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

Boosting the sensitivity of in vitro b-lactam allergy diagnostic tests

[EN] The synthesis of structurally new haptens and the development of suitable antigens are essential for boosting the sensitivity of drug allergy diagnostic testing. Unprecedented structural antigens for benzylpenicillin and amoxicillin are characterised and evaluated in a cohort of 70 subjects with a turnkey solution based on consumer electronics.This work was funded by the H2020 program (project COBIOPHAD, grant agreement No. 688448), an initiative of the Photonics Public Private Partnership; Agencia Estatal de Investigacion (CTQ2016-75749-R, FEDER) and program UPV-La FE 2019 (PI05 VALBIOAL). E. P. M. holds a FPU doctoral fellowship from Ministerio de Educación, Cultura y Deporte.Peña-Mendizábal, E.; Morais, S.; Maquieira Catala, Á. (2020). Boosting the sensitivity of in vitro b-lactam allergy diagnostic tests. Chemical Communications. 56(80):11973-11976. https://doi.org/10.1039/D0CC04903DS11973119765680Van Boeckel, T. P., Gandra, S., Ashok, A., Caudron, Q., Grenfell, B. T., Levin, S. A., & Laxminarayan, R. (2014). Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data. The Lancet Infectious Diseases, 14(8), 742-750. doi:10.1016/s1473-3099(14)70780-7Khan, D. A., & Solensky, R. (2010). Drug allergy. Journal of Allergy and Clinical Immunology, 125(2), S126-S137.e1. doi:10.1016/j.jaci.2009.10.028Blanca, M., Romano, A., Torres, M. J., Férnandez, J., Mayorga, C., Rodriguez, J., … Atanasković-Marković, M. (2009). Update on the evaluation of hypersensitivity reactions to betalactams. Allergy, 64(2), 183-193. doi:10.1111/j.1398-9995.2008.01924.xWorld Health Organization, WHO Report on Surveillance of Antibiotic Consumption: 2016–2018 Early implementation, Geneva, 2018Blanca-Lopez, N., Jimenez-Rodriguez, T. W., Somoza, M. L., Gomez, E., Al-Ahmad, M., Perez-Sala, D., & Blanca, M. (2019). Allergic reactions to penicillins and cephalosporins: diagnosis, assessment of cross-reactivity and management. Expert Review of Clinical Immunology, 15(7), 707-721. doi:10.1080/1744666x.2019.1619548Chang, C., Mahmood, M. M., Teuber, S. S., & Gershwin, M. E. (2011). Overview of Penicillin Allergy. Clinical Reviews in Allergy & Immunology, 43(1-2), 84-97. doi:10.1007/s12016-011-8279-6Baldo, B. A., & Pham, N. H. (2002). Immunoglobulin E binding determinants on β-lactam drugs. Current Opinion in Allergy and Clinical Immunology, 2(4), 297-300. doi:10.1097/00130832-200208000-00002Weltzien, H. U., & Padovan, E. (1998). Molecular Features of Penicillin Allergy. Journal of Investigative Dermatology, 110(3), 203-206. doi:10.1046/j.1523-1747.1998.00122.xBALDO. (1999). Penicillins and cephalosporins as allergens - structural aspects of recognition and cross-reactions. Clinical & Experimental Allergy, 29(6), 744-749. doi:10.1046/j.1365-2222.1999.00575.xZhao, Z., Baldo, B. A., & Rimmer, J. (2002). β-Lactam allergenic determinants: fine structural recognition of a cross-reacting determinant on benzylpenicillin and cephalothin. Clinical & Experimental Allergy, 32(11), 1644-1650. doi:10.1046/j.1365-2222.2002.01492.xZhao, Z., Baldo, B. A., Baumgart, K. W., & Mallon, D. F. J. (2001). Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects. Journal of Molecular Recognition, 14(5), 300-307. doi:10.1002/jmr.541Peng, J., Kong, D., Liu, L., Song, S., Kuang, H., & Xu, C. (2015). Determination of quinoxaline antibiotics in fish feed by enzyme-linked immunosorbent assay using a monoclonal antibody. Analytical Methods, 7(12), 5204-5209. doi:10.1039/c5ay00953gPeng, D., Ye, S., Wang, Y., Chen, D., Tao, Y., Huang, L., … Yuan, Z. (2011). Development and Validation of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Screening of Tylosin and Tilmicosin in Muscle, Liver, Milk, Honey and Eggs. Journal of Agricultural and Food Chemistry, 60(1), 44-51. doi:10.1021/jf2037449Brun, E. M., Garcés-García, M., Puchades, R., & Maquieira, Á. (2004). Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure. Journal of Immunological Methods, 295(1-2), 21-35. doi:10.1016/j.jim.2004.08.014Kim, Y. J., Cho, Y. A., Lee, H.-S., Lee, Y. T., Gee, S. J., & Hammock, B. D. (2003). Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Analytica Chimica Acta, 475(1-2), 85-96. doi:10.1016/s0003-2670(02)01037-1Montañez, M. I., Mayorga, C., Torres, M. J., Ariza, A., Blanca, M., & Perez-Inestrosa, E. (2011). Synthetic Approach to Gain Insight into Antigenic Determinants of Cephalosporins: In Vitro Studies of Chemical Structure−IgE Molecular Recognition Relationships. Chemical Research in Toxicology, 24(5), 706-717. doi:10.1021/tx100446gPastor-Navarro, N., Morais, S., Maquieira, Á., & Puchades, R. (2007). Synthesis of haptens and development of a sensitive immunoassay for tetracycline residues. Analytica Chimica Acta, 594(2), 211-218. doi:10.1016/j.aca.2007.05.045Chen, Z.-J., Fu, H.-J., Luo, L., Sun, Y.-M., Yang, J.-Y., Zeng, D.-P., … Xu, Z.-L. (2017). Development of competitive indirect ELISAs with a flexible working range for the simple quantification of melatonin in medicinal foods. Analytical Methods, 9(10), 1617-1626. doi:10.1039/c6ay03380fPastor-Navarro, N., Maquieira, Á., & Puchades, R. (2009). Review on immunoanalytical determination of tetracycline and sulfonamide residues in edible products. Analytical and Bioanalytical Chemistry, 395(4), 907-920. doi:10.1007/s00216-009-2901-ySingh, K. V., Kaur, J., Varshney, G. C., Raje, M., & Suri, C. R. (2003). Synthesis and Characterization of Hapten−Protein Conjugates for Antibody Production against Small Molecules. Bioconjugate Chemistry, 15(1), 168-173. doi:10.1021/bc034158vMas, S., Badran, A. A., Juárez, M.-J., Fernández de Rojas, D. H., Morais, S., & Maquieira, Á. (2020). Highly sensitive optoelectrical biosensor for multiplex allergy diagnosis. Biosensors and Bioelectronics, 166, 112438. doi:10.1016/j.bios.2020.112438Vultaggio, A., Matucci, A., Virgili, G., Rossi, O., Filì, L., Parronchi, P., … Maggi, E. (2009). Influence of total serum IgE levels on thein vitrodetection of β-lactams-specific IgE antibodies. Clinical & Experimental Allergy, 39(6), 838-844. doi:10.1111/j.1365-2222.2009.03219.

