Identification and Biosensing Application of Molecular Recognition Elements

Molecular recognition elements (MREs) are biomolecules such as single-stranded DNA (ssDNA), RNA, small peptides and antibody fragments that can bind to user defined targets with high affinities and specificities. This binding property allows MREs to have a wide range of applications, including therapeutic, diagnostic, and biosensor applications. The identification of MREs can be achieved by using the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This process begins with a large library of 109 to 1015 different random molecules, molecules that bind to the user defined target or positive target are enriched in the process. Subsequently, this process can be modified and tailored to direct the enriched library away from binding to related targets or negative targets, and thus increasing the specificity. Single-stranded DNA (ssDNA) MREs are particularly favorable for biosening applications due to their relative stability, reusability and low cost in production. This work investigated the identification and application of ssDNA MREs to detect different bacterial toxins and pesticide.;In Chapter 1, it begins by reviewing recent discovery and advancement in the SELEX technique for the identification and biosensing application of ssDNA MREs specific for bacteria, viruses, their related biomolecules, and selected environmental toxins. It is then followed by a brief discussion on major biosensing principles based upon ssDNA MREs. In Chapter 2, the pilot project of this work, ssDNA MRE specific for Pseudomonas aeruginosa exotoxin A was identified. In this chapter, a novel variation of SELEX called Decoy-SELEX, previously developed by our laboratory is described in greater detail. Additionally, the development of a ssDNA MRE modified enzyme-linked immunosorbent assay (ELISA) for the exotoxin A detection is also discussed. In Chapter 3, similar methodology was applied to identify a ssDNA MRE specific for the second target, Clostridium difficile toxin B. Subsequently, similar ssDNA MRE modified ELISA was developed for target detection in clinically relevant samples. In Chapter 4, ssDNA MRE specific for alpha toxin of Staphylococcus aureus was identified, and it was applied for sensitive detection of the target in clinically relevant samples. In Chapter 5, the overall conclusion and potential future studies as a result from this work is discussed. Lastly, in Appendix, the project of identifying and potential future application of ssDNA MREs specific for a pesticide, Fipronil is described.;Overall, this work has shown the proof-of-principle of using ssDNA MREs in biosensing application for target detections in clinically relevant samples. The work will be useful in the development of potential point-of-care diagnostic tools for rapid diagnosis of bacterial infections

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

Bioconjugated nanomaterials for monitoring food contamination

Maintaining food safety and hygiene standards is top priority and challenge for farmers, food industries, governments and food technologists working in the food supply chain. Pesticides, toxins, veterinary drug residues, foodborne pathogens and many other harmful chemicals that may be present in a vast array of food products, due to various stages of their production like packaging and transport, constitute a global health problem that requires powerful and innovative technologies allowing constant and accurate detection of food products from production to consumption. Recent progress in generation of specific synthetic oligonucleotides against food contaminants has provided a new insight into the current sensor technologies, where these functional synthetic oligonucleotides, so-called aptamers, have been successfully combined with nanomaterials for rapid and cost-effective detection of several substances related to the food contamination, such as antibiotics, mycotoxins, heavy metals, carcinogenic dyes, pesticides, pathogens and other plastic products used for food packaging. Unique characteristics of aptamers over antibodies, such as in vitro selection, chemical and thermal stability, small size and ease of labeling have laid the solid foundation for exploring aptamers further in multiplexed food monitoring systems. In this chapter, we reviewed the application of aptamer-conjugated nanomaterials in food safety surveillance as well as the conventional techniques used for food safety monitoring in order to provide a comprehensive and comparative approach

Aptamers for Diagnostics with Applications for Infectious Diseases

Aptamers are in vitro selected oligonucleotides (DNA, RNA, oligos with modified nucleotides) that can have high affinity and specificity for a broad range of potential targets with high affinity and specificity. Here we focus on their applications as biosensors in the diagnostic field, although they can also be used as therapeutic agents. A small number of peptide aptamers have also been identified. In analytical settings, aptamers have the potential to extend the limit of current techniques as they offer many advantages over antibodies and can be used for real-time biomarker detection, cancer clinical testing, and detection of infectious microorganisms and viruses. Once optimized and validated, aptasensor technologies are expected to be highly beneficial to clinicians by providing a larger range and more rapid output of diagnostic readings than current technologies and support personalized medicine and faster implementation of optimal treatments

Rapid and Sensitive Detection of Escherichia coli O157:H7 Using a QCM Sensor based on Aptamers Selected by Whole-Bacterium SELEX and a Multivalent Aptamer System

