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. 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Un aspecto de la polémica gongorina: la lengua de las Soledades y el Polifemo como discutido criterio de corrección

22 p.Maquieira Rodríguez, Marina A.. Un aspecto de la polémica gongorina: la lengua de las Soledades y el Polifemo como discutido criterio de corrección. En: Quaderns de filologia. Estudis lingüístics, 2008, No. 13. Dedicado a: Historiografía lingüística hispánica: 135S

Las gramáticas castellanas para extranjeros a lo largo del siglo XVI

A lo largo del s. XVI, y paralelamente a la construcción de los primeros tratados de español para hablantes nativos, surgen otros cuyo interés se centra en la enseñanza de esta lengua a extranjeros. Unos y otros, evidentemente, muestran distintas formas de estructuración y exposición de los contenidos gramaticales. El presenta articulo intenta analizar los diferentes procedimientos de ambos grupos de textos, desde la perspectiva de los tratados para extranjero

Revisión del sistema fonológico del español en el siglo XVI

Comentario de la obra clásica “Tractado de Orthographía y Accentos en las Tres Lenguas Principales”, de Alejo Venegas. Empieza dicho comentario resaltando la importancia que representa en el panorama de nuestra historia de la tingüística la publicación de este texto. Efectivamente, ta obra de Venegas supone una nueva e interesante aproximación a la tarea lingüística del siglo XVI, que todavía hoy es escasamente conocida, en parte por la dificultad que supone el acceso a textos de problemática localizació

Panorama gramatical entre 1771-1847

La autora realiza un profundo estudio de la obra "Gramática y categorías verbales en la tradición española (1771-1847). En ella su autor, José Jesús Gómez Asencio, presenta un análisis de la historia de la lingüística en España entre 1771 y 1847, y lo hace exclusivamente desde el punto de vista gramatica

Recovery of Shallow Charge-Trapping Defects in CsPbX3 Nanocrystals through Specific Binding and Encapsulation with Amino-Functionalized Silanes

[EN] We report a facile methodology to restore photoluminescence (PL) of CsPbBr3 nanocrystals (NCs) based on their postsynthetic modification with 3-aminopropyltriethoxysilane (APTES). By this methodology, a stark PL recovery factor of near 2-fold was obtained compared to their uncoated counterparts. H-1 NMR studies confirmed the presence of APTES on the NCs shell and provided more insight into the nature of the alkoxysilane passivation mechanisms. We further highlight that, contrary to expectations, preferential attachment of APTES does not take place through their amine terminal groups. The proposed surface-repair strategy can be extended to other halide compositions, yielding similarly effective 4-fold and 2-fold PL enhancements for CsPbCl3 and CsPbI3 NCs, respectively. Our work thus exemplifies that careful management of the perovskite NC interfaces and surface engineering is one of the most important frontiers in this emerging class of optoelectronic materials.This work was financially supported by FEDER projects BiHolog-CTQ2016-75749-R from MINECO and GVA PROMETEO II 2014/40. V.G-P. thanks Universitat Politecnica de Valencia for her postdoctoral fellowship (Grant-PAID-10-14) and for her research mobility grant (UPV- Research mobility Grant-PAID-00-15), and Miguel Cebrian for his help and valuable technical support. S.A.V., N.M., and S.M. acknowledge financial support by the National Research Foundation, Singapore under its Competitive Research Programme (NRF-CRP14-2014-03). S.A.V. thanks Alasdair Brown for help with the nanocrystal syntheses.González-Pedro, V.; Veldhuis, SA.; Begum, R.; Bañuls Polo, M.; Bruno, A.; Mathews, N.; Mhaisalka, S.... (2018). Recovery of Shallow Charge-Trapping Defects in CsPbX3 Nanocrystals through Specific Binding and Encapsulation with Amino-Functionalized Silanes. ACS Energy Letters. 3(6):1409-1414. https://doi.org/10.1021/acsenergylett.8b00498S140914143

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

Label-free piezoelectric biosensor for prognosis and diagnosis of Systemic Lupus Erythematosus

[EN] An autoantigen piezoelectric sensor to quantify specific circulating autoantibodies in human serum is developed. The sensor consisted on a quartz crystal microbalance with dissipation monitoring (QCM-D) where TRIM21 and TROVE2 autoantigens were covalently immobilized, allowing the selective determination of autoantibodies for diagnosis and prognosis of Systemic Lupus Erythematosus (SLE). The sensitivity of the biosensor, measured as IC50 value, was 1.51 U/mL and 0.32 U/mL, for anti-TRIM21 and anti-TROVE2 circulating autoantibodies, respectively. The sensor is also able to establish a structural interaction fingerprint pattern or profile of circulating autoantibodies, what allows scoring accurately SLE patients. Furthermore, a statistical association of global disease activity with TRIM21-TROVE2 interaction was found (n=130 lupic patient samples, p-value=0.0413). The performances of the biosensor were compared with standard ELISA and multiplex DVD-array high-throughput screening assays, corroborating the viability of piezoelectric biosensor as a cost-effective in vitro assay for the early detection, monitoring or treatment of rare diseases.We acknowledge financial support from the Generalitat Valenciana (GVA-PROMETEOII/2014/040) as well as the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund under award numbers CTQ2013-45875-R and CTQ2013-42914-R.Do Nascimento, NM.; Juste-Dolz, A.; Grau-García, E.; Roman-Ivorra, JA.; Puchades, R.; Maquieira Catala, A.; Morais, S.... (2017). Label-free piezoelectric biosensor for prognosis and diagnosis of Systemic Lupus Erythematosus. Biosensors and Bioelectronics. 90:166-173. https://doi.org/10.1016/j.bios.2016.11.004S1661739

Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once-living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules, making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L-aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1–10 ppb level and some even at the part-per-trillion level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (ca. 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside EarthThis work was supported by granted projects AYA2011-24803, ESP2014-51989-P, and ESP2015-69540-R, from the Ministry of Economy and Competitiveness (MINECO) of Spain, and by Grant No. ST/N000803/1 (United Kingdom). A. G-C was a fellow of ‘‘Plan de Formación from INTA.Peer reviewe