Novel biorecognition elements against pathogens in the design of state-of-the-art diagnostics

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020.info:eu-repo/semantics/publishedVersio

Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and of LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and of LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Electrochemical aptasensor for the detection of the key virulence factor YadA of Yersinia enterocolitica

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and of LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Projeto de materiais multifuncionais para capturar patogénicos de amostras biológicas

Tese de doutoramento em Chemical and Biological EngineeringDiagnosis of bacterial infections is primarily accomplished by culture and laboratory analysis, immunological methods and nucleic-acid amplification techniques. However, these methods have limitations related to affordability, accessibility and time to inform a result. Moreover, the emergence of antibiotic resistant bacteria comprises a global threat to health, strengthening the relevance of early detection of pathogens to provide adequate and timely treatments. To address these concerns, point-ofcare (POC) diagnostic platforms arose as a solution to deliver affordable, equipment-free and rapid accurate testing especially in resource-limited settings. The combination of cell-SELEX with highthroughput sequencing and bioinformatic analysis led to the identification of a novel ssDNA aptamer sequence (Apt1YadA) with high affinity and selectivity towards the adhesin YadA from Yersinia enterocolitica. An electrochemical aptasensor POC platform was assembled using Apt1YadA on a gold screen printed electrode which successfully detected recombinant YadA. This was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and square wave voltammetry techniques, attaining a limit of detection of 7.0 × 104 CFU mL−1. The selectivity of Apt1YadA was validated in the presence of non-target bacteria. A novel aptamer Apt1_RCUspA2 specifically targeting UspA2 adhesin from Moraxella catarrhalis was also herein identified using a similar workflow. Apt1_RCUspA2 was characterized, demonstrating high affinity and selectivity towards recombinant UspA2. Likewise, an electrochemical aptasensor assembled using Apt1_RCUspA2 successfully detected UspA2 with a limit of detection of 4.0 × 104 CFU mL−1. To further enhance the sensitivity of biosensors, a previous enrichment step of samples, is envisaged. Since both YadA and UspA2 have an affinity for collagen, magnetic nanoparticles (MNPs) functionalized with hydrolyzed collagen were synthesized by a quick, green co-precipitation method and were characterized by ATR-FTIR. The collagen MNPs specifically interacted with YadA and UspA2, with capture efficacies of 94.7 ± 5.2% and 87.4 ± 4.7%, respectively, providing a viable and efficient tool for bacterial capture. The results gathered in this thesis contributed to the goal of developing novel POC platforms and diagnostic workflows for pathogenic biomarkers (YadA and UspA2). Moreover, the innovative workflows developed, can serve as a prototype for developing similar POC platforms for other bacterial pathogens.O diagnóstico de infeções bacterianas recorre principalmente a métodos de cultura e análise laboratorial, métodos imunológicos e técnicas de amplificação de ácidos nucleicos. No entanto, estes métodos possuem limitações que se prendem com os seus custos, acessibilidade, e rapidez de resposta. Adicionalmente, o surgimento de bactérias resistentes a antibióticos representa uma ameaça global à saúde, reforçando a relevância da deteção precoce de organismos patogénicos para possibilitar a escolha de tratamentos adequados, atempados e oportunos. As plataformas de diagnóstico rápido (em inglês point-of-care (POC)) surgiram assim como uma solução para fornecer testes precisos, rápidos e acessíveis, sem equipamentos, particularmente relevantes em ambientes com recursos limitados. A combinação da técnica de cell-SELEX com sequenciação em escala e análise bioinformática possibilitou a identificação de um aptâmero novo de ssDNA (Apt1YadA) com alta afinidade e seletividade para a adesina YadA da Yersinia enterocolitica. Foi então desenvolvido um aptasensor eletroquímico usando o Apt1YadA num elétrodo impresso em folha de ouro que permitiu detetar com sucesso a adesina YadA. A deteção desta adesina foi confirmada por voltametria cíclica, espectroscopia de impedância eletroquímica e técnicas de voltametria de onda quadrada atingindo-se um limite de deteção de 7.0 × 104 UFC mL−1. A seletividade de Apt1YadA foi validada também na presença de bactérias não-alvo. De forma similar, foi selecionado um aptâmero Apt1_RCUspA2 visando especificamente a adesina UspA2 de Moraxella catarrhalis. Este aptâmero Apt1_RCUspA2 foi caracterizado, demonstrando alta afinidade e seletividade para a adesina UspA2. Desenvolveu-se então um aptasensor eletroquímico com o Apt1_RCUspA2 que permite detetar com sucesso a adesina com um limite de deteção de 4.0 × 104 UFC mL−1. Como estratégia para incrementar a resposta dos biossensores, é possível ter etapas prévias de enriquecimento das amostras. Tendo em conta que ambas as adesinas, YadA e UspA2, têm afinidade para o colagénio, usou-se colagénio hidrolisado para funcionalizar nanopartículas magnéticas (MNPs) sintetizadas por um método de co-precipitação rápida e verde. As MNPs modificadas foram caracterizadas por ATR-FTIR e verificou-se que as MNPs interagem especificamente com YadA e UspA2, com uma eficácia de captura de 94.7 ± 5.2% e 87.4 ± 4.7%, respetivamente e constituem uma ferramenta viável e eficiente para capturar bactérias de uma amostra mista. Os resultados reunidos nesta dissertação contribuíram para o seu objetivo global de desenvolver novos métodos de diagnóstico rápidos focados em biomarcadores específicos de organismos patogénicos (YadA e UspA2). Adicionalmente, o fluxo de trabalho e racional inovadores que foram aqui desenvolvidos podem no futuro servir como protótipo para o desenvolvimento de biossensores similares para outros organismos patogénicos.I would like to acknowledge my fellowship, the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. I also acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit

Selection and validation of DNA aptamers against Yersinia Enterocolitica and Moraxella Catarrhalis adhesins

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DNA aptamers towards Yersinia enterocolitica and Moraxella catarrhalis adhesins in novel POC diagnostics

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Design of new hydrolyzed collagen-modified magnetic nanoparticles to capture pathogens

Enrichment and diagnosis tools for pathogens currently available are time consuming, thus the development of fast and highly sensitive alternatives is desirable. In this study, a novel approach was described that enables selective capture of bacteria expressing hydrolyzed collagen-binding adhesins with hydrolyzed collagen-coated magnetic nanoparticles (MNPs). This platform could be useful to shorten the time needed to confirm the presence of a bacterial infection. MNPs were synthesized by a simple two-step approach through a green co-precipitation method using water as solvent. These MNPs were specifically designed to interact with pathogenic bacteria by establishing a hydrolyzed collagen-adhesin linker. The bacterial capture efficacy of hydrolyzed collagen MNPs (H-CollThe study received financial support from ViBrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie, Grant agreement no. 765042 and Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. DL and AS receive additional funding from the Research Council of Norway (grant no. 294605, Center for Digital Life). The authors acknowledge Diana Vilas Boas from Centre of Biological Engineering for technical assistance in confocal scanning laser microscopy. The TEM experiments were carried out at the INL Advanced Electron Microscopy, Imaging and Spectroscopy Facility.info:eu-repo/semantics/publishedVersio