Neo-antigens for the serological diagnosis of IgE-mediated drug allergic reactions to antibiotics cephalosporin, carbapenem and monobactam

[EN] New antigens deriving from -lloyl and -llanyl, major and minor determinants, respectively, were produced for beta-lactam antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem and aztreonam. Twenty beta-lactam antigens were produced using human serum albumin and histone H1 as carrier proteins. Antigens were tested by multiplex in vitro immunoassays and evaluated based on the detection of specific IgG and IgE in the serum samples. Both major and minor determinants were appropriate antigens for detecting specific anti-beta-lactam IgG in immunised rabbit sera. In a cohort of 37 allergic patients, we observed that only the minor determinants (-llanyl antigens) were suitable for determining specific anti-beta-lactam IgE antibodies with high sensitivity (<0.01 IU/mL; 24 ng/L) and specificity (100%). These findings reveal that not only the haptenisation of beta-lactam antibiotics renders improved molecular recognition events when the 4-member beta-lactam ring remains unmodified, but also may contribute to develop promising minor antigens suitable for detecting specific IgE-mediated allergic reactions. This will facilitate the development of sensitive and selective multiplexed in vitro tests for drug-allergy diagnoses to antibiotics cephalosporin, carbapenem and monobactam.This work has been funded by the H2020 programme (Project COBIOPHAD, grant agreement No. 688448), and is an initiative of the Photonics Public Private Partnership (www.photonics21.org); Agencia Estatal de Investigacion (CTQ2016-75749-R, FEDER) and programme UPV-LA FE 2019 (PI05 VALBIOAL). The authors acknowledge the Instituto de Investigacion Sanitaria La Fe, Valencia, Spain, which provided the samples from both allergic patients and controls. E.P.M acknowledges an FPU grant from the Ministerio de Educacion, Cultura y Deporte.Peña-Mendizábal, E.; Morais, S.; Maquieira Catala, Á. (2020). Neo-antigens for the serological diagnosis of IgE-mediated drug allergic reactions to antibiotics cephalosporin, carbapenem and monobactam. Scientific Reports. 10(1):1-12. https://doi.org/10.1038/s41598-020-73109-wS112101Klein, E. Y. et al. Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc. Natl. Acad. Sci. U.S.A. 115, E3463–E3470 (2018).Torres, M. J. et al. Controversies in drug allergy: Beta-lactam hypersensitivity testing. J. Allergy Clin. Immunol. Pract. 7, 40–45 (2019).Fontaine, C. et al. Relevance of the determination of serum-specific IgE antibodies in the diagnosis of immediate β-lactam allergy. Allergy Eur. J. Allergy Clin. Immunol. 62, 47–52 (2007).Romano, A. & Gue, R. Cross-reactivity and tolerability of cephalosporins in patients with immediate hypersensitivity to penicillins. Ann. Intern. Med. 141, 16–22 (2004).Caimmi, S., Bousquet-rouanet, L. & Arnoux, B. Safety of cefuroxime as an alternative in patients with a proven hypersensitivity to penicillins: A DAHD cohort survey. Int. Arch. Allergy Immunol. 153, 53–60 (2010).Chandel, A. K., Rao, L. V., Narasu, M. L. & Singh, O. V. The realm of penicillin G acylase in β-lactam antibiotics. Enzyme Microb. Technol. 42, 199–207 (2008).Gruchalla, R. S. Drug allergy. J. Allergy Clin. Immunol. 111, 548–559 (2003).Warrington, R. & Silviu-dan, F. Drug allergy. Allergy Asthma Clin. Immunol. 7, S10 (2011).Page, M. G. P. Beta-lactam antibiotics. In Antibiotic Discovery and Development (eds Dougherty, T. J. & Pucci, M. J.) 79–117 (Springer, Boston, 2012).World Health Organization. WHO Report on Surveillance of Antibiotic Consumption: 2016–2018 Early implementation (World Health Organization, Geneva, 2018).European Centre for Disease Prevention and Control. In Antimicrobial Consumption in the EU/EEA, Annual Epidemiological Report for 2018. (2019).Frumin, J. & Gallagher, J. C. Allergic cross-sensitivity between penicillin, carbapenem, and monobactam antibiotics: What are the chances?. Ann. Pharmacother. 43, 304–315 (2009).Baldo, B. A. Penicillins and cephalosporins as allergens—Structural aspects of recognition and cross-reactions. Clin. Exp. Allergy 29, 744–749 (1999).Adkinson, N. F., Mendelson, L. M., Ressler, C. & Keogh, J. C. Penicillin minor determinants: History and relevance for current diagnosis. Ann. Allergy Asthma Immunol. 121, 537–544 (2018).Blanca, M. et al. Side-chain-specific reactions to betalactams: 14 years later. Clin. Exp. Allergy 32, 192–197 (2002).Ho, H. P. et al. Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry. Rapid Commun. Mass Spectrom. 