y, Deng GH, Xia H, et al. Single-tubed wild-type blocking quantitative PCR detection assay for the sensitive detection of codon 12 and 13 KRAS mutations. PLoS One. 2015;10:1–23.Kim HR, Lee SY, Hyun DS, Lee MK, Lee HK, Choi CM, et al. Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping. J Exp Clin Cancer Res. 2013;32:50.Zhang S, Chen Z, Huang C, Ding C, Li C, Chen J, et al. Ultrasensitive and quantitative detection of: EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp. Analyst. 2019;144:1718–24.Tolnai Z, Harkai Á, Szeitner Z, Scholz ÉN, Percze K, Gyurkovics A, et al. A simple modification increases specificity and efficiency of asymmetric PCR. Anal Chim Acta. 2019;1047:225–30.Jia Y, Sanchez JA, Wangh LJ. Kinetic hairpin oligonucleotide blockers for selective amplification of rare mutations. Sci Rep. 2014;4:1–8.Vashist SK, Luppa PB, Yeo LY, Ozcan A, Luong JHT. Emerging technologies for next-generation point-of-care testing. Trends Biotechnol. 2015;33:692–705.Peterson AW. The effect of surface probe density on DNA hybridization. Nucleic Acids Res. 2001;29:5163–8.Falcomatà C, Schneider G, Saur D. Personalizing KRAS-mutant allele–specific therapies. Cancer Discov. 2020;10:23–5.McEvoy AC, Wood BA, Ardakani NM, Pereira MR, Pearce R, Cowell L, et al. Droplet digital PCR for mutation detection in formalin-fixed, paraffin-embedded melanoma tissues: a comparison with sanger sequencing and pyrosequencing. J Mol Diagnostics. 2018;20:240–52.Milbury CA, Li J, Makrigiorgos GM. Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic Acids Res. 2011;39:e2.Xiang Z, Wan R, Zou B, Qi X, Huang Q, Kumar S, et al. Highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA. Anal Bioanal Chem. 2018;410:6751–9.Huang T, Zhuge J, Zhang WW. Sensitive detection of BRAF V600E mutation by amplification refractory mutation system (ARMS)-PCR. Biomark Res. 2013;1:1–6.Matsuda K. PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement. Adv Clin Chem. 2017;80:45–72.Lázaro A, Yamanaka ES, Maquieira Á, Tortajada-Genaro LA. Allele-specific ligation and recombinase polymerase amplification for the detection of single nucleotide polymorphisms. Sensors Actuators B Chem. 2019;298:126877