Escherichia coli O157:H7 is one of the top five pathogens contributing to foodborne diseases, causing an estimated 2,138 cases of hospitalization in the US each year. The extremely low infectious dose demands for more rapid and sensitive methods to detect E. coli O157:H7. The objective of this study is to select aptamers specifically binding to E. coli O157:H7 using whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and to create a multivalent aptamer system by rolling circle amplification (RCA) with the selected aptamer sequence for sensitive detection of E. coli O157:H7 using a quartz crystal microbalance (QCM) sensor. Briefly, A total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of counter selection were performed for SELEX. One sequence S1 that appeared 16 (out of 20) times was characterized and a dissociation constant (Kd) of 10.30 nM was obtained. Using phi29 DNA polymerase, RCA reaction was performed, which produced a long ssDNA strand composed of thousands of repetitive aptamer sequences, termed as a multivalent aptamer system, on the electrode. The QCM sensor based on a multivalent aptamer system was able to quantitatively detect E. coli O157:H7. The limit of detection (LOD) of the QCM sensor was determined to be 34 CFU/ml, respectively, with the whole detection procedure in less than 40 min. The QCM sensor also showed high specificity for E. coli O157:H7 when it was cross-tested with five non-target bacteria. The QCM aptasensor in this study provided a common platform for detection of different foodborne pathogens

Optical methods for ultrafast screening of microorganisms

En aquesta tesi doctoral hem desenvolupat un mètode per la detecció i quantificació múltiple dels microorganismes més comuns que causen infeccions bacterianes amb una velocitat de detecció sense precedents a baix cost i alta sensibilitat. A més a més, fent servir fluids humans reals directament evitant així, els pretractaments tediosos de les mostres. El disseny del sistema està basat en augments d'intensitat del senyal obtingut per espectroscòpia SERS. Això s'aconsegueix utilitzant nanopartícules plasmòniques codificades i funcionalitzades amb elements de reconeixement biològics. D'aquesta manera, quan una mostra conté el patogen a identificar interactua amb els elements de reconeixement units a les nanopartícules, induint la seva acumulació en la superfície del microorganisme. Aquesta agregació de partícules a la membrana dels bacteris produeix espais molt petits entre les partícules fent que el senyal Raman s'amplifiqui en diversos ordres de magnitud respecte a les partícules soltes. Permetent així, la identificació de múltiples microorganismes a la vegada. La quantificació d'aquests, s'aconsegueix passant la mostra a través d'un dispositiu de micro-fluids amb una finestra de recol•lecció on un làser interroga i classifica els agregats a temps real. A més a més, també hem investigat els avantatges de fer servir aptàmers en lloc d'anticossos com a elements de reconeixement biològic. Aquest nou sistema de detecció de patògens obre interessants perspectives per al diagnòstic ràpid i econòmic d'infeccions bacterianes.En esta tesis doctoral hemos desarrollado un método para la detección y cuantificación múltiple de los microorganismos más comunes que causan infecciones bacterianas con una velocidad de detección sin precedentes a bajo coste y alta sensibilidad. Utilizando además, fluidos humanos reales directamente evitando así, pre-tratamientos tediosos de las muestras. El diseño del sistema está basado en aumentos de intensidad de la señal obtenida por espectroscopia SERS. Esto se logra utilizando nanopartículas plasmónicas codificadas y funcionalizadas con elementos de reconocimiento biológico. De esta manera, cuando una muestra que contiene el patógeno a identificar interactúa con los elementos de reconocimiento unidos a las nanopartículas, induce su acumulación en la superficie del microorganismo. Esta agregación de partículas en las membranas de las bacterias produce espaciados muy pequeños entre las partículas haciendo que la señal Raman se amplifique en varios órdenes de magnitud con respecto a las partículas sueltas. Permitiendo así la identificación de múltiples microorganismos a la vez. La cuantificación de los mismos, se logra pasando la muestra a través de un dispositivo de micro-fluidos con una ventana de recolección donde un láser interroga y clasifica los agregados en tiempo real. Además, también hemos investigado las ventajas de usar aptámeros frente a anticuerpos como elementos de reconocimiento biológico. Este nuevo sistema de detección de patógenos abre interesantes perspectivas para el diagnóstico rápido y barato de las infecciones bacterianas.This doctoral thesis intended to develop and optimize a method for multiplex detection and quantification of the most common microorganisms causing bacterial infections. This detection approach envisions to directly use different real human fluids avoiding thus, tedious pre-treatments of the samples with an unprecedented speed, low cost, and sensitivity. The design of the system is based on variations in the SERS intensity. This is accomplished using encoded plasmonic nanoparticles functionalized with bio-recognition elements. Consequently, when a sample containing the biological target to be identified interacts with the recognition elements attached to the nanoparticle, will induce an accumulation of them at the surface of the targeted microorganism. This particle aggregation on the bacteria membranes renders a dense array of inter-particle gaps in which the Raman signal is amplified by several orders of magnitude relative to the dispersed particles, enabling a multiplexed deterministic identification of the microorganisms. Quantification is achieved by passing the sample through a microfluidic device with a collection window where a laser interrogates and classifies the bacteria–nanoparticle aggregates in real time. Additionally, a comparison between two of the most common bio-recognition elements (antibodies and aptamers) was performed. This new pathogen detection system opens exciting prospects for fast inexpensive diagnosis of bacterial infections