25, 25–32 (2011).Bousquet, P. J., Co-Minh, H. B., Arnoux, J. P. & Daures, P. J. D. Importance of mixture of minor determinants and benzylpenicilloyl poly-l-lysine skin testing in the diagnosis of b-lactam allergy. J. Allergy Clin. Immunol. 115, 1314–1316 (2005).Anderson, J. A. Allergic and allergic-like reactions to drugs and other therapeutic agents. In Current Clinical Practice: Allergic Diseases—Diagnosis and Treatment (eds Lieberman, P. & Anderson, J. A.) 295–318 (Humana Press, Totowa, 2007).Romano, A. et al. Cross-reactivity and tolerability of aztreonam and cephalosporins in subjects with a T cell-mediated hypersensitivity to penicillins. J. Allergy Clin. Immunol. 138, 179–186 (2016).Geng, B., Eastman, J. J., Mori, K., Braskett, M. & Riedl, M. A. Utility of minor determinants for skin testing in inpatient penicillin allergy evaluation. Ann. Allergy Asthma Immunol. 119, 258–261 (2017).Atanasković-Marković, M. et al. Tolerability of meropenem in children with IgE-mediated hypersensitivity to penicillins. Allergy Eur. J. Allergy Clin. Immunol. 63, 237–240 (2008).Demoly, P. & Romano, A. Update on beta-lactam allergy diagnosis. Curr. Allergy Asthma Rep. 5, 9–14 (2005).Doña, I., Torres, M. J., Montañez, M. I. & Fernández, T. D. In vitro diagnostic testing for antibiotic allergy. Allergy Asthma Immunol. Res. 9, 288–298 (2017).Marwood, J., Aguirrebarrena, G., Kerr, S., Welch, S. A. & Rimmer, J. De-labelling self-reported penicillin allergy within the emergency department through the use of skin tests and oral drug provocation testing. Emerg. Med. Australas. 29, 509–515 (2017).Baldo, B. A., Zhao, Z. & Pham, N. H. Structural determinants of antibiotic allergy. Curr. Allergy Rep. 1, 23–31 (2001).Hermanson, G. T. Bioconjugate Techniques (Academic Press, Cambridge, 2013).Pham, N. H. & Baldo, B. A. β-lactam drug allergens: Fine structural recognition patterns of cephalosporin-reactive IgE antibodies. J. Mol. Recogn. 9, 287–296 (2002).Zhao, Z., Batley, M., D’Ambrosio, C. & Baldo, B. A. In vitro reactivity of penicilloyl and penicillanyl albumin and polylysine conjugates with IgE-antibody. J. Immunol. Methods 242, 43–51 (2000).Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).Wang, Y., He, C. H., Zheng, H. & Zhang, H. B. Characterization and comparison of fumonisin B1-protein conjugates by six methods. Int. J. Mol. Sci. 13, 84–96 (2012).Hawkes, R., Niday, E. & Gordon, J. A dot-lmmunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142–147 (1982).Badran, A. A., Morais, S. & Maquieira, Á. Simultaneous determination of four food allergens using compact disc immunoassaying technology. Anal. Bioanal. Chem. 409, 2261–2268 (2017).Dobosz, P., Morais, S., Bonet, E., Puchades, R. & Maquieira, Á. Massive immuno multiresidue screening of water pollutants. Anal. Chem. 87, 9817–9824 (2015).Belliveau, P. P. Omalizumab: a monoclonal anti-IgE antibody. MedGenMed 7, 27 (2005).Zhang, Z. et al. Enhanced sensitivity of capture IgE-ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens. Mol. Med. Rep. 19, 3497–3504 (2019).Baldo, B. A., Zhao, Z. & Pham, N. H. Antibiotic allergy: Immunochemical and clinical considerations. Curr. Allergy Asthma Rep. 8, 49–55 (2008).Takeuchi, Y., Inoue, T. & Sunagawa, M. Studies on the structures of meropenem (SM-7338) and it’s primary metabolite. J. Antibiotech. (Tokyo) 46, 827–832 (1992).Lambert, D. G. Drugs and receptors. Contin. Educ. Anaesth. Crit. Care Pain 4, 181–184 (2004).Torres, M. J. & Blanca, M. Diagnosis of immediate allergic reactions to beta-lactam antibiotics. Allergy 58, 961–972 (2003).Van Boeckel, T. P. et al. Global antibiotic consumption 2000 to 2010: An analysis of national pharmaceutical sales data. Lancet Infect. Dis. 14, 742–750 (2014).Gueant, J.-L., Gueant-Rodriguez, R.-M., Viola, M., Luigi Valluzzi, R. & Romano, A. IgE-mediated hypersensitivity to cephalosporins. Curr. Pharm. Des. 12, 3335–3345 (2006).Har, D. & Solensky, R. Penicillin and beta-lactam hypersensitivity. Immunol. Allergy Clin. N. Am. 37, 643–662 (2017).Romano, A., Gaeta, F., Poves, M. F. A. & Valluzzi, R. L. Cross-reactivity among beta-lactams. Curr. Allergy Asthma Rep. 16, 1–12 (2016).Madaan, A. & Li, J. T. C. Cephalosporin allergy. Immunol. Allergy Clin. N. Am. 24, 463–476 (2004).Lemke, T. L., Williams, D. A., Roche, V. F. & Zito, S. W. Foye’s Principles of Medicinal Chemistry (Lippincott Williams & Wilkins, Philadelphia, 2013).Blanca-Lopez, N. et al. Allergic reactions to penicillins and cephalosporins: Diagnosis, assessment of cross-reactivity and management. Expert Rev. Clin. Immunol. 15, 707–721 (2019)

Surface coupling of oligo-functionalized dendrimers to detect DNA mutations after blocked isothermal amplification

[EN] This study presents a useful strategy for the bioconjugation of probes for developing high-performance versatile chips applied to fast-response, low-cost DNA biosensing. We herein demonstrate that the high-density immobilization of dendrimer-oligonucleotide hybrids promotes the reliable sensitive sensing of single-nucleotide variants despite their low concentration. Carboxyl-terminated poly(amidoamine) dendrimers directly coupled to amine-derivatized oligonucleotides were anchored to the activated surfaces of thermoplastics (polycarbonate and cycloolefin polymer). Surface characterization techniques and hybridization assays reported that the multiple functional sites of the oligofunctionalized dendrimer facilitated the efficient immobilization of probes and the sensitive capture of DNA targets (5 pM detection limit). Array performance was better than that of the surfaces functionalized with linear crosslinkers, such as vinyltriethoxysilane or 3-(triethoxysilyl)propyl isocyanate, as well films with unconjugated dendrimer molecules. Based on oligo-dendrimers hybrids, disposable DNA-based biosensing platforms were developed for point-ofcare diagnostics. A selective high-sensitive hybridization assay was performed that included amplification products of blocked PCR and recombinase polymerase amplification (RPA) as an isothermal reaction. The chip results indicated that the single-nucleotide mutant variant of the BRAF oncogene was correctly discriminated in colorectal tissues from cancer patients.The authors acknowledge the financial support received from EU FEDER, the Spanish Ministry of Economy and Competitiveness (PID2019-110713RB-I00 and Technical Support Personnel PTA-2016) and Generalitat Valenciana (PROMETEO/2020/094).Martorell, S.; Tortajada-Genaro, LA.; González Martínez, MÁ.; Maquieira Catala, Á. (2021). Surface coupling of oligo-functionalized dendrimers to detect DNA mutations after blocked isothermal amplification. Microchemical Journal. 169:1-10. https://doi.org/10.1016/j.microc.2021.106546S11016

Chemical Surface Modifications development of Silicon Based Label Free Integrated Optical (IO) Biosensors: A Review