Bio-oligomers as antibacterial agents and strategies for bacterial detection

In this thesis I examined the potential of Bio-Oligomers such as peptoids, peptides and aptamers, as therapeutic and diagnostic entities. Therapeutic Bio-Oligomers; A series of peptoid analogs have been designed and synthesised using solid phase synthesis. These peptoids have been subjected to biological evaluation to determine structure-activity relationships that define their antimicrobial activity. In total 13 peptoids were synthesised. Out of 13 different peptoids, only one peptoid called Tosyl-Octyl-Peptoid (TOP) demonstrated significant broad-spectrum bactericidal activity. TOP kills bacteria under non-dividing and dividing conditions. The Minimum Inhibitory Concentrations (MIC) values of TOP for S. epidermidis, E. coli and Klebsiella were 20 μM, whereas Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) were 40 μM. The highest MIC values were observed for Pseudomonas aeruginosa (PAO1) at 80 μM. The selectivity ratio (SR) or Therapeutic index (TI) was calculated, by dividing the 10% haemolysis activity (5 mM) by the median of the MIC (50 μM) yielding a TI for TOP as 100. This TI is well above previously reported peptidomimetics TI of around 20. TOP demonstrates selective bacterial killing in co-culture systems and intracellular bacterial killing activity. Diagnostic Bio-Oligomers; In the second part of my thesis, I investigated aptamer and peptide-based molecular probes to detect MRSA. As well as screening aptamers and peptide probes against whole MRSA, I over-expressed and purified PBP2A protein. This purified protein was used as a target for aptamer and peptide probes to detect MRSA. Two different aptamer libraries were initially screened for utility. In-vitro conditions for SELEX were optimised. Biopanning with a phage derived peptides was also performed. Target sequences for both methods were identified and chemically synthesised. Evaluation of fluorescently labelled sequences with flow cytometry and confocal imaging showed no specificity for MRSA detection with either method. The Bio-Oligomers and the in-vitro selection methodology require further refinement to improve diagnostic utility

Aptamer-based optical biosensors

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Functional nucleic acids for detecting bacteria

The final publication is available from De Gruyter through http://dx.doi.org/10.1515/revac-2012-0027Bacterial infection represents one of the leading causes of disease and death, and as such, bacterial detection is an important step in managing infectious diseases. The current protocol requires growing cell cultures, which can take several days. Fast detection of low copies of bacterial cells has thus posed an analytical challenge. Among the new strategies developed to achieve this goal, functional nucleic acids (FNAs) have emerged to be a promising platform. FNAs include DNAzymes, aptamers, and aptazymes, all of which can recognize analytes other than complementary nucleic acids. FNAs are obtained using a combinatorial biology technique called systematic evolution of ligands by exponential enrichment (SELEX). FNAs have been isolated against not only purified proteins and surface markers from bacterial cells but also whole cells. A diverse range of signaling mechanisms including fluorescence, color, and electrochemistry-based detection has been reported. Although the majority of current sensors cannot achieve single-cell sensitivity, with improved combinatorial selection techniques and the incorporation of nanomaterials to realize multivalent binding and signal amplification, FNAs represent a feasible solution for bacterial detection.Canadian Institutes of Health Researc

Isolation of DNA Aptamers for Enteropathogenic Escherichia coli (EPEC) Detection using Bacterial-SELEX Approach

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative pathogenic bacterium that causes diarrheal disease, especially in infants and children. Aptamers are short chain oligonucleotides that have high affinity, specificity, and selectivity to their targets, which have potential to be developed as a method for diagnosing pathogens. In this study, aptamer was isolated through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method using whole cells bacteria (Bacterial-SELEX) for recognizing pathogenic E. coli EPEC K1.1 which was isolated from children with diarrhea in Indonesia. Ten rounds of bacterial-SELEX procedure were conducted with modification conditions by using Top10, DH5a E. coli cells, Listeria monocytogenes, and Lactobacillus plantarum S34 as counter-selections. The selection process was started with a pool of ssDNA random library consisting of a random base with 40-nucleotides long flanked with fixed primers sequence for aptamer amplification purpose. Short single-stranded DNA amplification was done by symmetric and asymmetric PCR. The highly enriched oligonucleotide pools (pooled 8, 9, and 10) were cloned and the resulting ssDNA aptamers were identified by Sanger DNA sequencing. Finally, twelve aptamers with unique sequences and various secondary structures including G-quadruplex sequence motif within aptamers were obtained as candidates specific aptamer for detection and capturing of EPEC K1.1