[EN] Increasing interest has been paid to label-free biosensors in recent years. 11 Among them, refractive index (RI) optical biosensors enable high density and the chip-12 scale integration of optical components. This makes them more appealing to help 13 develop lab-on-a-chip devices. Today, many RI integrated optical (IO) devices are made 14 using silicon-based materials. A key issue in their development is the 15 biofunctionalization of sensing surfaces because they provide a specific, sensitive 16 response to the analyte of interest. This review critically discusses the 17 biofunctionalization procedures, assay formats and characterization techniques 18 employed in setting up IO biosensors. In addition, it provides the most relevant results 19 obtained from using these devices for real sample biosensing. Finally, an overview of 20 the most promising future developments in the fields of chemical surface modification 21 and capture agent attachment for IO biosensors follows.This research has been supported by the Spanish Ministry of Science and Innovation through project CTQ2010-15943/BQU and by the Regional Valencian Government, through GVA/PROMETEO 2010/08. The authors thank Dr. Miguel Holgado, from the Universidad Politecnica de Madrid, for his helpful discussion about the classification of RI optical sensors.Bañuls Polo, MJ.; Puchades Pla, R.; Maquieira Catala, Á. (2013). Chemical Surface Modifications development of Silicon Based Label Free Integrated Optical (IO) Biosensors: A Review. Analytica Chimica Acta. 777:1-16. https://doi.org/10.1016/j.aca.2013.01.025S11677

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. 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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. 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Fluor-thiol Photocoupling Reaction for Developing High Performance Nucleic Acid (NA) Microarrays

Spatially controlled anchoring of NA probes onto microscope glass slides by a novel fluor-thiol coupling reaction is performed. By this UV-initiated reaction, covalent immobilization in very short times (30 s at 254 nm) is achieved with probe densities of up to 39.6 pmol/cm2. Modulating the surface hydrophobicity by combining a hydrophobic silane and a hydrophilic silane allows the fabrication of tuned surfaces where the analyte approaches only the anchored probe, which notably reduces nonspecific adsorption and the background. The generated substrates have proven clear advantages for discriminating single-base-pair mismatches, and for detecting bacterial PCR products. The hybridization sensitivity achieved by these high-performance surfaces is about 1.7 pM. Finally, this anchoring reaction is demonstrated using two additional surfaces: polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) membranes. This provides a very interesting pathway for anchoring thiolated biomolecules onto surfaces with C–F motifs via a quick clean UV reaction.Financial support from INTERBOINTER (project CTQ2013-45875-R) and BIHOLOG (Project CTQ2016-75749-R), FEDER, and GVA PROMETEO II 2014/040 is acknowledged. P.J.-M. acknowledges the Spanish Ministry of Economy, Industry and Competitiveness for the public FPI grant (Project CTQ2013-45875-R) and the cofinancing by the European Social Fund

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

Site-specific immobilization of DNA on silicon surfaces by using the thiol-yne reaction

[EN] Covalent immobilization of ssDNA fragments onto silicon-based materials was performed using the thiol-yne reaction. Chemical functionalization provided alkyne groups on the surface where the thiol-modified oligonucleotide probes can be easily photoattached as microarrays, reaching an immobilization density around 30 pmol cm(-2). The developed method presents the advantages of spatially controlled probe anchoring (by using a photomask), direct attachment without using cross-linkers, and short irradiation times (20 min). Hybridization efficiencies up to 70%, with full complementary strands, were reached. The approach was evaluated by scoring single nucleotide polymorphisms with a discrimination ratio around 15. Moreover, the potential applicability of the proposed methodology is demonstrated through the specific detection of 20 nM of a genomic target of bacterial Escherichia coli.This research was supported by Ministerio de Ciencia e Innovacion (CTQ2013-45875-R) and Generalitat Valenciana (PROMETEO/2010/008).Escorihuela Fuentes, J.; Bañuls Polo, M.; Puchades, R.; Maquieira Catala, Á. (2014). Site-specific immobilization of DNA on silicon surfaces by using the thiol-yne reaction. Journal of Materials Chemistry B. 2(48):8510-8517. https://doi.org/10.1039/c4tb01108bS8510851724